Antoine van Leeuwenhoek 64: 1-8, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands. Influence of growth conditions on production of capsular and extracellular polysaccharides by Rhizobium leguminosarum M.W. Breedveld ~'3,L.P.T.M. Zevenhuizen ~, H.C.J. Canter Cremers 2'4 & A.J.B. Zehnder 1'5 1Department of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands; 2Institute of Plant Molecular Biology, State University of Leiden, Nonnensteeg 3, 2311 VJ Leiden, The Netherlands," (Present addresses: S Department of Food Science, 101 Borland Laboratory, The Pennsylvania State University, University Park, PA 16802, USA; 4AHA, Zeemanstraat 35, 2912 BK Nieuwerkerk a/d IJssel, The Netherlands," 5 EAWAG/ETH, CH-8600 Diibendorf Switzerland) Received 29 September 1992; accepted in revised form 7 January 1993 Key words: capsular polysaccharide, extracellular polysaccharide, polysaccharide production, Rhizobium leguminosarurn Abstract The influence of growth rate and medium composition on exopolymer production by Rhizobiurn Ieguminosa- rum was studied. When grown in medium containing 10g/l mannitol and lg/1 glutamic acid, Rhizobium legu- minosarurn biovar trifolii TA-1 synthesized up to 2.0 g/1 of extracellular polysaccharide (EPS), and up to 1.6 g/1 of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8 g/l). Maxi- mal CPS-yield was 2.9g CPS/1 medium in a medium containing 20g/1 mannitol and 2g/1 glutamic acid. The EPS-deficientstrain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell pro- tein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h-~). The production rates were 50-100mg/g protein/h for EPS and 15-20mg/g protein/h for CPS. Only low amounts of cyclic J3-(1,2)-glucans were excreted (10-30mg/1) over the entire range of growth rates. Abbreviations: bv - biovar; CPS - capsular polysaccharide; EPS - extracellular polysaccharide; HM r - high molecular mass; LM r - low molecular mass; YEMCR - Yeast Extract-Mannitol-Congo Red agar Introduction Many Gram-negative and Gram-positive bacteria are capable of synthesizing a variety of polysaccha- rides (Sutherland 1988). Rhizobium leguminosa- rum strains produce exopolysaccharides such as high molecular weight anionic extracellular poly- saccharide (EPS), insoluble neutral capsular poly- saccharide (CPS) and cellulosic microfibrils. In ad- dition, cyclic ~3-(1,2)-glucans secreted from the pe- riplasm can be found in the medium (Zevenhuizen 1990). The EPS type commonly found with this group of organisms is composed of an octasaccha- ride repeating unit with sugar composition of gluco- se:glucuronic acid:galactose in the ratios of 5:2:1 (McNeil et al. 1986). The CPS from a number of R. leguminosarum strains, composed of neutral sugars without any non-sugar substituents, has a constant