Growth and accumulation of polysaccharide by Xanthomonas campestris

Zeitschrift fur Allgemeine Mikrobiologie 20 3 1980 209-213

Kurze Originalmitteilungen

(Akademie der Wissenschaften der DDR, Institut fur technische Chemie, Leipzig, Direktor : Prof. Dr. sc. M. RINGPFEIL)

Growth and accumulation of polysaccharide by Xanthomonas campestris

U. BEHRENS, M. KLIMA and S. FIEDLER

(Eingegangen am 25. 6.1979) The synthesis of bacterial exopolysaccharides is favoured by a high carbon : nitro-

gen ratio and generally stimulated by nitrogen limitation (SUTHERLAND 1972). For xanthan accumulation with Xanthomonas campestris in a medium containing both yeast extract and nitrate as nitrogen sources, BEHRENS et at. (1977) found diauxic growth. Separated by a transition period, the first period with logarithmic growth is followed by a second in which accumulation of the exopolysaccharide occurs. The authors proposed nonbalanced growth as one essential condition for xanthan over- production. We investigated whether diauxic growth is caused by the mixed nitrogen source, and attempted to elucidate the influence of growth on xanthan accumulation.

Fermentation: Strain and methods of cultivation were the same as described previously with the exception of the nitrogen source (BEHRENS et al. 1977). Instead of a mixture of nitrate and yeast extract, nitrate or peptone alone were used.

Analytical: Bacterial cells were counted. The bacterial mass was determined gravimetrically and by optical-density measurements as described previously (BEHRENS et al. 1977). Peptone and

t

Fig. 1. Growth of X . campestria in a medium with nitrate (2.18 g NO,- per 1) as sole nitrogen source

210 U. BEHRENS, M. KLIMA and s. REDLER

nitrate content in the cell-free broth were determined by the KJELDAHL-method and by the sodium salycilate method, respectively (MULLER and WIEDEMANN 1955).

For xanthan determination broth samples were separated from the cells by centrifugation (1.5 x 104 min-1, 15-30 min). When necessary viscous samples were diluted with water to obtain separable solutions. An aliquot of a quaternary ammonium salt solution (5% Quartolan, VEB Hydrierwerk Rodleben, in distilled water) was added to the supernatant under stirring. The precipi- tate was collected, washed twice with distilled water, dried in vacuo and determined gravimetri- eally. The samples had been calibrated against solutions with different concentrations (0.5-2%) of Kelzan (KELIX Company, USA).

As indicated in the previous paper (BEHRENS et at. 1977) the diauxic course of growth is not caused by the mixed nitrogen source, which consisted of both yeast extract and nitrate. In media with nitrate as the sole nitrogen source growth started after an excessively long incubation phase (ca. 40 hr), although precultivations had been performed in inoculum media, which also contained nitrate as the sole nitrogen source (Fig. 1). This observation and related unpublished data indicate that this pheno- menon is not due to a prolonged induction phase but presumably to the toxic effects

Fig. 2. Growth of X . cam- pestris in a medium with

peptone (3.0 g per 1) as sole nitrogen source.

cell number, o - tur- bidity, A - biomasses

Polysaccharide accumulation bei Xanthomoiias 211

of nitrogen-oxygen compounds. The ability of X . campestris to perform nitrate reduc- tion is assumed by FROBISHER (1958) and KRASSILNIKOV (1959). I n contrast to this HAYWARD (1966) rules out nitrate reduction. Diauxic growth also occurs in media with peptone as the sole nitrogen source (Fig. 2). I n the course of this investigation both gravimetrical and optical biomass determinations as well as cell counting were performed. I n the second period of growth the biomass increased twofold, while the number of cells rose only 1.5-fold. The reason for the difference between the two rates could be attributed to the adhering gum which is not completely removed by repeated washings.

MORAINE and ROGOVIN (1973), who adjusted the pH-value in xanthan fermentations with NH,OH, observed a twofold increase of cell size1) and therefore they used cell

Fig. 3. Influence of the amount of the nitrogen source on growth and xanthan accumulation. a - 0,5 g per 1 .peptone, A - 1.0 g per 1 peptone, 0 - 2.0 g per 1 peptone, - 3.0 g per 1 peptone (in each case initial concentration)

~- cell number, - - - - turbidity, -. - . - xanthan t .

*) during fermentation

212 U. BEHRENS. M. KLIMA and S. FIEDLER

numbers as growth indicators. As proposed in a previous paper (BEHRENS et al. 1977), the second period could be attributed to nonbalanced growth, Investigations per- taining to the variation of main cell constituents (protein, carbohydrates, DNA, RNA) support this hypothesis. During nonbalanced growth the content of RNA and protein increases in relation to the constant amount of DNA (FIEDLER and BEHRENS, in prep.). The connection of nonbalanced growth with xanthan accumulation is confirmed in Fig. 3. Xanthan yields increase as the supply of peptone rises. A higher concentration of nitrogen sources makes higher biomass concentrations possible which, in turn, lead to higher production rates, whereas nonbalanced growth is essential for gum accumu- lation. These findings are in contrast to general conceptions of exopolysaccharide synthesis, in which different limitations of growth promote synthesis and accumula- tion of slime (For a review see SUTHERLAND 1972). Furthermore the metabolite requi- rements for the synthesis of cellular polysaccharides are believed to be competitive with those for the synthesis of extracellular gums (HUSSEY and BADDILEY, cit. in SLODKI and CADMUS 1978). In X. carnpestris as in Klebsiella aerogenes (DUGUID and WILKINSON 1953) polysaccharide production is connected with growth. This special kind of overproduction may be attributed to the synthesis of capsular polysaccharides. According to SUTHERLAND (1975), xanthan could have been a capsular polysaccharide that has lost the ability to adhere to the cell surface. I n this context xanthan, which is a heteropolysaccharide produced by a gram-negative bacterium, could have been a part of the outer polysaccharide moiety of the cell wall, which was altered in struc- ture by mutation, thus losing contact with the cell surface.

The period of nonbalanced growth which shows a declining growth rate may be related to the conception of linear growth that is caused by a critical concentration of an essential nutrient (MULLER et al. 1978). While MONOD (1949) explained linear growth as an effect of a bottleneck enzyme, in the case of X . carnpestris the decreasing growth rate is instead caused by increasing viscosity which renders the influx of nutrients more difficult. The excretion of polysaccharide a t the beginning of nonbalan- ced growth may also be attributed to regulatory mechanisms as described for secon- dary metabolite synthesis (FORRESTER and GAUCHER 1972). The reduction of replica- tory growth could cause increased levels of CAMP or similarly acting substances which control m-RNA-initiation in different steps. So far, evidence leading to a uni- fied view of the role for CAMP during phases of limited growth is very sparse.

After logarithmic growth xanthan accumulation starts in appropriate carbohy- drate media. Xanthan synthesis obstructs the influx of nutrients which causes a shift from balanced to nonbalanced growth. Nonbalanced growth is prerequisite to xan- than accumulation. In the course of normal xanthan accumulation after logarithmic growth the biomass increases 2fold while the number of cells increases only 1.5fold.

The quantity of nitrogen source available after logarithmic balanced growth is proportional to the amount of xanthan which can be accumulated. The overproduc- tion of xanthan a t the onset of limited growth may be attributed to similar regulatory mechanisms as proposed for secondary metabolites.

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Mailing address: Dr. U. BEHRENS Institut fur technische Chemie der Akademie der Wissenschaften DDR 705 Leipzig, PermoserstraBe 15