Folia Microbiol. 37 (2), 102-104 (1992)

Use of Whey for Production of Exocellular Polysaccharide by a Mutant Strain of Xanthomonas campestris J. KONI(~EKand M. KONIt~KOVA-RADOCHOVA Institute of Microbiology, CzechoslovakAcademy of Sciences, 142 20 Prague4 Received June 5, 1991

ABSTRACT. Growth and kinetics of the production of exoceUular polysaccharide was studied in a mutant strain of Xanthomonas campestris lac + during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1A %. The results were comparable with those obtained during cultivation on a lactose medium.

Bacterial polymers of the polysaccharide type prepared by modern biotechnologies are widely used in many branches of industry. Special rheological properties of bacterial polysaceharides are exploited in food products, cosmetics, pharmaceutical products, geological engineering and in the production of paper and textile. In our previous work we reported on the preparation of a collection of mutants of Xanthomonas genus bacteria that produced an exocellular polysaccharide of glucomannan type (Las~ and StanEk 1963). The mutants capable of using lactose as carbon source and those having white colonies (Las~ and Konf~ek 1976; Konf~ek et al. 1977; KonfEek and Las~ 1978) were prepared by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The parental strain grows on a solid complete medium in the form of smooth, glossy and mucous yellow colonies and uses primarily glucose, sucrose, fructose, maltose and starch as carbon source. Having a very low level of fl-galactosidase, bacteria of this genus cannot use lactose as carbon source for growth. The idea to prepare lactose-fermenting mutant strains was motivated by the effort to utilize cheap carbon sources that contain a high proportion of lactose (e.g. whey) for the production of the polymer. It was for the first time that Xanthomonas bacteria capable of using lactose as carbon source were isolated. The growth characteristics of the mutant strains prepared that were measured in liquid cultures in a medium containing 2 % lactose were comparable with those of the parental strain. The lac § strains produced the maximum amount of the exocellular polysaccharide during the period of stationary growth phase and the yield related to the saccharide consumed as the carbon source was in the range of 9-20 %. Gas chromatography showed us that the exocellular polysaccharide contained, as the main components, a- and fl-D-mannose and a- and fl-D-glucose, minor components being D-ribose, 6-deoxy-L-mannose and a-D-galactose (Koni~ek et aL 1977). Last'k and Koni~ek (1989) studied the course of production of the exopolysaccharide of the glucomannan type by a lactose-utilizing strain in an SLF20 fermentor equipped with a special type of stirrer developed for cultivation during which large changes of the density of the cell suspension occurred (ICdov~ikand Salvet 1986). Significant growth of a mutant strain of X. campestris in a medium containing 8 % lactose was observable already after 2 d, the maximum polymer production being detected after 4 d. The viscosity of the culture medium rose up to 6 d when cultivation was stopped. In a control experiment, when the same type of fermentor was used but equipped with a classical Rushton turbine, the growth yield and the viscosity of the medium were lower. The lower production of the polysaccharide as well as the lower biomass yield were caused by uneven mixing of the microbial culture. In this work, growth and kinetics of the polymer production by a lactose-fermenting mutant strain were examined under the conditions of growth in a liquid medium containing whey.

M A T E R I A L S AND METHODS Bacterial strain. A mutant strain of Xanthomonas campestris lac +1 (previously designated as X. fuscans) prepared by treatment with N-methyl-N'-nitro-N-nitrosoguanidine was used (Koni~ek et al.

1977). Culture media. The microorganism was grown in a WH medium (mg/L): corn-steep 1 000, yeast extract (Oxoid) 750, K2HPO4 750, KNO3 375, MgSO4"7H20 150, NaC1 75, CaCl2 75, FeCl3 70; distilled water ad 800 mL. Fifty g of lactose dissolved in 200 mL of distilled water was added after ster-

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ilization. In case whey was substituted for lactose, dry nondeproteinated whey was used at an amount corresponding to the above-mentioned concentration of lactose. The whey was dissolved in distilled water, heated to 85 *C for 25 min three times. The whey solution was checked for sterility and added to the WH medium. The nutrient medium was solidified with 2 % agar (OxoM No. 1). Estimation of exocellular polysaccharide production in submerged cultivation. To prepare the inoculum, the liquid WH medium containing 2 % lactose was used, incubation was stationary at 28 *C. Five mL of a bacterial suspension growing exponentially was used for inoculation of 80 mL of the liquid WH medium in a 500-mL flask. The culture was incubated on a reciprocal shaker (frequency 1.6 Hz; amplitude 80 mm) for 7 d at 28 ~ Samples were removed at suitable time intervals and the culture growth was measured. Cell numbers were estimated by plating on a solid WH medium with 2 % lactose. The polymer was isolated according to Las~ et al. (1978). The product was dried under an IR lamp and the dry mass was weighed.

R E S U L T S A N D DISCUSSION The mutant strain of X. campestris lac + forms short Gram-negative rods, in contrast to the parental strain that is characterized by a coccobacillary shape with mostly single cells that sometimes produce small clusters. When grown in a liquid WH medium, the strain produces homogeneous turbidity. On a solid medium of the same composition, oval, bulging yellow colonies are formed 3 to 4 mm in size. Their surface is smooth, glossy and mucous. Strain growth and the formation of the exocellular 2.0 polysaccharide during sub% merged cultivation in a whey g WH medium were compared 1.6 with the situation when lactose was the carbon source used for Iogc growth. The kinetics of the polymer formation depended 1.2 on growth. The maximum production of the polymer was reached at the initial stage of 8 the stationary growth phase 0.8 (Fig. 1). No significant differences were found with the two carbon sources. In both cases, 0.4 a rapid division of the cells took place at the beginning of the cultivation, the cell number I I I I I I increased by about 1.5 orders 60 120 180 60 120 180 of magnitude during the first h 24h of cultivation. In the Fig. 1. Growth curve of X. campestris lac + (cell concentration c, 1/mL; 1) and course of 7 d of cultivation at production of exocellular polysaccharide (%; 2) in WH medium containing whey 28 ~ the highest production (left) or lactose (r/ght). of the polymer was attained at 96-110 h with the maximum growth yield occurring at about 76 h. The mean maximum production of the exocellular polysaccharide was about 1.4 %; if lactose was used as the carbon source, the production was slightly higher. If maltose served as the carbon source for growth, its utilization was significantly lower and the production of the polymer was decreased to 0.5 %. The xanthan polymer was successfully produced under the conditions of submerged cultivation on a whey medium; the utilizability of this carbon source and the production of the exocellular polysaccharide were comparable with those in the lactose medium. The producing strains were prepared by induced mutagenesis using a powerful alkylation mutagen. The mutation consisted in a change of the regulatory gene and derepression of the lac § operon (KoniEek et al. 1977). Other authors have also I

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been able to prepare X a n t h o m o n a s strains capable of lactose fermentation. Walsh et al. (1984) genetically manipulated X. campestris to obtain strains utilizing lactose. They transferred the pGC9114 plasmid from E. coli with the lactose transposon Tn951 by conjugation to X. campestris Nal r. The specific fl-galactosidase activity in the conjugants was more than 200-fold greater. Similarly, Fu and Tseng (1990), working with X. campestris, constructed a strain containing a recombinant plasmid that enabled the microorganism to utilize lactose. The recombinant molecule consisted of phage ~ L O and plasmid pKM005 that was derived from plasmid pBR322 bearing lacZ and lacY genes. As compared with the work of Walsh et al. (1984), the latter authors grew the constructed strain on 10 % whey and measured the production of xanthan that reached the value of 4.2 g / L of the culture medium. Schwartz and Bodie (1985) isolated a strain of X. campestris BB-1L whose production of xanthan in a whey medium was measured viscosimetrically. The authors obtained this strain by a spontaneous selection. It was capable of using lactose but was not genetically stable since, after 35 generations, the viscosity of the culture filtrate was already lower. After 85 generations, the capability of the polymer formation was completely lost. Baig et al. (1990) were using a strain ofX. cucurbitae PCSIR B-52 (Culture Collection Unit, P C S I R Laboratories, Lahore) that produced an exocellular polysaccharide when grown in a whey medium. The authors optimized the amount of lactose in the synthetic medium and found that the maximum polymer production in the submerged cultivation (7.8 g/L) was detected if the concentration of lactose was 4 %. During a cultivation in an 18-L fermentor, the production of the polymer in a whey medium increased to 16 g/L. By using a cyclic fermentation process, the production could be raised to 20 g/L. With our strain of X. campestris we assume that the production of the polymer attained in the submerged cultivation will be further increased by using a fermentor for growth. The authors thank Mrs Marta Fingerovfifor excellenttechnicalassistance. REFERENCES BAIGS., QADEERM2~., AKHTARM.S., AHMEDT.: Utilization of unhydrolyzedcheese whey for the production of extracellular polysaccharideby Xanthomonas cucurbitae PCSIR B-52.J.Ferment.Bioeng. 69, 345-349 (1990). Fu J.F., TsF~o Y.H.: Construction of lactose-utilizingXanthomonas campestris and production of xanthan gum from whey. Appl.Environ.Microbioi. 56, 919- 923 (1990). KON/(~EKJ., LAS[KJ.: Bacterialstrain Xanthomonasfuscans (BURKHOLDER)BURK.FXL. Czech.Pat. 185 394 (1978). KONfdEKJ., LASiKJ., WURSTM.: Production and characteristics of the exocellular polysaccharide in mutant strains of Xanthomonas fuscans. Folia Microbiol. 22, 12-18 (1977). KI~OVAKP., SALVErM.: Equipment for fermentationespeciallyviscose aerobic media. Czech.Pat. 228 221 (1986). LASiK J., KONi~EKJ.: Properties and production of the exocellular polysaccharide of some strains of the bacterium Xanthomonas ~uscans isolated from the rhizosphere. 12th Ann. Meet. Czech. Microbiol. Society, Ko~ice (Czechoslovakia) 1975;Folia Microbiol. 21, 218 (1976). LASiKJ., KoNidEKJ.: Fermentation of glukomannan exopolymerfrom a mutant strain Xanthomonas campestris pv. phaseoli (SMITH1897) DYE 1978on a new type of stirrer. Zbl.Mikrobiol. 144, 169-172 (1989). LASiKJ., STANI~KM.: Xanthomonas fuscans (BURKIIOLDER)BURK.in rhizosphere of beans (Phaseolus vulgaris L.). (In Czech) Rostlinnd v~roba 9, 715 (1963). ~ i K J., UnER J., AwroNv K.: The way of isolation of the exocellular carbon polymer of glucomannuronan type from a bacterial microorganism.Czech.Pat. 173 894 (1978). SCHWARTZR.D., BODIEE.A.: Production of high-viscositywhey broths by a lactose-utilizingXanthornonas campestris strain. Appl.Environ.Microb iol. 50, 1483-1485 (1985). WALSHP.M., HAASMJ., SOMKUTiG.A.: Genetic construction of lactose-utilizingXanthomonas carnpestris. Appl.Environ. Microbiol. 47, 253-257 (1984). Translated by (~. Novotn~