METHANE OXIDATION METHODS AND COMPOSITIONS
20170107479 ยท 2017-04-20
Inventors
Cpc classification
C12M29/04
CHEMISTRY; METALLURGY
C12M43/08
CHEMISTRY; METALLURGY
C12M29/00
CHEMISTRY; METALLURGY
Y02E60/50
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
C12M29/26
CHEMISTRY; METALLURGY
H01M8/1039
ELECTRICITY
H01M8/06
ELECTRICITY
H01M8/1009
ELECTRICITY
International classification
H01M8/06
ELECTRICITY
H01M8/1009
ELECTRICITY
H01M8/1039
ELECTRICITY
Abstract
The disclosure provides devices and methods to produce electrical energy from microorganisms capable of metabolizing methane.
Claims
1. A bioelectrochemical device comprising: a methane source to provide methane to the bioelectrochemical device; an aqueous solvent system that comprises an aqueous soluble electron acceptor that is re-circulated in the bioelectrochemical device; a bioreactor comprising one or more different types of C.sub.1-metabolizing microorganisms that are capable of metabolizing methane and reducing the electron acceptor to a reduced electron acceptor; a fuel cell comprising: an anode that can oxidize the reduced electron acceptor back to the electron acceptor, a cathode that can reduce an oxidant, and an ion conductor placed between the cathode and anode, wherein the ion conductor selectively transports positively or negatively charged ions.
2. The bioelectrochemical device of claim 1, wherein the bioelectrochemical device further comprises one or more of the following: a series of tubes that are fluidly in contact with the bioreactor and the fuel cell; a pump that powers the recirculation of the solvent system; a check valve that selectively allows the passage of methane into the bioelectrochemical device; a set of valves and inlets that are fluidly in contact with the aqueous solvent system that allow for addition or adjustment of the aqueous solvent system; and/or a CO.sub.2 extraction device that removes dissolved CO.sub.2 from the aqueous solvent system.
3. The bioelectrochemical device of claim 2, wherein the bioelectrochemical device comprises: the pump that powers the recirculation of the solvent system; the check valve that selectively allows the passage of methane into the bioelectrochemical device; the set of valves and inlets that are fluidly in contact with the aqueous solvent system that allow for addition or adjustment of the aqueous solvent system; the CO.sub.2 extraction device that removes dissolved CO.sub.2 from the aqueous solvent system; and/or the series of tubes that are fluidly in contact with the bioreactor, and the fuel cell, wherein the series of tubes are also fluidly in contact with the pump, the check valve, the set of valves and inlets/outlets, and the CO.sub.2 extraction device.
4. The bioelectrochemical device of claim 1, wherein the one or more different types of C.sub.1-metabolizing microorganisms are methanogens, methanotrophs, and/or a recombinantly engineered organism(s) that are capable of oxidizing methane.
5. The bioelectrochemical device of claim 1, wherein the one or more different types of C.sub.1-metabolizing microorganisms are anaerobic methanotrophs; and wherein the aqueous solvent is substantially free or devoid of dissolved oxygen.
6. The bioelectrochemical device of claim 1, wherein the methane source is a methane producing fermentation system utilizing biomass, coal, human waste, and/or animal waste as a fuel source, and/or a purified fuel gas stream that is enriched with methane.
7. The bioelectrochemical device of claim 1, wherein the electron acceptor is a single-electron acceptor with a standard reduction potential more positive than ca. 240 mV.
8. The bioelectrochemical device of claim 7, wherein a redox active molecule is used as electron acceptor is selected from the group consisting of 2,6-AQDS (9,10-anthraquinone-2,6-disulfonate), 2,7-AQDS (9,10-anthraquinone-2,7-disulfonate), 1,5-AQDS (9,10-anthraquinone-1,5-disulfonate), Fe(III)-citrate, Fe(III)-EDTA, humic acids, and melanin.
9. The bioelectrochemical device of claim 1, wherein the aqueous solvent system further comprises one or more of the following: buffers, salts, and/or nutrients.
10. The bioelectrochemical device of claim 1, wherein the bioreactor comprises: a housing with two surfaces an inner surface that comes into contact with the aqueous solvent and an outer surface that does not come into contact with the aqueous solvent; at least two ports to allow for the aqueous solvent to enter the bioreactor and to allow for the aqueous solvent to exit the bioreactor.
11. The bioelectrochemical device of claim 10, wherein the bioreactor comprises one or more C.sub.1-metabolizing microorganisms that are grown or contained within a bed of media or solid support(s).
12. The bioelectrochemical device of claim 11, wherein the media and solid support comprises a high surface area so that C.sub.1-metabolizing microorganisms can spread across the surface of the media or the solid support.
13. The bioelectrochemical device of claim 12, wherein the solid support is comprised of a porous or very porous material.
14. The bioelectrochemical device of claim 10, wherein the bioreactor further comprises filters or membranes that prevent the passage of C.sub.1-metabolizing microorganisms or cellular debris out of the bioreactor.
15. The bioelectrochemical device of claim 1, wherein the aqueous solvent flows into the top of the fuel cell and exits out the bottom of the fuel cell.
16. The bioelectrochemical device of claim 1, wherein the aqueous solvent flows into the bottom of the bioreactor and exits out the top of the bioreactor.
17. The bioelectrochemical device of claim 1, wherein the anode and cathode of the fuel cell comprise of electrodes made of carbon or metals such as titanium, and modified by coating with platinum or platinum ruthenium alloys as catalysts; and wherein the ion conductor includes but not limited to a sulfonated tetrafluoroethylene based fluoropolymer-copolymer membrane.
18. The bioelectrochemical device of claim 2, wherein the CO.sub.2 extraction device is a membrane degasifier device or CO.sub.2-precipitating chemical reactor.
19. The bioelectrochemical device of claim 3, wherein the pump, check valve and CO.sub.2 extraction device are electronically controlled directly and/or controlled remotely over a network or wireless communication using a computer, cell phone, and/or tablet.
20. A method to produce direct electrical current comprising providing methane to the bioelectrochemical device of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description, serve to explain the principles and implementations of the invention.
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DETAILED DESCRIPTION
[0026] As used herein and in the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a polynucleotide includes a plurality of such polynucleotides and reference to the microorganism includes reference to one or more microorganisms, and so forth.
[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.
[0028] Also, the use of or means and/or unless stated otherwise. Similarly, comprise, comprises, comprising include, includes, and including are interchangeable and not intended to be limiting.
[0029] It is to be further understood that where descriptions of various embodiments use the term comprising, those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language consisting essentially of or consisting of.
[0030] Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
[0031] As used herein, the terms C.sub.1-metabolizing microorganism or C.sub.1-metabolizing non-photosynthetic microorganism refers to any microorganism having the ability to use a C.sub.1-substrate as a source of energy or as its sole source of energy and biomass, and may or may not use other carbon substrates (such as sugars and complex carbohydrates) for energy and biomass. For example, a C.sub.1-metabolizing microorganism may oxidize a C.sub.1 substrate, such as methane, and methanol. C.sub.1-metabolizing microorganisms include Bacteria (such as aerobic methanotrophs in the phylum of Proteobacteria or Verrucomicrobia) and Archaea (such as methanogens and anaerobic methanotrophs in the phylum of Euryarchaeota and orders of Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanocellales, Methanopyrales and Methanomassiliicoccales, or potentially in the phylum of Bathyarchaeota or Verstraetearchaeota) and yeast. In at least some instances, a C.sub.1-metabolizing microorganism does not include a photosynthetic microorganism, such as algae. In certain embodiments, the C.sub.1-metabolizing microorganism could be an obligate methane metabolizing microorganism, meaning its sole source of energy comprises methane and nothing else.
[0032] As used herein, the term methylotrophic organism refers to any organism capable of oxidizing organic compounds that do not contain carbon-carbon bonds. In certain embodiments, a methylotrophic organism may be a methanotroph. For example, methanotrophic organism refers to any methylotrophic organism that has the ability to oxidize methane as its primary source of carbon and energy. Exemplary methanotrophic organisms include, but are not limited to, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, or Methanomonas in the Domain of Bacteria, and those in phylum of Euryarchaeota such as Anaerobic Methanotrophic Archaea (ANME) clade 1, 2 or 3 (or ANME-1, ANME-2, ANME-3 respectively), Methanoperedens (formerly known as ANME-2d), or in the phylum of Bathyarchaeota or Verstraetearchaeota in the Domain of Archaea. In certain other embodiments, the methylotrophic organism is an obligate methylotrophic organism, which refers to organisms that are limited to the use of C.sub.1 substrates for the generation of energy.
[0033] The term microorganism includes archaeal, bacterial and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms microbial cells and microbes are used interchangeably with the term microorganism.
[0034] The term prokaryotes is art recognized and refers to cells which contain no nucleus or other cell organelles. The prokaryotes generally refer to two of three Domains of life, namely the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA gene.
[0035] The term Archaea refers to a categorization of organisms formerly of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of 16s rRNA analysis, the Archaea consist of multiple phylogenetically-distinct groups, including but not limited to Euryarchaeota, Bathyarchaeota, Verstraetearchaeota and Crenarchaeota. Besides the unifying archaeal features that distinguish them from Bacteria (i.e., no murein in cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats.
[0036] Bacteria, or eubacteria, refers to one of three Domains of life. Bacteria include at least 74 distinct phylums widely distributed in the environment with a range of metabolisms.
[0037] As used herein, an activity of an enzyme is a measure of its ability to catalyze a reaction resulting in a metabolite, i.e., to function, and may be expressed as the rate at which the metabolite of the reaction is produced. For example, enzyme activity can be represented as the amount of metabolite produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.
[0038] The term biosynthetic pathway, also referred to as metabolic pathway, refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another. Gene products (e.g., enzymes or polypeptides) belong to the same metabolic pathway if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product. The disclosure provides recombinant microorganism having a metabolically engineered pathway for the production of a desired product or intermediate.
[0039] Accordingly, metabolically engineered or modified microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice thereby modifying or altering the cellular physiology and biochemistry of the microorganism. Through the introduction of genetic material, the parental microorganism acquires new properties, e.g. the ability to produce or consume a new, or greater quantities of, an intracellular metabolite. In an illustrative embodiment, the introduction of genetic material into a parental microorganism results in a new or modified ability to metabolize methane or methanol. The genetic material introduced into the parental microorganism contains gene(s), or parts of gene(s), coding for one or more of the enzymes involved in a biosynthetic pathway for the production or consumption of formylmethanofuran, 5-formyl-5,6,7,8-tetrahydromethanopterin, 5,10-methenyltetrahydromethanopterin, 5,10-methylenetetrahydromethanopterin, 5-methyltetrahydromethanopterin, methylcoenzyme M, coenzyme M 7-mercaptoheptanoylthreonine-phosphate heterodisulfide, acetyl phosphate, acetyl coenzyme A, and may also include additional elements for the expression and/or regulation of expression of these genes into functional enzyme products, e.g. promoter sequences, chaperones, post-translational modification enzymes, and cofactor and coenzyme synthesis and incooperation pathways and enzymes.
[0040] An enzyme means any substance, typically composed wholly or largely of amino acids making up a protein or polypeptide with possibly post-translational modifications that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.
[0041] A protein or polypeptide, which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds, and may include cofactors as well as post-translational modifications for their intended function and catalysis. A protein or polypeptide can function as an enzyme.
[0042] As used herein, the term metabolically engineered or metabolic engineering involves rational pathway design and assembly of polypeptides having a desired activity, biosynthetic genes, genes associated with operons, and/or control elements of such polynucleotides, for the production of a desired metabolite, such as an formylmethanofuran, 5-formyl-5,6,7,8-tetrahydromethanopterin, 5,10-methenyltetrahydromethanopterin, 5,10-methylenetetrahydromethanopterin, 5-methyltetrahydromethanopterin, methylcoenzyme M, coenzyme M 7-mercaptoheptanoylthreonine-phosphate heterodisulfide, acetyl phosphate, acetyl coenzyme A, higher alcohols or other chemical, in vitro or in a microorganism. Metabolically engineered can further include optimization of metabolic flux, particular reaction conditions, optimization of transcription, translation, protein stability and protein functionality using genetic engineering and appropriate culture condition including the reduction of, disruption, or knocking out of, a competing metabolic pathway that competes with an intermediate leading to a desired pathway. A biosynthetic gene can be heterologous to the host microorganism, either by virtue of being foreign to the host, or being modified by mutagenesis, recombination, and/or association with a heterologous expression control sequence in an endogenous host cell. In one embodiment, where the polynucleotide is xenogenetic to the host organism, the polynucleotide can be codon optimized.
[0043] A metabolite refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process that gives rise to a desired metabolite, chemical, alcohol or ketone. A metabolite can be an organic compound that is a starting material (e.g., methanol, methane, formaldehyde etc.), an intermediate in (e.g., formic acid), or an end product (e.g., carbon dioxide or dissolved inorganic carbon) of metabolism. Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
[0044] A native or wild-type protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.
[0045] A parental microorganism refers to a cell used to generate a recombinant microorganism. The term parental microorganism describes, in one embodiment, a cell that occurs in nature, i.e., a wild-type cell that has not been genetically modified. The term parental microorganism further describes a cell that serves as the parent for further engineering. In this latter embodiment, the cell may have been genetically engineered, but serves as a source for further genetic engineering.
[0046] For example, a wild-type microorganism can be genetically modified to express or over express a first target enzyme such as a methyl-coenzyme M reductase. This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or over-express a second target enzyme, e.g., a methyltetrahydromethanopterin:coenzyme M methyl-transferase. In turn, that microorganism can be modified to express or over express a third target enzyme, e.g., a heterodisulphide reductase. As used herein, express or over-express refers to the phenotypic expression of a desired gene product. In one embodiment, a naturally occurring gene in the organism can be engineered such that it is linked to a heterologous promoter or regulatory domain, wherein the regulatory domain causes expression of the gene, thereby modifying its normal expression relative to the wild-type organism. Alternatively, the organism can be engineered to remove or reduce a repressor function on the gene, thereby modifying its expression. In yet another embodiment, a cassette comprising the gene sequence operably linked to a desired expression control/regulatory element is engineered in to the microorganism.
[0047] Accordingly, a parental microorganism functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing one or more nucleic acid molecules into the reference cell. The introduction facilitates the expression or over-expression of one or more target enzyme or the reduction or elimination of one or more target enzymes. It is understood that the term facilitates encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term facilitates encompasses the introduction of exogenous polynucleotides encoding a target enzyme into a parental microorganism.
[0048] Polynucleotides that encode enzymes useful for generating metabolites (e.g., enzymes such as methyl-coenzyme M reductase, methyltetrahydromethanopterin:coenzyme M methyl-transferase, heterodisulphide reductase, F420-dependent methylenetetrahydromethanopterin reductase, F420-dependent methylenetetrahydromethanopterin dehydrogenase, methenyltetrahydromethanopterin cyclohydrolase, formylmethanofuran: tetrahydromethanopterin formyltransferase, formylmethanofuran dehydrogenase, ATP:acetate phosphotransferase, acetyl-CoA:phosphate acetyltransferase, acetate:CoA ligaseacetyl-CoA decarbonylase/synthase) including homologs, variants, fragments, related fusion proteins, or functional equivalents thereof, are used in recombinant nucleic acid molecules that direct the expression of such polypeptides in appropriate host cells, such as prokaryotic or eukaryotic cells.
[0049] Exemplary protein sequences encoded by polynucleotides can be found below from published genomes of methanogenic archaea and published incomplete genomes of methanotrophic archaea. These genes and their homologs are useful in the methods described herein, include but are not limited to the following listed in Table 1:
TABLE-US-00001 TABLE 1 Examples of protein sequences encoded by polypeptides from two methanogenic archaea. Methanosarcina barkeri str. Methanococcus Gene Full_Name Fusaro maripaludis X1 fmdA formylmethanofuran WP_011306299 WP_011170453 dehydrogenase subunit A fmdB formylmethanofuran WP_011306302 WP_013999131 dehydrogenase subunit B fmdC formylmethanofuran WP_011306300 WP_013999132 dehydrogenase subunit C fmdD formylmethanofuran WP_011306301 AEK19995 dehydrogenase subunit D fmdE formylmethanofuran WP_011306297 WP_013999133 dehydrogenase subunit E fmdF formylmethanofuran AAZ71499 AEK20270 dehydrogenase subunit F fmdG formylmethanofuran AAZ71816 dehydrogenase subunit G fmdH formylmethanofuran AEK20269 dehydrogenase subunit H ftr formylmethanofuran-- WP_011306001 WP_013999908 tetrahydromethanopterin N-formyltransferase mch methenyltetrahydromethan- WP_011307204 WP_013999597 opterin cyclohydrolase mtd methylenetetrahydromethan- WP_011306111 WP_011171660 opterin dehydrogenase mer 5,10- WP_011305293 WP_013998544 methylenetetrahydromethan- opterin reductase mtrA tetrahydromethanopterin WP_011306268 WP_013999872 S-methyltransferase subunit A mtrB tetrahydromethanopterin WP_011306269 WP_011171507 S-methyltransferase subunit B mtrC tetrahydromethanopterin WP_011306270 WP_011171506 S-methyltransferase subunit C mtrD tetrahydromethanopterin WP_011306271 WP_011171505 S-methyltransferase subunit D mtrE tetrahydromethanopterin WP_011306272 WP_013999871 S-methyltransferase subunit E mtrF tetrahydromethanopterin WP_011306267 S-methyltransferase subunit F mtrG tetrahydromethanopterin WP_011306266 WP_011171510 S-methyltransferase subunit G mtrH tetrahydromethanopterin WP_011306265 WP_011171511 S-methyltransferase subunit H mcrA methyl-coenzyme M WP_011305916 WP_013999870 reductase subunit A mcrB methyl-coenzyme M WP_011305920 WP_011171499 reductase subunit B mcrC methyl-coenzyme M WP_011305918 WP_011171501 reductase subunit C mcrD methyl-coenzyme M WP_011305919 WP_013999868 reductase subunit D mcrG methyl-coenzyme M WP_011305917 WP_013999869 reductase subunit G hdrA heterodisulfide AAZ71500 AEK19828 reductase subunit A hdrB heterodisulfide AAZ70892 WP_013999494 reductase subunit B hdrC heterodisulfide AAZ70891 WP_013999495 reductase subunit C hdrD heterodisulfide P96797.4 reductase subunit D hdrE heterodisulfide P96796.2 reductase subunit E mtaA [methyl-Co (III) Q48949 methanol-specific corrinoid protein]: coenzyme M methyltransferase mtaB methanol---5- Q46EH3 hydroxybenzimidazolyl- cobamide Co-methyltransferase mtaC methanol corrinoid AAZ72501 protein Examples of protein sequences encoded by polypeptides from archaea involved in or potentially involved in anaerobic oxidation of methane. Candidatus Candidatus Candidatus Bathyarchaeota Bathyarchaeota Methanoperedens archaeon archaeon ANME-1 ANME-2a nitroreducens BA1 (Evans BA2 (Evans (Meyerdierks (Wang et (Haroon al. et et al. et al. Gene et al. 2010) al. 2014) 2013) 2015) 2015) fmdA CBH38674 IMG KCZ72275 2566126780 fmdB CBH38673 IMG KCZ72274 2566124824 fmdC CBH38675 IMG KCZ72276 KPV62166 2566125475 fmdD CBH38672 IMG KCZ72273 2566124823 fmdE IMG KCZ73342 2566125238 fmdF IMG 2566123321 fmdG IMG 2566125853 fmdH ftr CBH38579 IMG KCZ73568 KPV63694 KPV63869 2566123292 mch CBH37751 IMG KCZ70764 KPV62158 2566124083 mtd CBH37899 IMG 2566126853 mer IMG KCZ71526 KPV65334 2566123633 mtrA CBH38255 IMG KCZ73426 2566123987 mtrB CBH38256 IMG KCZ73425 2566123988 mtrC CBH38257 IMG KCZ73424 2566123989 mtrD CBH38258 IMG KCZ73423 2566123990 mtrE CBH38259 IMG KCZ73422 2566123991 mtrF KCZ73427 mtrG CBH38253 IMG KCZ73428 2566125855 mtrH CBH38252 IMG KCZ71022 KPV64965 2566123984 mcrA CBH37407 IMG KCZ72673 KPV65186 KPV61791 2566125579 mcrB CBH37405 IMG KCZ72670 KPV65184 KPV61799 2566125583 mcrC CBH37386 IMG KCZ72448 KPV61780 2566125581 mcrD IMG KCZ72671 KPV61779 2566125582 mcrG CBH37406 IMG KCZ72672 KPV65185 KPV61800 2566125580 hdrA CBH38803 IMG KCZ70533 KPV63203 KPV64464 2566125169 hdrB CBH38352 IMG KCZ71427 KPV63202 KPV61761 2566124428 hdrC CBH37748 IMG KCZ71093 KPV63201 KPV61762 2566124427 hdrD IMG KPV65052 KPV63063 2566126466 hdrE IMG KCZ70770 2566126467
It is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence (e.g., poly HIS tags), is a conservative variation of the basic nucleic acid.
[0050] In addition to methane metabolizing genes, methanotrophic archaea or engineered host should contain a mechanism to interact with extracellular electron acceptors and transfer electrons to these redox active molecules. Potential proteins found in ANME-2 such as ANME-2a (IMG Gene ID 2566123487 and 2566125052), ANME-2b (NCBI Accession Number KR811028), ANME-2d or Methanoperedens nitroreducens (IMG Gene ID 2515322983, 2515320866) together with membrane and extracellular cytochrome b and/or c such as the one in ANME-2a (IMG Gene ID 2566125773 and 2566125774) and their homologs, as well as promoter sequences, chaperons and genes associated with their synthesis and operation with the reverse methanogenesis or methane oxidation genes list in Table 1 are useful in the methods described herein and represent one way to transfer electrons to electron acceptors. Other ways of transferring electrons to extracellular electron acceptors include but not limited to the systems described in bacterial genuses Geobacter, Shewanella, and Rhodopseudomonas involving genes omcB (NCBI Accession Number WP 010943369), cytochrome C (NCBI Accession Number AJY69163), ppcA (NCBI Accession Number AAN40982), mtrA (NCBI Accession Number NP_717386), mtrB (NCBI Accession Number NP_717385), mcrC (NCBI Accession Number NP_717387), cymA (NCBI Accession Number NP_720107.1), pioA (NCBI Accession Number ABL63052), pioB (NCBI Accession Number ABL63053), pioC (NCBI Accession Number ABL63054) and their homologs, as well as promoter sequences, chaperons and genes associated with their synthesis and operation with the reverse methanogenesis or methane oxidation genes list in Table 1.
[0051] It is understood that a polynucleotide described herein include genes and that the nucleic acid molecules described above include vectors or plasmids. For example, a polynucleotide encoding a methyl-coenzyme M reductase can comprise an mcrA gene or homolog thereof, or an mcrB gene or homolog thereof, or a mcrG gene or homolog thereof. Accordingly, the term gene, also called a structural gene refers to a polynucleotide that codes for a particular polypeptide comprising a sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter region or expression control elements, which determine, for example, the conditions under which the gene is expressed, in cooperation with cofactors such as Fe or Ni, synthesis and in cooperation with coenzymes such as heme or iron-sulfur clusters, as well as addition of post-translational modifications. The transcribed region of the gene may include untranslated regions, including introns, 5-untranslated region (UTR), and 3-UTR, as well as the coding sequence.
[0052] The term polynucleotide, nucleic acid or recombinant nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
[0053] The term expression with respect to a gene or polynucleotide refers to transcription of the gene or polynucleotide and, as appropriate, translation of the resulting mRNA transcript to a protein or polypeptide. Thus, as will be clear from the context, expression of a protein or polypeptide results from transcription and translation of the open reading frame.
[0054] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of codons differing in their nucleotide sequences can be used to encode a given amino acid. A particular polynucleotide or gene sequence encoding a biosynthetic enzyme or polypeptide described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes polynucleotides of any sequence that encode a polypeptide comprising the same amino acid sequence of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with alternate amino acid sequences, and the amino acid sequences encoded by the DNA sequences shown herein merely illustrate exemplary embodiments of the disclosure.
[0055] The disclosure provides polynucleotides in the form of recombinant DNA expression vectors or plasmids, as described in more detail elsewhere herein, that encode one or more target enzymes. Generally, such vectors can either replicate in the cytoplasm of the host microorganism or integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host microorganisms with increasing numbers of cell divisions). The disclosure provides DNA molecules in isolated (i.e., not necessarily pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form.
[0056] A polynucleotide of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques and those procedures described in the Examples section below. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
[0057] It is also understood that an isolated polynucleotide molecule encoding a polypeptide homologous to the enzymes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the particular polypeptide, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into the polynucleotide by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In contrast to those positions where it may be desirable to make a non-conservative amino acid substitution, in some positions it is preferable to make conservative amino acid substitutions.
[0058] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called codon optimization or controlling for species codon bias.
[0059] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res. 17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891, and the references cited therein.
[0060] The term recombinant microorganism and recombinant host cell are used interchangeably herein and refer to microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express non-endogenous sequences, such as those included in a vector. The polynucleotide generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite as described above, but may also include protein factors necessary for regulation or activity or transcription. Accordingly, recombinant microorganisms described herein have been genetically engineered to express or over-express target enzymes not previously expressed or over-expressed by a parental microorganism. It is understood that the terms recombinant microorganism and recombinant host cell refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism.
[0061] The term substrate or suitable substrate refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. Further, the term substrate encompasses not only compounds that provide a carbon source suitable for use as a starting material, but also intermediate and end product metabolites used in a pathway associated with a metabolically engineered microorganism as described herein.
[0062] Transformation refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including electroporation, microinjection, biolistics (or particle bombardment-mediated delivery), or agrobacterium mediated transformation.
[0063] A vector generally refers to a polynucleotide that can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are episomes, that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature.
[0064] The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell(s) in which the vector is intended to replicate or drive expression. Expression vector components suitable for the expression of genes and maintenance of vectors in E. coli, yeast, Streptomyces, and other cells including archaea that have been published in the literature and/or are available commercially. For example, suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host microorganism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433, which is incorporated herein by reference in its entirety), can also be used. For expression vectors such as those been demonstrated in E. coli, it is useful to include an E. coli origin of replication, such as from pUC, plP, p1, and pBR.
[0065] Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of a gene coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.
[0066] In addition, and as mentioned above, homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein. The term homologs used with respect to an original enzyme or gene of a first family or species refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
[0067] A protein has homology or is homologous to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have similar amino acid sequences. (Thus, the term homologous proteins is defined to mean that the two proteins have similar amino acid sequences).
[0068] As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid identity is equivalent to amino acid or nucleic acid homology). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0069] When homologous is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al., 1994, hereby incorporated herein by reference).
[0070] In some instances, isozymes can be used that carry out the same functional conversion/reaction, but which are so dissimilar in structure that they are typically determined to not be homologous.
[0071] A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0072] Sequence homology for polypeptides, which can also be referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.
[0073] A typical algorithm used to compare a target sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997). Typical parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
[0074] When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated herein by reference.
[0075] The disclosure provides accession numbers for various genes, homologs and variants useful in the generation of synthetic pathways and/or recombinant microorganism described herein. It is to be understood that homologs and variants described herein are exemplary and non-limiting. Additional homologs, variants and sequences are available to those of skill in the art using various databases including, for example, the National Center for Biotechnology Information (NCBI) access to which is available on the World-Wide-Web. Such homologs and variants can be identified by merely performing a BLAST search using a reference sequence from the present disclosure. For example, a BLAST search of ANME-1 mcrA gene (NCBI accession number CBH39484) using BLASTp algorithm yielded 9994 homologous or related protein sequences in about one minute performing the search, with identity ranging from 40% to 100%.
[0076] Culture conditions suitable for the growth and maintenance of a recombinant microorganism provided herein are known (see, e.g., Culture of Animal CellsA Manual of Basic Technique by Freshney, Wiley-Liss, N.Y. (1994), Third Edition). The skilled artisan will recognize that such conditions can be modified to accommodate the requirements of each microorganism. Appropriate culture conditions useful in metabolizing methane and/or methanol comprise conditions of culture medium pH, ionic strength, nutritive content, temperature and other culture conditions that permit production of dissolved inorganic carbon by the host microorganism, i.e., by the C.sub.1-metabolic action of the microorganism.
[0077] It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway or for the production of enzymes for use in a pathway suitable for the metabolizing methane and/or methanol. It is also understood that various microorganisms can act as sources for genetic material encoding target enzymes suitable for use in a recombinant microorganism provided herein.
[0078] The disclosure provides methods for the heterologous expression of one or more of the biosynthetic genes or polynucleotides involved in methane and/or methanol metabolism and recombinant DNA expression vectors useful in the method. Thus, included within the scope of the disclosure are recombinant expression vectors that include such nucleic acids.
[0079] Recombinant microorganisms provided herein can express a plurality of target enzymes involved in pathways for the production of dissolved inorganic carbon from a suitable carbon substrate such as, for example, methanol, methane, formaldehyde and the like. The carbon source can be metabolized to, for example, carbon dioxide. Sources of methanol, methane and formaldehyde are known. Of particular interest is methane gas, which occurs in nature and is a common by-product of waste degradation.
[0080] In another embodiment, a recombinant microorganism provided herein includes elevated expression of methyl-coenzyme M reductase (MCR) as compared to a parental microorganism. This expression may be combined with the expression or over-expression with other enzymes in the metabolic pathway for the production of carbon dioxide from methane. The methyl-coenzyme M reductase can be encoded by mcrA, mcrB, and mcrG genes, polynucleotides or homologs thereof. The mcrA, mcrB, and/or mcrG genes or polynucleotides can be derived from various microorganisms including methane-oxidizing archaea (ANME-1, 2 and 3).
[0081] In addition to the foregoing, the terms methyl-coenzyme M reductase or MCR refer to proteins that are capable of catalyzing the first step of the anaerobic oxidation of methane, and which share at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence similarity, as calculated by NCBI BLAST, using default parameters, to one of the following sequences: ANME-1 MCR (Meyerdierks et al. 2010), mcrA: NCBI Accession Number CBH39484, mcrB: NCBI Accession Number CBH37405, mcrG: NCBI Accession Number CBH39483; ANME-1 MCR crystalized from Black Sea microbial mat (Shima et al. 2012), mcrA: PDB ID 3SQG Chain A, mcrB: PDB ID 3SQG Chain B, mcrG: PDB ID 3SQG Chain C; ANME-2a MCR (Wang et al. 2014), mcrA: IMG Gene ID 2566125579, mcrB: IMG Gene ID 2566125583, mcrG: IMG Gene ID 2566125580; Candidatus Methanoperedens nitroreducens MCR (Haroon et al. 2013), mcrA: NCBI Accession Number WP_048089615, mcrB: NCBI Accession Number WP_048089608, mcrG: NCBI Accession Number WP_048089613; Candidatus Bathyarchaeota archaeon BA1 MCR (Evans et al. 2015): mcrA, NCBI Accession Number KPV65186, mcrB: NCBI Accession Number KPV65184, mcrG: NCBI Accession Number KPV65185; Candidatus Bathyarchaeota archaeon BA2 (Evans et al. 2015), mcrA: NCBI Accession Number KPV61791, mcrB: NCBI Accession Number KPV61799, mcrG: NCBI Accession Number KPV61800.
[0082] Biological methane oxidation in the absence of oxygen is restricted to anaerobic methanotrophic archaea (ANME) that are phylogenetically related to methanogens. These organisms evolved to metabolize methane to CO.sub.2 near thermodynamic equilibrium (E =245 mV for CH.sub.4/CO.sub.2) via the pathway of reverse methanogenesis or methane oxidation, which includes the chemically challenging step of methane activation without oxygen-derived radicals. Reported terminal electron acceptors for anaerobic oxidation of methane (AOM) include sulfate, nitrate, and metal oxides. Nitrate reduction coupled to methane oxidation is directly mediated by a freshwater archaeal methanotroph Ca. Methanoperedens nitroreducens ANME-2d; however, the electron transport mechanism coupling methane oxidation with other terminal electron acceptors (such as sulfate and metal oxides) is still debated.
[0083] Sulfate-coupled methane oxidation is the dominant mechanism for methane removal within marine sediments, preventing the release of teragrams per year of this greenhouse gas from the oceans:
CH.sub.4+SO.sub.4.sup.2HCO.sub.3.sup.+HS.sup.+H.sub.2O (G)=17 kJ mol.sup.1
Multiple methanotrophic archaeal lineages (ANME-1; ANME-2a,b,c; and ANME-3) form syntrophic consortia with sulfate-reducing bacteria (SRB) that drive AOM in areas of methane release at the seabed. The metabolism of AOM with sulfate appears to be partitioned between the two partners, requiring the exchange of electrons or intermediates. The mechanism underlying this syntrophic association has been studied using microcosm experiments [with AOM microorganisms exhibiting doubling times of 2 to 7 months], as well as through the application of stable isotope analyses, radiotracer rate measurements, metagenomics, metatranscriptomics, metaproteomics, and theoretical modeling.
[0084] Attempts to metabolically decouple the syntrophic association and identify the intermediate compound passaged between ANME archaea and their SRB partners have been unsuccessful when diffusive intermediates such as hydrogen, acetate, formate, and some redox active organic electron shuttles were used. Culture-independent evidence for direct interspecies electron transfer in sulfate-coupled AOM by members of the ANME and their SRB partners supports earlier genomic predictions of this process occurring in the methanotrophic ANME-1 and ANME-2.
[0085] Guided by the recent evidence of direct interspecies electron transfer from ANME-2 to SRB, it was determined whether artificial electron acceptors can substitute for the role of the SRB partner as a terminal oxidant or electron acceptor for AOM. Respiration of the artificial electron acceptor 9,10-anthraquinone-2,6-disulfonate (AQDS, E =186 mV) has been previously reported in methanogens. AQDS was tested as a sink for methane-derived electrons generated by the ANME archaea in incubations with deep-sea methane seep sediment. The stoichiometry of methane oxidation coupled to AQDS predicts the reduction of four equivalents of AQDS per methane:
CH.sub.4+4AQDS+3H.sub.2O.fwdarw.HCO.sub.3.sup.+H.sup.++4AQH.sub.2DS G=41 kJ mol.sup.1
[0086] By using a combination of rate measurements and single-cell stable isotope probing, it has been shown herein that ANME in deep-sea sediments can be catabolically and anabolically decoupled from their syntrophic SRB partners using soluble artificial oxidants or electron acceptor. The ANME still sustain high rates of methane oxidation in the absence of sulfate as the terminal oxidant or electron acceptor, lending support to the hypothesis that interspecies extracellular electron transfer is the syntrophic mechanism for the anaerobic oxidation of methane.
[0087] It was hypothesized and demonstrated that redox active compounds that have the ability to accept single electrons could replace the role of the SRB partners during AOM. Several compounds were shown in the Examples section below, including AQDS isomers, humic acids, and iron(III) complexes, that could accept electrons from AOM and become reduced. The total number of electrons that the electron acceptor Q can take vary depending on the compound, and equations below show the stoichiometry that changes based on the molecule:
CH.sub.4+8Fe(III)+3H.sub.2O.fwdarw.HCO.sub.3.sup.+9H.sup.++8Fe(II)
CH.sub.4+4AQDS+3H.sub.2O.fwdarw.HCO.sub.3.sup.+H.sup.++4AQH.sub.2DS
CH.sub.4+2 humic acid+3H.sub.2O.fwdarw.HCO.sub.3.sup.+9H.sup.++2 humic acid(reduced)
The humic acid is commonly prepared by extraction from soils and/or commercially available, and contains of a mixture of redox active organic molecules of high molecular weight; a molecule that can accept 4 electrons total is shown above as an example.
[0088] Mechanistically, extracellular electron transfer from ANME-2 to single electron acceptors can account for the findings herein. Large, S-layer-associated multi-heme c-type cytochromes such as those in ANME-2a (IMG Gene ID 2566123487 and 2566125052), ANME-2b (NCBI Accession Number KR811028), ANME-2d or Methanoperedens nitroreducens (IMG Gene ID 2515322983, 2515320866) together with membrane and extracellular cytochrome b and/or c such as the one in ANME-2a (IMG Gene ID 2566125773 and 2566125774) could putatively conduct electrons derived from reverse methanogenesis or methane oxidation from the archaeal membrane to the outside of the cell, where they can be taken up by a suitable electron acceptor. A congruent path of extracellular electron transfer has been proposed for the bacterium Geobacter sulfurreducens when oxidizing acetate coupled to the reduction of AQDS or humic acids. The similar catabolic and anabolic activities observed within ANME-2 archaea, independent of whether the terminal electron acceptor is AQDS or sulfate, suggest that the biochemistry within these organisms may follow the same pathway under AQDS conditions as when syntrophically coupled to SRB. The data therefore also lend experimental evidence in support of the hypothesis of direct interspecies electron transfer as the syntrophic coupling mechanism between methane-oxidizing ANME-2 and SRB in the environment.
[0089] The apparent ability of ANME-2 to oxidize methane via the release of single electrons constitutes a versatile half-metabolism. This physiology suggests that methanotrophic ANME-2 archaea should also be able to respire solid electron acceptors directly via extracellular metal reduction, such as methane oxidation coupled to soluble iron(III) as shown in the Examples section below and insoluble iron(III) and manganese(IV) reduction. Evolutionarily, methane oxidation with metal oxides could have served as a transient life style for ANME before the establishment of a syntrophic association with SRB. According to this hypothesis, methanogenic archaea first evolved the capability to conserve energy as a methanotroph coupled with the respiration of solid metal oxides as electron acceptors. In a subsequent evolutionary step, SRB developed a symbiosis with ANME archaea, gaining a direct source of electrons for sulfate reduction and leading to the highly structured syntrophic consortia common today in seep environments. This physiology of using extracellular electron transfer to enable syntrophic interaction has the advantage that intermediates cannot be lost via diffusion and that electrical conductance is much faster than diffusive transfer of reducing equivalents. Further, this described metabolism may have industrial utility, providing a mechanism for the conversion of methane to CO.sub.2 plus single electrons that can be catalyzed reversibly at low temperatures, with the potential to convert methane to electricity at high overall efficiencies.
[0090] Natural gas accounts for over 20% of the energy consumption worldwide. Its primary component, methane, is a renewable energy carrier, but it's conversion to electricity in fuel cells usually requires temperatures around 650 C. to cleave its inert CH bonds. Efficient conversion of chemical energy to electricity must avoid such high-temperature processes. Biosystems containing the enzyme methyl-coenzyme M reductase (MCR) are an attractive alternative to convert methane at low temperatures reversibly to a methyl-group that can be used for downstream reactions.
[0091] In certain embodiments, the disclosure provides for a bioelectrochemical device that utilizes a bioreactor comprising a MCR-containing biosystem that is capable of converting methane from sources such as biomass or a fuel gas stream to electricity via an electron carrier QH.sub.2 as shown in the scheme presented as
CH.sub.4+2H.sub.2OCO.sub.2+8e.sup.+8H.sup.+G17 kJ mol.sup.1
This reaction can be used to generate a reduced artificial electron acceptor QH.sub.2 according to the following formula, at a metabolic rate comparable to the rate when coupled to sulfate:
CH.sub.4+2H.sub.2O+4Q=CO.sub.2+4QH.sub.2
The results of Eq. 4 have two important consequences: First, ANME can now be grown in pure cultures for genetics and functional studies. Second, it demonstrates that the metabolism of ANME can be used for the generation of artificial electron acceptors that permit energy-relevant applications. Therefore, the free energy for methane oxidation with oxygen (G810 kJ mol.sup.1) can be partitioned into a biochemical step for energy conversion followed by an electrochemical step to harvest the energy. An example is shown here with the artificial electron acceptor Q being 2,6-AQDS:
CH.sub.4+4Q+2H.sub.2O.fwdarw.CO.sub.2+4QH.sub.2G=30KJ mol.sup.1
4QH.sub.2+2O.sub.2.fwdarw.4Q+4H.sub.2OG=780KJ mol.sup.1
An example of a bioreactor with internal cycling of the artificial electron acceptor Q is shown in
[0092] Various aspects of the disclosure are generally directed to a bioelectrochemical device which comprises a fuel cell or other devices that use similar operating principles, for example, devices that are able to oxidize fuel to produce electrons. A fuel cell is a device that converts fuel to electrical energy without combustion of the fuel (although a fuel cell could be used in conjunction with a device deriving energy from combustion of the same fuel; most fuel cells do not). A typical fuel cell includes two electrodes, an anode and a cathode, an electrolyte in contact with both the anode and cathode, and an electrical circuit connecting the anode and the cathode from which power created by the fuel cell can be drawn. The anode and the cathode are typically contained within separate compartments, which may be separated by an interface or a barrier. In a particular embodiment, a fuel cell disclosed herein utilizes or is based upon an organic-inorganic aqueous flow battery described in Huskinson et al. (Nature 505:195-200 (2014)), which is incorporated in-full in this disclosure.
[0093] In one set of embodiments, the barrier may be formed from a metal, such as gold, palladium, or platinum, and in some cases, the barrier may be backed on one or both sides by an inert film, for instance, comprising a hydrophobic polymer such as polytetrafluoroethylene (Teflon), and/or the barrier may be composed of anion/cation/proton exchange membranes. Such a barrier may be used, in some cases, to allow the exchange of hydrogen gas and/or ions, but generally impede the exchange of other dissolved gases.
[0094] In some cases, the fuel cell may contain a plurality of anodes and/or cathodes, e.g., in the same or different compartments, which may be operated in series and/or in parallel.
[0095] In typical operation, an oxidant (e.g., oxygen, such as the oxygen found in the air, or bromine) is provided to a cathode of a fuel cell where it is reduced, e.g., to form water or hydrogen bromide, while a reduced electron acceptor (QH.sub.2) in contact with the anode is oxidized, e.g., to produce an electron acceptor (Q). The electrons may be removed from the anode by a current collector, or other component of an electrical circuit, which results in an electrical current. The overall reaction may be energetically favorable, i.e., the reaction gives up energy in the form of electricity or power driving electrons from the anode, through electrical circuitry, to the cathode. This energy can be captured for essentially any purpose, e.g., for immediate use and/or for storage for later use.
[0096] The fuel cell may be fabricated from any suitable material. For example, in one set of embodiments, the fuel cell, or a portion thereof, such as an anode compartment, may be fabricated from non-conductive materials, for instance, from any polymer such as polyvinyl chloride, polyethylene, polypropylene, or polyethylene terephthalate. In another set of embodiments, the fuel cell (or portion thereof) may be formed from thermally insulating and/or non-conductive materials such as ceramics, glass, wood, and/or metals that may or may not be coated with thermal or electrical insulators, e.g. Teflon-coated aluminum, polymeric-coated steel, glass-lined stainless steel, etc. As discussed herein, in some embodiments of the disclosure, thermal insulators are useful for the management or retention of heat within the fuel cell, which may lead to higher power output.
[0097] In some aspects of the disclosure, the bioelectrochemical device of the disclosure comprises a fuel cell that is connected to bioreactor system comprising C.sub.1 metabolizing microorganisms (e.g., ANMEs) that produce dissolved inorganic carbon from CH.sub.4. In such a system, an electron acceptor/reduced electron acceptor can be cycled between the fuel cell and the bioreactor. Thus, the fuel cell uses the reduced electron acceptor produced from the bioreactor system to make electrical energy by oxidizing the reduced electron acceptor to an oxidized form, whereby the electron acceptor is then cycled back to the bioreactor to be reduced by the action of C.sub.1 metabolizing microorganisms to form a reduced electron acceptor. In one set of embodiments, the fuel cell contains an anode and a cathode, each within different compartments. The cathode may be placed in a compartment with an abundance of oxygen (i.e. an aerobic environment), and/or in the presence of a soluble oxidant or electron acceptor such as nitrate, sulfate, iron oxide, manganese oxide, or other redox active compounds, while the anode may be placed in a second compartment having an environment that is deficient or devoid oxygen (i.e., an anaerobic environment), and/or other oxidants or electron acceptor including, but not limited to, soluble oxidants or electron acceptor such as nitrate, sulfate, iron oxide, manganese oxide, etc. In one embodiment, the anode contains a percentage of oxygen that is less than atmospheric oxygen, i.e., less than about 21% by total volume. Typically, the anode is fluidly in contact with a solution which comprises a reduced electron acceptor (QH.sub.2). The source of methane that is converted to dissolved inorganic carbon by the microorganisms of the bioreactor can come from any number of sources, including from natural gas, syn gas, shale gas, town gas, etc. For example, the methane may be produced from materials such as chemical or industrial reactions, or biomass, i.e., matter derived from living biological organisms.
[0098] Biomass, as used herein, may arise from plants or animals. For example, plants such as switchgrass, hemp, corn, poplar, willow, or sugarcane may be used as a fuel source to provide methane to the bioreactor disclosed herein via fermentative pathways utilizing methanogens. The entire plant, or a portion of a plant, may be used as the methane source, depending on the type of plant. As another example, biomass may be derived from animals, for instance, animal waste or animal feces, including human sewage (which may be used raw, or after some treatment). Still other non-limiting examples of biomass include food scraps, lawn and garden clippings, dog feces, bird feces, composted livestock waste, untreated poultry waste, etc. The biomass need not be precisely defined. In some cases, the biomass does not necessarily exclude fossil fuels such as oil, petroleum, coal, etc., which are not derived from recently living biological organisms, nor does it exclude refined or processed materials such as kerosene or gasoline.
[0099]
[0100]
[0101] In a particular embodiment, methane source 5 is a methane producing system from a fuel source such as biomass. Methane production is the consequence of a series of metabolic interactions among various groups of microorganisms. The first group of microorganisms secrete enzymes which hydrolyze polymeric materials found in biomass to monomers such as glucose and amino acids, which are subsequently converted to higher volatile fatty acids, H.sub.2 and acetic acid. In the second stage, hydrogen-producing acetogenic bacteria convert the higher volatile fatty acids e.g., propionic and butyric acids, produced, to H.sub.2, CO.sub.2, and acetic acid. Finally, the third group, methanogenic archaea convert H.sub.2, CO.sub.2, and acetate, to CH.sub.4 and CO.sub.2. Methanogens are physiologically united as methane producers in anaerobic digestion. Although acetate and H.sub.2/CO.sub.2 are the main substrates available in the natural environment, formate, methanol, methylamines, and CO can also be converted to CH.sub.4 by methanogenic archaea. In an alternate embodiment, methane source 5 is from a methane containing fuel gas stream, wherein the fuel gas stream may be enriched for methane by purifying the methane from other components of the fuel gas stream. Examples of a fuel gas stream include, but are not limited to, a natural gas stream, a shale gas stream, or a town gas stream. Inlet tube 25 can be comprised of any material. For example, inlet tube 25 can be comprised of metal, plastic or rubber. As the fluid running through inlet tube 25 is pressurized, inlet 25 should have a maximum burst pressure that exceeds, preferably greatly exceeds, the pressure being exerted by the fluid on the walls of inlet tube 25. While inlet tube 25 typically is an elongated cylindrical shape, it should be understood that inlet tube 25 can have any shape (e.g., square, hexagonal, triangular, etc.) as long as fluid can freely pass through the inside space of inlet tube 25. Valve 20 is provided to control the flow of methane into inlet tube 25. Typically, valve 20 is a check valve that only allows methane to flow through it. The body of valve 20 can be made of any material, but plastic or metal is preferred. Valve 20 can be any type of valve, including a ball valve, diaphragm valve, swing valve, tilting disc valve, stop-check valve, a lift valve, in-line valve, duckbill valve, etc. Moreover, although not depicted in
[0102] For the exemplary bioelectrochemical device depicted in
[0103] Turing to bioreactor 10 depicted in
[0104] Turing to outlet tube 30 and CO.sub.2 separator 35 depicted in
[0105] Turing to fuel cell 40 depicted in
[0106] The aqueous solvent that exits anode compartment 45 of fuel cell 40 into fuel cell outlet tube 65 is enriched with an electron acceptor (Q), i.e., a product of the oxidation reaction at anode 50. The solvent in fuel cell outlet tube 65 can then be analyzed and supplemented/adjusted via the medium inlet and outlet valve 67. Typically, medium inlet and outlet valve 67 is a check valve that allows fluid (liquid or gas) to flow through it in only one direction. The medium inlet and outlet valve 67 can be made of any material, but plastic or metal is preferred. Medium inlet and outlet valve 67 can be any type of valve, including a ball valve, diaphragm valve, swing valve, tilting disc valve, stop-check valve, a lift valve, in-line valve, duckbill valve, etc. The aqueous solvent in fuel cell outlet tube 65 is then pumped into inlet tube 25 via pump 70. Fuel cell outlet tube 65 can be comprised of any material. For example, fuel cell outlet tube 65 can be comprised of metal, plastic or rubber. As the fluid running through fuel cell outlet tube 65 is pressurized, fuel cell outlet tube 65 should have a maximum burst pressure that exceeds, preferably greatly exceeds, the pressure being exerted by the fluid on the walls of fuel cell outlet tube 65. While fuel cell outlet tube 65 typically is an elongated cylindrical shape, it should be understood that fuel cell outlet tube 65 can have any shape (e.g., square, hexagonal, triangular, etc.) as long as fluid can freely pass through the inside space of fuel cell outlet tube 65. Pump 70 is used to drive the re-circulation of the aqueous solvent through the bioelectrochemical device. Pump 70 can be any type of standard pump, including, but not limited to, centrifugal pumps, ANSI process pumps, API process pumps, axial flow pumps, booster pumps, circulator pumps, magnetic drive pumps, diaphragm pumps, peristaltic pumps, piston pumps, plunger pumps, and screw pumps.
[0107] It should be noted that one or more of the components of a bioelectrochemical device disclosed herein may be manually and/or electronically controlled. Thus, the opening of values, operation of pumps, etc. may be controlled directly and/or remotely across a network or by wireless communication (e.g., bluetooth) via a computer, cell phone, tablet, terminal, and the like.
[0108] The invention is illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
Examples
[0109] Sediment collection and processing. Santa Monica basin seep sediments overlain by a white mat were collected from the Santa Monica Mounds site in a push core (PC 61) deployed by the ROV Doc Ricketts. Samples were collected in May 2013 during a research cruise organized by the Monterey Bay Aquarium Research Institute (MBARI) using the R/V Western Flyer. PC61 was collected during dive 463 at 860 m depth with an in situ temperature of 4 C. (lat. 33.78905, long. 118.66833). The intact sediment core was extruded shipboard and then heat-sealed in a large mylar bag flushed for 5 minutes with argon. Sediments were stored at 4 C. until processed in the lab (40 days after collection). The whole push core (ca. 12 cm, yielding 800 mL wet sediment) was suspended in 1600 mL filter sterilized N.sub.2 sparged bottom seawater from the site (1 in 3 ratio) in an anaerobic chamber (3% H.sub.2 in N.sub.2). The anaerobic sediment slurry was then distributed into three 1 L pyrex bottles, sealed with a large butyl rubber stopper, and pressurized with methane (0.25 MPa). Aggregate counts at the start of the experiment were determined by DAPI staining and epifluorescence microscopy, yielding approximately 9.710.sup.5 aggregates per mL wet weight sediment. The initial sulfate-coupled AOM activity of the sediment was assessed via sulfide production measurements, showing the generation of 2.8 mM sulfide within the first 15 days.
[0110] All manipulations of the sediment incubations were done anaerobically at 4 C. or on ice. Prior to establishment of the microcosm experiments, the seep sediment was maintained for 12 months at 4 C. under methane (0.25 MPa) in anoxic bottom seawater that was exchanged every 3 months. For all reported experiments in this study, the seawater above the sediment was exchanged with a modified artificial seawater (see below) that contained 10 less Ca.sup.2+, no sulfate, no sulfide, and 25 mM HEPES buffer at pH 7.5. The low Ca.sup.2+ concentration and lower pH prevent carbonate precipitation, which allows quantitative analysis of the .sup.13C-bicarbonate formed in solution during .sup.13C-methane oxidation. Methane was added (0.30 MPa), shaken and the sediment allowed to settle for 48 hours (sediment/total volume=1:3). The supernatant was exchanged 3 times with the described medium following the same procedure in order to obtain sulfate and sulfide-free sediment.
[0111] Medium Composition.
[0112] The final composition in the medium was: NaCl 457 mM, MgCl.sub.2 47 mM, Na.sup.+-HEPES (pH=7.5) 25 mM, KCl 7.0 mM, NaHCO.sub.3 5.0 mM, CaCl.sub.2 1.0 mM, K.sub.2HPO.sub.4 1.0 mM, NH.sub.4Cl 1.0 mM, SeO.sub.3.sup.2 0.01 M, WO.sub.4.sup.2 0.007 M, 0.1% trace element solution, containing per liter: nitrilotriacetic acid 150 mg, MnCl.sub.24 H.sub.2O 610 mg, CoCl.sub.26 H.sub.2O 420 mg, ZnCl.sub.2 90 mg, CuCl.sub.22 H.sub.2O 7 mg, AlCl.sub.3 6 mg, H.sub.3BO.sub.3 10 mg, Na.sub.2MoO.sub.42 H.sub.2O 20 mg, SrCl.sub.26 H.sub.2O 10 mg, NaBr 10 mg, KI 70 mg, FeCl.sub.36 H.sub.2O 500 mg, NiCl.sub.26 H.sub.2O 25 mg. No vitamins, indicators, reducing agents, or other substances were added.
[0113] The sulfate concentration of the final sediment slurry was below detection limit (<10 M). Before the start of the microcosm experiments, the sediment slurry was flushed with methane (ca. 20 min) to remove traces of sulfide. The presence of sulfide can chemically reduce AQDS, preventing methane oxidation with AQDS. It is possible that under these conditions, AOM is inhibited by the polysulfides formed from sulfide+AQDS rather than directly by reduced AQDS, as reduced AQDS was observed to accumulate in the experiments with no apparent inhibition of AOM (see TABLE 2A). For Table 2, the concentrations measured after a 21-day incubation (experiments in
TABLE-US-00002 TABLE 2 Final concentration of reduced electron acceptors AQH.sub.2DS and sulfide. A) Final concentrations of reduced AQDS (AQH.sub.2DS): DIC formed Experiment [mM] AQH.sub.2DS [mM] Stoichiometry* AQDS 10 mM 2.39 8.6 3.60 AQDS 10 mM 2.64 9.6 3.62 AQDS 10 mM + 2.82 9.8 3.45 MoO.sub.4.sup.2 25 mM AQDS 10 mM + 2.59 9.2 3.55 MoO.sub.4.sup.2 25 mM B) Final concentrations of sulfide: DIC formed Experiment [mM] sulfide [mM]* Stoichiometry.sup. Sulfate 28 mM (A) 6.44 5.9 0.92 Sulfate 28 mM (B) 7.77 6.5 0.84 Sulfate 28 mM (C) 7.51 7.0 0.93 Sulfate 28 mM (D) 7.29 6.1 0.84 *The lower amount of AQH.sub.2DS found than expected (4:1 stoichiometry) may be attributed to partial oxidation during the process of sampling and titration. *sulfide [mM] = sum of HS.sup. and H.sub.2S. .sup.The lower amount of aqueous sulfide measured relative to the expected 1:1 stoichiometry may be attributed to a combination of factors resulting in a loss of sulfide. These include some sulfide partitioning into the headspace as gaseous H.sub.2S and possibly escaping as gaseous H.sub.2S during sampling, partial sulfide oxidization during the sampling and centrifugation on the benchtop, or possibly precipitation with divalent cations during the incubation.
[0114] Sediment Characterization.
[0115] AOM rates with sulfate (1.5 mol methane (cm.sup.3 wet sediment).sup.1 d.sup.1) were comparable to active methane-seep sediments described previously. The dominant groups of archaea included ANME-2a and ANME-2c based on Illumina Tag sequencing using the Earth Microbiome primer set (
[0116] Chemicals and Reagents.
[0117] AQDS (=2,6-AQDS, >98% purity) and Fe(III)-EDTA was purchased from Sigma. Humic acids (sodium salt, tech. batch no. 10121HA) were obtained from Aldrich. 2,7-AQDS and 1,5-AQDS (>98% purity) were purchased from TCI chemicals. The different AQDS isomers were found to contain variable amounts of residual sulfate as determined by Ion Chromatography: 11 M sulfate per 10 mM AQDS; 176 M sulfate per 10 mM 2,7-AQDS; 344 M sulfate per 10 mM 1,5-AQDS. 1,5-AQDS was re-crystallized from boiling water to remove traces of sulfate present in the purchased product. Residual sulfate in the re-crystallized 1,5-AQDS: 13 M per 10 mM 1,5-AQDS. 2,7-AQDS, AQDS and all other chemicals were used as received. 50 mM Fe(III)-citrate stock solution was prepared by dissolving 2.0 mmol citric acid in a small amount of DI water, followed by the addition of 1.0 mmol FeCl.sub.36 H.sub.2O and pH adjustment to pH=7.5 with NaOH. The solution was then diluted to 50 mM ferric ions (20 mL final volume).
[0118] General Description of Methane Oxidation Measurements Via .sup.13C-Methane.
[0119] Methane oxidation was quantified by determining the production of inorganic carbon (CO.sub.2). Accurate quantification of the concentration of inorganic carbon formed from methane oxidation is challenging due to 4 main reasons:
(1) Inorganic carbon is present as a mixture of CO.sub.2 (g) in the headspace, and CO.sub.2 (aq.), H.sub.2CO.sub.3, HCO.sub.3.sup. or CO.sub.3.sup.2 in solution (dissolved inorganic carbon, DIC); (2) Inorganic carbon may also be produced from respiration of organic carbon sources other than methane (3) Inorganic carbon can also be slowly produced via dissolution of carbonates (a major component of seep sediments); and (4) Inorganic carbon may also precipitate with divalent cations as insoluble carbonates.
[0120] It was found in the experiments, that quantifying methane oxidation using the stable isotope tracer .sup.13CH.sub.4 in incubations with a known amount of unlabelled (dissolved inorganic carbon, DIC) was the most accurate in analyzing the .sup.13C enrichment in DIC (see
[0121] Incubation Conditions for AOM Rate Measurement.
[0122] Each incubation vial was set up with 1.0 cm.sup.3 wet sediment (wet sediment=volume of sediment after allowing the sediment slurry settle for 48 h) in total slurry volume of 5 mL as follows: Sterile serum vials were closed with butyl rubber stoppers (volume=12.9 ml after closing) and flushed with methane. 1.0 mL .sup.13CH.sub.4 (99% .sup.13C, Cambridge Isotope Laboratories, containing 0.05 vol % .sup.13CO.sub.2 as an impurity) was introduced anaerobically. 2.0 mL of artificial, anaerobic seawater containing 2.5 the target concentration of the corresponding electron acceptor was injected into the serum vial cooled on ice. For AQDS and 1,5-AQDS, this was a suspension corresponding to 25 mM (see TABLE 3).
TABLE-US-00003 TABLE 3 Solubility of different AQDS regioisomers in the incubation medium. Compound 4 C. 22.5 C. 2,6-AQDS (AQDS) 0.9 mM 1.9 mM 2,7-AQDS >25 mM >25 mM 1,5-AQDS 2.5 mM not determined
[0123] The 1 L pyrex bottle with the sediment in the sulfate-free medium (1 part wet sediment in 3 parts of slurry volume) was vigorously shaken each time and 3.0 mL of slurry immediately removed and injected into the individual serum bottles. Each stoppered serum vial was supplemented with unlabeled methane (0.250 MPa overpressure: pressure gauge SSI Technologies, Inc., Media Gauge) shaken and stored inverted at 4 C. (final headspace: 0.35 MPa methane, with ca. 4% .sup.13CH.sub.4). The exact fractional abundance of .sup.13C in the methane was quantified via .sup.1H-NMR spectroscopy for individual incubations.
[0124] AOM Rate Measurements (Quantification of Newly Formed DIC Based on .sup.13CH.sub.4).
[0125] For .sup.13C-DIC analysis, 0.25 mL of the medium above the settled sediment in the microcosm was sampled with a disposable needle and syringe at each time point (same intervals for all experiments) and centrifuged (16000 rcf, 5 min). The supernatant was transferred into 0.6 mL eppendorf tubes, flash frozen in N.sub.2 (l), and stored at 20 C. until measurement. 150 L of the thawed supernatant was then added to He-flushed vials containing 100 L H.sub.3PO.sub.4 (85%). The resulting CO.sub.2 was analyzed for the isotopic enrichment (.sup.13F(t.sub.n)) on a GC-IR-MS GasBench II (Thermo Scientific). The amount of DIC newly formed ([DIC](t.sub.n), see
[DIC](t.sub.n)=[DIC](t.sub.0)*(.sup.13 F(t.sub.n).sup.F(t.sub.0))/(.sup.13F(CH.sub.4).sup.13F(t.sub.n))(EQ. 1)
[0126] [DIC]=sum of carbonate, bicarbonate and CO.sub.2, [DIC](t.sub.0)=5.0 mM
[0127] .sup.13F(t.sub.0)=0.01153 (higher than medium due to .sup.13CO.sub.2-impurity in the .sup.13CH.sub.4 used)
[0128] .sup.13F(CH.sub.4)=.sup.13C in the methane used (measured via .sup.1H-NMR spectroscopy). The amount of DIC formed per vial (See
DIC.sub.total=5 mL*[DIC](t.sub.n)(EQ. 2)
[0129] Calculation of Specific AOM Rates Per Volume Sediment.
[0130] For each incubation, the methane oxidation rate per volume sediment slurry was determined via linear regression of the time points 1, 2, 3 and 4 (17 h, 42.5 h, 67 h and 142.5 h). The 95% confidence intervals were calculated. Rates per cm.sup.3 wet sediment are 5 higher than for the sediment slurry (sediment+modified HEPES-buffered seawater), as displayed in
[0131] Quantification of Absolute DIC Concentrations Via Standard Addition.
[0132] For two incubations with sulfate, quantified the absolute concentrations of DIC were quantified for the full time course of the incubations (see
[DIC](t.sub.n)=10 mM* (.sup.13 F.sub.mix(t.sub.n).sup.13 F.sub.std)/(.sup.13F(t.sub.n).sup.13F(t.sub.n).sup.13F.sub.mix(t.sub.n))(EQ. 3)
[0133] Quantification of Fractional Abundance of .sup.13CH.sub.4 in the Headspace Used.
[0134] The exact fraction of .sup.13CH.sub.4 (ca. 4.0%) was quantified for individual incubations at the end of the 21-day incubation period via .sup.1H-NMR spectroscopy (Varian 400 MHz Spectrometer with broadband auto-tune OneProbe). Methane in the headspace was passed through deuterated chloroform (99.8% D, Cambridge Isotope laboratories) via a long 23 G needle and acquired at 400 MHz with a repetition time of 10 s. Fractional abundances of .sup.13C in the methane were obtained via integration of the .sup.12CH.sub.4 signal and of the .sup.13CH.sub.4-satellites (iNMR version 4.3.0).
[0135] Quantification of Residual Sulfate.
[0136] Residual sulfate was quantified via Ion chromatography on a DX-500 or DX-2000 instrument (Dionex, Sunnyvale, Calif., USA) housed at the Caltech Environmental Analysis Center following the protocol outlined in Green-Saxena et al. (Isme J. 8:150 (2014)). The DI water used throughout this study contained <10 M residual sulfate. Incubations with 2,7-AQDS contained a maximum of 200 M residual sulfate, from traces of sulfate present in the purchased material (176 M per 10 mM 2,7-AQDS), which could not be removed via re-crystallization due to the high solubility of 2,7-AQDS (described in the chemicals and reagents section above). For all other incubations, the sulfate concentration remained below 50 M throughout the incubation period.
[0137] Quantification of Sulfide.
[0138] Supernatant was removed via syringe and centrifuged in 0.6 mL Eppendorf tubes (16000 rcf, 30 s). 20 L of the clear supernatant was removed and added to 400 L Zn(OAc).sub.2 (500 mM) to preserve the sulfide. Analysis was carried out in triplicate via the methylene blue method described in J. D. Clin (Waters. Limnol. Oceanogr. 14:454 (1969)), using standards of 0.1, 0.25, 0.5, 1, 2, 5, 10 and 25 mM sulfide added to Zn(OAc).sub.2 (500 mM) in the same ratios as for the samples. Quantification was carried out in a plate reader (TECAN Sunrise) by monitoring the absorbance at 670 nm.
[0139] Quantification of AQDS Solubility in the Incubation Medium.
[0140] Different regioisomers of AQDS (1,5-AQDS, 2,6-AQDS and 2,7-AQDS) were separately added to the modified artificial seawater described above, to a targeted final concentration of 25 mM. The suspensions were ultra-sonicated at room temperature to dissolve as much of the AQDS as possible. The tubes were kept at 4 C. or at 22.5 C. overnight. The tubes were centrifuged at 4 C. or 22.5 C. (16000 rcf, 30 min). The supernatant (300 L) was mixed with 300 L acetate (10.0 mM) as the internal standard and 100 L deuterated water (99.9% D, Cambridge Isotope Laboratories) was added. The concentration of AQDS was obtained from integration of the .sup.1H-NMR spectra by comparison relative to the acetate standard.
[0141] Identity of Reduced AQDS by UV-Vis and NMR.
[0142] Reduced AQDS was identified photospectrometrically matching the spectra reported previously by Gamage et. al. (J. Chem. Soc.-Faraday Trans. 87:3653 (1991)). Solutions containing reduced AQDS were paramagnetic (ca. 1000 Hz line width, .sup.1H-NMR spectroscopy), putatively due to the presence of the semiquinone-radical. Supernatant from an incubation that contained ca. 10 mM reduced AQDS was sparged with air for 30 min in order to re-oxidize AQH.sub.2DS back to AQDS. .sup.1H-NMR spectroscopy shows full conversion to AQDS, undistinguishable from the AQDS used initially. No signals other than AQDS or HEPES buffer were visible in the spectrum, proving reversible reduction and oxidation without detectable formation of side products.
[0143] Quantification of Reduced AQDS by Iodometry.
[0144] Reduced AQDS (2,6-AQH.sub.2DS) was quantified via iodimetric titration in the anaerobic chamber. A standard solution of ca. 20 mM KI.sub.3 was prepared as follows: iodine (254 mg, 2.0 mmol) and potassium iodide (1.66 g, 10 mmol) were dissolved in 100 mL DI water. After 2 days, the clear, dark solution was N.sub.2-sparged and brought into the anaerobic chamber. The exact concentration of the KI.sub.3 standard solution was quantified to be 19.5 mM via duplicate titration with sodium thiosulfate (exactly 101.5 mM, N.sub.2-sparged). Procedure for the quantification of reduced AQDS in the incubations: 1000 L anaerobic assay solution centrifuged in the anaerobic chamber+200 L anaerobic HEPES (1.0 M, pH=7.5)+KI.sub.3 standard solution as needed until color minimum, added in steps of 100 L first, then in steps of 10 L when closer to the equivalence point.
[0145] Control Experiments without Methane.
[0146] Sediment incubations with AQDS (10 mM) under nitrogen did not generate substantial amounts of reduced AQDS (less than 1 mM after 3 weeks). To confirm that the methanotrophic microorganisms in the sediment had not been killed during the nitrogen+AQDS incubation, methane was later injected into these incubation bottles after 3 weeks. Upon addition of methane, AQDS was reduced at rates similar to that shown in
[0147] Check for Methanogenic Activity.
[0148] Sediment slurries were incubated under a nitrogen headspace in duplicates without an added oxidant or electron acceptor to probe for endogenous methanogenic activity. After 6 days, methane in the headspace was quantified via GC-MS (Hewlett Packard 5890 Series II gas chromatograph with mass selective detector 5972) by triplicate injections. For the first biological replicate, methane was found at a concentration that was close to the detection limit, corresponding to a specific methanogenesis rate of 0.50.25 nmol methane (cm.sup.3 wet sediment).sup.1 d.sup.1. For the second replicate, the methane peak was too small for accurate quantification, however, an upper limit of 0.25 nmol (cm.sup.3 wet sediment).sup.1 d.sup.1 was established. Based on these measurements, the endogenous methanogenesis rate and/or enzymatic equilibration rate is ca. 3000 lower than methane oxidation with sulfate.
[0149] Thermodynamic Calculations.
[0150] The standard free energy for AOM with AQDS was calculated from the corresponding redox-potentials:
HCO.sub.3.sup.+4H.sub.2+H.sup.+=CH.sub.4+2H.sub.2OG=135.3 kJ mol.sup.1
E for CH.sub.4/HCO.sub.3.sup.=414 mVG/(nF)=239 mV
E for 2,6-AQDS=186 mV [E=+228 mv]
E=53 mV, G=nFE=41 kJ mol.sup.1
[0151] Incubations for FISH-nanoSIMS and RNA Analysis.
[0152] For AOM incubations with sulfate or AQDS that were used for nanoSIMS (
[0153] For the second independent set of experiments with iron citrate, humic acids and sulfate (
TABLE-US-00004 TABLE 4 List of oxidants or electron acceptors tested for AOM. Addition Rate relative to of 25 mM AOM with Oxidant [conc.], replicates E (mV) MoO.sub.4.sup.2 sulfate (%) Sulfate [28 mM] A, B, C*, D 220 100 Sulfate [28 mM] E, F, G + 20 AQDS [10 mM] A, B 186 74 AQDS [10 mM] C, D + 83 No oxidant A, B 1.5 .sup. No oxidant + H.sub.2CO (4%) A, B 0 2,7-AQDS [10 mM] A, B 185 97 1,5-AQDS [10 mM] A, B, C 175 38 Fe(III)-citrate [10 mM] A 372 53 Fe(III)-citrate [10 mM] B*, C, D + 46 Fe(III)-citrate [5 mM] A, B, C + 37 Fe(III)-citrate [2 mM] + 7 Fe(III)-EDTA [1.6 mM] A, B 96 8 Humic acids [1%] A*, B n.a..sup. 36 Humic acids [0.5%] 26 Melanin [8 mg/ml] n.a..sup. + 6.4 .sup. Melanin [2 mg/ml] + 4.3 Melanin [0.5 mg/ml] + 2.6 Fe(III)-citrate [25 mM] 372 + 2.5 Fe(III)-NTA [1 mM] 2.5 Fe(III)-NTA [10 mM] 385 1.5 .sup. Fe(III)-EDTA [10 mM] A, B 96 1.5 .sup. Phenazine methosulfate [1 mM] 80 1.5 .sup. Methylene blue [1 mM] 11 1.5 .sup. Indigo tetrasulfonate [10 mM; 46 1.5 .sup. 1 mM] Resazurin [10 mM] 51 1.5 .sup. 1-Hydroxynaphthoquinone 137 1.5 .sup. [10 mM; 1 mM] Phenosafranine [10 mM; 1.6 mM] 252 1.5 .sup. Safranine T [10 mM; 1.6 mM] 289 1.5 .sup. *Replicate that was analyzed via nanoSIMS (see FIG. 8B and FIG. 9). .sup. No net methane oxidation can be deduced, because incubations without oxidant show an apparent AOM rate of 0.023 mol cm.sup.3 day.sup.1 (1.5% relative to sulfate). This label conversion of .sup.13CH.sub.4 to .sup.13C-DIC arises via enzyme catalyzed isotope exchange between .sup.13CH.sub.4 and DIC without net methane oxidation. .sup. The midpoint reduction potentials of humic acids and melanin are not well defined. Both compounds can act as single electron acceptors due to their quinone moieties as shown experimentally for humic acids and for melanin. .sup. AOM occurs linearly, rates per wet sediment: with 8 mg/ml melanin: 0.096 0.020 mol cm.sup.3 day.sup.1; with 2 mg/ml melanin: 0.064 0.012 mol cm.sup.3 day.sup.1; with 0.5 mg/ml melanin: 0.039 0.015 mol cm.sup.3 day.sup.1
[0154] Sampling for FISH-nanoSIMS and RNA.
[0155] After an 18-day incubation in the presence of .sup.15NH.sub.4.sup.+, each incubation was shaken to suspend the sediment and 1.0 mL sediment slurry was sampled using a disposable sterile needle and syringe (ca. 0.3 mL wet sediment). The sediment aliquot was briefly centrifuged (4000 rcf) in an eppendorf tube and 0.5 mL of the supernatant was removed for DIC and metabolite analysis. 0.5 mL 4% paraformaldehyde in PBS (100 mM phosphate pH=7.4, salinity=10 g L.sup.1) was then added to the remaining 0.5 mL sediment slurry (2% formaldehyde final), resuspended and fixed for 120 min at 4 C. The fixed sediment was subsequently washed 3 times with 1PBS, followed by a single wash in EtOH/PBS (1:1) and then re-suspended in EtOH/PBS (1:1) to a final volume of 0.5 mL that was used for microscopy and nanoSIMS.
[0156] All remaining sediment from the identical incubations (ca. 0.7 mL wet sediment, see above) was removed for RNA analysis using a disposable sterile needle and syringe. The sediment slurry was immediately centrifuged (16000 rcf, 15 s), followed by removal of the supernatant and flash-freezing of the sediment pellet in liquid N.sub.2. Frozen sediment was stored at 80 C. until RNA extraction.
[0157] RNA Extraction, PCR, Clone Library Construction, and Phylogenetic Analysis.
[0158] RNA was extracted from the 0.7 mL frozen wet sediment described above using the RNA Powersoil Total RNA Isolation Kit (cat #12866-25; MO BIO Laboratories, Inc., Carlsbad, Calif., USA) with modifications. The eluted RNA was immediately DNase treated using the MO BIO RTS DNase Kit (cat no. 15200-50, MO BIO Laboratories, Inc., Carlsbad, Calif., USA), purified and concentrated to 24 L using the Qiagen RNeasy Plus Micro Kit (cat #74034, Qiagen, Valencia, Calif., USA), and converted to cDNA using the Invitrogen Superscript III First-Strand Synthesis SuperMix (cat no. 18080-400, ThermoFisher Scientific, Grand Island, N.Y., USA) with no reverse transcriptase (NRT) controls following manufacturer's instructions. cDNA was stored at 80 C. until further processing.
[0159] PCR conditions were as follows: 15 PRIME HotMasterMix buffer (cat #2200400; 5 PRIME, Inc., Gaithersburg, Md., USA), 0.4 M of each forward and reverse primer, and 1 l of cDNA in a final volume of 25 L. Methyl-coenzyme reductase alpha subunit (mcrA, primers ME1: GCMATGCARATHGGWATGTC (SEQ ID NO:1); ME2 TCATKGCRTAGTTDGGRTAGT (SEQ ID NO:2); and archaeal and bacterial 16S rRNA (primers Arc23F: TTCCGGTTGATCCYGCCGGA (SEQ ID NO:3) or Bac27F: AGAGTTTGATYMTGGCTC (SEQ ID NO:4) with U1492R: GGYTACCTTGTTACGACTT (SEQ ID NO:5) were amplified with 40 cycles as described in E. Trembath-Reichert et al. in Microbial Metagenomics, Metatranscriptomics, and Metaproteomics, E. F. DeLong, Ed., vol. 531, pp. 21-44. The cDNA from the sulfate microcosm was diluted 1/50 to have similar number of amplicons compared with other cDNA samples. 5 L of the PCR products was quantified in 1% (w/v) agarose electrophoresis with SYBR safe stain under ultraviolet light, and NRT controls did not show any amplification. The remainder of the PCR product for each sample was immediately cleaned by filtration (MultiScreen PCR Filter Plate #MSNUO3010, Millipore) and resuspended to the original volumes using Tris-HCl (pH 8). 1 L of the cleaned PCR product was used per ligation reaction and cloning according instructions in the TOPO TA Cloning Kit for Sequencing using the pCR4-TOPO Vector and One Shot Top 10 chemically competent Escherichia coli (Life Technologies). Recombinant clones were checked for inserts by PCR, gel electrophoresis and sequenced using primers M13F (5-GTAAAACGACGGCCAG-3 (SEQ ID NO:6)) and M13R (5-CAGGAAACAGCTATGAC-3 (SEQ ID NO:7)) on an ABI Prism 3730 DNA sequencer using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc., Foster City, Calif., USA).
[0160] Sequences were manually checked and contigs assembled using the Sequencer v4.1.4 software. Sequences were then compared against the SILVA 16S rRNA database [version 119] or GenBank DNA database by using the BLAST algorithm. Alignment against reference sequences was completed using the SILVA online aligner for 16S rRNA and ClustalO for mcrA, and aligned sequences were then imported into the ARB package version 6.0.2 and manually verified. An additional 80 16S rRNA sequences were retrieved from SILVA database and 44 mcrA sequences were retrieved from NCBI Refseq and used as references for the phylogenetic analyses. The Bayesian phylogeny was generated using 1421 aligned 16S rRNA nucleic acid positions (inverse gamma rates) and 248 aligned mcrA amino acid positions (mixed amino acid model) using MrBayes v3.2.2 until split frequencies were less than 0.01. Clone library sequences fall into several clades of high phylogenetic support. Clone library sequences fall into several groups of high phylogenetic support. One representative sequence is used to represent highly similar sequences; the number of sequences represented and the representative accession number are indicated in brackets in
[0161] Illumina Tag Sequencing.
[0162] DNA was extracted from 0.25 g of wet weight sediment using the PowerSoil DNA Isolation Kit (Cat#12888-05, Mo Bio Laboratories, Carlsbad, Calif.). PCR amplification and barcoding of the 16S rRNA gene were performed as described by O. Mason et al. (USA. Microb. Ecol. 70:766 (2015)). Amplicons for deep sequencing were outsourced to Laragen, Inc (Culver City, Calif.) and run on an Illumina MiSeq platform. Data was analyzed using QIIME 1.8.0 and processed sequences were assigned to phylotypes using a 99% similarity cutoff to the SILVA database version 115 following the procedures described in O. Mason et al.
[0163] Sample Preparation for Aggregate Embedding, Sectioning, FISH, and nanoSIMS.
[0164] Sample preparation was carried out following the recently optimized protocol outlined in S. E. McGlynn et al. (Nature 526:531 (2015)). Briefly: Paraformaldehyde fixed consortia were detached from the sediment particles via sonication on ice with a sterile remote-tapered microtip probe (Branson) inserted into the liquid. Aggregates were concentrated on a 3 m filter, covered in molten noble agar (2%) and embedded in glycol methacrylate (Heraeus KulzerTechnovit 8100). Sections of ca. 1 m thickness were cut and stretched on a water droplet on a polylysine coated slide with teflon wells (Tekdon Inc) and analyzed by fluorescence in situ hybridization (FISH). Images of the FISH-stained consortia were collected and the location of these consortia were mapped for subsequent nanoSIMS analysis as described herein.
[0165] FISH Conditions and Probes.
[0166] The FISH hybridization on thin sections of resin embedded microbial aggregates followed the recently optimized protocol described in S. E. McGlynn et al. The phylogenetic identity of microorganisms in the thin sections were determined using conventional FISH using oligonucleotide probes fluorescently labeled on both the 5 and 3 ends (dual labeled) as outlined herein. The FISH hybridization followed a standard protocol as described in B. M. Fuchs et al., (Methods for general and molecular microbiology 3:886 (2007)) and used a hybridization buffer containing 60% formamide and incubation at 46 C. for 120 min. Followed by a wash step at 48 C. for 10 min. to remove excess probes. Visualization via epifluorescence microscopy (light source EXFO, X-Cite, Series 120 Q) was accomplished by mounting the hybridized sample with a mixture of DAPI-Citifuor (5 g DAPI/mL) and imaging with a 100 objective (Microscope Olympus BX51, objective UPlan FL N, 100/1.30 Oil, /0.17/FN 26.5). The following FISH probes were used (final concentration 2.5 ng/L for each): S-D-Arch-0915-a-A-20 in FITC, dual labeled: 5 to 3=GTGCTCCCCCGCCAATTCCT (SEQ ID NO:8); S-*-ANME2c-0760-a-A-18 in Cy3, dual labeled: 5 to 3=CGCCCCCAGCTTTCGTCC (SEQ ID NO:9); and S-*-Dsb-0658-a-A-18 in Cy5, dual labeled: 5 to 3=TCCACTTCCCTCTCCCAT (SEQ ID NO:10). The overlay figures (
[0167] nanoSIMS Procedures and Parameters.
[0168] After FISH and mapping, the glass slides were scored and broken to size to fit in the Cameca Geology holder for the nanoSIMS, and coated with 50 nM gold to enhance conductivity. Secondary ion mass spectrometry analysis was carried out on a nanoSIMS 50 L instrument (Cameca) using a primary Cs.sup.+ ion beam. Pre-sputtering of target consortia was conducted with 100 pA current at D1=1 until 70,000-1,000,000 counts were reached on the .sup.12C detector. Analytical conditions included a 256256 raster (or 512512 raster for frames larger than 35 m), ES=2, D1=4 (0.5 pA of current). Chained analyses were set up using SIBC, EOS, and HMR automatic peak centering every 2 frames using the .sup.14N.sup.12C.sup. ion as reference. The following secondary ions were collected during the analysis: .sup.12C.sup., .sup.13C.sup., .sup.12C.sup.14N.sup., .sup.12C.sup.15N.sup., .sup.32S.sup., .sup.33S.sup. and .sup.34S.sup.. 1-4 frames were collected for each aggregate. The ions .sup.12C.sup.14N.sup. and .sup.12C.sup.15N.sup. were used for the fractional abundances of .sup.15N reported herein.
[0169] nanoSIMS Data Processing.
[0170] Raw nanoSIMS data files were initially processed in the Matlab-based program Look@NanoSIMS to align and accumulate frames and extract ion count data. FISH images of aggregates were used to define regions of interest (ROIs) on the nanoSIMS ion images that correspond to archaeal or bacterial biomass. Total ion counts in each ROI for .sup.12C.sup.15N.sup. and .sup.12C.sup.14N.sup. were used to calculate partner-specific relative biosynthetic activity (.sup.15N/(.sup.14N.sup.+15N)) for each aggregate (data for
[0171] Calculation of doubling times from average cellular .sup.15N after 18 days (.sup.15F.sub.18days): [0172] Fraction of new biomass: =B.sub.18 daysB.sub.0/ B.sub.18 days [0173] With exponential growth: =1exp(*18 days) [0174] With .sup.15F.sub.00: .sup.15F.sub.18 days=*.sup.15F.sub.NH.sub.
[0178] Quantification of AOM with AQDS.
[0179] To quantify AOM with AQDS, anaerobic microcosm experiments were performed using methane seep sediment from the Santa Monica basin that had been rendered sulfate- and sulfide-free and amended with 50 mmol AQDS and .sup.13C-labeled methane [0.35 MPa]. After a 21-day incubation at 4 C., approximately 12.5 mmol of dissolved inorganic carbon (DIC) formed from the .sup.13C-methane (see
[0180] The initial rates of AOM with AQDS were equivalent to the rates measured with sulfate over the first 6 days (see
[0181] To confirm that the observed methane oxidation with AQDS was not coupled to traces of sulfate, AOM was tracked in the presence of sodium molybdate, a competitive inhibitor for sulfate reduction. With the addition of 25 mM molybdate, rates of sulfate-coupled AOM decreased by approximately fivefold relative to controls, which is consistent with previous reports. The high rates of methane oxidation in the sulfate-free incubations containing AQDS showed no inhibitory response if molybdate was included, indicating a decoupling of AOM from sulfate reduction (see
[0182] Stimulation of AOM without sulfate is not restricted to AQDS. Regioisomers of AQDS (1,5-AQDS and 2,7-AQDS), humic acids, and soluble iron(III) complexes (ferric citrate and ferric-EDTA) also stimulated anaerobic oxidation of methane at rates that were at least 0.1 mmol cm.sup.3 day (see
[0183] The archaeal 16S ribosomal RNA (rRNA) gene diversity of the seep sediment used in the AOM microcosm experiments was dominated by ANME-2 of the subgroups ANME-2a and ANME-2c, with a low relative abundance of ANME-1 phylotypes (see
TABLE-US-00005 TABLE 5 Bacterial 16S rRNA diversity. Oxidant/electron acceptor Bacterial 16S cDNA sequences recovered* Sulfate AQDS.sup. none Proteobacteria/Deltaproteobacteria/ 18 2 0 Desulfobacterales_Desulfobacteraceae/SEEP-SRB1 Proteobacteria/Deltaproteobacteria/Desulfuromonadales/ 2 3 0 Desulfuromonadaceae_Pelobacter_2 Bacteroidetes/Sphingobacteriia_Sphingobacteriales_1/WCHB1-69 2 0 0 Chlorobi/Ignavibacteria_Ignavibacteriales/BSV26 2 0 0 Proteobacteria/Deltaproteobacteria/ 2 0 0 Desulfarculales_Desulfarculaceae/uncultured Proteobacteria/Gammaproteobacteria_1/ 1 3 0 Pseudomonadales_Pseudomonadaceae/Pseudomonas_1 Chloroflexi/Anaerolineae_Anaerolineales_Anaerolineaceae/uncultured 1 1 1 Proteobacteria/Deltaproteobacteria/Sva0485 1 1 0 Acidobacteria/Subgroup 22 1 0 0 Candidate division OP8 1 0 1 Proteobacteria/Deltaproteobacteria/Sh765B-TzT-29 1 0 0 Proteobacteria_Deltaproteobacteria_Desulfobacterales_Nitrospinaceae/ 1 0 0 uncultured Spirochaetae_Spirochaetes/Spirochaetales/Spirochaetaceae/Spirochaeta_2 0 9 1 Proteobacteria/Betaproteobacteria/Burkholderiales/ 0 4 0 Oxalobacteraceae/Herbaspirillum_1 Candidate division JS1 0 2 3 Proteobacteria/Epsilonproteobacteria/ 0 2 1 Campylobacterales_Helicobacteraceae/Sulfurimonas Bacteroidetes/Flavobacteriia_Flavobacteriales/ 0 1 0 Flavobacteriaceae_1/Maritimimonas Chloroflexi/Ardenticatenia/uncultured 0 1 1 Firmicutes_Clostridia_1/Clostridiales/Family XII/Fusibacter 0 1 0 Proteobacteria/Alphaproteobacteria/Rhizobiales_1/ 0 1 0 Brucellaceae/Ochrobactrum_1 Actinobacteria/Acidimicrobiia_Acidimicrobiales/OM1 clade 0 0 3 Bacteroidetes/BD2-2 0 0 2 Bacteroidetes/Bacteroidia_Bacteroidales/ 0 0 1 dPorphyromonadaceae_4/unculture Candidate division WS3 0 0 1 Chloroflexi/Dehalococcoidia/GIF9 0 0 1 Lentisphaerae/B01R017 0 0 1 Planctomycetes/Phycisphaerae/MSBL9 0 0 1 Planctomycetes/Phycisphaerae/Phycisphaerales/ 0 0 1 AKAU3564 sedimentgroup Proteobacteria/Gammaproteobacteria_2/ 0 0 1 Chromatiales_Ectothiorhodospiraceae_Acidiferrobacter Spirochaetae_Spirochaetes/Spirochaetales/Leptospiraceae/uncultured 0 0 1 TOTAL NUM OF CLONES 33 31 21 *Data based on 16S cDNA clone libraries. .sup.PCR amplification and cloning of bacterial cDNA from the AQDS treatment was challenging due to weak amplification, few insert containing clones, and chimeric sequences.
[0184] Cell-specific stable isotope analysis was used to quantify the anabolic activity of ANME-2 (including ANME-2c) and their co-associated syntrophic partners in consortia recovered from incubations supplied with different oxidants or electron acceptor (including sulfate, AQDS, humic acids, and ferric iron). Using .sup.15NH.sub.4.sup.+ stable isotope probing combined with fluorescence in situ hybridization and nanoscale secondary ion mass spectrometry [FISH-SIMS], the cell-specific anabolic activity (.sup.15N cellular enrichment) was measured in paired ANME and SRB populations in consortia. After 18 days of incubation with .sup.15NH.sub.4.sup.+, consortia were phylogenetically identified by FISH using ANME-2c and Desulfobacteraceae-targeted oligonucleotide probes and were analyzed by nanoSIMS to quantify the assimilation of .sup.15NH.sub.4.sup.+ for each paired population of ANME-2 and SRB.
[0185] In AOM microcosms containing sulfate, the .sup.15NH.sub.4.sup.+ assimilation by co-associated bacteria and archaea in consortia from two sets of experiments (n=20 and n=19 consortia) was positively correlated at a ratio of approximately 1:1, indicating balanced syntrophic growth during AOM (see
[0186] The ANME cells paired with SRB in consortia from AQDS incubations showed similar levels of anabolic activity [3.3 months doubling time based on average .sup.15N-incorporation] as those of ANME archaea conserving energy through conventional sulfate-coupled AOM [2.9 months doubling time] in parallel incubations, suggesting equivalent potential for growth (see
[0187] AOM incubations with iron(III)-citrate and humic acids as the alternative electron acceptors also demonstrated exclusive biosynthetic activity of ANME-2c and other ANME-2 cells (see
[0188] Certain embodiments of the invention have been described. It will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.