Functional Microbial Interactions And Metabolic Mechanism In Integrated Autotrophic-Heterotrophic Denitrification Process

Compared with physical and chemical processes,biological nitrogen removal process has the advantages of low energy consumption and low operating cost,occupying nearly 90% of the market in China's sewage treatment field.At present,heterotrophic denitrification and autotrophic denitrification processes are the most widely used for nitrogen removal,but they expose the problems of high carbon source demand,long start-up period,and low denitrification rate in the presence of dissolved oxygen.In response to this situation,our research team has developed the integrated autotrophic-heterotrophic denitrification(IAHD)process,which used sulfide and organic compounds as electron donors for the denitrification process,and can obtain satisfied removal efficiencies of sulfide,nitrate and organics under micro-aerobic conditions.Nitrogen and carbon removal efficiency.The efficient operation of IAHD process relies on the interaction of autotrophic and heterotrophic denitrifying microorganisms,but the discovery of heterotrophic sulfur-oxidizing nitrate-reducing bacteria breaks this conventional perception.Therefore,this paper focuses on the interactions amoung functional microorganisms during IAHD process,and analyzes the contributions of heterotrophic nitrate reducing bacteria(h NRB),autotrophic sulfur-oxidizing nitrate-reducing bacteria(a-soNRB)and heterotrophic sulfur-oxidizing denitrifying bacteria(h-soNRB)to the removal of sulfur,nitrogen and carbon under different sulfide concentrations and micro-oxygen conditions.Meanwhile,based on the genomic and transcriptomic analysis of representative h-soNRB strains,exploring the unique sulfur-nitrogen-carbon metabolism advantages of h-soNRB by co-cultured with a-soNRB,providing a theoretical basis for further expanding the application of engineering.Firstly,under the cyclical transformation of trophic conditionsb(autotrophic,heterotrophic and mixotrophic),the operational efficiency of IAHD system and the succession of microbial community structure were investigated.And the functional microorganisms were classified into three groups: a-soNRB,h NRB and h-soNRB.Based on this,we proposed two kinds of functional mechanism of IAHD process under different sulfide concentrations.After switching amoung autotrophic,heterotrophic,and mixotrophic conditions,the removal efficiencies of sulfide,nitrate and acetate at sulfide concentration of 200 mg/L and 400 mg/L were all 100% in the three IAHD bioreactors.The microbial community structure exhibited similar characteristics under different sulfide concentrations respectively.The co-work of a-soNRB and h NRB support the simultaneous removal of sulfide,nitrate and acetate under low sulfide concentration(200 mg/L).Under high sulfide concentration(400 mg/L),the h-soNRB substituted of a-soNRB as key functional bacteria for sulfide oxidation and cooperated with h NRB to support IAHD processSecondly,the removal of sulfur,nitrogen and carbon under micro-aerobic conditions was discussed from the perspective of functional microorganisms and functional genes.In the sulfur autotrophic denitrification process,the limited oxygen enhanced the desulfurization and denitrification efficiencies.According to the microbial community structure,the abundances of Azoarcus,Thauera,Alkaliflexus and Aliidiomarina showed a significant(p<0.05)positive correlation with sulfide concentration and aeration rate.And the enhanced autotrophic denitrification performace under micro-aerobic conditions was analyzed by 16 S metaproteomics functional composition prediction.In the IAHD process,the genes related to sulfur oxidation,denitrification and sulfur-containing amino acid metabolism were up-regulated under micro-aerobic conditions.The limited oxygen made IAHD bioreactor achieve a sulfur,nitrogen and carbon removal efficiencies of more than 90% under high sulfide concentration conditions,coresponding with h-soNRB such as Pseudomonas and Azoarcus occupied appraximately 90% in microbial community.Thus,h-soNRB were speculated as functional microorganism that maintains the efficient operation of the micro-aerobic IAHD bioreactor.After four stages of micro-aerobic cultivation,the sulfide tolerance of IAHD bioreactor under anaerobic conditions is doubled,and the removal efficiencies of sulfide,nitrate and acetate are about 2?5 times higher than the initial anaerobic condition.So in order to save energy and ensure the performance of IAHD process,batch micro-aerobic should be considered as an efficient strategy.In addition,based on the electron current analysis of the micro-aerobic IAHD process,it was found that there was a positive correlationship between oxygen content and electron deletion rate,and when the sulfur,nitrogen and carbon removal rate is the highest,the corresponding electron deletion rate also reached a maximum.As the dominant genus,the relative abundance of Thiobacillus increases with the increase of electron deletion rate.Finally,five h-soNRBs were isolated from the activated sludge of IAHD bioreactor.After comparing the metabolic characteristics of sulfur,nitrogen and carbon,Pseudomonas C27 was selected as a representative h-soNRB,and its genes network of sulfur,nitrogen and acetate metabolism was constructed.Based on transcriptomics analysis,the sulfur,nitrogen and carbon metabolism genes of Pseudomonas C27 under micro-aerobic condition(O2/S2-=0.5:1)were investigated.It was found that the limited oxygen was tend to function under high sulfide concentration,and compared with anaerobic condition,the micro-aerobic condition decreased the number of down-regulated(p<0.05)genes which inhibited by high sulfide concentration.When the sulfide concentration increased,pdo gene was significantly(p<0.05)up-regulated under anaerobic and micro-aerobic conditions,which was closely related to the Pseudomonas C27 high tolerance to sulfide.The co-work of Pseudomonas C27 and Thiobacillus denitrificans for sulfide removal was observed when co-cultured these two strains in high sulfide concentration(200?400 mg/L).Pseudomonas C27 and Thiobacillus denitrificans can achieve the quick removal rate on the basis of ensuring 100% removal of sulfide with biomass ratio of 3:1.Compared to Thiobacullus denitrificans,the different Sqr enzyme type and pdo and cys K genes containing characteristics in Pseudomonas C27 might be responsible for its ability to maintain high metabolic capacity at high sulfide concentrations.The high sulfide tolerance of h-soNRB and the synergistic desulfurization with a-soNRB have broad research and engineering application value.