Study Of Microbial Community Structure Involved In Methane And Nitrogen Cycles And Relationship Between Microbes And Environment Along Sediment Core From Qi'ao Island Of Pearl River Estuary

Qi'ao Island,located in the Lingdingyang of Pearl River,is the first outlet of Pearl River freshwater flow to the South China Sea.The water surrounding Qi'ao Island connects the river and open sea.The sediment here are rich in organic matters,which provide abundant carbon and nitrogen sources for microbes.The sediment cores were investigated by molecular ecology approaches and methanogen were enriched.The diversity of microbes which involved in methane,nitrogen and sulfate metabolism and the relationship between microbes and environment were analyzed.The community structure and depth distribution of microorganisms which involved in methane cycle in Pearl River Estuary sediment were analyzed by methanogen 16SrRNA clone library constructing,sequencing and T-RFLP approaches.The library was dominated by sequences related to Methanosarcinales/ANME groups(42%) followed by Methanomicrobiales(32%),Methanosaeta(19%).The analysis of methanogen 16S rRNA and mcrA clone libries implied that ANME-2a was responsible for methane oxidation in the non-seep Pearl River estuarine sediment and the sediment was dominated by the CO₂/H₂ utilizing methanogens.T-RFLP results reflected that there were clear changes in populations with depth.In top layers,CO₂/H₂ utilizing Methanomicrobiales group was predominant;in middle layers,the acetate utilizing Methanosaeta group was predominant,in bottom layers,the Methanosarcinales/ANME group was predominant.Based on dsrAB genes,we investigated the composition of sulfate-reducing microorganisms in Pearl River Estuary.Desulfosarcina,Desulfococcus and Desulfobulbus sequences that associated with ANME were not detected in this survey.We suggested that AOM was conducted by ANME associating with some unknown sulfate-reducing bacteria (SRB).Syntrophaceae and Desulfobacteraceae were only detected in SMTZ,occuping by 16.3%and 26.5%,respectivrly.They occupied 42.8%of total colones.It is likely that deltaproteobacterial species play an important part in anearbobic oxidization of methane and mineralization of organic matters in the Pear River Estuary. We used Q-PCR to quantify the abundance of the mcrA and dsrA genes.The resulting depth profiles were correlated with those of porewater sulfate and methane.Using amoA genes as a functional marker,we investigated the abundance and composition of ammonia-oxidizing organisms in Pearl River Estuary by using quantitative polymerase chain reaction,cloning and sequencing approaches.The quantification results showed that ammonia-oxidizing archaea were more abundant than ammonia-oxidizing bacteria,which may represent an evidence for numerical dominace of AOA in estuarine sediments,as well.In site7,bacteriai amoA library was dominated by Nitrosomonas-like sequences types.AOA diversity analysis of the top and bottom layers showed significant variation in community composition.The sequences from top layer(0-4 cm) fell into two groups:52.2%of clones fell into the "water column/sediment"cluster and 47.8%of clones fell into the "soil/sediment" cluster while most of sequences(93.3%) from bottom layer(16-30 cm) mainly fell into "soil/sediment" group and only two clones(6.7%) fell into "water column/sediment" group.The high sequence similarity to estuaries indicated that similar AOA communities might exist in similar estuaries environment,despite the geographical distance.The numerical dominace of amoA in anaerobic zone implied that some specific roles these archaea took part in.The diversity of microbes which involved in methane,ammonia and sulfate metabolism showed that these sequences were closely related to environmental clones from rich-particles,anaerobic environments and clones involved in mineralization of organic matters.These results reflected that zhujiang estuary was likely polluted in aspect of microbial ecology.We also tried to enrich methanogen to look into the relationship between microbes and Pearl River Estuary environment.In brief,the molecular ecology investigation and methanogen enrichment from the sediment samples of Qi'ao Island(Pearl River Estuary),southern China,uncovered the microbes' important roles in C,N,S cycles in this area from the biological aspect. These study filled up the deficiency on Pearl River Estuary researches,which were only limited to geochemisty.It is also a supplement to the knowledge of the composition and abundance of microbes what involve in methangenesis and methane oxidation in non-seep environment and ammonia oxidation in estuarine.