| Bacterial degradation of thiosulfate is always confronted by inhibitory effects caused by acetic acid during the treatment of polysulfide wastewater. Treatment efficiency would be significantly improved if acetate is removed prior to thiosulfate degradation by autotrophic Acidithiobacullus bacteria. This also leads to shorter HRT and thus lower operational cost. The objective of the present study was to isolate and characterize a halophilic acetate-degrading bacterium and explore the feasibility of removing acetate from polysulfide wastewater by using immobilized cells.A moderately halophilic acetate-degrading bacterium YT2 was successfully isolated from an oil contaminated paddy soil by using polysulfide wastewater as the enrichment medium. Strain YT2 formed white, round colony with smooth and moist surface on acetic acid medium. It was a strictly aerobic, Gram-negative, rod-shaped bacterium. Cells were motile and non-sporulating, measuring 2.08 μm×0.62μm. Strain YT2 showed positive reaction to catalase test but negative reactions to following biochemical tests:sugar fermentation, V.P. test, indole test, NH3 and H2S production, starch hydrolysis, gelatin liquefaction and litmus milk fermentation. It was preliminarily identified as Halomonas sp. according to its physiological, biochemical properties and similarity analysis of 16S rDNA gene sequence.Optimal conditions for bacterial growth and acetate degradation by strain YT2 in acetic acid medium were as follows:35℃, pH 9.0, liquid volume 50-200 mL/500 mL, inoculum size 5%-10%(v/v), respectively. Under these conditions 2 g/L of acetic acid could be degraded completely within 16 h. Strain YT2 could tolerate 75 g/L of NaCl or 150 g/L of Na2S2O3. The highest degradation efficiency was observed in the medium containing 30 g/L of NaCl and 60-90 g/L of Na2S2O3. Beyong this range bacterial growth and acetate degradation were both reduced. Results from the present study showed that strain YT2 grew well and could degrade acetic acid effectively present in raw polysulfide wastewater.However,removing acetic acid from polysulfide wastewater by using free YT2 cells will be challenged by low biomass and extreme difficulties in solid-liquid separation. Therefore the second purpose of the present study was to explore the feasibility of removing acetic acid by using immobilized YT2 cells. Freshly prepared YT2 cells were immobilized into agar gel by entrapment method. The conditions for acetate degradation by immobilized YT2 cells were optimized as follows:40℃～45℃,pH 7.0-9.0. Under these conditions 2 g/L of acetic acid could be degraded completely within 6 h. Degradation efficiency of immobilized YT2 cells could be improved to some extent by reducing particle size or increasing cell density in the agar beads with optimal bead size of 3 mm and cell density 5 g(wet weight)/200. Degradation efficiency was also influenced by the quantity of agar beads containing YT2 cells and satisfactory degradation rate could be obtained when ≥100 g/L agars beads were used in the synthetic wastewater. Immobilized YT2 cells in the form of agar beads were successfully applied in a continuous process designed for removing acetic acid from both synthetic and raw polysulfide wastewater. During the 2-month operation period acetate degradation rate stabilized at 97.6%, resulting in a low acetate discharge of≤50 mg/L。After acetic acid was removed by immobilized YT2 cells, thiosulfate could be degraded completely by Halothiobaillus sp.TX within 2 days whereas 6 days were required for complete thiosulfate degradation in the presence of acetic acid, confirming the feasibility of enhancing autotrophic thiosulfate degradation through removing acetic acid from polysulfide wastewater by using immobilized YT2 cells.Mechanisms and adaptations of strain YT2 to other salts had also been investigated in the present study. In addition to its tolerance to NaCl,strain YT2 could also tolerate 1 mol/L of KC1,1 mol/L of NaNO3,1 mol/L of Na2SO4 or 0.75 mol/L Na2S2O3, but could not tolerate a high concentrations of NaNO2, Na2SO3, NH4Cl or (NH4)2SO4 present in acetic acid medium. On Luria Broth salt tolerance of strain YT2 was increased to 2 mol/L of NaCl/KCl or 1.5 mol/L of NaNO3/Na2SO4/Na2S2O3. Further study showed that salt tolerance of this bacterium could be significantly improved by carbon source added in the liquid medium. For example, Halotolerance could be increased to 2 mol/L of NaCl in the medium with peptone or yeast extract as sole carbon source. Halotolerance of strain YT2 was also increased but to a lesser extent, namely 1.5 mol/L of NaCl when the following compounds were used as the sole carbon source:proline, aspartic acid, asparagine, glutamate and glutamine, glucose, fructose and maltose, valeric acid, citric acid and lactic acid, glycerol. In spite of better salt tolerance concentration of cellular protein at the end of incubation was significantly lower than that in the medium containing 1 mol/L of NaCl. Therefore there probably existed unknown compounds in peptone or yeast extract that help to enhance strain YT2’s salt tolerance.In order to elucidate the mechanisms on the bacterial adaptation to high salinity, compatible solutes were extracted and analyzed. The results from the present study showed that both betaine and ectoine existed in YT2 cells. The content of betaine remained almost unchanged when NaCl was added different concentrations in acetate medium. Meanwhile the content of ectoine showed an increase with the increase in the concentration of NaCl present in the liquid medium, indicating its correlation with the bacterial salt tolerance. In addition chemical analysis on Na+/K+content in YT2 cells was also made in response to different concentration of NaCl in the liquid medium. The results showed that the content of intracellular Na+ remained unchanged while the content of intracellular K+ exhibited linear increases as exposed to the increased NaCl concentrations, indicating its essential role in the bacterial salt tolerance. Therefore K+ uptake and ectoine synthesis constitute the major mechanism responsible for strain YT2’s survival and active growth in the hypersaline environments. |
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