Denitrifying Anaerobic Methane Oxidation Biofilm Responding To Differing Electron Acceptors

Anaerobic oxidation of methane(AOM)plays a crucial role in controlling the flux of methane from anoxic environments.Sulfate-,nitrite/nitrate-,and iron/manganese-dependent methane oxidation processes have been considered to be responsible for the AOM activities in anoxic niches.However,AOM coupled to microbial chromate/perchlorate/selenate reduction has not been reported so far.The discovery of denitrifying anaerobic methane oxidation(DAMO)has not only improved our understanding of global methane and nitrogen cycles,but also provided new technology options for removal of nitrate from nitrate-contaminated water.Previous studies have demonstrated DAMO organisms could remove nitrate and nitrite from wastewater under strictly anaerobic conditions.However,it is not clear whether the DAMO process enables to remove nitrate(or bromate)under oxygen-limited conditions.Here,a lab-scale membrane biofilm reactor(MBfR)was set up,followed by being inoculated with an archaeal and bacterial DAMO co-culture and provided with methane through bubbleless hollow fiber membranes.This study aimed to explore microbial chromate/perchlorate/selenate reduction coupled with AOM,to explore nitrate/bromate removal from the well-oxygenated groundwater,and to explore synergetic/competitive effects on DAMO archaea/bacteria by coexisting anammox bacteria.Long-term MBfR operation was continuously monitored and mass/electron-balance batch trials were conducted to trace the mass transformation.Global microbial community was characterized and functional microbial groups were disclosed to propose hypothetic pathways.The results herein are expected to not only advance our understanding of the biogeochemical element cycles but also of the diversity of microbially mediated oxyanions reduction in natural environments,as well as to support the development of alternative technologies for these contaminants removal using methane-based MBfR.The main outcomes are as follows.(a)The stimulated growth of DAMO biofilm was demonstrated by coexisting anammox bacteria.The activity enhancement of DAMO archaea and bacteria would be achieved after anammox addition,with the archaeal nitrate removal rate increased from 4.8±0.7 to 9.8±0.2 mg N/L/d.DAMO bacteria and anammox bacteria were responsible for about 91.8%and 8.2%of removing nitrite produced,respectively.To apply this DAMO-anammox process for nitrate-only-contaminated groundwater/wastewater,a portion of nitrate needs to be reduced to ammonium in a pre-treatment stage.FeO powder combined with activated carbon(AC),i.e.Fe0/AC micro-electrolysis system,was thus introduced to enhance nitrate reduction under near-neutral conditions in aqueous solution.(b)Nitrate removal from groundwater,which contains dissolved oxygen(DO)in addition to nitrate,was demonstrated when treating synthetic groundwater containing highly contaminated nitrate(50 mg N/L)and DO(7?9 mg O2/L),with a maximum volumetric nitrate removal rate of 45 mg N/L/d.Accumulations of acetate and propionate were observed in some transient periods,indicating their possible involvement as intermediates in methane oxidation.Candidatus Methylomirabilis(bacterial DAMO)was revealed as the dominant population,and no archaeal DAMO organisms were observed.However,a considerable amount of denitrifiers were developed in this system.It was assumed that,under oxygen-limited conditions,methane was oxidized into volatile fatty acids(VFAs),which then served as carbon sources for these heterotrophic denitrifiers to remove nitrate.Nitrate removal was achieved by DAMO bacteria and denitrifiers engaged in cooperative behaviour.(c)Microbial chromate reduction was demonstrated to be coupled with the AOM.The soluble chromate was indicated to be reduced to Cr(?)precipitates.After chromate-reducing intervention,the methane-oxidizing genus Candidatus Methanoperedens was still present in the microbial community,potentially responsible for the methane-driven microbial chromate reduction with Candidatus Methylomirabilis eliminated.Two pathways of the microbial AOM-coupled chromate reduction were proposed:i)independent Candidatus Methanoperedens or ii)synergistic chromate reducers and Candidatus Methanoperedens convert chromate to Cr(?)precipitates,both with DAMO archaea oxidizing methane as the electron.(d)Microbial perchlorate reduction was demonstrated to be coupled with the AOM.The perchlorate was indicated to be completely reduced to non-toxic chloride.The dominated perchlorate reducers,such as Dechlorosoma and Azoarcus,were found in the biofilm after perchlorate-reducing intervention,with methane-oxidizing genera Candidatus Methanoperedens and Candidatus Methylomirabilis still present.A pathway of the microbial AOM-coupled perchlorate reduction was proposed with these phylotypes engaged in a synergistic behaviour:methanotrophs activate methane and secrete some intermediate which then served as the electron donor for perchlorate reducers to respire perchlorate.(e)Microbial selenate reduction was demonstrated to be coupled with the AOM.The soluble selenate was indicated to be reduced to nanoparticle elemental selenium.Candidatus Methanoperedens and Candidatus Methylomirabilis were revealed as the only known methane-oxidizing genera after nitrate was switched to selenate,suggesting these organisms probably coupling AOM to selenate reduction.(f)Microbial bromate reduction was demonstrated to be driven by methane under oxygen-limited conditions.A complete reduction of bromate to bromide was achieved,with 100%of bromate removal efficiency under a volumetric loading of 1 mg Br/L/d.VFAs were produced in the reactor with a concentration ranging from 0.8?11.5 mg C/L.Methanosarcina was indicated to become the only dominated methane-oxidizing genus,and Dechloromonas increased from 0.9 to 18.0%after they had been fed bromate.It is hypothesized that,under oxygen-limited conditions,methane is oxidized into VFAs which then served as carbon sources for dissimilatory bromate-reducing bacteria(likely Dechloromonas)to reduce bromate.(g)With respect to archaeal and bacterial DAMO responding to differing electron acceptors,Candidatus Methanoperedens was selected with chromate/perchlorate/selenate intervened,yet suppressed by bromate/oxygen;while Candidatus Methylomirabilis was selected with perchlorate/selenate/oxygen intervened,yet suppressed by chromate/bromate.