Isolation And Identification Of Mixotrophic Denitrifying Thiobacterium Thiopseudomonas Denitrificans X2 And Genomic Research

With the rapid development of social economy,the wastewater discharge of pharmaceutical and petrochemical industries increased year by year.Sulfur,nitrogen and high concentrations of chemical oxygen demand(COD)are typical characteristics of industrial wastewater pollutants.Such wastewater is not up to discharge into the environment will cause eutrophication and other problems.Simultaneous desulfurization and denitrification(SDD)process includes sulfate reduction and organic carbon removal unit,autotrophic and heterotrophic denitrifying sulfide removal unit,sulfur reclamation unit and aerobic nitrification unit.SDD process,coupling sulfate-reduction and denitrifying sulfide removal,could achieve efficient removal of organics,sulfate and nitrogen oxides.However,there are some problems in the process of pollutant removal and resource recovery efficiency,which are usually disturbed by the load of influent water.The mechanism of microbial community in activated sludge is unclear,and the yield of elemental sulfur is rather low.In this study,a functional strain X2 capable of simultaneously metabolizing COD,sulfide and nitrogen oxides under anaerobic conditions was isolated from the reactor of SDD process.Phylogenetic analysis based on 16 S r RNA gene indicated that strain X2 belonged to Pseudomonasaceae family and had close genetic relationship with Pseudomonas caeni.They were distinctly isolated from other closely related species of the Pseudomonadaceae family in the phylogenetic tree,forming a novel evolutionary unit.Using polyphasic taxonomy,the strain X2 was identified with Pseudomonas caeni as a reference strain.The results showed that strain X2 had significant differences in morphology,physiology and biochemistry compared with Pseudomonas caeni.The strain X2 represents a type strain of novel genus in the Pseudomonidaceae family.It was named Thiopseudomonas denitrificans X2 according to its physiological characteristics of sulfur oxidation and denitrification.According to different energy and carbon source,metabolic characteristics of strain X2 were analyzed under different conditions.The strain X2 could grow under the conditions of heterotrophic denitrification,mixotrophic sulfur oxidation-denitrification and autotrophic sulfur oxidation-denitrification respectively.It can use organic or inorganic carbon source,and can also use oxidation of sulfide or organics as energy source.So strain X2 is facultative chemolithotrophic sulfur bacteria.In the process of metabolism,sulfide did not undergo over-oxidation in the presence of high concentration electron acceptor.The main product of the oxidation reaction was elemental sulfur particles.Functional gene expression analysis showed that the sulfur oxidizing gene fcc AB and the denitrifying gene nir S were all housekeeping genes.Under the conditions of mixotrophic sulfur oxidation-denitrification,the expression level of these two genes was significantly lower than that of heterotrophic denitrification.Sulfide has a negative regulation on the functional genes of sulfur oxidation and denitrification.The strain X2 was employed for bioaugmentation of elemental sulfur production under high nitrate load.In addition to the efficient removal of acetates and nitrates,bio-enhanced reactors can significantly increase the rate of elemental sulfur production.Microbial community analysis showed that strain X2 was successfully colonized in the reactor and became a dominant population.The complete genome sequence of strain X2 was obtained by DNA sequencing and data assembly.The sequence length is 2.86 Mb and contains 2703 open reading frames.Bioinformatics analysis of protein functional annotation was performed using the database.The functional genes associated with carbon,sulfide and nitrogen oxide metabolism were found from the results of protein function annotation.Strains X2 can metabolize organic matter using glycolysis and citric acid cycling.There are Fcc AB and Sqr coding genes in the genome.They are capable of catalyzing the oxidation of sulfide to elemental sulfur.The clusters of functional gene that participate in denitrification process were found.According to the functional genome analysis,the carbon,sulfur and nitrogen metabolic genes network was constructed.The genomic evolution of strain X2 and related species was analyzed by comparative genomics.The ANI of strain X2 and Pseudomonas aeruginosa PAO1 was highly consistent and they shared 1885 homologous genes.There was a widespread gene rearrangement among their genomes.