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Rainer Buchholz

3. Enzyme reactions

specific characteristics and activity is usually superior to that of chemical

catalysts. The term enzyme was introduced in 1877, but already in earlier

times the presence of biological catalysts was suspected. The first general

theory of biochemical catalysis was published in 1835 by J. J. Berzelius.

Pasteur observed that alcohol fermentation is catalyzed by enzymes,

however in 1860 he claimed that those enzymes are integrated in viable

yeast-cells. In 1897 Eduard Buchner succeeded in extracting enzymes for

alcoholic fermentation from yeast cells. In 1927 J. B. Sumner isolated the

first enzyme (Urease) in pure crystalline form. Today there are more than

2000 enzymes known, of which 300 were purified and crystallized.

Enzymes are usually named by adding the syllable -ase to the name of

the converted substrate. E. g. the enzyme Urease is catalyzing the hydrolysis

of urea into ammonia and carbondioxide. But there also exists a relatively

great number of meaningless and trivial names such as Pepsin, Trypsin or

Katalase. As proposed by the International Enzyme Commission,

enzymes are divided into six main groups, which again are divided into

subgroups.

- 33 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

1.1 attacking >CH-OH 4.1 addition at >C=C<

1.2 >C=O 4.2 >C=O

1.3 >CH=CH- 4.3 >C=N-

1.4 >CH-NH2

1.5 >CH-NH-

1.6 NADH;NADPH

2.1 transferring C1-Groups 5.1 Racemases

2.2 aldehyde- or keto-groups

2.3 acyl groups

2.4 glycosyl groups

2.5 phosphate groups

2.6 S-containing groups

3.1 ester 6.1 C-O

3.2 glycoside bindings 6.2 C-S

3.3 peptide bindings 6.3 C-N

3.4 other C-N bindings 6.4 C-C

3.5 acid anhydride

Classification of Enzymes

Fig.: 3-1

- 34 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

protease Bacillus unspecific endo- Washing enzymes,

Aspergillus hydrolysis of proteins, flour addition,

specific casein hydrolysis leather processing,

Mucor

cheese production

a-amylase Bacillus unspecific endo- starch

Aspergillus hydrolysis of starch to saccharification,

oligosaccharides textile and paper

processing, baking

and mash

processes

glycoamylases Aspergillus exo-hydrolysis of starch

oligosaccharides saccharification,

to glucose glucose and

ethanol production

glucose Streptomyces isomerisation of glucose starch syrup

isomerase Bacillus to fructose production

pectinase Aspergillus hydrolysis of processing of

polygalacturonic acid and juices, beer and

its methyl esters wine

cellulase Trichoderma hydrolysis of cellulose Drying of raw

resii plant material

lipase Candida sp. triglycerides, Fat hydrolysis,

Pseudomonas esterification of fatty acids improvement of

digestion,

Mucor

production of

emulsifiers and

special fats

lactase Aspergillus hydrolysis of lactose to Dairy waste water,

galactose and glucose improvement of

digestion

glucose oxidase Penicillium oxidation of glucose to glucose oxidase /

gluconic acid and katalase system for

hydrogenperoxide removal of oxygen

from canned fruit /

katalase Aspergillus removal of

vegetables and

hydrogenperoxide

beer

- 35 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

In principle the above discussed reaction kinetics are directly valid for

enzymatic reactions. But there is one remarkable difference to non

enzymatic reactions:

Enzymatic reactions exhibit the phenomenon of substrate saturation.

Vmax

V0

1/2 Vmax

Km substrate concentration

reaction A P

substrate concentration, it is a first order reaction in substrate.

b) For rising substrate concentrations the growth of reaction rate slows

down, the reaction has a mixed order in this region.

c) For high substrate concentrations the reaction rate is constant, the

reaction is zero order, the enzyme is saturated by the substrate.

- 36 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

L. Michaelis and M. L. Menten and later expanded by G. E. Briggs and

J. B. S. Haldane. For simplification, it is assumed, that the enzyme E and

the substrate S form a complex ES, which decomposes into enzyme E and

product P (in practical applications more than one transition stage occurs).

k1 k2

E S ES E P (3 1)

k 1 k 2

reaction from E and P) is:

dES

k1 ET ES S (3 2)

dt

[ES] = Conc. of enzyme-substrate complex

[ET] - [ES] = Conc. of free enzyme

[S] = Substrate concentration

however this part can easily be neglected at start velocity V0, especially if [S]

is extremely high and [P] is near zero.

equation applies:

dES

k 1 ES k 2 ES (3 3)

dt

- 37 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

exists:

k1 ET ES S k 1 ES k 2 ES (3 4)

ET ES S k 1 k 2 KM (3 5)

ES k1

complex follows:

ES Et S (3 6)

K M S

rate:

V0 k 2 ES (3 7)

Putting eq. (3 7) into eq. (3 6) one gets:

V0 k 2

Et S (3 8)

K M S

If the total amount of enzyme is bound, the maximum initial production rate

follows:

Vmax k 2 ET (3 9)

Vmax S

V0 (3 10)

K M S

This rate equation for enzyme catalytic reactions connects the initial reaction

velocity, the maximum of reaction velocity and the initial concentration of

- 38 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

enzyme is found in the expression Vmax.

equal to the substrate concentration at which the reaction rate is half of the

maximum reaction rate. In the case of a single substrate reaction the value

for KM has the dimension of a concentration (mol/l) and is not affected by

the enzyme concentration.

The value for KM should not be mixed-up with the dissociation constant KS!

The only exemption is given in the case for k2 << k1. In this case you find:

KM

k 1

and K M K S

E S

k1 ES

c .c / mol L -1

E ES c S .c P / mol L -1

0.020

0.10

0.015

0.08

P

0.06

0.010

0.04

S

0.005 ES

0.02

E

0.000 0.00

0 50 100 150 200 250 300 350

time / s

Fig.: 3-3

- 39 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

[Product]

[E]

[Substrate] [ES]

[Product]

Time

[E] [ES]

Time

In order to simplify evaluation of experimental data, eq. (3 10) is

linearized by inverting and rearranging:

1 K S

M

V0 Vmax S

or

1

KM

S

V0 Vmax S Vmax S

1 K 1 1

M (3 11)

V0 Vmax S Vmax

By plotting 1/V0 versus 1/[S] the Lineweaver-Burk plot results, this yields for

Michaelis-Menten kinetics a straight line.

- 40 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

1

V0

slope Km

Vmax

1

Vmax

1

-1 S

Km

- 41 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Eadie-Hofstee

V0

V0 Vmax K M (3 12)

S

Vmax

slope -Km

V0

Vmax

Km

V0

S

Fig. 3-6: Eadie-Hofstee plot

- 42 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Hanes

S KM

S (3 13)

V0 Vmax Vmax

1

slope

S Vmax

V

-Ksm S

S

activity curve is bell-shaped in most cases, with a narrow optimum. This

optimum pH however, is not necessarily the pH-value within the cell. The

assumption seems reasonable that there is an influence of activity control of

the enzymes by the pH inside the cells.

- 43 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

trypsin

pepsin

6 8 10 2 4 6

pH pH

papain (substrate =

cholinesterase benzoylargininamid)

6 8 10 4 6 8

pH pH

optimum due to an increase at low temperature ranges like chemical

reactions followed by a clear decrease at a higher temperature. This can be

viewed as a result of two processes:

reactions)

2) Decreasing activity caused by thermal denaturation of the enzyme

quite resistant however, especially enzymes from thermophilic bacteria.

- 44 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Some enzymes (e.g. ribonuclease) lose their activity during heating but

regain it after cooling.

kinetics, but deviations from this model can be observed. Typical deviations

from the simple model are shown below:

a b

rmax rmax

r r

S S

Fig. 3-9: Substrate activation without (a) and with (b) substrate inhibition

helps binding more substrate molecules (cooperative effect which

occurs if the enzyme-molecule consists of protein-groups of the

same structure. Such an oligomer can bind several substrate

molecules)

c.f. b) Activation at low substrate concentration: For high substrate

concentrations the reaction rate decreases (inhibition).

- 45 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

2) Two substrates react at the same enzyme to one product.

3) The enzyme has to be regenerated after formation of the product.

inhibition, the enzyme and the inhibitor form a stable complex; the

enzyme cannot be activated again. Such inhibitors are for example heavy

metals (e. g. lead, silver, mercury, cadmium) or ions which are capable of

forming stable metal complexes like the cyanide ion. This type of inhibition

is also called enzyme poisoning.

The reversible inhibition is characterized by an equilibrium for the

enzyme-inhibitor complex. Inhibitors can act on the enzyme, on the

enzyme-substrate complex, or both. Another possibility is a reaction of the

inhibitor with the substrate.

the active center of the enzyme. It is likely to happen if the molecular

structure of substrate and inhibitor are similar. Additionally, the product

may also act as a competitive inhibitor. A simple reaction scheme for

competitive inhibition is shown below.

- 46 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

k1 k2

S ES E P

k 1

E (3 14)

ki

I EI

k i

EI: Inactive enzyme-inhibitor complex

and inhibitor I, the inhibition-constant KI is applied, which is comparable to

the dissociation-constant KS of the enzyme-substrate complex.

KI

E I (3 15)

EI

The reaction rate for conversion of substrate in the presence of an inhibitor I

is:

V0 Vmax

S (3 16)

K M 1 I / K I S

increasing the substrate concentration, the competitive inhibition can be

decreased or completely suppressed. The linearized form of eq. (3 16) for

the Lineweaver-Burk plot (Fig. 3 10) is:

1 K 1 1

M 1 I / K I (3 17)

V0 Vmax S Vmax

- 47 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

inhibitor. Both bind at different sites, but the inhibitor influences the

substrate activation. This type of inhibition cannot be suppressed by

increasing the substrate concentration. The inhibitor may bind at the

enzyme, at the enzyme-substrate complex, or at both.

k1

ES

k

ES

k

EP

1 2

(3 18)

I I

kI k I

k I k I

EI ESI

For the reaction rate holds:

1

Vmax S

1 I / K I

V0 (3 19)

KM S

maximum reaction rate is decreased. For graphical representation, eq. (3

19) is linearized (c.f. Fig. 3 10 b):

1 K 1 1

M 1 I / K I 1 I / K I (3 20)

V0 Vmax S Vmax

- 48 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Uncompetitive inhibition means that the inhibitor may only react with the

enzyme-substrate complex, not with the free enzyme.

k1 k2

E S ES E P

k 1

(3 21)

kI

I ESI

k I

1

Vmax S

1 I / K I

V0 (3 22)

KM

S

1 I / K I

1 K 1 1

M 1 I / K I (3 23)

V0 Vmax S Vmax

rate are reduced.

- 49 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

competitive non-competitive

inhibition inhibition

inhibited reaction inhibited reaction

V0 V0

slope slope

1 1

[S] [S]

uncompetitive substrate

inhibition inhibition

Slope of the Slope of the

1 inhibited reaction 1 inhibited reaction

V0 V0

slope slope

1 1

[S] [S]

concentrations on the reaction. In this case the substrate binds at the ES

complex, forming an ESn complex, which cannot decompose into enzyme

and product.

- 50 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

k1 k2

E S ES E P

k 1

(3 24)

k3

S ES2

k 3

V0 Vmax

S (3 25)

K M S S2 / K 3

whereby

KM

E S k 1 k 2

ES k1

K3

ES S k 3

ES2 k 3

Linearization of eq.(3 25) results in (c.f. Fig 3 11 d):

1 K 1 1 1

M S (3 26)

V0 Vmax S Vmax K 3 Vmax

Although this will not be a straight line in the Lineweaver-Burk plot, there

are two parts of the curve. For low substrate concentrations it follows:

1 K 1 1

M Michaelis Menten (3 27)

V0 Vmax S Vmax

For high substrate concentrations:

1 1 1

S (3 28)

V0 Vmax K 3 Vmax

- 51 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

pH, temp., enzyme and design

substrate concentration

properties of

thermodynamics kinetics

biocatalyst

Keq Mr, mechanical fragility, Vm, Km, Ki

activity, stability

process reactor

Tau, XA, productivity, design

enzyme consumption

Fig 3-11

Definitions

(R)-Product

(R,S)-

Substrate (S)-Product

molS

Turn Over Frequency (TOF) =

molcat * time

1

Deactivation Number (kde) =

time

TOF

Total Turnover Number (TTN ) =

kde

- 52 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Definitions

(R)-Product

(R,S)-

Substrate (S)-Product

S

conversion (X) = 1-

S0

(R)-Product

Yield (Y) =

S0

(R)-Product

Space-Time-Yield (STY) =

Tau

(R)-Product

Selectivity =

S0 - S

S0 - S

S0 = initial substrate concentration

Tau = residence time

100 100

80

enantiomeric excess (ee) / %

(R)-product

(R,S)-substrate

(R) 50 (S)-product 10

ratio R/S / -

90

0 1

(R)-product

(S) 50 selectivity =

S0 - S 0.1

(R)-Product - (S)-Product

ee =

S0 - S

100 0.01

0 20 40 60 80 100

chemical selectivity / %

- 53 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

4. Growth Kinetic

4.1 Biomass

Target of a quantitative description of the growth rate of any kind of

organisms or cells is the estimation of the functional correlation of biomass

increase during time.

dX g (biomass) 1

dt lfermentation broth time

approximation possible. Furthermore, numerous parameters are not

detectable and an experimental proof of a model description is only rarely

possible. Whether there is a sensible use of expert systems and

mathematical models for the process description depends on the given

cultivation system. In some cases it was possible to develop the whole

process control by using process models.

For measuring the biomass in a biological culture two different methods can

be applied.

2) Cell number (cell counting, optical density)

weight of the cells and some ingredients of the cells is given in Fig. 4-1.

- 54 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

10

3

5

2

2

1 1

time

accurate method. One of the noticeable disadvantages of this method is the

fact, that it is non on-line. Furthermore, it is impossible to distinguish

between vital and non-vital cells, however, this method will fail, provided

substrates of solid consistency are used.

In order to determine the amount of yeast and bacteria the cell count as

well as the dry-weight can be used. For pellet or mycel forming organisms

(e.g. fungies, streptomyces etc.) the method of dry-weight estimation is the

most dependable procedure.

For the biomass estimation the DNA and RNA concentration can also be

applied. During the lag phase the RNA content increases slightly and the

DNA content decreases, but during the exponential growth the relation

between both is constant. At the end of the growth phase, the stationary

phase, the RNA content decreases and the DNA content increases.

- 55 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Exponential growth

different species.

V

IV

VI

III

II

I

time

I. Lag phase

N N 0 konst (4 1)

2

t t0

N N 0 N L N 0 (4 2)

tL t0

Cells proliferate by division, for the generation time g, the number of

generations n and time t holds:

- 56 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

t

g (4 3)

n

Is the initial number of cells N0, for n divisions it follows:

N N 0 2n (4 4)

The number of divisions per hour is defined as division rate v:

n log N log N 0 1

(4 5)

t log 2 t t 0 g

change of biomass is proportional to the amount of biomass. Defining the

specific growth rate , the growth rate increases after the lag phase and

reaches the value max, which is constant during the exponential phase.

Consequently, the growth is a first order reaction. Therefore from the mass

balance follows:

dX

max X (4 6)

dt

Integration with the initial condition X = X0 at t = t0 :

X

ln max t (4 7)

X0

X X 0 e max t (4 8)

The time for doubling the amount of biomass is called doubling time td .

It may be calculated by inserting X=2 X0 into eq. (4-8):

2X 0 X 0 e max t (4 9)

ln 2 0.693

td (4 10)

max max

The specific growth rate and the specific rate of division v are not

necessarily equal. Only in the case of a constant cell size and the division of

one cell into two cells the are values equal.

- 57 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Table 4-1: Maximum growth rates for different microorganisms (substrate: glucose)

Aspergillus nidulans 20 0.09 7.72

25 0.148 4.68

30 0.215 3.23

37 0.360 1.96

Penicillium

25 0.123 5.65

chrysogenum

Mucor hiemalis 25 0.17 4.1

Fusarium avanaceum 25 0.18 3.8

Fusarium

30 0.28 2.48

graminearum

Verticillium

25 0.24 2.9

agaricinum

Geotrichum candidum 25 0.41 1.7

Geotrichum candidum 30 0.61 1.1

Neurospora sitophila 30 0.40 1.73

Candida tropicalis 30 0.60 1.1

Bacillus

60 0.18 3.8

stearothermophilus

Escherichia coli 40 0.35 2.0

Bacillus subtilis 40 0.43 1.6

- 58 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

(Monod equation)

1.0

(5)

0.8

(4)

0.6

(3)

0.4

(2)

0.2

(1)

1 2 3 4

time t [h]

Fig. 4-3: Growth of E. coli under various initial glucose concentrations

(conc. In g/l: (1)=0, (2)=15, (3)=25, (4)=35, (5)=45)

In this simple case, the maximum specific growth max rate is independent of

a substrate concentration. The maximum biomass concentration at the end

of the process however, is a function of initial substrate concentration. This

leads to the definition of the yield coefficient YX/S :

X max X 0 g Biomass

YX / S g Substrate (4 11)

S0

In the preceding example the yield coefficient is constant at about 0.45, but

influences by other substrates or products may change the linear relation

between biomass produced and substrates consumed:

- 59 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

dX

dt

YX / S is not necessarily constant ! (4 12)

dS

dt

the substrate concentration is shown in Fig. 4 8.

max

1/2 max

1 2 3 4 5

-4

glucose concentration [10 mol/l]

Fig. 4-4: Specific growth rate vs. substrate concentration (E. Coli

cultivation using glucose as carbon source)

The observations are similar to the reaction rate of the Michaelis Menten

equation. Therefore, in analogy the equation is set up:

max

S (4 13)

K S S

The analogy between enzyme kinetics and microbial growth was first

described by Monod (1942). Provided there are two limiting substrates, the

specific growth rate is formulated accordingly:

- 60 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

max

S1 S2 (4 14)

K S S1 K S S2

1 2

Setting up the mass balances for microbial growth in a batch reactor using

the simple Monod equation, two coupled differential equations result:

dX

max

S X (4 15)

dt K S S

dS

1

max

S X (4 16)

dt YX / S K S S

simulations may be used to predict the time course of bioprocesses and to

control the optimal conditions during cultivation.

treatment, the problem of inhibitory or toxic substrates arises. In analogy to

substrate inhibition, Haldane expanded the Monod equation to include

such effects in the description of microbial growth:

max

S (4 17)

K S S

S

2

K1

The differential equations for modelling a batch process are:

dX

max

S X (4 18)

dt

K S S

S

2

K1

dS

1

max

S X (4 19)

dt YX / S

K S S

S

2

K1

- 61 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

maximum growth rate is only reached at the end of cultivation in a batch

process. Moreover, only at the end of the batch process the substrate

concentration is below the inhibitory level. It is therefore preferable to use

fed-batch or continuous processes to degrade inhibitory substrates.

media several exponential growth phases were found. Conclusively, more

than one substance from the yeast extract serves as a substrate and those

substrates were utilized after each other. Consequently, the Monod model

was extended by Tsao and Hanson.

dX 1l S1l 1 jS1 j

X .....

dt K S K1 j S1 j

1l 1l

2l S2l 2 j S2 j

..... (4 20)

K S K 2 j S2 j

2 l 2 l

il Sil ij Sij

.... .....

K S K ij Sij

il il

with:

ij specific growth velocity with Sij

Sij substrate concentration

Kij Monod constant at given substrate

different growth increasing substances j (j = 1 ... j). Those substrates j

appear as summands at the different Monod terms.

(sodium)-citrate is formulated very well using this model (i =1; j = 2):

- 62 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

dX S S

X 1 1 21 2 (4 21)

dt K1 S1 K 2 S2

The substrates S1 and S2 are consumed mainly one after the other.

mg/l

2,0

1,5

1,0

ln X

0,5 ln S1

10

ln S2

10

0 10 20 30 40 50 h

Fig. 4-5: Growth of Proteus vulgaris with glucose (S1) and Citrat (S2)

- 63 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

data received from a batch fermentation process correctly. For this reason

several empirical modified equations are found in literature. Most of them

can be reduced to the following power equation:

dX

K max X

p

(4 22)

dt

with

K = constant

p = exponent

In the case of fast growth an adaptation of the Monod equation compared

to experimental data is often not possible. Here the following equations are

used:

max

S (4 23)

K So S0 S

or

max

S (4 24)

K S K So S0 S

KSo and KS = constants

Using these models the experimental behaviour of the deviation from the

exponential growth by increasing start concentration (S0) of the substrate

instead of approaching max can be described.

- 64 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

This model starts out of the fact that the population is regulated by the

restricted space available. It is borrowing from the animal growth in nature.

In contrast to the pure exponential growth a mathematical function is

introduced which reduces the growth by increasing cell density. This

enhanced model is given by:

dX X

max X 1 (4 25)

dt X max

whereby Xmax is given by the maximum cell density in the stationary phase.

With increasing cell density the expression in the bracket decreases until the

change dX/dt becomes 0 by the maximum cell density. The equation (4

25) can be read as a combination of a growth term in

the 1st order and a dying term in the 2nd order.

dX

max X max X 2 (4 26)

dt X max

X max

exponential

growth evironment resistance

dX

= max X actual growth

dt

dX X

= max X 1-

dt X max

time t

- 65 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

and t and X0 and X leads to:

X0

max t

X max 1 e X max

X (4 27)

The logistic model itself describes only the part of the growth curve between

the exponential and the stationary phase.

1

Moser, H.: max (4 28)

1 K S B

S

(4 29)

S

Contois, D. E.: max (4 30)

BX S

using a transition complex, meaning the biological growth behaves similar

to the enzyme kinetic.

k1 k2

X S X...S XS P (4 31)

k 1

with:

(X...S) = transition complex

[XS] = new biomass

k1 k2

E S / X EXS XS E P (4 32)

k 1

- 66 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

The conversion of substrate is carried out in three steps.

1) Interaction between substrate and surface of the microorganism

2) Transport into the inside of a cell

3) Enzymatic reaction

Only the substrate which has direct contact with the cell can react. The

temporary change of the transient complex is given by:

dEXS S

k1 E k 1 k 2 EXS (4 33)

dt X

- 67 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

E S k 1 k 2 K (4 34)

EXS X k1

or:

EXS E S

KX

The specific growth rate is given by:

1 dX

k 2 EXS (4 35)

X dt

The maximum growth rate can be obtained, if the total amount of enzymes

are available as an EXS-complex:

max k 2 E t k 2 E EXS

k 2 E

E S (4 36)

KX

1 S

k 2 E 1

K X

Dividing by max leads to:

k2

E S/ X

(4 37)

max K k 2 E 1 S / X 1 / K

That means:

S/ X

max

K S/ X

or:

S

max (4 38)

KX S

This equation is formally equal to the Monod equation containing a

substrate limiting constant KS which depends on the cell density X .

K S f X (4 39)

- 68 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

This model can describe the influence of the initial cell density during the

lag phase in a certain qualified sense.

The decline phase is not describable because never becomes negative.

The Monod model can not be used without restrictions for fungi cultures,

due to the different rules compared to bacteria or yeast in growth

behaviour. E.g. the fungi Penicillium chrysogenum (belonging to the group

of Plectomycete) is growing filamenteously. The hyphes are equipped with

multiple branches. The active growth of this type of fungi is restricted to the

top of the hyphes. The growing area is small compared to the entire

biomass of mycelia, and for each top of the hyphes this area remains

constant. In literature one can find three to five stages of cell distinctions

with different nutrient consumptions, age distributions and metabolite

productions.

In submerged culture two extremes of morphology can be observed: the

filamenteous and the pellet form. If pellet behaviour is found, numerous

spherical compact cell clusters are formed. These clusters are only growing

on the outer surface. If filamentuous growth is observed a mycelia of low

density is formed. Ideally, in the case of filamenteous growth all hyphes are

homogeneously supplied with nutrients. Only then a kind of exponential

growth can be observed.

The growth of mycel-forming organisms is shown in the next graph.

- 69 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

apex

t0

X

t1

X

t2

X0 X1 X2

The hyphes consist of the cell membrane and the cytoplasma with its

inclusions. The hyphes grow in one dimension only at the top (apex), also

named apecal growth. Some organisms form so-called septa (Mycomycetes

like Aspergillus spec. and Penicillium spec.). Others grow without forming

septa (Phycomycetes like Mucor spec. and Rhizopus spec.).

The pellet growth can not be described by the Monod equation (Emmerson:

J. Bacteriol. 60, 221 (1950)), therefore the following expression was

developed:

dX 2

Rx X 3 (4 40)

dt

- 70 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

1

X X 03 t

3

(4 41)

2

6 3

k g const.

with

= pellet density

Kg = dRP / dt

RP = radius of pellet

Pirt found the same relations using the assumption that oxygen is

transported only by diffusion into the pellet. Consequently, growth can only

take place in a zone at the surface due to the lack of oxygen in the centre of

the pellet. It is often found that the pellets with a diameter of more than one

millimeter are hollow spheres.

- 71 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

4.2.1 Fundamentals of the product formation in batch processes

groups:

1) Production of biomass

2) Production of metabolites

The following tables give an incomplete summary on the spectra of

fermentation processes.

fermentation substrates condition type

fodder-, nutrient and other pulp, paraffin,

yeast yeasts sulfite liquors

bacteria species hydrolysate anaerobic

bacteria Bac. popilliae hydrolysate

Buc 052-1

hydrolysate

- 72 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

fermentation substrates condition type

hydrolysate

acetobutylicum hydrolysate

hydrolysate

Buc 051

fermentation substrates condition type

Brevibacter spec. molasses

5- ribonucleotide various bacteria salts + certain

purines

ca. 50 different Streptomyces sp. complex substrates

products Bacillus sp. a.o.

polyol Buc 049

- 73 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

fermentation substrates condition type

carotenoids trispora a.o.

shermanii hydrolysate

hydrolysate

fujik. hydrolysate

steroids ochraceus a.o. steroids

steroids a.o. appropriate

double bond

Buc 0501

fermentation substrates condition type

Bac. subtilis reduced germs

reduced germs

(mixtures) Trichoderma a.o.

6.

-galactosidase Aspergillus sp. lactose non sterile, aerobic

Saccharomyces sp. reduced germs

Buc 047

- 74 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

fermentation substrates condition type

reduced germs

8. acetic & neutral various fungi plant protein non sterile aerobic

protease various bacteria

(cleaning enzyme)

10. lipase fungi, bacteria plant oil, train oil reduced germs aerobic

and other hydrolysate

and other hydrolysate semi aerobic

Buc 048

fermentation substrates condition type

drinks raw materials, reduced germs

starch

milk products a.o. milk protein anaerobic

plant products microorganisms material anaerobic

Buc 043

- 75 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

The following tables show the compositions of the mainly used carbon

sources. For being competitive waste materials are often used.

Carbohydrates are the traditional substrates of the fermentation industry.

Pure glucose or saccharose are seldom used as substrate due to its price.

The only exception is the demand on pure and exactly defined substrates

for product validation.

Molasses

most inexpensive substrates. Other valuable compounds and tracers are

contained under a high concentration of saccharose. The main

disadvantage is a remarkable change in quality depending on the raw

material, the region, the climatic conditions, and last but not least on the

production processes of the sugar companies.

Hydrol-molasses, a by-product of the hydrolysis of starch, is also often

used in fermentations.

Malt extract

as the only carbon source. Carbohydrate, accounting for more than 90%,

contains hexoses like glucose and fructose, di-saccharides like maltose and

saccharose, tri-saccharides like maltotriose, and several dextrines.

Additionally, various proteines such as peptides, amino acids and vitamines

are contained in malt extract.

Other suitable raw materials for fermentations are starch, dextrines, and the

sugar containing wastes of the cellulose industry. Also vegetable oils,

alcohols and alkanes are used as substrates.

- 76 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

and sugar cane

Sugar Beet and Sugar Cane

(Rhodes and Fletscher, 1966; Imrie, 1969)

Composition Sugar beet Sugar cane

Dry substance % 78 - 85 77 - 84

Saccharose 48.5 33.4

Raffinose 1.0

Invert sugar 1.0 21.2

Organic non sugar 20.7 19.6

N 0.2 - 2.8 0.4 - 1.5

P2O5 0.02 - 0.7 0.6 - 2.0

CaO 0.15 - 0.15 0.1 - 1.1

MgO 0.01 - 0.1 0.03 - 0.1

K2O 2.2 - 4.5 2.6 - 5.0

SiO2 0.1 - 0.5

Al2O3 0.005 - 0.06

Fe2O3 0.001 - 0.02

Ash 4 -8 7 -11 Buc 045

g /100 g dry weight

Riboflavin 41 250

Pyridoxine 540

Buc 046

- 77 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

(dry)

Maltose 52.2

Saccharose 1.8

Dextrin 15.0

Ash 1.5

pH (10% solution) = 5.5 Buc 042

complete concentration of product

P

fermentation time

The volumetric rate is given by the change in concentration in time [mass

per volume and time]. The specific rate is the volumetric rate divided by

the biomass concentration [mass per time and biomass].

- 78 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

max.

productivity

overall productivity

productivity P/t

Maxon (Appl. Microbiol. 3, 110 (1955)); Deindoerfer (Adv. Appl.

Microbiol. 2, 321 (1960)); Gaden (Chem. & Ind. London, p 154 (1955)

and J. Biochem. Microbiol. Technol. Eng. 1, 413 (1959).

For the kinetic considerations the classification given by Gaden is preferred.

Classification by Gaden:

Type of fermentation I:

This type is issued if the product is deduced directly from the primary

metabolism. For example, ethanol, lactic acid and gluconic acid are some

potential products next to pure biomass. In this case, growth, substrate

consumption and product formation are running under nearly identical

conditions. Trophophase (exponential growth phase) and Idiophase

(production phase) can not be separated from each other.

Example: alcohol production with Saccaromyces cerevisiae.

- 79 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

a) Cell mass- and product concentrations 5 25 50

P Alcohol

S Substrate used

and substrate uptake,

4 20 40

b) Growth-, product formation- and X P S X

substrate uptake rates and 3 15 30 P

c) specific rates as function of time 2 10 20 S

1 5 10

0 0 0

a)

0 2 4 6 8 10 12 14

RX Growth, g/(lh)

RP Alcohol synthesis, g/(lh) 0.25 1.2 2.5

RS Sugar utilization, g/(l

h)

5 10 0.20 1.0 2.0

1

4 8 0.8

0.8 0.15 1.5

RP,RS 0.6 ,

0.6 3 6

RX 0.10 1.0

RX RP RS

2 4 0.4

0.4 Growth, g/(g h)

0.05 0.2 0.5 Alcohol synthesis, g/(g

h)

0.2 1 2 Sugar utilization, g/(g

h)

0 0 0 0

0 0

0 2 4 6 8 10 12 14

b) 0 2 4 6 8 10 12 14 c)

Time [t]

Buc 036

negative free energy.

A products

A B C products

It is typical for these types to have only one maximum in all rates. These

maxima coincide roughly.

Also, in this case the product is deduced from the primary metabolism for

energy uptake. The biochemical reaction is carried out by bypassing the

metabolism. It is then separated from the primary metabolism.

substrate A B C D primary metabolism

E F product

- 80 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

This type leads to two maxima in all volumetric rates during a batch

fermentation. In the beginning there is only growth in biomass, a high

consumption of substrates, and no or only a small product forming. Later

the growth is reduced, but by using a high consumption of substrate,

product formation is induced. Trophophase (exponential growth phase) and

Idiophase (production phase) are separated from each other.

Examples for this type of fermentation are the production of citric acid,

itaconic acid and some amino acids like glutaminic acid.

24 240 S

X Biomass dry wt, g/l

20 200 P Citric acid, g/l

Titrable acidity

Citric acid

a) Cell mass- and product concentrations 16 160 S Substrate

X

used

and utilized substrate,

X 12 P,S 120

b) Growth-, production and substrate

uptake rates and 8 80 P

a)

0 0

0 40 80 120 160 200 240 280

Time [t]

0.14 0.28

0.10 10 RX Growth, g/(lh) 0.12 0.24

RP Acid synthesis, g/(lh) Growth, g/(g h)

RS Sugar utilization, g/(l

h) Acid synthesis, g/(gh)

0.10 0.20 Sugar utilization, g/(g

h)

0.08 1.6

RX RP ,RS

0.08 0.16

0.06 1.2

,

0.06 0.12

0.04 0.8 RP

RX RS

0.04 0.08

0.02 0.4

0.02 0.04

0 0 b) c)

0 40 80 120 160 200 240 280 0 0

0 40 80 120 160 200 240 280

Time [t] Time [t]

Buc 037

- 81 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

Here the primary metabolism is also separated from the product formation.

Contrary to type II the product is not deduced from the primary metabolism

for energy uptake, but from the amphibolism (secondary metabolism). In

the beginning of these fermentations, a growth of biomass is found (primary

metabolism), while later on the product is formed by secondary metabolism.

Examples of this type of fermentations are the antibiotic productions such as

penicillin, streptomycin, etc. Also synthesis of vitamines, fats and some

biopolymers belong to this group.

25 1.25

S

20 1.00

a) Cell mass- and product concentrations

and utilized substrate, 15 0.75 X

X,S P

P

b) Growths-, production- and substrate 10 0.50

conversion rates and

X Biomass dry wt, g/l

c) specific rates as function of time 5 .25 P Penicillin, g/l

S Substrate us ed, g/l

0

a) 0 20 40 60

Time [t]

80 100 120 140

RP Penicillin synthesis, 0.25 0.0012 0.12 Growth, g/(g h)

g/(l

h) penicillin synthesis,

0.4 0.015 0.8 RS Sugar utilization,

0.10 g/(g

h)

g/(l

h) 0.20 0.0010 Sugar utilization,

QO2 Oxygen uptake, g/(l h) g/(g

h)

0.3 0.6 0.0008 0.08 Q2X-1 Oxygen

0.010 0.15 uptake, g/(g h)

, Q2,X-1

0.2 0.4 0.0006 0.06

RX ,RS RP QO2

0.005 QO2 0.10

0.1 0.2 RS 0.0004 0.04

RX RP Q2X-1

0 0 0 0.05 0.0002 0.02

-0.1 0

0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

b) Time [t] c) Time [t]

Buc 038-1

growth- and substrate consumption rate, and also another maximum of

product formation which is moved in time.

- 82 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

follows:

Time Time

4.3.1 Model developed by Luedeking and Priret

(Lit.:R.Luedeking; E.L.Piret; J.Biochem. Mikrobiol. Technol., 1, 393 (1959)

non growth related production rate . It is also based on the classification

given by Gaden.

The production rate is demonstrated by the following equation:

dP dX

X (4 42)

dt dt

divided by the biomass the following equation is valid:

1 dP

(4 43)

X dt

The coefficients and can be obtained by the graphic (5-14).

- 83 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

= 1/X dX/dt

First, this model was used to estimate the kinetic of the production rate of

lactic acid. It was shown that the production is neither alone proportional to

the growth rate nor to the biomass concentration. Using equation (4-42) the

following types of fermentation can be distinguished.

Type A: The production rate is not related to the growth rate, that means

=0

dP

X (4 44)

dt

Type B: The production rate is only related to the growth rate, that

means = 0

dP dX

(4 45)

dt dt

An example of this particular type is the fermentation of sorbose with

Acetobacter suboxidans.

the cell density

dP dX

X (4 42)

dt dt

- 84 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

delbrckii.

Type C

Type B

Type A

1/XdX/dt

Luedeking & Piret

dP 1 dX

dt dt

dP 1 dX

dt max dt

that means

dP dX

const

dt dt

In some cases the formed product is not stable, e.g. penicillin production.

Here the product hydrolysis is in an aqueous solution identical to a 1st order

reaction. This phenomena can be taken into account by using a destruction

term:

- 85 -

Bioprocess- and Bioreaction Engineering Prof. Dr. Rainer Buchholz

dP dX

X P (4 46)

dt dt

analogy to the enzyme kinetics.

Competitive inhibition:

dX 1

max X (4 47)

dt P

K S 1 S

K P

Noncompetitive inhibition:

dX S KP

max X (4 48)

dt KS S K P P

linear equation defined by Hinshelwood:

dX

X 1 a P (4 49)

dt

a: empiric proportional constant

- 86 -

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