Microbially mediated anaerobic oxidation of methane (AOM) is very important to the Earth’s climate because it consumes methane produced from natural sediments before this greenhouse gas escapes to the atmosphere. In marine environments, the microorganisms that are responsible for AOM consist of a consortium of anaerobic methanotrophic (ANME) archaea, and sulfate-reducing bacteria (SRB). It has been hypothesized that ANME archaea can oxidize methane anaerobically and shuttle electrons for sulphate reduction by SRB. Thermodynamically, oxidized nitrogenous compounds are more favourable electron acceptors than sulphate. However the microorganisms capable of coupling AOM to denitrification, noted as the denitrifying anaerobic methane oxidizing (DAMO) organisms, were enriched only very recently. In 2006, the first DAMO culture was enriched from canal sediments by Dutch scientists after 16 months incubation. The dominant bacteria (80% of the microbial population) in the culture belonged to the candidate division NC10, and the archaea (10% of the population) were distantly related to Methanosaeta and ANME –II. However, the archaea declined and disappeared after prolonged incubation, leading to the conjecture that it may not have been involved in methane oxidation. The study also showed that the NC10 bacteria prefer nitrite to nitrate as electron acceptors.
The objective of this work is to further improve our understandings of the DAMO process and the microorganisms involved.
Since the physiological and biochemical studies of the DAMO organisms were limited by the availability of DAMO cultures, the first research objective was to enrich these microorganisms in Australia, which is half-an-earth away from The Netherlands, where they were first discovered. Two DAMO cultures were enriched at 35°C and at 22°C, respectively, from a mixture of activated sludge and digester sludge from a wastewater treatment plant in Brisbane, Australia, and sediments from a lake also located in Brisbane. The culture enriched at 35°C contained bacteria and archaea close to the bacteria and archaea previously reported. The culture enriched at 22°C contained the same NC10 bacteria, but no archaea.
The comparison of the DAMO cultures enriched above and those reported in literature suggested that DAMO archaea may play an important role in nitrate reduction and DAMO bacteria contribute significantly to nitrite reduction in DAMO cultures. The second research objective of this thesis was to verify if nitrate and nitrite feeding has an effect on the selection of DAMO microorganisms. Two reactors were seeded with a DAMO culture containing both DAMO archaea and DAMO bacteria, and were fed with nitrate and nitrite, respectively. DAMO archaea were eliminated from the nitrite-fed reactor, while both archaea and bacteria continued to develop in the nitrate-fed reactor. The results suggest that DAMO bacteria are more competitive for nitrite reduction than DAMO archaea.
This work also investigated the potential interactions between the DAMO archaea, DAMO bacteria and anammox bacteria in anoxic environments. When DAMO archaea and bacteria were mixed with anammox bacteria, and fed with ammonium, methane and nitrate or nitrite, DAMO archaea formed a synergistic relationship with anammox bacteria, while DAMO bacteria were eliminated from the culture. The DAMO archaea coupled the anaerobic oxidation of methane with the reduction of nitrate to nitrite, which is then subsequently reduced to nitrogen gas by the anammox bacteria. During batch tests, DAMO archaea were found to reduce nitrate and nitrite simultaneously in the absence of ammonium, with the nitrate reduction rate being twice the nitrite reduction rate. Based on these findings, several hypotheses were proposed on the relationship between DAMO archaea, DAMO bacteria and anammox bacteria:
• DAMO archaea would need collaboration with other nitrite reducers such as DAMO bacteria or anammox bacteria to avoid the toxic effects of nitrite accumulation. This could potentially explain why DAMO archaea have always been found to co-exist with other nitrite reducers.
• Based on their preferences and abilities, DAMO archaea would be expected to mainly work on nitrate reduction while bacteria work on nitrite reduction in a DAMO culture containing both archaea and bacteria.
• The collaborations between DAMO and anammox microorganisms may exist in natural environment and affect the carbon and nitrogen cycles globally.
Based on the observation that DAMO archaea and bacteria have different preferences on temperature and oxidized nitrogenous compounds, the effects of temperature and nitrate and nitrite feeding on DAMO cultures and their interactions with anammox bacteria were investigated. The study showed that the presence of nitrate in conjunction with no or limited supply of nitrite would favour the growth of DAMO archaea.
This thesis contributes significantly to the understanding of the functions and eco-physiological properties of DAMO archaea and bacteria. However, there is much more work remains to be done. In particular, the environmental factors regulating the distribution of DAMO microorganisms and their interactions with other denitrifiers in aquatic systems, such as anammox bacteria, require further investigation. Also, more research is required to find out the possibility of applying DAMO process in wastewater treatment industry.