Enrichment Of Waste Activated Sludge-based Anammox Consortia And Enhanced Nitrogen Removal By Coupling With Methane Recirculation

With the rapid enhancement of industrialization and improvement of living standard,a large amount of nitrogen-containing wastewater is discharged continuously,which causes adverse effects on human health and the ecological environment.Anaerobic ammonium oxidation（Anammox）,as a novel technology for nitrogen removal,has been widely used due to its superiority of low cost,energy saving,and high efficiency.However,it still cannot achieve complete removal of nitrogen.Meanwhile,due to the shortages of low content,disordered emissions,difficulty to recycle and cause of the greenhouse effect,CH₄-containing biogas produced from artificial sources such as WWTPs（Wastewater treatment plants）and landfills has a serious impact on the global environment.The methane-oxidation coupled nitrogen removal system can simultaneously remove nitrogen while reducing methane emissions,which has a broad application potential in nitrogen removal of wastewater and mitigation of greenhouse effect.However,both single Anammox system,and methane-oxidation coupled nitrogen removal system still have many technical issues,such as long start-up time,high operating cost,susceptibility to environmental variations,and poor process stability.In this study,to overcome such problems,more functional bacteria with high bioactivity were enriched in upflow anaerobic sludge bed（UASB）inoculated with wastewater activated sludge（WAS）.Moreover,the external methane-rich biogas was used as electron donor to regulate the nitrogen removal process.A series of researches have been carried out to optimize the efficiency of nitrogen and methane removal system and analyze the evolution and enrichment of key functional bacteria during the operation process.The main conclusions were as follows:（1）For enriching WAS-based anammox consortia and optimizing nitrogen removal performance,nitrogen removal performance,element composition and micro-structure of granule and community evolution were investigated.The results showed that Anammox system was started up successfully when inoculated with WAS in UASB bioreactor.The whole process was divided into four phases:cell lysis phase（I）for elimination of nonfunctional bacteria,lag phase（II）for activation of functional bacteria,activity elevation phase（III）for multiplication of biomass,and stationary phase（IV）for enhancement of key functional organism.The results indicated that the removal rate of NH₄⁺-N,NO₂^--N and TN stabilized at 98.7±0.9%,90.6±1.9%and 75.1±2.6%,respectively.The stoichiometric parameter stepped up to R_i(NO^-_{2【consumed】}/NH⁺_{4【consumed】})=1.22±0.03 and R_a(NO₃^-_{【produced】}/NH⁺_{4【consumed】})=0.28±0.04.The element formula of the Anammox granule was CH_2.55O_0.72N_0.13S_0.06.SEM and FISH analysis confirmed that spherical and ellipsoid anammox cell clustered tightly.In addition,AOB（Ammonium oxidation bacteria）and An AOB（Anaerobic ammonium oxidation bacteria）were symbiotic and anammox bacteria dominated the whole granule while AOB were distributed in the outlayer.The interaction force between cells made them aggregate together to form a primary structure.Then,the EPS（Extracellular polymeric substances）enhanced the aggregation and polymerized primary structure into bigger cluster,namely secondary structure.Moreover,the EPS stimulated the cluster to adhere abiotic surface.Thus,close linkages and spatial structures were formed,which benefited cells’stability and expanded reaction area and thereby increased efficiency.In the Anammox granule,Planctomycetes,Proteobacteria,and Bacteroidetes were the major phyla,accounting for 43.7%,24.4%and 13.8%,respectively.During the cultivation process,richness of the microbial community showed a first decrease and then increase tend（2000→1400→2000）.After that,a clear drop to 600 was observed.The abundance of Planctomycetes increased to 47.1%and finally became dominant at the stationary phase.From start-up to stationary phase,the dominant Anammox bacteria in microbial community was shifted from Ca.Anammoximicrobium to Ca.Brocadia and then to Ca.Jettenia.Under high-nitrogen condition,the Anammox community was further shifted from Ca.Jettenia to Ca.Brocadia.（2）For developing and optimizing methane-oxidation coupled nitrogen removalprocess,nitrogen removal performance,element composition and micro-structure of granule and community evolution were investigated.The results showed that the advanced nitrogen removal coupled with CH₄circulation system was started up successfully when inoculated with WAS in UASB bioreactor.The whole process was divided into three phases:primary phase（I）,exogenous anammox effluent-intervention phase（II）and CH₄removal strengthening phase（III）.The result indicated that the removal rate of NH₄⁺-N,NO₂^--N and TN stabilized at95±0.9%,92.1±1.2%and 80±2.1%,respectively.The average yield of N₂was 109±11.9 m L/L/d and the nitrogen removal efficiency was 5%higher than that of the previous Anammox system.In addition,the average methane utilization and utilization rates were 175±21.5 m L/L/d and 71.3±3.1%,respectively.SEM analysis confirmed the existence of ellipsoid,spherical and rod-shaped cells,and ellipsoid and spherical cells usually existed in clusters,which was more conducive for cells to transfer mass and particles,while the rod-shaped cells existed alone.In the initial period of phase I,Denitratisoma（1.1%）was the main nitrogen removal bacteria while Methylococcales（1.0%）was key CH₄removal bacteria.In the phase II,introduction of exogenous Anammox effluent led to expansion of anammox consortia.Thus,anammox bacteria（14.0%）and denitrification bacteria（8.1%）took charge of nitrogen removal while NC10（0.09%）and Methylococcales（2.1%）were responsible for CH₄removal.In the phase III,denitrifying bacteria（Denitratisoma,4.0%;Methylophilaceae,3.0%）and anammox bacteria（1.2%）were major nitrogen removal bacteria,meanwhile Methylococcales（8.1%）and NC10（1.0%）were responsible for CH₄removal.A variety of functional microorganisms existed in the coupled system,such as AOB（Ammonium oxidizing bacteria）,NOB（Nitrite oxidizing bacteria）,DNB（Denitrification bacteria）,An AOB（Anaerobic ammonium oxidation bacteria）,AMOB（Aerobic methane oxidation bacteria）,Ni-DAMO（Nitrite-dependent anaerobic methane oxidation）and o AB（Other autotrophic bacteria）.AOB,NOB,DNB and An AOB took part in nitrogen removal.Moreover,the competitiveness of anammox bacteria was weaker than that of denitrifying bacteria.AMOB and Ni-DAMO participated in CH₄removal.