Distribution Of Denitrifying Bacteria And Factors Influencing Community Structure In Sediments Of Yellow River Estuary

Estuary which is strongly influenced by land-ocean interaction,is the most dynamic zone where kinds of biogeochemical processes occur.Nitrogen is the main element of organisms,also one of the key factors affecting the occurrence and evolution of eutrophication in the estuary.The Yellow River Estuary is the confluence of Yellow River,Bohai Sea and Laizhou Bay,and is close to the Shengli Oil Field.The influence of a large amount of sediments,terrestrial pollutants and surrounding anthropogenic activities makes the ecological environment extremely complicated and changeable.In the present,as anthropogenic nitrogen(N)overload input lead to a series of environment deterioration problems,Yellow River Estuary bears great ecological pressures.Denitrification is considered to be the critical process removing reactive nitrogen in estuarine ecosystem.In this study,we collected sediment samples from five different typical sites from the Yellow River Estuary.The clone libraries and quantitative PCR(qPCR)were used to estimate diversity,community structure and abundance of denitrifying bacteria,then combining with physical and chemical factors,to reveal the relationship between environmental factors and denitrifying bacterial communities.The main results are as follows:(1)Using the nirK and nirS genes as molecular markers,clone libraries were established and sequences were determined.Valid sequences obtained for nirK and nirS genes were 370 and 356,respectively.Based on a threshold of 95% similarity of sequences,we obtained 120 OTUs for nirK gene and 126 OTUs for nirS gene.The OTUs number of nirK gene at the sampling sites ranged from 19 to 55,while OTUs number of nirS gene was from 30 to 39.(2)OTU-based ?-diversity results showed that the overall diversity of nirS-type denitrifying bacteria was slightly higher than that of nirK.The ?-diversity of both nirK-and nirS-type denitrifying bacteria was higher in sample SE than other sample(except sample SC for nirK).The OTU-based ?-diversity results showed there were distinct species distribution pattern between nirK-type denitrifying bacteria and nirS-type denitrifying bacteria.That is,sample SE had more species similarity with sample SB for nirK,while sample SA had more species similarity with sample SE for nirS.(3)Phylogenetic analysis of nirK gene showed most OTUs were closely related to the sequences belonged to water column in theYellow River Estuary,San Francisco Bay Estuary sediment,Arable Soil with silver,Activated Sludge,Coastal Microbial Mat,as well as some agriculture soils.All the nirK gene sequences could be grouped into seven clusters(I to VII).The majority of sequences in Cluster I,III,IV,V were dectected at the lower DO sites(SB,SC and SE),whereas most sequences in other clusters were in higher DO sites(SA and SD).NirK gene phylogenetic analysis showed the nirK sequences were matched with that from Changjiang Estuary sediment,East Lake sediment,Haihe River Estuary sediment,Pearl River Estuary sediment,Chesapeake Bay sediment and other environments.All the sequences could be divided into five clusters(I-V).Unlike nirK-type denitrifiers,there was no similar distribution pattern for the spatial structure of denitrifying bacterial communities for nirS gene.(4)The abundance of nirK-and nirS-type denitrifying bacteria in this study was estimated through qPCR.NirK-type denitrifying bacteria abundance at the sampling sites ranged from 4.17×103 to 9.72×105 copies/g sediment(dry weight),and the nirS-type denitrifying bacteria abundance varied from 4.34×104 to 2.62×105 copies/g sediment(dry weight),respectively.One-way ANOVA analysis indicated that nirS-type denitrifying bacteria abundance in sample SA,SC and SD tended to be significantly higher compared with the other samples(P<0.05).While nirK-type denitrifying bacteria showed different distribution patterns,the gene abundance among the samples was significantly different(P<0.001).NirK-type denitrifying bacteria abundance showed higher than that of nirS in sites belonging to the area of tidal flats(SA,SC and SD),whereas nirS-type denitrifying bacteria outnumbered nirK-type denitrifying bacteria in sites located in the river confluence(SB and SE).(5)Spearman analysis showed the abundance and ?-diversity targeting nirS-type denitrifying bacteria were more easily influenced by environmental parameters.CCA based on predominant OTUs and all OTUs showed the nirK-type denitrifying bacteria community structure was more responsive than that of nirS to the environmental factors.This may be due to the fact that nirS-type denitrifying bacteria community structure may be affected by other undetected environmental factors.