Population Dynamics And Morphotypes Analysis Of PAOs In Denitrifying Phosphorus Removal Systems

Compared with the traditional phosphorus removal process,denitrifying phosphorus removal process using nitrate or nitrite as electron acceptor realizes the simultaneous denitrification and polyphosphate removal。It theoretically saves the carbon source,reduces the residual sludge disposal and energy consumption.As an important functional microorganism,abundance and activity of denitrifying polyphosphate accumulating organisms（DPAOs）determine the removal efficiency of nitrogen and phosphorus.But due to the understanding of composition and structure of this microbial community is not perfect,there always be unexplained low nitrogen and phosphorus removal efficiency,and sewage treatment plant process can not be timely and effective regulated.So,as theoretical support,the analysis of nitrogen and phosphorus removal microorganisms by molecular biological methods can be more conducive for the operation of sewage treatment plant.Accumulibacter was dominant population of Polyphosphate Accumulating Organisms（PAOs）in EBPR system.Its community structure and metabolic activity directly affect the efficiency of phosphorus removal.Researchers hold dififfenent opinions about distinction between PAOs and DPAOs.And theory of denitrification and polyphosphate removal with these bacteria is not clear.Correlations between the characteristics of Accumulibacter clades and the effect of denitrification phosphorus removal process remain to be further studied.Corrlations between microbial population dynamics and denitrifying phosphorus removal efficiency in SBR under different operating conditions were studied in this paper.Besides,morphological characteristics of Accumulibacter clades and interrelations between PAOs and denitrifying bacteria were analysised in this study.SBR was conducted under anaerobic-aerobic condition for the enrichment of PAOs with oxygen as electron acceptor and propionic acid as carbon source.The community structure of dominant PAOs in system was analyzed by molecular biology method.Two SBR reactors（SBR 1 and SBR2）were conducted under anaerobic-anoxic-aerobic condition,and DPAOs were enriched with nitrate and nitrite as electron acceptors respectively.Dominant Accumulibacter enriched in different systems was analyzed qualitatively and quantitatively by FISH and qPCR.After hybridized and labled,PAOs with different morphology was quantified by new developed FISH-Staining-Flow cytometry method.Compared with the relative abundance of Accumulibacter clades,the morphological characteristics of Accumulibacter clades were analysed.Finally,PAOs labled were separated and purified through Flow cytometry,and functional gene ppk1 encoding polymeric phosphate kinase and the denitrification gene narG、nirS、nirK were used as genetic markers for phylogenetic analysis.SBR using propionic acid as carbon source was operated under anaerobic-aerobic condition and qPCR quantitative results showed that Accumulibacter gradually became the dominant PAOs in system and dominant clade IIC（ppk1 excluding OTU NS D3）accounted for 82.11%of Accumulibacter at the end of operation.When activated sludge in SBR was partially lost,the system can still maintain good phosphorus removal efficiency.As a dominant clade,IIC（ppk1 excluding OTU NS D3）has good load resistance and efficient phosphorus removal ability.FISH showed that polyphosphate accumulated organisms are the dominant bacteria in system.And DAPI staining showed that PAOs containing polymeric phosphate granules were widely distributed in microbial community.SBR1 was operated for 112 days with nitrate as electron acceptor,and the abundance of PAOs increased from 6.02×10¹¹copies/dry sludge to7.27×10¹¹copies/g dry sludge.With the ratio of clades IIA,IIB and IIC（ppk1 excluding OTU NS D3）decreased gradually,clade IIC became dominant Accumulibacter in system at the 112^thd.When influent carbon source was sufficient,clades IA and IIC showed more competitive advantage and played an important role in denitrifying phosphorus removal.With insufficient carbon load,clades IID and IIF were more competitive and can efficiently utilize organic substances for growth and metabolism,playing the role of nitrogen and phosphorus removal.By comparing the quantitative results of qPCR with the Flow cytometry,it was found that clade IIC in SBR1 is coccus and belonged to Group1,besides,clade IA may be rod and belonged to Group3.In addition,the equivalence test of the quantitative results of Flow cytometry confirmed the stability of the quantitative result,and the more accurate Flow cytometry quantitative results were obtained.SBR2 was operated with nitrite as the electron acceptor,the concentration of influent carbon source has a serious effect on the synthesis of PHAs,which affects the normal operation of reactor.But when the concentration of COD was too high,the SS（Suspended solids）of effluent became higher and sludge was lost.Clades IIC（ppk1excluding OTU NS D3）and IIF were important microbial strains for denitrification and phosphorus removal in poor operation of SBR.Abundance of clades IIC and IID was relatively high and always being rising in entire operation.Both of them played a very important role in phosphorus removal and gradually became the dominant clades in SBR2.qPCR quantitative results showed that dominant clade of system was IIC and its abundance was 3.17×10¹⁰cels/g dry sludge,which accounted for 42.94%of total Accumulibacter.Besides,IID abundance was 2.28×10¹⁰cels/g dry sludge（30.89%）and IIF abundance was 1.1×10¹⁰cels/g dry sludge（14.95%）.The sum of these three clades accounted for more than 90%of Accumulibacter;By comparing the quantitative results of qPCR with Flow cytometry,it was found that clade IIC and IID corresponding to Group1 is coccus;clade IIF corresponding to Group2 is coccobacilli,and clade IA may be rod corresponding to Group3.Based on the genetic markers of ppk1 and denitrifying functional genes narG、nirS、nirK,the results of phylogenetic analysis of PAOs sorted by FISH-Staining-Flow cytometry method indicated that SBR1 has a richer diversity of Accumulibacter than SBR2.SBR2 contained none of type I or type II Accumulibacter and it belonged to a new group of evolutionary branch Group II,which was subordinate to the Actionbacteria.Propionibacterium Acnes may be PAOs containing the membrane-bound nitrate-reducing gene（narG）.The red bacterium subspecies Paracoccus denitrificans accounted for highest relative abundance ofβ-Proteobacteria and was suspected as PAOs containing nirS.The evolutionary relationship between S1-19 and Thauera Aromatica of Thauera aromatica in phylogenetic tree was nearly 100.Thauera Aromatica may be a kind of PAOs containing nirS,which is close to Accumulibacter in evolutionary relation.A lab-scale Modified University of Cape Town（MUCT）process was used to treat domestic sewage with low C/N ratio,and the relationship between microbial community structure and efficiency of denitrifying phosphrous removal under nitriation and nitrification was studied.The results showed that during nitrification,the phosphorus release efficiency in anaerobic zone was good.But it decreased with the accumulation of nitrite at the end of Phase II and Phase III.The ammonia removal efficiency along the process operation in anoxic zone was nearly 65%and there may be anaerobic ammonia oxidation（Anammox）with nitrate or nitrite as electron acceptors to achieve denitrification.During nitritation,abundance and ration of Type II clades increased.Clades IIC,IIC（ppk1 excluding OTU NS D3）and IIF had a faster growth rate and their ability to denitrification with nitrite was stronger than other clades,which played an important role in denitrifying and phosphrous removal.Clade IID has been dominant in both of nitriation and nitrification and was the most important clade in N and P removal throughout the entire operation.FISH-Staining-Flow cytometry quantitative results showed that PAOs accounted for 22%of the total bacteria in system at the 120^th day,close to qPCR quantitative results 12.3%（3.6 copies/cell）and 14.01%（4.1 copies/cel）.PAOs before and after sorted by Flow cytometry was studied through phylogenetic tree,which was based on genetic markers of ppk1 and denitrification gene narG、nirS、nirK.The results showed that Accumulibacter clades in MUCT process treating domestic sewage were very rich.Besides some Type I clades,there were 6 Type II clades and their relative abundance were relatively high.The phylogenetic analysis of the sorted samples showed that the evolutionary branches containing narG belonged to Actionbacteria,which was consistent with the analysis results of SBR.Bosea that belonged to subspecies of Bradyrhizobiaceae ofα-Proteobacteria may be PAOs with denitrification gene nirK.