| Aerobic methane oxidation coupled with denitrification?AME-D?would potentially be an attractive way to remove nitrate?NO3-?or nitrite?NO2-?from water and wastewaters requiring the addition of an external carbon source,which is a big challenge in the filed of wastewater treatment.For AME-D process,aerobic methanotrophs oxidize methane?CH4?in the presense of oxygen?O2?and excrete organics to supply carbon sources for denitrifers.However,nitrate removal capacity,including nitrate removal rate and efficiency of AME-D process,should be further increased and the microbial mechanism of this process is still unclear.It was reported that conditions with low O2:CH4 ratio?<1.0?,a critical factor for AME-D process,are beneficial for aerobic methanotrophs to release more organics,providing more carbon sources and oxygen-limiting conditions for denitrifiers.Therefore,it is postulated that nitrate removal capacity could be further increased under conditions with low 02:CH4 ratio.Nevertheless,this inferance still needs relevant studies to demonstrate.In this study,the effect of different O2:CH4 ratio?<1.0?on nitrate removal of AME-D process was investigated combing batch test with thermodynamic analysis.Meanwhile,nitrate removal capacity of AME-D process was increased using membrane biofilm reactor?MBfR?with low O2:CH4 ratio.Moreover,the combined molecular methods were adopted to investigate the microbial mechanism of AME-D process with increased nitrate removal capacity.Primary results were listed as follows:?1?Nitrogen removal of AME-D process under conditions with different O2:CH4 ratios?<1.0?was investigated through batch tests.The optimal O2:CH4 ratio for denitrification was found to be 0.25.At this point,denitrifying activity reached the highest level of 7.32 mmol NO2--N/g VSS/d and the amount of nitrous oxide accumulation was the lowest.When the O2:CH4 ratio was below the optimal ratio,nitrite removal was improved with the increased O2:CH4 ratio,presumably due to an increase in available substrates released by aerobic methanotrophs.When the O2:CH4 ratio was above the optimal ratio,nitrite removal was presumably inhibited by the excessive O2.These results indicate that adjusting the O2:CH4 ratio can improve the cooperation between aerobic methanotrophs and denitrifiers to obtain better nitrogen removal performance using the AME-D process.?2?Through thermodynamic analysis,it was found that consuming 1 mol CH3OH could reduce highest amount of NO3--N or NO2--N and release highest amount of Gibbs free energy among common organic intermediates excreted by methanotrophs.Based on the model of TEEM2 from McCarty,aerobic methanotrophs could provide no more than 60%CH4-C,in the form of methanol,for co-existing denitrifiers,resulting in the highest nitrogen removal efficiency of AME-D process.Under conditions with O2:CH4 ratio of 0.25,the percentage of carbon flow from methane to denitrification?4.3-13.0%?was much lower than the ideal flow?60%?.?3?MBfR with low O2:CH4 ratio could further increase nitrogen removal capacity of the activated sludge?parental sludge?used in the above batch tests.Although methane consumption rates of parental sludge and MBfR sludge did not show significant difference,nitrate removal rate of MBfR sludge was remarkably increased to 286±5 ?mol/g MLSS/d,almost 4 times higher than that of parental sludge.Denitrification was the main pathway for MBfR sludge to remove nitrogen.Moreover,aerobic methanotrophs may release almost 37.4-40.7%of total methane-derived carbon for denitrifiers to remove nitrate.For parental sludge,however,this percentage is only 3.8-8.0%.?4?Results from q-PCR revealed that copy numbers of key denitrifying genes in MBfR sludge significantly increased in comparison with that in parental sludge,indicating that denitrifiers were quickly enriched in MBfR sludge.These highly enriched denitrifiers were mainly distributed in the family of Methylophilaceae?11.6%?and Rhodocyclaceae?9.8%?,especially those methanol-utilized denitrifiers from Methylobacillus,Methylotenera,Methylophilus and Methyloversatilis.For aerobic methanotrophs,Type I methanotrophs was the predominant aerobic methanotrophs in MBfR sludge,with the percentage of 9.8%.More interestingly,many globular aggragates?5-10 ?m in diameter?including methanotrophs and methanol-utilized denitrifiers were observed in MBfR sludge and it may promote denitrifiers to efficiently capture methanol released by methanotrophs.Theses results provided a powerful theoretical guidance for further increasing nitrate removal capacity of AME-D process,finally promoting its application wastewater treatment. |
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