Biosynthetic Pathway and Genes of Chitin/Chitosan-Like Bioflocculant in the Genus Citrobacter.

Chitin/chitosan, one of the most abundant polysaccharides in nature, is industrially produced as a powder or flake form from the exoskeletons of crustaceans such as crabs and shrimps. Intriguingly, many bacterial strains in the genus Citrobacter secrete a soluble chitin/chitosan-like polysaccharide into the culture medium during growth in acetate. Because this polysaccharide shows strong flocculation activity for suspended solids in water, it can be used as a bioflocculant (BF). The BF synthetic pathway of C. freundii IFO 13545 is expected from known bacterial metabolic pathways to be as follows: acetate is metabolized in the TCA cycle and the glyoxylate shunt via acetyl-CoA. Next, fructose 6-phosphate is generated from the intermediates of the TCA cycle through gluconeogenesis and enters into the hexosamine synthetic pathway to form UDP-N-acetylglucosamine, which is used as a direct precursor to extend the BF polysaccharide chain. We conducted the draft genome sequencing of IFO 13545 and identified all of the candidate genes corresponding to the enzymes in this pathway in the 5420-kb genome sequence. Disruption of the genes encoding acetyl-CoA synthetase and isocitrate lyase by homologous recombination resulted in little or no growth on acetate, indicating that the cell growth depends on acetate assimilation via the glyoxylate shunt. Disruption of the gene encoding glucosamine 6-phosphate synthase, a key enzyme for the hexosamine synthetic pathway, caused a significant decrease in flocculation activity, demonstrating that this pathway is primarily used for the BF biosynthesis. A gene cluster necessary for the polymerization and secretion of BF, named bfpABCD, was also identified for the first time. In addition, quantitative RT-PCR analysis of several key genes in the expected pathway was conducted to know their expression in acetate assimilation and BF biosynthesis. Based on the data obtained in this study, an overview of the BF synthetic pathway is discussed.

Slurry bioreactor modeling using a dissimilatory arsenate-reducing bacterium for remediation of arsenic-contaminated soil.

A slurry bioreactor using a dissimilatory arsenate (As(V))-reducing bacterium is proposed for remediation of arsenic-contaminated soils. Bacterial As(V) reduction can cause arsenic extraction from the solid to the liquid phase because arsenite, As(III), is much less adsorptive than As(V). A mathematical model was developed incorporating the reversible sorption process of arsenic as well as bacterial growth and decay via As(V) reduction. A linear isotherm equation expressed the sorption process. The model included Haldane kinetics with high As(V) concentrations and cell inactivation by toxicity due to As(III). Extraction experiments used synthetic contaminated soils (forest soil, Soil SF, 1100 mg kg(-1); paddy soil, Soil SP, 1100 mg kg(-1)) and actual contaminated soils (Soil AH 2200 mg kg(-1) and Soil AL, 220 mg kg(-1)) at 5% w/v slurry concentration. Simulation results matched the observed changes of arsenic concentrations in the liquid phase. The respective extraction efficiencies of arsenic were 63%, 41%, 20%, and 55% for SF, SP, AH, and AL soils. Sensitivity analyses showed that the rate-limiting step was the desorption rate of As(V) from the solid to the liquid phase, rather than the As(V)-reducing rate. The proposed model provides a useful framework for understanding and predicting the extraction of arsenic from soil.

Bacillus selenatarsenatis sp. nov., a selenate- and arsenate-reducing bacterium isolated from the effluent drain of a glass-manufacturing plant.

A facultatively anaerobic, selenate- and arsenate-reducing bacterium, designated strain SF-1(T), was isolated from a selenium-contaminated sediment obtained from an effluent drain of a glass-manufacturing plant in Japan. The bacterium stained Gram-positive and was a motile, spore-forming rod capable of respiring with selenate, arsenate and nitrate as terminal electron acceptors. The major cellular fatty acids of the strain were iso-C(15 : 0), iso-C(17 : 1)omega10c and C(16 : 1)omega7c alcohol. The G+C content of the genomic DNA was 42.8 mol%. Though the nearest phylogenetic neighbour was Bacillus jeotgali JCM 10885(T), with a 16S rRNA gene sequence similarity of 99.6 %, DNA-DNA hybridization studies showed only 14 % relatedness between these strains, a level that is clearly below the value recommended to delimit different species. This, together with the phenotypic differences (utilization of electron acceptors, NaCl tolerance), suggests that strain SF-1(T) represents a novel species of the genus Bacillus, for which the name Bacillus selenatarsenatis sp. nov. is proposed. The type strain is SF-1(T) (=JCM 14380(T)=DSM 18680(T)).

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens.

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721(T). The enzyme activity increased around 4-fold by the addition of 0.2-2.0 M NaCl in 0.1 M glycine-NaOH buffer (pH 9.0). KCl, Na2SO4, NaBr, NaNO3, CH3COONa, LiCl, NH4NO3, and NH4Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7-9.4 and 60 degrees C with 0.2 M NaCl, but pH 6.5-7 and 50 degrees C without NaCl; enzyme activity increased approximately 6-fold at 60 degrees C with 0.2 M NaCl compared to that at 50 degrees C without NaCl in 0.1 M glycine-NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe2+ and Hg2+ ions), regardless whether NaCl was present or not.

Laboratory-scale continuous reactor for soluble selenium removal using selenate-reducing bacterium, Bacillus sp. SF-1.

A model continuous flow bioreactor (volume 0.5 L) was constructed for removing toxic soluble selenium (selenate/selenite) of high concentrations using a selenate-reducing bacterium, Bacillus sp. SF-1, which transforms selenate into elemental selenium via selenite for anaerobic respiration. Model wastewater contained 41.8 mg-Se/L selenate and excess lactate as the carbon and energy source; the bioreactor was operated as an anoxic, completely mixed chemostat with cell retention time between 2.2-95.2 h. At short cell retention times selenate was removed by the bioreactor, but accumulation of selenite was observed. At long cell retention times soluble selenium, both selenate and selenite, was successfully reduced into nontoxic elemental selenium. A simple mathematical model is proposed to evaluate Se reduction ability of strain SF-1. First-order kinetic constants for selenate and selenite reduction were estimated to be 2.9 x 10(-11) L/cells/h and 5.5 x 10(-13) L/cells/h, respectively. The yield of the bacterial cells by selenate reduction was estimated to be 2.2 x 10(9) cells/mg-Se.