Study On Screening,Application And Ammonia Oxidation Mechanism Of Heterotrophic Nitrifying Bacteria

Biological nitrogen removal has the advantages of low energy consumption,less pollution,high efficiency and low cost.Traditional biological nitrogen removal mainly depends on autotrophic nitrifying bacteria and denitrifying bacteria.However,autotrophic nitrifying bacteria grow slowly and are vulnerable to interference from other microorganisms and environmental conditions.Therefore,problems such as unstable ammonia oxidation efficiency often occur.In addition,toxic pollutants such as thiourea and phenol in the actual industrial wastewater of chemical plants,electronic plants,iron and steel coking plants can potentially inhibit the activity of autotrophic nitrifying bacteria and even lead to the collapse of nitrification system.Compared with autotrophic nitrifying bacteria,heterotrophic nitrifying bacteria have the advantages of rapid growth and reproduction,strong adaptability,and a certain tolerance to toxic pollutants and potential degradation ability,which has a good application potential in sewage denitrification system.At present,the researches on heterotrophic nitrifying bacteria mainly focus on the screening of bacteria with low C/N,low temperature tolerance and high salinity tolerance.Its functional mechanism in sewage system and practical application effect still needs to be further explored.In this paper,the directional screening of heterotrophic nitrifying bacteria,characterization of metabolic function,detoxification mechanism of thiourea,and nitrogen removal efficiency of industrial wastewater were studied.Firstly,in view of the toxicity of thiourea,a common nitrification inhibitor in the process of industrial wastewater treatment,a thiourea resistant heterotrophic nitrifying bacteria BT1 was selected from natural multi-source samples,and its potential metabolic function was characterized by whole genome sequencing;The detoxification mechanism of BT1 to thiourea in activated sludge denitrification system was further analyzed.Combined with the immobilization technology of biomass substrate,the enhanced biological nitrogen removal effect of heterotrophic nitrifying bacteria BT1 was studied and successfully applied to practical wastewater treatment projects.The main research results are as follows:（1）A heterotrophic nitrifying bacteria BT1,which can efficiently degrade ammonia nitrogen and is resistant to thiourea toxicity,was identified as Pseudomonas sp.BT1 by16S r RNA,and was deposited in the General Microbiology Center of China Microbial Species Conservation and Management Commission,with the reservation number of CGMCC 1.18862.Strain BT1 can grow rapidly in Vickers salt Medium.The optimum growth conditions are which culture temperature 30.0℃,shaking table speed 120.0-160.0 rpm,initial ammonia nitrogen concentration 100.0-200.0 mg/L,C/N 10.0-15.0,carbon source is sodium succinate and p H 7.0-9.0.The ammonia nitrogen removal efficiency and ammonia oxidation rate reached 98.2%and 6.0 mg-NH₄⁺/L·h respectively within 48.0 h.Under pure culture conditions,The thiourea concentration that Pseudomonas sp.BT1 can tolerate is 0.0-200.0 mg/L,and can only degrade a small amount of thiourea（5.6-13.8%）.（2）The whole genome size of strain BT1 was 5576102 bp,with a total of 5115genes,including 5018 protein coding genes,74 t RNA genes,7 r RNA genes and 3 gene islands.It was no plasmid and no CRISPR structure.A total of 2763 genes were annotated and involved in 41 metabolic pathways,including heterotypic biomass degradation and metabolic pathways and other secondary metabolite synthesis pathways.It was found that strain BT1 had the closest genetic relationship with Pseudomonas sp.asiatica,had complete C,N and S cycle pathways,and the C and S cycle had cross intermediates.Pseudomonas sp.BT1 can degrade ammonia nitrogen and nitrite nitrogen by assimilation.Sulfate,thiosulfate,taurine and alkane sulfonate can be used by strain BT1.（3）Pseudomonas sp.BT1 can play a biological synergistic role in the municipal activated sludge system containing 10.0 mg/L thiourea,and it can degrade most thiourea in sewage.Compared with the control group without Pseudomonas sp.BT1,the ammonia nitrogen degradation rate was lower than 20.0%or even collapsed.The experimental group with BT1 had more than 90.0%ammonia nitrogen degradation rate and 72.0%thiourea degradation rate.The analysis of microbial flora shows that the proportion of Pseudomonas sp.BT1 population has not increased,but the abundance of other denitrification microorganisms increased by 4.5 times.The reason may be that the medium brought by Pseudomonas sp.BT1 and the secondary metabolites produced by Pseudomonas sp.BT1 stimulate the growth of other denitrifying microorganisms in the activated sludge,thereby improving the degradation rate of ammonia nitrogen and thiourea.Transcriptome sequencing was used to sequence and analyze the m RNA of Pseudomonas sp BT1.The expression rules of genes related to adenosine phosphate sulfate reductase,cysteine synthase and o-succinylhomoserine mercaptolase were locked,and found that Pseudomonas sp.BT1 can transform thiourea through the reduction pathway to synthesize sulfur-containing amino acids,and produce succinate or acetate to enter the tricarboxylic acid cycle（TCA）,this is the basic reason of thiourea tolerance and partial degradation of Pseudomonas sp.BT1.（4）By immobilization of self-developed biomass substrate with Pseudomonas sp.BT1,it was found that the optimal soaking time was 48 hours,and the removal performance of COD,NH₄⁺-N,TP after immobilization was increased by 8.5%,13.7%and 26.9%respectively compared with volcanic rock.The microbial community analysis of the biomass substrate after the operation of film hanging showed that the denitrifying related bacteria were dominant（24.2%）,and contained a certain proportion of phosphorus accumulating bacteria（4.6%）and autotrophic nitrifying bacteria（1.3%）,which confirmed that the connecting pores（16.7%）of the biomass substrate and the residual biomass were more conducive to the enrichment and immobilization of different kinds of nitrogen and phosphorus removal microorganisms,The application of bacteria carrying biomass substrate to the simulated sewage treatment system can further purify and enhance the tailwater,so that the effluent NH₄⁺-N,TN and COD concentrations can meet the requirements of Class I A standard specified in the Discharge Standard of Pollutants for Municipal Sewage Treatment Plants（GB 18918-2002）.（5）Providing a new possibility for biological nitrogen removal under low temperature:BT1 has better biological efficiency than AOB at low temperature（8-10℃）,the average concentration of ammonia nitrogen in effluent is 16.1%lower than AOB group,and AOR is 20μg/L·h higher than AOB group.After biofilm is eluted by packing,the ammonia oxidation efficiency of BT1 group is higher than that of AOB group,and the ammonia oxidation rate of biomass substrate in BT1 group is 1.85 times that of volcanic rock.In the cooling process from 25.0℃to 8.0-10.0℃,the loss of Pseudomonas sp.BT1 synergist group was the lowest（14.8%）.The co-occurrence analysis of microbial networks showed that the abundance of heterotrophic nitrifying bacteria played a key role in maintaining the stability of ammonia nitrogen effluent under low temperature environment.（6）In order to solve the problem of the breakdown of the nitrification system in the sewage treatment plant of the circuit board production enterprise,it is confirmed by GC-MS and field data analysis that it is caused by the impact of nitrification inhibitor.The nitrification inhibitor in the circuit board wastewater is mainly thiourea and accompanied by other phenol compounds.The three treatment methods of activated carbon adsorption,Fenton oxidation and excess sludge reflux are compared.It is proved that the method of activated sludge reflux in A/O tank combined with microbial synergist has the best effect on ammonia nitrogen removal.The ammonia nitrogen content decreases to 0.4 mg/L,and the ammonia nitrogen removal rate can reach98.9%.After this method is applied to the sewage plant,the ammonia nitrogen removal rate of A/O biochemical system can be increased from-20.0-20.0%to 90.0-95.0%within 15.0 d,and save cost greatly for enterprises.