Study On The Characteristics Of Nitrogen And Phosphorus Removal From Low Carbon Source And Oxygen Sensing Regulation Mechanism Of Thauera Sp. RT1901 Bacteria

The low carbon source is a typical feature of municipal wastewater and an important factor restricting the efficiency of nitrogen and phosphorus removal in municipal wastewater treatment process.Simultaneous nitrogen and phosphorus removal can effectively improve the carbon source utilization rate of the wastewater treatment system and enhance the removal effect of nitrogen and phosphorus pollutants.However,the realization of this approach needs to strictly rely on the content and activity of simultaneous nitrogen and phosphorus removal microorganisms in the wastewater treatment system,which leads to the problems of complex process flow,high operational control requirements in the existing solutions.Therefore,it is particularly important to study new methods to enhance simultaneous nitrogen and phosphorus removal.In this paper,Thauera bacteria,a functional strain of simultaneous nitrogen and phosphorus removal was screened.Its characteristics of nitrogen and phosphorus removal in low carbon sources were analyzed,and its induction control mechanism of simultaneous nitrogen and phosphorus removal under different oxygen environments was revealed.A bio-enhanced activated sludge simultaneous nitrogen and phosphorus removal system was constructed,forming an application strategy of using Thauera bacteria as a functional strain to strengthen low carbon source sewage treatment.It provides theoretical and technical support for strengthening the simultaneous nitrogen and phosphorus removal of low carbon source wastewater.The main conclusions of the study are as follows:（1）A strain of Thauera with high nitrogen removal efficiency was obtained from the activated sludge of wastewater treatment plant through denitrification enrichment culture and screening purification.The strain was named Thauera sp.RT1901 and its nitrogen removal performance and genomic characteristics were analyzed.The results showed that the nitrogen removal process of this strain mainly depended on denitrification,and it could achieve stable denitrification under the conditions of p H7～9 and temperature 25～40℃,and could use various carbon sources such as aromatic compounds as electronic donors for denitrification,and the denitrification rate was not affected by C/N.During the denitrification process,the strain preferentially uses NO₂^--N as the electron acceptor,and the removal rates of NO₃^--N and NO₂^--N reach 3.17mg/L/h and 4.94 mg/L/h respectively.Genome sequencing showed that the strain contained 4642 coding genes,12 r RNA,62 t RNA and 47 nc RNA.（2）By analyzing the nitrogen and phosphorus removal performance of Thauera bacteria under the conditions of different electron donors and electron acceptors,the metabolic function of Thauera bacteria using microbial metabolites for nitrogen removal and denitrifying phosphorus accumulation was confirmed,and the metabolic pathway of its low carbon source synchronous nitrogen and phosphorus removal characteristics was revealed in combination with the genome.The results showed that the substrate utilization associated products（UAP）and biomass associated products（BAP）of unit mg COD reduced 0.45 mg and 0.10 mg NO₃^--N to gaseous nitrogen respectively,and UAP could be used simultaneously with exogenous organic carbon.The strain can use NO₃^--N,NO₂^--N or O₂ as electron acceptor to absorb phosphate to synthesize polyphosphate（Poly-P）during Polyhydroxyalkanoates（PHA）catabolism,and the phosphorus removal rate is 0.1134 mg/L/min,0.1164 mg/L/min and 0.1181mg/L/min respectively.During anaerobic phosphorus release,glycolysis has been proved to be the only way of glycogen metabolism of the strain,and glycolysis and glyoxylic acid cycle together provide Nicotinamide adenine dinucleotide（NADH）reduction equivalent for the synthesis of PHA.In the process of aerobic phosphorus absorption,the glyoxylic acid cycle provides the precursor for the gluconeogenesis pathway to synthesize glycogen,and denitrification-related proteins compete for the electrons from ubiquinone and cytochrome C to achieve simultaneous nitrogen and phosphorus removal.（3）By analyzing the synchronous nitrogen and phosphorus removal performance and gene transcription expression patterns of this strain in different oxygen environments,combined with protein sequence structure and binding site analysis,the oxygen sensing regulatory mechanism of synchronous nitrogen and phosphorus removal from low-carbon sources was explored.The results showed that the gene expression of PHA metabolism,tricarboxylic acid cycle,glyoxylate cycle,gluconeogenesis,phosphate transport and Ploy-P synthesis in the process of denitrification phosphorus accumulation of the strain was not affected by oxygen molecules,and the differential expression of respiratory chain genes caused the strain to shift from denitrification phosphorus accumulation to aerobic phosphorus accumulation.The FNR protein and the Fix LJ dual component system jointly regulate the expression of differential genes by sensing changes in oxygen concentration.The FNR protein forms dimers in anaerobic environments,responsible for activating the expression of nitrate respiration related genes,inhibiting the expression of aerobic respiration related genes,and inactivating the FNR protein in aerobic environments to achieve reverse regulation of transcriptional expression.The Fix LJ dual component system perceives the activation of Fix K gene transcriptional expression in hypoxic environments.Fix K and FNR have the same transcriptional regulation function and its activity is not affected by oxygen molecules.The FNR protein and Fix LJ two-component system sense the change of oxygen concentration,which can realize the regulation of denitrification phosphorus accumulation and aerobic phosphorus accumulation metabolic pathways under anaerobic and aerobic conditions,and under hypoxic and aerobic conditions.The results of nitrogen and phosphorus removal at different oxygen concentrations also indicate that the two oxygen sensing control systems enable the strain to achieve the same synchronous nitrogen and phosphorus removal effect in both low oxygen and anaerobic environments.（4）A strategy using Thauera sp.RT1901 as a functional strain to enhance synchronous nitrogen and phosphorus removal was proposed for the low-carbon source water quality characteristics of urban sewage.The practicality of this strategy has been proven through long-term operation of an activated sludge system for 180 days.The results showed that when operating under the condition of low carbon source and low oxygen,the characteristics of simultaneous nitrogen and phosphorus removal of low carbon source of the strain made it more competitive than traditional denitrification bacteria in terms of carbon source utilization,and could quickly become the dominant bacteria in denitrification functional bacteria.The oxygen sensing regulation mechanism of simultaneous nitrogen and phosphorus removal made it more competitive than traditional phosphorus accumulating bacteria in terms of absorption of PO₄^3-in low oxygen environment,gradually become the dominant genus of phosphorus accumulating bacteria.The enrichment of Thauera bacteria and ammonia-oxidizing bacteria and nitrite-oxidizing bacteria form a stable mechanism of microbial mutual promotion and synergism in carbon source utilization,oxygen environment and simultaneous nitrogen and phosphorus removal.The results showed that the bioaugmentation increased the abundance of Thauera phase pair from 1.4%～2.48%to4.72%～8.86%,the relative abundance of denitrifying bacteria such as Zoogloea and Haliangium,and traditional phosphorus accumulating bacteria such as Candidatus＿Competitor and Candidatus＿Accumulibacter decreased significantly.The relative abundance of nitrifying bacteria such as Nitrospira increased significantly.The loss of TN in the aerobic section of the system increased by 3.28 mg/L,and the loss of NO₃^--N in the anoxic section increased by 2.64 mg/L.The removal efficiency of COD,TN and TP in the system increased from 82.55%,71.19%and 95.51%on average to88.67%,77.71%and 98.28%respectively.