| The greenhouse effect caused by the increasing methane concentration in the atmosphere is currently one of the most important environmental issues in the world.The microorganism-mediated anaerobic oxidation of methane(AOM)process,as the main way to consume methane in aquatic systems,has a great contribution to the control of global methane emissions.As the ubiquitous substance in the natural environment,nitrate and iron oxides have been found to be widely involved in AOM bioprocess.The study of nitrate-dependent AOM(N-AOM)process and Fe(III)-dependent AOM(Fe-AOM)process is indispensable in exploring global methane sinks.Among these AOM processes,ANME-2d archaea,a branch of ANME(anaerobic methanotrophic archaea),is the most important functional microorganism.The metabolic mechanism of ANME archaea and its interaction with symbiotic microorganisms are very important scientific issues;the adaptability of ANME-2d archaea driven AOM process to environmental effect factors and the response to common pollutants in anaerobic wastewater are very important engineering application issue.However,the current understanding of above issues is still limited,due to the slow reaction speed of the above two AOM bioprocesses and the difficulty in enrichment of functional microorganisms.This thesis taking ANME-2d archaea mediated N-AOM process and Fe-AOM process as the main research object,and combined a variety of physics,chemistry and molecular biology analysis methods for experimental research.The main research contents and objectives of this study include:(1)Enrich the functional microorganism ANME-2d archaea of the N-AOM and the Fe-AOM processes by using nitrate as electron acceptor in laboratory conditions.And explore the effect of anaerobic ammonia oxidation(anammox)bacteria on N-AOM system.(2)Explore the influence of temperature change on the N-AOM process,and expand the understanding of the temperature adaptability of ANME-2d archaea.(3)Explore the effects of sulfide,a common pollutant in anaerobic wastewater,and coexisting nitrate-reducing sulfide oxidizing bacteria(NR-SOB)on the N-AOM and anammox coupling system.(4)Explore the feasibility of AOM process driven by different types of iron oxides as electron acceptors,and expand the understanding of the metabolic diversity of ANME-2d archaea.Through the study of the above aspects,the following results are obtained:1)Using anaerobic mixed sludge as the inoculum,methane as the only electron donor and the way to maintain an anaerobic environment,and nitrate as the only electron acceptor,the ANME-2d archaea was successfully enriched in the sequence batch reactor(SBR).And this system has a long-term stability.Under the condition of limited nitrite substrate,the introduction of anammox bacteria cannot help increase the total nitrogen removal rate of the system.The reduction of nitrate to nitrite led by ANME-2d archaea was the limiting step of the system.2)Controlled variable temperature condition(30-20℃)inhibited the activity of ANME-2d archaea in a short-time period,but the inhibition gradually relieved with the adaptation of ANME-2d archaea to temperature change.Besides,ANME-2d archaea exhibited similar nitrate removal rate at 30℃ and 20℃ separately,suggesting that ANME-2d archaea has stable biological activity in the temperature range of 20-30℃.This result extended the temperature range(30-35℃)used for the current laboratory enrichment of ANME-2d archaea.Also,uncontrolled temperature conditions(13–38°C)had a strong inhibitory effect on the activity of ANME-2d archaea,and this inhibition cannot be restored after long-term cultivation.Finally,based on the results of the habitat survey,it was speculated that ANME-2d archaea may be a facultative psychrophilic microorganism.And its stable nitrate removal rate at a constant temperature of 20°C revealed the possibility of applying the N-AOM process to anaerobic wastewater treatment in a medium temperature environment in the future.3)In the study of the sulfide effect on the N-AOM and anammox coupling system,it was found that the presence of sulfide,on the one hand,inhibited the coupling system and this inhibitory effect increased with the increase of sulfide concentration;on the other hand,it stimulated the growth of NR-SOB bacteria and increased the total nitrate removal of the system.Besides,the existence of the NR-SOB process not only detoxified the inhibition of ANME-2d archaeal activity by sulfide,but also improved the total nitrate removal of the system.Finally,when the anammox process was inhibited,the NR-SOB bacteria replaced the anammox bacteria and became the symbiotic microorganisms of ANME-2d archaea to complete the removal of nitrite.4)Different types of iron oxides can be used as electron acceptors in AOM process,but there were differences in microbial metabolic pathways in different Fe-AOM processes.ANME-2d archaea and iron-reducing bacteria were the functional microorganisms in AOM process driven by amorphous iron oxide.Expect ANME-2d archaea and iron-reducing bacteria,iron oxide bacteria and other symbiotic microorganisms were also found in(partial)crystalline iron oxides driven AOM processes.With the exist of these symbiotic microorganisms,Fe(II)is re-oxidized to Fe(III),resulting in a special "iron cycle" process in the system.Secondly,the bioavailability of amorphous iron oxides may be higher than that of(partial)crystalline iron oxides.Finally,excessive iron oxides may inhibit the bioactivity of ANME-2d archaea.Through this study,the current knowledge of the AOM process dominated by ANME archaea had been expanded,and the metabolic diversity of ANME archaea in the use of different electron receptors to complete the AOM process had been enriched.This thesis not only add new knowledge to the global methane sink process under the action of ANME-2d archaea and their symbiotic microorganisms,but also provide theoretical basis and scientific guidance for the application of AOM biological processes in wastewater treatment in the future. |
