Characterization And Metagenomic Analysis Of Nitrifying Biofilms With Different Survival Strategies

The nitrifying biofilms with different survival strategies were cultured and enriched under different operating modes,and the characteristics of nitrifying biofilms were studied from the perspectives of nitrifying bacteria,functional genes,quorum sensing and inter-species relationship by means of metagenomic techniques.In order to further analyze the formation of nitrifying bacteria survival strategy and the quorum sensing system existing in biofilm,and provide a theoretical basis for improving nitrogen removal efficiency.Influent composition:67 mg/L CaCl₂,22 mg/L MgSO₄,500 mg/L KHCO₃,500mg/L NaHCO₃ and 10 mg/L yeast extract,0.4 mL/L trace elements.The nitrogen in NOB biofilm reactor is 50 mg/L NaNO₂ and the nitrogen in the complete nitrification biofilm reactor is 50 mg/L NH₄Cl.Operating conditions:two sets of reactors were operated,one set was NOB-based biofilm reactor,and the other set was complete nitrification biofilm reactor.Each set of reactors had two modes of operation,sequencing batch reactor（SBR）and the continuous flow reactor（CFR）,the effective volume of the NOB-based biofilm reactor is 2 L,and the effective volume of the complete nitrification biofilm reactor is 3.8 L.The reaction temperature was maintained at 25±2°C by water bath heating.The hydraulic retention time during the stabilization phase is 6 h and there are four cycles per day.The nitrogen conversion rate of the reactor in the steady phase can reach more than 99%.The genus acclimated in the SBR mode is the r-strategy,and the genus acclimated in the CFR mode is the K-strategy.The nitrification kinetic parameters of r/K nitrifying bacteria in NOB biofilm reactor were:K_r=224.73μΜ,K_K=76.23μΜ.Nitrifying bacteria in the two reactors were determined by 16s rRNA and metagenomic techniques.The r-strategy and K-strategy nitrite oxidizing bacteria were mainly Nitrospira.The kinetic parameters of the r/K strategy nitrifying bacteria in the complete nitrification biofilm reactor were:ammonia oxidizing bacteria K_K=286.58μM,K_r=298.37μM;nitrite oxidizing bacteria K_K=181.19μM,K_r=456.86μM.The main ammonia oxidizing genus in the whole nitrifying biofilm is Nitrosomonas,and the main nitrite oxidizing genus is Nitrospira.The results showed that the K_m values of NOB in biofilms of different survival strategies were more different,and the difference of survival strategies was more obvious.The difference of K_m values of AOBs was smaller,and the difference of bacteria was not the reason for the difference of survival strategies.The results of the metagenomics showed that Nitrospira and Nitrosomonas undertook the task of major nitrogen metabolism and carbon metabolism.In the NOB-based biofilm reactor,the quorum sensing of AHL type mainly exists between Nitrospira and heterotrophic bacteria.Nitrospira is the main carrier of AHL synthesis and sensing genes,and heterotrophic bacteria have genes of quorum quenching,and the acylase gene abundance is much higher than that of the lactonase.Among the complete nitrification biofilms,the quorum sensing of AHL type mainly exists between Nitrosospira,Nitrobacter and Methyloglobulus,and is the main carrier of AHL synthesis and sensing genes.Nitrosomonas,Nitrospira and heterotrophic bacteria are the main carriers of AHL quorum quenching genes.In the carrier,the carrier of the AHL quorum sensing genes also has the quorum quenching genes,and both Nitrosomonas and Nitrospira have a lactonase and acylase gene.The quorum sensing system is one of the main forms of nitrification biofilm metabolism.The AHL signal molecule is detected in the reactor.The AHL is the major type type quorum sensing in the NOB-based biofilm reactor and the complete nitrification biofilm reactor,and the acylase gene is the major AHL quorum quenching gene in nitrifying biofilms.Other quorum sensing systems also exist in nitrifying biofilms,and genes related to quorum sensing system such as lepB,PQS,DSF and AI-2 are detected.These quorum sensing system may also play an important role in biofilm systems.Other major genus in the reactor,including some heterotrophic bacteria,denitrifying bacteria,etc.,also have AHL-related synthesis and sensing genes,which can generate a quorum sensing through AHL signal substances,regulate themselves,and form a complete biofilm structure.