Strategies for Optimizing Microbial Growth and Product Formation

8

Strategies for

Downloaded by UNIV OF MASSACHUSETTS AMHERST on February 26, 2016 | http://pubs.acs.org Publication Date: January 18, 1983 | doi: 10.1021/bk-1983-0207.ch008

and P r o d u c t

Optimizing

Microbial

Growth

Formation

CHARLES L. COONEY Massachusetts Institute of Technology, Department of Nutrition and Food Science, Biochemical Engineering Laboratory, Cambridge, MA 02139

An essential element for fermentation processes, wheth er for cell mass or product, is the efficient growth of the organism. Optimizing the environment for growth requires balancing the economic feasibility of maintain ing a suitable chemical and physical environment, while meeting the physiological needs of the organism. Using several examples of bacteria, yeast and mold fermenta tions, strategies for optimizing growth are examined and compared. In the first case, the objective of the fermentation is cell mass production; two examples are Baker's yeast (Saccharomyces cerevisiae) and single -cell protein (Hansenula polymorpha) production. In seeking to optimize productivity and conversion yields, carbon source and oxygen become key operating vari ables. The second case focuses on production of en zymes; in particular, the production of heparinase by Flavobacterium heparinum and α-glucosidase (maltase) by Saccharomyces italicus. The objective functions are maximum total activity and maximum productivity. Im portant variables are growth rate and composition and concentration of nutrients in the environment. The level of these enzymes is subject to metabolic regulation in response to the environment and the productivity de pends also on microbial growth. Lastly, the third case to be examined is metabolite production; the production of penicillin by Penicillium chrysogenum is discussed. Optimization of growth must consider not only rapid accumulation of cell mass, but also conditioning of the cell to maximize the rate and extent of product forma tion. Maintenance of adequate nutrients in the environ ment, with high levels of some and low levels of others, is essential. An analysis of strategies to meet the meta bolic demands provides insight into ways to improve the organism through genetic engineering, as well as the process through bioreactor design. 0097-6156/83/0207-0179$06.00/0 © 1983 American Chemical Society

In Foundations of Biochemical Engineering; Blanch, Harvey W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


180

BIOCHEMICAL ENGINEERING

T h e common theme i n the development of optimum fermentation strategies i s "environmental management". T h e examples des c r i b e d here cover a spectrum of fermentations i n c l u d i n g cell mass, enzyme a n d secondary metabolite p r o d u c t i o n , y e t i t will become apparent that there i s a common need for c a r e f u l e n v i r o n mental management with particular emphasis on carbon source management throughout the fermentation. T h e objective functions important to fermentation process optimization a r e : volumetric p r o d u c t i v i t y , p r o d u c t concentration, c o n v e r s i o n y i e l d a n d p r o c e s s r e p r o d u c i b i l i t y . Maintaining a h i g h p r o d u c t i v i t y will allow one to maximize r e t u r n on capital i n v e s t ment. T h i s i s p a r t i c u l a r l y important f o r the development o f new p r o c e s s e s , as a consequence of the capital i n t e n s i v i t y o f fermentation processes. T h e need to achieve a n d maintain h i g h p r o d uct concentrations reflects the importance of r e c o v e r y i n the cost of p r o d u c t i o n . Achievement o f h i g h c o n v e r s i o n y i e l d s i s necess a r y to minimize raw materials cost. F u r t h e r m o r e , a h i g h conv e r s i o n y i e l d from the carbon source will minimize the demand for o x y g e n a n d the need f o r heat removal, thus minimizing these ope r a t i n g costs ( 1 ) . L a s t l y , r e p r o d u c i b i l i t y i s essential to minimize both capital and o p e r a t i n g c o s t s , as well as meet p r o d u c t i o n goals. T h e s e objective functions are not equally important, however, i n all fermentations as i l l u s t r a t e d i n the following b r i e f case studies which a r e compiled here to illustrate the importance of e n v i r o n mental management i n d e v e l o p i n g strategies for optimizing microbial growth a n d p r o d u c t formation. Production o f C e l l Mass T h e manufacturing costs for the p r o d u c t i o n of microbial cell mass are dominated b y : (i) the raw materials cost, especially the c a r b o n source; (ii) capital investment, i n the case o f a new manuf a c t u r i n g facility; a n d , (iii) e n e r g y c o s t s , with most of the energy b e i n g used for o x y g e n t r a n s f e r a n d heat removal (1, 2). A s a consequence, it i s essential to achieve h i g h c o n v e r s i o n y i e l d s while maintaining h i g h p r o d u c t i v i t i e s from the b i o r e a c t o r . T w o approaches to these goals a r e i l l u s t r a t e d b y the following: Baker's Yeast P r o d u c t i o n . One of the oldest fermentation processes for the p r o d u c t i o n o f cell mass i s Baker's yeast p r o d u c t i o n . T h e actual p r o d u c t of the fermentation i s cell mass a n d i t s " b a k i n g power", o r ability to generate carbon dioxide u n d e r baki n g conditions. T h i s i n d u s t r y , which was modernized b y Pasteur i n the 1800's, i s c h a r a c t e r i z e d b y i t s h i g h s e n s i t i v i t y to raw material costs v a r i a b l e s o r seasonal demand a n d need to maintain a



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p r o d u c t acceptable to the baker. T h e objective i n process development i s to achieve h i g h c o n v e r s i o n y i e l d s of yeast from t h e c a r b o n source at a h i g h p r o d u c t i v i t y while maintaining the best b a k i n g power. T h e solution to meet these objectives has been the evolution of the fed-batch fermentation p r o c e s s . It has often been suggested that Baker's yeast could be most economically p r o d u c e d b y a continuous p r o c e s s , however, there is little i n c e n t i v e to c o n v e r t e x i s t i n g (and fully depreciated c a p i tal investment) from fed-batch to continuous operation. T h e fedb a t c h fermentation allows a plant c o n t a i n i n g many fermentors to operate with a variable p r o d u c t demand. F u r t h e r m o r e , the food i n d u s t r y p r o d u c i n g Baker's yeast i s not i n t e r e s t e d i n a process change which may alter their p r o d u c t i d e n t i t y a n d hence d e s i r ability b y the baker. F o r these reasons, fed-batch operation o f f e r s maximum plant operation f l e x i b i l i t y . It i s i n t e r e s t i n g to note, however, i n c o n s i d e r i n g new plant c o n s t r u c t i o n , there may be more i n c e n t i v e to go to a continuous process as a means of r e d u c i n g the e n t r y costs associated with this capital i n t e n s i v e i n dustry. T h e s t r a t e g y u n d e r l y i n g the operation of the fed-batch fermentation i s b a s e d on matching the s u p p l y of c a r b o n source with the demand b y the microorganism. T h e rationale f o r this i s i l l u s t r a t e d i n F i g u r e 1 ( 3 ) . In o r d e r to maximize the c o n v e r s i o n y i e l d , there i s a narrow r a n g e of growth rates between 0.075 a n d 0.25 h " that c a n be u s e d . It i s important to stay above 0.075 h"* to p r e v e n t excessive maintenance metabolism ( 4 ) . In a d d i t i o n , the growth rate must be below 0.25 h " to p r e v e n t ethanol p r o d u c t i o n which o c c u r s i n response to the C r a b tree effect. In addition, it i s imperative that sufficient o x y g e n be available to p r e v e n t anaerobic p r o d u c t i o n o f ethanol. Several i n v e s t i g a t o r s (3, 5) have p r o p o s e d the application o f computer process control to this p r o c e s s . U s i n g on-line measurements o f a i r flow rate, o x y g e n consumption a n d carbon dioxide p r o d u c t i o n i n combination with a set of material balance equations d e s c r i b e d b y Cooney et a l . ( 6 ) , it i s possible to calculate on-line the cell concentration ancT growth r a t e a n d a p p l y a modified feedf o r w a r d control s t r a t e g y to increase (or decrease) the s u g a r a d dition rate so that t h e specific growth rate does not exceed a c r i t i c a l value while at the same time maximizing the volumetric p r o d u c t i v i t y (3. 7). R e s u l t s of a t y p i c a l fermentation are shown i n F i g u r e 2 a n d a summary o f r e s u l t s from a v a r i e t y o f fermentations u n d e r v a r i o u s o p e r a t i n g conditions are shown i n T a b l e I. From an a n a l y s i s o f these fermentations, it i s clear that the k e y to a s u c c e s s f u l fermentation i s c a r e f u l management o f the c a r b o n source; this i s achieved b y continuous assessment of the 1

1



0.2 0.3 0.4 SUGAR CONC. ( g / I.)

0.1 j 0.2 0.3 0.4 j SUGAR CONC. ( g/1.)

Figure 1. Relationships in baker's yeast fermentation. Left, specific growth rate and cell yield; upper right, sugar concentration and specific growth rate; and lower right, sugar concentration and ethanol production rate. Reproduced, with permission, from Ref. 3. Copyright 1979, John Wiley & Sons, Inc.

0.1 02 0.3 0.4 05 CELLULAR YIELD (g/g)


00 Ν)

COONEY


Growth


8.

Figure 2. Kinetics of baker's yeast fermentation under feedback-modified feedforward computer control. Reproduced, with permission, from Ref. 3. Copyright 1979, John Wiley & Sons, Inc.


183

In Foundations of Biochemical Engineering; Blanch, Harvey W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983. 17.0 16.0 12.8 14.0 11.7 10.2

59.1 52.0 54.6 54.1 49.3 63.2

3.8 4.9 5.8 8.9 9.0 14.4

Source: Ref. 3.

Fermentation time (hr)

Final cell cone (g/liter) 0.49 0.49 0.50 0.51 0.49 0.50

Overall cell y i e l d (g cells/ g sugar)

DIFFERENT

3.3 3.1 4.0 3.4 3.7 5.2

Volumetric productivity (g/liter-hr)

O F C O M P U T E R - C O N T R O L L E D Y E A S T F E R M E N T A T I O N S WITH INITIAL C E L L C O N C E N T R A T I O N S

Initial cell cone ( g/liter)

COMPARISON

Table I


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demand for the c a r b o n source a n d a feedback modified w a r d c o n t r o l s t r a t e g y to match s u p p l y a n d demand.

feedfor-

S i n g l e - C e l l P r o t e i n . T h e concept of single-cell p r o t e i n , while not new i n that it was utilized b y both the A z t e c s a n d v a r ious c u l t u r e s i n c e n t r a l A f r i c a , has only r e c e n t l y (since 1960) been p r o p o s e d as a means for p r o d u c i n g l a r g e - s c a l e , h i g h quality protein to supplement traditional p r o t e i n resources ( 8 ) . T h e manu f a c t u r i n g cost for S C P i s dominated b y the raw material cost, as well as capital investment ( 1 ) . A s a consequence, the objective i n p r o c e s s development i s to maximize the c o n v e r s i o n y i e l d a n d the volumetric p r o d u c t i v i t y . In this way, the o p e r a t i n g a n d capital investment cost c a n be minimized. Continuous c u l t u r e o f f e r s a u s e f u l a p p r o a c h i n which cells c a n be grown u n d e r c a r b o n source to p r o t e i n u n d e r conditions o f h i g h volumetric p r o d u c t i v ity. Two strategies for optimization o f S C P p r o d u c t i o n are suggested i n F i g u r e 3. F i g u r e 3A shows a traditional continuous c u l t u r e isotherm. T h e maximum p r o d u c t i v i t y o c c u r s at a value D x ( 9 ) . However, this analysis does not lead to a n economic optimum ope r a t i n g condition. It assumes that the inlet feed concentration, S , i s a constant u n d e r all o p e r a t i n g conditions, a n d furthermore does not take into account the need to operate the fermentor at i t s maximum p r o d u c t i v i t y , the value o f which i s determined b y the o x y g e n t r a n s f e r rate (or i n the case o f v e r y large r e a c t o r s , the heat t r a n s f e r r a t e ) . T h i s i s i l l u s t r a t e d i n Equation 1: m a

0

W

where W

m

m

=

DX

m

m

= Y

R

n

0

2

m

0

a

x

(1)

2

i s the maximum p r o d u c t i v i t y

m

D

i s the dilution rate

X_ m Rj? 2

i s the maximum cell concentration

max allowed b y C>2 a x

i s the maximum o x y g e n t r a n s f e r rate

u

Plotting the maximum cell density permitted at a given o x y g e n t r a n s f e r rate ( a n d hence constant volumetric p r o d u c t i v i t y ) , gives the r e s u l t s shown i n F i g u r e 3B. In this a n a l y s i s , S , the feed concentration o f c a r b o n source, i s not constant. C o n s i d e r i n g the s t r a t e g y for operation, one will choose a dilution rate t h a t : (1) will minimize the r e s i d u a l limited n u t r i e n t concentration a n d t h u s achieve highest substrate utilization a n d minimum waste treatment load, (2) will maximize the conversion y i e l d o f the limiting n u t r i ent, the c a r b o n s o u r c e , a n d (3) will maximize the cell concentration to minimize r e c o v e r y c o s t s . T h e d e s i r e d dilution rate i s i n dicated b y the arrow on F i g u r e 3B. A g a i n , the s t r a t e g y relates to effective management o f the c a r b o n s o u r c e . In this r e g a r d , Q



186


A X

Dilution

Rate

Figure 3. Strategies for optimization of continuous culture for production of single-cell protein. Top, under continuous culture isotherm with a fixed value for carbon source feed concentration. D is the dilution rate of maximum productivity. Bottom, comparison of isotherm for fixed substrate feed concentration, S , with that for fixed oxygen transfer rate, OTR (curves show the maximum cell concentration for a given OTR and cell yield). m

0



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the use of continuous c u l t u r e i s an important tool f o r S C P p r o d u c tion. It i s important, however, to go one step f u r t h e r i s this analy s i s . One o f the major c a r b o n sources of interest f o r single cell protein p r o d u c t i o n i s methanol. T h e reasons f o r this relate prima r i l y to i t s low cost a n d availability. It has been shown that h i g h methanol concentrations are toxic to cell growth a n d more importantly lead to decreased cell y i e l d (10). T h e r e f o r e , a f u r t h e r objective i s to maintain the r e s i d u a l methanol concentration low at all times. T h e importance o f this i s i l l u s t r a t e d i n F i g u r e 4 ( 1 1 ) , which shows the response of Hansenula polymorpha to a p e r t u r b a tion i n methanol c o n c e n t r a t i o n . T h e p e r t u r b a t i o n was initiated b y a p e r i o d of o x y g e n s t a r v a t i o n (achieved b y r e d u c i n g the agitation r a t e ) . Because methanol metabolism i s obligately aerobic, u n d e r o x y g e n limiting conditions methanol r a p i d l y accumulates. When the o x y g e n limitation i s eliminated b y r e s t o r i n g agitation, the methanol i s r a p i d l y catabolized a n d some formaldehyde a n d formic acid accumulates. B o t h these compounds are extremely toxic a n d lead to instability i n c u l t u r e operation. T o overcome this problem, an on-line computer control s t r a t e g y that would c o n t i n u o u s l y assess r e s i d u a l methanol concentration a n d , should methanol accumulate, take c o r r e c t i v e action to p r e v e n t c u l t u r e i n s t a b i l i t y was developed (12). T h e s t r a t e g y f o r optimizing single-cell p r o t e i n p r o d u c t i o n i s based not only on a dilution rate that will give a low r e s i d u a l substrate concentration and h i g h c o n v e r s i o n y i e l d , b u t also that will operate with the maximum cell d e n s i t y permitted b y the o x y g e n t r a n s f e r rate. Simultaneously, it i s important to p r e v e n t accumulation of r e s i d u a l methanol to achieve both h i g h y i e l d s a n d process stability. Enzyme Production T h e economics of enzyme p r o d u c t i o n are dominated i n most cases b y r e c o v e r y c o s t s . F o r this reason, the objective functions for optimization a r e to maximize the specific activity (units o f enzyme a c t i v i t y p e r weight of cell mass) a n d volumetric p r o d u c t i v i t y (units p e r l i t e r - h ) . Maximization o f specific a c t i v i t y e n s u r e s both efficient c o n v e r s i o n o f raw materials to the d e s i r e d p r o d u c t as well as facilitating enzyme r e c o v e r y ; maximization of total volumet r i c p r o d u c t i v i t y helps to minimize r e c o v e r y costs a n d maximize economic r e t u r n on capital investment. T o achieve these objectives i n process development, one utilizes both genetic manipulations a n d environmental management to achieve a h i g h enzyme specific activity a n d a h i g h cell d e n s i t y . Two case studies a r e examined here to illustrate the important elements i n developing a s t r a t e g y f o r optimal enzyme p r o d u c tion.



188


Figure 4. Response of a steady state culture of H . polymorpha to an interruption in agitation causing oxygen limitation. Reproduced, with permission, from Ref. 11. Copyright 1981, American Society for Microbiology.


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Maltase P r o d u c t i o n T h e intracellular enzyme maltase (α-glucosidase E . C . 3.2.1.20) i s an important enzyme f o r analysis of blood amylase. T h e enzyme i s intracellular a n d i s subject to c a r b o n catabolite r e p r e s s i o n b y glucose as well as i n d u c t i o n b y maltose. A s t u d y was initiated with the objective of d e v e l o p i n g a lower cost p r o c e s s f o r maltase manufacture; the goals were to minimize the fermentation medium cost while maximizing the total a c t i v i t y a n d p r o d u c t i v i t y of maltase formation. T h e s t a r t i n g point was a fermentation with Saccharomyces italicus grown i n complex medium c o n t a i n i n g maltose as the sole carbon source. R e s u l t s from t h i s fermentation are shown i n F i g u r e 5. T h e problems with this fermentation are h i g h medium cost a n d the point of h a r v e s t o f maximum maltase activity was difficult to assess, as soon as maltose disappeared the spe cific activity of the enzyme r a p i d l y d e c a y e d . Medium optimization was achieved b y d e v e l o p i n g a defined medium without maltose. Saccharomyces italicus does not use s u crose because it l a c k s i n v e r t a s e , however, maltase will h y d r o l y z e sucrose a n d allow it to be u s e d for growth. A mutation a n d se lection program was initiated to select f o r mutants o f S. italicus that would grow on sucrose as the sole c a r b o n s o u r c e . It was h y p o t h e s i z e d that s u c h mutants would have a h i g h concentration of maltase. T h e r e s u l t s are shown i n Table II, i n which mutant 1-4 i s compared with the wild type (13). A s expected, the mu tant, when grown on s u c r o s e , has h i g h maltase a c t i v i t y . When grown i n d e f i n e d medium with sucrose, the volumetric a n d spe cific activity of maltase are greatly e n h a n c e d . Table II MALTASE

Carbon

P R O D U C T I O N ON

Source

Sucrose Maltose Glycerol Acetate Fructose Glucose

VARIOUS C A R B O N

Maltase Wild T y p e no

growth 870 10 10 10 10

SOURCES

(units/g-cell) Mutant

1-4

770 1100 770 380 320

A comparison of alternative methods f o r p r o d u c i n g this en zyme i n b a t c h a n d continuous c u l t u r e i s shown i n Table III. T h e r e s u l t s a r e somewhat s u r p r i s i n g . It was anticipated that i n con tinuous c u l t u r e u n d e r carbon-limited conditions the specific activ i t y of maltase would be h i g h e r than o b s e r v e d i n b a t c h c u l t u r e . However, f o r reasons that are not c l e a r , the specific a c t i v i t y i s substantially h i g h e r i n b a t c h c u l t u r e . T h e s e r e s u l t s illustrate




190

TIME (HOURS) Figure 5. Batch fermentation for maltase production by Saccharomyces italicus. Reproduced, with permission, from Ref. 13. Copyright 1982, American Society for Microbiology.


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again the importance of u s i n g good carbon source management a n d d e v e l o p i n g an optimal s t r a t e g y f o r enzyme p r o d u c t i o n . It i s also i n t e r e s t i n g to note that b y going from a complex to a defined medium one not only r e d u c e d the medium cost b u t also stabilized the enzyme to subsequent degradation.


Table III MALTASE

P R O D U C T I O N BY M U T A N T AND

Process

Specific Activity (units/ g-cell)

Batch (1-4)

Batch (wild type) Heparinase

Productivity ( unit s7 g-cell-hr) (units/1-hr)

2

890

1300

170

2200

Continuous (1-4)

WILD T Y P E

9

Q

70

3

4

0

0

470

Production

T h e enzyme heparinase i s o f interest as a means f o r d e g r a d i n g h e p a r i n i n blood a n d f o r d e g r a d i n g l a r g e molecular weight h e p a r i n into smaller fragments h a v i n g potential as novel anticoagulants (14). T h e p r o d u c t i o n of heparinase b y Flavobacterium heparinum i n a complex medium with h e p a r i n as an i n d u c e r i s shown i n F i g u r e 6. T h e requirement f o r h e p a r i n makes the medium expensive a n d it i s difficult to p i c k the best h a r v e s t time because the enzyme a c t i v i t y r a p i d l y decays late i n the fermentation. The first step i n process optimization was to develop a d e f i n e d medium; r e s u l t s are shown i n F i g u r e 7 (JL5). A l t h o u g h h e p a r i n i s still r e q u i r e d as an i n d u c e r , this lowers medium cost a n d i n creases the stability o f heparinase. It was expected that the p r o duction o f this enzyme, i n addition to b e i n g u n d e r i n d u c t i o n b y h e p a r i n , would be u n d e r control b y c a r b o n catabolite r e p r e s s i o n . T h e r e s u l t s showed that i n c r e a s i n g the initial concentrations of glucose leads to both decreased growth a n d heparinase p r o d u c t i o n (15). F o r this reason, we examined a fed-batch c u l t u r e designed to keep the glucose low throughout the fermentation. T h e r e s u l t s showed that it i s possible to achieve h i g h enzyme activity which is r e l a t i v e l y stable at the e n d of the fermentation. A g a i n , the s t r a t e g y of c a r b o n source management has p r o v e d to be v e r y effective i n optimizing this fermentation. C u r r e n t l y we are i n the p r o c e s s of looking f o r mutants which no longer r e q u i r e h e p a r i n as an i n d u c e r . We a r e also e x p l o r i n g the use of continuous c u l -



192


CE Lu I

O

10

20 HOURS

30

Figure 6. Batch fermentation for the production of heparinase by Flavobacterium heparinum on complex medium. Key: heparinase specific activity; Δ , dry cell weight; O , heparin concentration. Reproduced, with permission, from Ref. 15. Copyright 1981, American Society for Microbiology.

Figure 7. Batch fermentation for the production of heparinase by F . heparinum in defined medium. Key: O , dry cell weight, (D) glucose; Δ , heparin; and V , heparinase activity. Reproduced, with permission, from Ref. 15. Copyright 1981, American Society for Microbiology.


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t u r e as a means f o r maintaining low glucose concentrations with the hope of maximizing the specific activity of heparinase.


Metabolite Production T h e p r i m a r y objective f u n c t i o n s f o r p r o d u c t i o n o f metabolites are to maximize the concentration (to minimize r e c o v e r y costs) while maintaining h i g h c o n v e r s i o n y i e l d s o f costly raw materials to the p r o d u c t , u n d e r conditions o f h i g h volumetric p r o d u c t i v i t y (to r e d u c e capital c o s t ) . T h e u s u a l a p p r o a c h i s to r a p i d l y p r o d u c e a h i g h cell concentration u n d e r conditions that maximize the c o n v e r sion rate o f raw materials to the d e s i r e d p r o d u c t . The example c o n s i d e r e d here i s penicillin p r o d u c t i o n b y Pénicillium c h r y s o g e n u m . Penicillin i s u n d e r control of c a r b o n catabolite r e p r e s s i o n a n d is t y p i c a l l y formed as a secondary p r o d u c t after p r i m a r y growth i n the fermentation. T h e strategies developed i n i n d u s t r y f o r this fermentation generally r e v o l v e a r o u n d a fed-batch fermentation which r e s t r i c t s the flow o f c a r b o n to the c u l t u r e to slow down growth, t h u s p r e v e n t i n g c a r b o n catabolite r e p r e s s i o n . Mou a n d Cooney (16) initiated a p r o g r a m to examine the application of on-line computer control as a means f o r achievi n g improved c a r b o n source management f o r penicillin p r o d u c t i o n . The rationale was to utilize on-line monitoring o f growth to assess the demand o f the c u l t u r e for the c a r b o n source a n d i n t h i s way develop a means to control the specific growth rate i n the fermentation. It was reasoned that on-line computer c o n t r o l would p r o v e to be more flexible i n allowing one to balance s u p p l y a n d demand of the c a r b o n source d u r i n g this fermentation. A series o f studies were conducted i n which a computer cont r o l system was utilized to control both growth phase a n d p r o d u c tion phase growth rates a n d to a s k the question, "what i s the effect o f changes i n these growth rates on the specific p r o d u c t i v i t y of p e n i c i l l i n ? " . In F i g u r e 8 are shown r e s u l t s i n which the p r o duction phase growth rate was manipulated from 0 to 0.015 h " while the initial growth rate was manipulated constant at i t s maximum value o f 0.107. T h e specific rates of penicillin p r o d u c t i o n are shown i n F i g u r e 9. It i s clear that control o f the p r o d u c t i o n phase growth rate i s important to maintaining the ability of the cells to make p e n i c i l l i n . While it i s not clear from these r e s u l t s what the optimum value i s , this a p p r o a c h does allow one to b e g i n to design a set of experiments to answer that q u e s t i o n . In a second set o f experiments, two growth phase growth rates were compared. In the first case, the growth was controlled at 0.11 h " a n d i n the second case at 40% o f this value. T h e r e s u l t s , shown i n F i g u r e 10, indicate that cells grown more slowly d u r i n g the initial growth phase have a h i g h e r specific rate of penicillin p r o d u c t i o n . A g a i n , it i s not clear what the optimum values f o r growth phase growth rate are; i t i s clear, however, that c a r b o n source management d u r i n g both the growth a n d p r o d u c t i o n phase is important to maximize the specific rate of penicillin p r o d u c t i o n . 1

1



194


600Γ

HOURS Figure 8.

Growth kinetics of P. chrysogenum grown in computer controlled fedbatch culture fsee text for details).



8.

COONEY


Growth

6Γ

HOURS Figure 9.

Specific penicillin production rate for the fermentation shown in Figure 8.


195



196

HOURS Figure 10.

Growth and penicillin production by P. chrysogenum grown at two different growth phase growth rates.


8.

COONEY


197

Growth

T h e s e r e s u l t s suggest that f u r t h e r refinement o f this important fermentation c a n be achieved t h r o u g h the use o f on-line monitoring and control.


Conclusions F o r the p r o d u c t i o n of cell mass, enzymes a n d metabolites, it is clear from the above examples that c a r e f u l c a r b o n source management i s important to achieve h i g h c e l l concentrations with h i g h y i e l d , maximum specific enzyme activities a n d the efficient c o n v e r sion o f raw materials to p r o d u c t s . One approach to achieve good environmental management i s t h r o u g h the use of on-line p r o c e s s c o n t r o l . In this way, it i s possible to match the s u p p l y with the demand f o r c a r b o n source i n a wide v a r i e t y o f fermentations. A n o t h e r approach i s to use both traditional genetics as well as genetic e n g i n e e r i n g to alter regulation a n d p a t t e r n s o f c a r b o n source utilization to achieve b e t t e r c a r b o n source management. Genetics c a n be used to eliminate c a r b o n catabolite r e p r e s s i o n , side reactions to u n d e s i r e d p r o d u c t s a n d the need for i n d u c e r s of d e s i r e d enzymes. In addition, the use o f recombinant D N A to increase gene dosage c a n be important to i n c r e a s i n g the specific activity o f d e s i r e d enzymes; these may b e enzymes that a r e des i r e d as p r o d u c t s o r that a r e rate limiting enzymes i n metabolic pathways. Simultaneously with optimizing the genetic constitution and the application o f p r o c e s s control s t r a t e g i e s , it i s essential i n bioreactor d e s i g n a n d scale-up to take into account the organism's s e n s i t i v i t y to carbon source concentrations. In situations s u c h as methanol fermentations where the organism may b e v e r y sensitive to small variations i n c a r b o n source c o n c e n t r a t i o n s , mixing time i s a k e y parameter a n d n o v e l e n g i n e e r i n g d e s i g n s must often be cons i d e r e d to avoid this problem. Despite what appears s u p e r f i c i a l l y to be d i v e r s i t y among fermentation p r o c e s s e s , there are s e v e r a l common factors important i n the d e s i g n o f optimum process strategies. C a r b o n source management i s k e y among these.

Literature Cited 1. 2. 3. 4. 5. 6.

Cooney, C . L . Microb. Growth on C . Compounds, 1975, pp. 183-197. Cooney, C.L.; Rha, C . K . ; Tannenbaum, S.R. Adv. in Food Res., 1980, 26, 1-52. Wang, H . Y . ; Cooney, C . L . ; Wang, D.I.C. Biotech. Bioengr., 1979, 21, 975-995. Pirt, S . J . "Principles of Microbe and Cell Cultivation", 1975, Blackwell Scientific Publications, London. Aiba, S.; Nagai, S.; Nishizawa, Y. Biotechnol. Bioengr., 1976, 19, 1001. Cooney, C . L . ; Wang, H . ; Wang, D.I.C. Biotech. Bioengr., 1977, 19, 55-66. 1


198 7. 8. 9.


10. 11. 12. 13. 14. 15. 16.


Wang, H . Y . ; Cooney, C . L . ; Wang, D.I.C. Biotech. Bioengr., 1978, 19, 67-86. Cooney, C.L.; Rha, C . K . ; Tannenbaum, S.R. Adv. Food Res., 1981, 26, 1-47. Herbert, D . ; Elsworth, R . ; Telling, R . C . J . Gen. Microbiol., 1956, 14, 601-622. Books, J . D . ; Meers, J . L . J . Gen. Microbiol., 1973, 77, 513. Swartz, J . R . ; Cooney, C.L. Appl. Env. Microbiol., 1981, 41, 1206. Swartz, J . R . Ph.D. Thesis, M . I . T . , Cambridge, MA, 1979. Schaefer, E.J.; Cooney, C . L . Appl. Environ. Microbiol., 1982, 43(1), 75-80. Langer, R . ; Linhardt, R.J.; Hoffberg, S.; Larsen, A.K.; Cooney, C . L . ; Tapper, D.; Klein, M. Science, 1982, in press. Galliher, P . M . ; Cooney, C.L.; Langer, R . ; Linhardt, R . J . Appl. Environ. Microbiol., 1981, 41(2), 360-5. Mou, D - G . ; Cooney, C . L . Biotech. Bioeng., 1982, submitted.

RECEIVED

July 22, 1981


Strategies for Optimizing Microbial Growth and Product Formation

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