Xanthan gum production isolates of Xanthomonas
by wild-type campesfris
M. Nitschke* and R.W.S.P. Thomas Five newly-isolated strains of Xunthomanas campesfris when compared with the standard strain, NRRL B-1459, showed higher broth viscosity and xanthan gum production. Evaluation of polysaccharide rheology is a very important determinant for selecting new xanthan-producing isolates. Key words: Polysaccharide,
viscosity, xanthan gum, Xanthomonas cumpestris.
Xanthomonas campestris is a phytopathogenic bacterium which causes black rot in crucifers (Sutton & Williams 1970). Some strains produce copious quantities of xanthan as an exopolysaccharide. Xanthan gum is produced commercially because it has a high viscosity at low concentrations, is compatible with mineral salts and has good stability at extremes of pH, temperature and ionic strength (Rocks 1971; Cottrell & Kang 1978). Most research on xanthan gum production has been performed with strains from culture collections, especially Xanthomonas cumpestris NRRL B-1459 (Northern Regional Research Laboratories, USA). There has been relatively little recent work on production by wild strains (Torrestiana et al. 1990). One of the main problems in the selection of new strains is the lack of efficient criteria for the screening of potential xanthan gum producers. Cadmus et al. (1976) showed that colony morphology could be useful, whereas Ramirez et al. (1988) suggested virulence in plants was a good indicator of gum production. In the present study, wild-type strains of Xanthomonas campesfris were evaluated in terms of final broth viscosity, xanthan gum production and gum rheology and compared with the NRRL B-1459 standard strain in order to determine the yield and quality of gums produced by the new isolates.
The authors are with the LaboraMrio de Microbiologia. Dept Solos, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul. Av. Sento Gonoalves 7712, Cx Postal 776, 9CKlOl-970, Porto Alegre, RS, Brazil; fax: 51 336 1762. ‘Corresponding author. @ 1995 Rapid Science
Cabbage and raddish leaves showing the yellow necrotic lesions characteristic of X. campestris were collected locally. Isolation was carried out by the technique of Schaad & Stall (1988) using the glucose/yeast/calcium carbonate agar (GYCA) of Starr (1981). Wild-type isolates were selected by choosing yellow, convex, viscous colonies with a diam. of at least 4 mm after 72 h at 28°C on YM agar (Haynes et al. 1955) at pH 7.0. Isolated colonies were submitted to standard microbiological tests and identified following the scheme of Bradbury (1984). Isolates were stored on YM agar slants at 5°C and transferred every 14 days (Jeanes et al. 1976). Inocula
Inocula were prepared by transferring cells from a 24-h YM slant (28°C) to a tube containing 7 ml YM broth (pH 7.0) and incubating at 28°C and 160 rev/min for 24 h. The culture was then transferred to 250-ml (pH 7.0), and incubated
flasks, each containing 43 ml YM broth for a further 24 h at 180 rev/min. Subse-
quently 35 ml of this culture (containing approximately IO9 c.f.u./ ml) was inoculated into each of several 2000-ml flasks with 315 ml production medium containing (g/I): glucose, 20; K,HPO,, 5; MgS0,.7H,O, 0.1; yeast extract, 0.5; and urea, 0.4 (pH 7.3). During incubation at 180 rev/min and 28°C for 72 h, samples were taken every 24 h. Analyfical Procedures Turbidity was measured in using a red filter (640 to using a Brookfield viscosimeter 4, at 25°C and a shear rate measurements, 0.5% (w/v) were added to 0.5% (w/v) different shear rates. Viable
a Klett-Summerson photocolorimeter 700 nm). Viscosities were measured (model LVTDV-II), spindle number of 6.3/s (30 rev/min). For rheology aqueous solutions of xanthan gums NaCl and viscosities measured at cell counts were performed using the
spiral plating method (Spiral Plate Systems, USA).
Xanfhan production by wild isolates Table 1. Broth characteristics campesfris isolates after
and xanthan 72 h in production
Broth viscosity W)
NRRL B-1459A Cv2C8 Cv2Cl c7 Rl NRRL B-1459 c2 R3 c5
5.75 5.36 5.56 5.66 5.75 6.03 5.32 5.20 6.30
3626 3406 2626 2496 2223 2063 1789 1183 880
gum production medium.*
Gum productfon Wks b=W
Viscosifying abilityt W/gkg)
14.2 14.5 13.1 13.3 11.2 11.3 12.1 10.7 10.6
256 234 200 186 196 162 148 110 83
*Production medium contained (g/i): glucose, 0.5; K,HPO,, 5; and MgS0,.7H,O, 0.1. incubation min and 28°C. inoculum was 10% (v/v). Data three replicates. tRatio of broth viscosity to xanthan concentration.
20; yeast extract, was at 180 rev/ are the means of
NRRL B-1459A Cv2C8 Cv2Cl c7 Rl NRRL B-1459 c2 R3 c5
for X. cempestris viscosity Index
1.7 1.6 1.3 1.2 1.1 1.0 0.9 0.6 0.4
*indices are relative to standard strain NRRL used for their calculation are shown in Table
production index 1.2 1.3 1.1 1.2 1.0 1.0 1.1 0.9 0.9
specialcaseof selecting new isolatesfrom established strains. Table I shows that NRRL B-1459A as well as isolates Cv2C8, Cv2C1, C7 and Rl gave final broth viscosities higher than that of the standard strain, NRRL B-1459. Ramirez ef al. (1988) defined viscosifying ability as the viscosity generated per unit of polysaccharide and suggested that it be used as an index to determine the quality of xanthan gum. Table 1 shows that viscosifying ability varied with the isolate, suggesting that gum quality also varies between isolates. Moraine & Rogovin (1971) showed that strains of X. cumpestris produce acidic metabolites and that xanthan gum is anionic. It is possible that the differences in final broth pH (Table 1) are due to physiological differences between strains or to distinct polymer composition. Table 2 shows the indices of broth viscosities and gum dry wts of the isolates relative to NRRL B-1459. NRRL B1459A and CVZCS isolates were similar, giving the highest xanthan and viscosity indices (30% and 70% higher than the standard strain, respectively). Such high indices were not observed for all isolates, indicating different gum qualities. Xanthan gum production curves for isolate Cv2C8 and variant standard strain B-1459A are shown in Figure I. Increased turbidity was probably due to xanthan gum acc~ulation, with gum production cont~uing even when cell numbers were decreasing. Weiss & Ollis (1980) have noted that production of xanthan gum may or may not be associatedwith ceil growth. The initial decrease in viable cell number could reflect cell adaptation to differences in media composition. The inoculum was produced in YM broth which had high
The values f
performed after cell removal as describedby Moraine & Rogovin (1966), using 10% (w/v) NaCl and Polymer extraction
3 vol. 95% ethanol. 300
g 5 250-
Isolates selected from cabbage were coded as C2, C.5,CVZCE and Cv2C8 and those from raddish were coded RI and R3. The strain NRRL B-1459A, a variant of the NRRL B-1459 with a colony diam. of 6 mm, was also evaluated. After 72 h growth on YM agar, all wild-type isolates gave colony diam. of 4 to 5 mm. Colony diam. did not correlate with xanthan gum production, as shown by the different xanthan levels given in Table 1. Torrestiana ef al. (1990) suggested that there is little correlation between colony diam. and polysaccharide concentration. However, variant NRRL B-1459A had a colony diam. of 6 mm and showed high xanthan gum production. Colony diam. could be a useful criterion in the
IJOIO0 50 O-
Figure 1. Xanthan gum production B-1459A (-) and isolate Cv2C8 0-Xanthan dry wt; Cl-turbidity; the means of two replicates.
curve for X. campesfris NRRL (- - - -) in production medium. A-log c.f.u./ml. Data are
World ~ournul of Microbiology & Biotechnology. Vol I?, 1995
M. Nifschke and R. W.S.P. Thomas Table 3. Viscosities at 25°C xanthan gums (0.5% w/v), dlfferent shear ratss.’
of aqueous containing
solutions of purified 0.1% NaCl (w/v), at
this work. This work RHAE and MIRCEN.
References Polysaccharide from strain
(cP) at shear
641 341 421
301 300 240
200 200 170
1300 952 a52
“Data are means viscosity
of two replicates. The replicates was 200 cP.
Behrens, U., Klima, M. & Fiedler, S. 1980 Growth and accumulation of polysaccharide by Xanthomonus cumpestris. ZeitschriJt fiir allgemeine Mikrobiologie 20,209-213. Bradbury, J.F. 1984 Genus II ~n~~omo~s. In Betgey’s Macho of Systematic ~cferiofo~, Vol. 2, eds Holt, J.H. & Krieg, N.R. pp. X99-210. London: Williams & Wilkins. Cadmus, M.C., Rogovin, S.P., Burton, K.A., Pittsley, J.E., Knutson, C.A. & Jeanes, A. 1976 Colonial variation in Xanthomonus campestris NRRL B-1459 and characterization of a polysaccharide from a variant strain. C~~~iu~ ~0~~~ of Micr~&i~~ogy 22,
nutrient levels, whereas the production media was low in nutrients. It is known that low concentrations of N in the culture medium generally stimulate polymer synthesis (Sutherland 1972). The isolated X. cumpesfris may also not use urea as a N source. These bacteria could require a complex N source for rapid cell growth, as observed by Behrens et a/. (1980) using nitrate. The rheology of aqueous solutions of xanthan gum obtained from different isolates is shown in Table 3. AI1 purified xanthan gums showed pseudoplastic behaviour. The gums produced by isolates RI, Ii3 and C7 gave the highest viscosities. Although viscosifying ability was highest with NRRL B-1459A and CvZC8, the xanthan gums purified from these broths did not have the best solution rheology. Jeanes ef al. (1961) showed that the viscosity of >0.5% xanthan gum solutions increases with the addition of salts and Symes (1980) has shown that this effect depends upon the degree of ionization of the polyanion. The differences in xanthan viscosities before and after extraction suggest that NaCi addition had a distinct effect on the different gums, the variation possibIy reflecting differences in molecular weight, molecular conformation or chemical composition. Of the seven isolates tested, four (0, RI, Cv2C8 and CVZCI) showed higher xanthan gum production and better rheological behaviour than the standard strain NRRL B1459. This work shows the importance of gum rheology as a quality indicator and we recommend that this characteristic be considered when screening for industrially useful strains of X. catnvestris.
Acknowledgements We are grateful to Northern Regional Research Laboratories, USA, for the kind gift of the standard Xmthomoms campest& strain, and to C. Knauss for help in carrying out
Microbiology & Biotechnology. Vol I?, 1995
Cottrell, LW. & Kang, KS. 1978 Xanthan gum, a unique bacterial polysaccharide for food applications. Developments in Industrial Microbiology 19, 117-131. Haynes, W.C., Wickerham, L.J. & Hesseltine, C.W. 1955 Maintenance of cultures of industrially important microorganisms. Applied ~icrobiolo~ 3, 361-368. Jeanes, A.R., Pittsley, J.E. & Senti, F.R. 1961 A new hydrocolloid polyelectrolyte produced from glucose by bacterial fermentation. ]oumal of Applied Polymer Sciences 5, 5 19-526. Jeanes, A.R., Rogovin, S.P., Cadmus, MC., Silman, R.W. &Knutson, A.C. 1976 Polysacch~ide i~nt~n~ of Xanthomonas campestris NRRL B-1459: procedures for Caltare Ma~n~e~nce and ~of~~~cc~aride Production, Ptrrificafion and Analysis. ARS-NC-51. Peoria, IL: Agricultural Research Service, US Department of Agriculture. Moraine, R.A. & Rogovin, P. 1966 Kinetics of polysaccharide B1459 fermentation. Biotechnology and Bioengineering 8, 511-524. Moraine, R.A. & Rogovin, P. 1971 Xanthan biopolymer production at increased concen~ation by pH control. B;otech~o~ogy and ~io~gineeri~g 13, 381-391. Ramirez, M.E., Fucikovsky, L., Garcia-Jimenez, F., Quintero, R. & Galindo, E. 1988 Xanthan production by altered pathogenicity variants of Xanthomonas cumpestris. Applied Microbiology and Biotechnology 29, 5-10. Rocks, J.K. 1971 Xanthan gum. Fog-Technology 25, 22-29. Schaad, NW. & Stall, R.E. 1988 ~~fhomonas. In ~borafoy Guide for Identification of Plant Pafhogenic Bacteria, ed Schaad, N.W. pp. 81-94. Minnesota, MN: American Phytopathological Society. Starr, M.P. 1981 The Genus Xanthomonas. In The Procayotes, eds Starr, M.P., Stolp, H., Triiper, H.G.B., Balows, A. & Schlegel, H.G. pp. 742-763. Berlin: Springer-Verlag. Sutherland, I.W. 1972 Bacterial exopolysaccharides. Advances in Microbial Physiology 8, 143-213. Sutton, J.C. & Williams, P.H. 1970 Comparison of extracellular polysaccharide of Xnnthomonas cumpestris from culture and from infected cabbage leaves. Canadian journal of Botany 48,645-651. Symes, K.C. 1980 The relationship between the covalent structure of the ~~fhorn~~~ polysaccharide (xanthan) and its function as a thickening, suspending and gelling agent. Food Chemisfy 6, 63-76.
Torrestiana, B., Fucikovsky, L. & Galindo, E. 1990 Xanthan production by some Xanthomonus isolates. Letfers in Applied Microbiology lO,Sl-83. Weiss, R.M. & Ollis, D.F, 1980 Extracellular microbiai polysaccharides. I. Substrate, biomass and product kinetic equations for batch xanthan gum fermentation.^Biotechnology and^Bioengineering 22, 859-873.
(Received in revised form 3 March 1995; accepfed
6 March 1995)