Production And Migration Of Methylmercury In Typical Wetland Ecosystems

Mercury（Hg）is an extremely toxic heavy metal element.Its organic compounds（methylmercury,MeHg）have strong neurotoxicity,lipophilic and high bioaccumulation characteristics.Since the―Minamata disease‖broke out in Japan,the toxicity of MeHg had attracted the worldwide’s attention.MeHg in the environment is produced by the methylation process of inorganic mercury（iHg）,which is an important component of biological cycle process of Hg in ecosystems and is also a prerequisite for mercury entering into living organisms.Since the first observation of microbial methylation of mercury by Jensen in 1969,a great deal of research on microbial methylation has been carried out throughput the world,and a diverse group of microbial guilds,including sulfate reducing bacteria（SRB）,Iron reducing bacteria（FeRB）and methanogens,were proved to be the main methylators in anaerobic environment.In 2013,with the discovery of hgcA/B,a key gene cluster for anaerobic methylators,the investigation of mercury methylation entered the gene level.Therefore,the microbial methylation of Hg has become a focus in the research filed of Hg biogeochemistry.Wetland is an active mercury pool with high Hg accumulation and MeHg yield.It is the most important methylation site in the environment.Generally,natural wetlands include swamps,peatlands,lakes,rivers and marshes,while constructed wetlands include reservoirs,paddy and ponds.Reservoir is conductive to the activation,methylation and bioaccumulation of mercury,which make it become―a typical mercury sensitive ecosystem‖.The Three Gorges Reservoir（TGR）is the largest annual regulation reservoir in the world,seasonally water level fluctuation makes it formed a large water level fluctuation zone（WLFZ）with a total area of 350 km².From October to the next January,all soils below 175 m above the sea level（a.s.l.）are inundated by the river water,owing to the highest water level maintained by the TGR project（175 m）.After January,the water level starts to drop,and all the soil above 145 m a.s.l are exposed to the atmosphere when the water level drops to the lowest in June（145 m）.During the flooding period,a huge amount of organic matter and nutrients from the inundated soil and plant will be released,which can stimulate the growth of Hg methylating microorganisms,resulting an enhanced MeHg production and release.When soils exposed to the atmosphere,soil MeHg decreased gradually,while it showed an increasing trend with the prolongation of the drying time.The exposed WLFZ is not a strictly anaerobic environment because it was exposed to atmosphere for a long time.So,where does soil MeHg in the WLFZ come from?Are there any Hg methylating microbes other than the anaerobic bacteria such as SRB and FeRB?If so,what are their Hg methylation pathways?The unique water level regulation of the TGR project makes it different form other reservoir and aquatic ecosystems.Therefore,it is necessary to investigate the characteristics of Hg methylation by microorganisms in the WLFZ.The experiments of verifying the influence of water level fluctuations on soil mercury were conducted by a field investigation,the results showed that soil MeHg in seasonally drying and flooding areas（DFA）were significantly higher than those in non-inundated（NIA）and inundated areas（IA）,indicating that seasonally water level fluctuations could enhance the production of MeHg.However,the relative MeHg content（the ratio of MeHg to THg,%MeHg）was only 0.08%-0.99%,which was obviously lower than those in other aquatic environments,indicting the Hg methylation degree of the WLFZ was still under a low level.The distribution characteristics of total culturable bacteria（TCB）in soils with different water levels in the WLFZ showed that seasonally water level fluctuations could also enhance soil TCB content,and a significant positive relationship was found between soil TCB and MeHg in the 10-20 cm soil layer,indicating soil bacteria played an important role in Hg methylation in the WLFZ.The soil TCB was analyzed by standard plate count（CFU）method under aerobic condition,which represented the total living bacteria in the soil,thus we speculated that there might be some aerobic microorganisms that play a leading role in Hg methylation in the soil of the WLFZ.There are multiple types of microorganisms in the soils,and most of them are unculturable.However,soil TCB can only reflect the variation characteristics of culturable bacteria in soil.Therefore,454 high-throughput pyrosequencing was employed to investigate the effect of water level fluctuations on the microbial community structure and diversity in the WLFZ of the TGR.The results showed that the number of OTUs and alpha diversity index of bacteria and archaea in DFA were higher than those in NIA and IA,indicating that seasonally water level fluctuations could enhance the microbial diversity and abundance in the soils.In addition,6 families of Deltaproteobacteria,4 families of Firmicutes,and 2 families of Methanomicrobia,which might contain Hg methylators,were detected in the soils of the WLFZ.Among them,the relative abundance of Deltaproteobacteria and Methanomicrobia in DFA were higher than those in NIA and IA,which could explain the relative higher MeHg production in the soils suffered from seasonally water level fluctuations.Overall,those families in Deltaproteobacteria might contain strong Hg methylators but had a low abundance,whereas the families in Methanomicrobia had a high abundance but had a very weak Hg methylation rate.These might be the reason why Hg methylation activity in the soils of the TGR was under a low level.Considering the WLFZ of the TGR is not a strict anaerobic environment,we speculated that there might be other Hg methylating microorganisms in the WLFZ in addition to those anaerobic Hg methylators based on the above field results.Therefore,a screening experiment was carried out based on the soils at the altitude of 165-160 m a.s.l.in Shibaozhai（SBZ）WLFZ,and a total of four bacterial strains with Hg methylation ability under aerobic and/or facultative conditions were isolated.Identification of strains was carried out using both 16S rRNA gene sequencing and physiological and biochemical technology.Strain TGRB1 and TGRB4 are strictly aerobic bacteria,both of which belong to Pseudomonas putida.Strain TGRB2 is a micro-aerobic bacterium,which belongs to Pseudomonas fluorescens.However,since the difference between the results of 16S rRNA gene and physiological and biochemical identification,the taxonomic stats of TGRB5 could not be identified currently.In addition,an aerobic pure culture system was established and strain TGRB4 was chosen as representive to further study the Hg methylation ability of aerobic microorganisms.When the initial Hg²⁺concentration was 300 ng?l^-1,strain TGRB4 can transform1.45%of Hg²⁺into MeHg,with a methylation rate constant（k_m）of（8.73±0.64）×10^-9 pg?cell^-1?h^-1during the exponential growth stage（5-17 h）,which was equal to the well-known SRB strain Desulfobulbus propionicus strain 1pr3.In addition,an incubation experiment was carried out under alternate drying and flooding conditions to further detect the Hg methylation ability of strain TGRB4and its contribution to Hg cycling in the TGR.The results showed that strain TGRB4 had a rapid Hg methylation ability in the first drying stage.Compared to the control treatment（treatment B）,the MeHg content and%MeHg in the soil inoculated with TGRB4（treatment A）were significantly higher,suggesting that strain TGRB4 could promote the production of MeHg.In addition,in the treatment A,MeHg in soil showed a significant positive relationship with the amount of strain TGRB4 in the first drying stage（r²=0.68,p=0.02）,which further demonstrate the key role of strain TGRB4 on Hg methylation in soil.In the flooding stage,soil MeHg showed a decreasing tendency while water MeHg showed an upward trend with the extension of flooding time,indicating that the produced MeHg could be released into the water column.Since strain TGRB4 was isolated from the soil of the WLFZ,it is expected that Hg methylation will occur in the drying stages of TGR.After impoundment,the produced MeHg might enter into the aquatic environment of the TGR,which might increase the exposure risk both for human and ecosystems.Different types of microorganisms have different mechanisms for Hg methylation.Strain TGRB4 is an aerobic bacterium,thus there may be a different Hg-methylation pathway from those anaerobic Hg methylators.In order to reveal the Hg-methylation pathway under aerobic conditions,the draft genome sequence of strain TGRB4 was obtained by the Illumina Hiseq and PacBio sequencing technologies.Strain TGRB4 has a gene length of 6.01 Mb,which contains a total of5,504 genes,79 tRNAs,and 25 rRNAs.The results of protein functional annotation showed that there were two coenzymes related Hg methylation in strain TGRB4 genome,including 5-methyltetrahydrofolic acid（5-Methyl-THF）and S-adenosine methionine（AdoMet）.Combined with the KEGG metabolic pathway annotation,a possible Hg methylation pathway by strain TGRB4 was proposed:the methyl group utilized to methylate mercury by strain TGRB4 is probably transferred from methyl-THF through 5-methyl-THF or AdoMet,with formate or serine as the original methyl donors.This study is of great significance for the further study of aerobic Hg methylation mechanism and the role of aerobic microorganisms in the geochemical cycling of mercury.Recent studies have shown that periphyton is also an important site for Hg methylation in some wetlands,and its Hg methylation potential is even higher than that in sediment.Periphyton is a complex mixture of eukaryotic algae,cyanobacteria,heterotrophic microbes（including bacteria,fungi,protozoa and micrometazoa）,and fine particulate detritus.It’s a unique microenvironment with distinct redox potential gradient from outside to inside,abundant in organic matter,diverse microbial communities（including Hg methylators）,all of which are inductive to Hg methylation and Hg transformation in general.Within periphyton community,extracellular polymeric substances（EPS）and low molecular weight organic acids（e.g.,acetate,butyrate,propionate,lactate,and citrate）excreted from algae provide carbon substrates for bacteria,increasing bacterial physiological activities and enhancing Hg methylation.Florida Everglades is one of the largest freshwater swamps in the world,and environmental problems caused by Hg have become the research focus for a long time in this region.In this ecosystem,periphyton is responsible for over half of the primary production,and is the primary food source for small fish,crayfish,grass shrimp and other small consumers at the base of the food web.Besides,very high Hg methylation potential was found within periphyton,thus it played an extremely important role in Hg cycling.In addition to documenting the increased Hg methylation rates in periphyton,previous studies have made efforts to identify microbes that could methylate Hg within periphyton community,attempting to explain enhanced Hg methylation in periphyton from a biological perspective.However,the unique environmental conditions of periphyton itself are largely ignored.For Everglades,the THg concentration in surface water is within the background,but the relative concentration of MeHg（the ratio of MeHg to THg,%MeHg）is much higher than that of other water bodies.Since the Everglades water itself does not have the Hg methylation ability,where does the water MeHg come from?Based on the widespread distribution and the high Hg methylation potential of periphyton in Everglades,periphyton and water samples at different distance away from periphyton were collected,with the purpose to study the role of periphyton on the distribution of THg and MeHg.The results showed that the closer to periphyton,the higher THg concentration in water,which performed a distribution trend of periphyton pore-water（PPW）>periphyton overlying water（POW）>surface water（SW）.In addition,THg concentrations in tightly-bound EPS（TB-EPS）were higher than those in loosely-bound EPS（LB-EPS）,indicating THg was tightly bound to the bio-membranes of periphyton.These results suggested that periphyton is an important inorganic Hg sink in aquatic ecosystems.The distribution characteristics of water MeHg around periphyton was similar with THg,while%MeHg showed a distribution trend of POW>SW>PPW,indicating MeHg had a higher re-distribution potential.In addition,MeHg concentrations in LB-EPS were significantly higher than those in TB-EPS,indicating MeHg could be easily transferred into the water column.Therefore,we speculated that periphyton acts as an important MeHg source in aquatic ecosystems.Periphyton is a kind of three-dimensional matrix biofilm that supported by its extracellular polymeric substances（EPS）,which contains a large number of functional groups and plays an important role in the transformation and migration of heavy metals.Therefore,LB-EPS and TB-EPS were extracted from periphyton,with the purpose to further investigate the role of periphyton on Hg cycling.When 1.5 ng Hg²⁺was added to 30 ml EPS solution and incubated for 24 h,the reactive Hg（RHg）only accounted for 6%and 16%of THg in LB-EPS and TB-EPS,respectively.However,there was no significant difference between RHg and THg in ultrapure water without EPS addition.Thus we speculated that Hg was combined with EPS.To further verify our speculation,EPS-Hg solutions incubated for 24 h were ultra-filtrated,using an ultrafiltration centrifuge tube with a molecular weight cutoff（MWCO）of 3 kDa.The results showed that the free-Hg in LB-EPS and TB-EPS treatments only accounted for 9%and 8%of the total amount of Hg,respectively,while the majority of Hg was intercepted in the upper layer,which further supported the binding reaction between Hg and EPS.Three dimensional fluorescence spectra（3D-EEMs）analysis showed that both LB-EPS and TB-EPS contained only one fluorescence peak,which was identified as tryptophan-like protein group.Through fluorescence quenching titration test,it was found that EPS could form complex with Hg in a static quenching way,which thus would affect the speciation and migration of Hg.In addition,the results also showed that the tryptophan-like protein group in LB-EPS and TB-EPS could provide 0.65 and 0.54 binding site for Hg²⁺,with the binding constant（lgK）as 3.54 and2.42,respectively.Fourier Transform Infrared Spectroscopy-Attenuated Total Reflection（FTIR-ATR）results showed that the amideⅡand nitro groups were the main functional groups that involved in the binding of Hg²⁺in LB EPS,while aromatic amines and alcohols were the main functional groups that involved in the binding of Hg²⁺in TB-EPS.This research expands an important field for subsequent work on the role of periphyton and EPS in the biogeochemical cycling of mercury in wetlands.