Study On The Enrichment Of Denitrifying Anaerobicmethane Oxidation Microorganisms

Denitrifying anaerobic methane oxidation(DAMO) is a novel denitrification process with methane as the electron acceptor and NO3-/NO2- as the electron donor under anaerobic condition. It is a new energy-efficient way for nitrogen polluted wastewater treatment via oxidization of methane to carbon dioxide and reduction nitrate or nitrite to nitrogen use of microorganisms. Due to the dominant microorganisms involved in the process growth slowly and enrichment difficulty, its development restricted. In consideration of the above problems, we enriched denitrifying anaerobic methane oxidation microorganisms, analysed influence factors of DAMO process in detail and the impact of electrical stimulation. It provides a theoretical basis for the practically application of anaerobic oxidation of methane denitrification technology. The main results are as follows:1) The membrane-aerated biofilm reactors were set up to enrich the denitrifying anaerobic methane oxidation microorganisms successfully.The enrichment cultures showed high nitrate and nitrite removal rate. The average removal rate of nitrate is about 0.56 mmol /d and the average removal rate of nitrate is about 0.83 mmol /d. Denitrifying anaerobic methane oxidation activity of the enrichment cultures were proved by stable isotope tracer and the 13CO2 formation from 13CH4 in the presence of nitrite and nitrate indicated that the anaerobic methane oxidation could be coupled to denitrification. Simultaneously, the observed stoichiometry of methane consumption versus nitrite(nitrate) was close to the theoretical value of 3 CH4 : 8 NO2-(5 CH4 : 8NO3-), respectively.Analysed NC10 phylum by using fluorescence in situ hybridization, clone library targeting the 16 S r RNA gene of bacteria, realtime fluorescence quantitative PCR. The clone library targeting the 16 S r RNA gene of bacteria affiliated to the NC10 phylum confirmed the presence of the NC10 phylum bacteria. The fluorescence in situ hybridization(FISH) showed that with the enrichment of microorganisms, NC10 phylum got enriched, and at the thirteenth month, The bacteria had become dominant and the aboundance increased to 73%. The realtime fluorescence quantitative PCR showed that the copy number of NC10 bacteria per gram of dry soil sample was about 106 after 22 months.2) Analysed the influence factors of denitrifying anaerobic methane oxidation through anaerobic batch experiments.The main influences is about substrate concentration,temperature, methane pressure and p H value.Generally, the solubility of methane is one of the most important facrors affecting the DAMO process, and the solubility of methane is mainly affected by the gas phase pressure. This study showed that the methane phase pressure was less than 49.72 k Pa, the activity incraesed as the methane phase pressure was increased. But the phase pressure was higher than 49.72 k Pa, the activity of denitrifying anaerobic methane oxidation microorganisms was not limited by the solubility of methane. In this experimental conditon, the optimum initial NO2-concentration, environment temperature, p H were 1.5mmol/L, 25℃, 7.48.3) The first time study on electrical stimulation of denitrifying anaerobic methane oxidation microorganisms. The voltage used here wes 0V, 0.5V, 1V, 1.5V. Simultaneously compared about two difference way of continuous and discontinuous to applied voltage.The research showed that the electric can strengthen the activity of the denitrifying anaerobic methane oxidation microorganisms. As applied 1V continuously, the activity was highest, suggested that it was the optimum. But as applied discontinuously, the activity of DAMO was limited.