Efficacy Of Mercury Biomethylation And Demethylation Of ?-Proteobacteria Under Different Oxygen Concentrations

The mercury(Hg)bio-methylation and its environmental problems have long been a hot research fields in the biogeochemical cycle of mercury.Scholars have carried out a large amount of relevant researches and obtained the substantial achievement.However,most of the researches have some limitations:(1)The most experiments were conducted under high mercury concentration conditions,which were inconsistent with natural conditions;(2)Many studies have focused on?-Proteobacteria anaerobic bacteria,such as sulfate reducing bacteria(SRB),iron reducing bacteria(Fe RB),and methanogens(Methanogens),etc;(3)The most researches focused on the anaerobic aquatic ecosystems such as lakes and marine sediments,but ignored the aerobic terrestrial ecosystems;(4)The processes of Hg biomethylation and demethylation were split in the most experiments.The net increase of methylmercury were overly concerned,but the overall dynamic response of microbial mercury methylation and demethylation were ignored;(5)It has been proofed that the ability of Hg bio-methylation was determined by the hgc A/B gene clusters in the anaerobic?-Proteobacteria.However,it was not clear that some bacteria possessed the ability of Hg bio-methylation but without those gene clusters.In the early stage of our research group,several strains of?-Proteobacteria with the ability of aerobic/facultative mercury-phobic methylation were isolated and identified from the soils of water level fluctuation zone of the Three Gorges Reservoir area.Based on their full genomic sequencing and annotation,it was not found any hgc A/B gene cluster and/or mer A/B operon.These studies,therefore,carried out the related exploration experiments about Hg methylation and Me Hg demethylation of the?-Proteobacteria.The main experimental results are as follow:(1)Under the fully anaerobic cultivation system(0%oxygen concentration),Desulfomicrobium escambiense CGMCC 1.3481(SRB1)?Desulfovibrio vlugaris miyazaki SRB(SRB2)and Raoultella terrigena strain TGRB3(B3)could grow normal.The specific growth rate and maximum growth rate of B3 were lower than the SRB controls,respectively.The maximum amount of Hg bio-methylation production per unit of bacteria,i.e.[Me¹⁹⁹Hg]_max,was observed in the following order:SRB1>SRB2>B3.The maximum Hg bio-methylation rate was the following order:SRB1>SRB2>B3.The performance of mercury methylation rate(km)was the following order:SRB1>B3>SRB2.May be affected by genotype and growth status,there are significant differences in mercury methylation potential between strains.The[Me¹⁹⁹Hg]_max of the three strains were occurred in the bacterial logarithmic growth period.After the peak of[Me¹⁹⁹Hg]_max,the content of methylmercury were significantly decreased.The Me Hg bio-demethylation rate,i.e.Me²⁰¹Hg production rate,was the following order:SRB1>SRB2>B3.The methylmercury demethylation rate(km)was:SRB1<SRB2<B3,respectively.The maximum Hg bio-methylation production of B3 was relatively lagged behind,which might be related to its maximum growth amount and low specific growth rate.All the results showed that bacteria themselves were the leading factor causing Hg bio-methylation and Me Hg demethylation in the cultivation system.The capacities of bacterial Hg bio-methylation and Me Hg demethylation were dominated by strain-dependent,there are individual differences due to different strains.(2)Under the conditions of facultative bacteria cultivation(7%oxygen concentration),the unit bacteriaal[Me¹⁹⁹Hg]_max was observed in the following order:Pseudomonas fluorescens strain TGRB2(B2)>Pseudomonas putida strain TGRB4(B4)>Raoultella terrigena strain TGRB3(B3).The maximum Hg bio-methylation rate was the folloeing order:B2>B4>B3.The performance of km was the following order:B2>B3>B4,Me²⁰¹Hg production rate was the following order:B2>B3>B4,and kd was:B2>B3>B4,respectively.The results showed that the [Me¹⁹⁹Hg]_max of the three?-Proteobacteria strains were occurred in the bacterial logarithmic growth period.There was no direct relationship between the bacterial growth and the maximum Hg bio-methylation.In addition,the ability of Me Hg bio-demethylation in these three experimental strains showed basically the same.(3)Under completely aerobic incubation conditions(21%oxygen concentration):the unit[Me¹⁹⁹Hg]_max was observed in the following order:B2?B3>B4,The maximum Hg bio-methylation rate was the folloeing order:B2?B3>B4.The performance of km was the following order:B3>B2?B4,Me²⁰¹Hg production rate was the following order:B4>B2>B3,and kd was:B2>B4>B3, respectively.Compared with the corresponding treatments of 0%and 7%oxygen concentration,it was found that the growth of?-Proteobacteria were different under different oxygen concentrations.The optimum incubation was the aerobic conditions.The results showed that all the three experimental?-Proteobacteria strains possessed the both abilities of Hg bio-methylation and Me Hg bio-demethylation.Hg bio-methylation was most likely to occur at low redox potential.It was remarkable that the more oxygen concentration,the maximum amount of bacterial Me Hg production were appeared earlier.On the other hand,the Me Hg bio-demethylation ability of these?-Proteobacteria were individual differences,but the tendency of Me Hg bio-demethylation showed basically the same.The km of all experimental bacteria were less than kd in the each oxygen concentration treatments.It was speculated that there was a stable biological Me Hg bio-demethylation pathway in these?-Proteobacteria.According to the above results,it could be reasons to believe that there should be a metabolic mechanism of Hg absorption and transformation to maintain the dynamic balance of mercury and methylmercury in?-Proteobacteria.(4)Based on the plasmid of p K18mob Sac B,we used the prokaryotic homologous recombination technology to carry out the target gene,i.e.SH417.Using the B4 as expeirimental strain,the gene of SH417 was knockout of strain B4.Using the B4 genomic DNA as a template,primers were designed for the target gene,and the CDS gene fragment containing the target gene and its upstream and downstream sequences was obtained by PCR amplification.After the series of technical methods,which were contained enzymatic digestion,ligation,transformation,blue-white screening,gel electrophoresis separation,and sequenced,we successfully constructed the p KSd417 and p K18d417 plasmids.Up till now,we are still currently screening for secondary homologous recombination mutants.This study preliminary explored the target gene knockout experimential conditions of the wild-type?-Proteobacteria,and provided experimental ideas and methods for the follow-up researches of our group.The results of this studies would help us understand the interaction between?-Proteobacteria,mercury,methylmercury and environmental factors more clearly.It could also provide basic theoretical basis for further exploring the mechanism of migration and transformation of mercury and methylmercury by microorganisms.