Title:study Of Metabolic Mechanism Of Functional Microorganisms In The Side-steam Enhanced Biological Phosphorus Removal System

In view of the deterioration of phosphorus removal performance caused by insufficient influent carbon source often faced by sewage plants at present,a new type of enhanced biological phosphorus removal(EBPR)process,side-stream enhanced biological phosphorus removal(S2EBPR)process,has attracted more and more attention because it can supplement influent carbon source through anaerobic hydrolysis fermentation of side-stream sludge to produce acid.The key process of phosphorus removal in S2EBPR process is anaerobic fermentation of sidestream sludge,in which functional microorganisms play a decisive role in phosphorus removal in S2EBPR system.However,most of the previous conclusions were based on stoichiometric data,which could not distinguish the changes of cell aggregates metabolism of different microbial species in the biochemical reaction process of side-stream reactor at the microscopic level,so the support of population level and even cell level analysis tools was needed.In this study,singlecell Raman spectroscopy(SCRS)technique coupled with optical microscopy and fluorescence in situ hybridization(FISH)technique were applied.The differences in the position distribution of intracellular poly-P characteristic peaks of different kinds of phosphorus poly-bacteria(PAOs)and glycan bacteria(GAOs)under different process structure conditions of S2EBPR system were investigated.The difference of the location distribution of PO2-characteristic peak in different PAOs and S2EBPR wastewater treatment process indicates that poly-P phenotypic characteristics are correlated with the diversity of PAOs,and there is a potential relationship with the performance and stability of S2EBPR.Based on the scanning of poly-P and Gly standard products by SCRS technology,the calculation method of Raman quantitative analysis of functional microorganisms was established.The different detection methods of poly-P were compared,which showed the rationality and feasibility of the establishment of SCRS method.The relative abundance and intracellular polymer changes of PAOs and GAOs were compared in the longterm anaerobic experiment.It was concluded that the metabolic activity of PAOs was the highest and suitable for PAO enrichment when the anaerobic condition was 60h.Finally,Raman analysis was conducted on the relative abundance of PAOs and GAOs and intracellular polymers under different side-stream ratios in S2EBPR process.The changes of different PAOs and GAOs under different side-stream ratios were revealed at the single-cell level,and the most appropriate sidestream reflux ratio of S2EBPR system was found to provide positive feedback for the phosphorus removal function of S2EBPR system.The main research results are as follows:(1)SCRS technology was used to analyze the main intracellular polymers poly-P and glycogen(Gly)of PAOs,and the corresponding relationship between the average intensity of Raman characteristic peaks and the surface density of intracellular polymers was determined,and then the intracellular polymer content per unit mass cell was calculated.The quantification of poly-P and Gly in PAOs and GAOs is realized.Raman spectroscopy of intracellular poly-P in PAOs showed that the position of PO2-peak of poly-P in Tetrasphaera PAOs was between 1157 cm-1 and 1165 cm-1.The PO2-peak position of poly-P in Accumulibacter genus PAOs is between 1168 cm-1 and 1177 cm-1,all of which are unimodal distribution.The PO2-peak location of polyP in PAOs of different S2EBPR wastewater treatment plants could be divided into three phenotypes,with 1157 cm-1,1164 cm-1 and 1172 cm-1 as the center,respectively.Different phenotypes of PAOs may affect the phosphorus removal performance of S2EBPR system.The peak position of poly-P in PAOs is also affected by the addition of different carbon sources.When acetic acid is used as carbon source,most of the peak positions of PO2-are between 1165～1177 cm-1,which may be Accumulibacter phenotypes using acetic acid.When using actual sewage as carbon source,the peak position of PO2-is mostly between 1155～1163 cm-1,indicating that in addition to Accumulibacter bacteria there are other different genera or undiscovered PAOs involved in biological phosphorus removal.Experimental results in this chapter show that SCRS technique can be used as a method to explore and evaluate intracellular poly-P characterization of PAOs,and prove its feasibility in real EBPR system.(2)In the long-term anaerobic fermentation experiment of activated sludge,the abundance of PAOs and GAOs and intracellular polymer content were affected by different anaerobic fermentation time.The relative abundance of PAOs containing poly-P based on SCRS method increased to 9.4%in the first 60 h of fermentation and then decreased to 5.7%.The relative abundance of GAOs containing Gly decreased from 16.3%to 2.4%at 60 h before fermentation,indicating that longer anaerobic time could eliminate GAOs.The results showed that the phosphorus release/acetic acid utilization ratio(P/HAc)increased from 0.33 P-mol/C-mol to 0.80 P-mol/C-mol 60 h before fermentation,and then decreased to 0.37 P-mol/C-mol,which was speculated to be related to the decrease of PAOs activity.The average Raman peak intensities of poly-P and Gly cells decreased 60 h before fermentation,and then tended to be stable,which was consistent with the results obtained by conventional method,which proved the feasibility of SCRS method to determine the changes of polymer content in microbial cells.The results of this chapter show that the most suitable time for the metabolic growth and enrichment of PAOs is 60h by the long-term anaerobic fermentation experiment of activated sludge.(3)SCRS and Raman-FISH techniques are used to study the abundance of active PAOs and GAOs and intracellular polymers in the main and side-stream reactors of S2EBPR system.The results showed that under the condition of 20%and 15%side-stream ratio(SRTss were 72 and 96 h),PAOs was more active in the aerobic stage of the main stream reactor,and the relative abundance increase value of PAOs containing poly-P was 6.70%and 3.73%,respectively.The relative abundance of GAOs containing Gly was 6.72%and 4.23%,respectively,which had little change compared with other lateral flow conditions.Under prolonged anaerobic conditions(SRTss=96 h)in the side-stream reactor,the relative abundance of PAOs changed little,while the relative abundance of GAOs decreased by 10.59%,indicating that the side-stream condition could deplete the intracellular Gly of GAOs and enter the cell decay stage,while the intracellular PAOs still contained poly-P,which could be used for energy metabolism.It is in a competitive position with GAOs.Based on the above reasons,the aerobic phosphorus absorption activity of PAOs in the aerobic stage of the mainstream reactor is improved,and the poly-P Raman peak signal intensity is increased significantly,which is conducive to ensuring the phosphorus removal performance and stability of the system.