Distribution And Diversity Of Nitrite-dependent Anaerobic Methane-oxidizing Bacteria In Different Habitats

Objective The nitrite-dependent anaerobic methane oxidation(n-damo)is a newly discovered process,which is an important link between the carbon and the nitrogen cycle.In this study,the distribution and diversity of nitrite-dependent anaerobic methane-oxidizing bacteria were studied in the sediments of the West Lake,the Qiantang River and the Jiaojiang Estuary by molecular methods.The results of the present study coud be the microbiology theory basis for eco-environment quality evaluation of the traditional Chinese medicine production.Methods(1)Sampling:sediment samples of the West Lake,the Qiantang River and the Jiaojiang Estuary were collected by grab sampler.Part of the collected samples was stored at 4 °C for subsequent physicochemical analysis,and the remaining samples were stored at-80°C for later molecular analysis.(2)DNA extraction and PCR amplification:DNA was extracted from the sediment samples with a Power Soil DNA Kit(Mo Bio Laboratories,Carlsbad,CA)according to the manufacturer's instructions.The extracted DNA was examined by electrophoresis using a 1.0%agarose gel.The 16S rRNA and pmoA genes of the samples were amplified based on a nested PCR approach.(3)Cloning and sequencing:The PCR products were cloned using the pMD19-T vector(TaKaRa)according to the manufacturer's instructions.Randomly selected clones from each sample were subjected to sequencing(Shenzhen Huada Genomics Institute,Shenzhen,China).(4)Phylogenetic analysis of 16S rRNA and pmoA genes:The phylogenetic analyses of the 16S rRNA and pmoA gene sequences were conducted with MEGA 4.0 software with the BLAST method to search the similar sequence and with the neighbour-joining method to structure phylogenetic tree.(5)Real-time quantitative PCR(qPCR):The primers targeting the 16S rRNA gene were used to quantify the copy number of M.oxyfera-like bacteria.(6)Statistical analyses:DOTUR program was used to generate the number of OTUs of 16S rRNA and pmoA gene sequences of M.oxyfera-like bacteria.DOTUR was also used to generate the Chaol estimator and the Shannon index.The geographical distribution of M.oxyfera-like bacterial communities and their correlations with environmental factors were determined using a principal components analysis(PCA)and a canonical correspondence analysis(CCA),respectively,using the CANOCO software.Pearson correlation analyses were used to test the correlations between M.oxyfera-like bacterial diversity,abundance and different environmental factors using the SPSS 18.0 software(SPSS,Chicago,Illinois,USA).Results The 16S rRNA and pmoA gene sequences of M.oxyfera-like bacteria were detected in all the collected sediment samples using nested PCR approach.Phylogenetic analysis showed that the recovered M.oxyfera-like 16S rRNA gene sequences from the West Lake could be grouped into two distinct clusters that exhibited 93-97%identity to the M.oxyfera 16S rRNA gene;and the recovered pmoA gene sequences could be grouped into five distinct clusters that exhibited 86-95%identity to the M.Oxyfera pmoA gene.The recovered M.oxyfera-like 16S rRNA gene sequences from Qiantang River could be grouped into 15 distinct clusters that exhibited 90-99%identity to the M.oxyfera 16S rRNA gene,and the pmoA gene sequences could be grouped into 13 distinct clusters that exhibited 86-95%identity to the M.Oxyfera pmoA gene.The recovered M.oxyfera-like 16S rRNA gene sequences from the Jiaojiang Estuary could be grouped into three distinct clusters that exhibited 92-97%identity to the M.oxyfera 16S rRNA gene,and the pmoA gene sequences could be grouped into 16 distinct clusters that exhibited 85-95%identity to the M.Oxyfera pmoA gene.Real-time quantitative PCR showed that the abundance of the M.oxyfera-like bacteria from the sediment of the West Lake was 2.15×105 copies g(dry weight)-1.The abundance of the M.oxyfera-like bacteria in the Qiantang River and the Jiaojiang Estuary varied from 1.32×106 to 1.03×107 copies g(dry weight)-1 and 5.80×104 to 8.35×107 copies g(dry weight)-1,respectively.Correlation analyses demonstrated that the total inorganic nitrogen content,the ammonium content and the organic content of the sediment were important factors affecting the distribution of the M.oxyfera-like bacterial communities in the Qiantang River sediments;the organic content of the sediment was important factor affecting the distribution of the M.oxyfera-like bacterial communities in the Jiaojiang Estuary.Conclusions In this study,both the phylogenetic analysis and real-time quantitative PCR confirmed the presence of diverse n-damo bacteria related to M.oxyfera in the sediment of the West Lake,the Qiantang River and the Jiaojiang Estuary,greatly expanding the knowledge of the biogeography of M.Oxyfera.This study expanded new content for the carbon and the nitrogen cycles of aquatic systems.The phylogenetic analysis of the recovered 16S rRNA and pmoA genes showed a much higher diversity of the M.oxyfera-like bacteria in the Qiantang River and the Jiaojiang Estuary than in other freshwater habitats reported to date.Correlation analysis revealed that the total inorganic nitrogen content,the ammonium content and the organic content of the sediment were important factors affecting the distribution of the M.oxyfera-like bacterial communities in the Qiantang River sediments;the organic content of the sediment was important factors affecting the distribution of the M.oxyfera-like bacterial communities in the Jiaojiang Estuary.The results of the present study could be the microbiology theory basis for eco-environment quality evaluation of the traditional Chinese medicine production.