[Advances in Biochemical Engineering_Biotechnology 32] N. Kosaric, A. Wieczorek, G. P. Cosentino (Auth.) - Agricultural Feedstock and Waste Treatment and Engineering (1985, Springer-Verlag Berlin Heidelberg)

Advances in Biochemical Engineering/
32 Biotechnology
Managing Editor= A. Fiechter

Agricultural Feedstock
and Waste Treatment
and Engineering
With Contributions by
Y. J. Asher, G. P. Cosentino, Z. Duvnjak
N. Kosaric, H. C. Lim, R. Luttmann,
R. J. Magee, A. Munack, S. J. Parulekar,
M. Thoma, A. Wieczorek
With 65 Figures and 53 Tables
Springer-Verlag
Berlin Heidelberg New York Tokyo
1985
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I S B N 0-387-15490-6 S p r i n g e r - V e r l a g N e w Y o r k H e i d e l b e r g B e r l i n T o k y o
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Institut fiir Biotechnologie
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-
CH-8093 Ztirich
Editorial Board
Prof. Dr. S. Aiba Department of Fermentation Technology, Faculty of

Engineering, Osaka University, Yamada-Kami, Suita-
Shi, Osaka 565, Japan
Prof. Dr. H. R. Bungoy Rensselaer Polytechnic Institute,
Dept. of Chem. and Environmental Engineering,
Troy, NY 12181/USA
Prof. Dr. Ch. L. Cooney Massachusetts Institute of Technology,
Department of Chemical Engineering,
Cambridge, Massachusetts 02139/.USA
Prof. Dr. A. L. Demain Massachusetts Institute of Technology, Dept. of
Nutrition & Food Sc., Room 56-125,
Cambridge, Massachusetts 02139/USA
Prof. Dr. S. Fukui Dept. of Industrial Chemistry, Faculty of
Engineering, Sakyo-Ku, Kyoto 606, Japan
Prof. Dr. K. Kieslich Gesellschaft ftir Biotechnologie
Forschung mbH, Mascheroder Weg 1,
D-3300 Braunschweig
Prof. Dr. A. M. Klibanov Massachusetts Institute of Technology Dept. of Applied
Biological Sciences Cambridge, Massachusetts 02139/USA
Prof. Dr. R. M. Lafferty Techn. Hochschule Graz, Institut f~ir
Biochem. Technol., Schl6gelgasse 9, A-8010 Graz
Prof. Dr. B. S. Montenecourt Lehigh University, Biolog. and
Biotechnology Research Center,
Bethlehem, PA 18015/USA
Prof. Dr. H. J. Rehm Westf. Wilhelms Universit~tt, Institut fiir
Mikrobiologie, TibusstraBe 7-15, D-4400 Miinster
Prof. Dr. P. L. Rogers School of Biological Technology, The University
of New South Wales. PO Box 1,
Kensington, New South Wales, Australia 2033
Prof. Dr. H. Sahm Institut ftir Biotechnotogie, Kernforsehungsanlage
Jiilich, D-5170 Jiilich
Prof. Dr. K. Schiigerl Institut fiir Technische Chemie, Universifftt Haonover,
CallinstraBe 3, D-3000 Hannover
Prof. Dr. S. Suzuki Tokyo Institute of Technology,
Nagatsuta Campus, Research Laboratory of Resources
Utilization,
4259, Nagatsuta, Midori-ku, Yokohama 227/Japan
Prof. Dr. H.Taguchi Faculty of Engineering, Osaka University, Yamada-kami,
Suita-shi, Osaka 565/Japan
Prof. Dr. G. T. Tsao Director, Lab. of Renewable Resources Eng., A. A. Potter
Eng. Center, Purdue University, West Lafayette,
IN 47907/USA
Prof. Dr. E.-L. Winnacker Universit~it Mfinchen, Institut f. Biochemie, Karlsstr. 23
D-8000 Miinchen 2
Table of Contents
Industrial Processing and Products from

the Jerusalem Artichoke
N. Kosari¢, A. W i e c z o r e k , G. P. C o s e n t i n o , Z. D u v n j a k . . . 1
The Utilization of Cheese Whey and its Components

N. K o s a r i c , Y. J. A s h e r . . . . . . . . . . . . . . . . 25
Bioconversion of Hemicellulosics
R. J. M a g e e , N. K o s a r i c . . . . . . . . . . . . . . . . 61
Mathematical Modelling, Parameter Identification and

Adaptive Control of Single Cell Protein Processes in Tower Loop
Bioreactors
R. L u t t m a n n , A. M u n a c k , M. T h o m a . . . . . . . . . . 95
Modelling, Optimization and Control of Semi-Batch Bioreactors

S. J. Parulekar, H. C. Lira . . . . . . . . . . . . . . . 207
Author Index Volumes 1-32 . . . . . . . . . . . . . . . 259

Industrial Processing and Products
from the Jerusalem Artichoke
N . K o s a r i c , A . W i e c z o r e k , G . P. C o s e n t i n o a n d Z . D u v n j a k
Biochemical and Food Engineering, The University of Western Ontario, London,
O n t a r i o , N 6 A 5B9, C a n a d a
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Food and Fodder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1 Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2 Aerial Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Ethyl Alcohol Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.l Preparation of Raw Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.l.1 Juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1.2 Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1.3 M a s h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.2 Pretreatment of Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.1 Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.2 Acid Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.3 Thermal Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.3 Ethanol Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.3.1 The Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.3.2 p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l1
3,3.3 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l1
3,3.4 Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3,3.5 Carbohydrate Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3.6 Systems of Ethanol Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3.7 Ethanol Production from the Stalks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.4 Recovery of the Ethanol from "'Beer" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.5 By-Product (Stillage) Obtained after Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.6 Energetics and Economics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.6.1 Energy Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3,6.2 Economics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4 High-Fructose Syrup Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
High biomass yields per hectare, coupled with a favourable composition give the Jerusalem artichoke
a number of important applications. It can be used as a foodstuff for h u m a n s and livestock as well as
a carbohydrate source for m a n y industrial processes. In this article, considerable attention is given to
the potential utilization of this crop for ethanol and high-fructose syrup production, qZhe methods
of raw material and carbohydrate preparation for ethanol production as well as the parameters and
systems of processing are discussed in detail. Some aspects pertaining to the food and fodder value
of the Jerusalem artichoke are also presented.
2 N. Kosaric et al.
1 Introduction
High biomass yields per hectare, coupled with a favourable composition and sub-
stantial level of carbohydrates, give the Jerusalem artichoke a number of important
applications.
The simplest and original use of this crop was as a foodstuff for humans and
livestock. However, since about the turn of the century, many other industrial uses
have been suggested and studied. The greatest extent of these applications have been
reported to be in the production of ethyl alcohol and high-fructose or pure fructose
syrups. For this reason, these three applications will be discussed in detail separately.
Since a comprehensive review of high-fructose syrup production has recently been
published 1~, the discussion of this aspect will be less extensive, and special attention
will be given to the ethyl alcohol production from this raw material.
In addition to these major uses, the Jerusalem artichoke has also been studied
as a substrate for the production of acetone and butanol 2 -4) mixture acetone-butanol-
ethanol (ABE) s 8), fodder yeast 9,101, beer i1.12), lactic acid la, 14), propionic acid 15p,
mannitol 16, 17), and pectic substances 18)
2 Food and Fodder

The original impetus in the agricultural domestication of the Jerusalem artichoke
(JA) was for its consumption by humans or farm livestock. Though less commonly
grown at present (it was primarily superseded by the potato in the middle of the 18th
Century), the continued and potential uses of this crop as raw material in various
food and agricultural industries warrants some discussion. For a detailed examination
of the compositional characteristics and development of the Jerusalem artichoke,
the reader is referred to an earlier review 19~
2.1 Tubers
Either the fleshy tubers or the fibrous tops of the JA may be used as animal feed
whereas human consumption is primarily limited to the tubers. The JA tuber is a
good source of B vitamins, pantothenic acid, potassium, and phosphorous. Tubers
also contain large quantities of trace elements such as Vitamin A, iron, and calcium.
Total amounts of protein are approximately 5 ~o (w/w) of the tuber on a dry basis.
Of special interest to the food industry is the quality of protein, and this may be quan-
titated in terms of the protein score 2o~. This value is based on the content of each
essential amino acid compared to ideal protein (egg) which is defined as 100 ~o. The
amino acid present as the lowest percentage compared to egg is most limiting and is
equivalent to the protein score. This method gives an indication of protein utilization
after absorption by the organism. The limiting essential amino acid for the artichoke
is methionine (58 ~o of that in egg), while most other amino acids are present in excess
of 100~/o (exceptions are found for phenylalanine, tyrosine, isoleucine, and leucine
which range from 80--95 ~ In comparison, the protein score for the JA is found
to be greater than, or equivalent to, most other traditional food crops (i.e., soybean,
corn, wheat flour, and beans) 2o).
Industrial Processing and Products from the Jerusalem Artichoke 3
The carbohydrate portion of the JA tuber constitutes approximately 75 o; of the

dry matter (15-20 oo of wet weight) and is composed of polyfructans (termed inulides)
which exhibit a degree of polymerization from 3 to 30 units. It is generally understood
that these long-chain carbohydrates are not readily metabolized by humans, and thus,
as a "~non-food", this crop has importance to diabetics or obese individuals. This is
because caloric intake is reduced yet protein and mineral requirements are still satisfied.
The authors of this review could find no quantitative data as to the fate of ingested
inulides within the human gastrointestinal system: however, if injected intravenously,
the inulides are excreted in large quantities by the kidneys in a short time 2t~. In any
case, free fructose is metabolized without the need for insulin, and the high content
of B vitamins in the tuber is especially desirable for restricted diets due to diabetes.
The caloric value is extremely dependent upon storage time, however, and may range
from 42 to 420 kJ per kg 22~. This compares favourably to the potato which is approx-
inaately 420 kJ per kg.
In terms of food processing, the tubers of the Jerusalem artichoke have been utilized
in the manufacture of bread sticks, cookies, macaroni and noodles 221. However,
a number of disadvantages are inherent to the use of tubers in cooking. Though
often referred to as a potato substitute, the organoleptic properties of JA tubers are
considered to be much different 231. In addition, inulides do not swell like starch, and
cooked tubers remain extremely watery. A crisp brown coat does not develop upon
frying as it does for the potato, and required cooking times are much shorter (about
10 min) after which the tubers become transparent and soggy.
Farm experiences and feeding trials have not only substantiated a high feeding
value for tubers given to hogs, dairy cows, poultry, and other livestock 24-26., but
also show that the pulp which remains after carbohydrate extraction still has high
value as fodder. This pulp varies from 2-4- ~o of total fresh weight and contains about
26~ o protein. Stauffer et al. 27 studied the composition of carbohydrate extracted
pulp for several strains of JA (Table 1). These researchers found variability in the
N D F , A D F , ADL, and ether extracts of the samples: however, CP and digestible
energy remained relatively constant (see Table 1 caption for definitions). It should
Table I. Composition of carbohydrate extracted pulp for several experimental JA accessions 27~
Accession Digestible energy % of DM

(MJ per kg)"
ADe Ether
CP b NDF c ADF a Lig extract
Morden @5 14.703 26.9 47.8 34.4 1.43 3.9

Perron 14.878 26.7 45. l 42.8 2.62 2.9
Branching 15.096 25.6 47.6 38.1 0.92 3.3
Nonbranching 14.930 25.4 50.4 38. l 0.60 3.2
Digestible energy is derived from percent digestible dry matter content determined using in vitro
digestion of pulp:
b Crude protein;
c Neutral detergent fibre (theoretically equivalent to cellulose, hemicellulose, and lignin content);
d Acid detergent fibre (theoretically equivalent to cellulose and lignin content);
" Acid detergent lignin
4 N. Kosaric et al.
be noted that the accuracy of the analytical methods used in this study is sometimes
questionable 2s~, and this m a y account for the above variations.
In ethanol production, a non-volatile fraction is obtained after distillation known
as stillage o f distillery slops. Dietrich 29) was able to increase the nutritional value of
JA stillage by treating it further with various bacteria and thus substantially increasing
the vitamin B12 content for feeding to livestock (see also Sect. 3.5).
2.2 Aerial Parts

Since the yield of green mass and digestible protein per ha for the Jerusalem artichoke
is 2-4 times higher than that found in other plants 2s), much interest has been given
to the use of aerial parts as forage.
It has been found that forage composition changes with advancing plant maturity 271
While cellulose and ash content remain relatively constant after flower buds appear,
a quantitative increase in lignin occurs between weeks 7 and 8. Protein levels decrease
continually and a significant reduction is observed following the sixth week after
budding. It must also be noted that considerable variations exist for the composition
and yield data for forage among various strains of the plant 27~.
The quality of leaves and stems for silage purposes is highly dependent upon the
time of harvest, and this probably accounts for differing reports found in literature.
The quality has been described as inferior to corn silage in some cases 22, 30~ or of
equivalent fodder value to red clover in other studies 31~. Generally, as the plant
matures, increasing lignin and decreasing protein content lowers the silage value of
the upper parts. In most cases, the forage quality may be characterized as high in
roughage but lacking palatability and can be considered as comparable to corn
stover 32.33~. Data comparing the digestibility of the organic matter (OMD) of JA
forage against other reference crops is presented in Table 2. Field trials by Kazant-
seva 36) compared the weight gained by steers fed hay plus either corn silage (24 kg
per day) or Jerusalem artichoke silage (20 kg per day). It was found that from the
ages o f 16-20 months, the average daily weight gain of the control versus the JA fed
animals was 836 and 916 g, respectively.
Table 2. Organic matter digestibility of Jerusalem artichoke forage samples

compared to other forages 34.35~
Sample ~o OMD a
JA b whole forage 60-70

(budding) leaf fraction 70
Wheat straw c 39.8
Wheat straw
(treated with high pressure steam)c 57.6
Brome-alfalfa hayr 59.6
" Organic matter digestibility defined as percentage material solubilized using sheep rumen fluid
as broad source of enzymes:
u In vitro determination of OMD;
c In vivo determination of OMD
Encouraging data has been reported by Zitmane 37,3s~ regarding the growth of
single cell protein on stem and leaf hydrolyzates of the Jerusalem artichoke. Addition
of oat flour and salt to Torula utilis propagated on leaf and hydrolyzed stem fractions
resulted in a protein + mineral + vitamin product which constituted a complete
animal feed on an equal basis with other animal protein feeds. Mice and rats were
found to grow rapidly and maintain good health on a diet of this preparation.
3 Ethyl Alcohol Production

One of the first studies which dealt with the purification, hydrolysis, and microbial
conversion of the polyfructans contained in the Jerusalem artichoke to ethanol was
undertaken by Champy and Flis in 1885 391. Subsequent development over the course
of this century led to the commercial utilization of this crop for alcohol production
in France 45-43~, Russia 44-46~, Germany 47-49~, Poland so.51), the U.S.A. 52~ and
Japan 53)
Although, at present, there is a considerable amount of literature data available
on the JA and its industrial processing, confusion remains as to the optimum condi-
tions for its use in ethanolic production. This is because a number of these publications
contain quantitative information which is unsubstantiated by experimental data, or
ethanol yields per unit mass of tubers are given without specifying their carbohydrate
content. Information on the yeast strain or yeast preparation used is also generally
lacking. As well, most authors compare different methods in the preparation of raw
material and alcohol production, but in many cases, there is no agreement presented
on the merits of techniques used. For a meaningful comparison, one should consider
differences in the varieties of JA utilized, the prevailing agricultural conditions during
growth, and the storage methods used, in addition to the various parameters studied
for ethanol production.
Four major steps which must be undertaken to produce ethanol from feedstocks
are as follows:
A. Preparation of raw material.
B. Pretreatment of carbohydrates.
C. Ethanol production.
D. Recovery of the ethanol from beer.
In each type of ethanol production process using the JA, success depends on the
efficiency of the preliminary steps such as preparation of raw material and pretreat-
ment of carbohydrates. However, these steps are difficult to examine separately
in their effect on the overall process since they are highly dependent upon each other
and the method of tuber storage used. Thus, to aid in this discussion, the steps may
be further subdivided as follows.
Considering the preparation of raw material, the ethanol production can be per-
formed using:
1. The juice obtained by diffusion or expression (with or without maceration).
2. The pulp-ground tubers (with or without additional water).
3. The magh obtained by cooking the tubers (ground or whole).
6 N. Kosaricet al.
Taking into account conversion of the carbohydrate to forms easily utilized by the
microorganisms, three basic methods can be considered:
1. Enzymatic hydrolysis
a) by enzymes contained in the tubers
b) by enzymes produced by molds or yeasts
2. Acid hydrolysis
3. Thermal hydrolysis (under high pressure)
It is important to note that non-hydrolyzed polysaccharides from the JA may be
utilized directly by selected yeasts which exhibit inulase activity (an enzyme group
capable of inulide hydrolysis). This aspect will also be discussed.
3.1 Preparation of Raw Material
3.1.1 Juice
The carbohydrates from the Jerusalem artichoke may be obtained by expressing s4-591
or by diffusion ~s-63) using the same equipment as for sugar manufacture. Adding
water to ground or sliced tubers and macerating prior to each step of extraction can
improve the extraction efficiency. Countercurrent extraction or diffusion may also
be utilized to improve the diffusion efficiency and reduce the water requirement for
complete extraction.
It is reported that the rate of extraction increases with increasing temperature up
to about 60 ~ but no differences were observed with higher temperatures 11. This
temperature is also significant since the activity of [3-fructofuranosidases (inulases
which are naturally present in the tubers) is still considerable at 60 ~ but these
enzymes lose activity at higher temperatures 64)
Acidified water may be used for extraction or diffusion to improve the efficiency
of the process 46). Utilizing an atmosphere of sulfur dioxide will lower the pH of the
water, and as well, this method will prevent contamination ssl. It is important to note
that the water solubility of oligo- and polyfructans decreases with an increasing
degree of polymerization. Therefore, maturity and storage conditions of the JA
tubers will influence the extractability of the fructans.
3.1.2 Pulp
After being ground, the raw pulp can be fermented directly or after conversion of the
carbohydrates to a form easily consumed by the yeast.
Due to the physical consistency of this pulp, difficulty with the removal of CO2
has been observed which illicits a detrimental effect on microbial processing 52~.
This same problem also causes explosive gas disgorgement as reported by Williams
and Ziobro 65~. In addition, heat removal may be difficult on a large scale.
Special methods of agitation will probably be required to optimize CO2 removal,
heat transfer, and mass transfer of nutrients for the use of raw pulp in ethanol produc-
tion.
3.1.3 Mash
Cooking the tubers is primarily performed so as to hydrolyze the long-chain carbo-
hydrates. During the process of cooking, however, it is important to note that cell
rupture is extensive and non-soluble matter (such as cellulosic material) is suspended

in the liquid phase. Problems in the pumping of this mash to bioreactors and sub-
sequently to the distillation columns on a commercial scale have been reported 5o. 5~, 66)
Wlodarczyk 5~) has found that this non-soluble material (mostly the skin of tubers)
makes up 2.5M.0 ~ of total tuber mass depending on the temperature used for cooking.
This material should be removed from the mash prior to further processing.
3.2 Pretreatment of Carbohydrates
3.2.1 Enzymatic Hydrolysis
Hydrolysis by Inulases Present in Tubers

As previously stated, Jerusalem artichoke tubers contain native inulases which may
be used to break down poly- and oligofructans. This simple technique for carbo-
hydrate preparation was used and recommended by many investigators 47,48, 54.
6'7 - 7 5 )
In this method, the raw pulp obtained after grinding is heated for one or two hours
at 55:-56 ~ after which it is cooled and subsequently inoculated with yeast. This
same technique can also be effectively used for JA juice. Rfidiger 481 and Windisch 47>
reported that the ethanol yields obtained by this method were 10-15 ~o better than
those achieved using thermal hydrolysis (see Sect. 3.2.3).
Different conditions for optimal tuber inulase activity have been reported by
various workers. Rtidiger 48j states that 55~ ~C and a pH of 6.(~6.5 (pH of the
tubers) results in the greatest extent of polyfructan hydrolysis; however, Malsch 49)
indicates that these values are 40 ~ and pH 5.1, respectively. Activity at 56 ~ is
advantageous since this temperature will partially prevent contamination of the pulp ;
however, 40 '~C will not.
Also, it has been found by Bastin and Onltier 76) that the synthesis of inulases in
JA may be increased by slicing the tubers. This is due to oxidation processes which
occur at the exposed surface and serve to induce enzyrlae production, Thus, the method
and extent of tuber size reduction should also be studied since polyfructan hydrolysis
could be improved by taking advantage of this phenomenon.
Hydrolysis by Inulases Present in Filamentous Fungi

Some filamentous fungi, such as Aspergillus niger, Penicillium spp., Fusarium roseum,
and Citromyces spp., produce inulases. Asai vT> used extracts from Aspergillus,
Penicillium and Citromyces for hydrolysis of JA carbohydrates before alcohol produc-
tion. He found that some species of Penicillium hydrolyzed about 80 ~o of carbo-
hydrates during 14~24 h at an optimum temperature of 50 55 ~ and a pH of 4.1.
Enzymatic hydrolysis by inulases produced by Aspergillus niger was also reported
by Malsch 49, vs, v9~. He reported that the optimum pH for Aspergillus niger inulases
was 3.8 (lower than the optimum for the JA tuber inulases). Two important advantages
of this process include a reduction in the time required for subsequent ethanol produc-
tion (i.e. 30 h as compared to 4-5 days without prehydrolysis by this enzyme) and a
more complete utilization of sugars (residual sugars were only 0.25 ~o w/v as compared
to 1.0-1.5 ~ w/v when no hydrolysis by Aspergillus niger was employed).
8 N. Kosaric et al.
Some yeasts, such as Kluyveromycesfragilis 80,811, Saccharomyces lactis 827, Candida

kefvr s51, and Kluvveromyces marxianus 84~ also produce inulases. These yeasts may
be used for direct conversion of Jerusalem artichoke carbohydrates to ethyl alcohol.
The time for complete hydrolysis of the inulides is dependent upon the degree of
polymerization of polyfructans in the tuber, and this value is subject to a high degree
of variation.
3.2.2 Acid Hydrolysis

Fructans from JA have been hydrolyzed with hydrochloric acid or sulfuric acid 41, ,2,
45.48, 5o, 52.53,68, 85-911, oxalic acid 65,92) citric acid 93), phosphoric acid 55,941, and
lactic acid 52). These acid treatments are combined with temperatures from 70 ~
up to about 137 ~ at various pH values.
Some researchers have obtained a high yield of ethanol (about 8 ~ higher as com-
pared to enzymatically hydrolyzed fructans) by treating tubers with HC1 (4 L of
38 ~o HCI per 100 kg of tubers)48, v31. Tanabe and Kurihara 537 stated that addition
of HzSO~ to tubers heated at 0.3 MPa did not improve the ethanol yield. At an
industrial scale, they obtained a yield of about 80 ~o of theoretical.
Underkofler et al. 527 processed the juice of JA which had been hydrolyzed by
sulfuric acid under various conditions (pH from 1.8 to 5.4 and heated for 1 h at
80 ~ Different levels of hydrolysis were obtained but the yield of ethanol for each
of the tested conditions was almost the same, at about 90 o/0.
Blanc 87,88), Colin and Belva190), Mariller 95~ and Mayeras 421 adjusted the pH
of the juice to about 3.5 and heated it at 100~ ~ for 1 h; however, no data on
the efficiency of hydrolysis were reported. Wartarasiewicz 501 obtained 95-98%
hydrolysis by using sulfuric acid to adjust the pH to about 2 and heating the pulp
for 1 h at 80 ~ However, even though hydrolysis was almost complete, an ethanol
yield of only about 75/~ of theoretical was obtained in this process.
Microbial processing of the pulp hydrolyzed by oxalic acid gave low yields due to
toxicity of this acid to the yeast 651
3.2.3 Thermal Hydrolysis

This method of preparation of Jerusalem artichoke for bioconversion is very popular
in Eastern Europe, primarily as it is analogous to the production of ethanol from
potatoes, and the same apparatus, known as a batch Hinze cooker, is used.
Different temperatures and pressures (from 125 ~ to 145 ~ and 0.2 MPa-0.6 MPa)
have been used. Maess 66) steamed the juice and tubers at a pressure of 0.3 MPa
and obtained ethanol yields of 8 L and 9 k per 100 kg of tubers respectively. Rfidiger 45)
obtained 6-7 L of ethanol per 100 kg of tubers while Windiseh cv obtained 7-10 L
of ethanol per 100 kg of tubers by steaming tubers at 0.2 MPa. The carbohydrate
content of the original medium was not reported by any of the above authors.
A number of experiments were done during the 1930's in the U.S.S.R. by Obrosov ~"
96) and Bobkov 45,97,981. These researchers hydrolyzed the carbohydrates by steam
with or without addition of sulfuric acid. Some of the results obtained by Obrosov 44j
are shown in Table 3. Based on these results, Obrosov recommended steaming 40 min
at 0.3 MPa or steaming 30 min at 0.4 MPa for thermal hydrolysis o f J A fructans.
Systematic experiments on thermal hydrolysis have also been performed by
Table 3. Comparison of the ethanol yield obtained at different methods of treating of the J A tubers 44~
Method of treating the tubers Ethanol yield, L
Per 100 kg Per 100 kg ~ of theoretical from

of tubers fructose contained initial carbohydrate
l. Maintain the mash

tot 1.5-2.5 h at 55-56 ~
(enzymatic hydrolysis) 6.5-7.7 45-51 71 79
2. Cooking for I-2 h
at 100 ~ 7.3 9.0 50-55 78 85
3. Cooking for 0.5-1.5 h
at 100 ~ with added H2SO4
(0.5 o of tuber weight) 7.3-8.8 50-55 78-85
4. Steamed
0.25 MPa for 1.5 h 8.19 54.1 84.1
0.30 MPa for 125 h 9.70 54,2 82.2
0.35 MPa for 40 rain 9.00 54.5 84.6
0.40 MPa for 30 rain 8.50 54.7 84.9
0.45 MPa for 25 min 8.90 52.8 82.2
0.50 MPa for 15 min 8.95 53.0 82.3
5. Steamed at 0.40 MPa for
30 rain. with added HaSO4
(0.5 ~ of tuber weight) 8.92 54.7 84.9
W l o d a r c z y k 5~,99~. The extent of carbohydrate hydrolysis was found to be highly

dependent on the pressure applied to the tubers during treatment (10 ~0 o f hydrolysis
at 0.2 M P a as opposed to more than 90 % at 0.6 MPa), However, it was also shown
that carbohydrate losses became significant with increasing pressure above 0.4 M P a
(up to 21 ~o losses at 0.6 MPa). It is important to note that even though different
levels o f hydrolysis occurred for 0.2, 0.3, and 0.4 M P a treated mash, the same yield
of ethanol (about 8550 of theoretical based on initial carbohydrate content) was
achieved after microbial processing.
C o m b i n a t i o n s of the methods reported above have also been used for hydrolysis.
Vadas ' 1~recommended a combination o f thermal hydrolysis with enzymatic hydroly-
sis by enzymes contained in the tubers. F o r this purpose, 80 o o f tubers were cooked
at 0.25 MPa, and the remaining 20 ~o were ground and mixed with the cooked mash
at 56 ~ Asai and H a y a k a w a 100) used a combination o f acid hydrolysis under
pressure with enzymatic hydrolysis. After steaming the tubers at 0.24 M P a for 30 min
with the addition o f 5 parts 0.05 ~o HCI, additional hydrolysis was performed using
mold inulases. The best results obtained were 82.50o hydrolysis and an ethanol yield
o f 86.40o of theoretical after bioconversion with Saccharomycespombe. Usually
ethanol yields of up to 95 ~ o f theoretical can be obtained using this organism.
3.3 Ethanol Production

The efficiency o f product formation depends on the use of a vigorous strain o f yeast
with high ethanol yield and also exhibiting high ethanol tolerance. O f additional
importance is the use o f o p t i m u m pH, temperature, presence o f nutrients, and an
10 N. Kosaric et al.
optimal concentration of sugar. From an economic viewpoint, the ethanol process

used should also exhibit high ethanol productivity. Each of these points will be
discussed in detail.
3.3.1 The Yeasts

Up to the 1950's, brewery 47,48.67,68), distillery 44,45, 87,88) or bakery 73) yeasts were
the only types used for ethanol production. Underkofler et al. 52), Asai and Haya-
kawa 100), and Tanabe and Kurihara 53) recommended the use of Saccharomyces
pombe, while Sachetti 101~and Wlodarczyk 51) recommended the use of Saccharomy-
cesfragilis for conversion of Jerusalem artichoke carbohydrates to alcohol.
Christiansen to2) patented a method to use a selected yeast which converted the
diffusion juice of JA directly (without prior hydrolysis) and named this microorganism
Saccharomyces helianthus. After being pregrown in the juice (probably for the induc-
tion of inulases) this yeast could convert JA carbohydrates to ethanol at a yield of
about 95 % of theoretical during' a 4-day period.
Within the last few years, interest in this direct, one-step production of ethanol
from JA carbohydrates has greatly increased 84't~176 This approach would
simplify the process with a consequential saving in energy. The selected yeast strains
which have inulase activity and good fermentative capabilities such as Kluyveromyces
fragilis, K. marxianus, Candida pseudotropicalis, C. macedonienas, and C. kefyr have
been reported to produce a high yield of ethanol (up to about 95 % of theoretical)
from JA carbohydrates. It is interesting to note that for some of these species, better
kinetic parameters of ethanol production were obtained with non-hydrolyzed rather
than with hydrolyzed juice (Fig. 1). Opposite results were obtained when Saccharo-
myces species were used (Fig. 2) t~
It has also been found that the yeasts, K. fragilis and K. marxianus (as per studies
undertaken in our laboratories), have a better process capability when they are
pregrown in the artichoke medium. This observation agrees with that previously
found by Underkofler 52) or Christiansen 102), Nogoro and Kito 83) reported that
14
12
non-hydrot
1o
~8 4
2
0
c~ 2 1
0 i i i i i i i i
0 10 20 30 40 50 60
Time (h)
Fig. 1. Conversion of hydrolyzed and non-hydrolyzed juice with Kluyveromyces fi'agilis to7~
12 - 61X~ "
'x
~10 , 51 \ ~\ hydrolyzed
.~8- 4
e~
~2- 1
X
0- 0
' 10 20
' ' 3'0 ~0
' 5O
Time (h)
Fig. 2. Conversion of hydrolyzed and non-hydrolyzed juice with Saccharomyces cerev&iae 125 toT,
inulase activity was not detected in Candida kefyr which was cultured with media
containing glucose, fructose, or sucrose as. the carbon source.
The inulase of the yeast K. fi'agilis (previously Saccharomycesfi'agilis) was found
to be extra and intra-cellular am, but GrootWassink and Fleming s~) suggest the
possibility that soluble inulases had become detached from their original location
in the cell. This extensive solubilization of enzyme during microbial growth presents
a problem for continuous systems and could adversely affect the economies of ethanol
production.
3.3.2 pH
The optimum pH for ethanol production by Saccharomyces species is reported to be
5 lo9~; however, for processing of JA, an initial pH of 4.0-4.5 was recommended for
best results 4s. 51~.When these initial pH values were applied, the pH during the process
(without pH control) dropped only slightly; e.g., about 0.1-0.2 units. In the case
of an initial pH value of 6.3 (which is the normal value in the juice), the pH at the
end of the batch growth process dropped to about 3.4~3.7 45. tos,. A low pH of 3.5-3.8
is advantageous for control of contamination 45.56~. GrootWassink and Fleming 81~
found that inulase yields for K. fi'agilis were constant in the pH range 3.5-6.0.
3.3.3 Temperature
The production temperature depends mainly on the yeast strain used. For most
yeasts, the optimum temperature for anaerobic process is about 10 :C higher than
that for growth alo,. The specific productivity rate of ethanol is higher at increased
temperatures ( ~ 4 0 ~ but overall productivity of the process decreases due to
enhanced ethanol inhibition HI~. Commonly, temperatures of 28-35 ~ were used
for ethanol production sl,sz, xos) When operating at higher temperatures "foam
ferrrrentation" develops due to the presence of pectic substances in the JA 46.72)
Only a few yeast species can utilize these substances (e.g., Kho'veromycesfi'agilis ) ~12~
3.3.4 Nutrients
The requirements for nutrients which are not utilized in ethanol synthesis are in
relative proportion to the major components of the yeast cell. Nutrients may have to
be supplied to JA juice (i.e., N and P sources); however, it is not certain whether
additional nutrients are needed in all cases. Addition of barley malt for preparation
of the yeast inoculum for ethanol production was recommended by Maess 661 and
Fuks 46~, but Windisch 471 and Lampe 691 did not find this to be necessary. Wlodar-
czyk 511 found that addition of barley malt increased the rate of production at the
outset, but after two days, no differences were observed. This researcher also pointed
out that addition of barley malt could be a source of infection, and for this reason,
ammonium sulfate was used which improved the process rate by only about 3-8 ~'o.
According to Wlodarczyk 51~, no addition of phosphate was needed for ethanol
production from JA juice.
Asai 113~ showed that addition of peptone increased the rate and improved the
ethanol yield. Margaritis and Bajpai 1141 have supplemented the JA extract with
a small amount of Tween 80, oleic acid, and cornsteep liquor. From the work of
Underkofler 521 and from our experiments, JA jui~ce supplies all nutrients necessary
for yeast growth and no additional nutrients are needed 1081. It is also possible that
requirements for additional nutrients depends on agricultural conditions and varieties
of Jerusalem artichoke used as substrate in the bioprocess2
3.3.5 Carbohydrate Concentration

The rate of ethanol synthesis is considerably affected by the concentration of ferment-
able carbohydrates. Mante 115) and Wlodarczyk 511 reported an optimum carbo-
hydrate concentration between 10-16 ~ Blg.
This agrees with the latest data reported by Cysewski and Wilke 116)who found an
optimum glucose concentration of about 100 g L-1 gave the highest ethanol product-
ivity. GrootWassink and Fleming 81) reported that the activity of inulases at high
sucrose concentrations was followed closely with that of invertase activity. Sucrose
concentrations over 100 g L -1 have been shown to cause considerable deviation
from normal Michaelis-Menten kinetics ofinvertase action due to substrate inhibition.
Both enzymes showed a similar decrease in activity with increasing sucrose concentra-
tion.
It is important to note that 100-160 g g -1 of carbohydrate can be obtained in the
juice from JA tubers without pre-concentration.
3.3.6 Systems of Ethanol Production

Ethanol production from JA has been mainly carried out in batch mode systems.
Some data are available on ethanol production from JA juice using free yeast cells
in continuous stirred-tank bioreactor systems (CSTB) lo8,11~1 and by an immobilized
cell system 118~
A maximum volumetric productivity of 7.0 g L -1 h -1 at D = 0.15 h -1 (ethanol
yield 90 ~o of theoretical and 95 ~o sugar utilization) was obtained using free cells of
Kluyveromyces marxianus UCD (FST) 55-82 in non-hydrolyzed juice (supplemented
with nutrients) for a one-step CSTB 117t. Figure 3 shows total sugars, ethanol and
120 6
,....
100
E 80
x
o=
o_ 60- .3
ii1
=o
.ic
"~ ~0- -2 o~
tJ
i/1
E
o
; 20- -1 h5 Fig. 3. Total sugars, ethanol, and biomass
effluent concentrations as a function of
(.r dilution rate at 35 ~ Hs~: (R) total sugars;
0
( 0 ) ethanol; (A) biomass
0 0.1 0.2 0.3 0.4
Ditution rate D (h -~}
Table 4. Continuous ethanol production using free and immobilized cells of Kho veromyces
marxianus ~ts~
Parameter Free cells at Immobilized cells at

D = 0.2h -1 D = 2.9h -1
Max. ethanol productivity 7.0 104

(g ETOH per L per h)
Feed sugar concentration 110 101
(g sugars per L)
~o feed sugars utilized 82 ao 80 ~o
Effluent ethanol concentration 36 35
(g ETOH per L)
Biomass conc. 3 43 a
(g dry wt. per L bioreactor vol.)
Ethanol yield 0.40 0.44
(g ETOH per g sugars utilized)
( % theoretical) (78 Oo) (86 %)
Specific ethanol productivity 2.2 0.55
(g ETOH per g biomass per h)
Specific sugars uptake rate 5.6 1.21
(g sugars per g biomass per h)
Original biomass loading; Operating conditions for maximum volumetric ethanol productivities
b i o m a s s c o n c e n t r a t i o n s as a f u n c t i o n o f d i l u t i o n r a t e for this system. In t h e s a m e

m e d i u m a n d w i t h t h e s a m e yeast strain, a v e r y h i g h m a x i m u m e t h a n o l p r o d u c t i v i t y
o f a b o u t 100 g L -1 h -1 was o b t a i n e d in a n i m m o b i l i z e d cell r e a c t o r . H o w e v e r , t h e
e t h a n o l yield a t t a i n e d was o n l y 86 ~o o f t h e o r e t i c a l , c a r b o h y d r a t e u t i l i z a t i o n was o n l y
80~ o f total, a n d t h e final e t h a n o l c o n c e n t r a t i o n was 35 g L -1. T a b l e 4 s h o w s a
c o m p a r i s o n o f t h e p a r a m e t e r s for a C S T b i o r e a c t o r w i t h free cells o f K. marxianus
a n d a n i m m o b i l i z e d cell s y s t e m 1181
More research is required in order to obtain optimum parameters for operation

of the continuous free cell system and the immobilized cell system. Of special con-
sequence is the rapid decline of inulase yield with increasing dilution rate 81, lo8~
This decline in inulase yield (which subsequently affects ethanol yield and kinetics) is
probably a consequence of the high solubility of yeast inulases. GrootWassink and
Fleming 81) found that the total amount of inulase which was released into the medium
increased from 24 ~o in batch culture to 50 ~0 in continuous culture.
3.3.7 Ethanol Production from the Stalks

There is limited literature information on the use of Jerusalem artichoke stalks for
ethanol production 119. ~20~. Jandolo 1191 reported that depending upon variety, time
of harvest, and agricultural conditions, one can obtain up to 8 L of ethanol per 100 kg
of stalk. Surminski ~z0~ utilized the stalks of two varieties of.JA-white and red. A
higher yield of ethanol was obtained from the white varieties; however, this was only
2.4 L per 100 kg of stalks.
3.4 Recovery of the Ethanol from "Beer"
Alcohol concentration and purification is an expensive step in industrial alcohol

production as distillation consumes large amounts of energy. The preparation of
industrial alcohol from "beer" is further complicated as the reaction mixture contains
many by-products such as aldehydes and fusel oils. The distillation system must be
designed to remove these by-products.
Little information is available on the quantity and quality of rectified spirit made
from Jerusalem artichokes. According to Mante ~51, the yield of rectified ethanol
from JA is lower than from potato or corn. High levels of aldehydes and acetaldehyde
have been noticed in the crude spirit from JA. Also, higher levels of methanol were
reported when tubers were prepared by steaming at high pressure (0.6 MPa) 5~1 or
at lower pressures for longer periods of time 461. Kiippers 122) reported that crude
spirit from the JA does not contain HCN.
The quality of rectified spirit from Jerusalem artichokes seems to be very good~
In Germany, vodka produced from this crop in the 1950's was more expensive than
from other substrates 115,121). According to P~tzold 123), there is a general belief in
Bohemia that the composition of vodka produced from JA is good for the health.
3.5 By-Product (Stillage) Obtained after Distillation

Stillage consists of the non-volatile fraction of the material remaining after alcohol
distillation. The composition of stillage depends on the composition of the
raw materials (tuber) and on the method of its preparation (bioprocessing of the
whole tubers, mash, or juice). The stillage obtained after processing of the whole tuber
mash contains residual sugars, mineral salts and proteins. Approximate composition
of this stillage is given in Table 5. It is also important to note that the digestibility of
proteins in stillage is very high. The stillage has a high nutritive value and can be utilized
as animal feed 46, ~0.51~
Table 5. Composition of stillage obtained by the microbial processing

of whole tubers after steaming sl~
Fresh weight basis Dry Matter

(%) basis (DM)
Extract ~ 3.85 --
DM 3.10 100
Reducing sugar 0.24 --
Total sugar 0.46 14.8
Protein (N x 6.25) 0.89 28.7
Digestable protein 96.3 oo of total --
Ash 0.33 10.6
P (P205) 0.068 2.19
pH 4.3 - -
Composition of stillage obtained after microbial conversion of the juice depends

on the extraction method and its conditions. Using the diffusion method or extraction
by pressing, most of the protein content in tubers remains in the pulp which is the
major by-product and can be used as, animal feed 427
3.6 Energetics and Economics
Little information about the energetic and economic aspects of ethanol production
from JA is available 56. 124 126~. All of these authors agree that the production of
fuel ethanol from this high-yielding crop can be attractive from an energy and econom-
ics point of view. It must be noted that this balance can vary from country to country.
3.6.1 Energy Balance

Energy analysis of the farm-scale production of fuel ethanol (3.8 x 106 kg per year)
showed that the overall energy balance is positive t26~(The ratio of energy output/input
is higher than 1). This ratio can easily be improved up to 3.5 by utilizing all by-products.
Table 6 shows the total energy analysis in this study. Pasquier and de Valbray 1241
reported even higher values at 4.9. This difference is due mostly to the different
accounting methods of energy in the agricultural residue.
3.6.2 Economics
Since energetic balances can only provide a partial answer regarding either the
feasibility of ethanol production or relative merits of different systems, then they
must be coupled with a detailed economic appraisal.
The preliminary cost analysis for production of ethyl alcohol and the concurrent
generation of rich protein by-product from Jerusalem artichoke s6,124.125~ showed
that the cost for production of fuel ethanol can be presently acceptable if an appropriate
method for by-products utilization is chosen. Special attention must be given to the
use of carbohydrate extracted pulp as animal feed. The distance and size of the plant
that produces this pulp from the location of its final utilization (i.e., livestock location)
Table6. Total energy analysis for ethanol production from JA juice 126)
MJ per kg
of ethanol
l. Inputs
Agricultural subsystema 7.53
Alcohol plant subsystem 15.00
Total inputs 22.53
2. Outputs
Alcohol: HHV 29.87
Alcohol: LHV 26.64
Agricultural residue (50 5o of total) 13.87
By-products from alcohol plants
extracted pulp 9.93
stillage 2.53
Total outputs 56.20
52.97
HHV high heat value; LHV -- low heat value; " Estimated energy
inputs into Agricultural subsystem: 23.7 GJ ha -1
will illicit a great effect on the overall credit for by-product utilization. This would
-come about from both the costs o f transportation and drying of the wet by-product.
In the near future, the reality of these analyses will be tested as a pilot plant facility
is soon to be put into operation in France 43).
4 High-Fructose Syrup Production

High-fructose syrups are important to the food industry for a number o f reasons.
Fructose has a greater sweetening capacity than sucrose or D-glucose on an equivalent
weight basis 1277. The syrups may be stored at high fructose concentrations without
danger of crystallization (up to 80~o w/w) 11. They exhibit a low viscosity as well as
water holding and antioxidant properties 1287. In addition, better prevention against
microbial attack may be achieved with fructose over that of sucrose syrups since the
osmotic pressure is higher for the fructose syrups at equal concentrations 1291
These industrial advantages, coupled with the many favourable nutritional
characteristics of fructose (i.e., no insulin requirements for metabolism 1301and a low
cariogenicity 131~) has resulted in much interest in the study of novel low-cost sources
of fructose-rich syrups.
Since approximately 60 ~o of the dry matter of the Jerusalem artichoke is composed
o f fructose, many researchers have proposed to use tubers as a source of these syrups 1,
58.94. 1 3 2 - 1 3 7 1
Four major processing steps which must be undertaken in the preparation of syrup
from the JA are as follows:
a) extraction of the carbohydrates from tubers;
b) hydrolysis o f the carbohydrates to simple sugars;
c) purification of the extract;
d) concentration o f the liquor to high sugar concentrations.
Table 7. Composition of the syrup obtained from the JA under different methods of juice extraction 58~
Method of extraction Ash (as sulfate) Protein Carbohydrate

o; DM o; DM ~o DM pH
Expressing 7.5 5 75 about 5.0

Diffusion (normal) 6.0-6.5 4.6--4.9 80-85 5.8
Diffusion under SO_~ 5.9-6.0 4.2-4.3 88-92 l 2
atmosphere
The methods used in some of these steps can be the same as those employed for
ethanol production or they may be somewhat modified. These modifications are
necessary due to the requirement for high final product quality. The product should
be colourless, have no flavour other than sweetness, no objectionable odour, and
should not contain solid precipitates. It is also important that these characteristics
be stable for a relatively long time.
As an example, Table 7 illustrates the effect o f different methods of juice extraction
on final syrup quality. The best quality syrup was purified from juice which was
obtained by diffusion under an SO2 atmosphere.
McGlumphy et al. laai used the following method for fructose production. The
carbohydrates from Jerusalem artichoke were hydrolyzed with sulfuric or hydro-
chloric acid at a pH of approximately 1.5 for 1 h at 80 ~ Hydrated lime was sub-
sequently used for neutralization (pH 7.0-7.5). After filtration, lime levulate was
precipitated with lime, filtered, washed, and following suspension in distilled water,
the carbonation was carried out. A syrup with 88 90 ~0 soluble solids was obtained
after separation of calcium carbonate, decolourization and evaporation. Fructose
was crystallized from the syrup. Using this process, 5.5 kg of fructose was obtained
from 41 kg of desiccated tubers containing 5 6 o; of moisture. The production cost
was relatively high and yield of fructose was low. Sugar syrup containing more than
90 ~ of the total sugar in the tubers used as raw material was prepared in a similar
way by Ohira and Kobayashi 63i
Dykins et al. 941 prepared syrup with 82~ soluble solids on a semipilot scale.
A battery delivered each hour, about 90 kg of extract with 35 2o solids. The extract
was filtered through a filter press with the aid of Super-Cel and then acidified with
HC1 to a pH of 4.2. Hydrolysis was carried out for 20 minutes under a pressure of
about 0.28 MPa. Then the syrup was concentrated to 60 ~o solids and p H was adjusted
to 5.4 with soda. After decolourization with charcoal, the syrup was concentrated
to 82 ,~o solids.
Englis and Fiess 1341used Zeo-Karb H to reduce the p H of the extract to 3.4. The
extract was treated under a pressure of 0.17-0.22 MPa for 30 minutes. Active carbon
was used for decolourization and then extract was treated with Amberlite IR-4 in
order to raise the pH to 6.7. After the resin separation, syrup was concentrated to
80 ~o solids. The final syrup was of a light amber colour with excellent flavour.
Kierstan 135~ used Ca(OH)2 for clarification of Jerusalem artichoke extract and
then phosphoric acid or CO2 for removal of Ca § This procedure reduced the colour
of the extract. A subsequent treatment yeith cation exchange resin (at a pH about 2)
was followed by a delay of 30 min at 60 ~C for total hydrolysis of the inulin. An anion
18 N. Kosaricet al.
exchange resin step (at a pH about 6-7) was then carried out similar to the process
of Englis and Fiess 134~.The effluent of the anion exchange column (which contained
20 o/~solids) was concentrated to 60 o%w/w solids. The final traces ofcoloured material
were removed by activated charcoal treatment.
Conti 58) produced fructose through the extraction of juice from dried cossettes
at 35-40 ~C under a sulfur dioxide atmosphere. The purification of juice was then
achieved by filtration over activated charcoal and desalting by ion exchangers. The
final product was a pure syrup containing 70-80~ fructose and 20-30 ~'/o glucose.
Hydrolysis of long-chain carbohydrates in juice has been carried out at low pH
values (1-4.2) and at high temperatures. The pH was lowered wither with the addition
of acids (very often hydrochloric or sulphuric) or with ion exchange resins which
replace cations from juice with hydrogen ions. Stronger conditions (lower pH value
and higher temperature) gave a product of lower flavour quality. The influence of
the pH of hydrolysis on colour development is shown in Fig. 4. The purification of
such a product was reported to be difficult.
!
o 12
I
!
I
~0.2 I
I 3"
I
I
c
E 8 ~gl
Q.
O
"O "O
a o.1
O~
/
re"
s J
s, t
0
! I I
2 3 4
pH -
Fig. 4. Influence o f p H on the rate ofcolour development and on the time required for 95 ~o hydrolysis
of tuber fructans 1 (35 ~ total solids: pH adjusted with H C L ; hydrolysis measured by dinitrosalicylic
acid determination o f total reducing sugars, colour = (600 n m - - 450 nm) sugar concentration:
rate is in units o f colour development per h)
When hydrolysis was carried out with enzymes 1%reaction conditions were milder:
the pH was 5.0 and the temperature 50 ~ Tuber juice did not change colour for 3 h
after that treatment, and after 8 h, a slight change was noticed. The change in flavour
and odour of the product was minimal.
Fleming and GrootWassiuk i~ recommended a process for preparation of syrup
from JA tubers (Fig. 5). For carbohydrate hydrolysis, they used either an acid or
enzyme process. The prepared syrup contained at least 80 ~ (w/w) solids in which
Industrial Processing and Products from the Jerusalem Artichoke ]9
Tubers (1 kg*)
Slice into water containing 0.1% SO=. (2 1)-

70~ 15 to 30 mln contact time.
Tuber slices in raw juice

Re )eat
nt and/or filter
Pulp
Raw uice (95-100% extraction efficiency)
Filter through activated animal charcoal

(10 g powdered Darco G-60 or S-51) and filter aid
(5 g Celite)
Clear, colorless juice
pH2.Sto3.0,85~ / ~ Inulase, 50~ pH 5.0,

3toSh / 5to7h
Acid-hydrolyzed juice! Enzyme-hydrolyzed juice

Decolorize (3.0 g I Filter (1 g Celite)
Darco G-60 or S-51 I
and 1 g Celite) .I.
Clarified juice Clarified juice

Adjust pH to 5.0
with IRA 401S (100 g)
H to 9.0 with IRA 401S (180 g), filter
I adjust pH to 3.5 with IR 120 (80 g), adjust
. pH to 5.0 with IRA 68 (60 g)
Sweet, Colorless solution

Condense
HIGH-FRUCTOSE SYRUP ( 75% yield}
Fig. 5. Recommended process for preparing syrup from artichoke tubers 1~

* Quantities designated in brackets to be used only as guidelines due to wide variations inherent to
the process
fructose accounted for 85 to 92 o/0 of the total reducing sugars. Crude ash and nitrogen
content were 1.5 ~o and 0.5 ~o of total solids respectively. The clear, colourless appear-
ance of the syrup obtained was stable for several months at room temperature without
change in flavour or odour. The total yield of sugars was 75 o0, the major losses
occurring during ion-exchange treatment.
50 ,100
40
E
30 9 .2555-=- -=-=-=-!
r 20
m 10
'~-x-x-x- -~ .... x .... ,x . . . . . . . . . .x- . . . . . . x 0

0
0 60 120 180 240
Time (rain)
Fig 6. Time course of release of sugars from inulin or artichokes tuber extract t37b.
( ) from tuber extract; (. . . . . ) from inulin; (O) total reducing sugar; ( 0 ) fructose; ( x ) glucose
Table 8. Production of fructose from Jerusalem artichoke tuber by enzymatic hydrolysis ~3~
Total sugar Glucose Fructose Fructose yield

(g) (g) (g) ( oo)
Dried tuber (100 g) 52.1a 12.0" 40.1 100

Tuber extract 41.6a 9.6" 32.0 80
Hydrolyzate 37.5 8.7 28.8 72
Sugar separated 31.5 7.0 24.5 61
a The quantity of sugar was measured after acid hydrolysis
The production o f fructose on a l a b o r a t o r y scale by enzymatic hydrolysis o f juice

from J A was studied by Byun and N a h m 137). Inulase used for this purpose was
produced by Kluyveromyces fi'agilis 351. The time course o f hydrolysis o f higher
carbohydrates is shown in Fig. 6. When sugars from juice and pure inulin (dp > 30)
were used as substrates, 90 ~0 and 67 % o f reducing sugars were released respectively
after 4 h o f enzymatic hydrolysis. Applying the m e t h o d o f enzymatic hydrolysis,
the yield of pure fructose was 61% of its total content in tubers (Table 8).
The economic feasibility o f producing high fructose syrups from Jerusalem
artichoke depends on its ability to compete with similar products in the market
place, which are obtained mainly from hydrolyzed corn or beet carbohydrates.
In 1953, it was reported that the production o f sugars from Jerusalem artichoke
was more expensive than that o f sucrose from beets 58). This was mainly due to the
price o f the tubers. However, because o f the improved J A species presently available,
technological improvements, and large economic credit obtained from by-products,
this economic analysis will be more favourable.
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Vol. 2 (Rose, A. M., Harrison, J. S. eds.), N.Y.N.Y., p. 119, 197l
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und Hauswirtschaftlichen Auswertungs- und Informationsdienst e.V. (AID), Braunschweig-
V61kenrode, West Germany 1957
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130. Roch-Norlund, A. E., Hultman, E., Son Nilsson, k. H. : Metabolism of fructose in diabetics in:
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Acta. Med. Scand., Helsinki 1972
131. Grenby, T. H.: Chem. Br. 7, 276 (1972)
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133. Eichinger, J. W., McGlumphy, J. H., Buchanan, J. H., Hixon, R. M. : ibid. 24, 4l (1932)
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135. Kierstan, M. P. J.: Biotech. Bioeng. 3, 447 (1978)
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137. Byun, S. M., Nahm, B. H.: J. Food Sci. 43, 1871 (1978)
The Utilization of Cheese Whey
and its Components
N. K o s a r i c
Chemical and Biochemical Engineering, The University of Western Ontario, London,
O n t a r i o , C a n a d a N 6 A 5B9
Y. J. A s h e r
Ault F o o d s Ltd., A division o f J o h n Labatt Ltd., L o n d o n , Ontario, C a n a d a N 6 A 4M3
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2 Products Based on Whole Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.1 Alcohol from Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 Non-Alcoholic Beverages Based on Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3 Xanthan Gum from Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.4 Miscellaneous Uses of Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3 Lactose Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.1 Hydroid, sis of Lactose and its Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2 Single Cell Proteins from Lactose and Whey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4 Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
5 The Utilization of Whey Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
5.1 Recovery of Whey Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
5.2 Functional Properties of Whey Protein Concentrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.2.1 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.2.2 Whippability and Foaming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.2.3 Gelation Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.2.4 Water Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5.2.5 EmulsiL~ing Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.3 Applications of Whey Protein Concentrates in Food Manufacturing . . . . . . . . . . . . . . . . . 52
6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Annually, approximately 1.2 million tons of lactose and 200,000 tons of milk protein are transferred
into whey worldwide, of which less than 60 % are utilized for human food and animal feed. Thus,
a nutritionally valuable food resource is wasted. Some progress has been made in utilizing whey,
whey solids, and whey protein concentrates in the manufacture of dairy, bakery, and specialized
products. However, the potential of whey and whey derivatives is not being fully utilized.
In particular, various avenues of utilizing lactose are open. These possibilities include the
conversion of lactose into glucose, galactose and fructose to improve its sweetening power,
production of alcohol from lactose, production of single cell proteins from lactose, and synthesis
of gums from dairy products. There is a great potential to produce long shelf-life whey-based
drinks, but little progress has been made in the widespread commercialization of such products.
A need does exist to improve the functional properties of whey proteins. The use of whey
proteins to replace non-fat dry milk and casein would reouire :~n improvement in their water
absorption property. Aside from the development of new processes and products, there is also a
great need to economize and commercialize newly developed technology.
26 N. Kosaric, Y. Asher
1 Introduction
The worldwide production o f fluid whey by the cheese and casein industries runs
into millions o f tons, and yet effective utilization o f this material is not well-developed.
One o f the m a j o r problems faced by the dairy industry is to find proper uses for
the whey. W h e y is an important, and yet not sufficiently utilized, source o f proteins.
N o r m a l l y , whey contains a b o u t half o f the solids present in the original milk. The
world annual liquid whey production for 1974 was estimated to be 74 million
tons, 95 % o f which was cheese whey. In the United States alone, about I million
tons o f whey solids were produced in 1974 (which is equivalent to approximately
17 million tons o f liquid whey). According to Shahani and M a t h u r 115), only about
56 ~ o f the whey solids were utilized for h u m a n food and animal feed.
Total C a n a d i a n cheese production in 1978 a m o u n t e d to 1.83 x 10 61 pounds, which
resulted in a total equivalent amount o f 3.13 x 10 9) pounds o f liquid whey. A b o u t
35.6 o of the total whey, i.e. 1.1. billion pounds, was disposed o f 78). Canadian whey
production, disposal, and utilization are presented in Table 1.
The composition o f whey as c o m p a r e d to other dairy products is presented in
Table 2. This material also has g o o d nutritional value, containing a balanced
spectrum o f amino acids and vitamins (Table 3).
Table 1. Whey production, disposal and utilization in Canada (1973--1978)78)
Year Whey Whey utilized in Whey utilized in Disposed Disposal as o5

produced food and dry liquid animal whey of total prod.
feed feed
lb( • 106) lb( x 106) lb( • 106) lb( • 106)
1973 2,610 847 205 1,558 59.7

1974 2,906 1,004 205 1,697 58.4
1975 2,709 1,099 205 1,405 51.9
1976 2,806 1,293 205 1,328 47.0
1977 3,015 1,572 205 1,238 41.1
[978 3,130 1,810 205 1,115 35.6
Table 2. Composition of dairy and whey products 9o)
Product Component (%)
Lactose Protein Fat Ash Moisture
Whole milk 4.8 3.5 3.5 0.7 87.4

Skim milk 5.1 3.6 0.1 0.7 90.5
Sweetened, condensed
skim milk 16.3 10.0 0.3 2.3 28.4
Condensed whey 38.5 7.0 2.4 4.0 48. l
Non-fat dry milk 52.3 35.9 0.8 8.0 3.0
Dried sweet whey 73.5 12.9 0.9 8.0 4.5
Dried acid whey 66.5 13.0 -- 10.2 3.2
Modified whey solids 56.6 36.0 3.3 2.4 1.7
The Utilization of Cheese Whey and Its Components 27
Table 3. Minerals and vitamins present in liquid and dried skim milk and whey (per 100 g) 9o)
Component Unit Liquid Liquid Skim milk Whey

skim milk whey powder powder
Calcium mg 121 51 1293 646

Phosphorus mg 95 53 1005 589
Iron mg trace 0.1 0.6 1.4
Sodium mg 52 526 --
Potassium mg 145 1725 --
Vitamin A I,U. trace 10 30 50
Thiamin mg 0.04 0.03 0.35 0.5
Riboflavin mg 0.18 0.14 1.78 2.51
Niacin mg 0.1 0.1 0.9 0.8
Ascorbic acid mg ! 7.0
Food energy cal 36 26 359 349
2 Products Based on Whole Whey
Based on the nutritional value of whey, a number of conventional uses have been
reviewed by M a n n 66.67.68.69.70), Mathis vlj, McDonough'61, Robinson 105) and
others.
In many dairy products whey is utilized to replace egg products and part of the
non-fat dry milk. Probably the largest single use of whey in a dairy product is in ice
cream. U.S. Federal regulations permit the use of whey in ice cream for up to 25 o;
of the non-fat milk used 1191. Another large dairy use for whey is in cheese products.
Softer processed cheese can be manufactured with the partial incorporation of whey
solids, e.g. pimento cheese. Ricotta cheese, however, is made from precipitated
whey proteins. Ricotta-type bases have been utilized successfully in the manufacture
of dairy spread, imitation sour cream, cheese-flavoured dips, and dips with other
flavours 1,~.73. 130~. Whey is also utilized in the manufacture of whipping cream,
yogurt, dairy desserts, and frozen desserts 1o5). Other uses of whey include the
Table 4. The uses and functions of whey in various foods 68697~
Food ~o Whey Function

solids used
Baked goods--sweet 3.0 (of Flavour, texture, shorter dough time,

goods, bread, crackers flour wt.) improved keeping quality, colour
Dry mixes 10.00 Tenderizing, colour, flavour
Ice cream sherbert 2.7 Flavour, acid and fruit stability
Confections 10.0 Flavour, body, moisture
Frosting, icing 6.0 Retention, whipping properties
Jams, apple butter 4.0 Flavour
Batter mix (for frying) 5.0 Colour, flavour
Whey soy beverage,
citrus flavour 6.0 Flavour, lactic acid
Processed cheese food 10.00 Body flavour
Fig. l. Some proposed processes and products associated with the utilisation of whey 105~
manufacture of bakery products 69,105), confectioneries, and meat products 66.67.74.

105). In sausages and bologna, precipitated lactalbumin can be used as a meat
extender 105~
The uses and functions of whey solids in various foods are illustrated in Table 4 68.
69,-,o). Modified whey solids are utilized in many food products. These include soups,
cheese, bakery products, candy, infant foods, etc.
Depending on the whey treatment process, various categories of products are
possible, as presented in Fig. 1. The potential is great, particularly taking into account
the quantities of whey available for processing. As already mentioned, approximately
200,000 t of protein and 1.2 million t of lactose are transferred into whey annually on
the basis of worldwide cheese and casein production. Whey can be concentrated
and fractionated bv various t~rocesses such as evaporation, ultrafiltration-reverse
osmosis, ion exchange, gel filtration, electrodialysis, etc. Several large-scale uses of
whey solids already exist. However, most of these uses consume a greater part of the
protein fraction of the whey. Also, many such uses have been developed to replace
non-fat dry milk in various dairy and non-dairy processed foods. Bakeries and
dairies are the leading users of dried whey 61. This usage led to a reduction in the
amount of non-fat dry milk used in dairy and bakery products by 39 to 36 ~o in the
U.S. from the years 1973 to 1976. Such concentration of whey and reduction in the
utilization of non-fat dry milk, which contains 50 ~o lactose, leads to stockpiling of

non-fat dry milk as well as lactose.
Some of the newer development in whole whey utilization are in the production of
alcoholic and non-alcoholic beverages, Xanthan gum, and other products. A brief
review of these avenues is given below.
2.1 Alcohol from Whey
A major problem in using whey as a substrate has been in the fact that relatively few
organisms are able to use lactose. Lactose degradation is a slow process; thus it
increases processing cost 7,u. It has been reported that for acid whey containing 10 ?o
total solids, Saccharomycesfragilis was the most efficient lactose strain 93). However,
only 55 o of the available lactose was converted, possibly due to an inability of the
yeast to tolerate alcohol. If the lactose in whey could be hydrolyzed into its
constituent monosaccharides glucose and galactose, non-lactose-fermenting organisms
such as strains of S. cerevisiae that tolerate high concentrations of alcohol could be
used.
Roland and Alto 106), as well as O'Leary et al. 93), have utilized lactose-
hydrolyzed whey for ethanol production. Kho'veromyces fragilis N R R L Y-1109
(formerly classified as Saccharomycesfi'agilis) and Saccharono.'ces cerevisiae ATCC 834
were used for cottage cheese ~.hey, in which over 80 0'o of its lactose was hydrolyzed.
Saccharomyces cerevisiae produced alcohol from glucose more rapidly than K.~agilis,
but galactose was attacked only when S. cerevisiae was pregrown on galactose.
However, the maximum alcohol yield obtained was only 1.65 %. Even though this
was twice the yield obtained with glucose-pregrown S. cerevisiae, it was still
significantly below the 2 ~o yield obtained with glucose-pregrown K. fi'agilis under
similar conditions. K. fi'agilis was able to utilize more of the total available carbo-
hydrate than was S. cerevisiae. No significant amounts of acid products were formed
from the hydrolyzed or lactose-containing whey. Thus, no need of pH control is
necessary. It was recommended to use hydrolyzed whey over conventional whey
even though lactose-hydrolyzed whey required a longer reaction time as compared to
conventional whey. According to this source, the preparation of concentrate of
relatively high solids content would be possible due to better solubility of the
lactose-hydrolyzed whey. This would result in higher alcohol yields than is usually
possible with normal whey.
Roland and Aim 106) used various strains of S. cerevisiae var. ellipsoideus to ferment
mixes containing grape juice, hydrolyzed whey permeate, water, nutrients, yeast
energizer, acid blend, and Na2S205. The composition of the mixes is given in
Table 5. To obtain hydrolyzed whey permeate syrups, electrodialyzed whey permeates
(24 Oo total solids) were adjusted to pH 6.0 and treated with a commercial lactase,
"'Maxilact" (Enzyme Development Corporation, New York).
It was reported that all grape concentrate were successfully converted into 10-20 ~o
v/v alcohol wines. Similarly, hydrolyzed whey permeate syrup was yielded in a 12.5 ~o
v/v alcohol beverage with a wine-like aroma. These results are not in agreement,
however, with the results obtained by O'Leary et al. 93~ One of the major differences
between the procedures employed by O'Leary et al. 93) and Roland and Alm t06) was
Table 5. Composition winemaking recipes 106)
Wine components Table wine classes
Red White Ros6 Hydrolyzed

whey permeate
syrup
Grape juice, litre 3.78 3.90 3.78

Hydrolyzed whey permeate, litre 3.30 3.30 3.42 1.55
Water, litre 15.22 15.10 15.15 1.70
Yeast inoculum, litre 0.20 0.20 0.20 0.03
Yeast nutrients, ga 30.00 30.00 28.00 4.00
Yeast energizer, gb 4.00 4.00 1.00
Acid blend, gC 45.00 45.00 42.00 10.00
Tanning, g 4.00 -- 5.00 0.50
Na2S205, g 2.50 2.50 2.50
Start#Tg values, day 0
pit 3.75 3.75 3.25 3.50
Total acidity, g tartaric 0.56 0.57 1.30 0.07
acid per 100 ml
Specific gravity, B6 14.00 14.5 18.50 9.00
Volume, litre 20.00 20.00 20.00 2.75
S. cerevisiae var. Allpurpose Hock Ros6 Hock
ellipsoideus strain
" Urea-ammonium phosphate (50--50):

b Grey Owl Laboratories Ltd.. Bristol, England:
c Tartaric acid, malic acid, and citric acid (50 30--20)
related to the feeding procedures. Roland and Aim 106) reported that the utilization
of hydrolyzed whey permeate syrup was most efficient when interval feeding
procedures were employed, using incremental additions of hydrolyzed whey permeate
syrup at 24- to 28-h intervals. It appears that such interval feeding of hydrolyzed whey
permeate syrup may have minimized the diauxie process pattern observed, with
glucose being used before galactose. The results of sensory evaluations of wines
prepared with hydrolyzed whey permeate syrup indicated that palatable wines can be
produced from grape juice concentrates supplemented with hydrolyzed whey permeate
syrup 5.96). However, the alcoholic beverage made by hydrolyzing whey permeate
syrup had a light,.flowery aroma. It did not mature beyond two months of aging
and deteriorated both in flavour and character.
Presently under construction in Denmark is a plant expected to continuously
ferment whey into alcohol for fuel or beverages. A by-product of the process,
methane gas, will help power the operation *'28~ The whey is first concentrated by
reverse osmosis and then by ultrafiltration. The yield from the process corresponds to
approximately 80~ o of what is theoretically obtainable, calculated on the basis of
lactose content. About 421 of whey containing 4.4~ lactosq is required for the
production of 1 litre of 100~ alcohol. A strain of Kluyveromyces fragilis will be
utilized. The schematic of the processing operations is shown in Fig. 2.
Yang and co-workers 146) at Oregon State University have developed a process to
convert whey into a lightly carbonated, flavoured alcoholic beverage and high-
Fig. 2. Alcohol production from whey 8) 1 Store tank for whey permeate 2 Acid container 3 Plate
heat exchanger 4 Control Unit 5 Tank for antifoam 6 Tank for chemicals 7 Reaction vat 8 Separator
9 Storage tank for yeast cream 10 Buffer tank 11 Distillation plant 12 Storage tank for alcohol
13 Substrate reservoir 14 and 15 Propagation plant
protein material equivalent to current commercial products. The product depended

heavily on added dextrose; about 22 ~o of dextrose was added to the deproteinized
whey. Therefore, hydrolysis of lactose was not employed. No mention was made
concerning the species of yeast utilized. However, it appears that the strains suitable
for the manufacture of Montrachet, champagne, sherry, port and Burgundy were
evaluated. Whey wine was also blended with fruit and berry wines to produce fruit-
whey wines. It was also flavoured with synthetic flavours and carbonated to produce
effervescent, flavoured whey wine.
Ochi and Nakanishi 9t) used a strain of Saccharomyces sake Yabe IFO 0305
for trypsin-digested recombined skim milk. Vanossi a38~ produced a beer-type
product from cheese whey. Gawel 33.34~ obtained an alcoholic wine-type beverage
from whey by lactose hydrolysis, using lactose with simultaneous fermentation of the
resultant glucose and galactose by means of wine yeast. It was reported that in the
solutions containing 24 % lactose, =<10 % ethanol did not inhibit the activity of lactase
(WaIlerstein Co.). Additional research is required to establish a process which would
utilize lactose most efficiently and economically.
2.2 Non-Alcoholic Beverages Based on Whey
Considerable progress has been made in developing non-alcoholic beverages from

whey. Excellent reviews on this subject are published by Mann 657 and Holsinger
et al. 441 Most of the non-alcoholic whey beverages are prepared by mixing
pasteurized and deodorized whey with the appropriate proportion of water, fruit
juice concentrate, flavourings, colour, and preservatives. A drink based on cheddar
cheese whey was formulated at Mississippi State University 62. 117). This product was
prepared by mixing whey, sugar, orange concentrate, citric acid, and other ingredients
(pH of 3.8 and a total solids content of 16.5 ~ A product shelf-life of at least
14 days at 22 ~ was claimed. A total of 956 consumers of all ages sampled the
beverage; 76.5 o~oof the respondents rated the drink acceptable t t 7). Personal interviews
with 46% of the families showed that 90% would purchase the product for $ 0.38
per litre. A frozen whey concentrate was developed 129) and used as a punch base at
various dilutions with ginger ale. The product, in which at least half of the liquid was
fresh sweet whey, was similar to frozen orange juice concentrate and was claimed to
have no whey flavour.
A U.S. patent granted to The Coca-Cola Co. 65) covers a process for producing
a whey protein concentrate suitable for incorporation into beverages. A process from
India 6ol, suitable for manufacture of whey beverage on a small scale, involved addition
of sugar syrup, citric acid, and colouring to heated, filtered whey and incubation
with Saccharomyces cerevisiae at 22 ~ for about 15 h. The product was then flavoured
with pineapple, orange, mango, etc., and bottled for distribution. Vaidi and Pereira 134)
used liquid whey and whey powder as a base for the production of lemon, strawberry,
and chocolate drinks. The compositions are shown in Table 6. It was reported that
the products had acceptable organoleptic properties. However, the chocolate-
flavoured drink was not well received. N o change in the flavour and taste of the
products was reported during the one-month storage under refrigeration.
Fortification of.soft drinks 43) and orange juice t) with whey proteins has also been
reported. Soft drinks consumed in the United States amount to an estimated
380 8-oz bottles per capita per annum, producing nearly $ 5 billion for the soft
drink industry. It was reported that the beverages fortified with 1% dehydrated whey
protein concentrate maintained excellent clarity and colour during one year of storage
at room temperature. It was estimated that the fortification of soft drinks with 1%
protein by weight would cost about $ 0.075 per 8-oz (220-mi) bottle. It has been
reported that the acid whey powder blend would have all nutrients substantially
increased from the levels found in orange juice alone. Such a blend would contain all
the nutrients found in a glass of orange juice, plus all those found in an equal-sized
Table 6. Composition of some whey-based drinks 134)
Ingredients Weight (1bs.)
Strawberry drink Lemon drink Chocolate drink
Stabilizer 0 05 0.025 0.05

Cream (35 % fat) 5.7 5.7 --
Sugar 5.0 5.0 5.0
Non-fat dry milk 6.0 --
Flavour 0.17 0. l 5
Liquid whey 86.0 -- 85.0
Whey concentrate -- 90.0 --
Whey powder -- -- 8.0
Chocolate -- -- 2.0
glass of skim milk, except for protein. However, the blend would have 2.5 times the
protein found in orange juice alone ~1
Patents 2.3~ granted in England describe methods for manufacturing beverages
and concentrates containing whey proteins and fruit juices. One example as illustrated 21
contained as much as 60 ~o whey proteins by weight. A pfitent 8tl granted to General
Foods Corporation describes the production of a heat-and acid-stable whey protein
fraction which can be used for the manufacture of acid beverages and powdered acid
beverage mixes. Galzy and Moulin a21 used non-pathogenic protoprophic yeast and
bacteria possessing [3-galactosidase activity and a gel-character on a media containing
lactose, i.e. lactoserum, to produce galactose solution. The galactose solutions were
considered suitable for use in beverages. A very popular whey-based soft drink is
widely marketed in Switzerland under the name "'Rivella" and in Austria under the
name "'Almdudler".
2.3 Xanthan G u m from W h e y
Investigation carried out by Charles ~s) and Radjai ~9~indicates that Xanthan gum can
be produced from media containing deproteinized acid-set and culture-set cottage
cheese wheys. Xanthan gum is a polysaccharide product synthesized by Xanthomonas
campestris from a glucose-containing medium. Xanthan gum is used extensively in the
food industry as well as in other industries. It has been claimed that the use of
deproteinized acid whey to synthesize Xanthan gum is advantageous because of the
following reasons :
- - demineralization of the hydrolyzate is not reqnired
- - carbohydrate cost is competitive with glucose
process would utilize a high BOD x waste stream
- - enhances economics of whey protein recovery.
Xanthomonas campestris N R R L 1459A was grown on a medium prepared from

cottage cheese whey. The permeate obtained by ultrafiltration of the whey was
hydrolyzed by immobilized lactase and the hydrolyzate, which contained 2.05~
glucose, 2.05 % galactose, 0.3 % lactose, 0.3 ~o protein, 0.5 ~o lactic acid, and 0.5 ~o
ash, was sterilized at pH 3.5 and then supplemented with 0.5% KzHPO4 and
0.01% MgSO4 9 7 H20. A repeated batch mode was employed with cycle times of
approximately 90 hours and draw-off volumes adjusted to leave a seed of I0 go (v/v).
During each cycle, glucose was used more rapidly than galactose, but both were almost
completely consumed with diauxie. Each cycle ended with a Xanthan concentration
in the range of 3.4 o~ to 3.6 o . The culture pH did not decrease (as with conventional
media), but rose from 7.0 to a high of 7.5, to 7.8 near the mid-point of a cycle,
and then slowly returned to approximately 7.0. A direct pH control was not necessary.
Diluted medium (50.~ provided similar results, but with lower final Xanthan
concentration and somewhat improved productivity. A final gum concentration of
3.4~ to 3.6~ (in approximately 90 h) was obtained, which represented an 85%
yield from the assimilable sugars present in the medium.
l biological oxygendemand
2.4 Miscellaneous Uses of Whey
This segment of the review includes some of the isolated, newly reported uses of
whey. Very little progress has been made in these areas, but potential for expansion of
some of the uses does exist. It has been reported 47) that milk proteins and lactose play
an important role in providing desirable texture, flavour, and colour in confections
and bakery products. Various flavours are produced during heating of the milk
proteins (Table 7).
Table 7. Flavour descriptions applied to the products generated

by the thermal degradation of amino acids 47)
Probable amino acid precursors Typicalflavour description
Phenylalanine, glycine Caramel-like

Leucine Bread-like, toasted
Glycine, cystine Smokey, burnt
Alanine Nutty
d-Amino butyric acid Walnut
Phenylalanine Roasted nuts, almonds
Proline Bakery, cracker
Ornithine Cracker-like
Glmamine, lysine Buttery
Arginine Popcorn
Methionine Broth. beany
Leucine, arginine, histidine Bread-like
Fritzsche, Dodge and Olcott Laboratories have developed two new cocoa substitutes
using whey solids and natural or artificial f/avour components 7,~).It has been claimed
that these products could replace cocoa (kilo for kilo) at very high levels. New
applications of whey-based cocoa flavour include coatings, beverages, cakes, icings,
pudding, and syrups. Stauffer Chemical Company 3o) reports the development of
modified dried whey, which can enhance flavour in foods and improve binding,
flavour enhancement, and emulsification in comminuted meat products. It has been
reported that if permitted, the use of lactic acid and whey proteins in mayonnaise
could drastically reduce the necessity of oil in such products 123) Fermented whey
is also used in the development of oriental-type soy sauce flavour 63). A new cheese-
type product contained a mixture of milk proteins and vegetable proteins 39)
Vegetable proteins were isolated from soybeans, rapeseed, broad beans, sunflower,
and cottonseed.
Sialic acid, also known as neuraminic acid, is currently obtained from natural raw
materials such as the sub-maxillary glands of bovines 29). This production is extremely
costly. A process reported by Eustache 291 can recover sialic acid and glycoprotein
from whey at a lower cost. From 50 kg of powdered whey, 45 g of extremely pure
N-acetyl neuraminic acid (sialic acid) was recovered.
Like sialic acid, synthetic riboflavin is currently also expensive. Therefore, the need
for an inexpensive, facile way to isolate riboflavin from dairy products is of interest.
Brewington and Schwartz 13) passed whey over a column of the neutral resin Amberlite
XAD-2 onto which riboflavin was absorbed. Re-distalled acetone was used to elute
the riboflavin. Satisfactory recoveries (90 ~o) of riboflavin by absorption and desorp-
tion from the resin were reported. A patent 45 granted to Morinaga Milk industry
Co. Ltd. describes the use of activated carbon to absorb riboflavin.
3 Lactose Utilization
As mentioned earlier, lactose is the major ingredient of non-fat dry milk and whey
solids. Yet it is the least utilized as a food ingredient. Following are factors which
contribute to the weak marketing potential for lactose:
L a c t o s e m a h T u t r i t i o n - - A great proportion of the world population cannot digest
lactose due to a lack of lactase (beta-galactosidase), an enzyme that breaks lactose
into glucose and galactose.
L a c t o s e c o ' s t a l l i z a t i o n - - Excessive amounts of lactose in ice cream and condensed
milk causes undesirable crystallization which develops a sandy texture.
B u l k i n g a g e n t - - Since the sweetness produced by lactose is very low, it does not
serve as a good sweetening agent, even in those products which contain relatively
high proportions of it. Thus, it increases the calorie content of such food products
without contributing to the functional sweetening effect.
P o o r s o l u b i l i t y - - Lactose is not easy to dissolve (about 20 g per 100 g water at room
temperature). Particularly, alpha-lactose hydrate crystals are hard and dissolve slowly.
There are several alternatives for the effective utilization of lactose. These include
hydrolysis of lactose and the use of hydrolyzed lactose, single cell protein production
from lactose, and other newly developed uses of lactose and its derivatives.
3.1 Hydrolysis of Lactose and its Uses
The disaccharide lactose, 4-([3-D-galactopyranosyl)-D-glucopyranose, present in milk

and cheese whey, can be hydrolyzed to its constituent monosaccharides D-glucose
and D-galactose. This reaction is catalyzed by the enzyme lactase (/3-D-galactosidase).
gactose-hydrolyzed milk is suitable for lactose-intolerant individuals, and it would not
cause problems of sandiness in ice creams 1o71.In the preparation of cultured products,
advantages include increased rate of acid development, increased yield of cottage
cheese, and reduced aging time for cheddar cheese. The potential benefits of hydroly-
zing lactose in preparing dairy products are numerous 365. There are several means
of hydrolyzing lactose, the most popular being the use of ]3-galactosidase. Acid-
catalyzed hydrolysis of lactose in whey has also been investigated 21.22.75.103)
Sulphonated polystyrene polymerized with divinyl benzene ion exchange resin
beads 75. lO75is also utilized to carry out lactose hydrolysis.
Numerous reports concerning the use of [3-galactosidase to hydrolyze lactose are
found in the literature. It is beyond the scope of this report to discuss ]3-galactosidase
technology. Interested readers are advised to review the re?oft published by Shukla 1185
on this subject. Other related references are also cited in the bibliography of this report.
13-galactosidase can be obtained from plants, animal organs, yeasts such as S a c c h a r o -
myces lactis, S. fragilis, and Candida pseudotropicalis, bacteria, and fungi l~S).
13-Galactosidases from different sources have been immobilized by a variety of me-
thods. Ngo et al. 89) hydrolyzed 90 ~ of the lactose within 20h using an open tubular
lactase reactor, immobilized to the inner surface of nylon tubing. Porous glass particles
have also been utilized t o immobilize [3-galactosidase 99,100,141,142,143,145). Cattle
hide collagen has also been used to immobilize 13-galactosidase 35,49)
The use of immobilized whole cells in place of immobilized enzymes has also
been reported 92). The use of immobilized whole' cells as an enzyme source
generally eliminates the need for release of intracellular enzymes and for the
succeeding purification steps. In some cases, the enzymes are more stable if
immobilized within their natural environment, the cell. The authors reported that the
activity of Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel
beads was fairly stable, but that of E. coli and Lactobacillus bulgaricus decreased
gradually.
Blankenship and Wells lO) studied 13-galactosidase obtained from 125 identified
yeasts, molds, and bacteria. Among the organisms surveyed, only two produced
substantial levels of enzymes, and both were inhibited by galactose in excess of
53 %. The organisms Kluyverornyces lactis N R R L Y-1109 and K. fragilis are known
13-galactosidase producers, and the enzyme from Y-1109 has been well studied in dairy
product applications. Fluidized-bed processing with immobilized 13-galactosidase has
also been reported 21). The rate of reaction, processing requirements, enzyme stability,
and the degree of inhibition of enzyme are greatly affected by the source of
enzyme, the method of immobili(ation, and the properties of the substrate under
consideration.
Pastore and co-workers 983 conducted long-term pilot plant studies to evaluate
the feasibility of [3-galactosidase technology for commercial operations. [3-Galactosi-
dase was immobilized on cellulose acetate fibres. A total of more than 10,000 1 of milk
was processed, using 50 batches each consisting of 200 litres. Each processing lasted
about 21 h at 7 ~ 0.5 ~ After each processing, the fibres were washed with
0.01 M phosphate buffer at pH 7.2 and stored in a buffer. Quatern~iry ammonium salt
was used to prevent bacterial growth. After 50 processings, only a 9 % drop in the
activity of the enzyme was reported. With regards to the quality of the product
obtained, a slight increase in sweetness was evident. No significant changes in the
physical and microbial properties were reported when stored for 3 4 months at 4 ~
However, the samples stored at 17 ~ showed slight darkening after 30 to 60 days of
storage due to the interaction of glucose with proteins. Results of this study
indicate that industrial application of 13-galactosidase technology is possible without
excessive uncertainties about sealing-up problems regarding the activity of the catalyst
and bacterial contamination.
Once the lactose is hydrolyzed into glucose and galactose, the glucose can be
converted to fructose using immobilized glucose isomerase. The use of immobilized
glycose isomerase to prepare high-fructose corn syrup has received a great deal of
attention over the past several years. Sproull et al. 124) used a batch reactor to
study the reaction kinetics and enzyme deactivation of glucose isomerase. Valuable
information was also provided by Korus and Olson 58), who immobilized glucose
isomerase on anisotropic hollow fibre cartridges. The cartridges were made up of
co-polymers of vinyl chloride and poly-acrylonitrile and had a nominal molecular
weight cut-off of 50,000. Polysulfone polymer cartridges with a weight cut-off

of 10,000 were also used. The stability of glucose isomerase in the Amicon P10
hollow fibre cartridge was much less than that of the free enzyme. Polysulfone
fibres had to be pre-conditioned with an inert protein to minimize enzyme inactiva-
tion. No significant amount of enzyme was leaked except when Romicon XM50 fibres
were used, which resulted in a high leakage of the enzyme. Enzyme stability was
determined at 60 ~
As far as whey is concerned, a patent 1,~o)granted to Corning Glass Works describes
the simultaneous use of immobilized [3-galactosidase and glucose isomerase to convert
lactose into glucose, fructose, and galactose. A schematic of the process is shown
in Fig. 3.13-galactosidase was immobilized on zirconia-coated porous glass particles
having an average pore diameter of 550 .'~ + 10 %. Glucose isomerase was prepared in
a manner similar to that used for the preparation of the immobilized 13-galactosidase.
Glutaraldehyde was used as a coupling agent. Whey (15 % solids of which 70 ~ was
lactose) was processed through the system. The final product contained 1% lactose,
45 % galactose, 20 % fructose, and approximately 25 % glucose. It was also found that
the calcium ions from whey had to be removed by cation exchange to prevent
Whey ]
I Ultrofittrotion I
JImmobilized I Re erse osmosis I
lactosecolumn
I l JMi'k protein J
JSweeteners (wasteploducts)
Dry sweeteners Electrodiaiysis
I Immobilized Lac't!s tJ Fractional

crystallization
Lactosecolumn
solutionell J= I I~ i
exchange
Sweeteners
I Immobi(izedtactase
cOlUMn
I Glucose+ Golaclose.Lactose
I t
~ AddglucoseJ
l Immobilizedglucose
JUsefulsweeteners J
t
isomerasecolumn Ga[octose+ Lactose
Fig. 3. Production of glucose and fructose from whey t~,o)
Table 8. Relative sweetnessof various sweeteners(equivalentto 10~ aq. sucrosesolution z~8)
Sweetener Sweetness value Sweetener Sweetness value
Sucrose 100 Maltose 30

Dextrose 90 Lactose 15
Glucose 70--75 Fruitalose 200
Fructose 140-- 175 Aspertame 18,000
Invert sugar 100-- 130 Cyclamates 30,000
Corn syrup 60--80 Saccharine 30,000- 50,000
inactivation of glucose isomerase. The flow rate was maintained at 200 ml h - 1

Conversion of lactose into glucose and galactose would increase the sweetness of the
lactose syrup or milk. However, the syrup prepared in this manner cannot be used
on a kilo per kilo basis as a substitute for corn syrup. A further increase in
sweetness can be accomplished by converting the glucose into fructose, which is
sweeter than conventional sugars used as food-grade sweeteners. The relative sweet-
ness of various sweeteners is shown in Table 8.
Recent studies 37,112~ carried out to determine the relative sweetness of hydrolyzed
lactose indicated a synergistic enhancing of sweetness when sucrose, glucose, and
galactose were used in combination. It was also found that the relative sweetness of
hydrolyzed lactose was greater when used at a higher than 50 ~ substitution level.
At 30 ~ total solids, the sweetness of the syrup was equivalent to that of sucrose
containing 20 ~ - 2 2 ~ total solids. However, above 50 ~ total solids, the sweetness of
syrups was not significantly different from that of sucrose at equivalent solids.
Other properties such as stability, humectancy, and viscosity of lactose syrups were
also studied 37,112,113,1~4). It was reported that 95 ~ hydrolyzed syrup crystallized
fastest irrespective of solids. However, syrups of 60 ~ - 7 0 ~ total solids did not show
a problem of crystallization during storage. Heating of the syrup to 75 ~ and
sealing container prevented microbiological deterioration as well as increasing the
stability against crystallization. Increasing the total solids and decreasing the
extent of hydrolysis of syrup increased the viscosity of the syrup. The viscosity of the
75 o/~ hydrolyzed syrup was below that of sucrose at equivalent solids. At every
temperature lactose was least soluble, galactose next, and glucose most soluble.
However, considerable improvement in the solubility of the lactose syrup was found
upon hydrolysis of the lactose. Humectant properties of the hydrolyzed lactose syrup
and sucrose syrup having equivalent total solids were comparable at 20 ~ and
15 ~ - 9 3 ~ relative humidity.
Some of the applications of [3-galactosidase-treated milk and whey are discussed by
Mann 697, Ash 97, Bouvy 12), and Holsinger et al. ms). The major uses are based on the
increase in sweetness, improvement in solubility, improvement in humectant proper-
ties, and reduction in the problem of crystallization of lactose syrup upon hydrolysis
of lactose. In addition, glucose or galactose are degraded by many organisms, whereas
lactose is not a suitable substrate in many processes. Thus, milk containing hydrolyzed
lactose shortens the ripening time of cheddar cheese, causes faster acid production,
and sets firmer curd during the production of cottage cheese. Upon hydrolysis of
lactose, greater quantities of whey can be incorporated into ice cream due to
Table 9. Applications for hydrolyzed lactose 132~
Use Major products
Sweetener Ice cream, confectioneries, soft drinks, canned fruit,

honey substitutes, baked goods, food syrups
Dietary specialties for Hydrolyzed milk drinks, i. e. Zymil, kactalac
lactose-intolerant
Accelerated fermentation Yogurt, cheese, wine, other processes, i. e.
Xanthan gum synthesis
Prevention of lactose crystallization Condensed and evaporated milk, frozen milk concentrate,
ice cream, frozen desserts
Browning agent Baked goods, confectioneries
improved sweetening power and elimination of crystallization. The applications for

hydrolyzed lactose are outlined in Table 9. One dairy company in Holland produces
milk under the trade name "Lactalac" in which 99 ~o of the lactose is hydrolyzed.
Similar fluid or powdered products are under development by several companies xz.
61. 132 I
Several new uses of lactose, hydrolyzed lactose and lactose derivatives have also
been reported. Australian workers 133) report that hydrolyzed whey lactose prepared
from cheddar cheese whey is suitable for replacement of up to 50 04 of the sucrose
in canned fruit syrup. Hayashibara and Sugimoto 4t) synthesized lactitol from lactose.
kactitol is a polyhydric alcohol and has a mild, refreshing, sweet taste. Lactitol has
no caloric value as it is not digested or absorbed by the digestive organs of higher
animals. It has been claimed that lactitol can successfully replace the sucrose from
carbonated drinks, concentrated syrup, ice cream, confections, canned foods, etc.
In another development, improvement in the texture of fresh-pack dill pickles was
reported with the partial replacement of sucrose by lactose Sl). It has also been reported
that diluted liquid cottage cheese whey (1 water: 1 whey) is suitable as a colour-
improving agent in the production of French flied potatoes 53).
A Canadian patent t21) describes the use of lactose in the manufacture of candies
with reduced risk of tooth decay. Lactose is treated with milk alkali such as
calcium hydroxide to convert the glucose moiety into the levulose form. Galactose
has also been used to replace sorbitol, which is considerably more expensive than
many other sugars. Other sweeteners such as dulcitol and sorbitol can also be
produced from lactose t32~. Lactose monoesters, which act as surfactants, are com-
mercially manufactured 1321
3.2 Single Cell Proteins from Lactose and Whey
Recently, considerable efforts have been made to utilize various waste materials
(food, agricultural and industrial) through microbial fermentation.
Progress has been made in the utilization of whey to produce single cell
protein, with yeast being the predominant microorganism. Vananuvat and Kinsella t3s.
136.1371 used Saccharomycesfragilis for protein production from crude lactose, using
batch as well as continuous culture techniques. A maximum yield of 62~ o was
Table 10. Gross percentage composition of spray-dried Saccharomyces fragilis cells z3) compared
to commercial lactic yeast
Components S. fragilis "Bel" lactic yeast
Crude protein (TN x 6.25) 49.5 46.5

Non-protein nitrogen (NPN) 1.23 1.1
True protein (TN-NPN) • 6.25 41.8 39.5
Fat 8.4 9.6
Ash 7.6 7.0
Moisture 5.4 6.4
Carbohydrates by difference 29. I 30.5
Table 11. Amino acid composition of Saccharomyces fi'agilis biomass (g amino acid) 23)
Amino acid S. fragilis "Bel" Whole egg

whole cells lactic yeast FAO ref. protein
Asparagine 9.4 9.1

Threonine 5.3 5.9 5.1
Serine 4.7 5.4
Glutamine 13.8 14.7
Proline 4.2 4.2
Glycine 3.7 4.1
Alanine 5.8 5.5 7.3
Valine 5.6 5.3 6.6
Isoleucine 4.8 4.5 8.8
Leucine 8.1 7.4 4.2
Tyrosine 3.9 3.6 5.8
Phenylalanine 4.2 3.8 6.4
Lysine 8.0 7.3
Histidine 2.0 1.9
Arginine 4.9 4.8
Tryptophan 1.7 1.4 1.6
Methionine 1.5 1.2 3.1
Cysteine 1.7 1.0 2.4
Total amino acids 93.3 91.1

Total essential amino acids 44.8 41.4 51.3
Chemical score 58.2 40.0
Essential amino acid index 76.0 55.8
r e p o r t e d in batch culture, whereas a yield o f 72.5 ~o was r e p o r t e d in a c o n t i n u o u s

culture system. C o n t i n u o u s culture o f S. fragilis reduced C O D by a b o u t 60 ~o. In
these studies, S. fi'agilis yeast g r o w n on c r u d e lactose c o n t a i n e d a b o u t 42 o protein
and 12 ~o nucleic acid on a dry weight basis. D e l a n e y et al. z3~ r e p o r t e d that S. fi'agilis
N R R L Y-1109 g r o w n on ultrafiltered c h e d d a r cheese whey yielded a b i o m a s s with
37 2 ~o "'true p r o t e i n " and 5.7 ~o nucleic acid c o n t e n t on a dry weight basis. T h e
c o m p o s i t i o n s are s h o w n in Tables 10 and 11. R a t s fed on a diet c o n t a i n i n g S.fi'agilis
b i o m a s s as the sole source o f dietary protein s h o w e d a net weight increase o f
67.0 + 3.9 ( N P U carcass) for S.-fragilis and 73.5 + 3.0 for a casein control. H o w e v e r ,
the s u p p l e m e n t a t i o n o f S. fragilis b i o m a s s with D L - m e t h i o n i n e (2 g per 16 g N)
raised the N P U carcass value to 84.4 _ 2.8.
A pilot plant-scale tower fermenter has also been used to produce yeast protein
from lactose 15.87). Burgess reported lower yields (67 ~4 of the theoretical yield) using
the tower fermenter compared to the yield obtained using a stirred tank reactor
compared to the yield obtained using a stirred tank reactor (90 ~o of the theoretical
yield). Foda3] used Hansenula anomala, Candida lipolytica, Debaromyces hansenii,
and Candida utilis to synthesize proteins from sweet and salted cheese whey. The
addition of salt (sodium chloride) into the whey supernatants at the rate of 5.2~o
significantly decreased the yield of cells of all yeast strains. However, the supplemen-
tation of salted whey supernatants with ammonium sulfate at the rate of 0.5~o
significantly increased the yield of Debarono'ces hansenii. Under proper conditions,
D. hansenii could be used effectively to synthesize protein from salted cheese
whey.
It has been suggested 122~ that compared to yeast and bacteria, microfungi have
several advantages: they possess a better protein profile, recovery from the growth
medium is less difficult, and the fungi have a filamentous structure which facilitates
their use in the manufacture of textured foodstuffs without extraction and spinning.
Also, micro-fungi are already accepted as foods in many countries. The potential of
growing micro-fungi on whey and lactose permeate does exist. The use of bacteria
to ferment cheese whey has also been recommended 16). It has been reported that
bacteria have more rapid growth and metabolic rates than yeasts; they have simpler
nutritional requirements: and their use should eliminate the contamination which
often occurs in yeast propogations. Cell yields of 59.8~ o were obtained by using
Aeromonas hydrophila RH 726 with cottage cheese as a substrate. However, it should
be noted that the use of Aeromonas or Escherichia coli would be suitable for the
production of 13-galactosidase from whey rather than for single cell protein produc-
tion.
As stated earlier, methanol, methane, N-paraffins, gas, oil, ethanol, carbon dioxide,
and other waste materials in addition to cheese whey have been investigated for the
suitability as substrates for the production of single cell protein. A number of
problems associated with single cell protein have to be solved to render it a safe and
cheap source of protein for human use. Protein from microbial cells should be low in
cellular fragments to improve nutritional value, i.e. bioavailability of protein 12s)
The nucleic acid content should be reduced to minimize the intake of nucleic acid to
less than 2 g per day. If nucleic acid intake is too high, uric acid levels in the body
become elevated and may lead to gout formation in 10~ to 15~ of the population
that is susceptible to this condition. In addition, the protein should have acceptable
colour, flavour, and texture. As a food ingredient, the functional properties of single
cell protein are a prime consideration. For example, it has been reported that the
functional properties of protein isolates from the yeast S..~'agilis are inferior as
compared to soy protein isolates, The functional properties of single cell protein
would be affected by the choice of organism, substrate, and processing. Such factors
will also influence the costs.
Consumer attitudes towards the use of single cell protein in food will have to be
considered. Very little informa.tion exists on how consumers will react to single cell
protein as a human food. Some types of yeasts have been eaten for many years by a
small segment of the population, but acceptance has been limited. Labelling require-
ments and regulations have still to be established. The use of the term "Lactoprotein"
has b~en proposed for single cell protein derived from whey 94). It is beyond the
scope of this report to cover in detail information concerning harvesting techniques,
purification techniques, methods of reducing nucleic acid content, and economics in
relation to the specific organisms and substrates. Interested readers are referred to the
articles by Kihlberg 57), Waslien 139), Moo-Young 80), MacLaren 64), and Skinner 120)
4 Lactic Acid
Lactic acid is an established food acid. It is used mainly because it has a bland acid
taste, preserving properties, is naturally occurring in many foods, and is a liquid.
Some uses are:
a) in beverages such as soft drinks, mineral waters, and carbonated fruit juices, lactic
acid may be used to improve taste and flavour.
b) In beer manufacturing, lactic acid is used in various stages of operation for pH
adjustment and flavour improvement.
c) Lactic acid is used for the acidification of low-acid wines.
d) The use of buffered lactic acid solutions in sugar confectioneries has greatly
simplified manufacturing of high-boiled sweets. Direct acidification of the molten
confectionery syrups is feasible with buffered lactic acid solutions at high
temperatures, without the risk of increased sucrose inversion.
e) Lactic acid acts as a preservative of green olives and improves their flavour.
f) In sauerkraut, lactic acid is known to suppress undesirable processes and to
improve shelf life, colour and taste.
g) In dairy products, lactic acid has a number of applications. It can be used in
cheeses to adjust pH and improve flavour, texture, and stability. It is also used in
the production of directly acidified cottage cheese and other dairy products that
are normally manufactured using bacterial cultures. In unsalted butter, lactic
acid is used as a preservative.
h) Lactic acid and its salts are used in the curing of bacon to accelerate the
penetration of curing salts, thus reducing production time. It also acts a s a
preservative.
i) In egg white, lactic acid improves stability and whipping properties.
j) Lactic acid is used in the manufacturing of two types of food emulsifiers the
lactylated fatty acid esters of mono- and .di-glycerides (e.g. glycerolactopalmitate)
and the fatty acid lactylate (sodium and calcium-stearoly-2-1actylate).
Lactic acid is also used in a number of"0ther applications such as in condensed
milk; baking powder, puddings, jams, jellies, sugar confectioneries, and in Frankfurter-
type sausages. Ferrous lactate is used in infant foods and weaning foods, and in
animal feeds for calves, pigs, etc.
5 The Utilization of Whey Proteins

Casein, which makes up 78 ~o-85 ~ of milk proteins 25), is produced commercially by
acidifying skim milk to about pH 4.6 or to the isoelectric point of casein.
The manufacture of sodium caseinate involves the preparation of an aqueous colloidal
solution of acid casein at pH approximately 6.8 by addition of an alkali, usually sodium

hydroxide. The solution is then dried, preferentially by the use of spray driers, which
assure an improved flavonr and solubility of the final product 48~. Various uses of
casein and its derivatives are well established in the food industry. These include
the use of sodium caseinate as a fat emulsifier and water binder in processed
meats, powdered toppings, and cereals, and in the manufacture of milk substitutes
and coffee whiteners. Sodium caseinate's functionality is also applied in frozen desserts,
ice creams, filled and imitation milks, pie mixes, milk shakes, and other specialty food
products 11,48)
During the manufacture of cheese and casein, the coagulation fraction of milk is
recovered, which contains casein and most of the milk fat. The remaining liquid por-
tion, known as whey, contains water (about 93 ~ fat (0.1 ~o to 0.3 ~ whey proteins
o..
(0.9 5o to 1.0%), ash (0.5~ to 0.7/o), lactose (4.9% to 5.1 o{,), and lactic acid (0% to
0.2Oo) 90). Thus, the whey proteins, which make up 15~ to 22~ of the total milk
proteins, are recovered in the whey. The whey proteins are subdivided into two
major components, 13-lactoglobulin (7% to 1 ~o of total milk proteins), and ~-
lactalbumin (1.9 ~o to 3.3 %0 of total milk proteins). The typical composition of the
whey proteins is shown in Table 12.
Table 12. Composition of the whey proteins in

lactic casein whey 1o4~
Whey protein %
[3-Lactoglobulin 58
~-Lactalbumin 21
Immuno-globulin 10
Serum-albumin 5--8
Proteose-peptonefraction 5--6
5.1 Recovery of Whey Proteins
Lactalbumin can be recovered from whey by adjusting the pH to 4.5-5.2 and then
heating the whey to obtain a, coagulum containing lactalbumin 1~. ~o4~. However, the
products obtained by heating and denaturation are non-functional in their properties,
due to their poor solubility. A schematic of the process is shown in Fig. 4. The
resulting insoluble precipitates resemble casein in terms of composition and functional
properties. The product contains from 8%92 To protein and a variable amount of
ash, depending upon the washing steps of the preparation process.
Food applications for this protein product are similar to those for the other
insoluble milk protein products such as casein, Ca-caseinate, and coprecipitates.
It would be expected to have a slightly higher nutritional value than caseinates
and caseins, due to the high concentration of lysine and sulfur-containing amino
acids. However, the interaction of protein and lactose during the heating of whey
to form the co-precipitate could result in a product with minor colour and flavour
defects. Due to its low solubility and high water absorption and nutritional properties,
Sweet whey pH 6.0...6.5
~ . ~ ---- Residualwhey elimination

,[ (drinking water)
I .-- Residue[acid elimination

,earl g Cd}
60_. 7o~ ] cation
li I I eio,,i e woterl
exchange
L .,,,..Regeneration . . . . . .
], efftuenl ] i
L..,,.. Partially de- I i
eolian[zealwhey
1.0..ol.5
!
Acidulated whey pR 4.5
[ FlocculuSseparating ~ - SupernQtant
Fig. 4. lteat coagulation process for protein recovcry from cheddar whey ~o~
lactalbumin is being used in a variety of baked products, meat products, yogurt,

processed cheese, confectionery food and snack foods. Jelen s4) reported that a
lactalbumin-type whey protein product functions well as a ground meat extender.
These protein products have also been found to function well in fortifying macaroni
and similar pasta products which require that the protein remain insoluble ~ ' ,097
Panzer el al. 9v) developed a new. continuous lactalbumin process whereby whey is
adjusted to pH 6 and heated to 120 "C by steam injection to denature and precipitate
the whey proteins. The resulting lactalbumin contains 20- 25 }o ash, which is consider-
ably higher than for conventional lactalbumin. An optional modification was develop-
ed, in which the heated whey is adjusted to pH 4.6 to simultaneously aid in
protein precipitation and lower the ash content to 2-5 i~o.
Modlcr and Emmons 7~1developed a novel approach for preparing a lactalbumin-
type whey protein product which possesses a solubility that approaches that of whey
protein concentrate (WPC). The product was prepared by adjusting the pH of sweet
whey to 2.5-3.5 and heating at 90 :C for 15 minutes. Upon cooling the pH was
adjusting to 4.5 and the precipitated protein removed by centrift,gation. The
precipitate then was neutralized to pH 6.0 and spraydried. Recoveries of total protein
ranged from 63--743~ and from 35- 53 og for whey heated in the pH range of 2.5 to
3.5 with and without added iron (30 mg l-Z). WPC prepared by heating at pH 2.5
to 3.0 had a minimum solubility of 78~ This was reduced to 51 0;o when the pH
during heating was 3.5. Although the addition of iron to whole whey increased
protein recovery, solubility was reduced. Concentrating the whey prior to heating
greatly increased protein recovery but substantially reduced solubility. The viscosity
of spray-dried samples of acid-heat WPC reconstituted to 33 05 solids ranged from
4,000 to 36,000 centipoise, while commercial samples had viscosities of 400 to 1840
centipoise. Experimental samples of WPC gelled at protein concentrations of 2, 4, 6,
and 8 ~ when heated at 95 ~ for 20 minutes. All experimental samples had excellent
colour stability, while commercial samples darkened upon heating.
Heating below the isoelectric point appeared to enhance the waterbinding ca.pacity
and resulted in a more viscous product.
EIfagm and Wheelock 27) showed that at 74 ~ there is a greater degree of denatura-
tion of both ~z-lactalbumin and ]3-1actoglobulin in heated milk than in heated
whey. This fnding is consistent with the view that both ~.-lactalbumin and [3-1acto-
globulin are involved in complex formation with K-casein when milk is heated 116)
Another process to precipitate and recover lactalbumin on a continuous basis is the
centri-whey process, which recovers heat-precipitated whey proteins as a concentrate
for reincorporation into cheese milk. Heat-precipitated whey proteins have been used
for many years for the production of whey cheese in European and Scandinavian
countries ~o4)
Ultrafiltration is also used to recover whey proteins. The WPC's prepared by
ultrafiltration contained 68.40(, ]3-1actoglobulin, 21.3 o~ ~-lactoglobulin and 10.3~
serum proteins as separated on a polyacrylamide gel electrophoresis 24~. This is
approximately the same ratio of proteins as in whole milk. The ratio of individual
proteins in any ultra-filtered WPC is influenced by such factors as: (a) type and
source of whey, (b) rejection characteristics of the ultrafiltered membrane employed
and (c) degree of concentration achieved. The composition of WPC's prepared by
membrane filtration and related processes is shown in Tables 13 and 14. However,
commercially available WPC's contain 35 0.0-60 ~o protein.
5.2 Functional Properties of Whey Protein Concentrates

It is important for most food applications that WPC's be prepared with the
proteins in a soluble and functional state. Care is required in all processing steps
that involve heating, agitation, and pH adjustments to avoid protein denaturation
and concomitant loss of solubility.
After unfolding, denatured whey protein molecules undergo extensive protein-
protein interaction, which is a function of temperature, pH, and calcium content.
These interactions result in large molecular weight aggregates which impart excessive
turbidity and are, therefore, unsuitable for certain food and beverage applications 83).
WPC's prepared by mild processing conditions are essentially free of off-
flavours and odours. However, undesirable brown pigments and associated off-
flavours are produced during WPC processing and by Maillard interaction of
proteins with lactose if care is not taken to minimize heat treatment, especially in the
alkaline pH range. This Maillard browning reaction also reduces the nutritional value
of WPC through a reduction of available lysine.
46 N. Kosaric, Y. A~her
Table 13. Composition of whey protein concentrates 82)
Preparation process Number Lactose Ash NPN" Fat

of (~o) (%) (~.;) ( 0.4)
samples
Metaphosphate
complex
Range 3 54.1--58.0 12.7 1 3 . 2 10.6--15.5 1.1--1.3 3.3--7.2
Mean 55.7 13.0 13.7 1.2 5.3
Electrodialysis
Range 5 27.3--37.0 40.5--60.0 1.4-- 19.7 5.4--7.7 2.4--4.3
Mean 32.9 51.8 9.0 6.7 3.3
Ultrafiltration
Range 3 49.9--62.0 15.5--40.2 0.4--6.2 2.6--6.8 1.4--14.7
Mean 56.5 27.2 3.4 4.8 7.3
Sephadex
Range 2 38.7--45.1 -- --
Mean t41.9 24.9 11.5 4.9 0.8
Dialysis 1 66.0 26.2 2.0 1.5 2.0
CMC complex l 49.8 20.1 8.0 -- 1.2
Iron complex
Range 2 32.7--37.4 0.7--0.9 53.1--54.9 0.3--0.8
Mean 35.1 0.8 54.0 1.1 0.6
Expressed as percentage of total WPC N
Table 14. Elemental analysis of whey protein concentrates 82~
Preparation Number K Ca Na Fe AI Zn Cu
process of o; (ppm)
samples
Metaphosphate 1 b 1.05 -- 0.30 -- 34 14.1 47.9

complex
Electrodialysisa 2 1.03 2.20 0.69 3.22 144 16.0 16.0
Ultrafiltration 1 0.59 3.29 0.67 0.98 -- -- 59.l 80.7
Sephadex 2 0.94 2.89 0.75 1.91 -- 202 41.8 167.2
Dialysis 2 -- 2.54 0.39 0.63 -- 59 23.5 41.6
CMC complexa 1 -- 1.67 3.40 217 404 33.0 41.2
Iron complex~ 1 0.87 b 1.97 0.49 0.58 1079c 94 48.9 123.0
" Ash incompletely soluble in HCI/LiCI solution:

b Phosphorus content determined by the Sumner Colorimetric Procedure was 4.0 ~o (metaphosphate
complex) and 12.5 ~o tiron complex);
c Iron content by atomic absorption was 9.0~..0
A p r o t e i n a d d i t i v e s h o u l d m a i n t a i n o r e v e n e n h a n c e the q u a l i t y a n d a c c e p t a b i l i t y
o f the f o o d to w h i c h it is a d d e d . T h u s , a p a r t f r o m h a v i n g intrinsically s a t i s f a c t o r y
p r o p e r t i e s s u c h as n u t r i t i v e value, f l a v o u r , a n d c o l o u r , the p r o t e i n c o n c e n t r a t e s
s h o u l d p o s s e s s c e r t a i n f u n c t i o n a l p r o p e r t i e s w h i c h m a k e t h e m c o m p a t i b l e w i t h the
v a r i o u s f o o d s to w h i c h t h e y are to be a d d e d . T h e m o s t c o m m o n f u n c t i o n a l p r o p e r t i e s
a s c r i b e d t o W P C ' s are given in T a b l e 15.
Table 15. Important functional properties of whey protein concen-

trates 83~
Dispersibility/Solubility
Viscosity/Stabilization
Elasticity/Cohesion/Adhesion/Dough Properties
Emulsification
Foam expansion and stability
Water sorption
Gelation/Fibre fcrmation
5.2.1 Solubility
Solubility is an important functional property of WPC's. Complete solubility of
the proteins is a prerequisite for optimum functionality in foams, emulsions, beverages,
and similar applications. In some cases whey proteins may be soluble and in a non-
aggregated state so that they carl thoroughly mix with the other ingredients of the
formulation or orient at the interface of the emulsions or foams. WPC's prepared
by different processes exhibit a different protein solubility profile. Protein solubility
for iron complex, CMC complex and metaphosphate complex z4) WPC's were highly
dependent on pH, whereas protein solubility of other WPC's was essentially indepen-
dent of pH. WPC's prepared as metaphosphate and carboxymethyl cellulose complex
have serious limitations in functionality, especially at pH levels of 4.6-5.0. Reduced
solubility and excessive viscosity tend to limit their utility in a number of food applica-
tions83L The solubility of WPC's at different pH values is shown in Fig. 5.
12
Cr 8
f
=6
o
.E
I 9 Sephadex (U)
o
~ eLectrodTeLysis. 19
Fig. 5. SoLubility of WPC dispersed in
I * eledrodiQLysis, [10
I § electrodiQlysis, F35
pH 6.98 phosphate buffer (1.0 % protein
/ 9 meIQphosphate, Nc concentration). Dispersion was stirred
2 h, adjusted to pH from 2 to 9 with
o.._._.e~ " e / l o electrodiolysis, N250
1 N HCI or NaOH and centrifuged at
1,000 • g for 10 min. Supernatants were
examined for total N by micro-Kjel-
I I I I I I I I dahl
2 4 6 8 10
pft ,,-
Addition of sodium chloride markedly improved the solubility of metaphosphate

complex WPC by neutralizing the complex. Gel-filtered and ultrafiltered WPC's
have a solubility in the range of 90-98 ~o at a wide pH range (2-8). It has also
been reported that the addition of calcium chloride (0.3 M) improved the solubility
of WPC's at the iso-electric zone 42, 82,83) Hidalgo et al. 42) also reported that the
tryptic hydrolysis of the protein increased the heat stability of WPC's considerably.
5.2.2 Whippability and Foaming

Whippability is one of the attractive properties of WPC, and use of whey proteins as
egg white substitutes is greatly affected by this property. Richert, Morr and Cooney lo2)
reported on the effects of heat and other factors upon the foaming properties of
WPC. They found that WPC prepared by ultrafiltration had poor whipping properties.
They also showed that heating temperature and removal of the heat-acid coagulated
proteins greatly improved whippability.
Total solids content also influences foaming and foam stability 26.52). At a 10 ~o or
lower total solids level, whey protein produced foams with a lower drainage rate than
egg white used at the same level. Jelen reported an increase in foam stability, with
a decrease in overrum upon increase in total solids from 22.5~ to 38~. An
improvement in the foam stability with added sucrose or soluble starch was also
observed.
Hansen and Black 40) reported that adjustment of whey protein-CMC complex
solution to pH 9.5 improved whipping performance. They also found that treatment
Table 16. Comparison of foaming properties of different WPC products lo2)
WPC Product
Sephadex a Electro- Ultra- Polyphosphate Polyphosphate

dialyzed, filtration ~ precipitated * precipitated d
delactosed b
Protein content 40.3 35.0 66.0 54.2 76.2

(Vo)
Undenatured 88.6 94.4 55.8 30.0 102
protein ( ~)f
Whipping time 9--3 2.5--2.25 no foam no foam 8.0 no foam
(rain) g
Maximum 800 1290 1060-- 1300 -- 610----
overrun (~)~
Foam stability 4 15 10--17 -- 5----
(min)g
" Stauffer Chemical Co., Rochester, Minn;

b Foremost Foods Company, San Francisco, Calif;
Abcor, Inc., Cambridge, Mass;
d Borden Inc., Elgin, Ill. (sodium neutralized);
e Richert;
f ~ of total protein ;
g First number, unheated WPC dispersion value; second number, 60 ~ per 30 min heated WPC
dispersion value
of whey proteins with H2O 2 greatly improved the whippability. Jelen s2) reported a
retardation of the air uptake upon treatment of whey proteins with Ca(OH)>
Richert to2) reported that the overrum increased at low solids with increasing solids
content and it decreased at higher solids content. Foam stability generally appeared to
increase at higher solids content. A comparison of the foaming properties of different
WPC products is presented in Table 16.
It was also observed by Richert et al. lo2) that heating WPC at temperatures of
60 70 ~ greatly improved the foaming properties, while treatment at higher temper-
ature had a negative effect on the foaming properties. They also reported that the
optimum heat treatment appeared to be dependent upon pH. A statistically significant
interaction effect indicated that a mild heat treatment ( < 70 ~ was preferred at
pH 5.0, whereas higher temperatures ( > 80 ~ were preferred at pH 7.0. Cooney 2o~
found that ultracentrifuged WPC preparations did not improve in foaming properties
upon heating, which indicates possible lipid-protein association developed during the
heating process.
Mort et al. s2) reported that whipped topping mixes made with casein or non-fat
dry milk had higher overrum with better foam stability than those made with WPC.
It has also been reported that fat content over 30 ~ retards the whipping property of
WPC 247. Chang 17) studied the development of an egg white substitute from whey.
Liquid whey was reacted with sodium lauryl sulfate. The sodium lauryl sulfate content
was subsequently reduced to below 1.0 ~o by weight, to provide an edible product
having unique foam and heat setting properties that allow for its use in egg
meringues, as well as in other applications where egg white is traditionally used.
5.2.3 Gelation Properties

The property of foaming gel matrices capable of holding water is one of the
important functional properties of food ingredients. Gelation properties of WPC
are dependent on various factors such as ionic components, the physico-chemical
state of the protein as influenced by ionic environment and processing conditions,
and the degree of heat treatment employed to produce the gels ssl
Multiple regression analysis was used by Schmidt et al. t0s~ to obtain prediction
equations to measure individual and combination effects of CaCl2 and cysteine on gel
texture parameters (hardness, cohesiveness, and springiness) and on compressible
water of dialyzed WPC systems. Predicted maximum gel hardness occurred at
11.1 mM CaCl2 or 9.7 mM cysteine. Cysteine levels at 30 mM or above drastically
reduced gel hardness. Cohesiveness tended to decrease with addition of either reagent.
Reagent addition generally decreased gel springiness while compressible water
increased. Response surface contour predictions suggested that increases in CaCI2
or cysteine in reagent combination systems decreased hardness and increased com-
pressible water. Predicted cohesiveness and springiness maxima were at 13.9 mM
cysteine and 18.4raM CaC12 and at 10.3 mM cysteine and 10.0mM CaC12.
respectively. Significant x , ' x 2 interaction terms in the mathematical model for
combined reagent effects were observed on hardness, cohesiveness, and compressible
water in the WPC gel systems.
Delaney 24~ reported that skim milk containing 1.5 o whey protein produced a
custard-like gel which was free of leakage when heated at 85 ~ for 5 minutes.
Table 17. Water absorptive capacity of milk protein concentrates 24)
~/ o
HCl-casein 68
Na-caseinate 250
Low Ca-coprecipitate (insoluble) 75
Low Ca-coprecipitate (soluble) 260
Ultrafiltration whey protein concentrate 50
Gel filtration whey protein concentrate 62
Skim milk 65--75
Table 18. Effects of drying methods on the water-holding capacity of denatured whey protein
powders (25 ~ ss)
Whey protein Water-holding capacity
g H20 per 100 g Relative water Bulk density

protein a retention b, ~ g ml- 1
Freeze-dried 577 _+ 6 76.3 0.39

Roller-dried 253 + 7 33.5 0.55
Spray-dried 249 +_ 6 32.9 0.56
Air-dried 195 + 2 25.8 0.80
Fresh curd 756 • 2.5 100.0
a Mean and standard error of the mean quadruplicate determinations;

b As compared to the fresh curd
In contrast, almost twice as much egg albumin was required to achieve comparable
results. The W P C gels formed were not reversible either by temperature or pH.
5.2.4 Water Absorption

Water bound by denatured and undenatured protein is essentially the same 24)
The water absorptive capacity of W P C (and other milk protein concentrates) is
shown in Table 17. Due to lower water absorptive capacity, W P C ' s produce lower
viscosity even at higher concentrations (45 ~o-50 ~;) compared to sodium caseinate.
Thus W P C ' s at 50 ~o total solids can be spraydried without any difficulty 24~.
Modler 79~ reported that the water-holding capacity of reconstituted WPC ranged
from 0.95 to 1.68 (g g - l ) and was affected by two variables; heating after ultrafiltra-
tion increased water-holding capacity from the range of 0.95-1.55 to 1.42 1.68;
higher pH of heating (3.0 and 3.5) increased the water-holding capacity. Recovery
of protein nitrogen ranged from 79.3 to 86.00~; for samples heated at 90 ~ for
15 min (pH 2.5 3.5) prior to ultrafiltration.
Jelen et al. 55~ studied the effects of drying methods on the water-holding capacity
of denatured whey protein powders. The results are illustrated in Table 18. The
results o f this investigation confirmed the negative effect of drying on water-
]he Utilization of Cheese Whey and Its Components 51
holding capacity of heated whey proteins. Losses of water-holding capacity in spray-,

roller-, and air-dried powders were 67.1, 66.5, and 72.5 %, respectively, when compared
with the water-holding capacity of the fresh curd. However, freeze-drying resulted in
only a 23.7% 0 loss in the water-holding capacity. They also reported that the
microstructure of the freeze-dried powder resembled that of fresh curd. All other
drying processes were reported to have caused a severe compaction of the protein
network into a surface crust.
5.2.5 Emulsifying Capacity

In general, WPC's have a lower emulsion capacity than does casein under
identical conditions s2). However, the emulsifying capacity of different WPC's varied
considerably; in particular, CMC complex WPC showed significantly higher emulsify-
ing capacity than other formulations. Results are shown in Table 19. Delaney 24~
also reported that compared to soy proteins and skim milk proteins, WPC showed a
good emulsifying capacity. In addition, the emulsifying capacity of WPC was
maintained over a wide pH range.
Richert et al. 102) reported that the controlled denaturation of whey proteins at
specific points in the overall processing scheme, in the presence of polar lipids Ca + §
etc., could be used to markedly alter the functionality of WPC. Morr 84) stated
that the segregation of acidic polar groups from apolar groups within the caseinate
monomer units is undoubtedly the factor that permitted them to function better
than most proteins in those applications that require surface activity. Furthermore,
the globular conformation of whey proteins, which results from a uniform distribution
of polar and apolar groups along with the polypeptide chain and a relatively high
number of sulphhydryl disulphide groups, accounts for their susceptibility to
denaturation and intermolecular interactions that control their functionality 84)
Table 19. Emulsification and whipping properties of whey protein concentrates 82~
Preparation process Number of samples Emulsioncapacitya

(g)
Metaphosphate complex
Range 3 32--40
Mean 37
Electrodialysis
Range 5 39--42
Mean 41
Ultrafiltration
Range 3 36 49
Mean 40
Sephadex
Range 2 --
Mean 42
Dialysis 1 41
CMC complex 1 64
Iron complex
Range 2 26--41
Mean 34
a Gram corn oil required to produce infnite electrical resistance in the emulsion
According to M o r r 86) the following factors a p p e a r to be important in controlling

the emulsion properties of W P C :
a) conditions that control emulsification properties are similar to, but not identical
with, those that control whipping/foaming properties
b) experimental equipment for preparing the emulsion, e.g. Omni-mixer blender,
valve homogenizer type and pressure, and ultrasonic generator
c) time and intensity o f the emulsification process
d) ionic strength and composition o f the aqueous phase
e) type o f WPC, e.g. ultrafiltration W P C , chemical complex WPC, or other type
used.
The buffer capacities o f W P C ' s have been studied by M o r r et al. 82)
5.3 Applications of Whey Protein Concentrates in Food Manufacturing
W P C ' s are extensively produced by several major food companies. The chemical
composition o f selected commercial whey-based food ingredients is shown in Table 20.
The m a j o r processes developed on a pilot or commercial scale for recovering whey
proteins from whey include: ultrafiltration, gel filtration, metaphosphate complex,
carboxymethyl cellulose complex, and electrodialysis/lactose crystallization processes
to preferentially concentrate and fractionate the proteins from the lactose, minerals,
and other whey components.
Table20. Chemical composition of selected commercial whey-based food ingredients 55~

Product Typical analysis
%. Protein o/joLactose ~ Ash Suppliers
Products manufacturedfi'om whey alone

-Dried sweet whey 12 74 8.5 1,2,4,5,6
Partially demineralized whey 13 75 5.5 l
Demineralized whey 14 82 0.8 1
Demineralized/delactosed whey 36 56 2.4 1
Whey protein concentrate 53 36 4.0 2,3,4,5
Whey protein concentrate 85 4 1.2 4
Heated lactalbumin 80 5 2.5 4
Blends of whey with other materials

Whey, skim rnilk (and/or buttermilk) 22 54 10 2,6
Whey, caseinates 34 52 8.0 2,6
Whey, soy (and/or corn) solids 28 60a 8.0 2,6
Whey, soy protein isolate 35 52 8.0 2,6,7
a Total carbohydrate (contains CHO foam corn + soy).

1 = Foremost Foods Co.; 5 = Purity Cheese Co.;
2 = Dairyland Products; 6 = Land-O-Lakes Co.;
3 = Stauffer Chemical Co.: 7 = Ralston-Purina
4 = New Zealand Dairy Board;
Although WPC's generally have good solubility and function reasonably well in
certain food applications, they are not as functional in certain important food systems
as are caseinates and soy proteins 82~. For example, whey proteins do not function as
well as egg proteins in baked food applications because they do not form a sufficiently
strong foam structure to return loaf volume. However, they do contribute beneficial
attributes to such products as confections and related foods. Similarly, they are not
capable of stabilizing emulsions or foam as effectively as caseinates.
The relatively poor functional properties of WPC's in baked food applications are
probably due to their relatively low sulphydryl and disulphide content compared to
those of egg proteins. Thus, they may not be capable of forming a firm, 3-dimensional
structure to stabilize loaf or cake volume. Their inferior ability to stabilize emulsions
and foam is likely due to their compact, globular conformation, which does not
exhibit sufficient amphiphilic properties to orientate the water/oil or water/air
interface 84)
One potentially important area for WPC's in food formulations is in acid-type
foods, e.g. carbonated, fortified beverages. Further developments are possible in this
area,
WPC's prepared as metaphosphate and carboxymethyl cellulose complexes have
serious limitations in functionality, especially at pH levels approaching the isoelectric
point, e.g. at pH 4.6 5.0. Reduced solubility and excessive viscosity tend to limit
their utility in a number of food applications. WPC's prepared by electrodialysis/
lactose crystallization do not have a sufficiently high protein content for certain
food applications. However, such products do offer special utility in the formulation
of milk-based foods that compare closely with human milk.
Panzer et al. 9,-) obtained a low-moisture, free-flowing protein powder of high
nutritional value through the high temperature (120 :C) heat coagulation of cottage
cheese whey protein in a constant stirred reaction vessel using steam injection
heating. As stated earlier, heat recovered WPC's have limited use in the manufacturing
of many foods due to their insolubility and gritty character. On the other hand, the
native lactalbumin has some of the valuable functional properties required in functio-
nal protein ingredients. One of these properties includes its ability to coagulate and
to form complexes with other proteins when their solution is heated t44) The
coagulation temperature for this process can be lowered by the addition of reducing
agents such as sulphite.
Various uses of WPC are summarized in Table 21.
6 Summary
Annual whey production in the world is estimated to be 74 million tons, which
means that about 200,000 t of milk protein and 1.2 million t of lactose are transferred
into whey annually. Even through many uses of whey and whey solids have been
developed recently, only a little more than half of the available whey solids are utilized
in human food and animal feeds.
Considering the magnitude of this problem and the nutritive value of whey solids,
there is a need to develop new uses for whey and its derivatives in order to fully
realize the benefits of this wasted resource. This can be achieved through the develop-
Table 21. Uses of whey protein concentrate L/I
4:~
Product Level of utilization Method of recovery Functions and effect on the quality of the product Ref.
(%)
Ice cream 2-10 Ultrafiltration To replace 20 to 100% non-fat milk solids. At 100% ~2,s)
level of replacement cooked flavour and a higher
viscosity, as well as an overall adverse effect on
hardness, plasticity, flavour, colour and specific
gravity of the final product.
Meat products 5 % sodium and Heat/acid hnprove water/fat binding capacity without 46..s4. sb)
calcium WPC precipitation adversely affecting flavour and texture. Reduced
cooking losses.
Cheese products 0.3 to 0.6 % of Heat precipitation Stimulate proteolysis. Decreased firmness, density, 59)
cheese milk hardness. Enhanced organoleptic properties.
Ricotta cheese 25.1 to 35.9% Condensed whey producf To improve yield through the use of cheese whey 126~
total solids in condensed product.
whey product
Macaroni and pasta 3 20 % Heat precipitation Spray-dried Improve nutritional value. Bleached product. No 38.8a. J~o)
sweet whey significant effect on aroma, texture and flavour.
Snacks:
Tortilla 5-6% Heat precipitation overdrying Improve nutritional value. No adverse effect on Jo~)
organoleptic properties.
Cheese and bacon- 45.8 % WPC, calcium, sodium Improved nutritional value. No adverse effect on 50)
flavoured snacks and acid precipitates organoleptic properties.
Bases for chip dip, 65 82.1 ,~ Ricotta base Heat precipitation Improved nutritional value, reduced caloric content, l-~J) Z
salad dressings (moisture 74.9 %) Provide functional protein-rich base.
7~
Simulated or extended
meat products: 3'
Canned meat loaf- 14% Low-calcium milk Provide gel, emulsion and base for canned meat loaf- 127) ,.<
type product co-precipitates type product. No significant difference in general >
acceptability and improved sliceability. &
Extended beef patties 5 10~ Calcium milk Improved yield, reduced shrinkage. Improved 128)
co-precipitates appearance, flavour and texture with improved

acceptability.
Infant food formulations 25~,0~o Gel filtration Nutritional, biological 72)
O
Dried soups and 50 75 Flavour, body 7)
gravy bases
=r"
Dried culture media 85 97 ~,, m 72)

Nutrition. Rapid growth of cultures.
Bakery producl s 3-10 o4, High heat-treated, low-lactose" Flavour, texture, keeping quality 72)
whey solids
(..)
o
t.~t
ment of new products which would utilize a significant amount of whey or whey
solids, reformulating the existing products to incorporate whey and whey solids,
and fractionation and modification of whey solids to diversify their functional pro-
perties. Such modifications would render whey solids as potential ingredients in many
dairy and non-dairy processed foods. Several uses of whey proteins have been develop-
ed, which include production of softer cheeses, dairy spreads, chip dips, whipping
creams, manufacture of certain bakery products, enrichment of infant foods, etc.
However, lactose, which is approximately six times more concentrated in whey
than proteins, is not fully utilized. Factors which limit the utilization of lactose
fiaclude the lower level of sweetening power compared to fructose, sucrose and glucose,
poor digestibility, poor solubility, and tendency of crystallization. However, the
sweetening power of lactose can be increased considerably through its hydrolysis by
chemical, enzymatic or microbiological means. Further increase in the sweetness can
be achieved by converting glucose into fructose by glycose isomerase. Numerous
studies at laboratory levels and pilot-plant scale have been carried out. but wide-
spread commercialization of these processes is yet to be accomplished. Developments
could lead to new dairy and food products which would be sweetened partially or
fully with the hydrolyzed and isomerized lactose derivatives. Currently, most of the
products containing }nilk, non-fat milk solids or whey required additional conventio-
nal sugars because the sweetening power of lactose is not utilized. The above-stated
modifications would reduce the ingredient cost of the products containing lactose,
reduce the caloric content, and improve digestibility. Lactose can also be utilized as a
substrate for alcoholic fermentation. The potential of converting lactose into single
cell proteins is very good : however, the techniques of product processing and purifica-
tion are yet to be refined. Also, the development of food products based on single
cell proteins is in its infancy stage. Xanthan gum, which is utilized widely in many
food products as a stabilizer, is also synthesized from lactose. Further investigation
in this area would make it possible to produce naturally fermented dairy products,
which would contain microbiologically synthesized gums for use as food stabilizers.
As far as whey proteins are concerned, little progress has been in improving the
functionality of these proteins through chemical and enzymatic modifications. There
is a great need to improve the water absorption capacity of whey proteins and to
increase the viscosity of the systems at lower concentrations. Such developments would
permit the substitution of non-fat dry milk and casein derivatives by whey proteins.
Extensive research work has been carried out in developing whey-based drinks, and
some commercial products are available.
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Bioconversion of Hemicellulosics
R o b e r t J. M a g e e a n d N a i m K o s a r i c
C h e m i c a l a n d B i o c h e m i c a l E n g i n e e r i n g , F a c u l t y o f E n g i n e e r i n g Science,
T h e U n i v e r s i t y o f W e s t e r n O n t a r i o , L o n d o n , O n t a r i o , C a n a d a N 6 A 5B9
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2 Pentoses: Structure and Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.1 Structure of Hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.2 Potential Substrates and their Availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3 Bioconversion of Pentoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3. l Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.1.1 Mixed Acid-2,3-Butanediol Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.1.2 Acetone-Butanol-Ethanol Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2 Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2.1 Ethanol Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.2.2 Xylitol Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.3 Mycelial Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4 Isomerization and Pentulose Bioconversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.1 Xylose Isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.2 Xylulose Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.3 Two-Step Xylose Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
5 Hemicellulose Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.1 Hemicellulose Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.1.1 Autohydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.1.2 Alkaline Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.1.3 Selective Acid Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5.1.4 Enzymatic Hydrolysis: Hemicellulases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.2 Holocellulose Conversion Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.2.1 Ethanol from Corn Stover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5.2.2 Ethanol from Wheat Straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5.2.3 Ethanol from Wood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Hemicellulose carbohydrates comprise a significant proportion of available biomass resources such

as agricultural residues and wood wastes. These sugars, of which D-xylose is the most abundant,
can be converted to valuable fuels and chemical feedstocks by bacteria, yeasts and mycelial
fungi. Examination of the metabolism of pentoses by these microorganisms, reveals major differences
in both the initial reactions of sugar breakdown and the nature of the resultant endproducts.
Wide variability exists in the ability of microbes to utilize D-xylose and its ketose isomer,
D-xylulose. Methods for recovery of hemicellulose sugars include autohydrolysis, alkaline or dilute
acid extraction and enzymatic hydrolysis. Processes indicating the feasibility of ethanol manufacture
from corn stover, wheat straw and wood have been studied.
62 R.J. Magee, N. Kosaric
1 Introduction
While biomass has served as a substrate in microbial processes for the production
of alcoholic beverages for thousands of years, it has only been recently that
broader applications of this material have been envisaged. Thus it is that bio-
technologists are now developing efficient systems for the production of liquid fuels,
pharmaceuticals, foods and chemical feedstocks from "waste" organic materials.
Potential substrates include sugar crops (sugar cane, fodder beet, etc.), starchy
materials (corn, sweet potato, cassava, etc) and cellulosics (agricultural residues,
wood wastes, etc). Industrial processes using this latter material have traditionally
made use of only the hexose component of the holocellulose. Therefore the pentose
sugars, which may comprise as much as 40 ~o of the plant material 1), have in
most cases been wasted.
This study deals with the structure and availability of these hemicellulose carbohy-
drates, and with the biochemistry and process technology involved in their conversion
to valuable fuels and chemical feedstocks. Certain aspects of the above will be discussed
only briefly. In such cases, reference will be made to recent review articles which
deal extensively with the particular topic. For a review of ethanol production in
general, see Kosaric et al. 2)
2 Pentoses: Structure and Occurrence
2.1 Structure of Hemicellulose

The three major organic components of land-plant biomass are cellulose, lignin
and hemicellulose.
Cellulose is a high molecular weight, insoluble, linear polymer of [3-(1 ~ 4)-linked
D-glucose units. Sinapyl, coniferyl and p-coumaryl alcohols make up the random,
three-dimensional structure of lignin. This component is also insoluble and of high
molecular weight.
The hemicellulose component of biomass is composed of alkali-soluble, linear and
branched heteropolymers of D-xylose, L-arabinose, D-mannose, D-glucose, o-galac-
tose and D-glucuronic acid (homopolymers of xylose, mannose and galactose are
known to occur). In addition, the individual sugars may be methylated or
acetylated.
The majority of hemicelulose polysaccharides are derived from the cell wall middle
lamella. Playing no part in cellulose biosynthesis, they represent a distinct group
of plant polysaccharides 3)
Hemicellulose is commonly composed of two to six different sugar residues with
a degree of polymerization (d. p.) of approximately 200. Except for galactose-based
hemicelluloses with a [3-(1 ~ 3) linkage, most hemicelluloses are based on a [3-(1 ~ 4)
linkage of their constituent sugars. A D-xylose backbone with L-arabinose side chains
is the most abundant form. Wood xylans are characterized by a D-xylose backbone
with 4-0-methyl-D-glucuronic side chains. D-xylans comprise 15-30 % of annual
plants, 20-25% of hardwoods and 7-12~ of softwoods 3). "True" D-xylans,
composed exclusively of D-xylose subunits, are rare.
Bioconversion of Hemicellulosics 63
2.2 Potential Substrates and their Availability
F o r e c o n o m i c r e a s o n s , t h e s u b s t r a t e cost for a c o m m e r i c a l p r o c e s s m u s t b e r e d u c e d
to an absolute minimum.
F r o m T a b l e 1, it is e v i d e n t t h a t large q u a n t i t i e s o f c e r t a i n a g r i c u l t u r a l r e s i d u e s
are g e n e r a t e d a n n u a l l y o n a g l o b a l basis. I n m a n y o f t h e s e m a t e r i a l s , a s i g n i f i c a n t
p r o p o r t i o n o f t h e t o t a l c a r b o h y d r a t e c o n t e n t exists as p e n t o s a n s . T h e h e m i c e l l u l o s e
Table 1. Composition and availability of agricultural residues ""5,6)
Residue Global Composition (weight %)

availability
(1972) (106 t a -1) Glucose Xylose Arabinose Lignin Ash
Rice straw 180 39 13.9 4.3 9.9 12.4

Barley straw 53 37.5 15.0 3.96 13.8 10.8
Corn stover 150 37 13.9 3.0 15.1 4.3
Wheat straw 550 34.7 18.0 2.2 14.5 9.6
Wood residues 25 Pentosans
a) hardwood 60 23 28
b) softwood 53 7 23
Table 2. Carbohydrate composiuon of representative (North American) woods 7)
Wood species Composition ( % of extractive free wood)
Glucan Xylan Arabinan Uronic anhydride
Hardwood
Trembling aspen 57.3 16.0 0.4 3.3
Beech 47.5 17.5 0.5 4.8
White birch 44.7 24.6 0.5 4.6
Yellow birch 46.7 20.1 0.6 4.2
Red maple 46.6 17.3 0.5 3.5
Sugar maple 51.7 14.8 0.8 4.4
Sweet gum 39.4 17.5 0.3 --
White elm 53.2 11.5 0.6 3.6
Southern red oak 40.6 19.2 0.4 4.5
Softwood
Balsam fir 46.8 4.8 0.5 3.4
9Eastern white cedar 45.2 7.5 1.3 4.2
Eastern hemlock 45.3 4.0 0.6 3.3
Jack pine 45.6 7.1 1.4 3.9
White pine 44.5 6.3 1.2 4.0
Loblolly pine 45.0 6.8 1.7 3.8
Douglas fir 43.5 2.8 2.7 2.8
Black spruce 47.9 a 8.0 -- 4.1
White spruce 46.5 6.8 1.6 3.6
Tamarack 46.1 4.3 1.0 2.9
a including galactan
fraction of crop residues such as barley and wheat straw make up roughly 35 ~ of
the sugars present.
Logging wastes are also worthy of investigation as possible sources of biodegradable
sugars, as the pentosan content of many wood species is considerable. This is
especially true of hardwoods; white birch for example, containing 36 ~o of its total
carbohydrates as five-carbon sugars (Table 2).
An industrial waste of considerable potential as a biomass resource is waste
sulfite liquor (WSL). Some 100 x 106 metric tons of this material are produced
annually as a by-product of the world's pulp and paper operations s). From Table 3
it is apparent that hemicellulose sugars are a major component of WSL.
Thus it can be seen that biomass resources are available in sufficient quantity to
merit evaluation of their use in the manufacture of valuable products. The significant
proportion of these materials that are represented by hemicellulosic carbohydrates
in turn makes examination of pentosan conversion necessary.
Table 3. Chemical composition of organic dry substance in a

spent spruce sulfite liquor 91
Organic substance ~o
Lignosulfonic acids 43
Hemilignin compounds 12
Incompletely hydrolyzed hemicellulose compounds
and uronic acids 7
Monosaccharides
D-Glucose 2.6
D-Xylose 4.6
D-Mannose 11.0
D-Galactose 2.6
L-Arabinose 0.9
Acetic acid 6
Aldonic acids and substances not investigated 10
3 Bioconversion of Pentoses
3.1 B a c t e r i a
In contrast to processes conducted by yeasts, bacterial systems characteristically

generate multiple products. These may include alcohols (butanol, ethanol, isopropa-
nol, 2,3-butanediol); organic acids (acetic, butyric, formic and lactic acids); polyols
(arabitol, glycerol, xylitol); ketones (acetone); or. gases (methane, carbon dioxide,
hydrogen).
The metabolism of pentoses by bacteria has been reviewed by Horecker 101
The initial reaction may be one of three possibilities. In most species, the direct
isomerization of the aldopentose to its respective ketose occurs. Thus D-xylulose
is formed from D-xylose and L-arabinose is converted to L-ribulose. Oxidation-
reduction to the corresponding sugar alcohol (D-xylose ~ xylitol) is the first event
in conversions conducted by Enterobacter and Corynebacterium species. A third
possibility is the direct phosphorylation of the pentose, for example the reaction
to produce I>ribose-5-phosphate from I>ribose.
A summary of these initial routes of pentose metabolism and the key enzymes
involved is given in Fig. 1. It can be seen that D-xylulose-5-phosphate serves as a
common intermediate in the utilization of five-carbon sugars. Figure 2 shows how
this compound is subsequently converted into pyruvate through a combination of the
pentose phosphate and Embden-Meyerhof pathways.
The nature and relative yields of the bacterial products depend on both the
particular species involved and the precise conditions within the bioreactor.
D -Ribose kinase ~ D -Ribose-5 - Phosphclte
]somerase D-Ribu[ose kinase

D- Arclbinose 9 D-Ribu[ose-5-Phosphate
i
R[ bito[ q dehydrogenase T
D-Xylose isomerctse
D-Lyxose isomerose m- D-Xytulose kinase "l D-Xytu[~176

dehydroge~ l
D-Arabitol ~"
Xylito[ dehydrogenose
L-Arobinose isomerase ~ L-Ribulose kincase ~ L- Ribu[ose- 5- Phosphate
L- Xylose
isomerase
isomerase --'- L-Xytulose kinase

L- Lyxose ~ l_-• - 5 -Phosphate
L- Arabitol dehydrogenclse t
Fig. 1. Pathways of pentose and pentitol metabolism in Aerobacter aerogenes (Klebsiella pneumo-
niae) u~
3.1.1 Mixed Acid-2,3-Butanediol Formation
In addition to hexose, D-xylose and L-arabinose are also good substrates for the
mixed acid and 2,3-butanediol formation characteristics of the "enteric" group of
facultative anaerobes ,21, of which Aeromonas hydrophila, Bacillus polymyxa and
Klebsiella pneumoniae are representative species. The metabolic pathways and key
enzymes involved are illustrated in Fig. 3. Products of both o-xylose and D-glucose
degradations by these bacteria are listed in Table 4. Thus it is apparent that significant
yields of ethanol, butanediol and organic acids can be obtained from both five
and six carbon substrates. A review of butanediol production by K. pneumoniae
has been given by Jansen et al. lv~
2 Xylose 2Xytose 2 Xylose
1
2 Xylu[ose 2 Xylutose 2 Xylutose
2 AIP--~ 2 ATP ~ 2ATP--~J
2ADP~'~ 2ADP~T 2ADP-~'{
2 Xytutose- 5 -P 2 Xytu[ose- 5-P 2 Xy[u[ose- 5 - P
1
2 Ribulose-5-P
2 Ribose-5-P
I
_S
t
2 Sedoheptu[ose-7-P 2 Glycera[dehyde-3-P
I I
f
Z
2 Erythrose -4 - P
I
~C
T T
2 Glyceraidehyde-3-P 2 Fructose-6-P 2 Fructose- 6-P
I
4 Fructose-6-P
&A]'P--~
4- Fructose -1,6 - dip
4
10 G[yceratdehyde - 3 -P
10NAD + ~ 1 0 P i
10NADH2
10 1~3- Diphosphoglycerate
10 ADP'~
10ATP ~
10 3- Phosphog[yeerate
10 2 - Phosphogtycerate
10H20 4
10 Phosphoenolpyruvate
10ADP --~
IOATP -'~
10 Pyruvate
Fig. 2. Pathway for the conversion of xylose to pyruvic acid in bacteria

C02 NADH2 NAD
Phosphoenolpyruvate ~ 0 x a t o a c e t a t e ~ Z Matate
H2_ Ir ADP PI
CoA ~" NADH~ NAD L H20

C02""~ For m at e.~.. ~, l!' " ~ ATP (")l Fu m arate
--
/ dehydrogenase
r
Pyruvate
I
" ~ /_-Lactate
Lactate
I dehydrogenase
[/J'Pyr uvGte
~ NADH2
NAD
Acety[ - CoA /1 Succinate
CoA # I / ~ P' C O ~ ~,
" "~"~---NADH~[ to- Aceto[actate
Acetaldehyde [
dehydrogenase / ~
-~ I Acetotactate I
I
/ "NADj,~ decarboxyiase k
/ CoA-
/
//Acetyl- Phosphate Acetoin
~' [//~-ADP Acetoln /-" NADH2
AcetQ[dehyde [
AcetateI X reductase I "~ NAD
I kin~se ~ " ~ A T P
NADH2-.~]Ethanol Acetate 2,3-Butanedio[
NAD1 dehydrogenase
Ethanol Fig. 3. Pathways of mixed acid and 2,3-butanediol formation t3)
Table 4. Mixed acid and 2,3-butanediol from glucose and xylose (mmol product per 100 mmol sugar
used)
Product Aeromonas Bacillus Aerobacter

hydrophila ~4~ polymyxa xs~ indologenes t6j
Glucose Xylose Glucose Xylose Glucose Xylose
2,3-Butanediol 54.7 39.0 65.1 38.0 64.1 44

Acetoin 1.7 2.6 2.8 2.5 0.7 0.92
Ethanol 52.0 48.9 66.2 63.0 66.6 55.9
Acetic acid 4.6 9.3 2.9 7.7 1.0 11.4
Formic acid . . . . 27.9 26.3
Succinic acid 3.6 1.1 -- -- -- 5.5
Lactic acid 23.3 20.4 -- -- 3.0 5.2
Carbon dioxide 166.2 134.7 199.6 161.0 153 114
Hydrogen 57.5 53.9 70.9 82.0 27.6 19.0
% Carbon recovered 98.2 96.6 101.6 92.9 97.0 98.5
A n enteric bacterium isolated from decaying w o o d and tentatively named A U - l -

d3 181 also produces organic acids and 2,3-butanediol from pentoses. Results
o f initial studies completed with this organism are presented in Table 5. Later
investigations 19~ indicated that o p t i m u m conditions for alcohol (ethanol plus butan-
ediol) production were: initial xylose concentration of 7.5-8.0 %; p H 5.5 ; 32 ~ ; and
aeration at 0.01--0.02 V V M for batch culture. Sharp increases in acetic acid formation
leading to product inhibition were noted when p H exceeded 6.3. Acetic acid,
furfural and ethanol concentrations of 0.44, 0.15 and 1.62~163(w/v) respectively were
found to cause severe inhibition in the culture.
The effect of several cations (Mn 2+, Co 2+, Cu 2--, Ni z+ and Zn 2+) on product
distribution with AU-I-d3 was also examined. Manganese, added as MnSO4,
produced the most significant effect, improving both cell growth and product for-
mation. Comparison of Tables 5 and 6 reveals that supplementation of the culture
medium with manganese also resulted in a shift in microbial metabolism, with en-
hanced ethanol formation clearly evident.
Table 5. Products from 10% initial xylose with AU-I-d3

(after 75 h) 18~
Product wt o/of xylose consumeda
2,3-Butanediol 44.7
Ethanol 5.7
Acetic acid 4.9
Acetoin 5.5
" 81% of initial xylose was consumed in 75 h
Table 6. Effect of 10ppm MnSO4 on

product distribution of 7.8 os (w/v) initial
xylose by AU-I-d3 ~9~
Product Yield (~ w/v)
2,3-Butanediol 1.41
Ethanol 1.49
Acetic acid 1.05
Acetoin 0.04
When evaluating the benefits of 2,3-butanediol production, consideration must

be given to the following facts. Butanediol is less toxic to microbial systems than
other alcohols or ketones, and its energy content is comparable with other micro-
bially-produced liquid fuels. An equimolar mixture of ethanol and 2,3-butanediol
has an energy content of 27,700 kJ per kg, while pure ethanol, 2,3-butanediol and
methanol contain 29,100, 27,200 and 22,100 kJ per kg respectively 20). 2,3-Butanediol
may also be converted to methyl ethyl ketone by acid-catalyzed hydrolysis to yield
a higher grade fuel. In addition, this compound may serve as a feedstock for the
production of numerous valuable chemicals, including synthetic rubbers and poly-
urethane foams. A discussion of these various conversion possibilities is included
in a review by Long and Patrick 2l). Thus the production of 2,3-butanediol as an
alternative to traditional ethanol production has attracted considerable interest
in recent years 22-27~
Several species of the genus Closttqdium have been utilized for the conversion of
cellulose and hemicellulose to mixtures of ethanol plus lactic and actic acids. The
anaerobic thermophile C. thermosaccharolyticum, alone and in stable mixed culture
with C. thermoceltum, has been used for this purpose. While the xylanase activity of
C. thelvnocellum enables conversion of unhydrolyzed substrates, its inability to utilize
the pentose monomers formed makes co-culture necessar2~. Ethanol yields with the
latter system have been found to be 0.57 g per g of cellulose consumed zo)
Bacillus macerans (B. acetoethylicum) carries out a modified mixed acid process,
with ethanol, acetic acid, CO2, H 2 and acetone the major products. At a pH
optimum of approximately 6, 18~0 ~o of xylose consumed is converted to ethanol
with a further d~5 ~o broken down to acetone 5). Significant inhibition of both
rate and extent of product formation occurs when the initial xylose concentration
exceeds 4~ (w/v). At initial xylose levels of 2.3 and 1 ~ 91 and 98~ respectively
of the carbohydrate are consumed. At 0.8 g g 1 h-1, the specific ethanol productivity
of B. macerans is 10 times that of Fusarium oxvsporum, but only 1/4 that of
Saccharono, ces cerevisiae 5).
Semicontinuous processes with B. macerans are facilitated by the slime layer formed
by the organism which permits adherence of the cells to surfaces. This characteristic
thus enables the maintenance of large inoculums on inert materials within the bio-
reactor.
3.1.2 Acetone-Butanol-Ethanol Formation
Acetone, butanol and ethanol are the major products generated in "'ABE" formation,
of which bacteria of the genus Clostridia are the principle organisms involved.
Volesky and Szczesny 28) have recently published a review dealing specifically with
this subject.
In all of the metabolic categories discussed, pH plays a key role in directing
the nature of the end products. In butanediol processes, low pH favors the formation
of neutral products (ethanol, diol) while alkaline conditions lead to the accumulation
of organic acids in the broth. This trend is also evident in ABE formation:
high pH stimulating acetate production, solvents appearing under acidic contitions.
3.2 Y e a s t s
The bioconversion of xylose by yeasts has been extensively reviewed by Jeffries 291
Presented here is a brief overview of pentose utilization by yeasts in general, with
several specific examples which were not studied in the above paper or have
appeared since its publication.
Although many yeasts are capable of metabolizing pentose sugars via an oxidation-
reduction pathway (Fig. 4), of the more than 400 species examined by Barnett in
1976 30), none could consume these carbohydrates anaerobically. Two further studies
NADPH 2-dependent NADP-dependent

O-Xylose aldoreducta~e ' Xvlitol
" D-,:ylulose reductase
, O-Xylulose ki .... ~" D-Xvlulose-'~-P
- -
Fig. 4. Oxidation-reduction metabolism of D-xylose to D-xylulose-5-phosphate
have obtained similar observations 31.32~. Several strains of Schizosaccharomyces

pombe, however, have recently been identified by Gong et al. 33) that are capable of
ethanol formation under anaerobiosis. Further discussion of these organisms will
be given in Sect. 3.2.1 of this review.
The general routes of pentose metabolism by yeasts are shown in Fig. 5. Despite
the fact that several yeasts have been shown to possess the enzyme xylose isomerase 12),
the oxidation-reduction pathway appears to be obligatory in the breakdown of
A[doses Pentitols Ketoses Pentose phosphates
NADP NAD ?
L- Arab[nose dehydro- L-Arabinito[ dehydrogenase | Pentose cycle
genase
L-Xyiutose
~ I
NADP NAD(P} l
D- Xv[ose" dehydrogenase - Xytito[ ~,dehydrogenose l
NAD(P)
iso mere,se dehydrogenase ~'l l
NAD(P) kinase
D- Arabinose NADP
dehydro-
~- D-Arabi• dehydro_~ D-Xylutose ~ D-Xylulose-5-P
genase genase T
NADP 3- epimerase
dehydrogenose kinase
= D-Ribul.ose
IlL " D-Ribu[ose-5-P
NADP
dehydrogenase
NADP
D- Ri bose dehydrogenase = Ribito[ ]somerase
kinase
D - Ri bose - 5- Phosphate
Fig. 5. Pathways of pentose and pentitol metabolism in yeast ao)
five carbon sugars by these organisms. Aerobic conditions Would thus appear to be
mandatory for xylose utilization by yeasts, as the cofactor N A D P H required for the
initial oxidation or xylose to xylitol is most readily generated by isocitrate dehydro-
genase of the tricarboxylic acid cycle.
3.2.1 Ethanol Production
Several yeast species have demonstrated a capacity to produce ethanol from pentoses
under aerobic conditions. Examples include Candida tropicalis 34) and Candida sp. 35)
Direct conversion of pentoses to ethanol by a mutant strain Candida sp. XF217
has been studied by Gong et al. 35). A review of D-xylose metabolism by this and
other mutants of Candida sp. has been given by McCracken and Gong 36). Ethanol
production by XF217 and the native strain was greatest under "aerobic" conditions,
with the yield from xylose exceeding that from glucose in the case of the mutant
strain. Maximum ethanol production from xylose was, however, only 62 ,~ of that
obtained from glucose under anaerobic conditions. Formation of by-product xylitol
was considerably lower with the mutant strain. This is considered advantageous
in view of the fact that polyols have a negative effect upon ethanol yields with
Candida sp. There has been, however some interest expressed in optimizing production
of xylitol for commercial application (see Sect. 3.2.2).
In further studies of this mutant strain, Baillargeon et al. a7) examined the effect
of oxygen uptake rate (OUR) on ethanol productivity. By controlling dissolved
oxygen tension or agitation rate, these authors were able to vary the O U R between
2 and 32 mmol L -1 h - L Candida sp. XF217 was found to achieve a maximum
ethanol production rate of approximately 0.55 g L -a h -1 at an O U R of 9-12 mmol
Bioconversionof Hemicellulosics 71
L -1 h -1. It was also observed that at low OUR's, xylitol production rates were
increased, cell growth decreased and the organisms tended to become filamentous.
A yeast isolated from soil, Candida shetatae, has demonstrated the ability to
directly convert D-xylose to ethanol 381 under "oxygen-limited" conditions (1.5 L
medium in a 2 L bioreactor aerated at 0.5 L min -1, 350 rev min-l). Ethanol and
xylitol yields of 26 and 17.6gL -1, respectively were reported from 9~o (w/v)
O-xylose.
Margaritis and Bajpai 39) tested 8 strains of Kluyveromyces marxianus, and identi-
fied one of these as being able to effect 100 50 utilization of D-xylose within 48 hours.
The yield of ethanol obtained under aerobic conditions from 2 o(, (w/v) carbohydrate
was 5.6 g L -1 (55 ~ of theory).
The use of thermotolerant strains of microorganisms for the bioconversion of
cellulosics has several significant advantages: a) reduction of process cooling
requirements, b) enhanced product formation and substrate consumption rates, c)
reduced threat of contamination by mesophilic organisms, d) enhanced rates of
both cellulose hydrolysis and pentose isomerization. A thermotolerant strain of
Candida sp. has recently been isolated from sugarcane compost 40). This organism,
(named HT4), was found to consume 98.3 o / o f a 10 o(, (w/v) D-xylose solution within
4 days at a temperature of 45 ~ Yields of ethanol and xylitol were 1.27 and
3.29 ~ (w/v) respectively. HT4, in the presence of an immobilized whole-cell prepa-
ration of Bacillus sp., produced 19.7 g L -~ ethanol in 5 h from a hydrolyzate of
sugar cane bagasse hemicellulose (composition in weight o(,: D-xylose, 10.92;
D-glucose, 4.26; L-arabinose, 4.21).
A total of 49 strains from Candida, Saccharono,ces and Schizosaccharomyces
pombe were tested for product formation from D-xylose, L-arabinose, D-xylulose
and xylitol by Gong et al. 33). Two strains of S. pombe produced ethanol under anaero-
bic conditions (ATCC ~'s 2478 and 20130). Both produced 0.5% (w/v) ethanol
in 3 days from 5 }o D-xylose. Under aerobic conditions, no alcohol was produced
by 2478, and only 0.1 o; ethanol was formed by 20130. Aerobic metabolism of 5 ?/o
D-xylose by C. blankii (ATCC 18735) was found to result in the generation of 0.51 o/ "o
ethanol.
Pachysolen tannophilus has attracted great interest recently as a possible organism
of choice in hemicellulose conversion processes 41-5o). A brief review of ethanol
production by this organism has been given by Schneider et al. 51). Smiley and
Bolen so) screened five strains of this microbe for the presence of aldoreductase
(E.C. 1.1.1.21) and D-xylulose reductase (E.C. 1.1.1.9). Both enzymes were found in
all five strains, indicating that D-xylose metabolism in P. tannophilus occurs via the
oxidation-reduction pathway. A review of the biological and physiological properties
of this organism has been recently given by Kurtzman s2).
In batch studies, Slininger et al. 48) achieved ethanol yields of 0.34 g per g of pentose
consumed. The optimum pH for alcohol production by P. tannophilus was found to be
about 2.5 ; thus while culture contamination would as a result be minimized, corrosion
of process equipment could present a problem. These authors reported that severe
inhibition was produced by ethanol or xylose concentrations of 2.0 and 5.0 ~o (w/v)
respectiveley.
Veliky and Williams 531 reported that alcohol yields exceeding 90 % of theoretical
could eventually be achieved (after approximately 40 d) with immobilized cells of
P. tannophilus in a column reactor. Slininger et al. 4-9) found that 0.35 g of ethanol
per g of xylose consumed (69 ~ theoretical) was the best yield attainable with similarly
immobilized cultures in a Bellco continuous culture vessel. This latter group noted
that while the maximum specific ethanol production rate occurred when a feed
containing 2.8-3.5 ~o (w/v) was used, this substrate concentration resulted in a reduc-
tion of the ethanol yield to 42 ~ of theory. This observation could be explained in
part by the fact that the calcium alginate matrix employed became unstable at pH
values less than 3.5, so that the pH optimum of 2.5 could not be realized. In
addition, it was noted that only 40 ~ of the entrapped biomass was effective in
the formation of ethanol due to mass transfer limitations.
A mutant of P. tannophilus, selected for its ability to grow rapidly on D-galactose,
has been found to give high yields of ethanol from mixtures of five and six carbon
sugars 4x), In trials of the ability of this mutant to utilize synthetic media simu-
lating soft and hardwood spent sulfite liquors, ethanol yields of 90 and 83 ~o
respectivley were obtained. Lesser amounts of acetic acid, xylitol and arabitol were
also produced. When actual steam-stripped softwood SSL was employed as substrate,
ethanol yields were only slightly reduced, but the rate of the conversion was much
slower.
A comparison has been made of the abilities of P. tannophilus and C. tropicalis
to directly convert D-xylose to ethanol 42) This study found that while C. tropicalis
requires oxygen, P. tannophilus will produce ethanol under either aerobic or strictly
anaerobic conditions. P. tannophilus was able to yield ethanol in higher concentration
and over a wider range of culture conditions. When the two organisms were
immobilized in calcium alginate beads, continuous production of ethanol for a period
of 3 weeks from 0.5 M xylose was possible with P. tannophilus, while cells of C. tropi-
calis tended to leak from the support and clog the circulation lines of the bio-
reactor.
Mahmourides et al. 541 have isolated a mutant strain of P. tannophilus capable of
producing excess quantities of acetic acid from lignocellulosics (approximately 0.3 ~o
(w/v) from 1.5 ~o o-xylose).
Hsiao et al. 55) studied the sequential utilization of mixed monosaccharides by
several yeast species. In all cases, glucose was consumed first and was shown to inhibit
the metabolism of D-xylose, xylulose and xylitol. Following the addition of glucose,
a short transition period was observed before any inhibition of pentose metabolism
occurred. Thus it was believed that intracellular glucose (or a metaholite) was the
controlling agent. Competitive inhibition of the D-xylose transport system appears
to be the mechanism for this regulation 56)
Cell-free extracts of strains of brewers yeasts can convert D-ribose and o-ribose-5-
phosphate to ethanol and CO2 ~31, suggesting that the enzymatic potential to utilize
pentoses anaerobically is present in certain strains. The inability to do so in intact
cells must result from membrane impermeability and/or unfavourable patterns of
enzyme regulation. The former explanation is unlikely in view of observations that
xylose appears to enter S. eerevisiae by facilitated diffusion 57) and that active
uptake of o-xylose occurs in Rhodoturula gracilis 56,58-65).
Wang et al. 66) observed that Kluyveromyces lactis, Saceharono.'ees amureae and
Candida utilis were able to metabolize D-xylose aerobically but not in the absence
of oxygen, They proposed that some means of regulation prevented the conversion.
Bioconversion of Hernicellulosics 73
of D-xylose to D-xylulose and that this control might take the form of either (a)
regulation of the D-xylose transport system (by anoxic inhibition or via some mito-
chondrial factor), or (b) regulation of key enzymes (D-xylose reductase or xylitol
dehydrogenase) at the gene or gene product level.
Efforts are being made by several research groups to remove or circumvent this
metabolic control through genetic manipulation of the cultures. The inability of many
yeast species to utilize D-xylose anaerobically can be linked to a deficiency of the key
enzyme xylose isomerase. Based on evidence 67) that C. utilis contains this enzyme,
Suihko and Drazic 32) attempted to transfer it's activity to a yeast with higher ethanol
productivity (S. eerevisiae) via protoplast fusion techniques. The authors were
unsuccessful, however, in obtaining a stable fusion product. In a recent review article,
Schneider et al. 51~ discuss their attempts to transfer the gene for xylose isomerase
from E. coli to yeasts.
The advantages of simultaneous saccharification and bioconversion of lignocellu-
losics has lead to the search for microbes that possess both abilities. Cellobiase
activity is also desirable due to the fact that cellobiose has been shown to inhibit
cellulases 6sl. Of the more than 100 species of yeasts found by Barnett et al. 69~ to
be capable of assimilating cellobiose, only 8 could generate products from this
substrate, and none could convert D-xylose. Recently, however, Maleszka et al. 7o)
have discovered that Candida lusitaniae can produce 1.20 ~/(w/v) ethanol from a mix-
ture of D-xylose and D-cellobiose (2 o~ each) under semi-aerobic conditions. This
yield is greater than the sum of the products when the two substrates are separately
metabolized, and therefore indicates some form of synergistic effect.
3.2.2 Xylitol Production

While pentitols are usually treated as undesirable byproducts of five carbon sugar
utilization, and serious attempts at reducing their occurrence are made, systems aimed
at the production of xylitol may prove to be attractive.
Xylitol possesses certain properties that could make it valuable commercially. It is
already employed as a sweetener in some food products. Clinically, xylitol may be
useful as a sweetening agent suitable for diabetics. It can also be used in cases of
deficiency of the enzyme glucose-6-phosphate dehydrogenase 71). The anticariogenic
properties of this compound have been noted by Makinen 72)
While many yeast species produce xylitol in differing degrees as a by-product of
D-xylose metabolism (refer to Sect. 3.2.1), the level of its formation is generally
low. Several organisms have been identified, however, that produce xylitol in substan-
tial quantities.
Gong et al. 731discovered a mutant strain HXP2 of Candida tropicalis that generated
xylitol at greater than 90 o~ of the theoretical yield. In contrast, the native strain of this
species produces only 41.2~ o theoretical yield under identical conditions. Twelve
other species of yeasts were examined in this study. Species that were found to form
significant amounts of the pentitol include Trichosporon melibiosaceum ATCC 28580
(yields 42.9 ~o of theory) and Torulopsis candida ATCC 20214 (34.7 ~ yield).
In further studies 33), a strain of C. parapsilosis (ATCC 28474) was found to
achieve 100 ~o consumption ofa 5 }o D-xylose solution within 3 days, generating 3.32 o/
(w/v) xylitol, 0.42 O/o arabitol and no ethanol under aerobic conditions. Xylitol,
arabitol and ethanol yields when anaerobic growth was tested were 2.96, 0.28 and
0.27 ~ respectively.
A thermotolerant yeast, Candida sp. HT8, identified by McCracken et al. 4ol,
was found to consume 94.4 % of an initial 10 % D-xylose substrate within 4 days at
a temperature of 45 ~ End products of the process were xylitol (4.69 %) and
ethanol (0.76 %).
Several bacterial species have also been shown to produce o-xylitol as a major
product of pentose metabolism. Corynebacterium, for example, generated this polylol
when grown on a xylose-gluconate medium 74). A 3.33 % (w/v) yield of xylitol was
formed by Enterobacter liquefaciens when 10 ~ D-xylose was provided as the sole
carbon source 75). Further study 76) revealed that addition of N A D P H to the media
accelerated pentitol formation and repressed the isomerization of the xylose to
xylulose.
3.3 Mycelial Fungi

Fusarium oxysporum, alone and in combination with Saceharomyces cerevisiae, has
been studied as a potential producer of ethanol since the 1920's 77,78). Recent work
by several groups v9,so) has carried on this approach.
Ethanol and acetic acid are produced in equimolar quantities from pentoses by
F. oxysporum, as shown in Fig. 6. Growing cells of this organism have been shown
to generate larger quantities of ethanol and COz with much less acetate. Possible
explanations for this include (a) growing cells utilize a different metabolic pathway,
and (b) growing cells are capable of reducing acetate ~3)
..,acetyl-phosphate ~ Acetate
D-xylulose-5-P.~ Embden-
Glyceraldehyde-3-P ~Meycrhof
~ ~ Pyruvate ---,~ Ethanol
Fig. 6. Pentulose metabolism in Fusarium o.vysporum
In continuous culture with a 1 ~o xylose medium and a dilution rate of 0.0112 h,

F. oxysporum achieved 98.5 % utilization of the pentose. Increasing cell mass in the
reactor by a factor of 2.6 through cell recycle made increasing the dilution rate by
a similar degree possible. Ethanol productivity, however, was increased to only 0.07
from 0.04 g 1-1 h -1 (a 1.75 fold increase) by this action 5).
At 0.082 g g-~ h -1, the specific ethanol productivity of F. oxvsporum is less than
3 ~o that of S. cerevisiae: A further disadvantage of this organism is its extreme
sensitivity to lignins and phenolic compounds 12)
Other mycelial fungi of interest include Rhizopus sp. (which also converts pentoses to
mixtures of ethanol and acetic acid) and Mucor sp. (which produces ethanol only).
The preferred substrate of the Mueor species examined by Ueng and Gong 8o)
was glucose, although production of ethanol was seen from D-xylose, xylitol and
sucrose. No ethanol was formed from L-xylose, D-arabinose or c-arabitol, and only
trace amounts (less than 0.1 wt %) were detected from L-arabinose and D-arabitol.
These authors tested several species of Fusaria and Mucor for the ability to produce
ethanol from a hydrolysate of sugar cane hemicellulose. The sugar content of this
material was, ]fi % (w/v): D-xylose, 4.3; o-glucose, 1.4; and L-arabinose, 0.9. Best
yields were obtained by Mucor (approximately 1.8 ~o ethanol after 5 days). It was
concluded that the Fusaria were subject to inhibition by an unidentified component
of the hydrolysate.
It is hoped that the very slow rates of ethanol production with these organisms may
be overcome through the use of high cell density systems utilizing novel mycelial
columns.
A comparison of the kinetic parameters of the metabolism of xylose by representat-
ive species of yeast, bacteria and fungi is given in Table 7.
Table 7. Kinetic parameters of xylose degradation
Kinetic parameter Pachysolen Fusarium Bacillus

tannophilus oxysporum macerans
(RL-17I)
Specific growth rate,

(h -1) 0.017 0.024" 0.150"
Biomass yield,
Yx,s (g g-l) 0.169 0.41 0.050
Ethanol yield
Yp,'s(g g-l) 0.224 0.41 0.262
Specific ethanol
production rate, qp
(g g 1 h-l) 0.023 0.082 0.80
Reference Dekker s,) Batter and Wilke ~9) Rosenberg5)

a maximum specificgrowth rate (P,m~x)
4 Isomerization and Pentulose Bioconversion
4.1 Xylose Isomerase

The terms xylose isomerase and glucose isomerase are often used interchangeably in
the literature. According to Antrim s2), however, four separate enzymes have been
called "glucose isomerase". These four enzymes, their Enzyme Commission names
and their major characteristics are listed in Table 8.
The properties of the xylose isomerase isolated from Lactobacillus brevis have
been studied by, Yamanaka 83,s4~. Substrates for this enzyme were found to be
D-xylose, D-glucose and D-ribose, with Km values of 5 x 10 -3 M, 0.92 M and
0.67 M respectively. Activity depended upon the presence of Mn 2+ (Kin = 6.1
• i0 -6 M ) and the pH optimum was 6.0-7.0. Inhibitors included glucose, chelators
(EDTA), cations (Ni 2+, Zn 2+, Cu 2+, Cd 2+, Fe z+ and Fe3+), xylitol and o-sorbitol.
Xylose isomerase from Streptomyees albus requires M f + and Co 2 + (Kin values
of 3 x 10 -4 M and 3 • 10 -6 M respectively) 85). Optimum pH was found to be between
7.0 and 9.0 and temperature between 70 and 80 ~ Although this enzyme was
found to catalyse the isomerization of D-xylose, D-ribose, D-allose, L-arabinose,
L-rhamnose and D-glucose, only D-xylose was effective in inducing its synthesis.
76 R. J. Magee, N. Kosaric
Table 8. Enzymes frequently designated as glucose isomerase
Enzyme Characteristics
1) Xylose isomerase a) Absolute dependence on presence

(D-xylose ketol-isomerase, of a source of D-xylose for production of
E.C. 5.3.1.5) isomerase
b) Higher affinity for D-xylose than for
D-glucose
2) Glucose phosphate isomerase a) Possesses no xylose isomeras e activity
(D-glucose-6-phosphate ketoMsomerase,
E.C. 5.3.1.9)
3) Glucose isomerase a) Specific for D-glucose
(D-glucose ketol-isomerase,
E.C. 5.3.1.18)
4) Unclassified a) Mediates isomerization of both
glucose and mannose to fructose
The o p t i m u m p H and temperature for the isomerase from Candida utilis are
6.5 and 70 ~ respectively. M n 2+, Co 2+ and M g 2+ were found to enhance its
activity 67)
The equilibrium established by xylose isomerase is unfortunately far to the left.
Thus in a steady state condition, the m a j o r i t y o f the xylose will not be converted
to xylulose. Displacement o f the equilibrium in favor o f xylulose formation has
been found to be possible through the addition o f borate.
Thus Barker et al. s6) found that for aerobic conditions at a p H of 7.5, equilibrium
was established with isomerase from Pseudomonas hydrophila when the ratio o f
xylose to xylulose was 84:16. A d d i t i o n of borate shifted the equilibrium to 18.5:81.5
in favour o f xylulose. This effect o f borate is also evident in experiments conducted
with isomerase from Lactobacillus pentosus (Table 9).
Borate appears to exert its effect t h r o u g h preferential and stronger binding to
xylulose, thereby preventing reconversion to xylose. I f borate is a d d e d to a mixture o f
Table 9. Effect of borate on the formation of xylulose by xylose isomerase from Lactobacillus
pentosus 87)
Xylose concentration Borate concentration Xylulose formed
(gM) (gM) (/aM) %
5 0 0.36 7
5 2 1.23 24.5
5 5 2.0 40
5 20 2.25 45
20 0 2.9 14.5
20 2 4.3 21.5
20 5 5.0 25
20 20 6.4 32
80 0 12.7 16
80 20 17.5 22
xylose and xylulose in a bioreactor, the xylose will be preferentially utilized. Purifica-
tion of D-xylulose is thus possible for efficient conversion by yeasts to valuable
products.
Chiang et al. 88) have proposed such a process for the isomerization of D-xylose.
A schematic of their system is given in Fig. 7. The preferential assimilation of D-xylose
in the presence of D-xylulose characteristic of F. oxysporum is an important factor
in this purification method.
D-Xytose (700g [-1; 1000m[)

Enzymaticisomeriz~t[on[ 68~ pH7.0)
Immobilized whole cell Bacillus sp.
150m[ h-1 f[owrate
D-Xytose/Xylulose mixture {77:23)

I Vacuumevaporation
Ethanol extractiorr(750m[ EtOH, 4~
I
I n s ~ Vacuum~-
EthanoleVap~176 b'e
High- D-Xylose High- D-Xylulose

mixture mixture (25:75)
I
Isomerization " / Fusarium oxysporurn

(Bacillus sp.~0.3 M ]
sodiumtetraborate) D-Xytulose
|
Methano(extraction
D -Xytose / Xytulose ~l
mixture (77:23) D-Xytulose (95g)
Fig. 7. Schematic diagram for the preparation of D-xylulose from D-xylose 88)
4.2 Xylulose Utilization
While it is true that most yeasts are incapable of anaerobic utilization of D-xylose,
its ketose form is a good substrate for many species. Ueng et al. 891 tested 40 yeast
cultures for ethanol production from D-xylulose. Five metabolic types were identified
and are summarized in Table 10.
Schizosaccharomyces pombe grows rapidly on a 1 ~o (w/v) solution of D-xylulose,
achieving an optical density at stationary phase similar to that for growth on
D-glucose. In contrast, the optical density of S. cerevisiae and S. uvarum after 120 h
of growth on this substrate is only 1/3 to 1/4 that from D-glucose 9o1.
While growth rates may be similar, the rate of conversion of xylulose to ethanol
by Schizosaccharomyces pombe is slower than that of glucose, with completion in
48-72 vs. 3 6 4 8 h 66). The conversion efficiency, however, is found to be at least
85% if it is assumed that the net pathway is the formation of 10 moles of
ethanol and 10 moles of CO2 per 6 moles of xylulose-5-phosphate. Data from the
78 R. J. Magee, N. Kosaric
Table 10. Types of yeasts degrading D-xylose
Type Characteristics Example yeast(s)
A -- Good ethanol production from Saccharornyces

cerevisiae
D-glucose but low yield from
D-xylulose
- Good uptake of D-xylulose leading to
- Schizosaccharomyces pombe
ethanol production in good yield with
very small amounts of by-products
Good uptake of D-xylulose, C. melibiosica (ATCC 18738)
xylitol as major product S. cerevisiae (ATCC 24553)
S. rouxii (ATCC 32901)
D m
Major product: D-arabitol S. uvarum (ATCC 24556)
Saccharomyces sp. (ATCC 764)
E Major products: C. Magaii (ATCC 18364)
xylitol, ethanol and D-arabitol
Table ll. Batch of 5 5o D-xylulose in 250 ml shake flasks at 30 ~ by Schizosaccharomycespombe 89~
Strain Xylulose Ethanol Arabitol Xylitol

consumed produced produced produced
( ~ w/v) ( % w/v) (% w/v) (% w/v)
S. pombe ATCC 16979 4.2 1.1 0.1 0.2

S. pombe ATCC 24751 3.3 0.9 0.1 0.3
S. pombe ATCC 26192 3.3 0.9 0.1 0.1
breakdown of 5 ~ initial xylulose by three strains of S. pombe are presented in

Table 11.
The inability of petite mutants of S. cerevisiae to grow on D-xylulose 90), indicates
mitochondrial involvement in the metabolism of this pentulose. Phosphorylation of
D-xylulose to D-xylulose-5-phosphate may be the critical step determining which
yeasts can successfully catabolize pentose sugars 90). Phosphorylation may be mediated
by a kinase whose normal substrate is some other sugar. This theory is supported by
the observation that no lag phase occurs in the growth of cells on D-xylose that
have been started on D-glucose. It is also possible that certain strains possess a specific
D-xylulose kinase or that phosphorylation occurs during transport of the sugar. The
role of oxygen in the transport of pentoses is unclear; studies by Cahn 41~ have shown
that acylic polyols (e.g. xylitol) may enter S. cerevisiae by a rapid, non-active process.
The structural similarity of D-xylulose may enable its entry by the same route.
Cultivation of S. cerevisiae on a 50:50 mixture of D-xylose and D-xylulose has
been optimized by Chiang et al. 9~1. They found that a maximum ethanol production
rate was achieved with a cell density of 150 mg m1-1. The time required to effect
complete utilization of the pentose syrup was significantly prolonged at cell densities
below 100 mg ml 1. The optimum temperature and p H were found to be 37 ~
and 4 to 6 respectively. By-product formation was noticeably increased at temperatures
below 37 ~ and pH above 6. N o changes in the rate of ethanol production
were observed at D-xylose concentrations of up to 9~ (w/v). Thus there was no
evidence of D-xylulose transport inhibition, suggesting that different carriers exist for
xylose and xylulose. Ethanol, however, exerted strong end-product inhibition of the
system. The initial rate of ethanol production varied directly with D-xylose concen-
tration; however the amount of ethanol formed after 24 h remained essentially
constant at 13 g L-1.
The ability of several other yeasts to metabolize D-xylulose was also examined
in this study 91j. Thus K. lactis was found to produce 0.36~ (w/v) ethanol after
15-20d, S. amureae yielded 0.28% ethanoi in the same period, and C. utilis
formed 0.14 % alcohol in 30 d.
4.3 Two-Step Xylose Conversion

Processes which combine initial xylose isomerization with subsequent xylulose con-
version may provide solutions to the effective management of hemicellulose resources.
Such coupled processes could take the form of either single bioreactors or two vessels
in series.
Wang et al. 92) conducted 2-step conversions of D-xylose with K. lactis and
S. pombe. They tested isomerases from several sources: commercial free and immobiliz-
ed preparations (Maxazyme GI, Maxazyme GI Immobilized, Taka Sweet Immobiliz-
ed) and sonicates of Lactobacillus plantarum, L. brevis and Streptomyces albus.
Best results were obtained with the Maxazyme GI and the L. brevis sonicate. Ethanol
yield, however, was only 10 % of theoretical for a 5 % xylose solution at 29 ~ This
low yield may have resulted from (a) decreased cell viability after 2-3 d, (b) an increase
in proteolytic activity in the broth, (c) accumulation of Maillard Reaction products,
or (d) denaturation of the isomerase by the shaking of the flask. Increasing the
xylose concentration to 10-20 % did not enhance ethanol yields, largely as a result
of reduced cell viability. Some protection against proteolysis was afforded by the
presence of 1 o,
/O
bovine serum albumin in the media.
In a similar study 93), S. eerevisiae achieved complete utilization of D-xylose in
24 h, giving ethanol yields of approximately 80 o/ of theoretical. Data for ethanol
yields in trials with various sources of xylose isomerase are given in Table 12.
The ethanol produced by the system utilizing a whole cell preparation of Bacillus sp.
was increased to 1.8 % when the initial xylose and cell concentration were doubled at
a controlled pH and temperature of 40 ~ and 6.0 respectively.
Table 12. Effect of xylose isomerase on D-xyloseconversion by S. cerevis&e (ATCC 24860) 93~
Isomerase source Preparation Ethanol produced

(% w/v)
Control 0
A. missouriensis Cell free 1.81
Bacillus sp. Whole cell 1.30
Bacillus sp. Immobilized whole cell 1.26
Streptomyces sp. Cell free 1.95
S. olivaceus Immobilized whole cell 2.03
Conditions: flask cultures, 30 ~ 60 mg xylose per ml, 10 IU isomerase per ml, 48 h, non-aerated,
200 rpm, initial pH 5.8, initial cell density 2 x 108 cells per ml
Holocellulose
boil in 30 parts H20, 1 h precipitate = Fraction 1B
filter
filtrate ~ add 95 ~ ethanol ~ , filtrate add acetone ~ precipitate = Fraction IA
Residue 1
10 parts 2 ~ Sodium Carbonate, 48 h
filtrate filtrate _precipitate = Fraction H

filter add 95 ~ ethanol / " I ~ add H20 add e t h a n o l / ~
preeip, and HCI
and acetone.Nsalt s
Residue 2
10 parts 4 ~ N a O H at room temperature
l ./-,- filtrate
filter filtrate ~ add 95 ~o ethanol / , add H 2 0 add ethanol precipitate = Fraction III
l precip, and HC1 and acetone
Residue 3
10 parts boiling 1 0 ~ NaOH, 1 h
add 95 ~ ethanol precip. ~ add H20 , add ethanol precipitate = Fraction 1V

and HC1 and acetone
0
filtrate
Fig. 8. Flowsheet for alkaline extraction of hemicellulose

In experiments using sugar cane bagasse and wood chip hemicellulose as substrates,
Gong and Tsao 947 found that addition of xylose isomerase had no effect on the
amount of ethanol produced by Pachysoten tannophilus. This observation may be
explained by reports that P. tannophihts prefers D-xylose tO D-xylulose for ethanol
production 95).
5 Hemicellulose Utilization
5.1 Hemicellulose Extraction

5.1.1 Autohydrolysis
Due to its heterogeneous structure and relatively low degree of polymerization,
hemicellulose is much easier to hydrolyze than the crystalline cellulosic component of
biomass. Simple steam treatment, without the aid of acid catalysts ("autohydrolysis"),
has been found to be effective in many cases in liberating sugars from hemicellulosic
materials. As mentioned previously (Sect. 2.1), many of the sugars present in pentosans
are acetylated. In fact, approximately 60 % of xylose residues in typical hardwood
species contain an O-acetyl group 96) Autohydrolysis occurs at least in part via
acetic acid formed by the cleavage of these acetyl side chains. The weakly acidic
environment created in this manner appears to promote the observed hydrolysis.
5.1.2 Alkaline Hydrolysis
Separation of hemicellulose into two fractions, designated A and B, can be easily
achieved with an alkaline treatment 31. Extraction with 6 o/~alkali followed by neutrali-
zation results in the precipitation of hemicellulose A. This material is composed
mainly of L-arabino-D-xylans with some o-glucuronic acid residues. Fraction B,
with higher levels of lower molecular weight acidic polysaccharides, can be precipitated
from the remaining solution with ethanol.
Mitchell and Ritter 97) proposed a more complex scheme whereby five separate
fractions could be isolated from holocellulose. The procedure yielded lignin-free
extracts, thus eliminating the need for further refinement. By using increasingly
stronger solvents (Fig. 8), extraction of the hemicellulosics proceeded in order of
decreasing solubility. The composition of the five fractions obtained from sugar
maple holocellulose (74% of the extractive-free wood) is given in Table 13. It can
Table 13. Composition of hemicellulose fractions 97)
Fraction c',; of Uronic acid Xylan Methoxyl Acetyl Hexosan [~]o

hollo- anhydride ( ~ w/v) ( ~ w,;v) ( ?o w/v) ( ~o w/v)
cellulose ( % w/v)
IA 3.0 17.1 46.1 2.7 9.3 24./ .~

IB 1.6 15.8 48.7 2.3 9.2 24.0 --25
II 2.2 28.9 54.7 2.6 -- 13.7 --34
III 14.9 12.2 79.2 2.1 -- 6.5 --70
IV 3.5 9.3 80.9 2.3 -- 7.5 --83
be seen that 25.2 % of the holocellulose was extracted in total, with xylan accounting
for 71.4% of this material (18.0% of the holocellulose). This quantity of xylan
represents 13.3 % of the extractive-free wood (0.74 • 18 %). The process thus success-
fully removed 89.9 % of the xylan present in this wood species (Table 2).
In the University of Pennsylvania-General Electric Process, wood chips are
heated in an alkaline solution with water, sodium carbonate and butanol. Hemi-
cellulose dissolves into the aqueous phase, lignin is partitioned into the butanol phase,
and cellulose remains undissolved. Lignin can be precipitated from the butanol
upon cooling, and excess alcohol can be washed from the cellulose. While the
system is less corrosive than other alternatives and would therefore reduce equipment
costs, the expense of the butanol could prove prohibitive.
5.1.3 Selective Acid Hydrolysis

Current techniques of sugar extraction from biomass favour harsh conditions with
strong acids in which much of the hemicellulose content is lost, largely through
degradation to furfural. In attempts to increase the yield of valuable sugars, many
authors in recent years have stressed the need for selective removal of the hemicellulose
as a first step in the treatment of the substrate.
Initial, mild acid hydrolysis has several important advantages: it would (a) prevent
the decomposition of xylose to furfural (an inhibitory agent in microbial process),
(b) limit the production of microbial by-products (ie. xylitol, arabitol), (c) enhance
the susceptibility of cellulose to subsequent enzymatic or acid hydrolysis, (d) reduce
the requirement for expensive corrosion-proof equipment, and (e) avoid the environ-
mental problems incurred through the use of strong chemical treatments. Such a
process would thus serve both a recovery and a pretreatment function.
Ninety-four percent extraction of hemicellulose sugars (90 % D-xylose) was achieved
in 2 h from Southern Red Oak sawdust (12-40 mesh) by Lee et al. 98) using
0 20% H2SO4 at 170 ~ Better selectivity (i.e. decreased extraction of hexoses)
resulted from the use of 0.10% H2SO4. When 0.40~ acid was employed, the
maximum concentration of hemicellulose sugars was reached during the preheating
stage of the process (before 170 ~ achieved).
Limbaugh obtained an 83 % yield of xylose from oak hardwood within 90 min
using conditions of 0.20% H2SO4 and 150 ~ Glucose and furfural yields were
restricted to 17 and 8% respectively 99).
A general process for the separation and depolymerization of wood hemicelluloses
has been proposed Chambers et al. ~8). Isolation of pentose and lignocellulosic
solutions is achieved using mild acid treatment followed by washing and defibration
operations. A flowsheet of the scheme is presented in Fig. 9. Note that the positioning
of the defibration stage depends upon the operational characteristics of the diffusion
washing apparatus.
Sulfur dioxide can also be employed in the hydrolysis of hemicellulose sugars.
Saturation of a 1 : 10 solid: liquid ratio of hardwood hemicellulose with SOz at 10 psig
and 150 ~ resulted in 40 0/0 recovery of xylose while maintaining the glucose concen-
tration at near zero 98~. Increasing the reaction temperature to 170 ~ improved the
yield of xylose, but at the expense of selectivity. A major disadvantage of this
process is the high chemical cost.
The use of acetic acid for hemicellulose hydrolysis has also been tested 98). A 1 o/~,
Xylose Solution
t
~ Defibration II
wood -~ Pre- hydrolysis } I High I (option 1) I
hydrolysis 1-140 - 160~ -11-'1 temperature ---~ size reductionl
Water 10.02 - 0 06"/oHzS041 I washing I ~ increase mass II
L20 min JJl transfer rates J
Multistage t
cou ntercu rrent
diffusion
washing
....... L .....
I 1
I Defibration I
LLU on 21 JI
Fresh water -IFiItrat,oo

Lignocetlutose
(Ce[[ulose+Lignin matrix)
Fig. 9. Schematic of dilute acid hemicellulosehydrolysisand xylose-lignocelluloseseparation ~s~
solution at 170 ~ produced a 35% theoretical yield of xylose in 4 h. Increasing

the acetate concentration to 10 % reduced the yield, possibly due to decomposition of
the product.
The enhanced susceptibility of cellulose to enzymatic or acid hydrolysis following
mild hemicellulose extraction appears to result from structural changes produced
within the cellulose. The predominant effect of sulfuric acid is to increase the surf-
ace area of the cellulose. SO2 acts primarily to reduce the crystallinity of the
structure 98)
The kinetics of the acid hydrolysis of hemicellulose have been investigated by
several authors loo, lol). Three phases exist in the process. An initial random attack on
the hemicellulose chains by the acid results in the formation of oligomers of
varying degrees of polymerization. These are then split into monomers with
subsequent degradation to decomposition products (eg. furfural). The individual
reactions involved in the hydrolysis are generally first order loo~. From analysis of the
rate constants, the hydrolysis reactions occur much faster than the decomposition
steps, thus the observations of high sugar yields (frequently better than 80 %) and low
formation of degradation products are to be expected.
Compounds present in wood hydrolyzates that are inhibitory to microbial systems
can be produced from four potential sources, according to Leonard and Hajny lo21.
These are: a) sugar decomposition products (furfural, hydroxymethalfurfural, acetic
acid); b) soluble lignin products (phenols); c) wood extractives (resin acids, tannic
acid. terpenes, phenols); and d) heavy metal ions from equipment corrosion (iron,
chromium, nickel, copper).
Cahela et al. 103) maintain that a percolation reactor would be well suited for
hemicellulose hydrolysis. Advantages of this design (in which liquid flows through
a stationary bed of solids) include the following: sugars are removed from the
reactor as they are formed, minimizing the amount of degradation that can occur
and increasing yield; the high solid: liquid ratio characteristic of this reactor
configuration permits the production of a concentrated sugar solution; solid-liquid
separation is not required following hydrolysis. Burton gives a detailed description of
a pilot-scale percolation unit with a 220 L working volume lO4).The reactor can effect
the hydrolysis of 70 kg of wood per batch, giving an operational capacity of 0.5 t
per day. For a discussion of the composition of the resultant hydrolyzate and its
degradability, refer to Sect. 5.2.3.
5.1.4 Enzymatic Hydrolysis: Hemicellulases

Enzymatic hydrolysis is a fourth alternative for pentose recovery. The main advantages
of this approach are greater specificity and reduced formation of degradation
products. Important drawbacks include high cost and slow rates of hydrolysis.
Hemicellulases (glycan hydrolases, E.C. 3.2.1) effect the specific hydrolysis of hemi-
celluloses. Included in this group are the xylanases, which cleave the [3-(I~4)-D-
xylopyranosyl linkages of [3-(l~4)-D-xylans. Separation by gel permeation and ion
exchange chromatography has revealed the existence of at least 15 different enzymes
in the xylanase complex, nearly all of which break xylan down to xylose. The
molecular weights of these proteins range from 17,000 to 50,000 daltons lo5)
Hydrolysis of hemicellulosics by xylanase and [3-xylosidase isolated from Tricho-
derma reesei has been studied by Dekker 106). Optimum temperature and pH for both
enzymes was found to be 55-60 ~ and 4~5 respectively. Xylanase was completely
inactivated within 1 h at 65 ~ 30 to 40 ~o hydrolysis of heteroxylan was achieved in
24 h by these enzymes.
Linko 107) tested immobilized xylanases from Trichoderma reesei, Aspergillus
foetidus and Bacillus subtilis for their ability to hydrolize the hemicellulose component
of waste sulfite liquor (WSL). Immobilization was accomplished by adsorption and
cross-linking to phenol-formaldehyde resin. Almost complete hydrolysis was reported
with a plug-flow reactor at a flow rate of 1 bed volume per h at 40 ~ and
pH 4.5. Enzyme half-life in the reactor was found to be 30 days.
A strain of Aspergillus niger with enhanced xylanolytic activity has been isolated
by Conrad 10s>. Near complete hydrolysis of arabinoxylan was effected within 24 h
by a crude enzyme preparation from this species.
An organism capable of simultaneous production of xylanase and cellulase enzymes
would of course be desirable for the hydrolysis of holocellulose. Rickard and Peiris 109)
have identified a mutant Cellulomonas strain with enhanced activity in both classes.
5.2 Holocellulose Conversion Processes
The following examples show that utilization of the total carbohydrate content
of biomass is indeed feasible. In the first process, selective acid hydrolysis of corn
stover yields separate glucose and xylose-rich streams for ethanol formation. Three
pretreatment options for wheat straw are presented in the second example. Again,
utilization of resultant pentose and hexose-rich streams is conducted by separate

microbial systems. In the final investigation, conversion of wood hydrolysates (both
hard and softwood species) is examined. All of these substrates have been shown to
posses significant potential for the manufacture of liquid fuels and/or chemical
feedstocks, in terms of both composition and availability (Tables 1 and 2).
5.2.1 Ethanol from Corn Stover

Initial studies of a conversion scheme developed by Sitton et al. ttol indicate that
188 kg of ethanol could be produced per ton of corn residue. At a ~redicted
Prehydrolysis tank
~,, /+.4_~176
H2SOL
Cornstcflks ', [ ~]
220t/day .I ". I H qr~
Recyclewater / ' /~ -2 ~_ .
U00~ / I~I~Sk~h I I 0.8O~176
Xylose
\ / 4 _ ~ ~ 1 7 6 1 7 6 Gluc~
o,74~ xylose ~ ........
0.18~ I / / (129 m3h-1 )
dLZ, [ I/~ ~ /+.35"/o Glucose
Fi[ter Ps Elect rodi olysis ( 4-7.2 m 3 h-1 }
h ~ unit
~Rotary dryer
L.04~ Glucose
"~ 80cC 110oc
l ' Hydrolysis tank
",8.00/0 H2SQ '
Recycle
acid
(85~ Impregnat~ I X /
I ~/3.89~ f
Recycle water
Glucos,~
Fi [ter
L Sotids
[/+7.8m3h-1) J (2100kgh -1 )
Fig. 10. Process for the acid hydrolysisof cornstalks ii01
Cells to
recycle
C~centrifuge
Fixed film of ~ 125% ~ o

Eoxysporum I V I / ~ 95/o ETOH
I/XI ; ,~4. "1 c2.2m3 h-1
r-----7 0.80%~ ~35oc/~9ooc
Corr, stalks I Acid I Xyiose , ~ I I Distillation
~-I hydrotysis" ~ , ~ ~ '__-_-_~ . . . . . . . . . . . I
Recyclewoter Jprocess ~ ......... ',]uu t. ]0.5O/o i I column
:
| Fixed film of H v,, I 12-1%- ~ la

/ S-cerev[s/'oe/~ L'etts 25 C ETOH J~
/ ~ Water recycle
L.35% GlucoseI47.gm3 h-1 ) 100~

Fig. 11. Process for the conversion of cornstalk hydrolysate to ethanol ~10~
consumption rate of 220 t of cornstalks per day, the process would generate
41,360 kg of alcohol daily. Schematics o f the design, complete with material balances,
are given in Figs. 10 and 11.
In a two-stage acid hydrolysis, ground corn stover is first treated with 4.4 ~/o H2SO4
at 100 ~ for a period of 50 minutes. The resultant xylose-rich liquor is separated by
filtration, and then dialyzed to recover acid. The solids remaining from the first
hydrolysis step are dried before impregnation with 85 }~ sulfuric acid. Water is added
to achieve an acid concentration of 8 %, and second stage hydrolysis is carried out at
110 ~ for approximately 10 min. Dialysis of the glucose-rich stream removed by
filtration of the product permits further acid recovery. The solids generated
(2100 kg h - I ) can either be returned to the soil as fertilizer, or used as a source of
fuel.
Separate microbial systems are employed for alcohol production from the glucose
and xylose-rich streams. A fixed film reactor containing S. cerevisiae is suggested
for hexose conversion, while the pentose mixture is metabolized by immobilized
C. utilis. The feeds for both reactors are cooled to 25 ~ by exchanging heat with
the product streams. Yeast cells in the reactor effluent are recovered by centrifuga-
tion. Nutrients and vitamins released by the autolysis of these cells are then returned
to the culture. Cell overgrowth is removed every two weeks from the columns by
sparging with compressed (30 psig) CO2. Ethanol in the broths is concentrated to
Table 14. Capital investment for 1.2 x 10 6 L a - ' ethanol from cornstalks process 110~
Equipment Investment (1979)

$US
Hydrolysis tanks
1 -- 115,000 L 89,200
1 -- 17,000 L 55,800
Rotary vacuum filters
2 -- 1,300 m 394.000
Rotary dryer
8,200 kg h -1 277,700
Impregnator
309,000 kg d- l 334,500
Bioreactors
2 -- 3.8x 15.2 m 512,100
Centrifuges
2 -- Helical conveyor 389,900
Distillation column
Reboiler and condenser 670,200
Acid recovery (electrodialysis unit) 2,472.000
Miscellaneous
Heat exchangers 113,200
Pumps and piping 253,300
Heaters 203,500
Compressor and tanks 27,700
Sub-total $5,793,100
30 o~sContingency 1,737,900
Total $7,531,000
Table 15. Operating costs for ethanol production from corn-

stalks 110~
Item Dollars per year
Cornstalks $1,825,000
Acid recovery 300,700
Make up acid 421,700
Utilities 381,200
Neutralizer 265,000
Yeast extract 256,000
Labor 409,400
Maintenance 409,400
Depreciation, taxes, insurance 818,800
Total $5,087,200
Production (L a 1) 1.2 • 106

Breakeven price ($ L -1) $0.299 L-'
95 o~ in a single distillation column. Further concentration could be achieved

through the use of additional extractive distillation columns using benzene.
An economic analysis of this process was conducted by Sitton et al. tl0~
Principle findings are summarized in Tables 14 and 15. At a substrate cost of
$25 per ton (36%o of operational expenses), the breakeven price of ethanol for a
plant producing 1.19 • 106 L a -1, was found to be $0.299 L. This analysis does not
include the assignment of a credit for the biomass by-product of the operation
which could be sold as animal feed or fertilizer.
5.2.2 Ethanol from Wheat Straw

Detroy et al. 111.112) have designed a process for ethanol production from wheat
straw. Three methods of straw pretreatment were proposed: autohydrolysis with
subsequent ethanol extraction, autohydrolysis followed by ether extraction, and alkali
extraction of the substrate. Flowsheets of these options are presented in Figs. 12 and
13. Both autohydrolysis and alkali extraction generate a cellulosic pulp which contains
approximately 100 ~ of the cellulose found in the crude substrate. Slightly less than
half (45 Or/oand 43 o/~
o respectively) of the original pentosan is also found in this pulp.
41 o/,~of wheat straw pentosans are extracted by autohydrolysis and are recovered ,in
the liquor solids. The lignin content of this material is only 29 % of that of the initial
substrate. Ethanol extraction of the liquor solids successfully removes 71%0 of the
remaining lignins which might otherwise be inhibitory to the microbes involved in
sugar conversion. However, a 25 0% loss of pentosans is also incurred in this step.
Ether extraction, on the other hand, achieves only 70~ recovery of the pentose
sugars present in the liquor solids.
In further assessment of the suitability of each of these pretreatment methods,
trials were conducted to test the ability of Pachysolen tannophilusto produce ethanol
from the hemicellulosic hydrolyzates 111). Incomplete utilization of the sugars in the
product derived by autohydrolysis and ethanol extraction of wheat straw was noted.
63% of the xylose (initial concentration: 43 g L -~) remained after 8 d of growth.
Ethanol was, however, produced with a yield of 10 g L-1. Fermentation of the ether-
Wheat straw lOOg I

1
Cellulose 33g
Pentosans 29g
Lignin ILg
Ash 9g
Autohydrolysis
1 h~ 165~
7:1 ( [iq :sol)
f f
I CelluLosic pulp ,69g I I Liquor solids, 27g
Celtulose 33g
Pentosans 13g Pentosans 12g
Lignin 10g Lignin Lg
Ash 5g Ash 5g
Extraction with 5% H2SOz,
3h, 96~
70% ethanol
l
[ Hydro[yzate
I Resldue
I Ether
Pentoscms 9g wash
Lignin 1g
Ash 3g
I Extract, 2g ] t Xylose, 8g
5~ H2SO L
3 h, gT~
pH Gdjusted
~to 4.5 HCI)
] Sugar solution ]
(Precipitate + [ign[n)
Fig. 12. Autohydrolysisof wheat straw
extracted substrate did result in complete utilization of the xylose (initially 48 g L a)

after 7 d, but a lag period of 2-3 d was observed and ethanol yields were limited
to 6 g L 1. Complete consumption of the alkali-extracted sugars was achieved by
P. tannophilus within 6 d with no evident lag phase (initial xylose concentration:
47 g L-a). This was concluded to be the treatment of choice, yielding 8.2 g L -1
ethanol.
The cellulosic pulp obtained in the initial phase of the extraction methods was
treated with cellulase from Trichoderma viride, and the monosaccharides liberated
were subsequently converted to ethanol in a separate bioreactor with S. uvarum.
The combined yield of alcohol from both cellulosic and hemicellulosic fractions
was thus expected to be at least 104 g per kg of wheat straw m )
5.2.3 Ethanol from Wood

Deverell n3) has evaluated the ability of hydrolysates from a percolation reactor lO4)
to support ethanol formation. Two species of wood, a hardwood aspen (Populus
tremuloides), and a softwood (Pinus radiata), have been examined. A schematic of the
I Wheat straw,lOOg I
Wiley Mit~ ( 4 m m screen)
Extracted with &% NaCH (w/vl
overnight at room temperature
Fittered and washed
I Cellulosic pulp,60g J I Combined fittrate J

Cellulose 32.5 g pH adjusted to 5 (HC[)
Pentosans 12.5 g Ethanol added to
Lignin 5.0g give ethanol : solids
Ash 2.5 g =1.5:1
IPrecipitate J
Filtered
Dehydrated by
ethanol extraction
Dried
J Hemice[lulose precipitate , 18.3g
Dilute H2SOL
hydrotysis
Fig. 13. Alkaline extraction of wheat straw I Xylos%3.5g [
Wood F ~
~ Sutphuric acid
F ash& wa' r 9 i
E
_~f-]Geo- ~ T
IthermaFl~ IIIP
I t+ I----~
[~ 6 11
I Sulphuric acid tank 7 Flash vessel

2 Water tank 8 Vapourcondensor
3 Electric water heater 9 Condensatetank
Z, Geotherma[ heat exchanger 10 Flash vessel
5 Hydrolysisreactor 11 Hydro[ysatetank
5 Residuetank
Fig. 14. Forest Research Institute wood hydrolysis plant -- hydrolysis section ~o4~
Calcium
hydroxide
Sodium
Isu[phite
// Steam ~ i Flash ,! Carbon
tt ~ vapours oxide
coo,,
Hydrolysote -~~ 4, ~-~---~ Beer

Yeast
1
1 Hydrotysate tank ~ I I
2 Treatment vessel 7 Centrifuge I I
3 Flash vessel 8 Distillation column 6 steam ~
4 Filter 9 Ethanot tank 10~----~
5 Fermentation vessel 10 Stillage tank i i
6 Yeast tank 11 Filter residue
Fig. 15. Forest Research Institute wood hydrolysis plant - - fermentation and ethanol recovery
section to4)
hydrolysis process is presented in Fig. 14. During percolation, wood chips are exposed
to dilute sulfuric acid (approximately 0.5 %) at a temperature of approximately 185 ~
Prior to innoculation, the hydrolysate is first neutralized with calcium hydroxide to
a pH 5, then filtered and finally treated with sodium sulfite and heated for 20 min at
135 ~ (Fig. 15).
Because of the significant compositional differences of the hydrolysates obtained
from the two wood species, it was recommended that separate schemes for bioconver-
sion of the sugars be developed. As would be expected for hardwoods (Table 2),
pentose sugars made up a large proportion of the total sugar content of the
aspen hydrolysate (31.8 %; 27.3 % D-xylose and 4.5 % arabinose). Glucose accounted
for 57.4 % of the carbohydrate present, while minor quantities of galactose and
mannose were also detected. In contrast, only 13 % of the sugars in the softwood
hydrolysate were pentosans, and glucose made up the remaining 87 %.
A single treatment of the Populus tremuloides hydrolysate with Pachysolen tanno-
phihts was recommended in view of the high proportion of five carbon sugars in the
substrate. After 35 h, utilization of 96 % of the D-xylose, and almost all of the glucose
and mannose was achieved. The ethanol yield, at 7.1 g L-1, was 25 % better than
could be obtained using conventional brewer's yeasts. Ethanol productivity was 84 %
of theoretical (0.43 g per g of carbohydrate consumed). Xylitol and acetic acid were
also produced, both at a concentration of 0.6 g L -1.
A two stage process was implemented for more effective bioconversion of the
Pinus radiata hydrolysate. Initially, the substrate was innoculated with S. cerevisisae.
A 24 h batch at 30 ~ resulted in the accumulation of 16.5 g L -1 ethanol. Centrifuga-
tion of the broth produced a "beer" containing 7.8 g L-~ of unconsumed carbohydra-
tes. Pentosans and galactose accounted for 69.2 % and 26.9 % of these sugars, respecti-
vely. Innoculation o f the " b e e r " with P. tannophilus followed supplementation of the
solution with additional urea and sodium dihydrogen phosphate. A n improvement
in ethanol yield of 9 % resulted, giving a final ethanol concentration of 18 g L - I .
Productivity of the P. tannophilus was thus 0.33 g g - 1 (65 ~ of theoretical).
While the processes described here certainly require further development and opti-
mization, they do demonstrate the feasibility ofhemicellulose bioconversion programs.
6 Conclusions
It is becoming increasingly apparent that renewable energy sources must be developed
to compliment existing supplies. Biomass has been shown to represent an enormous
reservoir from which liquid fuels and chemical feedstocks can be generated in a
relatively simple and cost-effective manner. The high p r o p o r t i o n o f pentose sugars
in much of this material, makes utilization of the hemicellulose fraction an essential
factor in the management o f this resource. It has been demonstrated that micro-
organisms of widely diverse classifications posses the ability to produce valuable
chemicals from lignocellulosics. The integration o f new ideas with existing technologies
must soon result in the development of processes for the efficient manufacture of
chemicals from the total sugar content o f biomass.
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Mathematical Modelling, Parameter Identification and
Adaptive Control of Single Cell Protein Processes
in Tower Loop Bioreactors
Reiner Luttmann
G B F - - G e s e l l s c h a f l ffir B i o t e c h n o l o g i s c h e Forschung mbH.,
Mascheroder W e g 1, D - 3 3 0 0 B r a u n s c h w e i g - S t 6 c k h e i m , FRG
Axel Munack and Manfred Thoma

I n s t i t u t ffir R e g e l u n g s t e c h n i k der Universitfit Hannover,
A p p e l s t r . 11, D - 3 0 0 0 H a n n o v e r , FRG
1 Introduction'. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
2 Experimental Design and Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3 Modelling of Cultivations Using Distributed Parameter Models . . . . . . . . . . . . . . . . . . . . . . . . 99
3.1 System Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
3.2 Process Idealization and General Mass Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
3.3 Oxygen Mass Balances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
3.4 Substrate Balances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
3.5 Cell Mass Balances and Reaction Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
3.6 Normalized Model Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
4 Characterization of Model Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.1 Phase Velocities and Dispersion Coefficents in the Gas-Liquid System . . . . . . . . . . . . . . . . 113
4.2 Parameter Estimation with Exhaust Gas Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ll4
4.3 U n k n o w n Mass Transfer Parameters, Coalescence Function and Reaction Kinetic
Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.4 Definition of the Theoretically M a x i m u m Oxygen Transfer Rate . . . . . . . . . . . . . . . . . . 120
5 Simulation and Identification Techniques and their Application to Cultivations with Space
Dependent Oxygen Balances and Well Mixed Substrate and Biomass Conditions . . . . . . . . . 121
5.1 Extendend Culture Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
5.2 Discussion of Various Simulation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.3 General Remarks on Identification of Parameters in Distributed Parameter Systems 129
5.4 Quasi Steady State Identification of U n k n o w n Process Parameters . . . . ' . . . . . . . . . . . . . . 131
5.4.1 Hybrid Steady State Simulation and Identification-Procedure . . . . . . . . . . . . . . . . 131
5.4.2 Mass Transfer Analysis of a Batch/Fed-batch Cultivation . . . . . . . . . . . . . . . . . . . . . 135
5.5 Simulation of Fed-batch Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
5.5.1 Hybrid Dynamic State Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
5.5.2 Analysis and Simulation of Hansenula polymorpha Cultivation on Ethanol Substrate 141
5.5.3 Analysis and Simulation of Hansenula polymorpha Cultivation on Glucose Substrate 147
5.5.4 Comparison of Ethanol and Glucose Substrate Systems . . . . . . . . . . . . . . . . . . . . . 149
6 Application of Optimization Techniques to the Nonstationary Case (Fed-batch Process) .. 152
6.1 Discussion of Adaptive Control Algorithms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
6.2 Application of Open Loop Feedback Control to Distributed Parameter Processes . . . . . . 156
6.2.l Formulation and Solution of the Optimal Control Problem for Distributed
Parameter Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
6.2.2 Formulation and Solution of the Parameter Identification Problem . . . . . . . . . . . . 158
6.2.3 Applicational Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
6.3 Cost Optimization of Aeration for a Fed-batch Process . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
6.3.1 Model Reduction and Solution of the Reduced Equations . . . . . . . . . . . . . . . . . . . 160
6.3.2 Parameter Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
6.3.30LFO-Control ........................ 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
96 R. Luttmann et al.
7 Cultivation with Space Dependent Oxygen and Substrate Balances . . . . . . . . . . . . . . . . . . . . . 169

7.1 Process Behaviour and Model Extensions for Unlimited, Oxygen-Transfer Limited, and
Substrate Limited Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
7.2 Analysis, Identification of Kinetic Parameters. and Simulation . . . . . . . . . . . . . . . . . . . . . . 174
8 Set Point Optimization of Continuously Operated Pilot Plants . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8.1 Scale Up, Control Possibilities and Process Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8.2 Steady State Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
8.3 Formulation of the Performance Index and Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . 191
9 Conclusion and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
l0 Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
l l Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
In recent years a new type of bioreactors, the tower loop reactor has successfully been applied
for production of single cell protein (SCP). For an.improved economical operation of these
processes -- and other applications of this type of reactors -- a detailed mathematical modelling
is necessary. The large dimension of the production plants used leads to distributed-parameter models;
in other words, the balances are formulated as partial differential equations.
The treatise reviews modelling and optimization techniques for SCP processes in tower loop
reactors, considering substrate, cell, oxygen, and indirectly carbon dioxide balances.
Appropriate simulation and parameter identification methods are discussed and their application
is illustrated by means of experimental data.
A time variant behaviour of certain model parameters is observed during batch and fed-batch
cultivations. Since the course of these parameters cannot be predicted in every case, adaptive
control methods should be applied for biotechnological processes under these operating conditions.
A concept for adaptive control is presented, which separately incorporates identification of the
unknown time variant system parameters and optimization of the control inputs.
For continuously operating plants, the optimization can be restricted to a determination of the
optimal set points of the process. This method is demonstrated by means of a pilot plant and some
results are given.
1 Introduction
Since the desired p r o d u c t s and the c o r r e s p o n d i n g process c o n d i t i o n s o f b i o t e c h n o l o g i -
cal cultivations m a y differ c o n s i d e r a b l y b e t w e e n va~ ious applications, the goals and
m e a n s o f c o n t r o l s h o u l d be quite different, too. F o r a l o n g time, however, the
c o n t r o l o f processes in b i o r e a c t o r s was restricted to classical p a r a m e t e r s o f c h e m i c a l
engineering, such as t e m p e r a t u r e , p H - v a l u e , stirrer speed and a e r a t i o n rate.
Recently, due to a d v a n c e s in on-line m e a s u r e m e n t s but also by extensions o f bio-
reactor a c t u a t o r s for c o m p u t e r c o n t r o l and c o n n e c t i o n o f process c o m p u t e r s to
c u l t i v a t i o n processes, m o r e sophisticated c o n t r o l tasks have been e m p l o y e d 1.2~
A n i m p o r t a n t future trend in b i o t e c h n o l o g i c a l c o n t r o l will be the design o f c o n t r o l
strategies for o p t i m i z a t i o n o f p r o d u c t i o n rates. T h e decision variables will either
be o b t a i n e d by direct m e a s u r e m e n t s o f the p r o d u c t o r by c o m p u t e d p a r a m e t e r s
derived f r o m balance equations. These calculations will be carried out on-line. T h e
c o n t r o l o f v o l u m e t r i c or cell specific o x y g e n o r substrate u p t a k e rates can be m e n t i o n e d
as an example.
A n o t h e r future c o n t r o l task is the m i n i m i z a t i o n o f o p e r a t i o n cost in high energy
c o n s u m i n g processes. T w o different o p t i m i z a t i o n m e t h o d s will be investigated in
this article, n a m e l y on one h a n d the o p t i m i z a t i o n o f d y n a m i c a l l y c h a n g i n g cultivations
Mathematical Modellirtg, Parameter Identification and Adaptive Control 97
and on the other hand the optimization of processes with steady state behaviour.
Both methods will be presented and discussed for single cell protein (SCP) production
processes.
Recently, several new types of bioreactors have been developed for large scale SCP
production 3-6). Among these, tower and tower loop reactors have already attained
the level of production plants (50.000 t/a SCP of ICI in Billingham) or pilot plants
(1.000 t/a SCP of Hoechst and Uhde in Hoechst).
An almost accurate mathematical model of the process is a fundamental pre-
requisite for application of optimization methods in real plants. The modelling
generally results in a process description by means of differential equations for the
various balances. In contrast to well mixed stirred tank reactors, processes in tower
bioreactors are space- and time-dependent. Therefore the description of fluid dyna-
mical and growth kinetic behaviour of the processes leads to partial differential
equations (PDEs).
Papers dealing with modelling, parameter identification and simulation of distri-
buted-parameter processes in biotechnology are hardly found. In previous research
work only physical characteristics of tower reactors were investigated, or systems
with model media were treated 7-12) _ The first applications of mass transfer
parameter identifications during growth processes were based on quasi steady state
methods t3-18~ _ Models as well for the transient behaviour of cell growth as for
oxygen and substrate consumption in tower reactors, which consider only partial
problems of aerobic cultivation processes, are also to be found 19-31). _ A link
between identification of unknown process parameters of real (measured) processes
and the simulation of dynamical processes in bioreactors is given in32-39) _
Several papers deal with optimal control of cultivation processes, 40-4v) however,
more or less all authors concentrate on processes in well mixed stirred tank reactors.
- The application of modelling and optimal control techniques to distributed-para-
-
meter systems based on experimental data is very scarce 33,48 51 -
It is the purpose of this paper to review the present knowledge and to give an
extensive overview of mathematical problems dealing with modelling of cultivations
in tower loop reactors under different growth conditions. Further on, we will give
some ideas concerning simulation, identification of unknown system parameters,
and optimal control methods for distributed-parameter systems.
In case of dynamically changing fed-batch processes, adaptive control methods are
considered for cost optimization. However, for continuously working plants, cost
optimization is applied to steady state conditions.
Although the various algorithms presented in this article are illustrated by appli-
cations to SCP production processes, it is the opinion of the authors that in
principle the methods can also be successfully applied to other cultivations in tower
loop reactors.
2 Experimental Design and Measurements

The development of tower loop bioreactors for single-cell-protein production has been
carried out by various groups using extensive measurement methods. Previous work
was done by Lehmann et al. 52-561, cultivating Candida boidinii (DSM 70034) and
r MEASUREMENTS IN THE LIQUID PHASES
G,4S 1
(F: COLUMN; B: LOOP)
GG
"UO
POF(Xi,t) dissolved oxygen tension at 11
N points along the column
], o
COOLING W pOd(t) dissolved oxygen tension at the
OUT /4 .- end of the nongassed loop
XF(XK,t) cell (biomass) concentration at

the sampling point K
SF(XK,t) substrate concentration at the

sampling point K
EXHAUST GAS ANALYSIS
X~G(t) oxygen mole fraction

' P3 X~G(t) carbon dioxide mole fraction
, [22}-- S~(t) substrate (ethanol) concentration
SYSTEM PARAMETERS MEASUREMENTS
5O L s relative gas hold up in the column

CO-CURRENT Vu(t) volumetric liquid circulation rate
BUBBLE COLUMN V~(t) volumetric gas flow rate at the

COOLING WATE._~._._._.
IN entrance
LOOP BIOREACTOR
C RQ(t) (exhaust gas) respiratory quotient
?I "C /1.20ti2
F(t) volumetric substrate feed rate 7~
r--
Fig. 1. Schematical view of the single stage concurrent air lift tower loop reactor 58.62,63).
H A water supply for steam generator; B flow meter; C pressure reducing valve; D
E
D pressure gauge; E gas flow meter; F valve; G filter for air sterilization; H three-way-valve;
C [U .! condensation collector; K sampling; L alkali reservoir; M substrate reservoir; N 0 2-
electrode; O pH-electrode; P pump; R exhaust gas cooler; S recorder; T engine for
A I R IN I
mechanical foam destroyer; U porous plate; V amplifier; W heat exchanger; 0 2 O2-gas
LI(~
ro
DISCHARGE analyzer; CO z CO2-gas analyzer; pH pH-controller
Mathematical Modelling, Parameter Identification and Adaptive Control 99
Methylomonas M 15 (DSM 580) both with methanol as a carbon source. The results
of later work of Liicke 5v) with Candida boidinii on methanol and ethanol and
Buchholz 58)and Zakrzewski 59)with Hansenula polymorpha (CBS 4732) on glucose
and ethanol have already been reviewed in detail by Schiigerl et al. 60.61) in this series.
These measurements have provided the base for our mathematical investigations
on cultivation in tower loop bioreactors.
The experimental set up is shown in Fig. 1 together with the measured data used
in the following mathematical modelling. The measurement devices and methods are
not discussed in detail, the interested reader is refered to the literature 61-66)
3 Modelling of Cultivations Using Distributed Parameter Models
3.1 System Classification
In order to illustrate the mathematical description of the process, the tower-loop

bioreactor is shown schematically in Fig. 2. From the view of system theory, the
plant must be considered as a coupled reactor system. The main bioreactor, the
aerated tower is a multi-phase system and includes two subsystems, the liquid phase
(subscript F) and the gas phase, (subscript G). In contrast to the tower, the second
reaction system loop is nonaerated. It is the third subsystem and subscript with B
(bypass).
The cultivation processes in tower reactors are space- and time-dependent. A
mathematical description of the dynamic behaviour requires a set of partial differential
equations, thus causing some difficulties in modelling the processes, simulation
of the model equations and identification of the unknown parameters.
From the many variables influencing the process, only the main components can
be taken into account. The chosen model variables and parameters depend in
general on the tower coordinate x or loop coordinate x*. A summary of these is shown
in Fig. 2.
3.2 Process Idealization and General Mass Balance
Reactors usually are described by momentum, heat and mass balance equations 67-71).
To simplify the model development, the following assumptions are made:
1. Momentum balances need not necessarily be considered, since hydrodynamical
startup processes are not intended to be simulated. According to the authors'
experience, a new steady state is quickly established after a change in aeration rate.
Thus the hydrodynamical transient state does not have great importance for the
reactor operation.
2. Heat balances are not considered either, since effective temperature control allows
almost isothermic reactor operation.
Hence, in the following treatment only mass balances are noted. Considering
the mass balances for component i in a volume element, dVN, of a reactor
~/A ~HA#
6 ~'G
SYSTEM VARIABLES
dissolved oxygen concentration (column) OF(X,t)

(loop) OB(X*,t)
substrate concentration (column) SF(X,t)
(loop) SB(X*,t)
biomass concentration (column-loop) X(t)
oxygen gas phase mole fraction xoG(x,t)
carbon dioxide gas phase mole fraction xcG(X,t)
linear gas velocity uG(x,t)
SYSTEM PARAMETERS
linear liquid velocity (column) uF(t)

(loop) uB(t)
relative liquid hold up s
relative gas hold up EG(t)
longitudinal dispersion coefficient (liquid) DF(t)
(gas) DG(t)
volumetric mass transfer coefficient k L a(x,t)
respiratory quotient RQ(t)
dilution (feed) rate D(t)
maximum specific growth rate ~max
iI
specific death rate (containing maintenance) ~T
cDi
oxygen yield coefficient Yx/o(t)
substrate yield coefficient YX/S(t)
oxygen saturation constant K0
RESERVOIR
substrate saturation constant KS
R
longitudinal pressure profile P(x,t)
Fig, 2. Schematical view of the double reactor system with system variables and parameters used in
the mathematical model ~ 6 - 1 8 , 3 2 - 3 6 , 3 8 . 3 9 . 5 2 . 5 4 . 6 2 , 1 . 1 . 1 , 1 4 2 1
consisting of M phases the following generalized partial differential equation

(PDE) is valid v2. 731:
n
~QIN
- div(QNDiN grad giN) -- div(o~iNfi) + Y" RikNMi
(~t k=/
M-1
+ ~ kiNjaNj(Qi~j- QiN). (1)
In addition to assumptions 1 and 2 the following simplifications are made:

3. The reactor contains only two phases (gas-liquid-system), i.e. the microorganisms
are not considered as a separate phase. Because of their small size and density
similar to that of water, this is a fairly realistic assumption.
4. The two-phase flow is homogeneous in angular and radial directions. This also
holds true for the concentrations of components i in both phases. This is a fairly
good approximation as has been shown experimentally by Zakrzewski et al. 59,66,
"74)
5. For all components i in a phase N, the same transport velocities and longitudinal
dispersion coefficients are assumed.
6. The reactor consists of a tower section which is aerated (two phase flow) and a
loop which is not aerated (liquid flow alone). In the loop, backmixing is
negligible due to the high liquid velocity.
This model is based on mass balances for the component cells, substrates and
dissolved oxygen in the liquid phases of the tower section and loop. Mass balances
for oxygen, carbon dioxide and inert components are also applied in the gas phase
of the tower section.
Each of the phases in the tower section and loop is characterized by a single phase
velocity and a longitudinal dispersion coefficient. Phase velocities have non-
disappearing mean values only in the longitudinal directions. Because of the radial
and angular homogenities of the phases, gradients can only prevail in the longitudinal
direction.
The chosen system variables and process parameters are summarized in Fig. 2.
The fluid dynamical parameters are considered as quasi steady state values. Space
invariant process parameters are calculated from the measurements as follows:
VoCt)
cG(t) - , (2)
VR
Vv(t)
+v(t)- VR (3)
"Qu(t) + F(t)
uv(t) - , (4)
QReF(t)
%(0
uB(t)-- QB ' (5)
where:
VG(t) gas volume in tower section,
VR volume of two phase system in tower section,
Vv(t) liquid volume in tower section,
x)u(t) volumetric liquid recirculation rate,
F(t) volumetric substrate feed rate,
QR cross sectional area of tower section, and
QB cross sectional area of loop.
Pressure and gas phase velocity depend on the longitudinal coordinate x. Since
according to our assumption, av is invariant in x, there is a linear pressure drop along
the column,
P(x,t) = pA + QF av(t) g (LR - - X), (6)
where:
pA head pressure in tower section,
Ov liquid density,
g acceleration of gravity, and
LR height of two phase system in tower section (Fig. 2).
Of course, the pressure variation along the column also influences the volumetric
gas flow rate, 9G(x,t). This yields a spacial dependence of the linear gas velocity,
uG(x,t ) - VG(x,t)
QR ~,(t) ' (7)
3.3 Oxygen Mass Balances
Figure 3 shows the oxygen mass balances in gas and liquid phases for a volume
element of the tower section. In contrast to a constant liquid density, ~v, the gas
density, o~, is time- and space-dependent.
The oxygen balance in the liquid phase is given in terms of the concentration,
Or, and, in the gas phase, in mole fraction, x G. To develop the balance equations
in addition to the simplifications 1 to 6, further assumptions are made:
7. Henry's law prevails at the interface,
PoG(X, t) = RTOG(X, t) = Ho20~(X, t ) , (8)

where :
PoG(X, t) oxygen partial pressure in the gas phase,
R gas constant,
T absolute temperature,
_D;QRE F ___..8_(DFQR~:FSOF)d
80; x P ~ Q 8Xo~ 8 , P ~ O 8x_x~,
8X ax I ox --R~ UG RE:G cqx-~xxPR-TU i R~:G ax jdx
// f/J / / / .l i/q.u/i d.p/h/a/s/e// /// I1 gasphase

~///~.,(su b script F)~'//] (subscript G)
oxygen oxygen
reaction
(OUR)
.z////X4-kLdA(O~F-0F) - transfer
(OTR)
Q RE ~
X
9~!OG
aOF P DGOREG 8XoG
-DFQR gF " ~ RT ax
Fig. 3. Oxygen mass balances in the gas and liquid phases of a tower volume element 8, aa-a8,32 34 39,
52. I4-2 )
Mathematical Modelling,Parameter Identificationand AdaptiveControl 103
OG(x, t) molar oxygen concentration in the gas phase, and

O~(x, t) oxygen saturation concentration at the interface.
According to Eq. (8) it is assumed that there is a linear relationship between
oxygen partial pressure and oxygen saturation concentration in the liquid. The pro-
portionality constant, Ho2 (Henry constant for oxygen), depends only on temperature
and does not depend on medium components.
8. The mass flux of oxygen between gas and liquid phases, which is given by
riao(X, t) _ kO(x" t) [O*(x, t) -- Or(x, t)] (9)

A(x, t)
is not influenced by the mass transfer resistance in the gas phase v~. 76). In Eq. (9)
riao(X, t) is the oxygen mass flow across the interfacial area A(x, t) and k~ t)
is the mass transfer coefficient.
Because of the strong experimental evidence showing the variation of k ~ and A
along the column 77--79), and their variation during the cultivation, the time and
space dependences of k~ and A are not neglected. Equation (1) yields, when
combined with these eight assumptions, the following PDE's for the oxygen balances
in the liquid phase,
~Or(x, t) a2Ov(x, t) ~Ov(x, t)

at - Dr(t) ax 2 ur(t) ax R~ Sv' OF' x, t)
+ ke~ t) a(x, t) [O*(x, t) - Or(x, t)], (10 a)

and in the gas phase,
P(x, t)OXoG(X'
D G (t t) )=a t ~xxa(P(x, t) Ox~
O
- - - (P(x, t) XoG(X, t) uc(x, t))
0x
RT%(t)
MozeG(t~ kL~ t) a(x, t)[O*(x, t) - OF(x , t)].
(ll a)
The following initial and boundary conditions are assumed:
At the beginning of cultivation, before the medium is inoculated, the medium is
saturated with oxygen,
P(x, O) x~,o
O d x , 0) = O~(x, 0) = (10b)
Ho2
and the inlet oxygen mole fraction in the gas is preserved along the column,
XoG(X, 0) = xE~. (11 b)
During cultivation, 'Danckwerts' boundary conditions are valid at the bottom

boundary of the liquid phase,
OOF(0, t) uF(t)
[OF(0, t) -- OrE(t)], (10C)
~x Dv(t )
and of the gas phase,
~Xo~(0, t) uE(t)
[XoG(0, t) -- XgG]. (1 1C)
~x DG(t)
At the top boundary of the liquid phase,
fi~OF(L R, t)
-0, (10d)
~x
and of the gas phase,
OXoG(LR, t)
-0. (11 d)
~x
the gradients disappear 80-82).

In Eqs. (10) and (11), the specific interfacial area with regard to liquid volume,
A(x, t) dA(x, t)
a(x, t) - - (12)
Vv(t) Qg~v(t)dx '
and the oxygen mole fraction in the gas phase,
Xoo(X, t) - RTOa(x, t) (13)

P(x, t) '
are used.
The unknown dissolved oxygen concentration in the liquid phase at the entrance,
OvE, can only be calculated by means of a loop oxygen mass balance (Fig. 4). This
balance is given by a PDE for a plug flow reactor,
c~OB(x*, t) OOB(x*, t)
at -- UB(t) OX* Ro~(XB, SB, OB, x*, t). (14a)
In Eq. (14a) Oh(x*, t) is the dissolved oxygen concentration in direction

x*, the longitudinal distance along the loop, whereas
X B and SB, are the cell mass and substrate concentration in the loop.
For solution of Eq. (14a), the initial condition,
OB(X* , 0) ~- O~(LR, 0), (14b)

~BOB
x ////'.,, /////
///// loop ///~.
//Z IsobscriptSlU/.
• _
oxygen
reaction
(OUR)
/,
" / "/, "/, ///// /
x~dx*
VBOe+~--~x,(VaOa) dx*
Fig. 4. Oxygen mass balance in a loop volume element 3-, ,34)
and the boundary condition at the upper end (x* = 0) of the loop,
OB(0. t) = OF(LR, t) = O~(t), (14c)
are used. Both are controlled by the exit concentration of the tower section,
O~(t).
The entrance concentration of oxygen in the liquid which appears in the boundary
condition (14c) is given by a mixture of the loop exit concentration, 06, and the
dissolved oxygen concentration in the reservoir, O R. The feedback ratio,
y(t) = 9 ~?u(t) (15)

Vu(t ) + F ( t ) '
controls this liquid entrance concentration, OF(t ) . This yields from the boundary
condition (10c) at the lower end,
8Or(0 , t) _ _UF(!! [Or(0, t) - - ? ( t ) O~(t) - - {1 - - 7 ( 0 } Odt)] 9 (16)

8x l_)v(t)
Solution of the oxygen balance equations is still not possible due to the unknown
space variant gas velocity, u o 8.1 t. 34.35)
For all gas components, the sum of the mole fractions equals unity at each point
of the tower,
K 1
xio(x, t) - 1 . (17)
i=l
It is necessary to develop carbon dioxide (C G, XcG), nitrogen (N o, XNG) and trace

components (I G, xio) balances, which are similar to the oxygen balance (11), (Fig. 5).
P in Q z 8xl@-a--{ P r', n ~- axing,4,,
J-----x+dx
gesphose
l
,// (subscript F ) J / / J (subscript G)
mass
tronsfer
~QR EG~"tx -X
( 0 2, C 0 2 , N 2 .... )
0 X IG
ax
Fig. 5. Gas phase balances in a tower volume element s, 16-~8,32,34,54)
Addition of those K component balances yields a quasi steady state continuity

equation,
--~ (e(x, t) UG(X, t)) = - R T a ( x , t) ~v(t) {ki~

Ox f( ~ t) [O~(x, t) -- OF(x, t)]
keC(x, t) kN(x, t)
+ - - [C~(x, t) - CF(X, t)] + - - [N*(x, t) - Nv(..x, t)]
Mco2 MN2
K-3 k[(x, t) "~

+ Z - - [I~(x, t) - Iv(x, t)] . (18)
i=l M 1
In Eq. (18), kC(x, t), k~(x, t) and k[(x, t) are the mass transfer coefficients for carbon
dioxide, nitrogen and tracer components exchange, C~, N* and I~- are the interracial
saturation and C v, N v and I v are the bulk concentrations of dissolved carbon
dioxide, nitrogen and component I in the medium. To eliminate the dissolved
liquid phase balances for N z, CO 2 and tracer components, two further assumptions
are made in addition to the eight former assumptions:
9. Nitrogen as well as the tracer components are considered as inert gas components
in this system. Cyclic sorption processes due to liquid circulation are considered
to be negligible. By assuming the validity of
JF(X, t) = J~(x, t), J = N, I , (19)
mass transfer is neglected due to the assumption of equilibrium between the

phases with regard to nitrogen and tracer components.
Mathematical Modelling, Parameter Identificationand Adaptive Control 107
10. It is assumed that between desorbed CO 2 and adsorbed 02, a space invariant
equilibrium exists. When using a respiratory quotient,
kC(x, t) a(x, t) [C*(x, t) -- Cv(x, t)] Mo2

RQ(t) = -- kO(x' t) a(x, t) [O~(x, t) -- OF(X, t)] Mco2' (20)
which depends, according to this assumption, only on time, the CO2 balance
equations disappear from the system, since the CO2-exchange is indirectly taken
into account by means of oxygen exchange.
This assumption can be justified by the observed changes in the driving forces
of oxygen and CO2 along the tower section. At the bottom, the oxygen and CO2
driving forces, which are in opposite directions are large. At the top, both driving
forces are smaller and their ratio is nearly constant.
This permits us to take the 02 and CO2 exchange processes into account
simultanuously by means of RQ,
(P(x, t) uG(x, t)) =
RTeF(t)
k~ t) a(x, t) [O*(x, t) -- Or(x, t)] [1 -- RQ(t)]. (2l a)
Mo2e~(t)
From Eq. (21 a), the gas phase velocity, uG(x, t), can be calculated by means of the
initial condition,
9~ ( o ) P~(O)
UG(X, 0) -- QR~G(0)P(x, 0) ' (21b)
and boundary condition at the lower end,

V~(t)
uo(0, t) = - - = : uE(t), (21C)
QRe~(t)
together with the oxygen balance in the liquid phase (Eqs. (i0)), in the gas phase
(Eqs. (11)), and in the loop (Eqs. (14)).
3.4 Substrate Balances

It is assumed that the substrate is not volatile. Thus the substrate balances are only
considered in the liquid phase.
The substrate balances in the tower Section,
aSv(x, t) a2Sv(x, t) aSF(x, t)

at - Dr(t) aX2 uv(t) aX RsF(Xv' Sv' Or' x, t),
and in the loop, (22 a)

~Ss(x*, t) ~SB(x*, t)
St -- uB(t) Ox* RsB(Xs' $8' OB' x*, t), (23 a)
108 -R. Luttmann et al.
are coupled by means of their boundary conditions. These are similar in form to those
of the oxygen balances and yield in the tower,
OSF(0, t) _ % ( 0 [SF(0, t) -- "f(t)sA(t) - - {1 - - ?(t)} SR(t) ] (22b)

~x Dv(t )
OSF(LR, t)
- 0, (22c)
0x
and in the loop,
SB(0, t) = S)(t). (23b)
The substrate is assumed to be well mixed at the beginning of the cultivation,
SF(X, 0) = SB(X*, 0) = So , (24)
where So is the substrate concentration at t = 0 h.
3.5 Cell Mass Balances and Reaction Kinetics
The cell mass balances in the liquid phases of the tower and loop section are given by,
~Xv(x, t) O2Xv(x, t) 8Xv(x, t)

~t - Dr(t) 8x 2 uv(t) ~-~x---xq- RxF(XF' SF' Or' X, t),
(25a)
and
aX,(x*, t) ~XB(x*, t)
- uB(t ) + RxB(XB, SB, O B, x*, t). (26a)
8t ax*
Again boundary conditions link these two PDE's,
OXv(0, t) UF(t)
[Xv(O, t) -- 7(t)X~(t) - - {1 - - ?(t)) XR(t)] (25b)
~x - Dr(t)
~XF(LR, t)
- 0, (25c)
~x
XB(0, t) = X ) ( t ) . (26b)
When, as usual, sterile feed is used, in boundary condition (25b) XR(t) = 0 holds
true. The initial conditions are given by
XF(X , 0) = XB(X :~, 0) ~- X o , (27)

where X o is the initial cell concentration just after inoculation. It is assumed that the
inoculum is ideally mixed in the medium shortly after inoculation.
The usual calculation of endogenous metabolism with a specific substrate main-
tenance rate, qsmx, yields a maintenance substrate consumption rate,
R~(R, t) = q~xX,(R, t ) , (28)
which is of zero order with regard to substrate s3-s5 I. This often causes meaningless
negative values during simulation. In the model presented here the maintenance
is considered by a virtual loss of cell mass according to a specific death rate gT. Thus
the observed cell mass variation is the difference between growth rate and death rate,
gr -
Rxl(.~, t) = [gi(Si, O1, :~, t) -- g~] X1(s t) = Rxi(X , t) - - Rx,(X,t).
e - (29)
The specific growth rate is calculated according to a twofold substrate Monod-

kinetics 4t .86-90)
S~(L t) O,(K, t)
~i(Si, Oi, X, t) : la.... (t) KS -t- St(x , t) K O + Oi(~. , t) "
(30)
The virtual cell growth rate, Rgr~, is linked to the substrate consumption rate, Rsi ,
by the substrate yield coefficient,
R~,~,(~, t)
Yx"s(t) - ]~sT(2, t) (31)
and to the oxygen consumption rate, RoT, by the oxygen yield coefficient,
Yx,o(t) _ R~](:~, t)
"' Roi(2, t)" (32)
Equations (29) to (32) are valid in the tower liquid phase (I = F, .~ = x) and in the
loop (I = B, ~ = x*).
Variations of g .... Yx/s, and Yx..o with time, due to different cell adaptabilities
to the cell environment are taken into account. Because of the slow cell adaptation
rate in comparison with the high rates of environmental change due to medium
circulation, no spacial variations of g .... YxCs, and Yx~o are considered.
3.6 Normalized Model Equations
The simulation of the developed model is carried out using digital and hybrid
computers. It is usual therefore to normalize the model equations and their physical
dimensions into dimensionless form 17,91-93.1. The space coordinates, x and x*,
are related to the length o f the reaction system (tower or loop) and are now
represented as a dimensionless longitudinal coordinate in the tower section,
X
z - (33)
LR '
and a dimensionless longitudinal coordinate in the loop,
X*
z* = - - . (34)
LB
The cultivation time t is transformed into a dimensionless time,
T = --,
t (35)
TN
where T s is a fixed reference time.

F u r t h e r the m a x i m u m possible b o t t o m pressure is used as reference pressure,
E pA (36)
Pmax = + QFgLR 9
F o r the dissolved oxygen concentration, the m a x i m u m oxygen saturation concen-

tration is used as reference,
E E
Pmax XOG
O *E (37)
F max ~ . Ho 2
Other references are the entrance oxygen mole fraction, XOaE,for XoG, SO for S F and S B,
as well as X o for X v and X w
To simplify these mass balance equations, the following linear differential operators
are introduced:
Liquid phase operator,
~ClF(z' "~) BOF(.~)-I ~2CIF(Z' ~) ~ClF(Z'~) (38)

F{ClF} "-- ~'C ~Z2 + vF(~) az '
loop operator,
~ClB(Z$, 12) ~CIB(Z*, T)
B~c t : - + vB(~) (39)
t IBJ ~'17 ~Z* '
gas phase operator,
G{c,o} : = p(z, r) -&,o(z,

- az ~) Boo(Z)_1~~ ( p(z, ac,o(z,az
z) - -
+ ~ (p(z, 'r) Vo(Z, "0 c~o(z, 'r)}. (40)

The system variables and parameters used in Eqs. (38) to (40) are discussed in
detail in Chapter 4 and have the following meanings:
cm(L T), related system component I in subsystem K(F, G, or B) (2 = z or z*),
va(z, x), normalized linear velocity in the gas phase,
B%(,), modified Bodenstein-number in subsystem K,
vj(~), normalized liquid velocity in liquid phase J, and
p(z. "t). related pressure drop in tower section.
The three operators, (38), (39) and (40), form the components of a system (differential)
operator,
sr := diag [F, F, F, B, B, B, G, G], (41)
in a matrix form, in which only the main diagonal elements are different from
zero.
This operator is applied to a space- and time-variant state vector,
~e := [CodZ, v), CsF(z, x), CxdZ, ~), CoB(Z*,X), %B(z*, ~),

(42)
CxB(Z*,~), Coa(Z, X), 1] T ,
which includes as an 8th component the scalar quantity 1 (sum of the mole
fractions).
The system of PDEs for the tower and the loop section can be expressed in a
simple form, if two additional vedtors are introduced. The reaction vector,
~(cg) := [ror(C]p z, r), rsv(C,r, z, x), -- rxF(CIF, Z, Z),

(43)
roB(Cm, z*, x), rss(Cm, z*, r), -- rxB(Cm, z*, ~), O, O]s ,
in which system components ce4 are non-linearly connected with each other, and
the mass transfer vector,
y : = [p(z, ~) CodZ, r) - Coy(Z, ~)1

(44)
[Sty(z, *),0, O, O, O, O, -- StG(z, ~), -- StG(Z, v) rq(z)]T ,
include the parameters,

rij(Z r~ normalized reaction rate of component I in liquid phase J,
St~(z..) modified Stanton number in subsystem K, and
rq normalized respiratory quotient.
Equations (41) to (44) yield the matrix notation of the model equations,
~4{~g} = - - . ~ ( ~ ) + J - , (45a)
where the initial conditions are,
Coy(Z, 0) = p(z, 0),
CoB(Z*, 0) = pA(0) ,
CsF(Z, 0) = CsdZ*, 0) = 1 ,
(45b)
Cxv(Z, 0) = CxdZ*, 0) = I ,
Coo(Z, 0) = 1 ,
vc(z, 0) = v~(0) pC(0) p(z, 0) - t .
The boundary conditions for the three liquid phase components (I = O, S, X) are
at the lower end (z = 0),
&~v(0, z)
~z (45c)
BoF(z) vv(z) [qv(0, Z) - - y(Z) cA(z) - - {1 - - y(r)} C,R(~)],
and at the upper end (z = l ) ,
~CIF(I , Z)
Oz - 0. (45d)
At the lower end for the loop components (I = O, S, X),
cm(0, z) = cA('0 (45e)
is valid.
The boundary conditions at the lower and upper end for the (related) gas phase
oxygen mole fraction are,
8%o(0, z)
~z - B%(z) v~(O [Co~(0, z) - - 1], (45f)
and
0Coo(l, z)
- 0, (45g)
~z
whereas the boundary condition at the lower end for the gas velocity is,
vG(0, r) = rE(Z). (45h)

Mathematical Modelling, Parameter Identificationand Adaptive Control 1 13
The solution of the partial vectorial differential equation system (45) is unique
and is carried out in normalized (z, z) space. However, the results will be presented
further on in physical (x, t) space.
4 Characterization of Model Parameters
4.1 Phase Velocities and Dispersion Coefficients in the Gas-Liquid System
The differential operators for the liquid phases of the tower and the loop (Eqs. (38)
and (39)) and for the gas phase of the tower (Eq. (40)) include five system parameters,
Bov(z), VF(~), V,(~), BO~(~), and p(z, z), and the system variable vG(z, ~).
The normalized liquid velocity in the tower,
VF(r) = [Vu(zTN) + F('cTN)] T N

QR[1 _ aG('CTN)]LR , (46)
the normalized liquid velocity in the loop,
"Qu(ITN) T N
vB(z) - , (47)
QnLB
and the related pressure profile along the tower,
pA + Qvg[1 __ aG(ZTN)] [L R __ ZLR]

p(z, Z) = pE ' (48)
max
are known for the entire cultivation, since the following variable parameters are
measured on controlled as indicated:
liquid recycling rate (measured),

F('rTN) substrate feed rate (controlled),
~(~T~) mean relative gas hold up (measured).
The theoretical maximum bottom pressure, PEr..x,the liquid density, Qv, the accelera-
tion of gravity, g, the cross sectional areas of the tower and loop, QR and QB, the height
of the two phase layer, LR and the length of the loop, LB, are known as well as the
chosen reference time T N.
The operator parameters BOF and Boa are unknown. According to the simulation
results, the longitudinal oxygen concentration profiles depend only slightly on Bov
and Bo a. Thus these parameters are approximated by means of empirical relationships.
The modified Bodenstein numbers are given by
Bo,(z) - L2 , I = F, G . (49)
DI(rTN) Z N
DF(t), the longitudinal liquid dispersion coefficient is calculated by
DF(t) = D~(fi~o(t)) ~ , (50)
as recommended by Badura et al. 94), and De(t), the longitudinal gas phase dispersion
coefficient,
De(t) = D~(uo(t))
N -~ 3.0 , (51)
is as investigated by Mangartz et al. 95)

In Eqs. (50) and (51) the characteristic constants,
DrN = 2.4• 10 -4 m 2 s -1 (d R • 102 m - l ) 1"4 , (52)
and
D~ = 5.0x 10 -8 m 2 s -1 (d Rx 102 m - l ) 1"5 , (53)
are both dependent on the tower diameter dr ( = 0.15 m).

The controlling parameter in DF (Eq. (50)) is the dimensionless superficial gas
velocity,
V~(t)
u~~ - QR • 102 s m -1 , (54)
whereas D e (Eq. (51)) is controlled by the dimensionless (real) gas velocity,
c,~(t) (55)
ag(t)- %(0
The normalized gas velocity in the tower,
TN (56)
v~(z, z) = UG(zLR, xT N) L ~ '
is an unknown system variable and is only known at the gas entrance (Eq. (21 c)).
4.2 Parameter Estimation with Exhaust Gas Analysis
From the input-output balances for oxygen and carbon dioxide, the oxygen transfer
rate into the system,
Qoz(t) = Q~2(t) _ QA2(t), (57)

and the carbon dioxide production rate of the system,
Qco2(t) = QAo2(t) -- QcEoz(t), (58)

can easily be calculated 96, 97)
Qoz and Qco2 (g 1-1 h-1) are both related to the liquid volume in the tower section,
V F9
Qgz(t) and QcEoz(t) are the entrance and QAa(t ) and QAo2(t) are the exit
values. The overall respiratory quotient, RQ, is defined as the molar quotient between
Qcm and Q02,
Qcoz(t) Mo2
RQ(t) = Qoz(t) Mcoz ' (59)
where Mo2 and Mco 2 are the mole masses of O z and CO2, respectively.
RQ is calculable by means of a balance for the remaning inert gas components
excluding 02 and CO 2,
Q~(t) = ~ QA(t), I * 02, CO:, (60)

i=l i=l
which yields
x~G(t) [1 - x g G ] - x ~ [ 1 - xoA~(t)]
RQ(t) = --XoA~(t) [1 -- Xga] + XgG[1 -- XcAG(t)]" (61)
In Eq. (61) XoGEand XCEGare the known O 2- and CO2-mole fraction in the gas inlet,
whereas XO~
rA and XCGA are the measurements in the exhaust gas.
The theoretical upper limit tbr the transfer of oxygen into the system from the
gas phase, is the maximum oxygen supply rate,
Vg(t) pE(t)XgGMo2 (62)

Q g z ( t ) - Vv(t ) RT '
where the controlling parameters are,

vE(t), the volumetric gas flow rate at the inlet,
Vr(t ), the volume of the liquid phase in the tower section, and
pE(t), the pressure at the gas inlet.
In practice, due to the finite residence time of gas phase in the system, the actual
oxygen supply rate,
E
XoG - x~G(t)
Qo2(t) = Q~z(t) (63)
x ~ [ 1 - {1 -- RQ(t)} X~G(t)] '
is lower than QE02"
The carbon dioxide production rate can be calculated from RQ(t) and Qoz(t)
according to Eq. (59).
Under steady state conditions, (i.e. by neglecting dynamical O2-accumulation effects

in the gas and liquid phases), the integral oxygen transfer rate, OTR(t), from the
entrance, x = 0 to the exit x = L R,
L~ dx
OTR(t) = f k~ t) a(x, t) [O{-(x, t) - Or(x, t)] ~ , (64)
o
and the integral oxygen uptake rate,

LR
f OF(x, t) dx
OUR(t) = Roy m,x(Xv, Sv, x, t) Ko + OF(X, t) L~
0
LB
VB f Oa(x*, t) dx* (65)
+ V~ ROBmax(XB,SB, X*, t) Ko + OB(X*, t) L B '
o
are identical to the oxygen supply rate Qo2(t) for batch operation.
As previously mentioned in Sect. 3.5 simulation- and identification procedures are
carried out using normalized variables.
Normalization is given by
Q~)2(zTN) TN
q~2(~) -- *E (66)
O F max
for Q~)2(t) and similarly to Eq. (66), qoz('r) for Qo2(t), otr(z) for OTR(t) and
our(z) for OUR(t).
The system parameter RQ, used in the quasi steady continuity equation (Eq. (21 a)),
is normalized by
,E
rq(x) = [1 -- RQ(z" TN)] XOG, (67)
where RQ = MRQ is calculated from measurements by Eq. (61).
4.3 Unknown Mass Transfer Parameters, Coalescence Function and

Reaction Kinetic Parameters
The simulations indicated that the assumption of space independent mass transfer
coefficients and oxygen reaction rates according to Reuss et al. a. is. 52) was inadequate
to describe the measured longitudinal dissolved oxygen profiles in tower reactors with
porous plate aerators.
Both mass transfer influencing parameters, the (liquid side) oxygen mass transfer
coefficient k~ t) and the specific interfacial area a(x, t) are unknown space and
time dependent variables.
Furthermore they are related in one parameter, the volumetric mass transfer coef-
ficient
kLa(X, t): = k~ t) a(x, t). (68)
By using spatial constant kLa values and reaction rates significant deviations between
calculated and measured dissolved oxygen profiles in the tower were found 16,17)
This is shown by curves 2 and 3 in Fig. 6. Only curve 1 describes the course of the
measured concentration profile suitably. This curve was calculated by using oxygen
Monod-kinetics,
OF(x, t)
R o F ( X , [) ~- ROF max(XF, SF, t) (69)
Ko + Ov(x,t)'
and by assuming a variation of kLa along the tower reactor. Space variation of kLa is
described by
_ Kst(t ) ~L R
kLa(x, t) = 0 < x < ~LR,
kLaEIt) e (70)
[kLaE(t) e-~St (~, ~L a ~ x ~ LR,
I
I
~1 coalescence rate function
f'l~,." and oxygen Monodreaction
C 3 ,,3:. I
t
9 -2 constant volumetric mass

~ transfer coefficient and
] ~"~j'~
'~.oxygen Monodreaction .
: 3 coalescence rate function.-'~'".. 1 , ~ ~--~..~.,~0 '

oA , and constant oxygen reaction ~'-,~
,,I .... "-.. . . . . .
0 a 0.25 0.5 0,75 ~ 1.0

X
Z=-
LR
Fig. 6. Longitudinal dissolved oxygen profiles in the liquid phase of the tower: oxygen transfer rate
(OTR):4.9 gl l h - l .
(l) Coalescence function and Monod kinetics with regard to oxygen are considered. (2) Space
independent volumetric mass transfer coefficient and Monod kinetics with regard to oxygen are
assumed. (3) Coalescence function is taken into account but space independent oxygen reaction rate
is assumed 32.3r
i.e. kLa E holds true only for x = 0 (aerator). With increasing distance, x, kLa expo-
nentially decreases up to x = 0~Lg and in the range ~xLR < x ~ LR, kLa is constant.
The ratio kLa(x , t) t o keaE(t) is called the coalescence function,
kLa(zLR, zT N)
W(z,'0 .-- krar(zTu) , (71)
which is shown in Fig. 7.

The timevarying exponent in Eq. (70) is defined as the coalescence factor Ks,.
At first kLaE, Ks,, and cr were fitted parameters. Later on, for all identifications, a
constant ~ ( = 0.1 = 27.5 cm) was used. Thus only kLaE and Kst had to be identified.
Table 1 shows the identified mass transfer parameters for the example considered in
Fig. 6. Because of the best fit obtained, the parameters of curve 1 were accepted.
The unknown modified Stanton functions, Stl(z, ~), were calculated by
Sti(z, "0 = St~(kt aE, r) uxJ(Kst, z, "0, I = F, G , (72)
where
St~(z) = kLaE(zTN) T N , (73)
is the modified liquid Stanton number at the gas entrance and
RT[1 - - gG(ZTN)]
(74)
St~(z) = kLaE(zTN)T N Mo2Ho2~;G(ZTN)
is the modified gas Stanton number at the gas entrance.
i i i
E
1.0
"I '\ I I Kst
0.75
u~% e-v,st . . . .
,\\ 0.5
1.0
0.25
Fig. 7. Coalescence function W as function
of the distance from the aerator; parameter:
0 ' # I Coalescence factor Kst 32,347
0 e=0.1 0,2 0.9 --,,-1.0
z=~R [-1
Table 1. Identified mass transfer parameters for the examples

considered in Fig. 6
kLaE Kst kta ~ W~ Ko

(h -1) (--) (h 1) (_) (mgl-l)
1 2104. 0.55 1214. 0.58 0.25

2 1213. 0.00 1213. 1.00 0.25
3 1556. 0.37 1074. 0.69 0.00
In Eq. (74), Hoz, the Henry coefficient o f oxygen in the medium, is considered to
be constant during the cultivation time, which o f course is not at all a realistic
assumption 98).
For the evaluation of the mass transfer parameters, the dissolved oxygen concentra-
tions in tower and loop are used (Fig. 6).
The calculation of the measured concentrations from the measured relative local
saturation values of oxygen tension, pOEF(X~, t), causes some difficulties, since the
proposed linearly decreasing initial concentration along the tower (Eq. 10b) only
occurs in towers with the liquid at rest. Since the calibration of the O2 electrodes was
carried out under operation conditions in the tower reactor just before inoculation,
cyclic sorption processes yield a saturation concentration profile which slightly
deviates from those of Eq. (10b). These can be evaluated from the steady state
solution of the oxygen balance in Eq. (10a) by setting RoE = 0. The solution is defined
as
O~o(X ) : = OFstat(X , 0 ) . (75)
Using these O*o values and the measured pO2v values, the dissolved oxygen concentra-
tion values are calculated by
MOr(xi, t) = MPO2F(xi, t) O~-o(Xl) , (76)
where MOF(XI, t), is the calculated dissolved oxygen concentration and

MPO2(xi, t), the measured dissolved oxygen tension at
x i, i = 1, 2 ... 11, the discrete longitudinal positions at which these measure-
ments were carried out.
The normalized MOF(xi, t) is given by
MOF(ZiLR' "~TN) (77)

McodZ~, ~) = o,E ,
F max
where z~ = xi/LR, the dimensionless longitudinal coordinate for the pOz-electrodes in

the tower section.
The normalized dissolved oxygen concentration at the loop exit, CoaB,is calculated
in a similar way,
MCoAB(-) = MPOzB(Ls ' ~TN) O*o(L R)

O* E (78)
F max
The chosen growth kinetic parameters are also needed and unknown. They are related
by X o, So, O*EF
max and T N and used in simulation and identification as
- - normalized maximum specific growth rate,
~-lm(T) ~--- ~tmax(l:Ty) f N , (79)
-- normalized death rate,
~tt = PTTN, (80)
-- related oxygen yield coefficient
O *E
F max
Yx.o(~) = Yx/o(~TN) Xo , (81)
-- related substrate yield coefficient
So
Yx,,s('O = Yx/s(ZTN) Xoo (82)
-- related oxygen saturation constant,
Ko
k o - O.Em.x, (83)
-- and related substrate saturation constant,
Ks
ks - (84)
So
In the process analysis, Pm, YX/O' and Yx/s are defined as space independent, but time-
varying parameters, whereas p,, k o and k s are assumed as constant during the cultiva-
tions.
4.4 Definition of the Theoretically Maximum Oxygen Transfer Rate
The normalized oxygen supply r a t e qE2, the upper limit of oxygen transfer from the
view of the gas phase, can only be used as a standard for the aeration rate in
oxygen transfer units. However, the theoretical upper limit of oxygen transfer rate
from the view of the hquid phase, otrL~M, is a proper criteria to evaluate the mass
transfer capacity of a plant 32). By rearranging Eq. (63) in normalized parameters,
otruM is given by
1 - C~Gmin('l~)
otruM(r) = qoE2('C) 1 -- rq(z) CAGmin("~) ' (85)
In this equation, qE 02 is known from the aeration rate and rq from the exhaust gas
analysis, whereas the possible related minimum oxygen mole fraction is unknown.
Neglecting the longitudinal dispersion in the gas phase and assuming COF = 0,
i.e. that cells can consume dissolved oxygen completely, a steady state balance for the
normalized model (Eqs. (45)) yields a nonlinear ordinary differential equation for
C O G m in ~
1 - rq(~) dcoo min(Z, "~)

- - X ( z , z) (86)
[1 - rq(r) COGrain(Z, .~)]2 CoGmln(Z' .~) dz
On the right hand side of Eq. (86), the coalescence-pressure function,
Z(z, x) _ stE(x) p(z, r)

V~(X) pE(~) q~(z, r), (87)
only quantities appear whose spatial dependence is known (after identification of

kLaE and Kst). Thus an analytical implicit solution of Eq. (87) with
1 1 { 9 }
CoGmin(Z , T)" e I - rq ('0 c O G rain [z. "r) __ _ _1 e 1-~q(~) 1-oJ~(~'~)a~
1 - rq(~) CoG mi.(Z, X) 1 -- rq(r)
(88)
is possible. Since COGml. can be calculated iteratively for every arbitrary z in Eq. (88),
the minimum mole fraction in the exit gas can be determined by computing
CoGrainat the upper boundary (z = 1). When putting this minimum mole fraction into
Eq. (85), the theoretical upper limit of the oxygen transfer rate and oxygen utilization
rate of the organisms is found.
5 Simulation and Identification Techniques and their Application

to Cultivations with Space Dependent Oxygen Balances and Well
Mixed Substrate and Biomass Conditions
5.1 Extended Culture Model
Extended culture operation is a special fed-batch technique in which a constant

substrate concentration is maintained during the cultivation 99, loo). Measurements
and simulations using the general model (Eqs. (45)) indicated that the shbstrate and
cell mass are in an ideally mixed state in the tower-loop system, provided that high
substrate concentration (S ~> Ks) for both batch and fed batch operation is employed
35,64). Significant substrate concentration profiles prevail in fed batch operations
only under substrate limiting conditions (S ~ Ks). These systems will be discussed in
Chapter 7.
122 R. Lunmann et al.
Owing to the practically constant longitudinal substrate and cell mass profiles,
the substrate and cell mass balances of Eq. (22) to Eq. (27) can be disregarded. Thus
the related concentrations of substrate, CsF(Z, t), and cell mass, CxF(Z, t), in the tower
section, and Css(Z*, t) and CxB(Z*, t) in the loop are given by
Csv(Z, t) = CsB(Z*, t) = : Cs(r) (89)

Cxr(Z, t) = CxB(Z*, t) = : Cx(t ) . (90)
During non-transfer-limited growth for oxygen the maximum oxygen reaction reaches
its optimal value,
rOmax("c) = roopt(t) 9 (91)
Assuming the validity of the substrate Monod model, roopt is controlled by the
increasing cell mass concentration, c x, and the constant (extended culture) substrate
concentration, Cs, as given by
~[m(t) CS('IS)
roopt(V) - Cx(l:) . (92)
Yx/o(t) ks + Cs(~)
So r o opt only varies with respect to time in the whole tower-loop liquid system.
However, if the maximum oxygen demand of the cells is higher than the theoretical
oxygen transfer rate from the gas phase, the maximum oxygen reaction rate,
r o.,ax(z), cannot be satisfied by the transfer mechanism. In a first modelling step it
is assumed that r o m,x(O is limited by the theoretical upper limit of the integral
oxygen transfer rate, otrLim(t ).
In Chapter 7 we extend this model and discuss the influences of spacial varying
substrate- and oxygen transfer.
Under extended culture conditions, r o max(r) and OtrL~M(Z) are in equilibrium during
limited growth conditions for oxygen transfer,
roma,(Z ) --" otrLlm(t) if Otre,m(r) < roopt(t). (93)
Under these conditions, the following reduced oxygen balance system was developed
from Eqs. (45) in Chapter 3 for the oxygen balance in the tower liquid phase,
CoF(Z, :)
lq'/Cor, = --romax('C)ko + Coy(Z, t) + Stvlz, ~) [plz, t) CoOZ, r) - Coy(Z, t)],
(94 a)
with initial and boundary conditions,
CoF(Z, 0) = p(z, 0), (94b)
~CoF(0, r)
- B%(r) v d t ) [Cov(O, t) - - c ~ . ( t ) ] . (94c)
~z
~C~ z~) - O, (94d)

~z
IVIathematical Modelling, Parameter Identification and Adaptive Control 123
and for the oxygen balance in the loop,
B{CoB} = - - r o max(r) CoB(Z*, r) (95a)

k o + CoB(Z*, r) '
with
CoB(Z*, 0) = pa(0) , (95b)
CoB(0, r = c~,F(r). (95c)
Furthermore, for the oxygen balance in the gas phase holds
G{CoG} = --Stc(z, ~) [p(z, ~) CoG(Z, r) -- Coy(Z, Q] (96 a)
with initial and boundary conditions,
CoG(Z, 0) = 1 , (96b)
~CoG(0, r)
~z - BOG(Z) v~(r) [Co~(0, r) -- 11, (96c)
~Coo(0, r)
--0, (96d)
~z
and for the quasi steady state continuity equation of the gas phase,
f Ii = --StG(Z, r) [p(z, r) CoG(Z, ~) -- CoF(Z, r)] rq(r),

G,tl (97a)
with
VG(Z, O) = vg(O) pE(~

p(z, 0)' (97b)
VG(0, ~) = V~(Z). (97C)
Substrate balances are not taken into account for extended culture simulations,
whereas the lumped parameter behaviour of cell mass is described by a space integro
balance equation,
dcx(r) __ vsO:)
Yx,o(Z) our(r) -- ptCx(Z), (98 a)
dr vv(r) + vn(z )
with the initial condition
Cx(0) = 1. (98b)
The coupling of space independent growth of cell mass (Eq. '(98)) and the spacial varia-
tion of oxygen reaction (Eq. (94) and Eq. (95)) follows from the overall oxygen uptake
rate,
v~(z)f CoB(Z*,z) (99)

our(z) = r o max(Z) f i ko CoAZ,~) Z) dz + v ~
+ CoF(Z, ~ ko + CoB(Z.' "c) dz* .
Although ro max(Z)is an unknown parameter in Eq. (99), it is calculable by integration

of Eqs. (94) to (97) to yield
qo2(Z) - Vv(Z)[c%(r) - cA,(z)]

romax(Z) = 1 (loo)
f Coy(Z,Z) dz
k o + Coy(Z,z)
o
Hence, r o max(Z) is known after identification of K o and calculation of Cov(Z, z).

Several problems occurred in solving this (simplified) model for a growth process in
tower reactors.
The choice of proper simulation methods and the estimation of the unknown par-
ameters is discussed in general in the next two chapters. The practical application,
using the developed model, and the comparison of the simulated data with the
measured values follows in Sects. 5.4 and 5.5.
5.2 Discussion of Various Simulation Methods
Process simulation is required in many different fields; e.g. design and case studies,
identification, optimization, and control. Some of these applications are treated in
Sects. 5.5 and 6.3. For distributed-parameter processes, a great variety of methods is
known from the literature, depending on the particular type of PDE involved. In this
chapter, the problem will be restricted to systems which show spatial (concentration)
profiles in one spatial coordinate only, so that an abstract description -- including
the five effects of accumulation, diffusion/dispersion, convection, reaction, and inter-
phase mass transfer -- can be made by a parabolic partial differential equation of the
form
Oy(x, t) ~2y(x, t) ~y(x, t)

at - a2(x' t) Ox2 al(x, t) ~x ao(X, t) y(x, t) -- f(x, t) (101)
where a2(x, t) > 0 in the entire time-space domain. Appropriate initial and boundary
conditions have to be added.
The following discussion of simulation techniques for PDE's of type (101)
concerns three aspects which are imposed by applications.
Firstly, fast routines are required, because computations are needed in real time.
Secondly, the suitability of the various methods to spatial 6-functions and their first
derivatives in the disturbance fwill be discussed. This leads to buckles or jumps in the
spatial direction of the solution y(x, t). A demonstration of the reason for inclusion
of these distributions will be given in Sect. 5.5. Furthermore the procedure should be
simple, easy to program and easy to use. -- Thus the available simulation techniques
will be evaluated.
Many simulation procedures for parabolic PDE's are known from the literature.
A classification can be made in the way proposed by Schuchmann ,01), who divides
into classes by treating the space and time domain as continuous, discretized or trans-
formed. This leads to a maximum of nine classes, of which the most important ones
will be treated in the following.
The DSCT-method (discrete space, continuous time) is the classical analog
computer method for simulation of PDE's, cf. e.g. Bekey/Karplus 917. The spatial
domain of the system is divided into a certain number of intervals, and at the
boundaries between the intervals the spatial differential operator is substituted by a
difference operator, employing only values of the solution at the location under
consideration and the adjacent node points. After inclusion of the boundary condi-
tions one 'obtains a coupled system of ordinary differential equations in the time-
domain, the solution of which approximates the solution of the PDE at the node
points. Using analog computers the solution of the entire system of ordinary differen-
tial equations can be performed in parallel. For digital computers many approximate
procedures are known, cf. e.g.P. Rechenberg lo27. A valuation based on the above
formulated criteria yields the following results:
The method is
I. on analog computers extremely fast, but with fine discretization relatively
complicated to patch; on digital computers relatively slow,
2. scarcely suited for/5-functions in the disturbance function,
3. clear and flexible in the programming structure, especially where digital simula-
tion packages are available, e.g. FORSIM lo3, HoT
The CSDT-method (continuous space, discrete time) is applicable on pure analog
computers only if an analog memory is installed. Here the temporal differential
operator is approximated by a difference operator. In this way, a sequence of spatial
boundary value problems is formed, the solutions of which are approximations for
the solution of the PDE at the corresponding time-instants. Due to the continuous
treatment of the spatial variable the procedure is very well suited for problems with
spatially varying parameters.
The algorithm is readily implemented on a hybrid computer. For pure digital realiza-
tions, approximation routines are used for the ordinary differential equations as in the
DSCT-method. For problems of type 1o1~,direct integrations of the two-point bound-
ary value problems usually lead to severe difficulties, since the equations are not
integratable in the forward nor in the backward direction without unstable error-
propagation. A method proposed by Vichnevetsky lO~7circumvents this difficulty by
decomposing the problem into one forward integratable and one backward integrat-
able subsystem. The following formulas give a short outline of the procedure: Let the
(temporally discretized) two-point boundary value problem at a distinct time instant
be
d2a dy
dx 2 al dxx -- aoy = ?, in ] 0, 1 [, (102a)
with boundary conditions
dY / =d, (102 b)
bly(O) + b2 ~ x=O
cxy(l ) + c 2 -dY X= 1 =e. ( 102 c)

dx
The eigenvalues
)~1.2=~ - 1+ 1 + < 5- , kx<0, ~2 > 0, (103)

al
are of different sign. Now forming two Equations,
du
dx klu = ~' (104a)
dy
dx Zzy = u , (104b)
one can see by substitution, that by (104a/b) just the system described by (102a-c)
can be modelled. However, (104 a) can be integrated in a stable manner in the forward
(ascending x) -- direction, and (104b) can be integrated in the backward (descending
x)-direction.
If homogeneous initial conditions are taken for the integrations, the problem can be
solved on a hybrid computer by first integrating (104a), then making a convolution of
the solution UAp and then integrating (104b) in backward direction. After a further
convolution of the obtained solution YAP,*one part of the complete solution of problem
(102 a-c) is formed. After computing two more partial solutions which are calculated
by letting u ( 0 ) = 1 with f - - 0 in (104a) and then solving (104b) which yields
YDP and by letting y ( 1 ) = 1 and u - 0 in (104b) which gives Ynm the final
solution of boundary value problem (102a-c) is obtained by superposition:
Y = YAP -{- KlYDP + K2YnB " (lO5)
The factors K 1 and K 2 a r e uniquely determined by the requirement that the boundary
conditions (102 b/c) must be satisfied.
The overall procedure is demonstrated in Fig. 8. -- Coupled equations and equations
with nonlinear or spatially varying parameters usually must be solved by iterative
methods, cf. Munack and Luttmann 1~)
V a l u a t i o n ."
The method is
1. on a hybrid computer very fast, particularly in the case of coupled PDE's; on
digital computers relatively slow,
YDP
_uAp
[ ~ YAP| YHB
ANALOG COMPUTER DIGITAL COMPUTER
Fig. 8. Schematic diagram of solving two-point boundary value problems by the decomposition
method 1 6 , 3 2 , 1 4 2 , 1 8 2 )
2. after some modifications suited for 8-functions on the right hand side of the Equa-
tion.
3. The programming is relatively complicated: an appropriate simulation package
is not known.
As DSDT-method (discrete space, discrete time) the commonly used difference
approximations are known. Here the temporal and the spatial differential operator
are each approximated by a suitable difference operator. At every time-step one
obtains a system of algebraic equations, the solution of which approximates the solu-
tion of the PDE at this time-instant at the node points. Detailed information is provided
in the book by Richtmyer and Morton tos)
Valuation ."
The method is
1. relatively slow, particularly in the case of systems of coupled equations,
2. after some modifications suited for 5-functions on the right hand side of
the equation.
3. Programming is very easy and clear: programming packages are available, e.g.
DSS 1o6)
In applications relatively restricted are the Monte-Carlo-methods. These generally also
use a grid-like discretized time-space-domain. Due to the fundamental difference
in the basic idea of this method it is not classified as a DSDT-method.
T S C T / T S D T - m e t h o d s (transformed space, continuous/discrete time) are the final
methods considered here. The spatial profile of the solution of the PDE is approximat-
ed by a time-dependent (linear) combination of given coordinate or basis functions,
which are usually members of a set of orthogonal functions in the space domain.
Insertion of these basis functions into the differential equation results in a spatial
error function, the so-called equation residual. In the same manner a boundary and an
initial residual can be defined. Using the interio~ method lO7), the boundary residual
is identical to zero. The inner product o f the equation residual with a suitable
spatial weighting function is finally used to determine the temporal derivatives o f the
coefficient functions for the corresponding basis functions. In the case of the discre-
tized time domain, a system of algebraic equations for the coefficients of the next time
step is formed in the same way. - - Many well-known and widely used algorithms can be
assigned to this class o f 'functional approximation methods'. So the Galerkin-
methods use as weighting functions just the basis functions, and taking 6-functions
one gets the collocation methods. Other basis functions lead to the modal simula-
tion 1087 and the Spline- or finite-element-approximation methods 109)
Having in mind this variety of possibilities it is impossible to give a general
valuation; so the following statements may certainly not apply to every special
case.
V a l u a t i o n ."
The method is
1. on analog- or hybrid computers extremely fast, if implementable there (normaliza-
tion problems !); on digital computers relatively fast; but in the cases of nonlinear
or coupled problems the speed of solution may be greatly reduced,
2. after some modifications well suited for 5-functions on the right hand side o f the
Equation.
3. The programming is simple, if simulation packages are used, otherwise excepting
collocation methods it is relatively time-consuming.
The above stated valuations are summarized in Table 2.
From this one can draw the conclusion, that for a fixed problem, the TSCT- or
TSDT-method would be preferable for our purposes. Compared with the discretiza-
Table 2. Valuation of on-line simulation techniques for parabolic partial differen-

tial equations
SPACE
continuous discretized transformed
analog/digital
0'1:3
~ physical speed + +/-- speed+ +/0
"==~ system 5(x) -- 5(x) +
8 ~ impl. + impl. 0
V-
.~ speed +.,'-- speed -- speed 0
~(x) + 6(x) + ~(x) +
9~ impl. - impl. + + impl. 0
uz not used for on-line calculations

tion methods, the lower flexibility should be no disadvantage, whereas the higher
computation speed and the good suitability for ~-functions offer great advantages.
- - During tests, requiring many modifications in the employed model, however, it
may be preferable to use the simple and very flexible DSDT-methods which have
usually automatic mesh size control and other attractive characteristics when a simu-
lation package is available. Using a hybrid computer, the CSDT-method runs extreme-
ly fast. Therefore, in cases where such a system is available (as it was in our
research), this method should be one of the first to be considered.
5.3 General Remarks on Identification of Parameters in Distributed

Parameter Systems
The last stage in system modelling is usually the identification of unknown system
parameters. For distributed parameter systems, many identification procedures are
known from the literature, see the surveys by Goodson and Polis 111), Polis l~a) and
Kubrusly 113). In the following, we will make some general remarks on parameter
identification. Section 6.2.2 contains a direct approach to the identification problem,
employing some results of optimal control theory for distributed parameter systems.
Here, however, a more indirect path is taken which immediately makes use of the
simulation techniques discussed previously.
A systematic approach to the parameter identification problem was published by
Goodson and Polis 114~. Based on our practical experience, a short summary of this
approach is given in the following, with some remarks from an applicational point
of view, in order to provide an insight into the extensive problems.
1. Formulation of a mathematical description of the plant. This is done by writing
down the balance Equations in the way previously described. Usually we are
involved with PDE's rather than with integral equations if we proceed in this
way.
2. Selection of a solution technique for the system equations. Here the discussion and
valuation of simulation techniques leads to a choice of an approximation scheme.
Goodson and Polis consider in their paper only the indirect approaches to the iden-
tification problem, which first approximate and then determine unknown para-
meters in the approximation. Hence this point is included here and not later.
In contrast to this procedure, there are many scientists who prefer the alternative
direct approach where an approximation is left until all other problems are
resolved. An example of the direct approach is to be found in Sect. 6.2.2, as
already mentioned.
3. Decisions on measurements. Many compromises have to be made concerning the
theoretical requirements on one side and practical considerations on the other.
Usually it is not possible to position the measurement sensors exactly where re-
quired and, of course, the available sensors usually do not have the desired
characteristics. But if possible the measurement characteristics and positions should
be chosen to be consistent with the theoretical requirements. Goodson and Klein Hs)
define the observability for distributed parameter systems and also a so-called N-
mode observability for systems having eigenfunctions. Kitamura and Nakagiri 116)
give some results concerning the relationship between N-mode observability and
the identifiability of parameters. It is noteworthy that for the identification of
constant parameters complete observability is not required. On the contrary, it suf-
fices to ensure obser~ability of some of the eigenfunctions, if the initial values are
known. Partial observability is guaranteed if the measurement sensors are not
positioned at the zeros of the corresponding eigenfunctions.
4. Definition of a petformance criterion. In most cases a quadratic functional is
chosen which weights the squared difference of the system's and the model's
output with a positive definite weighting operator. The weighting may vary
temporally and spatially. Spatial characteristics are determined usually by physical
considerations, while the temporal course of the weighting should be an increasing
function. In this way the newer measurements are weighted stronger, which results
in identified parameters lieing closer to the actual values of the temporally varying
system parameters.
5. Computation of sensitivity. The sensitivity can be divided on one hand into the
sensitivity of the functional with respect to the state of the system, and,
on the other side, to the sensitivity with respect to the unknown parameters. The
first mentioned sensitivity clearly forms a necessary condition for the second. If
low sensitivity is observed one should repeat steps 3) or 4). For further details, cf.
Seinfeld 1171
6. Realization of an experiment. For identifications, measurement data are needed.
If the parameters are to be identified off-line, then the experiment can be carried
out when having made decisions concerning 1) to 5). If one-line identification is
needed, then a preliminary experiment should also be performed to test the al-
gorithms and to decide properly on 7) and 8).
7. Selection of an optimization procedure. In every case, whether approximation is
made in an early stage or whether it is made as late as possible, finally a
numerical solution has to be searched with numerical optimization techniques. If
the gradient of the functional with respect to the unknown parameters is known,
then there is no doubt that gradient techniques should be employed for
optimization. These are Newton descent (if the second derivative is also known)
or steepest descent techniques. Procedures which circumvent the direct computa-
tion of the second derivative are called quasi-Newton-techniques, e.g. the conjugate
gradient method or the Davidon-Fletcher-Powell method. Sometimes heuristic
procedures prove to be even faster than quasi-Newton-methods, so that they should
be considered in addition. Good results in solving practical problems have been
achieved with the method of Nelder and Mead 118~and an evolution strategy - -
Rechenberg ~19j
8. Evahlation of errors. These calculations are used to determine the various errors
that appear in the whole procedure. Particularly errors in the structure of the
mathematical model, approximation errors in the numerical solutions, and
measurement errors should be estimated. In general, it is a difficult task to evaluate
the effects of these errors in the determination of the unknown parameters.
Hence one should usually consider all these errors and attempt to keep them as
small as possible, consistent with the financial, temporal, and computational
restraints.
5.4 Quasi Steady State Identification of Unknown Process Parameters
5.4.1 Hybrid Steady State Simulation and Identification Procedure

In the following chapters, the practical application of the proposed mathematical
methods in Sects. 5.2 and 5.3 will be demonstrated for four typical cultivations.
The first task is the identification of unknown parameters in the oxygen mass balances
of the system.
To identify the unknown parameters kLa E, Ks~, R o ma• and Ko, the cultivation
process is considered to be in a quasi stationary state. Thus the PDE-system (94) to
(97) is modified into an ordinary nonlinear boundary value problem. In spite of
this simplification a pure digital, sequentially operating integration procedure requires
extreme computer time, since the systematic variation of coefficients which are to be
identified needs several repetitions of the iterative solution procedures.
For a fixed equation structure it is advantageous to use a hybrid computer, in which
simultaneously an analog computer carries out a rapid integration of the model equa-
tions and a digital computer takes care of coordinating the particular solution
procedures 91. ~19,12ol
In the following, the problems of hybrid simulation are discussed briefly for the
dissolved oxygen balance in the liquid phase.
In quasi steady state behaviour, the simulation time, ~, appears only as a par-
ameter in the model equations, whereas the independent space variables, z and z*, are
transformed into the analog computer times
~, = D.z, (106a)
~, = flz* (106 b)
where -Q is the analog computer time interval. Thus partial operators are converted
into ordinary differential operators,
d 1 d
- (107a)
d~, f~ dz
d 1 d
. . . . . 9' (107 b)
d~* f~ dz*
The quasi steady state transformation of the oxygen mass balance for the liquid
phase (Eqs. 94) into the analog domain leads to a nonlinear ordinary boundary value
problem of second order,
Cov(~, ~) B~ vr(T) Cor(~, ~) + - - COF(~, ~)

f~ -Q
B~ Vr('C) i COF(~,, "r) li08 a)

- f~ 2 " or~ max{r) ko ~- COF(~, 2j)
+ St~(~) [(~(~,, "c) - 1) Cot( ~, rl - W(~,, r) p(~,, r) COG(C,,r)]}

)
=: flCov, COG, ~,, "el,
with boundary conditions at the lower end (signified by 7 = 1),
Boy('0 vv(r) r ,~
~ov(0, ~) - ~ tCovtU,~) -- coB(tl, v)], (108b)
and at the upper end of the tower,
6ov(~, ~) = 0. (108c)
Equation (108 a) is already split into a linear differential term at the left hand side as
well as a perturbation function at the right hand side. The latter includes the
solution in the nonlinear reaction term and the spatially variable oxygen transfer
rate.
Several so-called 'shooting methods' are given in the literature to solve nonlinear
boundary problems 121 - 1231
However, these methods are unsuitable for a hybrid solution of the given problem
because the left side of Eq. (108a) describes a time independent nonoscillating
unstable system of second order with the eigenvalues,
1~1,2(~) = Bov(~)VF(~) 1 -T- 1+ . (109)

2fl Boy(r) VF(~)
The analog computation of Eq. (108 a) is possible neither in forward nor in backward
direction. The only suitable integration method is the decomposition method of
Vichnevetsky 104,124~, already demonstrated in Sect. 5.2.
In contrast to the given linear problem of Sect. 5.2, the solution of Eqs. (108)
causes some difficulties:
1. The boundary problem is nonlinear in the solution Coy, this requires an iterative
solution method.
2. The lower boundary condition is not fixed, since it depends on the exit value of the
loop, which is itself dependent on the solution at the upper boundary of the
tower.
3. The oxygen balance in the liquid phase is coupled with the balance in the gas phase
of the tower.
In some cases unstabilities in the iterative solution procedure occur, but they can be
handled by relaxation methods 12sl.
The application of this decomposition procedure on the reduced oxygen balance
model will not be discussed in this paper. For more extensive detail the reader is refered
to the work of Luttmann 321. As a result of simulation, the typical course of
particular variables in the decomposition procedure is shown with regard to the
analog value range, [--1, +1]. From the perturbation function, f, of the forward
constituent, only the reaction term roe is plotted in Fig. 9 for simplification.
The identification of the unknown parameters was carried out by repetitive solution
of the model equations with differing parameter sets.
As performance criterion at identification time '~i'
ij(.~i ' i1~La E, i Kst,

" i Ro
^ .... lifo)
t
iJ(xi, kLaE, Ks, Ro .... Ko) V kLaE, Ks,, Ro .... Ko, (1 lo)
where
11
i j(.) = ~-~ Wji(CoF(Zj ' "[i) -- MCoF(Zj, "[;i))2 4- O'i(qoz('ri) -- MqO2('rl)) 2
j=l
+ qi(C~G(~i) -- MCgG(rl}) 2 4- ui(rOmax('Cl) -- cromax(ri)) 2
+ vi(rom~,(q)- OtrLiM('Ci))2 + t i k o . (111)
was minimized by varying kLaE, Kst, R 0 m,x and K o.

The search for the optimal parameter set, indicated by A, was under control of a
heuristic optimization method, proposed by Nelder and Mead H8. t26-1281
The performance criterion includes six sub-criteria, which are weighted with time
varying weighting factors, w, c~, q, u, and v. In Eq. (111) the weighting of
CoF, q02 and CoG minimizes the deviation of the measured and simulated process
variables, whereas the maximum oxygen rate r 0 max is only an intermediate unknown
1.0 -- 0.75 0.5 0.25 0

1.0 ~ i
tJ _ , Co F
0.5 ~ ~ " ~ . . ~ . ~ ~
2 BACKWARDINTEGRATION ..~... /
0 ~,-'3"~,3HOMOGENEOUS
SOLUTIONS/ ~,
-0.5
~ 5 FORWARD INTEGRATION -rOF

-1.0 q -UAp ~ I
0 012~ 0.5 0.7~ i~- Ii0
Fig, 9. Example for the variation of the particular solutions: (1) Final solution profile, COF.
(2) Backward step, CAp.*(3) Homogeneous solution in backward direction, c% and c*p. (4) Ox,r
reaction, - r o y . (5) Forward step, --UA~ ~8.32.34~
parameter. After identifikation of K o and calculating the optimal profile 6or(Z, ~),
romax(~) is always correctable by Eq. (100). The fifth criterion is used to fulfilI
the modelling approach of the limitation of maximum reaction by otruM (Eq. (93))
during oxygen transfer limited growth. Finally, the weighting of K o is due to a
widely unchanged specific growth rate occuring during oxygen transfer unlimited
growth conditions. This requires a very low Ko-value.
The decoupling of the optimization problem enables the decomposition of the
general problem into three sub-problems.
These were solved by the multi level procedure 32,34), shown in Fig. 10.
In the base level kea E and Kst are identified by means of the performance criterion 1,
tt
jltm(l:i) = E ,m
Wji(CoF(Zj, Ti) __ MCoF(Zj" Ti)) 2 + O'i(ql2('Ci) -- Mqo2(ti)) 2
j=l
Arm (112)
4- 1]i(CoG (ri) -- MCAG(Ti)) 2 ,
with fixed ko and r o ~,ax (optimization time ~i, identification step m).
After reaching a minimum in optimization step 1, the maximum reaction rate
ro max is recalculated by
Mqo2(.[i ) -- VF(I:i ) LF ~OAF~,"lSi

I , J, __ MCAB(Ti)]
J',(T,) '+'
= rOmax('Ci) -- 1
ko + 6~v(z, 1:i) dz
o
1+i I
= rOmax['[ i) -- crlmax(Ti) --" 0 (113)
in a correction level.
J3: IOENTIFICATIONOF Ko MODEL-LEVEL

HIGHER IDENTIFICATION
METHOD
OtrLlM romax
7 7
I q J2" C
ORRECT
O
IN OF Roma• ~ - I CORRECTION- LEVEL
I I
LOWER IDENTIFICATION
I COF I METHOD
I [
~Jl: IDENTIFICATION OF kLOEKst i BASE-LEVEL
! ' I
]. J
C o g , COB, C o G , V G I StFE,~,roma•
ITERATIVE HYBRIDE SOLUTION

OF MODEL EQUATIONS SIMULATION-LEVEL
Fig. 10. Three levelidentificationmethod 32.34)

The lower identification method is finished after satisfying the stopping criterion,
1+1
roma• r/max(Zi)l
l-el < eR. (114)
r o max('f i )
The oxygen saturation constant K o is identified in the upper model level, using the
described lower identification method.
In the initial range of oxygen transfer limited growth phase (identification time
rK. . . . . rK+N), the performance criterion 3,
K+N
n
J3 (TK . . . . . ZK+N) =
n
E Vklroma• "Ok) --
n 2 n
otrLIM(rk)) + tkko, (ll5)
k-K
is minimized with respect to K o.

In optimization step n, the lower method is carried out for each identification
time z K of Eq. (115) with fixed k o. After identification of kLaE(zK) and Kst(zK),
otruM(rK) is calculated in the base level.
This procedure is repeated with changing ko, until a minimum of Eq. (115) is
found. After identification of Ko, the time course of kLa E and Kst is evaluable for
the whole cultivation using the lower identification method.
5.4.2 Mass Transfer Analysis of a Batch/Fed-batch Cultivation

Several cultivations were carried out in tower reactors by Buchholz 61-65) under
different operational conditions. They were analyzed in their oxygen mass transfer
behaviour by means of the quasi-steady state identification method 32-39). However,
in the present paper, only some typical examples will be discussed, whereas a survey
of the identification results is reported by Schtigerl 61).
In Fig. 11 measured and simulated longitudinal dissolved oxygen concentration
profiles in the tower liquid phase are shown for the cultivation on ethanol substrate
at different cultivation times.
The experiment began as a batch process with a substrate concentration of
So = 6 . 6 g l 1. After this amount of substrate was consumed at t = 14.5 h, a
substrate shift was carried out, and substrate was fed to the system continuously (fed
batch operation). With increasing biomass concentration, the oxygen requirement
increased and consequently the necessary dissolved oxygen driving force increased.
Hence, the dissolved oxygen concentration gradually diminished up to t = 13.5 h.
Thereafter, the cell respiration diminished due to lack of substrate. Thus the oxygen
requirement decreased and the dissolved oxygen concentration increased again at
t = 14 h. To investigate the influence of substrate concentration variation on mass
transfer behaviour, at t = 11.1 h a substrate pulse was added to the system. This
caused a short substrate concentration increase from 2.6 g 1-t to 5.2 g 1-1 and a
corresponding increase o f kea (Table 3 and Fig. 12).
In the upper part of Fig. 12 according to Eq. (70), ke a', the identified volumetric
mass transfer coefficient in the greater distance range from the aerator (0.1 < z < 1),
kLa~(ti) = ktaE(ti) q~(ti), (I 16)

t[h]
~6 0.0
"11.1 S-shift
N11.0
4
"14.0 end of
botch phase
"13.0
/13.5
~'15,0 extended
LR= 2.75 m culture
l l l l
0 ~ , , , , , , , , I , , , ,
0 A-~ 0 1.25 0.5 0.75 - 1.0

z= x
LR
Fig. II. Batch/fed-batch cultivation, Hansenulapolymorpha with ethanol as substrate. Measured and
calculated longitudinal profiles of dissolved oxygen concentration at different cultivation times,
Ko = 0.25 mg 1-1, (symbols refer to the measured values, curves to calculated ones) 32.36~
Table 3. Measured and simulated values and identified parameters for the cultivation considered in
Fig. 1I. M: measurements, S: simulation, I: identification
t Qo2 A
XoG X S kLa= qJ~ U~o
(h) ( g l - l h -1 (%) (g1-1) (gl -~) (h -1) (--) (cms-')
1 6.0 0.27 0.28 20.37 20.24 0.47 4.90 859. .37 1.89
2 9.0 0.61 0.60 19.67 19.60 1.00 3.70 1297. .43 1.89
3 10.0 0.80 0.80 19.25 19.22 1.12 3.30 1093. .45 1.89
4 11.0 1.08 1.07 18.64 18.51 1.50 2.60 814. .66 1.89
5 11.1 1.16 1.16 18.64 18.56 1.50 5.20 1286. .72 1.92
6 13.0 1.96 1.96 17.06 17.04 2.92 2.30 748. .50 1.92
7 13.5 3.12 3.12 16.80 16.71 3.38 1.30 825. .45 2.89
8 14.0 1.04 1.04 19.48 19.44 4.05 <0.08 393. .68 2.89
9 15.0 1.88 1.89 18.47 18.44 4.48 0.50 406. .44 2.89
M S M S M M I I M
where
qJ~(tl) = e -Ksdtil , (117)
is p l o t t e d a t i d e n t i f i c a t i o n t i m e s t i.
T h e values o f kL a~ w e r e c a l c u l a t e d f r o m the i d e n t i f i e d v o l u m e t r i c m a s s t r a n s f e r
c o e f f i c i e n t at tile gas e n t r a n c e , k~ean, a n d t h e i d e n t i f i e d c o a l e s c e n c e f a c t o r K.st.
E F[-] --.- ,' ,,

0.90 #.8~: 0.8~
1.89 ,1.92,
I , 2.89
U~o [c m s -1}..--i=~-,
1
T
k~ ~ ','1 . . . . . .
Tf 7
bo,~h 1 / qa f~d-bot~h
1000 f I' :ll ~- I 9 2000
L / ~l t II.AERATIONRATE
, ~ ~ ~,.CREASE I
/ ,H,FT , '! IJ ) I
500 :z'l&l L _,~ 1000
j . .-....wr, ~u~u~s~,~t~ :i :
_ . _
,, ,~ONT,.oou~,~ I
i i
100 /(m}FOT0 , :1, I I 2OO
r z.o;, , ', i l l t_ I 0
2 10 20 30
. . . . '' i' ' ' I ' '
-
"T
- 2.0 I0
s
:3 t~
1.0 5
e ~
. ]. L,,.,'.l~moyl .:., I
, 0
0 2 10 20 = 30
t [hi
Fig. 12. Variations of measured (M) and identified (I) parameters of the cultivation considered in
Fig. 11 and Table 3. C), volumetric mass transfer coefficient, kLa= (I); I . specific interfacial area, a
(M); O, superficial liquid velocity, UFo (M); U], substrate concentration, SExu (M); ~ , coalescence
factor, Kst (I) 32, 34)
To compare the identified values and the time course of kLa with other information
concerning mass transfer parameters, the specific gas/liquid interfacial area,
A(tk) 6~G(tk) (I 18)

a(tk) -- VF(tk) -- ds(tk) [1 - - e(tk)] '
is also plotted in Fig. 12.

The parameter of Eq. (118),
A(tK), the gas/liquid interracial area at cultivation time, tK,
Vv(t~), the liquid volume in the tower,
e6(tK), the mean relative gas hold-up, and
ds(tK), the local Sauter bubble diameter at z = 0.5,
were measured and evaluated using the methods of Buchholz et al. 13o) and
Zakrzewski sg~
According to the measurements of Zakrzewski, which were carried out using double
sensor electrical conductivity probes, the spacial distribution of d s is nearly
uniform in the tower with the exception of the aerator range. In the lower part of
Fig. 12, the identified Kst value and the measured substrate concentration, S, as well
as the measured superficial liquid velocity, UFo, are plotted.
Following a steep increase of kLa~ during the first six hours, a sharp decrease
due to decreasing alcohol concentration was observed. A short substrate shift at
t -= 11.1 h, causes a strong increase in a and kLa'. Owing to a significant increase in
biomass concentration and substrate consumption rate with time, the ethanol concen-
tration fell rapidly at the end of the batch phase. At low ethanol concentration, the
coalescence rate of small primary bubbles produced by the porous plate aerator
increased, thus the specific gas/liquid interfacial area, a, strongly decreased.
Even a considerable increase in the gas flow rate at t = 13.5 h, which usually yields
a significant increase in kLa in model systems, to, la0), could not compensate this
coalescence effect.
Figure 12 also indicates the strong dependence of the liquid circulation rate due to
the air lift effect on the two-phase flow properties due to a substrate concentration
change. With the exception of the initial cultivation time, kLa", Uvo and S are
clearly related in this run. Such a clear relationship, however, is not always present
and indicates the complexity of this system. For instance, a considerable increase of
kLa and a at the beginning of the cultivation was observed in several yeast cultiva-
tions 6o) and is caused probably by compounds secreted by the cells to improve their
environment.
During fed-batch cultivation, cell growth becomes oxygen transfer limited at times
greater than t = 16 h. Longitudinal dissolved oxygen concentration profiles exhibit
a steady state character during this oxygen transfer limited growth range, and kLa"
remains almost constant as long as the substrate concentration is kept constant
(extended culture). At the end of the cultivation, the substrate concentration and
kLa" change, too.
5.5 Simulation of Fed=batch Processes
The dynamic behaviour of fed-batch processes with constant substrate concentrations

(extended culture cultivations) is described by the distributed parameter oxygen mass
balances model and the lumped parameter cell mass model given in Sect. 5.1.
5.5.1 Hybrid Dynamic State Simulation
The chosen CSDT-method for the quasi-steady state identification of the mass transfer
parameters kLaE(t) und Ks~(t) as well as identification of the oxygen saturation
constant K o has been discussed briefly in the previous section.
Only the extensions of the decomposition method for nonstationary hybrid simula-
tion of the model equations will be considered here and demonstrated by means of
the oxygen mass balance in the liquid phase. The model equations are solved for
continuous space at discrete points of time
v~ = i A t . (119)
The time-differential operator is replaced by the backward difference quotient,
0CoF(Z, t) ~=i~ Cg~:(Z)-- C~I)(Z)

(120)
0z At '
and the remaining independent variable, z, is assigned to the analogous time, C,, by
means of Eq. (106a).
This yield for every discrete time, ~, in the spacial iteration step k, an ordinary
nonlinear boundary value problem of second order,
BO~)V~)"
I ],,
Bo~)v'~' [ cgg-1'(~)
- n~ rg'L', ko + cgr
+ coL
) EFl i ) rttI
f t LI/(i)[~%
~,J- 1) Cg'Vk- 1)ff,)- tp(1)(~,)p(1)(C,)CO(i'k)' ~J)
G ~t'z
At J
=: f"" k)(cgi:k-1), COG,"'k)r(~,

k,o
. . . . Cg{ 1), ~), (12t a)
with the boundary conditions,
dg.g)(o) _ Bo~'
- -
v<~) [Cgi=k'(0) -- Cgi3k)(~)], (121 b)
f~
dgi~k'(f2) = O. (12lc)
In addition to the quasi-steady-state treatment, the following changes are necessary:

a) The eigenvalues of the linear part of Eq. (121a),
?,, B@'v~' { l 4(1 + A~ Sty")))>

""- 2f~ 1 -T- 1+ X~Bo~'v~
~ J' (122)
depend on the discrete time interval, At, and their values increase considerably
with diminishing time interval A~.
b) Besides the superposed solution, c~'vk- ~1, of the preceding space iteration step
(k -- 1), the perturbation function, f,,k), also contains the solution, cg~7 t~, of the
preceding time step (i -- 1).
c) The maximum oxygen reaction rate, ,,. k~ in case of process conditions which
~o ....
are not limited by oxygen transfer (otrLiM(~) > roopt('0) is given by
rli, k) __ l'llmit c ~ , k ) ,
Omax- v(i~
CS>~> k s. (123)
ax/o
At time ~, the cell mass concentration can be calculated analytically by t h e

Monod-model,
= e (124)
The specific growth rate is calculated at xi-0.5, as a mean value for the tower-
liquid system,
oI.k~+ ~ . - ~
120- o.5.k) ~ (125)
2
where
v~' ,.,j. l)(., v~' cgi~"(~,*) d~,*
(126)
In the oxygen-transfer limited growth phase, (otrLIM(Z) < roopt('c)), the maximum
oxygen reaction is limited:
ti, k) (i) t c + E(i)

(127)
r o max = r o max = o t r L I M b 3 t F , tr*
~a_=lit) .
In this growth range a quasi-linear growth and an exponential cell mass loss
prevail:
c'~'
. = c~ . t ' e .ta~
. + .Ya)o + Y~/o1' ~trli)
L I M P ".(i.k) _}_ otr~lMl~pr
'- -"- 1~ A t . (128)
~/~m
i) @ ~l~mi-1} 2
Similar extensions of the quasi-steady-state oxygen mass balances in the gas phase
and in the loop are necessary.
5.5.2 Analysis and Simulation of Hansenula polymorpha Cultivation on Ethanol

Substrate
Figure 13 shows the longitudinal dissolved oxygen concentration profiles, both
measured and calculated by means of the presented model, in the tower medium
during the extended culture operation at constant ethanol concentration (S = 5 g 1-1)
and constant aeration rate (0.55 vvm) 58,62)
Flat longitudinal profiles exist due to the low cell mass concentration and oxygen
uptake rate at the beginning of cultivation. With increasing cell mass concentration
and oxygen uptake rate, the dissolved oxygen concentration gradually diminishes in
the upper part of the tower to maintain a high driving force for the oxygen
transfer rate. At the beginning of the oxygen transfer limited growth range (t = 14 h),
the longitudinal profiles exhibit the most significant nonuniformity. In the strong
oxygen transfer limited growth range the profiles again become flat (t-= 25 h),
since the maximum driving force is attained rapidly due to the high cell mass
concentration.
The corresponding measured and simulated process variables and identified model
parameters are listed in Table 4. Owing to the strong coalescence suppressing
substrate ethanol, high volumetric mass transfer coefficients, kLa, and high oxygen
10
S:5g1-1
l" [hi
8
~,...~1,:"-'0"-'0---.0~
2 "0-4:~,C~
\3.o
"11.0
"13 o
"""Q~7~ ~_. / ,16,0
0~" 0 0.25 0.5 0.75 -'-- 1.0

x [-I
LR
Fig. 13. Extended culture cultivation, Hansenulapolymorpha with ethanol as substrate, S = 5 g l-t.
Measured and calculated longitudinal profiles of dissolved oxygen concentration at different cultiva-
tieq times, Ko = 0.12 mg 1- ~, (symbols refer to the measured values, curves to calculated ones) 32.3v.38~
142 R. Luttmann et at.
Table 4. Measured and simulated values and identified parameters for the cultivation considered in
Fig. 13. M: measurements, S: simulation. I: identification
t Qo2 xoAo X S kLa~ qJ" UEo

(h) (gl - l h -1) (%) (gl -l) (gl-') (h -1) (--) (cms -1)
1 3.0 0.30 0.26 20.53 20.49 0.39 5.00 1315. .57 2.89
2 8.0 0.85 0.85 19.27 19.22 1.34 1630. .61 1.92
3 11.0 1.62 1.62 17.78 17.74 2.80 1555. .63 1.93
4 13.0 2.68 2,67 15.63 15.56 4.77 1314. .64 1.93
5 14.0 3.65 3.65 13.67 13.63 6.15 1147. .60 1.93
6 16.0 4,91 4.92 ll.29 11.18 9.93 1164. .50 1,94
7 19.0 5.11 5.11 11.16 11.11 17.60 1118. .51 1.94
8 25.5 4.73 4.72 11,87 11.85 29.30 5.00 951. .73 1.94
M S M S M M I I M
':
I i
:{
0.82 '&8' 0.78 ', 0.72
I ' 0.84
I I I
,1..941,
, 1.94 ' 2.961
1.92 1.93
1500 1.5
s
1000 1.0
500 0.5
100 0.1
0 0
0 2 10 20 ~ 30
t [h]
Fig. 14. Variations of measured (M) and identified (I) mass transfer parameters of the cultivation
considered in Figs. 13, 15, and Table 4. IN, specific interracial area, a (M); C), volumetric mass
transfer coefficient, kLa~ (I); A, coalescence factor, Ks~ (I)32.37. 381
supply rates, Qo2, are attained. In spite of the nearly constant operation conditions,
in the range t = 4.5 h to 32 h with regard to S and the aeration rate u Go~ E the
volumetric mass transfer coefficient, kLa", varies considerably during the cultivation.
The identified parameters kLa", Kst and the local specific gas/liquid interfacial area,
a, are plotted as functions of the cultivation time, t, in Fig. 14. The interfacial area a
was calculated by Eq. (118), where the mean gas hold-up to(t) and the Sauter bubble
diameter ds(t ) was determined by Zakrzewski 597
kLa" as well as a pass through a steep maximum and then gradually diminish:
The maximum is probably due to the cells controlling their environment by secretion
of surface active agents.
The significant increase of a in the transition range between nonlimited and oxygen
transfer limited growth coincides with strong foam formation. Under these conditions
large amounts of small bubbles are formed which remain in the medium for a long
time, thus they are no longer "mass transfer active".
kra ~ therefore does not follow the increase of a in the range t = 14 h to 18 h.
The diminution of k L due to the adsorption of proteins at the interface can also con-
tribute to this difference in the courses of kta" and a.
The model parameters kLa E and Kst were identified for a fixed time by the
quasi-steady-state method, discussed in the previous chapter. The time course of
kra E and Kst during the process was evaluated by a linear interpolation between
identified values.
The unknown growth parameters ~t.... gx, Yx/o and K o are necessary to simulate
the extended culture fermentation. K o is determined by the quasi-steady-state method
of Sect. 5.4., whereas information is available for the specific growth rate ~t and the
yield coefficient Yx,o from experiments.
The experimental specific growth rate,
In MX(tk + 1) - - In MX(tk _ 1)
la~xp(tk) : = , (129)
tk+ 1 -- tk_ 1
is evaluated from the exponential growth of the cell mass during non-oxygen-transfer
limited growth phase. The observed value of p includes the virtual (maintenance)
death rate gx. It is very difficult to evaluate gx from measurements. Assuming
a time varying yield coefficient Yx/o, the parameter jaT can be neclected and the
unknown maintenance is identified indirectly in the time course of Yx/o'
In the non-transfer limited growth range, the calculated oxygen transfer rate (with
regard to the entire amount of liquid) and the cell growth are coupled, as given by
dMX(tk) Vv(tk)
dt - MYx/o(tk) Vv(tk) + VB MQoz[tk), t130)
where the oxygen accumulation term and cell death rate are neglected. The oxygen
yield coefficient at measurement time, t K, can be calculated approximately from Eqs.
(129) and (130) :
Vv(tk) + VB btexp(tk)MX(tk)
MYx/o(tk) -- Vv(tk) MQo2(tk) (131)
In the oxygen-transfer-limited range the cell growth rate is controlled by the maximum
oxygen transfer rate, O T ~ M , and the parameters Yx,,o and K o.
Sifice the Jells grow quasi-linearly with time, Yx/o can be calculated by means
of the slope of the cell mass at the time tK:
VF(tk) + VB MX(tk+l) -- MX(tk_l)

MYx/o(tk) -- Vv(tk) [tk+ l __ tk-a ] MQo2(tk) . (132)
The maximum specific growth rate, ~t. . . . is calculated according to Eq. (133) in the
nonlimited growth range (up to t = 14 h):
1 N
- - ~ ~t~xp(tk)
N k=l
~s = LR (133)
TF(0) (' O~-o(X) dx TB(O) O~o(LR)
Tu(0) J K o + O~o(X) L R + Tu(0) K o + O~o(LR)
0
]s and MYx/o are shown in Fig. 15a as functions of cultivation time. MYx.,o varies
considerably during the cultivation, especially following the transition of growth
into the oxygen transfer limited range at t. > 14 h. In the strongly oxygen-transfer
limited growth range (t > 25 h).uYx! o is low due to the endogeneous reaction
term.
The inaccuracies of measurement analysis during the transition interval (16 h < t
< 25 h) are reduced by identification of the smoothed time course of Yx/o during
non-stationary simulation. It is assumed, that Yx/o changes linearly in an identifi-
cation interval T K -- [tk, tk + ~],
Yx/o(t) = Yx/o(tk) + AYx/o(TK) [t -- tk] . (134)
The course of Yx/o is given by the known final value of the previous interval and
the unknown slope AYx/o(TK).
The identification problem in the interval T~ is converted into an optimization
problem,
J~(rk, "Ok+l, A~Yx/o) "--<J~('Ck, Zk+ l, AYx/o) V AYx/o, (135)
with the extended culture model as constraint.

in the performance criterion,
Xk+l
cAm A 2
J~(Zk, rk+l, AYx/o) = {~K(q~2(T) --i~qoz(Z))2 + rlK( OG(Z)- MCoG(Z))
Tk
+ (c~(z) - MCx(X))z} 6(MZi) d ~ , (136)
the quadratic deviation of the measured and simulated values of the exhaust gas
analysis (qo2 and C~G) and cell mass (Cx) is weighted. During the oxygen limited growth
Mathematical Modelling. Parameter Identification and Adaptive Control 145
0.75 0.35
'~'i
i
i
~i "'
, ETHANOL ~Jmax
i
i i
i
k F~
I I
i r
\ ----'L__ ~
r-, ,_J
i
0.50 ! \ 025 ~' ~t~.~---~ ~_J .c
9 "r"
i i
; ' \.
0.25 0,15 i i i
0 10 20 = 30 0 10 2O
t [hi t [h]
EF [-1 -- :,,
0.84 0.82 :=o.8 i 0.78 ;0,72
0.7:: 2.89! 1.92 ,i 1.93 ~.94i 1.94 ', 2.96
uE ,, ,,
Go [cm s4] ', i
NONTRANSFER LIMITED TRAN- OXYGEN TRANSFER

GROWTH SITION LIMITATION
.12 ,, I '
r 1
~/i/
i
7.5 i/! 37.5

~ \.
OTRLIM X
,l~r V
1
\,/-~-..
,i T
s tel i /
5.0
! : x ;~ 25.0
j
A
x OG
-,, !/ Z
2.5
/
/4
~
"
~
~o']~~_~. ~ ~ 12.5
c~
' : 0
0 10 20 t [h]" 30
Fig. 15a. Oxygen yield coefficient. Yx:o, and specific growth rate, ~tew. during the extended culture
cultivation considered in Figs. 13, 14. 15b, and Table 4; - - - - , parameter identification with
methods discussed in Chapter 6 32,37); b. Courses of measured (symbols) and calculated system
variables and process parameters during an extended culture cultivation of HmTr nnh'mnroha
with ethanol as substrate, S = 5 g I 1. O, oxygen supply rate, Q02; A , cell mass concentration, X;
1~, oxygen mole fraction in exhaust gas, Xoa6; - - . - - , upper limit of oxygen transfer rate, OTRuM;
- - - - maximum oxygen reaction rate, R 0 max; - - - - , productivity (cell growth rate), PRD 32. 371
phase, t > 17 h, the exhaust gas data qo2 and cAG are known via pre-identification
of kLa and Ks1 and the proposed reaction limitation with OTRL~ u (Eq. (93)). In this
range the weighting operators ~K and qx are kept to zero.
In Fig. 15b, the results o f a nonstationary simulation are shown.
Four ranges can be distinguished:
1. Cultivation starts with a low aeration rate. In this lag phase the constant
oxygen yield coefficient is identified for the initial phase.
2. Nonlimited growth is begun with an increase of the aeration rate for a short
duration in order to start the medium recirculation. Owing to the high dissolved
oxygen concentration in the tower and loop and the low oxygen saturation
constant (K o = 0 . 1 2 m g l - 1 ) , up to t = 14h, no oxygen-transfer limitation
prevails.
3. In the time range from t = 14 h to 18 h, a transition between nonlimited growth
and oxygen transfer limited growth occurs. The oxygen uptake rate is limited
by the oxygen transfer rate, and the specific growth rate falls (Fig. 15 a).
At t = 17 h, the cell oxygen requirement depends on the maximum oxygen
transfer rate, OTRLI u. Thus the maximum reaction rate, Romax , is limited by
OTRLIM.
In Table 5 the comparison between calculated Roma~ (similar to Eq. (100)) and
the "identified" OTRLIM (Eq. 85)) shows quite a good agreement. A fitting with a
model which does not consider the CO2-backtransfer (RQ = 0) yields oxygen mole
fraction values which are too high in the exit gas at the same oxygen transfer
rates. This indicates that an increase in the gas flow rate due to CO 2 backtransfer
must be considered. In a model without CO2 backtransfer 14. ~5~exit glas flow rates,
u A, which were too low, and oxygen mole fractions, x'~G, which were too high
in the exit gas, were obtained.
4. In the strong oxygen-transfer limited growth range the oxygen uptake of the cells
is reduced with the diminishing volumet~:ic mass transfer coefficient. A considerable
increase o f the aeration rate at t = 31.5 h improves the oxygen supply rate, Qo2,
and the cell oxygen uptake rate.
The identified course of the yield coefficient (continuous line in Fig. 15 a) smooths
the strong fluctuations o f Yx/o evaluated for the identification intervals.
Figure 15b, shows the excellent agreement between the simulated (continuous
line) and the measured (symbols) values. P R D indicates the cell mass productivity
Table 5. Measured and simulated values and identified parameters of the cultivation considered
in Fig. 15b during the transition range of non limited and oxygen transfer limited growth phase.
M: measurements, S: simulation, I: identification
t Qo2 xAG kLa~ W" Ro may OTRLtM

(h) (gl - l h -1) (,%) (h -1) ( ) (gl - l h -1)(gl lh-1)
17.0 5.12 5.11 10.87 11.17 11.48 1202. 0.57 5.92 5.96
18.0 5.23 5.17 11.04 11.18 11.46 t151. 0.55 6.04 6.10
19.0 5.11 5.11 11.16 11.11 11.63 1118. 0.5l 6.01 5.94
20.0 4.9l 4.91 11.54 11.64 12.15 1061. 0.68 5.70 5.66
M S M S:MRQ S:RQ = 0 I I I I
which is identical to the cell growth rate in batch and fed-batch cultures. The
exponential increase of P R D in the nonlimited growth range follows a maximum
in the transient range and then P R D diminishes due to the reduction of the actual
oxygen supply rate, Qo2, and the yield coefficient, Yx/o.
5.5.3 Analysis and Simulation of Hansenula polymorpha Cultivation on Glucose

Substrate
The model and the simulation techniques described in Chapter 3 and 5 are also
used for the modelling and the simulation of cultivations with glucose, which acts as
a coalescence neutral substrate. An extended culture operation was again carried
out at a constant substrate concentration of 2 g l - t . During the cultivation, the
aeration rate was increased step by step. The quasi-steady-state evaluation with
regard to the mass transfer parameter exhibits a strong influence of the type of
substrate on the fluid dynamic behaviour of the process. In spite of the low
oxygen transfer rate in the initial range of the cultivation, the longitudinal dissolved
oxygen concentration profiles diminish considerably with increasing time (Fig. 16).
The low values of the identified volumetric mass transfer coefficients, kLa", (Table 6)
are due to the relative low aeration rate and high bubble coalescence. The gradual
S= 2 g [ 4
t [hl
~2--~ o --"----W
~1.0
3
2 ~3.0
~6~---..x3 o
/4.3
\12.0
LR: 2.75 m
~'5.0
o. 0.25 0.5 0.75 -- 1.0

x
LR [-]
Fig. 16. Extended culture cultivation. Hansenulapolymorpha with glucose as substrate, S = 2 g 1- t.
Measured and calculated longitudinal profiles of dissolved oxygen concentration at different
cultivation times, Ko = 0.23 mg 1-1, (symbols refer to the measured values, curves to calculated
ones) 32.37)
EF [-] i '', ,'

i i i
0.96 i I i
,0.94,.92,.88 0.82 0.80
0.57 10.9511.411.91 2.90 2.91
O~o[Cms-']!--i l I
! !
i f

GROWTI4 SITION LrMITATION
" " . ,, . . ,+.-.. .

500 ,i ', z , /~mO 1.0
i / f
I i, / ,, kL oct
i
,..C I i
'= ': Ii
t~
o i
,, ,
i
,
!
i
I+
250 0.5
i
" i,
50 ~4"~J" '--'~4 ETNA~NOL DEITECTiON L[MIT-.---~"~"EI - ~_____._~._ .o 0.1
0 i I 0
1 2.5 5 7.5 0
i i i
a
i i i = i
i
i
', ,~
2.0 L 2.0
9i" i
-'.% 1 r u
o
T ,i ^i / E
"A...~ 0~
<1
9A9 _ ". ~ IL'zN"
"A ""zX, ,~......~."
,.~176
i
1.0
,-*4,' i
i
I
i
"-.~."
1.0
i
i
:J
i
I i
i i i
i i i
i i
I i
i i i
i i
i i
i
0.2 i r a2
i i
0 I I I i i i i I , i
0 1 2.5 5 7.5 0 -- 1Z5
t [h]
Fig. 17. Variations of measured (M) and identified (I) process parameters of the cultivation considered
in Figs. 16, 18, and Table 6. O, volumetric mass transfer coefficient, kLa= (I): Fq, concentration
of product ethanol, PFT, (M); A, coalescence factor, Ks~ (I); 0 , superficial liquid velocity,
UFo(M) 3z, 3v)
increase o f the superficial gas velocity, UEo, causes an increase o f kLa '~ at a nearly
constant coalescene rate. (Table 6).
A t t = 5 h, ',he oxygen-transfer limited growth phase starts. It can only be
interrupted for a short period o f time by a considerable increase of the aeration
rate at t = 5.1 h (Fig. 17). This figure shows how kLa" and Uvo increase with
Table 6. Measured and simulated values and identified parameters for the cultivation considered
in Fig. 16. M: measurements, S: simulation, I: identification
t Qoz x~o X S kLa~ ~P~ Ugo

(h) (gl -~h -~) (~ (gl -~) (gl -~) (h -t) (--) (cms -~)
1 0.1 0.12 0.12 19.94 19.81 0.33 2.00 38. .14 0.57
2 1.0 0.26 0.25 18.74 18.79 0.51 75) .2l 0.57
3 3.0 0.82 0.83 16.89 17.06 1.73 198. .24 0.95
4 4.3 1.80 1.80 16.80 16.76 336. .20 1.90
5 5.0 2.19 2.19 15.88 15.85 5.61 380. ,25 1.90
6 12.0 2.44 2.46 17.47 17.48 24.02 2.00 437. ,2l 2.91
M S M S M M I I M
increasing superficial gas velocity, U~-o. The coalescence factor, Ks,, is much higher
in this system than in media with substrate ethanol (compare Figs. 14 and 17). This
means that the coalescence function, W~, is much lower in substrate glucose systems
than in systems with ethanol as substrate (compare Tables 4 and 6). In glucose systems,
the bubbles attain a much higher degree of coalescence (larger size) than in ethanol
systems. Since ethanol has already been produced during the nonlimited growth
range (product ethanol, PEx,), the value of the identified kLa" parameter during
this time is higher than that in ethanol-free systems with similar aeration rates 13o)
In Fig. 18 a, the simulated and measured process variables are shown as a function
of cultivation time, t, with glucose as substrate. During cultivation, the superficial
gas velocity, U~o,E is increased step by step to improve the maximum oxygen
transfer rate, OTRusv In spite of the considerably improved OTRr~M, the maximum
oxygen requirement of the cells, Rom,~ surpasses OTRrIM at t = 4.8 h. Thus a transi-
tion between nonlimited and oxygen-transfer-limited growth occurs. By means of a
considerable increase of UEo at t = 5.1 h, this transition can only be interrupted for a
short period of time. Thus, the cells grow under oxygen-transfer limited conditions
for the rest of the experiment.
The corresponding growth parameters, Yx,,o and gexp, are shown in Fig. 18b.
Ywo dimin!shes with increasing time. In the lag phase ~texp increases, attains a maximum
and then diminishes due to ethanol production.
5.5.4 Comparison of Ethanol and Glucose Substrate Systems
In Fig. 19, the three-dimensional plots of the courses of processes are shown for
ethanol and glucose substrate cultivations.
The base of the diagram consists of the dimenslonless longitudinal coordinates
x/k R for the tower and x*/L B for the loop, and the cultivation time coordinate t.
Perpendicular to the base, the concentrations of dissolved oxygen in the tower, O F,
and in the loop, On, and the cell mass, X, as well as the oxygen supply rate, Qo2, the
maximum oxygen transfer rate, OTRLI~V and the oxygen mole fraction in the gas
at the outlet, x~a, are plotted. For OF and On twodimensional (time and space)
variations and for X, Qo2, OTRLI~a and Xo~Aonly one-dimensional (time) variations
are possible. The changes of the superficial gas velocity, UOo,
E and the mean relative
liquid hold-up, ev, are indicated by means of arrows.
EF [-] ; ,,i :,, ,,i

0.96 ]0.94:0.92t0,88: 0.82 : 0.8
r
0.57 i0"95
' i'1 "41i 1"9 i 2.9 I 2.91
U~o[Cm s -~1~', ' , ' i
i
I t 1 " i

GROWTH SITION LrMITATrON
i i i i t i i
i
, ;
i i
i i
i
-C
5 ,, ; 25
%-
! i
, , : ,%-~ : /
20
l 3 , , ,
(b t 1 i .~
i i !K/" o~ ',
:; ~ / ~
; c'/~ /
2 ' i ,i lo
ll J7 2 i-
1
- OTRLIM /
"_ / / ~ i/4 :i / / 5 ":
0 , i , , ' ' 0
0 2.5 5 7.5 t[h]-- 10 12,5
a
1.5
_--r-AT ~ G A - , i t i ;--T--F-- 0.65 I I I
_Lv/
i
i
i '.c l~
o i
t
-- /
i i i
1.0
J \
i ~T a55 I
r
\
f0Loooj i
,
i
i L
___L_L__
1
i
i
i
i
1
a~sIll' Pma, = 0.65 h -1
I
0.5 I ] I i
3 6 12 0 3 -----"-
t[h] t[h]
Fig. 18a. Courses o f measured (symbols) and calculated system variables and process parameters
during an extended culture cultivation of Hansenula polymorpha with glucose as substrate,
S = 2 g 1-1. 9 oxygen supply rate, Qo2; A . cell mass concentration, X; 7q, oxygen mole fraction in
exhaust gas, x ~ : - - . - - , upper limit of oxygen transfer rate, OTRLI~,I ; - - - - , maximum oxygen reac-
tion rate, Rom,, 32,3~; b. Oxygen yield coefficient, Yx~o, and specific growth rate, #exp, during the
extended culture cultivation considered in Figs. 16, 17. 18a, and Table 6 32.37,39)
~OF[mg1-1]
0a [mg (-I]
8
[-I
t [h] ~ . ~ 0.84
~.82 ~.9~"7~
.80 1.93 2.Bg
, ', LOOP 1.94
I" -COLUMN .~, t , l,.,.t 1.94
0.72
~F[-]I 2.96
a U~o[Cm s'U
Q02[g[-lh-I1 IOF[rng [-1]
30 14 / 8 OB[mg t-l]
15"2 X
1 J 0.922 0.87
0,8 0.82 0 95
,'~"~COLUMN -!J LOOP
~ i ,EF[-] / / 1.91.41
2.91 2.9
1/
b u~o[cms-I ]
Fig. 19a and b. Comparison of time- and space behaviour of cultivations of Hansenula polymorpha,
(a) with ethanol as substrate (Figs. 13, 14 and 15), (b) with glucose as substrate (Figs. 16, 17 and
18) 32,37, 39). Base: x/LR, dimensionless longitudinal coordinate in the tower: x*/LB, dimensionless
longitudinal coordinate in the loop: t, cultivation time. Perpendicular to the base: O F and O B as
functions of space and time, Qo2, OTRuM, X, x~o as functions of the time. Arrows: variations of
superficial gas velocity, U~o, and liquid hold-up, e F
When using the strongly coalescence suppressing substrate ethanol, high volumetric
mass transfer coefficients exist. Hence, in the initial range of cultivation, low driving
forces (O~. -- Or) are sufficient to supply the necessary oxygen. Therefore the longi-
tudinal dissolved oxygen profiles are flat. With increasing cell mass concentration
these profiles gradually become nonuniform and a steep drop in the oxygen
concentration occurs. In the transition range between nonlimited and oxygen-transfer
limited growth the profiles attain the most significant non-uniformity. With increasing
oxygen-transfer limited growth the profiles again become flat, because the maximum
driving force is already needed at the bottom of the tower. In this growth range no
dissolved oxygen can be detected in the loop (Fig. 19a).
When using the coalescence neutral substrate glucose, the volumetric mass transfer
coefficients are low. The dissolved oxygen concentration drops to a fairly low value
immediately after the inoculation. By gradually increasing the aeration rate the
dissolved oxygen concentration, O F, can only be increased for a short period of time.
In the oxygen-transfer limited growth range, low dissolved oxygen concentration
exists in the tower. The oxygen transfer rate and the cell mass growth are low.
In the loop no dissolved oxygen can be detected (Fig. 19b). In both cultivations, the
oxygen transfer rate attains its maximum in the transition range. It can be increased
cons*iderably by increasing the aeration rate.
The two examples presented in this chapter indicate that it is possible to simulate
the cultivation of Hansenula polymorpha on ethanol and glucose substrates by means
of the distributed parameter model considered here. Furthermore, the simulations
indicate that the oxygen yield coefficient is not constant during cultivation. In addition,
it must be assumed that the timevarying volumetric mass transfer coefficient, kLa,
is space dependent in the lower section of the tower near the aerator. In the rest of
the tower it can be considered as space independent.
6 Application of Optimization Techniques to the Nonstationary Case

(Fed-batch Process)
6.1 Discussion of Adaptive Control Algorithms
In the following section, a method is proposed to enable the control of a batch process
using a type of reactor, which can be described by a set of partial differential
equations. A general discussion of control methods for distributed parameter systems
was given by Ray 132). As shown in Sect. 5.3, the plant exhibits time dependent
parameters. Partly this is due to the fact that the model used is somewhat too
simple and model inaccuracies have to be compensated by variations of the para-
meters. On the other hand, everyone who is involved in batch processes will have
had the experience that running a bioreactor twice with "exactly the same initial
conditions" may produce two entircly different courses for the process. For control
of these batch processes with respect to minimization of an economically formulated
cost functional therefore an adaptive type of control law is required.
The concept of adaptive control should be considered as "more an art than a
science" 133) since its application is mainly for plants whcre the model is partly
unknown of for which an -expensive- exact optimization is not made for reasons
of economy.
A very well-known type to adaptive controller is the self-tuning regulator, cf.
Astr6m et al. 134), the structure of which is shown in Fig. 20a.
The classical feedback scheme with reference inputs, controller, and plant is retained
entirely, with the exception that the controller parameters are adjusted using identified
values of the system parameters. Identification is performed with a model whose
outputs are compared with the system outputs. The difference is used to update
parameter estimates for the unknown or time-varying system parameters.
It is worth mentioning that this structure is not optimal, beca~use separation of
identification and control is presumed, but this is physically meaningful and far more
easy to calculate than the so-called dual controller, which is optimal -- Feldbaum
135). Hence the optimum of the combined problem (identification and optimization)
is not found in this way, and the self-tuning regulator is not dual in the sense of Feld-
baum.
Even this relatively simple structure, however, turns out to be too complicated for
our purposes. For distributed-parameter systems, operator-Riccati-equations would
have to be solved on-line if one would try to implement a controller with the
structure of Fig. 20a. But the control inputs turn out to be computable much
easier. Hence the already for lumped parameter systems well-known 136) heuristic
structure of 'open loop feedback optimal" control (OkFO) will be used here.
external influences
control inputs | [ systemoutputs

21 SYSTEM(PLANT)
t=. - 'Q"I
H MODEL ,, ~
Variable [[ [
1[ pa,rameters[ II I
variable
parameters
r 4.
),"
reference
inputs
L adaptive control unit
Fig. 20a. Structure ofa selFtuning regulator 33182~
] 54 R. Luttmann ct al.
external influences
control inputs | system outputs

>
~:: tt
Jl ~ variable ~ It
II ~ parameters rail
Iil1 ~> , | >ll
01~ IDENTIFICATION ~" ~ll
I, .2 !" ""
~.~ ~ variable .-
parameters
ix rLc_: ~ MODEL
reference
OPTIMIZATION ~ 4- "
adaptive control unit - - ]
Fig. 20b. Structure of the open loop feedback optimal controller (OLFO) 33.182)
The structure of this algorithm is shown in Fig. 20b and is explained via the
aid of Fig. 21, which shows the course of the procedure. A comparison of Figs. 20a
and 20b shows that the main difference in the two algorithms lies in the substitution
of the optimal controller by the combination of model and optimization algorithm
for the computation of the optimal control functions. The (finite) control interval
]0, T[ considered here is divided into N subintervals ; the so-called adaption intervals,
each of length ~. During each interval, say ]kz, (k + 1) x[, estimates of the system's
parameters are computed by the identification procedure using measurement data
of the preceding interval ](k -- 1) z, kz[. These parameters form the basis for the
calculations of the optimal control functions for the remaining interval ](k + 1) ~, T[.
They are imposed on the system from time t = (k + 1)~, but are valid only until
t = (k + 2) ~. This is because during the next interval, the procedure is repeated, to
yield new optimal control functions based on measurements during the interval
]k~, (k + 1) z[ and so on. So the procedure works in the whole interval ]z, T[. For
the first interval ]0, ~[, pre-computed control functions have to be used because of the
lack of system measurements. -- At the beginning of each optimization cycle, the model
used for optimization has to be set to the proper initial values. These can be taken
from system measurements, if the state of the system is completely measurable. If
IVlathematical Modelling, Parameter Identification and Adaptive Control 155
outputs:
control functions
computation:
optimal control ==~
---- I---II
o
identification
I
/I /
/
/
/
/
)
I
i
inputs:
system
observations
Fig. 21. Course of the OLFO-algorithm 33, ~s2~
this is not the case - - as in most applications - - estimations o f the system's states
have to be used. Here an observer is used, which is based on a simple extension o f
the algorithm provided for parameter identification, due to the very similar
structure o f both procedures.
Let us have a further look on the structure o f the O L F O controller. Altogether,
two feedback paths are evident: One path, cyclically d o s e d , contains the initial
values for optimization, and the other path, again cyclically closed but at different
external influences
control inputs | I system outputs

-~ SYSTEM (PLANT) .
i
variable
r parameters initial 1
l values I
MODEL ~'~ T I
I
I ~inputs-
OPTIMIZATION C (~"('l-
i
I
k adaptive control unit - - 2
Fig. 22. Structure of a state-adaptive feedforward controller a3, zs2~
time-instants, is a higher-level path containing the estimated parameters of the system.

So, in spite of the pure feedforward structure of the optimal control algorithm,
there are two feedback paths in this adaptively controlled system. Therefore the
paradoxically sounding name 'open loop feedback optimal control' seems to be
justified.
It should be noted here that from the literature, an adaptive controller for
distributed parameter systems is known which has only one feedback path, cf.
Yvon 13~1 and Bamberger/Saguez/Yvon 1381. This can be called 'state-adaptive
controller', because it works without parameter identification and only takes over
the state of the system at the beginning of each adaption interval. The structure there-
fore can be drawn as shown in Fig. 22. -- This algorithm, for which simulations
and practical applications are reported in 139.1401, forms the basis for the complete
OLFO-controller; in case of unknown, but only slowly varying system parameters,
however, the OLFO-controller can be shown to give a better performance 141.142~
This means that the control is closer to an optimal one than by using the state-adaptive
controller. Since usually biotcchnological processes show very slow variations in the
parameters, only the complete OLFO strategy will be discussed in the following
Sections.
6.2 Application of Open Loop Feedback Optimal Control to Distributed

Parameter Processes
In order to apply the OLFO strategy, outlined in the preceding chapter, to real
processes, the different sections of the complete algorithm have to be implemented.
This means that both optimal control of distributed parameter systems as well as
parameter identification and state observation techniques must be considered. In
both fields, simulation techniques are extensively used in order to solve the model
equations. Appropriate techniques have been discussed in Sect. 5.2.
6.2.1 Formulation and Solution of the Optimal Control Probiem tbr Distributed
Parameter Systems
In the design of algorithms for the optimization of control input functions, one
can make use of sophisticated mathematical results of optimization theory for distri-
buted parameter systems. Since modelling leads directly to partial differential equa-
tions, the theory used here will be that one developed by Lions ~43-~46), whereas
other optimization results by Butkovskiy 147) or Curtain and Pritchard ~48~, which
apply to models in integral or semigroup representation, will not be considered.
Detailed discussions of the underlying theory and extensions can be found in ~49-151~
and adequate numerical methods are discussed in 15z.153~
The basic mathematical results apply to cases, where the system equation is given
in the form
~y(x, t, v)
+ A(x, t) y(x, t, v) = f(x, t) + B(x, t) v(x, t)
~t
in ]0, T [ x ] 0 , L[, (137a)
with initial conditions
y(x, 0) = yo(x) in 10, L[, t = 0. (137b)
Here, all terms affecting the system's state y and its spatial derivatives are combined
in the (linear) system operator A. Extensions to quasilinear cases can easily be
derived. The action of the control v onto the system is performed through an input
operator B, and all disturbances are combined in the distributed disturbance function f.
Several assumptions have to be made concerning appropriate choices for the operator
and function spaces. We will not go into further details here and refer to the literature
cited.
Next, the control problem will be defined. In many cases of industrial applications,
the system's states should follow a prescribed trajectory, and this aim should be
achieved by using only a small amount of energy. It seems reasonable therefore to
define a cost functional which weights the error between the desired trajectory and the
system's state, as well as the cost of the control action. This leads to the commonly
used quadratic cost functional
T T
J{v) = j" llC(x, t) y(x, t, v) - zs(x, t)lt} dt + f (G(x, t)v(x, t), v(x, t))E d t ,
0 o
(138)
where C is an output operator and G gives the weighting of control energy; z S denotes
the desired trajectory. By II lit a n d - ( , )~ we denote norm and inner product in
function spaces F and E, respectively.
The aim is to find an optimal control u which minimizes the cost functional
and at the same time belongs to a set of admissible control functions U,a, so that
J(u) ~ J(v) gv ~ Uaa ; u ~ U,a. (139)
The solution of this problem is given by means of a variational inequality ~43} which
characterizes the minimizing element u. After definition of the adjoint state by
~p(v)
-- - - + A'p(v) = C'Av(Cy(v) -- zs) in ]0, T [ , (140a)
~t
p(t = T, v), = 0 , (140b)
this variational inequality reads
T
j'(A~tB'p(u)+Gu, v - u ) Edt > 0 Vx"~Uaa; u ~ U a a . (141)
0
Here the signifies the transpose ~5,~1 or dual ,491 of an operator and A is the
canonical isomorphism ~r
These results can directly be applied to the OLFO-controller. The single difference
lies in the fact that at time t = it, optimization is only needed in the remaining time
interval ]it, T[. So the optimization interval has to be changed and initial values
have to be defined at t = it. As pointed out before, these initial conditions for the
optimization model can be taken directly from the process or may be estimated by
means of process observations, using the model of the parameter identification
procedure.
6.2.2 Formulation and Solution of the Parameter Identification Problem

The second task to solve in the adaptive controller is the parameter identification
problem. This can be reformulated as an optimization problem by defining a functional
which weights the difference between the true measurements and the simulated
"measurements" computed with the identification model. Since it turns out that
by application of optimal control theory the solution of the parameter identification
problem is very similar to the solution of the -already implemented- optimal control
problem, it seems to be very advantageous to use just these algorithms here.
Moreover, this method has proven to converge pretty fast in several practical
tests. - - The theoretial results were published by Seinfeld and Chen 15s) and in a
mathematically very rigorous approach by Chavent t56)
Since these algorithms shall be employed in the OLFO-controller, the formulas
are formulated for one adaption interval only. - - Let the actual time be t = iz.
Then the system to be identified is given by
~y
Ot + Ay = f + Bu in [(i - - 1) r, it[, (142a)
with the initial condition
y((i - - 1) z) = Yi- 1 9 (142b)
It is assumed that k sensors are distributed along the spatial domain of the system.
Given are measurements
L
zj(t) =of Zj(x, t) y(x, t ) d x , j = 1 .... , k, t e ](i - i) z, i t [ , (143)
with sensor characteristics Zj(x, t). The model Equation is denoted
~Yu
~t + Ay~ = ~ + Bu in ](i - - 1) z, it[, (144a)
with initial condition
yM((i - - 1) z) = YM,i - 1 - (144b)
After application of the sensor characteristics onto the model's state, it is possible
to define the errors between system and model as
L
ej(t) = zj(t) - f Zj(x, t) yM(x, t) dx, j = 1..... k . (145)
0
A common weighting of all errors is given by the quadratic functional
iz k
J,,i = J" ~ wj(t) e~(t) dt, (146)
(i-Dr j = l
where
Wj(t) > 0 Vt e [0, T]. (147)
The parameter identification problem is now formulated as an optimization problem.

The aim is to determine estimated parameters Aop,, l]opt, f'op,, such that
JI, i(flkopt' ])opt' fopt) ~ J,.i( ~, 13, f) V A, 13, f . (148)
A necessary condition for optimality is that the first variation of the functional
becomes zero, that is
8JL i(Aopt, Bop, fopt) = 0 . (149)
For this first variation one can calculate in a formal way 33):
ix
8J,,i(,~, t3, f) = f (p. 6Ay - 613u - 6f) dt, (150)
(i- 1)
where the adjoint state p is given by

~p ^ k
- ~- + A'p = 2 ~ Wjxje j in ](i - 1) z, ix[, (151 a)
j=l
and the final condition
p(iQ = 0. (151b)
The bracket ( , ) in (150) denotes a generalized inner product or a so-called duality

pairing between elements of the solution space and elements of the dual space ~57)
In practical applications, it is advisable to calculate the variation of the functional
directly ~55). The adjoint state used for the solution of the parameter identification
problem has the same left-hand side of the underlying PDE as the adjoint state (140)
in the optimal control problem. Therefore both equations are of same type and
can be solved by one simulation procedure. This again illustrates the attractive features
of this identification algorithm for use in an adaptive controller.
The special aspect of pointwise measurements should be mentioned briefly. Here
the spatical characteristic Z(x) of the sensor is modelled by a 8-function. After
inserting this into the above formulas, the adjoint state exhibits a 8-function on the
right-hand side. So, even for problems that do not usually show buckles or jumps
in their solution profile, buckles in the adjoint state for parameter identification
may be obtained. That underlines the great importance of considering 8-functions
on the right-hand side of the equation as emphasized in Sect. 5.2.
6.2.3 Applicational Remarks

In the preceding parts of this chapter, we have collected, in brief, the theoretical
results needed for the implementation of the complete OLFO-controller. Thus for
computation of optimal control functions, respectively parameter estimates, one has
to run iteratively through the following steps :
1) Set the optimization, resp. identification, model to appropriate initial conditions
(measured or estimated functions) ;
2) solve the model equation with estimated control functions, resp. estimated model
parameters;
3) compute the optimization functional (138), resp. identification functional (146).
If the difference of the functional computed in this iteration to that of the preceding
iteration is below a given bound then stop else go to Step 4;
4) solve the corresponding adjoint equation;
5) compute the gradients by means of solutions to Steps 2 and 4, and feed these into
a numerical optimization routine in order to minimize the optimization, resp.
identification functional. With updated control functions, resp. parameters, con-
tinue with Step 1.
As numerical optimization procedures, quasi-Newton techniques 158)or modifications
of these have proven to work very effectively on these problems. In 33) and 159) also
a detailed discussion of gradient formulas can be found.
For the optimization procedure, the greatest restriction for general applications
lies in the fact, that a quadratic functional is needed. In problems of economical
optimization, however, cost or gain factors may be linear functions of the states or
the inputs. So in these cases one has to take another optimization technique, e,g. a
heuristic method like that one proposed by Nelder and Mead 118)
On the contrary, for parameter identification a quadratic functional is rather
suggestive, since in the optimum then the real measurements are approximated by
the model in a least-squares sense.
6.3 Cost Optimization of Aeration for a Fed-batch Process
In the following, we treat an application of open loop feedback optimal control

to a particular plant 32,33). For this purpose, the model equations developed in
Chapter 4 and reduced in Sect. 5.1 need to be simplified further in order to allow
treatment by OLFO-techniques at the present time. There is no doubt, however, that
the rapid development of microelectronics, particularly of parallel digital processors,
will provide the opportunity to treat such complex models as the complete one in the
near future.
6.3.1 Model Reduction and Solution of the Reduced Equations

For the investigations, Hansenula polymorpha is cultivated in a bench scale reactor.
The process works, as already discussed in Sect. 5.1, in an extended culture mode.
Furthermore, the cells can be treated as well mixed in the reactor and the loop.
These properties lead to several simplifications in the model equations, cf. (91)-(99).
For further simplification, it is assumed, that no limitation of the maximum oxygen

reaction rate occurs, such that - - cf. (91), (92) - -
rOmax('C) = roopt(z) ----qo/xm('C) CX('C), CS >> ks (152)
holds, where the maximum specific oxygen uptake rate qo. x~ (the metabolic quotient)
is given by
ltlmax(/:TN ) Tr~X0
(153)
q~ = Yx/o(zTr~) O*EFmax "
Furthermore the temporal derivative in the oxygen mass balance of the loop (Eq.
(95 a)) is also omitted. With boundary condition (95c), the abbreviation
F(z*, r) -
CoB(Z*, r) , (154)
CoB(0, r)
and
k0 qo/xm(l:) Z*
F(Z*, r) + Coy(l, z--~In IF(z*, ~)] = 1 vB(z) Cot(l, z) ' (155)
the course of CoB is easily computed for each point z*. CoB(l, r) is inserted into
the boundary condition (94b). However, it must be pointed out that for calculation
of CoB(Z*, r) the boundary value Coy(l, r) is needed, and for calculation of Coy(Z, r)
the value of CoB(l, "r) enters in the lower boundary condition. This coupling via the
boundary conditions reflects the physical couplings of dissolved oxygen in reactor and
loop, and leads to an iterative solution scheme for both equations.
Application of the O L F O concept requires much computing time. Therefore a
further (rough) simplification is needed in order to avoid exact calculation of the
gas phase balances (Eqs. (96a~t) and Eqs. (97a-c). Neglecting the backmixing in the
gas phase (D G = 0), the overall oxygen conversion,
qo2(Z) i - Co~(1, 1:)

U~ - q~z(Z) - 1 - rqfT) Co~(l, "c) ' (156)
is assumed to be caused by a spatially constant conversion rate, which leads to a

linear decrease of the oxygen concentration in the gas phase, described by
1 - Uo2(r) z
CoG(Z, "c) = 1 - rq('r) Uo2('r) ' (157)
In the following, only the special case rq -- 0 (RQ = 1) will be considered.

This case results in
CoG(Z, r) = 1 -- Uo2("0 z = " 1 -- AcoG('c) z . (158)

The solutions of the reduced model equations must be computed iteratively since
the mole fraction CoG is needed to solve the oxygen mass balance in the liquid phase
(Eq. (94a)). On the other hand, the unknown slope Aco~ can only be calculated with
the solution Coy of Eq. (94a) from
1
StvZ(~) t l
ACoG(~)- |
qoEz(Q a~/(z' "c) [p(z, Z) {1 -- ACo~(Z)z} -- CoF(Z,X)] dz. (159)
0
Summing up the simplifications which have been introduced in Sect. 5.1. and in the
above stated formulas of this chapter, the following fundamental assumptions are
made:
1) biomass and substrate are well mixed in the liquid phase,
2) the substrate concentration is nowhere growth-limiting (extended culture),
3) the oxygen mole fraction falls linearly in the gas phase of the reactor,
4) the respiration quotient is rq = 0, (RQ = 1); therefore the velocity of the gas
phase is reciprocal to the pressure profile in the reactor,
5) residence time in the loop is small in comparison with that one in the column.
Conditions in the loop are assumed to be quasistationary.
By these means the model is reduced drastically to a quasilinear PDE of parabolic
type, describing the dissolved oxygen concentration in liquid phase of the column,
an implicit algebraic equation for the corresponding concentration in the loop, and
an ordinary differential equation for the biomass. The simplified model equations
are summarized in Sect. 5.1.
Solution of these equations was accomplished by a DSDT-simulation package 16o),
cf. Sect. 5.2. The sequence for each time step is as follows (z = zl):
1) Estimate Cx(Zi)e.g. by linear extrapolation from the preceding time steps z~_1 and
"lii_ 2 .
2) Estimate CoB(l, zl).
3a) Estimate Acoc(Zi).
3b) Solve the boundary value problem, describing the DO in the reactor (Eqs. (94 a-c)).
3c) Calculate Acoo(Xi) by means of (159). If the difference to the estimated Aco~(Xi)
is not small enough, update the estimate and repeat from Step 3b.
4) Solve the loop equation. If the difference to the estimated CoB(l, "Ci)is not small
enough, update the estimate and repeat from Step 3 a.
5) Solve the cell-mass equation for the time interval from z~_1 to xi- If the difference
to the estimated cx(ri) is not small enough, update the estimate and repeat
from Step 2.
To speed up the three iteration loops involved in the calculation, several tricks may
be used. For the first run through, the relative differences tolerated may be set higher
than in the following steps. Or loops may be merged. This means that the error checks
in 3c), 4) and 5) are combined in 5), which results in only one loop with a threefold
('and'-type) stopping criterion. Furthermore, instead of the linear extrapolation,
higher-order extrapolation can be used, and the update of the estimates can be
weighted with an overrelaxation factor ~6~). All these more or less heuristical
changes in the principal procedure depend on the numerical experience of the pro-
grammer and the time-requirements, i.e. the maximal allowable length of the adaption
interval and the speed of the computer used.
6.3.2 Parameter Identification

Four parameters have turned out to be unknown and/or temporally varying: These
are the two fluid dynamical parameters kLaE and Kst, which describe oxygen
transfer from gas phase into liquid phase, and two biological parameters, the meta-
bolic quotient qo, Xmand the yield coefficient Yx;o- Measurements are provided of the
dissolved oxygen concentration in the liquid phase at ten distinct points along the
column and of the cell's concentration. During our identification analyses, we
found that - - even taking simulated values -- these measurements are not sufficient
to identify the unknown parameters if there is some noise imposed on the signals.
This is due to the fact that fitting the model to these measurements does not
guarantee that the overall oxygen balance is fulfilled, which makes the identification
meaningless. Therefore, the overall oxygen transfer rate, calculated from the outlet
gas analysis must be included as a further measurement.
The identification procedure works in a two level structure, determining kLaE,
Kst, and qo/xm by using the oxygen concentration equations and Yx,,o by using
the equation describing the cell mass. By use of coordination methods the overall
optimum parameter set is found iteratively. General theoretical remarks on the
application of decomposition/coordination methods to the parameter identification
problem for distributed parameter systems are given by Munack and Thoma 162)
It should be pointed out, that, due to the pointwise measurements, the adjoint
equation exhibits ten 6-functions on its right-hand side. This emphasizes again the
importance of a proper choice of solution techniques for this type ofPDE, cf. Sect. 5.2.
The adjoint system is given in Eqs. (160a-c) with PDE, final, and boundary
le adjoint state for parameter

1 ~i-1 identification
33, ,41,159j
conditions. A typical course of the adjoint state is shown in Fig. 23, where the
influences of the &functions on the right side are quite evident.
8~(z, "0 1 0^ 2 ~g(z, ~:) ~,(z, z)

PDE: 8 ~ = - -Bov(~) ~z 2 vv(~) ~--
I CxU) k~ 1
+ ~(z, ~) St~qqKst, z) + qo/xm (k ~ + Coy(Z, z))2
10
+ 2w(~) ~ ((Co~j(:) - Co~(Z, ~)) a(z - z j))
j=l
- 2W(~) o(otrM(~) --otr(~))
q~)2('0 St~W(Kst, z)
1
qoE2(~) + otr~('0 f (p(z, z) z~(Ks~, z))dz
0
"~:]"Ci_l, "lTi[, Z ~ ] 0 , 1 [ . ] (160a)
FC" ~,(z, zi) = 0, z e ] 0 , 1[, (160b)
BC' ~(z, "c) = 0, "c E ] zi-l, "ci[ (160c)

~Z z=0
O~(z,z) ~ c~ ~)(c~ ~) + k~ 1
z= = - B o A r ) vv(z) L~,(1,
F
)+ r z) codl, ~) (%,(1, z) + ko)J
~-z ~
The gradients are computed by the formulae listed below in Eqs. (161a-c), where
the abbreviations (161d-g) are used.
~JI, 1, i
= TN f ~(z, ~c) ~(Kst i, z) [p(z, 1;)CoG(Z, "C)-- CoF(Z, "C)]dz d~
aSt~, i
"q-1
- 2cYTN
i
ri-1
' qg2('C) II(z)
W(z) (otrM('(I -- mr(v)) (q~2(,c) + otr~(z) I2(z)) 2
dr;
(161 a)
~Jl, 1. i _ St~ [ r) OW(Ks"i'z) [p(z, z) Coo(Z, z) - Coy(Z,r)] d z } dz

~Kst,i .ri,_I 1 o ~Ks,. i
zi
-- 2~St~, i f I VV{z) (otrM(z) - otr('r))
E E j
{q~2('~) + Otrm(l:)I2(z)) I3(r) - OtrmU:) 11(T)Ia(zl
• ,161 b)
~Ji, 1.i _ Cx(0) TNSt~,i " "[)

Cov(Z, I:) Cx(Z)
;{z, dz
aqo/xm, i otrEm(Z) ko + Coy(Z, ~)
b
+ ,~(0, z) vv(z) Coy(l, z l

Cx(Z)l d't :
vB(z) ko + codl, ~)
(16l c)
with
1
Ii(z) = j' (p(z, ~) - CoF(Z, z)) ~(Kst, i, z) dz, (16t d)
o
I2(z) = f p(z, z) zqJ(Kst, i, z) dz , (161 e)

o
1 ~ i, zt
[3(I") = " (p(z, "C)-- Coy(Z, r)) OKst i dz, (161 f)
0
1
I OW(Kst i, z)
I4(z ) = (p(z, z) - Coy(Z, z)) z " dz . (161 gJ
o ~ i
The OTR-measurements cause some trouble in the adjoint equation and particularly
in the calculation of gradients, since these are measurements which involve a
spatial integral of the state and therefore the formulas become quite lengthy. On
the other hand, the procedure turned out to converge pretty fast 331 and to give
parameter estimates that were physically meaningful and comparable to other identi-
fications.
Figures 24a-d show identified parameters for an extended culture cultivation,
already treated in Figs. 13-15, and the corresponding text in Sect. 5.5.
An adaption interval length of three hours was found to be fast enough according
to the dynamics of the growth process. During the intervals, several measurements
166 R. k u t t m a n n et al.
"7r
|
oO
ill " o ' ~
E
_ _ / kLa
o
o
. . . . i/V [
i
, _o~oo 9
o
o 9 L--~ ~_o_9_
o 0
. . . . I . . . . i . . . . i
0 5 10 15 t [ h ] ~
0
I |
o~ 1 I--
. . . . i . . . . i . . . . i
0 5 10 15 t [h]---,~
QO
o" " " " ' ' l . . . . I . . . . I
L LO
qo"
IQ 9
*~
o
0
9 9
m
o
9
cq
o"
s- o s ~o ~5 t'[h]'--.--
9 - ' 9 9 i . . . . i . . . . i
eo Fig. 24 a and b. Identified fluid-

"ff~o dynamical parameters of the
cultivation considered in Figs.
, I 13, 14 and 15 s3, ~4t, t59~; c and
E
::3,.
| d. Identified biological para-
meters o f the cultivation con-
o"
sidered in Figs. 13, 14 and
1 5 33.1~-t. 159~
, , , , [ i i , , I , , , i t
0 5 10 15 , r ~ l . - - ~
were taken. Figure 25 shows the DO measurements in the reactor and the correspon-
ding identified states. - - As it can be seen from this figure, in the first 12 h the state
estimates are excellent. Then a disturbance occurs, which cannot be described by the
model (the measured DO-concentrations at times t = 14 h and t = 15 h are nearly
the same!). - - Afterwards, identification of measurements at t > 17 h, improves
again. The increase of DO at the top o f the reactor, however, cannot be described,
since it is presumedly due to a disturbance introduced by the formation o f some foam
at this part o f the reactor.
6.3.30LFO-Control
Until now, the complete adaptive control scheme has not been tested in on-line
connection with the real plant. The implementation is under progress, but up to now
only simulated results have been obtained. - - F o r optimization a cost functional
is minimized which weights the cost o f air and substrate fed into the reactor and the
yield of cell mass at the final cultivation time (t = 18 h).
Customary unit prices are taken as weighting factors. The aeration rate is used as
control input ; the flow rate o f substrate is controlled according to the condition that
substrate concentration should nowhere be growth-limiting.
Since the functional is linear, the heuristic optimization procedure by Nelder and
Mead 11s) is employed for minimization o f the cost. The course of the input variable
cO
E ~o
-4"
03
t"q
O
0 0,5 1.0 1.5 2.0 2.5
x [.1]
Fig. 25. Dissolved oxygen concentration in liquid phase of the column -- measurement data of the
cultivation considered in Figs. 13, 14 and 15 and corresponding identified state profiles 33, ~9~
168 R. L u t t m a n n et al.
is assumed to be a continuous, piecewise linear function of time, linear in each

adaption interval.
For comparison, three cases are simulated. The first is a feedforward optimal
control with constant parameters which have an initial deviation of about 10% to
the true initial parameters. The second is the adaptive control with the same initial
parameter set, and the third is an optimal control of the plant which is of course un-
realistic, since the course of all parameters over the complete time interval is
supposed to be known. But this latter control serves as a lower bound for the cost
(or an upper bound for the gain) one can achieve with the process. The trajectories
of the input variable the inlet gas stream, are shown in Fig. 26. The adaptive
control functions lie between the optimal and the non-adaptive. Variations are mainly
to be seen in the last six hours, since the cell concentration then is most sensitive
to the input strategy and the unknown parameters.
From Fig. 26 one can see, that the control functions computed by the OLFO-al-
gorithm tend to lie one adaption interval behind the optimal ones. This "time delay"
can be diminished by choosing a smaller adaption interval length. But, of course, the
OLFO-control does not converge to the optimal control in case of letting the interval
length go to zero. This is due to the fact, that the future parameter course is not known:
optimization is always based on the present knowledge about the system's par-
ameters.
But as the computed functionals
for open loop control with estimated parameters : --0.0076,

for adaptive control with identification: -0.0127,
and for the - - theoretically -- optimal control: --0.0137,
0 R . . . . I . . . . I . . . . I
......." ......... . .........
IiIO ~..
..."
co
.:"
0 ~ .::
r -
C',I ~ :": /// I

0~I-.. 9 / I \ /
0 Fig. 26. C o m p u t e d c o n t r o l func-

t i o n s 33,1,1~.
0 1
I/ /. . . . I , ,
',
~ , I , , ~ , I
/ ~ ,
........... w i t h o u t a d a p t i o n ;
- - OLFO-control;
o s lo 15 trh]--- - - optimal control
show, the adaptive control scheme in this case has turned out to come quite close to
the theoretical optimum. This is due to the very slow variations of the system
parameters in bioreactors.
Remarks. Some critical remarks should be made regarding the previous results.
One can make the objection that the cost of substrate usually is much higher than
the cost of air fed into the reactor. This is in general true, but if you think of waste
disposal, then you have exactly the situation considered above. On the other hand,
an inclusion of the substrate balance into the model gives the opportunity to carry
out optimizations with respect to substrate flow. This will involve more computation,
and the calculations of gradients become more difficult, but the principle of the
proposed algorithm does not need to be changed. - - The same answer can be given
to the objection that a linearly decreasing oxygen mole fraction does not apply to large
plants. - - An improvement of our results by means of a more accurate description is
just under research.
7 Cultivation with Space Dependent Oxygen and Substrate Balances
7.1 Process Behaviour and Model Extensions for Unlimited,

Oxygen-Transfer Limited and Substrate Limited Growth
Up to now we have dealt with extended culture processes under constant substrate
concentrations. These were simulated using both a distributed parameter oxygen
balance model and a lumped parameter cell mass balance model.
In this chapter, we will investigate the influence of temporally and spacially varying
substrate concentrations using an extended model.
To illustrate this a batch/fed-batch cultivation is analyzed in detail in order to
demonstrate the simultaneous interaction between nonlimited, substrate limited an~t
oxygen-transfer limited growth conditions 32,35,163). The simulations illustrate the
suitability of the model to handle SCP-processes, in all possible growth ranges.
In Fig. 27 the time course of some measured data (symbols) and the corresponding
simulated values are compared, whereas in Fig. 28 the comparison between the
measured and simulated longitudinal dissolved oxygen profiles is shown.
Cultivation was started in batch operation, at a high substrate concentration.
During these conditions the substrate S was ideally mixed in the tower-loop
system.
In the main part of the batch cultivation range, the cell mass concentration, X,
the oxygen demand, OUR, and the necessary oxygen supply rate, Qo2, increased
exponentially. The substrate concentration (measured at the end of the aerator range,
z = ~ = 0.1), S~, and the oxygen mole fraction in the exhaust gas, Xoac, diminished
exponentially. At time t = 7.7 h, the substrate concentration became of comparable
magnitude to the substrate limitation constant. Ks, of the Monod model. The oxygen
reaction rate was considerably reduced, Qo2 diminished and Xo~Aincreased due to
the decreased rate of substrate consumption.
' I I
SUBSTRATE FEEDRATE
INCREASE
2O :" 4.5~ .;o 3 . 0 ~ 18
I INCREASE
A
~ 17 3.0
I
I
2.0 " 12~
.,a
14 /~> 1.0
/
11 '~ 0
0 5 10 15 ~ 20
t [hl
e b c
batch substrate oxygen

transfer tronsfer
limited limited
Fig. 27. Batch/fed-batch cultivation, Hansenula polymorpha with ethanol as substrate. Measured
(symbols) and calculated process variables during the cultivation. A, cell mass concentration, X;
I , substrate concentration, Sr; O, oxygen mole fraction in exhaust gas, XoAc; 0 , oxygen supply rate,
Qo2 32, 35)
At the start of the process, the dissolved oxygen profiles were relatively flat (Fig. 28).
With increasing oxygen transfer rate the oxygen mole fraction in the gas phase decreases
more and more rapidly to the top of the column. In order to fullfill the uniform oxygen
demand of the cells along the column, the longitudinal dissolved oxygen concen-
tration profiles diminished and became increasingly nonuniform.
Shortly before the end of the batch range, at t = 7.75 h, cell respiration drastically
diminished due to the lack of substrate and the dissolved oxygen concentration
increased (profile t = 8 h).
In the second process period, starting at t = 8 h, ethanol was fed into the tower
bottom (fed-batch process). Since the cells immediately assimilated the substrate,
the alcohol concentration remained below the detection limit (80 m~l 1). The
dissolved oxygen concentration diminshed at first due to the commencement of cell
respiration, but later at time t = 10 h it increased in the upper half of the tower
since the oxygen uptake rate was reduced by the strong substrate limited cell growth
in this part of the tower. The ethanol feed rate and the aeration rate were then increased
step by step.
During the ranges of constant substrate feed rates, substrate supply (Qs) and
oxygen demand (Qo2) are in a quasi steady state equilibrium,
Vr(t) + VB dX(t)
Vv(t ) dt - Yx/o(t) Q o 2 ( S v , t) - Yx/s(t) Qs(t), (162)
i i
t [h]
2 ~
,Q ~ . . ~ g -
LR=2 , 7 5 m
0 i i i i i i I i I I I I I I
ct 0.25 0.5 0.75 = 1.0

-X[- I
LR
Fig. 28. Measured (symbols) and calculated longitudinal dissolved oxygen concentration profiles
during the different growth ranges in Fig. 27. a, batch operation; b, fed batch operation with
substrate limited growth; c, fed batch operation with oxygen transfer limited growth a2,3s)
where
F(t) SR rhs(t)
Qs(t) = D(t) S R - ~Vv(t - Vv(t) (163)
In the substrate limited growth range, cell growth increases with increasing feed rate.
This requires a higher OTR. Hence, at a constant aeration rate, XOG diminishes when
the feed rate is increased.
In the initial range o f the fed-batch operation, between t = 9 h and 11 h, significant
changes in the process variables occurred, due to the reduction o f the liquid
recycling rate at a constant aeration rate. Although O T R increased further, the cell
mass concentration, X, (measured at x - - 1.9 m) apparently remained constant.
The reduction in the liquid recycling rate was accompanied by considerable foam
formation and cell microflotation. Since the mathematical model employed assumed
a uniform cell mass concentration in the entire reactor, it was not able to simulate
the system behavior at this stage.
To eliminate cell flotation, the aeration rate was increased considerably at t = 11.1 h.
As a consequence, the liquid recycling rate improved and uniform cell concentration
was restored. This is confirmed by the agreement between the measured and simulated
data at t = 12 h.
After the final feed rate increase at t = 17 h the cultivation changed from a
substrate limited to an oxygen-transfer limited growth process. This is due to the fact,
that the possible oxygen transfer rate is not high enough to sufficiently convert the
offered substrate. The rate of consumption of substrate was less than the rate of
supply of substrate. Thus the substrate concentration in the medium increased.
The substrate concentration lost its spacial influence on the rate o f the oxygen
reaction and the longitudinal dissolved oxygen profiles again became flat (profile
t = 20 h).
With increasing ethanol concentration, the bubble coalescence rate diminished,
such that the kea value and the oxygen transfer rate increased until a new steady
state was attained between oxygen feed rate (Qo2) and substrate feed rate (Qs). Under
these conditions, the cell growth was controlled again by the substrate feed rate.
A mathematical description of these observations using the oxygen-cell mass
balance model of Sect. 4.1 failed, since it was not possible to represent the influence
of the substrate by means of a lumped parameter model.
The oxygen reaction in the tower (I = F, s = x) and in the loop (I = B, s = x*)
was extended by allowing for the influence of a spacial varying substrate concen-
tration,
OI(X , t)
R o g L t) = Roimax(X, Si, -~, t) (164)
K o + Ol(~,t)'
Under optimum conditions (K o = 0 and no transfer limitation) the oxygen reaction

rate in the tower is given by simple substrate-Monod kinetics,
~tmax(t ) SF(X, t)
RoF(X , t) = ROFopt(X, t) -- X(t). (165)
Yx/o(t) Ks + Sv(x, t)
However this optimal rate cannot be satisfied under every operational condition.
In some cases the oxygen reaction rate is limited by the local maximum oxygen trans-
fer rate,
P(x, t) Xoo(X, t)
OTRmax(X, t) = kra(S F, x, t) (166)
Ho2
In the oxygen-transfer limited growth range, the maximum oxygen reaction rate no
longer depends on the substrate and cell mass concentrations directly. However,
kca is a function of the substrate concentration. This influence of the substrate is
found by analysing the cultivation according to the quasi-steady state identification
methods of Sect. 5.5.
In Fig. 29 a, the identified volumetric mass transfer coefficient beyond the range of
the aerator (0.1 ( = ~ ) < X/LR _--<1),
kLa=(t) = kraE(t) e -Kstm , (167)

~'F [-] "--'"

0.88 l i 0.8/,
0.96 1,9 }2,281Z86 3.8/,
i i
U E [cm s 4 ] ---,,,- ,
i
,,
Go
i i i
• i i I I i I I 1 I ~
I
I I: i i i I i i [
fed- ,' botch

i
1200 ~ - f
r 3.0
i i i
S- Ilmfted 02-limited
i
f '%
,,:
..x Boo y - - - - x ,,\ 2.0
........ -V ........ -V ................ "V-
:
/,00 I---p-o-~c-'-'~,.-~-.~ ~ " ";" i, "t'--
4. ~..-'- ,v, , ,,n 1.0
f i~i ,:
-~
~ - -
I 0
'
......
,,
~...~.....~..'-~'
i , , ,
.-
.v
,
',J I
i
i
..,~-_-,5-_--~-_-z~,5.~L.~-_-d-;.~
i
0
0 /, 8 12 6 ~ 2 0 2/,
t [h]
r 1 500 i i i i
/
1200
/ Fig. 29 a. Measured (M) and iden-
tified (I) parameters of the cultiva-
tion considered in Figs. 27 and
28 32, 35). /k, substrate concentra-
go0
--ft. tion at z = ~ = 0.1, S~- (M); [~,
2. kLa o volumetric mass transfer coeffi-
BOO
/ ]_= I
I
I
l
u E - 0.96 cms -1-
Go-
cient, kLa= (I); 9 kLa-value in
substrate free medium, kra ~ (I);
V, coalescence factor, Ks, (I).
b. Determination of the volumetric
mass transfer ratio mL from batch
300
I ], 1 , l , data in Fig. 29a. Explanation of
0 1.0 S~ 2.0 3.0 --- /,.0 SD; kLa0 = 396 h -~, So =
1.39 g I-~ a2.3s~
S~" [g Iq )
the v o l u m e t r i c mass transfer coefficient calculated for an ethanol-free m e d i u m

36,61 , 164, i65 )
kLa'(t)
kLan(t) = mr(S r, ~LR, t ) ' (168)
the identified coalescence factor, Kst , and the m e a s u r e d substrate c o n c e n t r a t i o n ,

S~, are plotted.
In Eq. (168), mL(SF, ZtLR, t) is the ratio of the actual volumetric mass transfer
coefficient, kLa'(t), to that applying to a substrate-free medium,
kLan(t) SF(c~LR, t) S~(t)

mL(SF, ~LR, t) = - - 1+ - - -- 1 + - - (169)
kLan(t) SD So
m L was determined during batch cultivation by means of a linear regression (Fig. 29 b)

between kLa~ values and (uniform) substrate concentrations.
During substrate limited growth, the ethanol concentration was below the detection
limit of 80 mg 1-1. Thus kLa" and kLa~ were approximately equal. However, under
these process conditions, significant longitudinal substrate concentration profiles
prevailed, which considerably influenced the volumetric mass transfer coefficient.
Hence, kLa became space dependent beyond the range of the aerator as well,
I SF(x, t)~.
kLa(X, t) = kLan(t) 1+ SD j , aLR < X < LR. (170)
Within the range of the aerator, the following relationship holds true,
,
kLa(x,t)=kLa~(t)e " x I 1+
Kst")(1-~) sF(•SD J 0<X<etLR (171)
Thus the bubble coalescence near the aerator is described by its "natural" behaviour
(Kst) and the substrate influence.
The strong effect of the substrate on the volumetric mass transfer coefficient can
be recognized from Figs. 29 a and 29 b. After the initial increase in kLa" due to inocula-
tion effects, kLa, diminished with increasing cultivation time, t, due to diminishing
substrate concentration in the batch growth range (Fig. 29a). During substrate
limited growth, the medium has a coalescence promoting character, hence, kLa~ was
fairly low ('-200 h-l).
In the following process simulations, it is assumed that SD, which is characteristic
for the dependence of mL on the substrate concentration, is constant during the
entire cultivation. The time dependencies of kLa~ and Ks~ are taken into account.
7.2 Analysis, Identification of Kinetic Parameters, and Simulation
The simulation of the entire cultivation (Figs. 27 and 28) and the following simula-
tion and identification results were attained using the distributed parameter model of
Sect. 3.3 for the oxygen and substrate balances in the column and loop. The gas and
liquid balances are extended to allow for the coalescence depressing effect of the
substrate on the volumetric mass transfer coefficient kLa (Eq. (170) and Eq. (171))
as well as for the considered reaction behaviour. In the dissolved oxygen balance of
the liquid phase of the tower (Eq. (10a)) with initial and boundary conditions
Eq. (10b), Eq. (16) and Eq. (10d) the maximum oxygen reaction rate depends on the
relationship of the optimal demand (RoFop~(x, t)) and the possible maximum
oxygen transfer rate (OTRmax(X, t)),
RoFm,x(x, t) = ~'R~176 t); Rovop,(x, t) < OTRmax(X, t) (172 a)

[OTRmax(X, t)- Roy opt(x, t) > OTRm,x(X, t). (172 b)
It is assumed that in the nonaerated loop the Monod equation is valid for oxygen
and substrate. Hence, the maximum oxygen reaction rate in the dissolved oxygen
balance in the loop (Eqs. (14)) is given by
/3~,x(t) SBlx*, t)
RoB max(X*, t) -- - - X(t). (173)
Yx/o(t) K s + SB(x*, t)
The reaction rates in the distributed substrate balances of tower and loop (Eqs. (22)
and (23)) are controlled by the oxygen reactions and given with I = F, ~ = x for the
tower and with I = B, 2 = x* for the loop,
Yx/o(t) O1(i, t)
Rsl(~, t) = Rolm,x(.~, t) . (174)
Yx/s(t) K o + Ot(~, t)
These are related to the oxygen reaction rates by means of the cell mass reaction
rate, i.e., by the two yield coefficients, Yx..s and Yx/o- In contrast to the heavily
changing substrate and oxygen reaction rates during a liquid cycle in the tower-loop
system, the relative cell growth is very small.
Hence, the cell mass may be regarded as uniformly distributed in the tower-loop
system, and described by a spacial integro cell mass balance equation,
dX(t) TF(t )
-- - - Yx,o(t) OUR(t), (175 a)
dt Tu(t )
with the initial value (inoculum concentration),
X(0) = X o . (175b)
The connection between the lumped parameter cell mass balance and the distributed
substrate and dissolved oxygen balances is found in Eq. (175 a) via the overall oxygen
uptake rate OUR, cNculated with time- and space-dependent oxygen and substrate
concentrations,
LR
OUR(t) = f Rov max(x, t) OF(x, t) dx
K o + O v ( x , t) L R
0
LB
OB(X*, t) dx*
+ T.(t) ktma• X(t) f SB(X*, t)
Tv(t) Yx/o(t) Ks + SB(x*, t) K o + OB(X*, t) LB
(176)
In contrast to usual mathematical modelling in biotechnology, the kinetic parameters

in the described model are assumed to be temporally varying. The following analysis
of the measured data and the mathematical identification methods demonstrate that
this assumption is essential. In Fig. 30. the variations in the measured and identified
yield coefficients, Yx,o and Yx/s, and the specific growth rate gexp (exp = determined
experimentally) are plotted as functions of cultivation time, t. The symbols represent
the experimental results, the dash-dotted lines their connection, and the heavy lines
the course of the identified parameters.
During the first four hours of cultivation, an adaptation phase of the cells was
clearly recognized which was accompanied by a low specific growth rate, laexp, and a
low substrate yield coefficient, Yx/s- The oxygen yield coefficient, Yx:o, diminished
during this initial cultivation phase.
0Y5 , , , , , , , [
BATCH FED - BATCH
0,50
S-LIMITED O2-LIMITED
I i i i i , r , i
i r t i t i i
0,25
z, 8 12 16 2O
0.80
-gJ-, 43"
0.60
0.40
0.20
4 8 12 16 20
0.40
't-
~'mox l
::a. 1
0.30
0.20
0 4 8 12 16 ~ 20 24
t[h]
Fig. 30. Identified oxygen and substrate yield coefficients, Yx,o and Yx/s, and specific cell growth
rate, g~,p, of the cultivation considered in Figs. 27, 28 and 29 ~2,35.38)
The experimental values of la and Yx.,o are calculated at discrete times t k,
In MX(tk + 1) - - In MX(tk _ 1) (177)

]'texp(tk) = tk+l -- tk 1 '
Vv(tk) + Va I'texp(tk) MX(tk) (178)

MYx/o(tk) -- Vv(tk) MQo2(tk)
The analysis of the substrate yield coefficient Yx,,s is given in time intervals
TK = ]tk, tk+l[.
During batch and substrate limited fed batch growth (0 < t < 17 h) Eq. (179)
holds true.
MX(tk-- 1) - - MX(tk)
~Yx..s(rK) = -- (179)
MS(tk+ 1) - - MS(tk)
whereas in the oxygen transfer limited growth phase (17 h < t < 20 h) the substrate
accumulation is taken into account,
VF(TK) MX(tk+0 - - MX(tk)

Qs(TK) [tk+ 1 -- tk] =
VF(T K) + g B MYx/s(TK)
+ Mg(tk+l)- MS(t0. (180)
Between t = 4 h and 6 h, the cell growth rate, Pexp, and the yield coefficients,
Yx,.o and Yx.'s, increased. During this transient range, the cells had a higher specific
growth rate than during extended culture cultivation at S = 5 g 1-a. Hence, substrate
inhibition during the extended culture cultivation, discussed in Sect. 5.5.2, cannot be
excluded. As soon as the lack ofsubstrate became noticeable, for t > 7 h, the identified
yield coefficients diminished.
During substrate limited growth, the cell mass concentration increased approximate-
ly linearly with time. The identified oxygen yield coefficient gradually increased and the
substrate yield coefficient rapidly increased and approached a constant value. In the
oxygen-transfer limited range, the identified yield coefficients appeared to be constant.
Since the specific growth rate and the yield coefficients were calculated from the
difference in cell mass concentrations as well as from O T R and the difference in sub-
strate concentrations, the errors in the determination of the cell mass and the
substrate concentration were attenuated in these parameters.
In spite of these uncertainties, the experimentally determined and identified para-
meters agreed fairly well.
For the simulation of cell cultivations it was 5.ssumed that neither one of the limita-
tion constants, K s or K o, is influenced by the cultivation time; they are space
and time independent. ~'maxwas evaluated from the nonlimited growth range:
" t a m a x l = 0 . 2 2 3 h -1; 0h< t < t~

t -- t 4
llmax(t) = ~max, + [~max2 - /-t.... 1] t5 _ t4 ; t~ < t _< t 5 (181)
~max 2 = 0.350 h - a ; t5 < t

witht 4 =4hundt 5= h.
The oxygen limitation constant was evaluated from quasi steady state identification
during the strong oxygen-transfer limited growth range up to Ko = 0.20 mg 1-z.
The substrate limitation constant was difficult to determine, since K s is far below
the detection limit o f the substrate (80 mg 1-1).
The end o f the batch cultivation phase was used for identification of K s (between
t = 7 h and 8 h in Fig. 27), since in this range no spacial dependence of the substrate
concentration yet existed and the corresponding volumetric mass transfer coefficients
were identified by means of the longitudinal dissolved oxygen profiles.
The aim of the identification procedure for this period o f time was to describe the
measured rapid increase in the oxygen mole fraction in the outlet gas, x~G, by
varying the free parameters, Yx/o, Yx/s, and K s.
For the substrate limiting constant, K s = 11 mg 1-1 was determined. This value
was kept constant during the whole cultivation. The identification of K s from
time varying measurements was varified from the simulation of space varying variables
in Fig. 31.
There the spacial dependences of the oxygen mole fraction in the gas phase, Xo~,
the superficial gas velocity, U6o, the substrate concentration, SF, the dissolved oxygen
concentration, O F, and the cell mass concentration, X, are shown for t = 14 h.
The cell mass is calculated using the lumped parameter model (Eq. 173a). The
oxygen mole fraction XoG decreases up to the top o f the tower, due to oxygen loss by
oxygen transfer in the liquid phase. The gas velocity u~o, however, increases due to
the decreasing pressure in the tower. The substrate is fed at x/k R = 0. The concen-
tration is less than 35 mg 1-1 and diminishes rapidly along the column, causing
limitations in the substrate and oxygen reactions.
The increase of the dissolved oxygen concentration in the upper half of the tower
occurs when the substrate concentration is of the order of magnitude of K s. Its
decrease towards the top o f the tower is due to a lower oxygen demand (transfer)
from the gas phase and the self regulating character o f the driving force O~-O v.
t =lt, h
20.6 ~ 3 . 0 e " ' ~ ~5~" 7.5~

l-
"7
T_r iJ.
30 to 5.0~
O')
~" 17z ~ 2.0

S~, X/U"
_-----g---o 15 2.5--
t3)
Ks
LR= 2"75 m .[3

11.0 ~ ~0~ . . . . . . . . . . . 0 z
0 cc 0.25 0.5 0.75 x/--~--R[-] 1.0
Fig. 31. Longitudinal profiles of dissolved oxygen (O) and substrate concentration, Ov and SF, as well
as cell mass concentration, (A), X, in the medium, oxygen mole fraction in the gas phase ([Z]),
Xoo, and superficial gas velocity (V), Uco, at t = 14 h. Ko = 0,20 nag 1-1, Ks = 11 mg 1-1 a2,35.39~
The identification problem is defined for all unknown parameters. The mass trans-
fer parameters are known in batch and oxygen-transfer limited growth phase from
quasi steady state identification methods (Figs. 29a, 29b) as well as the oxygen
limitation constant K o.
The identified values of kea in the substrate limited growth phase had to be slightly
modified since the missing substrate kinetics caused slight deviations between measure-
ments and the simulations.
The deviations between the kLa values determined by the simplified and extended
models are within 6 90 163)
In each identification interval TK = [tk, tk + 1] the course of the yield coefficients
Yx,,o (I = O) and Yx/s (I = S) is approximated with the known value at the end
of previous identification step and an unknown slope AYx/I,
Yx.~(t) = Yx,l(tk) + A Y x / I ( T K ) [ t - tk]. (182)
In the ranges of constant aeration rates the volumetric mass transfer coefficient for
substrate free medium,
kLag(t) = kLag(tk) + AkLa~(TK) [t - - tk], (183)
is calculated in a similar manner.

The identification problem in the simulation interval T K is formulated as an optimi-
zation problem, as already shown in Sects. 5.4 and 6.2,
J~%. "c,,+1,A~Yx/o,s A~La;, Ks)

N J~.(Zk, "rk~_l,AYx/o, AYx/s, AkLa~, Ks) V AYx/o, AYx/s, Aki_a~, K s ,
(184a)
with the performance index of iteration step i,
j~(.) = . m 9 'I;) - MCoF(Zj, T))2 + tCoB('0

. Am - MCoRt0)-
A ;'
"~k J
+ ~-K(q~2(z) - Mqo2(~))z + qKt,'cAm,T,

oGt ~ -- ~,tCAodV))2
+ VK(C~(Z) -- MCx(Z))2 + tK(C~('r ) -- MC~F(Z))21 6(M'Ci) d7:,
(184 b)
and the proposed mathematical model as constraint.
This optimization procedure is used to identify Yx,'o and Yx:s during the batch
and substrate limited phase (Oh < t < 17 h), K s at the end of batch phase
(7 h < t < 8 h), and kLa during the substrate limited growth phase (8 h < t __< 17 h).
Yx,,o and Yx/s are kept constant during the oxygen transfer limited growth phase
(17 h < t < 20 h) in order to improve the model for the description of the interaction
between substrate limited and oxygen-transfer limited growth via the coalescence
depressing effect of the substrate.
180 R. guttmann et al.
In Fig. 27 it is shown, that the proposed coalescence model (Eqs. (170) and
(171)) describes the process behaviour very well.
The space- and time dependent behaviour of cell cultivations on tower reactors
is once more demonstrated in the interaction between longitudinal substrate profiles
and dissolved oxygen profiles during the substrate limited growth phase. This is
shown in two three dimensional plots in Fig. 32.
On the average, the substrate concentration was below 30 mg 1-I. Only during
the decrease in the liquid recycling rate, which occurred for a short period of time
between t = 9 h and t = 11 h, the substrate concentration increased to 50 mg 1-1 at
the tower bottom. In the upper half of the tower, strong reaction limitation
prevailed due to the reduction of SF to values below K s.
As a consequence of substrate limitation in the upper half of the tower, the
dissolved oxygen concentration increased and the longitudinal dissolved oxygen
concentration profiles passed through a minimum. The increase in the aeration
rate caused an improvement in the liquid recycling and a slight increase in liquid
dispersion.
During the increase in the substrate feed rate, the substrate concentration increased
at first, then gradually diminished due to the higher cell growth rate.
At t -- 15 h, oxygen-transfer limitation already began at the tower bottom. Now
the substrate reaction was limited in the lower part of the reactor and the
substrate concentration slightly increased in the upper part of the tower. This is ob-
servable in a smaller substrate limitation and the re-decrease of the longitudinal
dissolved oxygen profile at the top of the tower between t = 15 h and t = 17 h.
After the last feed rate increase at 17 h oxygen-transfer limited growth prevailed in
the entire reactor. As a consequence, the substrate concentration increased and its
longitudinal profile became uniform. The longitudinal dissolved oxygen concentration
profiles reacted very sensitively to variations in operational conditions. An increase
in the substrate feed rate causes an increase of the growth rate. This leads to a
higher oxygen demand, an increased OTR, and a diminished Xoo and O F.
An increase in the aeration rate does not influence OTR, but slightly increases kLa,
O F and XoG.
The interaction of substrate limited and oxygen-transfer limited growth along the
tower coordinate is observable in the longitudinal dissolved oxygen profiles. Feeding
pure ethanol at the bottom of the tower leads (dependend of feed rate) to unlimited
growth in the lower part of the column and to substrate limited growth in the
upper part. This yields a minimum in the dissolved oxygen profiles.
A spacial oxygen-transfer limitation reduces the spacial dependence of substrate
reaction. With increasing oxygen-transfer limitation the oxygen profiles become
more and more flat.
The profiles in the loop are influenced by the recycling rate of medium, by the
substrate concentration and by the dissolved oxygen concentration at the tower top.
All of these experimentally observed phenomena can be described by the model
presented.
The taller the tower, the greater is the probability that a longitudinal substrate
concentration profile prevails in the tower.
Furthermore, since SCP producing reactors are usually operated in the substrate
limited growth range, the use of a distributed parameter model with regard to
O _
S~- [mg [-I]

75
\
25
" ' --1.072.28

- - 1.3.'-
- - 2 . 8 6
--t.6 ,1:_,
UE S-
D-S R [gl-lh -1] Go [cm ]
Fig. 32. Longitudinal dissolved oxygen concentration profiles in tower and loop, O F and OB, and
substrate concentration profile in the tower, SF, during the substrate limited growth phase.
(1), substrate feed rate increase; (2), aeration rate increase 32.3s. 39~
dissolved oxygen and substrate concentration in the medium and O z- and C O :

concentration in the gas phase is necessary for the simulation of these processes in
both pilot and production plants.
8 Set Point Optimization of Continuously Operated Pilot Plants
8.1 Scale Up, Control Possibilities and Process Parameters

The agreement between measured and simulated data in the preceding chapter is
encouraging enough to allow some scale up predictions with the developed mathe-
matical model.
In this chapter in a first step the nonreduced model is used to simulate a non-
stationary cultivation of Methylomonas M 15 on methanol 166) in an air lift tower loop
reactor with a height of 40 m. The calculations are based on the measurements
gained with this bacterium 54 56) and on the data of the pilot plant air lift tower
loop reactor investigated in the previous chapters.
In a second step the model is used for cost optimization of the process. Based on
a proper cost function the cost optimal set point of cultivation process with the
same bacterium in a 20 m height (40 m 3 volume) pilot plant will be investigated. In
this example the plant works continuously, therefore a stationary optimization
procedure is employed.
For the scale-up calculations it is assumed that the general distributed parameter
model (Eq. (45 a)) and the cc;rresponding initial and boundary conditions (Eq. (45 b)
to Eq. (45h)) hold true. When using this model the spacial dependencies of model
parameters are neglected. According to the measurements on the bench-scale tower
the volumetric mass transfer coefficient, kLa, exhibits a spacial variation only within
30 cm above the aerator. In the rest of the tower kLa is constant. Thus the spacial
independence of the model parameters in the pilot plant is a fairly realistic
assumption.
The excellent agreement between the data which were measured in the bench-scale
tower loop reactor with Hansenula polymorpha and simulated with the distributed
parameter model by assuming space independent biological model parameters,
lamax, PT, Yx,'o' Yx,s, Ko, Ks and RQ, suggests to make the same assumption also
for the pilot plant reactor and the bacterium Methylomonas M 15.
Furthermore, it is assumed that these kinetic parameters remain constant in a wide
range of operational conditions, which is, of course, a fairly strong idealization.
When using Methylomonas M 15, the longitudinal dissolved oxygen profiles do
not approach a stationary limiting state in tower loop reactors t67 ) which is in contrast
to earlier results with Hansenula polymorpha. Therefore, the reaction mechanism
can be described by the double Monod model (Eq. (30)) in the entire cultivation range
without the proposed reaction limitation in Eq. (172).
In Fig. 33 the parameters of the kinetic model (p . . . . laT, Ks, Ko, Yx/s, Yx/o and RQ),
the reactor height, LR, the tower cross section, QR, the loop height, L R, and the cross
section, QB, as well as the operating parameters (U~-o, e6, Uvo, Tv, D, TD, and SR) are
S~.IQrl ~ KINETIC DATA M15
1o I lO
Hma* = 0.51 h -1
0.05 g 1-1
IJr = 0.01 h "1
~s = 0.15mgl-,
o
Yx/s = 0,5
Yx/o = 0.42
2. ~ RQ -- 0.4
OF Irng r 1}
219,
o,,7,
1
L B =/-.Am QB = 0 . 4 5 dm 2 ,i,,,,,"'~ ~ " ~
~ XF Igt-ll 3.27
~..s OPERATING DATA

,',15 ~ u Go
E --
7.83 cm s -1
4~
r = 0.41
UFo = 1.04 cm s -1
TF = 0.63 h
1.o D = 0.L h -1 t>3h
1.0 -- TD :2.5 h
t [hi
y = 0.75
S R = 10.0 g 1-1
Fig. 33. Process simulations, and kinetic, reactor, and operating data for an airlift tower-loop
reactor with a height of 40 m 32, ~,0)
compiled. The simulation started in batch operation with So = 10 g 1-1 initial substrate
concentration and Xo = 1 g l - t inoculum concentration.
The three system variables in the liquid phase o f the tower, cell mass concentration
Xv, substrate concentration Sv and dissolved oxygen concentration O v are shown in
their spacial and temporal behaviour in Fig. 33.
During the batch phase, cell mass is growing exponentially. Due to the extreme
pressure profile in the aerated tower the dissolved oxygen concentration shows
184 R. kuttmann et al.
distinct profiles. Cell mass concentration and substrate concentration are nearly
ideally mixed. At t = 3 h, the batch operation is switched to a continuous one.
Substrate is fed cell-free from a reservoir with SR = 10 g 1-1 at the bottom of the
tower, whereas cells and nonconsumed substrate leave the system with the same
liquid flow rate at the top. In contrast to the batch phase, cell mass and substrate
are no longer well mixed. The steady-state values depend on the control parameters
of the process and the kinetic parameters of the microorganismn.
In the following the profit maximization (cost minimization) is investigated for
steady-state conditions. Therefore the steady-state balance equations are used for
calculations. The procedure is carried out for a 40 m 3 plant and is based on the
assumption of constant kinetic parameters of Methylomonas M 15 (Table 7), but
varying control parameters. In contrast to the scale-up example, we are now
investigating a plant of 20 m in height. Figure 34 shows the four control possibilities
of the process. The variations of the control parameters, which are specific aeration
rate under standard conditions [3N,
~N -- ~ (185)
VF '
dilution rate D,
F
D - - TI; 1 (186)
VF
recirculation ratio 7,
fU
7 - F+ VU (187)
and substrate reservoir concentration SR are bounded.

In high air lift tower loop reactors the maximum recireulation ratio, y .... is con-
trolled by 13N and other unknown parameters 168.169)
Hence, y is generally not an independent variable. Only if the optimal 7 is less than
7max, Y is - - within some constraints - - an independently controllable par-
ameter.
Several parameters are influenced by the independent operating variables. Such
parameters are :
a) The coefficients of the longitudinal mixing in the liquid, D p and in the gas
phase, D G, in the tower. Both of them depend on the aeration rate. According
to our assumption the longitudinal liquid mixing in.the loop can be neglected.
b) The mean relative gas hold up,
EG = EGU + As U~o, (188)
which is as well controlled by the aeration rate as,

c) the volumetric mass transfer coefficient in substrate free medium,
kLao = kLau + AkLao Ugo. (189)

CONTROLLABLE OPERATION PARAMETERS

normalized aeration rate
~
xT* O ~ x:LR ~N = __vG [ wM
VF
~U ' ] F dilution rate
F
D =-- [ h -~ ]
VF
recirculation ratio
+dx ~U [-]
F + VU
substrate reservoir concentration

{v} sR [gH l
x*=LR~/ o o o
OPERATING VARIATIONS
,V-7 aeration rate
{r 0.083 V V M < ~N < 1.06 V V M
dilution rate
~u
0 h -I < D < Dcrit ( ~ 0.5 h -I)
m
recirculation ratio
{5R 0 < ~ ~ Ymax(UFomax,D)
substrate reservoir concentration
0 < S R < SRcri t (= 45.0 gl-I)
Fig. 34. Control possibilities ofan airlift tower-loop reactor 32. ls9.17o)
In Eq. (188) CG = CGu and in Eq. (189) k L a o = kLa U are the mathematical values at
U~o -+ 0.
In Fig. 35a the assumed linear relationships between ea and/or kLa0 and U~o are
shown, which are based on the idealization of experimental data 6o, 17o)
The gas flow rate,
E
[Pmax APmaxgG] T N
9 N = UEoQR , (190)
pNTE
J~
f
/
1000 1.0 1000 20

4-
"T
E
~" L~
W LO0
/ c~
5OO 0.5 / 500 10
r L'
/~.. y
100 0.1 ~ , , , , 100 2

0 0
0 1 5 = 0
uE~ [cm s -11
10 //...,..,,~ --
O OELS ETHANOL
zx OELS : METHANOL /
'~'~-mL :11 S.r:g.Ogl-1
5 k IDENTIFI- ~ _I
CAT~ON J I _.----"" Xmt.~=5.5 ST=5.7 gl q
1 mLoo=3.0 ST= 3.0 g [-1
0. . . . . ] . . . . I
0 1 5 10 15 20 -- 25
S [g [ -1]
Fig. 35a. System parameters and operational data as functions of the superficial gas velocity,
U~o. LR = 20 m, QR = 2 m2, kLau = 100 h -1, and %0 = 0.1 3z.t7o,. b. Comparison of volumetric
mass transfer coefficients as function of the substrate concentration using different alcohol substrates lo,
32,131,170)
and the u p p e r limit o f the o x y g e n supply rate,

E E
UGo[Pma x - - AP~a x %] Mo2XEG
Q~2 = (191)
LR[1 - - CO] R T E
where
APmax = QFgLR , (192)
are also p l o t t e d in Fig. 35a to illustrate the o p e r a t i o n a l conditions.

Table 7. Constant parameters of the sta-

tionary optimization procedure
Growth kinetic parameters

la=ax = 0.509 h -1
= 0.009 h - 1
Ks = 0.05g 1-t Yxts = 0.50
Ko = 0.15 mg 1-l Yx~o = 0.42
RQ = 0.4
Reactor- and loop dimensions
La = 20m dR= 1.6m
LB = 24m d B =0.1m
In Sect. 7.1 the dependence o f the volumetric mass transfer coefficient on the sub-
strate concentration and the aeration rate was given by
kLa(x) = kLao(U~o) m~.(SF, X). (193)
It is assumed that the aeration rate influences ktao, which is the k t a value in
absence of substrate. The coalescence effect o f the substrate is taken into account
in the mass transfer multiplier function m~, ,0. ~64.165). In Fig. 35b m~, is plotted as a
function o f the substrate concentration for methanol and ethanol. One can recognize
that a high substrate concentrations, m~ approaches a constant value. Hence, this
relationship can be represented by
~'~7
mL(S r , x ) = 1 +[mLo o-1] 1--e STJ. (194)
At low concentrations the curve can be approximated by a linear relationship.

The initial slope o f Eq. (194),
OmL(SF, X) _ meoo - 1 1 (195)

s ,x,=o SD'
is comparable With S~ ~, the slope o f Eq. 0 6 9 ) in Sect. 7.1.
In tower reactors, the substrate concentration, St, diminishes from its entrance
value at x = 0 (feed inlet at the b o t t o m o f the tower) to its exit value at x = LR (medium
outlet at the head of the tower). According to Eqs. (193) and (194), kLa also
decreases along x.
d) The longitudinal dispersion coefficients in the liquid and gas phase of the tower,
D v and D•, were correlated with the gas velocities by
D F = Dv~(dR'l(fi~o) ~ , (196)
and
DG = DN(d
G\
"~q]F,o.o
R j k G] " (197)
This correlations 94,95) have already been discussed in Sect. 4.1.

8.2 Steady State Model
The steady state balances of the continuous cultivation will be calculated using the
normalized operation and system parameters considered in the foregoing section.
In the following optimization procedure the model equations are solved repetitively
until a minimum is found. Since the model equations are nonlinearly coupled, the
iterative solution of this D.E. system has an extremely high expenditure of computer
time. Therefore, a reduction of the model is carried out with regard to the gas phase
and the loop balances.
Hereby the following assumptions are made:
1. The mean liquid residence time in the loop is negligibly small in comparison
with the overall recirculation time. Hence, the slight cell mass concentration
increase and substrate concentration decrease in the loop are neglected. This
means
A
CxB(Z*) = Cx~ (198)
and
CsB(Z*) = %A (199)
are assumed. Thus, the cell mass and substrate balances for the loop are
eliminated.
2. The oxygen balance in the loop is taken into account. It can be solved for
every point, z*, by means of Eqs. (198) and (199).
The implicit solution is given by
A A ,
ko [..[m C s F C x F Z
r(z*) + In F(z*) = 1 - (200)
Yx/o[ks + CA] VBC~F '
with
F ( z * ) - CoB(Z*) CoB(Z*) (201)

c,% cAF
However, only the exit concentration is of interest for the lower boundary condition
(Eq. (94b)) of the dissolved oxygen balance in the tower liquid phase,
A = F(1) COF
COB
A "
(202)
3. The three growth components in the liquid phase of the tower are calculated by
means of coupled stationary liquid phase balances,
I.tfCsv,Coy, z)
~[CsF} - CxF(Z), (203)
Yx/s
~(CsF, CoF, z)
Fs{c~ - Yx,'o CxF(Z)
+ Stv(kLao, m[, Csv, z) [p(z) Coa(Z) -- Cov(z)l , (204)
Vs{Cxv} = + [~(CsF, Cot, z) - ~,] Cx~(Z), (205)
with the corresponding boundary conditions (I = O, S, X),
ccIF(O) - Bov(fi~o) vF(D, 7) (clv(O) -- yci]~ -- [1 -- 7] c,R), (206)

~z
~ClF(1)
(207)
~z
and the following abbreviations:

-- stationary liquid phase operator,
f I __Bov(~o ) - 1 ~2ClFCZ) OClF(Z)

FstClF i := ~ OZ + VF(D, "{) ~-- (208)
modified Stanton number in the liquid phase,
Stv(kcao, mL,
' CSF, Z ) = kcao(u6o)
-E mL(CsFSo,
, zLR) T s , (209)
-- normalized spacially dependent specific growth rate,
CsdZ) Coy(Z)
latCsv, Coy, z) = ~m (210)
ks + CsF(Z) ko + CoF(Z)
4. Modelling of the gas phase by means of the steady-state gas phase operator,
Gs{CIG } --BOG(I~E) -1 =-oz

~ ( p(z) ~CIG(Z)"/
~z / + ~ (p(z) vo(z) Clo(Z)) (211)
yields the steady-state oxygen gas phase balance,
f ) i
Gs~coG~ = -- Stn(kLao, mc, CSF, Z) [p(z) COG(Z) -- COF(Z)] , (212a)
with the boundary conditions,
~Coo(O) Bo6(fi~-) rE[Coo(O) -- 1] (212b)

~z
~CoG(1)
-- O. (212c)
~z
and the steady-state continuity equation of the gas phase,
Gs{ 1} = Sto(ktao, m[, Csv, z) [p(z) Coo(Z) -- Coy(Z)]rq, (213 a)
with the lower boundary condition,
vo(0) = vE . (213 b)
This gas phase balances are used to calculate longitudinal oxygen mole fraction
in the gas phase. Equations (212) and (213) are rearrangeable by an analytical
expression.
To describe the longitudinal oxygen mole fraction in the gas phase different
approximations were used 32). The best approximation was achieved using a poly-
nomial relationship,
The three unknown coefficients Ki of the polynomial,
Coo(Z) = KI + K2 z + K 3 z 2 , (214)
are calculable from the boundary conditions (212b), and (212c) and the oxygen
conversion,
Uo 2 _ qo2 1 - CoAo (215)

qg2 1 -- rq CAG '
In the spacial dependence of the related oxygen mole fraction in the gas phase,
[1 - rq] Uo2 2 + BOovg[2z - - z 2]

CoG(Z) = 1 -- 1-rqU02 .2+Boav ~ ' 1216)
the modified Bodenstein number BoG, the normalized gas velocity vg and the norma-
lized respiratory quotient rq are known parameters from relationships of Sect. 4.2.
The unknown oxygen conversion Uoz is calculated from overall balances of the
system Eqs. (203) to (213),
I
'
~t ~ Cxv(Z) dz + Vv~CxF-- v c r r]Cx.}
o
Uo2
Yx/oqEo2
v {cg - v c a . - [ l - v] Co.}
+ E (217)
qo2
The simulation must be carried out iteratively, because Eqs. (216) and (217) are
coupled via the system Eqs. (203) to (205).
8.3 Formulation of the Performance Index and Optimization
The economy of the production process is influenced by the ratio of expenditure and
revenue. The investment, personal, operating and retail costs influence the expenditure
and the product quality and price the revenue ~71 1791
In the cost function (218a) used here, only two primary parameters of operating
and raw material costs, namely the costs of aeration and methanol substrate, are
taken directly into account.
The considerable cooling cost of biotechnological production processes is included
in the aeration cost 1801. Such a simplification is allowed for air lift tower loop
reactors in which no mechanical devices (impeller, pumps etc.) are used in the reactor
and hence the power input takes place only by aeration.
In the cost function, F J, which is to be minimized, the cost of aeration, PGAS, and
substrate, PsuR as well as the market price of biomass, PDCM,is included,
'
FJ = ,tPaAs 13TM + PsuBD(SR + • -- PDcMDX'~}eF(13~)
--+ Mi~([3N, D, 7, SR). (218a)
S~ and X AF are the substrate and cell mass concentrations in the medium at the
reactor outlet, • is an arbitrary "'penalty factor" which fixes the cost multiplier for the
nonconsumed substrate.
• takes into account the additive downstream and waste water cost caused by a
considerable amount of methanol in the medium at the reactor outlet 18~)
The following cost factors are used:
0.23 DM min
PGAS - m3 h , (218 b)
0.25 DM
PSUB ---- kg methanoi " (218c)
Since the produced cell mass consists only partly of the desired proteins, the price
of the dry biomass depends on the protein content of the microorganisms, gpROT:
0.80 DM
PDCM -- gPROT 9 (218 d)
kg protein
DCM is the dry cell mass and DM is German Marks. Thus the cost function, FJ,
has the dimension DM h - t i n -3.
Furthermore, a high growth rate is connected with a high RNA content which
yields a low protein content and a low cell mass quality. Thus, high cell productivity
and product quality are contradictory demands. The protein content of DCM
depends on the growth rate and thus on the RNA content.
The R N A range of Methylomonas M 15 is given by Lehmann et al. 541, from

~ t = 0 h -l,
RNA minimum content
gaNAmin ~--- DCM = 0.07, (219 a)
to p = ~max'
RNA maximum content
gRNA max = DCM = 0.20. (219 b)
The mean specific growth rate, ~t, in the tower reactor is calculated by
p = D + P'T- (220)
It is assumed that gRNA varies between gRNAmin and gRYAmax linearly with 15.
When disregarding the other cell components, the protein content is calculated by
protein content
gPROT = DC M = 1- gRNA min -- /gRNA max -- gRNA min} ]'in'lax
(221)
The optimization procedure is outlined in the flow diagram of Fig. 36. After setting
the operation parameters on chosen initial values,
p(o) = [[~N(O) D(O), 7(o), SR(0)]T (222)
I INITIF4LVFILUESOF I
OPERATIONPARAMETERS
F
SOLVINGOF
I MOC,EL EQUATIONS
I
I
I PERFORMFINC:E
I ON OF
C:FILC:LILFIT
INDEX I
HEURISTICHILLC:LIMSING
METHODOF NELDER/MEFID
EXAMINATIONIN[',EX
PERFORMANCE OF I I OPERF~TIDN
CHANGINGOF
PARAMETERSI
J.
' T ' E ~ NO I
.... ,,] Fig. 36. F l o w d i a g r a m o f the s t a t i o n a r y o p t i m i z a t i o n p r o c e d u r e 32. 159)

the iterative solution of the model equations (Eqs. (203) to (205) and Eq. (216)) is
carried out in step 1. The stationary solution depends on the control parameter
values.
In the second step, the value of performance index (Eq. (218a)) is calculatedwith
the substrate and biomass concentration at the exit of the column, S g and XVA,the
control parameter 13y and D, the model parameter ~v and the penalty factor •
The repetition of the procedure with a changed parameter set p~0 is under control
of a hill climbing method, cf. Nelder and Mead ns~ and Refs. 126-,281
In the examination part of the method it is checked, whether the minimum of
performance index is reached or the next iteration step is to be carried out.
To avoid loca[ minima, the nonlinear optimization problem requires a repetition
of the procedure with different initial values P(~
In Fig. 37 the result of an optimization run with a high penalty factor (• = 10)
is shown, i.e. the nonconsumed substrate is very expensive. Other examples are
to be found in 32.50)
In Fig. 37a the exit values of cell mass concentration X~, substrate concentration,
A
SaF~ dissolved oxygen concentration, OvA, oxygen mole fraction, XOG, as well as the
productivity, PRD, are plotted as functions of the ratio of the dilution rate, D, and
the maximum specific growth rate, lamax.
The productivity is defined
PRD = DX~. (2_a)
The optimum operational conditions (maximum profit) are marked as well as the
maximum cell mass productivity. For the calculations, at first the evaluation of the
optimum control parameter set (~N, I), ~, SR) was carried out by the discussed
optimization method. In the next step the entire X-D-diagram was calculated by
varying D.
More information on the operational conditions is supplied by the productivity/
conversion-diagram given in Fig. 37 b.
In this diagram once more the productivity PRD, the oxygen conversion,
QO2
Uo2 Qg2 (224)
the substrate conversion,
s. - svA
Us - , (225)
SR
the cost function, F J, and the volumetric mass transfer coefficient which holds true
in the main part of the tower and at the outlet, kLa A, are plotted as functions of the
dilution rate D.
It can be seen from this figure that the maximum profit isattained at the
boundary between oxygen-transfer limited and substrate limited growth.
NORMALIZED PLOT: - NORMALIZED PLOT:

OFA/10.0mg1-1 x~6/20.9% PRD/5.0g t-1 h-I PRD/2.0g I-th -1 / 336gl-lh q 1644h-t
1.0 X~'/15.0 g [-1 sA/30.0g [-t HLOAIIO00h-I ,~/,,,,.- ~ ~ ~
X A
F
r o~\ I.ubgt-'n-zI / - ~ 0.764

0.5
/ k / AI / / 0.70/, ~ . ~ |
PRD/
oy 0.5 0.75 1.0

O/#max f o t, . . . .
MAXIMUM PROFIT MAXIMUM PRODUCTIVI'~Y MAXIMUM PROEI MA• P.OD C VITu
D = 0 . t 3 8 h-1 D=0.30h-1 #,,ax=0.5Ogh-t b 0 = 0.138 hq D = 0,30 h-1 /r h-1
15.0 ! 5 0 ~ 0. 120.9
~oo~ !T
~7.5 125 i i l I~ !o~
. . . . . -D, K
KS ~ . . . . . .
0 o 0 J0
o lO 15 20
o x[rn],
Fig. 37a~. Optimum operational conditions, and oxygen, cell mass and substrate data of an
optimization run with x = 10 n.159, tTO). a) X-D diagram; b) Productivity/conversion diagram;
e) Longitudinal variation of process variables at optimum conditions
The maximum productivity is achieved in the strong oxygen-transfer limited growth

range. But the limitation of the substrate conversion leads to a high substrate con-
centration and a noneconomic substrate loss.
The increase of kLaA is due to the coalescence repressing influence of the
substrate.
In Fig. 37c the longitudinal variations of the cell mass concentration, X v, the
substrate concentration, Sv, the dissolved oxygen concentration, O F, and the oxygen
mole fraction in the gas phase, XOG are shown at the optimum conditions,
There is an interrelation between the two limitations in the reactor. In the lower
part of the tower, oxygen-transfer limited growth conditions and in the upper part
substrate limited growth conditions exist. The transition from oxygen-transfer limi-
tation to substrate limitation causes a renewed increase in the dissolved oxygen

concentration in the upper half of the tower as has been found experimentally in a
bench-scale column as shown in Chapter 7.
9 Conclusion and Outlook
The complex behaviour of spatially varying biotechnological processes in tower loop

reactors is hardly understood by measurements only. However, the knowledge of
a mathematical model leads to a better insight into the dynamical behaviour of the
processes. Moreover, an almost precise mathematical model offers the opportunity
to research the optimization of the plant's structure as well as of the control strategies
systematically, without the need of time consuming and expensive experimental
studies.
In this treatise, methods of modelling and optimal control of SCP production
processes in tower loop reactors are reviewed. The models and algorithms presented
are thoroughly discussed and demonstrated with the aid of measurements. These
experiments were carried out by co-operating groups of Prof. K. Schiigerl, Institute
of Chemical Engineering, University of Hannover, and Dr. J. Lehmann, GBF-In-
stitute for Biotechnological Research, Braunschweig/St6ckheim.
The analysis of the batch and fed-batch experiments shows a strong temporal
variation of some of the model parameters. Since these variations cannot be anti-
cipated, to our opinion only adaptive control strategies are well suited to control
this type of processes. The developed model therefore formed the basis to apply Open
Loop Feedback Optimal (OLFO) control strategies to the process under consideration.
For the optimization of continuously operating plants, the problem of cost-
optimization leads to the determination of optimal set points for the process.
The procedure and the results were demonstrated in detail, too.
In case of models with distributed parameters, the algorithms used for implemen-
tation of the given mathematical formulae require a large computer memory and a
high amount of arithmetical capability of the processor used. However, problems
of computer performance and memory capacity have attained less and less signifi-
cance in recent years. This is due to the rapid development of microelectronics. In
the near future, this progress in hardware will enable the users to implement even
more sophisticated control and optimization packages for technical processes,
instead of trying to generate condensed software for small hardware capabilities.
Of course, the application of the methods presented in this article to stirred tank
reactors (described by ordinary differential equations) is readily performed.
In consideration of the permanently increasing demands on product qualities
and purity and the encouraging results of simulations and pilot plant tests, the
authors are thoroughly convinced that application of models of biotechnological
processes is highly efficient in various fields, e.g. in estimation of system states
("indirect measurements"), identification of internal system parameters, optimal
design of (multistage) processes, and optimal control. -- Particularly in the field of
parameter and state identification, adaptive control, and optimal design of processes
with lumped and distributed parameters, further research is under progress in the
laboratories of the authors and other institutions.
I0 Symbols
L (length), M (mass), M~a (mole mass), T (time), K (temperature), P (profit, price),
-- (dimensionless)
A gas/liquid interfacial a r e a L2
A(x, t) spatial differential operator
a specific interfacial area L- 1
ai(x , t) coefficient functions
B(x, t) input operator
Boj modified Bodenstein-number in subsystem J --
bi coefficients in boundary conditions
C(x, t) output operator
Cj carbon dioxide concentration in subsystem J ML -3
r related concentration of component I in the
liquid system tower-loop
ClJ related concentration of component I in
subsystem J
D dilution rate,, T- 1
Dj longitudinal dispersion coefficient in L Z T -*
subsystem J
d1 diameter of reaction system I L
ds Sauter bubble diameter L
e error function
F liquid feed rate L3T - 1
FJ performance index (cost function) p L - 3 T -1
f perturbation function
G(x, t) weighting operator
g acceleration of gravity LT- 2
gPROT protein content of cells
gRNA RNA content of cells
Uo2 Henry coefficient of oxygen L2T -2
Ij concentration of component I in subsystem J ML -3
J performance index of parameter
identification
L! height of reaction system I k
Ki polynomial coefficients
KI saturation constant of component I ML-3
Kst coalescence factor
ki related saturation constant of component I --
k[ mass transfer coefficient of component I LT-t
kt a volumetric oxygen mass transfer coefficient T-x
kLao k L a a t S = 0 g l -~ T -~
Mj mole mass of component I MM
m E, m E mass transfer multiplier functions
ria1 mass flow rate of component I MT-
Nj nitrogen concentration in subsystem J ML -3
Oj oxygen concentration in subsystem J ML -3
OTR oxygen transfer rate M L - 3 T -~

OUR oxygen uptake rate ML-3T- 1
otr normalized oxygen transfer rate
our normalized oxygen uptake rate
P pressure ML iT-2
DDCM price of dry cell mass PM-1

PETH product (ethanol) concentration ML -3
DGAS aeration cost PL -3
DoG oxygen partial pressure in the gas phase M L - 1 T -2
PsuB substrate cost PM-*
PRD productivity ML-3T-
P . related pressure
p(u) adjoint state
PO2j dissolved oxygen partial pressure in
subsystem J
QI cross section area of reaction system I L2
Qo2 oxygen supply rate ML-3T

Qs substrate supply rate ML-aT -
qo2 normalized oxygen supply rate
qo,,x specific oxygen uptake rate T-
R gas constant L2T-2K -1
RIj reaction rate of component I in liquid ML-3T -
phase J
RQ respiratory quotient
rlj normalized reaction rate of component I
in liquid phase J
rq normalized respiratory quotient
So substrate concentration in m L ML -3
Sj substrate concentration in liquid system J ML -a
ST substrate concentration in m~. ML-3
Stj modified Stanton-number in subsystem J --
T temperature K
T upper bound of optimization interval T
Tj mean residence time in subsystem J T
TK identification interval T
TN normalization time T
Tu liquid recycle time in the tower-loop system T
t time T
ti, tk identification time T
U~d set of admissible control functions
Ut conversion of component I
U control function
Uj velocity of phase J LT-X
Ujo superficial velocity of phase J LT-1
fl normalized velocity
Vj volume of subsystem J L3
volumetric flow rate in subsystem J L 3 T -1
"Qu volumetric recycle rate L3T - 1

v control function
vj normalized velocity of phase J
w j/ weighting functions of dissolved oxygen
concentration in parameter identification --
X cell mass concentration in tower-loop system M L -3
Xj cell mass concentration in liquid system J ML -3
X longitudinal coordinate in tower L
X* longitudinal coordinate in loop L
XIG mole fraction of component I in the tower --
gas phase
Yxl~ yield coefficient of component I
Y state
Yxi~ normalized yield coefficient o f component I --
Z dimensionless longitudinal coordinate
in tower
Z* dimensionless longitudinal coordinate
in loop
Zs(X, t) desired trajectory
zj(t) measurements
Greek symbols
dimensionless aerator range (z = 0.1)
specific aeration rate T-l
F related dissolved oxygen concentration in --
the loop
7 recycle ratio
A parameter variation
6 variation
~j mean relative hold-up of phase J
normalized adjoint state
analog computation times T
rl,t weighting factors in parameter identification --
penalty factor of nonconsumed substrate --
A canonical isomorphism
k eigenvalue T- l
specific growth rate T-1
~[max maximum specific growth rate T-
specific death rate T
[Am normalized/am, x
normalized gr
V weighting factor in parameter identification --
integration variable
QJ density of phase J M L -3
tY weighting factor in parameter identification --
T normalized time
D weighting factor in parameter identification
Mathematical Modelling. Parameter Identification and Adaptive Control 199
)~ coalescence pressure function --

Xj(x, t) sensor characteristics
u? coalescence function
~2 analog computation interval T
Subscripts
S)'stem variables
C, CO2 carbon dioxide
N, N2 nitrogen
O, 02 oxygen
S substrate
X cell mass
Subsystems
B loop
F liquid phase of the tower
G gas phase of the tower
R bioreactor, reservoir
Parameters
crit critical value
K identification interval
k identification time
LIM upper limit
M modelled states
max maximum
rain minimum
0 forS=0
0 initial value (t = 0 h)
O superficial velocity
opt optimal value
U (mathematical) lower value for u~-o ~ 0
Pre-subseripts
C calculated value
M measured value
Superscripts
A outlet ( = top of the tower)
E inlet ( = bottom of the tower)
i time step
k space iteration step
1 optimization step
M measurement
m maintenance
IYl identification step
N normalized conditions
T transpose of a vector
O~ at the end of the aerator range
A optimal (estimated) parameter
!
transpose of an operator
* dissolved saturation concentration

Partial differential operators
B loop operator
F liquid phase operator
G gas phase operator
Matrices and vectors
~r system differential operator matrix
space and time dependent state vector
reaction vector
Y mass transfer vector
11 Acknowledgement
The authors acknowledge the financial support of the Ministry of Research and
Technology of the Federal Republic of Germany, Bonn, and thank Dr. H. Buchholz,
Prof. Dr. K. Schiigerl, and Dr. W. Zakrzewski, Institute of Chemical Engineering,
University of Hannover, Dr. W. Scheiding and Dr. H. Schlingmann, Institute of
Automatic Control, University of Hannover, as well as Dr.-Ing. J. Lehmann,
Dr. J. Ingham, and Prof. F. Wagner, GBF-Institute for Biotechnological Research,
Braunschweig-St6ckheim, for their excellent cooperation and support.
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Modeling, Optimization and Control
of Semi-Batch Bioreactors
S a t i s h J. P a r u l e k a r a n d H e n r y C. L i m
S c h o o l o f C h e m i c a l E n g i n e e r i n g , P u r d u e U n i v e r s i t y , W e s t L a f a y e t t e , I n d i a n a 47907,
USA
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -. . . . . . . . . . . . . . . 207
2 Justification and Advantages of Fed-Batch Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
3 Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
3.1 Unstructured Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
3.1.l Characteristics of Various Fed-Batch Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
3.1.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
3.2 Structured Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
3.3 On-Line Estimation of Bioreactor Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
4 Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4.1 Formulation of a General Optimization Problem and its Solution . . . . . : . . . . . . . . . . . . . . 226
4.2 Optimization of Fed-Batch Bioreactors Used for Biomass Production . . . . . . . . . . . . . . . . . 229
4.2.1 Solution for Constant Biomass Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
4.2.2 Solution lbr Non-constant Biomass Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
4.3 Optimization of Fed-Batch Bioreactors Used for Metabolite Production . . . . . . . . . . . . . . . 236
5 Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
5.1 Classification and Characterization of Control Schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
5.2 Instrumentatiuil lbr Monitoring the State of Fed-Batch Bioreactors . . . . . . . . . . . . . . . . . . . 242
5.3 Feed-on-Demand Control lbr Biomass Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
5.4 Control of Specific Growth Rate for Antibiotic and Enzyme Productions . . . . . . . . . . . . . . 248
6 Current Problems and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
7 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
8 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
1 Introduction
I n a f e d - b a t c h o p e r a t i o n (also r e f e r r e d to as t h e " ' z u l a u s process) t h e n u t r i e n t s
n e c e s s a r y for m i c r o b i a l g r o w t h are fed e i t h e r i n t e r m i t t e n t l y o r c o n t i n u o u s l y d u r i n g
t h e c o u r s e o f a n o t h e r w i s e b a t c h o p e r a t i o n . T h e r e a c t i o n m i x t u r e b r o t h is t h e n h a r -
vested e i t h e r fully o r p a r t i a l l y at the e n d o f t h e o p e r a t i o n a l p e r i o d a n d this w h o l e
p r o c e s s m a y be r e p e a t e d several times. T h e f e d - b a t c h o p e r a t i o n h a s b e e n f o u n d to
be p a r t i c u l a r l y effective l b r p r o c e s s e s in w h i c h effects s u c h as s u b s t r a t e i n h i b i t i o n ,
c a t a b o l i t e r e p r e s s i o n a n d glucose effect are i m p o r t a n t .
T h e c o n c e p t o f f e d - b a t c h c u l t u r e s h a s b e e n in use since t h e early 2 0 t h c e n t u r y 1)
w h e n it was r e c o g n i z e d t h a t in t h e p r o d u c t i o n o f y e a s t f r o m m a l t wort, t h e c o n c e n t r a -
t i o n o f m a l t w o r t m u s t b e k e p t low e n o u g h so t h a t t h e yeast is n o t g r o w n t o o fast
t h e r e b y p r e v e n t i n g o c c u r r e n c e o f a n a e r o b i c c o n d i t i o n s in t h e c u l t u r e a n d s u b s e q u e n t
208 S.J. Parulekar and H. C. kim
production of ethanol. Additional wort was added at a rate which was always less
than the rate at which the yeast cells coulduse it. This led to increased yeast yields
while obviating production of ethanol 21
The technique of nutrient feed additions to batch cultures was given its current
name by Yoshida et al. 37, who used it to refer to a batch culture to which substrate
is fed at a constant rate. Following successful application to yeast production, fed-
batch cultures have been tried in the industrial production of antibiotics, amino
acids, enzymes, vitamins, single-cell proteins, biomass, and various organic com-
pounds of commercial importance. Whitaker 11 provides an excellent documentation
of these applications. The nutrient which limits the growth of microorganism in
a fed-batch culture provides an excellent means of controlling the growth and meta-
bolism of the microorganism 41.
Unlike the batch and continuous Noreactors, the fed-batch bioreactors may be
operated in a variety of ways by regulating the nutrient feed rate in a predetermined
manner (feedforward control) or using feedback control. The most commonly used
fed-batch cultures are constant fed-batch culture, exponentially fed-batch culture,
extended culture and repeated fed-batch culture. The constant fed-batch culture,
as the name suggests, is a batch culture to which the substrate is fed at a constant
rate 3~. Exponentially fed-batch culture is a batch culture to which substrate is fed
at an exponentially increasing rate sj. An extended culture is a fed-batch culture to
which substrate is fed at a varying rate so as to maintain concentration of the limiting
substrate constant at all times 6~. Fed-batch cultures, where part of the reactor contents
are periodically withdrawn are known as repeated fed-batch cultures ~1 or cyclic
fed-batch cultures 8j
The variation of the culture volume in a semi-batch bioreactor must be properly
accounted for in the models used for optimization and control of these reactors.
Such variation in culture volume (dilution effect) can be neglected only when the feed
is concentrated in nutrients and when the volume change is small. The dilution effects
have been neglected in some of the previous studies on fed-batch cultures without
justification.
Modeling, optimization and control of fed-batch bioreactors have been subjects of
considerable interest in the literature in the past ten years. The purpose of the present
article is to present a comprehensive review on these topics. To the best of our knowl-
edge, such a review is not available at present. Computers have been extensively
used for control of fed-batch bioreactors. The reduced cost and improved reliability
of computer hardware in recent years have resulted in increased usage of computers
for process optimization and control. Sevdral reviews pertaining to these topics are
available 9 l~j
Fed-batch cultures can be broadly classified into two types : (1) processes in which
the products are strictly growth associated and (2) processes in which the desired
product is not necessarily growth associated (metabolite product). The second type
of processes, examples of which include enzyme and antibiotic production, involve
two distinct phases, a rapid growth phase followed by a production phase. In the
growth phase, it is desirable to maintain the growth rate at a maximum to achieve
rapid growth unless such a rapid growth rate results later in a lower product formation
rate. In the production phase, the cell growth rate is much lower than that in the rapid
growth phase. A performance index such as productivity, production rate or yield is
Modeling. Optimization and Control of Semi-Batch Bioreactors 209
maximized by maximizing the cell growth (or cell growth rate) for the first type of
processes and by maximizing the productivity (or production rate) of the metabolite
product for the second type of processes. Due to the obvious differences in the ob-
jectives of the two types of processes, the optimization and control strategies for these
are significantly different.
2 Justification and Advantages of Fed-Batch Operations
Fed-batch bioreactors are well suited for processes in which the cell growth and/or
product formation are significantly sensitive to the concentration of the limiting
substrate. These reactors offer enormous flexibility with respect to control of the
limiting substrate.
Overfeeding of nutrients often leads to formation of undesired by-products. For
example, in the production of baker's yeast with glucose as the substrate, when glucose
concentration in the aerobic culture medium exceeds a critical value (70 mg I- 1 15.16));
glucose is partly metabolized to ethanol 17). This is the so-called Crabtree effect
or the glucose effect. In production of Candida utilis grown on ethanol, an overfeeding
of ethanol leads to production of acetate as a by-product and a corresponding reduc-
tion in the single-cell protein yield 18~. Overfeeding of nutrients has been observed to
reduce production of non-biomass products (such as antibiotics and enzymes) due
to excessive cell growth 19~. An underfeeding of nutrients results in cell starvation
and reduced specific growth rates, thereby lowering the productivities of biomass
and metabolite products. The fed-batch mode of operation of bioreactors provides
an excellent means of regulating the nutrient feed rate to optimize the productivity
while at the same time obviating over- and under-feeding of nutrients. In the baker's
yeast process, the yeast productivity is maximized by controlling the respiratory
capacity of the cells at a maximum, i.e., by operating under conditions that correspond
to the switching of metabolism from pure oxidation to oxido-reduction z~
The productivity of a metabolite product is maximized by maintaining low specific
growth rates in the production phase by regulating the nutrient addition rates prop-
erly 4, 23 --26),
Ethanol and methanol, when present in the culture medium either as a substrate or
product, severely inhibit the growth of microorganisms 27-31 I. If the concentration of
ethanol or methanol in the culture broth is high, there is considerable loss of substrate
due to evaporation. In a fed-batch culture, concentrations of these species in the broth
can be kept low and the inhibition and evaporation problem can be overcome.
Synthesis of many metabolite products is repressed when the cells are grown
rapidly on a readily utilizable carbon source such as glucose. Such repression, the
catabolite repression, may be caused by the limiting substrate, an intermediate,
or the product. Examples of processes in which catabolite repression occurs include
1) synthesis of 13-galactosidase by Escherichia coli grown on glucose 32),

2) formation of cellulose in cultures of Pseudomonasfluorescens grown on sugars 33),
210 s.J. Parulekar and H. C. Lira
3) production of cephalosporin C by Cephalosporium acremonium grown on glucose

34 - 3 6 )
4) production of heparinase by Flavobacterium heparinum fed with glucose 37~,

5) penicillin production by Penicillium chrysogenum grown on glucose 25.26~
6) production of [3-amylase by Bacillus megaterium grown on sugar 3s) and
7) production of glucose by Trichoderma reseei with cellulose as substrate 39.4o~
In all these examples, catabolite repression is caused by glucose and other sugars.
One powerful way to circumvent the depressed formation of desired products is
to limit the growth rate by slow feeding of the carbon source, i.e., to operate the bio-
reactor in fed-batch mode 32"33'36-42). By operating the bioreactor in a ted-batch
mode, it is possible to supply the substrate at an optimal rate to obtain maximum
productivity while at the same time circumventing catabolite repression.
The fed-batch processes have received wide attention for their ability to produce
high density biomass ,,3-,,6) and dense metabolite products ,,7-,,9~ without inhibition
or catabolite repression. These dense cultivations have definite advantages of increas-
ing the productivities and facilitating the separation of products.
3 Modeling
The biological behavior has a complexity unparalleled in the chemical industry

and consequently, its prediction from information about the environmental con-
ditions is extremely difficult. In many cases, the objective of development of a mathe-
matical model is explicitly aimed at providing the basis for controlling the performance
of a bioreactor. Moreover, the mathematical models are absolutely indispensible
in the calculation of optimal operating conditions.
Depending on the level of complexity, the general models can be divided into
phenomenological (unstructured) models and mechanistic (structured) models ~2, 5o~
Phenomenological models are usually unstructured and are used to describe the overall
observed microbial response. No provision is made for changes in the internal com-
position of the cells when the metabolism is shifted. The mechanistic or structured
models take into account the various cellular processes which are important to the
process. The activities of specific enzymes in the cell as well as structures such as
ribosomes, mitochondria, macromolecular components such as proteins, D N A / R N A
and carbohydrates may all be included in a highly structured model. An advantage
of such models is the potential for modeling the interrelationship of the metabolic
processes and predicting the effects of disturbances on the overall microbial process 5~~.
The disadvantages of the highly structured model are the difficulty in determining
the various kinetic constants to describe the individual reactions and the computational
difficulty posed by large sets of equations that result 12). Although the extremely
complex nature of the biological system may require complicated models, over-
sophistication of models should be avoided since it tends to defy the very purpose
of modeling by obscuring the essence of the model and makes the prediction of
microbial behavior exceedingly difficult 5z~
Modeling, Optimization and Control of Semi-Batch Bioreactors 21 I
3.1 Unstructured Models
The most widely used state variables in the unstructured models are the concentra-
tions of substrate, biomass and product and the culture volume. The general mass
balance equations for a semi-batch bioreactor are written as 13,2v-29,531
dV
-- = F -- F o (1)
dt
d(VX)
-- FoX + g"e'XV (2)
dt
d(VS)
dt - FSv -- FoS - - o n e t x v GSg (3)
and
d(VP)
dt - FPv FoP -~ '/~netxv (4)
where F and Fo represent the inlet and outlet volumetric flow rates for the bioreactor
and V represents the bioreactor volume. X, S and P represent the concentrations of
the cells, limiting substrate and metabolite product, respectively. S F and PF refer to
the concentration of the limiting substrate and product respectively in the feed.
The feed does not contain any biomass (cells). G is the aeration rate, and Sg is the
concentration of the limiting substrate in the gas phase. The last term in-Eq. (3)
accounts for the loss of a volatile substrate such as ethanol due to evaporation.
Any change in density of the culture medium is assumed to be negligible while deriving
Eq. (1). ~1net, O"net and r~net are the net specific rates for cell growth, substrate con-
sumption and metabolite product formation, respectively and are related to the specific
rates for cell growth (g), substrate consumption (~) and metabolite formation (re) as
gnet ---- g __ mx _ kx (5)
~,,e~ = o- ms (6)
P
rc~t = ~ -- k p ~ (7)
~t
o- - (8)
YX~'S
and
rc = nl + ~z2~t (9)
212 S.J. Parulekar and H. C. Lim
where m s and m~ represent the maintenance requirements for the limiting substrate
and biomass, respectively, k x and kp are the specific aging rates for biomass and
metabolite product, respectively, rcI and rc2 are arbitrary constants and Yx,.s is the
cell-to-substrate yield (g cell{g substrate}-l). The specific growth rate la and the
yield coefficient Yx,.s are in general dependent only on the concentration of the
limiting substrate. The volumetric flow rates F and Fo may vary with time t.
Equations (1)-(4) are subject to the initial conditions
v(0) = Vo (10)
X(O) = X o (1 I)
S(O) = So (12)
p(0) = Po (13)
dX FX
- - = ~ne'X -- - - (14)
dt V
Substitution of Eq. (1) into Eqs. (2)-(4) yields
dS _ F (S v _ S) --c~n~tX - GSg
(15)
dt V V
and
dP F
dt - V (PF P) -~ "/'cnetx (16)
where F/V is the "dilution rate" for the semi-batch culture. Unlike in the case of
continuous culture, the dilution rate for the semi-batch culture varies with time due
to variations in F and V. For a fed-batch culture, F o is zero.
In derivation of the general mass balance equations, it is assumed that the specific
rates for cell growth (g) and substrate uptake (c0 do not depend on the cell age distri-
butions. If this assumption is not valid, an additional cell age distribution equation
as well as the precise dependence of g and cy on the cell age distribution must
be known 5)
The rate expression for growth of microorganisms due to M o n o d is applicable
only under steady or "slowly" changing conditions, i.e., in a state where balanced
growth will usually occur 54, 55). Despite this limitation, M o n o d equation has been
widely used to describe the growth of microorganisms in fed-batch bioreactors.
3.1.1 Characteristics of Various Fed-Batch Bioreactors

Fed-batch bioreactors in which the feed rates are maintained constant are referred
to as constant fed-batch reactors. For a constantly fed-batch culture, the dilution
rate (F/V) decreases continuously with time. Therefore, such a culture can be repre-
sented by an equivalent continuous culture with flow rate decreasing with time 5.55)
Using this equivalency, it can be established that "~dynamic" steady states could be
Modeling, Optimization and Control of Semi-Batch Bioreactors 213
attained for sufficiently low teed rates such that the net specific growth rate is essentially
maintained equal to the dilution rate ss). Such dynamic steady states are termed as
quasi-steady states (QSS) ~). A quasi-steady state is characterized by a constant cell
concentration. To maintain a quasi-steady state in a constant fed-batch culture,
the net specific growth rate must be decreased continually by varying the limiting
dS
substrate concentration, i.e., ~ is not zero. At quasi-steady state, the rate of change
of total biomass is constant.

In the exponentially fed-batch cultures the feed rate varies exponentially with
time. Variation in the culture volume with time in this case (F = Ae Bt, A and B are
arbitrary constants.) is expressed as 5~
A A eBt
V=Vo 17)
with
t _< ~ In (V m -- Vo) + 1 lig}
The upper limit on t is imposed in view of the limited capacity of the bioreactor.
(Vm is the maximum culture volume.) The dilution rate for an exponentially fed-batch
culture is expressed as
F B
D = -- = 119)
V [(V~ 1 ] e-13'+
~ -
It is interesting to note that exponentially fed-batch cultures can be represented by

equivalent continuous flow cultures with decreasing as well as increasing or constant
flow rates, depending on the magnitude of (VoB A) 5~.
A quasi-steady state will be attained in an exponential fed-batch culture if the net
specific growth rate is equal to the dilution rate. If (VoB -- A) is non-zero, then for
the maintenance of quasi-steady state the net specific growth rate must change with
time (in view of Eq. (19)) which implies that the substrate concentration will vary
with time. In the special case where VoB = A, the dilution rate is constant. Both
the cell concentration, X, and the limiting substrate concentration, S, will then be
constant under quasi-steady state conditions which will be established at all times
provided
F(t) = F(0) exp [~,2net(so)t] (20)
and
F(0) - V~162176 Xo (21)

( S F - So)
214 S.J. Parulekar and H. C. Lira
In derivation of Eqs. (20) and (21), the loss of substrate due to evaporation has been
assumed to be negligible. It is evident from Eq. (20) that the specific growth rate of
microorganisms in an exponentially fed-batch culture can be controlled by varying
externally the exponent on the feed rate 31,56)
An extended culture is a led-batch culture in which the feed flow rate is continually
manipulated so that the concentration of the limiting substrate, S, remains constant
at all times 5,,2, sv~. When the net specific growth rate and the net specific substrate
uptake rate depend exclusively on the concentration of the limiting substrate and the
loss of substrate due to vaporization can be neglected, it is evident from Eq. (2) that
the total biomass must increase exponentially with time
VX = VoXo exp {I,t"e~(sD)t} (22)
where SD is the desired substrate concentration. Substituting Eq. (22) in Eq. (15)
dS
and noting that d t = 0 for an extended culture, we arrive at the following relation
for the feed flow rate
~"et(SD) XoVo exp -[lan~t(SD) t}

F(t) = (23)
(Sv - - So)
Even with the feed rate defined in Eq. (23), there would be transients in the substrate
concentration for a shortwhile unless, of course, the initial substrate concentration
is also at the desired value SD. The extended culture with feed rate defined in Eq. (23)
is indeed an exponentially fEd-batch culture.
The variation in volume with time for an extended culture is estimated by solving
Eq. (1) with F being defined in Eq. (23)
V = Vo[1 - - k + k exp {I,t"e~(Sv)t}] (24)
where
k = ~n~t(SD)Xo (25)
I-t"~ (Sv -- So)
The cell concentration history is obtained by substituting Eq. (24) into Eq. (22)
X(t) = X o e x p ~t~t
r ..... tre
t~ (26)
[1 -- k + k exp {lanet(SD) t}]
F r o m Eq. (26), it is apparent that the cell concentration may increase, decrease or
remain constant at Xo depending on the magnitude of k. When k is negligibly small,
the cell concentration can increase exponentially since the increase in the culture
volume is neglibible over a short time interval. This situation can be realized in
practice when the initial dilution rate required to keep the substrate concentration
at the desired level is negligible in comparison with the initial net specific growth rate.
When the initial dilution rate is greater than the initial net specific growth rate, i.e.,
Xo
when k > 1, the cell concentration decreases and approaches the value - ~ asymptotic-
ally 5). If k = l, not only the substrate concentration but also the cell concentration
may be kept constant at its initial value Xo. In this case, the limiting substrate is supplied
at such a rate that the cells generated and the substrate consumed will keep up with
the increasing culture volume in such a way as to maintain the constancy of the cell
and substrate concentrations. The required flow rate is defined in Eq. (20) with
F(0) = g(So) Vo. Eq. (25) provides a constraint for the initial cell and substrate con-
centrations, X o and So. The culture volume increases exponentially in this case and
the dilution rate is constant. Thus, the extended culture of this type, when fed with
the exponential feed rate of Eq. (23), is equivalent to an exponentially fed-batch
culture which is maintained at a quasi-steady state.
So far, we have described the characteristics of single-cycle fed-batch bioreactors.
Typically, a cycle consists of three stages: filling stage (F > O, F o = 0), batch stage
(F = F o = 0) and harvesting stage (F = 0, F o > 0). In the filling and harvesting
stages, the bioreactor is operated in a semi-batch mode. In a repeated fed-batch
bioreactor, as the name itself suggests, the cycles described above are repeated. Ideally
the harvesting stages in a repeated fed-batch reactor should be carried out as rapidly
as possible (Fo should be maintained at the maximum permissible value 58)). If the bio-
reactor is properly operated, every cycle in a repeated fed-batch operation is reproduc-
ible. In the filling stage of a cycle (F > 0, Fo = 0, 0 < t < t0, the volume of the bioreac-
tor increases from its initial value Vo to the maximum permissible value V m and the cell
and substrate concentrations change from their initial values X o and So to Xri n and
Sfill , respectively. In the batch stage (F = F o = 0, tf < t < Tf), the cell growth
is continued so that the cell and substrate concentrations change from Xfill and
Sfm to Xf and Sf. The third stage is the rapid draw-off where the culture volume is
reduced from V m to Vo while the cell and substrate concentrations remain practically
unchanged at Xe and Sf 58-61). After a steady cyclic operation has been achieved,
the cell and substrate concentrations at the beginning of a cycle will be equal to those
at the end of the previous cycle 58 61); i. e.,
X o = Xf and So = Sf (27)
The feed rates for exponentially led-batch cultures and extended cultures may be
varied with time using feed programmers which are essentially automatic curve
tracers 31.38,62) Another way of programming the time varying rate is to divide
the cultivation period (t = 0 to t = Tf) into n time intervals of individual periods
At~ (i = 1, 2 .... , n). The flow rate can be considered to be constant in each of the
subintervals. Discretized version of the mass balance equations can then be used for
modeling the bioreactors zo, 63.64)
3.1.2 Applications
The simple but yet general model presented earlier and many complex versions of it
have been systematically tested with experimental findings for various processes.
These attempts are tabulated in Table 1. In what follows, we briefly review some of
the more complex unstructured models for fed-batch bioreactors.
In the baker's yeast process where Saccharomyces cerevisiae is grown on glucose
or other sugars, ethanol is formed as a byproduct. Similarly, in the single-cell protein
216 S.J. Parulekar and H. C. k i m
Table 1. Models proposed and experimentally tested for various processes carried out in fed-batch
bioreactors
No. Process Refs.
1 Baker's yeast production ethanol substrate 30,65-69)

2 Penicillin production glucose substrate 19.~o-so)
3 Cephalosporin C production - glucose substrate 3-*-36.s~)
4 Glntamic acid production ethanol substrate 27 29)
5 Streptomycetes production 63, 64, 82,831
6a Production of methylotroph L3 methanol substrate 58, 84-861
7" Growth of Candida utilis ethanol substrate 53)
8 Synthesis of lysine by auxotrophic mutant s~. 8s)
9 Glucoamylase synthesis corn steep liquor substrate sg~
a Used for production of single-cell proteins
production using Candida util& grown on ethanol, acetate is formed as a by-product.

In both of these processes, the microorganisms used have capability of growing on the
limiting substrate fed to the bioreactor and the by-product formed in the culture
medium. When there is appreciable by-product formation, the model used to describe
the process must account for
a) consumption of the substrate (S) for cell growth and by-product (P) formation,
b) formation of by-product from the substrate and consumption of by-product
for cell growth, and
c) cell growth owing to consumption of substrate and by-product.
Such models have been proposed by Watteeuw et al. s31 for fed-batch cultures
of Candida utilis grown on ethanol and by Okada et al. 6sl tbr fed-batch cultures
of baker's yeast grown on sugar. Dilution effects have not been accounted for in the
model by Watteeuw et al. s31. The net specific rates l.tn~t, c~net and ~net of Eqs. (14) (16)
are defined in this case as
]..[net = P S 21- [Ll,p - - m~ -- k, (28)
cy,et m Ps + ms (29)
Yx,,s Yp,,s
~net = ~ PP
Yx,e mp (30)
In Eqs. (28)-(30), Ps and p~ are the specific growth rates on the substrate and the
by.product, respectively. YP,s and Yx.,P are the by-product to substrate yield and
biomass to by-product yield, respectively, m v accounts for the maintenance require-
ments for the by-product and will, in general, depend on the substrate and by-product
concentrations.
For the baker's yeast process, it has been observed 66 68) that the cell growth is
inhibited by side-products accumulated in the culture broth. The balance for the
Modeling. O p t i m i z a t i o n a n d C o n t r o l of Semi-Batch Bioreactors 217
inhibitory species can be represented by Eq. (16) with P and rc"~' being replaced by
concentration of inhibitory species I and its specific formation rate rq. Fukuda et
al. 66.67) and Okada et al. 68~ have assumed rq to comprise of growth-associated and
non-growth associated terms
XI = A~ + Bxl.t (31)
where A1 and B 1 are arbitrary constants. Carbon dioxide produced by the microbes
during cultivation has been found to inhibit growth of several microorganisms 9o-95(
Metabolites such as organic acids are also known to be growth inhibitors 921
In the model developed by Bajpai and ReuB vo, v~ for penicillin production, the
specific growth rate of mycelia was modeled using Contois kinetics. Thus,
~tS
la - (32)
K~X + S
where la and K, are kinetic coefficients for the Contois kinetics. The specific rate of
product formation was represented by substrate inhibition kinetics. The Bajpai
and Reug model can be expressed in terms of the general balance equations (Eqs. (l),
(14)-( l 6)) derived earlier for a fed-batch bioreactor with p ne,, one, and rc"e' being defined
as
[aS
. . . . . (33)
KIX + S
~ - -
cyne, ~t + - -/'[ ms [34)
Yx/s Yp/s
~cS P
rc"e' - $2 kp ~- 135)
Kp+S+--
Kl
where ~, Kp and K~ are the kinetic coefficients for the substrate inhibition kinetics.
The maintenance energy term [msX } in the substrate balance (Eq. (15)) is independent
of substrate concentration. As Stutts 96) has pointed out, such a term is physically
unrealistic as it predicts the consumption of substrate for maintenance purposes
even when no substrate is present. To prevent such unreasonable behavior, Stutts 96)
used M o n o d form of expression to express the dependence of the organism's (Penicil-
lium chrysogenum) maintenance requirement on substrate concentration.
The models discussed so far did not incorporate nitrogen and oxygen balances.
For penicillin formation, it has been found 19.75)that an irreversible loss of productivity
occurs when the concentration of oxygen in the culture medium decreases below
a critical value even in a short time. It is necessary to guarantee the most favorable
proportions among oxygen, nitrogen and carbon sources for biosynthesis of penicillin
as an aerobic process 9vl. Hegewald et al. "76) have incorporated oxygen balance in
addition to the carbon source balance in the model they developed for benzyl-penicillin
production. Both sucrose (S), which is the carbon source, and dissolved oxygen (S~)
are consumed for the formation of biomass as well as for the synthesis of penicillin.
The product formation by Peniciltium chrysogenum occurs only if a sufficient supply
of oxygen, carbon source and precursor is available. The specific growth rate and
the specific production rate are expressed as
la = la,~,xfx(S) ~(S~) (36)
= '/I;maxf3(S) f4(Sl) 82 (37)
where S and S~ denote the concentrations of the two substrates, sucrose and dissolved
oxygen, respectively. $2 represents the concentration of ammonia nitrogen, fl is a
monotonically increasing function of S, f2 and f3 are single hump functions of Sx
and S, respectively, while f4 is a sigmoidal function of Sa. A m m o n i a nitrogen is formed
from amino nitrogen, whose concentration is denoted as $3. Hegewald et al. v6)
neglected the change in the culture volume due to addition of the carbon and nitrogen
sources. When the variation in volume is properly accounted for via Eq. (1), the
balances for biomass (Penicillium cho'sogenum ), sucrose and benzyl-penicillin are
expressed using Eqs. (14)-(16) with a) Pv = Sg = 0, b) lan~, being defined in Eq. (5)
with m,, c) rc"~ = ~, and d) o "~t being defined in Eq. (34) with ms = 0. The dissolved
oxygen balance is expressed as
dS1 laX reX FS1

- + kLa(S* - S ~ ) - - - (38)
dt Yx/~l Yp/sl V
where Yx.sl and Y~.sl are the biomass-to-oxygen yield and penicillin-to-oxygen yield
cr transfer coefficient and $1" is the equilibrium
respectively, ki, a is the volumetric oxy~,en
concentration of dissolved oxygen. The mass balances for ammonia nitrogen and
amino nitrogen are expressed as
dS 2 ~X FS 2
- - - + %1S3) ~tX - - - (39)
dt YP/s2 V
and
dS3 ~o($3) FS3 (401

- laX---
dt ~rs 2/s3 V
where %(S3) is the deamination coefficient.

Heijnen et al. 77) used simple kinetic expressions in combination with elemental
and enthalpy balances to develop a model for the fed-batch penicillin process.
Although the specific production rate of penicillin is assumed to be a function of the
specific growth rate, the simulations done using the model showed that the model
allows for the commonly observed lag between the growth phase and the production
phase. The model developed by Calam and Russell 72) for penicillin formation
divides the cell growth process into two phases. In the first phase, nitrogen is con-
Modeling, Optimization and Control of Semi-BatchBioreactors 219
sidered to be the limiting nutrient and dictates the initial growth of the mold. The
carbon source, sugar, dictates the growth of the culture in the second phase.
3.2 S t r u c t u r e d M o d e l s
In the models discussed thus tar, although the cell behavior was correlated and modeled
in terms of extracellular environment, it is actually the intracellular environment
that a cell responds to. Each cell can be viewed as a complex chemical reactor in
which thousands of enzymatically catalyzed reactions with intimate interactions take
place along with internal regulatory effects such as inhibition/repression/activation 9sl.
These reactions can be roughly divided into two categories: those which break up the
nutrient compounds to derive energy (catabolism) and those which assimilate carbon
sources to form cell mass (anabolism). The models that describe the intracellular
activities of the organism, the structured models, should be developed by selecting
the parameters which are most relevant for the description of the physiological state
of the organism.
The structured models used for description production of antibiotics and enzymes in
fed-batch bioreactors allow for cellular differentiation. The model proposed by Megee
et al. 99) for the production ofmetabolites in mycelial growth allowed for five stages of
cellular differentiation plus the production of growth associated as well as non-growth
associated products. The model accounts for the hyphal elongation and branching
occurring during mycelial growth. During the course of these processes, the mycelial
morphology often undergoes dramatic alterations, typically accompanied by decreases
in culture activities (respiration and product synthesis) loo). The model for penicillin
formation proposed by Nestaas et al. ~ot. ~o2)was based on the model ofMegee et al. 99 ~.
Three differentiation states were considered rather than five, the three being: growing
hyphae, non-growing hyphae that produced penicillin, and non-growing, non-
producing hyphae. Penicillin production was assumed to be proportional to the
concentration of penicillin producing hyphae, penicillin hydrolysis was accounted
for, and the glucose uptake rate was assumed to equal its feed rate. The modified
version of the model L03,104) also allows for three stages of cellular differentiation
with the three cell types being: hyphae which may grow (elongate) or branch (Ao),
penicillin producing hyphae (A0, and degenerate hyphae (Az). Hyphal growth
results in the production of penicillin producing hyphae while branching produces
additional hyphae capable of growth or branching. The degenerated cell mass A2
is formed due to loss of cytoplasm and cytoplasmic contents from the penicillin
producing hyphae AI. The model is divided into two sets of equations, one set de-
scribing the growth phase and the other set describing the production phase. The
lag seen prior to the initiation of penicillin production and the appearance of peni-
cillin is described by postulating the formation of an intermediate as being therate
limiting step in penicillin production.
Cagney los~ proposed a model similar to the model proposed by Nestaas 103)
Unlike Nestaas' model however, this model considers that branching to form growing
cells results from the penicillin producing cells rather than the growing cells 99).
Furthermore, the entire process is modeled by a single set of equations. Finally,
unlike the Nestaas" model, the Cagney model includes the substrate accumulation
and dilution effects. The hyphal tips A 0 are produced from the branching penicillin
producing hyphae At. The penicillin producing cell mass At can be produced by
the growth of Ao or from the differentiation of Ao. The degenerate hyphae A2 can
only be produced from the degeneration of cell mass A~. Cagney lo5) modeled the
penicillin synthesis using substrate inhibition kinetics and accounted for hydrolysis
of penicillin in the culture medium. The equations that describe the Cagney model lo5)
are shown below
dV
- F (411
dt
dao valS klao Fao

- (421
dt K+S L+S V
dat glaoS valS klao k2at Fal

- - - + 1431
dt K+S K+S L+S L+ S V
da2 k2at Faz

(44)
dt L+S V
dP hatS FP
(45)
dt
i Kp + S +
dS 1 plaoS 1 nalS
dt Yx/s K + S YP/s Kp + S +
moaoS mlatS F
Kmo -t- S Kml + S + V (Sv - S) (46~
where ao, at, and a2 represent the concentrations of Ao, At and A2, respectively,
and k 1, k2, K, Kmo, Kmt, L, too, mr, gt and v are constant coefficients in the various
rate expressions. Since the total cell (mycelium) concentration is the sum of the con-
centrations of thre cell types, i.e., ao, al, and a2, the total cell balance is obtained
by addition of Eqs. (42)-(44)-as
dX IataoS FX (47)
dt - K + S V
It must be noted that only Ao and At have a maintenance requirement. The cell
mass A2, being nothing but cell walls, does not have maintenance requirement.
From Eq. (47), it is worth noting that the specific growth rate is a modified Monod
equation. The maintenance requirements of Ao and At are substrate concentration
dependent, avoiding the infeasible situation created by the Bajpai and Reug model -o.
v~I. Using the empirical feeding policies used by Heijnen et al. vv~ Cagney 105~obtained
penicillin yields that are comparable to those obtained by Heijnen et al. 77~. Con-
siderable variation in penicillin yields was observed for different feeding policies.
The structured model developed by Matsumura et al. 35, 36~"for ted-batch cultures
allows for three stages of cellular differentiation. Cultures of Cephalosporium acremo-
nium show three main morphological cell types during production; hyphae (Xh),
swollen hyphal fragments (X~) and arthrospores (X~)as, 36,106,107). Methionine acts
as a regulator for the production of cephalosporin C. Catabolite repression by glucose
has been observed. The cellular differentiation was assumed to progress irreversibly
according to the sequence
'Xh ~ X~ --' X~
Experimental data ~ov~ suggested that assimilation of both glucose and methionine
enhanced the differentiation of hyphae to swollen hyphal fragments, while the exhaus-
tion of glucose in the medium accelerated the formation of arthrospores from swollen
hyphal fragments. Model simulations showed that production may be enhanced by:
(1) the successive formation of swollen hyphal fragments, (2) the maintenance of
a high endogeneous methionine concentration, and (3) the minimization of catabolite
repression by glucose.
A series of structured models for aerobic, glucose-limited growth of Saccharomyces
cerevisiae in batch and continuous cultures have been constructed by Hall and co-
woikers lOS-lllj. These and other structured models for S.cererisiae have been
reviewed by Lievense and Lim 1121.These models do not however describe the dynamic
aspects of growth, except for the lags observed in batch cultures ~,2~. Lievense n31
has proposed a structured, non-segregated dynamic model for aerobic growth of
S. cerevisiae in a glucose-limited culture. Particular attention is paid in this model to :
(1) the catabolic (energy-generating) pathways involved in the utilization of glucose,
the production of ethanol, and the utilization of ethanol, (2) the genetic level controls
(i.e., induction and repression) of the synthesis of the enzymes involved in the various
catabolic pathways, (3) control of the activities of enzymes for catabolic pathways
and (4) the influence of macromolecular synthesis on the response of the system under
transient conditions. Growth occurs through a two-step assimilation (catabolism
and anabolism) of nutrients b y the biomass. The model recognizes that catabolism
and anabolism may become uncoupled during unbalanced growth. During balanced
growth, of course, the specific rates (based on biomass concentration) of catabolism
and anabolism are equal. The glucose consumption to yield the precursors and energy
is accounted for by two catabolic pathways. The glycolytic pathway carries out the
degradation of glucose to ethanol and carbon dioxide with a low yield of precursors
and energy. The enzymes required for this pathway are referred to as the oxido-
reductive enzyme pool. The respiratory pathway results in oxidation of glucose to car-
bon dioxide with a relatively high yield of precursors and energy. This pathway also in-
volves oxidation of ethanol to carbon dioxide with a high yield of precursors and ener-
gy. The enzymes required for this pathway are collectively called the respiratory en-
zyme pool. The Crabtree effect is accounted for in this model 1~3~by the induction of
the oxido-reductive enzyme pool and the repression of the respiratory enzyme pool at
high specific glucose consumption rates. The dissolved oxygen concentration is assum-
ed to be high enough so that no oxygen limitation occurs. The consumption of glucose

for maintenance, endogenous metabolism and loss of cell viability are assumed to be
negligible. The intracellular compounds represented by precursors and energy are
assumed to constitute a negligible fraction of the total biomass and these thereby
do not enter quantitatively into the model. The model is fundamentally limited by its
inability to account for the dynamic nature of growth at the level of individual cells.
Esener et al. 114~presented a two-compartment model for the growth of Klebsiella
pneumoniae and tested it with experimental data obtained from the led-batch and
continuous culture data. In this model, RNA, carbohydrate and other small cellular
molecules are lumped into one compartment, the K-compartment. The other compart-
ment, the G-compartment, contains the genetic material and all the rest of the cell
constituents, i.e., proteins, DNA, structural material, etc. This approach assumes
the RNA concentration and its synthesis.to be the bottleneck. The extracellular
limiting substrate is assumed to be converted to K, from which G is formed. A third
reaction involves the turnover of macromolecules, i.e., the G-material, back to the
K-material (small molecules). Although this model predicted the cell concentration
transients in a fed-batch culture accurately, it failed to yield an accurate description
of the RNA fraction since no consideration was given in the model to any regulatory
mechanism operating inside the biotic phase la4~. Thus, unless the internal structure
data are considered during the verification of structured models, these may remain as
fits and not mechanistic models9
3.3 On-Line Estimation of Bioreactor Parameters

It is important to be able to estimate parameters which are not easily measured. One
9 ~'. .
approach ~s to extract the mformat~on about such parameters from appropriate

models. Most of the kinetic models for growth and product formation are empirical
and theretbre account for a limited number of phenomena under ideal situations9
These models contain parameters which either change during the course of cultivation
or have been determined under conditions that are different from the expected operat-
ing conditions. Extension of models developed for steady state situations to dynamic
situations is often not valid9 Simulations done with detailed structured models are
usually expensive and time consuming. The large number of parameters in these
models gives rise to further uncertainties9 Many of the bioreactor parameters must
therefore be estimated without a kinetic model using limited measurements. Several
schemes for estimation of bioreactor parameters have been proposed thus far 20.21,
1~5-123~. The use of elemental and enthalpy balances has been a popular approach
in estimation of various bioreactor parameters that cannot be directly measured 20, 2i,
58. 116.120,121. 124,125). This approach is supplemented by information on cellular
metabolism and fermentation conditions provided by indirect measurements. Oxygen
uptake rate (OUR), carbon dioxide evolution rate (CER) and respiratory quotient
(RQ) are useful indicators of the cellular respiratory activities. Other indirect measure-
ments frequently employed include metabolic heat evolution rate 121t, specific growth
rate, substrate utilization rate, secondary metabolite production rate and nitrogen
uptake rate (NUR). Wang et al. 21, a251 and Cooney et al. 116~used the CER, OUR
and N U R measurements in elemental and material balance calculations for fed-batch
cultivations of baker's yeast to obtain estimates of the cell mass, ethanol concentra-
tion, and cell yield. Hennigan 5s, 124j used a similar approach for methylotroph (L3)
production using methanol as the carbon and energy source to estimate the substrate
uptake and cell mass production rates.
The basic feature of the elemental balance method is to represent the biological
conversion of nutrients to cell mass and a metabolite product by an overall chemical
reaction. A typical reaction involving a single carbon and energy source is written as
aC~H~Oy + bO 2 -r- c N H 3 --~ CaH~Or n + d H 2 0

(48)
+ eCO 2 q- fC~H,pO,N
It is assumed that the compositions of the carbon source, cell mass and metabolite
product (i.e., the coefficients ~., [3, 7, 6, a, (,, 1"1, % q~, and A) are known and remain
constant throughout the fermentation. There are six unknown stoichiometric co-
efficients, a through f, and four elemental balance equations, viz.
C: ~ a = 8 +e +of (49)
H: 13~ + 3 c = e + 2 d +q0f (50)
O:Ta +2b =~ +d +2e +qtf (51)
N: c = rl + Af (52)
An additional relation is obtained from the overall oxygen and carbon dioxide balances
after applying a quasi-steady state approximation. The quasi-steady state assumption
is valid due to the short time constant associated with the gas stream relative to the
system time constant. The oxygen and carbon dioxide concentrations in the inlet
and outlet gas streams can be measured with gas analyzers or a mass spectrometer
to determine O U R and CER. The total cell growth rate is expressed as
mOUq - out in
R,, = Yx/oz[mio~ - ozA = ~x/c%[mco 2 - m c % ]
or
Yx/o2(OUR) = Yx/coz(CER) (53)
mAin and mAout denote the inlet and outlet mass flow rates of component A. From the
relation (53), we obtain
CER Yx/% e
RQ - - - (54)
OUR Yx/co 2 b
The sixth and final relationship needed to solve for the six unknowns could be obtained
from the measurement of one of the following: nitrogen source, substrate, product and
fermentation heat. San and Stephanopoulos 141 point out that the direct substrate
and product measurements cannot be used to solve for the stoichiometric coefficients in
Eq. (48) if their time rates of change cannot be simultaneously monitored with a certain
degree of accuracy. The use of process heat together with an enthalpy balance has also
been considered as another possible independent relation 77,122. 126-128). A sensitivity
analysis 14~ has revealed that, due to the close relationship between the heat evolution
and oxygen utilization 126-128) Eqs. (49)-(52), (54) and the enthalpy balance equation
form a nearly singular set of linear equations in most instances. If ammonia uptake
rate (AUR) is measured, it can be related to the total cell growth rate as
R~ = Yx/Sn3(AUR ) 155)
From relations (53) and (55), we obtain
AUR Yx/o 2 c
g - - - (56)
OUR YX/NH3 b
For the case of baker's yeast cultivation, where ethanol (C2HsOH) is the metabolite
product, Wang et al. 21) found a linear relation (as expected) between the rate of
ammonia addition and the total cell growth rate, i.e., constant Yx.Yn3. For a reaction
represented by Eq. (48) (for A r 0), YX,'NH3 may not necessarily be constant. The
stoichiometric coefficients a to f are obtained by solving Eqs. (49)-(52), (54) and (56)
simultaneously
f = [{~(g 2r 3rl) - - ([3 - - 27) 6} g + [4a(1 - - RQ) -- RQ([3 - - 2u

[{~(3A + 2qt qo) + ~([3 - - 27)} g + {4~A(RQ -- 1) + ([3 - - 27) RQA}]
(57)
(8 + Qf)g + RQ(rl + fzM

a = (58)
~.g
q+fA
b - (59)
c=rl +fA (60)
d =ya +2(i-- RQ) b r qtf (61)
RQ(rl + fA)
e - (62)
On-line determination of the stoichiometric coefficients from the A U R , C E R

and O U R data allows determination of the various yield coefficients during the course
of fermentation. In some cases the stoichiometric coefficient of the metabolite product
can be correlated with another stoichiometric coefficient if only O U R and C E R are
estimated. For synthesis of single-cell proteins (SCP) via the methylotroph L3 grown
on methanol, Hennigan 58) related the stoichiometric coefficient for the metabolite
(poly-saccharide C6Hlo05) to the stoichiometric coefficient for carbon dioxide in
Eq. (48) as f = he where h is a constant. If the metabolite is an organic acid, the
additional relation may be obtained by writing a proton balance and measuring the
rate of ammonia addition for pH control. It is interesting to note that Eqs. (57)-(62)
developed for processes in which a metabolite is produced in addition to the cell mass,
reduce to Eqs. (16) of Rolf and Lim ~3)when f = 0 (i.e., when the only product is the
cell mass itselt). In addition to elemental balances for carbon, hydrogen, oxygen and
nitrogen, phospherous and sulfur balances must be considered when the nutrients,
biomass, precursors, acids/bases added for pH control and metabolite products
contain these elements v;I
Having determined the stoichiometric coefficients on-line from the data on CER,
OUR and AUR, the changes in the cell, substrate and product concentrations can be
determined from the mass balance equations (1) (4) which are rewritten as (F o = 0)
dV
- F, V ( 0 ) = Vo (63)
dt
d(XV) (CER)
V(O) X(O) = VoXo (64)
dt e
d(VS) a
-- FSF -- • GS~ V(0) S(0) = VoSo (65)
dt e ~
d(VP) f
- FPF + (CER), V(0) P(0) = VoPo (.66)
dt e
The inaccuracies of the measuring instruments give rise to measurements that may
be corrupted by noise which in conjunction with process noise may lead to model
parameters which inevitably include some errors. Therefore, one should take into
account how much the errors in estimation of bioreactor parameters affect optimal
operation and control of fed-batch cultivations. Moreover, the effect of uncertainties
in the initial conditions on the subsequent operation must be properly accounted
for, especially when an optimal start-up is attempted 129). The sensitivity analysis
carried out for an exponentially fed-batch culture by Kishimoto et al. ~291provides
useful information concerning how precisely the parameters should be set or measured.
The various noise filtering algorithms have been reviewed in considerable detail by
San and Stephanopoulos 14) and are beyond the scope of this review.
4 Optimization
The optimization ofbioprocesses is extremely important because these processes utilize
expensive raw materials, require large capital-intensive plants and yield products
that are low in concentrations. Every small improvement in any of these variables
may result in considerable reduction in the production costs. The objectives of bio-
process optimization are frequently maximization of volumetric productivity, product
concentration and conversion yield and minimization of capital and operating costs 4~j.
Processes such as the yeast production processes can be optimized to increase the
yield of biomass. In the case of antibiotic production, the formation of secondary

metabolites must be maximized.
Vital to the success of optimization methods is the development of mathematical
models that describe adequately the behavior of the system under a variety of condi-
tions and give quantitative relationships between the state variables and the control
variables of the system. The model parameters must be estimated directly or indirectly
from experimental data. The optimal control is aimed at achieving process optimiza-
tion. A model being a tool for control system design, any increase in the complexity
of the model is justified only if it results in a significant improvement of the system
performance. In the light of the complex nature of the microbial cultivations, improved
on-line data acquisition methods are of central importance to optimization of these
processes. On-line optimization studies have been very scarce.
Processes such as batch and semi-batch fermentations, which involve inputs that
are functions of time, are optimized using the path or function optimization techniques.
Function optimization based on the maximum principle of Pontryagin 130)is particu-
larly useful when the manipulated variables and/or state variables are constrained.
Function optimization of a two state variable system can be achieved via the Green's
theorem 131). Miele's technique ~31) obviates the two-point boundary value problem
resulting from the usage of Pontryagin's maximum principle 10~.Pontrygin's maximum
principle has been extensively used 58'60'61'7r for finding the optimal
time profiles for control variables such as the feed rate of the limiting substrate.
There have been a few reviews that include accounts of dynamic optimization of
fermentation processes 10.80.135). Experimental studies on dynamic optimization of
fed-batch reactors have included optimization of cellular productivity 5s, 1,_4.136) and
metabolite productivity 137) by controlling the addition of the limiting substrate and
optimization of metabolite productivity 29) by calculating the optimal trajectory of
the substrate concentration.
4.1 Formulation of a General Optimization Problem and its Solution
The optimization is aimed at maximizing an objective function which is normally

based on some combination of the anaount of cells grown, the production ofmetabolites
and operating costs. The problem is generally to find the feed rate F(t), and in some
cases the initial culture volume Vo, that maximize the objective function. Weigand xo)
points out that much of the time spent in calculations related with dynamic optimiza-
tion will be wasted if care is not exercised in the formulation of proper objective func-
tions. For a given flow rate F(t), the conservation equations for the limiting substrate,
biomass, and metabolite product (e.g., Eqs. (14)-(16)) are solved in conjunction with
Eq. (1) (F 0 = 0) to estimate the objective function. In addition to the initial conditions
(10)-(13), these equations are subject to the following contraints
0 < F __< Fm (67)
and
0 <Vo_--<V_--<Vm (68)
where Fm and Vm refer to the m a x i m u m allowable feed rate and maximum permissible
bioreactor volume, respectively. The objective function (index of performance) is
generally of the form
1 f {Pc(V'X) + Pm(V'P)} dt - P~ ~ FS F dt - O P (69)

IP = Tf o
where Tr is the cycle time and Pc, Pm and P~ are unit prices associated with the cell
mass, metabolite product and limiting substrate, respectively. The first integral in
Eq. (69) represents the product value, while the second integral represents the substrate
cost. OP represents the operating costs. The superscript 9 is used to denote differentia-
tion with respect to time. Maximization of the index of performance may be achieved
through (a) maximization of the cell production and/or (b) maximization of the
metabolite production and/or (c) minimization of the substrate cost. In studies dealing
Table 2. Various objective functions used for optimization of fed-batch bioreactors
Product(s) Objective Objective function Refs.
(A) Cell mass Maximize [P V(Tf) X(Tf) -- V(0) X(0)

(1) maximum IP = sT}
production rate Tf
X(Tf), Tf not fixed
(2) maxinmm IP = V(Tf) X(Tf) V(0)X(0) t3s)
productivity Tt fixed
(3) time-optimal IP = --Yf 58-61,65,
V(Tf), X(Tt) , V(0), X(0) fixed I39)
(B) Metabolite Maximize IP

74.87, 132,
(1) maximum V(Tr) P(Tf) V(O)P(O) 14o~
production rate IP =
Tf
Tr P(Tr not fixed
(2) maximum
productivity IP = V(Tr) PITf) V(0)P(0) 27,29. 135~
T r fixed 14o.141)
(3) time-optimal IP = --Tf 140)
V(T0, P(T0, V(0), P(0) fixed
(C) Cell mass Maximizize IP 1

and metabolite (1) maximum IP = T [Pc{V(Tf) X(Tf) V(0)X(0)}
profit rate
+ Pm{V(Tr) P(T0 -- V(0) P{0)}]
Tf, X(Tf), P(Tr) not fixed
134)
(2) maximum IP = Pc{V(Tf) X(Tf)-- V(0) X(0)}
profit +Pm(V(T0 P(Fr) V(0)P(0)}
Tf fixed
(3) time-optimal IP = Tf
V(T0, X(Tf), P(Tt.), V(0), X(0).
P(0) fixed
with optimization of fed-batch bioreactors, various special forms of the general

objective function defined in Eq. (69) have been considered and these are listed in
Table 2.
The dynamic optimization problems can be solved by employing the optimal
control theory 1r The objective is to maximize the index of performance of the type
Tf
IP = Ll(x(Tf), Tf) + f Lz(Xlt), u(t), t) dt t701
0
where L1 and L 2 are some specified objective functions, x is the vector of state variables
and u is the vector of control variables. The system dynamics is expressed as
dx _ [(x, u, t) x(0) = ~ (71)

dt " '
The solution to the optimization problem proceeds through formulation of a Hamil-

tonian H composed of the Lagrangian L2 and a vector of adjoint variables ?z
H = L 2 q- ~ t f (72)
where )T is the transpose of vector k. The variation of ?v with time is expressed in

terms of the Euler-Lagrange equations as
dZ T = _ ~ , ~ H ~ ( T f ) = (~LI~ "r (73)

dt ox \ 5x Jt =Tf
where
5x ~x2
The necessary condition for an optimal solution is
5H
-- = 0 (74)
~u
Eqs. (70)-(74) form a two-point boundary value problem, the solution of which is
generally obtained numerically. Various boundary value iteration methods or control
vector iteration methods exist and must be used with caution in searching for the
optimal trajectory. The direction in which the solution moves is decided by the gra-
dient of the Hamiltonian. Rather frequently, however, flat plateaus of the Hamiltonian
exist resulting in insensitive profiles that may be incorrectly mistaken for the optimal
solution. If the final time Tf is not known, it may be included in the set of variables
to be optimized s8-61,139. 140).
4.2 Optimization of Fed-Batch Bioreactors Used for Biomass Production
In some situations analytical solution of Eqs. (70)-(74) is possible. To illustrate

this, we consider fed-batch cultures in which biomass is the major product. If the
initial and final values of the cell concentration and culture volume are known, the
production rate (see case A1 of Table 2) will be maximized by minimizing the operation
time Tf, i.e., by employing a time-optimal (case A3 of Table 2) policy. The index of
performance to be maximized in this case is
~f
IP = ( - 1) dt (751)
0
Eq. (75) is subject to the system dynamics described by Eq. (71) with x, u and f being
defined as
x = p c v s] T , x(0) = p c ( 0 ) s ( 0 ) v ( 0 ) ] 9
u=F
f FxI
and
[fil .net,S>X
-f = f~ = (S v - S)-cy"et(S) V (76)
f
F
Comparison of Eqs. (70) and (75) reveals that L~ = 0 and L 2 = 1 in the present
case. The Hamiltonian in this case is expressed as 6o)
H ~- ~ l -}- (P2F (77)
with
% = gn~ XX, ~"e'(s) X),_, -- 1 (78)
and
X (SF
q~2 = Vkl + S ) L 2 ~- X 3 (79)
V
The adjoint variables satisfy conditions (73), i.e.,
d>,l
d~- -
[ ~tL'~(S)- V ),t + o"~'(S) t'.2, ),l(Tr) = 0 (80)
dt (g.eL) X~'l q- (~net), X q- ~'2, ~.2(Tf) = 0 (81)

'x
d~ 3 V
dt - V 2 [ - X?,~ + (Sv - S) L2], ~,3(Tf)= 0 (82)
The primes denote differentiation with respect to S. By transversality, the Hamiltonian

vanishes identically to zero on the optimal path. Maximization of H by F(t) yields
an optimal feeding policy of the form
F m, q32(tl> 0
F(t) =
I F~,
~.0,
q32(t) = 0,
q32(t) < 0
tI < t < t 2 (83)
In the finite time interval (t~, t2), referred to as the singular control period, an inter-
mediate and variable flow rate F~ is used. The feeding policy described by Eq. (83) is
commonly referred to as bang-singular-bang policy if the policy consists of the max-
imum flow rate, the singular control and finally a minimum flow rate. The number of
state variables in this case is three and the Hamiltonian H is a linear function of the
control variable F. For' the linearized version of this system the optimal control
theory predicts that there will be at the most two switching points (t~ and t2 in Eq. (83)).
F~ is determined by taking advantage of the fact that q~2 and all its time derivatives
must vanish during the singular control period. This yields the following relations
X ( S v - S)
q~E(t) = - V ?~1 + ~ k~2 + ~3 -- 0 (84)
dq~z(t ) _ X (Sv _ S)[(cy"e')' ~-2 - 0-l"et)' ~-1] = 0 (85)

dt V
d-q32~ --
X (S F - S) [ {(o'net)' ? ~ , - (netj, ~'1} ~dS-
dt 2 V
(861
Substitution of Eqs. (15) (with Sg = 0) and (85) into Eq. (86) and further algebraic
manipulation leads to the following expression for the feed rate during the singular
control period
g [ (~net I' {~net(o-net)r -- (~net I' o'netl, ~
F, .
(S F Z S) (~ne'X
. . . . . . net~ --,et
{(o'ne') " [unet) ' -- ((5") (~.1 },,~, J (87)
The primes denote differentiation with respect to the substrate concentration S.

The necessary condition for existence of a singular control period can be shown to
be 142~
>-- 0 (88)
This requires that
(~lnet), (o-net),,__ (~net},, (o.net),

io-ne,)' ~t.et __ (lane~), o-net > 0 (89)
The existence of singular control period is thus dependent upon the kinetic model
under consideration. The overall optimal feeding policy therefore is 58 601
~p2(t) > O, 0 < t < t~

/ Fm~ Fs >__Fm
F(t) = ~= F~, Fs < Fm q~2(t) = O, t~ < t < t 2 (90)
I!m O, F~ < 0
(P2(t) < 0 , te < t < Tf
Eq. (90) has been obtained from Eq. (83) acknowledging the fact that the feed rate dur-
ing the singular control period is subject to constraint (67). The general feeding policy
therefore consists of three stages; the initial fillling stage (0 < t < h) in which the
feed rate is maintained at the maximum permissible value, Fm, followed by the second
stage (h < t < tf) in which the feed rate is varied and finally followed by the third
stage (t r < t < Tf), a batch operation where the cells are allowed to grow to the final
desired cell concentration. For certain initial conditions it is possible to have no initial
bang period, instead the optimal policy may begin with the singular control.
4.2.1 Solution for Constant Biomass Yield
dj.lnet
For the special case of constant biomass yield Yx/s, condition (85) reduces to
dS
-0 (91)
The substrate concentration therefore should be held constant at S = Sc (the value

at which the specific growth rate is maximum) during the singular control period and
the feed rate is obtained from Eq. (15) as
~~
F~ SF -- S' tl < t < t, (92)
Condition (91) is a necessary condition for the existence of singular control period.
For M o n o d kinetics, there is no singular control interval since the growth rate is
a monotonically increasing function of the substrate concentration. In this case
tl = t 2 = tf and the optimal feed policy is of "'bang-bang'" type
F(t) = ~ Fro" 0 < t < t1

Vm -- V 0 (93)
0, t 1 < t < T e, tl m
Fm
When the microbial growth is inhibited by the substrate or when there are catabolite
repression effects, the specific growth rate exhibits a maximum. The total amount
of cell mass at any time during the singular control period is obtained in this case by
solving Eq. (2) (F 0 = 0) as
X(t) V(t) = X(h) V(tO exp {gn"'(S~) (t -- h)} , t, < t < t2 (94)
We observe that exponential growth occurs in the singular control period. Substituting
Eq. (94) into Eq. (92), it is seen that in the singular control interval, the bioreactor
is operated in exponentially-fed batch mode
Fs = F(h) exp {la"e'(Sr (t -- h)} 9
[ { In (Vm/F(tt))~
t 1 -< t -< min tf, tI + ~ )j (95)
In this case, the bioreactor should be fed at the m a x i m u m permissible flow rate until
the substrate concentration reaches the value at which the specific growth rate is
maximum. After this time (switching time h), the feed rate is varied exponentially
according to Eq. (95) and this operation is continued until the reactor is full (V = Vm)
provided that the feed rate never exceeds the allowable maximum, Fm.I f F s reaches Fm
during the singular control interval, then the flow rate is set at Fm and the reactor is
fed at this rate until its volume reaches the m a x i m u m volume, V m. The reactor is then
operated in batch mode until the specified cell concentration Xf is realized (t = Tr)-
If the initial cell and substrate concentrations, Xo and So respectively, lie on the
hyperplane
X
Sv S - 0 (96)
Yx/s
then it can be shown that all the subsequent cell and substrate concentration transients
lie on this hyperplane provided !un~ = g a n d ~ "e' = c~. For this case, not only the sub-
strate concentration but also the cell concentration remain constant at S~ and Xc
respectively during the singular control period 6o~
In (Fm/F(t 1)) ~ (97)

X(t) = X ( q ) , tl < t < min tf, t~ + B(Sr J
For initial substrate and biomass concentrations satisfying the stoichiometric

relation (96), the dilution rate in the singular control period (h, t2) is seen to be constant
in view of Eqs. (94), (95) and (97).
The implementation of the optimal feed policy, given by Eqs. (90)-(92), (94) and
(95) requires a continuous monitoring of the state and control variables. In this sense,
the optimal feed policy described here is a kind of feedback policy. However. in prac-
tice, measurement of substrate concentration is difficult. In such instance, an open-
loop policy may be used with the state equations being integrated by a computer
with the known initial conditions Xo, So and V 0 so that the switching time tl. the
exponential feed rate (Eq. (95)). t2, tf and Tf can be generated. The optimal feeding
policy' formulated earlier (Eqs. (90)-(92), (94) and (95)) is dependent on the values
of S v, So, Xo and X t.
This optimal feeding policy has been applied to repeated led-batch bioreactors ss -61..
In a repeated led-batch bioreactor with reproducible cycles, Xo and Xr and So and
St must satis~' conditions (27). The average biomass production rate for a repeated
led-batch bioreactor therefore is expressed as
p - Xo(V~, -- Vo)
Tr (98)
Further, when t,tnet = g and crnet = ~, the biomass and substrate concentrations lie on
the hyperplane defined by Eq. (96) ss-6n. As the maximum permissible teed rate F m
increases, the filling time t~ and the total cycle time Tf decrease. The biomass produc-
tivity P is enh~mced by larger values of F m and feed substrate concentration Sv.
Weigafid et al. s9-6~)considered the case of instantaneous fill [F m = oo]. In practice,
this may happen if the maximum permissible feed rate is sufficiently large in com-
parison to the maximum growth rate so that essentially no growth occurs during the
filling period 6o). In the case of repeated-fed batch bioreactors, an additional opera-
tional parameter besides the feed rate of the limiting substrate is the initial culture
volume Vo.
4O
SF = 10 g [-1
35
30
25
k
~15
ca_ 10 ~Subop'dmo{
Fig. 1. Comparison of optimal and suboptimal feed
policies for constant biomass yield 6% Yx,~= 0.45,
5~ Vm = 101
S
I I 0.03 + S + 0.5S2
0 0.2 O. Z, 016 0.~8 1.0
Vo/V,. - - - - "
The maximum biomass productivity varies with the initial culture volume Vo as
demonstrated in Fig. 1. As Vo/V m becomes negligible, the cycle time increases without
bound and the time average productivity drops to zero. While operation with smaller
V0/Vm may appear to be an intuitive mode of operation since more fermentor volume
is available for utilization, the cell density is much lower after the rapid fill when
Vo/Vm is small thereby resulting in a lower cell production. With higher Vo/Vm more
cells are retained and therefore the cell concentrations after instantaneous fill are
higher so that the cell production rate per unit volume is higher. However, the bio-
reactor volume available for operation is less. Therefore, the time-average productivity
exhibits a maximum. The performance of the suboptimal feeding policy described
by Eq. (93) is compared with that of the optimal feeding policy in Fig. 1. The initial
volumes that result in maximum productivity for the two policies are close. At all
lower initial volumes, the suboptimal policy is considerably inferior to the optimal
policy.
4.2.2 Solution for Non-Constant Biomass Yield

The feed rate of the limiting substrate during the singular control period is in this
"case a complex exponential function that is obtained by substituting Eq. (87) into
Eq. (l) with Fo being zero
F~(t) = V(t0 G(tJ exp (,! G(r) dx) (99)
where
I (Hnet] ' ,fn,net / net]t /-nnnet~, net)7
G(t)--- r~"r (100)
(sF s) {(~"~176 - (~"~ (~"~ } d
The primes are used to indicate differentiation with respect to S. It can be seen that
the substrate concentration in this case varies during the singular control period.
Thus, unlike in the case of constant biomass yield in which the switching time tl was
predetermined by the fact that at t = tl, S = So; in this case no such information is
available and hence an iterative technique must be used to solve for the optimum tl.
One procedure is to integrate Eqs. (71) and (78) with F = F m starting from the known
X o and assumed So and Vo up to switching time tag. At this time, the singular control
(Fs defined in Eqs. (99) and (100) provided 0 < F~ < Fm) is implemented. If F, > F m
at t l g , F is set equal t o F m and there is no singular control. Similarly, ifF~ < 0 at t l g ,
F is set equal to zero and there is no singular control. IfF~ < F m at t = t x g , then the
singular control is implemented and integration of Eqs. (71) and (78) is continued as
long as F~ < Fn, and V < V m. The time at which F~ = F m (while V < Vm) is denoted
as t2, F is set equal t o F m after this time and integration is continued until V = V m.
The corresponding time is denoted as t r. The bioreactor is operated in a batch mode
after this time until the final biomass concentration is reached at time Tf. A new guess
value of h, hg, is selected and the entire procedure is repeated to evaluate a new final
time T~. The optimum value of tl is the one that minimizes Te.
For a repeated-fed batch bioreactor with reproducible cycles, Xo and Xr and So
and Sf must satisfy conditions (27). In general, the above procedure does not yield
the final substrate concentration, St, that agrees with the assumed initial value, So.
Therefore, we must iterate on So until it agrees with Sf. This procedure is to be repeated
for various values of tl until the best tl that results in minimum cycle time Tf is found.
Since this optimum value of h is for an arbitrary initial culture volume Vo, the entire
Modeling. Optimization and Control of Senti-Batch Bioreactors 235
procedure has to be repeated for different culture volumes Vo until the one that
gives the highest cell productivity is found. An extensive numerical effort is required
to obtain the optimum initial culture volume V o and the optimal teed policy for a
repeated fed-batch fermentation. An algorithm for this has been developed by Hen-
nigan _~8). The harvesting stage in each cycle is essentially ignored by assuming that
it is carried out instantaneously ss~. The optimal feed policy was implemented in the
operation of a repeated-fed batch culture of a methanol-utilizing bacterium L3
which is utilized for production of single-cell proteins. The growth kinetics of L3
determined by DiBiasio ~43) were used in the on-line optimization 58~
2.20 ------ SF=6g Iq ~ SF=30g L-1

k
92-- \
o.os / j-
o o, o o ,o
V0/V~
Fig. 2. Effect of Vo/Vm on biomass productivity for optimal and bang-bang feeding policies ss,
Vm = 4.5 1, So = 0.00l g 1-1
0.504S[1 - 0.0204SI
h-t
It = (0.00849 + S + 0.0406S2)
0.383(1 0.0204S)
Y'*" (1 t 0.296S - 0.0050IS2) {g cell} ~ot~substrate} -t
P
Normalized productivity = Vm(SF So) h -I
The effect of the initial culture volume V 0 on the biomass productivity for optimal
feeding policy is illustrated in Fig. 2 for the growth of methylotroph L3 ssI. The
results /'or the optimal feeding policy are compared with those for the suboptimal
bang-bang policy described by Eq. (93). The productivity P undergoes a maximum
as Vo/V m is changed. Alter undergoing the maximum, the time-average productivity
decreases and approaches the productivity of a constantly stirred tank bioreactor
in the limiting case of V o = V m. As seen earlier for the case of constant biomass
yield, the initial volumes that result in maximum productivity lbr the two policies are
close. At all lower initial volumes the suboptimal policy is considerably inferior to
the optimal policy; especially at high substrate feed concentrations. The qualitative
comparison between the results shown in Figs. 1 and 2 is remarkably good.
236 S.J. Parulekar and H. C. Lirn
The relationship between the average production rate P and V0/V m depicted in
Figs. 1 and 2 has also been predicted by Mori et al. 138)for repeated fed-batch cultures
of Candida brassicae grown on ethanol. The times necessary for (a) preparation for
cultivation such as cleaning and sterilization of the bioreactor, (b) filling up the initial
culture medium into the bioreactor, and (c) harvesting the cell mass were incorporated
in the definition of the time-average productivity 138)
Yamane et al. 139) considered the time-optimal fed-batch culture operation as a
means of start-up for a continuous culture. The optimal start-up policies are batch-
operation for cultures with specific growth rates that increase monotonically with
substrate concentration and exponential fed-batch operation followed by batch
operation for cultures with specific growth rates that exhibit maximum as a function
of substrate concentration. Similar conclusions were reached by Dunn et al. 14~ using
graphic analysis.
4.3 Optimization of Fed-Batch Bioreactors Used for Metabolite Production

The dynamic optimization problems arising in the case of metabolite production can
be analyzed within the framework of singular control theory using an approach very
similar to the one followed earlier in Sect. 4.2. The system dynamics in this case is
described by Eq. (71) with x, u, and [ being defined as
x = [X S P V]T , x(O) = [Xo So Po Vo] T
u = F(t) (I01)
and
f=
If1]
f2
f3
f4
=
F
un~
i-- ,~
~(s~ - s ) - ~ " ~
F
]
X
\~ ( P F - P) -t- /znetx
FX
-- - -
V
-
GSg
v
One of the earliest works in this direction has been that of Fishman and Biryukov 74).
The process under consideration, a penicillin fed-batch process was modeled with four
state equations, i.e., balances for biomass, substrate, inhibiting metabolite and product
(antibiotic). Change in the culture volume was neglected in the analysis. The specific
production rate of the antibiotic product was fit as a quadratic polynomial of the
mean culture age which was determined experimentally. In addition to the constraint
on the total glucose supplied, the feed rate was constrained as shown in Eq. (67).
The index of performance to be maximized was the antibiotic productivity. The
optimal feeding policy obtained via the maximum principle was shown to consist of
a rapid fill followed by batch mode to allow for optimum growth and ageing of the
cells so as to attain maximum antibiotic production rate. The batch mode is followed
by a singular control period to maintain the optimal antibiotic production rate until
the integral constraint on total glucose addition is reached after which the bioreactor
is operated in batch mode until the antibiotic production reaches its maximum.
Through various manipulations, Y inane et al. 1,~01were able to reduce the optimi-
zation problem for the fed-batch reactor whose dynamics are described by Eq. (101)
(with Sg = 0) to a problem which requires solution of only cell and product balance
equations. The idea was to eliminate the singular control problem by selecting as a
control variable one which does not appear linearly in the Hamiltonian. This manipula-
tion results in removal of feed rate as the control variable from the remaining state
equations and cause control of the state variables to occur through manipulation of
the trajectory of the specific cell growth rate. The optimal specific growth rate trajectory
then leads to the corresponding optimal teed rate through the relationship developed
during the process of reduction of the state equations. Despite its convenience and
simplicity, this approach however has certain severe limitations 10.96~ Since no
physical limitation was placed on the specific growth rate, the feed rate required to
achieve it is often physically unrealizable and a physically relizable suboptimal feed
rate must be substituted.
Optimization of fed-batch penicillin fermentations was also attempted by Stutts 96~
using/he models due to Bajpai and Reuss v0, 7~ Cagney ~osj and Heijnen et al. vT~
Conjugate gradient technique was used for obtaining the optimal feeding policies.
Since the specific penicillin production rate in the case of Bajpai and Reuss model 70, ,tl
and Cagney model ~051depends solely on the concentration of the limiting substrate,
Stutts 96) was able to show that the optimal feeding policies based on these models
are similar to the feeding policies for led-batch reactors used for biomass production.
Ohno et al. a7) used a method developed by Miele 13~1based on the use of Green's
theorem for maximization of product formation by manipulating the feed rate of
the limiting substrate. Although in principle the system dynamics will be described
by Eq. (101), the system dimension must be reduced from four to two if the Green's
theorem is to be used. In addition, the final states for both the state variables must
be specified. The method of Miele transforms the index of performance (see case B 1
of Table 2) to be optimized to a line integral and then the Green's theorem is used
to transform the line integral into a surface integral. The path which minimizes the
surface integral can then be found by examining the state space plot of possible
paths for the two-dimensional system. The state variables chosen in the analysis
were substrate concentration and culture volume. The optimal feeding policy was
applied to lysine fermentation by an auxotrophic mutant 881. For several values of
Vo/Vm, the operation patterns tha{ result in optimum index of performance were
classified. These are (1) bang (F = F r o ) - - bang (F = 0), (2) bang (F = Fro) -- singular
(F = Fs) bang (F = 0) and (3) bang (F = Fro) bang (F = 0) -- singular (F = F~)
- -bang (F = 0). The first two patterns are similar to those obtained earlier by Hen-
nigan 58~ and Weigand et al. s9-611, while the third pattern is similar to that obtained
by Fishman and Biryukov 7,a~.
In a subsequent study, Ohno et al. ~4~) have considered the optimization problem
for a general semi-batch reactor (F 4: 0, F 0 r 0) where the product once again is a
metabolite. The objective function to be optimized was the metabolite productivity
tbr a fixed operating time (case B2 of Table 2). Optimal solutions for a system whose
dynamics are expressed by Eqs. (1)-(4) were generated using a transformation due to
Kelley 145~. This transformation reduces the dimensionality of the state equations to
two, and in the process the singular control problem which is common for path optimi-
zation of bioreactors is eliminated so that the maximum principle can be more readily
applied. Of course, this approach may lead to a potential difficulty in realization as
mentioned earlier. The results generated by application of the optimal feeding policy
to lysine fermentation indicate that the mode of operation is dependent upon both
the ratio of the initial culture volume and final culture volume, V0/Vm; and the operat-
ing time T r. For long operating times, continuous operation is preferrable while for
large bioreactors, the fed-batch operation is recommended.
If the biomass that is simultaneously produced with metabolite products is market-
able, then it is justifiable to reclaim it and include it with metabolite production in the
index of performance. Choi and Park 134) analyzed optimization problems for such
processes using the index of performance defined for case C2 in Table 2. Choi and
Park 134) have followed the approach of Yamane et al. 140j in their work. Yamane
et al. 140} formulated the optimal policy as consisting of maintenance of the specific
growth rate at its maximum until a switching p o i n t a n d thereafter operation of the
bioreactor at a monotonically decreasing specific growth rate. The results of Choi
and Park 134) reveal that with increase in the relative price of metabolite (increased
Pro/Pc), the switching point moves toward smaller biomass concentration values and
the feed rate must be adjusted to obtain a lower specific growth rate. This is expected
since a higher price of metabolie would dictate lower cell growth. In the limiting case
of Pm = 0 (cell biomass is the only product), the recommended feed policy would be
exponential fed-batch with the substrate, concentration being kept constant at the
value which results in maximum specific growth rate. In this case, the switching point
coincides with the final time Tf. In the other limiting case of Pc = 0 (metabolite is
the only marketable product), there is no initial period of operation at maximum
specific growth rate.
Although formulation of the optimal control problem in terms of the maximum
principle is relatively easy, its solution cannot always be determined analytically and
numerical techniques must then be used for solution. Stutts 96) has reviewed the various
numerical search methods used for this purpose. Software packages for the solution
of singular as well as non-singular optimal control problems associated with fed-batch
processes have recently been developed 96.1461
In many cases of non-growth associated metabolite production, it is difficult to
construct deterministic models which cover the entire time courses of fed-batch
cultivations. In such cases, the optimization must be carried out on-line using a multi-
variable optimum seeking technique. Based on the observed responses, the next
control variable increments are chosen and this process is repeated until no further
improvement in the index of performance can be achieved. Kishimoto et al. 27,29)
employed regression analysis for estimation of the specific rate parameters in the
system equations and used dynamic programming to maximize the production of
glutamic acid in a fed-batch culture of Brevibacterium divaricatum with ethanol being
the limiting substrate. For a reliable optimization scheme, it is necessary to collect
experimental data which cover as wide ranges of operating conditions as possible.
The ethanol concentration in the culture broth was controlled in an on-off fashion
by comparison of the ethanol concentration on the optimal trajectory Sopt and that
in the culture broth (,S) measured by the porous Teflon-tubing method of Dairaku
and Yamane ~47~
When the optimal feeding policy is implemented experimentally, very.frequently
the control is open-loop, feed-forward control. Disturbances to the system and uncer-
tainties in the model and its parameters are likely to cause the actual transients to
deviate away from those predicted by the optimal policy. Therefore, the state of the
bioreactor must be monitored continuously and measurements of the key variables
must be used as the feedback information. Many of the recent accounts ~6,5~, 119,
125.14s-151) have indicated the use of empirical, suboptimal feeding policies in which
regulatory feedback control using the flow rate of the carbon source is utilized.
5 Control
Since the biological systems are characterized by a number of state variables and
process inputs that are highly interactive, any effective control policy must account
for these interactions. The complexity of accomplishing this task necessitates the
use of an on-line computer. The decrease in cost and improved reliability of computer
hardware in recent years have made the use of computers even more attractive. The
advent of microprocessors has had an enormous impact on the computer control.
Microcomputers can provide a relatively inexpensive and reliable means to integrate
computer control into the processes.
The control systems for bioreactors have been limited to the manipulation of
variables in the environment external to the microorganism. The term "environmental
control" is therefore often used in this context. The dominant control system of course
is the metabolic control mechanism inside the microorganism. Therefore, the goal
here is to develop a control system which maintains the environmental conditions in
a manner which drives the microbial metabolic control system to produce the desired
product in an optimal fashion. The lack of clarity in the relationships among the
system variables and the difficulties in state estimation make the design of an effective
control system an enormously interesting and challenging problem.
Empirical control methods have a variety of limitations 801. The control strategy
is often based upon a subjective treatment about the system states that yield optimal
performance. Empirical procedures are usually specific for a particular process and
cannot be readily extended to other processes. Control systems based on the optimal
control theory are not limited by the above mentioned factors. Established methods
are available to control multiple process inputs in an optimal sense with respect to
an objective function of integral square error. However, the resulting system depends
upon the objective function (integral square error) and the specification of quadratic
error criteria is difficult. Hence, in practice it becomes iterative. The quadratic error
criteria are picked, the system optimized and the responses are observed, and then
iterated on different parameters in the error criteria until the resulting responses are
satisfactory. The control policy is made specific for the desired process by the choice
of the kinetic model or the parameter values in a generalized model. In order to
implement optimal process control, it is necessary to monitor pertinent parameters.
Through such continuous monitoring it is possible to evaluate the effect of operating
variables on process performance and to select the desired set points for an optimal
240 S.J. Parulekar and H. C. kim
control strategy. Continuous assessment of the state of the fermentation is necessary

to anticipate the control actions. In the past, substrate concentration, biomass con-
centration, dissolved oxygen, RQ, dissolved carbon dioxide, concentration of carbon
dioxide in the effluent gas and other variables have been controlled at constant
values, in combination or alone, with the aid of computers. These variables in turn
have been used in some cases as feedback parameters for control of fed-batch bio-
reactors.
5.1 Classification and Characterization of Control Schemes
Computer control schemes for fed-batch bioreactors can be broadly classified into
three categories ~4~.The schemes belonging to the first category involve separate con-
trols for different variables with totally independent loops. In such control schemes,
which are at present the most widely used schemes, conventional on-off or PID con-
trollers are employed to regulate individual variables. In the second type of schemes,
the interactive effects of the manipulated variables on the controlled variables are
taken into account. The simultaneous computer control of biomass and substrate
concentrations effected through manipulation of substrate feed concentration and
dilution rate respectively constitutes one example of these schemes 56, x52). The third
category comprises of control schemes in which the interactions between different
controlled variables are also considered in addition to the interactive effects of the
manipulated variables on the controlled variables. Typically, many levels of cascaded
loops are contained in the nmch more sophisticated programmed controller. The
intracellular enviromnent and metabolic activities are directly measured or indirectly
estimated in the outer loop. They are then compared to the desired set points stored
in a computer and the outputs from the computer (master controller) serve as set
points for the inner loop. As was pointed out earlier, the primary objective of such
cascaded control configuration is to influence the intracellular environment through
manipulations in the extracellular environmental variables. An example of such
schemes is suppression of undesirable secondary metabolites by monitoring gas
exchange conditions, which act as indicators for the intracellular metabolic activities ;
the set points being generated for biomass, substrate and/or dissolved oxygen concen-
trations and the controls being attained by varying nutrient feed rate, aeration rate,
agitation speed, etc.
Control loops in a computer control scheme can be divided into low-level contro!
loops and high-level control loops ~3.85,s6). The low-level control loops are those
which feed back directly upon one of the basic process measurements ~s31. Most low-
level control loops can be handled by simple controller techniques such as PID and
on-off control. Examples of variables that can be controlled in this fashion include
agitation speed, liquid volume and air flow rate. To prevent oscillations that may
occur with two-sided on-off control, the supervisory program that decides the set
points for these controllers must decide how the control action must be exercised.
For example, pH may be controlled with addition of base using a base pump when
the set-point is positive while a negative set-point would cause the pH control by acid
addition. Similarly, in the case of temperature control, a positive set-point may be
used for controlling the temperature with a heater while a negative set-point would
Mode ing, Optimization and Control of Semi-Batch Bioreactors 241
result in control with cooling water. These on-off controllers have been found to
provide satisfactory control 5s. 15~. The major decision in setting the configuration
of the low-level control loops is whether to use set-point control (SPC) or direct digital
control (DDC). Economics frequently favors the use of DDC when the number of
low-level control loops is large 11,153, 1551. Some of the advantages of direct digital
control are tremendous versatility, decreased hardware requirements, improved
response, easy measurement of manipulated variables and automatic documentation
of control actions t3~: which explain the wide use of direct digital control for bio-
processes 2 9 , 8 6 . 1 5 6 - 1 5 8 )
Cascaded digital control (high-level control) allows the control of variables that
are not associated with a particular control element or biologically significant para-
meters that are calculated from basic measurements. It is possible to use any variable
or set of variables to feed back on any other variable or set of variables with the aid
of a computer. Because of this, the structure of the cascade control strategies can be
changed to meet the requirements for a particular application of the computerized
system s% Cascade control is commonly used for control of cell density (OD) measure-
ment upon the inlet substrate feed rate. The DO controller drives either or both the
air flow and agitator speed controllers using a cascade control scheme 159) which
greatly speeds up the response and overall quality of DO control s6). Nyiri et al. 1601
have also used both air flow and agitation speed to control the dissolved oxygen con-
centration.
The set-points for the cascade controllers and low-level controllers are determined
by a supervisory program. The micro-minicomputer hierarchial system described
by Hennigan et al. 58,851 and Rolf et al. ~3, 86,1541 can accommodate many computer-
coupled bioreactors (operated for different processes) at the same time. Incorporation
of microcomputer gives more versatility, aids in generation of an intelligent real time
interface and provides a convenient structure for control. Routine work being done
by the microcomputer, the minicomputer performs more efficiently tasks such as
supervisory control, advanced control, implementation of optimization strategies
and data analysis. The supervisory program, which is stored in the minicomputer is
used to define the bioreactor mode and objectives of the experiment. Advanced
control strategies such as on-line optimization and adaptive control are generally
handled within the supervisory program. The micro-minicomputer hierarchial system
has been sucessfully used for control of fed-batch culture of methylotroph L 3 ss~
and penicillin ,6a) and for control of continuous culture of baker's yeast ,s4)
and will be employed for control of fed-batch cultures of baker's yeast in the near
future.
The closed loop control of the manipulated variables is accomplished by combina-
tions of feedback and feedforward control. For control of most manipulated variables,
feedback control is generally desirable to maintain them at the desired values. Although
the computer may be used for direct control of various input variables, it is frequently
more desirable to use the computer in a supervisory mode with local analog controllers.
This minimizes the risk of process upset by computer hardware or software failure
and permits bioreactor operation without computer control when such failure
occurs ~2, 1~). As pointed out earlier, the use of set-point control however, increases
cost of the control system. Feedforward control is particularly useful when un-
controllable loads are placed on the system.
242 S.J. Parulekar and H. C. kiln
5.2 Instrumentation for Monitoring the State of Fed-Batch Bioreactors
Because of the generally poor understanding of biological processes, feedback infor-

mation must be utilized to obtain a satisfactory operation. Open-loop control schems
that do not utilize any feedback of information are subject to the effect of numerous
uncertainties and disturbances which eventually will lead to instabilities and economic
losses. An example of such situations is the exponential fed-batch culture 31). If the
dB net
substrate concentrations are such that ~ _< 0, the exponentially fed-batch culture
is unstable. Control strategies formulated from the solution to optimal control
problems cannot therefore be entrusted for the bioreactor operation unless a feedback
parameter adjustment mechanism is provided for.
The inherent complexity of the biological processes makes measurement problems ~
in these significantly different from and more difficult than those associated with
conventional chemical processes. Hatch 12} divides the measurable process variables
into three categories: environmental, physiological and cell culture composition.
Commonly measured environmental variables include temperature, pH, dissolved
oxygen, agitation speed, aeration rate and nutrient (substrate) concentration. Physiolo-
gical variables can be subdivided into two groups: (a) products of metabolism, such
as cell mass and extracellular metabolic products and (b) variables describing the
state of metabolism such as cell age, intracellular composition and cell viability.
For those variables which are not directly measurable, the concept of "gateway
sensors" 162) has played an important role. Using this approach, certain biologically
significant parameters can be extracted from a combination of available sensors.
The computer serves as an useful aid in establishing the necessary correlations, thus
providing "gateway" to predict the variable values for which process instrumentation
is not available.
Autoclavable sensors for pH, temperature and dissolved oxygen are readily available
and are being widely used. If the dissolved oxygen is not a limiting substrate, its
measurement may be less important. However, in many processes, the dissolved
oxygen is a very good indicator of a change in the cell metabolism. It is therefore a
key variable to monitor even though its control may not be necessary. Another
promising indicator of the status of aerobic growth is the redox potential, which
can be directly measured with Beckman redox electrodes 163 166). The redox potential
indicates the oxidative status of aerobic growth at low dissolved oxygen concentrations
( < 1% saturation).
If the measuring instrument (sensor) cannot be made aseptic, the component to
be measured must be withdrawn from the bioreactor without contamination. Further,
the non-biomass components must be free of cells. Measurements of oxygen, carbon
dioxide and other gaseous species which include volatile substrates/products such
as ethanol and methanol are fi'equently carried ou.t in a continuous manner in the
exit gas. Ethanol can be detected in the exit gas phase by a semiconductor sensor 16.22.
150.16;I. An implicit assumption here is that the ethanol in the culture medium is
in equilibrium with the ethanol in the gas phase. Ethanol (or methanol) concentrations
in the culture medium have been measured directly using the porous Teflon tubing
and silicon tubing s e n s o r s 29" 65'147'148'16s x72). The variables that are frequently
measured in addition to these are concentrations of oxygen and carbon dioxide in
the exit gas 21. 116,118,123.173) and nutrient or energy addition rates 21.116,174.175)
Mass spectrometry has been used to monitor low molecular weight compounds in
exit gases 176-178). A wide variety of electrodes which sense the concentrations of
dissolved ions other than the hydrated proton (ions such as potassium ion, sodium
ion. calcium ion and magnesium ion) are now commercially available 179). The
availability of numerous ion-selective electrodes allows for continuous measurement
and control of concentrations of various nutrients and other species. Unfortunately,
nutrients such as "inorganic phosphates cannot at present be measured using this
approach 12)
Off-line cell concentration measurements have often been made using a spectro-
photometer 58.15~1for unicellular microorganisms by developing correlations between
the optical density readings and the actual concentrations. A Spectronic 20 spectro-
photometer built into a computerized fermentor was used to provide an on-line
cell concentration measurement 58,154). A correlation between the on-line optical
density readings and off-line cell concentration measurements was used to obtain
the on-line cell concentrations 58.154). In penicillin processes, the filtration characteris-
tics of mycelial biomass can be directly related to the metabolic activity of the organ-
ism, Penicillium chrysogenum 100,~0a,t0,,161,180). The ratio of cell dry weight to cell
volume reflects the cytoplasmic contents of the cells. Cell mass is determined using
a filtration probe 100.~03.10~) which can be interlaced to a computer for continuous
on-line monitoring and control of penicillin production lOO.103.104,161.180)
Culture fluorescence has been reported 18, ~81.182) to provide a cumulative index
of culture activity and therefore may be important in control of a variety of processes.
Ristroph et al. ,8) used culture fluorescence measurements to control the addition
of ethanol to a fed-batch single-cell protein (SCP) process. The fluorometer can be
used to indicate the proper time for ethanol addition since substrate limitation is
indicated by a large drop in fluorescence 18,181)
The powerful technique of laser flow microfluorometry can provide for measure-
ment of DNA, RNA, specific proteins and possibly N A D H by flow of cells in a single
file through a beam of light 12. lS6,183 186). Flow microfluorometry can also be used
to determine cell size distributions and population distributions. This technique has
great potential in modeling of the cellular kinetics.
In what follows, we briefly review separately the control schemes used for fed-batch
bioreactors used for biomass production and metabolite production.
5.3 Feed-on-Demand Control for Biomass Production

Feed-on-demand type of control has been extensively used to manipulate the cellular
metabolic activities, especially in biomass production processes. To optimize a set
objective, the nutrient feed rate must be regulated so'that conditions in the fermentor
are favorable for cell growth. For various reasons mentioned earlier, both under-
feeding and overfeeding of nutrients should be avoided. Two types of feedback control
are frequently used to regulate the nutrient feed rate. In the first type, an indirect
control parameter that can be continuously monitored (parameters such as respiratory
quotient (RQ), carbon dioxide evolution rate (CER), oxygen uptake rate (OUR),
dissolved oxygen concentration (DO), and pH) is used as an indicator of the metabolic
activity. In the second type, the formation of an undesirable side product is directly
detected by monitoring its concentration in the culture medium or in the exit gas.
The substrate concentration in the bioreactor may also be directly monitored to
regulate the feed substrate rate.
Respiratory quotient measurements have widely been used as an indicator of ethanol
formation in growth of baker's yeast and Candida utilis using glucose as the main
carbon and energy source owing to the easy and reliable operation of the oxygen and
carbon dioxide analyzers, Additionally, the response of RQ to changes in the glucose
feed rate is very fast 22). Wang et al. 21j have shown that the respiratory quotient
(RQ) can be correlated to the fraction of sugar converted to ethanol as
EPR = [RQ -- (RQ)o] (OUR) = (CER) -- (RQ)o (OUR) (102)
where EPR denotes the ethanol production rate (moles h-l). (RQ) represents the
magnitude of RQ when no ethanol is being produced. It has been observed 14, 21.22,
120. 187.188) that RQ should be maintained near 1.0 to eliminate ethanol formation.
The first studies on computer control of substrate feeding in baker's yeast growth
using RQ as the control parameter are due to Aiba et al. 2o) and Nagai et al. 1s9~
For a led-batch culture, Aiba et al. 2o, 135) showed that the feed rate is explicitly dep-
endent on the total oxygen consumption rate. The relationship between the feed
rate and total oxygen consumption rate involved a constant which depended on the
product to substrate yield, Yp,.s, precise estimation of which may be difficult. Despite
this limitating and lack of true feedback control, Aiba et al. 2o) were able to control
RQ in the range 1.1-1.2.
A control strategy for a repeated fed-batch bioreactor aimed at reducing the cata-
bolite repression effects in baker's yeast growth was proposed by Peringer and
Blach6re 5;). The strategy was essentially to keep the substrate and dissolved oxygen
concentrations constant by operating the bioreactor in an exponential fed-batch
mode. The optimal feeding policy could be expressed as a function of RQ and OUR.
The relationship between the nutrient feed rate, RQ and OUR involved two para-
meters which have to be specified. These parameters, which relate the specific carbon
dioxide evolution rate to specific substrate uptake rate and specific oxygen uptake
rate, depend on the culture medium composition 19o). These parameters can be identi-
fied experimentally. The experimental work of Peringer and Blach~re 57) showed
that both RQ and OUR should be used for optimal control. In the repeated fed-batch
cultures, RQ was used 5v~ as a qualitative index and OUR as the quantitative index
of the energy yielding metabolism of the yeasts; RQ being maintained in the range
1.0 1.4.
Wang et al. i25. 191jinvestigated the possibility of using RQ as the control parameter
in baker's yeast cultures. The demand for substrate was expressed as a function of the
cell-substrate yield, Yx.s, specific growth rate ~t~r substrate feed concentration Sv
and amount of biomass as H6j
~lle[xv
F ~-- -- SF ~> S (103)
SvYx.'s
The above relation is obtained from Eq. (15) by neglecting (a) variation in the substrate
concentration with time, (b) substrate loss due to evaporation and (c) maintenance
requirement. The appropriateness of relation (103) is justified in view of the fact

that to prevent the occurrence of the Crabtree effect, the concentration of sugar
(glucose) in the medium (S) should be low and to maintain optimal performance,
S should be maintained constant at its optimal value. The substrate feed rate must
equal the cellular demand at all times during the process. In the absence of a sensor
for direct measurement of cell mass, a computer-aided material balancing technique
was developed to calculate continuously the cell concentration, the specific growth
rate, and the specific substrate consumption rate from measurements of the rates
of air flow, the carbon dioxide production and the oxygen consumption 116). The
ethanol production rate is related to the carbon dioxide evolution rate and the oxygen
uptake rate through Eq. (102). The use of RQ alone in feed-forward (or anticipatory)
control of feed rate was not adequate and a proportional feedback control law had
to be added based on Eq. (102). The corrected feed rate F c is expressed as
Vc = V[1 Kc{CER (RQ)oOUR}I (104)
K c is the controller gain and F is defined in Eq. (103). Despite various process pertur-
bations such as oxygen limitation, variations in the inoculum and variations in feed
compositions, Wang et al. 1251were able to maintain both high cell yield and high
volumetric productivity.
The respiratory quotient was also found to be a sensitive indicator of the growth
of Candida utilis 120. 187). The growth rate of Candida utilis and production of ethanol
were found to depend on the carbon to nitrogen ratio of the culture medium. It was
determined experimentally that the growth rate was maximized and the production
of ethanol minimized when the RQ was equal to 1.0. An empirical feedback control
policy was implemented in a computer-coupled fed-batch bioreactor using RQ as
the control parameter. Cell densities as high as 105 g 1-1 were obtained i20.187,188~
In aerobic fed-batch cultures, the dissolved oxygen tension in the culture medium
may become a growth-limiting factor as the microorganisms grow. Exposure to an
oxygen-limited state for a long time may cause damage to the microorganism and/or
some physiological changes, thereby retarding the growth of the microorganism.
Dissolved oxygen concentration has been used as a nutrient feed indicator for regula-
tion of the added carbon source 48. 168.192, 193). An apparatus which can maintain
DO level constant, DO-star, is often needed for fed-batch bioreactors. Various versions
of the DO-stat have been developed thus far 44.46.49.92.168,194,195). With these
DO-star systems, high cell densities have been obtained in the fed-batch cultures
oti (a) Protaminobacter ruber (on methanol)45), (b) Escherichia colt (on glucose) -~4)
and (c) Candida brassicae (on ethanol) 92,195). The overall oxygen balance for a led-
batch bioreactor can be represented as
C o 2 = Hpo~ Q~ (105)
- kLa
where Co2 represents the dissolved oxygen concentration in the culture medium,
H is the Henry's law constant for oxygen, po 2 its partial pressure in the gas phase,
Q02 the specific oxygen uptake rate and kca the mass transfer coefficient for oxygen
absorption. It is evident from Eq. (105) that at constant cultivation temperature,
po 2 and/or kLa must be changed as the microorganism grows to maintain DO constant.

These variables may be altered by varying the agitation speed and/or flow rate of
the feed gas (consisting often of air and pure oxygen). The earlier versions of the DO-
stat 45,46~ required manual operations to change the preset values for maximum and
minimum agitation speed and aeration rate during cultivation. These DO-stats were
useful only when air was supplied to the bioreactor. When biomass concentration
increases and consequently DO cannot be maintained constant even at the maximum
agitation rate and maximum air flow rate, a mixture of air and oxygen must be supplied
to the bioreactor. The latter versions 138. 194,195) of DO-stat are capable of controlling
DO throughout the cultivation whether air or a mixture of air and pure oxygen is
supplied. These DO-stats make use of a microcomputer for DO control. The DO
control system consists of a microcomputer, an interface, thermal mass flow controllers
for air and oxygen and a DO meter. D O levels have been maintained as low as 2.0 ppm
with DO-stats 92)
Due to poor efficiency of oxygen utilization in the conventional bioreactors, it is
u n c o m m o n for much more than 25%o of the oxygen in air supplied to the bioreactor
to be utilized by microorganisms even when DO is nearly zero and is the rate-limiting
variable. Bioreactors with the ability of self-induction of gas can be considered to be
suitable for usage of pure oxygen in place of oxygen-enriched air because internal
circulation of exhaust gas occurs by itself and high efficiency of oxygen utilization is
a t t a i n e d 1961. Supply of oxygen in such fed-batch bioreactors can be controlled by a
DO controller. Fed-batch cultures of Candida brassicae were grown on ethanol in
the gas entraining fermentor 196~. Matsumura et al. 196) suggest that usage of gas
entraining reactor may result in enhanced biomass productivity with a low energy
requirement if pressurized pure oxygen is used.
Another indirect indicator of the metabolic state of a microorganism is fluorescence
and its continuous measurement may be used as a potential means for fed-batch
monitoring 18,53,1821. A healthy active culture should display a relatively high con-
centration of N A D H and a large fluorescence signal. How readily a microorganism
utilizes a substrate therefore should have an effect on the fluorescence. An example
of such situations is the growth of Candida utilis on ethanol. Acetate is produced as
a by-product in the growth of Candida utilis on ethanol when ethanol concentration
is high s3). As the ethanol concentration decreases, the yeast utilizes the acetate and
the fluorescence level decreases. Further, when Candida utilis consumes ethan01,
the fluorescence is high and it is low when the cells utilize acetate. A large drop in
fluorescence, indicating substrate limitation, signals the need for addition of fresh
substrate. Zabriskie 1821 used culture fluorescence measurements to regulate glucose
addition in a baker's yeast process to prevent glucose repression effects and excessive
conversion of glucose to ethanol.
Hennigan et al. 8s) have suggested that in a fed-batch culture with constant biomass
to substrate yield, Yx,.s, the cell concentration can be used in a feedback mode to
regulate the substrate feed rate during the singular control period according to Eq.
(92). The cell concentration is maintained constant at X(h) (see Eq. (97)) using the
feedback control during the singular control period. The on-line measurement of
cell concentration was made by passing a recycle line through a Spectronic 20 spectro-
photometer which contained a dilution device 2os~
Boyle 56,197) examined the possibility of manipulating the dilution rate and the
feed substrate concentration/'or the simultaneous control of the biomass and substrate
concentrations at constant values in a fed-batch bioreactor maintained at a quasi-
steady state. Without feedback control, the rate of approach to quasi-steady state is
very slow, requiring large volume expansion (large Vm/V0), thereby effectively preclud-
ing observation of the quasi-steady state in laboratory reactors. The controller is
designed to manipulate the dilution rate D and feed substrate concentration Sv con-
dS
tinuously to maintain d t = 0 and S = S D. Error in the cell concentration is used to
manipulate Sv with a proportional control.
Ethanol concentration in the medium is often controlled when ethanol is either
a product or the limiting substrate. In baker's yeast growth, for example, optimal
feeding of the substrate is achieved when the respiratory capacity of the cells is at its
maximum, i.e., when metabolism tends to switch from respiration to oxido-reduction
and vice versa. The ultimate goal of the control system is to maintain the ethanol
production rate (EPR) at zero because the negative and positive values of EPR
mean deficiency and excess, respectively of glucose in the culture medium. The feed-
back control scheme that maintains ethanol concentration constant will also keep
the ethanol production rate zero. Ethanol concentrations in the fed-batch bioreactors
have been measured using the Teflon-tubing method 27.29,65.68,147,148,171,198)
Alternately, the ethanol concentration in the culture medium can be related to the
ethanol concentration in the gas phase which can be measured by a semiconductor
sensor 16,22.150.167) Huang and Chu 199) have used a solid-state electrolytic cell
for monitoring the gas phase ethanol concentration.
Ethanol concentration has been used as a feedback parameter for regulating
glucose feed rate using on-off control 68) and PID (proportional-integral-derivative)
control 22,65,148,150,198). The performance of the PID controller is better than the
on-off controller 148). Various constants for the PID controller can be estimated
using a material balance for the fed-batch bioreactor. The controller parameters
must be adjusted frequently to account for various disturbances such as increase
in cell concentration, sudden changes in feeding conditions and deviations in the
ethanol concentration. PID feedback controllers with automatic tuning algorithms
have been employed in control of baker's yeast fed-batch fermentation 16,171.198)
On-off control was used to regulate ethanol feed rate using ethanol concentration
as the feedback parameter in production of glutamic acid by Brevibacterium divari-
catum 29). The optimal trajectory for ethanol concentration was determined using
the computational algorithm for adaptive optimal control and ethanol concentra-
tion in the culture medium was controlled along the optimal trajectory by feedback
control. Automatic control of fed-batch culture of Candida utilis using ethanol as
the sole carbon source was achieved by Huang and Chu 199) employing the gas phase
ethanol concentration as the feedback parameter. A proportional-derivative (PD)
feed-back control system incorporating a porous Teflon tubing sensor for measuring
the ethanol concentration in the bioreactor was developed for yeast fermentation
using ethanol as the sole carbon source 171). The controller parameters were estimated
from the dynamic mass balance for ethanol.
5.4 Control of Specific Growth Rate for Antibiotic and Enzyme Productions
In the antibiotic and enzyme production processes, there are generally two phases:
a growth phase and a production phase; although the transition from growth phase
to production phase is sometimes characterized as an additional phase. In the growth
phase, the usual strategy is to keep the cell growth rate at a maximum so that the
desired growth will be achieved in the shortest time possible, although the cells grown
too rapidly may result in lower product formation rates. After the desired growth has
been attained, the production phase is initiated by reducing the specific growth
rate or by adding additional inducing agents. The control of the specific growth rate
is effected through manipulation of the nutrient feed rate. As in the biomass production
processes, the substrate feed rate must be carefully controlled to avoid catabolite
repression resulting from nutrient overfeeding and starvation (and severe irreversible
damage to production phase) caused by underfeeding of the substrate. Due to inability
to conduct direct measurements of the specific growth rate and substrate concentration
in the bioreactor, indirect correlations for these are often used.
Carbon dioxide concentration in the exit gas has been found to be a good indicator
of the growth of Trichoderma reesei using cellobiose or a mixture of cellobiose,
cellulose and glucose as the carbon source 23,24,4o.2oo). A linear relationship was
observed between the specific carbon dioxide evolution rate and specific growth
rate 4o( In the study by Waki et al. 40), the time variant set-point for carbon dioxide
concentration in the exit was preset to keep the specific growth rate constant for
optimum producti01~ of cellulase based on the results of continuous cultures on
cellobiose. A PDP-1 1 minicomputer was used to regulate the addition of substrate
by Allen and Mortensen 241. Proportional control was used to regulate the substrate
feed rate using the negative deviations from the set-point C02 concentration. No
control action was taken for positive deviations. The computer control ensured
carbon limitation by not allowing substrate addition if the DO fell below 30"o of
the saturation. This control strategy was also used for repeated led-batch cultures of
Trichodernm reesei using lactose as the carbon source.
Lundell 2011 used CER and RQ as control parameters on which the intermittent
feeding of the carbon source was based. The fed-batch run was either terminated or
additional nutrient was added to induce further enzyme production when the produc-
tion rate fell below the preset value. Lundell _~01)showed that a properly chosen feed-
ing strategy would improve productivity over the conventional batch process by more
than a factor of two, at the same time resulting in substantial energy savings.
In order to develop a rational cQntrol strategy for sugar addition in the production
of penicillin using a Penicillium cht3,sogenum strain, it is necessary to know both the
cell mass concentration and the growth rate 2o2-2o4). For accurate calculation of cell
concentration, glucose addition and CER must be followed as closely as possible.
Since measurement of glucose in the culture medium on a continuous basis is difficult,
carbon balancing is very useful under carbon-limiting conditions 202-204) In the
growth phase, CER was found to be indicative of the cell growth rate and the total
carbon dioxide evolved provided an accurate estimate of the amount of mycelium
present 25, 26,202-204). With this information, it was possible to develop an adaptive
control strategy for continuous addition of glucose in order to meet the demands of
the growing mycelium. The maximum specific growth rate of a mycelial culture
depends on the inoculum growth activity, cell morphology and culture age. Theretbre,
it is desirable not to fix the value of the growth rate set point ; rather let it be adaptive 2o3,
20,,1. Cell mass (XV) is the load variable for the entire control system. Instantaneous
specific growth rate (g,e,) and residual substrate concentration (S) are the state
variables and substrate feed rate is the control variable. Since the calculated load
disturbance is used to correct the output of the primary controller in addition to the
feedback of growth rate and residual substrate concentration, this control system is
a feedforward modification of feedback control for cell mass production 26,204)
The adaptive control strategy would add sufficient carbon source to maintain the
maximum (or any desired) growth rate, yet not overfeed the glucose 25,26,202-204)
Once a desired cell concentration was obtained, the computer could be used to change
the control strategy from rapid growth to slow, controlled growth required in the
production phase.
Since the cells are grown at a lower growth rate under carbon limitation during the
transition and penicillin production periods, the maintenance activity and endogeneous
metabolism are significant and it is not possible to use CER as the sole indicator of cell
growth. The specific cell growth rate and cell concentration in the production phase
can be continuously estimated by means of overall and instantaneous carbon balances.
Again, the supply of the carbon source can be controlled to meet the demands of
the organism with the added constraint of a desired specific growth rate 25.26.202 - 2 0 4 ~
The ability to achieve 90')0 recovery of the substrate and precursor carbon as carbon
dioxide, cell mass and penicillin carbon enabled consistent reproduction of the desired
cell growth patterns by controlling substrate feed rate 203.20~).
Alternately, the buildup and degradation of mycelial biomass can be quantitatively
characterized during a fed-batch penicillin process using the on-line filtration technique
of Nestaas and Wang 100-104) During the production phase, the hyphal density
and cell maintenance activity, as indicated by the respiratory rate, decrease due to
the loss of cytoplasmic material (mainly proteins) while the cell wall remains practically
unchanged. Nestaas and Wang 102)found that the rate of penicillin synthesis correlated
well with the hyphal density. A control strategy using nutrient feed manipulation
was formulated by Nestaas and Wang which followed predetermined desired growth
rate profile that made sure that the specific growth rate was in excess of 0.01 h -1
to prevent deterioration of penicillin synthesis rates zoo, io4) The open-loop control
based on the predetermined feed schedule failed under certain conditions. The devia-
tion from the desired growth rate was minimized by using a feedback control system
to manipulate the glucose feed rate. The difference between the desired cell mass
and the cell mass measured directly by the filtration probe was used for such manipula-
tion ioo-lo4). The biomass concentration estimation by material balance was based
on the assumption that all the carbon remaining in the broth was present either in cells
or in penicillin. In the presence of a large amount of residual complex nutrients, such
estimation fails and feedback of biomass concentration from the filtration probe is
necessary for following the desired growth rate profile.
6 Current Problems and Future Directions

A large number of products such as antibiotics, fine organic acids, amino acids,
proteins, cell mass and enzymes are produced by fed-batch cultures. These continue to
receive wide attention for the ability to achieve high cell concentrations which in turn
result in higher rates of product formation and higher metabolic concentrations by
overcoming effects such as substrate inhibition, glucose effect, catabolite repression
and auxotroph mutation. Since fed-batch cultures are run dynamically under various
modes and under quasi-steady state mode, they are very versatile and hence suitable
for various rate studies.
In spite of these attractive features, there are many problems associated with fed-
batch cultures. Many of the problems are not unique to fed-batch culture~, but indeed
are faced in operation of any bioreactor. Since these cultures are usually run dynami-
cally, accurate dynamic models are necessary to interpret the.experimental data for
these cultures. At present, very few models capafile of predicting the dynamics of
fed-batch cultures accurately are available. Unstructured models are frequently
inadequate to represent these cultures. Structured models have a build-in capability
to predict the dynamics of fed-batch operation more precisely for they are developed
by selecting the most relevant parameters for description of physiological state of
the organism. The structured models therefore can be used with more confidence
in optimization and control studies related with fed-batch cultures. Many physiological
states are however difficult or impossible to estimate/identify, especially on-line, and
therefore the structured models proposed in literature have seldom been rigorously
tested. Additionally, identification of physiological state is necessary for implementa-
tion of the optimization and control policies based on structured models. Current
capability in this area, especially the on-line identification of physiological state,
is very limited. For example, on-line cell mass measurements are difficult (almost
impossible) especially when the cell concentration is high, the cells are not unicellular
and there are insoluble solids in the medium. Biosensors and special instruments
capable of measuring the physiological states of microorganisms are therefore highly
desirable and are needed urgently. In many situations, the off-gas is analyzed for
various components such as oxygen, carbon dioxide and other volatile metabolites
and from these measurements the critically needed physiological state information
is inferred under specific assumptions which may or may not be valid in practice.
Availability of more biosensors along with extended estimation schemes is necessary
to predict with greater accuracy those parameters which are difficult-to- or impossible-
to-measure. On-line measurements and indirect estimations will continue to play an
important role in control of fed-batch reactors.
Reliable models needed for optimization of fed-batch bioreactors are seldom
a~vailable in practice and a major effort is involved in developing an elaborate rood_el.
Furthermore, one does not expect such a model to be valid under all operating con-
ditions such as those wherein microorganisms go through adaptation to a new environ-
ment and those wherein undefined medium components vary from batch to batch.
Further, any model without proper incorporation of the internal control structure
of the microorganisms would be expected to be inadequate. Hence, the available
model must often be updated during the course of fed-batch process. In view of the
arguments made earlier, it is logical to emphasize the need for developing on-line
optimization strategies, in which the dynamic response over a short time period is
used to update the model, the optimal control policy calculated and implemented.
The resulting dynamic response is then used to further update model and the process
is repeated several times during the course of growth and production.
Modeling, Optimization and Control of Semi-BatchBioreactors 251
7 Acknowledgment
This work was supported in part by a grant from the National Science Foundation,
CPE 7918902, to one of the authors (HCL).
8 Nomenclature
ho hyphae which elongate or branch

A1 penicillin producing hyphae
A2 degenerate hyphae
ao, a~, a2 concentrations of Ao, At, and Az, respectively
CER carbon dioxide evolution rate
Co 2 dissolved oxygen concentration
D dilution rate
DO dissolved oxygen
EPR ethanol production rate
F feed flow rate
Fo effluent flow rate
Fm maximum allowable flow rate
Fc corrected flow rate-defined in Eq. (104)
Fs feed rate during the singular control period
f~ defined in Eqs. (71) and (76)
G aeration rate
G(t) defined in Eq. (100)
H Hamiltonian
H Henry's law constant for oxygen
I int/ibitory species
IP index of performance
K, Kmo, Knal, kl, k2 rate constants for various rate expressions in Eqs. (42)-(46)
Kc controller gain
Kl, Kv kinetic coefficients for the substrate inhibition kinetics in the
Bajpai and Reuss model
K-~ kinetic coefficient for Contois kinetics
k defined in Eq. (25)
kp specific aging rate of metabolite product, h- a
kx specific aging rate of biomass, h-
kLa volumetric mass transfer coefficient for oxygen, h-1
L1, L2 defined in Eq. (70)
L, too, ml defined in Eqs. (42)-(46)
ms maintenance requirement for substrate
mp maintenance requirement for by-product
mx maintenance requirement for cell mass
OP operating costs
OUR oxygen uptake rate

P metabolite product concentration
Pv feed product concentration
1"o initial product concentration
Pc, Pro, P~ unit prices associated with cell mass, metabolite product and
limiting substrate, respectively
P average biomass production rate (defined in Eq. (98))
Po2 partial pressure of oxygen in gas phase
Qo 2 specific oxygen uptake rate
RQ respiratory quotient
(RQ)o value of RQ when no ethanol is produced
S limiting substrate concentration
Sr feed substrate concentration
So initial substrate concentration in the bioreactor
So desired limiting substrate concentration
Sg limiting substrate concentration in the gas phase
Sf substrate concentration at the end of batch stage
Sfill substrate concentration at the end of filling stage
S; concentration of dissolved oxygen in Eqs. (36), (37) and (38)
S~' equilibrium concentration of dissolved oxygen
$2 concentration of ammonia nitrogen
Sc constant (preset) substrate concentration
t time
tf filling time
Tf cycle period
t~ switching time
(tl, t2) singular control time interval
tlg guess value of switching time tl
U vector of control variables
V bioreactor (culture) volume
vo initial bioreactor (culture) volume
Vm maximum bioreactor volume
X biomass (cell) concentration
Xfili biomass (cell) concentration at the end of filling stage
Xo initial biomass" concentration
Xf biomass concentration at the end of batch stage (final biomass
concentration)
Xh hyphae
Xs swollen hyphal fragements
Xa arthrospores
Xc constant (preset) biomass concentration
X vector of state variables
Yx,'s biomass-to-substrate yield (g g-~)
Yp,s byproduct-to-substrate yield (g g-~)
Yx,'p biomass-to-byproduct yield (g g-l)
Yxcsl biomass-to-oxygen yield (g g- ~)
YR;S 1 penicillin-to-oxygen yield (g g- ~)
Greek Symbols
% d e s a m i n a t i o n coefficient in Eqs. (39) a n d (40)
vector o f a d j o i n t variables
~t specific cell g r o w t h rate
~net net specific cell growth rate
specific growth rate for growth o n substrate S
P~p specific g r o w t h rate for g r o w t h o n b y - p r o d u c t P
kinetic constants for C o n t o i s kinetics
t11, V rate c o n s t a n t s in Eqs. (42)-(46)
% , q~2 defined in Eqs. (78) a n d (79)
net specific p r o d u c t f o r m a t i o n rate
specific f o r m a t i o n rate o f i n h i b i t o r y substances
r kinetic coefficient for the substrate i n h i b i t i o n kinetics in the
Bajpai a n d Reuss m o d e l
(5 net specific substrate c o n s u m p t i o n rate
Supercripts
time derivative
derivative w.r.t, limiting substrate c o n c e n t r a t i o n
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159. Hennigan, P. J. : MS Thesis Purdue Univ. 1979
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[Advances in Biochemical Engineering_Biotechnology 32] N. Kosaric, A. Wieczorek, G. P. Cosentino (Auth.) - Agricultural Feedstock and Waste Treatment and Engineering (1985, Springer-Verlag Berlin Heidelberg)

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[Advances in Biochemical Engineering_Biotechnology 32] N. Kosaric, A. Wieczorek, G. P. Cosentino (Auth.) - Agricultural Feedstock and Waste Treatment and Engineering (1985, Springer-Verlag Berlin Heidelberg)

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Advances in Biochemical Engineering/

Managing Editor= A. Fiechter

With 65 Figures and 53 Tables

© by Springer-Verlag Berlin. Heidelberg 1985

Typesetting and Offsetprinting: Th. Mtintzer, Bad Langensalza

Professor Dr. A. Fiechter

Prof. Dr. S. Aiba Department of Fermentation Technology, Faculty of

Industrial Processing and Products from

The Utilization of Cheese Whey and its Components

Mathematical Modelling, Parameter Identification and

Modelling, Optimization and Control of Semi-Batch Bioreactors

Author Index Volumes 1-32 . . . . . . . . . . . . . . . 259

2 Food and Fodder

The carbohydrate portion of the JA tuber constitutes approximately 75 o; of the

Accession Digestible energy % of DM

Morden @5 14.703 26.9 47.8 34.4 1.43 3.9

2.2 Aerial Parts

Table 2. Organic matter digestibility of Jerusalem artichoke forage samples

JA b whole forage 60-70

3 Ethyl Alcohol Production

3.1 Preparation of Raw Material

rupture is extensive and non-soluble matter (such as cellulosic material) is suspended

3.2 Pretreatment of Carbohydrates

3.2.1 Enzymatic Hydrolysis

Hydrolysis by Inulases Present in Tubers

Hydrolysis by Inulases Present in Filamentous Fungi

Some yeasts, such as Kluyveromycesfragilis 80,811, Saccharomyces lactis 827, Candida

3.2.2 Acid Hydrolysis

3.2.3 Thermal Hydrolysis

Method of treating the tubers Ethanol yield, L

Per 100 kg Per 100 kg ~ of theoretical from

l. Maintain the mash

W l o d a r c z y k 5~,99~. The extent of carbohydrate hydrolysis was found to be highly

3.3 Ethanol Production

optimal concentration of sugar. From an economic viewpoint, the ethanol process

3.3.1 The Yeasts

3.3.5 Carbohydrate Concentration

3.3.6 Systems of Ethanol Production

Parameter Free cells at Immobilized cells at

Max. ethanol productivity 7.0 104

b i o m a s s c o n c e n t r a t i o n s as a f u n c t i o n o f d i l u t i o n r a t e for this system. In t h e s a m e

More research is required in order to obtain optimum parameters for operation

3.3.7 Ethanol Production from the Stalks

3.4 Recovery of the Ethanol from "Beer"

Alcohol concentration and purification is an expensive step in industrial alcohol

3.5 By-Product (Stillage) Obtained after Distillation

Table 5. Composition of stillage obtained by the microbial processing

Fresh weight basis Dry Matter

Composition of stillage obtained after microbial conversion of the juice depends

3.6 Energetics and Economics

3.6.1 Energy Balance

4 High-Fructose Syrup Production

Method of extraction Ash (as sulfate) Protein Carbohydrate

Expressing 7.5 5 75 about 5.0

Slice into water containing 0.1% SO=. (2 1)-

Tuber slices in raw juice

Raw uice (95-100% extraction efficiency)

Filter through activated animal charcoal

Clear, colorless juice

pH2.Sto3.0,85~ / ~ Inulase, 50~ pH 5.0,

Acid-hydrolyzed juice! Enzyme-hydrolyzed juice

Clarified juice Clarified juice

Sweet, Colorless solution

HIGH-FRUCTOSE SYRUP ( 75% yield}