The Production of Alginic Acid by

2

The

P r o d u c t i o n of

vinelandii

Alginic

Acid

by

Azotobacter

in Batch and Continuous Culture

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 7, 2014 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch002

L. DEAVIN, T. R. JARMAN, C. J. LAWSON, R. C. RIGHELATO, and S. SLOCOMBE Tate & Lyle Ltd., Group Research and Development, Philip Lyle Memorial Research Laboratory, P.O. Box 68, Reading, Berks., RG6 2BX, U.K.

The production of polysaccharides by fermentation has been heralded by some of the more optimistic microbial technologists as the next major fermentation area. It is now gaining similar treatment in public and private meetings to that offered to single cell protein some years ago. This optimism is based on the undoubted success of the one major product, xanthan gum, which has raised the tantalising prospect of a whole range of microbial gums which would not only reflect and improve upon the available plant gums, but also introduce novel properties for exploitation in existing and as yet undeveloped applications. About a dozen companies are thought to be developing on a large scale the production of microbial polysaccharides; some of them are already in the fermentation industry but others, like our own, are newcomers to this technology. Despite this enormous research and development effort the state of the technology, as judged from patents and the scientific literature, is relatively poorly advanced. There is little public literature on the production technologies used by industry and academic microbiology has for the most part ignored the physiology of exocellular polysaccharide synthesis and excretion. For this reason, we, along with other groups,have been studying the physiology of polysaccharide synthesis as a basis for developing production processes. In order to gain a greater understanding of the effects of individual environmental parameters on cell growth and polysaccharide synthesis continuous flow cultures(l) have been used wherever possible. For those unfamiliar with the methods of mass cultivation of microbes, the time honoured industrial and laboratory method is to inoculate a small amount of the microbe into a medium containing all of the necessary nutrients for growth and product formation. The microbes then grow until one or other substrate is exhausted and then growth stops. This is a simple batch culture system. In continuous flow culture, by contrast, the nutrient medium is continuously added to the culture and the culture continuously harvested. 14

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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T h e r a t i o of the flow rate of the medium to the c u l t u r e v o l u m e is c a l l e d the d i l u t i o n r a t e , a n d e x c e p t at the maximum growth rate of the m i c r o b e , the c o n c e n t r a t i o n o f o n e o f the substances i n the medium determines the c o n c e n t r a t i o n o f the m i c r o b e s . It is

This is c a l l e d the g r o w t h - l i m i t i n g substrate.

w e l l established that c h a n g e s in g r o w t h - l i m i t i n g substrate c a n


c o n s i d e r a b l y a f f e c t the p h y s i o l o g y of m i c r o b e s .

So too c a n changes i n the

d i l u t i o n r a t e , w h i c h in a steady state is e q u a l to the s p e c i f i c growth r a t e . In continuous cultures steady states c a n b e m a i n t a i n e d i n d e f i n i t e l y a n d changes i n i n d i v i d u a l parameters c a n r e a d i l y b e s t u d i e d .

By contrast

i n b a t c h c u l t u r e s , c o n c e n t r a t i o n of nutrients, c e l l s a n d p r o d u c t s , a n d a l l of these w i t h respect to c e l l a g e , c h a n g e c o n t i n u o u s l y , w h i c h makes the study o f c e l l p h y s i o l o g y and b i o c h e m i s t r y e x t r e m e l y c o m p l i c a t e d .

This is

i l l u s t r a t e d b y some b a t c h fermentation processes for e x o p o l y s a c c h a r i d e s . T h e best known is of course x a n t h a n p r o d u c t i o n b y Xanthomonas

campestris.

In the simplest fermentation d e s c r i b e d b y M o r a i n e a n d R o g o v i n (2), the c o n c e n t r a t i o n s of the major substrates c h a n g e throughout the f e r m e n t a t i o n . So too do the main products:

bacterial cells and polysaccharide.

Analysis

o f several b a t c h cultures l e d M o r a i n e & R o g o v i n (2) to c o n c l u d e that several f a c t o r s , i n c l u d i n g x a n t h a n c o n c e n t r a t i o n , a f f e c t e d the rate o f x a n t h a n p r o d u c t i o n , though the d e t a i l s o f the r e l a t i o n s h i p were not c l e a r . T h e c o m p l i c a t e d k i n e t i c pattern that emerged from these studies has b e e n of c o n s i d e r a b l e v a l u e in understanding the b a t c h fermentation process for x a n t h a n gum but does not e n h a n c e the understanding o f the control o f b i o synthesis, as it n e c e s s a r i l y deals p r i m a r i l y w i t h the e f f e c t of the c h a n g i n g fermentation parameters o n the environment of the c e l l s rather than d i r e c t l y w i t h the e f f e c t o f the environment on the c e l l s . In b a t c h cultures of a Pseudomonas sp . w h i c h produces an e x o p o l y s a c c h a r i d e composed of g l u c o s e and g a l a c t o s e i n the r a t i o 7 : 1 a n d contains both a c e t a t e and p y r u v a t e (3) p o l y m e r synthesis was d e t e c t a b l e in the later part of the e x p o n e n t i a l growth phase (Figure 1) a n d c o n t i n u e d m a x i m a l l y d u r i n g the p e r i o d of z e r o s p e c i f i c growth r a t e , the s o - c a l l e d stationary phase (4).

The

l i m i t i n g substrate, that is the substrate w h i c h

determined the c e l l mass that was f i n a l l y o b t a i n e d , was not established i n these c u l t u r e s . A n o t h e r e x a m p l e of b a t c h c u l t i v a t i o n for a n e x o p o l y s a c c h a r i d e is that of a l g i n i c a c i d production by Azotobacter v i n e l a n d i i .

W h e n the organism

was grown under p h o s p h a t e - d e f i c i e n t c o n d i t i o n s p o l y s a c c h a r i d e synthesis c o n t i n u e d throughout the growth phase but in contrast to the last e x a m p l e ceased when the microbes stopped g r o w i n g (Figure 2 ) . From the studies of b a t c h cultures of the types discussed it is d i f f i c u l t to draw a n y conclusions o n the w a y in w h i c h b a c t e r i a control the synthesis o f these e x o p o l y s a c c h a r i d e s .

It has b e e n supposed b y many

microbiologists

that such products w o u l d b e formed w h e n a c e l l has a n excess o f c a r b o h y d r a t e



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4


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substrate a n d its growth îs r e s t r i c t e d b y some other p a r a m e t e r .

N e i j s s e l and

Tempest (5) h a v e r e c e n t l y suggested from studies of A e r o b a c t e r aerogenes that t h e y a c t as A T P sinks and a r e p r o d u c e d m a x i m a l l y under c o n d i t i o n s w h i c h w o u l d cause the c e l l s to o v e r p r o d u c e A T P , c o n d i t i o n s such as nitrogen l i m i t a t i o n .

T h e observations o n A z o t o b a c t e r v i n e l a n d i i w o u l d


perhaps c o n t r a d i c t that p a r t i c u l a r hypothesis s i n c e h i g h p r o d u c t i o n rates w e r e observed under p h o s p h a t e - d e f i c i e n t c o n d i t i o n s (Figure 2 ) .

Measurement

o f the rates of synthesis under a v a r i e t y of e n v i r o n m e n t a l c o n d i t i o n s might shed some light o n the c e l l u l a r control a n d the r o l e of e x o p o l y s a c c h a r i d e production.

T h e major rate c o n t r o l l i n g process in a c e l l is its s p e c i f i c

growth r a t e .

A c o m p l e x network o f control mechanisms exist w h i c h permit

the m i c r o b e to assimilate substrates, synthesise intermediates a n d form polymers ( i . e . p r o t e i n s , n u c l e i c a c i d s , c e l l w a l l s , e t c . ) at rates w h i c h p r o d u c e more c e l l u l a r material of the same t y p e a n d i n similar ratios in the f a c e of enormous e n v i r o n m e n t a l changes.

It seems l o g i c a l , t h e n , to look

first at the e f f e c t of growth rate on e x o p o l y s a c c h a r i d e synthesis i n continuous c u l t u r e systems. S i l m a n a n d R o g o v i n (6) studied continuous cultures of Xanthomonas campestris

in cultures thought to b e l i m i t e d b y the nitrogenous component

i n the m e d i u m .

p H was not c o n t r o l l e d in these experiments so the d a t a has

b e e n redrawn t a k i n g o n l y the c o n d i t i o n s i n w h i c h the p H was b e t w e e n 6 . 3 and 7 . 2 , a range i n w h i c h it has b e e n found that p H has l i t t l e e f f e c t on x a n t h a n p r o d u c t i o n (Figure 3 ) .

A t growth rates b e t w e e n 0 . 0 5 a n d 0 . 2 0 h " ^

i . e . d o u b l i n g times between 14 a n d 3 . 5 h , there was l i t t l e c h a n g e in the s p e c i f i c rate of synthesis o f x a n t h a n .

T h e c o n c e n t r a t i o n o f x a n t h a n therefore

increased with decreasing dilution rate.

It is interesting to note that the

x a n t h a n p r o d u c t i o n rate i n these cultures v a r i e d o n l y 1 5 % e i t h e r side of the mean v a l u e .

This is q u i t e d i f f e r e n t from the b a t c h c u l t u r e analysis w h i c h

showed a t h r e e f o l d c h a n g e i n s p e c i f i c rate o f xanthan p r o d u c t i o n o v e r a similar c o n c e n t r a t i o n range

(2).

A similar i n d e p e n d e n c e o f the r a t e of e x o p o l y m e r synthesis on s p e c i f i c growth rate was found both w i t h the Pseudomonas p o l y s a c c h a r i d e (4) a n d a l g i n i c a c i d synthesis b y A z o t o b a c t e r v i n e l a n d i i .

O v e r an even wider

growth rate range the s p e c i f i c rate of synthesis o f Pseudomonas e x o p o l y m e r v a r i e d o n l y 2 5 % about the mean (Figure 4 ) , whilst the p o l y s a c c h a r i d e c o n c e n t r a t i o n i n c r e a s e d i n p r o p o r t i o n to the r e s i d e n c e time o f the c u l t u r e (the r e s i d e n c e time is the r e c i p r o c a l of the d i l u t i o n r a t e ) . l i m i t e d continuous cultures o f A z o t o b a c t e r v i n e l a n d i i

In p h o s p h a t e -

the rate of a l g i n a t e

synthesis was i n d e p e n d e n t of s p e c i f i c growth rate (Figure 5 ) . there was an i n c r e a s e i n biomass at lower d i l u t i o n rates.

In this case

This was almost

e n t i r e l y d u e to the i n t r a c e l l u l a r a c c u m u l a t i o n o f the storage compound poly-B-hydrosybutyrate.

W i t h these three p o l y s a c c h a r i d e s , then the rate

o f synthesis appears to be i n d e p e n d e n t of the rate of growth a n d h e n c e i n d e p e n d e n t o f the rate of most of the other i n t r a c e l l u l a r biosyntheses.


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Exopolymer


Ο mg/ml

>mg/OD/h χ 100

Biotechnology and Bioengineering

Figure 3. Effect of dilution rate on the production of xanthan by Xanthomonas campestris in con tinuous culture (6)

0.05

0.10

0.15 1

Dilution rate h

Exopolymer

Ο mg/ml

Figure 4. Effect of di lution rate on produc tion of an exopolysac charide by Pseudomonas sp in ammonia-limited continuous culture (Data from Williams A. G., 1975; Ph.D. Thesis Uni versity College, Cardiff, U.K.)

• mg/mg protein/h

0.25 Dilution rate h


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W e h a v e studied a l g i n i c a c i d synthesis b y A z o t o b a c t e r v i n e l a n d i i

in

some d e t a i l and w o u l d l i k e to pursue this argument w i t h that p a r t i c u l a r system.

A l g i n a t e as o b t a i n e d from the c o n v e n t i o n a l s o u r c e , the brown a l g a e ,

is a 1 , 4 - l i n k e d l i n e a r c o p o l y m e r of J i - D - m a n n u r o n i c a c i d and its 5 - e p i m e r « C - L - g u I u r o n i c a c i d (7)

(Figure 6).

T h e arrangement of monomers i n this


c o p o l y m e r has b e e n referred to as the b l o c k structure (8), the p o l y m e r h a v i n g b e e n shown to consist of regions o f h o m o - p o l y m e r i c b l o c k s of mannuronic a c i d and o f g u l u r o n i c a c i d together w i t h the s o - c a l l e d a l t e r n a t i n g or random s e q u e n c e s .

T h e properties of the polymer, e s p e c i a l l y w i t h respect

to its g e l l i n g in the p r e s e n c e o f c a l c i u m ions,depends both on the mannuronic a c i d to g u l u r o n i c a c i d r a t i o a n d the b l o c k structure, the higher the proportion of p o l y g u l u r o n i c a c i d blocks in the p o l y m e r the stronger and more b r i t t l e the gel formed in the presence of c a l c i u m ions (9).

The polymer

p r o d u c e d b y A z o t o b a c t e r v i n e l a n d i i has the same b a s i c structure as that from a l g a l sources e x c e p t that it is p a r t i a l l y a c e t y l a t e d , a p p r o x i m a t e l y o n e in ten of the C 2 α η σ / o r C 3 h y d r o x y l groups b e i n g e s t e r i f i e d w i t h a c e t a t e

(ίο, η.). T h e markets for a l g i n a t e s demand products h a v i n g a range of solution viscosities and g e l l i n g q u a l i t i e s .

A range o f a l g i n a t e types c o m p a r a b l e

a l g a l products c a n b e p r o d u c e d b y A z o t o b a c t e r v i n e l a n d i i c h o i c e o f fermentation c o n d i t i o n s .

with

by appropriate

H a u g a n d Larsen (12) showed that the

mannuronic to g u l u r o n i c a c i d r a t i o of A z o t o b a c t e r a l g i n a t e c o u l d b e i n f l u e n c e d b y the c a l c i u m i o n c o n c e n t r a t i o n o f the growth medium and they presented e v i d e n c e w h i c h suggested that mannuronic a c i d residues

were

epimerised to g u l u r o n i c a c i d residues b y a n e x t r a c e l l u l a r e n z y m e d e p e n d e n t on c a l c i u m ions for a c t i v i t y .

In a d d i t i o n

we h a v e b e e n a b l e to m a n i p u l a t e

the m o l e c u l a r w e i g h t and thus solution v i s c o s i t y of the p r o d u c t p r o d u c e d b y Azotobacter vinelandii. By a p p r o p r i a t e c h o i c e o f fermentation c o n d i t i o n s products w i t h a w i d e range of viscosities w e r e o b t a i n e d w h i c h c o m p a r e d f a v o u r a b l y w i t h c e r t a i n c o m m e r c i a l a l g a l a l g i n a t e s h a v i n g l o w , medium a n d h i g h viscosities (Figure 7 ) .

T h e results reported here a p p l y to products o b t a i n e d from

continuous cultures but products w i t h a similar range o f viscosities c a n also b e o b t a i n e d from b a t c h c u l t u r e s . T h e metabolism o f A z o t o b a c t e r v i n e l a n d i i in r e l a t i o n to p o l y s a c c h a r i d e biosynthesis is shown in F i g u r e 8.

Sucrose, the c a r b o h y d r a t e growth

substrate used , is transported into the c e l l , i n v e r t e d , a n d

glucose-6-phosphate

a n d f r u c t o s e - 6 - p h o s p h a t e formed b y their r e s p e c t i v e kinases.

Fructose-6-

phosphate enters the a l g i n a t e b i o s y n t h e t i c p a t h w a y w h i c h has b e e n shown to be v i a m a n n o s e - 6 - p h o s p h a t e / n a n n o s e - l - p h o s p h a t e

and

GDP-mannose

n u c l e o t i d e w h i c h is o x i d i s e d to G D P - m a n n u r o n i c a c i d (13).

Mannuronic

a c i d residues a r e then p o l y m e r i s e d to form p o l y m a n n u r o n a t e w h i c h is p a r t i a l l y e p i m e r i s e d e x t r a c e l l u l a r l y ( 1 2 ) , to y i e l d a l g i n a t e .

Azotobacter

is an o b l i g a t e a e r o b e , c a r b o h y d r a t e growth substrates are metabolised v i a



20

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0.05

0.10

0.Ï5

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0.25

Dilutionrate(h ) Figure 5.

Exopolysaccharide production by Azotobacter vinelandii at a range of dilution rates

Monomers

^5-D-Mannuronic acid

oL -L-Guluronic acid

Block Structure

-M-M-M-M-M-M-

Figure 6. The structure of alginic acid

-G-G-G-G-G-G-M-G-M-G-M-G-


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10,000 ρ

α

Lj

n

j

u

mxfc

Rate of shear (sec ^) Figure 7.

Apparent viscosity vs. rate of shear plots for Azotobacter algi nates and certain commercial algal alginates



EXTRACELLULAR

Figure 8.

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Metabolism of Azotobacter vinelandii in relation to alginate synthesis


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the E n t n e r - D o u d o r o f f p a t h w a y , pentose phosphate c y c l e and t r i c a r b o x y l i c a c i d c y c l e (14) a n d a r e o x i d i s e d to c a r b o n d i o x i d e *

T h e products of sucrose

metabolism are e s s e n t i a l l y a l g i n a t e , biomass and carbon d i o x i d e .

With

increases in o x y g e n tension A z o t o b a c t e r e x h i b i t a n increase in respiration rate (15);

the e f f i c i e n c y o f e n e r g y c o n s e r v a t i o n f a l l i n g until at v e r y high


respiration rates as much as 9 0 % o f the sucrose u t i l i s e d c a n b e burnt off as carbon d i o x i d e .

O n e o f the problems i n d e v e l o p i n g a process for

A z o t o b a c t e r a l g i n a t e p r o d u c t i o n has therefore been to control this adverse respiration.

This was a d i f f i c u l t proposition i n b a t c h c u l t u r e w i t h

c o n t i n u a l l y c h a n g i n g biomass and o x y g e n d e m a n d , e s p e c i a l l y as o x y g e n l i m i t a t i o n has p r o v e d to be a disadvantageous production.

c o n d i t i o n for polymer

T r i a l s i n b a t c h c u l t u r e under p h o s p h a t e - d e f i c i e n t c o n d i t i o n s

i n d i c a t e d maximal o b t a i n a b l e y i e l d s of sodium a l g i n a t e to b e a p p r o x i m a t e l y 2 5 % of the sucrose u t i l i s e d .

T h e e f f e c t of respiration rate on a l g i n a t e

p r o d u c t i o n in continuous c u t l u r e was therefore i n v e s t i g a t e d . T h e organism was grown at a range of s p e c i f i c respiration rates o b t a i n e d b y a l t e r i n g the fermenter i m p e l l e r speed thus c h a n g i n g the rate o f o x y g e n transfer into the c u l t u r e broth (Figure 9 ) .

W e chose p h o s p h a t e - l i m i t e d

growth c o n d i t i o n s , as a phosphate d e f i c i e n t m e d i u m , as discussed e a r l i e r was known to b e c o n d u c i v e to p o l y s a c c h a r i d e synthesis in b a t c h c u l t u r e . A l t h o u g h c e l l mass, w h i c h

r e m a i n e d e s s e n t i a l l y constant,was l i m i t e d b y

a v a i l a b i l i t y of phosphate, the s p e c i f i c respiration rate was d e t e r m i n e d b y oxygen a v a i l a b i l i t y.

P o l y s a c c h a r i d e c o n c e n t r a t i o n was a l s o e s s e n t i a l l y

constant, d e c r e a s i n g o n l y at v e r y low r e s p i r a t i o n rates.

T h e rate of

a l g i n a t e synthesis was therefore l a r g e l y i n d e p e n d e n t of both the rate at w h i c h sucrose e n t e r e d the c e l l , as i n d i c a t e d b y the amount of sucrose u t i l i s e d , a n d the rate at w h i c h intermediates e n t e r e d the c a t a b o l i c pathways and were respired to c a r b o n d i o x i d e .

T h e maximum y i e l d of sodium a l g i n a t e ,

which

o c c u r r e d at the lower respiration rates, was i n the r e g i o n of 4 5 % of the sucrose u t i l i s e d as compared w i t h the y i e l d s of 2 5 % o b t a i n e d in b a t c h c u l t u r e . A t higher respiration rates the y i e l d f e l l d r a m a t i c a l l y d u e to a greater p r o p o r t i o n o f the sucrose b e i n g o x i d i s e d t o c a r b o n d i o x i d e . T h e e f f e c t of d i f f e r e n t growth limitations o n a l g i n a t e p r o d u c t i o n has also been investigated.

S t e a d y state continuous cultures w e r e o b t a i n e d w i t h

d i f f e r e n t nutrients l i m i t i n g growth but c e l l mass and also s p e c i f i c respiration rate were c o n t r o l l e d to w i t h i n narrow ranges.

Polysaccharide, determined

as isopropanol p r e c i p i t a t e d material,was p r o d u c e d under a l l limitations tested ( T a b l e 1).

M o l y b d a t e l i m i t a t i o n f o l l o w e d b y phosphate l i m i t a t i o n ,

the c o n d i t i o n r o u t i n e l y used, g a v e the most f a v o u r a b l e s p e c i f i c rates of p o l y s a c c h a r i d e synthesis.

Surprisingly,

under sucrose l i m i t a t i o n , a

c o n d i t i o n w h e r e the c e l l w o u l d be e x p e c t e d to make the most e f f i c i e n t use possible of its a v a i l a b l e carbon and e n e r g y substrate, p o l y s a c c h a r i d e was still

p r o d u c e d at similar rates to other l i m i t a t i o n s .

It is d i f f i c u l t to

c o m p a r e o x y g e n l i m i t a t i o n , o n e c o n d i t i o n tested w h e r e the s p e c i f i c rate of



24

EXTRACELLULAR

0

10

20

MICROBIAL

30

POLYSACCHARIDES

40

Specific respiration rate (umol O^/h/mg cell)

Figure 9.

Exopolysaccharide production by Azotobacter vinelandii at a range of respiration rates

T a b l e 1. E f f e c t of g r o w t h - l i m i t i n g

nutrient o n e x o p o l y s a c c h a r i d e

production b y Azotobacter vinelandii Growth-limiting nutrient

C e l l Mass

S p e c i f i c Rate o f

(mg/ml)

polysaccharide production (mg/mg c e l l / h )

1.1

0.34

1.9

0.28

Fe-H-

1.4

0.25

C(sucrose)

1.3

0.25

N

1.5

0.22

1.2

0.20

1.9

0.16

1.2

0.06

M004

2

C a "

o D

2

=

0.15 + 0.01

h

_ 1


2.

DEAViN E T A L .

Production

of Alginic

Acid

in

25

Culture

p o l y s a c c h a r i d e p r o d u c t i o n was v e r y much l o w e r , w i t h other c o n d i t i o n s s i n c e under these c o n d i t i o n s the c e l l mass was p r o b a b l y less a c t i v e due to i n t r a c e l l u l a r a c c u m u l a t i o n of p o l y - j i - h y d r o x y b u t y r a t e (16).

With

the e x c e p t i o n of O 2 - I i m i t a t i o n the s p e c i f i c rate o f p o l y s a c c h a r i d e p r o d u c t i o n v a r i e d b y just a l i t t l e over t w o f o l d w h i c h c o n s i d e r i n g the large


#

changes i n the p h y s i o l o g y o f the c e l l w h i c h a r e l i k e l y under the various l i m i t a t i o n s i s not v e r y g r e a t . Some c h a n g e was found however i n the /

p h y s i c a l properties o f the p o l y s a c c h a r i d e p r o d u c e d under the various limitations.

T h e r e f o r e a l t h o u g h the s p e c i f i c rate of a l g i n a t e p r o d u c t i o n does

not v a r y g r e a t l y w i t h changes i n fermentation c o n d i t i o n s the y i e l d o f a l g i n a t e i n terms o f the amount o f sucrose u t i l i s e d is m a i n l y d e t e r m i n e d b y o x y g e n a v a i l a b i l i t y a n d thus the respiration rate o f t h e c u l t u r e .

Continuous

c u l t u r e studies h a v e g i v e n us s u f f i c i e n t information o n the control o f a l g i n a t e biosynthesis to choose conditions where improved y i e l d s of a l g i n a t e can be obtained. In summary, t h e rate o f a l g i n a t e synthesis per unit c e l l mass remains r e l a t i v e l y constant o v e r a range o f c o n d i t i o n s where the p h y s i o l o g i c a l

state

of the c e l l w o u l d b e e x p e c t e d to v a r y w i d e l y , that is o v e r a range o f growth rates, o v e r a range o f respiration rates a n d w i t h a v a r i e t y o f growth l i m i t i n g nutrients.

How this constant rate is o b t a i n e d i n terms o f control

remains u n c l e a r .

mechanisms

A s y e t w e a r e u n a b l e to distinguish whether it is a

r e l a t i v e l y u n c o n t r o l l e d process or whether f i n e controls a r e necessary to o b t a i n this constant r a t e .

From these findings a n d our observations o n other

e x o p o l y s a c c h a r i d e p r o d u c i n g organisms, n a m e l y Xanthomonas

campestris

a n d a Pseudomonas s p . the a b i l i t y to p r o d u c e e x o p o l y s a c c h a r i d e at similar rates under a v a r i e t y o f c o n d i t i o n s c o u l d b e much more general than has hitherto b e e n r e c o g n i s e d .

Literature Cited (1) (2) (3) (4) (5) (6) (7)

Herbert, C., Ellsworth, R. and Telling, R.C. J. Gen. Microbiol. (1965), 14, 601-622. Moraine, R.A. and Rogovin, P. Biotechnol. Bioeng. (1973), 14 225-237 Lawson, C.J. and Symes, K.C. Unpublished data. Williams, A.C. Ph.D. Thesis, University College, Cardiff, U.K. (1974) Neijssel, O.M. and Tempest, D.W. Arch. Microbiol. (1976), 107, 215-221 Silman, R.W. and Rogovin, P. Biotechnol. Bioeng. (1972), 14 23-31 Drummond, D.W., Hurst, E.L. and Percival, E. (1961). J. Chem. Soc., London, p. 1208-1216.


26

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES


(8)

Larsen, B.,Sandsrød,O . , Haug, A. and Painter, T. Acta Chem. Scand. (1969), 23, 2375-2388. (9) Smidsrød, O. Disc. Faraday Soc. (1974),57, 263-274. (10) Gorin, P.A.J. and Spencer, J.F.t. Can. J. Chem. (1966) 44, 993-998 (11) Larsen, B. and Haug, A.Carbohyd.Res.(1971), 17, 287-296. (12) Haug, A. and Larsen B. Carbohyd. Res. (1971 ),17, 297-308. (13) Pindar, D.F. and Bucke, C. Biochem. J. (1975), 152, 617-622. (14) Still, G.C. and Wang, C.H. Arch. Biochem. Biophys. (1964) 105, 126-132. (15) Downs, A.J. and Jones, C.W., FEBS Lett.(1975),60,42-46. (16) Dawes,Ε.A.and Senior, P.J. Adv. Microbiol. Physiol. (1973) 10, 135-266.


The Production of Alginic Acid by

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