Enrichment And Characteristic Research Of Denitrifying Anaerobic Methane Oxidation Microorganisms

Denitrifying anaerobic methane oxidation (DAMO) is an anaerobic process which uses methane as the electron donor and nitrate or nitrite as the electron accepter. In2006, this process was confirmed for the first time in a laboratory study, and then it became the research hotspot of the environment domain. The discovery of the DAMO process provides a new way for nitrogen removal from wastewater. This process can use CH4as the electron donor for denitrification. If DAMO process can be applied to treating wastewater, it can reduce the greenhouse emissions and energy consumption of wastewater treatment plant (WWTP). Meanwhile, the confirmation of DAMO process has significant meaning to improve the understanding of global methane and nitrogen cycles. And its unique reaction mechanism provides a new way of oxygen generating.The microbes involve DAMO process included ANME archaea and NC10bacteria, the extremely slow growth rates of these microorganisms seriously limit the obtaining of enrichment culture and further study of DAMO reaction. Therefore, dominant factors which effect the enrichment of DAMO microorganisms should be investigated to solve the growth restriction problem, in order to accelerate the research of DAMO process.Four reactors were estalished in this test, which were specially designed for the enrichment cultivation of DAMO microorganisms. All reactors were supplied CH4as the electron donor. Four different DAMO cultures (A, B, C, D) were enriched under different conditions. R1and R2started with the same mixed inoculum including sediment from Xixi river, West lake and paddy soil (Hangzhou, China), but the different nitrogen: NH4+and NO3-were added into R1to get Culture A after362days, which included DAMO archaea (39.4%) and Anammox bacteria (45.8%); Only NO2-was added as the electron acceptor into R2and the Culture B was obtained after202days, which consisted only of DAMO bacteria (88.2%). R3startd with the mixed inoculum including sediment from Xixi river, the activated sludge from the second sediment tank and the digested sludge from excess sludge storage tank in Qige WWTP (Hangzhou, China), NH4+and NO3-were added and the Culture C was obtained after232days, which consisted of DAMO archaea (77.7%) and Anammox bacteria (17.7%).The sludge collected from R3(227days) was added into R4as the inoculum, and only NO3-was added as the electron acceptor, Culture D was achieved successfully after326days, which included both DAMO bacteria (62.2%) and archaea (26.5%).The effects of different nitrogen on DAMO microbial community were also studied. NO3-and NO2-played an important role in choosing DAMO archaea and bacteria respectively. In Culture A and C, NH4+and NO3-were provided simultaneously, DAMO bacteria eliminated from the competition of NO2-with Anammox bacteria. At the same time, the effects of different inocula on DAMO microbial community were also found. In this study, the mixture of fresh water river sediment, activated sludge and digested sludge from WWTP was proved the better inoculum in comparison with the mixture of freshwater river sediment, freshwater lake sediment and paddy soil.The environmental factors included pH, temperature and dissolved oxygen were investigated based on Culture D. The results showed that, when temperature was below35"C, DAMO reaction rate dropped with the decrease of temperature; when temperature was above35℃, DAMO reaction rate decreased as temperatures rise; DAMO reaction rate reached a maximum at35℃. When pH was below6.5, DAMO reaction rate decreased with pH decreasing; when pH was above7.5, DAMO reaction rate decreased with pH rising; the maximum DAMO reaction rates appeared both at pH6.5and7.5. DAMO activity was inversely proportional to dissolved oxygen concentration, that the existence of dissolved oxygen inhibited DAMO process.N2O was detected in the system, and obvious trends of N2O accumulation and consumption were observed. Due to the lack of gene for reduction of N2O in DAMO bacteria, two hypothesizes were proposed: the consumption of N2O was completed by other bacteria; there was an unknown N2O invertase existing in DAMO bacteria. On the premise of the second hypothesis, the reason of N2O accumulation and consumption trends in this experiment were analyzed.