Isolation,Identification And Biotransforming Ability Of Alcaligenes Faecalis

Hydrogen sulfide(H₂S),as a kind of toxic and harmful gas released during livestock production,can cause harm to the body and pollution to the environment,so the removal of H₂S especially the oxidation of H₂S by biological methods has gradually become a current research hotspot.To screen high-efficiency sulfur-oxidizing strains,study the biodegradation,understand the characteristics and predict related functions of the strains,this study collected activated sludge from a wastewater treatment plant and feces from an avian farm,isolated and identified heterotrophic sulfur-oxidizing strains,optimized the growth conditions and measured the transformation performance,subjected the strains to whole genome sequencing and transcriptome sequencing,analyzed hydrogen oxide of key enzyme genes.The results of isolation,identification and transformation experiments showed that13 sulfide tolerant bacteria,named JLAF1-JLAF9,N1-N4,were isolated from the faeces of livestock farms and activated sludge of sewage treatment plants.Among them,JLAF9 has better removal effect on S^2-than other strains,and its SO₄^2-transformation ability is stronger.Finally,JLAF9 is taken as the next research object.The results showed that strain JLAF9 belonged to Alcaligenes faecalis.When the substrate concentration was 0.5 g/L,the temperature was 35°C and the initial p H was 7.0,the conversion rate of H₂S could reach more than 94%.Whole genome sequencing of A.faecalis JLAF9 showed that JLAF9 was composed of 1 chromosome without plasmid,and the total length of whole genome was4,039,445 bp,the average sequencing read length was 12,396 bp,the GC content was about 56.63%,the protein coding genes were 3794,and the total length of coding genes was 3,587,088 bp,which accounted for 88.8%of the whole genome.The predicted genes were checked against the gene ontology database(GO),Kyoto Encyclopedia of genes and genomes(KEGG),cluster of orthologous groups of proteins(COG),carbohydrate active enzymes database(CAZy)were annotated,in which the go database was annotated to 2784 genes,the KEGG database was annotated to 2,718 genes,the COG database was annotated to 3075 genes,and the CAZy database was annotated to86 genes.The three sulfur oxidase genes reported in the study were screened from the annotation results,divided into the sulfide quinone oxidoreductase sqr gene,the sox Y,and sox Z genes in the Sox sulfur oxidation system.The whole genome sequencing results of Alcaligenes faecalis JLAF9 showed that it was composed of one chromosome without plasmid.The total length of the whole genome was 4,039,445 bp,the average reading length was 12,396 bp,and the GC content was about 56.63%.There were 3,794 protein coding genes,and the total length of coding genes was 3587088 bp,accounting for 88.8%of the whole genome.The predicted genes were annotated in GO,KEGG,COG and CAZy databases,among which 3,075 genes were annotated in COG database,2,718 genes were annotated in KEGG database,2,784 genes were annotated in GO database and 86 genes were annotated in CZAy database.Three sulfur oxidase genes were screened from the annotation results,which were SQR gene and so gene Sox Y and Soxz genes in sulfur oxidation system.Transcriptome sequencing of gene expression changes of A.faecalis JLAF9 in an environment with and without sulfur induced growth showed that a total of 250 genes were differentially expressed between the strains in a sulfur containing versus no sulfur environment,including 27 up-regulated genes and 223 down-regulated genes.The results of enrichment analysis showed that the differentially expressed genes were concentrated in Biosynthesis of cofactors,ABC transporters,Sulfur metabolism,Toluene degradation,Thiamine metabolism,Benzoate degradation,Degradation of aromatic compounds and other related pathways.Gene qPCR validation results of three sulfur oxidase genes and log2Fold Change>2 genes annotated to the A.faecalis JLAF9 genome showed that the expression of fix O,nir K,sox Y,and sox Z genes were significantly elevated before and after sulfur source induction,and the validation results were in accordance with the RNA-seq results.However,the gene expression of sqr and if was not significantly different.