WO2007069899A1 - Oxydation anaerobie de methane et denitrification - Google Patents

Oxydation anaerobie de methane et denitrification Download PDF

Info

Publication number
WO2007069899A1
WO2007069899A1 PCT/NL2006/050319 NL2006050319W WO2007069899A1 WO 2007069899 A1 WO2007069899 A1 WO 2007069899A1 NL 2006050319 W NL2006050319 W NL 2006050319W WO 2007069899 A1 WO2007069899 A1 WO 2007069899A1
Authority
WO
WIPO (PCT)
Prior art keywords
methane
anaerobic
nitrogen
reactor
sediment
Prior art date
Application number
PCT/NL2006/050319
Other languages
English (en)
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by St. Katholieke Universiteit Radboud Universiteit Nijmegen filed Critical St. Katholieke Universiteit Radboud Universiteit Nijmegen
Publication of WO2007069899A1 publication Critical patent/WO2007069899A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/28—Anaerobic digestion processes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/341—Consortia of bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • 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.
  • nitrogenous waste streams i.e. waste streams containing nitrogen-oxygen compounds
  • waste streams containing nitrogen-oxygen compounds can be treated by micro-organisms wherein the organisms use organic compounds as source of carbon and energy.
  • undesirable materials polylutants such as nitrous oxide
  • expensive chemicals such as methanol
  • metabolic products such as nitrite
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 schematically depicts an embodiment of a reactor and process of the invention.
  • Figures 2a-b depict the CH4, NO 2 " and NO 3 " consumption and dinitrogen gas production of the consortium according to an embodiment of the invention
  • FIGS 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.
  • biological treatment 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.
  • 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, NO 2 ,
  • N-O compounds can be reduced to N 2 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 NH 4 + (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.
  • 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.
  • 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.
  • DAMO Disitrification and Anaerobic Methane Oxidation
  • methanotroph refers to micro-organisms like bacteria or archaea that are able to grow using methane as their main source of carbon and energy.
  • 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.
  • oxygen limitation e.g. about ⁇ 0.5% air saturation
  • CANON Comppletely Autotrophic Nitrogen removal Over Nitrite
  • 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.
  • DAMO anaerobic oxidation of methane coupled to denitrification
  • One or more of the following DAMO reactions may occur in the process 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.
  • methane diffuseusing upwards
  • nitrate diffusing downwards
  • 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.
  • 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.
  • 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 13 C labeled methane. In these tests 13 CO 2 was detected as the end product of methane oxidation.
  • 13 CH 4 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 ⁇ M. 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 ⁇ M.
  • 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.
  • a method for the determination of a suitable consortium for application in the process of the invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FISH fluorescence in situ hybridisation
  • 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 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 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 ⁇ mol methane-g ⁇ -protein-h "1 , corresponding to 0.4 fmol-cell ⁇ -day "1 for the archaea in the enrichment culture.
  • 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.
  • 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.
  • 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.
  • 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 0 C, preferably between about 10 and 70 0 C.
  • 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 ⁇ mol/1 O 2 , preferably less than 10 ⁇ mol/1 O 2 .
  • the gaseous methane feed preferably contains less than about 10 ppm O 2 , more preferably less than about 3 ppm or even less than about 1 ppm O 2 .
  • the DAMO process can be performed in the reaction in the essential absence of oxygen.
  • 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.
  • the DAMO-reaction takes preferably place in one single reactor, i.e. the reactor wherein the consortium is contained.
  • 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.
  • 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 CO 2 gas 11 and N 2 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).
  • reactor 1 further comprises anammox bacteria (anaerobic ammonium oxidation bacteria), known in the art, which are suitable for the conversion of the ammonium into N 2 .
  • 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.
  • a slight excess of nitrogen oxides is not harmful, whereas an excess of methane is to be avoided.
  • 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.
  • FIG. 3a 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.
  • 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. NH 4 + + 1.5 O 2 - ⁇ NO 2 " . 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 NO 2 " , 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.
  • the liquid feed to reactor 1, here feed 3 preferably contains less than about 20 ⁇ mol/1 O 2 , preferably less than 10 ⁇ mol/1 0 2 .
  • methane as feed 4 is fed into reactor 1 , but methane may also be present in the influent to reactor 1.
  • an influent may comprise ammonium and dissolved CH 4 .
  • 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) .
  • feed 4 containing methane is the product of for example an anaerobic BOD oxidation reactor like a fermentor, as known in the art.
  • a BOD feed 204 e.g. waste water containing organic material
  • 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, CO 2 , etc.), which is fed as feed 4 to reactor 1, described above.
  • the BOD-containing feed 204 may further comprise N-O compounds.
  • 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
  • the fermentation bacteria 202 provide methane, which is fed to reactor 1 via a line 4.
  • 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.
  • 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.
  • 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).
  • at least part of the total amount of the N-O compounds and methane gas is converted to carbon dioxide and dinitrogen gas.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.).
  • waste stream i.e. such as water, e.g. sewage, industrial wastewater, etc.
  • the consortium of the present invention is used.
  • 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.
  • the cells or micro colonies are in general in close proximation to each other in the reactor, i.e. less then about 10 ⁇ m.
  • 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
  • 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).
  • SBR sequencing batch reactor
  • 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.
  • CH4/CO2 As the sole carbon and energy source, CH4/CO2
  • 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.
  • effluent gas from the culture is stored in a separate 10-liter flask which is flushed continuously with helium (20 ml-min "1 ).
  • helium 20 ml-min "1 .
  • the composition of the mineral medium is (gT 1 ): KHCO 3 1.25, KaH 2 PO 4 0.05, CaCl 2 .2H 2 O 0.3, MgSO 4 .
  • the trace element solution contained (gT 1 ): EDTA 15, ZnSO 4 7H 2 O 0.43, CoCl 2 .6H 2 O 0.24, MnCl 2 .4H 2 O 0.99, CuSO 4 0.25, (NH 4 ) 6 MoO 24 .4H 2 O 0.22, MC1 2 .6H 2 O 0.19, Na 2 SeO 4 0.067, H 3 BO 4 0.014, Na 2 WO 4 .2H 2 O 0.050.
  • the separate components of the medium are autoclaved at 120 0 C. To adjust the pH and prevent entry of oxygen via the liquid medium, it is flushed continuously with Ar/CO 2 (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 ⁇ l 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 30 0 C. Anions were detected using a CD25 conductivity detector (Dionex GB). 16S rRNA gene sequence analysis and FISH.
  • PCR 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.
  • molecular diagnostic tools were developed based on the 16S rRNA genes retrieved from the enrichment cultures sequence listing (as described herein).
  • 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
  • the sand filter samples were amplified twice (first 25 cycles, then 20 cycles) with 3 ⁇ l product of the first reaction incubated in 50 ⁇ l 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.
  • 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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Microbiology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Hydrology & Water Resources (AREA)
  • Engineering & Computer Science (AREA)
  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention vise à traiter efficacement les eaux et les gaz résiduaires contenant des composés azotés et des matières organiques pour produire de l'azote et du dioxyde de carbone moléculaires par oxydation anaérobie du méthane et dénitrification anaérobie simultanées à l'aide d'un consortium de deux micro-organismes : une bactérie dénitrifiante représentant un embranchement jusqu'ici sans aucune espèce cultivée et une archéobactérie apparentée de loin aux archéobactéries méthanotrophes marines. Dans le nouveau processus, des composés azote-oxygène, de préférence obtenus par nitrification aérobie de l'ammoniac, l'oxygène moléculaire étant essentiellement absent, sont introduits dans un réacteur de dénitrification contenant le consortium de la bactérie dénitrifiante anaérobie et d'une archéobactérie méthanotrophe anaérobie dans un milieu liquide en présence de méthane.
PCT/NL2006/050319 2005-12-15 2006-12-15 Oxydation anaerobie de methane et denitrification WO2007069899A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP05112228.1 2005-12-15
EP05112228 2005-12-15

Publications (1)

Publication Number Publication Date
WO2007069899A1 true WO2007069899A1 (fr) 2007-06-21

Family

ID=36499501

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL2006/050319 WO2007069899A1 (fr) 2005-12-15 2006-12-15 Oxydation anaerobie de methane et denitrification

Country Status (1)

Country Link
WO (1) WO2007069899A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103172171A (zh) * 2013-04-08 2013-06-26 中国科学技术大学 反硝化厌氧甲烷氧化微生物富集的***及其方法
PT105738B (pt) * 2011-05-31 2013-10-29 Nac De En E Geol Lab Processo de tratamento de efluentes orgânicos inibidores/tóxicos e da produção de biocombustíveis através da obtenção de um consórcio microbiano adaptado
CN109354174A (zh) * 2018-12-05 2019-02-19 青岛思普润水处理股份有限公司 基于canon_mbbr的强化脱氮***的快速启动方法
CN112062271A (zh) * 2020-09-17 2020-12-11 重庆大学 一种反硝化厌氧甲烷氧化膜生物电化学反应装置及其方法
CN115520955A (zh) * 2022-09-23 2022-12-27 清华大学 生物填料及其制备方法、应用和水质中硝酸盐的去除方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2026462A (en) * 1978-05-31 1980-02-06 Stengelin V Method and apparatus for the biological removal of nitrogen from waste water
DE3121395A1 (de) * 1981-05-29 1982-12-16 Klaus Ing.(grad.) 5600 Wuppertal Kratzenstein "verfahren und anlage zum denitrifizieren von wasser"
EP0261516A2 (fr) * 1986-09-26 1988-03-30 Gelsenwasser AG Procédé de dénitrification microbiologique continue des eaux souterraines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2026462A (en) * 1978-05-31 1980-02-06 Stengelin V Method and apparatus for the biological removal of nitrogen from waste water
DE3121395A1 (de) * 1981-05-29 1982-12-16 Klaus Ing.(grad.) 5600 Wuppertal Kratzenstein "verfahren und anlage zum denitrifizieren von wasser"
EP0261516A2 (fr) * 1986-09-26 1988-03-30 Gelsenwasser AG Procédé de dénitrification microbiologique continue des eaux souterraines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ISLAS-LIMA S ET AL: "Evidence of anoxic methane oxidation coupled to denitrification", WATER RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 38, no. 1, January 2004 (2004-01-01), pages 13 - 16, XP004473124, ISSN: 0043-1354 *
KNOWLES R: "Denitrifiers associated with methanotrophs and their potential impact on the nitrogen cycle", ECOLOGICAL ENGINEERING, ELSEVIER, vol. 24, no. 5, 30 May 2005 (2005-05-30), pages 441 - 446, XP004928588, ISSN: 0925-8574 *
M. STROUS, M. S. M. JETTEN: "Anaerobic Oxidation of Methane and Ammonium", ANNUAL REVIEW OF MICROBIOLOGY., vol. 58, 2004, USANNUAL REVIEWS INC., PALO ALTO, CA., pages 99 - 117, XP002384239 *
THALASSO F ET AL: "The use of methane as a sole carbon source for wastewater denitrification", WATER RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 31, no. 1, January 1997 (1997-01-01), pages 55 - 60, XP004168708, ISSN: 0043-1354 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT105738B (pt) * 2011-05-31 2013-10-29 Nac De En E Geol Lab Processo de tratamento de efluentes orgânicos inibidores/tóxicos e da produção de biocombustíveis através da obtenção de um consórcio microbiano adaptado
CN103172171A (zh) * 2013-04-08 2013-06-26 中国科学技术大学 反硝化厌氧甲烷氧化微生物富集的***及其方法
CN109354174A (zh) * 2018-12-05 2019-02-19 青岛思普润水处理股份有限公司 基于canon_mbbr的强化脱氮***的快速启动方法
CN109354174B (zh) * 2018-12-05 2021-08-17 青岛思普润水处理股份有限公司 基于canon_mbbr的强化脱氮***的快速启动方法
CN112062271A (zh) * 2020-09-17 2020-12-11 重庆大学 一种反硝化厌氧甲烷氧化膜生物电化学反应装置及其方法
CN112062271B (zh) * 2020-09-17 2021-12-07 重庆大学 一种反硝化厌氧甲烷氧化膜生物电化学反应装置及其方法
CN115520955A (zh) * 2022-09-23 2022-12-27 清华大学 生物填料及其制备方法、应用和水质中硝酸盐的去除方法
CN115520955B (zh) * 2022-09-23 2023-08-25 清华大学 生物填料及其制备方法、应用和水质中硝酸盐的去除方法

Similar Documents

Publication Publication Date Title
Xi et al. Heterotrophic nitrification and aerobic denitrification process: Promising but a long way to go in the wastewater treatment
Aktan et al. Inhibitory effects of free ammonia on Anammox bacteria
Qin et al. Effect of glucose on nitrogen removal and microbial community in anammox-denitrification system
Terada et al. Presence and detection of anaerobic ammonium-oxidizing (anammox) bacteria and appraisal of anammox process for high-strength nitrogenous wastewater treatment: a review
Wang et al. Start-up of the Anammox process from the conventional activated sludge in a membrane bioreactor
Chamchoi et al. Anammox enrichment from different conventional sludges
Chen et al. Methane-driven microbial fuel cells recover energy and mitigate dissolved methane emissions from anaerobic effluents
Pijuan et al. Effect of COD on mainstream anammox: Evaluation of process performance, granule morphology and nitrous oxide production
Hu et al. Nitrogen removal by a nitritation-anammox bioreactor at low temperature
Kartal et al. Application, eco-physiology and biodiversity of anaerobic ammonium-oxidizing bacteria
Modin et al. Denitrification with methane as external carbon source
Li et al. Enrichment of denitratating bacteria from a methylotrophic denitrifying culture
Zhai et al. New nitrogen removal pathways in a full-scale hybrid constructed wetland proposed from high-throughput sequencing and isotopic tracing results
Gupta et al. Managing dissolved methane gas in anaerobic effluents using microbial resource management-based strategies
Huang et al. C2/C3 fatty acid stress on anammox consortia dominated by Candidatus Jettenia asiatica
Sun et al. Stoichiometric and molecular evidence for the enrichment of anaerobic ammonium oxidizing bacteria from wastewater treatment plant sludge samples
Gao et al. Heterotrophic denitrification strategy for marine recirculating aquaculture wastewater treatment using mariculture solid wastes fermentation liquid as carbon source: Optimization of COD/NO3−-N ratio and hydraulic retention time
Trzcinski et al. Performance of a three-stage membrane bioprocess treating the organic fraction of municipal solid waste and evolution of its archaeal and bacterial ecology
Stams et al. Understanding and advancing wastewater treatment
Contreras et al. Nitrate/nitrite-dependent anaerobic oxidation of methane (N-AOM) as a technology platform for greenhouse gas abatement in wastewater treatment plants: state-of-the-art and challenges
Manasa et al. Current perspectives of anoxic ammonia removal and blending of partial nitrifying and denitrifying bacteria for ammonia reduction in wastewater treatment
WO2007069899A1 (fr) Oxydation anaerobie de methane et denitrification
Liu et al. Simultaneous dissolved methane and nitrogen removal from low-strength wastewater using anaerobic granule-based sequencing batch reactor
Molina-Macías et al. Denitrifying anaerobic methane oxidation and its applications for wastewater treatment
JP4610374B2 (ja) 新規微生物、当該新規微生物を用いた廃水処理方法及び廃水処理装置

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06824314

Country of ref document: EP

Kind code of ref document: A1