Study On The Characteristics And Metabolic Mechanism Of Simultaneous Nitrogen And Phenol Removal By Cupriavidus Oxalaticus T2

Phenol is a common pollutant and phenolic industrial wastewater has a wide source,large quantity,and complex composition.This type of wastewater also contains excessive nitrogen pollution except phenolic pollutants.The safe and effective treatment of pollutants in wastewater using biological methods is an important protection for the ecological environment and human health.However,phenol is toxic to bacteria and inhibits biological denitrification,which severely hinders the removal of pollutants in wastewater.Therefore,the use of microorganisms to efficiently remove phenol and nitrogen pollution in wastewater simultaneously has become an urgent problem to be solved in the treatment of phenolic wastewater.However,the current study on the co-degradation of phenol and nitrogen by a single strain is limited,and the understanding of the metabolic mechanism of simultaneous removal of nitrogen and phenol is not sufficient.A bacterium Cupriavidus oxalaticus T2 which can remove nitrogen and phenol simultaneously,was isolated from the activated sludge of a refinery wastewater treatment in this study.The characteristics of simultaneous nitrogen and phenol removal by strain T2 were investigated and the metabolic characteristics and adaptive mechanism to phenol of strain T2 were explored though combining omics study.Moreover,the function of the phenol degradation regulatory protein in strain T2 was identified by molecular biology methods.The results were listed as followed:（1）A strain T2 that can degrade phenol and ammonia simultaneously was isolated and identified as Cupriavidus oxalaticus.Sequencing the complete genome of strain T2 showed multiple gene clusters which related to phenol degradation exist in strain T2 and have all genes of the phenol ortho-pathway as well as a complete denitrification pathway.Moreover,Genomic information revealed a variety of potential functional genes that were beneficial to resist phenol toxicity,including universal stress proteins,efflux pump proteins,as well as a variety of molecular chaperones,antioxidant proteins.（2）The optimal conditions of removal of nitrogen by T2 were the carbon source of sodium succinate,C/N ratio of 10,30℃,shaking speed of 150 rpm,and p H of 6.In addition,strain T2can completely degrade phenol with a concentration of 1000 mg/L and performed the optimal degradation efficiency of phenol under the conditions of initial p H of 7–8 and shaking speed of150 rpm.Strain T2 was confirmed to be able to simultaneously remove nitrogen and phenol.When NH₄⁺–N,NO₃^-–N or a mixture of both were used as the nitrogen source,the strain T2could simultaneously remove nitrogen and phenol,and the utilization of NH₄⁺–N took precedence over NO₃^-–N.NH₄⁺–N.The results of q RT-PCR determined the ortho-degradation pathway of phenol in strain T2 and inferred the removal pathway of nitrogen in strain T2 with NH₄⁺→NO₃^–→NO₂^–→NO→N₂O→N₂.（3）The data-independent acquisition（DIA）method was applied in the present study to identify and quantify the alteration in the proteome of strain T2 at different stages during simultaneous degradation of phenol and nitrogen.The key metabolic mechanisms of strain T2for simultaneous nitrogen and phenol removal were inferred as follows:the ortho-and para-pathways of phenol degradation were observed in strain T2,and the expression of proteins involved in phenol degradation increased with treatment time with phenol;In addition,nitrogen metabolism exhibited different characteristics.Increased denitrification and decreased synthesis of glutamate from ammonia were observed in the lag phase;The analysis of differentially expressed proteins also identified various phenol response mechanisms as follows:enhanced energy production by altering the expression of cytochrome oxidases with different oxygen affinity in the respiratory chain;improved cell motility to enhance the degradation efficiency of phenol;increased synthesis of lipoic acid and biotin to resist phenol toxicity;the combined action of multiple anti-stress enzymes,chaperones,and transcriptional regulators contributed to the preservation of cellular stability.（4）The function of the phenol degradation regulator protein Pht R was identified.By fusion with reporter genes of green fluorescent protein and electrophoretic mobility shift assays,the regulatory mechanism of Pht R on the phenol hydroxylase gene cluster was verified,and the binding sequence of Pht R on the promoter was determined.Moreover,it was found that aromatic compounds such as m-cresol,benzoic acid,o-chlorophenol,and p-nitrophenol can also activate the Pht R.The isolated Cupriavidus oxalaticus T2 that can simultaneously remove nitrogen and phenol enriches the application resources of wastewater treatment microorganisms.The study on the simultaneous removal of nitrogen and phenol mechanism of this strain and its phenol tolerance mechanism can provide an important theoretical basis for practical applications.