Microbial Mechanisms Of Arsenic Methylation,Demethylation And Volatilization In Paddy Soils

Arsenic(As)is a toxic metalloid.In recent decades,arsenic contamination is widespread in the environment,especially in some paddy soils in southeast China.Arsenic contamination in paddy field can reduce rice yield and increase arsenic concentration in rice grains,leading to serious problems in agricultural production and food safety.Dominant arsenic species in paddy soils include inorganic arsenic(iAs,including arsenite(As(?))and arsenate As(V)),methylated arsenic(monomethyl-arsenic(MMAs),dimethyl-arsenic(DMAs)and trimethyl-arsenic(TMAs))and volatile arsenic(Trimethylarsine(TMAs(?)),dimethylarsine(DMAs(?)H),nonomethylarsine(MMAs(?)H2)and arsenie(AsH3)).When paddy field is flooded,As(?)concentration in porewater increases,leading to increase As concentration in rice grains,and eventually posing a potential threat to human health.More seriously,some studies show that DMAs can cause the outbreak of rice straight-head disease in areas without As contamination.The major arsenic species in rice grains are As(?)and DMAs,which can be taken up by rice from paddy soils directly.Although there are many studies on the mobility and methylation of arsenic in the environment the microbial guilds involved in arsenic methylation and volatilization in paddy soils remain largely unknown.The first part of this thesis investigated the mobilization of arsenic in different paddy soils with the aim of idcntifying a simple extraction method to predict arsenic mobility in paddy soil.We then investigated microbial guilds that mediate the production and disappearance of DMAs in paddy soils.The third part of the thesis investigated the pathway of DMAs volatilization mediated by microbes in paddy soils.The main results are summarized as follows:(1)Nine As-contaminated paddy soils were collected from South-East of China and incubated under flooded condition.The As species,total As,Fe,Mn concentrations and pH in porewater as well as soil redox potential(Eh)were determined The results showed that soil Eh decreased and the soil porewater pH increased toward a value of 7.0 after flooding.The concentrations of total Fe,Mn and As in porewater increased.The magnitude of As mobilization in the porewater varied by>100 times among the nine soils.The porewater As concentrations showed a strong correlation with the oxalate-extractable As concentrations,suggesting that As associated with amorphous iron(oxyhydr)oxides represents the potentially mobilizable pool of As under flooded condition.The major arsenic species in porewater were As(?)and As(V),and the proportion of As(?)was 60%-100%.DMAs was detected in the porewater from three paddy soils(Chenzhou(CZ),Yongzhou(YZ)and Changde(CD)).To estimate the uptake of As by rice in nine paddy soils,a pot experiment was performed.The results showed that As concentrations in shoots and roots correlated with the As concentrations in porewater rather than with total soil As concentrations,suggesting that As mobilization under flooded conditions paddy soils controls As availability to rice plants.(2)During flooded incubation of the nine paddy soils,DMAs was produced in CZ and QY and SM paddy soils.Although XY paddy soil was not contaminated with As,rice crop growing at this site showed severe straight-head disease.Experiments were carried out to investigate the microbial groups responsible for As methylation in the three paddy soils.Previous studies showed that arsenite S-adenosylmethyltransferase gene(arsM)was abundant in sulfate-reducing bacteria(SRB)in paddy soils.To verify whether SRB mediate arsenic methylation in paddy soils,a specific inhibitor of SRB,molybdate(Mo),was added to the paddy soils.The results showed that DMAs production was suppressed with sulfate reduction and the abundance of dissimilatory sulfite reductase(dsr)transcripts were significantly suppressed after with the addition of Mo.Addition of Mo decreased the abundance of arsenite S-adenosylmethyltransferase(arsM)transcripts.These results indicated that SRB played a major role in arsenic methylation in paddy soils.Furthermore,the enrichment cultures of SRB which contain the ability of arsenic methylation were obtained.Although Mo was generally considered to be an inhibitor of SRB,there was a possibility that it may affect biochemical processes other than sulfate reduction and the inhibition of As methylation is unrelated to SRB.To address this question,both in vitro assay with purified ArsM proteins from Bacillus.sp CX-1,Desulfosporosinus meridiei DSM 13257 and Desulfolomaculum gibsoniae DSM 7213 and arsenic methylation ability of two non-SRB strains in vivo experiments were conducted to test the effect of Mo.The results showed that the addition of Mo produced none or only marginal effect on ArsMs activity and the ability of arsenic methylation of non-SRB strains,providing further evidence that the addition of Mo suppressed arsenic methylation by decreasing the abundance of SRB.(3)During the later stage of the soil incubation under flooded condition,DMAs disappeared rapidly.We hypothesized that methanogens contributed to the disappearance of DMAs.A specific inhibitor of methanogens,2-bromoethanesulphonate(BES),was selected and added to the soils.The results showed that the disappearance of DMAs was inhibited by addition of BES.To clarify the pathway of DMAs disappearance,DMAs spiked soil were incubated.DMAs disappeared with the production of MMAs at later incubation stage,which was suppressed by the addition of BES.Enrichment cultures of methanogens which contain the ability of DMAs demethylation were obtained.Furthermore,13C-labled DMAs was synthesized and added to the soils.13CH4 and 13CO2 were detected in the headspace after flooded incubation for 14 and 40 days.Taken together,the results demonstrate that methanogens mediate demethylation of DMAs in paddy soils.(4)With the addition of Mo and DMAs(V),As volatilization was enhanced,producing DMAs(?)H as the main species.This process was abolished in sterilized soils,indicating the involvement of microbes.The addition of Mo changed the bacterial community structure significantly.A strain which could produce DMAs(?)H from DMAs(V)was isolated from paddy soils and named by CZ-2.To investigate the possible mechanism of DMAs(?)H production by Bacillus sp.CZ-2,reductants including GSH,NADH and NADPH were used in the culture of CZ-2.The results showed that NADH increased the volatilization of DMAs(V).This process was possibly mediated by the functional genes encoding reductases in CZ-2 which was upregulated by Mo.The genes expression was analyzed in strain CZ-2 by RNA-seq and 10 genes encoding reductases or dehydrogenases which significantly upregulated by Mo by more than 2 fold were selected as candidate genes mediating this process.Because Bacillus sp.strain CZ-2 was able to volatilize DMAs to DMAs(?)H but not able to methylate As(?)or MMAs,the results suggest a new pathway of DMAs volatilization that is independent of As methylation pathway.In conclusion,the present study has identified key microbial groups responsible for As methylation,demethylation and volatilization in paddy soils under flooded conditions.These findings provide a scientific basis for developing practical strategies to mitigate arsenic contamination and straight-head disease of rice.