431-5

ADVANCES IN CARBOHYDRHE CHE~IISTRY AND BIOCHEMISTRY, VOL. 36

EXOCELLULAR, MICROBIAL POLYSACCHARIDES*

By PAUL A. SA.l~DFORD**

Northern Regional Research Center, Federal Research,

Science, and Education Administration, U. S. Department of Agriculture,
Peoria, Illinois 61604

I. Introduction 266
1. Scope of Article 266
2. General Background 267
II. Stimulants to Usage of Exocellular, Microbial Polysaccharides 268
1. Increasing Consumption of Natural Gums 268
2. Needs of Industry Not Met by Traditional Gums 270
3. Large Variety of Types and Sources of Microbial Exopolysaccharides 270
4. Success ofXanthan Gum and Dextran 271
III. Sources and Types of Exocellular, Microbial Polysaccharides 272
IV. Production of Microbial Polysaccharides 273
1. Culture Maintenance and Productivity 273
2. Biosynthesis of Exopolysaccharides 286
3. Methods of Fern1entation 289
4. Isolation of Polysaccharides 291
V. Acidic Sugar-containing, Microbial Polysaccharides of Commercial Interest . 292
1. Xanthan Gum 292
2. Erwinia tahitica Polysaccharide 297
3. Beijerinckia indica (Azotobacter indicum) Polysaccharide 297
4. Bacterial Alginate 298
5. Arthrobacter Polysaccharides 299
6. Other .Microbial Polysaccharides 300
VI. Neutral, i\licrobial Polysaccharides of Commercial Importance 303
1. Dextran 303
2. Scleroglucan 305
3. Curdlan 307
4. Pull ulan 310
5. Polysaccharides from Methanol-utilizing lllicro-organisms 312

" I thank Dr. M. E. Slodki for his helpful suggestions in preparing this text. Also, 1
thank Drs. C. J. Lawson and 1. W. Sutherland for a preprint of their review article on
polysaccharides.
** Present address: Research Laboratory, Kelco Division, Merck and Co., Inc., 8355
Aero Drive, San Diego, California 92123.

265
Copyright © 1979 by Academic Press, Inc.
All rights of reproduction in any fornl reserved.
ISBN O-12-007236-X
266 PAUL A. SANDFORD

1. INTRODUCTION

1. Scope of Article

This article will emphasize the microbial exopolysaccharides that

are produced commercially and those whose potential for commer-
cialization is very likely. The involvement of microbial exopolysac-
charides in the two economically important areas of plant
pathogenicityl,2 and nih'ogen fixation 3,4 will not be covered in detail
here. Microbial polysaccharides having antihlmor activity have been
discussed in this Series. 5 Articles covering other pertinent areas are as
follows: bacteria15- 11 and fungal polysaccharides,1l,12 microbial exo-
polysaccharides,13,14 bacterial exopolysaccharides,15,16 biosynthesis of
bacterial polysaccharides l i- 19 cell-wall polysaccharides,20 production

(1) Y. Hennis and 1. Chet, Adt'. Appl. Microbiol., 19,85-109 (1975).

(2) C. E. Bracker and L. J. Littlefield, in "Fungal Pathogenicity and the Plant's Re-
sponse," R. J. W. Byrde and C. V. Cutting, eds., Academic Press, New York, 1973,
pp. 159-318.
(3) J. S. Wolpelt and P. Albersheim, Biochem. Biophys. Res. Commlln., 70,729-737
(1976).
(4) F. B. Dazzo and W. J. Blill, Appl. Ent'iron. Microbiol., 33, 132-136 (1977).
(5) R. L. Whistler, A. A. Bushway, P. P. Singh, W. Nakahara, and R. Tokuzen, Adt'.
Carbohydr. Chem. Biochem., 32,235-27.5 (1976).
(6) T. H. Evans and H. Hibbert, Adt'. Carbohydr. Chem., 2,203-233 (1946).
(7) D. A. L. Davies, Adt'. Carbohydr. Chem., 15,271-340 (1960).
(8) M. J. How, J. S. Brimacombe, and M. Stacey, Adt'. Carbohydr. Chem., 19,303-3.57
(1964).
(9) S. A. Barker and P. J. Somers, in "The Carbohydrates: Chemistry and Biochemis-
try," W. Pigman and D. Horton, eds., Academic Press, New York, 1970, Vol. IIB, pp.
569-587.
(10) O. Liideritz, K. Jann, and R. Wheat, in "Comprehensive Biochemistry," .'vl. Florkin
and E. H. Stotz, eds., Elsevier, Amsterdam, 1968, Vol. 26A, pp. 105-228.
(11) M. Stacey and P. W. Kent, Adt:. Carbohydr. Chem., 3,311-336 (1948).
(12) P. A. J. Gorin and J. F. T. Spencer, Adt'. Carbohydr. Chem., 23,367-417 (1968).
(13) A. Jeanes, in "Encyclopedia of Polymer Science and Technology," N. M. Bikales,
ed., 1nterscience, New York, 1968, Vol. 4, pp. 693-711.
(14) K. L. Smiley, Food Technol., 20, 112-116 (1966).
(15) 1. W. Sutherland, Adv. Microb. Physiol., 8, 143-213 (1972).
(16) J. F. Wilkinson, Bacteriol. Ret'., 22,46-73 (1958).
(17) B. L. Horecker, AllIlli. Ret'. Microbiol., 20,253-285 (1966).
(18) H. Nikaido, Adv. Enzymol., 31,77 -124 (1968).
(19) P. H. Makela and B. A. D. Stocker, Annll. Rev. Genet., 3,291-322 (1969).
(20) S. Kirkwood, Annll. Rev. Biochem., 43,401-417 (1974).
 EXOCELLULAR, .\IICROBIAL POLYSACCHARIDES 267

of microbial polysaccharides,21 industrial gums,22,23 microbial polysac-

charides in foods,24-27 and microbial exopolysaccharides of practical
importance .28-32

2. General Background
A common feature of bacteria, fungi (yeasts and molds), and higher
living organisms is the production of polysaccharides. Morphologi-
cally, there are three types: inb'acellular polysaccharides located
inside or as part of the cytoplasmic membrane; cell-wall poly-
saccharides; and exocellular polysaccharides located outside the cell
wall. Some exocellular polysaccharides are found covalently attached
to tlle cell as a true capsule, whereas otllers are secreted unattached
into the growtll medium. In some cases, both capsular and unattached
exopolysaccharides can be found associated with tlle same microbe.
For practical reasons, micro-organisms that produce unattached exo-
polysaccharide were first explored, thereby avoiding tlle costly purifi-
cation procedures that would be necessary to free capsular, cell-wall,
or internal polysaccharides from cells. Also, for obvious reasons, exo-
polysaccharides from human pathogens have been avoided; fortu-
nately, there are many micro-organisms nonpatllOgenic to humans that
produce exopolysaccharides. Despite the large number of these poly-
saccharide producers, relatively few have been shldied thoroughly.
Only within the last - 20 years has the practical potential of these bio-
polymers been given serious consideration.

(21) M. E. Slodki and M. C. Cadmus, A.d!:. Appl. Microbiol., 23, 19-.54 (1978).
(22) L. Stoloff, A.dD. Carbohydr. Chem., 13,265-287 (19.58).
(23) "Industrial Gums-Polysaccharides and Their Derivatives," R, L. Whistler and J.
N. BeMiller, eds., Academic Press, New York, 2nd Edition, 1973, pp. 1-807.
(24) M. Glicksman, "Gum Technology in the Food Industry," Academic Press, New
York,1969.
(2.5) E. R. Morris, in "Molecular Structure and Function of Food Carbohydrate," G. G.
Birch and L. F. Green, eds., Wiley, New York, 1975, pp. 125-132.
(26) A. A. Lawrence, "Nahlral Gums for Edible Purposes, 1976," Noyes Data Corp"
Park Ridge, New Jersey, 1977.
(27) J. E. Hodge and E. Iv!. Osman, "Principles of Food Science. Part 1. Food Chemis-
try," Dekker, New York, 1976, pp. 41-138.
(28) A. Jeanes, in "Water-Soluble Polymers," N. M. Bikales, ed., Plenum, New York,
1973, pp. 227 -242.
(29) A. Jeanes,]. Polym. Sci. Polym. Sump., 45, 209-227 (1974).
(30) 1. Sutherland, Process Biochem., 7(Il), 27 -30 (1972).
(31) ACS Symp. Ser. No. 45 (1977).
(32) 1. W. Sutherland and C. J. Lawson, unpublished review.
268 PAUL A. SANDFORD

II. STIMULAj\rrS TO USAGE OF EXOCELLULAR,

MICROBIAL POLYSACCHARIDES

1. Increasing Consumption of Natural Gums

Worldwide consumption of water-soluble gums continues to in-
crease as new gum-technology is applied to the needs of industry
Tables I and II list the distribution of gums according to types of uses
and functionality.33 In the United States, the total consumption of all
gums is gradually increasing (by ~ 2% per year), whereas the con-
sumption of microbial gums is increasing by over 8% per year. 33 In
some cases, this increase in consumption of microbial gums is at the
expense of other, traditional, gums. Table III lists the consumption of
gums in the United States as repOlted by Whistler. 34
Of the ~ 300 million pounds of nonstarchy gums consumed in 1973,
about one-sixtl1 of this amount, or ~ 50 million pounds, was imported
into the United States. Of tl1e gums listed in Table III, only xantl1an
gum is obtained from a microbial source.
The expected rising cost of petroleum-derived, syntl1etic polymers
has now elicited interest in natural polymers from replenishable re-
sources. Microbial polysaccharides are also increasingly being used in
the recovery of crude oil. During 1975, ~ 1800 tons of xanthan were
used in oil-drilling operations; it is predicted tl1at, by 1980,3000 tons
of xantl1an will be used in oil-drilling muds. 33

TABLE I
Distribution of Gums According
to Industrial U sage33

Types of Use %

Detergents and laundry products 16

Textiles 14
Adhesives 12
Paper 10
Paint 9
Food 8
Pharmaceutical and cosmetic 7
Other 24

(33) J. Wells,in "Extracellular IVlicrobial Polysaccharides," P. A. Sandford and A. Las-

kin, eds., ACS Symp. Ser., No. 45, American Chemical Society, Washington, D. C.,
1977, pp. 299-313.
(34) R. L. Whistler, personal communication, May 16, 1977.
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 269

TABLE II
Distribution of Gums According
to Functional Properties33

Usage
Function (%)

Stabilizer, suspending agent, and dispersant 25

Thickener 23
Film-forming agent 17
'.Vater-retention agent 12
Coagulant 7
Colloid 6
Lubricant or friction reducer 5
Other 5

The greatest, single, potential market for such polysaccharides ap-

pears to be as mobility-control agents in enhanced oil-recovery. It has
been estimated33 that, of all the oil thus far discovered, approximately
two-thirds of the total still remains in place. As costs of all fom1s of
energy go up, enhanced oil-recovery becomes increasingly attractive.
Therefore, there are great interest and research effort in recovering
this large fraction of original oil left in sh'ata by conventional recovery-
methods. For one of the foremost candidates for enhanced oil-recov-

TABLE III
Consumption of Gums in the United States 3•

Millions of pounds

Gum Food use Industrial use Total

Corn starch 600 2500 3100

Agar 0.3 0.4 0.7
Arabic 24 7 31
Alginate 10 2 12
Carrageenan 9 0.2 9.2
Furcellan 0.3 0.3
Ghatti 10 1 II
Guar 20 40-45 60-65
Karaya 1 7 8
Locust bean 9 3 12
Pectin 12 12
O-Methylcellulose 2 53 55
O-(Carboxymethyl)cellulose 16 100 II6
Tragacanth 1.3 0.2 1.5
Xanthan 3 9 12
270 PAUL A. SANDFORD

elY, namely, micellar flooding, it was estimated by the National Peh'o-

leum Council (1976) that 2 to 8 billion pounds of such polymers as
xanthan gum will be needed between the years 1976 and 2000. The
requirements of high tolerance to salts, heat, pH, and shear, and of
high viscosity and proper flow-properties are not met by most of the
h'aditional polymers, and this has stimulated the search for new,
water-soluble polysaccharides.

2. Needs of Indushy Not Met by Traditional Gums

The commercial usefulness of polysaccharides is based on their
abilitv to alter the rheological properties of water. Therefore, the min-
imun~ requirements for a new gum are to (l) dissolve readily in water,
(2) have constant, functional properties better than (or as good as)
those of h'aditional gums, and (3) be available on a regular basis from a
stable source of supply. Although a great number of plants35 and sea-
weeds 23 have been screened for gums, indush'y still has a need for
new gums. Traditional gums may suffer from a lack of reproducibility
in their properties, purity, supply, and cost. Many plants exude such
gums as arabic, ghatti, karaya, and tragacanth that require much un-
skilled hand-labor for their collection, as does locust-bean gum. Labor
costs in areas of the world where these traditional gums are harvested
have been very low, but they are expected to rise greatly witl1 time.
Most of tl1e gum-providing plants and seaweeds also require very spe-
cial climates. Therefore, adverse weatl1er-conditions may result in al-
teration of the quantity and quality of gums, which, in hm1, makes
their supply and prices unpredictable. Microbial gums can be pro-
duced under conh'olled conditions, and could, therefore, free nonpro-
ducing areas of the world from exclusive reliance on the importation
of plant and seaweed gums.

3. Large Variety of Types and Sources

of Microbial Exopolysaccharides
Microbial sources are known for numerous classes of polysac-
charides. 6 ,36 Some are even comparable to such well-kno\'in plant-

(.3.5) F. Smith and R. Montgomery, "The Chemistry of Plant Gums," Reinhold, New
York, 1959.
(36) M. Stacey and S. A. Barker, "Polysaccharides ofiVlicro-organisms," Oxford Univer-
sity Press, London, 1960.
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 271

pn)d1uc1:s as starch-like polymers,37,38 cellulose,39 and algin. 4o Others,

as glycogen,37,38 chitin,41 and hyaluronic acid,42 are similar to ani-
However, the great majority of microbial polysac-
c11ar:ld(:;s are unlike products known from any other source. It is this
diversity of microbial sources and types that is appealing to the
"U'.'Ll~L"HU user of such gums. Of the relatively few, microbial exopoly-
Sa(;ctlarld(:;s that have been studied in detail, it is obvious that each has
properties that derive from their unique combinations of
and their linkages. Therefore, it is to be expected that new
of polysaccharides possessed of new combinations are yet to be
in microbial sources.
The ability of micro-organisms to synthesize exocellular polysac-
charides from simple and complex organic subsh'ates is widespread. It
is the availability of required nutrients that determines the type and
location of microbes found in Nahue. For several organisms, the avail-
ability of such carbon-containing subsh'ates as carbohydrates allows
their growth in such places as effluents and waste waters from paper-
mills,43,44 sugar-cane and sugar-beet industries,45 decaying vegetation
and soil,46 and food-processing waters. Several phytopathogens pro-
duce copious amounts of exocellular polysaccharide that cause the
blockage of water in the vascular systems of the plant, resulting in
hun, in wilting of the plant. Such is true for the cabbage (and related
plants) pathogen Xanthomonas campestrisY

4. Success of Xanthan Gum and Dextran

Between 1945 and 1955, the Northern Regional Research Center
(NRRC) of the U. S. Department of Agriculhlre ,vas extensively in-
volved in the development of the bacterial polysaccharide dexh'an as a
blood-plasma volume-expander. The dextran program was success-

(37) E. J. Hehre and D. M. Hamilton,}. Bacteriol., 55, 197-208 (1948).

(38) G. Okada and E. Hehre,]. Bioi. Chelll., 249, 126-13.5 (1974).
(39) J. K. Kjosbakken and J. R. Colvin, Can. j. Microbiol., 21, Ill-120 (1975).
(40) P. A. J. Gorin and J. F. T. Spencer, Can. j. Chelll., 44, 993-998 (1966).
(41) N. Sharon, "Distribution of Amino Sugars in lvlicroorganisms, Plants, and Inverte-
brates," in "The Amino Sugars," E. A. Balazs and R. W. Jeanloz, eds., Academic
Press, New York, 196.5, Vol. IIA, pp. 1-43.
(42) G. H. Warren, Science, Ill, 473-474 (19.50).
(43) J. R. Sanborn,]. Bacteriol., 26,373-378 (19.33).
(44) J. R. Sanborn, Ind. Eng. Chelll., 28, Il89-1190 (1936).
(4.5) A. Jeanes, Encyc/. Sci. Technol., 4, 80.5-824 (1966).
(46) N. C. l\lehta, P. Dubach, and H. Deuel, AdJ). Carbohydr. Chelll., 16, 33.5-355
(1961).
(47) J. C. Sutton and P. H. Williams, Can. j. Bot., 48, 391-401 (1970).
272 PAUL A. SANDFORD

fully completed in 1955. This cooperative effort of microbiologists,

carbohydrate chemists, and bioengineers at the NRRC was refocused
toward the production, from microbes, of new types of extracellular
polysaccharides that could utilize either starch or its hydrolysis prod-
ucts (such as D-glucose). Part of the justification for such a project was,
at that time, the need for the United States to develop a domestic sup-
ply of water-soluble gums. The initial publications48 ,49 on the polysac-
charide xanthan fromXonthomonos compestris NRRL B-1459 in 1961,
and subsequent work from NRRC, led several industrial organizations
to consider and actually to produce xanthan. Because ofthis \vorldwide
acceptance, xanthan is used as a model for the industrial production of
other microbial polysaccharides.

III. SOURCES At'l"D TYPES OF EXOCELLULAR,

MICROBIAL POLYSACCHARIDES

Polysaccharides may be grouped into three categories, according to

whether they contain acidic (see Table IV), neutral (see Tables V and
VI), or amino sugars (see Table VII). It is important to note that it is
the constituent sugars and acyl substituents, and the types of linkages
between them, that detenl1ine the confom1ation and specific proper-
ties of each polysaccharide.
Acidic polysaccharides (see Table IV) that contain uronic acid resi-
dues are, perhaps, the most prevalent type of exocellular polysac-
charide. Often, these acidic biopolymers contain other sugars,
including pentoses, hexoses, and heptoses, also found in neutral poly-
saccharides (see Tables V and VI). In many instances, these polymers
possess alkali-labile O-acyl substituents, such as acetic, formic, ma-
lonic, pyruvic, and succinic acids. Positively charged biopolymers
that contain free amino sugars are rare, but have been found (see
Table VII). More often, these amino sugars are N-acylated, generally
with acetyl groups.
It is difficult to state whether formation of exocellular polysac-
charides is more prevalent among the bacteria, the yeasts, or the
molds. However, with bacteria, polysaccharide fom1ation has been
studied the most thoroughly. Several yeasts are known to elaborate
exopolysaccharides and are excellent sources thereof. Polysaccharide
formation by fungi is less frequently observed. However, species of

(48) S. P. Rogovin, R. F. Anderson, and M. C. Cadmus,]. Biochem. Microbiol. Technol.

Eng., 3,51-63 (1961).
(49) A. Jeanes, J. E. Pittsley, and F. R. Senti,}. Appl. Polym. Sci., 5,519-526 (1961).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 273

r.1ucor, Fusarium, and Tremella are known to elaborate uronic acid-

containing polysaccharides. Several fungi are excellent sources for
(1 - 7 3)-f3-D-glucans.

IV. PRODUCTION OF MICROBL~L POLYSACCHARIDES

1. Culture Maintenance and Productivity19B

a. Selection of Microbes.-Many useful microbes are directly ob-
tainable fi'om tl1e soil, otl1er natural sources, or botl1. Nutritional and
physiological characterization of the organism sought dictates tl1e use
of selective enrichment or inhibition principles. Often, tl1e reactivity
of dyes and the presence of celtain enzymes act as indicators of spe-
cific biochemical events. There are protocols in the literahue199-203 for
tl1e isolation of specific nutritional types. Such selective procedures
for polysaccharides should exploit some specific property of the de-
sired product, for example, viscosity, gel f0D11ation, and ionic char-
acter.
Searching existing culture collections is frequently quicker,
cheaper, and easier tl1an isolation from Nature. Two guides to collec-
tions of industrially useful microbes have been published.203 ,204
b. Maintenance of Genotype.-Microbial productivity is based on a
very large store of genetic information. Therefore, it is most desirable
to preserve genotypes of industrially useful microbes. It should be
recognized tl1at microbes are inherently unstable,205-207 and tl1at no
method has yet been devised for the complete preservation of geno-
types. However, ways are known by which mutagenesis 207 may be
kept at a minimum. First of all, the causes of mutation in general must
be recognized. Whatever the condition leading to mutation, tl1e most
effective protection consists in minimization of exposure of tl1e cul-
ture to it. As growth in the number of mutants is a function of the num-
ber of replications, tl1e total number of replications should be
minimized. The next line of defense is limitation of the opportunity
for selection of mutants; this selection is a function of the culture con-
ditions and of tl1e number of generations of culture growth peD11itted.
Storage of lyophilized, frozen, and "L-dried" (dried from the liquid
state) cells are tl1e principal metl10ds used for long-term preserva-
tion. 20B Although there is widespread preference for lyophilization, re-
covery of many difficult-to-preserve organisms is 10 to 100 times as
great \vitl1 L-drying metl10ds20B-21o than witl1 lyophilization. Metl10ds
for short-teD11 storage (less than a few montl1s) should provide high re-
I --_._--

TABLE IV
Microbial, ExoccIlular Polysaccharides Containing Acidic Sugar Ucsiducs

Componcnt sugars

Acidic Ncutral

O-Acylor
Micro-organism GIcA ManA Othcr Gal Glc Man Othcr acctal groups Hcfcrenccs

A. Bactcrial polysaccharidcs
Arthml)(/eler viscosus x X x acetyl 29
x x x acetyl 50-52
L-.:>
Azotobllcter chroococcUIII x x X 5:3
-1 Azotobllcter indiculII
""-
(Be(ierinckill indiclI) X X rhanlnOSl" 54-58
Azotol)(u;ter sp. X X X 52,59,60
Azotobllcter vinelll I/(lii X GnlA 40,61-65
Bllcillus POIUIIIUXIl X X X X 28,6(j
acidic sugar X 67,68
Diplococcus plumlllouill several different types 15
Ellterobllcterill
including species of
ArizoulI, CiI.ro/Jlleler,
Escherichill, Klebsiellll,
SIIIIII.()nellll, and Shigellil several dilhm,nt typl"S 15,m)
Erwinill tilhilicil X X X fucose acetyl 70-74
Pseudo/nona,\' aenlgilJOSa X GulA acetyl 75-77
HhizobiulIl legulliinosllrlllll X X X acetyl, pyruvate :3,78,79
Hhizo/JiulII lIIelilufi X X X acetyl, pyruvate 80,81
HhizobiulII plwseuli X X X acetyl, pyruvate :3,78,79
0'1 0'1 to t- (C ::.c .0:::
t- t- O) L" 0
cc :is X 00 00, 00 c;; .0) :::n :::n :::5
00
I ~
C<") 00 00
I
d -£
:::n
- I
00, 00 0') 6 5

.-.. >
:."
~ r.

.-,
-..
:;..
~-
6 3- ;: ~ :!:
--
>. >. >. >. >. >.
6; 6 6 6 6
X x X X X X X X X X X X X X X

X X X X X X X X X X X X

X X X X X X x

<
CJ

X X X x X X X X X x x X X X X X X X X

't .~
'"
~t
z
t:..~
--:::
.~
:,- .:;..: :~
~ ~

1t~
"'" ........
}~~
~~
275
 TABLE V

Bacterial, Exocellular l>olysaceharides Containing Neutral Sugar Hesidues

Component sugars

Polysaccharide O-Acylor
type Micro-organism Gal Gle Other acetal groups Heferences

A. Glucans
"Amylosc-I ikc" Escherichia coli x 116
Pseudomollas sp. x 117
1'0
Psel/(lonwlws .!lllorescelis x 118
~
x GaiN, lilcosc 119
Dextran Acetolmcter capsulatuIII x 120
Ace/ohacter IJiSCOSUlI1 x 1.20
Betahacterium IJermi.!iJrme x 120
Leucollostoc dextrallicuJlI x 120
Leucollostoc lIIesellteroides x 120
Streptohact.erium dextrallicum x 120
Streptococcus sp. x 1.20
Streptococcus lIIutalis x 121
Streptococcus IJiridalis x 120
"Glycogen-like" Agrol}(/cleriuJlI tUIII(~fiICielis x 122
(3-( 1 -, 2) AgrohacteriulII Sp. x 12:3,124
(3-(1 -, 2) AgrohacteriulII pseudotsugae x 123
(3-(I -, 2) AgrohacleriulII tUII/(~fi/(:ielis x 12:3,124
(3-(1 -, 2) x x acctyl, pyruvate 79
(3-(1 -, :3) AgrohacleriulII sp. x 12.'5,12f:i
 (:1-(1 -> :3) Agro/;act.eriu 11/ radiohacler x 125,126
(:1-(1 -> :3) Agrohact.eriulII rhizoge//es x 125,126
(:1-(1 -> :3) A[caligeues j'aecalis x 125,126
val'. IIII/xogeues
(:1-(1 -> 4) Ace!o/;acler aceNge//IlJll x 6
(:1-(1 -> 4) Acelo!Jacler pasleuria//us x 127
(:1-(1 -> 4) Acelo/;acter ra//ce//s x 127
{3-(1 -> 4) Acelo/;acter xl/lill//III x 127-133
B. Levans
Acelo/;acter sp. frudose lUi
Bacillus suhlilis li'uctose 116
Corl/ue/;acleriulII sp. li'udose 116
Pseudolllo//as sp. fructosc 116
Sireplococclls sp. frudose 116
C. Miscellaneous
SlIeeilloglueans Achrolllo/;acier [aclo[I/Ncus x x succinyl 134
W
'1
~
A[caligelles .Iiwcalis x x succinyl 135,136
val'. 11lyX()genes
Arlhro!Jacter sla/;ilis x x succinyl, acetyl, 28
pyruvate
Baderial sp. x rhamnose 137
Corl/lle!Jaclerilllll illSidioSll1ll x x fucose 138
C or1/11 e!)(/cleri 11111 sepedo//iclIlII x x fucose 138
Rhizo!Jilllll lIIeWoN x x acetyl, pyruvate 78,79
Hhizo!JiulII japo//iclIlII x X lnannose 82
X X 3
 TABLE VI

Fungal, Exoccllular Polysaccharidcs Containing Ncutral Sugar Hcsiducs

t'" Componcnt sugars

-1
00
Polysaccharidc Othcr
typc Micro-organism Gal Glc Man substitucnts Hcfcrcnccs

I'ullulan All reohasidilllli plllllllal/s x I:3H-141

,,-(I -> :3)-G Iu ca n Fungal sp. x 142
"Glycogen-like" A rill ilia ria lIIellae x 14:3
{3-(I -> :3)-Gluc<tn x 14:3
Fungal sp. x 144
FlIsarilllll solalli x 14.'>
llelolilllll sp. x 146
Pleclal/ia occidellialis x 146
Sclerogl"ca" C/aoiceps pllrpllrea x 147,148
Sclerotilllll glllcalliclI/1I x 147,14f),I.'>0
Sclerotillill m({:\'ii x 147,150
Sierelllll sallgllillolellill/I/ x 147
 l!) c.c
iji; iji; }j;;i; L') t- X .~ L') It) (,C t- .:J) 1>1 It)
l1"' L~ t6 L') ~. L') L') L') ::;; c.c ::;; c.c cC
(,C cD t-
~
I I I
6 1.
It)
1>1
L~ (,C ::;;
1>1
lD t:::: r:

x X X X X X X X X X X X

X X X X

X X X X X X X

.::2
.:';.;
~
~~

.~ .]
~- ~

~
6'"'
g -'"' '"''6
:"j l

~ -
; @:
:::::::
::: :::: :::
.::;

'"' '"'
-
j~~
l!
t"""\.
V "'"
'-"
~~~~
r", ,"" ....'-" .-.
v V "-' V

279
 TABLE VII
Microbial, Exoccllular Polysaccharidcs Containing Amino Sugar Rcsiducs

Componcnt sugars

Micro-organism GalNAc GaIN GlcNAc GlcN Other Ucfcrenccs

Achroll/o!Jacter georgiopo/ita/l/lll/ GlcNAcA, 2,6-didcoxy-GlcNAc 176

Aspergillus spp. x x Man 177
Aspergillus /lidu/aus x Gal 178
ASJIl'l'gillus pal'llsilicllS x x 17H
Aspergillus sldae x x KDO" 180-18:3
Bacillus cercus x x 184,185
Bacillus lI/egateriulI/ x l-IexN, Cal 186,187
Bacillus su!Jlilis x x 184
Chrolllo!JacteriulIl vio/aceuill HexN, CIe, nnmic acid 188,18H
Cutophaga eO//lII//laris x x WO
l'seudou/Ouas so/a/laccwrulI/ x tletltral stlgar WI
Hhi/locladiella e/a lior CIeNAeA W2
Hhi/locladiella 1/lIII1SO/lii x GIeNAcA 1!J:3-1H6
Streptococcus (grotlp A hClllolytic) x GIeA W7

" :3-Deoxy-lHlla/l/lo-oc!tllosotlie acid.

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286 PAUL A. SANDFORD

c. Improvement of Genotype.-Often, a desirable feature may be

enhanced, or an undesirable feature eliminated, by alteration of the
genotype. Various methods for altering genotypes include: (l) in-
duced mutation, (2) spontaneous mutation, and (3) transfer of existing
genes. Although induced mutation has been used successfully, un-
wanted mutations may also occur. Perhaps the most attractive
methods of improvement of the genotype involve transfer of genetic
material; this method offers the advantage of specificity, stability, and
relative freedom from unwanted changes in other genes. With bacte-
ria, genetic transfer by (1) conjugation, (2) b'ansduction, (3) h'ansfec-
tion, and (4) h'ansformation has been accomplished, and these
procedures are extensively used. 213 However, these operations have
only rarely been applied in the production of polysaccharides (for ex-
ample, see Ref. 214).

2. Biosynthesis of Exopolysaccharides 15 ,215

Most microbial exopolysaccharides are apparently synthesized in-
tracellularly. However, with various Leuconostoc, Streptococcus, and
Bacillus species, such exopolysaccharides as dexh'an and levan can be
fonned by adding proper substrates that do not penetrate the cell
membrane. 216,2li Surprisingly little infom1ation is available about the
biosynthesis ofbiopolymers of commercial value. However, as most of
them are probably formed intracellularly, the process by which sub-
sh'ates enter microbial cells, where they are modified by various enzy-
mic reactions and finally excreted in polymerized fom1 into the
medium, bears attention. Even with a lack of complete biosynthetic
information, the results of research on related micro-organisms may
be eXhapolated to form a reasonable hypothesis for the biosynthesis of
polysaccharides.
Several fundamental features of polysaccharide biosynthesis are ap-
parently present in all microbes. However, the fate of a subshate sup-
plied to an exopolysaccharide-producing, microbial cell depends on
the microbial species chosen. Although studies have been made with

(213) W. Hayes, "The Genetics of Bacteria and Their Viruses," Blackwell, Oxford, Eng-
land, 1968.
(214) A. Markovitz and N. Rosenbaum, Froc. Nat/. Acad. Sci. USA, 54, 1084-1091
(1965).
(21.5) 1. W. Sutherland, Ref, 33, pp. 40-57.
(216) R. J. Gibbons and M. Nygaard, Arch. Oral Bioi., 13, 1249-1262 (1968).
(217) E. E. Smith, FEBS Lett., 12,33-37 (1970).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 287

yeast,218-220 more studies deal \vith the synthesis of exopolysac-

charides (including lipopolysaccharides) by bacteria. 18 ,216,217,221,222
a. Uptake of Substrate.-The uptake of subsh'ate is one of the first
limitations of the production of exopolysaccharides. A specific sub-
sh'ate may enter the cell by one, or all, of three mechanisms: facili-
tated diffusion, active h'ansport, or group translocation. 223 Differences
are to be expected with different types of micro-organisms.
• b. Intermediary Metabolism.-A subsh'ate that has entered the cell
is usually phosphorylated either by a group-h'anslocation mechanism
or by a hexokinase that utilizes adenosine 5 '-triphosphate (ATP). This
• phosphorylated substrate can then be used either for energy (catabo-
lism) or for the formation of such anabolic products as intracellular
polysaccharides (such as glycogen), lipopolysaccharides, cell-wall
polysaccharides, or expolysaccharides. Thus, there is competition for
subsh'ate among the various anabolic products whose synthesis is con-
trolled by the cell. Control can be achieved by regulation of the
synthesis224-227 and hydrolys'is 228,229 of nucleoside 5 ' -(glycosyl diphos-
phates) ("sugar nucleotides") that are involved in the biosynthesis of
the polysaccharide. Examples are also known of genetic regulation of
precursors specific to a particular polysaccharide.230-232
c. Formation of Exopolysaccharide.-Several bacterial polysac-
charides are known to be constituted of repeating units of sugar resi-
dues. The consh'uction of such units is performed by the transfer of

(218) L. P. Kozak and R. K. Bretthauer, Biochemistry, 9, 1115-1121 (1970).

(219) R. K. Bretthauer, S. Wu, and \V. E. Irwin, Biochim. Biophys. Acta, 304,736-747
(1973).
(220) C. E. Ballou, Adv. Ellzymol., 40, 239-270 (1974).
(221) E. C. Heath, All/HI. Rev. Biochem., 40,29-56 (1971).
(222) P. H. Makela and B. A. D. Stocker, AIlIl!l. Rev. Genet., 3,291-320 (1969).
(223) S. Roseman, in "Metabolic Pathways," L. E. Hokin, ed., Academic Press, London
and New York, 1972, pp. 41-89.
(224) J. Preiss, in "Current Topics in Cellular Regulation," B. L. Horecker and E. R.
Stadtman, eds., Academic Press, New York, 1969, Vo!. 1, pp. 125-160.
(225) W. D. Grant, 1. W. Sutherland, and J. F. ·Wilkinson,]. Bacterial., 103, 89-96
(1970).
(226) R. L. Bernstein and P. W. Robbins,]. Bial. Chem., 240,391-397 (1965).
(227) R. H. Kornfeld and V. Ginsburg, Biachi11l. Biophys. Acta, 117,79-87 (1966).
(228) J. B. Ward and L. Glaser, Biache11l. Biaphys. Res. Call1mlln., 31,671-676 (1968).
(229) J. B. Ward and L. Glaser, Arch. Bioche11l. Biophys., 134,612-622 (1969).
(230) Ivl. M. Liebernlan and A. Markovitz,}. Bacterial., 101, 965-972 (1970).
(231) L. R. Rothfield and D. Romeo, Bacterial. Rev., 35, 14-38 (1971).
(232) C. J. Waechter and W. J, Lennarz, Anllll. Rev. Biache11l., 45, 95-112 (1976).
288 PAUL A. SAJ'iDFORD

the appropriate glycosyl group from a "sugar nucleotide" to a carrier, a

lipid isoprenoid alcohol phosphate. 231 ,232 For two Enterobacter aero-
genes systems,233,234 the reaction sequences have been well character-
ized. Troy and coworkers 233 found that the following series of
reactions is involved.

UMP
UDP-o-Galactose + P-lipid __
E L""-__ - - l__ o-galactose-PP-lipid

F
~~;~;e •
UDP UDP-o- GDP
\ glucuronic acid

o-Glucuronic acid -D-mannose- E ~ / D-mannose-o-

D-galactose- PP-lipid galactose-PP-lipid

UDP _O -
galactose

F UDP

D-Galactose-D-mannose-o-galactose-PP-lipid polysaccharide

o-glucuronic acid

where P = phosphate, and DP or PP = diphosphate.

The exact mechanisms involved in further elongation of the chain
and in the release of the exopolysaccharides are still unknown, but it
is suspected that the site of exopolysaccharide biosynthesis lies in the
cytoplasmic membrane,235 where lipopolysaccharide is known to be
synthesized.
As there is competition for isoprenoid lipid between lipo- and exo-
polysaccharide, peptidoglycan, and teichoic acids, the availability of
isoprenoid lipid phosphate is one of the most critical factors affecting
the synthesis of exopolysaccharide.236

(233) F. A. Troy, F. A. Frennan, and E. C. Heath,]. BioI. Chem., 246, 118-133 (1971).
(234) 1. W. Sutherland and M. Norval, Biochem.]., 120,567-576 (1970).
(235) M. J. Osborn, J. E. Gander, and E. Parisi,]. Bioi. Chem., 247,3973-3986 (1972).
(236) 1. W. Sutherland, "Surface Carbohydrates of the Prokaryotic Cell," Academic
Press, London and New York, 1977.
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 289

d. Modification and Release of Polysaccharides.-Many polysac-

charides contain acyl and acetal substituents. As the proportion of
acyl groups in exopolysaccharides is variable, and as the propeliies of
these polymers can also vary \vith their acyl content,96 more attention
needs to be given to factors governing the biosynthesis of acyl groups.
Although preliminary evidence has been acquired indicating that acyl-
ation occurs while the growing oligosaccharide is attached to a lipoi-
dal carrier, 15,237 fuliher studies are needed in order to verify this result.
The nonsugar groups most commonly found in exopolysaccharides
are the O-acetyl and O-(l-carboxyethylidene) (pyruvic acid acetal)
groups. Other acyl groups, such as formyl,137 succinyl,28,135 and mal-
onyl,159,160 have also been found in exopolysaccharides. Loss of acetyl-
ation by a strain, without loss of biosynthesis of an exopolysac-
charide, has been demonstrated. 238 It also appears that pyruvylation is
not essential for polysaccharide synthesis.215
The way in which a polysaccharide is released from the isoprenoid
lipid is not yet known. Most probably, enzymes are present that
cleave the terminal, phosphate-linked, monosaccharide residue. The
chain length of a polysaccharide may depend on the growth rate; that
is, a higher growth-rate might lead to a faster turnover of the carrier
lipid and release of polysaccharide of lower molecular weight. In-
stances are known where variable chain-length results from the action
of depolymerases. 239

3. Methods of Fermentation
The growth environment of micro-organisms is impoliant for maxi-
mal production of an exopolysaccharide. Almost all exopolysac-
charide-synthesizing microbes are either aerobes or facultative
anaerobes. Production of a polysaccharide is normally highest when
oxygen is not a limitation. Pure cultures of microbes are used, and
aseptic conditions are generally needed in order to prevent unwanted
organisms from growing in the medium.
a. Culture Media.-The sugar composition of most exopolysac-
charides is independent of the carbon and energy sources for
growth.240.241 However, where more than one exopolysaccharide is

(237) 1. W. Sutherland,]. Gen. Microbiol., 65, v (1971).

(238) P. J. Garegg, B. Lindberg, T. Onn, and T. Holme, Acta Chem. Scand., 25, 1185-
1194 (1971).
(239) E. N. Davis, R. A. Rhodes, and H. R. Shulke,Appl. Microbiol., 13,267-271 (1965).
(240) J. F. Wilkinson, W. F. Dudman, and G. O. Aspinall, Biochem.]., 59,446-451
(1955).
(241) P. A. Sandford and H. E. Conrad, Biochemistnj, 5, 1508-1517 (1966).
290 PAUL A. SANDFORD

fOID1ed, the relative amounts of each may vary with the conditions of
growth. 'With the synthesis of a number of homopolysaccharides, such
as dextrans and levans, very specific subsh"ates are needed by the exo-
cellular enzymes evolved in polysaccharide production. Unless a pho-
tosynthetic or nih"ogen-fixing organism is involved, a culture medium
always requires: (1) a source of carbon, usually a carbohydrate, such as
D-glucose or sucrose; (2) a source of nitrogen, usually added as inor-
ganic salts of NH 4+ and N0 3- ions, or as more-complex, natural prod-
ucts, such as yeast hydrolyzates or autolyzates, casein hydrolyzate,
distiller's dried-solubles, or soybean meal (the complex type often
contains other growth-factors, such as vitamins and amino acids that
may be favorable for growth by certain micro-organisms); and (3) such
cations as iron, magnesium, manganese, potassium, and sodium, which
are often added separately. Phosphorus is also required, and is gen-
erally added as potassium or sodium phosphate, \vhich may also be
used for their buffering action. Often, trace minerals are necessary,
but their need may not be recognized, as they may be present as part
of the organic components of the medium. To achieve maximal pro-
duction of the polysaccharide, the components most favorable for
growth, and their proportions, must be established experimentally.
Use of defined media, where possible, allows determination of the ef-
fect of a specific ingredient on the production of a polysaccharide.
Complex media, however, sometimes contain unknown growth-
factors that stimulate polysaccharide formation. 242
b. Optimal Growth-conditions for Synthesis.-Optimal conditions
for growth and for polysaccharide production are also affected by pH,
temperature, proportion of air, presence or absence of agitation, and
the size of the inoculum. For each organism, the optimal conditions
must be determined experimentally on a small scale in the laboratory,
and then scaled up for pilot and indush"ial operations.
c. Fermentation Methods.-In the traditional, batch-fermentation
method of mass cultivation, a small amount of the microbe is inocu-
lated into a medium containing all of the necessary ingredients, and
growth usually continues until one of the substrates has been ex-
hausted. In continuous felmentation, continuous addition of fresh me-
dium is made to the culhlre, with simultaneous harvesting of the
desired products (and a portion of the cells). More conh"ol may be ex-
erted with continuous culturing, and cell physiology and biochemis-
try may be more readily studied. Continuous fermentation is of

(242) R. A. Moraine and (S.) P. Rogovin, Call.]. Microbial., 17, 1473-1474 (1971).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 291

practical interest, in that more-efficient use of nuh'ients may be rea-

lized.
vVith xanthan gum, continuous fermentation243-245 resulted in lower
estimates of cost for its production. Also, with the production of "bac-
terial alginate" by Azotobacter vinelandii, serious loss of subsh'ate by
respiration could be minimized by proper selection of the conditions
in the continuous fem1entor. 62 vVith some micro-organisms, the de-
sired metabolites are not produced, owing to selection ofless-produc-
tive strains. Apparently, tl1is selection occurred with Xanthomonas
campestris 246 when Rogovin and coworkers243-245 found that produc-
tion of xanthan decreased after ~8-10 fermentor turnovers.

4. Isolation of Polysaccharides

The metl10d used for isolating an exopolysaccharide depends on tl1e

characteristics of the organism that produced it, tl1e type of polysac-
charide, and the desired grade of purity. Crude-grade products may be
obtained by drying tl1e entire fem1entation-brotl1. Unattached exo-
polysaccharides may be separated from cells eitl1er by differential
centrifugation or by filtration, botl1 of which require low viscosity to
be effective. The removal of water may be accomplished by spray- or
drum-drying, or by addition of a nonsolvent miscible with water (such
as acetone, metl1anol, etl1anol, or isopropyl alcohol) to precipitate tl1e
polymers. Often, addition of an elech'olyte (salt) helps in tl1e precipi-
tation by neutralizing charges on the polysaccharides. Recovery of tl1e
nonsolvent is essential for economic reasons.
Purification of exopolysaccharides is generally a difficult matter,
owing to tl1e high viscosity of most polymers. Quatemmy ammonium
compounds, which precipitate acidic polysaccharides, have been
used successfully247 to separate acidic from neuh'al polysaccharides.
Pretreahnent witl1 enzymes that preferentially cleave such undesir-
able contaminants as protein, nucleic acid, and cells results in isola-
tion of improved products. Selection eitl1er of polysaccharide or of
contaminating material on either ion-exchange or affinity-chromatog-

(243) R. A. Moraine and (5.) P. Rogovin, Biotechnol. Bioeng., 8,511-524 (1966).

(244) R. W. Silman and (5.) P. Rogovin, Biotechnol. Bioeng., 12,75-83 (1970).
(245) R. W. Silman and (5.) P. Rogovin, Biotechnol. Bioeng., 14,23-31 (1972).
(246) M. C. Cadmus, S. P. Rogovin, K. A. Burton, J. E. Pittsley, C. A. Knutson, and A.
Jeanes, Can.]. Microbiol., 22, 942-948 (1976).
(247) W. J. Albrecht, S. P. Rogovin, and E. L. Griffin, Nature, 194, 1279 (1962).
292 PAUL A. SANDFORD

raphy materials has thus far found little application, even though such
methods have been shown to be successfu1,248-25o.

V. ACIDIC SUGAR-CONTAINING, MICROBIAL POLYSACCHARIDES

OF COMMERCIAL INTEREST

1. Xanthan Gum28 ,29,116,251

a. Properties.-Xanthan gum is the name given to the exocellular
polysaccharide produced by the bacterium Xanthomonas campestris
NRRL B-1459 (a plant pathogen causing diseases of some plants). Xan-
than gum is composed ofD-glucosyl, D-mannosyl, and D-glucosyluronic
acid residues, and differing proportions of O-acetyl (-4.5%) and
pyruvic acid acetal (~2 to 6%). The main structural features 91 - 96 ,252-256
are shown in Fig. 1.
The technological importance of xanthan gum rests principally on
its unusual and distinctive properties25 ,28,29,49,116,251,257-260 in aqueous
solution. Some of these properties are: (1) remarkable emulsion-stabi-
lizing and particle-suspension ability, (2) low concentrations yield
high viscosities, (3) recoverable shear-thinning (extremely large shear
dependence of viscosity), (4) little variation in viscosity with tempera-
ture under normal conditions of industrial utilization, and (5) gel for-
mation \vhen mixed with certain other, nongelling polysaccharides.
Several of these properties can be explained in terms of a large mol-

(248) M. A. Jermyn, Allst.]. Bioi. Sci., 15, 787-791 (1962).

(249) R. G. E. Guy, M. J. How, M. Stacey, and M. Heidelberger,]. Bioi. Chern., 242,
5106-5111 (1967).
(250) E. E. B. Smith, G. T. Mills, H. P. Bernheimer, and R. Austrian,}. Bioi. Chern.,
235, 1876-1880 (1960).
(251) "Xanthan GumlKeltrollKelzanJA Natural Biopolysaccharide for Scientific Water
Control," Kelco Company, San Diego, California, 2nd Edition, 1975.
(252) J. H. Sloneker and A. Jeanes, Can.]. Chem., 40, 2066-2071 (1962).
(253) J. H. Sloneker and D. G. Orentas, Can.]. Chem., 40,2188-2189 (1962).
(254) D. G. Orentas, J. H. Sloneker, and A. Jeanes, Can.]. Microbiol., 9,427-430
(1963).
(255) 1. R. Siddiqui, Carbohydr. Res., 4, 284-291 (1967).
(256) R. Moorhouse, M. D. Walkinshaw, and S. Arnott, Ref. 33, pp. 90-102.
(257) M. Glicksman, "Polysaccharide Gums in Food Technology," Academic Press,
New York, 1970.
(258) E. R. Morris, Ref. 33, pp. 81-89.
(259) 1. C. 1,,1. Dea and E. R. Morris, Ref. 33, pp. 174-182.
(260) G. Holzwarth, Biochemistry, 15,4333-4339 (1976).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 293

CHpH

CH.OH ~O

o-
~~
CH 20H °
CHpH )--L(0 ° OH OH

r-0~~~ OH

~ 0 OR Ao~OCH' °
00
HO
00 °

/g
OH

CH. °
Me,
/C
°
H0 2C \ OH HO

FIG. I.-Apparent Repeating-unit of Xanthan Gum. 93 •94

ecule (molecular weighf61 of 1-10 million) having a rodlike, ordered

conformation.28,256,258,26o,262-265 In solution at moderate temperatures,
xanthan gum apparently exists in a native, ordered conformation. Evi-
dence has been acquired266 suggesting that native xanthan gum as-

(261) F. R. Dintzis, G. E. Babcock, and R. Tobin, Carbohydr. Res., 13,257-267 (1970).

(262) E. R. Morris, D. A. Rees, G. Young, M. P. Walkinshaw, and A. Darke,]. Mol.
Bioi., 110, 1-16 (1977).
(263) 1. C. M. Dea, E. R. Morris, D. A. Rees, E. J. Welsh, H. A. Barnes, and J. Price,
Carbohydr. Res., 57,249-272 (1977).
(264) D. E. Woessner and B. S. Snowden, Jr., Ann. N. Y. Acad. Sci., 204, 113-124
(1973).
(265) A. Darke, E. G. Finer, R. Moorhouse, and D. A. Rees,j. Mol. Bioi., 99,477--486
(1975).
(266) G. Holzwarth and E. B. Prestridge, Science, 197,757-759 (1977).
-------------
294 PAUL A. SANDFORD

sumes a multistranded, helical fonn, but, at low concentrations of a

salt, this order can be "melted out." It has also been suggested 256 that,
in the ordered conformation, the charged, trisaccharide side-chains
fold back around the cellulose backbone to give a rigid, rodlike struc-
ture. As the pylUvic acetal groups are located on the D-mannosyl end-
groups of side chains, it is not surprising to find that xanthans of dif-
fering pylUvate levels (1.0-6.0%) display differing rheological proper-
ties.96.267 The temperature at which the confom1ation can be "melted
out" is higher for xanthan samples low in pYlUvate than for those high
in pylUvate. 268 Of practical value in industrial processes, where fluid
flow or pumping is involved, is the ability of xanthan in low concentra-
tions to modify turbulent friction (friction reduction).29,269-271 Xanthan
shows a marked tendency to interact with f3-D-(l ~ 4)-linked polysac-
charides, especially locust-bean gum (LBG), guar gum, and konjac
mannan. 251 ,259,263,272,273 Although, under normal conditions, neither
xanthan gum nor LBG will gel alone, gels can be formed having total
polysaccharide concentmtions at or below 1%.
b. Preparation.-The initial studies on the feasibility of using such
microbes asX. campestris to produce industrial gums were conducted
at the NRRC, USDA. A summary of the procedures developed and
used at the NRRC for analysis and production (including media com-
position, culture maintenance, and inoculum buildup methods) of
xanthan gum produced by X. campestris NRRL B-1459 has been pub-
lished. 274 Methods for continuous culturing of xanthan were also de-
veloped. 245 ,275 Because of its unique properties and the ease with
which it can be produced in good yield (~60% of the sugar added)
from simple media containing common sugars and nitrogen sub-
strates, several industrial fim1s are now producing xanthan gum. Com-

(267) M. C. Cadmus, C. A. Knutson, A. A. Logoda, J. E. Pittsley, and K. A. Burton, Bio-

technol. Bioeng., 20, (1978).
(268) G. Holzwarth and P. A. Sandford, unpublished results.
(269) P. R. Kenis,]. lippl. Polym. Sci., 15,607-618 (1971).
(270) P. R. Kenis, Nature (London), 217, 940-942 (1968).
(271) J. W. Hoyt,]. Polym. Sci., Part B, 9,8.51-862 (1971).
(272) 1. C. .\1. Dea and A. Morrison, lidv. Carbohydr. Chem. Biochem., 31, 241-.312
(1975).
(27.3) P. A. Sandford, J. E. Pittswley, A. Jeanes, and C. A. Knutson, Abstr. Pap. Am.
Chem. Soc. (Great Lakes Regional) Meet., 11, 49 (1977).
(274) A. Jeanes, S. P. Rogovin, M. C. Cadmus, R. W. Silman, and C. A. Knutson, U. S.
Dep. ligric., ARS-NC-.51, 1-14 (1976).
(27.5) M. Charles and M. K. Radjai, Ref. 33, pp. 27 -.39.
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 295

mercial production of xanthan gum has been canied out in the United
States since 1967, and more recently in Europe. 2i6
In 1975, worldwide production of xanthan gum was estimated33 at
5000 tons, and it may reach 18,000 tons per year by the end of 1979.
Increasing knowledge of the biosynthesis and nutritional require-
ments that affect the yield and acyl composition of xanthan
gum should allow production of an improved and unvarying prod-
UCt. 96 ,198,2ii

c. Applications. 29 ,111,230,23i-Xanthan gum has numerous applica-

tions in both the nonfood and food indush-ies. Table VIn lists some of
the uses of xanthan gum in the food indushy.26,29,116,251,2i8,2i9 The gen-
eral use of xanthan gum in foods where "Standards of Identity" regu-
lations do not preclude such use has been cleared by the U. S. Food
and Drug Administration. 28o It is permitted in french dressing,281
cottage-cheese creaming-emulsions,282 cold-pack cheese-foods,283
poultry nonmeat-ingredients,284 paper and paperboard intended to
contact food,251 and pesticides to be applied to growing crops and raw
agricultural commodities.285 Xanthan has been used to stabilize tooth-

TABLE VIII
Current Uses of Xanthan in Foods

Salad dressings-pourable and spoonable; n0I111al and low-calorie

Relishes and tart sauces
Cheeses and cheese products
Egg substihlte (cholesterol-free)
Gelled meats
Gelled salads and desserts
Puddings-canned, dry cold-mix, milk-gel
Dense symps and dessert toppings
Beverages-fruit, nonElt dry-milk
Dehydrated gravies, sauces, and soups

(276) P. Godet, Process Biochem., 8(1),33-34 (1973).

(277) 1. W. Sutherland, Ref. 33, pp. 40-57.
(278) A. Jeanes, Food Technol., 28,209-227 (1974).
(279) A. A. Lawrence, "Edible Gums and Related Substances," Noyes Data Corp., Park
Ridge, New Jersey, 1973, pp. 234-279.
(280) Fed. Regist., 34, 5376 (March 19, 1969).
(281) Fed. Regist., 36, 17333 (August 28, 1971).
(282) Fed. Regist., 38, 1218 (January 10, 1973).
(283) Fed. Regist., 38, 6883 (March 14, 1973).
(284) Fed. Regist., 39,4466 (Febmary 4, 1974).
(285) Fed. Regist., 36, 24217 (December 22, 1971).
296 PAUL A. SANDFORD

TABLE IX
Industrial Applications of Xanthan Gum

Abrasives Petroleum
Adhesives drilling muds
Agricultural sprays flooding
Ceramics mobility-control buffers
Cosmetics Phannaceuticals
Emulsions Pigments
Gels Polish
Ink Suspending agent
Mining, ore separation Textile
Paint Wallpaper
Paper Welding rods

paste, canned gravy-type pet-foods, liquid cattle-feed supplements,286

and calf milk-replacers. 286 The gum may have potential as a gluten
substitute in baking. 287 ,288
Table IX lists some applications of xanthan in nonfood indus-
tries. 29 ,1l6,251 It is used to thicken aqueous solutions and dense, salt so-
lutions or dispersions, suspend heavy particles, stabilize suspensions,
provide pseudoplastic flow and enhanced gelation either by complex-
ing with trivalent metal ions or by synergistic interaction with galacto-
mannans,272 and to make, and stabilize, oil-water emulsions. Xanthan
gum is currently used in agricultural sprays, cutting-oil emulsions,
textile-print pastes, and rust removers. One of the more important
present uses is in the recovery of crude oi1,33,289 The flow characteris-
tics of xanthan, coupled with its stability to salts and extremes of pH,
give it a technical advantage over most polymers in drilling needs.
The greatest potential for xantllan gum appears to lie in enhanced, oil-
recovery operations.33 ,289 Such processes have potential for producing
oil that is unrecoverable by normal means. The function of xanthan
gum in polymer water-flooding and micellar-polymer flooding is to
lower tlle mobility of the water injected. It is anticipated that xanthan
gum will be used in several, enhanced oil-recovery projects. If
projections33 are correct, in 1985, the annual demand for the polymer
could be as high as 200-250 million pounds for enhanced, oil-recov-
ery operations that could yield 300-400 million barrels of oil. Un-
doubtedly, there will be much competition among various polymers

(286) T. R. Andrew, Ref. 33, pp. 231-241.

(287) K. Kulp, F. N. Hepburn, and T. A. Lehmann, Baker's Dig., 48, 34-37 (1974).
(288) D. D. Christianson, Baker's Dig., 50,34-36 (1976).
(289) E. J. Sandvik and J. lVI. Maeker, Ref. 33, pp. 242-264.
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 297

for this application. Xanthan gum has also been successfully used as a
stabilizing agent in the "heavy media separation" of various min-
erals. 290

2. Erwinia tahitica Polysaccharide

a. Properties.-Zanflo®-lO is the trade name for the exocellular,
high-molecular-weight polysaccharide produced by a bacterium des-
ignated Erwinia tahitica. The polysaccharide consists of glucose, ga-
lactose, glucuronic acid, and fucose residues in the molar ratios of
6:4: 3: 2 and has an O-acetyl content of4.5%. Zanflo-10 is readily solu-
ble in hot or cold water, and can be used to thicken, suspend, and sta-
bilize aqueous systems. Zanflo exhibits pseudoplasticity and high
viscosity (even higher than &at of xanthan gum), especially at tem-
peratures between 0 and 60°, at high polysaccharide concentrations,
and at pH values between 3 and 10. Zanflo is resistant to cellulase and
other common enzymes. It is shear-stable, and compatible with many
salts. Even though it contains ~ 20% of monic acid, Zanflo is compati-
ble with most cationic agents and such dyes as Methylene Blue. It has
been claimed that Zanflo does not precipitate cationic dyes at any pH.
b. Preparation. 74 -Zanflo-10 is a development of the Kelco Divi-
sion of Merck and Co., Inc. Various biochemical characteristics of the
Gram-negative bacterium have been listed. 70 The organism is quite
specific with regard to the carbon source for optimal production of the
biopolymer. Sucrose or maltose is converted more efficiently than
n-glucose into polysaccharide in a typical medium composed of alpha-
amylase-treated starch preparations, phosphate buffer, ammonium ni-
trate, soy protein, and magnesium sulfate. Maximum viscosity is ob-
tained within 64 h. The product is isolated by precipitation with such
organic solvents as isopropyl alcohol.
c. Applications.-Zanflo-10 apparently has established applica-
tions in paint, and shows potential in otl1er products that contain cat-
ionic dyes.

3. Beijerinckia indica (Azotobacter indicum) Polysaccharide57 - 60

a. Properties.-PS-7, a development oftl1e Kelco Division of Merck

and Co., Inc., is tl1e exocellular polysaccharide produced by a strain of
soil bacterium Beijerinckia indica (Azotobacter indicum) var. myxo-
genes. The biopolymer is composed of glucose, rhamnose, and a

(290) L. Valentyik and J. T. Patton, TrailS. Soc. Mill. Eng. AIME, 113-118 (1976).
298 PAUL A. SANDFORD

uronic acid in the approximate ratios of 13.2: 3: 2.0 and has an O-acetyl
content of -8-10%. PS-7 has an unusually high viscosity (higher than
that of xanthan gum) that is stable over a wide range of temperature
and pH. The gum shows outstanding, recoverable shear-thinning
(pseudoplasticity), and is soluble in hot or cold tap-water, brine, or sea
water. It is also compatible with a wide variety of salts, but is incom-
patible with cationic or polyvalent ions at high pH.
b. Preparation.58 -PS-7 is produced by aerobic, submerged fenllen-
tation. Potassium phosphate, or tih"ation with potassium hydroxide, is
used to maintain the pH at 7. The material is recovered from the fer-
mentation medium by precipitation with isopropyl alcohol.
c. Applications.-The high viscosity and excellent, recoverable-
shear properties of PS-7 should make it a highly useful agent in oil-
\vell drilling-muds. In tests for suspending ability,55 PS-7 was by far
the most efficient of the polymers tested in suspending particles
(sand). Other potential applications may be in dripless, water-based,
latex paint, waterflooding systems for enhanced oil-recovery, wall-
joint cement adhesives, and textile printing.

4. Bacterial Alginate 62 ,63

a. Properties.-The glycuronan [poly(glycosiduronic acid)] alginic
acid is a well known and commercially important constituent of the
brown algae. 35 ,291,292 Alginic acid is a linear copolymer of J3-D-manno-
syluronic acid and a-L-gulosyluronic acid residues, the relative
amounts of which vary greatly for alginic acids from different species
of algae. 293 The bacteria Pseudomonas aeruginosa 75,77,294-296 andAzoto-
bacter vinelandii 40 ,297 have been shown to secrete exocellular polysac-
charides similar to the alginic acid from algae. For commercialization,
P. aeruginosa was avoided, because of its association with pathogenic
conditions in humans. A. vinelandii "alginate" is very similar to its
algal counterparts in its chemical and physical properties, including

(291) E. Percival and R. H. McDowell, "Chemistry and Enzymology of Marine Algal

Polysaccharides," Academic Press, New York, 1967.
(292) W. H. McNeeley and D. J. Pettitt, Ref. 23, pp. 49-81.
(293) A. Haug, B. Larsen, and O. Smidsr0d, Carbahydr. Res., 32,217 -225 (1974).
(294) H. O. Bouveng, 1. Bremner, and B. Lindberg,Acta Chern. Scand., 19, 1003-1004
(196.5).
(295) T. Murakawa, lpn.]. Microbiol., 17, 513-520 (1973).
(296) L. R. Evans and A. Linker,}. Bacterial., 116, 915-924 (1973).
(297) G. H. Cohen and D. B. Johnstone,}. Bacterial., 88,329-338 (1964).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 299

having portions of its sugar residues in the block arrangement,298 The

behavior of aqueous solutions of A. vinelandii alginate toward multi-
valent metal cations is velY similar to that shown by aigaimateriaF92
The molecular weight of the bacterial alginate is repOlied299 to be
5 x 105. Early studies indicated that the polydispersity.300 and lower
molecular weight of bacterial alginate were responsible for its inferior
solution-rheology and gel behavior as compared to those of algal prod-
ucts. However, further studies 62 have led to much improved quality.
b. Preparation.-Large-scale production of bacterial alginate by A.
vinelandii has been investigated by Tate and Lyle Ltd. 62 - 64 ,301 The se-
quence of steps by which bacterial alginate is biosynthesized from su-
crose has been established by characterization of the individual,
enzymic reactions. 301 These studies indicated that D-mannuronan
[poly(D-mannosiduronic acid)] is f0D11ed prior to partial, enzymic epi-
merization of the D-mannosyluronic acid residues to L-gulosyluronic
acid residues at the polymer level. Use of this information, coupled
with the finding of Haug and Larsen65 that the ratio of D-mannuronic
to L-guluronic acid in Azotobacter alginate could be influenced by
calcium ion, which is necessary to an exocellular epimerase, allowed
selection of fermentation conditions that gave products having a wide
range of viscosities. Some of these products compared favorably to
algal material.
Shldies comparing batch \vith continuous fermentations illustr'ated
the utility of continuous culturing to avoid high rates of respiration en-
countered in batch culturing.
c. Applications.-Applications for bacterial alginate should be
those of ordinmy alginate; these applications include use as an emul-
sion stabilizer, a gelling agent, a thickener, a foam-stabilizer, and a su-
spending agent.

5. Arthrobacter Polysaccharides
a. Properties.-The structure and properties of exopolysaccharides
from three Arthrobacter species were found to differ significantly.
The exopolymer from 50- 52 ,302 A. viscosus NRRL B-1973 contains D-
glucosyl, D-galactosyl, and D-mannosyluronic acid residues in the

(298) A. Penman and G. R. Sanderson, Carbohydr. Res., 25,273-282 (1972).

(299) O. Smidsr¢d and A. Haug, Acta Chem. Scand., 22, 797 -810 (1968).
(300) C. Bucke,J. Chromatogr., 89, 99-102 (1974).
(301) D. F. Pindar and C. Bucke, Biochem. ]., 152,617-622 (197.5).
(302) C. A. Knutson and A. Jeanes, Anal. Biochem., 24,470-481 (1968).
300 PAUL A. SANDFORD

molar ratios of 1: 1 : 1, and contains a considerable proportion of 0-

acetyl groups (25% by weight). The polysaccharide from another52,303
A. viscosus, NRRL B-1797, was found to contain D-glucosyl, D-galacto-
syl, and D-glucosyluronic acid residues in the molar ratios of 3:3: 1,
with 7.8% of O-acetyl groups and 5.5% of pyruvic acid (acetal).
A. stabilis NRRL B-3225 was found28 ,29,304 to contain D-glucose: D-
galactose in the molar ratio of 2: 1, together with O-(l-carboxyethyli-
dene) (pyruvic acid acetal) (5.1%), O-acetyl (3.7%), and half-ester 0-
succinyl (5.9%) groups. Both the acetic and succinic acid ester groups
can be removed by mild base, to give a deacylated polysaccharide that
is a galactoglucan having pyruvic acid acetal groups.
b. Preparation.-Methods for the production5 of PS B-1973 and52
PS B-1797 have been patented.305-307 PB-3225 has been produced on a
similar medium. 308 A most critical factor for production is the presence
of magnesium ion, which is required for good yields.
c. Applications.-As each of the Arthrobacter polysaccharides dif-
fers in composition, structure, and rheological properties, each would
be expected to have different applications. All of them produce a high
viscosity, and may be useful as thickening and suspending agents.
Deacylation greatly alters the rheological characteristics of tlle gums.
Modified PS B-3225 has been found309 to react synergistically with
both guar and locust-bean gums,273 to give viscosity enhanced over
tllat of the individual gums.

6. Other Microbial Polysaccharides

a. Bacillus polymyxa.-Two strains of B. polymyxa, discovered in-
dependently in Japan66 and in the United States,310 appear to give tlle
same product, \vhich is composed of D-glucosyl, D-mannosyl, D-galac-
tosyl, and D-glucosyluronic acid residues in the molar ratios of
(303) C. A. Knutson, J. E. Pittsley, and A. Jeanes, Abstr. Pap. Am. Chem. Soc. Meet.,
161, CARB-28 (1971).
(304) J. W. Gill, U. S. Pat. .3,632,570 (1972); Chem. Abstr., 76, 111,69511 (1972).
(305) M. C. Cadmus and R. F. Anderson, U. S. Pat. 3,228,855 (1966); Chem. Abstr., 64,
1O,371d (1966).
(306) ~v1. C. Cadmus and R. F. Anderson, U. S. Pat. 3,314,801 (1967); Chem. Abstr., 67,
81,144h (1967).
(307) 1\1. C. Cadmus, M. O. Bagby, K. A. Burton, and 1. A. Wolff, U. S. Pat. 3,565,763
(1971); Chem. Abstr., 74, 139,56:lf (1971).
(308) C. A. Knutson, A. Jeanes, and J. E. Pittsley, Carbahydr. Res., in press.
(309) C. A. Knutson, unpublished results.
(310) M. C. Cadmus, K. A. Burton, A. A. Logoda, and K. L. Smiley, Bacterial. Proc.,
A102 (1967).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 301

3: 3: 1 :2. Aqueous solutions give a soft gel, and have exceptionally

high water-binding capacity.
b. Cryptococcus and Tremella.-Cryptococcus laurentii var. fla-
vescens NRRL Y-1401, a nonpathogenic yeast, produces an exopoly-
saccharide composed 104 of D-mannosyl, D-xylosyl, and D-glucosyl-
uronic acid residues, and O-acetyl groups, in the molar ratios of
4: 1: 1: 1.7. PS Y-1401 has been shown to suspend laundry soil311 and
to stabilize emulsion paints.312 A 1.0% solution of purified PS Y-1401
has a viscosity of - 9 Pa.s (- 9,000 centipoises), and displays plastic,
rheological character.28 PS Y-1401 was produced in good yield with a
medium containing D-glucose, yeast-autolyzate paste, and manganese
sulfate. 105 Exopolysaccharides structurally related to PS Y-1401 were
characterized from species of Tremella (haploid, yeast stage).113,114 A
more-detailed structural analysis of a Tremella mesenterica exopoly-
saccharide, conducted by Fraser and coworkers,313,314 permitted com-
parison with earlier structural studies315 on PS Y-1401.
c. Rhinocladiella.-An exocellular polysaccharide composed193 ,194
of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-
glucuronic acid in the molar ratio of - 2: 1 is produced by a nonpatho-
genic, black, yeast-like fungus designated NRRL Y-6272, later identi-
fied as Rhinocladiella mansonii,316 Difficulties in removing the black
pigment from the high-viscosity polysaccharide were overcome by
use of an improved medium and of pink mutants of the parent, black
strain that no longer evolved the undesirable, black pigment. 195 Even
with refinements of scale (20-liter fermentation),196 the polysaccharide
conversion-efficiency from D-glucose (5%) was only -20%. The puri-
fied polysaccharide gave a high viscosity of 9.4 Pa.s (9,400 cP) at 1%
concentration, 3.84 sec-I.
Another black, yeastlike fungus, NRRL YB-4163, identified as Rhino-
cladiella elatior,316 produces an exopolymer composed exclusively of
2-acetamido-2-deoxY-D-glucuronic acid residues 192 when grown on
the medium developed195 for PS Y-6272.

(311) A. Jeanes, R. G. Bistline, and A. J. Stirton,]. Am. Oil Chem. Sac., 49, 610-612
(1972).
(312) Heyden Newport Corp., Fr. Pat. 1,395,294 (1965); Chem. Abstr., 62, 133,896
(1965).
(313) C. G. Fraser, H. J. Jennings, and P. Moyna, Can.]. Biachem., 51,219-224 (1973).
(314) C. G. Fraser, H. J. Jennings, and P. Moyna, Can.]. Biachem., 51,225-230 (1973).
(315) }V!. J. Abercrombie, J. K. N. Jones, M. V. Lack, M. B. Perry, and R. J. Stoodley,
Can.]. Chem., 38, 1617-1624 (1960).
(316) K. A. Burton, L. K. Nakamura, and M. C. Cadmus, Myca/agia, 68,685-688 (1976).
302 PAUL A. SANDFORD

Both PS Y-6272 and PS YB-4163 show similar, non-Ne\vtonian vis-

cosity behavior, can be cast into flexible films, and stabilize oil-water
emulsions.317 Attempts to N-deacetylate PS Y-6272 and PS YB-4163
with aqueous alkali to produce an amphoteric polymer failed, owing
to gross decomposition of the products. 318
d. O-Phosphonomannans. 319-For some time, the Northern Re-
gional Research Center has studied the exocellular O-phosphonoman-
nans elaborated by yeasts of the genus Hansenula and related
genera. 155 ,320,321 In Type I O-phosphonomannan,319 the phosphate
groups occur exclusively in repeating, phosphoric diester Sb'Llctures
in which a phosphate group links the anomeric hydroxyl group of a
D-mannose oligosaccharide unit to the primary hydroxyl group of an-
other D-mannosyl residue. 322 ,323 In Type II O-phosphonomannan,319
phosphate occurs exclusively as hexosyl phosphate diester at nonre-
dueing end-groups of polysaccharides.324 Of the O-phosphonoman-
nans, the polymer from H. holstii NRRL Y-2448 has attracted the most
industrial attention. PS Y-2448 has a D-mannose to phosphate ratio of
- 5: 1. Aqueous dispersions ofPS Y-2448 display unusual clarity, plas-
tic rheological behavior that is characterized by rapid shear-thin-
ning,261,325 marked loss of viscosity in the presence of salts, and
formation of cohesive gels when complexed with borax. The funda-
mental conditions for the fermentative production21 of several O-phos-
phonomannans have been determined. 320 Fifty percent conversion of
D-glucose into the biopolymer was achieved in 4 days.
PS Y-2448 was found to be innocuous in acute toxicity, skin, and
feeding tests. 326 It also rated high in organoleptic tests. 327 PS Y-2448
stabilizes beer foam,328 and suspends laundry soil. 311

(317) P. A. Sandford, J. E. Pittsley, P. R. Watson, K. A. Burton, 1v1. C. Cadmus, and A.

Jeanes,]. App/. Po/ym. Sci., 22, 701-710 (1978).
(318) P. A. Sandford, unpublished results.
(319) M. E. Slodki, R. M. Ward, J. A. Boundy, and 1v1. C. Cadmus, in "Fem1entation
Technology Today," Proc. Int. Ferment. Symp., 4th, 597 -601 (1972).
(320) R. F. Anderson, M. C. Cadmus, R. G. Benedict, and M. E. Slodki,Arch. Biochem.
Biophys., 89,289-292 (1960).
(321) M. E. Slodki, L. J. Wickerham, and 1v1. C. Cadmus,]. Bacteriol., 82, 269-274
(1961).
(322) A. Jeanes and P. R. Watson, Can.]. Chem., 40,1318-1325 (1962).
(323) M. E. Slodki, Biochim. Biophys. Acta, 57, 525-533 (1962).
(324) M. E. Slodki and J. A. Boundy, Del). Ind. Microbiol., 11,86-91 (1970).
(325) A. Jeanes and J. E. Pittsley,]. App/. Po/ym. Sci., 17, 1621-1624 (1973).
(326) A. N. Booth, A. P. Hendrickson, and F. DeEds, Toxicol. Appl. Pharmacol., 5,478-
484 (1963).
(327) A. S. Szczesniak and E. Farkas,]. Food Sci., 27,381-385 (1962).
(328) E. Segel, U. S. Pat. 2,943,942 (1960); Chem. Abstr., 54, 2.5,565a (1960).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 303

VI. NEUTRAL, MICROBLAL POLYSACCHARIDES

OF COMMERCLAL IMPORTAt~CE

1. Dextran 13 ,141,329-331
a. Properties.-Dextran is the name given to a large class of exocel-
lular, bacterial polysaccharides composed of a-D-glucopyranosyl resi-
dues. An extensive bibliography of researches on dextran, exclusive
of clinical studies, has been compiled. 332 Although each bacterial
sh'ain produces a unique D-glucan,12o a feature common to all dextrans
is the preponderance of (1 -i> 6)-linkages. Some dextrans are com-
posed almost exclusively of (1 -i> 6)-linkages, whereas others may
contain as little as 50% of (1 -i> 6)-linkages. As the non-(l -i> 6)-link-
ages may be either (1 -i> 2)-, (1 -i> 3)-, or (1 -i> 4)-, a series of a-D-
linked D-glucans that contain a variety of linkage types is available.
Differences in solubility and rheological characteristics are ap-
parently due to proportions and types of linkage, and how these are
arranged in each dextran molecule. Most dextrans are considered to
have high molecular weights (in the millions); a potentially useful ex-
ception333 was described by Hehre. 334
b. Preparation.-Almost all producers of industrial dextran use the
NRRL 512(F) strain of Lcuconostoc mcscntcroidcs or a similar org-
anism.335-33i B-512(F) dextran contains ~959'0 of a-D-(l -i> 6)- and 5% of
a-D-(l -i> 3)-linkages, and has a molecular weight of 40-50 x 106 • All
of the a-D-(l -i> 3)-linkages are involved in chain branching; one and
two D-glucosyl residues constitute ~859'0 of the side chains. There is
much evidence for the presence of some velY long branches (see ref-
erences cited by Sidebotham330 and by Seymour and coworkers338 ).

(329) W. B. Neely, Adv. Carbohydr. Chem., 15,341-369 (1960).

(330) R. L. Sidebotham, Adv. Carbohydr. Chem. Biochem., 30,371-444 (1974).
(331) P. T. Murphy and R. L. Whistler, Ref. 23, pp. 513-542.
(332) A. Jeanes, "Dextran Bibliography: Extensive Coverage of Research Literature
(Exclusive of Clinical) and Patents, 1861-1976," U. S. Dep. Agric., Misc. Pub/.
1355 (1977).
(333) S. P. Rogovin, F. R. Senti, R. G. Benedict, H. M. Tsuchiya, P. R. Watson, R. Tobin,
V. E. Sohns, and M. E. Slodki,j. Biochem. Microbiol. Teclmo/. Eng., 2,381-399
(1960).
(334) E. J. Hehre,j. Bioi. Chem., 222, 739-750 (1956).
(335) A. Misaki, S. Yuk,nva, T. Asano, and .M. Isono, Annu. Rep. Takeda Res. Lab., 25,
42-54 (1966); Chem. Abstr., 66, 54,255t (1967).
(336) E. L. Rosenfeld, Biokhimiya, 23, 635-638 (1958); Biochemistry (USSR), 23, 597-
600 (1958).
(337) R. A. Ewald and W. H. Crosby, Transfusion (Philadelphia), 3, 376--386 (1963).
(338) F. R. Seymour, M. E. Slodki, R. D. Plattner, and A. Jeanes, Carbohydr. Res., 53,
153-166 (1977).
304 PAUL A. SANDFORD

Strain NRRL B-512(F) produces large proportions of the exh'acellu-

lar enzyme dextransucrase,339 which is responsible for the synthesis
oflinear sequences of a-D-(l --';> 6)-linked D-glucosyl residues. The en-
zyme transfers the D-glucosyl group from a sucrose molecule to an en-
larging dextran chain and liberates the D-fmctose portion. As dexh'an-
sucrase is an extracellular enzyme, production of dextran by cell-free,
culture filtrates can result in enhanced yield and quality, and ease of
purification of the product. By suitable adjustment of the conditions,
products in a chosen molecular-weight range can be obtained. Forma-
tion of branches is not yet well understood, but the enzymes responsi-
ble will certainly be found.
c. Applications. l41 ,322-B-512(F) dextran derives its useful proper-
ties from its composition and primary structure. The high proportion
of (1 --';> 6)-linkages gives it an unusually flexible backbone on which
the 3-hydroxyl groups in consecutive positions are available for com-
plexing metal ions. Probably the largest outlet for dextran and its de-
rivatives is through the pharmaceutical and fine-chemical industries.
Methods for the depolymerization of dexh'an to uniform fractions of
lower molecular weight have led to the use of two dextran fractions
that are suitable for parenteral adminish'ation. 13 ,3o In the United
States, a dextran fraction of lvlW 70,000 is used as a blood-volume ex-
pander. "Clinical dextran" is used to restore blood volume in the
treatment of patients who have either lost considerable amounts of
blood or are in shock. A dextran fraction of MW 40,000 is used to im-
prove the flow in capillaries, to prevent or treat vascular occlusion,
and to perfuse organs artifically. B-512(F) dextran is completely
metabolized 141 in man when fi'actions are administered parentally.
Various dextran fractions have been used to prepare numerous deriva-
tives,29 such as the sulfates, and O-(2-diethylaminoethyl) (DEAE)-
dextran, and complexes with various metals. Dextran sulfates have
anticoagulant,340 antilipemic,340 and anti-ulcer 341 activity. A soluble,
iron-dextran complex 342 of MW 5000 is used to alleviate iron-defi-
ciency anemia, and a calcium complex332 alleviates hypocalcemia of
cattle.

(339) H. J. Koepsell and H. M. Tsuchiya,]. Bacterial., 63,293-295 (1952).

...
(340) E. Morii, K. Iwata, and H. Kokkoku, U. S. Pat. 3,141,014 (1964); Chem. Abstr., 61,
14,478a (1964).
(341) E. Morii, T. Numasawa, K. Iwata, H. Hanai, H. Yamagata, and A. Ishimori, Jpn.
Pat. 71 25,020 (1971); Chem. Abstr., 75, 121,411a (1971).
(342) J. S. G. Cox, R. E. King, and G. F. Reynolds, Nature (London), 207, 1202-1203
(1965).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 305

Crosslinked dextran gels (for example, Sephadex®) have been used

extensively for the purification and fractionation of various biological
substances, including the recovery of proteins from liquid wastes and
effluent streams.343-347
Dextran is used in gel precipitation for purifying, separating,347,348
and concentrating metals for use as nuclear-reactor fuels,349,35o cata-
lysts,350-352 ceramic coatings,349 refractories, ferro-electric materi-
.. als,351,352 and pigments.353
Many uses of dextran in petroleum production have been proposed.
It has been tested successfully for use in drilling muds,354,355 and in
viscous water-flooding. 356 ,357 The patent literature332 suggests that the
native, high-molecular-weight dextran is now used in various proprie-
tmy, X-ray and photographic emulsions.

2. Scleroglucan147,358
a. Properties.-Scleroglucan is the term given to a class of exocellu-
lar polysaccharides secreted by certain fungi, particularly the genus
Sclerotium. Gums of similar or related structures have been found in
species from the genera Corticium, Sclerotinia, and Stromatinia. Al-

(343) J. C. Davis, Chem. Eng., Il4-Il5 (July 24, 1972).

(344) E. J. Forsum,J. Dairy Sci., 57,665-670 (1973).
(345) W. H. Wingerd,J. Dainj Sci., 54, 1234-1236 (1971).
(346) E.-G. Samuelson, P. Tibbling, and S. Holm, Food Technol., 21, 121-124 (1967).
(347) K. T. B. Scott, J. H. Grimes, and P. W. Ball, Br. Pat. 1,325,870 (1973); Chem.
Abstr., 75, 120,4Ile (1971).
(348) K. T. B. Scott, J. H. Grimes, and P. W. Ball, Br. Pat. 1,346,295 (1974); Chem.
Abstr., 77, Il6,897s (1972).
(349) W. Dress and J. H. Grimes, Br. Pat. 1,175,834 (1969); Chem. Abstr., 70, 120,434m
(1969).
(350) J. H. Grimes and E. S. Lane, Br. Pat. 1,231,385 (1971); Chem. Abstr., 73, 7,693u
(1970).
(351) J. H. Grimes and E. S. Lane, Br. Pat. 1,286,257 (1972); Chem. Abstr., 73, 27,Il4j
(1970).
(352) J. H. Grimes and W. Dress, Br. Pat. 1,286,871 (1972); Chem. Abstr., 73, 104,778h
(1970).
(353) J. H. Grimes, K. T. B. Scott, and N. J ..McKenna, Br. Pat. 1,350,389 (1974); Chem .
• Abstr., 77, 22,420d (1974).
(354) W. L. Owen, U. S. Pat. 2,602,082 (1952); Chem. Abstr., 46, 9,250b (1952).
(355) P. H. Monaghan and J. L. Gidley, Oil Gas j., 57, 100-103 (1959).
(356) W. J. Sparks, U. S. Pat. 3,053,765 (1962); Chem. Abstr., 58,8,838h (1963).
(357) G. P. Lindblom, G. D. Ortloff, and J. T. Patton, Can. Pat. 654,809 (1962); Chem.
Abstr., 63, Il,213d (1965).
(358) N. E. Rodgers and H. R. Goffette, "Polytran: Trademark ofScleroglucan," CECA
S.A., Velizy-Villacoublay, France, July 1976.
306 PAUL A. SANDFORD

though the polysaccharide from Sclerotium glucanicum l06 has been

studied extensively, it is the exopolymer from Sclerotium rolfsii that
is produced commercially.359
Scleroglucan was initially marketed by the Pillsbury Co., Minneap-
olis, under the trade name Polyh·an®. In 1976, a French company,
(CECA, S.A.) obtained worldwide rights to Polyh'an, and is now man-
ufacturing scleroglucan under the trade name Biopolymer CS®.
Scleroglucan from S. glucanicum is a neuh"al glucan. Its sh"ucture is
that of a linear chain of f3-D-(l - 3)-linked D-glucopyranosyl residues,
with single D-glucopyranosyl groups linked f3-D-(l - 6) to about evelY
third residue of the main chain. l49 The polysaccharides from other cul-
tures are similar, but differ in the number and length of side chains,
and the molecular size. Scleroglucan from S. glucanicum has l49 an es-
timated degree of polymerization (d.p.) of ~ 1l0. Related scleroglu-
cans are considerably larger, with some in the range of 200-500 d.p.,
but the majority are in the range of 500-1600 d.p. Commercial
scleroglucan has a d.p. of -800.
Descriptions of the solution properties of scleroglucan can be con-
fusing, in that more than one grade exists. Biopolymer CS-6 con-
tains 358 - 60-75% of scleroglucan, whereas biopolymer CS-ll is 358 a
refined product and has a content of polysaccharide of -85-90%. Re-
fined grades of scleroglucan dissolve readily in water, to give pseudo-
plastic solutions that tolerate high temperature, a broad range of pH,
and a variety of elech·olytes. Scleroglucan complexes with borate in
alkaline solution to fonn a stable gel. Various derivatives l4i ,358,36o have
been prepared. Dried films cast from solution are somewhat brittle,
but are pliable when plasticized with glycerol.
In concentrations of 0.1-0.2%, scleroglucan effectively stabilizes 5-
10% aqueous suspensions of fine powders, such as those of zinc oxide.
Combinations of scleroglucan with suspensions of Bentonite display
marked synergism.
Scleroglucan has the caloric equivalent of starch in tests with rats.
Studies with dogs, guinea pigs, humans, and rabbits demonsh'ate no
significantly adverse reactions. VVitll chicks and dogs, scleroglucan
lowered the cholesterol levels and increased the excretion of lipid.36l
Otller polysaccharides also elicit tllis effect. 36l
b. Preparation,146,147,358-Scleroglucan is produced by submerged,
aerobic fermentation of D-glucose by pellet growtll of the desired spe-

(359) P. Delest, personal communication.

(360) S. A. Williams, U. S. Pat. 3,373,810 (1968); Chem. Abstr., 68, 88,806e (1968).
(361) P. Griminger and H. Fisher, Proc. Soc. Exp. Bioi. !lIed., 122,551-553 (1966).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 307

cies. \Vith crude grades, the entire fermentation-liquor is spray-dried,

but, with refined grades, mycelia are filtered out before the polysac-
charide is precipitated with isopropyl alcohol.
c. Proposed Applications. 147 ,358-Scleroglucan appears to be gen-
erally useful in many areas now occupied by other industrial gums.
Suggested uses include porcelain and ceramic glasses and binders,362
water-based paints,362 paper coatings, printing inks, agriculhlral
sprays and seed coatings, liquid animal-feed concenb'ates,363 as a
source of gentiobiose,364 in hair sprays, hand lotions, tablet coatings,365
and ophthalmic solutions,366 and as a bodying, suspending, coating, and
gelling agent in the food industry. Perhaps the largest potential use
• will be in the area of enhanced oil_recovely.360,367-369

3. Curdlan 125
a. Properties.-During studies on Alcaligenes faecalis var. myxo-
genes (lOC3), Harada and coworkers369 found a spontaneous variant
(lOC3K) that no longer produced a succinoglucan135 ,37o-374 characteris-
tic of the parent sb'ain, but, instead, produced an insoluble exopoly-
saccharide composed entirely127,375-378 of D-glucosyl residues

(362) F. Halleck, U. S. Pat. 3,447,940 (1969); Chem. Abstr., 71, 51,304a (1969).
(363) Fed. Regist., 34, 13,162 (1969).
(364) F. E. Halleck and F. Smith, U. S. Pat. 3,423,288 (1969); Chem. Abstr., 70, 86,288e
(1969).
(365) P. Sheth and L. Lachman, U. S. Pat. 3,421,920 (1969); Chem. Abstr., 67, 111,442y
(1967).
(366) L. Lachman and P. Sheth, U. S. Pat. 3,415,929 (1968); Chem. Abstr., 70, 50,467y
(1969).
(367) S. A. Williams, U. S. Pat. 3,372,749 (1968); Chem. Abstr., 68, 106,634d (1968).
(368) J. D. Westover and R. B. Ferguson, U. S. Pat. 3,436,346 (1969); Chem. Abstr., 71,
.37,51Oh (1969).
(369) M. Hisamatsu, A. Amemura, T. Harada, 1. Nakanishi, and K. Kimura, Abstr. A1lI11l.
Meet. Agric. Chem. Soc. ]pn., 289 (1976).
(370) T. Harada and T. Yoshimura,]. Ferment. Technol., 42, 615-622 (1964).
• (371) T. Harada and T. Yoshimura, Biochim. Biophys. Acta, 83,374-376 (1964) .
(372) T. Harada, T. Yoshimura, H. Hidaka, and A. Koreeda, Agric. Bioi. Chem., 29, 757-
762 (1965).
(37.3) A. Misaki, H. Saito, T. Ito, and T. Harada, Biochemistry, 8,4645--4650 (1969).
• (.374) H. Saito, A. Misaki, and T. Harada, Agric. Bioi. Chem., 34, 1683-1689 (1970).
(37.5) T. Harada, "'I. Masada, H. Hidaka, and M. Takada,j. Ferment. Techno/., 44, 20-
24 (1966).
(376) T. Harada, M. Masada, K. Fujimori, and 1. Maeda, Agric. Bioi. Chem., 30, 196-
198 (1966).
(:377) H. Saito, A. Misaki, and T. Harada, Agric. Bioi. Chem., 32, 1261-1269 (1968).
(:378) J. Ebata,AIlstr. Int. Symp. Carbohydr. Chem., 8th, Kyoto, 112 (1976).
 308 PAUL A. SANDFORD

connected almost exclusively by ,6-D-(1 --'" 3)-linkages. This and simi-

lar glucans are termed curdlan. 127 Subsequently, curdlan, when
heated in water, was found to form thermally irreversible, resilient
gels. An improved strain was developed for production of gel-form-
ing (1 --'" 3)-,6-D-glucan379 by treatment with mutagens. 369
In an investigation of color-complex formation of various polysac-
charides with several dyes, it was found that such (1 ---i> 3)-,6-D-glucans
as curdlan, pachyman, and yeast glucan are specifically stained with
Aniline Blue. 126 ,38o By use of this Aniline Blue method,126 four strains
of Agrobacterium radiobacter, a strain of Agrobacterium rhizogenes,
and one unidentified strain of Agrobacterium were found to produce a
curdlan type of exopolymer. Other D-glucans, such as callose, pachy-
man, laminaran, and D-glucans from fleshy fungi 146 ,381 and Armillaria
mellea,382 are all similar in structure, but contain other linkages377 ,383
that apparently prevent them from fom1ing gels when heated. The
ready ability to form elastic and resilient gels has attracted the atten-
tion of the indusny to curdlan. When a 2% suspension of curdlan is
heated, it becomes clear at ~54°, and a gel fom1s at higher tempera-
tures. 384 Studies385-387 on the effect of temperature on gel formation
show that, from 54 to 60°, swelling occurs due to breakage of hydrogen
bonds, and that, on cooling to -40°, the viscosity rapidly increases
and a low-set gel is obtained. 360 Curdlan gels can be formed by dialyz-
ing alkaline solutions thereof,369 by cooling heated solutions in 0.2-
0.63 M dimethyl sulfoxide,388 or by adding calcium ions to weakly al-
kaline solutions.369 Studies 125 of curdlan gels, alone and in the
presence of urea,385 ethylene glycol, and salts,385 suggest that, at low
temperatures, gelation is due to formation of hydrogen bonds and that,

(379) 1. Nakanishi, T. Kanamaru, K. Kimura, A. Matsukura, M. Asai, T. Suzuki, and S.

Yamatodani, Meet. Kansai B"GlICh Agl'ic. Chem. Soc., Jpn., 284th, Osaka (1972).
(380) 1. Nakanishi, K. Kimura, S. Kusui, and E. Yamazaki, Carbohydr. Res., 32,47-52
(1974).
(381) L. L. Wallen, R. A. Rhodes, and H. R. Shulke, Appl. Micl'Obiol., 13,272-278
(1965).
(382) J. Jeisma and D. R. Kreger, Carbohyd,'. Res., 43, 200-203 (1975).
(383) G. O. Aspinall and G. Kessler, Chem. Ind. (London), 1296 (1957).
(384) 1. Maeda, H. Saito, ~I. Iv!asada, A. Misaki, and T. Harada, Agric. Bioi. Chern., 31,
1184-1188 (1967).
(385) T. Harada, Ref. 319, pp. 603-607.
•
(386) H. Kimura, S. Iv!oritaka, and M. Misaki,j. Food Sci., 38,668-670 (1973).
(387) A. Konno, Y. Azeti, and H. Kimura, Abstr. Anml. Meet. Agric. Chem. Soc. jpn.,
310 (1974).
(388) M. Aizawa, M. Takahashi, and S. Suzuki, Chem. Lett., 193-196 (1974).

j
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 309

at the starting temperature for gel formation, some or all of the hydro-
gen bonds must be broken. Fmther studies 389 , 390 (optical rotatOly dis-
persion, viscosity, and flow birefringence) on the conformational
behavior of curdlan in alkaline solutions may be interpreted as indi-
cating that, at low concenh'ations of alkali, curdlan has an ordered con-
formation, whereas, at higher concenh'ations, it consists of random
coils. Studies on curdlans having different degrees of polymeriza-
tion391-394 showed that cm,dlan takes an ordered form at low' concentra-
tions of alkali, when d.p, = 25, until it reaches a maximum and
constant value at d.p, values of ~ 200. X-Ray diffraction studies 395 sug-
gesting that curdlan has a simple, helical stmcture have been sup-
pOlted by the results of 13C-n.m.r. analysis. 390
b. Preparation.-Curdlan can be produced in good yields
(~50%) from extraordinarily high concentrations of carbohydrate sub-
strate in a simple, defined medium if the pH is maintained at neuh'al-
ity.376,379,396 Cell suspensions in a nih'ogen-free medium containing
only D-glucose and calcium carbonate 397 also elaborate the bio-
polymer. Curdlan is readily isolated by taking advantage of its unique
insolubility in water. When the medium is made sufficiently alkaline
to dissolve the curdlan, cells can be removed by centrifugation. Neu-
tralization of the base witll acid then precipitates curdlan in a purified
foml.
c. AppIications.-Although curdlan is not yet in full production, it
has been shown to have several potential uses in foods, as a gelling
agent for jelly products and as an additive in baking. 125 Other potential
uses for cm'dlan are as a film, a fiber, or a support for immobilized en-

(389) K. Ogawa, T. Watanabe, J. Tsurugi, and S. Ono, Carbohydr. Res., 23, :399-405
(1972).
(:390) H. Saito and T. Sasaki. Abstr. Ann!l. Meet. lpn. Biochem. Soc., 651 (1976).
(391) K. Ogawa, J. Tsurugi, and T. Watanabe, Carbohydr. Res., 29,397 -403 (1973).
a (:392) K. Ogawa, J. Tsurugi, and T. Watanabe, Chem. Lett., 689-692 (1972).
(393) K. Ogawa and M. Hatano, Meet. Kansai Branch Agric. Chem. Soc., lpn., 288th,

. Osaka (1974).
(394) A. Koreeda, T. Harada, K. Ogawa, S. Sato, and N. Kasai, Carbohydr. Res., 33, .396-
399 (1974).
(395) S. Suzuki and .\1. Aizawa, Abstr. lnt. Symp. Carbohydr. Chem., 8th, Kyoto, 76
(1976).
(396) T. Harada, K. Fujimori, S. Hirose, and M. Masada, Agric. Bioi. Chem., 30, 764-
769 (1966).
(.397) T. Harada, K. Fujimori, and M. Masada,). Ferment. Tee/lIlol., 45,145-150 (1967).
310 PAUL A. SANDFORD

zymes. 398,399 As studies indicated that curdlan has no caloric value, it

should be useful in low-calorie foods.

4. Pullulan 141
a. Properties.-Pullulan is the generic name 400 given to any extra-
cellular a-D-glucan elaborated by the yeastlike fungus Aureobasidium
pullulans, fODnerly called Pullularia pullulans. It is one of the com-
monest and most \videspread of fungi.401-408 Most isolates produce co- .
pious amounts of polysaccharides, each of which differs slightly.
Pullulan is a water-soluble, neutral glucan. The feature most com-
monly observed is tl1at of a linear polysaccharide made up of malto-
triose repeating units that are polymerized through a-D-(l -lo 6)-
linkages. Also, a-maltotetraose units 140 ,409,410 and (1 -lo 3)-linked resi-
dues 41 1,412 have been found in some preparations. Pullulan readily dis-
solves in water, to give colorless, adhesive solutions. Both the molec-
ular weight4 13 and the rheological properties 414 ,415 of pullulan differ
with the conditions and length of fermentation, and tl1e strain used. 405
The molecular weight of pullulan gradually increases during tl1e

(398) K. Takahashi, Y. Yamazaki, K. Kato, and T. Takahashi, Abstr. Anntl. Meet. Agric.
Chem. Soc. jpn., 401 (1976).
(399) Y. Murooka, T. Yamada, and T. Harada, All/HI. Meet. Ferment. Technol. jpn.,
Osaka, 1976.
(400) H. Bender, J. Lehmann, and K. Wallenfels, Biochim. Biophys. Acta, 36,309-316
(1959).
(401) W. B. Cook, Mycologia, 52,210-230 (1960).
(402) \V. B. Cook, 'Alycopathol. tVlycol. Appl., 12, 1-45 (1959).
(403) B. Bernier, Can.]. Microbiol., 4, 195-204 (1958).
(404) E. Merdinger, W. S. Guthmann, and F. W. Mangine, Appl. Microbiol., 18,365-
368 (1969).
(405) K. Wallenfels, H. Bender, G. Keilich, and G. Bechtler, Angew. Chem., 73,245-
246 (1961).
(406) E. T. Reese and A. Maguire, Can.]. Microbial., 17,329-332 (1971).
(407) J. E. Zajic, U. S. Pat. 3,320,136 (1967); Chem. Abstr., 67, .31,565a (1967).
(408) E. Merdinger,}. Bacterial., 98, 1021-1025 (1969). •
(409) B. J. Catley and W. J. Whelan, Arch. Biochem. Biophys., 143, 138-142 (1971).
(410) R. Taguchi, Y. Kikuchi, Y. Sakano, and T. Kobayashi, Agric. Bioi. Chem., 37,
1583-1588 (1973). ...
(411) W. Sowa, A. C. Blackwood, and G. A. Adams, Can. ]. Chem., 41, 2314-2319
(1963).
(412) N. P. Elinov and A. K. Matveeva, Biakhimiya, 37,255-257 (1972); Biochemistry
(USSR), 37, 207-209 (1973).
(413) B. J. Catley, FEBS Lett., 20, 174-176 (1972).
(414) A. LeDuy, A. A. Marsan, and B. Coupal, Biotechnol. Bioeng., 16,61-76 (1974).
(415) S. Yuen, Process Biochem., 9(9), 7 -9,22 (1974).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 311

fermentation, until it reaches a maximum, after which it starts to de-

crease. By proper selection of strain, pH, and phosphate content of
the medium, pullulan of definite molecular weights lying between
5 x 104 and 250 x 104 may be obtained. 416 Even though pullulan
is structurally related to amylose, neither alpha-amylase,417 nor iso-
amylase 418 from Pseudomonas sp., attacks it. However, there are
various microbial pullanases 410 ,419-422 and bacteria423 that readily de-
grade pullulan.
b. Production.-Pullulan can be produced from a variety of carbo-
hydrate substrates, such as sucrose,400 D-glucose,400 D-fructose,400 mal-
tose,400,417 D-mannose,417 D-xylose,417 L-arabinose,417 and leaf or litter
extracts. 403 No production4oo ,417,424 of pullulan is observed with acetate,
D-galactose, glycerol, lactose, or D-mannitol as the carbon source.
Thiamine and slightly acid conditions promote growth and higher
yields of pullulan.4oo,407 The best results are obtained by fermentation
at room temperature. 407
High conversion (70%) of substrate (sucrose) into pullulan makes
the polysaccharide attractive economically. Pilot-plant, batch ferrnen-
tations of D-glucose to yield pullulan have been repOlted. 425 Large-
scale, pilot fermentations by Hayashibara Biochemical Laboratories 423
are expected to lead to full-scale production of pullulan.
c. Applications.-Numerous applications of pullulan and its deriva-
tives have been proposed and patented, but apparently pullulan is not
yetin use. 415,423 Pullulan has been shown to flocculate clay slimes from
aqueous suspensions resulting from the beneficiation of uranium, pot-
ash, and other ores .407,425,426
Films formed from pullulan have excellent physical properties, are

(416) K. Kato and M. Shiosaka, U. S. Pat. 3,912,.591 (197.5).

(417) H. O. Bouveng, H. Kiessling, B. Lindberg, and J. McKay, Acta Chem. Scalld., 16,
615-622 (1962).
(418) K. Yakobayashi, A. Misaki, and T. Harada, Agric. Biol. Chem., 33,625-627 (1969).
(419) K. Wallenfels and 1. R. Rached, Biochem. Z., 344,524-526 (1966).
(420) S. Veda and R. Ohba, A.gric. Biol. Chem., 36,2381-2391 (1972).
(421) Y. Sakano, N. Masuda, and T. Kobayashi, Agric. Biol. Chem., 35, 971-973 (1971).
(422) Y. Sakano, M. Higuchi, and T. Kobayashi, Arch. Biochem. Biophys., 153, 180-
187 (1972).
(423) S. Yuen, "Pullulan and Its New Applications," Hayashibara Biochemical Labora-
tories, Inc., Okayama, Japan, 1974.
(424) B. J. Catley, Appl. Microbiol., 22,641-649 (1971).
(425) J. E. Zajic and A. LeDuy,Appl. Microbiol., 25, 628-635 (1973).
(426) M. B. Goren, U. S. Pat. 3,406,114 (1968); Chem. Abstr., 70, 14,26.5x (1969).
312 PAUL A. SANDFORD

water-soluble, and are impervious to oxygen. Pullulan films are suit-

able for coating or packaging foods and pharmaceuticals, where pre-
vention of oxidation is desired. 415 ,423 Fibers from pullulan appear to be
as strong (after stretching) as those of rayon or nylon. 416 •423 These fibers
can be used with natural fibers to make special papers and products. 42i
Pullulan and its derivatives make suitable adhesives, and can be com-
pression-molded to make articles that have characteristics similar to
those made of poly(vinyl alcohol) or poly(styrene).415·423 Pullulan im-
parts to foods and drinks satisfactory texture, structure, viscosity, dis-
,.
persibility, and moisture-retention properties. It is apparently
nontoxic and nondigestible.409.423 Further work is, however, needed in
order to verify the claim that it is noncaloric.

5. Polysaccharides from Methanol-utilizing Micro-organisms

a. Properties.-Two sources of potentially useful biopolymers
appear to be (1) the methanol-utilizing bacteria,428 such as
Methylomonas mucosa (NRRL B_5696)429-431 and Methylomonas
methanolica,432 and (2) the activated-sludge system433-435 that utilizes
methanol. The viscous material produced by both systems appears to
be a mixture of polymers. The M. mucosa polymer is composed of glu-
cose (10-30%), mannose (3-15%), and galactose (3-15%) residues,
pyruvic acid groups (5-35%), and inorganic matter (10-40%).
The polymer produced from methanol by activated sludge is also
composed mainly of the neutral sugars glucose, galactose, and man-
nose, but no pyruvic acid has been found in it.
b. Preparation.-Production of the Methylomonas polymer was
studied in batch and continuous fermentations. 431 The average yield,
based on the methanol used, was - 20-40%. Comparable yields were
obtained with the sludge system.433-435 Unifoml results are obtained

(427) T. Nomura, U. S. Pat. 3,936,347 (1976); Chem. Abstr., 83, 12,639s (1975).
(428) D. Ballerini and D. Parlouar, Fr. Pat. 2,231,748 (1974); Chem. Abstr., 83,41,541 •
(1975).
(429) R. K. Finn, A. L. Tannahill, and J. E. Laptewicz, U. S. Pat. 3,923,782 (1975);
Chem. Abstr., 84, 57,403x (1976).
(430) R. K. Finn, A. L. Tannahill, and J. E. Laptewicz, U. S. Pat. 3,923,218 (1977);
Chem. Abstr., 84, 103,858 (1976).
(431) K. T. Tam and R. K. Finn, Ref. 33, pp. 58-80.
(432) L. Haggstrom and N. Molin, Abstr. lnt. Ferment. Symp., 5th, Berlin, 398 (1976).
(433) E. N. Davis and L. L. Wallen, Appl. Environ. Microbial., 32,303-305 (1976).
(4.34) E. N. Davis, Trans. Ill. State Acad. Sci., 70,80-85 (1977).
(43.5) W. R. Roth, U. S. Pat. 4,065,287 (1977).
 EXOCELLULAR, MICROBIAL POLYSACCHARIDES 313

either by use of fresh sludge or by back-seeding from fermentations.

The Methylomonas sp. deposited as NRRL B-5696 is apparently not a
pure culture, eitller.
c. Potential Applications.-There are as yet no known applications
of the Methylomonas polysaccharide, but various uses have been sug-
gested. 429 ,430 Polysaccharide produced from methanol by sludge is
being tested as an encapsulating agent for slow-release pesticide and
herbicide preparations. 435

"