Reprinted From: ADVANCES IN APPLIED MICROBIOLOGY Vol. © 1978, Academic Press, Inc. .N Yorlf. San FrancisCo London Production of Microbial Polysaccl'1anaes M. E. SLODKI A:\D M. C. CAm,IUS Northern Regional Research Center, AgriCllltural Research Service, U.S. Department ofAgriculture! Peoria, Illinois I. Introduction..................................... 19 II. Xanthan Gum. ......................................... 21 A. Background........................................ 21 B. Strain Variability. .................................. 22 C. Production 2.3 D. Other Xanthomonas Polysaccharides 25 III. Arthro!Jacter Polysaccharides. ............................ 26 IV. Microbial Alginic Acids 27 V. Succinoglucan and Curdlan .............................. 29 A. Succinoglucan...................................... 29 B. Curdlan........................................... 31 C. Other ,B-(1~3)-Linked Glucans .. ..................... 32 VI. Other Bacterial Polysaccharides 33 VII. Phosphomannans and ~Iannans. .......................... 34 VIII. Cryptococcus Heteropolysaccharide ....................... 37 L'\. "Black Yeast" Polysaccharides. ........................... 38 X. Pullulan............................................... 40 XI. Polysaccharides from Hydrocarbons and Low-Molecular-Weight Petrochemical Substrates. .. .. ...... 44 A. n-Alkanes 44 B. Lower Polyhydric Alcohols. .......................... 45 C. Methanol.......................................... 46 XII. Concluding Remarks. ................................... 47 References 49 I. Introduction Interest in extracellular microbial polysaccharides as possible industrial fermentation products received its major impetus from a program of research and development initiated in 1955 at the Northern Regional Research Center of the Agricultural Research Service. Prior to that time, this labora­ tory had successfully completed a massive effort which developed both 'The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other finns or similar products not mentioned. 19 20 M. E. SLODKI AND M. C. CADMUS fermentative and enzymic processes for the production of dextran suitable for clinical use. The concept of employing corn sugar as a substrate for fermentative production of polysaccharide gums followed naturally from the Center's mission to develop industrial processes for utilization of surplus agricultural commodities. A further consideration was the desirabilitv of finding substitutes for imported plant gums of possibly limited supply. The polysaccharide elaborated by Xantholllonas campestris NRRL B-1459 is a major outgrowth ofthe ARS research program Qeanes, 1968), and its produc­ tion has been the basis of a new fermentation industry (McNeely, 1967; McNeely and Kang, 1973). This successful development has stimulated oth­ ers to search for extracellular polysaccharides ofpotential utility in foods and in industrial processes Qeanes, 1973). It is difficult to ascertain whether extensive replacement ofplant gums has indeed occurred; there is no doubt, however, that new applications and markets have arisen to exploit xanthan gum's unique properties. Our experience has been that the various polysac­ charides offer different physical behaviors in solution and different degrees of stability. Consequently, it is to be expected that any lle\vly developed gums will also find new applications based on their unusual properties. It is our purpose here to review the factors involved in fermentative production of extracellular polysaccharides, i.e., the environmental factors that lead to optimum fermentation yields and, furtl1er, the effects of these conditions on chemical composition, macromolecular structure, and physical properties of solutions of these polysaccharides. Sutherland (1972) has reviewed in detail what is known about tl1e biosyntheses and genetic control of the production of bacterial exopolysac­ charides. Actually, little knowledge has been added since tl1at particularly relates to extracellular polysaccharides. In general, tl1e mechanisms involved in cell-wall, membrane, lipopolysaccharide, and extracellular polysaccharide biosyntl1esis from simple sugars, or even noncarbohydrate substrates, are similar. Hexose phosphates are converted in tl1e cells to sugar nucleotides. Conversion to various precursor monosaccharide components takes place at the sugar nucleotide level (Cabib, 1963). Enzymes involved in the synthesis of exocellular polysaccharides are associated \Vith the membrane underlying the cell wall and are generally obtained as particulate complexes (Markovitz and DOIfman, 1962). Lipoidal transport components, notably polyprenols (Lennarz, 197,5), perhaps serve to transport polar monosaccharide and oligosaccharide repeat unit precursors through tl1e nonpolar cell membranes to tl1e exterior where polymerization might occur. Hussey and Baddiley (1976) have recently discussed the possible regulatory role of polyprenyl phosphates in biosynthesis of exocellular polysaccharides. From a practical standpoint, this review \ViII be concerned mainly \Vith polysaccharides found in submerged liquid cultures throughout the medium PRODUCTION OF MICROBIAL POLYSACCHARJDES 21 away from the cells. Only products of this type can be obtained in sufficiently high yields to be considered for industrial production. A large body oflitera­ ture describes polysaccharides that (1) can be extracted from nonmucoid cell-wall membrane complexes by either chemical or enzymic treatment or (2) can occur as discrete capsules that surround cells. Often, depending on the conditions ofgrowth, small amounts of these capsular materials are found free in the medium. Such capsular polysaccharides or slimes are usually detached from cells by either physical, e. g., mechanical agitation, or en­ zymic treatment. Sutherland (1972) deals with these questions more exten­ sively. For now, it is noteworthy that such isolated capsular and cell­ associated polysaccharides (e.g., Ballou and Raschke, 1974) can contain sig­ nificant amounts of covalently bound peptide material. In contrast, it has been our experience that tmly extracellular polysaccharides can be obtained virtually free of protein by mild purification procedures that do not involve alkaline conditions which bring about cleavage of glycoproteins through j3-elimination reactions. It is tempting to speculate that lack of covalently bound protein in extracellular polysaccharides is related to their occurrence in the medium rather than in a cell-bound state. The finding of Dudman and Wilkinson (1956) that both capsular and free extracellular polysaccharides from variants of a Klebsiella strain were chemically and physically alike accords \\ith this view. This review will not be concerned with glucans and fructans whose biosyn­ thesis directly from sucrose or dextrin substrates is catalyzed by either cell­ bound or extracellular transglycosylases. Instead, polysaccharides will be considered that are synthesized through phosphate ester intermediates from monosaccharides and simpler substrates. These examples have potential for commercial development either because of their unusual physical properties or because they are rapidly formed in high yields from inexpensive sub­ strates. II. Xanthan Gum A. BACKGROUND Xanthan gum was first reported by Rogovin et al. (1961a) as a product of fermentation with Xanthomonas campestris NRRL B-1459 (a plant pathogen causing diseases ofsome plants) and was later determined to have a stmcture (Fig. 1) consisting ofa j3-(1~4)-linked D-glucosyl backbone chain \vith alter­ nate residues having appended a three-unit-Iong side chain of D-mannose and D-glucuronic acid in the molar ratio 2:1 (Jansson et al., 1975). Halfof the side-chain D-mannosyl residues are attached directly to the main chain 22 M. E. SLODKI AND M. C. CADMUS FIG. 1. Repeating unit ofxanthan gum as determined by Jansson et al. (19i.5) and confirmed by Melton et al. (Wi6); distributions of side-chains and of pyruvate ketal reflect average values. through a-(1-;>3) linkages; the remaining D-mannosyl components of the side chains occur as nonreducing end groups. Approximately half of these D-ri1annosyl end groups carry pyruvic acid as the di-O-4,6-ketal. This ex­ tracellular anionic heteropolysaccharide is now produced industrially in both the United States (Kelco Co., 1972) and Europe (Godet, 1973) and has numerous applications in food and nonfood industries (Jeanes, 1974). B. STRAIN VARIABILITY Although X. campestris is not difficult to cultivate on standard laboratory media (Jeanes et al., 1976), certain strain variations have been observed in both continuous (Silman and Rogovin, 1972) and batch-type (Cadmus et al., 1976) fermentations which affect the quality and the yield of the polysac­ charide produced. Variation was first associated with the formation of large (L) and small (S) colony types in which the L-type produced biopolymer in normal yields (1.4 gm per 100 ml ofglucose medium) and with apparently nor­ mal rheological characteristics. The S-colony type produced a polysaccharide in low yield (0.9 gm per 100 ml) with undesirable properties. The only detect­ able difference was a lower concentration of pyruvate in the S-tyVe poly­ saccharide. Sandford et al. (1976) demonstrated that viscosities of dilute xantl1an solutions are related to tl1e pyruvic acid content ofthe gum. Analysis of polysaccharide from tl1e S-tYVe showed it to contain about half (2%) the pyruvic acid content of