Extracellular Microbial Polysaccharides

Extracellular Microbial Polysaccharides In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium...

Author: Paul A. Sandford | Allen Laskin

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Extracellular Microbial Polysaccharides

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


Extracellular Microbial Polysaccharides Paul A . Sandford, EDITOR U. S. Department of Agriculture A l l e n Laskin, EDITOR Exxon Research and Engineering Co.

A symposium co-sponsored by the Division of Carbohydrate Chemistry and the Division of Microbial and Biochemical Technology at the 172nd Meeting of the American Chemical Society, San Francisco, Calif., August 3 0 - 3 1 , 1976

ACS SYMPOSIUM SERIES 45

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1977


Library of Congress CIP Data Extracellular microbial polysaccharides. (ACS symposium series; 45 ISSN 0097-6156) Includes bibliographical references and index. 1. Microbial polysaccharides—Congresses. I. Sandford, Paul Α., 1939- . Π. Laskin, Allen I., 1928. III. American Chemical Society. Division of Carbohydrate Chemistry. IV. American Chemical Society. Division of Microbial and Biochemical Technology. V. Series: American Chemical Society. ACS symposium series; 45. QR92.P6E97 ISBN 0-8412-0372-5

Copyright ©

660'.62 ACSMC 8

77-6368 45 1-326

1977

American Chemical Society All Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photo copying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E UNITED STATES O F AMERICA


ACS Symposium Series Robert F. G o u l d , Editor

Advisory Donald G. Crosby Jeremiah P. Freeman E. Desmond Goddard Robert A. Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold


FOREWORD The A C S

S Y M P O S I U M

SERIES

was founded in 1974 to provide

a medium for publishin format of the IN

CHEMISTRY

SERIES

parallels that of the continuing

SERIES

except that in order to save time the

papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form.

As a further

means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book.

Papers published in the A C S

S Y M P O S I U M

SERIES

are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.


PREFACE

A

new fermentation industry, the production of extracellular microbial water-soluble polysaccharides, arose in the late 1950's and early

1960's and is now expanding rapidly. Several factors have

accelerated

the use of microbial polysaccharides as well as the search for new sources of water-soluble polysaccharides. Although hydrocolloids obtained from plants and seaweed have been used successfully for numerous applications in the food, textile, agricultural, paint, and petroleum industries, increasing labor costs, limited sources, adverse climate conditions, and increased demands have several of these traditionally used plant and seaweed gums. Also industry has demands for water-soluble polymers that are not met by the traditional plant and seaweed gums. Extracellular polysaccharide production is a widespread characteristic of microorganisms.

Several of these polymers have proven to be

commercially significant. T h e usefulness of these microbial polysaccharides primarily results from their unique physical and chemical properties which are determined by their individual component sugars and their mode of linkages. Their constant chemical properties and constant supply also increase their desirability. Other reasons for industry's interest in microbial gums are their potentially diverse sources and types. This symposium focuses on the production and properties of extracellular microbial polysaccharides that are currently being used by industry or which have potentially useful industrial properties. Special emphasis is placed on new areas of research that would improve or stimulate industrial production and use of this valuable class of water soluble hydrocolloids. U.S. Department of Agriculture

P A U L A . SANDFORD

Peoria, Ill. 61604 Exxon Research and Engineering C o .

A L L E N I. L A S K I N

Linden, N.J. 07036 January 12, 1977

ix


1 Culture Maintenance and

Productivity

DENIS K. KIDBY Department of Soil Science and Plant Nutrition, The University of Western Australia, Nedlands, Western Australia, 6009

Microbial productivity is based upon a very large store of genetic information. I than one million items encoded build-up, i t is a common practice to transfer approximately 10 cells to a fresh medium. To retain the complete genetic identity of such an inoculum, for even a single generation, 10 base pairings must occur with complete f i d e l i t y . However, examination of such a c e l l population would reveal that thousands of errors had occurred. The f i d e l i t y of DNA replication is nevertheless impressive, and given s k i l f u l management, microbes can approach the r e l i a b i l i t y of solution chemistry in terms of product reproducibility. While genetic change may be a disaster when uncontrolled, i t is also the means of improving productivity. Genetic alterations were once achieved more or less by chance. However, the possibility now exists for the deliberate, and specific, alteration of genotype to yield productive chimeras limited only by the imagination. One can envisage the real possibility of producing a bacterial c e l l which could extract i t s energy and growth requirements from a few simple salts, the a i r and sunlight, producing a bacterial product such as Xanthan Gum or, an algal product such as agarose. However, despite such advances in the manipulation of genes, i t seems certain that the inherent genetic i n s t a b i l i t y of microbes w i l l remain an important problem for many years; and it is largely to this type of d i f f i c u l t y that the present paper is addressed. Before discussing i n s t a b i l i t y , the origins of industrial cultures w i l l be briefly considered. 9

16

Sources of Microbes Natural Sources. Many useful microbes are directly obtainable from the s o i l or other natural sources. It is often possible to employ unusual or extreme conditions as selective agents in the search for microbes with special a b i l i t i e s . Bacteria isolated from hot springs, can be grown near the temperature of boiling water (1). Acid mine leachings harbour 1


2

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

b a c t e r i a able to grow at high concentrations of s u l f u r i c a c i d (2). Microbes f r e e of toxins or e s p e c i a l l y a l l e r g e n i c substances may be sought i n f o o d s t u f f s i n which they are known to r e g u l a r l y occur i n h i g h c o n c e n t r a t i o n s . I s o l a t i o n Procedures. The p r i n c i p l e s employed are those of s e l e c t i v e enrichment or i n h i b i t i o n . The r e q u i r e d , or suspected, n u t r i t i o n a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the organism sought w i l l d i c t a t e and a c t u a l procedure. The o x i d a t i o n , r e d u c t i o n , b i n d i n g , or r e l e a s e , of dyes are p a r t i c u l a r l y adapt a b l e f o r s e r v i c e as i n d i c a t o r s of s p e c i f i c biochemical events. The possession of a p a r t i c u l a r enzyme, or s e r i e s of enzymes, may be l i n k e d to e i t h e r the a b i l i t y , or i n a b i l i t y , to grow on a p a r t i c u l a r medium. B i o l o g i c a l i n d i c a t o r s such as the growth of an i n d i c a t o r organism ar p a r t i c u l a r l s e n s i t i v t h func t i o n s as the e x c r e t i o n o methods have been devise which are by t h e i r nature c r y p t i c and seemingly i n a c c e s s i b l e f o r s e l e c t i o n . For example Okanishi and Gregory Ô ) were able to devise a simple method to r e v e a l yeast c o l o n i e s possessing higher than normal methionine l e v e l s . Protocols f o r the i s o l a t i o n of s p e c i f i c n u t r i t i o n a l types may be sought i n the taxonomic l i t e r a t u r e (4, 5). Specific procedures f o r various groups of organisms are a v a i l a b l e i n the recent l i t e r a t u r e (6>, J7> 8). However, the seeker of d e s i r a b l e microbes must o f t e n r e l y upon h i s own r e s o u r c e f u l n e s s . A fairly thorough biochemical understanding of the event of i n t e r e s t can b e a most u s e f u l guide to i s o l a t i o n procedures. In the case of e x t r a c e l l u l a r products, such as polysaccha r i d e s , there may or may not be c h a r a c t e r i s t i c a l l y mucoid colonies. S e l e c t i v e procedures should, i f p o s s i b l e , e x p l o i t some s p e c i f i c property of the d e s i r e d p o l y s a c c h a r i d e . However, there are p o s s i b i l i t i e s f o r i n d i r e c t s e l e c t i o n using a s s o c i a t e d c h a r a c t e r i s t i c s . For example, many c h a r a c t e r i s t i c s , s u i t e d to r e p l i c a - p l a t i n g methods, are a s s o c i a t e d with polysaccharide producing Xanthomonas campestris ( 9 ) . In the case of mucoid E s c h e r i c h i a c o l i , there appears to be a s s o c i a t e d UV s e n s i t i v i t y (10) . R e p l i c a - p l a t i n g procedures are f r e q u e n t l y the most u s e f u l technique s i n c e one can s e l e c t f o r c e l l s which e i t h e r grow or do not grow. D i a g n o s t i c procedures which are d e s t r u c t i v e may a l s o be used since a l l m a t e r i a l under i n v e s t i g a t i o n i s r e t a i n e d on the r e p l i c a s . The employment of s p e c i f i c enzymes f o r the recogn i t i o n of c e r t a i n types of polysaccharides i s an i n t e r e s t i n g p o s s i b i l i t y f o r the development of screening programmes. In t h i s connection i t i s i n t e r e s t i n g to note that r e c o g n i t i o n systems based upon enzyme s p e c i f i c i t y may already occur i n bacteriophage (11) . C u l t u r e C o l l e c t i o n s . Searching f o r microbes i n e x i s t i n g c u l t u r e s w i l l f r e q u e n t l y be quicker, cheaper and e a s i e r than i s o l a t i o n from nature. As an a i d to such a search, H e s s e l t i n e and Haynes (12) have w r i t t e n a guide to c o l l e c t i o n s containing


1.

KiDBY

Culture

Maintenance

and

3

Productivity

i n d u s t r i a l l y u s e f u l microbes. However, there can be no f o r thorough searching of the current l i t e r a t u r e .

substitute

Maintenance of Genotype Nature of the Problem. An i n d u s t r i a l l y u s e f u l microbe i s an asset which may range from being moderately valuable to almost p r i c e l e s s . The p r e s e r v a t i o n of such an asset deserves a p r i o r i t y which i t seldom r e c e i v e s . The greatest b a r r i e r to s u c c e s s f u l p r e s e r v a t i o n of genotype may be a f a i l u r e to appreciate that: ( i ) microbes are i n h e r e n t l y unstable, ( i i ) there i s no method yet devised f o r the complete p r e s e r v a t i o n of genotype. Inherent I n s t a b i l i t y of Microbes. The p o t e n t i a l f o r genotype v a r i a b i l i t y has been i n d i c a t e d i n the i n t r o d u c t o r y remarks. I t i s now necessary to discus th a c t u a l mechanis f chang d how these r e l a t e to phenotype A l l regions of a gen gene mutable than others because they have i n t r a g e n i c regions of high m u t a b i l i t y , are i n f l u e n c e d by some other gene which i s i t s e l f mutable or, are under the c o n t r o l of genes which promote mutation. A l l of these mechanisms are known to occur, i n c l u d i n g some i n which the m u t a b i l i t y i s e f f e c t e d by an extrachromosoma1 element o r , an i n f e c t i o u s agent (13). I t i s these more h i g h l y mutable genes, and e s p e c i a l l y those cases i n v o l v i n g i n f e c t i o u s agents, that are most troublesome. Mutations may be e i t h e r r e p l i c a t i o n dependent or r e p l i c a t i o n - i n d e p e n d e n t . I t i s speculated (14) that replication-dependent mutations r e f l e c t e r r o r s i n DNA r e p l i c a t i o n , and replication-independent mutations r e f l e c t error-prone r e p a i r systems, Mutations may i n v o l v e : ( i ) frame-shift; ( i i ) deletion; ( i i i ) i n s e r t i o n ; ( i v ) base p a i r s u b s t i t u t i o n . The e f f e c t on the code may be e i t h e r the production of missense, nonsense, or a non-code f u n c t i o n may be l o s t . The r e s u l t i n g phenotypes may i n c l u d e : ( i ) a l t e r e d RNA base sequence; ( i i ) a l t e r e d amino a c i d sequence; ( i i i ) premature termination; ( i v ) degenerate s i l e n c e . A c e r t a i n p r o p o r t i o n of these mutants w i l l be c r y p t i c , p a r t i c u l a r l y those i n v o l v i n g missense. Mutations which lead to the i n s e r t i o n of a s i m i l a r amino a c i d o r , because of code degeneracy, the w i l d type amino a c i d , w i l l u s u a l l y not be revealed. I t has been c a l c u l a t e d that 25% of 549 base p a i r s u b s t i t u t i o n s i n v o l v e degeneracy (15) . I t i s a l s o i n t e r e s t i n g to note that there i s a greater than random p r o b a b i l i t y that base p a i r s u b s t i t u t i o n s w i l l lead to s u b s t i t u t i o n of a s i m i l a r r a t h e r than a d i s s i m i l a r amino a c i d (16). L e t h a l mutations w i l l a l s o be c r y p t i c since these w i l l not p e r s i s t , unless they are c o n d i t i o n a l . Intragenic mutations are non-random. S i t e s which are h i g h l y mutable are hot spots (17) . Evidence on the nature of hot spots has been reviewed by Clarke and Johnston (1976) and w i l l be merely summarized here. High M u t a b i l i t y Regions, ( i ) Frameshift mutations tend to occur i n regions of repeated base p a i r s . Runs of e i t h e r AT or GC base p a i r s have been a s s o c i a t e d with f r a m e s h i f t s . ( i i ) Base


4

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p a i r s u b s t i t u t i o n s are i n f l u e n c e d by neighbouring bases. The AT-GC s u b s t i t u t i o n induced by 2-aminopurine at the second p o s i t i o n of a t r i p l e t has been demonstrated to occur 23 times more f r e quently when an AT base p a i r was present i n the t h i r d p o s i t i o n (18). ( i i i ) Mutator polymerase acts p r e f e r e n t i a l l y on s p e c i f i c regions of the gene, ( i v ) The frequency and l o c a t i o n of d e l e t i o n s i s non-random and such s i t e s are considered d e l e t i o n hot spots, (v) U l t r a - v i o l e t induced mutations are most frequent i n t r a c t s of p y r i m i d i n e s . Development of a S t a b l e Mutation. Most mutations are formed from pre-mutational l e s i o n s . The l e s i o n may or may not be r e p a i r e d o r , the r e p a i r process i t s e l f may lead d i r e c t l y to mutat i o n . F a i l i n g r e p a i r , the pre-mutational l e s i o n may be e s t a b l i s h ed as a mutation by DNA development of a mutatio these steps may be subject to the i n f l u e n c e of adjacent base pairs. In the l i g h t of these o b s e r v a t i o n s , one might ask what avenues e x i s t f o r the a m e l i o r a t i o n or removal of hot spots? I f the mutation i s e f f e c t e d by a mutagen, i t may be p o s s i b l e to e i t h e r remove or suppress the c o n d i t i o n l e a d i n g to the presence of the mutagen or n e u t r a l i z e i t s a c t i v i t y with an antimutator. Precedents f o r t h i s l a t t e r approach are now w e l l documented (14). Antimutagenesis. I t has been q u i t e p r o p e r l y s t a t e d (14) that one cannot understand mutagenesis or the r e g u l a t i o n of mutation frequency without c o n s i d e r i n g antimutagenic e f f e c t s . Antimutagenesis may be d e f i n e d as a decrease i n the a c t u a l r a t e of mutation. Decreased apparent rates may be caused by e i t h e r a l t e r e d s u r v i v a l or dose r e d u c t i o n , and these e f f e c t s are termed apparent antimutagenesis. A mutation or premutation may a r i s e by: ( i ) r e a c t i o n between a mutagen and DNA; ( i i ) i n c o r p o r a t i o n of a mutagen-altered precursor or base analogue; ( i i i ) r e p l i c a t i o n e r r o r ; ( i v ) recombination e r r o r ; (v) r e p a i r e r r o r ; ( v i ) t r a n s c r i p t i o n e r r o r ; ( v i i ) t r a n s l a t i o n e r r o r . The l a s t two mechanisms i n v o l v e the p r o d u c t i o n of error-prone RNA or p r o t e i n s which a l t e r the base sequence of DNA e i t h e r d i r e c t l y or i n d i r e c t l y (19, 20, 21). C l a r k e and Shankel (14) have d i s t i n g u i s h e d between genetic antimutagenesis, which i s the antimutagenic e f f e c t of r e p l i c a t i o n genes, r e p a i r genes, or other genetic determinants, and p h y s i o l o g i c a l antimutagenesis which i s achieved by added chemicals or a l t e r e d c e l l c o n d i t i o n s . The p h y s i o l o g i c a l mechanism would appear to o f f e r c o n s i d e r a b l e p o t e n t i a l f o r the r e d u c t i o n of mutation r a t e s f o r c e r t a i n c l a s s e s of mutation. For example, adenosine appears to be capable of v i r t u a l l y a b o l i s h i n g the mutagenicity of purine mutagens (14). Spontaneous mutation rates have a l s o been d r a m a t i c a l l y reduced by the use of a c r i d i n e s (22). An o b s e r v a t i o n of c o n s i d e r a b l e i n t e r e s t i s that genes are more l i k e l y to mutate when being t r a n s c r i b e d (14). Thus the r e p r e s s i o n of gene a c t i v i t y i s antimutagenic. I t might be expected,


1. KiDBY

Culture

Maintenance

and

WILD GENOTYPE

WILD PHENOTYP

ι

5

Productivity

LESION REPAIR

PREMUTATION

SUPRESSION SILENT MISSENSE DEGENERACY

REPLICATION

MUTATION SELECTION

Figure 1.

Sequences of events in mutation and selec tion


6

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

t h e r e f o r e , that i n maintenance and inoculum build-up c u l t u r e s , the r e p r e s s i o n of the productive f u n c t i o n would help to a r r e s t v a r i a b i l i t y by decreasing the r a t e of mutation. I t may a l s o be the case that r e p r e s s i o n of product formation w i l l help prevent s e l e c t i o n against producer c e l l s . There i s some evidence (23^ 24) that product r e p r e s s i o n may be of use i n reducing v a r i a b i l i t y i n Xanthomonas campestris. There seems l i t t l e reason to doubt that DNA which i s not being t r a n s c r i b e d should be r e l a t i v e l y s t a b l e . I t would be of considerable i n t e r e s t to see i f mutations i n derepressed genes are i n f a c t p r o p o r t i o n a l to t r a n s c r i p t i o n r a t e s . I t may w e l l be that c e r t a i n microbes with high product y i e l d s are i n h e r e n t l y unstable because of high t r a n s c r i p t i o n a l a c t i v i t y . L i m i t i n g the Opportunit f o Mutation Mutatio be a f u n c t i o n of r e p a i r mutagen or antimutagen , physica such as r a i s e d temperature, low water a c t i v i t y , or i c e c r y s t a l s . Whatever the c o n d i t i o n l e a d i n g to mutation, the most e f f e c t i v e p r o t e c t i o n i s to minimise the exposure of the c u l t u r e to the conducive c o n d i t i o n . The growth i n mutant numbers i s a f u n c t i o n of the number of r e p l i c a t i o n s (Table I ) . I t f o l l o w s , t h e r e f o r e , that the t o t a l number of r e p l i c a t i o n s should be minimized. If r e p l i c a t i o n - i n d e p e n d e n t mutations are taken i n t o account, then i t a l s o follows that the t o t a l residence time i n c u l t u r e should be minimized. I f , as seems to be the general case, mutation i s p r o p o r t i o n a l to t r a n s l a t i o n a l a c t i v i t y , then the productive f u n c t i o n should be repressed u n t i l needed. The e x c l u s i o n or r e d u c t i o n of potent mutagens may seem too obvious to r e q u i r e f u r t h e r comment. However, many commonly o c c u r r i n g mutagens such as metal i o n s , adenine, c a f f e i n e , ozone, to name a few, seem o f t e n to escape a t t e n t i o n . The number of base analogues generated by chemical, or high temperature, treatment of concentrated sources of purine and pyrimidine bases must o f t e n be c o n s i d e r a b l e . The frequent proximity of c u l t u r e s to e l e c t r i c motors and, i n p a r t i c u l a r , atmospheres r e c e n t l y i r r a d i a t e d with u l t r a - v i o l e t l i g h t must s u r e l y produce l a r g e numbers of ozone-induced mutants. Extremely high l e v e l s of mutation have been observed i n E. c o l i exposed to as l i t t l e as 0.1 ppm ozone f o r 60«minutes (10). The question of l i m i t i n g the opportunity f o r mutation w i l l be f u r t h e r discussed i n connection w i t h p r e s e r v a t i o n techniques. L i m i t i n g the Opportunity f o r S e l e c t i o n . The s e l e c t i o n of a mutant, i n the present context, may be taken to mean the increase of any given mutant to a s i g n i f i c a n t p r o p o r t i o n of the t o t a l p o p u l a t i o n . The extent of t h i s s e l e c t i o n w i l l be a f u n c t i o n of the c u l t u r e c o n d i t i o n s and the number of generations of c u l t u r e growth permitted. S e l e c t i v e media may be employed to remove p a r t i c u l a r c l a s s e s of mutant. N u t r i t i o n a l l y r i c h media w i l l tend to preserve and o f t e n concentrate auxotrophs while a poorer medium may s e l e c t f a i r l y e f f i c i e n t l y against auxotrophs, unless



a

m = mutation r a t e

Mutant: T o t a l

Mutant C e l l s

64mN 4m

24mN 3m

8mN 2m

2mN m

0

0

16N

8N

4N

2N

Ν

Total

Cells

4

3

2

1

0

THE PROPORTION OF MUTANTS IN A GROWING CULTURE

Generations

TABLE I

8

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

high r a t e s of c r o s s - f e e d i n g occur. Short-term P r e s e r v a t i o n . The p r e s e r v a t i o n of c e l l v i a b i l i t y f o r periods of l e s s than a few months might a r b i t r a r i l y be termed short-term p r e s e r v a t i o n . While there can be no doubt as to the d e s i r a b i l i t y of long-term p r e s e r v a t i o n , methods of a c h i e v i n g t h i s u s u a l l y provide r e l a t i v e l y i n a c c e s s i b l e i n o c u l a and, i n some cases, may be of l i m i t e d success. In order to be u s e f u l , a short -term p r e s e r v a t i o n method must provide a high recovery of v i a b l e c e l l s which grow with a minimum l a g phase. The inoculum should be e a s i l y a c c e s s i b l e and of a standard and s u i t a b l e s i z e . Subc u l t u r e to achieve v i g o r o u s l y growing and r e p r o d u c i b l e c u l t u r e s should not be necessary. I f these c r i t e r i a cannot be met, i t may be b e t t e r to consider the r o u t i n e use of i n o c u l a preserved by long-term methods. U s e f u l short-term p r e s e r v a t i o t i o n s of d r y i n g procedures. A p a r t i c u l a r l y s u i t a b l e method i s the d r y i n g of c u l t u r e s onto paper (2_5, 26). Paper s t r i p s have the advantage of being e a s i l y handled and are r e a d i l y adjusted i n s i z e to y i e l d an appropriate inoculum s i z e . The method has been used w i t h success f o r X. campestris NRRL B1459 ( 9 ) . Other s h o r t term p r e s e r v a t i o n methods have been reviewed elsewhere (26). The repeated t r a n s f e r of c u l t u r e s f o r r o u t i n e maintenance must be considered an unwise p r a c t i c e and i s d i f f i c u l t to j u s t i f y where a l t e r n a t i v e non-propagative methods e x i s t . Long-term P r e s e r v a t i o n . Storage of l y o p h i l i z e d , frozen, or L - d r i e d c e l l s are the p r i n c i p l e means of long-term p r e s e r v a t i o n (26). There i s an extremely widespread b e l i e f that the method of choice i s l y o p h i l i z a t i o n . This b e l i e f i s not j u s t i f i e d by e i t h e r f a c t or theory. The reasons f o r the widespread preference f o r l y o p h i l i z a t i o n are: ( i ) t h i s was the f i r s t g e n e r a l l y s u c c e s s f u l method of longterm p r e s e r v a t i o n ; ( i i ) the product has an " a t t r a c t i v e " appearance; ( i i i ) i n j u r y from concentrated solutes i n the l i q u i d s t a t e seemed a reasonable s u p p o s i t i o n ; ( i v ) p r o t e c t i o n against i n j u r y by d r y i n g at f r e e z i n g temperatures seemed an a t t r a c t i v e advantage. I t i s now c l e a r that h i g h l y concentrated solutes are not as i n j u r i o u s as has been formerly supposed and may i n f a c t exert s i g n i f i c a n t p r o t e c t i o n (27). In the l i g h t of extensive i n v e s t i g a t i o n s of the L-drying methods of Annear (28-33) by other workers (26, 34, 35), i t seems that t h i s procedure i s to be p r e f e r r e d since recovery of many d i f f i c u l t to preserve organisms i s t y p i c a l l y 10 to 100 times higher than i s achieved with l y o p h i l i z a t i o n . I t has a l s o been observed that l a r g e increases i n mutants can accompany l y o p h i l i z a t i o n (36, 3_7, 38) . While no proper comparison appears to have been made between mutant y i e l d s from l y o p h i l i z a t i o n and L - d r y i n g , i t seems reasonable to expect that the higher r e c o v e r i e s obtained by L-drying would be accompanied by l e s s damage and t h e r e f o r e fewer mutants. There are a number of steps i n p r e s e r v a t i o n and subsequent recovery procedures which may cause genetic damage ( F i g u r e 2 ) .


1.

KiDBY

Culture

Maintenance

and

Productivity

9

F r e e z i n g i s i n i t s e l f i n j u r i o u s (39) . The extent of d r y i n g a l s o appears to i n f l u e n c e the y i e l d of mutations (40, 4l)· Prophage may a l s o be induced by d e s i c c a t i o n (42, 43). The r e h y d r a t i o n procedure i s a l s o of importance and there appears to be some evidence of c e l l leakage l e a d i n g to poor recovery (27). The r e covery medium i s an important s e l e c t i v e agent and can c l e a r l y i n f l u e n c e the recovery of c e r t a i n types of mutants. For example, some medium components can i n h i b i t recovery of nonsense suppressors i n Saccharomyces c e r e v i s i a e , w h i l e other components can r e l i e v e t h i s i n h i b i t i o n (44). Storage i n the f r o z e n s t a t e has l i t t l e to recommend i t except convenience. Storage i t s e l f i s not considered to be i n j u r i o u s provided that i c e c r y s t a l damage i s precluded by h o l d i n g the temperature below -130°C (45) I t i s suggested tha procedures f o r both long requirement y particularly successful. I t i s not c l e a r how low a temperature should be employed f o r storage of d r i e d m a t e r i a l , but i n the absence of evidence to the c o n t r a r y , as low a temperature as i s a v a i l a b l e would seem des i r a b l e . For long-term p r e s e r v a t i o n , the m a t e r i a l i s normally h e l d under vacuum while f o r short-term p r e s e r v a t i o n , l e s s s t r i n gent, and t h e r e f o r e more convenient, c o n d i t i o n s may be employed. When r e h y d r a t i n g , a low c e l l r c u l t u r e volume r a t i o should be employed. The c u l t u r e medium should be as n u t r i t i o n a l l y r i c h as i s c o n s i s t e n t w i t h good growth. This procedure w i l l to some degree s e l e c t f o r auxotrophs. However, i t i s p o s s i b l e to screen these out i n subsequent c u l t u r e i f necessary. No c e l l population i s g e n e t i c a l l y i d e n t i c a l to i t s parent c u l t u r e . The change i n i d e n t i t y can, however, be minimized by the use of methods which lead to high recovery r a t e s . The p r e s e r v a t i o n of f r e s h i s o l a t e s should not be delayed and i t i s worth adopting a standard p r o t o c o l to deal with t h i s s i t u a t i o n ( F i g u r e 3 ) . Improvement of Genotype. C o n t r o l Mutants. One of the most u s e f u l types of mutant i s the c o n t r o l mutant where feed-back i n h i b i t i o n or r e p r e s s i o n i s absent. In the case of p o l y s a c c h a r i d e production such mutants are most l i k e l y to be recognized by t h e i r production of l a r g e mucoid c o l o n i e s . C o n d i t i o n a l mutants. The c o n d i t i o n a l mutant has great potent i a l f o r c o n t r o l l i n g complex c e l l f u n c t i o n s by such simple means as r a i s i n g or lowering of temperature. Such mutants are r e l a t i v e l y easy to o b t a i n . For example, p o l y s a c c h a r i d e production which i s c o n d i t i o n a l may be switched on and o f f or, c o n d i t i o n a l growth may be switched o f f to permit polysaccharide production i n the absence of growth. C o n d i t i o n a l l y s i s i s a l s o of c o n s i d e r a b l e a p p l i c a t i o n where i t i s d e s i r a b l e , and i t u s u a l l y i s , to remove the c e l l s from the completed fermentation. L y s i s may be achieved by the i n d u c t i o n of bacteriophage. B a c t e r i o c i n s a l s o


EXTRACELLULAR

10

LYOPHILIZATION

F.EEEZJ_N6

[FREEZING!

IFREEZINGI

MICROBIAL

POLYSACCHARIDES

DRYING STORAGE

STORAGE

L-DRYING

DRYING I STORAGE

I

REHYDRATION ITHAWINGI • i GROWTH GROWTH GROWTH Figure 2. Comparisons between sequences of events involved in preservation of cells and their subsequent recovery REHYDRATION

ENRICHMENT SELECTION

PURIFICATION

CHARACTERIZATION

REPEATED TRANSFER

VIABLE COUNT

L-DRYING

CHARACTERIZATION

Figure 3. Selection and preservation of microbes. The scheme described incorporates tests of irdried cultures to determine viability and any alteration of characteristics as a result of the preservation procedure.


1.

KiDBY

Culture

Maintenance

and

Productivity

11

o f f e r great p o t e n t i a l f o r l y s i n g of c u l t u r e s . S t a b i l i z e d Genes. The p o t e n t i a l f o r s t a b i l i z a t i o n v a r i e s according to the o r i g i n of the i n s t a b i l i t y . . In the case of hot spots, the breaking up of runs of base p a i r s might be expected to be e f f e c t i v e . An increase i n the number of genes may be e f f e c t i v e and may, i f t r a n s l a t i o n i s the r a t e - l i m i t i n g step i n production, a l s o lead to higher production l e v e l s . I t may be p o s s i b l e to t r a n s f e r genes from a r e l a t e d organism e x h i b i t i n g a more s t a b l e genotype. Stable genotypes may be f a i r l y r e a d i l y revealed by employing the s e l e c t i v e pressure of chemostat c u l t u r e (46). Methods f o r Genotype A l t e r a t i o n . Genotypes are a l t e r e d by: ( i ) induced mutation; ( i i ) spontaneous mutation; ( i i i ) t r a n s f e r of e x i s t i n g genes. The first.method i s r a p i d and some degree of s p e c i f i c i t y i s p o s s i b l e as f o r example i n the case of ozone and UV induced mutants (10) wanted mutations may a l s are, of course, slower, but are capable of producing the r e q u i r e d mutants i n a s u r p r i s i n g l y short time. The s e l e c t i o n pressure to o b t a i n p a r t i c u l a r types of spontaneous mutants should be a p p l i e d i n a continuous, r a t h e r than a discontinuous, manner. This permits a more complete range of p o s s i b i l i t i e s to be expressed and i s l i k e l y to lead to a more s t a b l e mutant s i n c e the d e s i r e d character can be acquired by a s e r i e s of small steps rather than one l a r g e step which could, f o r example, be due to a s i n g l e point mutation. For example, s t a b l e and high l e v e l a n t i b i o t i c r e s i s t a n c e has been achieved i n Xanthomonas by u s i n g gradient p l a t e s but was not r e a d i l y achieved when using d i s c r e t e steps (24). A p a r t i c u l a r l y h e l p f u l account of methods of mutant i s o l a t i o n i s given by Hopwood (47). Perhaps the most a t t r a c t i v e methods of genotype improvement i n v o l v e t r a n s f e r of genetic m a t e r i a l . The advantage of t h i s method i s s p e c i f i c i t y , s t a b i l i t y , and r e l a t i v e freedom from unwanted changes i n other genes. Some very e x c i t i n g a l t e r a t i o n s can be attempted by t h i s means. I t i s d e s i r a b l e f o r the organisms to be c l o s e l y r e l a t e d because the t r a n s f e r r e d gene i s more l i k e l y to behave c h a r a c t e r i s t i c a l l y i n the r e c i p i e n t . However, genes c e r t a i n l y are t r a n s f e r a b l e between d i s t a n t l y r e l a t e d species and genetic engineering may be expected to r e v o l u t i o n i z e the synthesis of n a t u r a l products. The methods of genetic t r a n s f e r among b a c t e r i a are: ( i ) conjugation; ( i i ) t r a n s d u c t i o n ; ( i i i ) t r a n s f e c t i o n ; ( i v ) t r a n s formation, and (v) i n v i t r o recombination and t r a n s f e r from divergent species or genetic engineering. The f i r s t four methods are conventional and are e x t e n s i v e l y described (48). However, genetic engineering i s a combination of methodologies and the t o t a l procedure may be v a r i e d c o n s i d e r a b l y . One r e c e n t l y described method (49) c o n s i s t s of i s o l a t i o n of the gene as i t s RNA t r a n s c r i p t i o n product, r e t r a n s c r i p t i o n back to DNA and synt h e s i s of a complementary s t r a n d . These strands are elongated w i t h homopolymer t a i l s of o l i g o - ( d G ) . This double stranded gene


12

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

i s then mixed w i t h a plasmid which has been prepared as f o l l o w s . A n i c k i s placed i n the c i r c u l a r plasmid to provide l i n e a r DNA which i s r e p a i r e d then extended w i t h a homopoIyer t a i l o f o l i g o (dC) which i s , o f course, complementary to the a r t i f i c i a l t a i l on the copied gene. The plasmid p i c k s up the gene by the complementary t a i l s e c t i o n s and, i n doing so, becomes c i r c u l a r and thus i n f e c t i v e . F o l l o w i n g i n f e c t i o n , the plasmid i s c o v a l e n t l y l i n k e d to the copied gene by host enzymes. This gene may be t r a n s f e r a b l e to a wide range o f b a c t e r i a . Furthermore, i n t h i s p a r t i c u l a r example, the gene may be removed again from the plasmid, using a s p e c i f i c r e s t r i c t i o n nuclease, and t r a n s f e r r e d to some other plasmid. Thus i t i s p o s s i b l e to conceive o f n a t u r a l products which are e i t h e r i n a c c e s s i b l fermenters w i t h i n hours o n l y f o r production cost production volumes can be r e g u l a t e d .

Abstract Sources of microbes and procedures for their selection, isolation and maintenance are discussed. Maintenance of genotype is considered in terms of the nature of genetic variability, antimutagenesis, inoculation schedules, growth media and preservation methods. The improvement of genotype is discussed in terms of control mutants, conditional mutants, and methods of genotype alteration. Some common practices which may be conducive to culture degeneration are discussed and suggestions are made as to alternative procedures. Literature Cited 1. Brock, T . D . , Ann. Rev. Ecology System (1970) 1, 191. 2. Lundgren, D . , et al., "Water Pollution Microbiology", John Wiley, New York (1972) 69-88. 3. Okanishi, M . , Gregory, K.F., Canad. J. Microbiol. (1970) 16, 1139. 4. "Bergey's Manual of Determinative Bacteriology" Williams and Wilkins. 5. "Abstracts of Microbiological Methods", John Wiley, New York (1969). 6. "Methods i n Microbiology" 3A, Academic Press, New York (1970) 7. "Methods in Microbiology" 3B, Academic Press, New York (1970) 8. "Methods i n Microbiology" 4, Academic Press, New York (1971) 9. Kidby, D . K . , et al., unpublished. 10. Hamelin, C., Chung, Y . S . , Mutat. Res. (1975) 28, 131. 11. Sutherland, I.W., J. gen. Microbiol. (1976) 94, 211. 12. Hesseltine, C.W., Haynes, W.C., Progress in Industrial Microbiology (1973) 12, 3. 13. Clarke, C.H., Johnston, A.W.B., Mutat. Res. (1976) 36, 147. 14. Clarke, C.H., Shankel, C.M., Bacteriol. Rev. (1975) 39, 33.


1.

KiDBY

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

Culture

Maintenance

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Productivity

13

Drake,J.W.,"TheMolecular Basis ofMutation",Holden-Day, San Francisco, 1970. Vogel, F., J. Molec. Evoln. (1972) 1, 334. Benzer, S., Proc. Natl. Acad. Sci. (1961) 47, 403. Koch, R.E., Proc. Natl. Acad. Sci. (1971) 68, 773. Lewis,C.M.,Tarrant,G.M.,Mutat. Res. (1971) 12, 349. McBride, A.C., Gowans, C.S., Genet. Res. (1969) 14, 121. Talmud, P., Lewis, D., Nature (1974) 249, 563. Puglisi, P.P., Mutat. Res. (1967) 4, 289. Cadmus,M.C.,et al., Can. J. Microbiol. (1976) in press. Kidby, D.K., unpublished. Coe, A.W., Clark, S.P., Mon. Bull. Minist. Hlth. (1966) 25, 97. Lapage, S.P. et a l . "Method i Microbiology" 3A Academi Press, New York, (1970 Leach, R.H., Scott, W.J., J . gen. Microbiol. (1959) 21, 295. Annear, D.I., Nature (1954) 174, 359. Annear, D.I., J. Hyg. Camb. (1956) 54, 487. Annear, D.I., J. Path. Bact. (1956) 72, 322. Annear, D.I., J. Appl. Bact. (1957) 20, 17. Annear, D.I., Aust. J . exp. Biol. med. Sci. (1958) 36, 1. Annear, D.I., Aust. J . exp. Biol. med. Sci. (1962) 40, 1. Hopwood, D.A., Ferguson, H.M., J . appl. Bact. (1969) 32, 434. Muggleton, P.W., Progr. Ind. Microbiol. (1962) 4, 191. Hieda, Κ., Ito, T., "Freeze-drying of biological Materials" International Institute of Refrigeration, Paris (1973) 71. Webb, S.J., Tai,C.C.,Canad. J . Microbiol. (1968) 14, 727. "Cryobiology", Academic Press, N.Y. (1966) 213. Mazur, P., Science (1970) 168, 939. Webb, S.J., Nature (1967) 213, 1137. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1968) 14, 841. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1967) 13, 33. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1967) 13, 303. Queiroz, C., Biochem. Genet. (1973) 8, 85. Martin, S.M., Ann. Rev. Microbiol. (1964) 18, 1. Veldkamp, H., "Methods in Microbiology" 3A Academic Press, New York (1970) 305. Hopwood, D.A., "Methods in Microbiology" 3A Academic Press, New York (1970) 363. Hayes, W., "The Genetics of Bacteria and their Viruses" Blackwell, Oxford (1968). Rougeon, F . , Kourilsky, P., Mach, B., Nucleic Acids Res. (1975) 2, 2365.


2

The

P r o d u c t i o n of

vinelandii

Alginic

Acid

by

Azotobacter

in Batch and Continuous Culture

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 optimisti fermentation area. It is no 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


2.

DEAVIN

E T AL.

Production

of Alginic

Acid

in

15

Culture

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 fermentatio 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


16

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

4


2.

DEAVIN

Production

E T AL.

of Alginic

Acid

in

17

Culture

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

synthesis

intermediate

d for

polymers ( i . e . p r o t e i n s , n u c l e i 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.


18

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

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


2.

DEAVIN

E T AL.

Production

of Alginic

Acid

in

19

Culture

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 th produced by Azotobacter vinelandi 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

EXTRACELLULAR

0.05

0.10

0.Ï5

0.20

MICROBIAL

POLYSACCHARIDES

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-


DEAVIN

E T A L .

Production

of Alginic

Acid

in

Culture

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.

MICROBIAL

POLYSACCHARIDES

Metabolism of Azotobacter vinelandii in relation to alginate synthesis


2.

DEAVIN

Production

E T AL.

of Alginic

Acid

in

Culture

23

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 2 5 % of the sucrose u t i l i s e d 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 thu c u l t u r e studies h a v e g i v e n 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 10, 135-266


3 Xanthan G u m from A c i d W h e y MARVIN CHARLES and MOHAMMED K. RADJAI Department of Chemical Engineering, Lehigh University, Bethlehem, PA 18015

Xanthan gum (fro 1459A) has been produced from media containing deproteinized acid-set or culture-set cottage cheese wheys, the lactose contents of which were hydrolyzed to glucose and galactose by means of immobilized lactase. Both glucose and galactose were used almost completely to give gum yields, productivities and final concentrations which were generally as good as, and in some cases better than, those obtained with comparable conventional media.With the exception of an anomalous pH history (the pH increased rather than decreased) when culture-set whey permeate was used, the fermentations followed courses typical of those previously reported. Details of media preparation, fermentation conditions, and experimental results w i l l follow a brief discussion of cottage cheese whey and whey permeate. C o t t a g e Cheese Whey and Whey Permeate A c i d whey i s t h e h i g h BOD waste r e s u l t i n g from t h e manufacture o f c o t t a g e cheese. I t s composition {!) (see T a b l e I) v a r i e s somewhat w i t h t h e c u r d - s e t t i n g p r o c e s s employed (and w i t h m i l k c o m p o s i t i o n , e t c . ) b u t i n g e n e r a l i t c o n t a i n s around 4% t o 5% l a c t o s e , 0.8% t o 1.0% p r o t e i n ( l a c t a l b u m i n ) , and l e s s e r quant i t i e s o f a c i d s , m i n e r a l s , v i t a m i n s , e t c . Most o f t h e a c i d whey produced each y e a r i s r u n t o waste r e s u l t i n g i n c o n s i d e r a b l e c o s t s t o d a i r i e s and communities. Furthermore, such d i s p o s a l r e s u l t s i n y e a r l y l o s s e s o f o v e r 100 m i l l i o n l b s . o f v a l u a b l e and m a r k e t a b l e whey p r o t e i n ( l a c t a l b u m i n ) , which has e x c e l l e n t n u t r i t i o n a l and f u n c t i o n a l p r o p e r t i e s , and o v e r 500 m i l l i o n l b s . of lactose along with l e s s e r but s i g n i f i c a n t 27


EXTRACELLULAR

28

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POLYSACCHARIDES

q u a n t i t i e s o f o r g a n i c a c i d s and v i t a m i n s . Therefore t h e r e i s c o n s i d e r a b l e economic i n c e n t i v e f o r t h e development o f p r o c e s s e s f o r d i r e c t u t i l i z a t i o n o f a c i d whey o r f o r r e c o v e r y and subsequent use o f i n d i v i d u a l a c i d whey components b u t t h e l a t t e r approach appears t o have g r e a t e r p o t e n t i a l i n t h e f o r s e e a b l e f u t u r e . Table

I.

A c i d Whey C o m p o s i t i o n

L a c t o s e (wt %) P r o t e i n (wt %) Ash (wt %) L a c t i c A c i d (wt %) Glucono-6-Lactone(wt% C a l c i u m (G/L) Phosphorous (G/L) T o t a l S o l i d s (wt %) PH

Culture Set 4.3-4.4 0.8-1.0 0.7-0.8

1.2-1.3 0.7-0.8 6.9-7.0 4.3-4.7

(Typical) Acid Set 4.6-4.9 0.9 0.8-0.9

—

1.3-1.4 1.9-2.1 7.0-7.2 4.1-4.5

Recovery o f l a c t a l b u m i n by t h e proven t e c h n o l o g y o f u l t r a f i l t r a t i o n o f f e r s c o n s i d e r a b l e economic promi s e t h r o u g h o u t most o f t h e c o u n t r y and a l r e a d y has been operated commercially. However, an i m p o r t a n t f a c t o r i n f l u e n c i n g t h e economics o f t h e r e c o v e r y i s t h e u l t i mate use o f whey permeate which i s t h e b y - p r o d u c t o f u l t r a f i l t r a t i o n and which c o n t a i n s a l a r g e q u a n t i t y o f l a c t o s e , some low m o l e c u l a r weight p r o t e i n , o r g a n i c a c i d s , m i n e r a l s , v i t a m i n s , and some o t h e r minor comp o n e n t s . We r e q u i r e , t h e n , e c o n o m i c a l uses f o r whey permeate (2) . Many s u g g e s t i o n s have been made f o r d i r e c t u t i l i z a t i o n o f permeate i n c l u d i n g c o n v e r s i o n t o y e a s t a n d / o r a l c o h o l (_3) · F e r m e n t a t i o n t e c h n o l o g i e s f o r b o t h a r e w e l l known and i t seems r e a s o n a b l e t o e x p e c t t h a t t h e r e may be some c a s e s i n which such p r o c e s s e s w i l l be e c o n o m i c a l l y f e a s i b l e a l t h o u g h i t must be r e c o g n i z e d t h a t t h e r e l a t i v e l y low economic v a l u e s o f t h e p r o d u c t s might be a d e t e r r e n t t o i n v e s t m e n t . However, i n t h e absence o f r e c e n t well-documented economic s t u d i e s i t i s d i f f i c u l t t o make a s a t i s f a c t o r y a n a l y s i s p a r t i c u l a r l y i n l i g h t o f t h e p o t e n t i a l , b u t somewhat u n c e r t a i n , l a r g e - s c a l e use o f e t h y l a l c o h o l as a f u e l . A n o t h e r approach i n v o l v e s t h e h y d r o l y s i s o f whey permeate l a c t o s e t o g l u c o s e and g a l a c t o s e by means o f i m m o b i l i z e d l a c t a s e (£,!5,£, 1) . W i d e l y d i s c u s s e d food r e l a t e d a p p l i c a t i o n s o f t h e "sweet permeate" so p r o duced a r e based on t h e d e s i r e t o r e c y c l e whey permeate so as t o e l i m i n a t e d i s p o s a l c o s t s , t o d e c r e a s e sweetener c o s t s , and t o c i r c u m v e n t n u t r i t i o n a l and


3.

CHARLES

A N D

RADjAi

Xanthan Gum from Acid Whey

29

f u n c t i o n a l problems a s s o c i a t e d w i t h l a c t o s e . However, d e s p i t e t h e f a c t t h a t t h e h y d r o l y s i s c a n be performed f o r w e l l under ΙΟΦ/lb o f l a c t o s e (5) , t h e "sweet p e r meate" may s t i l l meet w i t h s t i f f c o m p e t i t i o n from a v a i l a b l e c o r n and h i g h f r u c t o s e s y r u p s s i n c e g a l a c t o s e i s n o t as sweet as g l u c o s e and hence on a pound f o r pound ( s o l i d s ) b a s i s t h e h y d r o l y z a t e i s n o t a s sweet as t h e a l r e a d y a v a i l a b l e s y r u p s . Furthermore, i t appears t h a t d e m i n e r a l i z a t i o n w i l l be r e q u i r e d t o make t h e h y d r o l y z a t e a c c e p t a b l e as a food i n g r e d i e n t and t h i s w i l l add c o n s i d e r a b l y t o i t s c o s t (15) . These f a c t s , c o u p l e d w i t h t h e d e c l i n e i n sugar p r i c e s have c a s t some doubt on t h e v e r y p r o m i s i n g economic progno s i s which e x i s t e d f o t h f "sweet permeate" food i n g r e d i e n t j u s An a l t e r n a t i v e use o f t h e h y d r o l y z a t e i s as a f e r m e n t a t i o n medium. There a r e many organisms which w i l l m e t a b o l i z e b o t h g l u c o s e and g a l a c t o s e (but n o t l a c t o s e ) t o p r o d u c t s c o n s i d e r a b l y more v a l u a b l e than y e a s t o r a l c o h o l and whose n i t r o g e n r e q u i r e m e n t s a r e s a t i s f i e d p a r t i a l l y o r c o m p l e t e l y by t h e low m o l e c u l a r weight permeate p r o t e i n s . T h i s i s p a r t i c u l a r l y t r u e i n c a s e s where p r o d u c t i o n o f l a r g e q u a n t i t i e s o f c e l l mass i s n o t r e q u i r e d o r even p a r t i c u l a r l y d e s i r a b l e (e.g., i n p r o d u c t i o n o f x a n t h a n ) . F u r t h e r m o r e , demin e r a l i z a t i o n o f the hydrolyzate i s g e n e r a l l y not r e quired f o r this application. Thus, i n s o f a r as use as a f e r m e n t a t i o n medium i s c o n c e r n e d , h y d r o l y z e d p e r meate has t h e f o l l o w i n g advantages: • carbohydrate cost competitive with glucose • adequate n i t r o g e n and o t h e r growth f a c t o r s f o r many a p p l i c a t i o n s » u t i l i z e s a h i g h BOD waste stream •enhances economics o f whey p r o t e i n r e c o v e r y . I t s h o u l d a l s o be n o t e d t h a t even i f c o n d e n s a t i o n i s required to f a c i l i t a t e t r a n s p o r t a t i o n , the cost o f h y d r o l y z a t e would s t i l l be c o m p e t i t i v e w i t h commercial dextrose. The m i c r o b i a l p r o d u c t i o n o f xanthan gum i s a p a r t i c u l a r example o f an a l r e a d y s u c c e s s f u l commercial f e r m e n t a t i o n which uses a c o n v e n t i o n a l g l u c o s e - c o n t a i n i n g medium b u t which c a n be conducted as w e l l o r b e t t e r w i t h a h y d r o l y z e d whey permeate medium. The

Fermentation

Process

Medium F o r m u l a t i o n . The medium c a n be produced from e i t h e r c u l t u r e - s e t o r a c i d - s e t c o t t a g e cheese whey by means o f t h e p r o c e s s i l l u s t r a t e d i n F i g u r e 1: (a) Whey i s f i l t e r e d t h r o u g h a h o l l o w - f i b e r


30

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(b)

(c)

(d)

MICROBIAL POLYSACCHARIDES

u l t r a f i l t e r h a v i n g a m o l e c u l a r weight c u t o f f o f 50,000 (HF 26.5-45 - XM50 c a r t r i d g e , Romicon, I n c . , Woburn, Ma s s . ) . The permeate, which has a pH o f 4.1-4.6, i s hydrolyzed i n a p i l o t - p l a n t f l u i d i z e d - b e d r e a c t o r c o n t a i n i n g A.niger l a c t a s e (Lactase, L.P., W a l l e r s t e i n , C h i c a g o , IL) i m m o b i l i z e d on alumina p a r t i c l e s (£,7). The h y d r o l y z e d permeate Ts then s t e r i l i z e d and supplemented w i t h s t e r i l e K 2 H P O 4 and MgS04«7H20 t o y i e l d a medium whose composit i o n i s given i n Table I I . The pH o f t h e medium i s a d j u s t e d t o 7.0.

Table I I .

Hydrolyze ( F u l l S t r e n g t h - C u l t u r e Set)

G l u c o s e (wt %) Galactose Lactose K Mg 2

HPO4 S 0 . 7 H 4

2

0

P r o t e i n (Lowry) Whey A c i d pH

2.05 2.05 0.30 0.50 0.01

0.20 0.70 7.0

(a) Medium a l s o c o n t a i n s whey a s h , a c i d s , vitamins, e t c . W h i l e e i t h e r a c i d - s e t o r c u l t u r e - s e t whey may be used, i t i s i m p o r t a n t t o note t h a t t h e two a r e n o t e q u i v a l e n t as w i l l be i l l u s t r a t e d below. In some c a s e s we have used t h e media d e s c r i b e d as i s w h i l e i n o t h e r s t h e y have been d i l u t e d t o a p p r o x i m a t e l y h a l f - s t r e n g t h , F u r t h e r m o r e , we o c c a s i o n a l l y have added s m a l l q u a n t i t i e s o f supplemental n i t r o g e n i n t h e form o f e n z y m i c a l l y - h y d r o l y z e d l a c t a l b u m i n (Edamin, S h e f f i e l d C h e m i c a l , Union, N J ) . T h i s was p r o v e n t o be p a r t i c u l a r l y v a l u a b l e when a c i d - s e t whey was u s e d . Sterilization. H y d r o l y z e d whey permeate i s a complex medium c o n t a i n i n g sugars and low m o l e c u l a r weight p r o t e i n a l o n g w i t h a c i d s and v a r i o u s m i n e r a l s and hence some c a u t i o n i s n e c e s s a r y d u r i n g steam s t e r i l i z a t i o n , p a r t i c u l a r l y when an a u t o c l a v e i s used as i t was i n o u r c a s e . We found t h a t i f t h e permeate was s t e r i l i z e d a t i t s n a t u r a l pH (4.1-4.6) t h e r e was obs e r v a b l e browning b u t t h e r e was almost no l o s s o f n u t r i e n t s and i n h i b i t o r y p r o d u c t s were n o t formed t o any a p p r e c i a b l e e x t e n t . Indeed, medium steam


3.

C H A R L E S A N D RADJAI

Xanthan

Gum

from Acid

Whey

31

s t e r i l i z e d a t t h e n a t u r a l permeate pH behaved a s w e l l as f i l t e r - s t e r i l i z e d medium. On t h e o t h e r hand, steam s t e r i l i z a t i o n a t pH 6.0 o r g r e a t e r r e s u l t e d i n s e v e r e browning, c o n s i d e r a b l e p r e c i p i t a t i o n , l o s s o f n u t r i e n t s , apparent formation o f r e l a t i v e l y high l e v e l s o f i n h i b i t o r y compounds and a g e n e r a l l y i n f e r i o r medium. Fermentation C o n d i t i o n s . Bench-scale fermentat i o n s were conducted i n 7 l i t e r a e r a t e d , n o n - b a f f l e d f e r m e n t o r s equipped w i t h t h r e e p i t c h e d - b l a d e t u r b i n e i m p e l l e r s h a v i n g tank diameter t o i m p e l l e r d i a m e t e r r a t i o s o f 1.8 t o 1.0. We found t h a t t h e use o f m u l t i p l e l a r g e i m p e l l e r s and t h e i n t e n t i o n a l removal o f baffles resulted i bette mixing transfer and p r o d u c t i v i t y whe viscous, p a r t i c u l a r l y g r e a t e r t h a n 1% (8_) . The f e r m e n t o r s were a l s o equipped w i t h a u t o m a t i c foam c o n t r o l l e r s , d i s s o l v e d oxygen m o n i t o r s , and pH c o n t r o l systems which added e i t h e r 4 N KOH o r gaseous N H 3 . The seed c u l t u r e was d e v e l o p e d as suggested by Moraine and h i s coworkers (9,10,11) and a 5% (V/V) seed was used t o i n o c u l a t e t h e main f e r m e n t o r s i n a l l c a s e s . Temperature was always m a i n t a i n e d a t 28°C and pH a t 7.0 e x c e p t when t h e pH remained above 7 as was t y p i c a l l y t h e c a s e when c u l t u r e - s e t whey was used. Analytical G l u c o s e , g a l a c t o s e , and xanthaa c o n c e n t r a t i o n s were measured a t r e g u l a r i n t e r v a l s . G l u c o s e was d e t e r m i n e d by means o f a g l u c o s e - o x i d a s e impregnated membrane and g a l a c t o s e by means o f a g a l a c t o s e - o x i d a s e impregnated membrane. Both were used i n c o n j u n c t i o n w i t h a YSI Model 23A g l u c o s e a n a l y z e r (YSI I n s t r u m e n t s , Y e l l o w S p r i n g s , O h i o ) . The l a c t o s e c o n t e n t o f unhydrol y z e d whey was u s u a l l y d e t e r m i n e d by f i r s t c o m p l e t e l y h y d r o l y z i n g i t w i t h e x c e s s A . n i g e r l a c t a s e ( L a c t a s e LP, W a l l e r s t e i n , C h i c a g o , IL) and t h e n measuring t h e r e s u l t i n g g l u c o s e o r g a l a c t o s e . In some c a s e s t h e g a l a c t o s e o x i d a s e membrane, which responds t o l a c t o s e t o an e x t e n t o f 10-15% o f i t s r e s p o n s e t o g a l a c t o s e , was used t o determine whey permeate l a c t o s e d i r e c t l y . The l a c t o s e c o n c e n t r a t i o n i n whey permeate used f o r f e r m e n t a t i o n s was c a l c u l a t e d from t h e h y d r o l y z a t e g l u c o s e c o n c e n t r a t i o n (which i s e q u a l t o t h e g a l a c t o s e c o n c e n t r a t i o n p r i o r t o i n o c u l a t i o n ) and t h e known i n i t i a l permeate l a c t o s e c o n c e n t r a t i o n . The l a c t o s e c o n c e n t r a t i o n remained e s s e n t i a l l y c o n s t a n t t h r o u g h o u t a l l t h e f e r m e n t a t i o n s performed as i t was n o t


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32

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POLYSACCHARIDES

m e t a b o l i z e d by X . c a m p e s t r i s under t h e c o n d i t i o n s employed. Xanthan was d e t e r m i n e d by f i r s t f i l t e r i n g f e r m e n t a t i o n samples t o remove a l l suspended s o l i d s , p r e c i p i t a t i n g the xanthan i n t h e f i l t r a t e by a d d i t i o n o f KC1(2%) and methanol (50-60%) and f i n a l l y d e t e r m i n i n g t h e d r y weight o f t h e p r e c i p i t a t e d gum. F e r m e n t a t i o n Modes Both b a t c h and r e p e a t e d - b a t c h f e r m e n t a t i o n s were performed. In r e p e a t e d - b a t c h o p e r a t i o n a g i v e n f e r m e n t a t i o n c y c l e was t e r m i n a t e d when t h e g a l a c t o s e conc e n t r a t i o n dropped t o a p p r o x i m a t e l y 0.1% o r when t h e xanthan p r o d u c t i o n t h a t time, approximatel t e n t s were r e p l a c e d w i t h f r e s h medium and a new c y c l e was i n i t i a t e d . R e s u l t s and D i s c u s s i o n G l u c o s e / G a l a c t o s e Medium. F e r m e n t a t i o n s were c o n d u c t e d u s i n g media based on 50/50 m i x t u r e s o f pure g l u c o s e and g a l a c t o s e t o p r o v i d e b a s e - l i n e d a t a f r e e o f a m b i g u i t i e s t h a t might a r i s e as a r e s u l t o f t h e complex n a t u r e o f whey-based media. The h i s t o r y o f a t y p i c a l f e r m e n t a t i o n i s g i v e n i n F i g u r e 2 and t h e comp o s i t i o n o f t h e medium used i n T a b l e I I I . Table I I I .

G l u c o s e - G a l a c t o s e Medium

G l u c o s e (wt %) Galactose Edamin K2 HP04 Mg S 0 . 7 H 0 pH 4

2

1.3 1.3 0.06 0.50 0.01 7.0

The most i n t e r e s t i n g p o i n t i l l u s t r a t e d by t h e s e r e s u l t s i s t h e s i m u l t a n e o u s use o f b o t h s u g a r s . A l though g a l a c t o s e was used l e s s r a p i d l y t h a n g l u c o s e t h e r e was c l e a r l y no d i a u x i e . Furthermore, both sugars were u t i l i z e d f o r gum p r o d u c t i o n . Otherwise t h e c o u r s e o f the f e r m e n t a t i o n was t y p i c a l o f t h o s e r e p o r t e d by Moraine and h i s coworkers (£, 10/11.) · The f i n a l gum c o n c e n t r a t i o n o f a p p r o x i m a t e l y 2% which r e p r e s e n t e d a 77% y i e l d was a c h i e v e d i n about 50 h o u r s . C u l t u r e Set Whey; B a t c h F e r m e n t a t i o n . The h i s t o r y o f a t y p i c a l b a t c h f e r m e n t a t i o n based on c u l t u r e - s e t


CHARLES

AND

Xanthan

RADJAI

Gum from Acid

Whey

Mgso

4

IME

K HPO 2

4

Reactor

UltraFilter Raw Whey "

KOH

Permeate

Concentrate S?Sterilize

Fermentor Figure 1.

Medium preparation 8.0 Ck

4J

U

20

30

Time (Hrs) Figure 2. Batch fermentation; glucose-galactose medium


EXTRACELLULAR

34

MICROBIAL

POLYSACCHARIDES

whey permeate medium i s i l l u s t r a t e d i n F i g u r e 3 and t h e c o m p o s i t i o n o f t h e medium used i s g i v e n i n T a b l e I . The most s i g n i f i c a n t p o i n t t o be noted here i s t h a t the pH b e h a v i o r was v e r y d i f f e r e n t from t h a t observed by o t h e r s u s i n g c o n v e n t i o n a l g l u c o s e media o r by o u r s e l v e s when we used t h e g l u c o s e / g a l a c t o s e medium. We w i l l return to this later. The o t h e r p o i n t worth n o t i n g i s t h a t t h e f i n a l gum c o n c e n t r a t i o n o f 3.5% ( i n a p p r o x i m a t e l y 90 hours) r e p r e s e n t s an 85* y i e l d from t h e a s s i m i l a b l e sugars which was c o n s i d e r a b l y g r e a t e r t h a n would have been expected on t h e b a s i s o f p r e v i o u s r e p o r t s . A g a i n , we w i l l r e t u r n t o t h i s l a t e r . A c i d - S e t Whey h Fermentation Result f batch fermentation usin medium supplemented w i t h Edamin and h a v i n g t h e compo s i t i o n g i v e n i n T a b l e IV a r e p r e s e n t e d i n F i g u r e 4. T a b l e IV.

Hydrolyzed-Permeate/Edamin M e d i u m ^ (Half Strength-Acid Set)

G l u c o s e (wt %) 1.3 Galactose 1.3 Lactose 0.2 Whey P r o t e i n (Lowry) 0.1 Edamin 0.06 K HP0 0.25 Mg S 0 . 7 H 0 0.005 pH 7.0 (a) Medium a l s o c o n t a i n s whey a s h , a c i d s , vitamins, e t c . 2

4

4

2

I n g e n e r a l , t h i s h i s t o r y i s t h e same a s t h a t f o r t h e f e r m e n t a t i o n i n which t h e g l u c o s e / g a l a c t o s e medium was used a l t h o u g h i t d i d p r o c e e d somewhat more r a p i d l y . In p a r t i c u l a r , t h e pH b e h a v i o r was t y p i c a l and t h e y i e l d was w i t h i n t h e range e x p e c t e d . I t should a l s o be noted t h a t media c o n t a i n i n g a c i d s e t whey b u t no Edamin gave somewhat lower y i e l d s and l o n g e r fermentations. The r e a s o n s f o r t h e enhanced gum p r o d u c t i o n and anomalous pH b e h a v i o r o b s e r v e d when c u l t u r e - s e t whey permeate was used a r e n o t c l e a r . A t t h i s time we c a n o n l y s p e c u l a t e t h a t d i f f e r e n c e s i n whey permeate comp o s i t i o n s must be r e s p o n s i b l e the primary d i f f e r e n c e s b e i n g i n t h e c o n c e n t r a t i o n s o f low m o l e c u l a r weight whey p r o t e i n , and i n t h e c o n c e n t r a t i o n s and composit i o n s o f t h e whey a c i d f r a c t i o n s . However, we c a n n o t r u l e o u t o t h e r f a c t o r s such as d i f f e r e n c e s i n v i t a m i n content. f


CHARLES

AND

0

Xanthan

RADJAI

10

Gum from Acid

©

Glucose

• Δ 0

Galactose Xanthan Viscosity

20

30

40

50

60

70

80

Whey

90 100

Time (Hrs) Figure S.

Batch fermentation; full-strength culture-set whey medium


36

EXTRACELLULAR

0

Glucose

•

Galactose

Δ

Xanthan

Ο

0

10

MICROBIAL

POLYSACCHARIDES

Viscosity

20

30

40

50

Time (Hrs) Figure 4.

Batch fermentation; half-strength acid-set whey medium


3.

CHARLES

AND

RADJAI

Xanthan

Gum from Acid

Whey

Time (Hrs)

Figure 5.

Repeated batch fermentation; full-strength culture-set whey medium


37

38

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Repeated-Batch F e r m e n t a t i o n . Results of a threec y c l e repeated batch fermentation with f u l l - s t r e n g t h c u l t u r e - s e t whey medium (no Edamin) a r e i l l u s t r a t e d i n F i g u r e 5. Other t h a n t h e anomalous pH b e h a v i o r and g r e a t e r - t h a n - e x p e c t e d y i e l d s t h e most n o t a b l e f e a t u r e o f t h e s e r e s u l t s i s t h a t t h e r e was l i t t l e change i n f e r m e n t a t i o n h i s t o r y from c y c l e t o c y c l e . However, i t s h o u l d be o b s e r v e d t h a t t h e r e i s a p e r c e p t i b l e i n c r e a s e i n l a g time from one c y c l e t o t h e n e x t . A t t h i s time we c a n n o t say w i t h c e r t a i n t y t h a t t h i s was a c t u a l l y a t r e n d n o r , i f i t was, c a n we p r e d i c t t h e number o f c y c l e s which c o u l d be performed b e f o r e t h e l a g would become p r o h i b i t i v e l long However s h o u l d note t h a t becaus fermentor t h e c u l t u r next c y c l e always came from t h e v e r y bottom o f t h e v e s s e l where m i x i n g and a e r a t i o n were p a r t i c u l a r l y poor d u r i n g t h e l a s t hours o f each c y c l e . T h i s may have been t h e cause o f t h e i n c r e a s e d l a g t i m e s . Conclusion H y d r o l y z e d whey permeate has been shown t o be a s u i t a b l e and c o m p e t i t i v e medium f o r t h e p r o d u c t i o n o f xanthan gum by X . c a m p e s t r i s . I t supports e x c e l l e n t y i e l d s and h i g h f i n a l c o n c e n t r a t i o n s i n b o t h b a t c h and r e p e a t e d b a t c h o p e r a t i o n p a r t i c u l a r l y when modif i e d n o n - b a f f l e d a g i t a t i o n systems employing m u l t i p l e l a r g e p i t c h e d - b l a d e t u r b i n e i m p e l l e r s a r e used.

The a u t h o r s w i s h t o e x p r e s s t h e i r g r a t i t u d e t o t h e P e n n s y l v a n i a S c i e n c e and E n g i n e e r i n g F o u n d a t i o n f o r s u p p o r t i n g t h i s work under PSEF Agreement #273 and t o Romicon, I n c . f o r t h e i r generous g i f t o f u l t r a f i l t r a t i o n c a r t r i d g e s used i n t h i s work.

Literature Cited 1. 2. 3.

Personal communication, Lehigh Valley Dairy, Allentown, PA. Melicouris, N . , paper presented at Enzyme Technology Transfer and Utilization Conference, Lehigh University, Bethlehem, PA, May 27, 1976. Goulet, J., paper presented at the First International Congress on Food and Engineering, Boston, Mass., August 10, 1976.


3.

CHARLES

A N D RADjAI

Xanthan

Gum from Acid Whey

4.

Si

Coughlin, R. W., Charles, Μ., in "Enzyme Engin eering" ed. Oye, Ε. Κ., and Wingard, L . Β., Plenum Press, N.Y., 1974. 5. Pitcher, W. H., in "Immobilized Enzymes for Indus trial Reactors" ed. R. A. Messing, Academic Press, New York (1975). 6. Charles, Μ., Coughlin, R. W., paper presented at NSF/RANN Grantees Conference, University of V i r ginia, Charlottesville, VA, May 19-21, 1976. 7. Charles, Μ., Coughlin, R. W., Allen, B. R., Paruchuri, Ε. Κ., Hasselberger, F. X . , in "Immobilized Biochemicals and Affinity Chromatography", ed. Dunlay, R. Β., Plenum Press, N . Y . , 1974. 8. Charles, Μ., Zmuda J., paper presented at AIChE Meeting, Nov. 28-Dec 9. Moraine, R. Α . , Rogovin, , Bioeng., , 511 (1966). 10. Moraine, R. Α . , Rogovin, P . , Biotech. Bioeng., 13, 381 (1971). 11. Moraine, R. Α . , Rogovin, P . , Biotech. Bioeng., 15, 225 (1973).


4 Microbial Exopolysaccharide Synthesis I. W. SUTHERLAND Department of Microbiology, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JG, Scotland

The fate of a carbohydrate (or other) substrate supplied to an exopolysaccharide-producin microbial species chosen from bacterial species, this review will be concerned essentially with the synthesis of exopolysaccharides by bacteria. In some bacteria, given the correct substrate, exopolysaccharide may be formed without penetration of the cell membrane by the substrate. This is seen in dextran and levan-forming cells supplied with sucrose or several of its analogues. Examples are to be found in Leuconostoc mesenterioides, Streptococcus or Bacillus species. Although this process has been studied by various workers, (1,2) the polysaccharides formed are more limited in their applications and current interest is centred rather on species which form their polymer intracellularly then excrete it into the medium. The aim is therefore to consider a series of processes by which substrates enter the microbial cells, are modified by a series of enzymic processes and finally are excreted in polymeric form from the microbial surface. Much of the information about these reactions has been gained from strains producing polymers which have little or no commercial value, but it is nevertheless possible to extrapolate many of the results and thereby obtain a reasonable hypothesis for the mode of synthesis of a polymer of given structure and to propose mechanisms for the regulation of its biosynthesis. Substrate Uptake The s u b s t r a t e may enter the c e l l by one of three mechanisms - f a c i l i t a t e d d i f f u s i o n , a c t i v e t r a n s p o r t or group t r a n s l o c a t i o n . The l a t t e r two processes, both of which a r e endergonic, are of p a r t i c u l a r i n t e r e s t i n the present context. In a c t i v e t r a n s p o r t , the substrate enters the c e l l u n a l t e r e d , but the group t r a n s l o c a t i o n process i n v o l v e s the phosphorylation o f the s u b s t r a t e , the o v e r a l l process being represented by: X

+

PEP

• X-P

+

pyruvate

40


4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

41

The i n i t i a l f a t e of the substrate i s summarised i n F i g . l . In E s c h e r i c h i a c o l i , the r a t e at which the b a c t e r i a grow on v a r i o u s substrates i s dependent on substrate uptake, i r r e s p e c t i v e of whether a c t i v e t r a n s p o r t or group t r a n s l o c a t i o n systems are involved ( 3 ) . Thus substrate uptake i s one of the f i r s t l i m i t a t i o n s on exopolysaccharide production. As y e t , no attempts to increase c e l l growth and hence exopolysaccharide production by d u p l i c a t i o n of the genes concerned with a c t i v e t r a n s p o r t or with group t r a n s l o c a t i o n appears to have been made. In many b a c t e r i a , t h i s might not even be necessary, as s e v e r a l uptake mechanisms may e x i s t f o r each substrate i . e . Although a s p e c i f i c substrate may be transported by d i f f e r e n t mechanisms i n d i f f e r e n t microorganisms b a c t e r i a such as E* c o l i possess various mechanisms f o r uptake of a s i n g l e substrate such as g a l a c t o s e D i f f e r e n c e s can c e r t a i n l y be expected between Gra between pseudomonads an The group t r a n s l o c a t i o n mechanisms i n v o l v i n g phosphorylation from PEP have been studied by Roseman and h i s colleagues (4) but i t i s not c l e a r whether the u t i l i z a t i o n of r e l a t i v e l y l a r g e amounts of PEP f o r substrate uptake lead to a r e d u c t i o n i n the amount of PEP a v a i l a b l e f o r other purposes. I f t h i s does r e s u l t under c o n d i t i o n s i n which growth i s l i m i t e d by substrate uptake and where high growth r a t e s are used, the r e s u l t might be a r e d u c t i o n i n the degree of p y r u v y l a t i o n observed i n the polymer excreted. Intermediary Metabolism and D i r e c t i o n to Polymer Synthesis F o l l o w i n g the entry of the substrate i n t o the c e l l and i t s phosphorylation by e i t h e r the group t r a n s l o c a t i o n mechanism or by a hexokinase u t i l i z i n g ATP, the substrate can be committed to e i t h e r anabolic processes or to m i c r o b i a l catabolism ( F i g . 2). I f i t s u f f e r s the l a t t e r f a t e , i t i s i n e f f e c t wasted as f a r as polymer production i s concerned, although i f i t enters the TCA c y c l e i t may be converted to pyruvate or to acetate and thus incorporated at a l a t e r stage i n t o polymer. The c o n t r o l of c a t a b o l i c processes w i l l not be considered here. The a n a b o l i c f a t e of the substrate can s t i l l take one of s e v e r a l l i n e s at t h i s stage. I f the m i c r o b i a l species under c o n s i d e r a t i o n i s a Gram negative species, forming exopolysaccharide, l i p o p o l y s a c c h a r i d e and glycogen, the carbohydrate may be converted to any one of these. In the p r o l i f e r a t i n g bacterium, glycogen i s r a r e l y synthesized, but i t s production i s a l s o d i f f e r e n t i a t e d from w a l l polymer or e x t r a c e l l u l a r polymer synthesis through the lack of involvement of i s o p r e n o i d l i p i d s . The c o n t r o l of glycogen synt h e s i s i s exerted through a l l o s t e r i c r e g u l a t i o n of ADP-glucose synthesis (5), the f i r s t enzymic step i n the pathway, which i s unique to glycogen synthesis ( F i g . 3). I t may thus be worth c o n s i d e r i n g the i s o l a t i o n of ADP-glucose pyrophosphorylase mutants i f the b a c t e r i a l s t r a i n i n which we are i n t e r e s t e d produces large


42

EXTRACELLULAR

SUBSTRATE

MICROBIAL

SUBSTRATE

POLYSACCHARIDES

SUBSTRATE

EXTRACELLULAR ENZYMES f POLYMER

(DEXTRANS, L E V A N S , ETC.)

f

>

HISTIDINYL+ PEP +

PERMEASE

PROTEIN MEMBRANE

ENZYME M SUBSTRATE

SUBSTRAT

j KINASE + ATP SUBSTRATE -

+

PHOSPHATE + ADP Figure 1.

HEXOSE-

Initial pathways for extracellular substrates

•HEXOSE

6 Ρ—-HEXOSE

1 P- -CATABOLISM ENERGY

ANABOLISM POLYMERS Figure 2.

GLUCOSE

Fate of hexose substrate

G L C - 6 P — - G L C - 1 P UDP-GLUCOSE

PYROPHOSPHORYLASE

UDP-GLUCOSE

ADP-GLUCOSE PYROPHOSPHORYLASE

ADP-GLC

(GLC1^4GLC) GLYCOGEN EXOPOLYSACCHARIDE Figure 3.

LPS Anabolic fate of glucose


4.

SUTHERLAND

Microbial

Exopolysacchande

Synthesis

43

amounts of glycogen and thus converts s u b s t r a t e to an unwanted product. T h i s would e l i m i n a t e the " d r a i n " of glucose-l-phosphate i n t o glycogen synthesis and away from the d e s i r e d product. Such mutants would be p a r t i c u l a r l y v a l u a b l e i f a two-stage p r o d u c t i o n process was envisaged i n which the second stage contained c e l l s i n an e s s e n t i a l l y n o n - p r o l i f e r a t i n g environment, i . e . c o n d i t i o n s under which l a r g e q u a n t i t i e s of glycogen are normally s y n t h e s i z e d . ( S i m i l a r arguments would apply i f the micro-organism produce p o l y hydroxybutyric a c i d or t r e h a l o s e r a t h e r than glycogen.) The next precursor through which c o n t r o l can be exerted i s the sugar n u c l e o t i d e such as UDP-glucose. UDP-glucose pyrophosphorylase i s a key enzyme producing i n many micro-organisms a precursor f o r both w a l synthesis. The l e v e l appears to be almost u n a l t e r e d i n mutants d e f e c t i v e i n these polymers and t h i s i s r e f l e c t e d at l e a s t i n the E n t e r o b a c t e r i a c e a e , i n the l e v e l of UDP-glucose found i n n u c l e o t i d e pools of s e v e r a l strains (6). The s t r i c t c o n t r o l exerted by such enzymes as UDPglucose pyrophosphorylase or TDP-glucose pyrophosphorylase (7) enables some micro-organisms to channel intermediates to one p o l y mer or another. Thus, TDP-glucose i s a precursor of TDP-rhamnose: f o r i n c o r p o r a t i o n i n t o one or more polymers. I n species poss e s s i n g both enzymes mutual cross i n h i b i t i o n was observed, UDPglucose i n h i b i t i n g TDP-glucose pyrophosphorylase and TDP-glucose i n h i b i t i n g UDP-glucose pyrophosphorylase (7). T h i s could perhaps be p r e d i c t e d , as l o s s of s y n t h e s i s of p o l y s a c c h a r i d e would lead to the accumulation of both g l u c o s e - c o n t a i n i n g sugar n u c l e o t i d e s . T h i s double c o n t r o l i s apparently r e s t r i c t e d to micro-organisms i n which polymers c o n t a i n i n g both sugars are found and i s absent from micro-organisms l a c k i n g rhamnose-containing p o l y s a c c h a r i d e s . S i m i l a r c o n t r o l mechanisms are found i n the formation of fucose as GDP-fucose from GDP-mannose. T h i s was s t u d i e d i n b a c t e r i a l species c o n t a i n i n g ( i ) D-mannose i n t h e i r p o l y s a c charides; ( i i ) c o n t a i n i n g L-fucose; and ( i i i ) c o n t a i n i n g both D-mannose and L-fucose ( 8 ) . In the f i r s t type, c o n t r o l of the r a t e of GDP-mannose s y n t h e s i s occurred through GDP-mannose pyrophosphorylase. In those b a c t e r i a i n which GDP-mannose i s s o l e l y a precursor i n fucose s y n t h e s i s , GDP-fucose c o n t r o l l e d both GDPmannose pyrophosphorylase and GDP-mannose hydrolyase through feedback i n h i b i t i o n . When both mannose and fucose are present i n polysaccharides produced by a s i n g l e bacterium, each sugar nucleot i d e c o n t r o l l e d i t s own s y n t h e s i s ( F i g . 4 ) . Xanthomonas campestris i s of p a r t i c u l a r i n t e r e s t because GDP-mannose and UDPglucose most probably both serve as precursors f o r l i p o p o l y s a c charide and exopolysaccharide. Further c o n t r o l o f the n u c l e o t i d e pool can occur through UDPsugar hydrolases (9,10), although, as these enzymes i n E. c o l i are


44

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p e r i p l a s m i c , they may not n e c e s s a r i l y have access to a l l the sugar n u c l e o t i d e formed by a c e l l but may be s p a t i a l l y separated from i t i n normal c e l l s . As s e v e r a l of the enzymes i n v o l v e d i n sugar n u c l e o t i d e synthesis are membrane-bound, i t i s by no means c l e a r whether t h e i r products occur f r e e l y w i t h i n the cytoplasm or whether they are produced i n c l o s e proximity to the enzymes which r e q u i r e them f o r polymer s y n t h e s i s . There i s a l s o the p o s s i b i l i t y of genetic r e g u l a t i o n of precursors s p e c i f i c to a p a r t i c u l a r polymer. The example of t h i s which has probably r e c e i v e d most study, through the work of Markovitz and h i s colleagues (11,12,13), i s c o l a n i c a c i d synthesis i n c e r t a i n b a c t e r i a of the Enterobacteriaceae. Knowledge of the s t r u c t u r e of c o l a n i c a c i c h a r i d e s , D-glucose and and to w a l l polymers and two others, L-fucose and D-glucuronic a c i d , unique to the polymer. C o n t r o l of the exopolysaccharide synthesis involved r e g u l a t o r genes; mutations i n these genes l e d to derepression and increased polysaccharide s y n t h e s i s . As a r e s u l t of the derepression, increased production of the three enzymes l e a d i n g to GDP-fucose synthesis and under the c o n t r o l of one r e g u l a t o r gene, was detected; increased formation of UDPglucose dehydrogenase ( r e s p o n s i b l e f o r conversion of UDP-glucose to UDP-glucuronic acid) occurred from mutation i n another r e g u l a t o r gene. As y e t , the concept of such r e g u l a t o r genes as those found i n c o l a n i c a c i d formation, dominant on episomes but r e c e s s i v e when located on the b a c t e r i a l chromosome, i s confined to a few s t r a i n s of E. c o l i , Salmonella e t c . One should not discount the p o s s i b i l i t y that polysaccharide production i n other genera and species i s under s i m i l a r genetic c o n t r o l , e s p e c i a l l y as so l i t t l e i s known about the genetic systems of most exopolysacchar ide-producing micro-organisms. Formation of Exopolysaccharide The c o n s t r u c t i o n of the r e p e a t i n g u n i t s of the polymer i s dependent on t r a n s f e r of the appropriate monosaccharides from sugar n u c l e o t i d e s to a c a r r i e r l i p i d i s o p r e n o i d a l c o h o l phosphate. The sequence of r e a c t i o n s has been w e l l c h a r a c t e r i z e d through i s o l a t i o n of the products at each t r a n s f e r step (16) and through i s o l a t i o n and i d e n t i f i c a t i o n of mutants (17) i n two Enterobacter aerogenes systems. The s e r i e s of r e a c t i o n s f o r the s t r a i n studied by Troy et ail. (16) was: UDP-Gal

+ P-lipid « = ±

+

UMP

Gal-P-P-lipid

+ GDP-Man

• Man-Gal-P-P-lipid

+

(GDP)

Man-Gal-P-P-lipid

+ UDP-GlcA

• GlcA-Man-Gal-P-P-lipid +

(UDP)

y Gal-Man-Gal-P-P-lipid

(UDP)

GlcA-Man-Gal-P-P-lipd + UDP-Gal

Gal-P-P-lipid

GlcA


+

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

f" — s Man - I - Ρ — - — - G D P - M a n ii)

Man- l-P J-GDP-Man

iii)

Man- I - P-^GDP-Man

- POLYMER

(-Man-)

n

GDP - Fuc - * P O L Y M E R ( - F u c - ) ^

-i

GDP - F u c — P O L Y M E R S (-Man-)

n

1 = GDP-mannos 2=

GDP-mannos

3= G D P - f u c o s e s y n t h e t a s e Figure 4. Control of mannose and fucose synthesis (after Kornfeld Gloser, 1966)

and

Ί

P Y R U V = ^ Gal 4 β GIcA 1 '3 Gal

3 \ β Glc 1

I

»· F u d

Figure 5

t

*4Fuc 1

Ac


n

46

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

In the other s t r a i n studied (17), the f i r s t r e a c t i o n a l s o involved t r a n s f e r of a hexose-l-phosphate. The methods employed i n these studies l i m i t e d the s i z e of fragment which could be i d e n t i f i e d as being attached to the l i p i d . The l a r g e s t o l i g o s a c c h a r i d e charac t e r i z e d was an octasaccharide equivalent to two r e p e a t i n g u n i t s (16). The exact mechanisms involved i n f u r t h e r chain e l o n g a t i o n and e x t r u s i o n of exopolysaccharides i s s t i l l unknown. Recently, two of the enzymes involved i n Ε. aerogenes have been shown to be extremely l i p o p h i l i c p r o t e i n s e x t r a c t a b l e from membrane prepara t i o n s with a c i d butanol. In t h i s they resemble the i s o p r e n o i d a l c o h o l phosphokinase p u r i f i e d e a r l i e r from Staphylococcus aureus (18) and a s i m i l a r but not i d e n t i c a l p r o t e i n prepared from E. aerogenes (19,20). The s i t e of s y n t h e s i requirement f o r c a r r i e r l i p i d and a l s o f o r c e r t a i n of the sugar n u c l e o t i d e s , has been i d e n t i f i e d as the cytoplasmic membrane (21). P r e l i m i n a r y experiments i n our labo .ratory have shown that i t i s a l s o the s i t e of exopolysaccharide synthesis (Table 1). Attempts to p u r i f y the t r a n s f e r a s e enzymes by detergent s o l u b i l i z a t i o n were u n s u c c e s s f u l ; membrane p r o t e i n s were s o l u b i l i z e d but the procedure u s u a l l y l e d to p a r t i a l or complete i n a c t i v a t i o n . Although studies of t h i s k i n d have only been a p p l i e d to a l i m i t e d number of micro-organisms, the general mechanisms appear to be the same. In the synthesis of the phosphorylated mannan °f Hansenula capsulata, both mannose and phosphate were derived from GDP-mannose (22). Although i n t h i s p a r t i c u l a r study there was no attempt to demonstrate the involvement of l i p i d i n t e r mediates, they f u n c t i o n i n the formation of s i m i l a r polymers i n m i c r o b i a l w a l l s (23). As the enzyme preparations used i n these studies were crude membranes, nothing i s known about t h e i r r e g u l a t i o n , although i n a s e r i e s of non-polysaccharide-forming Ε. aerogenes mutants, the amount of t r a n s f e r a s e a c t i v i t y appeared to be lower than that found i n w i l d type b a c t e r i a (17). Isoprenoid L i p i d s i n Exopolysaccharide Synthesis The requirement f o r i s o p r e n o i d l i p i d s f o r exopolysaccharide synthesis i s a l s o common to other repeating u n i t - c o n t a i n i n g glycan polymers l o c a t e d e x t e r n a l to the c e l l membrane i . e . the same c a r r i e r l i p i d s are used f o r synthesis of peptidoglycan, t e i c h o i c a c i d s , l i p o p o l y saccharide and exopolysaccharides. Considerable i n d i r e c t evidence suggests that the a v a i l a b i l i t y of i s o p r e n o i d l i p i d phosphate i s one of the most c r i t i c a l f a c t o r s a f f e c t i n g exopoly saccharide synthesis (24). Any mutation a f f e c t i n g i s o p r e n o i d l i p i d synthesis w i l l thus a f f e c t exopolysaccharide production. Various authors have i n d i c a t e d that b a c t e r i a c o n t a i n 6.5-20 mg i s o p r e n o i d l i p i d % dry weight ( c a l c u l a t e d from r e s u l t s i n 25,26). I t has a l s o been suggested that i t s a v a i l a b i l i t y could be c o n t r o l led through phosphorylation of the f r e e a l c o h o l and dephosphory-



r~~

GO

U

Table 1.

Location of Sugar Transferase A c t i v i t i e s

72

Ο

68

3

Cytoplasmic

Outer membrane

membrane

81

lOO*

75

100*

Gal Transfer (%)

Spheroplast membrane

Crude membrane

G l c - l - P Transfer (%)

techniques.

* A c t i v i t i e s were of the order of 0.154 nmol/mg p r o t e i n / h and 0.282 nmol r e s p e c t i v e l y .

ci

η

£J£. aeroqenes type 8, G l c - l - P and Gal I + I I t r a n s f e r a s e s assayed by standard

^

is

CO

I

2. 8CO

Ci

ο "Ρ

Ci 3 ft

48

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

l a t i o n of the a l c o h o l phosphate and pyrophosphate (27). Unfortu n a t e l y , Gram negative b a c t e r i a do not take up mevalonic a c i d and i t i s not p o s s i b l e to l a b e l the l i p i d precursors and thus o b t a i n more accurate e s t i m a t i o n of the amount present i n c e l l s than can be found from d i r e c t e x t r a c t i o n . However, one p o s s i b l e way of i n c r e a s i n g the i s o p r e n o i d l i p i d content appeared to be through s e l e c t i o n f o r b a c i t r a c i n r e s i s t a n c e , s i n c e t h i s a n t i b i o t i c binds very s t r o n g l y to i s o p r e n o i d l i p i d s and e f f e c t i v e l y removes them from b i o s y n t h e t i c processess. Mutants with c o n s i d e r a b l y elevated b a c i t r a c i n r e s i s t a n c e have been i s o l a t e d i n our l a b o r a t o r y and some undoubtedly y i e l d more exopolysaccharide and show increased t r a n s f e r of monsaccharides to l i p i d . (Other mutants were l i t t l e d i f f e r e n t from w i l d type i n a l l respects tested or had l o s t the a b i l i t y to synthesize exopolysaccharide. I t i s a l s o p o s s i b l e that some mutants d e f e c t i v e i n p e p t i d o glycan synthesis might r e q u i r e l e s s i s o p r e n o i d l i p i d than w i l d type c e l l s , thus r e l e a s i n g more f o r exopolysaccharide s y n t h e s i s . A mutant of t h i s type has r e c e n t l y been i s o l a t e d from E. c o l i Β and, u n l i k e the parent b a c t e r i a , produces exopolysaccharide (R.W. North, unpublished r e s u l t s ) . S i m i l a r observations have a l s o been reported during attempts to prepare mutants f o r genetic engineering. The reverse s i t u a t i o n , reduced i s o p r e n o i d l i p i d content, i s a l s o d i f f i c u l t to study and can only be checked i n d i r e c t l y . Mutants with l e s s l i p i d than w i l d type b a c t e r i a have not been c h a r a c t e r i z e d , but a group of CR (crenated) mutants i s o l a t e d from E. aerogenes have c h a r a c t e r i s t i c s which i n d i c a t e that they may be c o n d i t i o n a l mutants of t h i s type (28). These b a c t e r i a have rough c o l o n i a l appearance at lowered i n c u b a t i o n temperature and t h i s has been a s c r i b e d to a reduced content of l i p o p o l y s a c c h a r i d e . Exopolysaccharide i s not synthesized u n t i l growth has ceased. The enzymes f o r p o l y s a c c h a r i d e synthesis are present i n the b a c t e r i a grown a t low temperature and on t r a n s f e r to washed c e l l suspensions ( n o n - p r o l i f e r a t i n g c o n d i t i o n s ) exopolysaccharide i s immediately formed i n the presence or absence of chloramphenical. Thus no new enzymes have to be formed but at low temperature the synthesis of peptidoglycan - e s s e n t i a l f o r c e l l v i a b i l i t y appears to take precedence over exopolysaccharide production and, to a l e s s e r extent l i p o p o l y s a c c h a r i d e s y n t h e s i s . At 37°C, the mutants are i d e n t i c a l i n a l l respects t e s t e d to w i l d type b a c t e r i a . The mutants are not l i k e c l a s s i c a l membrane mutants, d e f i c i e n t i n membrane p h o s p h o l i p i d and s u s c e p t i b l e to various detergents. S i m i l a r c h a r a c t e r i s t i c s were observed i n a polysaccharide-forming pseudomonad (29). The e x t r a c e l l u l a r polymer was only produced l a t e i n the l o g phase of growth and i n the s t a t i o n a r y phase, having s e v e r a l of the a t t r i b u t e s of a secondary m e t a b o l i t e . Could t h i s too be due to i n s u f f i c i e n t i s o p r e n o i d l i p i d i n the growing and peptidoglycan-forming b a c t e r i a ?


4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

49

In the l i t e r a t u r e , frequent r e p o r t s of exopolysaccharide production being favoured by growth a t low temperature a r e to be found; a l t e r n a t i v e l y the polymer i s s a i d to be a product of the c e l l s a f t e r growth has ceased. No s a t i s f a c t o r y explanation f o r these observations has been provided, y e t b a c t e r i a from the l o g or e a r l y s t a t i o n a r y phases of growth appear to produce exopolysaccharide i n washed suspension a t s i m i l a r r a t e s . T h i s could be due to l i m i t a t i o n of exopolysaccharide synthesis during a c t i v e growth through the a v a i l a b i l i t y of i s o p r e n o i d l i p i d ; i t would be needed f o r the formation of w a l l polymers u n t i l l a t e i n the l o g phase of growth. L i m i t a t i o n of c a r r i e r l i p i d a l s o occurs i n c e r t a i n Salmonella mutants d e f e c t i v e i n l i p o p o l y s a c c h a r i d e formation. Mutants forming the l i p i d - l i n k e d O-antigen but unable to t r a n s f e r i t to the appropriate accepto p a r t of the normal i s o p r e n o i other processess, e f f e c t i v e l y reducing the t o t a l present i n the bacteria. Mutants of t h i s type could not produce exopolysaccharide although others d e f e c t i v e i n l i p o p o l y s a c c h a r i d e synthesis but not accumulating l i p i d - l i n k e d glycans, had t h i s c a p a c i t y (31). Several d i f f e r e n t types of mutations can thus a f f e c t i s o p r e noid l i p i d a v a i l a b i l i t y and consequently exopolysaccharide production. These are summarized i n F i g . 6. The i n d i r e c t evidence suggests a d i s t i n c t s e r i e s of p r i o r i t i e s f o r i s o p r e n o i d l i p i d utilization. The e s s e n t i a l w a l l polymer peptidoglycan has p r i o r i t y over l i p o p o l y s a c c h a r i d e which i n turn has p r i o r i t y over exopolysaccharide synthesis ( F i g s . 7 and 8 ) . T h i s could to some extent be achieved through s p a t i a l s e p a r a t i o n of the polysaccharide s y n t h e s i z i n g systems w i t h i n the m i c r o b i a l membrane but obviously requires further elucidation. The f i n a l stages - m o d i f i c a t i o n and e x t r u s i o n As already discussed, the o l i g o s a c c h a r i d e r e p e a t i n g u n i t s accumulate on the c a r r i e r l i p i d and t h i s type of mechanism probably a p p l i e s to a l l exopolysaccharides other than dextrans, levans and r e l a t e d polymers (24). The mechanism could accommodate b a c t e r i a l a l g i n a t e synthesis i f i t i s regarded i n i t i a l l y as a homopolymer of Dmannuronic a c i d and i s probably a l s o v a l i d f o r the glucans secreted by Agrobacterium species. However, many exopolysaccharides c o n t a i n a c y l and k e t a l s u b s t i t u e n t s . Are these added while the repeating u n i t s are attached to l i p i d or at some l a t e r stage? (Fig.9). P r e l i m i n a r y evidence suggests that a c y l a t i o n occurs while the o l i g o s a c c h a r i d e i s s t i l l attached to the l i p i d , but f u r t h e r s t u d i e s are needed. This might i n d i c a t e the lower degree of p y r u v y l a t i o n o c c u r r i n g i n polysaccharide produced a t higher growth r a t e s (and higher r e s u l t a n t l i p i d turnover rates) reported i n some s p e c i e s . The carbon source probably has no d i r e c t e f f e c t (Table 2). Considerable v a r i a t i o n s i n a c y l a t i o n are found w i t h i n a s i n g l e polysaccharide. Thus, a c e t y l groups may occur on each r e p e a t i n g u n i t or on every second repeating u n i t i n one E. aero gene s


50

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Wild-type bacteria (lipopolysaccharides, capsules, slime)

/ i

CR mutants* (decreased lipopolysaccharides, no slime or capsule until growth ceases)

SL mutants (lipopolysaccharides, slime)

\ Ο mutants (lipopolysaccharides)

« • Bacitracin-resistance

CRO mutants (decreased lipopolysaccharides)

SR mutants (1 repeat unit of lipopolysaccharid

R mutants* (core lipopolysaccharides, side chains unattached)

R mutants (inner core only)

* Mutations affecting isoprenoid lipidsfdirectly or indirectly) Biochemical Society Transactions

Figure 6.

How mutations affect the production of exopolysaccharides (31)

\

GROWING

CELLS

E N D OF L O G P H A S E -

Figure 7.

EXOPOLYSACCHARIDE

OR L O W I N C U B A T I O N

TEMPERATURE

Carrier lipid utilization


Microbial

SUTHERLAND

Exopolysaccharide

IPP-

C 5 5 - ISOPRENYL

IPA -

C 5 5 - ISOPRENOID ALCOHOL

PYROPHOSPHATE

ISOPENTENYL

1 1

IP-

PYROPHOSPHATE +

51

Synthesis

C - I S O P R E N Y L PHOSPHATE 5 5

FARNESYL

PYROPHOSPHATE

POLYMER -

MUCOPEPTIDE LPS orTEICHOIC ACID EXOPOLYSACCHARIDE

INTRACELLULAR and MEMBRANE - BOUND PRECURSORS

IPA

Figure 8.

Regulation of carrier lipids

L I P I D - P - P - GIc-GIc I Man I GIcA I Man • A c e t y l CoA

[GIC- Glc] I Man I GIcA I Man

n

OR

• A c e t y l CoA

+ PEP

I

• PEP

r

1

L I P I D - P - P - G I c - GIc [Glc-GlcJ I I Man-O-Ac Man-O-Ac I I GIcA GIcA I I Man = Pyr Man = Pyr Figure 9. Possible exopolysaccharide acylation mechanisms n


In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977. 1.92 1.74

5.48

3.61 3.67

14.5

72.8

Galactose

17.1

79.4

78.8

Sodium pyruvate

Sodium succinate

18.5

18.0

74.0

Raffinose

3.40

6.10 6.22

3.46 3.52

1.30

1.55

0.60

6.96

3.90

2.65 2.87

6.28 5. 81

3.75

16.9

73.6

Sucrose

Maltose

4.03

17.4

79.7

Lactose

1.89

3.78

5.20

5.36

3.16

16.7

75.7

17.9

72.1

Ribose

1.14 5. 70

3.46

16.5

70. Ο

Arabinose

8.75

3.36

0.43

2.20

6.46

3.84

18.2

74.4

Rhamnose

15.8

67.3

Xylose

3.39

6.64

Fructose

3.37

18.5 15.8

74.0

69.4

Mannose

3.23

5. 55 6.32

3.52

15.2

Exopolys acc har ide mg/ml

75. Ο

Carbon Source

Glucose

of

Pyruvic A c i d %

r

Acetate %

R e s u l t s

of a Pseudomonas Exopolysaccharide Derived from Growth on Various Substrates ^ Williams, 1974)

Deoxyhexose %

The Composition

Hexose %

Table 2.

Ω Ω

>

F *d Ο F *

>

W

ο

§

M F F

> Ω

αϊ to

4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

53

s t r a i n (32). I t has a l s o been demonstrated that a c e t y l a t i o n can be l o s t from a s t r a i n without l o s s of exopolysaccharides y n t h e s i z i n g capacity (33) . In c o n t r a s t , loss of any enzyme c o n t r i b u t i n g to the polysaccharide s t r u c t u r e would lead to a non-mucoid v a r i a n t . S i m i l a r l y , p y r u v y l a t i o n a l s o appears i n e s s e n t i a l f o r polysaccharide synthesis as, under c e r t a i n growth c o n d i t i o n s pyruvate groups can be l o s t but polysaccharide of apparently normal carbohydrate composition produced. The exopolysaccharides studied so f a r , have mainly comprised repeating u n i t s with a s i n g l e attached monosaccharide s i d e - c h a i n . I t i s p o s s i b l e that c o n s t r u c t i o n of the longer s i d e chains- found i n xanthan gum or c o l a n i c a c i d might r e q u i r e some other mechanism such as c o n s t r u c t i o n o separate c a r r i e r l i p i d found i n l y s o g e n i c conversion, 34.) The mode of f i n a l r e l e a s e from the i s o p r e n o i d l i p i d has not yet been demonstrated. I t i s u n l i k e l y that the process occurs through non-enzymic r e l e a s e of the i n c r e a s i n g l y h y d r o p h i l i c elongating polysaccharide chain. T h i s would probably leave the c a r r i e r l i p i d u n a v a i l a b l e f o r f u r t h e r polysaccharide s y n t h e s i s . In capsuleproducing s t r a i n s , a l i g a s e r e a c t i o n may remove the polymer chain and attach i t to the c e l l s u r f a c e . It is unlikely that h y d r o l y s i s of the polysaccharide chain occurs at t h i s stage unless a h i g h l y s p e c i f i c enzyme cleaves the t e r m i n a l , phosphate l i n k e d monosaccharide: L i p i d - Ρ - Ρ - Glucose - Galactose

etc.

Enzymes reducing the degree of polymerization have been i d e n t i f i e d i n alginate-producing b a c t e r i a (35) but the f u n c t i o n of the enzyme i s probably unconnected with polymer r e l e a s e of t h i s type. Mutants unable to attach to the c e l l surface (SI mutants) have been widely found, presumably through l o s s of the capsule a t t a c h ment s i t e s on the c e l l s u r f a c e ; other micro-organisms always produce exopolysaccharide as e x t r a c e l l u l a r s l i m e . The chain length of the polymer may a l s o depend on the growth rate i n a manner analogous to l i p o p o l y s a c c h a r i d e side-chains (36), but t h i s needs f u r t h e r study. Higher growth r a t e might lead to more r a p i d turnover of the c a r r i e r l i p i d and r e l e a s e of polymer of lower molecular weight. This i s obviously important to the commercial producer. I t may a l s o be advantageous to use rough mutants ( i . e . s t r a i n s with surface defects) which autoagglutinate of f l o c c u l a t e and lead to e a s i e r polymer recovery. Thus exopoly saccharide production should be examined along with the synthesis of other polysaccharides and not i n i s o l a t i o n .


EXTRACELLULAR

54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Hexokinase Phosphoglucomutase Phosphoglucose Isomerase Phosphomannose Isomerase Phosphomannomutase UDP-Glc Pyrophosphorylase GDP-Man Pyrophosphorylase UDP - Gal Epimerase UDP-Glc Dehydrogenase Glc Transferase I Glc Transferase 1 Man Transferase I Man Transferase Π GIcA Transferase Polymerase (s) Ketalase Acetylase Figure 10.

MICROBIAL

POLYSACCHARIDES

- [Glc - Glc] | Man - Ο - Ac QJ ^ J_ _ ipc Man « ryr ^ ^ ^* / \ \ • ' / _ \ \ " GDP-Man UDP - Glc A « j UDP- Glc ^ UDP-Gal F7 T* Man-I-P Glc-l-P N

C

%

Î5

Man-6-P

T*

Glc-6-P

Fruct J3

Biosynthesis of Xanthomonas polysaccharides

REACTION

CONTROL

Substrate

Substrate entry

Membrane CYTOPLASM

Hexose-phosphate

Hexose-phosphate level

X D P - hexose

XDP-hexose pyrophosphorylase X D P - hexose hydrolase

i

Lipid - Ρ - Ρ - hexose Isoprenoid lipid availability Lipid-P - Ρ - oligosaccharide

Ψ Polysaccharide Figure 11.

?

The control of ρ dysaccharide synthesis


4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

55

Summary The production of m i c r o b i a l exopolysaccharides i n v o l v e s a r e l a t i v e l y l a r g e number of enzymes, some of which are i n v o l v e d i n the formation of other polysaccharides while others are unique to exopolysaccharide s y n t h e s i s . By e x t r a p o l a t i o n from r e s u l t s obtained with other s p e c i e s , a b i o s y n t h e t i c pathway f o r X. campestris polysaccharide can be constructed ( F i g . 10). Loss of most of these enzymes leads to l o s s o f polysaccharide product i o n , but v a r i a t i o n s i n a c y l a t i o n or k e t a l a t i o n occur and may be of importance to the i n d u s t r i a l m i c r o b i o l o g i s t . C o n t r o l of p o l y s a c c h a r i d e synthesis probably occurs at a number of l e v e l s ( F i g . 11), and some mutations with a l t e r e d p o l y s a c c h a r i d e r e g u l a t i o n may have advantageou

Literature Cited 1. Gibbons, R.J. and Nygaard, M. Arch. oral Biol., 13, 12491249 (1968). 2. Smith, E.E.

FEBS Letters, 12, 33-37 (1970).

3. Herbert, D. andKornberg,H.L. Biochem. J., 156, 477-480 (1976). 4. Roseman, S. In'MetabolicPathways'Ed. Hokin, L.E., 6, 41-89 (1972). Academic Press, London and New York. 5. Preiss, J . In'CurrentTopics in Cellular Regulation' 1, pp. 125-160 (1969). 6. Grant, W.D., Sutherland, I.W. and Wilkinson, J.F. J . Bact., 103, 89-96 (1970). 7. Bernstein, R.L. and Robbins, P.W. J . Biol. Chem., 240, 391-397 (1965). 8. Kornfeld, R.H. and Ginsburg, V. Biochim. Biophys. Acta, 117, 79-87 (1966). 9.

Ward, J.B. and Glaser, L. Biochem. Biophys. Res. Commun., 31, 671-6 (1968).

10. Ward, J.B. and Glaser, L. Arch. Biochem., 134, 612-622 (1969). 11.

Lieberman, M.M. and Markovitz, A. J . Bact., 101, 965-972 (1970).


56

12.

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Lieberman, M.M., Shaparis, A. and Markovitz, A. J. Bact., 101, 959-964 (1970).

13. Markovitz, A. In 'Surface Carbohydrates of Prokaryotes', Ed. Sutherland, I.W., Academic Press, London and New York (In press). 14.

Lawson, C.J., McCleary, C.W., Nakada, H.I., Rees, D.A., Sutherland, I.W. and Wilkinson, J.F. Biochem. J., 115, 947-958 (1969).

15.

Sutherland, I.W. Biochem. J., 115, 935-945 (1969).

16.

Troy, F.A., Frerman F.A 246, 118-133

17.

Sutherland, I.W. and Norval, M. Biochem. J., 120, 567-576 (1970).

18.

Sandermann, H. and Strominger, J.L. J . Biol. Chem., 247, 5123-5131 (1972).

19.

Poxton, I.R., Lomax, J.A. and Sutherland, I.W. J . Gen. Microbiol., 84, 231-233 (1974).

20.

Lomax, J.Α., Poxton, I.R. and Sutherland, I.W. FEBS Letters 34, 232-234 (1973).

21.

Osborn, M.J., Gander, J.E. and Parisi, E. J . Biol. Chem.,

d Heath E.C

J Biol Chem.

247, 3973-3986 (1972). 22.

Mayer, R.M.

23.

Lennarz, W.J. and Scher, M.G. Biochim. Biophys Acta, 265, 417-441 (1972). Sutherland, I.W. In"SurfaceCarbohydrates of Prokaryotes", pp. - , Academic Press, London and New York (In press).

24.

Bio chim. Biophys. Acta, 252, 39-47 (1971).

25.

Umbreit, J.N., Stone, K.J. and Strominger, J.L. J . Bacteriol. 112, 1302-1305 (1972).

26.

Dankert, M., Wright, Α., Kelley, W.S. and Robbins, P.W. Arch. Biochem., 116, 425-435 (1966).

27.

Willoughby, E . , Higashi, Y. and Strominger, J.L. J . Biol. Chem.,247, 5113-5115 (1972).


4.

28.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

57

Norval, M. and Sutherland, I.W. J . Gen. Microbiol., 57, 369-377 (1969)

29.

Williams, A. Ph.D. Thesis, University College, Cardiff. (1974)

30.

Kent, J.L. and Osborn, M.J. Biochemistry, 7, 4396-4408 (1968). 31. Sutherland, I.W. Biochem. Soc. Trans., 3, 840-843 (1975). 32. Sutherland, I.W. In "Surface Carbohydrates of Prokaryotes", pp. - , Academic Press, London and New York, (In press). 33.

Garegg, P.J., Lindberg Scand., 25, 1185-1194 (1971).

34.

Wright, A. J . Bacteriol., 105, 927-936 (1971).

35.

Madgwick, J., Haug, A. and Larsen, B. Acta Chem. Scand., 27, 711-712 (1973). Collins, F.M. Aust. J . Exp. Biol. Med., 42, 255- 2 (1964).

36.


5 Polysaccharide Formation by a Methylomonas ΚΑΙ T. ΤΑΜ and R. K. FINN School of Chemical Engineering, Cornell University, Ithaca, NY 14853

Extracellular microbial polysaccharides show great diversity as well as novelty in thei applications of some o fiers, or thickeners in foods; as additives for recovery of pe troleum by water flooding; as plasma extenders or as selective adsorbents in laboratory research, are well documented (2,3,4). A new polysaccharide-producing bacterium called Methylomonas mucosa NRRL B-5696, was isolated from s o i l as an obligate methylotroph and the batch production of polymer and some of its prop erties have been described (5,6). Kinetics for growth of the cells and for polymer production in shake flasks and chemostats are reported here. Materials and Methods The bacteria were maintained on agar plates with a 3% (v/v) methanol basal medium which contained 3.0 g ΚΗ ΡO , 3.7 g Na HPO , 2.5 g NaNO , 0.4 g MgSO ·7 H O, 0.07 g Fe (NH SO ) , 0.025 g Ca (NO ) ·4 H O, 0.001 g ZnSO ·H O, in one liter of d i s t i l l e d water. Methanol concentration was determined by a gas chromatograph with a flame ionization detector using ethanol as the internal standard. Cell dry weight was calibrated against a modified Lowry's protein assay (7), and the latter was used for routine measurements. Polysaccharide concentration was expressed as glu cose equivalent by the phenol-sulfuric acid method of Dubios et. al. (8) with D-glucose as standard. Effluent gas composition was analyzed by a Fisher-Hamilton gas partitioner, model 29, using helium as a carrier gas. Dissolved oxygen measurements were made with membrane probes constructed as described by Johnson and Borkowski (9, 10). Polymer was recovered by acetone precipita tion (11). Viscosity was measured in a Brookfield SynchroLectric Viscometer, model LVT with U. L. adaptor. Fermenter broths diluted in the range 1:5 to 1:10, were f i r s t degassed in vacuum, and then viscosity measurements for each dilution were made at various shearing rates. In some cases, viscosity of the 2

3

3 2

2

4

2

4

4

4

2

4 2

2

58


4

5.

ΤΑΜ

Polysaccharide

A N D FINN

Formation

by a Methylomonas

59

f i n a l b r o t h was d e t e r m i n e d a t a s h e a r i n g r a t e o f 30 RPM w i t h a No. 3 s p i n d l e u s i n g 150 m l o f b r o t h c o n t a i n e d i n a 200 m l b e a k e r . M e t h a n o l i s a t o x i c s u b s t r a t e f o r b a c t e r i a ; e v e n f o r metha n o l u t i l i z i n g o r g a n i s m s a c o n c e n t r a t i o n b e l o w l.O/ may i n h i b i t t h e g r o w t h o f many s t r a i n s (12,13). T h e r e f o r e t h e e f f e c t o f meth a n o l c o n c e n t r a t i o n o n t h e g r o w t h o f M. mucosa was s t u d i e d i n shake f l a s k s . To do t h i s , 250 m l p o r t i o n s o f l o w p h o s p h a t e m e d i um ( b a s a l medium b u t w i t h o n l y h a l f t h e amount o f p h o s p h a t e ) i n 1 - l i t e r i n d e n t e d f l a s k s were i n o c u l a t e d w i t h s e e d f r o m a chemos t a t o p e r a t i n g a t a d i l u t i o n r a t e o f 0.25 h r " a n d a t a s t e a d y s t a t e e f f l u e n t m e t h a n o l c o n c e n t r a t i o n o f 1.0 v/v$>. M e t h a n o l c o n c e n t r a t i o n s i n t h e r a n g e 0.1k t o 2.0/o ( v / v ) were i n v e s t i g a t e d . S p e c i f i c g r o w t h r a t e s a t 30°C a n d 350 RPM r o t a t i o n o f t h e s h a k e r i n c u b a t o r were d e t e r m i n e d i n t h e t i m e p e r i o d when t h e maximum change i n t h e m e t h a n o l i n i t i a l value i n the f l a s k t i o n o f m e t h a n o l c o n c e n t r a t i o n was t h e n p l o t t e d . 0

1

R e s u l t s and

Discussion

K i n e t i c s o f Growth. The e x p o n e n t i a l g r o w t h d a t a f r o m shake f l a s k s i n d i c a t e t h a t m e t h a n o l c o n c e n t r a t i o n s above 1$ v / v a r e i n h i b i t o r y ( F i g u r e 1). F u r t h e r m o r e , a L i n e w e a v e r - B u r k p l o t shows t h a t a t c o n c e n t r a t i o n s l e s s t h a n 1$ t h e d a t a f i t a Monod m o d e l f o r s u b s t r a t e - l i m i t e d g r o w t h . The e x t r a p o l a t e d m a x i m a l s p e c i f i c g r o w t h r a t e , \x ., f r o m F i g u r e 2 i s 0.725 h r " , ( e q u i v a l e n t t o a g e n e r a t i o n t i m e o f 0.956 h r ) . T h i s i s a b o u t 3 t i m e s h i g h e r t h a n t h e a v e r a g e v a l u e f o r most o f t h e m e t h a n o l u t i l i z i n g b a c t e r i a r e p o r t e d i n t h e l i t e r a t u r e (11), a n d i s a b o u t t w i c e t h a t o f P s e u domonad C ( ώ ) , t h e f a s t e s t g r o w i n g m e t h a n o l b a c t e r i a r e p o r t e d . S u c h a f a s t g r o w t h r a t e makes M. mucosa a t t r a c t i v e a s a n o t h e r bacterium f o r s i n g l e - c e l l p r o t e i n production. The h i g h s p e c i f i c g r o w t h r a t e o b s e r v e d i n s h a k e f l a s k s was l a t e r c o n f i r m e d b y a c e l l w a s h o u t e x p e r i m e n t i n a c h e m o s t a t , where t h e m a x i m a l s p e c i f i c g r o w t h r a t e was m e a s u r e d a s 0.719 h r " . The o t h e r k i n e t i c c o n s t a n t , Ks i n t h e Monod m o d e l , was f o u n d t o b e 0.20 M m e t h a n o l . T h i s v a l u e i s two o r d e r s o f m a g n i t u d e l a r g e r t h a n t h e v a l u e o f 0.00375M (120 m g / l ) r e p o r t e d f o r H a n s e n u l a p o l y m o r p h a - a t h e r m o p h i l i c m e t h a n o l - u t i i i z i n g y e a s t whose g r o w t h k i n e t i c s a l s o f i t t h e Monod m o d e l (15). Recent s t u d i e s on the growth o f Candida b o i d i n i i , another m e t h a n o l - u t i l i z i n g yeast, show a K v a l u e a s h i g h a s 0.02M ( l 6 ) . No o t h e r l i t e r a t u r e v a l u e s o f K s f o r m e t h a n o l - a s s i m i l a t i n g b a c t e r i a a r e a v a i l a b l e f o r compar ison. The v a l u e o f K o b t a i n e d i s a l s o much h i g h e r t h a n v a l u e s o b t a i n e d f o r m i c r o b i a l g r o w t h o n o t h e r c a r b o n s o u r c e s , w h i c h gen e r a l l y r a n g e f r o m 1 t o 50 m g / l (17). S i n c e M. mucosa i s sub c u l t u r e d i n yjo m e t h a n o l - s a l t s medium w h i c h i s i n h i b i t o r y f o r most o t h e r m e t h a n o l - a s s i m i l a t i n g b a c t e r i a , t h e b a c t e r i u m must h a v e d e v e l o p e d a t r a n s p o r t mechanism t h a t r e g u l a t e s a slow p e r m e a t i o n of substrate i n t o the c e l l i n order t o reduce the i n h i b i t o r y 1

m

1

s

s


60

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Figure 1. Specific growth rate at different initial sub strate concentrations

Figure 2. Lineweaver-Burk plot for the specific growth rate data

-L

( % Μ · ! Η α η · Ι )r l


5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

61

e f f e c t o f t h e methanol. A l s o t h e p o l y s a c c h a r i d e s l i m e i s an a d d i t i o n a l b a r r i e r f o r t h e d i f f u s i o n o f m e t h a n o l i n t o t h e c e l l . The h i g h K i m p l i e s t h a t a low o v e r a l l a f f i n i t y f o r methanol should be e x p e c t e d . The good agreement o f t h e e x t r a p o l a t e d μ w i t h t h e w a s h o u t datum adds c o n f i d e n c e t o t h e a c c u r a c y o f t h e k i n e t i c c o n stants. The i m p l i c a t i o n o f s u c h a h i g h v a l u e f o r K g i s t h a t a s t a b l e r e a c t o r c a n be o p e r a t e d a t a d o u b l i n g t i m e as s h o r t as 1.8 h r f o r M. mucosa i n a c a r b o n - l i m i t e d c h e m o s t a t . F i g u r e 3 shows t h a t d a t a f o r t h e s u b s t r a t e - i n h i b i t o r y r e g i o n f i t t h e model, s

ηι

μ

where

μ

=

1 + S/K.

(

1

)

= 6.05 h r "

Κ. = 1 8 Λ m M o l a r ι The f a c t t h a t d a t a f i t t h e t w o - p a r a m e t e r m o d e l s does n o t t e l l u s t h e e x a c t mechanism o f i n h i b i t i o n o r growth s t i m u l a t i o n a t t h e molecular l e v e l . However, n o f u r t h e r e x p e r i m e n t s were done t o e l u c i d a t e t h e mechanism o r s i t e o f i n h i b i t i o n b e c a u s e t h e p r i m e o b j e c t i v e o f d e t e r m i n i n g t h e safe o p e r a t i o n range f o r a carbonl i m i t e d c h e m o s t a t was o b t a i n e d i n t h i s s e t o f e x p e r i m e n t s . Respiration Kinetics. From t h e d e p l e t i o n r a t e o f d i s s o l v e d o x y g e n a n d a n a v e r a g e c e l l mass o f 0.152 mg i n t h e Y e l l o w S p r i n g s D i s s o l v e d Oxygen m o n i t o r i n g chambers, 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 s were c a l c u l a t e d f o r d i f f e r e n t i n i t i a l m e t h a n o l c o n c e n t r a tions. I n t h e absence o f s u b s t r a t e i n h i b i t i o n , M i c h a e l i s - M e n t e n k i n e t i c s f i t t h e r e s p i r a t i o n r a t e d a t a as i n d i c a t e d b y t h e s t r a i g h t l i n e i nt h e Lineweaver-Burk p l o t (Figure 4). The c e l l s demonstrate a h i g h a f f i n i t y f o r m e t h a n o l as s u g g e s t e d b y t h e l o w v a l u e o f Κ , 8.1 pmolar methanol. The maximum r e s p i r a t i o n r a t e ( e x t r a p o l a t e d ) i s 33 mMole 0 / ( g c e l l , h r ) , w h i c h i s s l i g h t l y h i g h e r t h a n t h e a v e r a g e v a l u e o f 2 6 . 6 mMole 0 / ( g , h r ) o b t a i n e d from an oxygen balance i n t h e chemostat o p e r a t i n g w i t h a s t e a d y s t a t e e f f l u e n t m e t h a n o l c o n c e n t r a t i o n b e t w e e n 0.6$ a n d 1.5$ (ν/ )· The l o w e r c h e m o s t a t v a l u e o f V m i g h t be due t o s u b s t r a t e i n h i b i tion. Compared w i t h l i t e r a t u r e v a l u e s ( T a b l e I ) , M. mucosa h a s a K i n t h e same o r d e r o f m a g n i t u d e a s H y p h o m i c r o b i u m . The m a x i m a l s p e c i f i c r e s p i r a t i o n r a t e i s about t w i c e t h e h i g h e s t r a t e l i s t e d i n t h e Table. A h i g h e r r e s p i r a t i o n r a t e i s e x p e c t e d f o r M. mucosa because o f i t s v e r y h i g h s p e c i f i c growth r a t e . Another piece o f evidence t h a t agrees w i t h t h e e x t r a p o l a t e d s p e c i f i c r e s p i r a t i o n r a t e comes f r o m s e p a r a t e b a t c h e x p e r i m e n t s . A t t h e p o i n t when t h e d i s s o l v e d o x y g e n r e a c h e s z e r o , t h e o x y g e n demand c a l c u l a t e d f r o m 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 s h o u l d j u s t equal t h e o x y g e n 2

2

ν

m

r


EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES


5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

63

supplied. The o x y g e n s u p p l y , b a s e d o n g a s c h r o m a t o g r a p h i c a n a l y s i s o f t h e i n f l u e n t a n d e f f l u e n t g a s , was k6k0 mg 0 / h r , a n d t h e p r e d i c t e d o x y g e n demand b a s e d o n t h e above r e s p i r o m e t e r d a t a ( 33 mMole 0 p e r g c e l l p e r h r ) was 4780 mg Cg/hr. The endogeneous r e s p i r a t i o n r a t e i n m e t h a n o l - f r e e medium i s 1.21 ± 0.05 mMole 0 / ( g c e l l , h r ) w h i c h a g r e e s w i t h t h e a v e r a g e endogeneous r a t e o f 1.3 ± 0.3 mMole 0 / ( g c e l l , h r ) o b t a i n e d i n t h e Y e l l o w S p r i n g s D i s s o l v e d - O x y g e n M o n i t o r Chamber, u s i n g c e l l s grown i n d i f f e r e n t shake f l a s k s . 2

2

2

2

Table I Michaelis-Menten K i n e t i c Constants f o r the R e s p i r a t i o n o f B a c t e r i a Growing i n Methanol Reference Harrison

(l8)

Harrison

(l8)

Pseudomonas e x t o r q u e n s

20Λ

methane u t i l i z i n g

50.0

10.5 4.15

Pseudomonad Wilkinson

(19)

Hyphomi c r o b i u m

Wilkinson

(19)

mixed c u l t u r e

T h i s work Kim

& R y u (20)

8.53 29400

M e t h y l o m o n a s mucosa

8.1

0.0215 0.024 33.0 18.0

Methylomonas sp.

C a r b o n - l i m i t e d Chemostat. The s t r a i g h t l i n e i n t h e L i n e weaver-Burk p l o t f o r t h e s p e c i f i c growth r a t e i n t h e chemostat, w i t h m e t h a n o l as t h e l i m i t i n g s u b s t r a t e , s u g g e s t s t h a t Monod-type growth k i n e t i c s f i t t h e data ( F i g u r e 5).

s = 1.43 h r

where Κ

.1

=0.583 Molar

However, t h e s e c o n s t a n t s do n o t a g r e e w i t h t h e s h a k e f l a s k d a t a , where ^ = 0.725 h r " , a n d K = 0.20 M o l a r . Such a d i s c r e p a n c y i s t o o l a r g e t o be e x p l a i n e d b y t h e s h o r t - c i r c u i t o f t h e f l o w o r o t h e r e x p e r i m e n t a l e r r o r s . The o n l y l o g i c a l e x p l a n a t i o n i s t h a t b y c o n t i n u o u s l y c u l t i v a t i n g M. mucosa i n t h e c h e m o s t a t f o r o v e r a week, some v a r i a n t was s e l e c t e d t h a t h a d a l o w e r a f f i n i t y f o r methanol and a f a s t e r growing r a t e . When t h e s p e c i f i c m e t h a n o l u t i l i z a t i o n r a t e (Q^) i s p l o t t e d a g a i n s t d i l u t i o n r a t e (D), a s t r a i g h t l i n e i s o b t a i n e d ( F i g u r e 6) 1

s


Figure 6.

Specific substrate utilization rate correlation

0.1

0.2

Dilution

0J

Rat* ,

1

Hr*


5.

Polysaccharide

Τ Α Μ A N D FINN

Formation

by a

65

Methylomonas

This r e s u l t confirms t h e v a l i d i t y o f the e m p i r i c a l equation b y P i r t (17) a n d N a g a i e t a l . (21 ) :

used

(3) be

F o r s t e a d y - s t a t e i n a chemostat w i t h no r e c y c l e , e q u a t i o n comes

x/s where

m = maintenance c o e f f i c i e n t f o r methanol = 0.26 g methanol/( Υ ^ = 0.3*1-5 g c e l l / χ

3

The e x t r a p o l a t e d m i s t h e same o r d e r o f m a g n i t u d e as t h a t r e p o r t e d f o r A. v i n e l a n d i i (22 ), b u t a n o r d e r o f m a g n i t u d e h i g h e r t h a n t h a t f o r other microorganisms (Table II). U n l i k e most o f t h e m i c r o o r g a i s m s l i s t e d , ( w i t h t h e e x c e p t i o n o f A. v i n e l a n d i i w h i c h f o r m s p o l y - b e t a - h y d r o x y b u t y r i c a c i d ) M. mucosa p r o d u c e s e x t r a c e l l u l a r polysaccharides i n a d d i t i o n t o c e l l t i s s u e s and carbon d i o x i d e . The f o r m a t i o n o f e x t r a s t o r a g e p r o d u c t o r p o l y m e r r e q u i r e s more carbon uptake, and t h e r e f o r e a h i g h e r value o f t h e c e l l mainte n a n c e c o e f f i c i e n t s h o u l d b e e x p e c t e d f o r A. v i n e l a n d i i a n d M. mucosa a s i n d i c a t e d i n T a b l e 2. The e x p e r i m e n t a l y i e l d c o e f f i c i e n t Y / = 0.3^5 g c e l l / g CH3OH i s q u i t e r e a s o n a b l e , b e c a u s e M a t e l e s e t a l . r e p o r t e d Y / = 0.31 f o r t h e i r polymer-producing Pseudomonad C i n shake f l a s k s (24) a n d Y / = 0 . 5 4 i n a c h e m o s t a t t h a t f a v o r e d c e l l p r o d u c t i o n (iJT). The c e l l y i e l d s o f o t h e r m e t h a n o l - u t i l i z i n g b a c t e r i a , w i t h no polymer p r o d u c t i o n , range f r o m 0 . 2 t o 0 . 4 (11). H a g g s t r o m (2£) e s t i m a t e d t h a t f o r h i s m e t h a n o l - u t i l i z i n g b a c t e r i a , t h e e f f i c i e n c y o f transforming the carbon from methanol i n t o t h e c a r b o n i n c e l l s w o u l d be 4 l $ ( C M c - m a s s / m e t h a n o l ) · we assume t h e c o m p o s i t i o n o f M. mucosa i s C 5 H 8 O 3 N a n d c a l c u l a t e the e f f i c i e n c y o f carbon t r a n s f o r m a t i o n t o biomass from the exper i m e n t a l y i e l d Y / = Ο.345, t h e e f f i c i e n c y i s 42.7$, about t h e same a s t h e number a s o b t a i n e d b y H a g g s t r o m . The e m p i r i c a l f o r mula C 5 H 8 O 3 N i s used i n s t e a d o f the formula o f C H80 N based on Hamer a n d J o h n s o n s d a t a b e c a u s e t h e l a t t e r p r e d i c t s 13-7$ i n t h e c e l l , w h e r e a s t h e n i t r o g e n c o n t e n t o f M. mucosa i s 11.0 + 0 . 5 $ a v a l u e i n c l o s e r agreement w i t h t h e f o r m u l a C 5 H 8 O 3 N (Table 3). The a v e r a g e y i e l d c o e f f i c i e n t s Y n / = 0.175 a n d Y"c0 /s = 0.483 a r e c o n s t a n t w i t h i n t h e r a n g e o r d i l u t i o n r a t e s s t u d i e d (Figure 7). I f t h e hypothesis i s c o r r e c t , t h a t t h e carbon i n t h e methanol o n l y t u r n s i n t o c e l l s , polymer and carbon d i o x i d e then a c a r b o n b a l a n c e b a s e d o n t h e sum o f t h e t h r e e y i e l d c o e f f i c i e n t s x

S

x

S

x

S

c

x

I

S

4

2

1

S

2


f


methane

methanol

Methane m i x b a c t e r i a (23)

Methylomonas mucosa

oxygen

glycerol

glucose

(22)

(22)

Saccharomyces c e r e v i s i a e (22)

Azotobacter vinelandii

Aerobacter aerogenes

Organism

Limiting Factor

methanol

methane

glucose

glucose

glycerol

Substrate

0.26

0.12

0.02

0.15

0.08

m g sub — cell, hr

Growth Y i e l d and Maintenance C o e f f i c i e n t s and O t h e r M i c r o o r g a n i s m s

Table I I

0.345

0.7

O.5O

0.26

0.56

x/s g cell g sub.

f o r M^ mucosa

ο 2

0.039

0.06

0.02

0.18

0.10

g c e l l , hr

g o

0.425

Ο.38

1.10

0.4l

2

cell

g o 0.94

g

M

o

>

η

>

CO

o

S W >

53

>

r r

ο M

>


Haggstrom

Harrison et a l .

2.4

7.1

6.15

11.4

11.0

io.9

io.8

45.Ο

47.5

46.2

methanol

average :

methanol

36.9

32.12

ash

48.0

4.0

methanol

30.1

7.0

11.0

5

8

3

(28)

(27)

(23)

(26)

assuming a f o r m u l a of C H 0 N

(25)

Hamer e t a l .

Sheehan e t a l .

47-9

Ρ

methane

1.62

29.44

7.14

11.7

50.1

Vary & Johnson

Reference

methane

-

36.72

7.1

Other Elements

9.48

0

Η

Ν

46.7

C

methane

Substrate

E l e m e n t a l A n a l y s e s o f Methane a n d M e t h a n o l U t i l i z i n g Bacteria

Table I I I

68

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s h o u l d add up t o u n i t y . I n o r d e r t o do t h e c a r b o n b a l a n c e , t h e f o l l o w i n g two a s s u m p t i o n s were made: l ) t h e r e i s U6.2# c a r b o n i n t h e c e l l s as i n d i c a t e d b y t h e e m p i r i c a l f o r m u l a C5H8O3ÏÏ and 2 ) t h e r e i s 4θ$> c a r b o n i n t h e p o l y m e r . Since the polymer i s a h e t e r o p o l y s a c c h a r i d e , t h e g e n e r a l f o r m u l a f o r c a r b o h y d r a t e CH 0 i s a good a p p r o x i m a t i o n . The o v e r a l l c a r b o n b a l a n c e comes o u t t o b e (Figure 7): 2

ν fys Ô 3 7 5 ο

Λ

ν 0^62 . V s Ô375

ο

+

v Y

0.2725 C 0 / s 0.375

n =

2

s °* Q

R

9 6 5

S t r i p p i n g o f t h e v o l a t i l e m e t h a n o l o r a t r a c e amount o f b y p r o d uct f o r m a t i o n d u r i n g f e r m e n t a t i o n , s u c h as t h e y e l l o w p i g m e n t , w i l l a c c o u n t f o r t h e 3· 5$ d i s c r e p a n c y i n t h e c a r b o n b a l a n c e . Thus t h e d a t a a r e i n t e r n a l l c o n s i s t e n t To a c c o u n t f o r a l m e t r i c e q u a t i o n can be (ll) 22

CH3OH+I5.5 0 + 2 N 0 ~ 2 H 2

^ 2 C H 0 N + 4 CH 0+33 H 0

+

3

5

8

2

3

2

T h i s e q u a t i o n p r e d i c t s Y / = 0.171, Y / = Ο . 3 6 9 , Y c o / s = 0 . 5 0 . These numbers a g r e e w i t h t h e Y / = 0.175, Y / = 0.3^5, Y C 0 / s = 0.483 o b t a i n e d f r o m t h e e x p e r i m e n t . The e x p e r i m e n t a l p o l y m e r y i e l d o f 17· 5$ i - "too l°w £ ° a n y p r a c t i c a l polymer p r o d u c t i o n process. However, p r e v i o u s s h a k e f l a s k experiments performed w i t h n i t r o g e n l i m i t a t i o n suggested t h a t t h e p o l y m e r y i e l d c o u l d be i m p r o v e d a t t h e e x p e n s e o f c e l l yield (ll). The f e a s i b i l i t y o f a c o n t i n u o u s p o l y m e r p r o d u c t i o n scheme w i t h n i t r o g e n a s t h e l i m i t i n g s u b s t r a t e w i l l be i n v e s t i gated i n the f o l l o w i n g section. p

S

x

p

S

S

2

x

S

2

s

r

N i t r o g e n - l m i t e d C h e m o s t a t . To a c h i e v e n i t r o g e n - l i m i t e d g r o w t h , 1 g/L N a N 0 was u s e d i n t h e f e e d and t h e f l o w r a t e o f medium was a d j u s t e d s o t h a t t h e c e l l d e n s i t y was 1.63 ± 0 . 0 3 g c e l l / L f o r a l l t h e d i l u t i o n r a t e s ( 0 . l 4 t o Ο . 3 2 h r " i ) . The g r o w t h o f t h e c e l l s was n o t o x y g e n l i m i t e d s i n c e t h e d i s s o l v e d oxygen,D. 0., was a l w a y s more t h a n t h a t e q u i v a l e n t t o 30$ a i r s a t u ration. The s p e c i f i c m e t h a n o l u t i l i z a t i o n r a t e , Qj^, r e m a i n e d c o n s t a n t i n s t e a d o f i n c r e a s i n g l i n e a r l y w i t h d i l u t i o n r a t e as was t h e c a s e f o r c a r b o n - l i m i t e d g r o w t h ( F i g u r e 6 ) . The a v e r a g e i s 0.97 + 0.015 g m e t h a n o l / ( g c e l l , h r ) . S i n c e t h e c e l l c o n c e n t r a t i o n a n d t h e Çfa were e s s e n t i a l l y c o n s t a n t f o r a l l t h e d i l u t i o n r a t e s i n v e s t i g a t e d , an i n c r e a s e i n r e s i d e n c e time i m p l i e d t h a t more m e t h a n o l w o u l d be c o n v e r t e d i n t o p o l y m e r . Thus t h e s p e c i f i c p o l y m e r p r o d u c t i o n r a t e , Qp, s h o u l d i n c r e a s e l i n e a r l y w i t h r e s i d e n c e t i m e , and t h i s i s shown i n F i g u r e 8 where p o l y m e r f o r m a t i o n i s e x p r e s s e d b o t h as g l u c o s e e q u i v a l e n t , Qg, and a l s o as d r y w e i g h t , Q ,. The d a t a f o r Q a r e s c a t t e r e d b e c a u s e o f e r r o r s i n d r i e d weight determinations. However, t h e s l o p e s o f Qg and Q s h o u l d b e t h e same, and a t z e r o r e s i d e n c e t i m e , n o 3

p

p

p


5.

Τ Α Μ AND

Polysaccharide Formation

FINN

.3 Dilution

Figure 7.

.4 rat» ,

Hr."

RISIDENCI

Figure 8.

by a Methylomonas

TIM!

69

Yield coefficients for the car bon-limited chemostat

(hr)

9

Polymer production in nitrogen-limited chemostat


70

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p o l y m e r s h o u l d be p r o d u c e d . A s t r a i g h t l i n e w i t h s l o p e p a r a l l e l t o Qg a n d w i t h z e r o i n t e r c e p t i s drawn t h r o u g h t h e d a t a f o r t h e s p e c i f i c polymer p r o d u c t i o n r a t e . F o ra given c e l l population (X g / l ) , t h e t o t a l amount o f p o l y m e r f o r m e d u n d e r n i t r o g e n - l i m i t ed c o n d i t i o n s i s g i v e n b y : t

2

Op d0 = 0.035X ( t | - t f ) ( g p o l y m e r / 1 )

X

Jtx The y i e l d s o f c a r b o n d i o x i d e b a s e d o n m e t h a n o l consumed d i d n o t v a r y w i t h d i l u t i o n r a t e , D. The a v e r a g e v a l u e o f Y c o / s °.47 agrees w e l l w i t h t h e averag v a l u f 0.48 f o t h c a r t o n - l i m i t i n g c a s e . However Yp/ as d i l u t i o n r a t e was c h a n g e ( F i g u r 9) , extrapolate Υχ/s i s z e r o a n d Yp/s i s Ο . 5 6 , w h i c h s u g g e s t s t h a t t h e maximum y i e l d f o r t h e p o l y m e r i s a b o u t 56$ o f t h e m e t h a n o l consumed. This a p p a r e n t h i g h p r o j e c t e d y i e l d m i g h t n o t be a t t a i n a b l e i n p r a c t i c e because o f d i s s o l v e d oxygen l i m i t a t i o n d u r i n g t h e polymer forma t i o n phase. E v e n i f t h e s y s t e m were o p e r a t e d a t h a l f t h e maximum y i e l d , s a y a t Y / = 0 . 2 8 , t h e p e r f o r m a n c e w o u l d s t i l l be b e t t e r t h a n f o r t h e c a r o o n - l i m i t e d c a s e where Y / s 0-175The y i e l d data s t r o n g l y suggest use o f a n i t r o g e n - l i m i t i n g process f o r p o l y mer p r o d u c t i o n . A c h e c k f o r c o n s i s t e n c y o f t h e d a t a was made b y t a k i n g a c a r b o n b a l a n c e w i t h t h e same a s s u m p t i o n s a s b e f o r e , i . e . 4 6 . 2 $ c a r b o n i n c e l l s a n d 40$ c a r b o n i n p o l y m e r . =

2

p

S

=

p

Y

p / s

(i.o 8) 5

+

x

x / s

(i.2 2) 5

+

Y

C

0

2

/

S

(O.T26)

=c

The a v e r a g e v a l u e f o r C t u r n e d o u t t o be 0.990 i n s t e a d o f 0. 965 as i n t h e c a r b o n - l i m i t i n g c a s e . I n o t h e r w o r d s , t h e r e was o n l y 1$ e r r o r i n t h e c a r b o n b a l a n c e . When Yp/s i s z e r o , t h e e x t r a p o l a t e d maximum c e l l y i e l d Y / s i s 0-555Assuming t h a t t h e carbon d i o x i d e y i e l d remains c o n s t a n t a t 0.47 i n t h e a b s e n c e o f p o l y m e r f o r m a t i o n , a c a r b o n b a l a n c e g i v e s a v a l u e o f C a s 1.02, i . e . 2$ e r r o r i n t h e c a r b o n b a l a n c e when o n l y c e l l s a n d C 0 a r e formed. The c o i n c i d e n c e o f t h e m a x i mum v a l u e s f o r t h e y i e l d c o e f f i c i e n t s Υχ/s = 0.56 a n d Yp/s = 0.555 s u g g e s t s t h a t t h e e n e r g y d e r i v e d f r o m c a t a b o l i c p r o c e s s e s i s u s e d w i t h a p p r o x i m a t e l y t h e same maximum e f f i c i e n c y f o r t h e b i o s y n t h e s i s o f e i t h e r c e l l s o r polymer. I n f a c t , these y i e l d d a t a agree c l o s e l y w i t h t h e p r e d i c t i o n s based on " t h e o r e t i c a l " m o l a r g r o w t h y i e l d s f r o m A T P (29). From gas c h r o m a t o g r a p h i c a n a l y s i s , t h e e f f l u e n t a i r h a d a n a v e r a g e c o m p o s i t i o n o f Ο . 6 7 ± 0.02$ c a r b o n d i o x i d e a n d 19-4 ± 0.15$ oxygen. By an oxygen and carbon d i o x i d e b a l a n c e , t h e r e s p i r a t o r y q u o t i e n t (R.Q.) was f o u n d t o be 0 . 4 l 8 m o l e C 0 / m o l e 0 . The a v e r a g e s p e c i f i c o x y g e n c o n s u m p t i o n r a t e was 2 6 . 6 m m o l e x

2

2


2

5.

ΤΑΜ

Polysaccharide

A N D FINN

Formation

by a Methylomonas

71

°2/(g c e l l , h r ) , w h i c h comes c l o s e t o t h e e x t r a p o l a t e d maximum v a l u e o f 33 m m o l e 0 / ( g c e l l , h r ) f r o m t h e p r e v i o u s r e s p i r a t i o n study. An i n t e r e s t i n g f l o c c u l a t i o n phenomenon was o b s e r v e d a t t h e h i g h d i l u t i o n r a t e s (F>1.2 l / h r ) . C e l l s tended t o f l o c c u l a t e and s e t t l e much f a s t e r u p o n s t a n d i n g i n a t e s t t u b e a t room t e m p e r a ture. However, a f t e r a s h i f t t o l o w d i l u t i o n r a t e where more p o l y m e r was p r o d u c e d , t h e f l o c c u l a t i n g phenomenon d i s a p p e a r e d . There are two p o s s i b l e e x p l a n a t i o n s : e i t h e r a mutant i s formed o r t h e f l o c c u l a t i o n i s due t o a c o n c e n t r a t i o n e f f e c t o f t h e p o l y m e r . Only a t a p a r t i c u l a r c o n c e n t r a t i o n o f the a n i o n i c polymer t h a t t h e i n t e r a c t i o n b e t w e e n t h e f i x e d amount o f c e l l a n d t h e c o l l o i d a l p h o s p h a t e c a t i o n c o m p l e x i n t h e b a s a l medium w o u l d b r i n g t h e system t o the i s o e l e c t r i p o i n i agglomeratio precipitation. A g a r p l a t e s i n o c u l a t e d w i t h t h e p r e c i p i t a t i n g c e l l s gave t h e same t y p e o f c o l o n y as t h e n o r m a l c e l l s . A l s o the r a p i d r e v e r s i b i l i t y o f t h e c o a g u l a t i n g phenomenon a c h i e v e d b y c h a n g i n g t h e d i l u t i o n r a t e ( i . e . t h e amount o f p o l y m e r f o r m e d ) s u g g e s t s t h a t the second reason provides a b e t t e r e x p l a n a t i o n . 2

Hon-growth A s s o c i a t e d C o e f f i c i e n t . I t i s apparent from the n i t r o g e n - l i m i t e d growth data t h a t polymer p r o d u c t i o n i s nongrowth a s s o c i a t e d . I n order t o t e s t the e x t r a p o l a t e d polymer y i e l d d a t a (Yp/s °·56) f o r a non-growth s i t u a t i o n ( Y / = θ ) and to f i n d c o e f f i c i e n t f o r non-growth a s s o c i a t e d polymer p r o d u c t i o n i n t h e L u e d e k i n g (30 ) e q u a t i o n , d P / d t = a d X / d t + bX, a shake f l a s k e x p e r i m e n t was. done u s i n g w a s h e d c e l l s . D i f f e r e n t amounts o f w a s h e d c e l l s s u s p e n d e d i n p h o s p h a t e b u f f e r were u s e d t o i n o c u l a t e n i t r o g e n - f r e e b r o t h i n i n d e n t e d f l a s k s c o n t a i n i n g 1.29$ m e t h a n o l . The p o l y m e r p r o d u c t i o n r a t e s were l i n e a r f o r t h e f i r s t s i x t o e i g h t h o u r s b u t d e c r e a s e d when t h e t i m e o f i n c u b a t i o n i n c r e a s e d beyond f o u r generation times. The i n i t i a l p o l y m e r p r o d u c t i o n r a t e was p l o t t e d a g a i n s t t h e d r i e d c e l l w e i g h t . A s t r a i g h t l i n e was o b t a i n e d as shown i n F i g u r e 10. The n o n - g r o w t h a s s o c i a t e d c o e f f i c i e n t , b, o b t a i n e d from t h e s l o p e i s Ο.39 g polymer ( g c e l l , h r ) . The a v e r a g e p o l y m e r y i e l d f o r t h e f i v e f l a s k s was 0.59 ± 0.15 w h i c h a g r e e s w e l l w i t h t h e e x t r a p o l a t e d v a l u e o f 0.56 f r o m F i g u r e 9. The r e l a t i v e l y l a r g e e r r o r i n t h e Yp/s c a l c u l a t i o n i s due t o t h e s m a l l q u a n t i t i e s o f m e t h a n o l consumed i n t h e f i r s t s i x h o u r s ; a d i f f e r e n c e o f 0.01$ m e t h a n o l c o n t e n t w o u l d g i v e 1 0 $ e r r o r i n =

x

S

V*· N i t r o g e n - l i m i t i n g Batch. Based on the p r e v i o u s o b s e r v a t i o n s , a p o l y m e r p r o d u c t i o n scheme w i t h p e r i o d i c n i t r o g e n s t a r v a t i o n was investigated. A b a s a l medium c o n t a i n i n g 1.5 g/L N a N 0 was u s e d and t w o p u l s e s o f a d d i t i o n a l c a r b o n a n d n i t r o g e n (65 m l m e t h a n o l and 2.15 g ammonium s u l f a t e ) were a d d e d a t 9 l / h o u r s a n d a t 23 hours a f t e r the i n i t i a t i o n o f the batch run. For these e x p e r i ments t h e Magnaferm f e r m e n t o r h a d a n a e r a t i o n r a t e o f 5 l i t e r s o f 3

2


72

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Cproducts Cmethanol

Figure

9.

Yield

coefficients in nitrogen-limited chemostat

.25,

Figure

10. Polymer formation washed cell suspension

rate in

Dried Cell

(g/i)


5.

ΤΑΜ

AND

Polysaccharide

FINN

Formation

by

a Methylomonas

73

a i r p e r m i n u t e , and a s t i r r e r s p e e d o f 800 REM. For t h e e x p o n e n t i a l growth phase, the s p e c i f i c c e l l growth r a t e was 0.278 h r " (ta. = 2.5 h r ) w h i c h s h i f t e d t o 0.102 h r " u p o n the a d d i t i o n o f t h e f i r s t p u l s e o f carbon and n i t r o g e n . The l o w c e l l p r o d u c t i o n r a t e was l a r g e l y due t o d i s s o l v e d o x y g e n l i m i t a t i o n , as shown i n F i g u r e 11. The c o n c e n t r a t i o n o f d i s s o l v e d o x y gen r e m a i n e d z e r o a f t e r t h e c o n s u m p t i o n o f lfo m e t h a n o l . The f i r s t - o r d e r r a t e c o n s t a n t f o r g l u c o s e p r o d u c t i o n i s 0.24 hr" . A l a g o f a b o u t two h o u r s was o b s e r v e d b e f o r e p r o d u c t i o n o f polymer resumed a f t e r t h e p u l s e a d d i t i o n o f c a r b o n and n i t r o g e n . S i n c e ammonium s u l f a t e was u s e d as a n i t r o g e n s o u r c e , no n i t r i t e should accumulate t o i n h i b i t polymer p r o d u c t i o n . Perhaps i t t a k e s t i m e f o r M. mucosa t o a d j u s t t o t h e c o n c e n t r a t i o n s h o c k p r o d u c e d by a step increase of methanol inhibitin level Th impor t a n t t h i n g t o note her does n o t d e c r e a s e a p p r e c i a b l y non-growt t h e l 4 t h a n d 23rd h o u r s . A f t e r t h e d i s s o l v e d o x y g e n c o n t e n t r e a c h e d z e r o , t h e mass t r a n s f e r c o e f f i c i e n t Kjja, as c a l c u l a t e d f r o m o x y g e n b a l a n c e , was about c o n s t a n t . The a v e r a g e K a was 165 h r " i . With the aeration a n d s t i r r i n g p a r a m e t e r s k e p t c o n s t a n t , t h e e f f e c t o f t h e foam b r e a k e r on t h e o x y g e n mass t r a n s f e r c o e f f i c i e n t c o u l d be s e e n b y a sudden d e c r e a s e i n Kâ f r o m 165 t o 120 h r " when t h e s u r f a c e o f t h e b r o t h f a i l e d t o r e a c h t h e foam b r e a k e r ( a t t h e 34th h o u r ) . The t e r m i n a t i o n o f p o l y m e r p r o d u c t i o n c o i n c i d e d w i t h t h e d r o p i n Kâ v a l u e . T h i s o b s e r v a t i o n s u g g e s t s t h a t mass t r a n s f e r o f d i s s o l v e d o x y g e n may l i m i t p o l y m e r p r o d u c t i o n o r e l s e t h e r e i s a c c u mulation of t o x i c by-products i n the batch. A n o t h e r i n t e r e s t i n g p o i n t was t h e c o n t i n u e d m e t h a n o l consump t i o n a t a l i n e a r r a t e o f Ο.332 g m e t h a n o l / ( 1 , h r ) a f t e r b o t h g r o w t h and polymer s y n t h e s i s had stopped. T h i s decrease i n methanol c o u l d p e r h a p s be a c c o u n t e d f o r b y t h e c e l l m a i n t e n a n c e r e q u i r e m e n t and t h e s t r i p p i n g l o s s . The f i n a l y i e l d d a t a a t t h e e n d o f t h e 56 h o u r s w e r e : Y / = 0.118, Y / = 0.4θ8, a n d 1.897fo s o l i d p r o d u c e d f o r 4.55$> m e t h a n o l consumed. The maximum y i e l d c o e f f i c i e n t s f o r p o l y m e r p r o d u c t i o n i n t h e b a t c h s h o u l d be c a l c u l a t e d a t t h e 38th h o u r when p o l y m e r p r o d u c t i o n was t e r m i n a t e d . Thus, d i s r e g a r d i n g t h e methanol l o s s i n c e l l maintenance and s t r i p p i n g d u r i n g t h e l a s t 18 h o u r s o f i n c u b a t i o n , t h e y i e l d c o e f f i c i e n t s s h o u l d be Υχ/s =.0.122 a n d Y / = 0.452 w h i l e t h e t o t a l s o l i d y i e l d s h o u l d be 0.574. The p o l y m e r y i e l d a p p r o a c h e d t h e e x t r a p o l a t e d maximum o f Ο.56 ( F i g u r e 8 ) . 1

1

1

L

1

x

S

p

p

S

S

S e m i - c o n t i n u o u s F e r m e n t a t i o n . An a t t e m p t t o c u l t u r e t h e b a c t e r i a c o n t i n u o u s l y a t l o w d i l u t i o n r a t e was n o t v e r y s u c c e s s f u l . The y i e l d o f p o l y m e r d e c r e a s e d a f t e r one week o f c o n t i n u o u s f e r mentation. C o n t a m i n a t i o n a t l o w s t e a d y - s t a t e m e t h a n o l l e v e l and/ o r p o s s i b l y c u l t u r e d e g e n e r a t i o n became t h e m a j o r o b s t a c l e s t o s u c c e s s f u l o p e r a t i o n at low d i l u t i o n rate. Operation o f a carbonl i m i t e d chemostat a t a h i g h e r d i l u t i o n r a t e had p r e v i o u s l y r e -


EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES



RPM

dt

dS

jo

hr

CH3OH

dP g g l u c o s e d t 1, h r

dX g c e l l d t 1, h r

Time, h r

.37 t o

500

.065

.033

.16

0 t o 32

Semicontinuous

First

0.4 t o

.175

500

500

350

.175

hO

.018

.014

30 t o

.038

.057

.Oik

3 t o 47

.054

hi

Semi - c o n t i n u o u s

Second C y c l e Batch

.175

550

.045

.031

.044

42 t o 64

Fermentor

.049

.109

0

33 t o

Shake flask

Cycle

Rate Constants f o r the Semi-continuous Operation i n the l4 L

Table IV

76

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s u i t e d i n s e l e c t i n g a f a s t g r o w i n g v a r i a n t t h a t was a p o o r p o l y m e r former. I n o r d e r t o a v o i d p u t t i n g s e l e c t i v e p r e s s u r e on M. mucosa and t o i m p r o v e t h e p r o c e s s e c o n o m i c s , a s e m i - c o n t i n u o u s o p e r a t i o n scheme was t h e r e f o r e c o n s i d e r e d . The s e t - u p c o n s i s t e d o f t w o t a n k s i n s e r i e s ; t h e f i r s t f e r m e n t o r was t h e Magnaferm w h i c h s e r v e d as a c o n t i n u o u s c e l l p r o p a g a t o r o p e r a t i n g a t r e l a t i v e l y h i g h d i l u t i o n r a t e (0.42 l / h r ) . I n t h i s t a n k t h e s t e a d y s t a t e m e t h a n o l c o n c e n t r a t i o n was k e p t above lfo s o a s t o p r e v e n t g r o w t h o f c o n t a m i n a n t s . B a s a l medium w i t h 3$ m e t h a n o l a n d 2.5 g / l N a N 0 was f e d t o t h e Magnaferm c o n t i n u o u s l y . The e f f l u e n t f r o m t h e f i r s t t a n k was d i r e c t e d i n t o t h e l 4 - l f e r m e n t o r where n i t r o g e n - l i m i t e d g r o w t h began. The n i t r o g e n l i m i t a t i o n not o n l y f a v o r e d polymer formation but a l s o h e l p e d t o prevent the growth o f contaminants A f t e r a f i x e d volume h a d a c c u m u l a t e d i n the second tank, e f f l u e n d i v e r t e d i n t o another fermento t h e s e c o n d t a n k was a l l o w e d t o r u n a s a b a t c h p r o c e s s . A pulse o f 1.7 g N a N 0 a n d 84 m l m e t h a n o l was a d d e d t o t h e l 4 - l i t e r f e r m e n t o r when c o n t i n u o u s f e e d s t o p p e d ( F i g u r e 12). A t t h e 33**d h o u r o f o p e r a t i o n , t h e s e c o n d t a n k was e m p t i e d a n d 250 m l o f t h e b r o t h was p u t i n a n i n d e n t e d f l a s k a n d i n c u b a t e d a t 350 REM i n a c o n s t a n t t e m p e r a t u r e (30°C) s h a k e r . Then t h e s e c o n d c y c l e o f t h e c o n t i n u o u s f e e d t o t h e s e c o n d t a n k b e g a n a n d no a d d i t i o n a l n i t r a t e was a d d e d i n t h i s c y c l e . The r e s u l t s o f t h e t w o c y c l e s o f s e m i - c o n t i n u o u s o p e r a t i o n f o r t h e s e c o n d t a n k a r e shown i n F i g u r e s 12 a n d 13 a n d t h e r a t e c o n s t a n t s a r e summarized i n TableIV. I n t h e f i r s t c y c l e , 2.776 g / l N a N 0 was consumed i n 33 h o u r s a n d 4.22 g c e l l / l was p r o d u c e d . I f a l l t h e a v a i l a b l e n i t r o g e n h a d ended up i n t h e c e l l s , t h e p e r c e n t a g e o f n i t r o g e n i n t h e c e l l s w o u l d be 10.82$> w h i c h a g r e e s w i t h t h e v a l u e o f 10.8ofo p r e d i c t e d b y t h e e m p i r i c a l f o r m u l a C H s 0 N . The m e a s u r e d c e l l a n d p o l y m e r p r o d u c t i o n r a t e s were a l l l i n e a r (Table 4). I f t h e b r o t h were a l l o w e d t o i n c u b a t e l o n g e r t h a n 30 h o u r s i n t h e a e r a t e d t a n k , t h e r a t e o f p o l y m e r p r o d u c t i o n s h o u l d s l o w down t o a b o u t o n e - t h i r d o f t h e i n i t i a l v a l u e as s u g g e s t e d b y d a t a i n t h e s e c o n d c y c l e ( F i g u r e 12). However, t h e r a t e o f p o l y m e r p r o d u c t i o n a c t u a l l y i n c r e a s e d f r o m Ο.Ο65 t o 0.109 g g l u c o s e / l , h r i n t h e shake f l a s k ( a t 350 RFM). T h i s i n d i c a t e d t h a t f o r t h e v i s c o u s b r o t h , a shake f l a s k h a d b e t t e r a e r a t i o n , a n d t h a t a K L a v a l u e o f 98 h r " i n t h e a e r a t e d t a n k was n o t h i g h enough t o meet t h e o x y g e n demand. A n o t h e r p i e c e o f e v i d e n c e f o r o x y g e n l i m i t a t i o n was f o u n d i n t h e s e c o n d c y c l e o f t h e o p e r a t i o n i n the second tank. A 10fo i n c r e a s e i n t h e a g i t a t i o n r a t e o f t h e i m p e l l e r , f r o m 500 t o 550 RPM, r a i s e d t h e p o l y m e r f o r m a t i o n r a t e f r o m 0.018 t o 0.031 g g l u c o s e / ( L , h r ) , a n d t h e c e l l p r o d u c t i o n r a t e f r o m 0.014 t o 0.044 g c e l l / ( L , h r ) . However, t h e K a showed l i t t l e o b s e r v a b l e change a n d r e m a i n e d c o n s t a n t a t 120 h r - i . The r e s p i r a t o r y q u o t i e n t (R.Q.) i s a v e r y s e n s i t i v e p a r a m e t e r t h a t t e l l s t h e age d i s t r i b u t i o n o f t h e p o p u l a t i o n b e c a u s e t h e d e mand f o r o x y g e n a n d t h e e v o l u t i o n o f c a r b o n d i o x i d e a r e n o t c o n 3

3

3

5

1

L


3

5.

Polysaccharide

ΤΑΜ A N D FINN

Formation

by a Methylomonas

HOUR Figure 12.

I

Ο

.

ι

10

First cycle semi-continuous fermentation

J—Λ

20

,

ι

30

,

i _ U

40

1

50

.

•

60

•

I

70

HOUR

Figure 13.

Second cycle semi-continuous fermentation


77

78

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s t a n t d u r i n g t h e d i f f e r e n t phases o f the b a c t e r i a l growth c y c l e . For example, i n t h e f i r s t c y c l e o f t h e s e m i - c o n t i n u o u s o p e r a t i o n , a p r e s s u r e l e a k was d e v e l o p e d i n t h e f i r s t t a n k r e s u l t i n g i n no o v e r f l o w i n t o t h e second t a n k and b r o t h a c c u m u l a t i o n i n t h e c e l l p r o p a g a t o r . A b o u t a n h o u r l a t e r (10-1/2 h o u r i n F i g u r e 1 2 ) t h e p r e s s u r e was r e a d j u s t e d a n d a b o u t 0 . 6 l i t e r o f c e l l s f r o m t h e e x p o n e n t i a l g r o w t h p h a s e was f o r c e d i n t o t h e s e c o n d t a n k . The R. Q. i m m e d i a t e l y jumped f r o m 0.2 t o 0-31 as i n d i c a t e d b y t h e d a s h e d l i n e i n F i g u r e 12. The maximum i n t h e R. Q. c u r v e a l s o i n d i c a t e d t h a t a r e l a t i v e l y l a r g e p o r t i o n o f e x p o n e n t i a l l y growing c e l l s was i n t h e p o p u l a t i o n d u r i n g t h e c o n t i n u o u s f e e d i n g s t a g e i n the second c y c l e ( F i g u r e 12). T h e o r e t i c a l l y , t h e maximum r e s p i r a t o r y q u o t i e n t (R. Q. = 0 . 6 6 ) o c c u r s when c a r b o n i n m e t h a n o is completely oxidised t 2

CH3OH

+

3 0

> 4H 0 + 2 C 0

2

2

3^7 K c a l / m o l e CH 0H

2

3

However, when p a r t o f t h e c a r b o n i s d i v e r t e d t o c e l l a n d p o l y m e r s y n t h e s i s , l e s s c a r b o n d i o x i d e s h o u l d be f o r m e d a n d t h e R. Q. s h o u l d be l e s s t h a n 0.66. S i n c e p o l y m e r i z a t i o n r e q u i r e s l e s s energy than c e l l s y n t h e s i s , the r e s p i r a t o r y quotient s h o u l d de c r e a s e m o n o t o n i c a l l y as more p o l y m e r a n d f e w e r c e l l s a r e formed. The e x p e r i m e n t a l R. Q. d a t a f a l l b e t w e e n 0 . 4 t o 0.1 a n d t h e d e c r e a s e i n t h e r e s p i r a t o r y q u o t i e n t w i t h t h e i n c r e a s e i n t h e amount o f p o l y m e r f o r m e d does i n f a c t a g r e e w i t h t h e p r e d i c t e d g e n e r a l trend. The f i n a l y i e l d d a t a f o r t h e s e m i - c o n t i n u o u s e x p e r i m e n t a r e s u m m a r i z e d i n T a b l e V. Table V F i n a l Y i e l d Data f o r the Semi-continuous

Yield Constant Y

t o t a l solid/s

Y

,

T

a

n

k

Experiment

2

F i r s t Cycle

Second C y c l e

·3

·3*

2

(-W

.121». ( . ι 8 )

.128 (.160)

.196 (.229)

.212 (.264)

5

Χ/ S

Y^

g

Numbers i n t h e b r a c k e t s show t h e y i e l d c o n s t a n t s , c o r r e c t e d f o r l o s s due t o m e t h a n o l s t r i p p i n g a t a r a t e o f 0.12 g M e t h a n o l / ( 1 , h r ) f o r 60 h o u r s . The y i e l d d a t a a r e l o w e r t h a n t h e b e s t o b s e r v e d v a l u e s o f Y / = 0.122, Y p / = ΟΛ52 a n d Y t o t a l s o l i d / s = Ο . 5 6 i n t h e n i t r o g e n - l i m i t e d b a t c h ( F i g u r e 11). The p o o r e r y i e l d i s due t o t h e x

S

S


5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

79

i n f e r i o r o x y g e n t r a n s f e r c a p a c i t y o f t h e s e c o n d t a n k and, more i m p o r t a n t l y , t o t h e n i t r o g e n dosage scheme. I n t h e n i t r o g e n - l i m i t e d b a t c h c u l t u r e , a d d i t i o n a l n i t r o g e n s o u r c e was a d d e d i n a way t h a t avoided p r o l o n g e d p e r i o d s o f n i t r o g e n exhaustion, and conse q u e n t l y t h e r e was a r e l a t i v e l y l a r g e p o p u l a t i o n o f y o u n g c e l l s i n the broth. The f a c t t h a t t h e R. Q. was b e t w e e n 0.4 a n d 0.33 i n t h e f i r s t 50 h o u r s o f t h e b a t c h o p e r a t i o n a s compared t o t h e a v e r a g e R. Q. o f l e s s t h a n 0.2 i n t h e s e m i - c o n t i n u o u s o p e r a t i o n , s u p p o r t s t h e above argument. I n c o n c l u s i o n , a s e m i - c o n t i n u o u s o p e r a t i o n seems f e a s i b l e b e cause i t i s r e p r o d u c i b l e and because i t m i n i m i z e s problems o f contamination o r c u l t u r e degeneration. The p r e s e n t o p e r a t i o n a l p r o c e d u r e i s n o t t h e o p t i m a l one. Improvements i n a e r a t i o n b y i n s t a l l i n g a foam b r e a k e w i l l help t o bring the n i t r o g e n dosage c a n be large portio o f y o u n g c e l l s i n t h e s e c o n d f e r m e n t o r (R. Q. b e t w e e n 0.3 t o 0 . 4 ) .

Literature Cited 1. Bikales, Ν. M. (ed.) in "Water Soluble Polymers", pp 227-42, Plenum Publishing Corp., New York, N.Y., 1973. 2. McNeely, W. H. in "Microbial Technology", H. Peppler (ed.), 381-402, Reinhold Publishing Corp., New York, N.Y., 1967. 3. MacWilliams, D.C., Rogers, J. Η., and West, T. J. in "Ency clopedia of Polymer Science and Technology", Vol. II, pp 105-126, Wiley-Interscience, New York, N.Y., 1973. 4. Moraine, R. A. and Rogovin, P., Biotechnol. Bioeng. (1971), 13, 381-91. 5. Tannahill, Alex L. and Finn, R. Κ., U.S. Patent 3,878,045, April 15, 1975. 6. Finn, R. Κ., Tannahill, Alex L . , and Laptewicz, J. E. Jr., U.S. Patent 3,923,782, Dec. 2, 1975. 7. Herbert, D., Phipp, P. J., and Strange, R. E. in "Methods in Microbiology", Norris, J. R. and Ribbons, D. W. (eds.), Vol. 5B, pp 249-51, Academic Press, London, 1971. 8. Dubios, M. et al., Anal. Chem. (1956), 28, 350-56. 9. Johnson, M. J., Borkowski, J., and Engblom, C., Biotechnol. Bioeng. (1964), 6, 457-68. 10. ibid. 9, 635-39. 11. Tam, K. T., Ph.D. Thesis, Cornell University, Ithaca, Ν. Υ., 1975. 12. Van Dijken, J. P. and Harder, W., J. Gen. Microbiol. (1974), 84, 409-11. 13. Whittenburg, R., Phillips, K. C., and Wilkinson, J. F., J. Gen. Microbiol. (1970), 6 1, 205-18. 14. Battat, E . , Goldberg, I . , and Mateles, R. I., Appl. Microbiol. (1974), 28, 906-11. 15. Levine, P. W. and Cooney, C. L . , Appl. Microbiol. (1973), 26, 982-90.


80

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MICROBIAL

POLYSACCHARIDES

16. Pilat, P. and Prokop, Α., Biotechnol. Bioeng. (1975), 17 1717-28. 17. Pirt, S. J., "Principles of Microbe and Cell Cultivation", 10-12, Blackwell Scientific Publications, Oxford, England, 1975. 18. Harrison, D. E. F., J. Appl. Bacteriol. (1973), 36, 301-8. 19. Wilkinson, T. G. and Harrison, D. E.F., J. Appl. Bacteriol. (1973), 309-13. 20. Kim, J. H. and Ryu, D. Y . , J. Fermentation Technol. (1976), 54, 427-36. 21. Nagai, S., Mori, T., and Aiba, Α., J. Appl. Chem. Biotechnol. (1973), 23, 540-62. 22. Nagai, S. and Aiba, S., J. Gen. Microbiol. (1972), 73, 531. 23. Sheehan, Β. T. and Johnson M J., Appl Microbiol (1972), 21, 511-15. 24. Mateles, R. I. and Appl (1972) 135-40. 25. Haggstrom, L . , Biotechnol. Bioeng. (1969), 11, 1043-54. 26. Vary, P. S. and Johnson, Μ. Η., Appl. Microbiol. (1967), 15, 1473. 27. Wilkinson, T. G., Topiwala, Η. Η., and Hamer, G., Biotechnol. Bioeng. (1974), 16, 41-59. 28. Harrison, D. E. F., Topiwala, Η. Η., and Hamer, G., pp 4915, "Fermentation Technology Today: Proc. IVth Int'l Ferm. Symp.", G. Terui (ed.), Soc. Ferm. Technol., Osaka, Japan, 1972. 29. Abbott, B. J. and Gledhill, W. Ε., Adv. Appl. Microbiol. (1971), 14, 249-60. 30. Luedeking, R. and Piret, E. L . , J. Biochem. Microbiol. Technol. Eng. (1959), 1, 393.


6 Molecular Origin of Xanthan Solution Properties E. R. MORRIS Unilever Research, Colworth/Welwyn Laboratory, Sharnbrook, Bedford. MK44 1LQ., Great Britain

The technological importance of Xanthan gum rests principally on the following unusua properties in aqueous solution 1) Remarkable emulsion stabilising and particle suspending ability. 2) Extremely large shear dependence of viscosity, leading to pronounced thixotropy. 3) L i t t l e variation in viscosity with temperature under normal conditions of industrial utilisation. 4) High salt tolerance. The aim of this paper is to provide a unified explanation of the origin of these properties, at a molecular level. Solution Viscosity. Normally polyelectrolytes adopt a highly expanded conformation under conditions of low ionic strength, but collapse to a more compact coil on addition of salt, due to charge screening. Since polymer solution rheology i s c r i t i c a l l y dependent on molecular shape, these variations i n coil dimensions are normally reflected in large changes i n solution viscosity (4). Since the xanthan molecule is a polyanion, its maintenance of viscosity with increasing ionic strength is therefore particularly surprising, and indicates a considerable departure from normal random coil behaviour. The temperature dependence of i t s solution viscosity i s also complex. In the presence of moderate amounts of salt xanthan viscosity shows virtually no variation with temperature, i n contrast to the normal marked decrease i n polymer solution viscosity on heating. Under low ionic strength conditions, such as exist when the polymer i s dissolved i n distilled water, the temperature dependence of xanthan rheology i s even more unusual, showing an anomolous increase i n solution viscosity on heating, over a specific fairly narrow temperature range (l), suggesting a sharp change i n molecular conformation over this range. 81


EXTRACELLULAR

82

MICROBIAL

POLYSACCHARIDES

Optical Activity Changes i n polysaccharide conformation are f r e q u e n t l y accompanied by l a r g e changes i n o p t i c a l a c t i v i t y ( 5 - 1 3 ) , and i n p a r t i c u l a r s i n g l e wavelength o p t i c a l r o t a t i o n provides a s e n s i t i v e and convenient index of chain conformation. We have t h e r e f o r e used t h i s approach t o i n v e s t i g a t e f u r t h e r the o r i g i n of t h i s p e c u l i a r temperature p r o f i l e (14,15). As shown i n F i g u r e 1 the anomolous v i s c o s i t y behaviour c o i n c i d e s e x a c t l y with a l a r g e sigmoidal i n c r e a s e i n o p t i c a l r o t a t i o n , such as has been shown to accompany o r d e r - d i s o r d e r t r a n s i t i o n s i n other polysaccharide systems (6-10). Indeed a simple q u a n t i t a t i v e r e l a t i o n s h i p has been developed (5) t o p r e d i c t changes i n o p t i c a l r o t a t i o n a r i s i n g from changes i sugar r e s i d u e s i n the polyme I n t e r p r e t a t i o n of xanthan o p t i c a l r o t a t i o n i s , however, complicated by the presence of a c e t a t e , pyruvate, and uronate groups, a l l of which absorb l i g h t a t l o n g e r wavelengths than the polymer backbone and might t h e r e f o r e dominate o p t i c a l r o t a t i o n measurements i n the v i s i b l e r e g i o n . To explore t h i s p o s s i b i l i t y we have used c i r c u l a r dichroism t o monitor d i r e c t l y the temperature dependence of the o p t i c a l a c t i v i t y of these chromophores. As shown i n F i g u r e 2, there i s a l a r g e negative s h i f t i n c.d. on h e a t i n g . The observed o p t i c a l r o t a t i o n s h i f t t o l e s s negative values a t h i g h temperatures i s opposite i n sense, and must t h e r e f o r e a r i s e from changes i n the f a r - u l t r a v i o l e t where the e l e c t r o n i c t r a n s i t i o n s of the polymer backbone are known t o occur ( l 6 , 1 7 ) . N.m.r. R e l a x a t i o n C h i r o p t i c a l and r h e o l o g i c a l evidence t h e r e f o r e i n d i c a t e s that the xanthan molecule e x i s t s i n s o l u t i o n a t moderate temperatures i n an ordered conformation which, under s u i t a b l e c o n d i t i o n s , can be melted out. To f u r t h e r t e s t t h i s c o n c l u s i o n we have used time-domain pulsed n.m.r. t o probe molecular m o b i l i t y . N.m.r. r e l a x a t i o n by energy t r a n s f e r between adjacent n u c l e i provides a s e n s i t i v e index of polymer f l e x i b i l i t y , being extremely r a p i d f o r r i g i d molecules, but much slower f o r f l e x i b l e c o i l s , where t h e r mal motions i n t e r f e r e w i t h the exchange. At e l e v a t e d temperat u r e s , s a l t - f r e e xanthan s o l u t i o n s show o n l y the m i l l i s e c o n d r e l a x a t i o n processes normal f o r d i s o r d e r e d p o l y s a c c h a r i d e s . At ambient temperatures, however, a much more r a p i d r e l a x a t i o n i s observed i n the microsecond range t y p i c a l of r i g i d , ordered structures (18). High r e s o l u t i o n n.m.r. l i n e w i d t h i s i n v e r s e l y r e l a t e d t o the rate of decay of magnetisation, and so f r e e l y moving molecules show sharp n.m.r. spectra, while f o r r i g i d polymers the l i n e w i d t h i s so great that the high r e s o l u t i o n spectrum i s so f l a t t e n e d


6.

Molecular

MORRIS

Origin

of Xanthan

Solution

Properties

83

,4-0

t

3Ό VISCOSITY (CPSxlO" ) 3

Η2Ό

20

AO 60 TEMP C O

80

10

with temperature for Xanthomonas polysaccharide ("KeltroY')

arc

5CfC \ \

3x10 (deg)

2

Λ

· 2

S

iJfll Xlnm) 10·

Figure 2.

Keltrol. Effect of tem perature on CD.


EXTRACELLULAR

84

MICROBIAL

POLYSACCHARIDES

that no peaks are v i s i b l e . Thus normal h i g h - r e s o l u t i o n n.m.r. can be used to monitor o r d e r — d i s o r d e r t r a n s i t i o n s (9-10). At temperatures above the d i s c o n t i n u i t y i n o p t i c a l r o t a t i o n and s o l u t i o n v i s c o s i t y , xanthan s o l u t i o n s show n.m.r. spectra t y p i c a l of a normal d i s o r d e r e d polysaccharide c o i l . On c o o l i n g through the t r a n s i t i o n r e g i o n , however, the spectrum g r a d u a l l y c o l l a p s e s , u n t i l f i n a l l y no d i s c e r n a b l e h i g h r e s o l u t i o n spectrum can be detected. This decay i s conveniently monitored q u a n t i t a t i v e l y by measuring the area of the acetate and pyruvate resonances, which occur as w e l l r e s o l v e d s i n g l e t s at 2.1 and 1.5 ppm r e s p e c t i v e l y (Figure 3 ) . As shown i n F i g u r e 4, the n.m.r. r e l a x a t i o n behaviour f o l l o w s the same sigmoidal temperature course as o p t i c a l a c t i v i t y and s o l u t i o n v i s c o s i t y (see F i g u r e l ) . The Ordered State R h e o l o g i c a l evidence ( l ) i n d i c a t e s that xanthan conformation i s c r i t i c a l l y dependent on the presence or absence of s a l t . To i n v e s t i g a t e t h i s we have followed the o r d e r - d i s o r d e r t r a n s i t i o n at v a r i o u s i o n i c strengths, using o p t i c a l r o t a t i o n as a convenient index of conformational change. As shown i n F i g u r e 5, the t r a n s i t i o n s h i f t s to higher temperature with i n c r e a s i n g s a l t l e v e l , u n t i l f o r i o n i c strengths above about 0.15 M, the ordered c o n f o r mation p e r s i s t s up to 100 C. S i m i l a r s t a b i l i s a t i o n of ordered s t r u c t u r e s by a d d i t i o n of s a l t i s observed i n other charged polysaccharides ( 6 - 8 ) , and i s presumably due to the r e d u c t i o n of e l e c t r o s t a t i c r e p u l s i o n s between neighbouring charged groups i n the compact, ordered s t a t e . At constant i o n i c strength the temperature course of the t r a n s i t i o n appears to be independent of polymer c o n c e n t r a t i o n (Figure 6 ) . This i n d i c a t e s that e i t h e r the o r d e r - d i s o r d e r process i s unimolecular, or that i t i s extremely co-operative, as i n the case of DNA (19). The breadth of the xanthan t r a n s i t i o n argues against the l a t t e r explanation, and suggests i n t r a m o l e c u l a r order. The covalent s t r u c t u r e of xanthan has only r e c e n t l y been d e t e r mined ( 2 0 , 2 1 ) , and c o n s i s t s of a c e l l u l o s e backbone s u b s t i t u t e d on a l t e r n a t e residues with charged t r i s a c c h a r i d e sidechains, as shown i n F i g u r e 7· We suggest that i n the ordered conformation the sidechains are a l i g n e d with the main chain to give a r i g i d s t r u c t u r e s t a b i l i s e d by i n t r a m o l e c u l a r non—covalent bonding. D e f i n i t i v e d e s c r i p t i o n of the ordered n a t i v e conformation, however, must await X-ray evidence. Such work i s at present i n progress i n Purdue u n i v e r s i t y , and i s d e s c r i b e d i n the f o l l o w i n g paper. Molecular I n t e r p r e t a t i o n of S o l u t i o n P r o p e r t i e s Whatever the d e t a i l of the ordered s t a t e , i t s existence i n s o l u t i o n o f f e r s a s a t i s f a c t o r y u n i f y i n g explanation of the unusual and v a l u a b l e r h e o l o g i c a l p r o p e r t i e s of xanthan. In most


6.

MORRIS

Molecular

Origin

of Xanthan

Solution

Properties

Figure 3.

Figure

4.


85

EXTRACELLULAR

—ι

ι

j

INCREASING

40

1

/

1 \ CONSTANT IONIC STRENGTH

,

OR

POLYSACCHARIDES

A / ι

POLYMER CONC

f

MICROBIAL

60

80

100

T*C

Figure

6.


6.

MORRIS

Molecular Origin of Xanthan Solution Properties

-> 4 ) - p - B - G l c p - ( l - > 4 ) - p - D - G l c p - 0 - >

ι p-D-GlcAp

J, I

p-g-Man£

6

/ C

H C 3

/

COOH

Figure 7.


87

EXTRACELLULAR

88

MICROBIAL

POLYSACCHARIDES

t e c h n o l o g i c a l l y important a p p l i c a t i o n s , s u f f i c i e n t s a l t i s present t o maintain the ordered conformation a t a l l temperatures. The r e l a t i v e i n s e n s i t i v i t y of s o l u t i o n v i s c o s i t y t o a d d i t i o n of f u r t h e r s a l t i s then a d i r e c t consequence of molecular r i g i d i t y . The emulsion s t a b i l i s i n g , p a r t i c l e suspending and t h i x o t r o p i c behaviour a l l point t o the existence of a p p r e c i a b l e i n t e r molecular s t r u c t u r e i n xanthan s o l u t i o n s . Such an i n t e r p r e t a t i o n i s e n t i r e l y c o n s i s t e n t with the known tendency o f r o d - l i k e molecules i n s o l u t i o n t o a l i g n (22). Indeed, b i r e f r i n g e n c e s t u d i e s (2) give d i r e c t evidence of c o n s i d e r a b l e molecular o r i e n t a t i o n i n xanthan s o l u t i o n s . We t h e r e f o r e suggest that weak non-covalent a s s o c i a t i o n s between a l i g n e d molecules b u i l d up a tenuous g e l - l i k e network capable of supporting s o l i d p a r t i c l e s , l i q u i d d r o p l e t s , or a i r bubbles P r o g r e s s i v e breakdown of t h i s network with i n c r e a s i n of the remarkable t h i x o t r o p p r o p e r t y of the p o l y s a c c h a r i d e .

Abstract Xanthan exists in solution at moderate temperatures in a native, ordered conformation. At low salt levels this order may be melted out, as monitored by n.m.r. relaxation, optical rotation, circular dichroism, and intrinsic viscosity. We suggest that in the ordered conformation the charged trisaccharide side -chains fold back around the cellulose backbone, to give a rigid, rod—like structure. Increasing salt concentration stabilises this conformation by minimising electrostatic repulsions between the sidechains. At the salt levels encountered in most industrial situations, the ordered form is stable to above 100°C, hence the relative insensitivity of xanthan solution viscosity to tempera ture or further increase in ionic strength. Stacking of the rigid molecules in solution builds up a tenuous intermolecular network, giving rise to the other commercially attractive properties, such as suspending ability, emulsion stabilisation, and thixotropy. Literature Cited 1. 2. 3. 4. 5. 6.

Jeanes, Α., Pittsley, J.E. & Senti, F.R. J. Applied Polymer Sci. (1961). 5, 519-526. Jeanes, A. In"Proceedingsof the ACS Conference on Water Soluble Polymers" (Bikales, N.M., ed.), pp. 227-242, Plenum Press, New York, (1973). Glicksman, H."PolysaccharideGums in Food Technology", Academic Press, New York, (1970). Smidsrød, O. & Haug, A. Biopolymers. (1971). 10, 1213. Rees, D.A. J . Chem. Soc. (B). (1970). 877-884. Rees, D.A. & Scott, W.E. J . Chem. Soc. (B). (l971). 469-479.


6.

MORRIS

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Molecular

Origin

of Xanthan

Solution

Properties

89

Rees, D . A . , Scott, W.E. & Williamson, F . B . Nature. (1970). 227, 390-393. Rees, D . A . , Steel, I.W. & Williamson, F . B . J . Polymer S c i . (C). (1969). 28, 261-276. Bryce, T . A . , McKinnon, Α . Α . , Morris, E . R . , Rees, D.A. & Thom, D. Faraday Discuss. Chem. Soc. (1974). 57, 221. Dea, I.C.M., McKinnon, A.A. & Rees, D.A. J . Mol. B i o l . (1972). 68, 153-172. Grant, G . T . , Morris, E . R . , Rees, D . A . , Smith, P . J . C . & Thom, D. Febs. Lett. (1973). 32, 195-197. Morris, E . R . , Rees, D.A. & Thom, D. J . Chem. Soc. Chem. Commun. (1973). p. 245. Morris, E.R. & Sanderson, G.R. In "New Techniques i n Biophysics and Cell Biology" Joh Wiley London (1972) Morris, E . R . , Rees Darke, A. J . Mol. B i o l . Submitted. (1976) Rees, D.A. Biochem. J. (1972). 126, 257-273. Balcerski, J . S . , Pysh, E . S . , Chen, G.C. & Yang, J . T . J . Am. Chem. Soc. (1975). 97, 6274-6275. Pysh, E . S . Ann. Rev. Biophys. Bioeng. (1976). 5, 63-75. Darke, Α . , Finer, E . G . , Moorhouse, R. & Rees, D.A. J . Mol. Biol. (1975). 99, 477-486. Zimm, B.H. J . Chem. Phys. (1960). 33, 1349-1356. Jansson, P . E . , Kenne, L . & Lindberg, B. Carbohyd. Res. (1975). 45, 275-282. Melton, L . D . , Mindt, L., Rees, D.A. & Sanderson, G.R. Carbohyd. Res. (1976). 46, 245-257. Flory, P . J . Proc. Roy. Soc. ser. A. (1956). 234, 50-73.


7 Xanthan Gum—Molecular Conformation and Interactions R. MOORHOUSE, M. D. WALKINSHAW, and S. ARNOTT Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Xanthan Gum, the extracellular the microorganism Xanthomonas campestris has found widespread industrial use (1,2,3) because o f its unique r h e o l o g i c a l properties. The p o l y s a c c h a r i d e forms homogeneous aqueous d i s p e r s i o n s and s o l u t i o n s e x h i b i t i n g high viscosity, as w e l l as having characteristics of both p s e u d o p l a s t i c and plastic polymer systems ( 4 , 5 ) . Of particular s i g n i f i c a n c e is the a t y p i c a l insensitivity of s o l u t i o n viscosity to s a l t e f f e c t s and to heat, e s p e c i a l l y at h i g h ionic s t r e n g t h . Molecular weight measurements (6) i n d i c a t e p o l y d i s p e r s e systems of h i g h molecular weight (>2x10 ). The primary s t r u c t u r e of xanthan has r e c e n t l y been r e i n v e s t i g a t e d (7,8) and found to c o n s i s t of pentasaccharide r e p e a t i n g u n i t s (I). 6

Pyruvate is attached on average to about o n e - h a l f of the t e r m i n a l mannose r e s i d u e s ; 0 - a c e t y l groups correspond to one residue f o r each pentassaccharide r e p e a t i n g u n i t . When p r e v i o u s l y detected in bacterial p o l y s a c c h a r i d e s , pyruvate has u s u a l l y been observed on every r e p e a t i n g u n i t ( 9 , 1 0 ) . However, the c l o s e l y r e l a t e d 90


7.

MOORHOUSE ET

AL.

Xanthan

Gum

Conformation

and

Interactions

91

polysaccharides from other Xanthomonas species (11,12) also show differing pyruvate contents (13). We have prepared f i b e r s of both xanthan and the r e l a t e d p o l y s a c c h a r i d e from Xanthomonas p h a s e o l i (14). Using e s t a b l i s h e d techniques f o r f i b e r d i f f r a c t i o n and computer aided model b u i l d i n g (15,16,17,18,19) we have been able to examine the p o s s i b l e molecular conformations of xanthan. The almost i n d e n t i c a l X-ray d i f f r a c t i o n patterns,from a l a r g e number of p o l y s a c c h a r i d e samples from both X. campestris and X. p h a s e o l i , i n d i c a t e s an o v e r a l l s i m i l a r i t y of molecular conformation and primary sequence. R e s u l t s and D i s c u s s i o n It i s usually possibl t specimen f lon helical polymers i n which the molecule parallel. Often f u r t h e degree of a t h r e e - d i m e n s i o n a l l y ordered s i n g l e c r y s t a l . The xanthan X-ray d i f f r a c t i o n p a t t e r n (Figure 1) showing both c o n t i n uous i n t e n s i t y d i s t r i b u t i o n and Bragg maxima, i s c h a r a c t e r i s t i c of an ordered a r r a y of h e l i c e s which have t h e i r axes p a r a l l e l but are not f u r t h e r ordered (20). The presence of continuous d i f f r a c t i o n along the l a y e r l i n e s i n d i c a t e s that the i n d i v i d u a l molecules have random t r a n s l a t i o n s along and r o t a t i o n s about t h e i r axes and are not packed i n t o a w e l l developed c r y s t a l l a t t i c e . However, d e s t r u c t i v e i n t e r f e r e n c e has occurred near the center of the equator, l e a v i n g one broad Bragg r e f l e c t i o n of spacing 1.9 nm, the a r r a y of molecules t h e r e f o r e has some order when viewed down a molecular screw a x i s at s u f f i c i e n t l y low r e s o l u t i o n . The l a y e r l i n e spacing i s c o n s i s t e n t with a h e l i x of p i t c h 4.70 nm; the m e r i d i o n a l r e f l e c t i o n s (0,0,£) o c c u r r i n g only when Z=5n, suggests a 5-fold helix. T h i s gives a r i s e per backbone d i s a c c h a r i d e of 0.94 nm (Figure 2). The s t e r i c e f f e c t of the branching mannose r e s i d u e together with the consequent removal of the c e l l u l o s e 0(3)A—0(5) hydrogen bond across a l t e r n a t e 8-1,4 l i n k a g e s (u9ing the n o t a t i o n i n Figure 2) means that the backbone can no longer have the 2^ screw symmetry of c e l l u l o s e . Instead of the usual extended β-1,4 ribbon (21), a more sinuous h e l i x of the type shown i n F i g u r e 3 i s obtained. A p r i o r i we could have no preference f o r any of the four p o s s i b l e 5 - f o l d h e l i c a l models. The 5/1 and 5/4 conformations are r i g h t and left-handed r e s p e c t i v e l y and have a s i n g l e t u r n per h e l i x p i t c h while the two other (5/2 and 5/3) models a l s o d i f f e r by being r i g h t and left-handed and have two turns per h e l i x p i t c h . I n i t i a l l y molecular models f o r each of these f o u r s i n g l e h e l i c a l p o s s i b i l i t i e s , were examined assuming standard bondlengths, bond-angles and sugar r i n g conformation angles (15). The models were f u r t h e r c o n s t r a i n e d to e x h i b i t symmetry and p e r i o d i c i t y c o n s i s t e n t with the d i f f r a c t i o n p a t t e r n . On the b a s i s of a minimum s t e r i c compression comparison, the 5/1 (Figure 3) and 5/2 (Figure 4) right-handed h e l i c e s were most favored,


92

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Figure 1. Diffraction pattern typ ical for both Xanthomonas cam pestris and Xanthomonas phaseoli polysaccharides showing five-fold helical symmetry. The sharp Bragg reflection on the equator has a spacing of 1.9 nm.

Figure 2. The pentasaccharide repeat ing unit of xanthan showing atom label ing and aisaccharide backbone height. The unlettered residue and residue A are Ό-glucose, Β and Ε are O-mannose, and C is O-glucuronate.


7.

MOORHOUSE

Figure 3.

E TA L .

Xanthan

Gum Conformation

and

Interactions

The isolated 5/1 xanthan helix viewed perpendicular to the helix axis


93

Figure 4.

The isolated 5/2 xanthan helix viewed perpendicular to the helix axis


7.

MOORHOUSE E T A L .

Xanthan

Gum

Conformation

and

Interactions

95

having g l y c o s i d i c conformation angles w i t h i n the normal o l i g o saccharide ranges (Table 1) and no overshort non-bonded separat i o n s . With the left-handed h e l i c e s (5/3 and 5/4), minimization did not r e l i e v e a l l of the unacceptably short i n t e r a t o m i c cont r a c t s even a f t e r o p t i m i z a t i o n . In i s o l a t i o n there i s no d r i v i n g f o r c e to h o l d the s i d e chains c l o s e to the backbone and the i s o l a t e d chain models suggest a diameter of 3.8 nm as opposed to a value of 1.9 nm obtained from l a t e r a l p e r i o d i c i t i e s i n the d i f f r a c t i o n p a t t e r n . Studies on other branched polysaccharides favor the s i d e chains l y i n g roughly p a r a l l e l to the backbone (18), and we have t h e r e f o r e undertaken a second study i n which both packing and conformational v a r i a t i o n s were considered f o r each of the models. The most symmetrical and commonly observed c l o s e packing of polymeric molecules, havin hexagonal packing i n whic 6 nearest neighbors but not n e c e s s a r i l y f u r t h e r r e l a t e d . We t h e r e f o r e placed one xanthan h e l i c a l chain i n a hexagonal u n i t c e l l of s i d e a. = 2.19nm, c_ = 4.70nm, that i s c o n s i s t a n t with the e q u a t o r i a l Bragg r e f l e c t i o n indexed as (100). Minimizing s t e r i c r e p u l s i o n i n t h i s environment causes the side chain to f o l d down against the backbone. Stereochemically both the 5/4 and 5/3 h e l i c e s are u n l i k e l y as an unacceptable number of i n t r a m o l e c u l a r overshort contacts p e r s i s t a f t e r refinement. This r e i n f o r c e s our previous c o n c l u s i o n of right-handedness f o r the i s o l a t e d chains. Although the 5/1 and 5/2 h e l i c e s are s t e r i c a l l y acceptable, the 5/1 e x h i b i t s the more f a v o r a b l e comparison with o l i g o s a c c h a r i d e conformation angles. I t i s of i n t e r e s t to note that the backbone conformation angles shown i n Table I have v a r i e d l i t t l e during the process of wrapping the s i d e chains around the backbone. Further, the 5/1 packed' h e l i x (Figure 5) shows a number of p o t e n t i a l intramolec u l a r hydrogen bonds (Table I I and Figure 6). Relaxing the a t t r a c t i v e i n t e r a c t i o n (hydrogen bond) terms i n the refinement did not a l t e r the molecular conformation. Only the a d d i t i o n a l i n f l u e n c e of small p e r t u r b a t i o n s to the conformation angles about the branching mannose l i n k a g e caused the s t a b i l i s i n g i n f l u e n c e of the hydrogen bonds to be l o s t . The 'packed 5/2 h e l i x presents a much t i g h t e r s t r u c t u r e than the 5/1 model while e x h i b i t i n g some overshort i n t r a m o l e c u l a r contacts and few p o t e n t i a l hydrogen bonds and was considered u n l i k e l y on the b a s i s of t h i s a n a l y s i s . Our reasoning so f a r has been based on the premise that the e q u a t o r i a l Bragg r e f l e c t i o n on the d i f f r a c t i o n p a t t e r n (Figure 1), a r i s e s from the packing of s i n g l e molecular e n t i t i e s , the p a t t e r n does not t e l l us what form these take. In our examination of i n t e r - c h a i n i n t e r a c t i o n s , we have thus considered those i n t e r a c t i o n s that can a r i s e from some side-by-side arrangement of the 5/1 h e l i c e s and a l s o the case of c o a x i a l m u l t i p l e h e l i c e s . 1

1


96

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

TABLE I Comparison o f backbone c o n f o r m a t i o n a n g l e s i n t h e i s o l a t e d and ' p a c k e d ' 5/1 a n d 5/2 h e l i c a l m o d e l s

Angle

-100+ -161

(a)

-78+

(b)

(d)

-78+

(a) - e [ c

( 1 ) A

"

e t 0

-

Using

9 [ 0

helices 5/2

-121

-148

-119

-111

-99

-98

-97

-92

-22

-81

-61

-98

-6

-98

( 4 )

,c

( 4 )

,c

C

( 5 )

]

, C

(5)A, (l)A'°(4) (4)

(c) - 6 [ C (d)

,o

'Packed' 5/1

-136

-10O>- -161

(c)

(b)

Isolated helices 5/1 5/2

Range

0

{ 1 )

> (4 C

) A

>

C

]

C

( 4 ) A

> (5) ] A

C

(5)' (1),°(4)A, (4)

] A

atom n o t a t i o n i n F i g u r e 2.


7.

MOORHOUSE E T A L .

(a)

Xanthan

Gum Conformation

and

Interactions

97

(b)

Figure 5. The 'packed' 5/1 helix viewed (a) perpendicular to and (b) down the helix axis



Figure 6. Possible hydrogen bonds ( ) that may stabilize the molecule. Some adjoining residues are omitted for clarity, the backbone having solid bonds. See also Table II.


Xanthan

MOORHOUSE ET AL.

Gum Conformation

and

Interactions

TABLE I I P o s s i b l e a t t r a c t i v e i n t e r a c t i o n i n the X and 5/2 h e l i c e s

Model

Overshort

5/1

aampestris

3

5/1

Potential Hydrogen bonds

contacts (nm)

non 0

( 2 ) ~ -

°(8a)

D

°(6)

°(5)C

°(2)A

** °(7)B

°(3)B—* °(6) [

o

r

°(3)B

°(2)D 5/2

Η

°(5)Α··· (4)Α (0.195 nm)

°(3)~

Υ

" °(5)C

°(6b)C

> 0

(5)A

°(6)A

* °(5)

°(3)B

' °(5)C

°(3)C

°(5)D


]

100


P l a c i n g a s i n g l e 5/1 h e l i x i n o u r h e x a g o n a l c e l l r e v e a l s few i n t e r a c t i o n s w i t h i t s nearest neighbors. This suggests that t h e h e l i c e s a r e s l o t t i n g i n t o some g r o o v e t h a t i s w i d e enough t o a c c o m o d a t e them w i t h o u t s t e r i c c l a s h e s . A l t e r n a t i v e l y t h e molec u l e c o u l d be c o n s i d e r e d a s a r i g i d r o d o f p o l y s a c c h a r i d e s u r rounded by a c y l i n d e r o f w a t e r , i n which case v e r y few p o l y s a c c h a r i d e - p o l y s a c c h a r i d e i n t e r a c t i o n s would be a p p a r e n t . Furthermore, a s s u c h a s i t u a t i o n c l o s e l y m i m i c s t h e s o l u t i o n s t a t e , t h e unusual s o l u t i o n p r o p e r t i e s would p r o b a b l y a r i s e from i n t e r a c t i o n s b e t w e e n r e g i o n s o f O r d e r e d ' w a t e r some o f w h i c h may b e t i g h t l y bound t o t h e p o l y s a c c h a r i d e . Current X-ray f i b e r d i f f r a c t i o n t e c h n o l o g y c a n n o t e n a b l e u s t o l o c a t e t h i s amount o f w a t e r ( 1 8 ) , p o s s i b l y NMR s t u d i e s o n s o l u t i o n s may be a b l e t o l o c a t e 'ordered water but without t h d e t a i l t h a t i sometime a v a i l a b l e from d i f f r a c t i o ment f o r p r o l o n g e d p e r i o d c e l l volume c o n s i s t e n t w i t h a s h r i n k a g e i n t h e Bragg s p a c i n g on the e q u a t o r w h i l e t h e f i b e r a x i s d i m e n s i o n r e m a i n s u n a l t e r e d . Apparently the molecular conformation o f the xanthan molecule s u r v i v e s d r y i n g w i t h l i t t l e change a n d a s u b s t a n t i a l q u a n t i t y o f w a t e r w h i c h f i l l s o u t t h e s t r u c t u r e i s n o t f i r m l y bound. W h i l e i t i s p o s s i b l e t o c o n s t r u c t a double h e l i c a l model, u s i n g t h e 5/1 s i n g l e h e l i x a s p r e c u r s o r , i n w h i c h t h e s e c o n d c o a x i a l s t r a n d i s p a r a l l e l t o , a n d r e l a t e d t o , t h e f i r s t b y 180 r o t a t i o n some a p p a r e n t l y u n r e s o l v a b l e o v e r s h o r t i n t e r - s t r a n d contacts exist. I t i s p o s s i b l e t h a t r e l a x i n g t h e summetry s o t h a t the p a r a l l e l c o a x i a l s t r a n d s a r e n o t r e l a t e d b y a 180 f i b e r a x i s r o t a t i o n o r a r e a n t i p a r a l l e l t o one a n o t h e r , c o u l d r e s u l t i n a c c e p t a b l e i n t e r a c t i o n s between c h a i n s . Should t h i s be t h e case i t w i l l s t i l l be n e c e s s a r y t o o b t a i n s u p p o r t i n g e v i d e n c e from other sources t o demonstrate t h e e x i s t e n c e o f double h e l i c e s . N o r m a l l y t h i s would t a k e t h e form a comparison o f t h e model w i t h the X - r a y i n t e n s i t y d a t a f r o m a c r y s t a l l i n e d i f f r a c t i o n p a t t e r n (e.g. 16,17,18) p l u s e v i d e n c e f r o m s o l u t i o n s t u d i e s o f b i - m o l e c u l a r i t y ( e . g . 2 2 ) . We w o u l d s t r e s s however t h a t t h e r e i s no e v i d e n c e o f d o u b l e h e l i c e s e i t h e r i n s o l u t i o n (27) o r t h e s o l i d s t a t e . R e c e n t l y , we h a v e b e e n a b l e t o o b t a i n a d i f f r a c t i o n p a t t e r n t h a t e x h i b i t s i n c r e a s e d c r y s t a l l i n i t y and which h a s been t e n t a t i v e l y i n d e x e d o n a t e t r a g o n a l c e l l i n w h i c h f o u r 5/1 s i n g l e h e l i c e s w i l l p a c k w i t h t h e minimum o f s t e r i c c o m p r e s s i o n . A r e finement u s i n g b o t h s t e r e o c h e m i c a l and X-ray i n t e n s i t y d a t a has not y e t been completed. 1

Conclusions T h i s p r e l i m i n a r y s t u d y shows t h a t t h e o r d e r e d c o n f o r m a t i o n of xanthan i n t h e condensed s t a t e , and p r o b a b l y i n s o l u t i o n , i s r e l a t e d t o t h e 5/1 h e l i x o u t l i n e d h e r e .


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The Interactions t h a t o c c u r i n solution, giving rise t o viscosity effects showing t h e characteristic o f both flexible a n d stiff cross-linked r e g i o n s (4,5) must arise f r o m associations of t h e o r d e r e d 5/1 helical regions. The order/disorder transition s e e n with change o f t e m p e r a t u r e in solution ( 2 3 , 2 4 ) , w o u l d seem l i k e l y t o a r i s e f r o m c o n f o r m a t i o n a l changes p r i m a r i l y w i t h i n t h e s i d e c h a i n a s i t moves away f r o m i t s c l o s e a s s o c i a t i o n w i t h t h e o r d e r e d backbone e i t h e r accompanied b y , o r b e f o r e , c o n f o r m a t i o n a l changes i n t h e b a c k b o n e . T h i s s p r e a d i n g o f t h e 'arms o f t h e p o l y s a c c h a r i d e would cause a n i n c r e a s e d hydrodynamic volume and hence p r o v i d e t h e v i s c o s i t y s t a b i l i t y noted a t e l e v a t e d tempera tures (1,2,3,4). A s s o c i a t i o n , i n s o l u t i o n o f s i n g l e h e l i c e s does n o t r e q u i r e gel formation, a fact tha h e l i c a l xanthan,which doe Weak g e l a t i o n o b s e r v e d temperature probably due t o a n a g g r e g a t i o n phenomenon. I t i s i n t e r e s t i n g t o n o t e t h a t t h e 5/1 h e l i x p r e s e n t s two d i s t i n c t f a c e s ; one h a v i n g t h e s i d e c h a i n s a n d c h a r g e d g r o u p s , the o t h e r e s s e n t i a l l y t h e c e l l u l o s e backbone. As xanthan i n t e r a c t s s y n e r g i s t i c a l l y w i t h t h e 3-1,4 l i n k e d g a l a c t o m a n n a n s l o c u s t b e a n a n d g u a r gums, i t i s p o s s i b l e t h a t t h i s t a k e s p l a c e a t t h e c e l l u l o s e ' g r o o v e ' i . e . b e t w e e n s i m i l a r 8-1,4 l i n k e d g l y c a n s . I t i s t h o u g h t t h a t 'smooth' u n s u b s t i t u t e d r e g i o n s o f t h e g a l a c t o mannan a r e i n v o l v e d i n t h e a s s o c i a t i o n ( 2 3 , 2 5 ) . More d e t a i l e d i n t e r p r e t a t i o n s o f t h i s c o n t i n u i n g w o r k w i l l be p u b l i s h e d e l s e w h e r e ( 2 6 ) . 1

Acknowledgement s We w i s h t o t h a n k D r s . A. J e a n e s a n d P.A. S a n f o r d , U.S.D.A,, P e o r i a a n d D r . I.W. C o t t r e l l , K e l c o , San D i e g o , f o r t h e i r g e n e r ous g i f t s o f s a m p l e s .

Literature Cited 1. Jeanes, A. (1973) In "proceedings of the ACS Conference on Water Soluble Polymers", ed. N.M. Bikales, Plenum Press, New York. pp. 227-242. 2. Jeanes, A. (1974) J . Polymer S c i . , Symp. No. 45, 209-227. 3. McNeely, W.H. and Kang, K.S. (1973) In "Industrial Gums" R.L. Whistler and J.N. BeMiller eds., pp. 473-497, Academic Press, New York. 4. Jeanes, Α., Pittsley, J.E. and Senti, F.R. (1961) J . Appl. Polymer S c i . , 5,519-526. 5. Jeanes, A. and Pittsley, J.E. (1973) J . Appl. Polymer S c i . , 17,1621-1624. 6. Dintzis, F.R., Babcock, G.E. and Tobin, R. (1970) Carbohyd. Res. 13,257-267.


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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

EXTRACELLULAR MICROBIAL

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Jansson, P.E., Kenne, L. and Lindberg, B. (1975) Carbohyd. Res. 45,275-282. Melton, L.D., Mindt, L., Rees, D.A. and Sanderson, G.R. (1976) Carbohyd. Res., 46,245-257. Choy, Y.M. and Dutton, G.G.A. (1973) Can. J. Chem. 51,198-207. Choy, Y.M., Fehmel, F . , Frank, N. and Stirm, S. (1975) J . Virology 16,581-590. Gorin, P . A . J . , and Spencer, J.F.T. (1961) Can. J . Chem. 39, 2282-2289. Gorin, P.A.J. and Spencer, J.F.T. (1963) Can. J . Chem. 41, 2357-2361. Orentas, D.G., Sloneker, J.H. and Jeanes, A. (1963) Can. J . Microbiol., 9,427-430. Lesley, S.M. and Hochster R.M (1959) Can J Physiol 37 513-529. Arnott, S. and Scott, (1972) (Perki ) 324-335. Guss, J.M., Hukins, D.W.L., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R. and Rees, D.A. (1975) J . Mol. Biol. 95,359-384. Winter, W.T., Smith, P.J.C. and Arnott, S. (1975) J . Mol. Biol. 99,219-235. Moorhouse, R., Winter, W.T. and Arnott, S. (1976) J . Mol. Biol, in press. Smith, P.J.C. and Arnott, S. (1976) Acta Crystallogr., in press. Arnott, S. (1973) Trans. Amer. Crystallogr. Assoc., 9,31-56. Rees, D.A. (1973) In ΜΤΡ International Review of Science: Organic Chemistry Series 1, vol. 7, G.O. Aspinall, ed. 251283. Arnott, S., Fulmer, Α., Scott, W.E., Dea, I.C.M., Moorhouse, R, and Rees, D.A. (1974) J . Mol. Biol., 90,269-284. Morris, E.R. and Rees, D.A. (1976) J . Biol. Chem., in press. Holzworth, G. (1976) J . Biol. Chem., in press. Dea, I.C.M., McKinnon, A.A. and Rees, D.A. (1972) J . Mol. Biol. 68,153-172. Moorhouse, R. and Arnott, S., J . Mol. Biol., in preparation. Morris, E.R. personal communication.


8 Infrared and Raman Spectroscopy of Polysaccharides JOHN BLACKWELL Department of Macromolecular Science, Case Western Reserve University, Cleveland, OH 44106

During the last 30 years, infrared spectroscopy has been used to obtain informatio chain conformations of Raman spectra have also been available, and have provided useful complementary data. These techniques have mainly been applied in conjunction with other structural methods, especially x-ray diffraction, where the vibrational data have often given information on hydrogen bonding networks and side-group orientations. This work for polysaccharides can be discussed in two general areas. Firstly, there are the direct structural investigations, which have utilized the identifiable group frequencies. The 0-H, C-H, and carboxyl stretching frequencies, as well as some of the amide modes ,can be identified and their infrared dichroisms determined. Hence, Marrinan and Mann (1) and subsequently Liang and Marchessault (2,3) showed that the four polymorphic forms of cellulose had different spectra in the 0-H stretching region, indicative of different hydrogen bonding in their crystal structures. Based on the dichroisms of the 0-H and C-H stretching bands, these authors discussed the possibilities for hydrogen bonding and selected what they considered the most likely structures. Similarly for chitin, (4,5) the orientation of the amide side chain relative to the fiber axis was determined from the dichroisms of the amide I and II bands. Secondly, known conformations of polysaccharides can often be differentiated by their I.R. and Raman spectra. Apart from the stretching frequencies listed above, most of the bands in polysaccharide spectra are due to complex molecular motions and structural interpretation of their dichroisms is not possible at this time. Nevertheless, despite this lack of understanding, changes in frequency or intensity can be used to follow polymorphic transitions. For example, the transition from cellulose I to cellulose II during mercerization has-been followed by monitoring four intensities in the1500-800cm range (6,7). -1

103


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T h i s second aspect: i d e n t i f i c a t i o n of known conformations, i s probably the major area f o r p o t e n t i a l s t r u c t u r a l work on polysaccharides using t h i s technique. Raman spectroscopy and the r e c e n t l y developed F o u r i e r transform I.R. method, allow the s p e c t r a of polysaccharides i n s o l u t i o n t o be recorded at r e s o l u t i o n s comparable to the s o l i d s t a t e s p e c t r a . As a r e s u l t , i t i s p o s s i b l e to compare the s o l u t i o n s p e c t r a with those of known s o l i d s t a t e s t r u c t u r e s and hence a s s i g n a conformation to the p o l y saccharide i n s o l u t i o n or i n g e l s , i n a manner analogous to i d e n t i f i c a t i o n of polypeptide conformations i n s o l u t i o n using c i r c u l a r dichroism. In t h i s paper I w i l l review some of the progress we have made i n the l a s t few years i n a n a l y s i s o f a v a r i e t y of p o l y saccharide systems. Ou amylose l e d on to s t u d i e connective t i s s u e glycosaminoglycans and hence t o our present i n t e r e s t i n b a c t e r i a l polysaccharides i n s o l u t i o n . In a d d i t i o n , we have made t h e o r e t i c a l p r e d i c t i o n s of polysaccharide s p e c t r a using normal coordinate a n a l y s i s . Amylose Amylose (a(l,4)-D-glucan) i s the simplest polysaccharide which can be c r y s t a l l i z e d i n d i f f e r e n t chain conformations. P r e c i p i t a t i o n from organic s o l v e n t s leads to the s o - c a l l e d V-amylose s t r u c t u r e , (8,9) where the chains form compact h e l i c e s with s i x glucose residues per t u r n r e p e a t i n g i n 8.0Â. A v a r i e t y of chain packings are p o s s i b l e , depending on the degree of h y d r a t i o n of the presence of organic solvent molecules, but the b a s i c chain conformation i s b e l i e v e d t o be the same. When Vamylose i s maintained at high humidity f o r a p e r i o d of time, conv e r s i o n occurs to one or other of the s t r u c t u r e s found i n n a t i v e s t a r c h , A- and B-amylose, which again a r e b e l i e v e d t o be d i f f e r ent packings of a common chain conformation. The proposed conformation f o r B-amylose (10) i s a more extended 6^ h e l i x , repeating i n 10.4Â. Double h e l i c e s have a l s o been considered, t i l ) but such s t r u c t u r e s w i l l a l s o i n v o l v e more extended chains than occur i n V-amylose. The Raman spectrum o f V-amylose (12) i s shown i n Figure 1. The spectrum f o r B-amylose i s very s i m i l a r , except f o r four small but s i g n i f i c a n t d i f f e r e n c e s , which are shown i n Figure 2 : _ - l i n e s at 946 and 1263cm" f o r V-amylose s h i f t t o 936 and 1254cm~ r e s p e c t i v e l y i n the^B-form, and the r e l a t i v e i n t e n s i t i e s of l i n e s at 1334 and 2940cm are decreased with respect t o t h e i r neighbors (12). Based on our own C-H and 0-H deuterium exchange experiments, three of^the l i n e s i n question can be assigned as f o l l o w s . The 2040cm _ ^ i s probably a CH^ antisymmetric s t r e t c h i n g mode; those at 1334cm and 12£3cm are mixed -CHÔH deformation modes. For the mode at 946cm , from a study of tne s p e c t r a of glucose monomers and oligomers t h i s i s assigned as a l i n k a g e mode,


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Figure 1.

Raman spectrum of V -amylose in the region 1500-300 cm' (12) 1

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CM Carbohydrate Research Figure 2. Comparison of regions of the Raman spectra of V -, V,,-, and B-amylose. V - and V - are different hydrates of V-amylose; their spectra are identical (12). a

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i . e . a complex mode i n v o l v i n g a s i g n i f i c a n t c o n t r i b u t i o n from motion of the g l y c o s i d i c C-O-C. The V - s t r u c t u r e has compact h e l i c e s i n which residues on successive turns ( i . e . residues i and i+6) are l i n k e d by i n t e r turn hydrogen bonds i n v o l v i n g the -CHÔH s i d e chains. On conversion to the B-form, the chain becomes more extended and these i n t e r t u r n hydrogen bonds w i l l be broken. T h i s w i l l probably l e a d to a r e o r i e n t a t i o n of the s i d e chains and the formation of other hydrogen bonds, e.g. to water molecules. Such changes would be l i k e l y to a f f e c t the frequency and i n t e n s i t y of the -CHÔH modes and would account f o r the changes seen. At the same time, expansion of the chain w i l l be e f f e c t e d by r o t a t i o n of the residues about the g l y c o s i d i c l i n k a g e s , which^would f i t i n with the observed frequenc mode. In the s t r u c t u r turn bond i s broken an throug , the f i b e r repeat i s increased by r o t a t i o n of the residues about the g l y c o s e d i c bonds, which i s compatible with the observed Raman changes. Normal coordinate a n a l y s i s of the i s o l a t e d Vamylose chain p r e d i c t s complex deformation modes which are i n accord with the above assignments. (13) Increase i n the f i b e r repeat of the h e l i x to_J0.4Â reduces the frequency of the " l i n k a g e " mode by 4 cm . The above Raman c h a r a c t e r i s t i c s f o r V- and B- amylose can be used to i n t e r p r e t the s p e c t r a of t h i s polymer i n s o l u t i o n . Figure 3 shows the Raman spectrum of amylose i n deuterated DMSO.(12) Only a short region of the spectrum can be recorded, but the s p e c t r a l c h a r a c t e r i s t i c s are those of the B-form, with the observed frequency at 1254cm_^ and r e l a t i v e l y low r e l a t i v e i n t e n s i t y f o r the l i n e at 1334cm . These r e s u l t s are against the presence of the V - h e l i x i n s o l u t i o n , which i s i n t e r e s t i n g s i n c e the V - s t r u c t u r e i s formed when f i l m s are cast from t h i s solvent. This i s not to say that B - h e l i c e s are present i n s o l u t i o n s i n c e we b e l i e v e that random, s o l v a t e d amylose may show the same c h a r a c t e r i s t i c s . However, i t i s l i k e l y that the CHÔH groups are hydrogen bonded to solvent molecules r a t h e r than being involved i n i n t e r t u r n bonds on compact V - h e l i c e s . Glycosaminoglycans We are i n the process of extending t h i s type of work to the glycosaminoglycans of connective t i s s u e , each of which can be prepared as o r i e n t e d f i l m s i n a number of d i f f e r e n t chain conformations, depending on the r e l a t i v e humidity and type of counter ions. In c o l l a b o r a t i o n with E.D.T. Atkins and coworkers at U n i v e r s i t y of B r i s t o l , we have prepared c r y s t a l l i n e f i l m specimens of h y a l u r o n i c a c i d , c h o n d r o i t i n 4- and 6 - s u l f a t e s , and dermatan s u l f a t e . Raman s p e c t r a could not be obtained due to fluorescence of the specimens i n the l a s e r beam. However, using F o u r i e r transform techniques we have been able to record the


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i n f r a r e d s p e c t r a o f the same o r i e n t e d f i l m s (14) as were prepared f o r x-ray work. Figure 4 shows the p o l a r i z e d i n f r a r e d s p e c t r a of c h o n d r o i t i n 6 - s u l f a t e , prepared i n the 8^ h e l i c a l conformation. T h i s polymer approximates t o a repeating d i s a c c h a r i d e of N - a c e t y l D-galactosamine 6 - s u l f a t e and D-glucuronic a c i d , with a l t e r n a t i n g 8(1,4) and 8(1,3) l i n k a g e s , and~has e i g h t d i s a c c h a r i d e s r e p e a t i n g i n three turns, with a r i s e per residue of 9.8Â (15). The s p e c t r a i n Figure 4 show perpendicular dichroism f o r the amide I and I I modes a t 1650 and 1560cm r e s p e c t i v e l y , i n d i c a t i n g that the plane of the amide group i s approximately perpendicular t o the chain a x i s . S i m i l a r l y the antisymmetric and symmetric carboxyl s t r e t c h i n g frequencies a t 1620 and 1420cm respectively, both have s l i g h t perpendicular dichroism and the plane of the carboxyl group i s more n e a r l y perpendicular t o the c h j i n a x i s The bands with p a r a l l e complex C-0 and C-C s t r e t c h i n to that f o r c e l l u l o s e i n the same range, and i s c h a r a c t e r i s t i c of extended chain polysaccharides. We have a l s o prepared c h o n d r o i t i n 4 - s u l f a t e and dermatan s u l f a t e , each i n the 3- conformation, (16,17) and two forms of h y a l u r o n i c a c i d , both 4^ conformations with d i f f e r e n t f i b e r repeats (18,19). These give s i m i l a r r e s u l t s t o those f o r c h o n d r o i t i n 6 - s u l f a t e f o r the amide o r i e n t a t i o n . For the two forms of h y a l u r o n i c a c i d , and c h o n d r o i t i n 4 - s u l f a t e however, the carboxyl symmetric s t r e t c h i n g band has p a r a l l e l dichroism. These conformations a r e l e s s extended than the 8^ form of C6S, and the C-C0Ô bond can be o r i e n t e d so that i t i s more n e a r l y p a r a l l e l t o the chain a x i s . The same band has perpendicular dichroism f o r dermatan s u l f a t e , which i s c o n s i s t a n t with the CI chain f o r the L - i d u r o n i c a c i d residue of t h i s polysaccharide (17). So f a r we have only examined h y a l u r o n i c a c i d prepared i n two d i f f e r e n t conformations, both 4^ with d i f f e r e n t f i b e r repeats. These specimens do not show any s p e c t r a l d i f f e r e n c e s which can be a s c r i b e d t o the d i f f e r e n c e i n conformation. T h i s i s disappoint i n g , but such d i f f e r e n c e s are more l i k e l y when there are l a r g e r d i f f e r e n c e s i n conformation, e.g. between 3-, 8^, and 4- h e l i c e s . These i n v e s t i g a t i o n s are continuing, and w i l l be a p p l i e d t o s o l u t i o n s i f the d i f f e r e n t conformations can be s u c c e s s f u l l y differentiated. Bacterial

Polysaccharides

More r e c e n t l y we have examined the b a c t e r i a l polysaccharide xanthan, working with specimens obtained from Drs. A. Jeanes and P.A. Sandford at U.S.D.A., P e o r i a . T h i s polysaccharide i s b e l i e v e d t o be a repeating p o l y s a c c h a r i d e , the backbone i s a 8 ( l , 4 ) - g l u c a n with a l t e r n a t i n g residues having a t r i s a c c h a r i d e of mannose 6-acetate, g l u c u r o n i c a c i d , and mannose; approximately 50% of the t e r m i n a l mannose residues have a peruvate residue attached at the 4 and 6 p o s i t i o n s .


108


Carbohydrate Research

Figure S. Raman spectrum of amylose in deuterated DMSO solution (Me SO — d ) in the 1500-1200 cm' re gion. ( ) indicates the approximate base scattering by the 2

6

i

1

Carbohydrate Research

Figure 4.

Polarized infrared spectra for the 8 conformation of chondroitin 6-sulfate. ( )A ;(—)A (U). S

ll

1


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This p o l y s a c c h a r i d e has a very i n t e r e s t i n g property i n that the v i s i o s i t y of an aqueous s o l u t i o n undergoes a sudden i n c r e a s e as the temperature r i s e s (20). I t i s argued that the polysacchar i d e has a compact conformation at low temperatures and undergoes a t r a n s i t i o n to an expanded form at a s p e c i f i c temperature, reported at 55°C. This t r a n s i t i o n has been followed by Rees and coworkers (21) by N.M.R., which i n d i c a t e s an ordered conformation below 55°C and a d i s o r d e r e d random c o i l at higher temperatures. These workers have a l s o followed the change by c i r c u l a r dichroism spectroscopy . We have used F o u r i e r transform i n f r a r e d spectroscopy to i n v e s t i g a t e these thermal changes (22). The specimens were d i a l i z e d thoroughly against d i s t i l l e d water p r i o r to r e c o r d i n g the s p e c t r a . The s p e c t r f 1% xantha s o l u t i o t different temperatures are show seen i n the frequencie general broadening at higher temperatures, i n d i c a t i n g development of a l e s s ordered s t a t e . T h i s broadening can be quantized i n a v a r i e t y of ways; one convenient method i s to measure the areas of the peaks above the unresolved background. P l o t s of these " i n t e n s i t i e s " against temperature f o r three of the bands are shown i n Figure 6. A l l three show a sigmoidal t r a n s i t i o n , with midpoint at 40°C, i n d i c a t i n g development of a more random conformation above t h i s temperature. A d d i t i o n of s a l t s to the xanthan s o l u t i o n i s known to p r e vent the t r a n s i t i o n i n the v i s c o s i t y (20). F i g u r e 7 shows the i n f r a r e d s p e c t r a of a 1% xanthan s o l u t i o n i n 1% KC1 over the same temperature range as i n Figure 5. The c o n t r a s t between Figures"5 and 7 i s q u i t e s t r i k i n g i n that the s p e c t r a of the s a l t s o l u t i o n s show very l i t t l e change with temperature. Our observations of a t r a n s i t i o n at 40°C i s p u z z l i n g s i n c e other workers have reported 55°C. We have a l s o performed v i s c o s i t y and CD measurements on s o l u t i o n s of t h i s polysacchar i d e , and observe t r a n s i t i o n s with midpoints of 38° and 40°. I t i s p o s s i b l e that our specimen of xanthan i s d i f f e r e n t from those used by other workers, perhaps due to mutation or degradation, or that we have achieved a lower i o n i c s t r e n g t h when the specimen was d i a l y s e d against water. Acknowlegements T h i s work was supported by N.S.F. Grant No. GB 32405. am indebeted to my c o l l a b o r a t o r s i n Cleveland and B r i s t o l , e x p e c i a l l y J . J . C a e l , J . Southwick, and J.L. Koenig f o r t h e i r part i n the work described above.


I

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_J 1400

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ι

ι

of

ι

β 0 0 1200 1100 FREQUENCY (CM" )

Polysaccharides

I — 1000

1

Figure 7. Fourier transform infrared spectra of a 1% solution of xanthan in 1% aqueous potassium chloride solution at 22* 35* 45°, and 55°C (22)



112

Abstract Progress in several areas is described in the application of vibrational spectroscopy to investigate the structure and confor mation of polysaccharides. Infrared and Raman spectroscopy pro vides information on the orientation of side groups and the type of hydrogen bonds formed in crystalline polysaccharide structures. In addition, spectral characteristics of polysaccharides prepared in different known crystal structures can be used to investigate the conformation in solution. These methods have been applied to investigations of amylose, where differences in the Raman spectra of the V- and B- forms have been interpreted in terms of the change in conformation, and indicate that the V-conformation is not present in solution Fourier transform infrared spectra of oriented crystallin aminoglycans have been amide and carboxyl groups for the various crystal structures. Finally, infrared spectra of xanthan in solution show that an order-disorder transition occurs as the temperature is increased, which is correlated with the sharp increase in viscosity in the same temperature range. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Marinan, H.J. and Mann, J., J. Polymer Sci. (1958), 32, 357. Liang, C.Y. and Marchessault, R.H., J. Polymer Sci., (1959), 37, 385. Marchessault, R.H. and Liang, C.Y., J. Polymer Sci., (1960), 43, 31. Darmon, S.E. and Rudall, K.M., Disc. Farad. Soc., (1950), 9, 215. Carlstrom, D., J. Biophys. Biochem. Cytol.,(1957),3,669. McKenzie, A.W. and Higgins, H.G., Svensk Papperstidn., (1958) 61, 893. Hurtubise, F.G. and Krassig, Η., Anal. Chem., (1960), 32, 177. Rundle, R.F. and French, D., J. Amer. Chem. Soc., (1943), 65, 558. Zobel, H.F., French, A.D., and Hinkle, M.E., Biopolymers, (1967), 5, 837. Blackwell, J., Sarko, Α., and Marchessault, R.H., J. Molec. Biol., (1969), 42, 379. Kainuma, K. and French, D., Biopolymers, (1972), 11, 2241. Cael, J . J . , Koenig, J.L., and Blackwell, J., Carbohydrate res., (1973), 29, 123. Cael, J . J . , Koenig, J.L., and Blackwell, J., Biopolymers, (1975), 14, 1885. Cael, J . J . , Isaac, D.H., Blackwell, J., Koenig, J.L., Atkins, E.D.T., and Sheehan, J.K., Carbohydrate Res. in press. Arnott, S, Guss, J.M., Hukins, D.M., and Mathews, M.B., Science, (1975), 180, 743.


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16.

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Isaac, D.H. and Atkins, E.D.T., Nature (London), New Biol. (1973), 244, 252. 17. Atkins, E.D.T. and Isaac, D.H., J . Molec. B i o l . , (1973), 80, 773. 18. Dea, I.C.M., Moorhouse, R., Rees, D.A., Guss, J.M., and Balazs, E.A., Science, (1973), 179, 560. 19. Guss, J.M., Hukins, D.W., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R., and Rees, D.A., J . Molec. B i o l . , (1975), 95, 359. 20. Jeanes, Α., Pittsley, J . E . , and Senti, Α., J . Appl. Polymer S c i . , (1961), 17, 519. 21. Rees, D.A. and Morris, Ε., (in press). 22. Southwick, J., Koenig, J.L., and Blackwell, J., (in press).


9 Nuclear Magnetic Resonance and Mass Spectroscopy of Polysaccharides FRED R. SEYMOUR Baylor College of Medicine, Marrs McLean Department of Biochemistry, Houston, TX 77030

Various physical method determination have been developed. This discussion will be primarily limited to gas-liquid chromatography/mass spectrometry (g.l.c.-m.s.) and nuclear magnetic resonance spectrometry (n.m.r.). The equipment employed for these determinations has either been recently developed, or recently brought to the degree of sophistication necessary for carbohydrate studies. The procedures are rapid compared to previous methods of obtaining analogous data. Though most of the techniques may be applied to any carbohydrate containing compound, this work has been done with extra-cellular polysaccharides. The extracellular polysaccharides have proven to be valuable materials as they are readily obtainable homogeneous polymers which can be produced in relatively large quantities. These large amounts of uniform polysaccharides provide material to compare the g.l.c.-m.s. data to the much less sensitive (in terms of amount required) n.m.r. data. The wide variety of mannans and glucans available provide a considerable range of structures for this correlation. Though data is available from a number of different sources, the selected examples will be chosen from studies in which I have participated. Five general methods have been employed: a) polymer hydrolysis followed by g.l.c.-m.s., b) polymer permethylation followed by hydrolysis and then g.l.c.-m.s., c) recording the polymer's C-13 n.m.r. spectra, d) recording the polymer's P-31 n.m.r. spectra, and e) employing selective hydrolysis combined with high pressure chromatography (h.p.c.) These methods are complimentary in duplication and confirmation of data, but each method yields specific unduplicatable information. G a s - 1 i q u i d chromatography A v a r i e t y o f d e r i v a t i v e s , column p a c k i n g s , and oven

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c o n d i t i o n s have been employed f o r s a c c h a r i d e g . l . c . séparât i o n 0 ) . In o u r h a n d s , t h e p e r a c e t y l a t e d a l d o n o n i t r i l e s (PAAN) have p r o v e n t o be t h e most u s e f u l d e r i v a t i v e s . The b a s i c r e a c t i o n f o r c o n v e r s i o n o f s a c c h a r i d e s t o t h e i r PAAN d e r i v a t i v e s i s w e l l known. D i f f e r i n g g . l . c . c o n d i t i o n s have been r e p o r t e d f o r t h e s e p a r a t i o n o f PAAN d e r i v a t i v e s (_2, 3., k) . We f i n d a most s a t i s f a c t o r y s y s t e m i s t o u s e n e o p e n t y l g l y c o l s u c c i n a t e . The r e s u l t i n g n a r r o w peaks a l l o w good compound s e p a r a t i o n , t h e columns c a n e a s i l y h a n d l e t h e amounts o f m a t e r i a l r e q u i r e d f o r m.s. d e t e r m i n a t i o n s , and t h e base l i n e r e m a i n s f l a t , not a f f e c t i n g t h e h y d r o g e n f l a m e d e t e c t o r o r t h e mass s p e c t r o m e t e r . The a d v a n t a g e s o f t h e PAAN d e r i v a t i z a t i o n p r o c e d u r e a r e , a) i t i s f a s t and e f f i c i e n t , b) t h e a n o m e r i c c e n t e r o f asymmetry i s d e s t r o y e d , each s a c c h a r i d yieldin singl derivative the r e s u l t i n g s t r a i g h t - c h a i a b l e mass s p e c t r a . I t i s assumed t h a t a s u c c e s s f u l n o n - d e g r a d i n g h y d r o l y s i s has p r e c e d e d t h e s a c c h a r i d e d e r i v a t i z a t i o n and g . l . c . - m . s . determination. Carbohydrate h y d r o l y s i s i s e s s e n t i a l l y a subject i n i t s e l f , b u t i t c a n be n o t e d t h a t t h e C-13 n.m.r. d a t a p r o v i d e a u s e f u l n o n - d e s t r u c t i v e c h e c k on g . l . c . - m . s . s t r u c t u r a l determinations which hypothesize a non-degrading h y d r o l y s i s . TABLE 1 R e t e n t i o n Times o f P e r a c e t y l a t e d Aldononîtri l e D e r i v a t i v e s of Aldoses (a) Parent

Aldose

DL-glyceraldehyde D-erythrose D-digitoxose L-rhamnose 2-deoxy-D-r i bose D-ribose L-fucose D-Lyxose D-arabinose D-xylose (a)

Retention Time (min) 1.2 5.6 9.6 10.6 12.2 12.8 12.8 13.6 14.2 15.6

Parent

Aldose

Retention Time (min)

D-allose 2-deoxy-D-glucose D-mannose D-talose 2-deoxy-D-galactose D-glucose D-galactose 5-thio-D-glucose D-glucoheptose N - a c e t y 1 - D - g l u c o s a m i ne

19.0 19.2 19.6 19.6 20.2 21.0 21.8 24.6 26.8 3^.2

3% N e o p e n t y l g l y c o l s u c c i n a t e on 60/80 mesh Chromosorb W i n a p a c k e d g l a s s column 2 mm by 4 f t .

The r e t e n t i o n t i m e s o f 20 s a c c h a r i d e PAAN d e r i v a t i v e s a r e shown i n T a b l e 1. O n l y two p a i r s c a n n o t be r e s o l v e d and o n l y o n e d i f f e r s o n l y i n terms o f s t e r e o c h e m i s t r y (mannose and t a l o s e ) a n d c a n n o t be s e p a r a t e d by g . l . c . - m . s . T h i s a l l o w s o n e r e a s o n a b l e c o n f i d e n c e i n e s t a b l i s h i n g which sugars are not p r e s e n t . Partial


116


l i s t s o f t h e r e t e n t i o n t i m e s o f t h e s e PAAN d e r i v a t i v e s on d i f f e r e n t c o l u m n s ( p a c k e d L A C - 4 R - 8 8 6 (3) and open t u b u l a r S E - 3 0 (k)) i n d i c a t e t h a t t h e r e t e n t i o n t i m e o r d e r i s t h e same f o r t h e s e compounds. The r e t e n t i o n t i m e s o f t h e s e PAAN d e r i v a t i v e s a p p e a r t o be a f u n c t i o n o f t h e i n t e r a c t i o n between t h e a c e t y l g r o u p s and t h e s t a t i o n a r y p h a s e . The d e g r e e o f a c e t y l - s t a t i o n a r y p h a s e i n t e r a c t i o n i s a p p a r e n t l y d e p e n d e n t on t h e number o f a c e t y l g r o u p s p e r m o l e c u l e , and t h e a v a i l a b i l i t y o f t h e s e g r o u p s t o i n t e r a c t w i t h t h e s t a t i o n a r y phase. F o r t h e PAAN d e r i v a t i v e s o f the u n s u b s t i t u t e d s a c c h a r i d e s ( t r i o s e , t e t r o s e , pentoses, e t c . ) t h e o r d e r o f emergence o c c u r s i n g r o u p s : f i r s t triose (glycera l d e h y d e ) , then t e t r o s e s (e.g. e r y t h r o s e ) , pentoses ( e . g . r i b o s e ) , h e x o s e s ( e . g . m a n n o s e ) , and h e p t o s e ( g l u c o h e p t o s e ) PAAN d e r i v a t ives, i n a d d i t i o n , f o r t e t r o s e s t h e e r y t h r o s e PAAN d e r i v a t i v e ' s r e t e n t i o n t i m e has bee d e r i v a t i v e , f o r pentose t i m e i s s m a l l e s t and t h e x y l o s e PAAN d e r i v a t i v e ' s r e t e n t i o n t i m e i s t h e l a r g e s t C4 , 5)*. F o r each c l a s s o f s t e r e o i s o m e r s ( e . g . t h e p e n t o s e s ) t h e s t e r e o i s o m e r c o n t a i n i n g t h e g r e a t e s t number o f p a i r s o f c i s a c e t y l groups ( o r hydroxyl groups i n t h e u n d e r i v a t i z e d s u g a r ) has t h e s m a l l e s t r e t e n t i o n t i m e , and t h e s t e r e o i s o m e r c o n t a i n i n g t h e s m a l l e s t number o f p a i r s o f c i s a c e t y l g r o u p s t h e largest retention time. I t i s p o s s i b l e t h a t t h e c i s a c e t y l groups promote s a c c h a r i d e c h a i n b e n d i n g , and t h e s e l e s s l i n e a r m o l e c u l e s p r o v i d e l e s s o p p o r t u n i t y f o r a c e t y l groups t o i n t e r a c t w i t h t h e s t a t i o n a r y phase. On t h e b a s i s o f l i m i t e d d a t a , i t a p p e a r s t h a t the replacement o f a f u n c t i o n a l groups u n i f o r m l y changes t h e r e t e n t i o n time o f a s e r i e s o f stereoisomers. For example, t h e 2 - d e o x y - D - g l u c o s e and 2 - d e o x y - D - g a l a c t o s e PAAN d e r i v a t i v e s have r e t e n t i o n t i m e s o f 1 . 7 m i n u t e s l e s s t h a n t h e i r r e s p e c t i v e Dg l u c o s e and D - g a l a c t o s e PAAN d e r i v a t i v e s and t h e 6-deoxy-L-mannose and 6 - d e o x y - L - g a l a c t o s e PAAN d e r i v a t i v e s have r e t e n t i o n t i m e s 9 . 0 m i n u t e s l e s s t h a n t h e i r c o r r e s p o n d i n g mannose and g a l a c t o s e PAAN d e r i v a t i v e s . I t i s p o s s i b l e t h a t on 6-deoxy h e x o s e s u b s t i t u t i o n t h e r e m a i n i n g k a c e t y l g r o u p s have t h e same g e n e r a l a c e t y l - s t a t i o n a r y phase i n t e r a c t i o n a s t h e p e n t o s e s w i t h t h e 6 methoxy g r o u p n o t p a r t i c i p a t i n g . However, i n t h e c a s e o f 2 methoxy h e x o s e s u b s t i t u t i o n ( o r any n o n - t e r m i n a l s u b s t i t u t i o n ) t h e methylene u n i t a c t s as a c h a i n e x t e n d e r w i t h t h e a c e t y l - s t a t i o n a r y phase i n t e r a c t i o n s t i l l a p p r o x i m a t i n g t h a t o f a normal h e x o s e PAAN d e r i v a t i v e . Under t h e s t a n d a r d PAAN d e r i v a t i z a t i o n p r o c e d u r e t h e 5 ~ t h i o D - g l u c o s e r e s u l t s i n a w e l l d e f i n e d g . l . c . peak. However, m.s. shows t h a t t h i s g . l . c . peak i s n o t t h e PAAN d e r i v a t i v e , b u t t h e peracetylated 5-thio-D-glucopyranoside. N-acety1-D-glucosamine y i e l d s t h e c o r r e s p o n d i n g PAAN d e r i v a t i v e w i t h a r e t e n t i o n t i m e much l o n g e r t h a n t h e D - g l u c o s e PAAN d e r i v a t i v e — i n d i c a t i n g i n c r e a s e d N - a c e t y l i n t e r a c t i o n w i t h t h e s t a t i o n a r y phase.


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Polysaccharides

Spectrometry

The ammonia c h e m i c a l i o n i z a t i o n ( c . i . a . ) - m.s. o f t h e above PAAN d e r i v a t i v e s a r e v e r y s i m p l e . The most p r o m i n e n t , a n d u s u a l l y o n l y , m/e peaks a r e M + 18 and M - 5 9 , r e p r e s e n t i n g a d d i t i o n o f t h e ammonium i o n , o r t h e a d d i t i o n o f a p r o t o n and s u c c e s s i v e loss o f a c e t i c a c i d . The d e r i v a t i z a t i o n p r o c e d u r e i s n o r m a l l y d u p l i c a t e d , f i r s t w i t h N-15 h y d r o x y l a m i n e and t h e n w i t h p e r d e u t e r a t e d a c e t i c a n h y d r i d e . The N-15 i n t r o d u c t i o n s h i f t s M by 1 a.m.u. f o r e a c h a l d e h y d e o r i g i n a l l y p r e s e n t a n d t h e d e u t e r i u m s h i f t s M by 3 a.m.u. f o r e a c h h y d r o x y l g r o u p o r i g i n a l l y p r e s e n t . T h e r e f o r e , t h e m o l e c u l a r w e i g h t , number o f h y d r o x y l g r o u p s , and number o f a l d e h y d e g r o u p s p r e s e n t i n a l d o s e s c a n r a p i d l y be e s t a b l i s h e d The e l e c t r o n impac carbon-carbon cleavage o T h i s backbone c l e a v a g e i s e q u a l l y l i k e l y between any c a r b o n s , e x c e p t t h e C-1 and C-2 p o s i t i o n s , and d i f f e r e n t l e n g t h f r a g m e n t s are generated from both ends. The g l y c e r a l d e h y d e PAAN d e r i v a t i v e g i v e s v e r y f e w m/e f r a g m e n t s ; t h e s e same m/e a l s o a p p e a r i n t h e e r y t h r o s e PAAN s p e c t r a w i t h a new s e t o f m/e f r a g m e n t s . As t h e m o l e c u l e i s l e n g t h e n e d , more f r a g m e n t s a r e p o s s i b l e , and c o m p a r i son o f t h e s p e c t r a o f d i f f e r e n t l e n g t h m o l e c u l e s i n d i c a t e s t h e o r i g i n a l c a r b o h y d r a t e p o s i t i o n o f each fragment. The f r a g m e n t a t i o n pathways a r e a l s o i d e n t i f i e d by N-15 n i t r i l e s u b s t i t u t i o n and by d e u t e r o a c e t y l s u b s t i t u t i o n . Upon t h e s u b s t i t u t i o n o f a f u n c t i o n a l g r o u p a t a s p e c i f i c p o s i t i o n i n t h e c a r b o h y d r a t e m o l e c u l e , a l l m/e f r a g m e n t s o r i g i n a t i n g f r o m t h a t p o s i t i o n w i l l be s h i f t e d . T h e r e f o r e , t h e f u n c t i o n a l g r o u p s mass and p o s i t i o n c a n be e s t a b l i s h e d . F o r an a l d o s e PAAN d e r i v a t i v e , a c o m b i n a t i o n o f g . l . c . m.s. u s i n g c . i . a . and e . i . c a n e s t a b l i s h t h e m o l e c u l a r w e i g h t , t h e number o f a l d e h y d e and h y d r o x y l g r o u p s , t h e t y p e and p o s i t i o n o f f u n c t i o n a l g r o u p s , a n d p r o v i d e an e s t i m a t e o f t h e s t e r e o chemistry o f the molecule. Methane c h e m i c a l i o n i z a t i o n ( c . i . m . ) - m.s. o f PAAN d e r i v a t i v e s have been e x a m i n e d and t h o u g h interprétable s p e c t r a a r e o b t a i n e d f o r e a c h compound, i f t h e e . i . a . - m . s . and t h e e . i . m.s. a r e known, l i t t l e a d d i t i o n a l i n f o r m a t i o n i s o b t a i n e d . In g e n e r a l , c . i . m . mass f r a g m e n t s r e s u l t f r o m t h e p r o g r e s s i v e and e x t e n s i v e l o s s o f f u n c t i o n a l groups ( t h e 0 - a c e t y l groups a r e l o s t as a c e t i c a c i d and k e t e n e ) and t h e b a c k b o n e c h a i n r e m a i n s i n t a c t . F o r t h e PAAN d e r i v a t i v e o f N - a c e t y l g l u c o s a m i n e , t h e N - a c e t y l group i s n o t e a s i l y l o s t a n d t h e r e f o r e t h e s p e c t r u m o f t h i s compound i s v e r y s i m i l a r t o t h e c o r r e s p o n d i n g g l u c o s e PAAN d e r i v a t i v e — w i t h each m/e f r a g m e n t d e c r e a s e d by 1 a.m.u. P e r m e t h y l a t i o n G a s - L i q u i d Chromatography/Mass

Spectrometry

P o l y s a c c h a r i d e permethy1 a t i o n , f o l l o w e d by h y d r o l y s i s and


118


a n a l y s i s o f t h e r e s u l t i n g m e t h y l e t h e r s a c c h a r i d e s has t r a d i t i o n a l l y been employed f o r d e t e r m i n i n g s u g a r - s u g a r l i n k a g e t y p e and degree o f branching. The p r o c e d u r e i s s i m p l e i n c o n c e p t , b u t has proven d i f f i c u l t t o apply. T h i s i s due t o t h e d i f f i c u l t y o f i d e n t i f y i n g and q u a n t i t a t i n g t h e r e a c t i o n p r o d u c t s , o r t h e i r d e r i v a t i v e s , and has been used i n c o n j u n c t i o n w i t h p e r a c e t y l a t e d c y c l i c derivatives or the peracetylated a l d i t o l s . Lance and J o n e s (6) s e p a r a t e d m e t h y l e t h e r s o f x y l o s e PAAN compounds and Dmitrîev e t a l . (2) r e p o r t e d t h e m a j o r m/e f r a g m e n t s o f t h e e . i . m.s. o f s e l e c t e d PAAN d e r i v a t i v e s . The t e t r a - 0 , t r i - 0 , and dî-0-methyl e t h e r s o f D-mannopyranoside were s y n t h e s i z e d and g . l . c . c o n d i t i o n s f o u n d f o r t h e s e p a r a t i o n o f t h e s e compounds ( 7 ) . T h i s g . l . c . s e p a r a t i o n , combined w i t h p r e v i o u s l y developed e f f i c i e n t m e t h y l a t i o d h y d r o l y s i methods allowed the r a p i d p e r m e t h y l a t i o i d e n t i t y o f t h e g . l . c . peak m.s. The a v a i l a b i l i t y o f t h e p u r e m e t h y l e t h e r s o f m e t h y l a-Dm a n n o p y r a n o s i d e and t h e e s t a b l i s h m e n t o f a g . l . c . column c a p a b l e o f r e s o l v i n g t h e PAAN d e r i v a t i v e s , a l l o w e d a p r e c i s e d e t e r m i n a t i o n o f t h e f r a g m e n t a t i o n pathways. Di-O-methyl d e r i v a t i v e s o f m e t h y l α-D-mannopyranoside were s u b j e c t e d t o random p a r t i a l m e t h y l a t i o n and t h e r e s u l t i n g r e a c t i o n m i x t u r e h y d r o l y z e d and d e r i v a t i z e d t o PAAN d e r i v a t i v e s . On g . l . c . a n a l y s i s t h e s e m i x t u r e s gave a s e r i e s o f p e a k s , t h e r e t e n t i o n t i m e s i n d i c a t i n g t h e p o s i t i o n o f t h e c o m b i n e d m e t h y l - 0 - and deuteromethy1-0- e t h e r groups. The p o s i t i o n o f new e t h e r g r o u p s i d e n t i f i e d t h e s e a s d e u t e r o m e t h y 1 g r o u p s , and on c o m p a r i s o n o f t h e e . i . - m . s . o f t h e s e compounds t o t h e e . i . - m . s . o f t h e c o r r e s p o n d i n g n o n - i s o t o p i c a l l y s u b s t i t u t e d PAAN d e r i v a t i v e s , t h e o r i g e n o f each m/e f r a g m e n t c o u l d be e s t a b l i s h e d ( 7 ) . The e x t e n s i v e k n o w l e d g e o f g . l . c . - m . s . a l l o w e d t h e s t r u c t u r e o f s i x t e e n mannans t o be e s t a b l i s h e d i n terms o f l i n k a g e t y p e and d e g r e e o f b r a n c h i n g . T h i s method y i e l d e d e s s e n t i a l l y i d e n t i c a l d a t a f o r s e v e r a l o f t h e mannans. S i x c l a s s e s o f mannans w e r e o b s e r v e d , t h e s e p o l y s a c c h a r i d e s d i f f e r i n g i n b o t h l i n k a g e t y p e s and d e g r e e o f b r a n c h i n g . T h i s d a t a i s summarized i n T a b l e 2. The d a t a i n T a b l e 2 a l l o w s t h e c o n s t r u c t i o n o f an a v e r a g e r e p e a t i n g u n i t f o r each polymer c l a s s . F o r e x a m p l e , D-mannan p r o d u c e d by P a c h y s o l e n t a n n o p h i l u s Y-2460 c a n be e x p r e s s e d a s :

-{Μ - 0 - * ) } -

v

I 3 f

I M - (1+2) - M - (1+2) - M


9.

SEYMOUR

NMR

and MS of

119

Polysaccharides

TABLE 2 M o l e P e r c e n t a g e o f M e t h y l a t e d D-mannose Components i n H y d r o s y l a t e s o f P e r m e t h y l a t e d D-mannans ( a ) NRRL Number

M e t h y l e t h e r s o f D-mannose 2,3,4,6-•

3,4,6-

2.5 3.9 26.4 19.2 27.7 22.8

51.1 48.3 42.9 54.1 20.4

Y-1842 Y-2448 Y-2460 Y-2023 YB-2097 YB-1344 (a)

Data t a k e n

2,4,6-

2,3,444.3

44.0

2.1 9.7

25.2

3,42.0 3.9 16.9

2,4-

26.1 23 8

from r e f e r e n c e 8.

As a f i r s t a p p r o x i m a t i o n , i t was assumed t h a t t h e (1+6)l i n k a g e s were e x c l u s i v e l y c o n f i n e d t o t h e mannan b a c k b o n e c h a i n . T h i s a s s u m p t i o n was l a t e r t e s t e d by a c e t o l y s i s ( s e e b e l o w ) . T h i s g . l . c . - m . s . t e c h n i q u e was t h e n a p p l i e d t o g l u c a n s . The g e n e r a t i o n o f r e f e r e n c e compounds was n o t n e c e s s a r y . The mass s p e c t r a a r e n o t a f f e c t e d by s t e r e o c h e m i s t r y c h a n g e s , and t h e c o r r e s p o n d i n g mannose and g l u c o s e m e t h y l - e t h e r s y i e l d i d e n t i c a l s p e c t r a . T h e r e f o r e , each g l u c o s e m e t h y l e t h e r PAAN d e r i v a t i v e g . l . c . peak c o u l d be i d e n t i f i e d by c o m p a r i s o n t o t h e known mannose compounds. On t h e b u t a n e d i o l s u c c i n a t e columns employed f o r m e t h y l - e t h e r s a c c h a r i d e PAAN s e p a r a t i o n t h e r e t e n t i o n times a r e g e n e r a l l y , but not n e c e s s a r i l y , d i f f e r e n t f o r c o r r e s p o n d i n g g l u c o s e and mannose compounds. A g r o u p o f d e x t r a n s , p r e v i o u s l y suspected o f c o n t a i n i n g unusual s t r u c t u r a l f e a t u r e s , was a n a l y z e d by g . l . c . - m . s . and t h e r e s u l t s (9) a r e summarized i n T a b l e 3 . The d a t a i n T a b l e 3 a g a i n a l l o w s t h e c o n s t r u c t i o n o f average r e p e a t i n g u n i t s f o r t h e v a r i o u s p o l y s a c c h a r i d e s . F o r e x a m p l e , f r a c t i o n L o f t h e d e x t r a n p r o d u c e d by L e u c o n o s t o c m e s e n t e r o d i e s NRRL Β-1299 c a n be e x p r e s s e d a s h a v i n g a g e n e r a l repeating unit o f : -{G

- (1+6) - G -

(1+6)}-

I G where "G" i s t h e D - g l u c o p y r a n o s i d e

unit.


EXTRACELLULAR

120


TABLE 3 M o l e p e r c e n t a g e s o f M e t h y l a t e d D - g l u c o s e Components i n H y d r o l y z a t e s o f P e r m e t h y l a t e d Dextrans (a) Methyl e t h e r s o f D-glucose

NRRL

Number (b) 2 , 3 , 4 , 6 B-1351 Β-1399 6-1254 Β-1299 B-1355 (a) (b)

S L L S S

5.8 12.8 22.1 39.1 6.9

2,3,483.3 74.5 55.0 26.0 46.9

2,3,6-

2,4,6-

2,3-

2,410.5 5.9

3.4

3,40.3 6.8

19.5 35.0

Data t a k e n f r o m r e f e r e n c e 9 . The d e x t r a n p r o d u c i n g NRRL s t r a i n number. polymer f r a c t i o n s .

11.2

34.9

S and L r e f e r t o

A t t h i s p o i n t i t w i l l be seen t h a t g . l . c . - m . s . has been used t o c o n f i r m t h e u n i q u e p r e s e n c e o f g l u c o s e o r mannose a s t h e a l d o s e u n i t o f a s e r i e s o f d e x t r a n s and a s e r i e s o f mannans. In c o n j u n c t i o n w i t h p e r m e t h l y l a t i o n , g . l . c . - m . s . has been employed f o r e s t a b l i s h i n g t h e g e n e r a l r e p e a t i n g u n i t f o r t h e s e d e x t r a n s and D-mannans. A number o f D-mannans, n o t l i s t e d i n T a b l e 2, were shown t o have e s s e n t i a l l y i d e n t i c a l l i n k a g e t y p e s t o t h o s e shown. I t i s p o s s i b l e , i n p r i n c i p l e , t o perform t h e s e o p e r a t i o n s on a s u b - m i l i g r a m b a s i s . For ease o f m a t e r i a l s h a n d l i n g , a f e w mg o f e a c h p o l y m e r were employed f o r t h e mannan d e t e r m i n a t i o n s , and due t o i n c r e a s e d permethy1 a t i o n d i f f i c u l t y , a p p r o x i m a t e l y 10 t o 15 mg o f d e x t r a n s w e r e u s e d . T h i s data then provided t h e b a s i s f o r comparison w i t h t h e remaining t e c h n i q u e s , w h i c h r e q u i r e l a r g e r amounts o f m a t e r i a l . High-Pressure

Chromatography

P r e v i o u s work has shown t h a t a c e t o l y s i s o f mannans r e s u l t s i n s e l e c t i v e h y d r o l y s i s , w i t h t h e ( 1 + 6 ) - 1 i n k a g e s c l e a v e d much more r a p i d l y t h a n o t h e r s u g a r - s u g a r l i n k a g e s ( 1 0 ) . E m p l o y i n g t h i s s e l e c t i v e h y d r o l y s i s , f o l l o w e d by a d e a c e t y l a t i o n s t e p , yielded a mixture o f oligosaccharides. I t had p r e v i o u s l y p r o v e n p o s s i b l e t o employ h.p.c. t o s e p a r a t e a m i x t u r e o f oligosaccharides. By c a l i b r a t i n g t h e s y s t e m a g a i n s t known o l i g o s a c c h a r i d e s , t h e r e t e n t i o n t i m e s and d e t e c t o r r e s p o n s e s c o u l d be e s t a b l i s h e d . The h.p.c. s y s t e m was t h e n employed t o s e p a r a t e and q u a n t i t a t e t h e a c e t o l y s i s o l i g o s a c c h a r i d e s a c c o r d i n g to degree o f p o l y m e r i z a t i o n ( d . p . ) . An e x a m p l e o f t h i s d a t a f o r t h e mannan o f P a c h y s o l e n t a n n o p h i l u s , NRRL Y-2460 i s shown i n the f o l l o w i n g t a b l e .


9.

SEYMOUR

NMR

and MS of Polysaccharides

121

TABLE 4 O l i g o s a c c h a r i d e s f r o m A c e t o l y s i s o f NRRL Y - 2 4 6 0 D-mannan Degree o f Polymerizat ion Mole

ratio

1 10.0

2

3 3.7

4

3.6

11.9

5

6

7

8

1.3

0.7

1.0

0.9

T h i s d a t a i s a n a l y i z e d by making two b a s i c a s s u m p t i o n s , a) o n l y t h e ( 1 + 6 ) - 1 i n k a g e s have been b r o k e n , a n d b) a l l t h e ( l + 6 ) - l i n k ages a r e i n t h e c a r b o h y d r a t e b a c k b o n e . If this iscorrect, t h e n s e q u e n c e s o f ( 1 + 6 ) - 1 i n k a g e s w i l l y i e l d monomers and t h e s i d e c h a i n s w i l l remai backbone s a c c h a r i d e u n i t r e p r e s e n t a s i d e c h a i n non-reducing end group, a b r a n c h i n g end g r o u p , and non-(1+6)-1 i n k e d s a c c h a r i d e s . T h i s d a t a may t h e n be a n a l y z e d t o y i e l d an a v e r a g e r e p e a t i n g u n i t w h i c h c a n be e x p r e s s e d i n t e r m s o f m e t h y l e t h e r s -- an e x a m p l e i s shown b e l o w . TABLE 5 C o r r e l a t i o n o f M e t h y l a t i o n and A c e t o l y s i s Data f o r NRRL Y-2460 D-mannan ( a ) Data S o u r c e

C a l c u l a t e d percentages Tetra

Methylation

26.4

Acetolys i s

23.0

(a)

Data t a k e n

2,3,4-Tri

2.1 10.1

o f methyl e t h e r s

Non2,3, 4 - T r i

Di

42.9

26.1

44.5

23.0

from r e f e r e n c e 8.

I t c a n be seen t h a t t h e a c e t o l y s i s d a t a c l o s e l y p a r a l l e l s the m e t h y l a t i o n data. The m a j o r d i s c r e p a n c y i s t h e amount o f ( l + 6 ) - l i n k a g e s ( 2 , 3 , 4 - t r i - 0 - m e t h y l e t h e r ) as a c e t o l y s i s g e n e r a l l y g i v e s a h i g h e r v a l u e than m e t h y l a t i o n . The (1+6)l i n k a g e a c e t o l y s i s v a l u e comes f r o m t h e amount o f monomeric u n i t s o b s e r v e d by h . p . c , a n d i t i s p o s s i b l e t h a t n o n - ( l + 6 ) l i n k a g e c l e a v a g e c o n t r i b u t e s t o i n c r e a s e t h i s v a l u e . Two general r e s u l t s a r e o b t a i n e d from comparison o f t h i s d a t a : f i r s t l y , the assumption that the (1+6)-1inkages a r e confined t o t h e backbone i s c o n f i r m e d ; s e c o n d l y , .there a p p e a r s t o be a d i s t r i b u t i o n o f s i d e c h a i n lengths around t h e average o f t h r e e s a c c h a r i d e u n i t s p e r s i d e c h a i n . Good agreement between a c e t o l y s i s and m e t h y l a t i o n d a t a was o b t a i n e d f o r f o u r o f t h e s i x mannan t y p e s s t u d i e d . Mannan Y-1842 showed g r e a t d i f f e r -


122


POLYSACCHARIDES

e n c e s i n d e g r e e o f b r a n c h i n g a s d e t e r m i n e d by m e t h y l a t i o n a n d acetolysis. The d a t a s u g g e s t t h a t a l a r g e number o f ( 1 + 6 ) l i n k a g e s must o c c u r i n t h e s i d e c h a i n s . For p o l y s a c c h a r i d e s c o n t a i n i n g ( 1 + 6 ) - l i n k e d s a c c h a r i d e s , t h e c o r r e l a t i o n o f m e t h y l a t i o n and a c e t o l y s i s d a t a i s a u s e f u l method to e s t a b l i s h t h e p o s i t i o n o f t h e (1+6)-1inkages. I f t h e s e (1+6)1 i n k a g e s f o r m t h e backbone c h a i n , t h e s i d e c h a i n l e n g t h d i s t r i b u t i o n c a n be e s t a b l i s h e d . C-13 nurn.jr.

Spectroscopy

This technique i sa l o g i c a l step a f t e r the c o n s t i t u e n t s u g a r s , l i n k a g e t y p e s , and d e g r e e o f b r a n c h i n g have been e s t a b l i s h e d by g . l . c . - m . s . I principle h carbo i different chemical environment w i l C-13 s p e c t r a l r e g i o n . Th -13 s h i f t s i s d e p e n d e n t on t h e m a g n e t i c f i e l d s t r e n g t h . When compared t o H-1 n.m.r., C - 1 3 n.m.r. g i v e s much b e t t e r s e p a r a t i o n s i n an equivalent f i e l d . In a d d i t i o n , due t o improved r e l a x a t i o n t i m e s , C-13 n.m.r. c a n g i v e q u i t e s h a r p s i g n a l s f o r l a r g e p o l y m e r s . It has been e s t a b l i s h e d t h a t s i m p l e s a c c h a r i d e s ( e . g . m e t h y l a-Dg l u c o s e ) w i l l y i e l d s i x C-13 n.m.r. s a c c h a r i d e p e a k s ; t h e a n o m e r i c c a r b o n i n t h e 95 t o 105 ppm ( r e l a t i v e t o TMS) r e g i o n , t h e C - 2 , C - 3 , C - 4 , and C-5 peaks i n t h e 7 0 - 7 5 ppm r e g i o n , and t h e C-6 peak a t a p p r o x i m a t e l y 60 ppm ( 1 1 ) . On c o n v e r s i o n o f a h y d r o x y l g r o u p t o an a l k y l e t h e r g r o u p , t h e c h e m i c a l s h i f t o f t h e c o r r e s p o n d i n g s a c c h a r i d e carbon i s s h i f t e d d o w n f i e l d ( t o l a r g e r ppm v a l u e s ) . F o r m e t h y l e t h e r f o r m a t i o n t h i s change i n c h e m i c a l s h i f t has been shown t o be a u n i f o r m 10 ppm d o w n f i e l d s h i f t f o r each s a c c h a r i d e carbon p o s i t i o n (12). T h e r e f o r e , a c o n v e n i e n t approach t o p o l y s a c c h a r i d e a n a l y s i s i s t o c o n s i d e r t h e polymer as an agrégation o f i n d e p e n d e n t a l k y l e t h e r m o n o s a c c h a r i d e s . F o r e x a m p l e , m e t h y l a t i o n d a t a ( T a b l e 3) and t h e i m p l i e d g e n e r a l r e p e a t i n g u n i t have been p r e s e n t e d f o r d e x t r a n B-1299 f r a c t i o n S. F o r p u r p o s e s o f C - 1 3 n.m.r. t h i s p r o p o s e d b a s i c u n i t may be c o n s i d e r e d as e q u i v a l e n t t o an e q u a l m o l a r m i x t u r e o f m e t h y l Dm a n n o p y r a n o s i d e ( t h e end g r o u p ) , m e t h y l 2 , 6 - d i - 0 - m e t h y l - D - m a n n o p y r a n o s i d e ( t h e b r a n c h i n g g r o u p ) , and m e t h y l 6-0-methy1-D-mannop y r a n o s i d e ( t h e backbone e x t e n d i n g g r o u p ) . For three saccharides, a maximum o f e i g h t e e n (6x3) s a c c h a r i d e C-13 c h e m i c a l s h i f t s c o u l d be o b s e r v e d . Two l i m i t a t i o n s o f C - 1 3 n.m.r. s h o u l d be r e c o g n i z e d . First, t h e r e l a t i v e l y low s e n s i t i v i t y o f t h e C - 1 3 n u c l e i r e q u i r e s l a r g e s a m p l e s (100 t o 200 mg) w i t h F o u r i e r t r a n s f o r m d a t a p r o c e s s i n g . S m a l l e r s a m p l e s may be u s e d , b u t t h e d a t a a c q u i s i t i o n t i m e s t e a d i l y i n c r e a s e s . Secondly, t h e s i g n a l i n t e n s i t y o f each c l a s s o f c a r b o n n u c l e i i s n o t d e p e n d e n t on t h e t o t a l number o f e a c h species present. C-13 n u c l e i w i t h g r e a t e r d e g r e e s o f f r e e d o m of motion y i e l d l a r g e r s i g n a l s . However, i t has p r e v i o u s l y been shown t h a t f o r s a c c h a r i d e s , t h e c o n t r i b u t i o n o f e a c h c a r b o n


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9. SEYMOUR

123

s p e c i e s t o t h e C - 1 3 n.m.r. s p e c t r a i s a p p r o x i m a t e l y e q u a l ( 1 3 ) » F o r t h e C - 1 3 n.m.r. s p e c t r a o f d e x t r a n Β-1299 f r a c t i o n S ( F i g . 1 ) , and a n a l a g o u s d e x t r a n s p e c t r a , s e v e r a l p o i n t s become apparent. For the dextrans d e s c r i b e d i n Table 3 , a s e r i e s o f s i x c h e m i c a l s h i f t s ( d e s i g n a t e d a s A t h r o u g h F) a r e p r e s e n t i n each spectrum. T h e s e s i x peaks d o m i n a t e t h e s p e c t r a o f t h e more l i n e a r α-linked d e x t r a n s and r e p r e s e n t t h e c o n t r i b u t i o n o f a methyl 6 - 0 - m e t h y l - D - g l u c o p y r a n o s i d e analog. As p o l y m e r s w i t h g r e a t e r d e g r e e o f b r a n c h i n g were e x a m i n e d , t h e c o n t r i b u t i o n o f t h e o r i g i n a l s i x peaks d e c r e a s e d and o t h e r c h e m i c a l s h i f t s become prominent (14). B-1299 f r a c t i o n S d e x t r a n ( F i g . 1) p r o v i d e s an e x a m p l e o f a h i g h l y b r a n c h e d d e x t r a n w i t h t h e c o n t r i b u t i o n o f t h e o r i g i n a l s i x peaks i n d i c a t e d by l e t t e r s A t h r o u g h F. The 7 0 - 7 5 ppm r e g i o n (B t h r o u g s h i f t s , but o n l y seven a r detection. A p p a r e n t l y a number o f t h e s e c h e m i c a l s h i f t s a r e n o t resolved. In t h e a n o m e r i c r e g i o n t h e e x p e c t e d t h r e e peaks a r e o b s e r v e d , t h e d o w n f i e l d peak r e p r e s e n t i n g t h e ( 1 + 6 ) - 1 i n k e d u n i t . A l l a n o m e r i c p r o t o n s a r e l o c a t e d i n t h e 9 6 t o 101 ppm r e g i o n , d e m o n s t r a t i n g t h a t each o f the observed l i n k a g e s i s a. F o r l i n e a r d e x t r a n t h e 7 5 * 8 5 ppm r e g i o n d i s p l a y s no c h e m i c a l shifts. For dextrans c o n t a i n i n g 1+2, 1+3, o r 1+4-1inkages (as d e m o n s t r a t e d by g . l . c . - m . s . ) t h e 7 5 - 8 5 ppm r e g i o n c o n t a i n s t h e g l y c o s y l l i n k e d c a r b o n s ( C - 2 , C - 3 , o r C - 4 ) w h i c h upon s u b s t i t u t i o n have had t h e i r c h e m i c a l s h i f t s moved d o w n f i e l d f r o m t h e 7 0 - 7 5 ppm r e g i o n . We have o b s e r v e d a c h e m i c a l s h i f t o f 7 6 . 5 ppm f o r a - ( 1 + 2 ) - 1 i n k a g e s , 7 9 . 5 ppm f o r a - ( 1 + 3 ) - 1 i n k a g e s , and 8 1 . 6 ppm f o r a-(1+4)-1inkages (14). T h e o n l y c h e m i c a l s h i f t i n t h e 7 5 - 8 5 ppm

C

Ε

Ε

C

J

78

58

69 I05

PPM Figure 1. C-13 NMR spectra of dextran B-1299 fraction S recorded at 27° in D 0; ppm relative to TMS. Inset (78-69 ppm) recorded at 70°. 2


124


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r e g i o n o f t h e d e x t r a n Β-1299 f r a c t i o n S s p e c t r a i s a t 76.42 ppm, i n agreement w i t h t h e g . l . c . - m . s . d a t a . T h i s n.m.r. method t h e r e f o r e a l l o w s i d e n t i f i c a t i o n and rough q u a n t i t a t i o n o f t h e l i n k a g e t y p e s p r e s e n t by a n o n - d e s t r u c t i v e t e c h n i q u e n o t d e p e n d e n t on h y d r o l y s î s. In g e n e r a l , t h e d e x t r a n C-13 n.m.r. s p e c t r a w e r e r e l a t i v e l y simple, t h i s being e s p e c i a l l y n o t i c a b l e i n the anomeric region. A p r e v i o u s C-13 n.m.r. s t u d y o f p u l u l l a n s o b s e r v e d t h r e e w e l l d e f i n e d a n o m e r i c c h e m i c a l s h i f t s and employed t h i s a s a v e r y p l a u s i b l e argument f o r t h e o r d e r e d r e p e a t i n g - (1+4) - (1+6) - (1+4) g l u c o p y r a n o s i d e s u b u n i t (15)» Many o f t h e s e d e x t r a n s a l s o show s i m p l e C - 1 3 n.m.r. s p e c t r a w h i c h i n t u r n i m p l i e s a b a s i c o r d e r e d repeating sub-unit. Another point o f i n t e r e s i C-13 n.m.r. s p e c t r a on p o l y s a c c h a r i d e s had b r o a peak "sharpened y r a i s i n g t h e t e m p e r a t u r e . A h i g h t e m p e r a t u r e (70 ) i n s e t o f t h e 7 0 - 7 5 ppm r e g i o n i s shown i n F i g u r e 1 . The g e n e r a l s p e c t r u m p r o f i l e r e m a i n s t h e same, b u t e a c h peak i s n a r r o w e r . A number o f t e m p e r a t u r e d e p e n d e n t e f f e c t s were o b s e r v e d , t h e most i n t e r e s t i n g b e i n g t h a t a l l o f t h e c h e m i c a l s h i f t s w e r e tempe r a t u r e d e p e n d e n t , moving d o w n f i e l d on i n c r e a s i n g t e m p e r a t u r e . In a d d i t i o n , d i f f e r e n t c h e m i c a l s h i f t s d i s p l a y e d d i f f e r e n t t e m p e r a t u r e d e p e n d e n c i e s , i n t h e r a n g e o f Δδ/ΔΤ o f 0.01 t o 0 . 0 3 ppm/C° ( r e l a t i v e t o TMS). T h e s e d i f f e r e n t Δδ/ΔΤ e x c l u d e b u l k m a g n e t i c s u s c e p t i b i l i t y as t h e m a j o r f a c t o r and s u g g e s t t h a t t h e m a g n i t u d e o f Δδ/ΔΤ i s s t r u c t u r e r e l a t e d . In f a c t , t h e l a r g e s t Δδ/ΔΤ o b s e r v e d a r e g e n e r a l l y a s s o c i a t e d w i t h c a r b o n s involved i n sugar-sugar linkages (14). I t i s n e c e s s a r y t o c o n s i d e r t h e Δδ/ΔΤ e f f e c t , e s p e c i a l l y when c o m p a r i n g C - 1 3 n.m.r. c a r b o h y d r a t e s p e c t r a i n t h e c l o s e l y packed 7 0 - 7 5 ppm r e g i o n . The c h e m i c a l s h i f t s o f t h e C - 2 , C - 3 , C-4, and C-5 carbons f a l l i n g i n t h i s r e g i o n a r e a p p a r e n t l y d i a g n o s t i c f o r s a c c h a r i d e s o f s p e c i f i c l i n k a g e t y p e s ; however, a t e m p e r a t u r e change o f 50 c a n c a u s e a r e s o n a n c e change s o g r e a t as t o a l l o w c h e m i c a l s h i f t s t o i n t e r c h a n g e p o s i t i o n s . P-31 n_.m.£.

Spectroscopy

A d d i t i o n a l n.m.r. d a t a has been g e n e r a t e d by e m p l o y i n g a F o u r i e r t r a n s f o r m n.m.r. w i t h a P-31 p r o b e . The P-31 n u c l e i a r e r e l a t i v e l y i n s e n s i t i v e t o n.m.r. a n d , a s w i t h C - 1 3 s t u d i e s , l a r g e r amounts o f c a r b o h y d r a t e s were n e c e s s a r y . A variety of e x t r a c e l l u l a r y e a s t 0 - p h o s p h o n o h e x o s a n s were a v a i l a b l e f o r study. T h e s e compounds c a n be d i v i d e d , on a c h e m i c a l b a s i s , i n t o two g r o u p s ( 1 6 ) . Type I i s e x e m p l i f i e d by p o l y ( p h o s p h o r i c d i e s t e r s ) o f D-mannose o l i g o s a c c h a r i d e s . Type I I a r e p o l y s a c c h a r i d e s i n which t h e g l y c o s y l phosphate residues occur as n o n - r e d u c i n g e n d - g r o u p s — e i t h e r a s D-mannose, D - g l u c o s e , o r as d i s a c c h a r i d e s . Many o f t h e mannans and p h o s p h o n o h e x o g l u c a n s


9.

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NMR and M S of Polysaccharides

125

a r e r e l a t e d i n s o f a r a s t h e y a r e p r o d u c e d by t h e same y e a s t s t r a i n u n d e r d i f f e r i n g amounts o f o r t h o p h o s p h a t e i n t h e c u l t u r e m e d i a . The P-31 n.m.r. s i g n a l s f r o m t h e s e O-phosphonohexosans w e r e q u i t e sharp. The n a t i v e polymers a p p a r e n t l y c o n t a i n a l l phosphate g r o u p s a s t h e d i e s t e r , but i s o l a t i o n p r o c e d u r e s can r e s u l t i n p a r t i a l h y d r o l y s i s t o the mono-ester. P-31 n.m.r. p r o v i d e s an e x c e l l e n t method o f s u r v e y i n g f o r t h i s h y d r o l y s i s a s t h e n.m.r. s i g n a l s o f t h e m o n o - e s t e r and t h e d i - e s t e r p h o s p h a t e s a r e w i d e l y s p a c e d , the m o n o - e s t e r f a l l i n g a t a p p r o x i m a t e l y -4. ppm ( r e l a t i v e t o 85% o r t h o p h o s p h o r i c a c i d ) . In g e n e r a l , e a c h O - p h o s p h o n o h e x o g l y c a n gave a s i n g l e s h a r p P-31 s i g n a l , t h e c h e m i c a l s h i f t b e i n g u n i q u e f o r e a c h p o l y m e r . The two t y p e s o f O - p h o s p h o h e x o g l y e a n s can be r e p r e s e n t e d a s : (

M -

M -

M -

Ρ -

Μ )

χ

G

Type I

Type I I

Where M r e p r e s e n t s a m a n n o p y r a n o s i d e u n i t , Ρ r e p r e s e n t s a p h o s p h o d i e s t e r u n i t , and G r e p r e s e n t s a n o n - r e d u c i n g mannose, glucose, o rdisaccharide unit. Though e a c h O-phosphonomannan s t u d i e d has d i s p l a y e d a d i f f e r e n t P-31 c h e m i c a l s h i f t , t h e r e i s no o b v i o u s d i f f e r e n c e between Type I and Type II P-31 n.m.r. s p e c t r a ( s e e T a b l e 6 ) . TABLE 6 P-31 C h e m i c a l S h i f t s f o r O-phosphonomannans and M a t e r i a l s (a) Anomeric sugar phosphate

mannose M

mannose ·' glucose n

galactose

NRRL p r o d u c i n g strain

Related

Orthophosphate d i e s t e r chemical s h i f t

Type I Y-1842 YB-1443 Y-2448 Y-2461

1.94 1.74 1.84 1.72

Type I I Y-411 YB-2079 YB-2194 Y-2579 Y-2023 Y-6493

I.78 1.74 and 1.90 1.07 1.16 1.10 and 1.28 1.06


126


(a) d a t a

în T a b l e 6 t a k e n

from reference

17.

However, T a b l e 6 does show t h a t a s i g n i f i c a n t change i n t h e c h e m i c a l s h i f t o c c u r s when t h e a n o m e r i c s u g a r p h o s p h a t e i s g l u c o s e ( a t a p p r o x . 1.1 ppm) r a t h e r t h a n mannose ( a t a p p r o x 1.8 ppm). The a n o m e r i c s u g a r p h o s p h a t e i s b e l i e v e d t o be α-linked i n a l l c a s e s and t h e changes i n c h e m i c a l s h i f t a r e t h e r e s u l t o f s t e r e o i s o m e r e f f e c t s a t t h e d i s t a n c e o f s e v e r a l atoms f r o m t h e p h o s p h o r u s atom. R e l a t i v e l y s u b t l e changes i n p o l y m e r s t r u c t u r e a r e r e f l e c t e d i n t h e P-31 c h e m i c a l s h i f t v a l u e s . P o l y m e r s Y-2448 (δ = 1.84 ppm) and Y-1842 (6 = 1.74 ppm) a r e a p p a r e n t l y s t r u c t u r a l l y i d e n t i c a l e x c e p t t h a t f o r Y-1842 t h e s u g a r s a r e a - 1 i n k e d ( n o t t h e s u g a r p h o s p h a t e a n o m e r i c l i n k a g e ) and f o Y-2448 t h e s u g a r s a r e 3-1 i n k e d . O-Phosphonomanna di-saccharide residues a l s For e x a m p l e , i n p o l y m e r s f r o m YB-2097 and Y-2023, where b o t h t y p e s o f r e s i d u e a r e p r e s e n t , two d i e s t e r r e s o n a n c e s a r e observed. In YB-2097 O-phosphonomannan t h e mannose 6 - p h o s p h a t e r e s i d u e s a r e i n a n o m e r i c l i n k a g e w i t h r e s i d u e s o f mannopyranose and 6-0-a-D mannopyranosy1-D-mannopyranose, i n Y-2023 0-phosphomannan t h e l i n k a g e i s t o r e s i d u e s o f D - g l u c o p y r a n o s e and 2-0α-D-mannopyranosy1-D-glucopyranose. P r o t o n - c o u p l e d s p e c t r a f o r t h e d i e s t e r s showed q u a r t e t p a t t e r n s t h a t c o u l d be a n a l y z e d by c o m p u t e r s i m u l a t i o n t o o b t a i n the c o u p l i n g - c o n s t a n t s . These data are i n accord w i t h the i n t e r p r e t a t i o n t h a t most o f t h e l i n k a g e s i n t h e 0-phosphonomannans a r e o f t h e D-mannopyranose 6-(D-mannopyranosy1 p h o s p h a t e ) type. Conclus ions Examples have been p r e s e n t e d t o d e m o n s t r a t e how v a r i o u s forms o f g . l . c , h . p . c , m.s., and n.m.r. a r e employed i n e x t r a c e l l u l a r polysaccharide structure determination. These s t r u c t u r a l d e t e r m i n a t i o n s have p r o v e d f r u i t f u l i n p r o v i d i n g i n s i g h t s i n t o the r e l a t i o n s h i p of a wide v a r i e t y o f e x t r a c e l l u l a r polysaccharides. In t u r n , t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e s have p r o v i d e d m a t e r i a l s t o c o r r e l a t e t h e v a r i o u s s t r u c t u r a l d e t e r m i n a t i o n t e c h n i q u e s , and t h e s e methods may now be a p p l i e d t o more c o m p l e x s a c c h a r i d e c o n t a i n i n g p o l y m e r s .

Literature cited 1. 2.

Dutton, G.G.S., Advan. Carbohyd. Chem. Biochem., (1974) 30, 9-110. Dmitriev, B.A., Backinowsky, L.V., Chizhov, O.S., Zolotarev, B.M., and Kochetkov, N.K., Carbohyd. Res., (1971) 19 432-435.


9. SEYMOUR

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17.

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Polysaccharides

127

Varma, R. Varma, R.S., and Wardi, A.H., J . Chromatogr. (1973) 77, 222-227. Szafranek, J., Pfaffenberger, C.D., and Horning, E.C., Anal. Lett. (1973) 6, 479-492. Seymour, F.R., Chen, E.C.M., and Bishop, S.H., Carbohyd. Res. in press. Lance, E.G., and Jones, J.K.N., Can. J . Chem. (1967) 45, 1995-1998. Seymour, F.R., Plattner, R.D., and Slodki, M.E., Carbohyd. Res. (1975) 44, 181-198. Seymour, F.R., Slodki, M.E., Plattner, R.D., and Stodola, R. Μ., Carbohyd. Res. (1976) 48, 225-237. Seymour, F.R., Slodki, M.E., Plattner, R.D., and Jeanes, Α., Carbohyd. Res. in press Rosenfeld, L . , an 32, 287-298. Perlin, A . S . , Casu, B . , and Koch, H . J . , Can. J . Chem. (1970) 48, 2596-2606. Usui, T., Yamoka, N., Matsuda, K., Tuzimura, K., Sugiyama, H. and Seto, S., J . Chem. Soc. Perkin, I, 1973, 2425-2432. Gorin, P.A.J., Can. J . Chem., (1973) 51, 2375-2383. Seymour, F.R., Knapp, R.D., Bishop, S.H., Carbohyd. Res. in press. Jennings, H . J . , and Smith, I.C.P., J . Am. Chem. Soc. (1973) 95, 606-608. Slodki, M.E., Ward, R.M., Boundy, J.Α., and Cadmus, M.C. in Terui, G. (Ed.), Ρroc. Int. Ferment. Symp. IVth: Ferment. Technol. Today, Soc. Ferment. Technol., Osaka, 1972 pp 597-601. Costello, A.J.R., Glonek, T., Slodki, M.E., Seymour, F.R., Carbohyd. Res. (1975) 42, 23-37.

Acknowledgements T h i s work was s u p p o r t e d , i n p a r t , by a R o b e r t A. W e l c h F o u n d a t i o n G r a n t (Q 2 9 4 ) , a N a t i o n a l S c i e n c e F o u n d a t i o n G r a n t ( B M S - 7 4 - 1 0 4 3 3 ) , and N a t i o n a l I n s t i t u t e s o f H e a l t h G r a n t s (HL-05435, HL-14194, HL-17372). S p e c i a l t h a n k s a r e due t o D r s . Aliène J e a n e s and Morey E. S l o d k i o f t h e N o r t h e r n R e g i o n a l R e s e a r c h L a b o r a t o r y , ARS, USDA, P e o r i a , I l l i n o i s , f o r p r o v i d i n g t h e d e x t r a n s , mannans, and O-phosphonohexosans d e s c r i b e d i n t h i s paper.


10 Polysaccharide Polyelectrolytes W. M. PASIKA Chemistry Department, Laurentian University, Sudbury, Ontario, Canada

Macromolecules which possess a large number of some functionality an called polyelectrolytes function aids in the solubilization of the polyelectrolyte substance and is responsible for its unique properties. Although the ionogenic function may be regarded as a salt, dissolution of the polyelectrolyte substance is not comparable to the dissolution of a simple salt. A simple salt such as sodium chloride in solution produces a cation and an anion of comparable size. Each ion has independent mobility. A polyelectrolyte dissolves to yield a polyion and counter ions. The polyion holds a large number of charges in close proximity because they are attached to the macromolecular backbone. Although the polyion has mobility, the individual charges attached to the chain do not. They remain within the domain of the macromolecular c o i l . Not all the gegions or counterions are completely mobile. Anionic polyelectrolytes have positive counter ions whereas cationic polyelectrolytes have negative counter ions. Polyampholytes can acquire either positive or negative charge along the macromolecular backbone depending upon the composition of the solution. P i c t o r i a l l y , one has the following

128


10. PAsiKA

Polysaccharide

Polyelectrolytes

129

Because o f f r e e energy r e s t r i c t i o n s , n o t a l l the i o n o g e n i c groups " i o n i z e " . Many e x i s t as i o n p a i r s . A l a r g e number of p o l y s a c c h a r i d e p o l y e l e c t r o l y t e s can be i s o l a t e d from a v a r i e t y o f n a t u r a l s o u r c e s , h e p a r i n , h y a l u r o n i c a c i d , c h o n d r o i t i n and k e r a t i n , t o name a few, are i s o l a t e d from a n i m a l s o u r c e s . The more f a m i l i a r examples s u p p l i e d by the p l a n t w o r l d a r e p e c t i n i c a c i d s , a l g i n a t e s and carageenan. A number of p o l y s a c c h a r i d e p o l y e l e c t r o l y t e s , such as Xanthan, can be o b t a i n e d from nonpathogenic m i c r o organisms (1) . The common c h a r a c t e r i s t i c i s t h a t the macromolecular backbone i s composed o f s a c c h a r i d e r e s i d u e s c a r r y i n g i o n o g e n i c groups. The l a t t e r are more o f t e n than not "Synthetic" polysaccharid o b t a i n e d by s u i t a b l y d e r i v a t i z i n g p o l y s a c c h a r i d e s . The e n s u i n g d i s c u s s i o n w i l l focus on d e r i v a t i z e d d e x t r a n i n an attempt t o i l l u s t r a t e some o f the f a c t o r s which i n f l u e n c e the c h a r a c t e r i s t i c s o f p o l y saccharide p o l y e l e c t r o l y t e s . Viscosity. A l l macromolecular substances i n s o l u t i o n enhance the v i s c o s i t y o f the s o l v e n t c o n s i d e r a b l y . The l a r g e r the m o l e c u l a r weight o r macromolecular s i z e , the g r e a t e r the enhancement. In c h a r a c t e r i z i n g the macromolecular s i z e through the v i s c o s i t y enhancement, i t i s more c o n v e n i e n t l y done w i t h the v i s c o s i t y f u n c t i o n s l i s t e d i n F i g . 1. The dependence o f reduced v i s c o s i t y on c o n c e n t r a t i o n o f n e u t r a l macrom o l e c u l a r s u b s t a n c e s ( i . e . , dextran) i s l i n e a r as d e p i c t e d i n F i g . 1. E x t r a p o l a t i o n o f the v i s c o s i t y d a t a t o " z e r o " c o n c e n t r a t i o n y i e l d s the i n t r i n s i c v i s c o s i t y , which measures the hydrodynamic volume p e r a gram o f macromolecular s u b s t a n c e a t i n f i n i t e dilution. The reduced v i s c o s i t y which p e r t a i n s t o s o l u t i o n s o f f i n i t e c o n c e n t r a t i o n has the same u n i t s o f volume p e r gram o f s u b s t a n c e . P o l y e l e c t r o l y t e s ( i . e . , d e x t r a n s u l f a t e ) i n water do not e x h i b i t l i n e a r reduced v i s c o s i t y curves over the c o n c e n t r a t i o n range t h a t m a c r o m o l e c u l a r subs t a n c e s are u s u a l l y s t u d i e d ( 1%). The reduced v i s c o s i t y curve i s a c o n t i n u o u s l y i n c r e a s i n g f u n c t i o n with d i l u t i o n ( F i g . 2). The c o n t i n u a l i n c r e a s e w i t h d i l u t i o n does n o t o c c u r i n d e f i n i t e l y . At extremely low c o n c e n t r a t i o n s ( 10" ) the reduced v i s c o s i t y f u n c t i o n decreases very r p i d l y with f u r t h e r d i l u t i o n . S h o u l d the d i l u t i n g aqueous s o l v e n t c o n t a i n an e l e c t r o l y t e such as NaC3. e t c . , the reduced v i s c o s i t y a


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dl/g

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cone, polymer g/dl Figure 2.


10.

PAsiKA

Polysaccharide

Polyelectrolytes

131

curves e x h i b i t maxima a t f i n i t e c o n c e n t r a t i o n s . The l a r g e r the e x t e r n a l s a l t c o n c e n t r a t i o n , the s m a l l e r the reduced v i s c o s i t y v a l u e s and t h e f u r t h e r t o t h e r i g h t t h e maximum reduced v i s c o s i t y v a l u e tends t o appear ( F i g . 2 ) . A l i n e a r dependence o f reduced v i s c o s i t y on p o l y e l e c t r o l y t e c o n c e n t r a t i o n i s o b t a i n e d i n the presence o f a s u f f i c i e n t l y high e x t e r n a l s a l t concentration. The v i s c o s i t y b e h a v i o u r o f p o l y e l e c t r o l y t e s i s governed by the f i r s t , second and t h i r d e l e c t r o v i s c o u s e f f e c t (2) ( F i g . 3 ) . The 1 s t e l e c t r o v i s c o u s e f f e c t a r i s e s because o f the d i f f e r e n c e i n s i z e o f t h e macro i o n and t h e c o u n t e r i o n s . I n an hydrodynamic gradient, the small r a p i d l y than t h e muc s e p a r a t i o n o f t h e c o u n t e r i o n c l o u d from t h e macro i o n occurs. Because t h e two a r e c o u p l e d by a coulombic type i n t e r a c t i o n , t h e l a r g e r macro i o n a c t s as a b r a k e on t h e c o u n t e r i o n movement. This increases the v i s c o s i t y of the s o l u t i o n . I n s o l u t i o n , as t h e l i q u i d f l o w s , macro i o n s w i l l be d r i v e n p a s t each o t h e r because o f t h e hydrodynamic g r a d i e n t . Should the h i g h l y charged macro i o n s pass c l o s e l y , c o u l o m b i c r e p u l s i v e f o r c e s w i l l come i n t o p l a y . The f a s t e r moving macro i o n w i l l d e v i a t e from i t s i n i t i a l l i n e a r pathway. A g a i n , excess energy i s expended and t h e v i s c o s i t y o f t h e medium i s i n c r e a s e d . The l a r g e r the charge on t h e macro i o n , t h e s t r o n g e r w i l l be t h e 2nd e l e c t r o v i s c o u s e f f e c t . The 3 r d e l e c t r o v i s c o u s e f f e c t a r i s e s because o f t h e i n t e r a c t i o n o f t h e charges t h a t a r e a t t a c h e d t o t h e macromolecular backbone. I n t h e case o f a f l e x i b l e m a c r o m o l e c u l a r c o i l , t h i s i n t e r a c t i o n expands t h e c o i l t o an average c o n f o r m a t i o n which m i n i m i z e s t h e r e p u l s i v e i n t e r a c t i o n s . A t t h e new e q u i l i b r i u m c o n f o r m a t i o n ( l a r g e r than t h a t of the n e u t r a l macromolecule), the c o n t r a c t i l e f r e e energy o f t h e m a c r o m o l e c u l a r backbone i s e q u a l t o t h e e x p a n s i v e coulombic f r e e energy a r i s i n g from i o n i zation. The i n c r e a s e d m a c r o m o l e c u l a r c o i l s i z e enhances t h e v i s c o s i t y o f t h e s o l u t i o n . The v i s c o s i t y b e h a v i o u r t o t h e l e f t o f t h e maxima i n F i g . 2 i s p r i m a r i l y due t o t h e 2nd e l e c t r o v i s c o u s e f f e c t , w h i l e t h a t t o t h e r i g h t i s p r i m a r i l y due t o t h e 3 r d e l e c t r o viscous e f f e c t . Not a l l o f the c o u n t e r i o n s o f a p o l y e l e c t r o l y t e a r e f r e e t o move about. The f r e e i o n s form a counteion cloud about t h e p o l y i o n , whereas t h e i m m o b i l i z e d i o n s a r e bound t o a s p e c i f i c s i t e o r p o i n t o f t h e macromolecular backbone. T h i s model was p r e s e n t e d e a r l i e r i n the p o l y e l e c t r o l y t e d i s s o l u t i o n equation.



132

As t h e p o l y e l e c t r o l y t e s o l u t i o n i s d i l u t e d more and more o f t h e s i t e bound c o u n t e r i o n s a r e r e l e a s e d . T h i s b u i l d s up t h e charge on t h e macro i o n which expands, which i n t u r n i n c r e a s e s t h e reduced v i s c o s i t y . E x p a n s i o n on d i l u t i o n , however, cannot o c c u r i n definitely. When t h e c o n c e n t r a t i o n o f t h e e x t e r n a l i o n s o f t h e s o l u t i o n become e q u a l t o o r g r e a t e r than t h a t of the counterions o f the p o l y e l e c t r o l y t e , i o n i z a t i o n of the p o l y e l e c t r o l y t e ceases. Further d i l u t i o n d e c r e a s e s t h e reduced v i s c o s i t y because e x p a n s i o n o f t h e c o i l has c e a s e d and t h e charged p a r t i c l e s a r e p l a c e d f u r t h e r and f u r t h e r a p a r t , c a u s i n g a r e d u c t i o n i n t h e 2nd e l e c t r o v i s c o u s e f f e c t . This i s t h e o r i g i n o f th curves. Dextran P o l y e l e c t r o l y t e

Behaviour.

A s u f f i c i e n t l y large external s a l t concentration w i l l y i e l d l i n e a r reduced v i s c o s i t y - c o n c e n t r a t i o n plots. L i n e a r i t y , however, does n o t i n s u r e t h a t t h e v i s c o s i t y b e h a v i o u r i s t h a t o f t h e n e u t r a l macromolecule. F i g . 4 shows t h e r e d u c e d v i s c o s i t y b e h a v i o u r o f a B-512 l i n e a r d e x t r a n ( Jjt^"] 0.164 d l / g ) and a b r a n c h e d d e x t r a n B - 7 4 2 ( f j \ J - 0.158 d l / g ) and the s u l f a t e d e r i v a t i v e s d e r i v e d from them. Despite l i n e a r i t y , the reduced v i s c o s i t i e s o f the s u l f a t e s are h i g h e r than t h o s e o f t h e n e u t r a l m o l e c u l e s by a f a c t o r o f about two. The d i f f i c u l t y i n c o l l a p s i n g t h e s u l f a t e macromolecular c o i l t o t h e s i z e o f t h e n e u t r a l macromolecule may stem from one o f two f a c t o r s or a combination o f both. Introduction of the s u l f a t e group may d e c r e a s e t h e f l e x i b i l i t y o f t h e macrom o l e c u l a r backbone. A r i g i d backbone tends t o produce a more extended m a c r o m o l e c u l a r c o n f o r m a t i o n which would e x h i b i t h i g h e r r e d u c e d v i s c o s i t i e s . Alternately, a l t h o u g h s t r o n g l o n g range coulombic i n t e r a c t i o n s have been e l i m i n a t e d by t h e e x t e r n a l s a l t , i t may be t h a t s h o r t range i n t e r a c t i o n s o f t h e i o n p a i r s e x i s t . E f f e c t o f Degree o f S u b s t i t u t i o n . The r e d u c e d v i s c o s i t i e s o f a number o f p o t a s s i u m d e x t r a n s u l f a t e s o f d i f f e r i n g degree o f s u b s t i t u t i o n d e r i v e d from B-742(CnJ]*0.158) a r e shown i n F i g . 5. Increasing the degree o f s u b s t i t u t i o n enhances t h e reduced v i s c o s i t y and s h i f t s t h e p o s i t i o n a t which t h e maximum r e d u c e d v i s c o s i t y appears t o t h e l e f t . I n c r e a s i n g t h e number o f i o n o g e n i c groups produces more charge on t h e macro i o n , c a u s i n g g r e a t e r expansion o f the c o i l . On d i l u t i o n , f u r t h e r i o n i z a t i o n


PASiKA

Polysaccharide

O.304.

Polyelectrolytes

POTASSIUM DEXTRAN SULFATE 9/o S DS3 0.65 e

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134


Figure 5.


10.

PAsiKA

Polysaccharide

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Polyelectrolytes

and e x p a n s i o n o c c u r i n each c a s e . The h i g h e r the degree o f s u b s t i t u t i o n , the f u r t h e r must the p o l y e l e c t r o l y t e s o l u t i o n be d i l u t e d t o match the e x t e r n a l s a l t c o n c e n t r a t i o n w i t h the c o u n t e r i o n c o n c e n t r a t i o n of the p o l y e l e c t r o l y t e . S i m i l a r v i s c o s i t y behaviour i s o b s e r v e d f o r l i n e a r d e x t r a n s u l f a t e s and f o r branched and l i n e a r carboxymethyl d e x t r a n s . The t y p i c a l p o l y e l e c t r o l y t e v i s c o s i t y c u r v e s e x h i b i t e d by d e x t r a n s u g g e s t t h a t the macromolecular backbone i s f a i r l y f l e x i b l e and t h a t the c o i l can undergo e x p a n s i o n on a c q u i r i n g c h a r g e . E f f e c t o f M o l e c u l a r Weight. F i g . 6 i n d i c a t e s the e f f e c t of molecula weight potassiu carboxymethyl d e x t r a n reduced v i s c o s i t s u b s t i t u t i o n i s constan weigh v a r i e s from 73,000 t o 135,000. The r e d u c e d v i s c o s i t i e s i n c r e a s e w i t h m o l e c u l a r weight and the c o n c e n t r a t i o n at which the reduced v i s c o s i t y maximum appears i s i d e n t i c a l f o r a l l three molecular weights. I t would appear t h a t the m o l e c u l a r w e i g h t does n o t i n f l u e n c e the e x t e n t o r degree o f i o n i z a t i o n and t h a t the e x p a n s i o n i s d i r e c t l y p r o p o r t i o n a l t o the number o f s u b s t i t u t e d a n h y d r o g l u c o s e u n i t s i n the macromolecule £ ( \sf>/ )>τ*χχ 135,000 m o l e c u l a r weight sample a p p r o x i m a t e l y 2x ( T\* / c )VH*X o f 73,000 m o l e c u l a r weight samplej . T h i s s u g g e s t s t h a t the i n t e r a c t i o n of the i o n o g e n i c groups i s a l o c a l i z e d o r n e a r e s t neighbor i n t e r a c t i o n . S h o u l d i t be o t h e r w i s e , then each charge o f p o l y e l e c t r o l y t e would i n t e r a c t w i t h e v e r y o t h e r , compounding the i n t e r a c t i o n s . The h i g h e r m o l e c u l a r weight macromolecule c a r r y i n g more charge would r e g i s t e r a n o n - p r o p o r t i o n a t e reduced viscosity. The l i n e a r p r o p o r t i o n a l i t y between m o l e c u l a r w e i g h t and the maximum reduced v i s c o s i t y would n o t e x i s t . To show more q u a n t i t a t i v e l y t h a t the same i o n i z a t i o n and e x p a n s i o n p r o c e s s i s o c c u r r i n g w i t h the d i f f e r e n t m o l e c u l a r w e i g h t s , the d a t a o f F i g . 6 can be p l o t t e d i n terms o f a r e l a t i v e e x p a n s i o n f a c t o r R vs the c o n c e n t r a t i o n o f p o t a s s i u m carboxy methyl d e x t r a n as i n F i g . 7. The numerator o f R i s the maximum reduced v i s c o s i t y and the denominator i s the r e d u c e d v i s c o s i t y a t a p o l y e l e c t r o l y t e c o n c e n t r a t i o n g r e a t e r than t h a t a t which the maximum v i s c o s i t y appears. The c o i n c i d e n c e o f the l i n e a r p l o t s f o r the t h r e e m o l e c u l a r w e i g h t s i n d i c a t e s an i o n i z a t i o n e x p a n s i o n mechanism t h a t i s i d e n t i c a l f o r the t h r e e p o l y e l e c t r o l y t e samples. o

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estimated the diameter of a xanthan r o d t o be i n t h e r a n g e o f l 6 t o kOA the length calculation i s quite i n s e n s i t i v e t o diameter, 9

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LENGTH MICRONS

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0.96

Table IV gives the r e s u l t s of the r o d length calculation. I t would appear that a xanthan " r o d " has a l e n g t h b e t w e e n 0.7 a n d 1.0 m i c r o n s . This i s i n g o o d a g r e e m e n t w i t h H o l z w a r t h s (l_3) membrane c h r o m a tography measurements. He f o u n d t h a t e s s e n t i a l l y a l l x a n t h a n p a r t i c l e s c a n p a s s t h r o u g h a membrane w i t h 1.0 m i c r o n p o r e s b u t a r e b l o c k e d by a membrane w i t h 0.8 micron pores. f

Nomenclature D Κ L M MWn Ν O.D. R Τ β t η Ποη

οο-

[η]

Rod d i a m e t e r , (microns) Power l a w c o n s t a n t , i n t e r c e p t Rod l e n g t h (microns) Torque (gm-cm) Molecular Weight Power l a w c o n s t a n t , s l o p e A v o g a d r o number Optical Density Cone r a d i u s (cm) T e m p e r a t u r e (°C) Cone a n g l e (radians) Shear r a t e (sec ) Viscosity (poise) Zero shear r a t e v i s c o s i t y (poise) (poise) I n f i n i t e shear rate v i s c o s i t y Intrinsic viscosity (deciliters/gm) - 1


WHITCOMB E T A L .

12.

τ τ oo

Rheology of Xanthan Gum Solutions

173

2

1

2

Shear s t r e s s (dyne/cm ) Yield stress (dyne/cm ) Angular speed (radians/sec , 1 2

y

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

Jeanes, A., P i t t s l e y , J.E., Senti, F.R., J. Applied Polymer S c i , (1961), 5, p. 519-526. Van Wazer, J.R., et al, "Viscosity and Flow Measurement", p. 113-116, Wiley, Ν.Υ. 1963. Macosko, C.W., S t a r i t a , J., S.P.E. Journal,(1971), 27, p. 38-42. Middleman, S., "The Wiley Willey, S.J. Ph.D. Thesis, University of Minnesota, 1976. Ostwald, W., Kolloid-Zeitschrift, (1925), 36, p. 99-117. Metzner, A . B . , Reed, J.C., A.I.Ch.E. J., (1955), 1, p. 434-440. Mishra, P., Mishra, I., A.I.Ch.E. J.,(1976), 22, p. 617-619. Sheffield, R.E., Metzner, A . B . , A.I.Ch.E. J., (1976), 22, p. 736-744. Wilkinson, W.L., "Non-Newtonian Fluids", Pergamon, New York, 1960. Abdel-Khalik, S . K . , B i r d , R.Β., Biopolymers, (1975), 14, p. 1915-1932. D i n t z i s , E . R . , Babcock, G.Ε., Tobin, R., Carbohydrate Research. (1970), 13, p. 257-267. Holzwarth, G . , "Polysaccharide from Xanthomonas Campestris: Rheology, Solution Conforma t i o n , And Flow Through of Petroleum Chemistry, A.C.S., New York Meeting, April 4-9, 1976.


13 Synergistic Xanthan Gels I. C. M . D E A

and

E . R.

MORRIS

Unilever Research, Colworth/Welwyn Laboratory, Sharnbrook, Bedford, MK44, 1LQ, Great Britain

Although showing considerable evidence of strong i n t e r molecular i n t e r a c t i o n gel. On mixing with anothe bean gum, however, f i r m rubbery g e l s are formed at low polymer l e v e l s , t y p i c a l l y around 1% t o t a l p o l y s a c c h a r i d e , with most e f f e c t i v e xanthan u t i l i s a t i o n a t locust-bean gum: xanthan r a t i o s of about 3:1 (1/2/3.)· The molecular o r i g i n of the synergism has u n t i l r e c e n t l y (4_,5_,(5) remained obscure. In t h i s paper we w i l l attempt to answer two questions:1) What i s the mechanism o f formation o f these mixed g e l s ? 2) Why should two p o l y s a c c h a r i d e s of such d i v e r s e o r i g i n s interact: T e c h n o l o g i c a l Relevance o f B i o l o g i c a l Function Many o f the i n d u s t r i a l uses o f p o l y s a c c h a r i d e s r e s t s o l e l y on t h e i r water b i n d i n g c a p a c i t y and high v i s c o s i t y a t low concentrations. I n c r e a s i n g l y , however, more s o p h i s t i c a t e d a p p l i c a t i o n s depend on d e t a i l e d molecular s t r u c t u r e , and e x p l o i t i n v i t r o the s p e c i f i c f u n c t i o n o f the p o l y s a c c h a r i d e i n v i v o . T h i s i s p a r t i c u l a r l y t r u e i n g e l l i n g systems. Thus agar, carrageenan, f u r c e l l a r a n , p e c t i n and a l g i n a t e a l l have a s t r u c t u r a l r o l e i n nature, which g i v e s r i s e d i r e c t l y to t h e i r g e l l i n g behaviour. A l g i n a t e , f o r example, i s the major s t r u c t u r a l p o l y s a c c h a r i d e of Brown Seaweed. Chemically i t i s a block co-polymer of D-mannuronic and L - g u l u r o n i c a c i d , i n which the homopolymeric polyguluronate sequences are capable o f forming very strong i n t e r molecular c r o s s - l i n k s , while polymannuronate or a l t e r n a t i n g sequences show f a r l e s s tendency t o a s s o c i a t e (5-11). The r e l a t i v e amount o f the v a r i o u s block types i s under enzymic cont r o l a t the polymer l e v e l (12), p r o v i d i n g s u b t l e b i o l o g i c a l cont r o l o f the mechanical p r o p e r t i e s o f d i f f e r e n t p a r t s of the p l a n t at d i f f e r e n t stages of maturation. These s t r u c t u r a l v a r i a t i o n s are r e f l e c t e d d i r e c t l y i n the g e l a t i o n p r o p e r t i e s of the p o l y -

174


13.

DEA AND MORRIS

Synergistic

Xanthan

Gels

175

saccharide i n v i t r o . Thus a l g i n a t e e x t r a c t e d f r o m g r o w i n g fronds g i v i n g a p p r e c i a b l y l e s s r i g i d g e l s than m a t e r i a l from mature s t i p e s . Knowledge o f t h e n a t u r a l r o l e o f i n d u s t r i a l l y i m p o r t a n t polysaccharides can therefore provide valuable i n s i g h t i n t o t h e i r e f f e c t i v e commercial u t i l i s a t i o n . Thus a n y u n d e r s t a n d i n g we c a n g a i n o f t h e b i o l o g i c a l u t i l i t y o f x a n t h a n s y n e r g i s m may w e l l be of i n t e r e s t from a t e c h n o l o g i c a l a s w e l l a s an academic s t a n d point . Polysaccharide

Gel Structure

An u n d e r s t a n d i n g o f t h e mechanism o f s y n e r g i s t i c g e l a t i o n i s perhaps best approache s i n g l e p o l y s a c c h a r i d e system s t a b l e h a l f - w a y house between t h e s o l i d s t a t e , w i t h m o l e c u l e s i n r e g u l a r ordered conformations packed together w i t h l i t t l e hydrat i o n , and the s o l u t i o n s t a t e , w i t h e x t e n s i v e l y hydrated polymer m o l e c u l e s i n random c o n f o r m a t i o n s . The s t r u c t u r a l i n t e g r i t y o f polysaccharide gels i s maintained by intermolecular a s s o c i a t i o n i n t o l o n g , s t r u c t u r a l l y r e g u l a r j u n c t i o n zones, i n which the m o l e c u l e s a d o p t t h e same o r d e r e d c o n f o r m a t i o n a s i n t h e s o l i d state. These j u n c t i o n z o n e s a r e t h e r e f o r e e s s e n t i a l l y c r y s t a l l i n e , a l t h o u g h t h e y may o n l y i n v o l v e t w o p o l y m e r c h a i n s , a n d a r e terminated t y p i c a l l y by an i n t e r r u p t i o n i n the regular covalent s t r u c t u r e (e.g. i n a l g i n a t e t h e o c c u r r e n c e o f a mannuronate residue would terminate a s s o c i a t i o n o f polyguluronate sequences). Such j u n c t i o n s a r e h e l d t o g e t h e r b y a r e g u l a r a r r a y o f n o n c o v a l e n t i n t e r m o l e c u l a r b o n d s , whose e n e r g y o f f s e t s t h e l o s s o f conformational entropy i n forming such a r i g i d assembly, and whose c o - o p e r a t i v e a c t i o n e l e v a t e s t h e l i f e t i m e o f t h e j u n c t i o n s to a macroscopic timescale. The j u n c t i o n z o n e s a r e t h e n l i n k e d b y r e g i o n s o f t h e m o l e c u l e which are s t r u c t u r a l l y incapable o f forming stable a s s o c i a t i o n s , or a r e prevented from doing so by network c o n s t r a i n t s . These n o n - a s s o c i a t e d r e g i o n s p r e s u m a b l y m a i n t a i n e s s e n t i a l l y t h e same disordered conformation as i n s o l u t i o n , and s o l u b i l i s e the g e l network by extensive h y d r a t i o n . Thus b o t h a s s o c i a t i n g a n d n o n associating molecular regions are e s s e n t i a l f o r g e l a t i o n , too much a s s o c i a t i o n l e a d i n g t o p r e c i p i t a t i o n , a n d t o o l i t t l e preventing formation o f a cohesive network. Aggregation o f R i g i d

Structures

I n g e l s o f c a r r a g e e n a n o r a g a r t h e p r i m a r y mechanism o f i n t e r m o l e c u l a r a s s o c i a t i o n i s b y double h e l i x formation. F u r t h e r development o f t h e g e l network, however, i n v o l v e s a s s o c i a t i o n o f h e l i c e s i n t o l a r g e r a g g r e g a t e s ( 1 4 - 1 6 ) . The extent o f aggregation increases as e l e c t r o s t a t i c r e p u l s i o n b e tween t h e m o l e c u l e s d e c r e a s e s , b e i n g g r e a t e s t f o r t h e n e u t r a l


176


agarose h e l i x . Aggregation of r i g i d r o d - l i k e species i s a h i g h l y favourable process, s i n c e , u n l i k e t h e formation of ordered j u n c t i o n s between f l e x i b l e m o l e c u l e s , l i t t l e l o s s o f c o n f o r m a t i o n a l e n e r g y i s i n v o l v e d . M o r e o v e r , above a c r i t i c a l c o n c e n t r a t i o n , a l i g n m e n t o f e x t e n d e d s t r u c t u r e s becomes a g e o m e t r i c a l n e c c e s s i t y (17) p r o v i d i n g a n a d d i t i o n a l d r i v e t o a g g r e g a t i o n . S i m i l a r considerations apply t o thea s s o c i a t i o n of poly s a c c h a r i d e c h a i n s w h i c h , w h i l e n o t t o t a l l y r i g i d , have s e v e r e l y r e s t r i c t e d m o b i l i t y about t h e g l y c o s i d i c l i n k a g e s between a d j a c e n t r e s i d u e s , a n d t h e r e f o r e t e n d t o f a v o u r e x t e n d e d conford i n a t i o n s c l o s e t o t h a t f o u n d i n t h e s o l i d s t a t e . L o c u s t - b e a n gum f a l l s i n t o t h i s category. Chemically i t i s a galactomannan, w i t h a ρ 1-4 l i n k e d mannan b a c k b o n e a n d l i n k e d galactose subs t i t u e n t s , which occur i Fractions of varying galactos c o m m e r c i a l l o c u s t - b e a n gum s a m p l e s b y u t i l i s i n g t h e g r e a t e r s o l u b i l i t y o f t h e more h i g h l y s u b s t i t u t e d c h a i n s (4)· A s o u t l i n e d i n F i g u r e 2, t h e s o l i d s t a t e c o n f o r m a t i o n o f t h e mannan c h a i n i s an extended, t w o - f o l d , r i b b o n - l i k e s t r u c t u r e , v i r t u a l l y i d e n t i c a l t o t h a t o f c e l l u l o s e , s i n c e b o t h h a v e a 1-4 d i e q u a t o r i a l l y l i n k e d hexopyranose backbone, and d i f f e r o n l y i n t h e o r i e n t a t i o n o f 0(2). Under normal c o n d i t i o n s t h e r e i s no evidence o f a g g r e g a t i o n of galactomannan molecules i n s o l u t i o n . F r e e z i n g and thawing c o n c e n t r a t e d l o c u s t - b e a n gum s o l u t i o n s , h o w e v e r , y i e l d s s t a b l e g e l s whose g e l s t r e n g t h i n c r e a s e s w i t h d e c r e a s i n g g a l a c t o s e content. On f r e e z i n g , t h e f o r m a t i o n o f i c e c r y s t a l s must p r o g r e s s i v e l y r a i s e t h e c o n c e n t r a t i o n o f polymer i n t h e remaining u n f r o z e n s o l u t i o n , t o t h e p o i n t where a l i g n m e n t o f t h e c h a i n s becomes s t e r i c a l l y e s s e n t i a l , u n t i l f i n a l l y t h e c h a i n s p a c k together as i nthe s o l i d state. Once f o r m e d t h e c h a i n - c h a i n c o n t a c t s b e t w e e n t h e S m o o t h u n s u b s t i t u t e d mannan b a c k b o n e r e g i o n s a p p e a r t o be s u f f i c i e n t l y e n e r g e t i c a l l y f a v o u r a b l e t o h o l d t h e m o l e c u l e s t o g e t h e r on thawing. I n t h e r e s u l t a n t g e l network, t h e s u b s t i t u t e d h a i r y regions presumably a c t as t h e s o l u b i l i s i n g i n t e r c o n n e c t i n g regions which prevent p r e c i p i t a t i o n of the associated chains. Xanthan i s n o t alone i n showing synergism w i t h galactoman nan s, b u t s h a r e s t h i s p r o p e r t y w i t h b o t h a g a r a n d c a r r a g e e n a n . F o r b o t h o f t h e s e i t h a s b e e n e s t a b l i s h e d t h a t t h e mechanism o f g e l a t i o n i n v o l v e s a s s o c i a t i o n o f u n s u b s t i t u t e d backbone r e g i o n s of t h e galactomannan, i n an ordered conformation, w i t h t h e r i g i d , o r d e r e d , h e l i c a l s t r u c t u r e o f t h e p o l y s a c c h a r i d e (19). We must t h e r e f o r e c o n s i d e r w h e t h e r a s i m i l a r mechanism o p e r a t e s f o r xanthan - galactomannan i n t e r a c t i o n s . 1

1

Xanthan N a t i v e

1

Conformation

An e s s e n t i a l r e q u i r e m e n t o f t h i s m o d e l i s a r i g i d , r e g u l a r , o r d e r e d s t r u c t u r e w i t h w h i c h t h e mannan c h a i n c a n a l i g n . Until


13.

DEA AND MORRIS

Synergistic

Xanthan

Gels

177

recently no such r i g i d conformation was suspected for xanthan. Spectroscopic and rheological studies now show, however, that the native conformation of the molecule i s a r i g i d rod, which i s only melted out to the expected random c o i l under conditions of very high temperature and low ionic strength (4*6,20,21). The orderdisorder transition i s conveniently monitored as a sharp sigmoidal discontinuity i n a single-wavelength optical rotation (Figure 3). Detail of the ordered structure i s s t i l l the subject of X-ray studies (22), but i t i s known to involve the charged t r i saccharide side-chains aligning with the cellulose backbone of the xanthan molecule, i n a 5-fold h e l i c a l structure. The solution properties of xanthan are entirely consistent with extensive orientation and aggregation of the r i g i d molecular rods (20-21), analogous to the previousl carrageenan double helices Molecular Origin of Xanthan Synergism The strength of interaction between xanthan and galacto— mannans i s closely correlated with the degree of substitution of the mannan chain. Guar gum, i n which the ratio of mannose to galactose i s close to 2:1 does not gel with xanthan i n any concentration, although a slight viscous interaction i s observed ( l ) . Soft, f a i r l y weak gels are obtained with gum tara, where the mannose to galactose ratio i s around 3:1, as against 4 Ï1 i n locust—bean gum, which gives far stronger and more r i g i d gels. Even greater enhancement of gel properties i s found for hot water soluble locust-bean gum fractions i n which the ratio i s 5 î1 or more. Thus, once more, unsubstituted regions of the mannan backbone are implicated i n junction formation. Optical rotation studies of synergistic xanthan gelation provide strong evidence that the native ordered xanthan conformation i s present i n the mixed gel. As shown i n Figure 4y the characteristic sigmoidal curve which accompanies the orderdisorder transition persists i n the presence of galactomannan, and i s essentially complete before the onset of gelation. This interpretation i s confirmed by X-ray studies on oriented films prepared from synergistic xanthan gels (23), which show v i r t u a l l y the same diffraction features as for xanthan alone. We therefore conclude that xanthan — galactomannan gels are crosslinked by cooperative association of unsubstituted mannan regions i n a regular ribbon-like conformation, to the native ordered xanthan structure, as outlined schematically i n Figure 5· Biological U t i l i t y Xanthan i s the extracellular polysaccharide from Xanthomonas campestris which causes blight i n cabbage crops. Other related Xanthomonas species are parasitic upon a wide variety of other plants, and a l l appear to synthesise ordered polysaccharides 5


178


Figure 1. Schematic for a galactomannan such as locust bean gum. (O) 1-^4 linked β-Ό-mannopyranose residues; (Φ) α-Ό-gahctopyranose residues.

Figure 2.

Schematic

05*

-isoh

' /Λ,ϊν^.,.... -2501-

'

80

60 TEMPERATURE

40 «C

^

20 KELTROL 365nm

Figure 3.


Synergistic

DEA AND MORRIS

COOL

-ΛΑΑΑΑ

HEAT RANDOM COIL

Xanthan

Gels

GALACTOMANNAN >

XANTHAN NATIVE CONFORMATION

ΛΑΑΑΑ MIXED GEL

Figure 5



180

POLYSACCHARIDES

capable o f s y n e r g i s t i c i n t e r a c t i o n (24). Xanthan i s a complex m o l e c u l e ( 2 5 , 2 6 ) , a n d a l a r g e number o f s t e p s a r e i n v o l v e d i n i t s b i o s y n t h e s i s T27)» I t w o u l d be s u r p r i s i n g (28,29) i f t h e amount o f g e n e t i c i n f o r m a t i o n i n v o l v e d were s t o r e d s i m p l y t o d i s c h a r g e the r e l a t i v e l y t r i v i a l f u n c t i o n s w h i c h have so f a r been proposed for extracellular bacterial polysaccharides. The e x i s t e n c e o f a n ordered n a t i v e xanthan s t r u c t u r e , and i t s a f f i n i t y f o r s p e c i f i c sequences i n p l a n t p o l y s a c c h a r i d e m o l e c u l e s a l s o argue f o r a sophisticated biological function. To e x p l o r e t h i s f u r t h e r we h a v e e x a m i n e d t h e scope a n d s p e c i f i c i t y o f xanthan synergism. Enhancement o f m i c r o c r y s t a l l i n e c e l l u l o s e g e l s (7) shows t h a t x a n t h a n c a n i n t e r a c t w i t h g l u c a n a s w e l l a s w i t h mannan s e q u e n c e s . I n d e e d a v e r y s t r o n g i n t e r a c t i o n i s observe p o l y s a c c h a r i d e from Amorphophallu l i n k e d l i n e a r c o n t a i n s b o t h g l u c o s e a n d mannose r e s i d u e s . This m a t e r i a l shows a b o u t t h e same s t r e n g t h o f i n t e r a c t i o n w i t h t h e a g a r d o u b l e h e l i x a s d o e s l o c u s t — b e a n gum. W i t h x a n t h a n , h o w e v e r , i t s i n t e r a c t i o n i s v e r y much s t r o n g e r , a n d i n d e e d r e c o g n i s a b l e g e l s a r e formed a t t o t a l 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 s as l o w a s

0.05$. There i s e v i d e n c e t h a t a t t h i s c o n c e n t r a t i o n x a n t h a n c a n b i n d d i r e c t l y t o t h e c e l l w a l l s o f l i v i n g p l a n t t i s s u e (21,30331)· I t t h e r e f o r e a p p e a r s t h a t s y n e r g i s m w i t h g a l a c t o m a n n a n s may be a c o - i n c i d e n t a l by-product o f a n a t u r a l r o l e which i n v o l v e s i n t e r a c t i o n w i t h c e l l u l o s i c m a t e r i a l s on t h e c e l l w a l l s u r f a c e s o f t h e h o s t p l a n t . S u c h i n t e r a c t i o n s may p e r h a p s be i n v o l v e d i n r e c o g n i t i o n o f appropriate s i t e s f o r eventual c o l o n i s a t i o n by the b a c t e r i a , o r i n p r e p a r a t i o n o f t h e c e l l surface f o r attachment o f the p a r a s i t e .

Abstract Although neither xanthan nor locust-bean gum will gel alone under normal conditions, mixed gels can be formed at total poly saccharide concentrations well below 1%. Chemically locust-bean gum i s a galactomannan, with a mannose to galactose ratio of around 4:1. The 1,6 linked galactose residues occur i n long blocks, ('hairy regions'), interspersed by unsubstituted B 1,4 mannan backbone. Gelation occurs by co-operative association of these 'smooth regions' with the xanthan molecule i n its ordered conformation, while the 'hairy regions' act as connecting seg ments which solubilise the gel network and prevent precipitation. Xanthan shares this synergistic behaviour with the ordered conformations of carrageenan, furcellaran, and agar, but i s unique in showing a marked preference for interaction with B 1,4 glucose containing polysaccharides (including derivatised cellu lose) rather than mannan. This suggests a possible biological role for the polymer i n substrate recognition by the synthesising bacterium, Xanthomonas campestris.


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Synergistic

Xanthan

Gels

181

Literature Cited 1. "Xanthan Gum, a Natural Biopolysaccharide for Scientific Water Control", Kelco Co., San Diego, California, 1972. 2. Rocks, J.K. Food Technol. (1971). 25, 476-483. 3. Federal Register, U.S. Government Printing Office, Washington D.C., Xanthan Gum, Section 121.1224 5376-5377, March 19th. 1969. 4. Dea, I.C.M. & Morrison, A. Advan. Carbohyd. Chem. Biochem. (1975). 31, 241-312. 5. Rees, D.A. Biochem. J. (1972). 126, 257-273. 6. Morris, E.R. i n "Molecular Structure and Function of Food Carbohydrate". (Eds Birch G.G & Green L.F.) 125-130 Applied Science Publisher 7. Rees, D.A. Advan. Carbohyd. Chem. Biochem. (1969). 24, 267-332. 8. Morris, E.R., Rees, D.A. &Thom,D. J. Chem. Soc. Chem. Commun. (1973). p. 245. 9. Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C. & Thom, D. FEBS Lett. (1973). 32, 195-197. 10. Morris, E.R., Rees, D.A., Sanderson, G.R. &Thom,D. J . Chem. Soc. Perkin II. (1975). pps. 1418-1425. 11. Morris, E.R., Rees, D.A. &Thom,D. In preparation. 12. Madgwick, J., Haug, A. & Larson, B. Acta Chem. Scand. (1973). 27, 3592-3594. 13. Rees, D.A. i n "Biochemistry of Carbohydrates". (Ed. Whelan, W.J.) 1-42, Butterworths, London, 1975. 14. Rees, D.A., Steele, I.W. & Williamson, F.B. J . Polymer S c i . (C). (1969). 28, 261-276. 15. McKinnon, Α.Α., Rees, D.A. & Williamson, F . B . , J. Chem. Soc. Chem. Commun. (1969). pps. 701-702. 16. Arnott, S., Fulmer, Α., Scott, W.E., Dea, I.C.M., Moorhouse, R. & Rees, D.A. J. Mol. Biol. (1974). 90, 269-284. 17. Flory, P . J . Proc. Roy. Soc. ser. A. (1956). 234, 50-73. 18. Baker,C.W.& Whistler, R.L. (1975). Carbohyd. Res. 45, 237-243. 19. Dea, I.C.M., McKinnon, Α.Α., & Rees, D.A. (1972). J . Mol. Biol. 68, 153-172. 20. Morris, E.R. This Symposium. 21. Morris, E.R., Rees, D.A., Young, G., Walkinshaw, M. & Darke, A. J. Mol. Biol. Submitted. 22. Moorhouse, R. This Symposium. 23. Moorhouse, R. Personal Communication. 24. Schuppner, H.R. J r . Australian Patent, 401,434. (1966). 25. Jansson, P.E., Kenne, L . & Lindberg, B. Carbohyd. Res. (1975). 45, 275-282. 26. Melton, L.D., Mindt, L., Rees, D.A. & Sanderson, G.R. Carbohyd. Res. (1976). 46, 245-257.


182


POLYSACCHARIDES

27. Sutherland, I.W. This Symposium. 28. Sutherland, I.W. Advan. Microbiol. Physiol. (1972). 8, 143-213. 29. Sutherland, I.W. & Norval, M. Biochem. J. (1970). 120, 567-576. 30. Leach, J.G., Lilly, V.G., Wilson, H.A. & Purvis, M.R. J r . Phytopathology. (1975). 47, 113-120. 31. Lesley, S.M. & Hochster, R.M. Canad. J. Physiol. (1959), 37, 513-529.


14 X a n t h a n Gum—Acetolysis as a T o o l for the Elucidation of Structure

C. J. LAWSON and K. C. SYMES Tate and Lyle Ltd., Group Research and Development, P.O. Box 68, Reading, U.K.

The development of microbial gums is now moving at an ever increasing pace and it appears likely products will be available upon those found in many plant gums, but also of a novel nature to be exploited in as yet undeveloped applications. The most successful microbial gum to date is undoubtedly xanthan gum produced by Xanthomonas campestris, and this polymer is now commanding a market of several thousand tons per annum. The market position for xanthan gum has been developed through the unique physical properties which it shows, which are exploited for example in oil recovery and food applications. These properties are briefly, high viscosity, extreme pseudoplasticity stability to extremes of pH, salt tolerance and synergistic gelation in the presence of locust bean gum. The above properties are of course dictated by the primary, secondary and tertiary structures of the gum and it is necessary to determine these if any real understanding of the relationship between function and structure is to be obtained. Early reports on the structure of xanthan gum, presented the repeating unit as being made up of glucose, glucuronic acid, mannose and the substituents pyruvate and acetate, in a 14 or 16 residue repeating unit. (1) (2) A repeating unit as large as this is unusual as most microbial gums have tri, tetra or pentasaccharide repeats. Also some of the chemical evidence was somewhat ambiguous, for example the assignment of the pyruvate as being linked to a glucose residue when it could equally have been associated with mannose. More recently two papers have been published, revising the structure and proposing a new pentasaccharide repeating unit containing the same sugar residues as before. We now provide further supporting evidence for the revised structure and suggest an approach to a rapid and convenient qualitative analysis of aspects of covalent structure of this and similar polysaccharides. The interest of Tate and Lyle in microbial gums was originally connected only with microbial alginate, (3) but as a natural consequence of involvement with gums generally, it was decided to examine 183


184


POLYSACCHARIDES

the possibilities of d e v e l o p i n g other m i c r o b i a l p o l y s a c c h a r i d e s for i n c o r p o r a t i o n into a possible range of p r o d u c t s .

O n e c a n d i d a t e for e x a m i n a t i o n was

Xanthomonas campestrls as the properties shown by xanthan gum were c o n s i d e r e d to be c o m p l e m e n t a r y to m i c r o b i a l a l g i n a t e . T r a d i t i o n a l l y , three b a s i c lines of a p p r o a c h c a n be a d o p t e d in the e l u c i d a t i o n of p o l y s a c c h a r i d e structure.

These are m e t h y l a t i o n a n a l y s i s ,

p e r i o d a t e o x i d a t i o n a n d i s o l a t i o n of fragments w h i c h c a n be c h a r a c t e r i s e d ; the last a p p r o a c h o n l y b e i n g of use when the p o l y s a c c h a r i d e s have a r e p e a t ing u n i t .

The first two a p p r o a c h e s had b e e n reported in the previous papers

a n d therefore the third was the l o g i c a l c h o i c e .

A c e t o l y s i s was used because

aqueous a c i d hydrolysis often g i v e s a c i d i c oligomers from u r o n i c a c i d c o n t a i n i n g p o l y s a c c h a r i d e s a n d these are more d i f f i c u l t to c h a r a c t e r i s e than neutral fragments.

Also a

g i v e a c o m p l e m e n t a r y resul consideration.

A c e t o l y s i s i s , p r a c t i c a l l y , a r e l a t i v e l y straightforward

process performed a t room temperature in a p p r o x i m a t e l y two days using commonly a v a i l a b l e reagents.

A sample of xanthan fermentation broth

o b t a i n e d in b a t c h c u l t u r e of NRRL B1459 was t a k e n .

P u r i f i e d x a n t h a n gum

was r e c o v e r e d after b a c t e r i a l c e l l s were r e m o v e d using high speed c e n t r i f u g a t i o n a n d trypsin d i g e s t i o n , by a l c o h o l p r e c i p i t a t i o n . The c a r e f u l l y d r i e d gum was shaken w i t h the a c e t o l y s i s mixture o f reagents used b y M o r g a n a n d O ' N e i l l , (4) in studies on desulphated carrageenan.

λ-

The a c e t o l y s a t e was then poured into w a t e r , the a c e t y l a t e d

products e x t r a c t e d into c h l o r o f o r m , a n d d e a c e t y l a t e d using m e t h a n o l i c sodium m e t h o x i d e in the usual w a y .

T h e p a l e y e l l o w syrup o b t a i n e d in

h i g h y i e l d r e v e a l e d , on c h r o m a t o g r a p h i c e x a m i n a t i o n , a number o f spots in a d d i t i o n to the e x p e c t e d m o n o s a c c h a r i d e s .

The p r o d u c t was then

r e s o l v e d into a c i d i c a n d neutral fractions b y separation on ion e x c h a n g e resin in the a c e t a t e f o r m .

(5)

A s h o p e d , the major proportion o f o l i g o -

m e r i c m a t e r i a l was in the neutral f r a c t i o n .

The oligomers Β to Ε were then

o b t a i n e d in a p u r i f i e d state from the neutral f r a c t i o n by a c o m b i n a t i o n of c e l l u l o s e c o l u m n a n d t h i c k paper c h r o m a t o g r a p h y .

(Figure 1) (Figure 2)

A t this p o i n t in the work it was l e a r n e d from Professor Rees o f results o b t a i n e d b y his group (6) a n d o f Professor Lindbergs group (7) proposing the r e v i s e d structure o f xanthan gum w h i c h has b e e n m e n t i o n e d e a r l i e r . The r e v i s e d r e p e a t i n g unit is based upon a c e l l u l o s i c b a c k b o n e w i t h t r i s a c c h a r i d e side c h a i n s o c c u r r i n g on a l t e r n a t e g l u c o s e residues.

Analysis

o f the a c e t o l y s i s oligomers was therefore c o n t i n u e d in order to a s c e r t a i n , whether they were consistent w i t h the a b o v e structure. T h e structure o f o l i g o s a c c h a r i d e C w i l l be used as a n e x a m p l e o f the a p p r o a c h a d o p t e d , a n d the other o l i g o s a c c h a r i d e s w i l l o n l y be m e n t i o n e d for the purpose o f m e n t i o n i n g s p e c i f i c points of d i f f e r e n c e in their a n a l y s i s . This o l i g o s a c c h a r i d e was found to consist of glucose a n d mannose in a 2:1 r a t i o after hydrolysis a n d g l c o f the d e r i v e d a l d i t o l a c e t a t e s .

This was


14.

LAWSON A N D SYMES

Structure

Elucidation

of Xanthan

Gum

185

XANTHAN G U M Ac 0/AcOH/H S0 2

2

4

t ACETYLATED

PRODUCTS NoOMe/MeOH

PRODUCTS Acetate resin

NEUTRAL

FRACTION

Cellulose column/PC OLIGOSACCHARIDES B,C,D&E

ACIDIC FRACTION

electrophoresis ALDOBIOURONIC ACID A

Figure

1.

Figure

The

acetolysis of gum

xanthan

2. Xanthan acetolysate neutral oligosaccharides



186

POLYSACCHARIDES

c o n f i r m e d by α c o l o r î m e t r î c assay o f the r a t i o o f carbohydrates to g l u c o s e w h i c h a l s o showed that 5 0 % g l u c o s e was lost on r e d u c t i o n with b o r o h y d r i d e . T h e o l i g o s a c c h a r i d e was therefore shown to be t r i s a c c h a r i d e h a v i n g g l u c o s e as the r e d u c i n g m o i e t y .

T h e mass spectrum of the T . M . S . ether o f r e d u c e d

C h a d ions a t 451 as e x p e c t e d for fission of the substituted terminal hexose a n d 525 from the a l d i t o l m o i e t y , thus p r o v i d i n g e v i d e n c e that the t r i s a c c h a r i d e is not b r a n c h e d .

Further more the series o f fragments o b t a i n e d a t

m / e ratios 1 0 3 , 205 a n d 3 0 7 were those p r e d i c t e d from a 4 - l i n k e d r e d u c e d g l u c o s e residue a n d this was c o n f i r m e d w i t h a deuterium l a b e l l i n g e x p e r i ment.

(Figure 3)

The r e d u c e d o l i g o s a c c h a r i d e was then c o n v e r t e d into

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

The retention times o f the three

resulting peaks were c o m p a r e substitution pattern of the lished as e i t h e r 2-substituted mannose or 3-substituted g l u c o s e .

(Figure 4)

The s e q u e n c e o f sugar residues in the t r i s a c c h a r i d e a n d their a n o m e r i c c o n f i g u r a t i o n was then c l e a r l y shown by the use o f the e n z y m e sidase.

a-manno-

This e n z y m e c l e a v e d the sugar into mannose a n d e e l I o b î o s e d e m o n -

strating that it is i n d e e d

a-mannosyl eel I o b i ose o f the structure s h o w n .

(Figure 5) U s i n g a s i m i l a r a p p r o a c h the structures o f the other mannose c o n t a i n i n g o l i g o s a c c h a r i d e s were e l u c i d a t e d .

In the case o f the mannose c o n t a i n i n g

d î s a c c h a r î d e (E) the position of the mannosyl substituent was d e t e r m i n e d using the l e a d t e t r a a c e t a t e o x i d a t i o n method d e s c r i b e d b y Perl i n . (8) O n hydrolysis o f the o x i d i s e d d î s a c c h a r î d e , arabinose was d e t e c t e d , s h o w i n g that mannose was l i n k e d to O 3 . (Figure 6) The b r a n c h e d t r i s a c c h a r i d e (B) was u n e x p e c t e d l y resistant to the a c t i o n of both

a-mannosidase

and

β - g l u c o s î d a s e presumably through s t e r i c h i n d e r a n c e o f a d j a c e n t hexoses on O 3 a n d O 4 a n d therefore p a r t i a l a c i d hydrolysis was used for this f a c e t o f the structural i n v e s t i g a t i o n .

The a c i d i c d î s a c c h a r î d e (A) was shown to

c o n t a i n g l u c u r o n i c a c i d a n d mannose in r o u g h l y e q u a l proportions a n d was assumed to be the a l d o b i u r o n i c a c i d p r e v i o u s l y i s o l a t e d from the g u m . O l i g o s a c c h a r i d e D was shown to be c e 11 o b i ose by c o - c h r o m a tography w i t h a n a u t h e n t i c sample on paper a n d gas c h r o m a t o g r a p h y .

(Figure 7)

A l l o f the sugars in the n e w l y proposed r e p e a t i n g unit o f the p o l y s a c c h a r i d e w i t h the s i n g l e e x c e p t i o n o f the terminal mannose residue a r e represented in a t least one o f the o l i g o m e r s , a n d our results are e n t i r e l y consistent w i t h the r e v i s e d s t r u c t u r e .

(Figure 8)

It is possible that the c o v a l e n t structures o f gums p r o d u c e d under d i f f e r e n t c o n d i t i o n s may vary i n some w a y , for e x a m p l e , a f t e r c h e m i c a l treatment.

(10)

T h e r e is e v i d e n c e a l s o that structural v a r i a t i o n may o c c u r

in gums from d i f f e r e n t species o f xanthomonas (9).

V a r i a t i o n in structure

is l i k e l y to be a s s o c i a t e d w i t h v a r i a t i o n in p h y s i c a l properties a n d it is possible that a range o f xanthan gum types c o u l d be d e v e l o p e d to g i v e a


14.

Structure

LAWSON AND SYMES

Elucidation

of Xanthan

187

Gum

CH OTMS 2

TMSOHEX .

HEX -

307(308) -OTMS

_205

ITMSO-

103 CH OTMS 2

451 (452)

451

Figure 3. Mass spectroscopy of the perO-trimethyhilyl ether of the derived glycitol from oligosaccharide C (Figures in parentheses are after NaBD reduction) h

[Man]1or >3[Glc]1->

[GlclH CH OAc

ÇH OAc

2

2

-Ο Me

+

AcO-OMe

OR

-OAc CH OMe 2

CH OMe 3

Ο Me MeO-OAc -OMe CH OMe

CH OMe

2

2

•2[Man]1->

•UGlucitol)

Figure 4. Partially methylated alditol acetates possible from gas chromato graphic evidence

oc-Mannosidase Ψ MANNOSE +

CELL0BI0SE

Figure 5. Action of a-mannosidase on oligosaccharide C


188


-OHpH

OH

|Pb(OAc)

4

O H C ^ Î Î )

OA^CHO

H CT 3

HOH C 2

HO? OH Η Figure 6.

OH

Lead tetraacetate oxidation of disaccharide Ε

Arabinose

A.

p-P-GlcAp-(l->2)-P-Mang

B.

B-D-Glcp-(B4)-D-Glcp

i< - D - M a n p

C.

B - D - G l c p - ( l-*4> - D - G l c p

3

Î l I a. - D - M a n p

D. Figure 7. Oligosaccharides from acetolysis of xanthan gum

Ε.

p-D-Glcp-(l->4)-D-Glcp

* -D-Manp-(l->3) - D - G I cp


14.

LAWSON

AND

SYMES

Structure

Elucidation

of Xanthan

Ε

189

Gum

C

- ——ι

• GA«

11

D

1

— M-

»GA-

•Μ ι

M

JL

1

I

!

•M

-,

G-4i

— .

> GA-

A Figure 8.

GA
.98 0. 74 2 22

S o u r c e : I n t e r n a t i o n a l T r a d e C e n t e r , 1972 u p d a t e d f r o m C h e m i c a l Market Reporter.

TABLE I V WORLD PRODUCTION OF SELECTED GUMS Gum

Year

Agar 1 Alginate 1 Arabic 2 Carrageenan 1 Furcellaran 1 L o c u s t Bean 2 Methylcellulose 2 Pectin 2 Carboxymethylcellulose Xanthan

1973 1973 1966 1973 1973 1970 1972 1971 1969 1975

Sources:

2

INDUSTRIAL

Production

(tons)

7,950 17,000 60,000 8,000 1,200 15,000 25,000 9,000 60,000 5,000

1) J . N a y l o r FA0 P r o d u c t i o n , T r a d e and U t i l i z a t i o n o f Seaweed P r o d u c t s (1976). 2) R . L . W h i s t l e r

I n d u s t r i a l Gums ( 1 9 7 3 ) .


304


As p r e v i o u s l y m e n t i o n e d the f o l l o w i n g c l a s s e s :

t h e m a i n gums a r e c l a s s i f i e d

into

N a t u r a l P r o d u c t s . The f o u r most i m p o r t a n t h y d r o c o l l o i d s i n t h i s c l a s s a r e g e l a t i n , p e c t i n , d e x t r a n and x a n t h a n . I n t h e food i n d u s t r y over twenty thousand tons o f n a t u r a l p o l y s a c c h a r i d e s w e r e s o l d i n t h e U n i t e d S t a t e s i n 1975. T h i s was b r o k e n down i n t o 16,000 t o n s g e l a t i n e , 2,000 t o n s x a n t h a n and 3,000 tons p e c t i n . G e l a t i n i s p r e f e r r e d i n g e l a t i n d e s s e r t s , meat p r o d u c t s s u c h a s ham and l u n c h e o n meat and d a i r y p r o d u c t s . The p h o t o g r a p h i c and p h a r m a c e u t i c a l i n d u s t r i e s a r e t h e l a r g e s t u s e r s of h i g h grade g e l a t i n e . P e c t i n , because o f i t s g e l forming p r o p e r t i e s w i t h sucrose i s u s e d i n jams a n d c o n f e c t i n e r y in salad dressings, citru ducts as w e l l as o i l d r i l l i n g recovery Seaweed E x t r a c t s . Seaweed e x t r a c t s a r e o b t a i n e d f r o m two groups o f a l g a e , r e d algae which i s t h e source o f carrageenan and a g a r a n d b r o w n a l g a e w h i c h i s t h e s o u r c e o f a l g i n a t e s . The r e c e n t FA0 Seaweed R e s o u r c e s o f t h e W o r l d r e p o r t s l a r g e p o t e n t i a l f o r expansion i n t h i s group. P r e s e n t h a r v e s t s o f r e d and brown a l g a e a r e p u t a t 0.807 a n d 1.315 m i l l i o n t o n s r e s p e c t i v e l y w i t h p o t e n t i a l o u t p u t s l i s t e d a t 2.66 and 14.6 m i l l i o n t o n s of algae. E a c h o f t h e s e gums, h a s u n i q u e p r o p e r t i e s g i v i n g them e x c e l l e n t market p o t e n t i a l . Because o f i t s l o w g e l l i n g and heat r e s i s t a n c e , agar has a wide usage i n f o o d s . A l g i n a t e s a r e t h e most e x t e n s i v e l y u s e d gum o f t h e g r o u p and a r e u s e d i n d a i r y p r o d u c t s , c i t r u s b e v e r a g e s , b a k e r y f i l l i n g s , l i q u i d a n i m a l f e e d s , p h a r m a c e u t i c a l and many i n d u s t r i a l applications. Carrageenan has t h e l a r g e s t usage o f t h e group i n d a i r y , beverages and bakery p r o d u c t s . I t i s h e a v i l y used i n d a i r y products because o f i t s r e a c t i o n w i t h c a s e i n . Starch Derivatives. N a t u r a l starches are normally processed t o g i v e them p r o p e r t i e s f o r s p e c i a l a p p l i c a t i o n s . B e c a u s e of t h e i r s t r o n g adhesives p r o p e r t i e s , they are used i n adhesives and a r e h i g h l y c o m p e t i t i v e w i t h gum a r a b i c . Other uses i n c l u d e c e r a m i c s , f l o c c u l a t i o n , w e l l d r i l l i n g muds a n d p h a r m a c e u t i c a l s . Seed E x t r a c t s . The two m a j o r gums i n t h e g r o u p a r e g u a r and l o c u s t b e a n gum. G u a r i s o b t a i n e d i n I n d i a a n d P a k i s t a n , w h i l e l o c u s t s b e a n i s h a r v e s t e d i n S p a i n and t h e M e d i t e r r a n e a n a r e a . They a r e b o t h u s e d i n t h e d a i r y i n d u s t r y f o r c h e e s e m a k i n g and i c e c r e a n p r o d u c t i o n . G u a r gum i s t h e p r e f e r r e d i c e c r e a m s t a b i l i z e r . L o c u s t b e a n gum i s a v i s c o s i f i e r a n d b i n d e r o f f r e e w a t e r . Cellulose Derivatives. F o o d , drum, c o s m e t i c and d e n t i f r i c e p r o d u c t s a r e t h e f a s t g r o w i n g u s a g e o f CMC ( s o d i u m c a r b o x y m e t h y l c e l l u l o s e ) a n d MC ( m e t h y l c e l l u l o s e ) . MC c o s t s more t h a n CMC a n d h a s a US p r o d u c t i o n o f 30 m i l l i o n pounds a g a i n s t a US p r o d u c t i o n o f 74 m i l l i o n pounds o f CMC.


22.

WELLS

Extracellular

Microbial

Polysaccharides

305

Tree Exudates. The t r e e e x u d a t e s , gum a r a b i c , gum t r a g a c a n t h a n d gum K a r a y a h a v e a l l l o s t m a r k e t s h a r e s t o t h e s y n t h e t i c s a n d p r o c e s s e d gums. However, gum K a r a y a b e c a u s e o f i t s l a x a t i v e p r o p e r t i e s m a i n t a i n s a market. Gum t r a g a c a n t h h a s a w i d e r a n g e o f a p p l i c a t i o n s i n f o o d , t e x t i l e s , c o s m e t i c s and c e r a m i c s . However b e c a u s e o f s u p p l y d i f f i c u l t i e s and p r i c e d i f f e r e n t i a l i t h a s been r e p l a c e d t o a l a r g e e x t e n t b y x a n t h a n gum. Gum a r a b i c i s u s e d m o s t l y t o p r e v e n t c r y s t a l l i z a t i o n o f s u g a r s and a s a n e m u l s i f i e r t o k e e p f a t s u n i f o r m l y d i s t r i b u t e d . CMC, PVA and m o d i f i e d s t a r c h e s have t a k e n a l a r g e s h a r e o f t h e gum a r a b i c m a r k e t . MICROBIAL POLYSACCHARIDE At t h i s time o n l y m e r c i a l s i g n i f i c a n c e are i n commercial p r o d u c t i o n , dextran, p o l y t r a n a n d x a n t h a n . F i v e o t h e r s show p r o m i s e i n d e v e l o p m e n t and a r e a v a i t i n g d e c i s i o n s o n p o t e n t i a l c o m m e r c i a l e x p l o i t a t i o n . Dextran. Dextrans a r e p o l y g l u c a n s and have been produced i n t h e U n i t e d S t a t e s , C a n a d a , H o l l a n d a n d Sweden. They c a n be s y n t h e s i s e d f r o m s u c r o s e m i c r o b i a l l y f r o m many s t r a i n s o f c e l l f r e e c u l t u r e f i l t r a t e s o f l e u c o n o s t o c m e s e n t e r i d e s , though d e x t r a n s from o t h e r s t r a i n s w i l l d i f f e r both i n s t r u c t u r e and p r o p e r t i e s . M o l e c u l a r w e i g h t s may r a n g e w i d e l y . U s u a l p r a c t i c e i s t o o b t a i n a h i g h m o l e c u l a r weight m a t e r i a l and degrade i t by h y d r o l y s i s , s i n c e the d e x t r a n s t h a t are used i n the food i n d u s t r y must h a v e m o l e c u l a r w e i g h t s b e l o w 100,000, s i n c e o n l y t h e y a r e i n c l u d e d i n t h e GRAS l i s t o f t h e FDA. D e x t r a n s o l u t i o n s a r e c l o s e l y s i m i l a r t o l o c u s t b e a n gum. X a n t h a n . X a n t h a n gum i s p r o d u c e d f r o m g l u c o s e s o l u t i o n i n g r o w i n g c u l t u r e s o f xanthomonas c a m p e s t r i s . Commercial p r o d u c t i o n has been c a r r i e d o u t i n the U n i t e d S t a t e s s i n c e 1967 b y t h e K e l c o Company, who a r e c u r r e n t l y t h e m a i n m a n u f a c t u r e r a n d who p r o d u c e d a n e s t i m a t e d 5,000 t o n s o f x a n t h a n i n 1975. L o c a l e x p a n s i o n o f t h e San D i e g o p l a n t , t o g e t h e r w i t h a g r a s s r o o t s p l a n t i n Oklahoma f o r 10,000 t o n s a t an e s t i m a t e d c o s t o f 35 m i l l i o n d o l l a r s p l u s p l a n t b y Rhone P o u l e n c o f F r a n c e a n d G e n e r a l M i l l s c o u l d make a v a i l a b l e b e t w e e n 35-37J m i l l i o n pounds x a n t h a n b y 1978. T a t e a n d L y l e L t d . and H e r c u l e s I n c . h a v e announced a j o i n t - v e n t u r e t o enter i n t h i s market. C u r r e n t development s t a t u s 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 i s l i s t e d i n A p p e n d i x A. The u n i q u e p h y s i c a l p r o p e r t i e s o f x a n t h a n h a v e f o u n d many i n d u s t r i a l a p p l i c a t i o n s i n such d i v e r s i f i e d i n d u s t r i e s as t e x t i l e p r i n t i n g , d r i l l i n g muds, s u r f a c t a n t f l o o d i n g , r u s t r e m o v e r s , and l i q u i d t y p e o f a n i m a l f e e d s . Most i m p o r t a n t p r e s e n t u s e i s t h e r e c o v e r y o f c r u d e o i l . The f l o w c h a r a c t e r i s t i c s o f x a n t h a n , c o u p l e d w i t h i t s s t a b i l i t y


306


t o pH, g i v e s i t a t e c h n i c a l a d v a n t a g e o v e r o t h e r p o l y m e r s i n d r i l l i n g muds. T h e r e i s a n e s t i m a t e d w o r l d u s a g e o f 1,800 t o n s in drilling applications. S c l e r o g l u c a n ( P o l y t r a n ) . T h i s p o l y s a c c h a r i d e has been dev e l o p e d by t h e P i l l s b u r y Company a n d m a r k e t e d u n d e r t h e t r a d e name o f P o l y t r a n . P i l l s b u r y c l a i m i m p o r t a n t f l o w c h a r a c t e r i s t i c s o v e r a w i d e r a n g e o f pH a n d t e m p e r a t u r e and s t a b i l i t y i n t h e presence of s a l t s . P o l y t r a n w i l l s t a b i l i z e bentonite c l a y s d u r i n g s t o r a g e , o v e r r a n g e s o f t e m p e r a t u r e a n d pH. I t i s u s e d i n t h e c e r a m i c , d r i l l i n g mud, and i n k s a n d c o a t i n g s i n d u s t r i e s . P r i c e s i n t h e r e g i o n o f 9-10,000 d o l l a r s p e r t o n p u t i t i n comp e t i t i o n w i t h x a n t h a n gum. OTHER MICROBIAL POLYSACCHARIDE Pullulan. This polysaccharid H a y a s h a b a r a Company i J a p a n C o m m e r c i a l i n t e r e s t h a s b e e n shown o n a c c o u n t o f i t s a b i l i t y t o f o r m s t r o n g r e s i l i e n t f i l m s and f i b e r s a n d t h e e a s e i t c a n be m o l d e d i n t o s h a p e s . A t p r e s e n t p u l l u l a n i s o n l y i n t h e p i l o t plant stage. C o n s t r u c t i o n o f a p r o d u c t i o n p l a n t was r e p o r t e d t o h a v e s t a r t e d i n 1975. P a t e n t s c l a i m i n g b o t h f o o d a n d i n d u s t r i a l a p p l i c a t i o n s have been f i l e d . M i c r o b i a l A l g i n a t e . A l g i n i c a c i d a n d a l g i n a t e s a r e most i m p o r t a n t gums w i t h many a p p l i c a t i o n s i n f o o d , t e x t i l e , pharmac e u t i c a l a n d p a p e r i n d u s t r i e s . P r o d u c t s o b t a i n e d f r o m seaweed v a r y i n b o t h , q u a l i t y and s t r u c t u r e . S e v e r a l m i c r o o r g a n i s m s produce m i c r o b i a l a l g i n a t e s v e r y s i m i l a r t o a l g a l a l g i n a t e . The c o m p o s i t i o n o f t h e s e p o l y m e r s f o r m e d i s r e p o r t e d t o b e u n e f f e c t e d by t h e c a r b o h y d r a t e source used, and o f c o n s t a n t quality. Most o f t h e main development i n m i c r o b i a l a l g i n a t e s has b e e n c l a i m e d b y T a t e a n d L y l e . Curdlan. C u r d l a n h a s b e e n d e v e l o p e d b y t h e T a k e d a Company i n Japan from a c h e m i c a l mutant o f A l c a l i g e n e s f a e c a l i s v a r myxogenes IOCS. I t s i n d u s t r i a l d e v e l o p m e n t depends o n t h e g e l s t r e n g t h o f h i g h s e t g e l s n o t r e v e r t i n g g r e a t l y when c o o l e d . Aqueous s u s p e n s i o n s o f t h e p o l y m e r r e m a i n s o f t and r e s i l i e n t when c o o l e d , a f t e r h e a t i n g . A p p l i c a t i o n s a r e i m m o b i l i z e d enzymes a s w e l l a s b e i n g u s e d i n p r e p a r a t i o n o f f i l m s and g e l . E r w i n a ( Z a n f l o ) . E r w i n a was d e v e l o p e d b y t h e K e l c o Company s p e c i a l l y f o r c a r p e t p r i n t i n g a p p l i c a t i o n s , due t o i t s c o m p a t i b i l i t y t o c a t i o n i c dyes. I t i s produced from a s t r a i n o f Erwina t a h i t i c a . I t has been c l a i m e d t h a t t h i s p o l y s a c c h a r i d e possesses p s e u d o p l a s t i c i t y , pH s t a b i l i t y and f r e e z e - t h a w s t a b i l i t y . The e x c e l l e n t r e s i s t a n c e t o enzyme a t t a c k , a n d i t s f l o w and l e v e l l i n g q u a l i t i e s h a v e a l r e a d y made i t f i n d a p p l i c a t i o n in thepaint industry.


22.

WELLS

Extracellular

Microbial

Polysaccharides

307

INDUSTRIAL DEVELOPMENT OF MICROBIAL POLYSACCHARIDES. D e x t r a n was f i r s t p r o d u c e d c o m m e r c i a l l y i n Sweden i n t h e e a r l y f o r t i e s , t h e n l a t e r i n E n g l a n d , Canada and t h e U n i t e d States. X a n t h a n was f i r s t p r o d u c e d c o m m e r c i a l l y i n t h e U n i t e d States i nthe early s i x t i e s f o r i n d u s t r i a l applications. I n 1969 FDA c l e a r e d t h e g e n e r a l u s e o f x a n t h a n gum i n f o o d s where t h e s t a n d a r d s o f i d e n t i t y do n o t p r e c l u d e i t s u s e . I n 1973 FDA a l l o w e d u s e s i n p r o c e s s a n d cream c h e e s e s a s a t h i c k e n i n g and s t a b i l i z i n g a g e n t . I n 1974 MID/PID I n s p e c t i o n D i v i s i o n o f USDA i n c l u d e d x a n t h a n gums o n t h e i r a u t h o r i z e d l i s t o f n o n meat i n g r e d i e n t s . One c a n c o n v e n i e n t l y d i v i d e up t h e m a i n m a r k e t d e v e l o p ment o f m i c r o b i a l gums - Food a p p l i c a t i o n - P e t r o l e u m and o i l i n d u s t r y a p p l i c a t i o n s - Other a p p l i c a t i o n s . Food A p p l i c a t i o n s . Over 60% 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 s a l e s go t o t h e f o o d i n d u s t r y . I n 1975, K e l c o Company a r e s a i d t o have s o l d o v e r 5 m i l l i o n pounds o f x a n t h a n gums i n t o t h e US f o o d i n d u s t r y . X a n t h a n gums have g a i n e d r a p i d acceptance i n t o t h e U n i t e d S t a t e s f o o d i n d u s t r y a n d a p p l i c a t i o n s a r e now b e i n g d e v e l o p e d i n b o t h E u r o p e a n d J a p a n . Denmark, E n g l a n d , I r e l a n d , H o l l a n d , S p a i n a n d Canada h a v e g i v e n r e g u l a t o r y a p p r o v a l . A p p r o v a l s i n F r a n c e , Sweden a n d B e l g i u m a r e e x p e c t e d b e f o r e t h e end o f 1976. I n i t i a l l y the applications followed i n the United States f o r u s e i n s a l a d d r e s s i n g s , meat a n a l o g s , p e t f o o d , b a k e r y p r o d u c t s , c a r b o n a t e d b e v e r a g e s a n d f r o z e n f o o d s w i l l be s t u d i e d . New d e v e l o p m e n t s b y t h e USDA, announced d u r i n g 1 9 7 5 , f o r p r o d u c i n g m a t r i x t e x t u r e s f o r f o o d s and s n a c k f o o d s c o u l d open up v e r y l a r g e d e v e l o p m e n t s . J o i n t p a t e n t s b e t w e e n K e l c o Company and DCA g i v e p r o m i s e o f c h a n g e s i n t h e b a k e r y i n d u s t r y . I t i s r e p o r t e d t h a t a j o i n t - v e n t u r e i n Japan w i t h t h i s group and N i s s h i n F l o u r c o u l d open p o t e n t i a l m a r k e t s i n d o n u t s , o n i o n r i n g p r o c e s s i n g , s n a c k i t e m s a n d c e r t a i n new b a k e r y p r o d u c t s . I n o r d e r t o s e e how t h e s e m i c r o b i a l p o l y s a c c h a r i d e s f i t i n t o t h e o v e r a l l m a r k e t i t i s n e c e s s a r y t o s t u d y t h e t o t a l US c o n s u m p t i o n o f gums i n 1 9 7 5 , w h i c h i s g i v e n i n T a b l e V I . ( X a n t h a n w i t h US s a l e s o f 2,500 t o n s i s c l a s s i f i e d a s a n a t u r a l product. I n i t i a l l y x a n t h a n gums h a v e o b t a i n e d t h e i r m a i n m a r k e t s by r e p l a c i n g gum t r a g a c a n t h . However t h i s m a r k e t h a s p r a c t i c a l l y d i s a p p e a r e d i n t h e US. Though t h e r e e x i s t s s e v e r a l t h o u s a n d tons p o t e n t i a l elsewhere. The w o r l d m a r k e t f o r a l g i n a t e s i s o v e r 17,000 t o n s w i t h about f i v e thousand tons u t i l i z e d i n t h e food i n d u s t r y . About 2,000 t o n s a r e u s e d i n t h e U n i t e d S t a t e s and n e a r l y 1,500 t o n s i n Europe. L a r g e s t a p p l i c a t i o n s a r e i n d a i r y and bakery p r o d u c t s , where c o n s u m p t i o n i s e x p e c t e d t o expand. T h i s c o u l d



308

TABLE V I US CONSUMPTION OF HYDROCOLLOIDS IN FOODS IN Thousand tons

Product

1975 Million dollars

Natural products Starch derivatives Seaweed e x t r a c t s Tree exudates Seed e x t r a c t s Cellulose derivatives

20.5 124.2 4.1 1.4 5.9 5.9

113 64 19 7 10 12

TOTAL

162.0

225

Source:

James H i c k e y -

open e x c e l l e n t m a r k e t s t o m i c r o b i a l a l g i n a t e s i f t h e y c o u l d be produced at e q u i v a l e n t p r i c e s t o the a l g a l product. In g e n e r a l the shortages of n a t u r a l l y o c c u r r i n g hydroc o l l o i d s i n 1974 showed t h e v u l n e r a b i l i t y o f t h i s m a r k e t t o l e s s c o s t l y m a n u f a c t u r e d gums. I n g e n e r a l t h e m a i n f o o d p r o c e s s o r s w i l l t e n d t o p r e f e r t o f o r m u l a t e w i t h t h e s e manuf a c t u r e d gums, whose s u p p l y , q u a l i t y and p r i c e a r e n o t s u b j e c t t o v a g a r a n c i e s o f s u p p l y , w e a t h e r , p o l i t i c s and l a b o u r c o s t s . The E u r o p e a n and J a p a n e s e m a r k e t s a r e e x p e c t e d t o o f f e r l a r g e p o t e n t i a l f o r d e v e l o p m e n t . I t must be remembered t h a t o v e r 17,000 t o n s o f x a n t h a n w i l l be a v a i l a b l e p e r y e a r a f t e r 1978 and o v e r h a l f o f t h i s must be a b s o r b e d by t h e F o o d I n d u s t r y . Hence t h e m a i n m a r k e t i n g e f f o r t s o f x a n t h a n must n e c e s s a r i l y move t o E u r o p e . P r o b a b l y a d i f f e r e n t r a n g e o f a p p l i c a t i o n s w i l l e v e n t u a l l y d o m i n a t e o u t s i d e t h e US s i n c e use o f s a l a d d r e s s i n g s , meat a n a l o g s , and c a r b o n a t e d b e v e r a g e s i s n o t so d e v e l o p e d . F o r example f r u i t y o g h o u r t m a r k e t s a r e many t i m e s l a r g e r t h a n i n t h e US. The l a r g e s t E u r o p e a n h y d r o c o l l o i d f o o d u s a g e i s i n t h e m o d i f i e d s t a r c h f i e l d . O v e r 150,000 t o n s u s a g e has b e e n r e p o r t e d t o be u s e d i n t h e E u r o p e a n Community. P r o b a b l y t h e b e s t g r o w t h r a t e comes f r o m c e l l u l o s e dérivâtes. CMC a t 60 c e n t s p e r pound has made g r e a t i n r o a d s i n t o t h e s e e d gum m a r k e t . L a r g e v o l u m e s o f CMC a r e r e p o r t e d t o be g o i n g i n t o i n s t a n t soups and c a k e m i x e s , a m a r k e t t h a t x a n t h a n i s also t r y i n g to penetrate. I n summary t h e f o o d i n g r e d i e n t s m a r k e t i s v e r y c o m p l i c a t e d and o n l y t h e most t e c h n i c a l l y c o m p e t e n t and t e c h n i c a l l y m a r k e t ed o r i e n t e d w i l l s u r v i v e . To s e l l gums i n t o new f o o d p r o d u c t s demands a s o p h i s t i c a t e d t e c h n i c a l i n p u t . I t i s e s s e n t i a l t o u n d e r s t a n d p o t e n t i a l a p p l i c a t i o n . Gums a r e m u l t i f u n c t i o n a l . X a n t h a n added t o r e p l a c e an e m u l s i f i e r , w i l l a l s o i n c r e a s e viscosity. T h i s can c a u s e p r o b l e m s i f t h e o t h e r t h i c k e n e r s a r e not reduced.


22.

WELLS

Extracellular

Microbial

Polysaccharides

309

Petroleum and O i l I n d u s t r y A p p l i c a t i o n s . Polymers a r e f i n d i n g i n c r e a s i n g usage i n the o i l i n d u s t r y and developments a r e f o r e c a s t w h i c h c o u l d open u n l i m i t e d p o t e n t i a l . A t t h i s t i m e the market remains i n e x p l o r a t i o n usage. Market developments h e r e h a s b e e n d i v i d e d i n t o two s e g m e n t s : o i l d r i l l i n g muds a n d enhanced o i l r e c o v e r y . O i l d r i l l i n g muds. The f o u r m a j o r p o l y m e r s i n u s e a t t h i s time are xanthan, p o l y a c r y l a m i d e s , m o d i f i e d s t a r c h e s and c e l l u l o s e d e r i v a t i v e s p a r t i c u l a r l y CMC. D e x t r a n a n d p u l l u l a n a r e a l s o trying to get into this industry. B e c a u s e o f i t s s t a b i l i t y t o pH, h e a t , c a t i o n s a n d d i v a l e n t i o n s coupled t o i t s p s e u d o p l a s t i c behaviour under c o n d i t i o n s o f h i g h s h e a r , x a n t h a n gums a r e t h e t e c h n i c a l l y p r e f e r r e d p o l y m e r for l u b r i c a t i o n o f b e n t o n i t mud d t d r i l l o i l wells D u r i n g 1^75 a b o u t 1,80 ing operations w i t h a p o t e n t i a usag predicte y 1980. However r e c e n t p r i c e i n c r e a s e s h a v e c a u s e d s e v e r a l o f t h e m a j o r s t o s w i t c h some o f t h e u s a g e t o CMC e v e n t h o u g h i t t a k e s a l m o s t d o u b l e t h e amount o f CMC t o a c h i e v e t h e same p e r f o r m a n c e effects. X a n t h a n i s a l s o much u s e d i n s u m u l t a n e o u s w a t e r f l o o d i n g and p u s h i n g t e c h n i q u e s u s e d i n t h e N o r t h Sea, where s e a w a t e r c o n t a i n i n g s m a l l q u a n t i t i e s o f x a n t h a n (100 ppm) a r e p u s h e d into injection wells. However, p o l y a c r y l a m i d e s a r e a l s o b e i n g c o n s i d e r e d f o r t h i s a p p l i c a t i o n due t o l o w e r p r i c e . The d r i l l i n g s e r v i c e i n d u s t r y i s c o n t r o l l e d b y a s m a l l number o f s e r v i c e c o m p a n i e s i n c l u d i n g B a r o i d , M i l c h e m , Imco, D r e s s e r i n t h e US w i t h C r o d a and C e c a f r o m E u r o p e . They r e s e l l x a n t h a n o b t a i n e d f r o m K e l c o , G e n e r a l M i l l s o r Rhone Poulenc. Enhanced o i l r e c o v e r y . The g r e a t e s t f u t u r e p o t e n t i a l f o r p o l y s a c c h a r i d e s , l i e s i n enhanced o i l r e c o v e r y . Great i n t e r e s t and r e s e a r c h e f f o r t i s b e i n g c e n t e r e d on r e c o v e r i n g the l a r g e f r a c t i o n o f o r i g i n a l o i l remaining i n p l a c e i n o i l s t r a t a a f t e r c o n v e n t i o n a l r e c o v e r y methods have been u t i l i z e d . A l a r g e i n c e n t i v e f o r d e v e l o p i n g r e c o v e r y enhancement methods e x i s t s i n t h e U n i t e d S t a t e s a s t h e p e r c e n t a g e o f i m p o r t ed o i l f o r d o m e s t i c p u r p o s e s i s i n c r e a s i n g v e r y r a p i d l y . A c c o r d i n g t o the American Petroleum I n s t i t u t e , o f t h e 440 b i l l i o n b a r r e l s o f o i l d i s c o v e r e d i n t h e U n i t e d S t a t e s b y t h e e n d o f 1974, 295.8 b i l l i o n b a r r e l s w o u l d h a v e t o b e r e c o v e r e d b y e n h a n c e d r e c o v e r y , o r a d v a n c e d enhanced r e c o v e r y techniques. I n 1973 t h e G u l f U n i v e r s i t i e s R e s e a r c h C o n s o r t i u m i n a s t u d y w i t h many o f t h e l a r g e o i l c o m p a n i e s s t a r t e d a s e r i e s o f e n h a n c e d r e c o v e r y . I n 1974 v e r y h i g h l y o p t i m i s t i c p r e d i c t i o n s o n s u r f a c t a n t f l o o d i n g gave r i s e t o e s t i m a t e s o f r e c o v e r i n g 500,000 s t o c k t a n k b a r r e l s (STB) p e r day. However s i n c e


310


t h a t t i m e , r e d u c e d e s t i m a t e s and l o n g e r r e a l i z a t i o n t i m e s have been p r e d i c t e d . A t t h e e n d o f 1975, t h e c o n s o r t i u m were p r e d i c t i n g a n n u a l p r o d u c t i o n r a t e s f o r 1985 o f enhanced o i l b e t w e e n 300-400 m i l l i o n STB, w h i c h w o u l d c a l l f o r a n a n n u a l p o l y m e r demand o f b e t w e e n 200-250 m i l l i o n pounds b a s e d o n 5 8 % E0R b y s u r f a c t ant f l o o d i n g . However i n 1976, t h e G u l f C o n s o r t i u m h a s how l o w e r e d i t s 1986 p r e d i c t i o n t o a r e a l i s t i c g o a l o f 200,000 STB p e r d a y , w i t h a s t a r t i n g d a t e f o r l a r g e s c a l e d e v e l o p m e n t i n 1979. Table 7 gives t h e polymer requirements f o r both cases s t u d i e d . T h i s s t u d y h a s assumed t h a t t h e m a r k e t i s e q u a l l y s h a r e d between p o l y s a c c h a r i d e s and p o l y a c r y l a m i d e s .

POLYMER DEMAN (Thousands o f pounds p e r d a y ) Year

Case A

1979 1980 1981 1982 1983 1984 1985 1986

7.3 14.6 32.8 51.1 76.7 11.0 120.5 138.5

Source:

Case Β 18.3 40.2 79.0 131.5 175.2 215.2 233.6 248.2

G u l f U n i v e r s i t i e s R e s e a r c h C o n s o r t i u m , M a r c h 1976 1. P o l y m e r s demand assumed t o b e 5 0 % p o l y s a c c h a r i d e s , 50% p o l y a c r y l a m i d e . 2. C a s e A - assumes d e v e l o p m e n t t o 200,000 STB/day by 1986. C a s e Β - Assumes d e v e l o p m e n t t o 500,000 STB/day by 1986.

Hence t h i s d e l p h i t y p e e x e r c i s e p r e d i c t s m a r k e t s o f b e t w e e n t o 140 t h o u s a n d pounds p o l y m e r demand p e r d a y b y 1986 s t a r t a t b e t w e e n 7,300 t o 18,300 pounds d a i l y i n 1979. A s s u m i n g t h i s p o t e n t i a l t o be c o r r e c t , t h e n e x t p r o b l e m t o be r e s o l v e d b y t h o s e d e v e l o p i n g p o l y s a c c h a r i d e p o l y m e r s , w i l l b e t h e p o t e n t i a l s p l i t b e t w e e n p o l y s a c c h a r i d e and p o l y a c r y l a m i d e s . The d i f f i c u l t y i n p r e d i c t i n g f u t u r e t r e n d s ( a c c o r d i n g t o t h e Gulf Consortium) i s the general d i s s a t i s f a c t i o n w i t h the current generation of materials. P o l y a c r y l a m i d e s a r e i n t h e r i g h t p r i c e range b u t a r e d e s c r i b ed a s u n d u l y s h e a r s e n s i t i v e a n d s a l t s e n s i t i v e . T a b l e V I I I shows d e l p h i a n a l y s i s o f v a r i o u s f i g u r e s d i s c u s s e d i n numerous s t u d i e s s i n c e 1974. 250 ing


22.

Extracellular

WELLS

Microbial

Polysaccharides

311

TABLE V I I I USE OF POLYACRYLAMIDE AND POLYSACCHARIDE FOR EOR Polyacrylamide Viscosity

thickening

S a l i n i t y maximum o f f l u i d Maximum r e s e r v o i r t e m p e r ature Divalent

i o n maximum

Polysaccharide

Ideal

10-15 cp a t 10-15 c p a t 20 c p a t 500 ppm 100 ppm 500 ppm 1500-2000 ppm

10000 ppm

15000 ppm

175-200°F

200-225°F

up t o 250°F

200 ppm

5000 ppm

5000 ppm

Permeability o i l pluggin C o s t t/lb

1.30+0.3

T h i s i s b a s e d o n t h e G u l f C o n s o r t i u m recommend c h a r g e o f 10 pounds o f s u r f a c t a n t s , 3 pounds o f a l c o h o l s a n d a b o u t 1 pound of polymer per b a r r e l . Though t h e u s e o f a l c o h o l i s i n some d o u b t , s i n c e i t may be b e t t e r t o i n c r e a s e t h e s u l p h o n a t e r a t i o a t t h e expense o f t h e alcohol. I t s h o u l d a l s o be p o i n t e d o u t t h a t s u r f a c t a n t f l o o d i n g i s n o t t h e o n l y method o f e n h a n c e d r e c o v e r y . B a s i c r e c o v e r y p r o c e s s e s have b e e n a p p o r t i o n e d a s f o l l o w s : - Surfactant recovery 58% - Thermal recovery 29% - Carbon d i o x i d e processes 8% -Hydrocarbon m i s c i b l e processes 5% A recent development i s s t u d y i n g f e a s i b i l i t y o f d e v e l o p i n g s m a l l s c a l e u n i t s t o produce s u r f a c t a n t charges i n c l u d i n g p o l y s a c c h a r i d e s a t t h e o i l f i e l d s i t e . I t has been n o t e d t h a t EOR h a s s t i l l many t e c h n i c a l p r o b l e m s t o s o l v e b e f o r e i t w i l l be a c o m m e r c i a l r e a l i t y . However t h e i n c r e a s i n g f i n a n c i a l s u p p o r t by Energy R e s e a r c h and Development A d m i n i s t r a t i o n i s most w e l l c o m e a n d i n d i c a t e s p o l i t i c a l b a c k i n g w h i c h i s e s s e n t i a l t o make t h i s d e v e l o p m e n t a r e a l i t y . O t h e r A p p l i c a t i o n s . The o t h e r a p p l i c a t i o n s 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 h a v e come f r o m t a k i n g t h e m a r k e t o f t h e n a t u r a l p l a n t gums w i t h more r e l i a b l e o r t a i l o r made p r o d u c t s . Z a n f l o has o b v i o u s l y been developed f o r p a i n t and d y i n g applications. Other p o l y s a c c h a r i d e s have found markets i n t e x t i l e s , c o s m e t i c s , p h a r m a c e u t i c a l s and l i q u i d f e e d s . S e v e r a l p a t e n t s have r e c e n t l y been i s s u e d i n Japan f o r p r o d u c t a p p l i cations i n anticancer preparations. The i n d u s t r i a l u s e s a r e more c o m p l i c a t e d t h a n f o o d u s e s and a r e due t o r h e o l o g i c a l p r o p e r t i e s a n d w i d e r a n g e s o f s t a b i l i t y and c o m p a t a b i l i t y w i t h c o n v e n t i o n a l t i c k e n i n g agents and s u r f a c e a c t i v e a g e n t s .


312


The s y n e r g i s t i c e f f e c t s o f x a n t h a n w i t h l o c u s t bean gums is well exploited. However o n a c c o u n t o f t h e c o m p l e x i t y , most o f t h i s know-how r e m a i n s t h e c o n f i d e n t i a l p r o p e r t y o f t h e p r o c e s s o r and s u p p l i e r o f t h e gum. CONCLUSION T h i s paper has t r i e d t o e s t a b l i s h t h e p l a c e o f m i c r o b i a l l y p r o d u c e d gums i n a n e x p a n d i n g i n d u s t r i a l gum m a r k e t . I t i s c l e a r t h a t x a n t h a n gum p r o d u c t i o n w i l l r e m a i n a t a p l a t e a u u n t i l t h e the new p r o d u c t coming i n t o p r o d u c t i o n i s a b s o r b e d . The w h o l e i n d u s t r y r e q u i r e s a v e r y h i g h l e v e l o f t e c h n i c a l e x p e r t i s e and m a r k e t i n g s k i l l t o d e v e l o p i n d u s t r i a l u s a g e . The f u t u r e l a r g e p o t e n t i a l s i n enhanced o i l r e c o v e r y a r e s t i l l a l o n g way o f f a n d much the o i l p r o d u c e r s a n d cial reality. P r o d u c t i o n problems o f f e r m e n t a t i o n d r y i n g and r e c o n s t i t u t i n g d i l u t e s o l u t i o n must be s o l v e d . On a c c o u n t o f t h e l a r g e d e v e l o p m e n t c o s t s n e c e s s a r y f o r b o t h t e c h n i c a l a n d m a r k e t d e v e l o p m e n t i t must b e c o n c l u d e d t h a t o n l y companies d e v e l o p i n g whole range o f m i c r o b i a l p r o d u c t s w i l l predominate. APPENDIX "A" BIOPOLYMER CAPACITY SUMMARY ( M a i n l y Xanthan) tons/year) Date

Capacity

Company

Affiliates

Location

KELCO

Merck subsidiary

San Diego

KELCO

Merck subsidiary

Oklahoma

10,000

End 1976

BIOSYNTHESEMELLE

Rhone P o u l e n c Melle General M i l l s (France)

2,000

Existing

GENERAL MILLS

Rhone P o u l e n c

Iowa

2,500

Mid

TATE & LYLE

Hercules Inc.

NA°

NA°

NA°

TATE & LYLE

Hercules Inc.

NA°

NA°

NA°

T o t a l known c a p a c i t y b y end 1979

3,500

x

Existing

1977

18,000+ M e t r i c

Tons

E s t i m a t e d " c o n v e n t i o n a l " m a r k e t s b y end 1979 15-16,000 M Tons x B e i n g expanded t o 5 , 0 0 0 t / y r b u t i n c l u d e s d e v e l o p m e n t facilities. ° NA = n o t announced.


22.

WELLS

Extracellular

Microbial

Polysaccharides

313

BIBLIOGRAPHY A r n o l d C.W, " C h e m i c a l C h a l l e n g e s i n t h e Q u e s t f o r E n h a n c e d O i l R e c o v e r y " - A m e r i c a n C h e m i c a l S o c i e t y ( A p r i l 4-9 1976). G o g a r t y W.B. " S t a t u s o f S u r f a c t a n t o r M i c e l l a r Methods S.P.E." N a t i o n a l M e e t i n g , D a l l a s , T e x a s ( S e p t . 28 - O c t . 1 1975). H i c k e y J.R. " T h i c k e n e r s a n d S t a b i l i z e r s f o r F o o d ECMRA M e e t i n g of M a r k e t " - Development A n a l y s t s M e e t i n g , London ( A p r i l 1976). K a n g K . S . a n d K o v a c s P. " I n t . C o n g r e s s o f F o o d S c i e n c e " M a d r i d (1974). J e a n e s A. "Food T e c h n o l o g y " (May 1974) 34-39. K e l c o Company " T e c h n i c a K i m u r a H. " A b s t r a c t 3 2 M i n n e a p o l i s (1972). Lawson J . , S u t h e r l a n d I.W. " P o l y s a c c h a r i d e s f r o m M i c r o o r g a n i s m s i n Economic M i c r o b i o l o g y ; ed. by A.H. Rose - A c a d e m i c P r e s s ( i n p r e s s ) . " M a r k e t i n g o f P r i n c i p l e W a t e r S o l u b l e Gums i n P r o d u c i n g C o u n t r i e s " I n t e r n a t i o n a l T r a d e C e n t e r , Geneva ( 1 9 7 2 ) . N a y l o r J . " P r o d u c t i o n Trade and U t i l i z a t i o n o f Seaveeds and Seaweed P r o d u c t s " FAO, Rome ( 1 9 7 6 ) . " R e p o r t o n C h e m i c a l Demand and S u p p l y S t u d y " . " R e l a t i n g on M i c r o e m u l s i o n F l o o d i n g G u l f U n i v e r s i t i e s Research C o n s o r t i u m " Houston, Texas (March 26, 1976). S h a r p J.M. "The p o t e n t i a l o f E n h a n c e d R e c o v e r y P r o c e s s e s " S.P.E. M e e t i n g D a l l a s , T e x a s ( S e p t . 2 8 - O c t . 1 1975) Umland C.W. " P r e s e n t a t i o n f o r F e d e r a l E n e r g y A d m i n i s t r a t i o n " E n h a n c e d O i l and Gas R e c o v e r y Symposium, W a s h i n g t o n (Dec. 1 9 7 5 ) . W h i s t l e r R.L. " I n d u s t r i a l Gums" 2nd E d i t i o n A c a d e m i c P r e s s , New Y o r k ( 1 9 7 3 ) .


INDEX Azotobacter (continued) A vinehndii Acetolysis alginic acid production by 14 for the elucidation of structure 183 exopolysaccharide production by 20,24 of D-mannan 121 in relation to alginate synthesis, oligosaccharides 189 metabolism of 22 of xanthan gum 183,185 Acetyl, monosaccharide content Β pyruvic acid 196 Acid whey, xanthan gum from 27 Backbone conformation angles 96 Acylation mechanisms, exopolyBacteria growing in methanol, saccharide 5 Aerobacter aerogenes 17,28 Bacteria, methane and methanol Agrobacterium utilizing 67 radiobacter 267 Bacterial heteropolysaccharides 211,220 rhizogenes 267 Bacterial polysaccharides 107 species 49 Bacterium, soil 211,220 Alcaligenes faecalis 265,306 Batch Alditol acetates, partially methylated .. 187 culture, production of alginic Aldoses, peracetylated aldononitrile acid in 14 derivatives of 115 fermentation 32-38 Alginate(s) 174 kinetics, nitrogen-limiting 74 apparent viscosity vs. rate of shear nitrogen-limiting 71 plots for Azotobacter 21 Berea sandstone cores 246,253,261 microbial 306 Binding, ion 140 synthesis, metabolism of Bingham body, stress vs. shear Azotobacter vinelandii in rate for a 153 relation to 22 Biological function, technological Alginic acid by Azotobacter relevance of 174 vinehndii, production of 14 Biological utility 177 Alginic acid, structure of 20 Biopolymer capacity summary 312 Ammonia chemical ionization 117 Biopolymer injectivity tests, test-well.. 254 Amorphophallus konjac tubers 180 Birefringence 205 Amylose («(l,4)-D-glucan) 104 Block structure 19 Raman spectrum of V 105 Bragg reflection 92 Anabolic fate of glucose 42 Branching, degree of 118 Antimutagenesis 4 Application C field 260 in food products 278 Calcium ion concentration of the growth medium 19 in industry 281 238 Arthrobacter 220 Calf milk replacers 59 Ash separation vs. time 237 Candida boidinii Atom labeling 92 Carbohydrate C-13 N M R spectra on temperature, dependence of 124 Attractive interaction in the X. campestris helices 99 Carbohydrate composition of Zanflo .. 214 Carbon-limited chemostat 63 Aureobasidium ( Pullularia ) monod growth kinetics in 64 pullulans 285 yield coefficients for 69 Azotobacter 155 alginates 21 K-Carrageenan-milk-sugar system 154,155 indicus 220 Casson equation a

317


318


Cations on viscosity, effect of mono- and divalent 218 Cattle feed supplements, liquid 235 C D , effect of temperature on 83 Cell suspension, polymer formation rate in washed 72 Cellobiose 190 Cells, preservation of 10 Cellulose derivatives 304 Cheese spread, pasteurized processed 233 Chemical shifts for O-phosphonomannans and related materials, P-31 .. 125 Chemostat carbon-limited 63,64,69 nitrogen-limited 68, 69, 72 Chondroitin 4-sulfate 107 Chondroitin 6-sulfate 10 Chromatography gas 187 gas-liquid 114,117 high-pressure 120 C M C (sodium carboxymethylcelluluose) 151 Coefficient, non-growth associated 71 Compatibility 257 Concentration behavior, polymer viscosity246 viscosity vs 215 for xanthan solutions, 147 Cone and plate, flow between 165 Conformation 271 angles, backbone 96 of xanthan gum, molecular 90 Continuous culture, production of alginic acid in 14 Core test, injectivity 254-256 Cores, Berea 253,261 Cottage cheese dressing 233 Cottage cheese whey and whey permeate 27 Counter ion, effect of 140 Culture collections 2 maintenance and productivity 1 production of alginic acid in batch and continuous 14 set whey 32 Curdlan 306 gel 270 as microfibrils 269 production properties and application of 265 -type polysaccharides 268

D Darcy's law Degradation, mechanical

156 250

Derivation, peracetylated aldonitriles ( P A A N ) 115 Dermatan sulfate 107 Deuteromethyl groups 118 Dextran(s) 284,286,305 C-13 N M R spectra of 123 derivated 129 in industry 289 from Leuconostoc mesenteroides .... 292 methylated D-glucose components in hydrolyzates of permethylated 120 naturally occurring 285 polyelectrolyte behavior 132 Diffraction pattern for Xanthomonas campestris and Xanthomonas phaseoli 92

Azotobacter vinehndii at various on production of an exopolysac charide by Pseudomonas species, effect of on production of xanthan by Xanthomonas campestris, effect of Dioctyl phthalate Disaccharide backbone height Disaccharide E , lead tetraacetate oxidation of Dynamic viscosity

20

18

18 165 92 188 148

Ε Edamin medium, hydrolyzedpermeate/ 34 Effluent concentration for xanthan gum solutions 249 Elastic modulus of gels 280 Electron impact-M.S. yields 117 Electroviscous effect 131 E n d uses, rheological properties in .... 151 Enterobacter aerogenes 44 Enterobacteriaceae 43 E O R , polyacrylamide and poly saccharide for 311 Erwina tahitica 306 Erwina (Zanflo) 306 Escherichia coli 2,41 Ethylene glycol on gel formation, effect of 273 Exopolysaccharide acylation mechanisms 51 formation of 44 production by Azotobacter vinehndii 20,24 effect of mutations on 50 by Pseudomonas species 16,18


319

INDEX Exopolysaccharide ( continued ) synthesis, effect of growth rate on . synthesis, isoprenoid lipids in Extinction angle, effect of sodium hydroxide on Extracellular polysaccharide of Xanthomonas campestris Extracellular substrates, initial pathways for Extrusion

17 46 274 197 42 49

F Feed supplements, liquid cattle 235 Fermentation batch 32-34 conditions 31 modes 3 process 2 repeated-batch 38 semi-continuous 73, 77 Zanflo 213 Fermentor, rate constants for the semi-continuous operation in 75 Field application 260 Field project, evaluating proposed .... 261 Flavobacterium 268 Flood evaluation, polymer 261 Flooding, micellar-polymer 242 Flow behavior, transient 248 between cone and plate 165 through porous media 156 Food(s) additive 231 applications 307 industry 290 products, application in 278 and related products, xanthan gum in 231 systems 151 U.S. consumption of hydrocolloids in 308 Formation of mixed gels 174 Fragments, isolation of 184 Friction reduction 154,156 Fucose synthesis, control of 45 Fuel, atomic 290

G Galactomannan conformation interaction, xanthan gum and locust bean α-D-Galactopyranose residues Galactose medium, glucoseGel(s) breaking strength vs. elastic modulus of

176,178 178,234 234 178 32,33

280

Gel(s) (continued) curdlan formation effect of ethylene glycol on effect of urea on .... mechanism, mixed precipitation process properties, role of polysaccharide molecules in -sol transition, reversible strength, effect of heating temperature on structure, polysaccharide synergistic xanthan unctuous Genes, stabilized

270 234 273 272 174 292 234 149 272 175 174 152 11

improvement of 9 maintenance of 3 Glucans 119 α-D-Glucans, industrially significant.... 284 /M,3-Glucan microfibrils 277 « ( 1 , 4 ) - D - G l u c a n (amylose) 104 Glucose anabolic fate of 42 components in hydrolyzates of permethylated dextrans, methylated 120 -galactose medium 32,33 /M,3-Glucosidic linkages 265 Gluten substitute 241 Glycosaminoglycans 106 Glycitol from oligosaccharide C , pero-trimethylsilyl ether of derived .. 187 Growth kinetics 59 in carbon-limited chemostat, monod 64 -limiting nutrient on exopolysac charide production by Azoto bacter vinehndii, effect of 24 -limiting substrate 15 medium, calcium ion concentra tion of the 19 rate data, specific 60 on exopolysaccharide synthesis, effect of 17 inhibitory specific 59-60 yield for M . mucosa and other microorganisms 66 Guluronic acid 19 Gum(s) consumption of industrial 301 locust-bean 176,178 market for water soluble 299 natural and synthetic water-soluble 300



320

Gum(s) (continued) solutions, rheological properties of aqueous 144,146 structure 183 world production of selected industrial 303 xanthan 27 H Hansenula capsuhta Hansenula polymorpha

46 59

Heat on viscosity of PS-7 and xanthan gum solutions, effect of 223 Hele-Shaw models 242,244 Helical models 91,96 Helices, attractive interaction in the X. campestris

Ionization ammonia chemical constants of sodium dextran sulfate methane chemical Ionogenic function, effect of nature of the Ionogenic groups Isolation procedures Isoprenoid lipids i n exopolysaccharide synthesis

117 142 117 137 129 2 46

J

Junction zones

175

Κ

9

Helix formation, double 17 Helix, xanthan 93,94,97 Heteropolysaccharide, PS-7 220 Heteropolysaccharide, Zanflo 211 Hexose substrate, fate of 42 HPXan 199 dispersions 204,206 Hyaluronic acid 107 Hydrocolloids in foods, U.S. consumption of 308 Hydrogen bonds, possible 98 Hydrolysates of permethylated dextrans, methylated D-glucose components in 120 Hydrolysates of permethylated D-mannans, methylated D-mannose components i n 119 Hydrolysis, polymer 114 Hydrosulfite on polymer degrada tion, effect of 258 Hyphomicrobium

61

Hystersis loop treatment

150

I Industry application i n 281 applications, petroleum and oil 309 dextran and dextran derivatives 289 pharmaceutical 289 photographic 293 Injection values for various well completions, scaled 253 Injection well pressure response 256 Injectivity behavior 250 Injectivity core test 254-256 Instability of microbes, inherent 3 Instrument ranges 164 Ion(s) counter 128,140 binding 140 macro 131

in carbon-limited chemostat, monod growth constants for the respiration grow ing of bacteria in methanol, Michaelis-Menten of growth nitrogen-limiting batch respiration of substrate inhibition for M. mucosa

64

63 59 74 61 62

L Lead tetraacetate oxidation of disaccharide Ε 188 Length, calculated 172 Leuconostoc mesenteroides 119,284,292 Limitation, molybdate, oxygen, and phosphate 23 Lineweaver-Burk plot 60,62 «-l,6-Linkages, α-maltotriose poly merized through 292 Lipids, carrier 50,51 Lipids, isoprenoid 46 Liquid supplement formulation 236 Locust bean galactomannan inter action, xanthan gum234 Locust bean gum 176,178 LPXan 199 dispersions 204,206

M Macromolecular structure, effect of .... Maintenance coefficients for M. mucosa and other microorganisms a-Maltotriose polymerized through «-l,6-linkages Mannans, structure of D-Mannan, methylation and acetolysis data for


135 66 292 118 121

321

INDEX

D-Mannans, methylated D-mannose components in hydrolysates of permethylated 119 β-D-Mannopyranose residues 178 Mannose containing oligosaccharides, structures of 186 Mannose synthesis, control of 45 D-Mannose components in hydro lysates of permethylated D-mannans, methylated 119 α-Mannosidase on oligosaccharide C, action of 187 Mannuronic acid 19 Market for water soluble gums 299 Medium(a) calcium ion concentration of the growth 1 flow through porous 15 formulation 2 glucose-galactose 32,33 hydrolyzed permeate 30 hydrolyzed permeate/edamin 34 set whey 35-37 Metabolism of Azotobacter vinelandii in relation to alginate synthesis .. 22 Metabolism, intermediary 41 Metallurgy 290 Methane chemical ionization 117 Methane utilizing bacteria 67 Methanol effect of the 61 Michaelis-Menten kinetic constants for the respiration of bacteria growing in 63 utilization rate 63 utilizing bacteria 67 Methylation data for D-mannan 121 Methylomonas mucosa

growth yield and maintenance coefficients for kinetics of substrate inhibition for .. respiration rate data of Methylomonas, polysaccharide forma tion by a Micellar-polymer flooding Micellar/surfactant projects testing U.S. reservoirs Michaelis-Menten kinetic constants for the respiration of bacteria growing in methanol Microbes inherent instability of selection and preservation sources of Microbial alginate exopolysaccharide synthesis polysaccharides of commercial significance

58

66 62 62 58 242 262

63 3 10 1 306 40 305

Microbial (continued) polysaccharides, industrial develop ment of 307 Microfibrils, curdlan as 269 Microfibrils, 0-1,3-glucan 277 Microorganisms, growth yield and maintenance coefficients for 66 M i l k replacers, calf 238 Milk-sugar system, Casson plots for /(-carrageenan155 Mobility definition of 244 reduction(s) 243 in Berea sandstone cores 246 for xanthan gum solutions 249 Modification 49

Molybdate limitation Monod growth kinetics in carbonlimited chemostat Monosaccharide content, pyruvic acid acetyl M.S. yields-electron impact Mutability regions, high Mutagens on mutation of strain, effects of Mutants conditional control in a growing culture, proportion .... Mutation(s) development of a stable limiting the opportunity for on production of exopolysaccharides, effect of sequence of events in

23 64 196 117 3 267 9 9 7 266 4 6 50 5

Ν Natural products 304 Newtonian region 169 Newtonian viscosity 145 Nitrogen-limited chemostat 68 polymer production in 69 yield coefficients i n 72 Nitrogen-limiting batch 71,74 N M R of polysaccharides 114 N M R relaxation 82 Nomenclature 172 Nutrient on exopolysaccharide pro duction by Azotobacter vine landii, effect of growth-limiting .. 24 Ο Oil drilling muds industry applications


309 309

322


O i l (continued) production response, polymer and waterflood 262 recovery, enhanced 242,309,310 Oligosaccharide ( s ) from acetolysis of D-mannan 121 from acetolysis of xanthan gum 188 C, action of α-mannisodase on 187 C, per-o-trimethylsilyl ether of derived gylcitol from 187 in repeating unit of xanthan gum, acetolysis 189 structures of mannose containing .... 186 xanthan acetolysate neutral 185 Optical activity 82 Ordered state 84 Origin of xanthan solution properties molecular 8 Origin of xanthan synergism, molecular 177 Ostwald, power law of 169 Oxygen limitation 23

Ρ Pachysolen tannophilus .118,120 Pentasaccharide repeating unit 90, 92,183 Peracetylated aldonitriles ( P A A N ) derivation 115 Permeability reduction 245 Permeate cottage cheese whey and whey 27 /edamin medium, hydrolyzed34 medium, hydrolyzed30 Permethylation gas-liquid chroma tography /mass spectrometry 117 Permethylation, polymer 114 Per-o-trimethylsilyl ether of derived glycitol from oligosaccharide C .. 187 Petroleum industry applications 309 Petroleum production 291 H of H P X a n and L P X a n dispersions, viscosity vs 204 on PS-7 and xanthan gum solutions, effect of 224 viscosity vs 194,203 on Zanflo viscosity, effect of 217 Pharmaceutical industry 289 Phosphate limitation 23 O-Phosphonohexosans, extracellular yeast 124 O-Phosphonomannans, P-31 chemical shifts for 125 Photographic industry 293 Physiology of polysaccharide synthesis 14 Plate, flow between cone and 165 P

Polyacrylamide for E O R intrinsic viscosity of partially hydrolyzed partially hydrolyzed viscosity vs. shear rate for Polyampholytes Polyelectrolyte behavior, dextran Polyelectrolytes, polysaccharide Polymer degradation, effect of hydrosulfite on demand for enhanced oil recovery .. flood evaluation flooding, micellarformation rate in washed cell suspension

311 258 243 251 128 132 128

258 310 261 242 72

permethylation 114 production in nitrogen-limited chemostat 69 production rate, specific 68 retention 247 -sulfonate interaction 259 synthesis, direction to 41 viscosity-concentration behavior .... 246 and waterflood oil production response 242,262 Polymerization, specific rotation vs. degree of 276 Polysaccharide ( s ) bacterial 107 biosynthesis of Xanthomonas 54 C N M R spectra of 276,279 of commercial significance, microbial 305 concentration, viscosity vs 195,197 curdlan type 268 effect of heating temperature on transmittance and viscosity of 273,274 for E O R 311 formation by a Methylomonas 58 gel structure 175 industrial development of microbial 307 infrared and Raman spectroscopy of 103 molecules in gel properties, role of .. 234 optical rotation and viscosity variations w. temperature for Xanthomonas 83 polyelectrolytes 128 synthesis, control of 54 synthesis, physiology of 14 of Xanthomonas campestris, struc ture of extracellular 197 x-ray diffraction analysis of 279 Polyion 128 Polytran, scleroglucan 306 1 3


323

INDEX Pore volume, inaccessible 247,249 Potassium carboxymethyl dextran 135 Potassium dextran sulfate 132 Power law constants 170 fit of data 171 of Ostwald 169 region 145 Precipitation process, gel 292 Preservation of cells 10 long-term 8 of microbes 10 short-term 8 Pressure response, injection well 256 Productivity, culture 1 Products, natural 304 PS-7 effect of heat on viscosity o a new bacterial heteropolysaccharide 220 effect of p H on 224 viscosity of 222, 226 Pseudomonas C 59 Pseudomonas species 15,289 effect of dilution rate on production of an exopolysaccharide by 18 exopolysaccharide production by .. 16 Pseudoplastic system, viscosity vs. shear rate for 147 Pseudoplasticity (shear thinning ability) 160 Pseudoplasticity, xanthan gum 161 Pullulan(s) 284,288,306 proposed uses 293 Purification procedure 163 Pyruvate, xanthan products with intermediate levels of 204 Pyruvic acid acetyl monosaccharide content 196 content, xanthan products of differing 192 content of xanthan, viscosity vs 207

R Rate constants for the semi-continuous operation in fermentor Repeating unit, pentasaccharide Reservoirs, micellar/surfactant projects testing U.S Respiration of bacteria growing in methanol .... kinetics rate data for M . mucosa rates exopolysaccharide production by Azotobacter vinelandii at various

75 183 262 63 61 62 23

24

Respiratory quotient, maximum 78 Retardation vs. shear rate of H P X a n and L P X a n dispersions 206 Retardation vs. temperature 206 Retention times of peracetylated aldononitrile derivatives of aldoses 115 Retorting on viscosities of xanthan gum, effect of 240 Reynolds number 154 Rheological properties of aqueous gum solutions, effect of salts on the 146 in end uses 151 of gum solutions 144 Rheology 268 of xanthan gum solutions 160,167,168 vs. degree of polymerization, specific effect of sodium hydroxide on optical variations w. temperature for Xanthomonas polysaccharide optical

276 274

83

S Saccharomyces cerevisiae 9 Salmonella mutants 49 Salt on viscosity, effect of 200,202,205 Salts on the rheological properties of aqueous gum solutions, effect of .. 146 Sandstone cores, mobility reductions in Berea 246 Scleroglucan (Polytran) 306 Seaweed extracts 304 Seed extracts 304 Selection limiting the opportunity for 6 of microbes 10 sequence of events in mutation 5 Semi-continuous experiment, final yield data for the .. 78 fermentation 73 operation in the 14 L fermentor, rate constants for the 75 Shear plots for Azotobacter alginates, apparent viscosity vs. rate of .... 21 rate for a Bingham body, stress vs 153 vs. retardation of H P X a n and L P X a n dispersions 206 vs. stress for thixotropic material 150 vs. viscosity 198 for polyacrylamide 251 for pseudoplastic or shear thinning system 147 for xanthan solutions ...147,199,251


324


Shear (continued) thinning ability ( pseudoplasticity ) 160 thinning system, viscosity vs. shear rate for 147 Sodium alginate 23 carboxymethylcellulose ( C M C ) .... 151 dextran sulfate, ionization constants of 142 hydroxide on optical rotation, effect of 274 Soil bacterium 211,220 Solution(s) properties, molecular interpre tation of 84 properties, molecular origin of xanthan 8 rheological properties of gu rheology of xanthan gum 160 viscosity 81 Spectra C NMR 123,124,276,279 Fourier transform infrared 110, 111 polarized infrared 108 Raman 105 Spectrometry, mass 117 Spectroscopy infrared and Raman 103 mass 114,187 nuclear magnetic resonance 114 C-13 122 P-31 124 Staphyhcoccus aureus 46 Starch derivatives 304 Sterilization 30 Strains, colonies of 269 Strength vs. elastic modulus of gels, breaking 280 Streptococcus mutans 286 Stress response, effect of substitution degree on 153 vs. shear rate for a Bingham body .. 153 vs. shear rate for thixotropic material 150 yield 149,169 Structure(s) aggregation of rigid 175 effect of macromolecular 135 polysaccharide gel 175 of xanthan gum, acetolysis for the elucidation of 183 Substitution, effect of degree of 132 on stress response 153 Substrate(s) concentrations, inhibitory specific growth rate at different initial.. 60 fate of hexose 42 1 3

Substrate ( s ) ( continued ) growth-limiting 15 inhibition for M . mucosa, kinetics of 62 initial pathways for extracellular . . . 42 uptake 40 utilization rate correlation, specific 64 Sugar-sugar linkage type 118 Sugar system, Casson plots for K-carrageenan-milk155 Sulfonate-polymer interaction 259 Supermolecular structure 149 Surfactant projects testing U.S. reservoirs, micellar/ 262 Surfactant slug 259 Symmetry, helical 92 Synergism, molecular origin of xanthan 177 Synergistic xanthan gels 174

Τ Temperature on C D , effect of 83 dependence of carbohydrate C-13 N M R spectra on 124 dependence of viscosity 205 on gel strength, effect of heating . . . 272 retardation vs 206 on transmittance and viscosity of polysaccharide, effect of heating 273,274 vs. viscosity 193,198 for Xanthomonas polysaccharide, optical rotation and viscosity variations with 83 on Zanflo viscosity, effect of 215 Thermal changes 109 Thermal processing 238 Thixotropic material, stress vs. shear rate for 150 Thixotropy 149 Time, ash separation vs 237 Tom's effect 154 Transmitance of polysaccharide, effect of heating temperature on 273,274 Tree exudates 205

U Unctous gels Urea on gel formation, effect of

152 272

V Viscosity (ies) 129 commercial vs. purified 163 vs. concentration 215 -concentration behavior, polymer .... 246 determination, intrinsic 164


325

INDEX Viscosity ( ies ) ( continued ) dynamic 148,150 effect of mono- and divalent cations on 218 effect of p H o n 203,217 effect of salt on 200,202 effect of sodium hydroxide on intrinsic 274 effect of temperature on Zanflo 215 functions 130 intrinsic 171,194,203,258 measurement of xanthan 193 Newtonian 145 vs. p H 194 of H P X a n and L P X a n dispersions 204 vs. polysaccharide concentration 195,197 of polysaccharide, effect of heating temperature on 273,27 ofPS-7 222 effect of heat on 223 vs. pyruvic acid content of xanthan 207 vs. rate of shear plots for Azotobacter alginates, apparent.. 21 ratios, displacements in Hele-Shaw model at different 244 reduced 129 vs. shear rate 198 and concentration for xanthan solutions 147 for polyacrylamide 251 for pseudoplastic or shear thinning system 147 for xanthan solutions 199,251 solution 81 steady shear 150 temperature dependence of 193,198,205 xanthan gum 161 effect of retorting on 240 for Xanthomonas polysaccharide .... 83 W

Waterflood oil production response .... 262 Waterflooding, polymer 242 Well completions, scaled injection values for various 253 Whey acid-set 34 composition, acid 28 culture set 32 medium, set 35-37 and whey permeate, cottage cheese 27 xanthan gum from acid 27 X Xanthan 107,305 acetolysate neutral oligosaccharides 185 Fourier transform infrared spectra of 110, 111

Xanthan (continued) gels, synergistic 174 gum(s) 243 acetolysis for the elucidation of structure 183,185 acetolysis of oligosaccharides in repeating unit of 189 from acid whey 27 effect of retorting on viscosities of 240 for enhanced oil recovery 242 in foods and related products 231 injectivity core test 256 -locust bean galactomannan interaction 234 molecular conformation and interactions 90

pseudoplasticity 161 rheology 167,168 solutions effect of heat on viscosity of .... 223 effect of p H on 224 effluent concentration and mobility reduction for 249 rheology of 160 structure 233 viscosities 161 viscosity vs. shear rate for 251 helix, 5/1 93,97 helix, 5/2 94 laboratory purification of 193 native conformation 176 pentasaccharide repeating unit of .... 92 products of differing pyruvic acid content 192 products with intermediate levels of pyruvate 204 solution properties, molecular origin of 81 solutions, viscosity vs. shear rate for 147,199 synergism, molecular origin of 177 viscosity measurement of 193 viscosity vs. pyruvic acid content of 207 by Xanthomonas campestris, effect of dilution rate on production of 18 Xanthomonas campestris 2,15,27,43,90,160, 177,183,192,231 diffraction pattern for 92 effect of dilution rate on production of xanthan by 18 helices, attractive interaction in .. 99 structure of extracellular polysaccharide of 197 jughndis 190


326


Xanthomonas (continued)

phaseoli 91,190 diffraction pattern for 92 polysaccharide, optical rotation and viscosity variation with tem perature for 83 polysaccharides, biosynthesis of 54 X-ray diffraction analysis of poly saccharide 279 Xylose 190

Yield (continued) coefficients in nitrogen-limited chemostat data for the semi-continuous experiment, final stress

Yield coefficients for carbon-limited chemostat

69

78 149,169

Ζ

Zanflo carbohydrate composition of Erwina

Y

72

214 306

fermentation 213 a novel bacterial heteropoly saccharide 211 viscosity, effect of p H on 217 viscosity, effect of temperature on .. 215


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