Co-removal Of Nitrogen And Sulfur By NR-SOB From Black-Odors Creek Sediment

Contaminated with nitrogenous and sulfur compounds,the creek sediment has been facing the circumstances of black-odors phenomenon.Researches have found that,the creek sediment is widely distributed with nitrate-reducing,sulfur-oxidizing bacteria?NR-SOB?with nitrate-reducing faction including DNRA,denitrification or nitrate reduction to nitrite.Specifically,sulfur-oxidizing denitrifying bacteria could reduce nitrate to nitrogen gas through denitrification and simultaneously oxidize toxic sulfide to elemental sulfur or sulfate.Furthermore,the production of sulfate would stimulate the multiplication of sulfate reducing bacteria which promote the removal of organics by oxidizing to CO2.As a result,the carbon nitrogen and sulfur could simultaneously remove from the creek sediment.There is a great amount of nitrogen and sulfur contaminant in black-odors creek sediment of Pearl River Delta,however,the NR-SOB species and its metabolism character in this kind of sediment is seldom understand.In this research,we have carried on the research of NR-SOB from the creek sediment which was contaminated by persistent organic pollutants?POPs?to determine the property of NR-SOB in synchronous removal of carbon nitrogen and sulfur.The cultures were controlled to be chemolithoautotrophic and anaerobic conditions by adding sodium thiosulfate as electron donor,nitrate as electron acceptor in sealed bottles.A microbial community?namely F1?was obtained which exhibited strong nitrate reducing sulfur oxidizing ability.Some factor tests,including p H and the presence of Fe²⁺,Mg²⁺ and NH4+ were conducted for the influence on the activities of the microbial community.Through enrichment,another three various communities?namely F2,F3,and F4?were obtained from different generations which exhibited different activities.Microbial community structures were analyzed by using high-throughput sequencing which demonstrate that the F1 community was significantly different from other three communities.Ultimately,the isolation and purification of cultivable microorganisms were carried out to determine the activity characteristic of pure culture.?1?NR-SOB microbial community activity experiment results showed that the concentration of nitrate decreased coupling to the consumption of thiosulfate in the F1 community,due to the reduction of nitrate to N2 and the oxidation of thiosulfate to sulfate.Nitrite and elemental sulfur were slightly accumulated through the process of nitrate reducing sulfur oxidizing demonstrating that the F1 community was positive in co-removal of nitrogen and sulfur.The F1 community exhibited the strongest nitrate reducing sulfur oxidizing activity in pH 7.5 with less nitrite accumulation.Meanwhile,the F1 community could tolerant a low pH of 5.5 at which the nitrate reducing sulfur oxidizing activity was yet carried on.Both Fe²⁺ and Mg²⁺ had significant impact on community activity whereas NH4+ had little effect on it.Mg²⁺ was a necessity to the strong nitrite reducing activity of the F1 community.The presence of Fe²⁺ decreased the activity of the F1 community and produced more nitrite in the meanwhile.Whether there was NH4+ in the medium or not,the nitrate reducing sulfur oxidizing property of F1 community was by sulfur oxidizing denitrification,demonstrating that the F1 community was greatly different from those NR-SOB carried on DNRA from other sediment.?2?High-throughput sequencing consequences of the F1 community have revealed that Thiobacillus,an obligate chemolithoautotrophic anaerobic typical NR-SOB,was dominant in F1 community with a high abundance?38.62±2.25%?,which meant that Thiobacillus dominated the faction of communities.Heterotrophic genus Alicyclobacillaceae?8%?presented in F1 might consume the organics which presumably released the inhibition of organics to Thiobacillus.OTU34034 was classified to be Gammaproteobacteria with an abundance of 10.8%.Hence,most NR-SOB in black-odors creek sediment was unclassified microorganism.Correlation analysis between nitrate reducing sulfur oxidizing index and microbial community construction revealed that,on one hand,OTU34034 belonged to Gammaproteobacteria,OTU44535 and OTU14246 belonged to Betaproteobacteria and Alicyclobacillus contributed to the strong activity of F1 community with less nitrite and elemental sulfur accumulation.On the other hand,the increase of Thiobacillus genus and Rhizobiaceae family would decrease the activity of F1 community.?3?129 pure NR-SOB strains were obtained by isolation and identified to be Agrobacterium,Castellaniella,Desulfovibrio,Rhizobium and Thiobillus by using 16 S rRNA gene analysis.Oninly Rhizobium was isolated in F1 commuty whereas others NR-SOB and Rhizobium were isolated in enriched commuty meaning that the enrichment process improved the culturability of NR-SOB in black-odors creek sediment.All five strains exhibited nitrate reducing sulfur oxidizing faction with Thiobillus possessing the highest activity.Agrobacterium,Rhizobium,Desulfovibrio and Thiobillus had more nitrite accumulation which consistent to High-throughput sequencing consequences.The kinds of strains of Agrobacterium and Rhizobium with factions of nitrate reducing sulfur oxidizing were seldom reported.Hence,the two might be novel species which deserved future comprehensive study.The research research would provide scientific theoretical guidance to exert the faction of NR-SOB and accelerate the remediation of black-odors creek sediment.