Characteristics And Gene Regulation Mechanism Of 1,4-Dioxane Degradation By Xanthobacter Dioxanivorans Sp. Nov.

1,4-Dioxane is widely used as a solvent and solubilizer in industry,and is also a common unwanted byproduct.Due to inefficient wastewater treatment,the 1,4-dioxane contamination risk in the water environment has become more and more severe in the past decades.Because of its special physicochemical properties,1,4-dioxane is reluctant to biodegrade,which causes the accumulation in the environment,risking the safety of the water environment potentially.Biotreatment has great advantages on 1,4-dioxane,but its practical application performance is hindered by the lack of consistently dominant highly efficient degraders.In this study,a dominant1,4-dioxane degrader with high efficiency was isolated from active sludge.The strain was identified via taxonomy analysis.The growth and degradation characteristics of the strain were investigated,and the strategies to respond to 1,4-dioxane,maintain the dominant niche,and achieve high degradation performance were revealed by genome-and transcriptome-scale analysis.From the active sludge acclimatized using 1,4-dioxane,degrading bacterium strain YN2 was achieved by separation and purification.Through phylogeny and microbiology classification methods,the strain was considered to represent a novel species,designated Xanthobacter dioxanivorans sp.nov.（=CGMCC 1.19031=JCM34666）.According to the investigation of disturbance factors,the optimal growth and degradation conditions of YN2 were 30°C,p H=7,shaken at 180 r/min with initial biomass of OD₆₆₀=0.007.Strain YN2 was able to degrade 200 mg/L 1,4-dioxane in34 h under the optimal conditions,which was much better than the highest degradation performance of other reported degraders.Effects of different 1,4-dioxane concentrations on growth and degradation of strain YN2 were investigated,and both growth and degradation kinetics of strain YN2were described by the Monod.The maximum specific growth rateμ_max was 0.03/h,with a cell yield of 0.27 mg-protein/mg-1,4-dioxane;the maximum specific degradation rate k_max was 1.10 mg-1,4-dioxane/h·mg-protein,and the half-saturation concentration K_s was 410.91 mg/L.Comprehensively,strain YN2 had a better maximum specific growth rate and a higher cell yield compared with other reported degraders,and showed higher tolerance to 1,4-dioxane of 1000～3000 mg/L,meaning that the strain can be applied in a wide area.The whole genome of strain YN2 was sequenced by third-generation technology and was annotated using several databases,including KEGG（Kyoto Encyclopedia of Genes and Genomes）,COG（Cluster of Orthologous Groups of Proteins）,GO（Gene Ontology）,and Nr（Non-redundant protein sequence）.The YN2 genome was found to have a size of 6,650,818 bp,with a 67.95%G+C content spread over a chromosome and four plasmids,the sizes of which were 147 kb,144 kb,51 kb,and30 kb,respectively.1,4-Dioxane degradation-related genes were proposed by comparative genome analysis based on the annotation results of the YN2 genome.Differentially expressed genes strain YN2 grown on 1,4-dioxane compared with citrate were analyzed using transcriptome sequencing technology,and the essential intermediate was confirmed to be glyoxylate,with the key genes of YN2 related to1,4-dioxane metabolism and the degradation pathway as follow:1,4-dioxane was initially oxidized by Thm ABCDEF,and then transformed to glycolate through five alcohol dehydrogenase genes,one methanol dehydrogenase gene cluster,three glyoxalase genes,one aldehyde reductase/ketoreductase,and three aldehyde dehydrogenase genes,after that glycolate was catalyzed into glycolate via two glycolate oxidase clusters;glyoxylate was metabolized through three pathways:a glyoxylate degradation pathway,a malate synthesis pathway,and an anaplerotic ethylmalonyl-Co A pathway that specially belongs to YN2 as a methylotroph,by which glyoxylate was transformed into pyruvate and malate,entering the TCA cycle to complete the mineralization.The number of YN2 genes related to 1,4-dioxane metabolism,the expression level of these genes,and the number of metabolism pathways of the key intermediate of YN2 were all higher than reported degraders,which led to a higher degradation efficiency of YN2.By following the transcriptome of YN2 during different 1,4-dioxane degradation stages and after the stimulation of 1,4-dioxane,the mechanism of the high 1,4-dioxane degradation efficiency of YN2 was revealed,as well as the mechanism of YN2 responding to 1,4-dioxane and keeping a stable degradation efficiency.Degradation related genes of strain YN2 were constitutive,therefore the expression of the degradation enzymes would not be inhibited by non-inducible substrates,and did not possess any lag phase,which allowed the strain YN2 to start the degradation of 1,4-dioxane quickly;these genes had a high level of expression,and multiple of these genes were involved in a same pathway,resulting in a high transcription level in total,offering a better degradation performance to the strain YN2;genes related to quorum sensing,transporters,and two-component systems were dynamically regulated at different degradation stages,helping the strain adapt to the environment,adjust the biomass,and enhance the upregulation of key genes,maintaining the efficiency and stability of degradation performance.