Synergistic Symbiosis Of Double PAOs To Enhanced Biological Phosphorus Removal Based On Different Types Of Organic Substrates

The water eutrophication caused by nitrogen and phosphorus nutrients has always been the focus of our country’s control,especially the control of phosphorus has become more and more strict in recent years.The influent carbon source is extremely important for the stable operation of biological nitrogen and phosphorus removal,due to the generally low COD of our country’s municipal sewage influent,how to use effective carbon source and what kind of carbon source can improve the efficiency of biological phosphorus removal is a key problem.In the past,the biological phosphorus removal theory believed that traditional PAOs led by Accumulibacter used VFAs in influent to remove phosphorus,with the study went deep into EBPR system,the fermented PAOs which can utilize a variety of organic substrate types and have the ability to ferment,were gradually emerging in people’s eyes.This experiment used different types of organic substrates as the research objects,the characteristics of fermented PAOs to ferment larger molecular weight carbon sources（glucose,protein,amino acids,etc.）into VFAs was adopted to achieve"dual PAOs"synergistic phosphorus removal,and it provided a new idea for simultaneous nitrogen and phosphorus removal in our country’s low VFAs concentration wastewater treatment plants.In the first stage,SBR reactor was operated with glucose as a single carbon source,keep the influent COD concentration at 400mg·L^-1,NH₄⁺-N concentration at 20mg·L^-1,PO₄^3--P concentration at 15mg·L^-1,and the change of nitrogen and phosphorus removal performance in EBPR system during PAOs enrichment was monitored.The results showed that the EBPR system became stable after 40 days,and the average phosphorus removal rate increased from29.6%to 43.3%,but the overall phosphorus removal rate was not high due to the limitation of acclimation time.The typical cycle monitoring of EBPR system showed that the anaerobic phosphorus release reached the peak at 90min,followed by the phenomenon of anaerobic phosphorus uptake and continued to the end of aerobic phase,indicating that glucose as a single carbon source triggered the"anaerobic phosphorus uptake"metabolism of Tetraphaera-related PAOs.In the aerobic stage,the phosphorus uptake was completed by the fermented PAOs and the typical PAOs which depended on its growth,the synergistic metabolic mechanism of"dual PAOs"was preliminarily observed.The removal performance of NH₄⁺-N was good during the whole enrichment process,and the use of glucose as carbon source had no effect on sludge properties.In the second stage,the influent PO₄^3--P concentration was 30 mg·L^-1 and casein hydrolysate was used as a single carbon source to operate EBPR system,it was found that after40 days,the average phosphorus removal rate of the system remained stable at 41.8%and did not increase further,therefore,the concentration of PO₄^3--P was adjusted to 15 mg·L^-1.After changing the working conditions,the phosphorus removal rate decreased rapidly,and the lowest value was 16.1%after 80 days of operation,indicating that the impact load resistance of EBPR system with casein hydrolysate was poor,and adjustment of influent PO₄^3--P concentration did not significantly improve the phosphorus removal performance of the system.Then reactor was restored to the initial working condition,but the effluent quality was still not recovered and became worse.The typical cycle monitoring showed that the EBPR system had obvious"anaerobic phosphorus uptake"metabolism,and the PAOs with fermentation ability absorbed PO₄^3--P through the energy produced by amino acid fermentation.The change of influent PO₄^3--P concentration had little influence on the denitrification performance of the system,but the overall removal rate was not high,indicating that the NH₄⁺-N generated after amino acid hydrolysis cannot be completely removed by the oxidation of dissolved oxygen.In the three conditions,the poly-P content in PAOs tended to decrease,and the level of poly-P particles in activated sludge system decreased,the mean value of MLSS decreased from 2615 to 1981mg·L^-1,and the amount of PAOs decreased.In the third stage,the nitrogen and phosphorus removal performance of EBPR system under the conditions of mixed carbon source HAc:Cas aa being 4:0,3:1,1:1,1:3 and 0:4 was investigated,keep the influent COD concentration at 400mg·L^-1,NH₄⁺-N concentration at20mg·L^-1,PO₄^3--P concentration at 15mg·L^-1.At first,the EBPR system with HAc as the initial carbon source operated after 50 days,the phosphorus removal efficiency reached about90%,then Cas aa was gradually added to the system at 3:1,1:1 and 1:3,the system recovered phosphorus removal performance after 50 days,10 days and 6 days,respectively,and maintained the phosphorus removal efficiency above 80%,indicating that PAOs in the system had a good adaptability to Cas aa.After Cas aa completely replaced the mixed carbon source,the phosphorus removal performance of the system decreased slightly to 77.5%,at this time,PAOs with fermentation capacity mainly relied on carbon source fermentation to provide VFAs to traditional PAOs.The typical cycle monitoring of each working condition showed that with the gradual addition of Cas aa to SBR,the duration of anaerobic phosphorus release gradually increased,indicating that the fermented PAOs absorbed amino acids for fermentation and degradation into VFAs in the anaerobic environment,thus provided necessary carbon source for the anaerobic phosphorus release of traditional PAOs,therefore,with the increased of enrichment degree of fermented PAOs,the anaerobic phosphorus release time increased gradually.During the changed of five working conditions,due to the increased of the addition ratio of Cas aa,the groups in the amino acid react with water to generated more NH₄⁺-N,and the removal rate of NH₄⁺-N gradually decreased.The MLSS,MLVSS and f value showed an upward trend in each stage of carbon source replacement.In the forth stage,the microbial population structure characteristics of four activated sludge samples with influent carbon source:Glucose,HAc:Cas aa=4:0,1:1 and 0:4 was investigated.The results of high-throughput sequencing showed that the abundance and uniformity of species in each sample were high enough,the chao1 index and ace index showed that the total number of bacteria was the largest when the influent mixed carbon source HAc:Cas aa=1:1,but from the shannon index and simpson index,the community richness decreased and the system was more likely to enrich the dominant population.Unifrac distance matrix analysis,NMDS analysis and UPGMA tree analysis of the samples showed that the difference between the system using Glucose as carbon source and the system with HAc:Cas aa of 0:4 was the smallest,when the mixed carbon source HAc:Cas aa changed from 4:0 to 0:4,the species types of HAc:Cas aa of 4:0 and HAc:Cas aa of 1:1 were similar.The bacteria at the phylum level in the Glucose samples were mainly Bacteroidota,Proteobacteria,and Patescibacteria,when the mixed carbon source HAc:Cas aa changed from 4:0 to 0:4,the main bacteria at the phylum level were Proteobacteria and Patescibacteria.The genus-level community distribution analysis of samples under mixed carbon source changes in various conditions showed that the dominant species of microbial genus level in HAc:Cas aa=1:1 condition with the highest phosphorus removal rate were mainly as follows:Thauera（14.7%）,Zoogloea（5.1%）,Acinetobacter（6.0%）,Flavobacteriaceae＿Unclassified（16.9%）,Candidatus＿Accumulibacter（6.5%）.