Kuniho Nakata

High production of rhamnolipids by Pseudomonas aeruginosa growing on ethanol

Pseudomonas aeruginosa IFO 3924 produced 32 g rhamnolipid biosurfactant/l when grown on 55.3 g ethanol/l in fed-batch culture for 7 days. The conversion rate was 58%.

High production of pyoluteorin and 2,4-diacetylphloroglucinol by Pseudomonas fluorescens S272 grown on ethanol as a sole carbon source

Pseudomonas fluorescens S272 newly isolated from a soil sample produced a considerable amount of pyoluteorin and 2,4-diacetylphloroglucinol when grown on ethanol as a single carbon source. The coproduction of approximately 150 μg/ml of pyoluteorin and approximately 500 μg/ml of 2,4-diacyl-phloroglucinol was achieved by flask cultutivation in a medium containing approximately 2% ethanol. A high CN ratio and inorganic phosphate limitation in the medium were also important factors to be considered for optimization of production of the antibiotics.

Structural Analysis of an Extracellular Polysaccharide Bioflocculant of Klebsiella pneumoniae

The glycoside composition and sequence of an extracellular polysaccharide flocculant of Klebsiella pneumoniae H12 was analyzed. GC and HPLC analysis of the acid-hydrolysate identified its constituent monosaccharides as D-Glc, D-Man, D-Gal, and D-GlcA in an approximate molar ratio of 3.9:1.0:2.3:3.6. To analyze the glycoside sequence, the polysaccharide was partially hydrolyzed by acid and enzyme treatment. GC, HPLC, TLC, MALDI-TOF/MS, and 1H- and 13C- NMR spectroscopy characterized the obtained oligosaccharides. The results clarified the partial structure of H12 polysaccharide as a linear polymer of a unit of pentasaccharide with a side chain of one D-GlcA to D-Glc moiety (see below). Although the existence of other sequences or other constituent glycosides could not be fully excluded, H12 polysaccharide must be a novel types as such a complicated unit for a polymer has not so far been reported. The partial structure of a H12 polysaccharide flocculant is also discussed in this report. →4)- α-D-Glcp-(1→2)-α-D-Manp-(1→3)-4,6-Pyr-β-D- 3 Galp-(1→4)-β-D-Galp-(1→ ↓ 1 β-D-GlcpA

Enhancement of Plant Stem Growth by Flocculation of the Antibiotic-producing Bacterium, Pseudomonas fluorescens S272, on the Roots

The antibiotic-producing bacterium, Pseudomonas fluorescens, is assumed to be important in protecting plants from soilborne diseases. S. fluorescens S272, a hyper-producing strain of pyoluteorin (PT) and 2,4-diacetylphloroglucinol (DG), had previously been isolated from soil. The present paper reported that the growth of water-cultivated Kaiware radish was promoted to 120-140% of its normal level by the coaddition of an S272 culture broth (0.01-1% v/v) and a polysaccharide flocculant (1-100 ppm) from Klebsiella pneumoniae H12. Tight adhesion of S272 cells to the root tissue was microscopically observed. The growth promotion is assumed to have been caused by antibiotic effects for the following two reasons: 1) PT (4 mg/l) and DG (24 mg/l) addition to a radish culture enhanced stem growth to 130% of the normal level; 2) a culture solution containing the S272 culture broth (0.01-1% v/v) markedly inhibited the decomposition of hypersensitive chrysanthemum leaves. A soil-cultivation experiment with Gomphrena globosa under natural conditions also exhibited enhanced stem length (160%) by coaddition of the S272 culture broth and H12 polysaccharide. These results suggest that polysaccharide-enhanced adhesion of P. fluorescens S272 cells might be useful for promoting plant growth through the increased antibiotic effect.

Correlation between autoinducers and rhamnolipids production by Pseudomonas aeruginosa IFO 3924

High rhamnolipid productivity (32 g/l) was obtained in an ethanol fed-batch culture of Pseudomonas aeruginosa IFO 3924. Examination of the autoinducer level and exogeneous autoinducer addition tests indicated that in the fed-batch system high autoinducer activity, which was about ten-fold that obtained in an unfed system, was thought to be the cause of the high rate of rhamnolipid production. Both N-(3-oxohexanoyl)-l-homoserine lactone (OHHL) and N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL) enhanced rhamnolipid productivity in the unfed system.

Two glycolipids increase in the bioremediation of halogenated aromatic compounds

Two bacterial glycolipids were applied for the bioremediation of mono-halogenated aromatic compounds in soil. 4-Chloro-1-naphthol (CN) and 3-chlorodiphenylamine (CDA) were rinsed from a polluted soil sample using a biosurfactant, rhamnolipid. Deemulsification by pH adjustment to 4.0 resulted in about 90% recovery of CN and CDA in an organic solvent fraction which was finally solidified by evaporation. For the bioremediation of the aqueous phase, a bacterial glycoglycerolipid, di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol was used to decrease the level of cellular damage induced by the effects of oxygen radical of the intermediate metabolites of CN and CDA. The glycoglycerolipid at a concentration of 100 mg/l increased the cellular resistance to those CN and CDA approximately three fold. Instead of the addition of purified glycoglycerolipid, a 0.1%-volume addition of the culture supernatant of a glycoglycerolipid producer to active sludge was also effective.

Regulation by Organic Acids of Polysaccharide-mediated Microbe-plant Interactions

A polysaccharide flocculant of Klebsiella pneumoniae H12 has been suggested to mediate microbe-plant interactions with the aid of Ca2+ [K. Nakata et al., Biosci. Biotechnol. Biochem., 64, 459-465, 2000]. Here, two-way regulation of polysaccharide-mediated interactions between K. pneumoniae and Raphanus sativus was studied using organic acids. Namely, 10 mM equivalents of organic acids promoted production of the polysaccharide by the bacterium, but inhibited flocculation of bacterial cells by the polysaccharide. These phenomena were counterbalanced by equi-molar equivalents of Ca2+, suggesting competition for Ca2+ between the carboxylic residues of the polysaccharide and those of the aliphatic acids. By electron microscopy observations, bacterial cell aggregates were sparsely distributed over the main roots and root hairs, had various sizes, and seemed to tightly adhere to root tissues. Their shapes seemed to be distorted and abundant in cavities. In brief, these microscopical observations may be explained by a two-way regulation system of bacterial adhesion to a plant by organic acids.