Periodic Reporting for period 5 - GREENT (Greenhouse Gas Mitigation through Advanced Nitrogen Removal Technology)
Période du rapport: 2020-12-01 au 2021-08-31
Human activities have severe impacts on the biological carbon and nitrogen cycles. The most important consequences of these are global warming and water pollution. Wastewater treatment technology, in particular nitrogen removal systems, improved considerably in the last decade. The application of anaerobic ammonium oxidizing (anammox) bacteria in partial-nitritation/anammox reactors has the potential to turn wastewater treatment plants into energy-efficient systems with minimal greenhouse gas (GHG) emissions of carbon dioxide, nitrous oxide and methane. Recently, microorganisms that couple the anaerobic oxidation of methane to nitrate or nitrite reduction were discovered. An innovative integration of these microorganisms into partial nitritation-anammox reactors for wastewater treatment offers an elegant and efficient solution to combat GHG emissions from wastewater treatment plants. In the GREENT project the feasibility of an innovative bioreactor, which converts ammonium and methane simultaneously into harmless compounds by ammonium- and methane-oxidizing microorganisms, was explored and its GHG emission potential was examined. To this end, stable cocultures of anammox and nitrite- and nitrate-dependent anaerobic methane-oxidizing microorganisms were achieved in bioreactors. Under conditions relevant for wastewater treatment, these microorganisms removed ammonium and methane efficiently without producing significant amounts of nitrous oxide, providing the proof of concept for the proposed novel bioreactor. The results of the GREENT project showed that it is possible to use the otherwise harmful methane as a resource for efficient nitrogen removal. Physiological and molecular analyses revealed that the combined activity of aerobic and anaerobic methane and ammonium-oxidizing microorganisms under the continuous supply of limiting amounts of oxygen is feasible and can be implemented for the removal of methane and ammonium from wastewater. The technological advances made in the GREENT project is of paramount importance and can be used to design wastewater treatment plants that emit less GHG. The insight gained into the interplay of the microorganisms that govern the turnover of the greenhouse gases nitrous oxide and methane will help in understanding the reasons underlying their emission from natural ecosystems, and the impact of human activities on greenhouse gas emissions.
Research Line 1
- Determining nitrous oxide and methane emission from the nitrogen removal stage of a municipal wastewater treatment plant (wwtp), and its metagenome sequencing
Nitrogen removal at the studied wwtp (Seehausen, Germany) consists of two steps. Nitrate reduction to dinitrogen gas occurs in the anaerobic chamber. Then wasterwater is led into the aerobic chamber where ammonium oxidation to nitrate occurs. The highest nitrous oxide (N2O) emission was measured in the aerobic nitrification zone suggesting that in this wwtp, aerobic ammonia-oxidizing bacteria were responsible for N2O production (project 1.1). The highest methane concentration was measured in the anaerobic denitrification zone (project 1.1). The metagenome of the wwtp revealed a diversity of microorganisms that could carry out the complete or different parts of the denitrification process, whereas the diversity of ammonia- and nitrite-oxidizing bacteria were lower (project 1.2 & 1.3). These results are not published yet.
Research Line 2
- Detailed description of nitrite and nitrate dependent anaerobic methane-oxidizing microorganisms (project 2.1)
Growth rate, doubling time, methane and nitrite affinities of the nitrite-dependent methane-oxidizing bacterium M. lanthanidiphila was determined and published in Applied and Environmental Microbiology. The complexes involved in nitrite-dependent methane oxidation were identified in M. lanthanidiphila, including the putative nitric oxide dismutase enzyme and published in BBA-Bioenergetics. The physiology of nitrate-dependent methane-oxidizing archaea M. nitroreducens was determined and revealed that they reduce nitrate to ammonium via nitrite and do not produce the greenhouse gas (GHG) N2O. Surprisingly, we showed that this biomass coupled the anaerobic oxidation of methane to the reduction of Fe3+ and Mn4+. This unprecedented discovery furthered our understanding of anaerobic methane-oxidizing pathways. This was published in PNAS and gathered press attention, and was even included in popular German quiz show (Wer Weiß Denn Sowas) as a question.
Investigation of cocultures of anammox and nitrite- and nitrate-dependent anaerobic methane-oxidizing microorganisms (project 2.2)
The growth of anammox bacteria on nitric oxide and ammonium, a new growth mode, was discovered. Under these conditions anammox bacteria did not produce N2O. This was published in Nature Communications and attracted press interest in the project. A new nitrite reductase, unrelated to known nitrite reductases was discovered in anammox bacteria and published in JBC. Proteins related to this new nitrite reductase was also detected in databases indicating it is prevalent in natural and engineered ecosystems. The structure of nitrite oxidoreductase from anammox bacteria was revealed and characterized, which was published in Nature Microbiology and attracted press attention.
Stable cocultures of anammox and nitrite & nitrate dependent anaerobic methane-oxidizing microorganisms were achieved, and the results were published in Applied Microbiology and Biotechnology. The coculture could effectively remove ammonium and methane simultaneously as proposed.
Investigation into the effect of oxygen on the cocultures of anammox and nitrite- & nitrate-dependent anaerobic methane-oxidizing microorganisms (project 2.3)
Cocultures of anammox and nitrite & nitrate dependent anaerobic methane-oxidizing microorganisms were subjected to oxygen and growth of aerobic methane and ammonia oxidizers were achieved under O2 limitation. This reactor contained aerobic and anaerobic methane and ammonium oxidizers, and functioned under conditions relevant for wastewater treatment providing the proof of concept for the wastewater treatment plant suggested in the grant proposal. Physiological and molecular analyses showed that the combination of N-damo and anammox activity under the continuous supply of limiting oxygen concentrations is feasible and can be implemented for the removal of methane and ammonium from wastewater. These results were published in Applied and Environmental Microbiology. A new protein (mHAO) capable of oxidation of hydroxylamine to NO, active up to at least 80 °C was purified from an aerobic methane oxidizer This novel protein mHAO enables methanotrophs to cope with high ammonia concentrations, leading to reduced methane emissions. However, this activity simultaneously contributes to ammonia loss and nitrite production, and potentially leads to N2O emissions. This was published in PNAS and attracted press attention.
The GREENT team also contributed an invited review to Current Opinion in Biotechnology covering the state-of-the-art and the future of ammonium and GHG removal from wastewater, covering all aspects of the GREENT project.
- Determining nitrous oxide and methane emission from the nitrogen removal stage of a municipal wastewater treatment plant (wwtp), and its metagenome sequencing
Nitrogen removal at the studied wwtp (Seehausen, Germany) consists of two steps. Nitrate reduction to dinitrogen gas occurs in the anaerobic chamber. Then wasterwater is led into the aerobic chamber where ammonium oxidation to nitrate occurs. The highest nitrous oxide (N2O) emission was measured in the aerobic nitrification zone suggesting that in this wwtp, aerobic ammonia-oxidizing bacteria were responsible for N2O production (project 1.1). The highest methane concentration was measured in the anaerobic denitrification zone (project 1.1). The metagenome of the wwtp revealed a diversity of microorganisms that could carry out the complete or different parts of the denitrification process, whereas the diversity of ammonia- and nitrite-oxidizing bacteria were lower (project 1.2 & 1.3). These results are not published yet.
Research Line 2
- Detailed description of nitrite and nitrate dependent anaerobic methane-oxidizing microorganisms (project 2.1)
Growth rate, doubling time, methane and nitrite affinities of the nitrite-dependent methane-oxidizing bacterium M. lanthanidiphila was determined and published in Applied and Environmental Microbiology. The complexes involved in nitrite-dependent methane oxidation were identified in M. lanthanidiphila, including the putative nitric oxide dismutase enzyme and published in BBA-Bioenergetics. The physiology of nitrate-dependent methane-oxidizing archaea M. nitroreducens was determined and revealed that they reduce nitrate to ammonium via nitrite and do not produce the greenhouse gas (GHG) N2O. Surprisingly, we showed that this biomass coupled the anaerobic oxidation of methane to the reduction of Fe3+ and Mn4+. This unprecedented discovery furthered our understanding of anaerobic methane-oxidizing pathways. This was published in PNAS and gathered press attention, and was even included in popular German quiz show (Wer Weiß Denn Sowas) as a question.
Investigation of cocultures of anammox and nitrite- and nitrate-dependent anaerobic methane-oxidizing microorganisms (project 2.2)
The growth of anammox bacteria on nitric oxide and ammonium, a new growth mode, was discovered. Under these conditions anammox bacteria did not produce N2O. This was published in Nature Communications and attracted press interest in the project. A new nitrite reductase, unrelated to known nitrite reductases was discovered in anammox bacteria and published in JBC. Proteins related to this new nitrite reductase was also detected in databases indicating it is prevalent in natural and engineered ecosystems. The structure of nitrite oxidoreductase from anammox bacteria was revealed and characterized, which was published in Nature Microbiology and attracted press attention.
Stable cocultures of anammox and nitrite & nitrate dependent anaerobic methane-oxidizing microorganisms were achieved, and the results were published in Applied Microbiology and Biotechnology. The coculture could effectively remove ammonium and methane simultaneously as proposed.
Investigation into the effect of oxygen on the cocultures of anammox and nitrite- & nitrate-dependent anaerobic methane-oxidizing microorganisms (project 2.3)
Cocultures of anammox and nitrite & nitrate dependent anaerobic methane-oxidizing microorganisms were subjected to oxygen and growth of aerobic methane and ammonia oxidizers were achieved under O2 limitation. This reactor contained aerobic and anaerobic methane and ammonium oxidizers, and functioned under conditions relevant for wastewater treatment providing the proof of concept for the wastewater treatment plant suggested in the grant proposal. Physiological and molecular analyses showed that the combination of N-damo and anammox activity under the continuous supply of limiting oxygen concentrations is feasible and can be implemented for the removal of methane and ammonium from wastewater. These results were published in Applied and Environmental Microbiology. A new protein (mHAO) capable of oxidation of hydroxylamine to NO, active up to at least 80 °C was purified from an aerobic methane oxidizer This novel protein mHAO enables methanotrophs to cope with high ammonia concentrations, leading to reduced methane emissions. However, this activity simultaneously contributes to ammonia loss and nitrite production, and potentially leads to N2O emissions. This was published in PNAS and attracted press attention.
The GREENT team also contributed an invited review to Current Opinion in Biotechnology covering the state-of-the-art and the future of ammonium and GHG removal from wastewater, covering all aspects of the GREENT project.
- A new N2O-removing culture has been achieved is currently characterized.
- A new nitrite-oxidizing microorganism was enriched from a wwtp and is being characterized
- Molecular pathways of N2O production and consumption are currently characterized
- A new nitrite-oxidizing microorganism was enriched from a wwtp and is being characterized
- Molecular pathways of N2O production and consumption are currently characterized