Characteristics Of Microbial Mercury Methylation In The Soils And Sediments Of Three Gorges Reservoir And Nam Co Lake

Mercury?Hg?has been listed as a global pollutant.Mercury mainly exists in the environment as the form of elemental Hg?Hg?0?,Hg⁰?,divalent Hg(Hg?II?,Hg²⁺)and organic Hg compounds,such as monomethyl Hg?CH₃Hg+,methyl mercury,MeHg?and dimethyl Hg?DMHg?.Methylmercury is the form of most concern,because it can be enlarged biologically in the food chain of aquatic ecosystems,resulting in high concentrations in aquatic products such as fish,which poses a threat to human health.Studies have shown that both Hg?0?and Hg?II?can convert into MeHg through biotic and abiotic processes.However,the current mainstream view is that MeHg is mainly produced by microorganisms,especially a group of anaerobic microorganisms existing in the natural environment.A large number of studies have shown that sulfate reducing bacteria?SRB?,iron reducing bacteria?IRB?,methanogens,syntrophic,acetogenic,and fermentative Firmicutes are principle Hg-methylators.The key gene cluster hgcAB is found to be a reliable predictor of Hg methylation capabilities in microorganisms,which determines whether they have the methylating ability or not.Therefore,the study of Hg-methylators in the environment is of great significance for understanding the formation of MeHg and the risk for human exposure.A large number of studies have found that the sediment of aquatic ecosystem is the main source of MeHg.Therefore,it is very important to study the involving microorganisms in Hg methylation in the sediment of aquatic ecosystems.Therefore,the sediment of two Hg sensitive aquatic ecosystems,water-level-fluctuating-zone?WLFZ?of Three Gorges Reservoir?TGR?and Nam Co Lake of the Tibetian Plateau?TP?,were seleted.Total Hg?THg?and MeHg concentrations,MeHg/THg ratios of the water,soils and sediments,as well as the difference of microbial compositions,abundance and diversities between sediments and soils of the two aquatic systems were analyzed in this research.Methods used for analysis of microbial communities include terminal restriction fragment polymorphism?T-RFLP?,clone library construction,q-PCR,qRT-PCR and 16S rRNA amplifier high-throughput sequencing.We hope to predict the microorganisms associated with Hg methylation in the two typical Hg-sensitive aquatic ecosystems,so as to further understand the mechanisms of Hg methylation by microorganisms.Results showed that MeHg/THg ratios of the inundated soil,semi-inundated soil and non-inundated soil ranged from 0.18%to 2.81%in Shibaozhai?S?,Tujing?T?and Fuling Zhenxi?Z?of the WLFZ of TGR.While for the Nam Co Lake,MeHg/THg ratios of the sediment and bank soil ranged from 1.12%to 2.46%,which was comparable to the Hg methylation abilities of the inundated soil of the WLFZ of TGR.MeHg/THg ratios of the two aquatic systems were far lower than others.When the water levels changed from 155m to 175m,the inundated soil of Shibaozhai?SI?turned into sediment?SSe?.The ratio of MeHg/THg in SSe?2.05%?was significantly higher than that in SI?1.76%?,which confirmed that sediments had higher Hg methylation abilities.Low MeHg/THg ratios also showed that biotic Hg methylation abilities in sediments of the two aquatic systems were both at a relatively low level.The q-PCR analysis of absolute quantities of hgcA?Hg-methylators?,dsrB?SRB?and mcrA?methanogens?genes in the inundated,semi-inundated and non-inundated soil of the WLFZ of TGR showed that MeHg concentrations in the soils were positively correlated with the existing quantities of dsrB,and MeHg levels in soils of the WLFZ of site S and T were positively correlated with the presence of hgcA gene.Moreover,the abundances of dsrB in the soils and sediments of the WLFZ of TGR were significantly higher than that of hgcA?p<0.001?,which might indicate that only a small part of SRB was Hg methylating microorganisms.Phylogenetic analysis of dsrB-containing microorganisms in SI of the TGR showed that the sequences were mainly distributed in Desulfosarcina,Desulfofaba,Desulfonema,Desulfococcus,Desulfotignum,Desulfovibrio,Desulfomicrobium and Desulfotomaculum,as well as some unidentified genera.As for SN,dsrB-containing microorganisms mainly included Desulfomonile and Syntrophobacter under the class of?-proteobacteria,as well as some unidentified genera.The number of genera containing dsrB gene in SN was much smaller than that in SI.In addition,the abundances of dsrB and hgcA in SI were higher than those in SN?p<0.001?.At the same time,microbial diversity of dsrB-containing microorganisms analyzed by T-RFLP showed that the highest diversity was found in SI.It was thus evident that the MeHg/THg ratio,hgcA and dsrB abundances,and microbial diversity were all high in SI.The soil of SI in the dry period became sediments?SSe?when it came to the wet period.Therefore,the WLFZ of Shibaozhai was selected to further analyze the difference of Hg-methylating related microorganisms,as well as dsrB,hgcA and mcrA abundances between SI?dry period?and SSe?wet period?of the TGR.Results showed that the abundances of hgcA and dsrB in SI and SSe were both positively correlated with MeHg concentrations,indicating that microorganisms containing hgcA and dsrB might participate in Hg-methylating reactions in the WLFZ of TGR.The hgcA-containing microorganisms found in SI included bacteria from Geobacteriaceae,?-proteobacteria,and archaea from Methanobacteria,Euryarchaeota.The hgcA-containing microorganisms found in SSe were from Geobacteriaceae,Methanobacterium and Clostridium.This indicated that microorganisms with Hg methylation abilities in the WLFZ of TGR may be bacteria and archaea with weak Hg-methylating abilities in the family of Geobacteriaceae,Methanobacteria and Clostridium under the class of?-proteobacteria.The specific microorganisms that participated in the Hg-metnylating process need to be further studied.Based on the 16S rRNA amplifier high-throughput sequencing of sediments and bank soils of Nam Co Lake by Illumina MiSeq PE300 platform,the differences of abundance,diversity and community composition between sediments and bank soils were analyzed.Results showed that microorganisms from the genus of Proteiniclasticum was probably Hg-methylators in the sediments of Nam Co.The abundance of Proteiniclasticum in the sediment were 4.73%,but was sharply decreased to 0.02%in the bank soil.However,Hg methylation ability of Clostridium is believed to be the weakest on average,and it is not a dominant genus in the sediment of Nam Co.Therefore,the higher concentration of MeHg in fish living in the water of Nam Co may not be highly related to microbial Hg methylation in the sediment.It is thus predicted that biotic or abiotic Hg methylation in the water column or periphyton might be the potential sources of MeHg.Absolute quantitative analysis of the existing abundances of hgcA?Hg-methylators?,dsrB?SRB?,mcrA?methanogens?,pmoA?methanotrophs?,bacterial 16S rRNA?all the bacteria?and archaeal 16S rRNA?all the archaea?genes in the sediments and bank soils by q-PCR showed that the quantity of dsrB gene in the sediments was significantly higher than that in the bank soils.However,qRT-PCR analysis of hgcA?Hg-methylators?and dsrB?SRB?found that both the existing and active abundances of hgcA were lower than that of dsrB.This probably meaned that most SRBs in the sediments of Nam Co may not be Hg-methylating microorganisms and had no Hg methylation effect.The effects of biogeochemical factors,including the concentrations of THg,MeHg,OM,NH₄⁺,SO₄^2-,Fe²⁺,base saturation?BS?,cation exchange capacity?CEC?,MeHg/THg ratios,and pH on microbial community distribution in the sediment and bank soils of Nam Co were analyzed by distance-based redundancy analysis,db-RDP.Results showed that CEC was the most important variable affecting microbial composition in the sediment and bank soil of Nam Co Lake,followed by OM,pH,MeHg,Fe²⁺and THg,while BS and NH₄⁺had the minimum effect on microbial communities.THg concentrations were positively correlated with CEC,OM and pH,while MeHg concentrations were positively correlated with BS and NH₄⁺.MeHg/THg ratios were also positively correlated with MeHg,BS and NH₄⁺concentrations.The composition of microbial community in the sediment of Nam Co in the cold season was positively correlated with the concentration of CEC,OM,pH,SO₄^2-and Fe²⁺,while it was negatively correlated with BS,MeHg/THg ratios,MeHg and NH₄⁺.It was found that the correlation results of the composition of microbial community in the bank soil were quite different from that in the sediments.Comparing the WLFZ of TGR with Nam Co Lake,similar characteristics of the two aquatic ecosystems were:1)the methylation efficiency of Hg in the sediment was lower than other aquatic ecosystems worldwide.2)the probable Hg-methylators found in the sediments of the two aquatic ecosystems distributed in the families and classes with relatively lower Hg-methylating abilities.The differences between the two aquatic ecosystems were as follows:1)The Hg input to the TGR and Nam Co was similar,but the latter had extremely higher MeHg levels in fish,which indicated that Hg methylation efficiency of Nam Co was higher than other aquatic systems.This research proved that the high methylation efficiency did not mainly come from the Hg-methylating capacities of the sediment.Due to the high altitude and strong illumination intensities of the natural anaerobic lake,it was predited that the high MeHg levels in the fish may come from high biotic?microorganisms?and abiotic?photoinduced methylation?methylation efficiency of water column and/or periphyton.2)The probable Hg-methylators in the sediment of Nam Co Lake,TP may come from the genus of Proteiniclasticum and Clostridium of the class of Firmicutes,not?-proteobacteria.While Hg-methylators in the sediment and soil of the WLFZ of TGR may come from strains in the genus of Geobacteriaceae,Methanobacteria and Clostridium under the class of?-proteobacteria with weak capacities.In summary,this study provides basic data for understanding the characteristics of microbial methylation of Hg in two typical Hg-sensitive aquatic ecosystems,the biggest reservoir and plateau lake.Results of this research may provide theoretical basis for understanding the mechanisms of microbial methylation of Hg in the natural environment.