Study On The Effect And Mechanism Of Enhanced Denitrification By A Nitrate-removing Microbial Agent

Anoxic-anaerobic/oxic-anaerobic/oxic（Anoxic-A/O-A/O）is a common used process of waste water treatment for intensive livestock and poultry breeding.However,the excessive discharge of nitrate nitrogen is long-term unsolved problem.In this research,after combining process operation characteristics and effect analysis,it was proposed to use aerobic denitfrifying bacteria intensive treatment technology to achieve in-situ deep nitrogen removal.This technology realize simultaneous nitrification and denitrification in the same tank by adds high-efficiency aerobic denitrificying bacteria immobilized bacteria agent to the oxic treatment tank,which solved the problem of nitrate nitrogen accumulation and discharge exceeding standards.Moreover,this technology was easy to operated and low in cost of transformation and operation.It is expected to be used to solve the problem of deep nitrogen removal under low C/N ratio conditionsIn this study,an aerobic denitrifying bacteria suitable for the low C/N ratio environment was isolated from the sludge of the first-class aerobic tank of the livestock and poultry wastewater treatment system.The bacteria is gram negative.16S r RNA gene sequence analysis identified it as pseudomonas mendocina,and named it P.mendocina LYX.The results of single-factor condition experiments and orthogonal experiments indicated that the optimal denitrification conditions of P.mendocina LYX at p H=7.5,C/N=5,temperature 35℃and dissolved oxygen 3.86 mg/L.under the above conditions,95.21%of nitrate was removed in 24 h.The results showed that P.mendocina LYX had a good adaptability under the condition of low C/N ratio.At C/N 3,the removal rate of nitrate nitrogen can still be maintained at 60%.In the experiment,P.mendocina LYX was made into immobilized microbial agent through membrane culture,and shake flask culture and simulated aerobic tank were adopted to explore the treatment effect of the microbial agent on duck wastewater with C/N 2.2.The results showed that the removal rates of nitrate and COD_Cr were increased by 40%and 20%,respectively,with the addition of the bacteria.The nitrite concentration in the reactor peaked earlier which indicated that the denitrification process was significantly improved.The results of 16S r RNA gene high-throughput sequencing showed that the relative abundance of the aerobic denitrifying bacteria Pseudomonas in the reactor increased from 0.03%to 8.5%in the first 10 days after inoculated the bacterium agent,but decreased to 0.59%on the 22^nd day,meanwhile,the removal rates of nitrate and COD_Cr increased to 86%and 79%on the 10^th day respectively,and then decreased to the similar level as the time before addition microbial agent.With the addition of microbial agent,the nitrogen removed quickly for the enrichment of aerobic denitrifying bacteria,but the function disappeared owing to the washing out of aerobic denitrifying bacteria,the strengthen period was suggested does not exceed 22 days.A series of analysis on the denitrification pathway of the bacteria revealed that37.9%of the bacteria were assimilated into intracellular substances,33.1%were reduced to nitrous oxide,16.6%were reduced to nitrogen as the end product,and less than 0.5%were in the form of ammonia nitrogen and nitrite nitrogen,and the total removal rate of nitrate nitrogen was 90.5%.Experiments such as PCR and RT-q PCR revealed that the bacteria possessed key enzyme genes（nap A,nir K,nor B,nos Z）,and the expression level of nap A is the highest during the whole denitrification process,followed by nir K and nor B,and the expression level of nos Z is quite low.Therefore,it is inferred that the main ways of nitrate removal by P.mendocina LYX are assimilation and alienation.The main path of alienation is （?）,and the reduction of nitrous oxide is bottleneck link in the entire denitrification pathway.The contribution of N₂O pathway need to be controlled by debugging during the application process.As a strain that can simultaneously remove nitrogen through aerobic denitrification and assimilation,P.mendocina LYX is expected to achieve convenient and rapid in-situ deep nitrogen removal in wastewater systems which were nitrogen accumulation exceeding the standard.It has a good prospect in the treatment of low C/N ratio wastewater particularly.