Diversity Of Microorganisms In Jiulong River Estuary And Characteristics Of Nitrogen Removal Of A Strain Of Heterotrophic Nitrifying Bacteria

Nitrogen cycle promoted by microorganisms is one of the most important global biogeochemical cycles.In recent years,studies on microbial nitrogen metabolism are booming,including nitrogen transformation pathways,nitrogen metabolism mechanisms,nitrogen removal microorganisms and biological nitrogen removal process.Jiulong River estuaries,with rich microbial resource due to the mixture of freshwater and seawater,are major conduits for the transport of anthropogenically derived nitrogen from land to sea.However,the pollution caused by human activities,especially nitrogen pollution,has made eutrophication more serious and endangered the ecological environment of estuary.In this study,the spatiotemporal variation in the distribution and abundance of microbes as well as the response to anthropogenic environmental and the microbial community of denitrifying bacteria were analyzed.The characteristics of nitrogen removal of a strain of heterotrophic nitrifying bacteriawere studied,which could enhance our understandings of nitrogen removal microorganisms and nitrogen transformation pathways.Seven sampling sites at south and north tributaries around Zini Town were selected in Jiulong River estuary,which has experienced intensive human perturbation in recent years.The changes in the diversity and structure of microbial community were analyzed by 16 S rRNA gene clone libraries while the environmental factors were determined both in rainy and dry seasons.The study demonstrated that the microbial community in Jiulong River estuary displayed a distinct spatiotemporal pattern in response to human disturbance such as overuse of fertilizers,discharge of waste of livestock and domestic sewage.For understanding the diversity of denitrifying bacteria,sediment sample of one sampling sites at the downstream of the estuary was selected and nirS gene clone library was constructed for analyzing the phylogenetic diversity of nir S-type denitrifying bacteria,which domenstrated that there were still many unknown denitrifying bacteria in the area worth further excavation.So strains were isolated and screened from the sediment,including 18 strains of autotrophic nitrifiers,16 strains of heterotrophic nitrifying bacteria and 8 strains of denitrifying bacteria.Two strains of purple bacteria isolated by our laboratory in the past,namely Marichromatium gracile YL28 and Rhodopseudomonas palustris CQV97,were selected as control strains to test their removal ability of inorganic nitrogen.The results showed that two control strains had stronger ability to remove inorganic nitrogen with high concentration,which had the value for further study,while the strains isolated from the sediment had a certain nitrogen removal ability,but not so prominent.So we selected Marichromatium gracile YL28 for further study.We investigated the removal and transformation of ammonium,nitrate,and nitrite by Marichromatium gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium,sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen(DO)levels.In the sodium acetate-ammonium system under aerobic conditions,denitrification was suppressed by high concentration of oxygen,so we detected a continuous accumulation of nitrate and nitrite.However,under semi-anaerobic conditions,we observed a temporary accumulation of nitrate and nitrite because of the dynamic balance of nitrification and denitrification.Interestingly,under anaerobic conditions,nitrate and nitrite might be immediately transformed by denitrification,which resulted in the absence of nitrate and nitrite,but an increase in nitrous oxide production.In the sodium acetate-nitrite system,nitrite levels declined slightly under aerobic conditions,and nitrite was completely removed under semi-anaerobic and anaerobic conditions.Taken together,the data indicated that Marichromatium gracile YL28 performed simultaneous heterotrophic nitrification and denitrification.We further studied the nitrogen tolerance and removal ability of the growing cells of Marichromatium gracile YL28 and its resting cells and found in the light,the growing cells have strong tolerance and removal ability of inorganic nitrogen in semi-anaerobic and anaerobic conditions,and the resting cells can still remove nitrate and nitrite in the dark.Besides,we studied the growth pattern of the strain using nitrite as the sole nitrogen source and found that it could grow through nitrogen fixation in the light as well as grow using nitrite as the sole nitrogen source both in the light and in the dark.In summary,in this study we analyzed the spatiotemporal patterns of the bacterial community in Jiulong River estuary in response to human disturbance and showed the diversity of nirS-type denitrifying bacteria,as well as studied a strain of Marichromatium gracile YL28 for its heterotrophic nitrification,denitrification,nitrogen fixation and assimilatory nitrate reduction using nitrite as the sole nitrogen source.Our study is the first to demonstrate that purple sulfur bacteria perform denitrification and anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization under anaerobic condition.