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21 Production of Industrially Important Gums with Particular Reference to Xanthan Gum and Microbial

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Alginate CHRISTOPHER J. LAWSON Tate & Lyle Ltd., Group R & D, P.O. Box 68, Reading, Berks, RG6 2BX, England The p r o d u c t i o n o f p o l y s a c c h a r i d e gums b y f e r m e n t a t i o n h a s b e e n d e s c r i b e d b y some o f t h e m o r e o p t i m i s t i c m i c r o b i a l t e c h n o l o g i s t s as t h enext major ferment a t i o n area. I t now i s g a i n i n g t r e a t m e n t i n p u b l i c and p r i v a t e m e e t i n g s s i m i l a r t o t h a t o f f e r e d t o s i n g l e c e l l p r o t e i n some y e a r s a g o . This optimism i s based on t h e u n d o u b t e d s u c c e s s o f t h e m a j o r p r o d u c t i n t h i s a r e a , x a n t h a n gum, w h i c h h a s r a i s e d t h e t a n t a l i s i n g p r o s p e c t o f a w h o l e r a n g e o f m i c r o b i a l gums w h i c h c o u l d n o t o n l y r e f l e c t and improve upon t h e a v a i l a b l e p l a n t gums, b u t a l s o i n t r o d u c e n o v e l p r o p e r t i e s f o r e x p l o i t a t i o n i ne x i s t i n g and as yet undeveloped a p p l i cations. About a dozen major companies a r e thought to be c o n d u c t i n g s u b s t a n t i a l r e s e a r c h and development programmes i n t o t h e p r o d u c t i o n o f m i c r o b i a l p o l y s a c c h a r i d e s ; some o f t h e m a l r e a d y a r e i n t h e f e r m e n t a t i o n i n d u s t r y , b u t o t h e r s , l i k e Tate and L y l e and H e r c u l e s , a r e newcomers t o t h i s t e c h n o l o g y . Despite t h i s heavy r e s e a r c h a n d d e v e l o p m e n t e f f o r t w h i c h , i n some i n s t a n ces has a h i s t o r y o f a t l e a s t a decade, t h e s t a t e o r at l e a s t p u b l i c knowledge o f t h e t e c h n o l o g y a s judged from the p a t e n t and s c i e n t i f i c l i t e r a t u r e , i s low, t o say t h e l e a s t . There i s l i t t l e l i t e r a t u r e on t h e p r o d u c t i o n technologies used by i n d u s t r y . Academic m i c r o b i o l o g y , f o r the most p a r t , h a s i g n o r e d t h e phys i o l o g y o f e x o c e l l u l a r p o l y s a c c h a r i d e s y n t h e s i s and excretion. The p h y s i o l o g y o f p o l y s a c c h a r i d e s y n t h e s i s has been s t u d i e d a s a b a s i s f o r d e v e l o p i n g p r o d u c t i o n processes. A number o f t h e r e s u l t s o b t a i n e d o n m i c r o b i a l a l g i n a t e i nthe Tate and L y l e l a b o r a t o r i e s w i l l be d i s c u s s e d . As a b r i e f i n t r o d u c t i o n t o m i c r o b i a l gum p r o d u c t i o n , i t w i l l b e u s e f u l t o c o n t e m p l a t e a number o f more g e n e r a l q u e s t i o n s w h i c h w o u l d be p o s e d by a n i n d i v i d u a l o r g r o u p o f i n d i v i d u a l s c o n s i d e r i n g this field for possible exploitation. 282

In Sucrochemistry; Hickson, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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1. W h a t a r e b i o p o l y m e r s o f i n d u s t r i a l s i g n i f i cance? 2. Why s h o u l d s u g a r c o m p a n i e s show a n y i n t e r e s t ? 3. W h a t i s t h e c o m m e r c i a l s t a t u s o f m i c r o b i a l gums? 4« W h a t a r e t h e p r o p e r t i e s o f i n t e r e s t ? 5. W h a t a d v a n t a g e s h a s f e r m e n t a t i o n t o o f f e r ? 6. What d i s a d v a n t a g e s d o e s f e r m e n t a t i o n h a v e ? From t h e p o i n t o f v i e w o f c o m m e r c i a l u s e f u l n e s s , m i c r o b i a l p o l y s a c c h a r i d e s a r e found as s l i m y o r g e l a t i n o u s m a t e r i a l s s e c r e t e d i n t o t h e aqueous environment u p o n w h i c h a r e g r o w n many b a c t e r i a , f u n g i a n d y e a s t s . The r e a s o n why t h i s g r o u p o f c o m p o u n d s h a s r e c e i v e d a t t e n t i o n i s c o n n e c t e d w i t h t h e r h e o l o g i c a l and g e l f o r m i n g p r o p e r t i e s w h i c h t h e y show, d e f i n e d b y c o m p a r i s o n w i t h t h e w e l l - k n o w n , i n d u s t r i a l gums f r o m p l a n t sources. I n T a b l e I t h e m a j o r a r e a s o f gum c l a s s i f i c a t i o n a r e p r e s e n t e d , showing comparisons o f b o t h p l a n t a n d m i c r o b i a l gums. The i n t e r e s t o f T a t e a n d L y l e i n m i c r o b i a l gums was s t i m u l a t e d b y t h e e f f e c t i v e i n c r e a s e i n v a l u e w h i c h m i g h t be g i v e n t o s u c r o s e i f used a s a f e r m e n t a t i o n substitute. G e n e r a l l y , gums c a n b e c l a s s e d a s h i g h Table I . Classification of Water Soluble

Examples

Origin (i)

Polysaccharides

Unmodified Gums gum

'trees

arabïc

[

locust bean gum

seeds

guar gum

agar

seaweeds

alginate

plants

Lcarrageenan cornstarch

cereals

potato starch

tubers ^citrus

microorganisms (ii)

bacteria

- L

x a n t h a n gum

[

c a r b o x y m e t h y I c e 11 u i o s e methyl

grasses cottons

cellulose

hydroxymethyl

cereals

rdextrins

tubers

*-carboxymethyl

seaweeds ^citrus

microorganisms

dextran

C

Modified Gums ^trees

plants

pectin

fruits

fruits

bacteria

cellulose starch

propylene glycol low methoxy D.E.A.E.

alginate

pectin

dextran

(Diethyl amino ethyl)



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v a l u e c h e m i c a l s , a n d i t was c o n s i d e r e d t h a t t h e i r p r o d u c t i o n by f e r m e n t a t i o n processes c o u l d l e a d t o r e t u r n s on c a p i t a l e m p l o y e d b e t t e r t h a n t h o s e o b t a i n e d , f o r example, by s t r a i g h t f i n a n c i a l investment. There a l s o was a n a t u r a l a t t r a c t i o n t o w a r d s a p o t e n t i a l i n v o l v e m e n t w i t h t h e new a r e a o f d i v e r s i f i c a t i o n , w h i c h p r o d u c t i o n o f m i c r o b i a l gums w o u l d o f f e r . Glucose and g l u c o s e s y r u p s may b e o b t a i n e d m o r e c h e a p l y t h a n r e f i n e d cane sugar a n d , i n any l o g i c a l p r o c e s s d e v e l o p ment e x e r c i s e , i t w o u l d be n e c e s s a r y t o a c k n o w l e d g e this. F o r c e r t a i n p o l y s a c c h a r i d e f e r m e n t a t i o n s , howe v e r , t h e use o f raw sugar, r e f i n i n g syrups o r molasses may b e c o n t e m p l a t e d s u b s t a n t i a l l y i m p r o v i n g t h e e c o n o mics i n comparison w i t h glucose. A l s o , many s u g a r companies have s t r o n g i n t r i n s i c i n t e r e s t s i n carbohydrates other than sucrose. Again, this i s a possible r e a s o n f o ran i n t e r e s t i n f e r m e n t a t i o n s u b s t r a t e s o t h e r than sucrose. One o b v i o u s d i s a d v a n t a g e f o r a s u g a r company c o n t e m p l a t i n g t h e m a n u f a c t u r e o f m i c r o b i a l gums i s t h e p r o b a b l e l a c k o f b o t h a p p l i c a t i o n s know-how and m a r k e t i n g e x p e r t i s e . One o f t h e m a i n m o t i v a t i n g r e a s o n s f o r T a t e and L y l e ' s e n t h u s i a s m a t a j o i n t v e n t u r e w i t h a company l i k e H e r c u l e s , was i n t h e s t r e n g t h t o be g a i n e d from an o r g a n i s a t i o n a l r e a d y m a r k e t i n g gums. A v a s t number o f m i c r o o r g a n i s m s p r o d u c e e x o c e l l u l a r p o l y s a c c h a r i d e s , a n d many p u b l i c a t i o n s h a v e a p p e a r ed, i n which they a r e d e s c r i b e d . T h e m a j o r i t y , howe v e r , have n o t been e x p l o i t e d c o m m e r c i a l l y . I n Table I I some p o l y s a c c h a r i d e - p r o d u c i n g m i c r o o r g a n i s m s a r e shown t o g i v e a n i d e a o f t h e w i d e o c c u r a n c e o f t h e polysaccharide producers. I n Table I I I are l i s t e d t h o s e gums w h i c h a p p e a r t o b e t h e m o s t a d v a n c e d i n terms o f t h e i r commercial development. T h i s demons t r a t e s t h e c o m m e r c i a l s t a t u s o f t h e gums a n d g i v e s a n indication of possible future trends. To g i v e a n i d e a o f t h e s o r t o f p r o d u c t i o n volumes o f m i c r o b i a l gums, i t h a s b e e n e s t i m a t e d t h a t t h e p r o d u c t i o n o f x a n t h a n gum b y a l l m a n u f a c t u r e r s a m o u n t e d t o some 6,000 t o n n e s i n 1 9 7 5 . Table IV presents f o r comparis o n t h e c o n s u m p t i o n o f gums i n t h e U n i t e d S t a t e s i n 1973. P r o d u c t i o n c o s t s f o r m i c r o b i a l gums a r e v e r y h i g h , m a i n l y due t o t h e h i g h c o s t o f p l a n t . A number of reasons f o rt h i s a r e given l a t e r . Kelco, f o r i n s t a n c e , h a s a n n o u n c e d t h a t t h e i r new p l a n t f o r x a n t h a n gum p r o d u c t i o n i n O k l a h o m a , w i l l c o s t $35 m i l l i o n . The p r i c e s c h a r g e d r e f l e c t t h i s h i g h p r o d u c t i o n c o s t and t h e r e f o r e , i t i s n o t s u r p r i s i n g t h a t t h e p r i c e o f x a n t h a n r a n g e s f r o m $ 3 . 5 p e r l b t o $4 p e r l b , d e p e n d i n g upon grade.


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Table I I . Some Gram

Positive

Polysaccharide-Producing

Bacteria

Yeasts Rhodotorula

Bacillus spp. Leuconostoc

Pichia

spp.

Streptococcus

mutans

Pachysolen

spp.

Lipomices

negative

H.

Crypto

Rhizobium

Torulopsis

£p_.

SD£.

Escherichia

coli

Acetobacter

xylinum

Arthrobacter

viscosus

Pseudomonas

aeruginosa

Xanthomonas

campestris

X.

T.

aerogenes

capsulata

Holstii

A z o t o b a c t e r si

Klebsiella

tannophilus spp.

Hansenula

Bacteria

spp.

spp.

Streptococcus Gram


Microorganisms

_sp£.

molischiana

Pinus

Aureobasidium Other

pullulons

Fungi

Pénicillium Tremella

spp.

mesentaria

phaseoli

Achromobacter spp. Alcaligenes

f a e c a l is v a r .

Agrobacterium Erwinia

myxogenes

spp.

spp.

Sphaerotilus

mutans

Table V g i v e s an i n d i c a t i o n o f t h e type o f p r o c e s s e i t h e r b e i n g operated o r i n development. I t i s t h o u g h t t h a t a l l c o m m e r c i a l i s e d p r o c e s s e s , a t t h e moment, a r e b a t c h , and a l l s u b s t r a t e s a r e c a r b o h y d r a t e s , m o s t l y based on g l u c o s e o r s u c r o s e . I n t h e s e l e c t i o n o f m i c r o b i a l gums f o r c o m m e r c i a l exploitation,inevitably the conclusion i s reached,that, f o r a gum t o b e s u c c e s s f u l , i t m u s t h a v e some u n i q u e physical property. As a l r e a d y mentioned, p r o d u c t i o n costs are high and, i t i s u n r e a l i s t i c t o contemplate d e v e l o p i n g gum s y s t e m s w h i c h show n o n - s p e c i f i c t h i c k e n ing and suspending p r o p e r t i e s . I n Table VI are l i s t e d some p o l y s a c c h a r i d e s , b o t h p l a n t a n d m i c r o b i a l , i d e n t i f y i n g t h e p h y s i c a l p r o p e r t i e s which a r e unique, and uses which a r e s p e c i f i c and n o t e a s i l y c o p i e d by o t h e r gums. Both t h e advantages and disadvantages o f t h e f e r m e n t a t i o n a p p r o a c h t o p o l y s a c c h a r i d e p r o d u c t i o n now w i l l be examined (Table V I I ) . Advantages o f fermentat i o n o v e r t r a d i t i o n a l methods a r e f i r s t l y i n medium preparation. The raw m a t e r i a l s such a s c a r b o h y d r a t e s u b s t r a t e s , n i t r o g e n s o u r c e s and i n o r g a n i c s a l t s normall y a r e r e a d i l y a v a i l a b l e a n d , i n many f e r m e n t a t i o n s , i t


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286

Table

III.

Microbial

Polysaccharides of Commercial

Name

State of

Dextran

Present

- In

Future

- Static

Present

- In

Future

- Expanding

Xanthan

Gum

Importance

Development production

production

(Commercial Trade N a m e

Information) Company Dextran

Various

Products

Pol y d e x

Keltrol

Kelco

Co.

Rhone

Poulenc/

Kelzan Rhodigel

23

General P r e s e n t - In Future

Involved

Tate &

development

Mills Lyle/

Hercules

- Commercialisation announced

Pullulan

Present

- In

Future

- Commercialisation

development Pullulan

Hayashibara

Zanflo

Kelco

Corp.

announced Erwinia

Present

- In

production

(U.S.A.) Exopolysaccharide

Future

- Not

P r e s e n t - In Scleroglucan Microbial alginate

development

Future

- Not

Present

- In

Future

-

Co.

known Polytran

F.S.

Pillsbury

known

development Tate & L y l e

Promising

Ltd./

Hercules Bakers GI

yean

Curdlan

Yeast

Present

- In

Future

- Not

development

Present

- In

Future

- Not

known

development known

BYG

A n h e u s e r - Busch

Inc.

Takeda Chemical

Ind.


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Table IV. The C o n s u m p t i o n o f Industrial G u m s

in T h e U n i t e d States (1973)*

Gum

Uses

Corn

Food sugars

Industrial

Total

-

?

2,232,142

Cornstarch

(Tonnes)

223,214

1,116,071

1,339,285

6,696

43,303

50,000

900

23,660

24,553

6,696

15,625

22,321

Arabic

10,267

3,125

13,392

Pectin

5,357

0

5,357

4,017

1,785

5,803

Alginate

4,017

4,017

8,034

Ghatti

4,464

446

4,910

Carrageenan

4,017

89

4,106

Xanthan

1,000

2,678

3,678

Karaya

446

3,125

3,571

Tragacanth

580

89

669

Agar

133

178

311

C a r b o x y m e t h y l eel1 ulose M e t h y l e e l lui ose Guar

Locust

bean

Furcellaran

*

R.L.

89

Whistler

1974 (unpublished

0

results)


89


gum

Yeast

yean

Curdlan

GI

Bakers

alginate

Microbial

Scleroglucan

Exopolysaccharide

Erwinia

Pullulan

Xanthan

Alcaligenes

free enzyme

bacterial

fungal

bacterial

fungal

bacterial

yeast

(Process

of Process

bacterial

cell

Type

Importance

f a e c a l is

A g r o b a c t e r i u m sp

cerevisiae

Saccharomyces

vinelandii

Azotobacter

glucanicum

Sclerotium

tahitica

Erwinia

pullulons

Aureobasidium

campestris

Xanthomonas

mesenteroides

Leuconostoc

Dextran

of Commercial

Organism

Polysaccharides

Name

Microbial

T a b l e V.

syrup

glucose

glucose

sucrose

glucose

glucose

glucose? syrup?

glucose syrup

glucose

glucose

(sucrose)

glucose

Substrate

Information)

glucose

mannose

glucose

(acetate)

guluronic

acid acid

mannuronic

glucose

(acetyl)

(pyruvate)

uronic acid

fucose

galactose

glucose

glucose

mannose

acid

(acetate)

glucuronic

glucose

glucose

C o m p o n e n t sugar of


polymer

to

o

o

oo

00

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Table V I ,


Some Important Physical Properties of Industrial Gums and their Applications Physical Property

Gum

Application

Cold set, clear, gel formation with divalent cations

Alginate

Re-formed fruit pieces Dental gels

Gel formation with sucrose

Pectin

Jam manufacture

Heat reversible gel formation

Agar

Microbiological solid media Synthetic meat gels

Heat reversible gel formation in the presence of potassium ions

Carrageenan

Non-reactivity with 'reactive' (Procion) dyestuffs

Alginate

Stability in the presence of strong acids

Xanthan gum

Pseudoplastic behaviour under conditions of high shear

Xanthan gum

Synergistic gel formation with carob and guar gums

Xanthan gum

Retardation of sugar crystallisation Gum Arabic at low moisture contents

Synthetic meat gels Instant desserts Textile print paste thickener In rust curing gels containing phosphoric acid As a lubricant for the ben ton i te muds used to drill oil wells Synthetic meat gels In pastilles and jujubes

i s p o s s i b l e t o use p r e c i s e l y d e f i n e d media compositions. In t h ea c t u a l process o f fermentation, ongoing param e t e r s such a s pH, t e m p e r a t u r e , f e r m e n t a t i o n t i m e , d i l u t i o n r a t e and a e r a t i o n c a n be c o n t r o l l e d and manipulated. Two i n t e r r e l a t e d r e s u l t s p o t e n t i a l l y a r e p o s s i b l e through these c o n t r o l s . The f i r s t i s t h e maintenance o fproduct s p e c i f i c a t i o n s w i t h i n d e f i n e d l i m i t s , and t h i s i s p a r t i c u l a r l y v i t a l i n polysacchar i d e f e r m e n t a t i o n s where even s m a l l changes i n c e r t a i n p r o c e s s v a r i a b l e s c a n have d r a m a t i c e f f e c t s on polymer s t r u c t u r e and, t h e r e f o r e , p h y s i c a l b e h a v i o u r . This, i n c i d e n t a l l y , i s o n e r e a s o n why t h e u s e o f c o n t i n u o u s c u l t u r e i sfavoured. Under steady s t a t e c o n d i t i o n s , the v a r i a b l e s o f fermentation a r eh e l d constant w i t h r e s p e c t t o e a c h o t h e r , t h u s a f f o r d i n g a much b e t t e r understanding and c o n t r o l o f the process. The s e c o n d e f f e c t i s t h e p o t e n t i a l a b i l i t y t o mani p u l a t e product type and y i e l d . F o r example, t h e a b i l i t y through s p e c i f i c changes i n f e r m e n t a t i o n cond i t i o n s t o p r o d u c e r a n g e s o f m i c r o b i a l gums o f a p a r t i cular type, d i f f e r i n g i n molecular weight. Another advantage o f fermentation i s t h a t , i nproduct recovery, harsh e x t r a c t i o n techniques normally a r en o tr e q u i r e d a n d , v e r y o f t e n , s o l v e n t p r e c i p i t a t i o n may b e c o n t e m plated which, o f course, i s a very m i l d treatment, not l i k e l y t o l e a d t o product degradation. The c h o i c e


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Table VII. Benefits O b t a i n e d

in P r o d u c i n g

by


1 .

Medium

Fermentation

Preparation

(a)

Raw

(b)

Precisely

2.

Polysaccharides

materials

in plentiful

defined media

supply

possible

Fermentation (a)

H i g h degree of process control

(b)

Continuous culture possible/, high

Product

3.

(a)

Mild

possible productivity

Recovery

conditions can

be u s e d . M i t t l e

product

degradation.

Table

VIII. Technical

Problems

in M i c r o b i a l

H i g h broth viscosities,

(i)

Polysaccharide

r e s u l t i n g in

Low product concentration,

Production

:

therefore

l a r g e v o l u m e s of w a t e r large fermenter

(ii)

High energy requirements oxygen bulk water

(iii)

capacity

for

transfer

mixing removal

Difficulties

in c e l l

removal.



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of p r o d u c t i o n s i t e s can be f a i r l y f l e x i b l e , l i m i t e d mainly by the a v a i l a b i l i t y o f water. F i n a l l y , the use of c o n t i n u o u s c u l t u r e can enhance p r o d u c t i v i t i e s over t h o s e o b t a i n e d i n b a t c h c u l t u r e s , a s c e l l s a r e grown always under c o n d i t i o n s most conducive t o e f f i c i e n t product formation. Turning t o the disadvantages o f fermentation (Table V I I I ) , they can be summarised as b e i n g caused l a r g e l y by the h i g h l y v i s c o u s fermentation broths encountered. This s e v e r e l y l i m i t s the concentrations of polymer i t i s p r a c t i c a b l e t o s y n t h e s i z e . The r a n g e 3 0 - 4 0 , 0 0 0 c p s i s n o t uncommon a n d n o c o n c e n t r a t i o n s o f gum a b o v e a b o u t 4% c a n b e c o n t e m p l a t e d . It follows, t h e r e f o r e , t h a t very l a r g e fermenters are r e q u i r e d i n order t o s y n t h e s i s e economically s e n s i b l e tonnages o f gum. F e r m e n t e r s i z e s o f 5 0 - 2 0 0 m^ a r e p r a c t i c a l . Very l a r g e volumes o f both water and product recovery s o l v e n t are r e q u i r e d , s o t h a t e f f l u e n t problems must be c o n t e m p l a t e d , u n l e s s r e c y c l i n g i s t o b e u n d e r t a k e n . Other problems l i e i n t h e h i g h power r e q u i r e m e n t s needed t o o b t a i n s a t i s f a c t o r y b r o t h m i x i n g and a e r a t i o n w h i c h , i f not adequate,can l e a d q u i c k l y t o oxygen l i m i t a t i o n and lowered p o l y s a c c h a r i d e p r o d u c t i v i t i e s . F i n a l l y , c e l l r e m o v a l , p a r t i c u l a r l y b y m e c h a n i c a l means ( c e n t r i f u g e , f i l t e r s ) , i s d i f f i c u l t and, a l t h o u g h metho d s b a s e d o n e n z y m e d i g e s t i o n o f c e l l s (1) a n d a l k a l i d i g e s t i o n h a v e b e e n p u b l i s h e d (2^) ,many p r o b l e m s h a v e y e t t o be s o l v e d . I n summary, i t i s i m p o r t a n t t o p o i n t o u t t h a t , a l though f e r m e n t a t i o n as an approach t o p o l y s a c c h a r i d e p r o d u c t i o n i s not w i t h o u t problems, the concept c o u l d , i n t h e f u t u r e , r e v o l u t i o n i z e many a s p e c t s o f i n d u s t r i a l gum production. A s p e c t s o f t h e work b e i n g u n d e r t a k e n i n t o the p r o d u c t i o n o f m i c r o b i a l a l g i n a t e a t the Tate and L y l e Labo r a t o r i e s now w i l l b e e x a m i n e d . Briefly, alginate i s b e s t known a s t h e p o l y s a c c h a r i d e o b t a i n e d f r o m b r o w n algae such as species o f Laminaria and M a c r o c y s t i s (Figure 1). I t i s a l i n e a r polymer o f ß-D-mannuronic a c i d and a - L - g u l u r o n i c a c i d . The a r r a n g e m e n t o f t h e monomers h a s b e e n s t u d i e d b y H a u g a n d c o - w o r k e r s (3) a n d R e e s a n d c o - w o r k e r s (4_) a n d r e f e r r e d t o a s t h e block structure. That i s , homopolymeric b l o c k s o f mannuronic and g u l u r o n i c a c i d comprise the so c a l l e d , "alternating regions". The f l o w and g e l - f o r m i n g p r o p e r t i e s o f the polymer i n aqueous s o l u t i o n depend on the p r o p o r t i o n s o f the monosaccharide r e s i d u e s , on t h e i r arrangement and on the polymer molecular weight. T h i s a p p l i e s v e r y much t o g e l f o r m a t i o n i n t h e p r e s e n c e of d i v a l e n t metal c a t i o n s as, f o r example, a l g i n a t e s


SUCROCHEMISTRY

292 Monomers


^-D-Mannuronic

Block

acid

oC

-L-Guluronic

acid

Structure

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

Figure 1.

Structure of alginic acid

having a high proportion of guluronic a c i d p a r t i c u l a r l y as h o m o p o l y m e r s f o r m t h e s t r o n g e s t a n d most b r i t t l e gels. The e x o c e l l u l a r p o l y s a c c h a r i d 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 h a s b e e n shown i n i t i a l l y b y G o r i n a n d S p e n c e r (5») a n d b y t h e l a t e A r n e Haug a n d c o - w o r k e r s (6) t o h a v e t h e same b a s i c s t r u c t u r e a s t h a t f r o m a l g a l s o u r c e s e x c e p t t h a t a m a l l number o f hydroxy1 groups were a c e t y l a t e d . I n t h e Tate and L y l e study o f A z o t o b a c t e r a l g i n a t e the o b j e c t i v e has been t h e development o f a p r o d u c t w h i c h would compete b o t h i n b e h a v i o u r and economic terms w i t h t h e a l g a l m a t e r i a l s on s a l e i n world markets. E a r l y s t u d i e s i n batch c u l t u r e under t h e c o n d i t i o n s d e s c r i b e d b y G o r i n a n d S p e n c e r (5), g a v e p o o r p r o d u c t s , obtained i n very low y i e l d . Subsequent improvements w e r e made b y g r o w i n g t h e o r g a n i s m u n d e r 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 l u s o t h e r m o d i f i c a t i o n s w h i c h i n c r e a s e d t h e c o n s i s t e n c y i n d e x (a measure o f f l o w b e h a v i o u r i n a q u e o u s s o l u t i o n ) f r o m ^ 3 0 c p s t o 4,000 cps, thus c o v e r i n g t h e range o f commercially a v a i l a b l e , algal alginates. This wide range o f product v i s c o s i t i e s a l s o has been o b t a i n e d from c o n t i n u o u s c u l t u r e s (Figure 2 ) . The most v i s c o u s p r o d u c t h a s a c o n s i s t e n c y i n d e x o f 6,000 c p s ( 1 % c o n c e n t r a t i o n ) . The g e l forming p r o p e r t i e s o f m i c r o b i a l a l g i n a t e a l s o were d e m o n s t r a t e d t o be s i m i l a r t o t h e i r a l g a l c o u n t e r p a r t s . f


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In batch c u l t u r e s , the h i g h e s t y i e l d s o f sodium a l g i n a t e p o s s i b l e t o o b t a i n under phosphate d e f i c i e n t c o n d i t i o n s , were f o u n d t o a p p r o x i m a t e 25% o f t h e s u crose u t i l i s e d (Figure 3)• High r e s p i r a t i o n i n A.vinel a n d i i i s a w e l l - k n o w n phenomenon a n d , a s a r e s u l t und e r c e r t a i n c o n d i t i o n s , much o f t h e s u c r o s e c a n b e u t i l i z e d i n a n u n c o n t r o l l e d manner and l o s t a s c a r b o n dioxide. It,therefore,was decided to i n v e s t i g a t e the e f f e c t of r e s p i r a t i o n on alginate production i n continu o u s c u l t u r e ; a t e c h n i q u e h a v i n g t h e p o t e n t i a l o f much

10,000p

1

10

10Ö

TTürfo

Rate of shear (sec ^)

Figure 2.

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


SUCROCHEMISTRY

294

Cells


o

Alginate

• -^iginate/Cells

Figure 3. Effect of phosphate concentration on alginic acid synthesis by Azotobacter vinelandii in batch culture

greater control. Phosphate-limited growth c o n d i t i o n s were c h o s e n , a s a p a u c i t y o f p h o s p h a t e was i n d i c a t e d b y the work i n b a t c h c u l t u r e . R e s p i r a t i o n r a t e was c o n t r o l l e d by manipulating fermenter i m p e l l e r speeds, r e s u l t i n g i n a l t e r e d oxygen t r a n s f e r i n t o t h e fermentation broth. T h e r e f o r e , a l t h o u g h c e l l mass was c o n t r o l l e d a t an e s s e n t i a l l y constant l e v e l by t h e phosphate a v a i l a b i l i t y , t h e s p e c i f i c r e s p i r a t i o n r a t e was d e t e r m i n e d b y t h e a v a i l a b i l i t y o f o x y g e n ( F i g u r e 4 ) . Under t h e above c o n d i t i o n s , a t lower r e s p i r a t i o n r a t e s , t h e maximum y i e l d o f s o d i u m a l g i n a t e w a s i n t h e r e g i o n o f 45% o f t h e s u c r o s e u t i l i s e d . A thigher r e s p i r a t i o n r a t e s , t h e y i e l d f e l l d r a m a t i c a l l y due t o a g r e a t e r p r o p o r t i o n o f sucrose being burned o f f a s CO2. In c o n c l u s i o n , t h e production o f m i c r o b i a l polys a c c h a r i d e s o f c o m m e r c i a l s i g n i f i c a n c e i s now a w e l l e s t a b l i s h e d f a c t a n d a new g e n e r a t i o n o f w a t e r s o l u b l e polymers i s being i d e n t i f i e d . However, t h e p r o d u c t i o n t e c h n o l o g i e s a r e e x p e n s i v e , r e l a t i v e t o many o f t h e p l a n t gums. Biopolymers w i l l only gain a f u l l y established competitive position leading t o the use o f i n d u s t r i a l scale continuous c u l t u r e s through the succ e s s o f c u r r e n t r e s e a r c h programmes i n t o t h e p h y s i o l o g y of exopolysaccharide s y n t h e s i s .

Abstract In recent years, the exocellular polysaccharide elaborated by the bacterium Xanthomonas compestris has emerged as a product with significant industrial appli—



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cation, and the present annual world consumption is several thousand tons. This has demonstrated the potential of fermentation for producing polysaccharides having unusual solution and gel properties. At the present time there is an increasing demand for xanthan gum and new applications appear regularly in the patent literature and elsewhere. It is likely that other microorganisms w i l l be capable of producing commercially valuable polysaccharides and, as a consequence, a number of systems are under investigation with several apparently at an advanced stage of development. Tate and Lyle became interested in microbial gums through reports that the bacteria Azotobacter vinelandii and Pseudomonas aeruginosa produce polysaccharides similar to the polyuronide, alginic acid. The sole source of this polysaccharide at present is the brown algae from which approximately 20,000 tons of alginate are extracted per annum. Pseudomonas aeruginosa was rejected for study due to its association with pathogenic conditions in man. As initial assessments indicated that the Azotobacter polysaccharide could be commercially valuable if sufficiently high yields were

1.5,-

0

10

20

30

40

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

Figure

Exopolysaccharide production by Azotobacter vinelandii at a range of respiration rates



296 SUCROCHEMISTRY obtained, the latter was selected for further study. Subsequent developments to pilot plant level have involved improving yield and controlling and manipulating the physical properties of the polymer produced by appropriate choice of growth media and fermentation conditions, using both batch and continuous culture and also by strain selection. Literature Cited 1. Kelco Co., British Patent, 1,443,507, (1975). 2. Patton, J.T., United StaTes Patent, 3,729,460, (1970). 3. Larsen, B . , Smidsrod, O., Haug, A. and Painter, T. Acta. Chem. Scand., (1969), 23, 2375. 4. Rees, D.A., Biochem. J., (1972), 126, 257. 5. Gorin, P.A.J, and Spencer, J.F.T., Canad. J. Chem. (1966), 44, 993. 6. Larsen, B. and Haug, A . , Carbohyd. Res., (1971), 17, 287. 7. Imrie, F.K.E., British Patent, 1,331,771, (1973). Biographic Notes Christopher J . Lawson, Ph.D., Project Leader Microbiological Polysaccharides. Educated at the Univ. of Edinburgh. Joined Tate and Lyle, Ltd., in 1969, specializing in microbiological polysaccharides by fermentation. Tate and Lyle, Ltd., Philip Lyle Memorial Research Laboratory, P.O. Box 68, Reading, Berkshire RG6 2BX, England.


Production of Industrially Important Gums with ... - ACS Publications

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