.... TIBTECH - SEPTEMBER 1987 [Vol.

5]

fix themselves to surfaces as an

adhesive which will function in a
wet environment. Both a cloned
gene product and the material
extracted from natural sources are
under investigation.
Polymer synthesis by micro- Recombinant DNA techniques are
being used to produce silk-like pro-

organisms: technology and teins which have desirable fibre

properties. They are likely to enter
the high priced composites market
economics rather than emerge as an alternative
to the natural fibre made in fabric.
It is obvious from the above brief
D. Byrom survey of only a limited number of
applications, that there is a large
Industrial polymers are, in the main, petrochemical based. The amount of industrial interest in
textiles, gums, films, adhesives and coatings w h i c h comprise all or biopolymers. A recent forecast pre-
part of manufactured articles in daily use have been developed over dicts a market value of about £200
m a n y years of research. They are produced cheaply and in very million by the 1990s (Table 1). The
large volume, two features w h i c h go hand in hand. success or otherwise of biopolymers
In what form is the challenge from biopolymers? Many b i o l o g i c a l in the market, will depend on both
polymers undoubtedly have useful properties but is it possible to the properties of the material and the
economics of manufacture. This arti-
produce them efficiently enough to create volume markets? T h i s cle discusses some of the influences
article will concentrate largely on the production o f poly-~- of biology on the technology and
h y d r o x y b u t y r a t e where some of the most advanced technology has economics of the process to make
been a p p l i e d . poly-~-hydroxybutyrate on the large
scale.
The opportunity for biology to pro- Ribonucleic acid (RNA) is a poly-
vide novel routes from non-petro- mer that is used as the starting What is PHB?
chemical renewable resources to use- material for the manufacture of the Poly-~-hydroxybutyrate is a bio-
ful materials is very large, but the flavour nucleotides 5' guanosine degradable, biocompatible, thermo-
challenge faced in doing so is monophosphate and 5' inosine plastic made by microorganisms.
substantial. Nevertheless, biologic- monophosphate. RNA isolated from The material can be formed into
ally produced polymers can have yeast is specifically degraded using a films, fibres and sheets and moulded
advantages in biodegradability, per- 5' phosphodiesterase and the nuc- into shapes and bottles. PHB, and
formance, cheapness of substrate leotides separated. Adenosine mono- its copolymer with hydroxyvaleric
and in defined structural variability. phosphate is deaminated to inosine acid, is under development in a wide
Polysaccharides were probably monophosphate with a second en- range of applications by a number of
among the first microbially pro- zyme 5. companies using product supplied
duced polymers to be considered for Poly-~-hydroxybutyrate, a bio- by Marlbrough Biopolymers, a sub-
industrial use. A large number of degradable plastic produced by a sidiary established by ICI plc and
these materials have been described 1 microorganism s is another example MTM to exploit the material.
but only one, xantham gum, is on the of a biopolymer which is finding a PHB has been known since 1926".
market 2. It is sold as thickener for market. It is an intracellular storage polymer
the food industry or as an additive in Companies are developing the whose function is to provide a
enhanced oil recovery. anchor protein used by mussels to * Lemoigne, M. (1926) cited in Re[. 8.
Hyaluronic acid (HA), another
polysaccharide, has been used in --Table 1
ophthalmic surgery for a number of
years. The source of the material was Projected biopolymer market 1990s
cocks combs or umbilical cords but
Use Price r a n g e Value
recently several processes for pro- £ kg -1 £ M y r -1
duction of HA from microorganisms
have been reported 3,4. Adhesives/coatings 30-60 30
Fib res 15-60 35
Gums 1.5-3.0 100
D. Byrom is at the Biological Products Plastics 3-30 35
Business, Imperial Chemical Industries
PLC Billingham, Cleveland, TS23 1LB, See Ref. 7 for source.
UK.
C~ 1987, Elsevier Publications, Cambridge 0166-9430/87/$02.00
TIBTECH - SEPTEMBER 1987 [Vol. 5]
-Fig, 1
Electron microscope view of the accumulation of polymer granules, PHB in
cells of the species Alcaligenes eutrophus. Bar = 1 #m
reserve of carbon and energy 8'9. The
polymer accumulates as distinct
granules in the cell and can usually
be seen by light microscopy and is
certainly visible by electron micro-
scopy (Fig. 1). A wide range ot
organisms have been reported te
store PHB or PHB-like materials.
They include Gram positive and
Gram negative species and cyano-
bacteria. It will be noted from Table
2 that the organisms listed are all
prokaryotic. There have been no
substantiated reports of PHB in
eukaryotes.
The materials are polyesters. In
PHB, a homopolymer, the repeating
unit is 3-hydroxybutyric acid, the
formula being:
CH3 H O Azospirillum
I 1 II
--O--C--C--C-
I I
H H
13
where n = 23 000 to 25 000. Copoly-
mers with hydroxyvaleric acid can be
made by some organisms 1°. These are
usually random arrangements of
hydroxyvaleric and hydroxybutyric
acid residues. The structure of poly-
hydroxyvaleric acid (PHV) is:
-
cH3 1
CH2
IH
-O-C-C-C-
~)
HH n
In a fermentation, PHB/PHV copoly-
mers are made by supplying mixed
glucose/propionic acid substrate.
It is probable that a range of PHB-
like polymers can be made in nature.
There are reports of poly-~-hydroxy-
alkanoates from Bacillus species la
and from activated sludge 12, and
polyoctanoates from Pseudomonas
oleovorans 13.
Regulating PHB storage
Since the polymer is a storage
material, it is appropriate to consider
its metabolism as a cycle of synthesis
and breakdown. The enzymes of the
cycle (Fig. 2) have been examined in
a range of organisms and found
to be very similar. A key regulatory
enzyme in PHB metabolism is
acetyl-CoA acyltransferase 15, which
is inhibited by high concentrations of
free coenzyme A. Under balanced
growth conditions, CoASH levels are
high and the synthesis of PHB is
inhibited. In nutrient limitation but
carbon excess, build up of NADH
inhibits citrate synthase, and acetyl-
CoA levels rise to the point where
inhibition by CoASH is overcome.
The condensation reaction to aceto-
acetyl-CoA is possible and PHB is
synthesized.
Polymer degradation is controlled
through the oxidation of monomeric
3-hydroxybutyrate by the enzyme 3-
hydroxybutyrate dehydrogenase.
This enzyme is subject to product
inhibition by acetoacetate and
NADH.
These main regulatory elements of
the cycle are supplemented by a
second level of control: intermediates
of the tricarboxylic acid cycle cause
feedback inhibition of the enzymes.
Thus synthesis and breakdown of
PHB is linked to the metabolic state
of the cell and to the carbon flux
through intermediary metabolism.
PHB o n a n i n d u s t r i a l s c a l e
The economics of a fermentation
process are governed by several
factors including product yield. The
complexity of the technology and
hence the capital cost of the plant,
and the ease or difficulty of product
separation are also extremely impor-
tant so the selection of a particular
production organism and substrate
will have a critical influence on
costs.
-Table 2
Occurrence of poly-fl-hydroxy-
butyrate in microorganism
species
Actinomycetes Methylo-
Alcaligenes bacterium
Azotobacter Micrococcus
Nocardia
Bacillus Pseudomonas
Beijerinckia Rhizobium
Chlorogloea Rhodo-
Chromatium pseudomonas
Chromo- Rhodospirillum
bacterium Sphaerotilus
Derxia Spirillum
Ferrobacillus Streptomyces
Hypho- Vibrio
microbium Zoogloea
Lampropaedia
 TIBTECH - SEPTEMBER 1987 [Vol. 5]
-- Fig. 2

(a) Substrate

Balanced Carbon excess

growth AcetylCoA
Choice of organism an d s u bs tra te
PHB is made by a wide variety of
organisms (Table 2) but which one
Energy~ ' - ~ - ~ ~ o x y g ~
should be the basis for an industrial PHB
process? Some of the organisms in Cellmaterials Phl°p~ph°rusF limitation
Table 2 are slow growing (Rhizo-
bium, Ferrobacillus), or require light
(Chromatium) or only accumulate
small amounts of polymer. Others
are known to make substantial (b) TCA
citrate synthase
amounts of polysaccharide as well as
PHB. After consideration of these NADH OAA ~ ~ citrate
and other factors such as growth
yields, it seemed that the most likely Acetyl CoA
candidates were an Azotobacter sp, a
Methylobacterium sp or Alcaligenes ~ll J~ Acetyl-CoAacyltransferase
eu&ophus. NADH AcetoacetylCoA
These three organisms can use a
variety of substrates. Azotobacter
can use glucose and sucrose; Methy-
lobacterium, methanol and acetate;
Acetoacetate/~ ~ areductase
cetoaceCtyloA
A. eutrophus, ethanol, glucose and
hydrogen/carbon dioxide/air. The
yield values for cell biomass and 3-hydroxybutyrylCoA
PHB can be determined by experi-
ment or by calculation derived from
the metabolic pathways concern- roxybutyrate PHBsynthetase
ed 16. This gives an estimate of the NAD÷
substrate cost per tonne of PHB
produced (Table 3).
However, the basic substrate cost Poly-13-hydroxybutyrate
is not the only consideration to be I
weighed when choosing a substrate/ Depolymerization I Polymerization
organism combination. From Table 3
methanol would appear to be an
attractive substrate, especially since
ICI has experience in methanol
fermentation technology. However (a) Products of acetyl-CoA under balanced growth conditions and carbon
the amount of PHB accumulated by excess. (b) Biosynthesis and breakdown of PHB. Under conditions of carbon
methylotrophs was only moderate (energy) excess, NADH levels inhibit both the depolymerization of PHB and
meaning that more biomass has to be the tricarboxylic acid cycle (TCA). The resulting reduced levels of free CoA
processed per tonne of product; means that acetyl-CoA acyltransferase can catalyse the condensation of acetyl
CoA to acetoacetyl CoA, and that PHB can be synthesized. In energy (carbon)
there were severe difficulties with
deficiency, the control is reversed.
polymer extraction and the mol-
ecular weight of the recovered poly-

--Table 3 mer was low, restricting its applica-

tion as a useful materiaP 7'18.
Substrate prices, PHB yields, and substrate costs for PHB p r o d u c t i o n Azotobacter growing on sucrose
Yield t Substrate or glucose was another possibility:
Approximate PHBt -1 costs there was a substantial literature and
Substrate price £ t -1 substrate £ t -1 PHB body of experience on growing the
organism and producing high poly-
Methanol 90 0.18 504
mer yields. Closer examination,
Sucrose 200 0.33 600
Glucose (world market) 200 0.33 600
however, revealed that the PHB-
Glucose (EEC) 360 0.33 1080 producing strains were unstable and
Hydrogen 500 (estimate) 1.0 500 that they also synthesized poty-
Ethanol 440 0.5 880 saccharide, thereby wasting sub-
Acetic Acid 370 0.33 1220 strate.
Alcaligenes eutrophus therefore
TIBTECH - S E P T E M B E R 1987 [Vol. 5]
appeared to be the most satisfactory
candidate. It was easy to grow,
accumulated high levels of high
molecular weight polymer (up to
80% of cell dry weight) 19 and used a
range of substrates, some of which
were potentially very attractive for
PHB production.
At first sight, the use of hydrogen/
carbon dioxide/air as a fermentation
substrate looks an economically
viable alternative. An investiga-
tion of the engineering problems
involved in building plant and oper-
ating a fermentation with flammable
hydrogen/air mixtures, however,
forces the conclusion that the high
capital cost of such an installation
would make the process unecono-
mic.
Ethanol gives good yields but is
more expensive than carbohydrates,
as is acetic acid. Therefore, sucrose
as either cane sugar or molasses is
apparently the best carbon and
energy source.
Additional processing of these
carbohydrate sources would be
necessary: in both cases the sucrose
would have to be 'inverted' (con-
verted into glucose and fructose
sugar monomers) and molasses
might have to be purified to reduce
the nitrogen and phosphorus con-
centration so that fermentations
could be run under nitrogen- or
phosphorus-limited conditions. Dex-
trose would also be an attractive
substrate - it can be obtained in a
pure form, in large quantities and
requires no additional processing.
Unfortunately for those companies
operating in the EEC, the Common
Agricultural Policy has meant that
carbohydrate substrates are more
expensive for the EEC chemical
industry than for their competitors
who can obtain sugar at world prices
(Table 3). Nevertheless, growing
Alcaligenes eutrophus on glucose
still seems the best alternative for a
production process.
Production technology
ABcaligenes eutrophus is cur-
rently grown in a glucose-salts
medium in a fed batch reactor. The
phosphate content of the medium is
such that it will support only a
certain amount of cell growth: all
other nutrients are in excess. As the except that both glucose and prop-
culture grows, the phosphate supply ionic acid are fed in the second
becomes depleted and the culture stage. The hydroxyvaleric content of
enters phosphate limitation; little the polymer is controlled by varia-
PHB has been accumulated in the tion of the ratio of glucose to
cells up to this point in the fermenta- propionate in the feed. Good control
tion. During the next 48 hours, of propionate feed rate is essential,
glucose is added to the culture and since a supernatant concentration in
the dry weight of the biomass rises excess of 0.1% is toxic and prevents
considerably due to massive PHB polymer synthesis.
accumulation by the cells. Concen- The copolymers have superior
trations of PHB up to 75% of the total mechanical properties to the homo-
dry weight of the culture can be polymer. They are more flexible and
achieved. tougher which extends their versati-
The initial growth phase of the lity in end use to applications such
fermentation to the point where as bottle and film manufacture
phosphate limitation is reached which are not possible with PHB.
takes approximately 60 hours. The
total fermentation time, including Product recovery
the glucose feeding phase, is in the Polymer can be removed from the
order of 110 to 120 hours. biomass in a number of ways.
-Fig. 3 Through solvent extraction, recover-
Cells ies of greater than 90% of the
from fermenter polymer in cells can be achieved.
The cells are refluxed in hot meth-
I anol to remove lipids and phospho-
lipids and then extracted with chloro-
Cell ]
rupture form or methylene chloride. PHB is
soluble in these solvents. The solu-
I tion is filtered hot to remove cell
debris and cooled to precipitate
I 'nz'°e
treatment I PHB. The PHB, a low bulk density
white powder, is finally vacuum
I dried 2°. The process is expensive,
requires large inventories of solvent
[ Was.ing ] and heavy capital investment in
solvent recovery plant.
Development of an alternative
I non-solvent-based extraction pro-
Drying cess has been necessary to make the
Extrusion overall PHB production process
Comminution more economically attractive. Cells
are ruptured and the resultant debris
I is treated with an enzyme cocktail to
Polymer chips solubilize the non-PHB components.
Recovery and separation of poly-
After washing and flocculation, PHB
mer (PHB) from cell fermentation is recovered as white powder. The
process and subsequent conver- powder is converted to polymer
sion to polymer chips. chips in a final blending, melt
extrusion, comminution process
Two fermenter types have been (Fig. 3).
successfully used for PHB produc-
tion: a 35 000 litre air-lift fermenter The future
and various sizes of conventional The number of microbially pro-
stirred vessels up to 200 000 litres. duced biopolymers exploited by
industry is small. They are, in the
Copolymer synthesis main, the obvious products excreted
If a copolymer of PHB and PHV is or accumulated by a range of rela-
required, the fermentation is essen- tively well-known microorganisms.
tially similar to that described above It seems likely that there are other
 TIBTECH - SEPTEMBER 1987 [Vol. 5]

materials waiting to be discovered in
nature - one has only to draw the
analogy with the vast range of
antibiotics w h i c h were discovered
once the first few h a d been
characterized. Modern genetic tech-
niques enable copies or analogues of
some natural materials to be synthes-
ized and the biosynthetic p a t h w a y to
others to be m a n i p u l a t e d to advan-
tage. The future for biopolymers
looks exciting provided that t h e y can
fulfil requirements of price and
performance.
References
1 Kang, K. S. and Cottrell, I. W. (1979)
in Microbial Technology (2nd edn)
(Peppier, H. J. and Perlman, D. eds),
Academic Press
2 Jarman, T. R. (1982) European Patent
0 066 377
3 Bracke, J. W. and Thacker, K. (1985)
US Patent 4 517 295
[] [] [] []
4 Kennedy, J.F. and Bradshaw, L.J.
(1984) in Progress in Industrial Micro-
biology (Vol. 19), (Bushell, M.E.,
ed.), pp. 319-371, Elsevier
5 0 g a t a , K. (1975) in Advances in
Applied Microbiology (Vol. 19) (Perl-
man, D., ed.), pp. 209-247, Academic
Press
6 Holmes, P. A. (1985) Phys. Techno].
16, 32-36
7 Genetic.. Technology News (1987)
(March), 6-7
8 Dawes, E. A. and Senior, P. J. (1973)
Adv. Microb. Physiol. 10, 135-266
9 Martin, A., Veldhuis, C., Stegeman,
V. and Veenhuis, S. (1979) ]. Gen.
Microbio]. 112,249-355
10 Holmes, P.A., Wright, L.E. and
Collins, S. H. (1981) European Patent
0 052 459
11 Findlay, R.H. and White, D.C.
(1983) App]. Environ. Microbio]. 45,
71-78
12 Wallen, L. L. and Rohwedder, W. K.
(1974) Environ. Sci. Technol. 8, 576-
579
[] [] [] [] [] []
13 De Smit M., Eggink, G., Witholt, B.,
Kingman, J. and Wynberg, H. (1983)
J. Bacteriol. 154, 870-878
14 Ajsenjo, J. A. and Snk, J. S. (1986) J.
Ferment. Technol. 64, 271-278
15 Oeding, V. and Schlegel, H. G. (1973)
Biochem. J. 134, 239-248
16 Collins, S. H. (1987) in Carbon Sub-
strates in Biotechno]ogy (Vol. 21),
(Stowe]l, J. D., Beardsmore, A. J.,
Keevil, C. W. and Woodward, J. R.,
eds), pp. 161-169, IRL Press
17 Susuki, T., Yamano, T. and Shimigu,
S. (1986) Microbio]. Biotechno]. 23,
322-329
18. Holmes, P. A. in Developments in
Crystalline Polymers (Vol. 2) (Basset,
D. C., ed.), Applied Science Pub-
lishers (in press)
19 Heinzle, E. and Lafferty, R. M. (1986)
Eur. J. Appl. Microbio]. Biotechno].
11, 8-16
20 Stageman, J.F. (1983) European
Patent Application 124 309
[] [] [] []

Perfluorochemicals in produced from butyltetrahydrofuran

by electrolysis in a n h y d r o u s hydro-
gen fluoride (HE). In this process,
biotechnology side reactions such as isomerization
and degradation combined w i t h
incomplete fluorination result in a
Be Mattiasson and Patrick Adlercreutz complex mixture of c o m p o u n d s .
Perfluorodecalin (Fig. lb) and F-
Gas transport often is a limiting factor in biotechnology. Perfluoro- tripropylamine (Fig. 1@ are the
chemicals provide a new vehicle for the transport of gases. perfluorochemical c o m p o n e n t s of
the commercial blood substitute
Perfluorochemicals are organic the main constituent of FX-80 (later F l u o s o l DA p r o d u c e d by the Green
c o m p o u n d s in w h i c h all hydrogen called FC-75) produced by the 3M Cross Corporation. Perfluorodecalin
atoms have been replaced by company. This liquid, the first per- is prepared by fluorination of napht-
fluorine atoms (Fig. 1). Carbon- fluorochemical used for oxygen aline by CoF3 and is p r o d u c e d in a
fluorine bonds are strong, so per- transport in biological systems, was pure form by ISC Chemicals Ltd. Bis-
fluorochemicals are quite stable --Fig. 1
c o m p o u n d s and highly resistant to
heat and chemical reagents. Per-
fluorochemicals are also quite apolar F F
c o m p o u n d s which, since the solubil- F F
E F N(C3F7) 3
ity of gases increases with decreas-
ing polarity of the solvent, makes o F9
t h e m useful vehicles for the trans-
port of gases. They have, for exam- a b C
ple, been used in blood substitutes
as carriers of oxygen 1.

Properties ofperfluorochemicals C 4 F_9 C H = C H C 4 F 9 F F

F-butyltetrahydrofuran (Fig. la) is
F F

Bo Mattias,~on and Patrick Adlercreutz d e

are at the Department of Biotechnology, Perfluorochemicals. F-butyltetrahydrofuran (a), perfluorodecalin (b), F-tripro-
Chemical Center, University of Lund, PO pylamine (c), bis±(F-butyl)-ethene (d) and FHQ (e).
Box 124, S-221 O0 Lund, Sweden.
© 1967, Elsevier Publications, Cambridge 0166- 9430/87/$02.00