The Research On The Mechanism Of Microbial Community Interaction And Regulation In SNAD-IFAS Process

In recent years,energy neutral or energy positive have been proposed as the development target of municipal sewage(mainstream sewage)treatment in domestic and foreign countries.The main idea is first to recycle organic carbon and convert it into methane,and then utilize low energy consumption technology for biological nitrogen removal.Simultaneous partial nitrification,anammox and denitrification-integrated fixed film activated sludge(SNAD-IFAS)process has been listed as an important process for biological nitrogen removal in the future because of its many advantages,such as simultaneous removal nitrogen and carbon,low energy consumption,operational savings,and concise process.However,the stability of the SNAD-IFAS process is difficult to control due to the variety of microorganisms and the complex relationship between microorganisms in the process.Moreover,the mechanism for the formation of autotrophic denitrification biofilms and the regulation and interaction of microbial communities within the process is not yet clear.Therefore,in this study,the role of cyclic diguanylate(c-di-GMP)in the formation of autotrophic denitrifying biofilms and the influence of microbial community was analyzed by molecular biology techniques combined with secondary signaling molecules and microbial resource management concepts in the SNAD-IFAS process.Simultaneously,the spatial heterogeneity,shifts and interaction mechanism of microbial communities were also elucidated under different operating conditions in the SNAD-IFAS process.To understand the mechanism of autotrophic denitrification biofilm formation,the second signal molecule concept was used to analyze the relationship between c-di-GMP and extracellular polymeric substance(EPS)during the process of autotrophic denitrification biofilm formation.The results showed that the tightly bound EPS(TB-EPS)content of autotrophic denitrification biofilms was higher than that of aerobic ammonia-oxidizing bacteria(AOB)and anaerobic ammonium-oxidizing bacteria(AnAOB),whereas the loosely bound EPS(LB-EPS)content of biofilm was significantly lower than AOB and AnAOB.Simultaneously,the contents of polysaccharides and c-di-GMP were correlated in AOB,AnAOB,and biofilm,and the AnAOB secreted more EPS(especially polysaccharides)than AOB with a markedly higher c-di-GMP.During the autotrophic denitrification biofilm formation,the increased intracellular c-di-GMP concentrations in AnAOB enhanced the regulation of polysaccharides to promote the adhesion of AOB and formation of the autotrophic denitrification biofilm.To further understand the microbial community in the SNAD-IFAS process,in this study,molecular biology techniques were used to conduct in-depth research on the microbial species,the spatial heterogeneity and interaction relationship of microbial communities in the SNAD-IFAS process.The results illustrated that AnAOB,AOB and denitrifying bacteria(DNB)wasCandidatus Kuenenia stuttgartiensis,Nitrosomonas europaea and Denitratisoma oestradiolicum,respectively in the SNAD-IFAS process.AnAOB and DNB were mainly distributed on biofilms,accounting for 51.49%and 2.07%,respectively,whereas AOB was mainly distributed in suspended sludge,accounting for 18%.Candidatus Kuenenia occupied a dominant position in the microbial network,and other microbes were unfolding around its life activities.Candidatus Kuenenia displayed significant positive correlations with some heterotrophic bacteria,including Caldilinaeaceae,Ignavibacterium,Bryobacter and Denitratisma.In addition,AnAOB tended to form a coupling relationship with DNB rather than AOB.In the SNAD-IFAS process,some heterotrophic bacteria(Caldilinaeaceae,Bryobacter and Ignavibacterium)could be an important associated species with AnAOB,and there was a close interaction between them and AnAOB.pH is a major factor influencing the structure and diversity of microbial communities.One regulatory mechanism has been omitted for long time about the response mechanisms of microbial communities to pH,that is the function of c-di-GMP.Based on the above research results,the intrinsic mechanisms of the community structure shifts regulated by pH were studied.The results showed that when the pH increased from 7.5 to 8.5,the relative abundance of Nitrospira(nitrite-oxidizing bacteria,NOB)in the suspended sludge increased from 7.1%to 38%,whereas the relative abundance of Candidatus Kuenenia(AnAOB)decreased from 10.9%to 0.8%in the SNAD-IFAS process.The reason for this phenomenon was that the activity of NOB is still highly at a pH of 8.5.As the pH increased,some bacteria in the suspended sludge began to switch from the attached state to the moving state.Because excessively high pH(≥8.5)may cause the decrease of c-di-GMP content in these bacteria,and then enhancing their motility,so that they would wash out of the reactor with the effluent.This would lead to the shifts of the microbial community structure in suspended sludge and increased the proportion of NOB in suspended sludge,which would affect the activity of AnAOB on biofilm.The SNAD-IFAS process was applied to the treatment of simulated mainstream wastewater,and the effects of the C/N ratios on the performance of the mainstream SNAD-IFAS process and the microbial community structure were analyzed.The results illustrated that the optimal C/N ratios for mainstream wastewater treatment by SNAD-IFAS process was 1.2± 0.2,and the average concentration of COD,NH4+-N,NO2--N,NO3--N was 7.2,0.4,1.1 and 13.4 mg/L in the effluent,respectively.Excessive C/N ratios(≥ 2.0)would lead to the proliferation of heterotrophic bacteria(Hydrogenophaga)and NOB(Nitrospira)in the suspended sludge,thus reducing the nitrogen removal efficiency of mainstream SNAD-IFAS process.There were two different nitrogen removal pathways in the mainstream SNAD-IFAS process,that was,the nitrogen removal process in the biofilm was accomplished with AnAOB and DNB,and the nitrogen removal pathway in the suspended sludge was completed with AOB,NOB,AnAOB and DNB.In addition,the contents of polysaccharides and c-di-GMP were correlated in biofilm and suspended sludge.The reason why AnAOB(Candidatus Kuenenia)could survive in environment with low concentrations of organic matter was that AnAOB displayed significant co-occurrence through positive correlations with some heterotrophic bacteria(Limnobacter,Bryobacter)which could protect anammox bacteria from hostile environments(oxygen,organic matter).The SNAD-IFAS process has great potential to be integrated with the high-rate activated sludge and anaerobic hydrolysis denitrification technology to upgrade the municipal wastewater treatment plant.