Cu(2+) ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO4.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10(-8)-10(-6) M Cu(2+) ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu(2+) ion concentrations exist at low copper loads, there was competition of Cu(2+) for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO4, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu(2+), up to 10(-3) M.

In standard liquid medium containing 5 g mamitoll-' and 1 g glutamic acid 1-l, Rhizobium mefifoti SU-47 cells accumulated 350 mg cyclic 1,2-Q-glucans (g protein)-'. The cyclic glucans were 36 % glycerol-1-phosphatesubstituted and 64% were uncharged. In the same medium with 10 g mamitoll-' , repeating units of succinoglycan (1110 mg 1-l) were found as extracellular carbohydrates, and only low amounts of the succinoglycan polymer (up to 300 mg 1-l) were excreted. By raising the osmotic pressure of the medium by the addition of NaCl or other ionic and non-ionic osmolytes, succinoglycan production could be stimulated: up to 2.4 g 1-1 at 0.2 M-NaCI was produced at the expense of the repeating units. Above 0.2 M-NaCl growth was slowed down, and succinoglycan excretion diminished. At 1 M-NaCl growth stopped completely. In standard medium containing 0.6 M-NaCl the amount of cellular cyclic l,2-Q-glucans was lowered to 150 mg (g protein)-l out of which the glycerol-1-phosphatesubstituted glucan fraction was reduced to 15%. Instead, high amounts of oligosaccharides were synthesized as osmoprotectants, with trehalose as the major component [up to 200 mg (g protein)-']. Glycogen synthesis was completely suppressed at this salt concentration, while p l y Q-hydroxybutyric acid synthesis was unaffected.

Synthesis of acidic exopolysaccharides, neutral cellular polysaccharides and poly-beta-hydroxybutyric acid (PHB) by Rhizobium is strongly dependent on cultural conditions and the strains used. Exopolysaccharide production by R. leguminosarum, R. Phaseoli and R. trifolii closely parallels growth, whereas R. meliloti mainly excretes (low mol wt) polysaccharides when cell propagation is limited by lack of a necessary growth element (nitrogen) and an excess of carbon source is still present in the medium. In all strains, accumulation of cellular glycogen, beta-1,2-glucan and PHB is initiated only under growth-limiting conditions. When the external carbon source is exhausted, glycogen and PHB are metabolized by the cells, sustaining their longevity and thus act as true reserve materials; on the other hand, beta-1,2-glucan and excreted polysaccharides are not utilized on further incubation of the culture. Differences exist in the nature and relative amounts of the products synthesized by strains of different species of Rhizobium. R. leguminosarum, R. phaseoli and R. trifolii synthesize a uronic containing exopolysaccharide, PHB and/or glycogen, non-metabolizable capsular polysaccharide and low amounts of beta-1,2-glucan. R. meliloti synthesizes a uronic acid-free exopolysaccharide, PHB and /or glycogen and high concentrations of beta-1,2-glucan. Exopolysaccharides, beta-1,2-glucan and glycogen preparations were obtained by isolation and purification from cells of fast-growing species of Rhizobium and chemically characterized.

Methanogenic sludge granules grown on waste water from a sugar refinery consisted of several bacterial morphotypes embedded in a matrix of extracellular material. Comparison of critical point drying and freeze-drying methods for preparing samples for scanning electron microscopy to determine the presence of extracellular material indicated that the former method permitted observations of extracellular material and intact cells. The effects of different extraction methods used for isolation of these extracellular polymers was also investigated by scanning electron microscopy. Of the various extraction procedures (EDTA, NaOH, autoclaving, water–phenol), water–phenol left most of the cells intact and was found to be a very efficient method of extraction. Extracellular polymers equivalent to 10–20 mg hexose/g of granules were extracted. The high resistance of the granules against disintegration by various chemical methods suggested that different extracellular polymers and probably different groups of organisms contributed to the matrix in which the bacteria were embedded. The chemical composition of the granules did not differ from the composition of bacteria in general. The buoyant density of 1.00–1.05 g∙g−1of the granules indicated that a simple agglomeration was the mechanism by which these methanogenic consortia improved their settling characteristics.

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