System Trajecotry Analysis For Spatial Community Succession And Its Application In A～2/O Fixed Biofilm Process For Coking Wastewater Treatment

Wastewater treatment plant (WWTP) is an engineered microbial ecosystem often consisted of several reactors with complementary functions to each other, such as the widely used Anaerobic-Anoxic-Oxic (A1-A2-O) process. Relationships and interactions among reactors determine spatial or temporal community succession patterns of WWTP, which is closely related to the performance of whole systems. Previous studies focus on analysis of community structure of individual reactors in WWTPs, but few of them give insights to interactions and relationships among communities of all reactors in one system. Analysis of spatial or temporal succession patterns in systems can help us optimize the design and operation of WWTPs. One industrial system with A1-A2-O fixed biofilm process was built for treating coking wastewater but with poor performance ever since its completion, of which the COD and NH3-N removal efficiency was only 54% and -106% due to unknown malfunction. To diagnose the problem in the industrial system and identify conditions for recovering its normal functions, a lab system based on the same process was set up as a reference whose COD and NH3-N removal efficiency were 85% and 95% respectively. System trajectories for spatial community succession patterns can be displayed in two-dimension space by PCA analysis of the 16S rDNA V3 PCR-DGGE fingerprints of all reactors in A1-A2-O systems. This strategy based on system trajectories for spatial community succession patterns has been used as a powerful tool for ecological restoration of the malfunctioning industrial system.There are four parts in this dissertation:In the first part, we considered the malfunctioning industrial system as a damaged ecosystem that calls for ecological restoration interventions. The lab system operated with or without effluent recirculation was considered as a reference system or a system with known system fault. System trajectories of the reference lab system showed a continued succession pattern in which each reactor has a community structure different from its adjacent neighbors. But the industrial system revealed disrupted succession patterns in which the communities in the A2 and O reactors shared almost identical structures, which was most likely due to inadequate aeration in O reactor. Engineers finally found the fault in design and installment of the aeration system in O reactors. After renovation of all malfunctions, the industrial system recovered its full function and the spatial community succession pattern very similar to that of the reference system was concurrently established in it as well. Identification of key populations based on PCA loadings analysis of system trajectory of the reference system suggested that Thauera and Nitrospira populations were the most important in driving community succession from the A2 to the O reactor. The results of real-time PCR indicated that the gradual replacement of heterotrophic Thauera population by autotrophic Nitrospira population in spatial succession from the A2 to the O4 reactor is the governing process for this system. System trajectory analysis of the spatial community succession in WWTP can be used to diagnose where and why malfunctions occur and help us identify key conditions to prompt the ecological restoration of malfunctioning engineered microbial systems.According to the system trajectory analysis, the spatial succession of the lab system without effluent recirculation was obviously delayed after the A2 reactor. Therefore, in the second part, effect of effluent recirculation on community structures of the lab system was studied by comparison of them with (LR mode) and without effluent recirculation (LNR mode). Cluster analysis of 16S rDNA V3 PCR-DGGE fingerprinting profiles indicates that effluent recirculation changes the relationship among communities of individual reactors of the system, especially the A2 reactor. The community structure of the A2 reactor was shifted from one like the A1 reactor community to one like O reactor community in response to effluent recirculation. Further, the different composition of 16S rDNA clone libraries of the A2 reactor under LR and LNR modes revealed that during the shift of community structure of the A2 reactor, denitrifying populations related to Thauera aromatica, Thiobacillus denitrificans and etc. outcompete sulfate reducing bacteria mainly belonging to Desulforhabdus genera as the most predominant functional group . Thauera aromatica-like populations were unique in their versatile ability to degrade aromatic compound under anoxic condition, so they were assumed to be active in the significant improvement of contribution to total COD removal of the A2 reactor in response to effluent recirculation. Because the relationship among communities involving WWTP changes under different operational modes, the system trajectory analysis for spatial or temporal community succession is a good tool in investigation of effects of operations on these communities in a global mode and thus has great potential in sludge optimization of WWTP. To further understand the reason of the malfunctioning of the industrial system, 16S rDNA clone libraries were established for the O reactors of the industrial system (I O2) and its lab reference (LR O2). The composition of LR O2 clone library indicated Nitrosomonas europaea-Nitrosoccus mobilis and Nitrospira genus sublineage I as dominant ammonia oxidizer and nitrite oxidizer in this process respectively,but clones related to nitrifying population was undetected in I O2 clone library indicating low nitrifying population level in the industrial system and thus led to low NH3-N removal efficiency of this system. In addition, although COD removal efficiency of the lab system was far more than that of the industrial system, the proportion of clones related to Thauera genus was lower in LRO2 library than that in IO2 library. The industrial system with lower COD removal efficiency but high abundance of Thauera genus (functionally important populations for COD removal) was due to that low oxygen supply to the biofilm in the O2 reactors made these populations fail to remove COD in high efficiency. Therefore using the abundance of single population (group) as an indicator for the functional status of system may lead to wrong conclusion and spatial community succession pattern of the whole system are more reliable to reflect the functional status of WWTP.Finally, LP-RAPD (Long Primer-Randomly amplified polymorphic DNA) fingerprinting-based community DNA hybridization was used to monitor community structural dynamics and identify genomic fragments whose abundance shifts were concomitant to changes of function of WWTP. The hosts of these genomic fragments were functionally relevant populations and can be used as indicator organisms for systems monitoring. The contribution to total COD removal by the anoxic reactor increased from 4 % in LNR mode to 26 % in LR mode. DNA hybridization revealed one signature band of 2.1kb shared by the A2 and O reactors in LR mode but not LNR mode. Clone library profiling of this band resulted in one predominant 2.1kb genomic fragment (B3) with no homologous sequences in GenBank,which indicates this genomic fragment may be specific for the host microbe. Real-time PCR indicated that copy numbers of B3 in the A2 reactor under LR mode were 69 times higher than that under LNR mode, concomitant to significant increase of COD removal capacity in this reactor, which suggested B3 host is a functionally relevant population in A2 reactor. The different patterns of distribution of B3 in the lab system and the comparable malfunctioning industrial system demonstrated the potential of this genomic fragment as physical markers in systems monitoring. In addition, this genomic fragment may allow sequence-guided isolation of the host microbe.In this study, the malfunction of the industrial system was diagnosed and its function was recovered based on comparison of spatial community succession pattern between the industrial system and the reference system via trajectory analysis. As an important attribution of engineered ecological system, system trajectory analysis for spatial or temporal community succession pattern has significant potential in optimization and control of WWTP.