Exploration And Application Of Transformation Mechanism Of Sulfide By Cellulosimicrobium Sp. Strain

Sulfide(S^2-,H₂S)are one of the toxic pollutants in the aquaculture environment.The treatment cost of sulfide removal by physicochemical methods is relatively high and the operation is difficult.Some microorganisms can use sulfide as life energy to carry out redox,converting toxic and harmful sulfide into harmless products,and microbial methods for sulfide removal are gradually coming into the attention of researchers.However,microbial sulfide removal has been faced with various biochemical challenges,and therefore there is a need to develop a product with high conversion capacity and environmental friendliness.In this study,a strain L1 with high sulfur removal capacity was isolated from culture wastewater,and after optimizing its S^2-transformation conditions,it was applied to a biological trickling filter（BTF）to test its H₂S removal effect.The key enzyme genes for sulfide conversion in the strain were analyzed by whole genome sequencing to further investigate its mechanism of action.Finally,the active component of the strain transforming S^2-was located and isolated and purified to obtain an enzyme product that can improve the farming environment and is friendly to the environment,and the enzyme has promising applications in agroenvironmental remediation.In this experiment,12 strains with sulfur removal ability were isolated from the culture wastewater,and the strain L1 with the strongest sulfur removal ability was selected for the next study.The strain L1 was identified morphologically and molecular biologically as Cellulosimicrobium sp.strain,gram-positive and short rod-shaped.Single-factor and response surface methods were used to optimize the conditions for S^2-conversion of Cellulosimicrobium sp.L1.The results showed that the S^2-transformation rate of strain L1 was 76.20%with 500 mg/L S^2-concentration as the substrate concentration,3 g/L sucrose as the carbon source and 1 g/L NH₄Cl as the nitrogen source,at the temperature of 35℃,initial p H=4.45 and salt concentration of 1.13%,and 36 h incubation.The dynamic changes of S^2-transformation of strain L1 showed that S^2-was rapidly oxidized to S₂O₃^2-at the beginning of the transformation,and then further oxidized to SO₄^2-.The conversion rate of the strain could reach more than 86%by 48 h.The sulfide conversion performance was good.The experimental application results of biological trickling filter cell showed that strain L1 could successfully hang film on polyurethane foam and remove 359.53 mg/m³of H₂S,and the maximum removal capacity（EC）of H₂S was 5217.71 g/（m³h）.The whole genome sequencing results of strain L1 showed that Cellulosimicrobium sp.L1consisted of 1 chromosome without plasmids,with a total genome length of 4,301,405 bp and a GC content of about 74.67%.3,847 coding genes were predicted,and the total length of all coding genes was 3,898,119 bp,accounting for 90.62%of the whole genome.The predicted genes were annotated and analyzed in the database,and sulfur conversion-related genes were screened from the annotation results,including sulfide quinone oxidoreductase SQR,cysteine desulfurase（suf E,suf S,NSF1）,and thioredoxin reductase,which provide references for the study of the regulatory mechanism of sulfur conversion in heterotrophic bacteria.Based on the genome-wide results analysis,the sulfide conversion of strain L1 is dependent on various enzymes for completion.This strain has the potential to develop a sulfur convertase for application in the remediation of pollutants in the environment.In this study,the transforming active component of strain L1 was localized and the active component was determined to be present in the extracellular enzyme.The sulfur convertase was purified by ammonium sulfate graded precipitation,dialysis,and Sephadex G-75 gel exclusion chromatography with a molecular weight of 70 KDa.The physicochemical properties showed that the sulfur convertase is a thermally and storage-stable oxidase with good inhibitor and organic solvent adaptability.The purified sulfur convertase was applied to the culture wastewater environment and could completely remove S^2-from the culture wastewater,as well as have remediation effects on the remaining pollution indicators.In conclusion,this study developed an environmentally benign sulfur convertase that can be used for farm wastewater remediation,providing a basis for the study of using enzymes for environmental remediation.