Study On The Community Structure Of Bathyarchaeota And Methanogen In Shenzhen River-bay

Bathyarchaeota is a kind of uncultivated microorganism with high population diversity,abundant biomass and wide distribution.Bathyarchaeota has discovered up to 25 subgroups with diverse distinct physiological and biochemical functions,which can degrade organic matter such as proteins,polymeric carbohydrates,fatty acids,aromatics and methyl compounds,participate in methane metabolism cycle,produce acetic acid,and dissimilate reduction of nitrites and sulfates,indicating that they are one of the most important drivers in carbon cycling on the Earth.Recently,The new research found that Bathyarchaeota may be a potential new type of methanogenic archaea.In the previous investigation,a certain number of methanogens were found in the surface sediments of Shenzhen river-bay,and the methanogens has an inestimable contribution to global methane flux and carbon cycle.Therefore,studying the community structure of Bathyarchaeota and methanogens and their relationship with environmental factors have important ecological significance for protection in Shenzhen river-bay.This research is to study the abundance,diversity and distribution of Bathyarchaeota and methanogens,and the relationship between their community structure and environmental factors in Shenzhen river-bay.For these purposes,the surface sediments of Shenzhen Bay and Dasha River were collected as experimental samples.DNA was extracted from the sediment samples.The archaeal 16 S rRNA and mcrA genes were amplified from the extracted DNA by specific primers and subsequently sequenced by high-throughput sequencing.Community structure information(e.g.abundance and diversity)was obtained by analyzing sequences of 16 S rRNA and mcrA genes.Together with the environmental parameters(pH,salinity,ammonia concentration,nitrate concentration,nitrite concentration,total nitrogen,total organic carbon,total carbon and inorganic carbon)detected in Shenzhen river-bay,community structure was further analyzed by statistical methods,including Alpha diversity(OUT diversity and Shannon diversity),redundancy analysis(RDA analysis),Pearson correlation analysis,multivariate regression analysis,co-occurrence network analysis.In addition,in order to investigate the exact abundance of Bathyarchaeota and methanogens in Shenzhen river-bay,q-PCR of bathyarchaeal 16 S rRNA and mcrA genes was carried out.The relationships of microbial communities and environmental factors were also analyzed by statistical methods.The results of this study are as follows:1)In the surface sediments of Shenzhen river-bay,10 different archaea phyla were detected,Bathyarchaeota and Euryarchaeota were the dominant ones.We found that Bathyarchaeota are more abundant in the marine environment of Shenzhen Bay than Euryarchaeota,while the abundance of Euryarchaeota is higher in the freshwater of Dasha River.Statistical analysis revealed that the relative abundances of Bathyarchaeota and Euryarchaeota are completely opposite to the environmental factors,suggesting that the preferences of environmental factors are very different.Co-occurrence network analysis also revealed a negative correlation between the coexistence of Bathyarchaeota and Euryarchaeota,indicating that Bathyarchaeota and Euryarchaeota have different niches.2)In the surface sediments of Shenzhen river-bay,a total of up to 20 Bathyarchaeota subgroups were detected.Compared to the 25 subgroups of Bathyarchaeota that have been identified,almost all of the major Bathyarchaeota subgroups are covered in the surface sediments of Shenzhen river-bay.This result indicates that Bathyarchaeota are diverse in Shenzhen river-bay.Among them,Bathy-6,-8,-15 and-17 are the dominant subgroups of Bathyarchaeota in Shenzhen Bay.Bathy-8,which is the indicator of saline environment,is the most abundant subgroup in the seawater environment.In addition,Bathy-6,-15 and-17 are also salt-tolerant.3)Based on the redundancy analysis of the community structure of Bathyarchaeota and Pearson analysis of the relationship between absolute abundance of 16 S rRNA gene of Bathyarchaeota and environmental factors,it was revealed that salinity was positively correlated with the relative abundance(R=0.861,P<0.01)and the absolute abundance(R=0.832,P<0.01)of Bathyarchaeota,while ammonia concentration was negatively correlated with relative abundance(R=-0.821,P<0.01)and absolute abundance of Bathyarchaeota(R=-0.696,P<0.01).Thus salinity and ammonia concentration are the main factors affecting the community structure of Bathyarchaeota.It is worth noting that ammonia concentration may be a physicochemical factor inhibiting the survival of Bathyarchaeota,which could contribute to the further work of enrichment and isolation of Bathyarchaeota.In addition,pH and nitrate concentration are also important factors affecting the community structure of Bathyarchaeota.4)This study revealed that subgroup Bathy-6 has a unique environmental preference and distinct niche distribution.Bathy-6 was positively correlated with ammonia concentration(R=0.726,P<0.01)and nitrate concentration(R=0.849,P<0.01),and negatively correlated with salinity(R=-0.896,P<0.01).The significant correlation between Bathy-6 and environmental factors was different from that of other subgroups of Bathyarchaeota,suggesting that Bathy-6 has a unique environmental preferences and distinct niche distribution.Besides,the analysis of the coexistence network between Bathyarchaeota and the other archaea groups further proved the specialty of Bathy-6.5)1 phylum of methanogen from non-Euryarchaeota,6 orders of methanogens and 1 anaerobic methane oxidizing order from Euryarchaeota are detected in the surface sediments of Shenzhen river-bay.They are Verstraetearchaeota,Methanosarcinales,Methanomicrobiales,Methanobacteriales,Methanocellales,Methanofastidiosa,Methanomassiliicoccales and ANME-1,respectively.Among them,Methanosarcinales,Methanomicrobiales and Methanobacteriales are the dominant methanogenic archaea groups in Shenzhen river-bay.6)According to the quantification of methanogens,the average abundance of methanogens in Dasha River is significantly higher than that in Shenzhen Bay.Meanwhile,Pearson correlation analysis of absolute abundance of mcrA gene and environmental factors also reveals the positive correlation between methanogens and total organic carbon(TOC)/ total carbon(TC)(R = 0.733,P < 0.01).It was revealed that TOC was the most important factor effecting the abundance and distribution of methanogens.