Mechanism Of Rapid Start-up And Metabolic Pathway Reconstruction Of Anammox

The start-up of anaerobic ammonia oxidation(Anammox)process is a bottleneck for autotrophic biological nitrogen removal process.Granular sludge and biofilm are two common forms of functional microorganisms in wastewater biological treatment processes,which are often studied separately.The pre-immobilized biofilm is beneficial to maintain the retention of biomass in the Anammox reactor.The loss of Anammox cells during the early start-up of the Anammox reactor is particularly serious,which is one of the restrictions for fast start-up of Anammox process.In this study,the Anammox biofilm and particles were rapidly enriched by the internal circulation immobilized blanket(ICIB)and the sequential immobilization and granulation approach(SIGA)technologies,and the abundance of Anammox bacteria in the microbial community of the Anammox reactor was evaluated.After 100 days of bacteria pre-enrichment by the carrier,the relative abundance of Anammox bacteria reached 4-5%.The ICIB reactor,inoculated by the carrier biofilm,operated for 180 days.The nitrogen removal rate of the ICIB reactor was 1.11 kg·N/(m~3·d),while the nitrogen removal rate of the conventional UASB reactor in the control group was only 0.29 kg·N/(m~3·d).Illumina Hi Seq sequencing based on the 16S r RNA gene showed that the relative abundance of Anammox bacteria(Candidatus Kuenenia,Candidatus Brocadia,and Candidatus Jettenia)on the biofilm reached 42.5-50.6%,and the relative abundance of the granular sludge reached 45.3?47.1%in the ICIB reactor.In contrast,Anammox bacteria were not enriched in the sludge of the UASB reactor.These results indicated that the high relative abundance of Anammox bacteria were formed in both particles and biofilms forms in ICIB reactor.The existence of Anammox biofilm and its internal circulation promoted the formation of Anammox granular sludge,and free Anammox cells escaping from the carrier biofilm provided parent cells that can form Anammox granules under a suitable hydraulic shear condition.This study provided a new strategy of fast start-up of anaerobic ammonia oxidation based on ICIB reactor with SIGA-based activated sludge as inoculant.A new Anammox reactor was started within 180 days.The total nitrogen removal efficiency reached 92%,and the nitrogen removal rate(NRR)reached1.21 kg·N/(m~3·d).The second-generation Illumina sequencing based on 16S r RNA genes,third-generation SMRT sequencing and metagenomics methods were used to explore the changes of microbial communities before and after the reactor was started up simultaneously.The differences in the bacterial detection,abundance characterization,relative abundance characterization,interspecies interaction,and functional gene characterization of Anammox were compared with FISH data as a reference.Meanwhile,the related functional genes of Anammox were identified and analyzed,and the metabolic pathway of Anammox was reconstructed based on the results of metagenomics.The second-generation Illumina Hiseq results were most similar to the FISH detection results in detecting the relative abundance of Anammox bacteria in the sample,which was most suitable for rapid detection of their presence and relative abundance in the community;third-generation sequencing SMRT provided more accurate(species level)information in community species level and bacterial classification,and provided more specific community structure and inter-species interaction information,which could accurately reveal the mechanism of microbial interaction between Anammox and various bacteria existed in wastewater treatment process.The relative abundance of Anammox bacteria characterized by metagenomics was much smaller than the other two sequencing methods,and the dominant flora was not suitable for characterizing the abundance of Anammox bacteria in the community.Metagenomic sequencing could provide direct evidence for the metabolic pathway of Anammox,which could be used to reveal the metabolic pathway and potential of Anammox.Partial nitrification-Anammox process was performed to analyze the efficiency of low C/N wastewater treatment.Meanwhile,a draft genome of the pure Anammox bacterium Candidatus Kuenenia stuttgartiensis was constructed with the core methods of precise single cell sorting,high-throughput sequencing,and micro-genome sequencing,and metabolic activity of Candidatus Kuenenia stuttgartiensis was analyzed.The partial nitrification SBR reactor was successfully started within 150 days,and the community structure analysis based on the 16S r RNA gene showed that AOB(Nitrosomonas)was the dominant population in the granular sludge of partial nitrification,and AOA was the dominant archaea in the granular sludge of partial nitrification.However,the dominant flora of AOA changed from Candidatus Nitrososphaera(before start-up)to Candidatus Nitrosotenuis(after start-up).Besides,the influence of different aeration methods and aeration rates on the partial nitrification process was explored,and the low-concentration aeration(50 L/h)was beneficial to the ammonia nitrogen accumulation rate.The partial nitrification-Anammox process was successfully operated,and the total nitrogen removal rate in the coupled system exceeded 90%,and the concentration of nitrite and nitrate in the effluent was less than 5 mg/L.A single-cell sorting technique was developed based on Raman spectroscopy clustering,and was applied successfully obtaining biological samples of 14 cells of the pure Anammox bacterium Candidatus Kuenenia stuttgartiensis and its characteristic Raman spectra.The metabolic pathways of Candidatus Kuenenia stuttgartiensis such as carbon,nitrogen,and respiratory chain were reconstructed,and the EMP pathway was found in the genome,indicating the ability of Candidatus Kuenenia stuttgartiensis to metabolize organic matter.A high-abundance nitrogen-fixing gene nif U was found during the reconstruction of the nitrogen metabolic pathway,which indicated that Candidatus Kuenenia stuttgartiensis has the potential nitrogen-fixing function.A Na~+-dependent respiratory chain was observed in the metabolic pathway of the respiratory chain of Candidatus Kuenenia stuttgartiensis.This project developed a new method for the rapid start-up of the Anammox process,revealed the Anammox microbial population interaction mechanism,reconstructed the gene sketch of Candidatus Kuenenia stuttgartiensis,and provided an important theoretical basis and technical reference for the practical application of Anammox.