| In the past century,the contaminations of nitrogen and perchlorate in waters have become a worldwide problem,posing a strong threat to humans and the natural environment.Traditional biological processes for wastewater treatment often require the addition of carbon sources which lead to high operating costs.And they have become an important source of greenhouse gas methane.The newly discovered denitrifying anaerobic methane oxidizing microorganisms can sim ultaneously realize methane utilization and biodegradation of pollutants,providing a new solution for the development of sustainable and efficient water treatment technologies.In view of the nitrogen pollution and perchlorate pollution in waters,the anaerobic cultivation of denitrifying anaerobic methane oxidation?DAMO?microorganisms in both batch serum bottles and sequencing batch reactors were carried out.And then the combination of anaerobic ammonium oxidation?Anammox?and DAMO was further explored in a moving bed biofilm reactor?MBBR?.While there are many similarities between the bioreduction of perchlorate and denitrification process,the feasibility study of anaerobi c methane oxidation coupled with perchlorate reduction process is still limited and controversial.Thus,the performance,metabolic mechanism and microbial community characteristics of anaerobic methane oxidation coupled with perchlorate reduction w ere investigated in a membrane biofilm reactor?MBfR?.Moreover,the effects of hydraulic retention time and water temperature on anaerobic methane oxidation coupled with perchlorate reduction were discussed.The main findings are as follows:?1?The anaerobic cultivation of DAMO microorganisms was conducted by using mixed sludge from an anaerobic biofilm reactor.The activity of nitrate-acclimating DAMO microorganisms was improved,with the averaged reduction rate of nitrate increased by 1.022 mgN/g VSS·d-11 as time went on.The DAMO microbial growth accord ed with Monod equation with NO3-as substrate,and the maximum specific removal rate(qmax)was 10.08 mgN/g VSS·d-1,and the half saturation coefficient?Ks?was 12.88 mgN/L.Batch tests without methane confirmed the significance of CH 4 as a carbon source and electron donor for biological denitrification of DAMO microorganisms.A large number of cylindrical rod-shaped cells were observed in the DAMO enrichment,which was similar to the morpholo gy of anaerobic methane-oxidizing archaea ANME-1 reported in previous literatures.The microbial community was mainly composed of methanotrophs such as Methylobacter,Methyloversatilis,and denitrifying bacteria Denitratisoma and Hydrogenophaga.The abundances of the key microbial populations were significantl y different between the DAMO enrichment with different electron acceptor.The nitrate-acclimating DAMO enrichment contained more methanotrophs and methylotrophic bacteria.The experimental results showed that different electron acceptors had great influence on the activity of DAMO microorganisms and microbial community structure.Nitrate was more suitable for the long-term cultivation of DAMO microorganisms,while relatively high concentration of nitrite had an inhibitory effect on the activity of DAMO microorganisms.?2?The DAMO process could be achieved in sequencing batch reactors at the normal temperature of 20°C,with continuous supply of CH4 and NO3-as substrates.The DAMO activity was improved with the average d reduction rate of nitrate increased by 2.59 mgN/g VSS·d-1,compared with the previous anaerobic batch cultures.NO2-was accumulated during the reaction but was rapidly reduced when the concentration of NO3-decreased by 10 mgN/L,indicating that both of the nitrate-and nitrite-dependent DAMO process existed in the reactor.Fluorescence in situ hybridization analysis confirmed that DAMO archaea and DAMO bacteria were obviously enriched.The microbial community mainly included denitrifying bacteria,methanotrophs,methylotrophic bacteria,and archaea,such as Denitrasoma,Rhodocyclaceaeunclassified,Chitinophagaceaeunclassified,Methylocystis and Methylotenera,Methanobacterium,which played important roles in the DAMO process of the sequencing batch reactor.?3?Coupling of anaerobic ammonium oxidation?Anammox?with anaerobic methane oxidation?DAMO?was achieved in a moving bed biofilm reactor?MBBR?,inoculated with anammox microorganisms in biocarriers and DAMO flocs.The nitrate reduction rate increased to 83.66 mg N/L·d-1,and the ammonium oxidation rate was kept at 62.67 mgN/L·d-1,and nitrite was not present in the MBfR.The nitrite conversion rate for anammox reaction accounted for over 74%.The results showed that the biological denitrification could be realized by combining anammox bacteria and DAMO archaea.Fluorescence in situ hybridization analysis confirmed that there was a significant coexistence of DAMO archaea and a large number of anammox bacteria,as well as DAMO bacteria and met hanotrophs in the rector,which were the key microbial populations responsible for simultaneous nitrogen and methane removal.High-throughput sequencing results showed that the microbial communities consisted of anammox bacteria?Candidatus Scalindua?,methanotrophs?Methylobacter and Methylomonas?,methylotrophic bacteria?Methylotenera and Hyphomicrobium?,and denitrification bacteria?Denitratisoma and Rhodocyclaceaeunclassified?.?4?Perchlorate bioreduction coupled to methane oxidation was successfull y achieved without the addition of nitrate or nitrite in a membrane biofilm reactor?MBfR?inoculated with a mixture of freshwater sediments and anaerobic digester sludge as well as return activated sludge.The reactor was operated at different methane pressures?60,40 and 20 Kpa?and influent perchlorate concentrations?1,5 and 10 mg/L?to evaluate the biochemical process of perchlorate bioreduction coupled to methane oxidation.Perchlorate was completely reduced with a h igher removal flux of 92.75 mg/m2·d using methane as the sole carbon source and electron donor.Quantitative real-time PCR showed that bacteria prevailed over archaea and the abundances of mcrA,pMMO,pcrA,and nirS genes were correlated with the influent perchlorate flux.?5?The microbial community in the MBfR for anaerobic methane oxidation coupled with perchlorate bioreduction reactor were further investigated.The results show ed that the microbial community richness and diversity were slightly reduced due to microbial domestication and selection.However,it recovered and improved with the increase of the influent ClO 4-concentration over time.With the continuous introduction of high concentrations of methane and perchlorate,a specific complex microbial community has formed,which was an important reason for anaerobic methane oxidation coupled perchlorate in the reactor.It mainly included:methanotrophs?Methylocaldum,Methylobacteriaceae and Methylosinus?;methylotrophic bacteria?Hyphomicrobium,Pseudoxanthomonas and Methylophilus?;perchlorate-reducing bacteria?Azospira,Pseudomonas,and Bacillus?;and common denitrifying bacteria?Clostridium and Rhodobacter?.?6?Both HRT and water temperature had a significant effect on the bioreduction of perchlorate in the methane-based MBfR.The optimal HRT was 18h.When the reactor was stabl y operated,the removal efficiency of perchlorate reached 100%,and the abundances of key functional genes nirS,prcA,mcrA and pMMO increased,showing the growth of key populations responsible for the anaerobic methane oxidation coupled with perchlorate reduction.When the water temperature was graduall y lowered from 30?to10?,the perchlorate removal rate decreased from 100%to 66.63%;but it recovered when the water temperature was restored.The the abundances of genes nirS,prcA and mcrA was impacted by decreasing water temperature,reflecting the growth of perchlorate reducing ba cteria,denitrifying bacteria and methanotrophs were limited.However,high throughput sequencing analysis showed the functional populations still dominated the reactor,which was the key reason for methane-driven perchlorate reduction under relativel y low temperature. |
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