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Calcium nutrition of Sphaerotilus growing in a continuous-flow apparatus.

Sphaerotilus natans required calcium for the production of sheaths and probably requires calcium for growth as well, though at a lower concentration. Neither strontium nor barium substituted for calcium. S. natans grew attached to the culture vessels of the continuous-flow apparatus even when no sheaths were produced. Tentative evidence showed that the requirement for calcium is shared by the manganese-oxidizing species S. discophorus.

Bacteriological Techniques↗

Growth rate of Sphaerotilus in a thermally polluted environment.

In situ growth of Sphaerotilus on microscope slides immersed in a thermally polluted stream was studied. Ultraviolet radiation was used to differentiate between passive attachment of organisms from the water and growth of the organisms on the slides. Colonization of the slides in this environment took place solely by means of swarmer cells. A generation time of 2.3 hr was obtained at a water temperature of 18-22 C.

Bacteriological Techniques↗

Isolation and chemical composition of the sheath of Sphaerotilus natans.

A sheathed bacterium, Sphaerotilus natans, was cultured with vigorous shaking in a medium containing peptone. Then the biomass was harvested and treated with lysozyme, sodium dodecyl sulfate, and protease. With treatment, 1.6 mg of sheaths was obtained from 15 mg of biomass. For the preparation of sheaths of high purity, cultivation must be in the absence of glucose with sufficient aeration to prevent poly(3-hydroxybutyrate) accumulation. Carbohydrate (54.1%), protein (12.2%), and lipid (1-3%) were detected in the sheaths by colorimetric reactions and solvent extraction. Gas-liquid chromatography showed glucose and galactosamine to be present in the molar ratio of 1:4. The most abundant amino acids in the sheath protein were glycine (49.2 mol%) and cysteine (24.6 mol%). The sheaths were resistant to agents that reduce disulfide bonds (dithiothreitol and 2-mercaptoethanol) and to protease. However, sheathes were degraded completely by hydrazine, and a heteropolysaccharide composed of glucose and galactosamine (1:4) was released. The weight-average molecular weight of the polysaccharide was estimated to be 1.2 x 10(5) by gel filtration chromatography with a low-angle laser-light scattering photometer and a rotation index detector. A ladder of 1.5-kDa peptides separable by sodium dodecyl sulfate gel electrophoresis was obtained by partial hydrolysis of sheaths, suggesting the sheath protein has repeating units of 1.5 kDa.

Amino Acids↗

Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans.

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.

Carbohydrates↗

Commensal relationship between a sheath-forming bacterium, Sphaerotilus natans, and a sheath-degrading bacterium, Paenibacillus sp.

Paenibacillus sp. strain TB is capable of degrading the sheath prepared from a sheathed bacterium, Sphaerotilus natans. S. natans was able to grow alone on casamino acids but strain TB was not. Cocultivation of strain TB and S. natans was examined in a medium supplemented with casamino acids as a growth substrate. The growth of strain TB was observed when the sheath was supplied to the medium or in cocultivation with S. natans. The phospholipid amount reached a maximum after 24 h of cocultivation and subsequently kept almost the same level for 96 h. The sheath amount also reached a maximum after 24 h and then gradually declined. The cell concentration of strain TB increased throughout the cocultivation. By competitive PCR targeted for amplification of a part of 16S rDNA, the abundance ratio (S. natans/strain TB) of 6.7 was obtained at 72 h. Almost no growth of strain TB was detected in a coculture with a sheath-less mutant of S. natans. The evidence allows the conclusion that strain TB grew by utilizing the intact sheath in coculture with S. natans.

Bacillus↗

Sphaerotilus natans isolated from activated sludge and its production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Sphaerotilus natans is a sheathed bacterium existing in the activated sludge of wastewater treatment plants. It is one of the filamentous bacteria causing the bulking and foaming difficulties of activated sludge. Isolating the strain and culturing it in an axenic environment could not only provide the metabolic knowledge of the strains that would be useful in the development of wastewater treatment methods, but also could enable us to gain an understanding of the mechanism by which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly[3-HB-co-3-HV]) is produced by this strain. This article reports the screening and isolation of the strain from the activated sludge using the Nile blue staining method together with Fourier transform infrared analysis. We investigated the ability of the selected strain to produce poly(3-HB-co-3-HV) copolymer using glucose and peptone, or by adding valeric acid or sodium propionate as precursor. Proper precursor feeding could dramatically enhance its 3HV content in the copolymer P(3HB-co-3HV). By controlling the different feeding times in fed-batch fermentation, different desired copolymers were obtained with 15, 40, and 70% 3HV mole fraction of the copolymer. Polymer properties were analyzed by gas chromatography, differential scanning calorimetry, thermo-gravimetry, and nuclear magnetic resonance analysis.

3-Hydroxybutyric Acid↗

Competitive growth kinetics of Sphaerotilus natans and Acinetobacter anitratus.

Growth kinetics of Sphaerotilus natans and Acinetobacter anitratus (strain E932) in pure and mixed cultures were analysed. In order to determine mixed cultures biomass composition, a quantitative image analysis technique was developed. Pure culture studies showed that for dilution rates less than 0.188 h(-1), the filamentous micro-organism will predominate leading to bulking phenomena. By using the developed technique to determine biomass composition, mixed culture experiments showed that changes in the dilution rate modify the microbial composition of the biomass determining which micro-organism predominate. The stated equations that predict the predominance of S. natans at low dilution rates agree satisfactorily with the obtained results.

Acinetobacter↗

Antitumor activity of Sphaerotilus natans and its slime fraction in mice.

Antitumor activity of the whole cell and slime of an aquatic sheathed bacterium, Sphaerotilus natans IAM 12068, against ascites form of Ehrlich carcinoma in ddY mice was investigated. Intraperitoneal injection of whole cells and the slime fraction showed remarkable antitumor activity against mice inoculated with 10(4) to 10(5) tumor cells, the slime fraction being more effective. To examine the chemical nature of the active principle in the slime fraction, separation by Sepharose 4B gel filtration was carried out and two fractions designated as GF-P-1 and GF-P-2, which are mainly composed of protein, carbohydrate, and lipid, were obtained. GF-P-1 fraction, which contains large amounts of fucose and unidentified sugar as neutral sugar, showed marked antitumor activity at half the dose of the slime fraction, whereas the antitumor activity of GF-P-2, which is composed mainly of protein, was weak. This finding indicates that GF-P-1 fraction of S. natans slime may be a main active principle. The consistently demonstrable antitumor activity of GF-P-1 was abrogated by treatment of mice with silica, an anti-macrophage agent, suggesting that the antitumor activity of GF-P-1 depends on the activation of macrophages.

Animals↗

Biocumulation of copper and lead by Sphaerotilus natans.

The accumulation of copper and lead by the filamentous bacteria Sphaerotilus natans was studied. An increase in the amount of accumulated metal depending on the concentration of copper and lead in the medium was observed. The extent of accumulation also depended on the chemical form of the metal in the medium and time of its contact with the bacteria. Electron micrographs of S. natans revealed the presence of copper and lead in the mucous capsule, cell wall, cytoplasmic membrane and cytoplasm of the bacteria. Both copper and lead altered the growth curves of S. natans.

Cell Wall↗