Isolation And Identification Of Two Novel Species Capable Of Nitrate Reduction And Preliminary Study On Their Capability Of Dissimilatory Nitrate Reduction To Ammonium

With industrial wastewater,domestic sewage and nitrogen fertilizer effluent discharged into surface water,groundwater and seawater,the problem of nitrate contamination is becoming more and more serious.The dissimilatory nitrate reduction to ammonium(DNRA)has been paid more and more attention by researchers,which may as an important way of nitrogen cycling.In recent years,it has been found that dissimilatory nitrate reduction to ammonium(DNRA)pathway has an important effect on nitrate removal.The DNRA process in strict anaerobic environment is different from that in the reality of the environmental conditions of water and sediment,which may be contacted with air.This article takes the exploration of DNRA function as the core,carries on the exploratory study of Isolation and identification of aerobic novel bacteria-Exploration of influence factors of DNRA-Analysis of functional genes of DNRA.Firstly,two strains of new bacteria were isolated from the sediment of salt lake.The results based on characteristics of morphological,physiological,biochemical,cytochemical and molecular biology showed that the two strains were novel species.Then,under the condition of shaking culture,the influence of different environmental factors on the DNRA was explored by spectrophotometry.Finally,through gene cloning and sequencing,the dissimilatory nitrate reductase genes of functional strains were obtained,and bioinformatics analysis was used to provide theoretical basis for DNRA function.The main research contents and conclusions are as follows:1.Isolated and purified a DNRA of Gram-negative aerobic bacterium GSS12 from the sample of Shanxi Yuncheng Salt Lake sediment.GSS12 can be classified as Roseovarius genus by multiphase taxonomy,but not any species known to the genus,so it is determined as a novel species of the genus Roseovarius and named Roseovarius lacus sp.nov.GSS12~T.2.Another Gram-negative and anaerobic bacterium GSS14 was isolated from the samples of Shanxi Yuncheng Salt Lake sediment.The results of multiphase taxonomic identification showed that the strain GSS14 had capable of Nitrate Reduction,and belonged to a novel species of the genus Nitratireductor,which was named Nitratireductor lacus sp.nov.GSS14~T due to separation source and strain characteristics.3.The results of DNRA showed that:(1)Dissolved oxygen promotes the DNRA process when GSS12 with nitrate or nitrite as substrate.(2)The nitrite inhibits the DNRA process when nitrate is the substrate.(3)The nitrate inhibits the GSS12 DNRA process when nitrite is the substrate.(4)The ammonium nitrogen inhibits DNRA process when GSS12 with nitrate or nitrite as substrate,which was performed by different environmental factors such as dissolved oxygen,nitrate,nitrite and ammonium.4.The gene cloning technique was used to detect the dissimilatory nitrate reductase gene,and the NirS gene was obtained.The results of Basic Local Alignment Search Tool of nucleotide blast and protein blast showed that sequence of NirS of GSS12 had the highest similarity of more than 95%with NirS13,R-64 and ST3-nirS40,which indicated that NirS might be the gene response for the dissimilatory nitrate reduction in GSS12.