Effects Of Microbial Communities On The Accumulation Of Methylmercury In Paddy Soils

Mercury?Hg?is a globally recognized heavy metal pollutant that can be transported over long distances,with strong toxicity and easily biological enrichment.Methylmercury?MeHg?,being the most toxic compound in all forms of Hg compounds,can be accumulated and magnified into organisms through the food chain,and then endangers the human health.Rice consumption by residents in Hg Mining area is the main pathway for MeHg exposure,and paddy soils have been proved to be one of the major places responsible for the generation of MeHg in the terrestrial ecosystems.Presently,how to reduce the risk of MeHg exposure derived from rice in Hg mining area has become an urgent problem to be solved,but the mechanisms on production and accumulation of MeHg in paddy soils are still unclear.In this study,the distribution characteristics of microbial functional genes?hgcA?dsrA?dsrB?mcrA?pmoA?related to Hg transformation were studied,the microbial mechanism on accumulation of MeHg in paddy soil was studied based on the combined routine of field survey and indoor incubation.Firstly,soil samples were collected from typical Hg mining areas in China,and then the contents of soil total mercury?THg?and MeHg were measured to determine the key factors affecting soil MeHg content.Subsequently,the paddy soil with higher MeHg concentration was used in the following incubation experiment.To investigate the potential contribution of several functional microorganisms to MeHg degradation,an indoor soil incubation experiment with addition of microbial inhibitors was conducted.Nitrogen fertilizer application is an indispensable measure to ensure a high rice production in this area.Therefore,the effects of nitrogen fertilizer application on the accumulation of MeHg in the paddy soil was further investigated through an indoor soil incubation experiment with addition of NH₄Cl and NaNO₃.This study is capable of revealing the main factors controlling the MeHg accumulation,and provides direct evidence for MeHg pollution remediation in paddy soils.The main results were as follows:?1?A total of 47 soil samples including 5 areas of cropland and paddy soil were collected from Wanshan?WS?,Tongren?TR?in Guizhou Hg mining area and Qiliangqiao?QLQ?,Xinchangxiang?XCX?,Chatian?CT?in Hunan Hg mining area in China,and the Hg concentrations?THg and MeHg?and abundance of hgcA gene were measured in each soil sample.Results showed that there was a significantly positive correlation between soil MeHg and THg content?P<0.01?,and the concentrations of MeHg in the paddy soils were generally higher than these in the cropland soils.The results of qPCR showed that the hgcA gene could be hardly detected in the cropland samples,whereas which could be detected in all the paddy soil samples.In addition,in each paddy soil sample,the abundance of hgcA-containing archaea was higher than that of hgcA-containing bacteria,and the hgcA-containing archaea abundance was significantly correlated with soil MeHg concentration?R²=0.05,P<0.05?,implying that archaea might play an essential role in the process of mercury methylation in those paddy soils.These results suggest that the microbial communities may play an important role in MeHg accumulation in paddy soils.?2?Based on the result of field survey in section?1?,a paddy soil with relatively higher MeHg content in Taoyuan,Hunan,was chosen in the subsequent incubation experiment.Two microbial inhibitors,sodium molybdate?Na₂MoO₄,sulfate-reducing bacteria inhibitor?and sodium 2-bromoethylsulfonate?BES,methanogenic inhibitor?were selected to investigate the influence of the typical microorganisms on MeHg accumulation in paddy soils with the indoor soil incubation experiment under waterflooding?Y?.In addition,the potential role of aerobic Methanotrophs in MeHg removal in paddy soil was explored by adding Methanotrophs inhibitor?CuCl₂?incubation experiment under aerobic?H?conditions.Three treatments denoted as no inhibitor addition?CY?,MoO₄^2-addition?M?,and BES addition?B?were conducted in the waterflooding incubation experiment,and two treatments(no Cu²⁺addition,CH;Cu²⁺addition,Cu)were set up in the aerobic incubation experiment.All treatments were incubated at 25°C in the dark.Soil samples were taken at 0,2,4,7,14,30,60,120,150d,and then the concentration of MeHg was measured.Moreover,for 0,60,and 120 d samples,the abundance of functional genes and bacterial community composition were examined by qPCR and Illumina MiSeq sequencing,respectively.Results showed that there was no significant difference about the MeHg concentration between M and CY treatments during the whole incubation period;the abundances of hgcA in M treatment at 120 d were significantly lower than that at 0,60 d?P<0.05?,whereas the abundance of hgcA had no changes in CY treatment;based on genus,the M and CY treatments had similar bacterial community structure.The concentration of MeHg in the B treatment was significantly higher than that in the CY treatment at day 4,14,120,and 150 d?P<0.05?,and the mcrA gene abundance in B treatment showed a decreasing tendency from day 0 to 120 d;based on genus,B treatment had higher abundance of Azospirillum than CY.For the aerobic incubation experiment,the concentration of MeHg in the Cu treatment during the incubation of 120 days was significantly lower than the CH treatment?P<0.05?.Meanwhile,except the hgcA,the abundances of the other functional genes at day 60 and 120 were significantly lower than those at day 0.Compared to the CH treatment,relatively higher abundances of Proteobacteria and Firmicutes were detected in the Cu treatment;based on genus,Cu treatment had higher abundance of Dyella than CH.?3?Application of nitrogen?N?fertilizer is a common measure adopted by human to ensure a stable and high rice production,and thus the effect of N nutrient on the accumulation of MeHg in this paddy soil were studied by the soil incubation experiment with addition of NH₄Cl and NaNO₃.Here,two groups of treatments including waterflooding?Y?group and aerobic?H?group were set up,and each group contained 3treatments?no fertilizer,ammonia added,and nitrate added treatments?.In total,there were 6 treatments?waterflooding and no fertilizer,CY;waterflooding plus ammoni,NHY;waterflooding plus nitrate,NOY;aerobic and no fertilizer,CH;aerobic plus ammoni,NHH;aerobic plus nitrate,NOH?in this incubation experiment.All treatments were incubated at 25?in the dark for 150 days,and soil samples were taken at 0,2,4,7,14,30,60,120,150 d,and then the concentration of MeHg was measured.Moreover,for 0,60,and 120 d samples,the abundance of functional genes were examined by qPCR.The results indicated that under the waterflooding condition,the concentration of MeHg in all the three treatments decreased obviously with incubation time,and no significant difference was observed between the CY treatment and other N-added treatments?NHY and NOY?at each sampling day;the abundance of dsrB in CY and NHY treatments at 120 d were significantly lower than that at 0 d?P<0.05?,the abundances of dsrA in CY and NOY treatments at 120 d were significantly higher than those at 0 d?P<0.05?.Under the aerobic condition,the concentration of MeHg in each treatment dropped to the minimum value at 120 d,and then slightly increased from day120 to 150;the abundances of dsrA,dsrB,pmoA in NHH treatment at 60 d were significantly higher than those at 0 d,whereas the abundance of all genes had no changes in CH treatment;the abundance of hgcA in NOH and CH treatments at 120 d were significantly lower than those at 0 d?P<0.05?,while there was no significant varations for the abundances of dsrA?dsrB?mcrA?pmoA.