Study On The Reduction Of Divalent Mercury Catalyzed By Methanobactin

Methanotrophs are important multi-functional biocatalysts with very broad application.Methanotrophs also show great potential in pollution treatment,such as the treatment of Hg(II)in the environment.Methanobactcin(Mb)is a small chromopeptide secreted by Methanotrophs that has the characteristics of metal reduction and chelating.Mb can chelate and reduce Hg(II)in nature to Hg(0)and further adsorb Mb-Hg(0)on the cell surface and prevent Hg(0)volatilize.In this thesis,through the study on the ability of Mb chelating and reducation mercury,the detoxification of mercury,the molecular basis of methanotrophs for mercury tolerance was explained.The potential of methanotrophs to remove Hg(II)from the environment was explored.In the first section,the detection method of Mb was established.A standard curve about the Hg(?)concentration of 1?5ug/mL was established(y=0.1081 x+ 0.2681,R2=0.9932,with a good linear relationship).Use this as a analysis tool,the chelation and catalytic reducation of divalent mercury(Hg(?)by Mb extracted from Methylosinus trichosporium 3011 has been studied.The effects of different reaction time,reaction temperature,addition amount of Hg(?),addition amount of Mb on the reduction of Hg(?)catalyzed by Mb were investigated.It has been found by the UV Vis spectra and fluorescence spectra that Mb can reduce Hg(?)and the optimum reaction conditions were established as following:the reaction time was 5min,the reaction temperature was 30?,and Mb was bound to Hg(?)with a ratio of 2:1;The fluorescence spectrum analysis showed that Mb has three main characteristic emission peaks and the fluorescence quenching has been found at the excitation wavelength of 254nm after the addition of HgCl2.This also demonstrated that methanotrophs can capture Hg(II)by secreting Mb and Mb plays a key role in reducing Hg(?).In the second section,the detoxification of Mb against Hg(?)was studied.Origin8.0 was used to manage the experimental data,and the growth trend of methanotrophy was simulated by Logistic growth kinetics equation,and the bacterial growth curve was obtained.The lag phase was 20.1h and after 20.1h was logarithm phase while maximum growth rate was 0.0142h-1.The presences of Hg(II)extend the lag phase of the cell growth,The presences of Hg(II)also result in the decrease of maximum specific growth rate.Compare with the addition of Hg(II),Mb and Hg(II)co-addition at the same time shortened the lag to 15.3h,the maximum specific growth rate increased to 0.0138h-1.The maximum OD value increased to 1.185.It can be concluded that Mb has ability of mercury detoxification which may be the molecular level of mercury tolerance of methanotrophs.The last part of section discussed the effect of the presence of copper ions on Mb the chelation and catalytic reducation of Hg(?).Adding Cu(?)at a lower concentration can promote cell growth.When the copper ion concentration was 30?mol/L the maximum cell growth rate was 0.015h-1,the maximum OD600 value was 1.043.The cell density was the highest at this time.Although adding Cu(?)at a higher concentration has a certain toxic effect on the cell growth,Cu(?)constructed the pMMO active center.Cu(?)has promoted the growth of the cell itself and shorten the lag phase to 16.8 hours.In the fermentation broth,pMMO activity was mainly expressed in cells when Hg(?)was added after the Cu(?)addition.The activity of pMMO was gradually increased in the range of 0?20?mol/L,the highest value of the pMMO activity was 3.25nmol/(min·mg dwc).When Cu(?)was added followed by Hg(?)addition,the absorbance at 600nm gradually increased to 0.13 within 0-120h,and a small amount of sMMO activity was expressed.When Cu(?)and Hg(?)were added at the same time,the highest intracellular activity of pMMO was 3.15 nmol/(min·mg dwc).At this time,the absorbance of cells at 600 nm gradually increased to 0.1 at 0-120 h.Therefore,when Cu(?)and Hg(?)were added at the same time,the intracellular pMMO and sMMO activities remained unchanged with little effect.The activity of sMMO was determined by the naphthol method.The addition of mercury to the Mb-Cu had little effect on the methanogenic bacteria pMMO(no sMMO activity),indicating that Mb has a higher affinity to copper.However,Cu(?)can not bind to Mb after Mb was bound to mercury,This demonstrated that the binding of Mb to mercury is irreversible.