WO2007069899A1

WO2007069899A1 - Anaerobic oxidation of methane and denitrification

Info

Publication number: WO2007069899A1
Application number: PCT/NL2006/050319
Authority: WO
Inventors: Marc Strous; Ashna Anjana Raghoebarsing; Katinka Theodora Van De Pas-Schoonen; Adrianus Pol; Hubertus Johannes Marie Op Den Camp; Michael Silvester Maria Jetten
Original assignee: St. Katholieke Universiteit Radboud Universiteit Nijmegen
Priority date: 2005-12-15
Filing date: 2006-12-15
Publication date: 2007-06-21

Abstract

Waste water and waste gas containing nitrogen compounds and organic matter can be effectively treated to produce molecular nitrogen and carbon dioxide by simultaneous anaerobic methane oxidation and anaerobic denitrification using a consortium of two micro-organisms: a denitrifying bacterium representing a phylum without any cultured species so far and an archaeon distantly related to marine methanotrophic archaea. In the new process, nitrogen-oxygen compounds, preferably obtained by aerobic nitrification of ammonia, in the essential absence of molecular oxygen, are fed into an denitrifying reactor containing the consortium of the anaerobic denitrifying bacterium and an anaerobic methanotrophic archaeon in a liquid medium together with methane.

Description

Anaerobic oxidation of methane and denitrification

Field of invention

The present invention relates to a process for the conversion of methane and nitrogen- oxygen compounds, a microbial consortium capable of oxidising methane and reducing nitrogen-oxygen compounds for application in the process, the anaerobic denitrifying bacterium capable of reducing nitrogen-oxygen compounds and the anaerobic methano- trophic archaeon capable of oxidising methane per se. The invention further relates to a method for the production of the microbial consortium for application in the process of the invention.

Background of invention

It has been known that nitrogenous waste streams, i.e. waste streams containing nitrogen-oxygen compounds, can be treated by micro-organisms wherein the organisms use organic compounds as source of carbon and energy. However, such systems may be difficult to manage since undesirable materials (pollutants such as nitrous oxide) can be emitted and expensive chemicals (such as methanol) have to be used. Additionally, metabolic products (such as nitrite) may build up which are toxic to the metabolising micro-organisms. Removal of such toxic products appeared to be difficult and expensive. Furthermore, to maintain a viable population of such organisms, nutrients often had to be supplied to the systems and special conditions maintained, especially when commonly occurring organic acids were depleted by anaerobic pre-treatment. At low ambient temperatures the methane that is produced in the anaerobic pre-treatment partly remains dissolved and this fraction is discharged with the effluent. As a consequence, it can no longer be recovered as an energy source. Besides, after discharge, this dissolved methane will escape to the atmosphere where it contributes to global warming.

The conversion of methane and nitrate into molecular nitrogen and carbon dioxide is for instance mentioned in DE3121395 and Islas-Lima et al, Water Research, Elsevier, Amsterdam, NL, vol. 31, no. 1, 1997, p 55-60. However, the former relates to a two- stage process, wherein in a first part of the reactor in the presence of oxygen methane is removed by methane oxidizing bacteria, and subsequently in a second part of the reactor denitrification takes place by denitrifying micro-organisms. The latter describes results which, according to the authors, show the "evidence" of anoxic methane oxidation coupled to denitrification in waste water sludge pretreated with acetate. The rate they reported was about 5 mg nitrogen converted per gram volatile suspended solids per day. Which organisms are used is not described in these documents.

Summary of the invention

Therefore, it is an object of the invention to provide a process for the conversion of methane and nitrogen-oxygen compounds.

The invention shows for the first time that direct anaerobic oxidation of methane can be coupled to denitrification. A microbial consortium, enriched from anoxic sediments, was demonstrated to oxidise methane to carbon dioxide coupled to denitrification in the complete absence of oxygen.

Hence the invention provides a process for the conversion of methane and one or more nitrogen-oxygen compounds to molecular nitrogen and carbon dioxide, comprising feeding the nitrogen-oxygen compounds and methane, in the essential absence of molecular oxygen, into an denitrifying reactor containing a consortium of an anaerobic denitrifying bacterium and an anaerobic methanotrophic archaeon in a liquid medium, wherein preferably the 16S-rRNA of the anaerobic denitrifying bacterium or its DNA copy comprises a nucleotide sequence exhibiting at least 86% sequence identity with the nucleotide sequence of SEQ ID No. 1, and wherein preferably the 16S-rRNA of the archaeon or its DNA copy comprises a nucleotide sequence exhibiting at least 88% sequence identity with the nucleotide sequence of SEQ ID No. 2.

In another aspect, the invention provides a method for the production of a microbial consortium capable of simultaneous anaerobic denitrification using methane, comprising providing an anoxic sediment, checking the capability of the sediment to reduce nitrate and/or nitrite in the presence of methane, and enriching a culture of the sediment in the consortium by feeding methane and nitrate and/or nitrite.

The invention further provides an anaerobic denitrifying bacterium capable of reducing nitrogen-oxygen compounds in cooperation with an anaerobic methanotrophic archaeon, the bacterium exhibiting at least 86% sequence identity in its 16S rRNA nucleotide sequence with SEQ ID No. 1. The invention also provides an anaerobic methano trophic archaeon capable of oxidising methane in cooperation with an anaerobic denitrifying bacterium, the archaeon exhibiting at least 88% sequence identity in its 16S rRNA with the nucleotide sequence of SEQ ID No. 2 is provided. Furthermore, the invention pertains to a microbial consortium capable of oxidising methane and reducing nitrogen-oxygen compounds comprising the archaeon referred to above and a denitrifying bacterium, and/or the bacterium referred to above and a methanotrophic archaeon.

Brief description of drawings

Figure 1 schematically depicts an embodiment of a reactor and process of the invention. Figures 2a-b depict the CH4, NO₂ ^" and NO₃ ^" consumption and dinitrogen gas production of the consortium according to an embodiment of the invention

Figures 3a-d schematically depicts further embodiments of the reactor and process of the invention.

SEQ ID No. 1 gives the 1557 bp nucleotide sequence of the 16S rRNA section of the novel anaerobic denitrifying bacterium of the invention.

SEQ ID No. 2 gives the 916 bp nucleotide sequence of the 16S rRNA section of the novel methanotrophic archaeon of the invention.

SEQ ID No.'s 3-6 give the 18 b oligonucleotide probes used for identifying the novel anaerobic denitrifying bacterium and methanotrophic archaeon of the invention.

Description of invention

The term "biological treatment", is known to the person skilled in the art and means to be ingested or eaten by a living organism and converted or metabolised by the organism to a more environmentally friendly or manageable substance.

The term "nitrogen-oxygen compound" or "N-O compounds" refers to any neutral or charged nitrogen oxide in any form, be it gaseous, dissolved, solvated, hydrated, hydroxylated, protonated, or otherwise associated. It especially refers to NO, NO₂,

NO₂ ^", NO₃ ^" and N₂O. These N-O compounds can be reduced to N₂ according to the process of the invention. The N-O compounds will mainly be present in liquid, such as water, e.g. sewage, industrial wastewater, etc. Such sewage, wastewater, etc., may also comprise other compounds, amongst others NH₄ ⁺ (ammonium), BOD (biological oxygen demand), etc. Biochemical Oxygen Demand (BOD) refers to the amount of oxygen that is consumed by bacteria and protozoa by oxidising organics in water, or the material which consumes this oxygen.

The term "waste material" means a waste which is in a gaseous or liquid phase at ambient conditions. Examples of such materials are nitrous oxide, nitric oxide, nitrogen dioxide, ammonia, nitrite and nitrate containing liquids. Specific concentrations of such waste material may vary, e.g. from less than about 10 mg/1 to above 5000 mg/1.

The term "micro-organism" as used herein means an organism, normally unicellular, not visible to the unaided eye. Micro-organisms suitable for use in accordance with the present invention for metabolising waste materials, e.g., nitrogen containing components, include bacteria and archaea.

The term "DAMO" herein refers to "Denitrification and Anaerobic Methane Oxidation".

The term "methanotroph" refers to micro-organisms like bacteria or archaea that are able to grow using methane as their main source of carbon and energy.

The term "CANON process" or "CANON" refers to a process where under oxygen limitation (e.g. about <0.5% air saturation), a coculture of aerobic and anaerobic ammonium oxidisers is obtained, which culture converts ammonium directly to dinitrogen gas, with nitrite as the intermediate. Application of this concept in wastewater treatment can lead to complete ammonia removal in a single, autotrophic reactor. This concept has been named CANON, meaning "Completely Autotrophic Nitrogen removal Over Nitrite", and referring to the way the two groups of microorganisms interact: performing two sequential reactions simultaneously.

Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale. Brackish and especially freshwater sediments often receive increased downward fluxes of nitrate and upward fluxes of methane generated by anaerobic decomposition.

Global biogeochemical cycles are largely driven by micro-organisms known as chemo- lithotrophs (organisms which obtain their energy from the oxidation of inorganic compounds). These organisms derive energy for growth from the favourable transfer of electrons from an inorganic electron donor to an acceptor. The anaerobic oxidation of methane coupled to denitrification (DAMO; see equations below) was considered missing in nature. The lack of indications for the occurrence of this process can be explained because the gradients around the chemocline are generally very steep, masking the process from geochemical detection. Chemocline is the boundary in a meromictic lake separating less saline oxygen-rich upper layer from the more saline anoxic bottom layer. Furthermore, the laboratory enrichment of the responsible microorganisms could be difficult because of their potentially very slow growth. Since DAMO coupled to denitrification is thermodynamically possible (ΔG < -464 kJ-mol^"1 or -673 kJ-mol^"1 CH4 at 25°C, pH 7, substrate concentrations 0.1 mM, for equations 1 and 2, respectively) suitable micro-organisms might exist and the understanding of biogeo- chemical methane cycling would necessarily be incomplete.

One or more of the following DAMO reactions may occur in the process according to the invention:

5 CH₄ + 8 NO₃ ^" + 8 H^{+ ■}* 5 CO₂ + 4 N₂ + 14 H₂O (Eq. 1) 3 CH₄ + 8 NO₂ ^" + 8 H^{+ ■}* 3 CO₂ + 4 N₂ + 10 H₂O (Eq. 2)

CH₄ + 4 NO -^ CO₂ + 2 N₂ + 2 H₂O (Eq. 3)

CH₄ + 2 NO₂ -^ CO₂ + N₂ + 2 H₂O (Eq. 4)

CH₄ + 4 N₂O -^ CO₂ + 4 N₂ + 2 H₂O (Eq. 5)

CH₄ + NO₂ ^" + NO₃ ^" + 2 H^{+ ■}* CO₂ + N₂ + 3 H₂O (Eq. 6) CH₄ + 4 NH₄ ⁺ + 4 NO₃ ^{" ■}* CO₂ + 4 N₂ + 10 H₂O (Eq. 7)

According to the invention, consortia of micro-organisms capable of DAMO have been enriched. Anoxic sediment of a canal (Twentekanaal, the Netherlands) was used as a typical example of the inoculum for the enrichment culture. This canal contained nitrate at concentrations up to 1 mM and the sediment was saturated with methane, typical for freshwater habitats receiving agricultural run-off. In a steep (< 20 mm) chemocline in the top of the sediment, methane (diffusing upwards) and nitrate (diffusing downwards) were consumed. A one-litre sample from the top of the sediment was incubated anaerobically in the laboratory for 16 months. Mineral medium containing nitrate, nitrite, bicarbonate and trace elements was supplied continuously (300 ml-day^"1). Spent liquid culture was removed twice a day after a settling period to retain biomass and sediment. Methane was also supplied continuously as the only electron donor (14 1-day^"1), while the culture was well mixed by stirring to ensure good mass transfer of methane to the microbial cells. The nitrate concentration was maintained above 3 mM at all times to prevent the occurrence of sulphate reduction.

After an initial period of nitrite and nitrate consumption, four months of incubation led to stable concentrations of nitrite and nitrate, indicating that most readily biodegradable organic substances present in the inoculum had been mineralised. During the next two months the nitrite concentration in the effluent gradually decreased from 1 mM to 0.15 mM, indicating that a new substrate was being used to drive denitrification by the microbial population. No significant nitrate consumption was observed yet. From six months after inoculation onwards, the influent nitrite concentration could gradually be increased to 6 mM, while the effluent concentration remained around 0.1 mM, indicating growth of a microbial population consuming nitrite. During the latter period nitrate was also consumed. After 14 months, the culture consumed 1.8 mmol nitrite -day^"1, 0.3 mmol nitrate-day^"1 and contained approximately 100 mg protein.

The methane consumption by the culture was measured by removing excess methane and stopping the medium and methane supply. At this time the nitrite and nitrate concentrations were 0.24 mM and 3.8 mM, respectively. Methane, nitrite and nitrate were consumed in parallel and dinitrogen gas evolved (Figures 2a and 2b). Nitrite consumption alone accounted for 85% of the methane consumption, according to Eq. 1. This could indicate that methane consumption was mainly dependent on nitrite. However, after longer incubation in the absence of nitrite (10-20 h) methane consumption was also coupled to nitrate consumption. Apparently, the culture could adapt to nitrate and both nitrite and nitrate were suitable substrates for anaerobic methane oxidation (Eq. 1 and 2). Carbon dioxide production from methane was measured in separate experiments with ¹³C labeled methane. In these tests ¹³CO₂ was detected as the end product of methane oxidation. Furthermore ¹³CH₄ was incorporated in both bacterial and archaeal lipid biomarkers after 3 and 6 days.

Figures 2a-b also show that the affinity of the culture for methane is very high. The methane consumption rate only decreased to 50% of the initial rate at a methane concentration of approximately 0.6 μM. Since mass transfer limitation could not be ruled out at these low concentrations and the nitrite concentration was also very low, the affinity constant of the micro-organisms for methane must be less than or equal to 0.6 μM. Thus, these experiments show unambiguously that methane was oxidised anaerobically, and that this oxidation was coupled to denitrification. Participation of oxygen in the oxidation of methane can be excluded; firstly, oxygen was measured continuously in the liquid and periodically in the headspace of the culture and was not detected. Secondly, all the detected dinitrogen gas in the headspace of the culture was accounted for by the consumption of nitrite and nitrate. If air would have leaked into the culture, more dinitrogen gas would have been detected in the headspace. Thirdly, the stoichiometry of methane consumption versus denitrification was in good agreement with Eq. 1 and 2. If oxygen would have been involved, at least 50% less nitrite or nitrate would have been consumed per mol of methane. Fourthly, in the absence of nitrite, no methane consumption was observed. The addition of sulphate did neither stimulate nor inhibit anaerobic methane oxidation in this case.

Hence, according to an aspect of the invention a method for the determination of a suitable consortium for application in the process of the invention is provided, comprising obtaining an anoxic sediment, feeding nitrite and methane to a reactor containing the anoxic sediment under anaerobic conditions, and determining based on a decrease in nitrite and methane, whether the consortium is suitable. In a preferred embodiment, a consortium for the process of the invention is isolated from any one of the following anoxic sediments of brackish or fresh water receiving downward fluxes of nitrate (or other N-O compounds) and upward fluxes of methane. Sediments, especially anoxic sediments, receiving an upward flux of methane and a downward flux of N-O compounds may comprise the consortium of the invention. Hence, in an embodiment the anoxic sediment comprises brackish or fresh water sediment receiving a downward flux of N-O compounds (i.e. one or more of the above-mentioned N-O compounds) and an upward flux of methane, such as in the Twentekanaal (as described herein). Further sediments wherein one or both of the organisms of the consortium may be found are defined below.

When a suitable consortium is found, the sediment can be further enriched with the consortium. Hence, a further aspect of the invention concerns a method for the production of a microbial consortium capable of anaerobic denitrification using methane, comprising providing an anoxic sediment, checking the capability of the sediment to reduce nitrate and/or nitrite in the presence of methane, and feeding methane and nitrate and/or nitrite to the sediment under anaerobic conditions until a nitrite consumption of at least 0.1 mmol nitrite/day/liter sediment or a nitrate consumption of at least 0.02 mmol nitrate/day/liter sediment, and a methane consumption of at least 0.05 mmol/day/liter sediment are obtained. The anoxic sediment preferably comprises a fresh water anoxic sediment and/or brackish anoxic sediment.

Preferably, the sediment is further enriched until a nitrite consumption of at least 0.5 mmol nitrite/day/liter sediment or a nitrate consumption of at least 0.1 mmol nitrate/- day/liter sediment is obtained, and a methane consumption of at least 0.1, more preferably at least 0.3 mmol/day/liter sediment is obtained. This means that the sediment at least shows methane oxidation coupled to one or more of nitrate and nitrite reduction: i.e. the sediment comprises the consortium of the invention

The phylogenetic identity of the members of the consortium responsible for DAMO was determined in a 16S rRNA approach. Hereto genomic DNA was isolated from the biomass in the enrichment culture and bacterial and archaeal 16S rRNA gene libraries were constructed. Sequence analysis of the bacterial clone library showed one dominant group of sequences that are related to a novel subdivision with no relationship to known isolated organisms thus far.

The archaeal clone library contained one dominant sequence which formed a separate cluster from the of marine anaerobic methano trophic archaea (sequence identity 86- 87%) and cultivated methanogens (sequence identity 86-88%). This is consistent with the presence of hydroxylarchaeol as bio marker which is also biosynthesised by anaerobic methanotrophic archaea. Both the bacterial and archaeal 16S rRNA gene sequences were used to design specific probes for fluorescence in situ hybridisation (FISH).

The microbial community of the enrichment culture was hybridised with probe EUB338-mix, targeting all bacteria, with three new probes targeting specifically the dominant bacterial sequence, with probe ARCH915 targeting all archaea and with a new probe targeting specifically the dominant archaeal sequence. Approximately 5-10% of the community consisted of archaea all of which hybridised with the specific probe targeting the dominant archaeal sequence. The remainder of the culture consisted of bacteria, of which approximately 80% hybridised with the three specific probes targeting the dominant bacterial sequence simultaneously. Alpha, Beta and Gamma proteobacteria together made up <5% of the community. The sulphate reducers known to be involved in consortia with marine anaerobic methano trophic archaea were not detected after hybridisation with the relevant probes consistent with the observation that sulphate was not converted in the culture.

The data show that a consortium of newly discovered denitrifying bacteria and a new group of archaea capable of reverse methanogens could be responsible for DAMO. The ratio of denitrifying cells to methanotrophic cells (approximately eight to one) differs from the ratio of sulphate reducing cells to methanotrophic cells in case of sulphate dependent anaerobic oxidation of methane (approximately two to one). This difference is consistent with the higher energy yield of denitrification compared to sulphate reduction. The denitrifier apparently benefits more from the extra energy than the methanotroph. The high affinity for methane observed (affinity constant for methane less than 0.6 μM) is also consistent with this larger amount of energy. The methane affinity of sulphate-dependent anaerobic methane oxidation is four orders of magnitude lower (affinity constant > 16 mM). The nitrite-dependent DAMO rate was 140 μmol methane-g^-protein-h^"1, corresponding to 0.4 fmol-cell^-day^"1 for the archaea in the enrichment culture. For sulphate-dependent anaerobic oxidation of methane a similar rate was reported for the archaeal partner (0.7 fmol-cell^-day^"1). This indicates that also for DAMO the growth rate of the archaea could be extremely low, with a doubling time in the order of several weeks, consistent with the long duration of the enrichment procedure.

Thus, the anaerobic denitrifying bacterium of the invention, capable of reducing nitrogen-oxygen compounds in cooperation with an anaerobic methanotrophic archaeon, comprises a 16S rRNA nucleotide sequence which exhibits at least 86% sequence identity with SEQ ID No. 1, preferably at least 90%, more preferably at least 94%, most preferably at least 98%. The genetically closest bacterium at this moment is believed to be a Thermacetogenium phaeum strain, which exhibits a degree of identity of between 84 and 85% with SEQ ID No. 1. The new bacterium appears to belong to a novel division for which the exact taxonomic position is yet to be resolved.

The anaerobic methanotrophic archaeon of the invention, capable of oxidising methane in cooperation with an anaerobic denitrifying bacterium, comprises a 16S rRNA nucleotide sequence which exhibits at least 88% sequence identity with SEQ ID No. 2, preferably at least 92%, more preferably at least 98%, most preferably at least 99.6%. The closest archaeon at this moment is believed to be Methanosaeta soehngenii , which exhibits a degree of identity of 85.6 with SEQ ID No. 2, while Methanomethylovorans hoHandica and Methanosarcina acetivorans appear to be the next closest organisms thus far, even though they have a slightly higher degree of identity of 87.5 and 86.8%, respectively.

The sequences, RNA and DNA identities etc. herein are compared, as known to the person skilled in the art, using pairwise alignment with software and default settings from http://pir.georgetown.edu/pirwww/search/pairwise.shtml, which uses the Smith- Waterman full-length alignments between two sequences.

DAMO is a newly discovered microbial lifestyle and its potential contribution to the biogeochemical cycling has so far been overlooked. The process is mediated by a new group of methanotrophic archaea assisted by a bacterial partner, the latter belonging to a bacterial division without any cultivated representatives so far. The detection of related 16S rRNA gene sequences in multiple freshwater sediments at different locations indicates that the newly discovered process may contribute significantly to methane oxidation in freshwater habitats. Potentially it could counteract the increases in methane production associated with nitrate run-off resulting from fertilisation in agricultural areas worldwide. With the biomarkers and probes for the responsible micro-organisms which are now available, this possibility can be assessed.

Therefore, the invention provides a process for the conversion of methane and one or more nitrogen-oxygen compounds to molecular nitrogen and carbon dioxide, comprising feeding the nitrogen-oxygen compounds and methane, in the essential absence of molecular oxygen, into a denitrifying reactor containing a consortium of an anaerobic denitrifying bacterium and an anaerobic methanotrophic archaeon in a liquid medium. The liquid medium may be a dedicated liquid for carrying out the DAMO process, e.g. in case the nitrogen oxides originate from a gaseous source, but usually the liquid medium will be constituted by a liquid waste water containing N-O compounds, dissolved methane, and optionally other compounds or classes of compounds like ammonium, BOD, ammonium, etc.

The temperature of the liquid medium in the reactor will preferably be maintained between 5 and 90 ⁰C, preferably between about 10 and 70 ⁰C. Preferably, a process for the conversion of methane an nitrogen-oxygen compounds is applied wherein the pH of the liquid medium in the denitrifying reactor is maintained between 5 and 10, preferably between 6 and 9, if necessary by adding acid or alkaline additives. The nitrogen-oxygen compounds are preferably fed in a liquid feed, e.g. waste water, sewage, etc. This feed preferably contains less than about 20 μmol/1 O₂, preferably less than 10 μmol/1 O₂. The gaseous methane feed preferably contains less than about 10 ppm O₂, more preferably less than about 3 ppm or even less than about 1 ppm O₂. In this way, the DAMO process can be performed in the reaction in the essential absence of oxygen. As will be clear to the person skilled in the art, an inert gas like nitrogen may be fed to the reactor before or during start up of the reactor wherein the DAMO process takes place.

In the invention, the DAMO-reaction takes preferably place in one single reactor, i.e. the reactor wherein the consortium is contained.

Some embodiments of the invention are now described in more detail, with reference to the figures.

Figure 1 schematically depicts the process for the conversion of methane and nitrogen- oxygen compounds to molecular nitrogen and carbon dioxide according to the invention. Nitrogen-oxygen compounds are fed, e.g. in a liquid waste stream, as feed 3 into a reactor 1 , which is a denitrifying reactor. Methane may be fed as separate feed 4 into reactor 1 but feeds 3 and 4 may also consist of one liquid feed 3/4 containing N-O compounds and dissolved methane. Methane feed 4 may for example be biogas of an anaerobic BOD digester (vide infra). Methane feed 4 especially indicates a gaseous feed, but may also refer to a liquid feed containing dissolved methane or both.

The feeds 3 and 4 are fed to reactor 1 in the essential absence of molecular oxygen. According to a preferred embodiment, a process is applied wherein the nitrogen-oxygen compounds are fed in liquid feed 3. The denitrifying reactor 1 contains a liquid medium 10, which contains a consortium 2 of an anaerobic denitrifying bacterium and an anaerobic methanotrophic archaeon. This consortium 2 may be comprised in an anoxic sediment (preferably after enrichment), as described above. A liquid effluent 15 is provided, with a decreased N content, relative to N-O feed 3. Further CO₂ gas 11 and N₂ gas 12 will leave reactor 1. Mixing of the consortium 2 (for instance as enriched sediment) and the N-O-containing liquid and methane may be achieved by using up and down risers, mixers, etc, as known in the art.

Feed 3 containing N-O compounds may be e.g. sewage, waste water, etc. This liquid feed may also contain ammonium (not depicted in figure 1). Hence, in a specific embodiment, reactor 1 further comprises anammox bacteria (anaerobic ammonium oxidation bacteria), known in the art, which are suitable for the conversion of the ammonium into N₂.

In general, the total amount of methane and the total amount of N-O compounds fed to the reactor is adjusted such that stoichiometrically at least 80 mol % of the total amount of methane can be converted or at least 80 mol % of the total amount of N-O compounds can be converted. In most cases, a slight excess of nitrogen oxides is not harmful, whereas an excess of methane is to be avoided. With undetermined nature of the nitrogen-oxygen compounds, the preferred rate of methane supply is between 0.25 to 0.5 mole, more preferably between 0.3 and 0.45 mole of methane per mole of nitrogen compound.

A further embodiment is schematically depicted in Figure 3a. The same reactor 1, feed 4 containing CH4, etc. are depicted, but now the N-O compound containing feed 3 is at least partially the product of the nitrification of ammonia. Hence, this specific embodiment provides a process wherein the nitrogen-oxygen compounds of feed 3, preferably comprising nitrite and/or nitrate, are produced by aerobic nitrification of ammonia in an aerobic nitrification reactor 100.

An ammonium-containing feed 103 and an oxygen-containing feed 104 (such as air and/or oxygen dissolved in liquid feed 103) are fed to this reactor 100, which further comprises a liquid medium 110 containing aerobic nitrifying bacteria 102. The bacteria may be present as such, or may be fixed on a solid carrier such as beads, sand or the like. The reaction that may take place can be e.g. NH₄ ⁺ + 1.5 O₂ -^ NO₂ ^". Both nitrate and nitrate may be formed in reactor 100, but preferably substantially nitrite is formed. Reactor 100 may for example be a conventional nitrifying reactor, or a CANON reactor wherein the CANON process is partly performed, i.e., the conditions are chosen such that ammonium is not completely oxidised to nitrate nor completely converted to dinitrogen gas, but substantially only to the intermediate NO₂ ^", for example by applying limited oxygen conditions or adjusting the sludge retention time as known in the art e.g. in EP-A 1113997. Consequently the amount of dissolved oxygen in 3 is very low due to activity of the nitrifying bacteria in 100. As mentioned above, the liquid feed to reactor 1, here feed 3, preferably contains less than about 20 μmol/1 O₂, preferably less than 10 μmol/1 0₂.

Preferably methane as feed 4 is fed into reactor 1 , but methane may also be present in the influent to reactor 1. For example, such an influent may comprise ammonium and dissolved CH₄.

In a specific embodiment not shown in Figure 3a, a second nitrification reactor 100 is provided downstream of reactor 1, in addition to the upstream nitrification reactor 100. The influent of this second nitrification reactor is effluent 15, and this second reactor may be used to nitrify ammonium left in effluent 15. The nitrified product from the second reactor may be returned to reactor 1 (see also the description of the embodiment related to figure 3 d) .

A further embodiment is schematically depicted in Figure 3b. Herein, feed 4 containing methane is the product of for example an anaerobic BOD oxidation reactor like a fermentor, as known in the art. In this embodiment, at least part of the methane fed into the anaerobic reactor is produced by anaerobic treatment of BOD. A BOD feed 204, e.g. waste water containing organic material, is fed to reactor 200, which contains a liquid medium 210 which comprises fermenting bacteria 202 (such as Bacteroides, Clostridia, Ruminococci etc.). Reactor 200 produces methane or biogas (i.e. a gas comprising methane, CO₂, etc.), which is fed as feed 4 to reactor 1, described above.

In addition to or instead of the N-O compounds in feed 3, the BOD-containing feed 204 may further comprise N-O compounds. In this variant, 204 refers to a feed containing BOD and N-O compounds and 4 refers to one or more feeds containing N-O compounds (as liquid feed) and methane (dissolved in the N-O compound containing feed and/or as separate gas feed) (see also the description of figure 3 c).

Figure 3c schematically depicts an embodiment wherein anaerobic BOD fermentation and ammonium nitrification are combined, such that methane and nitrite (and/or nitrate) are provided for the DAMO process of the invention. This figure schematically depicts a variant of an integrated process of the embodiments as depicted in figures 3a and 3b. An ammonium-containing feed 103 and a BOD-containing feed 204 (e.g. containing sewage) are fed to an anaerobic fermentation reactor 200. Within reactor 200, the fermentation bacteria 202 provide methane, which is fed to reactor 1 via a line 4. A liquid feed 215 from reactor 200, containing at least ammonium, is fed to reactor 100, wherein aerobic oxidising bacteria 102 convert at least part of the total ammonium amount to nitrite and/or nitrate. Oxygen may be supplied as separate feed 104. Then feed 3 from reactor 100, containing N-O compounds, is fed to reactor 1. The DAMO process can take place in reactor 1 due to the presence of methane from feed 4, the N-O compounds of feed 3 and the consortium 2 according to the invention. The amount of dissolved oxygen in feed 3 is very low due to activity of the nitrifying bacteria in reactor 100 (see also above).

Figure 3d also depicts an embodiment wherein anaerobic BOD oxidation and ammonium nitrification are combined, such that methane and nitrite (and/or nitrate) are provided for the DAMO process of the invention. Here, the nitrification reactor 100 is positioned downstream of DAMO reactor 1. The ammonium-containing feed 103 and BOD-containing feed 204 (which may be one feed containing both ammonium and BOD), are fed to reactor 200, containing BOD fermenting bacteria. BOD is at least partially converted to methane/biogas (mixture of methane, carbon dioxide and other gases) under anaerobic conditions. This gas is preferably fed as feed 4 into reactor 1. Feeds 103 and 104 may for instance be a sewage feed or a liquid industrial waste stream, etc. The outflow of reactor 200, indicated with reference number 215, comprises a liquid which contains ammonium, and which may also contain some dissolved methane. N-O compounds are provided by reactor 100, indicated with feed 3, wherein at least part of the liquid feed 215 is transported via reactor 1. This is indicated with feed 16 from reactor 1 to reactor 100. Within reactor 100, the aerobic nitrifying bacteria nitrify at least part of the total amount of ammonium contained in liquid feed 16 into the desired N-O compounds, which are then fed with feed 3 into reactor 1. The amount of dissolved oxygen in feed 3 is very low due to activity of the nitrifying bacteria in reactor 100 (see also above). Here, in reactor 1, at least part of the total amount of the N-O compounds and methane gas is converted to carbon dioxide and dinitrogen gas.

As will be clear to the person skilled in the art, the embodiments of figures 3c and 3d may also be combined: referring to figure 3 c, liquid feed 15 may be fed to a second nitrification reactor (not depicted), which may have a return to reactor 1 as additional N-O compound containing feed.

Hence, according to yet a further aspect of the invention, there is provided an apparatus comprising at least 3 reaction sections, which sections may be 3 separate reactors, wherein

• a first section (for example reactor 200) is designed and arranged such that BOD may at least partially be converted anaerobically to methane thereby providing a methane feed 4;

• at least one second section (for example reactor 100) is designed and arranged such that ammonium is at least partially converted aerobically to N-O compounds, thereby providing feed 3 containing N-O compounds;

• a third section (for example reactor 1) is designed and arranged to perform the process according to the invention; and wherein the first section 200, the second section 100 and the third section 1 are designed and arranged such that feed 3 and feed 4 are provided to section 1.

In yet another embodiment, an anaerobic denitrifying bacterium capable of reducing nitrogen-oxygen compounds is used for denitrification of a liquid comprising one ore more N-O compounds, such as a waste stream, etc., wherein the 16S-rRNA of the bacterium or its DNA copy comprises a nucleotide sequence exhibiting at least 86% sequence identity with the nucleotide sequence of SEQ ID No. 1.

In yet a further embodiment, an anaerobic methano trophic archaeon capable of oxidising methane is used for oxidising methane in a liquid comprising methane, such as a waste stream, etc., wherein the 16S-rRNA of the archaeon or its DNA copy comprises a nucleotide sequence exhibiting at least 88% sequence identity with the nucleotide sequence of SEQ ID No. 2.

In again another embodiment, above anaerobic denitrifying bacterium and anaerobic methano trophic archaeon are used for the conversion of methane and one or more nitrogen-oxygen compounds to molecular nitrogen and carbon dioxide in a liquid comprising methane and one or more N-O compounds, such as waste stream (i.e. such as water, e.g. sewage, industrial wastewater, etc.). Preferably, the consortium of the present invention is used. As will be clear to the person skilled in the art, the consortium can be present in the reactor as individual micro organisms, but the micro organisms, i.e. the archeon an the bacterium can also be present as micro colonies (randomly) distributed in aggregates or flocks, etc. They can also be present as cells. The cells or micro colonies are in general in close proximation to each other in the reactor, i.e. less then about 10 μm. The aggregates and flocks can vary in since from about 0.01 to about 10 mm. As known to the person skilled in the art, this may vary on the feeding regime and biomass retention.

The bacterium or archeon or both may be found in the following sediments: Mid Atlantic ridge number AY225657; Sedimentary Rock AB 179508; Uranium mine tailing

AJ519670; Contaminated soil AF529103; Shallow sediment AB198831; Contaminated aquifer DQ664101; Contaminated ground water AF351217 and AF351214; Sediment lake Biwa Japan ABl 16934; Black Sea Ukraine AJ578033; Lake Pluss see Germany

PMID: 16332891; Eel river basin USA AJ578121, AY324376; Hakoon Mud Vulcano, Artie Sea AJ579330; Mud volcanoes in the Carpathian Mountains, Romania PMID:

16584470; and the Twentekanaal, Netherlands DQ369741 and DQ369742.

Examples

Sampling. Samples for inoculation were taken from the sediment of Twentekanaal (52° I T 04" N en 6° 24' 40" E, The Netherlands). The sediment was sampled at different water depths and samples were collected from the top 15 cm of the sediments. At the time of sampling the methane concentration at 15 cm sediment depth was 0.8 mM. The nitrate concentration at 0 cm sediment depth was 0.1 mM.

Enrichment of DAMO consortium.

Sediments samples of appropriate origin can be used and 1.0 liter should be sufficient as source of biomass in a sequencing batch reactor (SBR; Figure 1). The SBR is maintained in a 2 1 (height, 0.22 m, diameter 0.125 m) vessel without baffles. The vessel is stirred at 350-500 rpm (6-bladed turbine stirrer, diameter one third of the vessel diameter). The temperature is at 25°C. As the sole carbon and energy source, CH4/CO2

(95/5 % vol.) is added continuously by sparging (10 ml-min^"1). The oxygen concentration is monitored using an O₂ electrode and the absence of oxygen from the headspace of the culture can be verified periodically by gas chromatography. The CO₂ present in the gas will be sufficient to buffer the solution and to keep the pH between 7.0 and 7.5. The SBR is filled continuously with mineral medium with nitrate, nitrite, and bicarbonate (see below) at 0.2 ml-min^"1 during 11.5 hours. After the filling period, the stirrer, influent and gas supply are stopped and the aggregates are allowed to settle for 15 minutes. In the remaining 15 minutes of the total cycle, part of the liquid is purged by an effluent pump. To prevent entry of air and loss of anaerobiosis in the purge period, effluent gas from the culture is stored in a separate 10-liter flask which is flushed continuously with helium (20 ml-min^"1). When the liquid is removed from the culture during purging, it is replaced by gas from this flask. The minimum liquid volume, after the liquid is purged, was 1.5 1. The maximum volume at the end of the filling period, is 1650 1. The composition of the mineral medium is (gT¹): KHCO₃ 1.25, KaH₂PO₄ 0.05, CaCl₂.2H₂O 0.3, MgSO₄. 7H₂O 0.2, FeSO₄ 0.00625, EDTA 0.00625, trace elements solution 1.25 ml- 1^"1. NaNO₂ is gradually increased reaching 6 mM after 18 months. NaNO₃ was kept around 5 mM. The trace element solution contained (gT¹): EDTA 15, ZnSO₄7H₂O 0.43, CoCl₂.6H₂O 0.24, MnCl₂.4H₂O 0.99, CuSO₄ 0.25, (NH₄)₆MoO₂₄.4H₂O 0.22, MC1₂.6H₂O 0.19, Na₂SeO₄ 0.067, H₃BO₄ 0.014, Na₂ WO₄.2H₂O 0.050. The separate components of the medium are autoclaved at 120⁰C. To adjust the pH and prevent entry of oxygen via the liquid medium, it is flushed continuously with Ar/CO₂ (95/5% vol.).

Dinitrogen gas, oxygen, methane and carbon dioxide were analysed using gas chromatography. Gas measurements were performed on a HP Agilent 6890 Series GC System equipped with a Thermo Conductivity Detector and a Porapak Q column. HP 5890 gas chromatograph equipped with a flame ionisation detector and a Porapak Q column (80/100 mesh). Nitrite and nitrate were measured with high performance liquid chromatography (HPLC). Liquid samples were centrifuged and 10 μl from the supernatant was injected with a Hewlett Packard 1050 series auto sampler. A sodium hydroxide solution was used as the liquid phase at a flow rate of 2 ml-min^"1. The gradient was formed by increasing the concentration of sodium hydroxide from 1 mM to 15 mM in 9 minutes. Separations were performed on a 4x250 mm Ionpac ASI l-HC (Dionex, GB) column at 30⁰C. Anions were detected using a CD25 conductivity detector (Dionex GB). 16S rRNA gene sequence analysis and FISH.

Chromosomal DNA from 1 ml reactor biomass, was isolated and used as a template for PCR amplification of 16S rRNA genes. PCR was performed with general bacterial primers (616F & 630R), and general archaeal primers (AR20F & AR958R). Cloning, sequencing and phylogenetic analysis were performed as described previously. Based on the bacterial and archaeal 16S rRNA gene sequences new oligonucleotide probes were designed. The bacterial probes were S-*-Dbact-0193-a-A-18 (5'-CGC TCG CCC CCT TTG GTC-3') (SEQ ID No. 3), S-*-Dbact-0447-a-A-18 (5'-CGC CGC CAA GTC ATT CGT-3') (SEQ ID No. 4), S-*-Dbact-1027-a-A-18 (5'-TCT CCA CGC TCC CTT GCG-3') (SEQ ID No. 5) and the archaeal probe was S-*-Darch- 0872-a-A-18 (5'-GGC TCC ACC CGT TGT AGT-3') (SEQ ID No. 6). We also used the general archaeal probe S-D-Arch-0915-a-A-20, two anaerobic marine methanotrophic archaea probes (ANME- 1-3507, ANME- 1-8628) and a probe for group-2 marine anaerobic methanotrophic archaea (EelMS9328). FISH was performed as described previously. The consortium could also be detected in sediments at other places than described above. Preferably, brackish and especially freshwater sediments which receive downward fluxes of nitrate or other N-O compounds (for instance due to agriculture) and upward fluxes of methane (generated by anaerobic decomposition) are used as source of the consortium (or of the individual micro-organisms.

Detection of microbes involved in anaerobic oxidation of methane and denitrification in sediment samples with molecular diagnostic tools In order to detect the presence of the microbes involved in anaerobic methane oxidation and denitrification in sediment samples, molecular diagnostic tools were developed based on the 16S rRNA genes retrieved from the enrichment cultures sequence listing (as described herein).

Several sediment samples were tested. The three samples were from the Ooijpolder (near Nijmegen, Netherlands), and were collected from fine, organic rich sediment in three ditches along a cornfield ([NO₃ ^"]: 240 μM). Another sample was from a sand filter from a wastewater treatment facility in Schiedam the Netherlands. Two samples were taken from a freshwater intertidal floodplain in Appels, Belgium, along the river Schelde ([NO₃ ^"]: 300 μM). The last sample was from a fresh water sediment in Rønbjerg, Northern Denmark. To sample 5, a small quantity of microbes from our enrichment containing both the archaeon and bacterial partner was added as an internal control.

Genomic DNA from all samples was isolated with the phenol extraction method, and precipitated with isopropanol. To remove humic acids, the DNA was purified with the Sephaglass extraction method using a FlexiPrep kit. A 830 bp fragment of the 16S rRNA of the micro organisms present in the anaerobic methane and denitrifying consortium was then PCR-amplified using specific primers based on the specific probes used for FISH: 193f (sequence identical to probe S-*-DBACT-0193-a-A-18) and 1027r (sequence identical to probe S-*-DBACT-1027-a-A-18) and The primers used for amplification of the archaea were IAFOR (59-TCYG[G/T]TTG ATCCYG[G/C]CRGAG-39) and ARC915 (59-GTGCTCCCCCGCCAATTCC T-39) or more specifically S-*-Darch- 0872-a-A-18 (5' GGC TCC ACC CGT TGT AGT-3').

In the PCR reaction, 1 μl of a 1/10 dilution of the gDNA solution was added at 80⁰C (hotstart) to a 50 μl mixture containing 5 μl 1Ox BSA solution (Idaho Technology #1777), 5 μl 1Ox PCR buffer, 20 mM MgCl₂), 0.5 μl 20 pmol/ml 193f and 1027r, 2 μl 5mM dNTPs and 0.2 μl 5U/μl Taq polymerase. The reaction conditions were 3 min 94°C, then 35 cycles of 1 min 94°C, 1 min 69°C and 1 min 72°C, and a final 10 min 72°C. The sand filter samples were amplified twice (first 25 cycles, then 20 cycles) with 3 μl product of the first reaction incubated in 50 μl fresh reaction mixture during the second amplification, and three PCR-products were pooled and purified by a gel excision and subsequent gel extraction using the QIAEXII gel extraction kit (Qiagen), fully according to the manufacturer's instructions. The consortium DNA was used as a positive control in all PCR reactions. Products from the specific reaction from the Ooijpolder samples were cloned into a pGEM T-easy vector and prepared for sequencing. Specific products from the sand filter samples were cloned into a pJETl/blunt vector (Fermentas) according to the manufacturer's prescriptions, and were prepared for sequencing with the pJETl sequencing primer set. The archaeal per product were also cleaved with specific restriction enzymes (i.e. Hindlll). Results

Using PCR-primers based on the DAMO-specific micro organism partial 16S rRNA gene sequences similar to the DAMO partners were amplified from DNA extracted from sediment samples from the Ooijpolder and from a wastewater treatment facility sand filter. The 193f / 1027r primer set was found to be able to specifically amplify bacterial 16S rRNA gene sequences from the phylogenetic clade of the DAMO bacterium. The IAFOR and Darch- 0872 needs to be optimized further. Maximum parsimony, neighbour-joining, and minimum evolution phylogenetic trees were then created, and general topology was identical for all of them. This analysis showed that, Ooijpolder clone C4 clustered closely with the DAMO bacterium indicating the presence of this micro-organism in this methane and nitrate rich sediment.

Furthermore using the 16S rRNA gene sequence of the microbes as a diagnostic tool to identify other places where these microbes can be found in clone libraries, we were able to document that close relatives of the microbes involved in anaerobic methane oxidation and denitrifcation can be found in:

Mid Atlantic ridge number AY225657

Sedimentary Rock AB 179508 Uranium mine tailing AJ519670

Contaminated soil AF529103

Shallow sediment AB 198831

Contaminated aquifer DQ664101

Contaminated ground water AF351217 and AF351214 Sediment lake Biwa Japan AB 116934

Black Sea Ukraine AJ578033

Lake Pluss see Germany PMID: 16332891

Eel river basin USA AJ578121, AY324376

Hakoon Mud Vulcano, Artie Sea AJ579330 Mud volcanoes in the Carpathian Mountains, Romania PMID: 16584470

Comparison with prior art

A comparison was made with the nitrogen conversion described in Islas-Lima et al (see above) and the nitrogen conversion obtained with the anaerobic denitrifying bacterium according to the invention alone or comprised in the consortium. The comparison was made with substantial equal conditions. It appears that the conversion of nitrogen (from for instance NO₃ ^" or NO₂ ^") of the prior art is about 5 mg per gram VSS (volatile suspended solids) per day whereas the present bacterium is able to convert nitrogen with a conversion rate of about 130 mg N per dw (dry weight) per day. The amounts of volatile suspended solids and dry weight are in the comparison substantially the same (i.e., the both essentially refer in this comparison to the biomass amount in the reactor). Hence, the present invention provides an anaerobic denitrifying bacterium and a consortium comprising the anaerobic denitrifying bacterium which bacterium is able to convert nitrogen at a rate of at least about 50 mg per gram VSS per day, even more preferably at least about 100 mg per gram VSS per day, yet even more preferably at least about 120 mg per gram VSS per day.

Claims

1. A process for the conversion of methane and one or more nitrogen-oxygen compounds to molecular nitrogen and carbon dioxide, comprising feeding the nitrogen-oxygen compounds and methane, in the essential absence of molecular oxygen, into a denitrifying reactor containing a consortium of an anaerobic denitrifying bacterium and an anaerobic methanotrophic archaeon in a liquid medium, wherein the 16S-rRNA of the anaerobic denitrifying bacterium or its DNA copy comprises a nucleotide sequence exhibiting at least 86% sequence identity with the nucleotide sequence of SEQ ID No. 1, and wherein the 16S-rRNA of the archaeon or its DNA copy comprises a nucleotide sequence exhibiting at least 88% sequence identity with the nucleotide sequence of SEQ ID No. 2.

2. The process according to claim 1, wherein the nitrogen-oxygen compounds are selected from the group consisting of NO, NO₂, NO₂ ^", NO₃ ^" and N₂O, preferably from NO₂ ^" and NO₃ ^".

3. The process according claim 1 or 2, wherein the nitrogen-oxygen compounds are fed in a liquid feed and the liquid feed contains less than 20 μmol/1 O₂.

4. The process according to any one of claims 1-3, wherein the amount of methane and the total amount of N-O compounds fed to the reactor is adjusted such that stoichiometrically at least 80 mol % of the total amount of methane can be converted or at least 80 mol % of the total amount of N-O compounds can be converted into molecular dinitrogen gas.

5. The process according to any one of claims 1-4, wherein methane is at least partially fed in a gas feed, which contains less than 10 ppm O₂.

6. The process according to any one of claims 1-5, wherein the pH of the liquid medium in the denitrifying reactor is between 5 and 10, preferably between 6 and 9.

7. The process according to any one of the preceding claims, wherein at least part of the methane fed into the anaerobic reactor is produced by anaerobic treatment of BOD.

8. The process according to any one of the preceding claims, wherein the nitrogen- oxygen compounds comprise nitrite and/or nitrate, which are produced by aerobic nitrification of ammonia.

9. The process according to claim 8, wherein the aerobic nitrification of ammonia is performed on a part of the liquid effluent of the anaerobic denitrifying reactor.

10. An anaerobic denitrifying bacterium capable of reducing nitrogen-oxygen compounds in cooperation with an anaerobic methano trophic archaeon, the 16S- rRNA of the bacterium or its DNA copy comprising a nucleotide sequence exhibiting at least 86% sequence identity with the nucleotide sequence of SEQ ID

No. 1.

11. An anaerobic methano trophic archaeon capable of oxidising methane in cooperation with an anaerobic denitrifying bacterium, the 16S-rRNA of the archaeon or its DNA copy comprising a nucleotide sequence exhibiting at least 88% sequence identity with the nucleotide sequence of SEQ ID No. 2.

12. A microbial consortium capable of oxidising methane and reducing nitrogen- oxygen compounds, comprising an archaeon according to claim 11 and a denitrifying bacterium.

13. A method for the production of a microbial consortium capable of simultaneous anaerobic denitrification using methane, comprising providing an anoxic sediment, checking the capability of the sediment to reduce nitrate and/or nitrite in the presence of methane, and feeding methane and nitrate and/or nitrite to the sediment under anaerobic conditions until a nitrite consumption of at least 0.1 mmol nitrite/day/liter sediment or a nitrate consumption of at least 0.02 mmol nitrate/day/liter sediment, and a methane consumption of at least 0.05 mmol/day/liter sediment are obtained.

14. The method according to claim 13, wherein the anoxic sediment comprises a brackish or fresh water sediment receiving a downward flux of N-O compounds and an upward flux of methane, wherein the nitrogen-oxygen compounds are selected from the group consisting of NO, NO₂, NO₂ ^", NO₃ ^" and N₂O, preferably from NO₂ ^" and NO3^".