Exploration Of The Mechanism Of Cr(Ⅵ) Reduction And Resistance Of Bacillus Cereus BC4

Hexavalent chromium has strong biological toxicity and poses a great danger to human health and the stability of the ecosystem.The conversion of hexavalent chromium to trivalent chromium is an important method to solve chromium pollution in the environment,and its mechanism is also the focus of attention of scholars at home and abroad at present.However,the current research on Cr(Ⅵ)reduction reaction is mostly limited to the optimization of the reduction reaction condition parameters and the determination of the position of the reductase,and there is still a lack of in-depth understanding of the function of Cr(Ⅵ)reductase and the mechanism of the reduction and resistance action of microorganisms,so that the chromium pollution in the environment can not be controlled and controlled accurately and efficiently.In this study,the Cr(Ⅵ)-removing bacterium Bacillus sp.BC4 strain was isolated by screening,and the effects of different influencing factors and external electron donors on Cr(Ⅵ)removal by Bacillus sp.BC4 were firstly investigated;then the Cr(Ⅵ)removal sites of Bacillus sp.BC4 were determined by analyzing the Cr(Ⅵ)removal ability of resting cells,permeabilized cells and subcellular fractions,combined with the relative fluorescence quantitative PCR for changes in expression levels of genes associated with Cr(Ⅵ)tolerance and reduction mechanisms in Cr(Ⅵ)stress environments,the chromium removal mechanism of the strain was analyzed.Thereafter,the heterologous expression of specific genes was used to characterize the action of the proteins expressed by the specific genes on Cr(Ⅵ).The main results were as follows:(1)A Cr(Ⅵ)removal bacterium BC4 strain was screened from activated sludge and identified as Bacillus cereus after whole genome sequencing.This bacterium can tolerate500 mg/L of Cr(Ⅵ)at p H 8 and has a good ability to remove Cr(Ⅵ).(2)The results of different influencing factors to remove Cr(Ⅵ)showed that the optimum temperature of strain BC4 to remove Cr(Ⅵ)was 37℃,the optimum p H was 8,and the optimum shaking speed was 120 rpm.The comparative study showed that the removal of Cr(Ⅵ)by Bacillus sp.BC4 did not depend on the metabolic substances inside and outside the cell,but on the active substances produced by the bacterium itself,and the removal of Cr(Ⅵ)was not The removal of Cr(Ⅵ)was not limited by the transport of Cr(Ⅵ).By comparing the Cr(Ⅵ)removal ability of each subcellular fraction,it is clear that the Cr(Ⅵ)removal by the strain is almost entirely caused by the extracellular fraction,which indicates that the Cr(Ⅵ)removal process of Bacillus BC4 takes place extracellularly and is produced by extracellular enzymatic action.(3)One gene,nit R1,encoding a nitroreductase associated with Cr(Ⅵ)reduction,and one gene,chr A,encoding a chromate transporter protein,were analyzed by relative fluorescence quantitative PCR technique in Bacillus BC4.The results showed that the expression of chr A gene was 1.45-fold and that of nit R1 gene was 25-fold higher than that of the control under 100 mg/L Cr(Ⅵ)stress,but the expression of chr A gene was lower than that of the control and that of nit R1 gene decreased to 4.93-fold of the control under 300mg/L Cr(Ⅵ)condition.Gene sequence analysis indicated that nit R1 is an NADPH-dependent chromium reductase of Bacillus cereus BC4.(4)Heterologous expression of chr A and nit R1 genes in the engineered strain was constructed,and the results showed that the chr A gene could significantly enhance the Cr(Ⅵ)tolerance of the engineered strain under Cr(Ⅵ)induction,and the nit R1 gene could significantly enhance the Cr(Ⅵ)reduction ability of the engineered strain,indicating that the chr A protein was associated with the Cr(Ⅵ)tolerance mechanism of BC4 strain.Nit R1 protein was associated with the Cr(Ⅵ)reduction mechanism of BC4 strain.This study can provide a theoretical basis for the application of microorganisms in the treatment of environmental chromium pollution through the study of Cr(Ⅵ)removal characteristics and reduction and resistance mechanisms of chromium-reducing bacteria.