Mercury Biomethylation Efficiency Of Facultative Aerobic Bacteria At Different Oxygen Concentrations

Inorganic Hg can be converted into more toxic MeHg through biotic and abiotic pathways,which is mainly mediated by microorganisms.Multiple studies have been shown that methylmercury in the environment is primarily produced by anaerobic microorganisms.However,for the Three Gorges Reservoir?TGR?,the periodical water storage and discharge make it form a large water level fluctuation zone?WLFZ?,which is not a strictly anaerobic environment.After impoundment of the TGR,the physical and chemical properties of soil such as oxygen concentration in the WLFZ were significantly altered by the water level fluctuations,making the WLFZ conducive to the activation of mercury.More importantly,the periodical water level fluctuations leads the TGR hold the characteristics of newly-built reservoirs every year.Previous researches found that the soil methylmercury content in the WLFZ gradually decreased in the early drying stage of the TGR,whereas it showed an increasing trend with the extension of the drying time.In the late drying stage,the WLFZ soil is not a strict anaerobic environment due to its long exposure to the atmosphere.In addition,previous studies found that there was a significant or extremely significant positive correlation between culturable microorganisms and methylmercury in the 10²0 cm soil layer in the WLFZ of the TGR,and a facultative anaerobic strain with mercury methylation ability was successfully isolated from the soil of the TGR.These results indicated that there might be some guilds of mercury methylators other than anaerobes in the WLFZ soils.Oxygen content is an important factor affecting the mercury methylation process.During the flooding period of the TGR,the oxygen concentration in the WLFZ soil is very low or even anaerobic,while the oxygen concentration in the soil during the dry period is gradually increased..Thus,the WLFZ becomes an aerobic and anaerobic alternating environment,owing to the periodic water level fluctuations.Since a specific variation of soil methylmercury was also found during the drying and flooding alternation of the TGR,we speculated that there should be some mercury methylators that have good adaptation to oxygen changes in the WLFZ of the TGR.Based on this,a strain with the ability to produce methylmercury was isolated from the WLFZ soil of the TGR,which was then identified through morphology,physiological,biochemical characteristics and 16S rRNA gene sequence.Subsequently,the biological characteristics of this isolated strain were explored under different initial Hg²⁺concentration,pH,temperature,salinity conditions,as well as anaerobic and aerobic conditions,respectively.Then,the mercury methylation ability of this isolated strain as well as a facultative anaerobic Klebsiella?klebsiella.sp.TGRB5?,isolated previously by our lab,were investigated under different oxygen concentrations.Finally,a kinetics model was used to simulate the conversion rate of inorganic mercury to methylmercury,and then,the role of oxygen in the process of methylation and demethlation would be clarified by comparing the rate of methylation and demethylation of those strains.This study could provide a data basis for further exploring the mechanisms of mercury methylation under different oxygen conditions.The main results are as follows:?1?A strain capable of converting inorganic mercury(Hg²⁺)into methylmercury under aerobic conditions was isolated from the WLFZ soil of the TGR.Under an initial Hg²⁺concentration of 300ng/L,the maximum methylmercury amount?[MeHg]?_maxax produced by the strain was 3.41±1.90ng/L,with a methylmercury conversion rate of1.14%.Physiological and biochemical characteristics showed that this isolated strain is a Gram-negative bacterum.The tests of contact enzyme,starch hydrolysis and hydrazine were all positive,while MR,VP,sodium malonate,citrate utilization,gelatin and oxidase tests were all negative.After 15h of incubation,gas was produced by this strain.Combined with the 16S rRNA gene sequence analysis,this isolated strain was identified as RaouLtella terrigena,which was named as RaouLtella terrigena TGRB3?GenBank accession number:MK102091?.?2?Under Hg²⁺concentration of 1000?g/L,the growth of strain TGRB3 was significantly inhibited.In the treatment of pH=4⁹,CO₂ was produced when strain TGRB3 was under stable growth phase,resulting the pH value stable at 3.65±1.57 after27h of incubation.This results indicated that strain TGRB3 had a strong acid-base adaptability.Besides,this strain is a medium-high temperature bacterium,with an optimum growth temperature of 25°C,and it can grow normally in the salinity range of0.2%⁴%.More importantly,it can grow normally under both aerobic and anaerobic conditions,belonging to a facultative anaerobic bacterium.?3?To explore the methylation and growth characteriatics of TGRB3 and TGRB5strains under aerobic/facultative aerobic conditions,strains were incubated under 0,7%,14%and 21%oxygen concentration in KB medium,with an initial Hg²⁺concentration of 300 ng/L.Results showed that the growth of these two strains was not restricted by the oxygen concentration.With the increase of oxygen concentration,the growth rate of strain TGRB3 accelerated,which were faster than that of strain TGRB5.Under different oxygen concentration conditions,the methylmercury production abilities of these two strains were different.The mercury methylation ability of strain TGRB3 was stronger and stable under anaerobic or low oxygen concentration conditions,while strain TGRB5was stronger under aerobic condition.After analyzing the methylmercury amount and the mercury methylation kinetics model,it is showed that the mercury methylation of strains TGRB3 and TGRB5 performed a typical two-stage reaction process:the methylation process of inorganic mercury,and followed by a demethylation process of methylmercury.Among them,mercury methylation ability has significant individual differences,while methylmercury demethylation trend is consistent,and the demethylation rate increased with the increasing oxygen concentration.Therefore,we speculate that it is a mechanism of mercury tolerance to maintain the dynamic equilibrium of mercury-methylmercury concentration in the strains,with the purpose to survive.