Study On The Microbial Community Structure Of A Polluted River And The Screening And Construction Of Indigenous Nitrogen Removal Bacteria Consortium

Microbial communities play an important role in reducing pollutants in polluted rivers.Understanding the response of bacterial communities to the spatio-temporal changes of different environmental factors is of great significance for the monitoring,protection and restoration of the water environment.The Maozhou River is located between modern cities of the Guangdong-Hong Kong-Macao Greater Bay Area,which is a typical black and odorous river with serious pollution in China.The removal of ammonium in black and odorous water is a key indicator of the treatment of black and odorous water.Using the metabolism of nitrogen removal microorganisms to remove ammonium in black and odorous water has the advantages of cost-effective,simple process,and no secondary pollution.Moreover,Heterotrophic nitrification-Aerobic denitrification(HN-AD)bacteria are a type of bacteria with both nitrification and denitrification capabilities,and have attracted widespread attention in the treatment of wastewater nitrogen removal.At present,the research on HN-AD bacteria and its nitrogen removal technology at home and abroad was mainly focused on the laboratory stage,and the reports about its practical application were limited.As the addition of microbial inoculants may have negative impacts on river ecosystems,the isolation of indigenous microbial groups from contaminated rivers is of significant importance,and their application in the degradation of nitrogen pollutants in rivers could have stronger application feasibility and market development prospects.In this study,microbial DNA was extracted from surface water and mud samples collected from Maozhou River,Shenzhen,and the V3-V4 region of the bacterial 16 S r RNA was amplified.Illumina Hi Seq 2500 high-throughput sequencing technology was used to analyze the microbial compositions.Heterotrophic nitrification strains were then isolated from contaminated water and mud samples using a heterotrophic nitrification enrichment medium and a dilution plate method.BTB plates were used torescreen for the aerobic denitrification strains.Species were identified by morphology and 16 S r RNA gene sequences.Then,strains were cultured with ammonium sulfate,sodium nitrite,and potassium nitrate as sole nitrogen sources,to explore the nitrogen removal capabilities of the strains under the conditions of TC/TN(w/w)=7.5,p H=7.0-7.4,30oC?150 r·min-1.Response surface methodology was used to optimize the nitrogen removal conditions of efficient nitrogen removal strains.Finally,the strains were mixed based on their similar or different nitrogen utilization abilities with same proportions,and the nitrogen removal bacteria consortium that performed better was added to the wastewater for preliminary research.The results of high-throughput sequencing analysis showed that the dominant phyla in the water and mud samples of Maozhou River were Proteobacteria(58.9%),Bacteroidetes(8.5%),Actinobacteria(5.5%),and Chloroflexi(5.2%).Alpha diversity analysis showed that the diversity of the microbial community in the mud samples were significantly higher than that of the water samples.Additionally,the water samples at GH was lower than YC in microbial community diversity,while the mud samples showed the opposite pattern.Moreover,various environmental factors were related to microorganisms found in the water samples in varying degrees.Mn and fluoride were the environmental factors that significantly affected the composition and structure of microbial communities.PICRUSt analysis showed that a large number of species was associated with nitrogen metabolism such as Rhodococcus and Acinetobacter existed in the lower reaches of Maozhou River,and a large distribution of the classified species related to nitrogen metabolism was found in the water samples of Gonghe Village.Based on the results of high-throughput data analysis and the high ammonium concentration of the Maozhou River,the nitrogen-removing bacteria in the water samples and surface mud samples of the Maozhou River were screened.Nine heterotrophic nitrifying bacteria isolated from Gonghe Village were identified as Ochrobactrum sp.MZ1,Acinetobacter sp.MZ4,Acinetobacter sp.MZ5,Kerstersia sp.MZ9,Achromobacter denitrificans MZ14,Acinetobacter sp.MZ16,Pseudomonas sp.MZ17,Alcaligenes faecalis A2,and Delftia sp.B7.In the medium provided in thisexperiment,the MZ4,MZ5,MZ16,MZ17,and B7 could use ammonium sulfate,sodium nitrite,and potassium nitrate as the sole nitrogen sources for nitrogen removal.The MZ1,MZ9,and A2 could only use ammonium sulfate as the sole nitrogen source for nitrogen removal.MZ14 could use ammonium sulfate and sodium nitrite as the nitrogen sources for nitrogen removal.Except for A2,other strains had no accumulation of nitrite and nitrate during the stable period of heterotrophic nitrification.MZ4,MZ5,MZ16,MZ17,B7,and MZ14 were identified as HN-AD bacteria,while the HN-AD characteristics of MZ1,MZ9,and A2 need to be further verified.In addition,the optimal nitrogen removal conditions of B7 obtained by response surface method were 30oC,initial p H 7.2,C/N ratio 9.3,and rotation speed155 r·min_-1.Subsequently,the isolated bacteria were mixed in equal proportions to obtain mixed bacteria consortiums of different combinations.Compared with other combinations,F5(strains MZ5,MZ16,MZ17 and B7)with similar nitrogen removal characteristics showed obvious advantages.The ammonium,nitrite and nitrate degradation efficiencies were 92.22%,44.44% and 57.54%,respectively,and the total nitrogen degradation rate was 3.32 mg·L_-1·h_-1.The optimum C/N ratio of F5 for nitrogen removal was 16.And then,The F5 was directly added into wastewater to study its nitrogen removal performance.The bacterial group coexisted well with the indigenous microorganisms in the wastewater,and the concentration of ammonium was significantly reduced from 4.75 mg·L__-1 to 0.46 mg·L_-1,the ammonium degradation efficiency was 90.32%.Furthermore,it still had a good performance after15 days,with a ammonium degradation efficiency of 89.56%.After adding carbon source,total nitrogen and nitrate were significantly removed by 47.23% and 44.17%,respectively.The F5 has strong adaptability,high nitrogen removal efficiency,and good stability.The ammonium concentrations of wastewater and sterilized wastewater had reached the China national surface water quality criteria of category II and category IV after treatment,respectively.