Xanthan production by Xanthomonas campestris NRRL B-1459 and interfacial approach by zeta potential measurement

Xanthan production by Xanthomonas campestris NRRL B- 1459 and interfacial approach by zeta potential measurement Ph. Thonart, M. Paquot, L. Hermans and H. Alaoui Ddpartement de Technologie, Facultd des Sciences Agronomiques de l'Etat, B 5800 Gembloux, Belgium

and P. d'Ippolito lnstitut Sup~rieur Industriel Lidgeois, B-4000 Libge, Belgium

(Received 5 November 1984) Dissociation of cell growth and metabolite production allows the creation of continuous reactors where conversion yields of carbon to product are very high. This is especially true for products such as xanthans, where physical parameters change rapidly, especially viscosity. At pH 5, cell growth and biomass production are increased. A t pH 7, the opposite situation is observed, xanthan production is increased. Moreover, production is promoted at 30°C, while 25°C is the optimum temperature for cell growth. The composition of the medium (nitrogen source, citrate concentration, D-glucose concentration) has also been studied. Two-stage fermentation is an effective method, yielding up to 90% in the bioconversion of 1% D-glucose to xanthan. The xanthan/cell ratio is ~10. The major difficulty in carrying out continuous production is the anionic characteristics of the gum. Keywords: Bacteria; Xanthomonas campestris; xanthan; zeta potential measurement

Introduction Xanthomas campestris is a bacterial source for the anionic polysaccharide xanthan. 1 Xanthan gum is composed of o-glucose, o-mannose and o-glucuronic acid assembled in pentasaccharide repeating units. 2 Half of the terminal D-marmopyranosyl groups are substituted at positions 4 and 6 by pyruvic acid acetal residues, and the D-mannopyranosyl residues are O-acetylated at position 6. In vitro this polysaccharide is synthesized in two steps: a pentasaccharide-P-P-lipid is formed from UDP-D-glucose, GDP-D-mannose and UDP-D-glucuronic acid, followed by polymerization into xanthan gum. 3 Achieving optimum fermentation conditions "for polysaccharide production is particularly important because of the wide range of application of xanthan gum. Xanthan is extensively used by the petroleum industry, the food industry, and as a suspending and stabilizing agent for herbicides, pesticides, fertilizers and fungicides.4-6 Although Xanthomonas campestris exhibits no growth problems on standard laboratory media, both small and large colony strains have previously been observed in continuous 7 and batch-type fermentation. 8 The level of pyruvate substitution appears to be the best indicator of gum quality. 9 Xanthan production has been intensively studied with a batch fermentation taking ~ 5 0 h and giving 85% yield. 1° The viscosity of the medium 0141--0229/85/050235--04 $03.00 © 1985 Butterworth & Co. (Publishers) Ltd

and the relatively short time for biomass development limit the performance of the fermentation in a continuous process. The purpose of this paper is to show that a continuous fermentation yields high amounts of gum when it is realized in two steps. First, the strain is grown so that the polymer concentration in the medium is maintained at a low enough level, allowing the biomass to increase rapidly. When the cell mass has reached the desired level, gum production is promoted. As a result, the fermentation time is shorter, and the yields higher. Moreover, a cell recycle process or an immobilized cell reactor are possible. This dissociation of growth and gum production was made possible by knowledge and control of the physiological conditions of Xanthomonas campestris strain NRRL B-1459. The zeta potential of the cells was also studied, this factor being essential to the concept of an adsorbed cell continuous reactor. 11

Materials and methods Biomass estimation was carried out by measuring absorbance at 540 nm and gravimetrically after Millipore filtration (0.45/~m). The strains were cultivated in 250 ml baffled flasks, incubated at 29°C for 72 h. Xanthan estimation was performed by precipitation with KCl-saturated ethanol as

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described by Stauffer and Leeder 12 and viscosity measured with a Brookfield viscometer, model LVT. The zeta potential was measured with a Laser Zee Meter, model 500, as described by Thonart et aL 11

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Influence o f corn steep liquor concentration The optimum nitrogen concentration was 0.042 mg/ 100 ml with (g/100 ml): MgS04, 0.02; K2HP04, 0.5; and D-glucose, 4. This was obtained by adding 0.5% corn steep liquor. Higher levels rapidly inhibited xanthan production, which was practically zero for nitrogen concentrations >0.17 mg/100 ml.

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Influence o f n-glucose concentration With a medium containing (g/100ml): citrate, 0.2; MgSO4, 0.02; phosphate buffer, pH 7, Na2I-IPO4, 0.69 and KH2PO4, 0.37; and corn steep liquor, 0.5, polymer production was highly dependent on sugar concentration, while growth was not. Production decreased with increasing D-glucose concentration and the polymer/biomass ratio dropped rapidly with sugar concentrations >3%.

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Influence o f citrate concentration With the above medium and 4% glucose, the results obtained for various citrate concentrations are given in Figure 1. Xanthan biomass and yield were highest when the citrate concentration was 0.24%. The influence of citrate became negative when its concentration exceeded 0.33%.

Figure 2 biomass,

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Xanthan and biomass production are plotted against temperature in Figure 2. The xanthan/biomass ratio reached 19.9 at 33°C, which was a four-fold increase over the value at 20°C.

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Influence o f pH ~'ith the described medium at 29°C, the biomass/ xanthan ratio was optimum at pH 5, while the reverse ratio was optimum at pI-I 7. The pH variation during xanthan production (pH 7) must not exceed 0.5 units.

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E n z y m e Microb. Technol., 1985, vol. 7, May

Xanthan is an acidic polysaccharide that can influence the electric charge of cells, and as a result the membrane mass transport and the adhesiveness in an immobilized cell reactor. Cells of Xanthomonas were centrifuged, washed, and the zeta potential measured as described by Thonart et a/xl Various concentrations of xanthan were added and the zeta potential varied between - 3 4 mV and - 7 4 mV as shown in Figure 3. The same experiment was carried out with the addition of cationic starch (see Figure 4). Cationic starch brings the potential up to positive values, while xanthan increases the negative value. During experimental xanthan production,

Xanthan production by Xanthomonas campestris: Ph. Thonart et aL

the zeta potential remained constant during the biomass development (pH 5, 29°C) and increases its negative value during the production phase (pH 7, 33°C), as illustrated in Figure 5. Table I shows the change in zeta potential of Xanthomonas cells after addition ofxanthan, followed by cationic starch, and Table 2 the same when starch is added first,

40

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Table 1 Effect on zeta potential

of xanthan addition followed by cationic starch

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followed by xanthan. It can be seen that some of the differences dependent on the first added hydrocolloid were measurable. This is particularly true when small concentrations of hydrocolloids were used.

Dissociation o f growth and production The xanthan production was carried out in two separate steps. First, biomass increase was promoted while polymer production was depressed. This was done at pH 5 and 29°C in a medium containing Ogl-1): yeast extract, 3; malt extract, 9; peptone, 0.5; and D-glucose, 13, with phosphate buffe~ at pH 5. At the beginning of the stationary phase the pH was adjusted to 7, the temperature was raised to 33°C over 48 h, and various D-glucose additions (1,3 and 5%) were tested. The yield in the bioconversion of D-glucose to xanthan reached 90% with 1% D-glucose, 55% with 3% and 38% with 5%. The biomass may be recycled twice; the yields decrease significantly but this is probably due to a slackening of the metabolism and not biomass production.

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The cell should be considered as a tool and not as an endproduct. To dissociate cell growth and metabolite production allows us to conceive continuous reactors where conversion yields of carbon into product are very high. This is especially true for products like xanthan, where physical parameters change rapidly, viscosity being a demonstrative example. By controlling the culture conditions, one can promote either growth or the metabolite production for Xanthomonas campestris separately. A nitrogen concentration higher than 0.042% seems to inhibit polymer production, but pH and temperature are the easiest controlling factors. At pH 5, cell growth and biomass increase are promoted at the expense of xanthan production. At pH 7, the opposite situation is achieved. Production is promoted at 30°C, while 25°C is the optimum temperature for cell growth. A two-stage fermentation is a powerful method, yielding up to 90% in the bioconversion of D-glucose to xanthan.

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Papers The xanthan/cell ratio is ~10. The values found in the literature 13 are between 60 and 85% in a 50 h process. The fermentation time can be reduced, but only at the expense of conversion rate, and the optimum choice has to be made for a cost-effective industrial process. The major point of interest of this study, however, is the possibility for continuous reactor design. Biochemically xanthan production is complex, and the physical properties of the product (molecular weight, viscosity) are unfavourable to cell immobilization. Gel encapsulation is inappropriate considering the mass transfer, but cell absorption has the advantage of allowing cell growth and having good mass transfer characteristics. We previously demonstrated xl that the important factor for this type of immobilization is the zeta potential of the cell. This potential changes to highly negative values during gum production (see Figure 5) and the addition of cationic polymers is probably ineffective in keeping the value around zero. Xanthan, which is an anionic polymer, thus prevents immobilization on natural supports. We failed to immobilize Xanthomonas on negative matrices, so we now turn to positive ones, which are unfortunately more expensive. However, cell recycle assays showed real possibilities in this area. We are presently studying the equilibrium between xanthan production and maintenance of the biochemical potential o f the cell.

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Acknowledgement The authors thank Mrs Gregoire and Mr Maquet for excellent technical assistance.

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