Microbial Community Analysis And Characterization Of Sulfur-oxidizing Bacteria

Sulfur-oxidizing bacteria are the main participant in sulfur biological oxidation, which pay an important role in the sulfur geochemical cycle and biological treatment of industrial wastewater from tanneries, textile dying and printing factories, paper mills, petrochemical refineries, liquefied petroleum gas plants and so on. The microbial ecology analysis of sulfur-oxidizing bacteria in sulfide-rich wastewater treatment, and isolation of bacteria with high biodegradation ability, and characterization of the sulfur-oxidation for sulfur-oxidizing bacteria are helpful for revealing the microbial community and abundance of sulfur-oxidizing bacteria in the environment and optimizing the wastewater treatment process. In this Ph.D research project, we first constructed a viable cells based microbial ecology method to overcome the limitation that traditional PCR-based molecular techniques cannot discriminate viable cells from dead cells in microbial community analysis, and used this method to study the bacteria, archaea, and sulfur-oxidizing bacteria communities in printing and dyeing wastewater treatment. Then, we enriched and isolated sulfur-oxidizing bacteria by aerobic and anaerobic enrichments. Finally, the physiology, biochemistry, and biological characteristics of a sulfur-oxidizing bacterium with high sulfur-oxidizing ability were studied.Propidium Monoazide （PMA） is a membrane impermeant dye that selectively penetrates into dead cells with compromise membranes, and covalently cross-linked to DNA strain after intercalating into it, which strongly inhibits the PCR amplification of DNA from heat-killed and isopropanol-killed cells. However, PMA treatment can not completely suppress dead cells from PCR amplification when the targeted gene is too short （<200 bp）. Then, a method that PMA treatment in combination with two-step nested PCR was designed to overcome this problem. The results suggested that the PMA-nested PCR-DGGE method could detect only viable cells in microbial ecology analysis, and enhance the detection sensibility, which also useful for analysis of complicated environmental samples.BLAST analysis and RFLP analysis of bacterial and archaeal 16S rRNA gene clone libraries showed that the bacteria groups in printing and dyeing wastewater treatment mainly consisted of Gammaproteobacteria （73 %）, Anaerolineae （6 %）, Bacilli （5 %）, Deltaproteobacteria （7 %）, Clostridia （4 %）, Bacteroidetes （1 %）, Chlorobia （1 %）; Methanomicrobia （99 %） and Thermococci （1 %） were the only two lineages of the archaeal domains. The result also suggested that the bacteria domains have a higher microbial diversity than archaeal domains. Functional genes dsr、sox and sqr based clone libraries suggested that sulfur-oxidizing bacteria related to Alphaproteobacteria, Betaproteobacteria, Chlorobi and Gammaproteobacteria were the main sulfur-oxidizing bacteria groups in printing and dyeing wastewater treatment, which were Halothiobacillaceae （17 %）, Hydrogenophilaceae （14 %）, Rhodocyclaceae （13 %） and some uncultured bacteria （10 %）. The abundance, nutritional diversity, growth characteristics, and ecophysiological flexibility of Halothiobacillaceae, Rhodocyclaceae, and Hydrogenophilaceae families indicate that these bacteria mainly contribute to the sulfide oxidizing in the sulfide-rich wastewater treatment process. The sulfur-oxidizing bacteria community in the environment was steady, which not changed after time.The DGGE analysis suggested that in anaerobic enrichment, the main bacteria groups including Acinetobacter, Paracoccus, Alcaligenes, Rhodopseudomonas, Clostridium and Enterobacteria, and the Rhodopseudomonas was the predominant group, whose abundance increased after enrich time. In aerobic enrichment, the main bacteria groups including Pseudomonas, Halothiobacillus, Ochrobactrum, Paracoccus, Thiobacillus and Alcaligenes, in which, Thiobacillus and Halothiobacillus had a higher bacteria abundance, and mainly contributed to the thiosulfate oxidation. Based on the enrich cultures, six isolates were isolated from the aerobic enrichment and four isolates were obtained from the anaerobic enrichment, respectively. Phylogenetic analysis of 16S rRNA gene of isolates showed that they belong to the genus Acinetobacter, Rhodopseudomonas, Pseudomonas, Halothiobacillus, Ochrobactrum, Paracoccus, Thiobacillus, and Alcaligenes, respectively. The thiosulfate oxidation test showed the Halothiobacillus and Thiobacillus had higher thiosulfate oxidizing ability than others.The identification by 16S rRNA gene suggested that the strain JFA2 was closely related to the genus Halothiobacillus, and in good agreement with Halothiobacillus neapolitanus （99 %）. The cells of strain JFA2 were bacilliform （0.8-1.0×2.0-4.0μm）, Gram negative. The optimal pH, temperature, and rotation speed for bacteria growth were 4.0-6.0, 35℃-40℃, and 180 rpm, respectively. The limit conditions of pH and T for strain JFA2 growth were 3.0-9.0 and 15℃-50℃, respectively. Fe³⁺ is helpful for bacteria growth. The strain JFA2 grown quickly in medium with 0-0.05 mol/L NaCl, and could tolerant of high concentrations of solutes （e.g. 1 mol/L NaCl; 0.6 mol/L sodium thiosulfate）. CO₂ is fixed by strain JFA2 and used as carbon source for chemolithoautotrophic growth; strain JFA2 can use glucose, sucrose, acetate, succinate, and starch as carbon source for chemoheterotrophic growth. In aerobic, ammonium, nitrite, and nitrate were used as nitrogen source. The strain JFA2 was able of using thiosulfate to reduce nitrite anaerobically. In low concentration of thiosulfate, thiosulfate was oxidized as sulfate directly, and in high concentration of thiosulfate, element sulfur was produced as intermediate product in thiosulfate oxidation. Sulfide, element sulfur, sulfite, thiosulfate, rhodanates, SDS, tetrathionate, sulfanilic acid, and methionine were used as unique substrate and energy source for growth. Functional genes soxB、dsrA and sqr were detected in strain JFA2, which are possible for the sulfur-oxidizing.