METHODS TO STIMULATE BIOGENIC METHANE PRODUCTION FROM HYDROCARBON-BEARING FORMATIONS

Info

Publication number: 20120138290
Type: Application
Filed: Jun 3, 2011
Publication Date: Jun 7, 2012
Applicant: SYNTHETIC GENOMICS, INC. (La Jolla, CA)
Inventors: Brian G. Clement (Houston, TX), James G. Ferry (State College, PA), Stuart Underwood (Roanoke, VA)
Application Number: 13/153,120

Abstract

The present invention describes methods of stimulating the biogenic production of methane in hydrocarbon-bearing formations. The present application provides various stimulants which, when contacted with a hydrocarbon deposit in situ or ex situ, induce or enhance coalbed methane production.

Description

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No. 61/351,709, filed on Jun. 4, 2010, the disclosure of which is incorporated by reference herein in its entirety.

This application is related to U.S. application Ser. No. 12/464,832, filed May 12, 2009, now publication no. US20100047793A1.

BACKGROUND OF THE INVENTION

Coalbed methane (CBM) is a source of natural gas produced either biologically or thermogenically in coal deposits. Biogenic production of CBM is the result of microbial metabolism and the degradation of coal with a subsequent electron flow among multiple microbial populations. Thermogenic production of CBM is the result of thermal cracking of sedimentary organic matter or oil, occurring later in coalification when temperatures rise above levels at which the methane-producing microorganisms can live. In coalbeds, pressure from overlying rock and surrounding water cause the CBM to bond to the surface of the coal and be absorbed into the solid matrix of the coal as free gas within micropores and cleats (natural fractures in the coal), as dissolved gas in water, as adsorbed gas held by molecular attraction on surfaces of macerals (organic constituents that comprise the coal mass), micropores, and cleats in the coal, and as absorbed gas within the molecular structure of the coal.

Coal is a sedimentary rock with various degrees of permeability, with methane residing primarily in the cleats. These fractures in the coal act as the major channels to allow CBM to flow. To extract the CBM, a steel-encased hole is drilled into the coal seam, which allows the pressure to decline due to the hole to the surface or the pumping of small amounts of water from the coalbed (dewatering). CBM has very low solubility in water and readily separates as pressure decreases, allowing it to be piped out of the well separately from the water. The CBM is then sent to a compressor station and into natural gas pipelines.

CBM represents a significant portion of the natural gas produced in the United States, estimated as providing approximately 10% of the natural gas supplies, or about 1.8 trillion cubic feet (TCF). International reserves provide enormous opportunity for future CBM production. Among the most productive areas is the San Juan Basin, located in Colorado and New Mexico. Based on such enormous reservoirs of CBM, minimal improvements in CBM recovery could thus result in significantly increased production from a well, and accordingly, a variety of methods are being developed to improve the recovery of CBM from coal seams.

Purely physical interventions can include optimizing drilling and fracturing methods. Other improvement methods involve the application of external factors directly onto the coalbeds. These include, for example, the injection of gases such as nitrogen (see, e.g., Shimizu et al., (2007) Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan. Geobiology 5(4):423-433; U.S. Pat. No. 4,883,122) and CO₂(see, e.g., U.S. Pat. No. 5,402,847); and the injection of hot fluids such as water or steam (see, e.g., U.S. Pat. No. 5,072,990). Various methods are intended to increase the permeability of the coalbed seams either physically (see, e.g., U.S. Pat. No. 5,014,788) or chemically (see, e.g., U.S. Pat. No. 5,865,248).

SUMMARY OF THE INVENTION

There remains a need to effectively stimulate biogenic production in hydrocarbon-bearing formations such as coal and to enhance the CBM productivity of existing wells.

The present invention provides methods and processes for the use of compositions comprising stimulants for biogenic production of methane in hydrocarbon-bearing formations. The present invention provides methods for tailored interventions, such as the use of compositions comprising stimulants that can be introduced into an in situ environment to enhance the biogenic production of methane. The present invention also provides methods for tailored interventions, such as the use of compositions comprising stimulants that can be introduced into an ex situ environment to enhance the biogenic production of methane.

In one embodiment, one or more microorganisms from the hydrocarbon-bearing formation are enriched by selecting for the ability to grow on coal as the sole carbon source.

In another embodiment, the methods comprise in vitro testing of compositions comprising stimulants at more than one concentration to monitor and optimize methane production in a culture system comprising at least one microorganism isolated from said hydrocarbon-bearing formation, further wherein said culture system provides coal as the sole carbon source.

At least one microorganism is a bacterial species or an archaeal species capable of converting a hydrocarbon to a product selected from the group consisting of hydrogen, carbon dioxide, acetate, formate, methanol, methylamine, or any other methanogenic substrate; one or more hydrocarbon-degrading bacterial species, one or more methanogenic bacterial species or one or more methanogenic archaeal species that can convert substrates to methane.

In one embodiment, the methods are performed with a functional microbial subcommunity (enrichment) that is developed methods described in Example 1 below. The members of the functional microbial subcommunity act in concert to produce methane; and further wherein said culture system provides coal as the sole carbon source.

In an alternative embodiment, the methods are performed with a defined microbial assemblage that combines a culture of microorganisms from a hydrocarbon-bearing formation, such that members of said defined microbial assemblage act in concert to produce methane; and further wherein said culture system provides coal as the sole carbon source.

A hydrocarbon-bearing formation to be treated can be any formation containing hydrocarbons. Hydrocarbon-bearing formations include, but are not limited to: coal, peat, kerogen, oil, tar, heavy oil, oil shale, oil formation, traditional black oil, viscous oil, oil sands and tar sands. In one embodiment, the formation is coal in a coal seam or coalbed. The term “coal” as used herein refers to any rank of coal ranging from lignite to anthracite. The members of the various ranks differ from each other in the relative amounts of moisture, volatile matter, and fixed carbon contained in the matrix. The lowest in carbon content, lignite or brown coal, is followed in ascending order by subbituminous coal or black lignite (a slightly higher grade than lignite), bituminous coal, semi-bituminous (a high-grade bituminous coal), semi-anthracite (a low-grade anthracite), and anthracite. Coals for use in the present methods can be of any rank; representative examples of coal include, but are not limited to, lignite, brown coal, subbituminous coal, bituminous coal, coking coals, anthracite, and combinations thereof.

In various embodiments, the stimulant involved in the conversion of hydrocarbon to methane is yeast extract, sulfur, an oxyanion of sulfur (e.g., thiosulfate (S₂O₃), sodium thiosulfate (Na₂S₂O₃), potassium thiosulfate (K₂S₂O₃), sulfuric acid, disulfuric acid, peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid, thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid or polythionic acid), NH₄Cl, KCl, vanadium, VCl₃, VCl₂, VCl, Na₂SO₃, MnCl₂, Na₂MoO₄, FeCl₃or Na₂SO₄. In a preferred embodiment, the stimulant is vanadium, VCl₃, VCl₂, VCl, sulfur, thiosulfate or sodium thiosulfate.

The invention provides processes for enhancing biogenic production of methane in a hydrocarbon-bearing formation, said method comprising introducing a composition comprising a into a hydrocarbon-bearing formation.

In one embodiment, the process introduces the composition comprising the stimulant into the hydrocarbon-bearing formation. In a preferred embodiment, the hydrocarbon-bearing formation is coal.

The invention further provides processes for enhancing biogenic production of methane from coal by introducing one or more microorganisms, consortiums, functional microbial subcommunities, or a DMA into a coalbed. Microorganisms can be indigenous or exogenous to the formation to be treated. Compositions can include microorganisms that are naturally-occurring, genetically-engineered, or a combination thereof. Where more than one population of microorganisms is to be introduced, one or more populations can be genetically engineered and one or more populations can be genetically unmodified. In such embodiments, such processes comprise introducing compositions comprising one or more microorganisms, consortiums, functional microbial subcommunities, or a DMA into a coalbed together with a composition comprising a stimulant.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the present application can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are utilized, and the accompanying drawings of which:

FIGS. 1A and 1B illustrate methane production from coal after stimulation of a functional microbial subcommunity with a composition comprising vanadium (III) chloride (VCl₃) and a composition comprising sodium thiosulfate (NaS₂O₃), respectively. Each sample was run in four replicates; standard error bars are shown for each time point. Week 5 methane production is shown for three concentrations of stimulants tested in the presence or absence of coal.

FIGS. 2A and 2B illustrate methane production from coal after stimulation of a functional microbial subcommunity with a composition comprising ammonium chloride (NH₄Cl) and a composition comprising sodium sulfite (Na₂SO₃), respectively. Each sample was run in four replicates; standard error bars are shown for each time point. Week 5 methane production is shown for three concentrations of stimulants tested in the presence or absence of coal.

FIGS. 3A and 3B illustrate methane production from coal after stimulation of a functional microbial subcommunity with a composition comprising manganese chloride (MnCl₂) and a composition comprising sodium molybdic (Na₂MoO₄), respectively. Each sample was run in four replicates; standard error bars are shown for each time point. Week 5 methane production is shown for three concentrations of stimulants tested in the presence or absence of coal.

FIGS. 4A and 4B illustrate methane production from coal after stimulation of a functional microbial subcommunity with a composition comprising potassium chloride (KCl) and a composition comprising ferrous chloride (FeCl₃), respectively. Each sample was run in four replicates; standard error bars are shown for each time point. Week 5 methane production is shown for three concentrations of stimulants tested in the presence or absence of coal.

FIG. 5 illustrates methane production from coal after stimulation of a functional microbial subcommunity with a composition comprising sodium sulfate (Na₂SO₄). Each sample was run in four replicates; standard error bars are shown for each time point. Week 5 methane production is shown for three concentrations of stimulants tested in the presence or absence of coal.

FIG. 6 illustrates methane production from coal at weeks 1, 3 and 5 following stimulation of a functional microbial subcommunity with an intermediate concentration of VCl₃.

FIG. 7 illustrates methane production from coal at weeks 1, 3 and 5 following stimulation of a functional microbial subcommunity with an intermediate concentration of NaS₂O₃.

FIG. 8 illustrates a variety of potential enzymatic pathways in the conversion of coal to methane.

FIG. 9 illustrates a process for introducing an external factor such as a stimulant to a coalbed via injected formation water to increase methane production.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

The singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of cells and reference to “a compound” includes a plurality of compounds, etc.

As used herein, the terms “about” or “approximately” when referring to any numerical value are intended to mean a value of plus or minus 10% of the stated value. For example, “about 50 degrees C.” (or “approximately 50 degrees C.”) encompasses a range of temperatures from 40 degrees C. to 60 degrees C., inclusive. Similarly, “about 100 mM” (or “approximately 100 mM”) encompasses a range of concentrations from 90 mM to 110 mM, inclusive. All ranges provided within the application are inclusive of the values of the upper and lower ends of the range.

The term “substantially purified”, as used herein, refers to a molecule separated from substantially all other molecules normally associated with it in its native state. More preferably a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule may be greater than 60% free, preferably 75% free, more preferably 90% free, and most preferably 95% free from the other molecules (exclusive of solvent) present in the natural mixture. The term “substantially purified” is not intended to encompass molecules present in their native state.

As used herein, the term “yield” refers to the amount of harvestable product, and is normally defined as the measurable produce of economical value of methane. Yield may be defined in terms of quantity or quality. The harvested material may vary from hydrocarbon deposit to hydrocarbon deposit. The term “yield” also encompasses yield potential, which is the maximum obtainable yield. Yield may be dependent on a number of yield components, which may be monitored by certain parameters. These parameters are well known to persons skilled in the art and vary from deposit to deposit.

The present invention provides novel methods and processes to stimulate biogenic methane production in hydrocarbon-bearing formations, such as coal seams and coalbed methane wells, by stimulating cultivated microorganisms derived from the formation with various amendments. The present application also relates to further stimulating biogenic methane production in a hydrocarbon-bearing formation by exposing the formation by further exposing the formation to one or more microorganisms. The microorganisms can be consortiums, isolated cultures, genetically modified microorganisms.

The methods of the present invention provide an approach for the use of stimulants, functional microbial communities, and/or DMAs useful for increasing biogenic production of methane. Briefly, in the examples provided herein, formation water samples were collected from a coalbed methane well in the San Juan Basin, where previous studies indicated an age of 70 million years resulting from an isolation from the surface and no evidence of subsurface mixing events. The water could be collected from the well head, the separation tank (knock out drum) or reservoir tank as these water samples are the most readily available materials. The water samples containing living microorganisms were then visualized via light microscopy, and microorganisms were cultivated using formation water as mineral base. Cultures of microorganisms were enriched for methane-producing microbes using coal as sole carbon source. Various combinations of amendments were tested as stimulants for microbial respiration. The microbial enrichments (functional functional microbial communities) were then screened for methane production using gas chromatography.

The power of the methods of the present invention can be seen in the use of compositions comprising vanadium or thiosulfate to stimulate biogenic production of methane from coal.

Sources of Microorganisms and Their Characterization

As used herein, the term “hydrocarbon-bearing formation” refers to any hydrocarbon source from which methane can be produced, including, but not limited to, coal, kerogen, peat, oil shales, oil formations, heavy oil, traditional black oils, viscous oil, oil sands and tar sands. In the various embodiments discussed herein, a hydrocarbon-bearing formation or even a hydrocarbon-bearing formation environment may include, but is not limited to, coal, coal seam, waste coal, coal derivatives, peat, kerogen, oil formations, oil shale, tar, tar sands, hydrocarbon-contaminated soil, petroleum sludge, drill cuttings, and the like and may even include those conditions or even surroundings in addition to oil shale, coal, coal seam, waste coal, coal derivatives, peat, oil formations, tar sands, hydrocarbon-contaminated soil, petroleum sludge, drill cuttings, and the like. In some embodiments, the present invention may provide an in situ hydrocarbon-bearing formation sometimes referred as an in situ hydrocarbon-bearing formation environment or in situ methane production environment. Embodiments may include an ex situ hydrocarbon-bearing formation sometimes referred to as an ex situ hydrocarbon-bearing formation environment or an ex situ methane production environment. In situ may refer to a formation or environment of which hydrocarbon-bearing sources may be in their original source locations, for example, in situ environments may include a subterranean formation. Ex situ may refer to formations or environments where a hydrocarbon-bearing formation has been removed from its original location and may perhaps even exist in a bioreactor, ex situ reactor, pit, above ground structures, and the like situations. As a non-limiting example, a bioreactor may refer to any device or system that supports a biologically active environment.

Using coal as an exemplary hydrocarbon-bearing formation, there are numerous sources of indigenous microorganisms that may be playing a role in the hydrocarbon to methane conversion that can be analyzed. Coal is a complex organic substance that is comprised of several groups of macerals, or major organic matter types, which accumulate in different types of depositional settings such as peat swamps or marshes. Maceral composition, and therefore coal composition, changes laterally and vertically within individual coal beds. Once microorganisms are identified as involved in a conversion step, different functional microbial subcommunities, defined microbial assemblages and/or stimulants identified herein may work better on specific maceral groups and therefore, each coal bed may be unique in what types of microorganism and stimulant are most efficient at the in situ bioconversion of the coal.

There are numerous naturally-occurring microbes that are associated with coal and other organic-rich sediments in the subsurface. Over time, these microbial species may have become very efficient at metabolizing organic matter in the subsurface through the process of natural selection. The relatively quick adaption of bacteria to local environmental conditions suggests that microorganisms collected from basins, or individual coal seams, may be genetically unique. Once collected, these microorganisms can be grown in laboratory cultures as described herein to evaluate and determine factors enhancing and/or limiting the conversion of coal into methane. In some cases, a key nutrient or trace element may be missing, and addition of this limiting factor may significantly increase methane production. When bacteria are deprived of nutrients, physiological changes occur, and if the state of starvation continues, all metabolic systems cease to function and the bacteria undergo metabolic arrest. When environmental conditions change, the bacteria may recover and establish a viable population again. Therefore, it is possible that some bacteria in organic-rich sediments have reached a state of metabolic arrest and the addition of nutrients is all that is required to activate the population under the present invention. By specifically analyzing the effect of various amendments on such populations, we can identify compounds that methane production being carried out by one or more members of these microbial populations.

Anaerobic bacteria from a subsurface formation can be collected by several different methods that include (1) produced or sampled formation water, (2) drill cuttings, (3) sidewall core samples (4) whole core samples, and (5) pressurized whole core samples. Pressurized core samples may present the best opportunity to collect viable microbial populations, but we have found collection of microbial populations from formation waters has provided a representative sample of the microbial populations present. Methanogens are obligate anaerobes, but can remain viable in the presence of oxygen for as much as 24 hours by forming multicellular lumps. Additionally, anoxic/reducing microenvironments in an oxygenated system can potentially extend anaerobic bacterial viability longer. In some cases, drill cuttings collected and placed in anaerobic sealed containers will contain microorganisms that are capable of converting the coal to methane within a few hours, thereby giving erroneous gas content measurements.

Methods of on-site collection have been optimized to provide optimal recovery of anaerobic populations of microorganisms therein. The present invention involves anaerobic microbial populations previously described by PCT Application No. PCT/US2008/057919 (WO 2008/116187), and the cultivation of indigenous microorganisms residing in the hydrocarbon-bearing formation environment, such formation water or coalbed methane wells.

The methods provided herein also afford the opportunity for genetically altering microorganisms. By identifying stimulants that may be used to increase methane production, microorganisms can be genetically engineered to have abilities that can be tied to increased methane production. Selections of microorganisms by the methods described herein enrich for the ability to efficiently metabolize coal and other organic-rich substrates. Various possibilities to enhance methane production from wells comprise introducing compositions comprising stimulants, microorganisms, defined assemblages of organisms, genetically-modified organisms, or any combinations thereof into the formation.

According to the present methods, a functional microbial community is stimulated to transform hydrocarbons to methane. Microorganisms naturally present in the formation are preferred because it is known that they are capable of surviving and thriving in the formation environment, and should provide components of various pathways proceeding from hydrocarbon hydrolysis through to methanogenesis. However, this invention is not limited to use of indigenous microorganisms. When analyzing enzymatic profiles of indigenous microorganisms, it may be advantageous to combine such information with that of exogenous microorganisms. This information may come from known microorganisms, preferably those that are suitable for growing in the subterranean formation, and by analogy, have similar potential processes.

The terms “functional microbial community” or “microbial enrichment” as used herein, refers to a culture of more than one microorganism wherein the community has been developed by culturing a sample under specific conditions. The community may not necessarily remain static over time, but may continue to evolve depending upon nutrient supplements or substrates added to the culture.

The term “defined microbial assemblage” or “DMA” as used herein, refers to a culture of more than one microorganism, wherein different strains are cultured or intentionally combined to convert a hydrocarbon to methane. The microorganisms of the assemblage are “defined” such that at any point in time we can determine the members of the population by use of genetic methods, such as 16S taxonomy as described herein. The DMA does not necessarily remain static over time, but may evolve as cultures flux to optimize hydrocarbon hydrolysis and methane production. Optimally, the DMA is prepared to provide microorganisms harboring strong capacity to convert hydrocarbon to methane. The DMA may consist of 2 or more microorganisms, in any combination, to provide bacterial or archael species capable of converting a hydrocarbon to any intermediate leading to the production of methane, and/or any methanogenic species. For example, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more organisms present in a DMA. The members of the DMA act synergistically to produce methane, amongst themselves, or together with microorganisms present in the hydrocarbon-bearing formation.

The term “microorganism” is intended to include bacteria and archaea organisms, as well as related fungi, yeasts and molds. It will be understood that bacteria and archaea are representative of microorganisms in general that can degrade hydrocarbons and convert the resulting products to methane. The dividing lines between classes of microorganisms are not always distinct, particularly between bacteria and fungi. It is preferred, therefore, to use the term microorganisms to include all microorganisms that can convert hydrocarbons to methane, whatever the commonly used classifications might be. Of these microorganisms, those usually classified as bacteria and archaea are, however, preferred. If exogenous bacteria and archaea are used in the methods described herein, other microorganisms such as fungi, yeasts, molds, and the like can also be used.

The term “anaerobic microorganism” as used herein, refers to microorganisms that can live and grow in an atmosphere having less free oxygen than tropospheric air (i.e., less than about 18%, by mol., of free oxygen). Anaerobic microorganisms include organisms that can function in atmospheres where the free oxygen concentration is less than about 10% by mol., or less than about 5% by mol., or less than about 2% by mol., or less than about 0.5% by mol.

The term “facultative anaerobes” as used herein, refers to microorganisms that can metabolize or grow in environments with either high or low concentrations of free oxygen.

The conversion of hydrocarbons to methane requires the active participation of methanogens. A “methanogen” as used herein, refers to obligate and facultative anaerobic microorganisms that produce methane from a metabolic process. The presence of methanogens within the samples indicates the high likelihood of in situ methane formation. Methanogens are typically classified into four major groups of microorganisms: Methanobacteriales, Methanomicrobacteria and relatives, Methanopyrales and Methanococcales. All methanogenic microorganisms are believed to employ elements of the same biochemistry to synthesize methane. Methanogenesis is accomplished by a series of chemical reactions catalyzed by metal-containing enzymes. One pathway is to reduce CO₂to CH₄by adding one hydrogen atom at a time (CO₂-reducing methanogenesis). Another pathway is the fermentation of acetate and single-carbon compounds (other than methane) to methane (acetate fermentation, or acetoclastic methanogenesis). The last step in all known pathways of methanogenesis is the reduction of a methyl group to methane using an enzyme known as methyl reductase. As the presence of methyl reductase is common to all methanogens; it is a definitive character of methanogenic microorganisms. The method for identifying the presence of methanogens is to test directly for the methanogen gene required to produce the methyl reductase enzyme. Alternatively the presence of methanogens can be determined by comparison of the recovered 16S rDNA against an archaeal 16S rDNA library using techniques known to one skilled in the art (generally referred to herein as 16S taxonomy).

Classes of methanogens include Methanobacteriales, Methanomicrobacteria, Methanopyrales, Methanococcales, and Methanosaeta (e.g., Methanosaeta thermophila), among others. Specific examples of methanogens include Methanobacter thermoautotorophicus, and Methanobacter wolfeii. Methanogens may also produce methane through metabolic conversion of alcohols (e.g., methanol), amines (e.g., methylamines), thiols (e.g., methanethiol), and/or sulfides (e.g., dimethyl sulfide). Examples of these methanogens include methanogens from the genera Methanosarcina (e.g., Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina siciliae, Methanosarcina acidovorans, Methanosarcina mazeii, Methanosarcinafrisius); Methanolobus (e.g., Methanolobus bombavensis, Methanolobus tindarius, Methanolobus vulcani, Methanolobus taylorii, Methanolobus oregonensis); Methanohalophilus (e.g., Methanohalophilus mahii, Methanohalophilus euhalobius); Methanococcoides (e.g., Methanococcoides methylutens, Methanococcoides burtonii); and/or Methanosalsus (e.g., Methanosalsus zhilinaeae). They may also be methanogens from the genus Methanosphaera (e.g., Methanosphaera stadtmanae and Methanosphaera cuniculi, which are shown to metabolize methanol to methane). They may further be methanogens from the genus Methanomethylovorans (e.g., Methanomethylovorans hollandica, which is shown to metabolize methanol, dimethyl sulfide, methanethiol, monomethylamine, dimethylamine, and trimethylamine into methane).

As described herein, it is a feature of the present embodiments that microbial communities obtained from a variety of environmental samples are amenable to study using genomic tools as provided herein; in addition, microbial populations can be cultivated and optionally isolated and/or enriched in the laboratory using invention methods. By applying these approaches at the genomic level, and by specifically characterizing the enzymatic profiles of microorganisms involved in the conversion of hydrocarbons to methane, it is possible to develop a fundamental understanding of the metabolism of the microbial communities and, more specifically, the methanogenic degradation of coal in the formation water and coal seams. As such, we are then able to elucidate the ecological niche of each population and ultimately develop stimulants, functional microbial subcommunities and/or DMAs that could yield an enhancement in the biological methane production.

According to the present methods, microorganisms present in the hydrocarbon-bearing formation environment (indigenous microorganisms) are stimulated or modulated to transform hydrocarbons to methane. Microorganisms naturally present in the formation are preferred because it is known that they are capable of surviving and thriving in the formation environment. However, this invention is not limited to use of indigenous microorganisms. Exogenous microorganisms suitable for growing in the subterranean formation may be identified and such microorganisms introduced into the formation by known injection techniques before, during, or after practicing the process of this invention. For example, if the formation contains only two microorganisms of a desired three-component consortia, then the missing microorganisms, or a stimulant for such a microorganism could be injected into the formation. Microorganisms, indigenous or exogenous, may also be recombinantly modified or synthetic organisms.

Stimulants

The term “stimulant” as used herein refers to any factor that can be used to increase or stimulate the biogenic production of a metabolic product with increased hydrogen content from a hydrocarbon material. Metabolic products with increased hydrogen content include, but are not limited to, methane, hydrogen, acetate, formate, butyrate, propionate, substituted and un-substituted hydrocarbons, such as ethers, aldehydes, ketones, alcohols, organic acids, amines, thiols, sulfides, and disulfides, among others, substituted and unsubstituted, mono- and poly-aromatic hydrocarbons, and the like. In one embodiment, the metabolic product with increased hydrogen content is methane.

A stimulant can be a substrate, reactant or co-factor for a pathway that is involved in the conversion of a hydrocarbon to methane. The function of the stimulant is to boost existing production by increasing the level of activity or growth of a microorganism, or to increase, decrease or modulate by any means the enzymatic activity of an enzyme involved in a pathway involved in the conversion of a hydrocarbon to methane in order to optimize the end production of methane from the hydrocarbon-bearing formation.

Stimulants may provide for enhancement, replacement, or addition of any nutrient that is not optimally represented or functional in the hydrocarbon-bearing environment. The goal is to optimize and/or complete of the pathway from hydrocarbon to methane. Generally this requires representation of microorganisms that are capable of converting a hydrocarbon to a product such as hydrogen, carbon dioxide, acetate, formate, methanol, methylamine or any other methanogenic substrate. Microorganisms include those capable of low rank coal hydrolysis, coal depolymerization, anaerobic or aerobic degradation of polyaromatic hydrocarbons, homoacetogenesis, and methanogenesis (including hydrogenotrophic or CO₂reducing and acetoclastic), and any combinations thereof to achieve conversion of a hydrocarbon to methane.

Examples of stimulants include, for example, yeast extract, sulfur, an oxyanion of sulfur (e.g., thiosulfate (S₂O₃), sodium thiosulfate (Na₂S₂O₃), potassium thiosulfate (K₂S₂O₃), sulfuric acid, disulfuric acid, peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid, thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid or polythionic acid), NH₄Cl, KCl, vanadium, VCl₃, VCl₂, VCl, Na₂SO₃, MnCl₂, Na₂MoO₄, FeCl₃or Na₂SO₄. In a preferred embodiment, the stimulant is vanadium or thiosulfate.

Incorporation of Stimulants to Increase Methane Production

The methods and processes of the present invention can be readily used for field applications and the enhancement of in situ or ex situ methane production from any hydrocarbon-bearing formation such as coal. There are several methods or combination of injection techniques that are known in the art that can be used in situ. Stimulants, functional microbial subcommunities, DMAs, and/or microorganisms can be injected directly into the fractures in the formation. The stimulant components are to be injected as a composition in an aqueous solution such as, but not limited to, formation water, water or media. Fracture orientation, present day in situ stress direction, reservoir (coal and/or shale) geometry, and local structure are factors to consider. For example, there are two major networks (called cleats) in coal beds, termed the face cleat and butt cleat system. The face cleats are often more laterally continuous and permeable, whereas the butt cleats (which form abutting relationships with the face cleats) are less continuous and permeable. During the stimulation of coal bed methane wells, the induced fractures intersect the primary face cleats that allow greater access to the reservoir. However, when the present day in situ stress direction is perpendicular the face cleats, then stress pressure closes the face cleats thereby reducing permeability, but at the same time in situ pressures increase permeability of the butt cleats system. Under these conditions, induced fractures are perpendicular to the butt cleat direction, providing better access to the natural fracture system in the reservoir. The geometry of the injection and producing wells, and whether or not horizontal cells are used to access the reservoir, depend largely upon local geologic and hydrologic condition.

The objective of hydraulic fracture stimulation of coal bed methane, as in conventional oil and gas wells, is to generate an induced fracture network that connects with the naturally occurring fracture network of the reservoir. Stimulants, functional microbial subcommunities, DMAs, and/or microorganisms can be introduced into the naturally-occurring and artificially-induced fractures under pressure to drive the mixture into naturally-occurring fractures deep into the reservoir to maximize bioconversion rates and efficiency. During fracture stimulation of reservoirs, sand proppant and various chemicals may be pumped into the formation under high pressure through a drill rig.

Stimulants, functional microbial subcommunities, DMAs, and/or microorganisms may be injected into the reservoir at the same time as fracture stimulation and/or after the hydraulic fractures are generated. Most in situ microbial applications are expected to occur after fracture stimulation and removal of completion fluids when subsurface anaerobic conditions are reestablished. However, under simultaneous in situ microbial and fracture stimulation, the use of stimulation fluids under anoxic or suboxic conditions is preferred so that anaerobic conditions in the reservoir are maintained, or can be readily attained after stimulation. The injection of aerobic bacteria during simultaneous stimulation would result in the rapid consumption of oxygen and return to anaerobic conditions.

In some cases, pretreatment fluids that modify the coal, carbonaceous shale, or organic-rich shale for bioconversion may be used with the fracture fluids. However, the preferred method for encouraging in situ bioconversion of organic matter is to inject compositions comprising stimulants, functional microbial subcommunities, DMAs, and/or microorganisms under pressure and anaerobic conditions after hydraulic fracture stimulation and subsequent flushing of the well.

Stimulants, functional microbial subcommunities, DMAs, and/or microorganisms can be introduced by re-introduction of the formation water to the subsurface as depicted in FIG. 9. Briefly, methane and formation water are pumped from the well casing 1 into the separation tank 2 (also known as the knock out drum) to remove the gas from the water. The formation water is stored in the reservoir tank 3, from which it can be forwarded to a consolidation station or directed for re-injection to the subsurface. Stimulants, functional microbial subcommunities, DMAs, and/or microorganisms can then be added to the preparation tank 4 and mixed with the recovered formation water. A compressor 5 or pressurized system can then be used to introduce the stimulants, functional microbial subcommunities, DMAs, and/or microorganisms in the formation water to the subsurface.

The introduction of compositions comprising stimulants, functional microbial subcommunities, DMAs, and/or microorganisms, or the delivery of gases, liquids, gels or solids can provide an environment suitable for enhanced methane, including strains capable of aerobic degradation of hydrocarbons. For example, in an exemplary embodiment an inoculum composed of the suitable strains such as described herein at a cell number of 10⁷cells per ml can be mixed with a gel composed of organic substrates such as glycerol than can be used as nutrients stimulating growth through fermentation and secretion of metabolites, including hydrogen, that can be used by methanogens. Once the gel has been assimilated, it will slowly release the optimal amounts of stimulant that will be used by the strains with the capacity for hydrocarbon degradation. These amendments and resulting metabolism can stimulate the electron flow to methane producing a higher amount and yield compared to control wells in the same seam that are not intervened. This is particularly advantageous for strains with the capacity to grow aerobically or anaerobically and can adapt their metabolism for hydrocarbon degradation. In a separate embodiment, an aqueous composition (e.g., formation water, milliΩ water, buffered water, etc.) containing one or more stimulants is injected in a well in order to dispense stimulants needed for conversion of a hydrocarbon to methane reactions. One or more additional elements can be further added to the aqueous composition; such further elements include, for example, one or more of vitamins, trace elements, minerals, or a combination thereof. Exemplary additions include, but are not limited to, Wolfe's vitamin solutions, Wolfe's trace elements, trace element solution SL-7, trace element solution SL-10, etc. The concentration of such additional nutrients can be empirically optimized to the material to be treated and the conditions of the treatment (e.g., in situ vs. ex situ treatment conditions).

In an alternative embodiment, a particle-based method can be used to distribute compositions comprising stimulants, functional microbial subcommunities, DMAs, and/or microorganisms (collectively, the intervention agents) during a fracturing process. The goal is to introduce these interventions in order to produce an enhancement of methane production. A delivery system injects the agents deep into the well fissures and enables a time-released deployment. For example, the well intervention agent can be formulated as either a time-released coating over the sand grains used in the fracturing process or as hard particles which slowly dissolve with time; the size is envisioned as roughly the same as the sand grains used in the fracturing process, and could be mixed together before added to the guar gum solution known as the proppant. In either format, once the proppant and particles are pumped into the well and pressured, the coated sand grains or hard particles mixed with the sand are pressure-injected in the well fractures, keeping them open to facilitate gas or oil release. Since the intervention agents are formulated in a time-release manner not dissimilar to some pharmaceutical agents, the compounds and/or microbes would dissolve slowly and diffuse into the surrounding formation water and into the coal cleats (or fine rock cracks in the case of oil) where adhered bacteria presumably reside. In this fashion, the dissolving agents continuously stimulate the biogenic conversion of coal to methane. The formulations could be fashioned to release the intervention agent over a period of hours, days, weeks or months in order to optimize the methane stimulation process. The coatings or particles could be prepared in the absence of oxygen in order to maintain the viability of strict anaerobic microbes, or they could also harbor gases which stimulate methane production.

The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and embodiments will be apparent upon review of this disclosure. The following examples are offered to illustrate, but not limit, the disclosed embodiments.

EXAMPLES Example 1 Sampling and Enrichment of Methane-Producing Microorganisms from a Coalbed Methane Well (Upflow Reactor 2 L-245)

An inoculum containing a functional methane producing community enriched from formation water was collected from the San Juan Basin.

Formation water was collected from a coalbed methane well located in the San Juan Basin, Colo., USA. The water was then filtered with a series of sterile sieves from 1 mm to 45 μm to remove large pieces of coal and oils that came with the formation water. Subsamples were transferred into a 2 L plastic bottle and a 1 L sterile glass bottle. The glass bottle sample was sparged with N₂using a portable tank and a glass pipette and then sealed with a sterile butyl stopper. Both bottles were transferred to the laboratory in less than 12 hours and the 2 L volume was sterilized by filtration using a 0.2 micron sieve.

Sterile filtered formation water was used as the base for a growth medium. This base was supplemented with 10 ml/L each of trace metal and vitamin solutions and 200 μg sodium resazurin. A 1 L volume of this solution was sparged with N₂gas for 20 minutes, then transferred into an anoxic glove box and sterile-filtered through a 0.2 micron sieve. The resulting sterile solution was then dispensed in 5 ml volumes into Hungate tubes containing 0.5 g coal, the tubes were sealed with screw caps over butyl rubber septa and removed from the glove box.

An electron acceptor stock solution was made by combining 7 g of sodium sulfate and 1.7 g of sodium nitrate in a serum bottle and adding 50 ml of freshly boiled water. This solution was then immediately sparged for 15 minutes with N₂gas, capped with a butyl rubber stopper, sealed with an aluminum crimp seal and autoclaved at 121° C. for 20 minutes.

A yeast extract stock solution was made by combining 2.5 g yeast extract and 50 ml of freshly boiled water in a serum bottle. This solution was then immediately sparged for 15 minutes with N₂gas, capped with a butyl rubber stopper, sealed with an aluminum crimp seal and autoclaved at 121° C. for 20 minutes.

To create the primary enrichment culture, an anoxic Hungate tube with coal and base medium was supplemented with 0.05 ml of the electron acceptor and yeast extract stock solutions using N₂sparged syringes and needles and inoculated with 1 ml of anoxic formation water. The primary enrichment was incubated at 50° C., sampled occasionally for headspace gases and eventually found to produce 6.65% methane after 6 weeks. The enrichment was then transferred by adding 1 ml to an identical, previously uninoculated, Hungate tube using an N₂sparged syringe and needle. This tube, the secondary enrichment, was found to have 10.2% headspace methane after four weeks and transferred again in the same manner to create a tertiary enrichment.

The tertiary enrichment culture was maintained in an anaerobic reactor system as described by (Lettinga, G. 1995. Anaerobic digestion and wastewater treatment systems. Antonie van Leeuwenhoek 67:3-28). The reactor was a 2000-mL laboratory-scale glass reactor equipped with a heat jacket equilibrated to 50° C. The reactor was fitted with ports to take liquid and gas samples close to the reactor outlet. The effluent was recycled in a relationship of 4:1 (80% recirculation) relative to the inlet flow. Production of methane was monitored by GC-FID. Before start-up of the reactor system was filled with coal and autoclaved (30 minutes) and sparged with anaerobic gas (80% N₂/20% CO₂). The reactor was started DMY-media with a hydraulic retention time (HRT) of 48 h.

Example 2 Stimulation of Methane Production

The availability of an inoculum of a functional microbial subcommunity capable of producing methane from coal in vitro prompted laboratory experiments where various stimulants were tested for their effect on methane production.

Materials

Below is the detailed description of growth media composition, coal sample used and microbial inoculum for the batch experiments.

Inoculum 2L-245 (10% vol./vol.) Growth Medium DM Incubation Temp 50° C. Head-space atmosphere 20% CO₂/Bal. N₂ Coal Sterility Autoclave 30′ Liquid

Additives/Stimulants:

The experiments were conducted at three different levels of additives (0.5×, 1× and 2×): additives (stimulants) were added in the following three concentrations.

Concentration (μM) Additive/Stimulant High Medium Low KCl 8000 4000 2000 FeCl₃ 74 37 18.5 Na₂MoO₄ 10 5 2.5 MnCl₂ 10 5 2.5 VCl₃ 10 5 2.5 Na₂SO₄ 500 250 125 Na₂S₂O₃ 200 100 50 NH₄Cl 21 10 5 Na₂SO₃ 500 250 125

Media Preparations

DM media: the media composition in milli-Q water contains 1.2 g/L NaCl, 0.4 g/L MgCl₂×6 H₂O, 0.2 g/L CaCl₂×2 H₂O, 0.3 g/L NH₄Cl, 0.3 g/L KCl, 2.4 g/L NaHCO₃, 0.25 g/L Na₂S, and 0.2 g/L K₂HPO₄(Dibasic), 10 ml/L of Wolfe's trace elements and 10 ml/L of Wolfe's vitamins (as described below). After mixing, the solution is sparged with a N₂/CO₂mixture (80%/20%) to make the solution anaerobic.

DMY media: DM media composition in milli-Q water is made as described above with the addition of 0.5 g/L yeast extract.

DMSC media: DM media in milli-Q water is made as described above with the addition of 100 g/L sterile, anaerobic, sub-bituminous coal from the San Juan Basin.

DMSCY media: DM media in milli-Q water is made as described above with the addition of 100 g/L sterile, anaerobic, sub-bituminous coal from the San Juan Basin and 0.5 g/L yeast extract.

Trace Element and Vitamin Solutions (Concentration in MilliQ Water)

Wolfe's Trace elements (100X) EDTA 0.5 g/L MgSO₄•7H₂O 3 g/L MnSO₄•H₂O 0.5 g/L NaCl 1 g/L FeSO₄•7H₂O 0.1 g/L Co(NO₃)₂•6H₂O 0.1 g/L CaCl₂(anhydrous) 0.1 g/L ZnSO₄•7H₂O 0.1 g/L CuSO₄•5H₂O 0.01 g/L

Wolfe's Trace elements (100X) AlK(SO₄)₂(anhydrous) 0.01 g/L H₃BO₃ 0.01 g/L Na₂MoO₄•2H₂O 0.01 g/L Na₂SeO₃(anhydrous) 0.001 g/L Na₂WO₄•2H₂O 0.01 g/L NiCl₂•6H₂O 0.02 g/L A one liter solution is made: EDTA is added first to the milliQ water and each element is added individually thereafter. The solution is sparged with 80% N₂/20% CO₂and added to samples anaerobically.

Wolfe's vitamins (100X) Folic acid 2 mg/L Pyridoxine hydrochloride 10 mg/L Riboflavin 5 mg/L Biotin 2 mg/L Thiamine 5 mg/L Nicotinic acid 5 mg/L Calcium pantothenate 5 mg/L Vitamin B12 0.1 mg/L p-Aminobenzoic acid 5 mg/L Thioctic acid 5 mg/L Monopotassium phosphate 900 mg/L

Coal

The coal used in these experiments originated from the San Juan Basin (BHP Billiton, Tex.). The coal was stored, pulverized and dry-sieved under anaerobic conditions (>99.5% N₂). Coal used in the experiments described herein had a particle size of 150 μm to 250 μm.

Methods

Each reaction tube contained a 10% volume/volume sample from the upflow reactor described above in Example 1. Each stimulant was tested in the presence or absence of coal. The experiments were conducted at three different concentrations of additives (0.5× (low), 1× (medium), and 2× (high)) as described above. Each sample type was conducted in 4 replicates. Samples were incubated anaerobically for 5 weeks at 50° C. and headspace samples were taken at weeks 1, 3 and 5. Control sample types were media alone, media plus coal, media plus yeast extract and media plus coal and yeast extract.

At each time point, headspace measurements were taken using gas chromatography (microGC 3000A, Agilent Technologies, Inc.). Tubes were sampled for H₂, CO₂and CH₄at weeks 1, 3 and 5. Each experiment was performed in quadruplicate. Methane at time 0 was assumed to be 0%. Although methane production was not measured at time 0 in this experiment, multiple experiments have been run in prior experiments, and in each instance, methane production was 0%.

Results

FIGS. 1-5 show the results of stimulation of the culture system with three concentrations of various additives at week 5. FIGS. 6 and 7 show the results of stimulation of the functional microbial subcommunity.

Methane production from each additive experiment is provided in the Tables below.

TABLE 1 Control samples Week RepA RepB RepC Rep D Average DM 1 0 0 0 0 0.00 3 0 0 0 0 0.00 5 0.01 0.01 0.01 0.01 0.01 DMSC 1 0.02 0 0.02 0.02 0.01 3 0 0 0 0.08 0.02 5 0.08 0.053 0.25 2.98 0.84 DMY 1 0.19 0.22 0.20 0.23 0.21 3 0.69 0 0.64 0.75 0.52 5 0.71 0.67 0.72 0.77 0.72 DMSCY 1 0.28 0.28 0.26 0.28 0.27 3 0.73 1.01 1.02 1.07 0.96 5 1.26 1.30 1.23 1.31 1.28

TABLE 2 VCl₃ Week RepA RepB RepC Rep D Average DMY 1 0.22 0.20 0.21 0.21 0.21 2.5 μM VCl₃ 3 0.75 0.69 0.72 1.10 0.81 5 0.70 0.66 0.64 0.69 0.67 DMSCY 1 0.41 0.32 0.27 0.31 0.33 2.5 μM VCl₃ 3 1.70 1.45 1.12 1.18 1.36 5 2.93 2.40 1.37 1.50 2.05 DMY 1 0.21 0.20 0.20 0.20 0.20 5 μM VCl₃ 3 0.78 0.69 0.67 0.80 0.71 5 0.75 0.70 0.68 0.67 0.70 DMSCY 1 0.30 0.26 0.25 0.26 0.27 5 μM VCl₃ 3 1.51 1.40 1.21 1.88 1.50 5 2.41 2.28 1.95 3.13 2.44 DMY 1 0.21 0.25104 0.19 0.22 0.22 10 μM VCl₃ 3 0.85 0.749507 0.70 0.78 0.77 5 0.76 0.82 0.70 0.77 0.76 DMSCY 1 0.29 0.25 0.24 0.24 0.25 10 μM VCl₃ 3 1.15 1.04 1.04 1.01 1.06 5 1.49 1.31 1.30 1.33 1.36

TABLE 3 Na₂SO₄ Week RepA RepB RepC Rep D Average DMY 1 0.24 0.21 0.20 0.19 0.21 125 μM Na₂SO₄ 3 0 0.71 0.69 0.22 0.40 5 0.78 0.65 0.62 0.17 0.55 DMSCY 1 0.35 0.32 0.29 0.34 0.33 125 μM Na₂SO₄ 3 1.164 1.07 1.03 1.07 1.08 5 1.30 1.27 1.20 1.26 1.26 DMY 1 0.24 0.20 0.24 0.24 0.23 250 μM Na₂SO₄ 3 1.11 0.70 0.82 0.33 0.74 5 0.92 0.71 0.83 0.31 0.69 DMSCY 1 0.26 0.24 0.29 0.29 0.27 250 μM Na₂SO₄ 3 1.21 1.01 1.89 1.45 1.39 5 0.51 1.63 0.94 1.85 1.23 DMY 1 0.21 0.21 0.20 0.19 0.205 500 μM Na₂SO₄ 3 0.71 0.45 0.67 0.19 0.51 5 0.70 0.71 0.69 0.22 0.58 DMSCY 1 0.27 0.24 0.24 0.24 0.25 500 μM Na₂SO₄ 3 1.11 1.08 1.02 1.38 1.15 5 1.39 1.28 1.29 1.32 1.32

TABLE 4 Na₂S₂O₃ Week RepA RepB RepC Rep D Average DMY 1 0.24 0.24 0.22 0.23 0.23 50 μM Na₂S₂O₃ 3 0.75 0.66 0.73 0.74 0.72 5 0.75 0.68 0.68 0.72 0.70 DMSCY 1 0.30 0.29 0.29 0.27 0.29 50 μM Na₂S₂O₃ 3 1.16 1.02 1.04 1.09 1.08 5 1.47 1.32 1.36 1.34 1.37 DMY 1 0.21 0.19 0.21 0.23 0.21 100 μM Na₂S₂O₃ 3 0.70 0.69 0.34 0.74 0.62 5 0.71 0.76 0.75 0.77 0.75 DMSCY 1 0.24 0.22 0.25 0.26 0.24 100 μM Na₂S₂O₃ 3 1.32 1.18 1.28 1.01 1.20 5 1.81 1.58 1.87 1.30 1.64 DMY 1 0.18 0.18 0.17 0.17 0.18 200 μM Na₂S₂O₃ 3 0.66 0.63 0.68 0.63 0.65 5 0.70 0.66 0.63 0.64 0.66 DMSCY 1 0.20 0.17 0.17 0.18 0.18 200 μM Na₂S₂O₃ 3 1.18 0.85 0.82 0.93 0.95 5 1.14 1.13 1.11 1.27 1.16

TABLE 5 NH₄Cl Week RepA RepB RepC Rep D Average DMY 1 0.26 0.27 0.27 0.27 0.26 5 mM NH₄Cl 3 0.74 0.68 0.67 0.74 0.71 5 0.69 0.65 0.62 0.69 0.66 DMSCY 1 0.35 0.27 0.31 0.30 0.31 5 mM NH₄Cl 3 1.24 1.03 1.13 1.03 1.11 5 1.67 1.41 1.48 1.43 1.50 DMY 1 0.22 0.21 0.15 0.22 0.20 10 mM NH₄Cl 3 0.73 0.61 0.21 0.73 0.57 5 0.76 0.70 0.17 0.70 0.58 DMSCY 1 0.25 0.25 0.22 0.28 0.25 10 mM NH₄Cl 3 1.22 1.43 1.41 1.14 1.30 5 1.98 2.70 2.67 1.54 2.22 DMY 1 0.23 0.22 0.23 0.21 0.22 20 mM NH₄Cl 3 0.80 0.78 0.76 0.76 0.77 5 0.80 0.79 0.76 0.77 0.78 DMSCY 1 0.24 0.22 0.21 0.22 0.22 20 mM NH₄Cl 3 1.30 20 1.08 1.15 1.18 5 1.63 1.48 1.37 1.36 1.46

TABLE 6 Na₂SO₃ Week RepA RepB RepC Rep D Average DMY 1 0.20 0.20 0.21 0.22 0.21 125 μM Na₂SO₃ 3 0.71 0.68 0.69 0.75 0.71 5 0.66 0.69 0.66 0.71 0.68 DMSCY 1 0.26 0.24 0.25 0.32 0.27 125 μM Na₂SO₃ 3 1.04 0.97 0.99 1.06 1.02 5 1.21 1.22 1.25 1.31 1.25 DMY 1 0.14 0.15 0.13 0.15 0.14 250 μM Na₂SO₃ 3 0.67 0.63 0.65 0.72 0.67 5 0.62 0.59 0.62 0.65 0.62 DMSCY 1 0.23 0.18 0.19 0.20 20 250 μM Na₂SO₃ 3 1.04 1.02 1.06 1.18 1.07 5 1.26 1.24 1.45 1.74 1.42 DMY 1 0.09 0.08 0.08 0.10 0.09 500 μM Na₂SO₃ 3 0.71 0.64 0.64 0.74 0.68 5 0.83 0.68 0.68 0.76 0.74 DMSCY 1 0.14 0.13 0.14 0.15 0.14 500 μM Na₂SO₃ 3 1.01 0.98 1.07 1.04 1.04 5 1.34 1.26 1.37 1.41 1.35

TABLE 7 Control samples Week RepA RepB RepC Rep D Average DM only 1 0.00 0.00 0.00 0.00 0.00 3 0.00 0.00 0.00 0.00 0.00 5 0.00 0.00 0.00 0.00 0.00 DMSC 1 0.02 0.03 19.05 0.04 4.78 3 0.00 0.00 0.00 0.00 0.00 5 0.17 0.25 0.056 0.11 0.15 DMY 1 0.41 0.37 0.35 0.42 0.38 3 1.13 1.00 0.99 1.09 1.05 5 1.11 0.98 0.98 6 1.03 DMSCY 1 0.53 0.41 0.38 0.49 0.45 3 3.82 1.37 1.30 1.47 1.99 5 4.70 1.71 1.47 1.65 2.38

TABLE 8 MnCl₂ Week RepA RepB RepC Rep D Average DMY 1 0.40 0.32 0.35 0.36 0.36 2.5 μM MnCl₂ 3 0.96 0.96 0.93 0.94 0.95 5 0.94 0.87 0.91 0.91 0.91 DMSCY 1 0.36 0.46 0.41 0.42 0.41 2.5 μM MnCl₂ 3 1.40 1.70 1.41 1.50 1.50 5 1.59 1.90 1.59 1.73 1.70 DMY 1 0.34 0.33 0.34 0.36 0.34 5.0 μM MnCl₂ 3 0.92 0.90 0.90 0.94 0.92 5 0.89 0.86 0.85 0.88 0.87 DMSCY 1 0.24 0.51 0.49 0.52 0.44 5.0 μM MnCl₂ 3 1.51 1.55 1.53 1.64 1.56 5 1.94 1.81 1.70 1.85 1.82 DMY 1 0.39 0.38 0.35 0.39 0.36 10 μM MnCl₂ 3 1.00 1.05 1.04 1.01 1.02 5 0.89 1.03 0.96 1.01 0.97 DMSCY 1 0.44 0.41 0.41 0.43 0.42 10 μM MnCl₂ 3 1.45 1.49 1.43 1.48 1.46 5 1.63 1.82 1.71 1.70 1.72

TABLE 9 Na₂MoO₄ Week RepA RepB RepC Rep D Average DMY 1 0.38 0.36 0.38 0.35 0.37 2.5 μM Na₂MoO₄ 3 1.00 0.98 0.96 0.82 0.94 5 1.01 0.95 0.97 0.82 0.94 DMSCY 1 0.43 0.37 0.36 0.44 0.40 2.5 μM Na₂MoO₄ 3 1.43 1.10 1.14 1.41 1.27 5 1.61 1.32 1.32 1.57 1.45 DMY 1 0.25 0.27 0.30 0.33 0.29 5.0 μM Na₂MoO₄ 3 0.77 0.85 0.79 0.91 0.83 5 0.76 0.78 0.74 0.87 0.79 DMSCY 1 0.39 0.39 0.28 0.31 0.34 5.0 μM Na₂MoO₄ 3 1.13 1.09 1.14 1.40 1.19 5 1.39 1.31 1.29 1.63 1.41 DMY 1 0.30 0.26 0.30 0.32 0.29 10 μM Na₂MoO₄ 3 0.85 0.71 0.81 0.90 0.82 5 0.77 0.72 0.74 0.83 0.77 DMSCY 1 0.43 0.43 0.47 0.44 0.44 10 μM Na₂MoO₄ 3 1.03 1.15 1.14 1.07 1.10 5 1.11 1.14 1.15 1.13 1.13

TABLE 10 KCl Week RepA RepB RepC Rep D Average DMY 1 0.29 0.30 0.32 0.29 0.30 2.0 mM KCl 3 0.99 0.79 0.78 0.70 0.81 5 0.78 0.75 0.79 0.75 0.77 DMSCY 1 0.35 0.30 0.32 0.39 0.34 2.0 mM KCl 3 1.12 1.25 1.04 1.16 1.14 5 1.32 1.49 1.30 1.47 1.39 DMY 1 0.23 0.23 0.22 0.24 0.23 4.0 mM KCl 3 0.24 0.26 0.26 0.29 0.26 5 0.23 0.26 0.26 0.40 0.29 DMSCY 1 0.44 0.43 0.43 0.46 0.4 4.0 mM KCl 3 1.35 1.46 1.60 1.57 1.50 5 1.59 1.59 1.87 1.70 1.69 DMY 1 0.200534 0.203707 0.194967 0.206776 0.201496 8.0 mM KCl 3 0.221062 0.211906 0.220571 0.217698 0.217809 5 0.217698 0.208193 0.223103 0.211229 0.215056 DMSCY 1 0.387142 0.336361 0.421862 0.429047 0.393603 8.0 mM KCl 3 1.345807 1.300728 1.359635 1.303668 1.327459 5 1.564226 1.477874 1.556977 1.648504 1.561895

TABLE 11 FeCl₃ Week RepA RepB RepC Rep D Average DMY 1 0.42 0.43 0.38 0.42 0.41 18.5 μM FeCl₃ 3 1.01 1.03 0.94 1.03 1.00 5 0.96 1.04 0.93 1.05 0.99 DMSCY 1 0.40 0.41 0.46 0.42 0.42 18.5 μM FeCl₃ 3 1.47 1.38 1.40 1.28 1.38 5 1.69 1.61 1.57 1.47 1.59 DMY 1 0.33 0.31 0.30 0.29 0.31 37.0 μM FeCl₃ 3 0.88 0.89 0.86 0.82 0.86 5 0.83 0.86 0.79 0.75 0.81 DMSCY 1 0.47 0.35 0.35 0.43 0.40 37.0 μM FeCl₃ 3 1.20 1.33 1.14 1.47 1.28 5 1.612 1.44 1.26 1.53 1.46 DMY 1 0.43 0.37 0.36 0.29 0.36 74 μM FeCl₃ 3 0.84 0.89 0.85 1.01 0.90 5 0.80 0.83 0.81 0.94 0.85 DMSCY 1 0.44 0.06 0.42 0.53 0.36 74 μM FeCl₃ 3 1.31 0.05 1.30 1.94 1.15 5 1.51 0.06 1.48 2.26 1.33

VCl₃: A statistically significant positive effect on the production of methane was observed at the intermediate concentration of VCl₃(P=0.0084 at 5 μM VCl₃, P=0.0771 at 2.5 μM VCl₃); methane production was only observed in the presence of coal (FIG. 1A).

Sodium thiosulfate: A statistically significant positive effect on the production of methane was observed at the intermediate concentration of sodium thiosulfate; P=0.079); methane production was only observed in the presence of coal. (FIG. 1B).

NH₄Cl: A statistically significant positive effect on the production of methane was observed at the intermediate concentration of NH₄Cl (P=0.0007); methane production was only observed in presence of coal (FIG. 2A).

Molybdate (Na₂MoO₄): There was a negative correlation in methane production when molybdate was added to the cultures (R²=0.69). Effects with or without coal were not statistically significantly different (P=0.063) (FIG. 3B).

KCl: A decreasing trend in methane production was observed at week 5 in KCL-containing samples. KCl in the absence of coal (DMY) greatly decreased methanogenesis at >2 mM (FIG. 4A).

Overall, with the exception of KCL, trends with increasing or decreasing concentrations of methane production were not observed (see, e.g., FIGS. 2, 3, 4B and 5).

Thus, vanadium and thiosulfate compositions were observed to increase methane production using the present methods.

Example 3 Stimulation of Methane Production by Vanadium in Cultures Utilizing Formate

Formate is a likely product of coal matrix degradation and acts a substrate for methanogenic microbes.

The anaerobic reactor system described above in Example 1 contains formate-utilizing, methane-producing species in the enrichment culture. Methanogens in the culture were previously identified by 16sRNA sequencing.

A 10% vol./vol. inoculum was taken from the anaerobic reactor system described above in Example 1, and the effect of vanadium on methane production by methanogens utilizing formate as a carbon and energy source was determined.

Cultures, established with DM media (prepared as described above) containing 50 mM formate as the sole carbon and energy source, were sub-cultured with the same media (in the absence of vanadium) or media supplemented with either 5 or 10 μM VCl₃. Methane production was measured after 48 hours of growth at 50° C.

Replicate Cultures Methane (% (μM VCl₃added in final concentration) in head space volume of 21 ml) none 0.7 none 0.5 5 2.0 5 1.2 10 1.9 10 1.4

The VCl₃-supplemented cultures showed an average of approximately 2-fold greater methane formation than cultures without VCl₃. The increase in methane production was observed when VCl₃was added to the cultures whereby the vanadium stimulated methanogens could utilize formate as the carbon and energy source, thereby inducing methane production. The data are consistent with replacement of molybdenum or tungsten with vanadium as a co-factor for key enzymes (e.g., formate dehydrogenase and/or formyl-MF dehydrogenase) by formate-utilizing methane-producing species. The results are also consistent with the reported metal content of San Juan basin coal showing several fold more vanadium vs. molybdenum and tungsten available for evolution of vanadium-dependent enzymes. These results are also consistent with the observed decrease in methane production by 2 L-245 with increasing concentrations of molybdate, a suspected antagonist of vanadium-dependent enzymes (see FIG. 3B).

Example 4 Stimulation of Methane Production by Vanadium in Cultures Utilizing Acetate

Acetate is found in coal formation water, is a likely product of coal matrix degradation and acts a substrate for methanogenic microbes.

The anaerobic reactor system described above in Example 1 contains acetate-utilizing, methane-producing species in the enrichment culture. Methanogens in the culture were previously identified by 16sRNA sequencing.

A 10% vol./vol. inoculum is taken from the anaerobic reactor system described above in Example 1, and the effect of vanadium on methane production by methanogens utilizing acetate as a carbon and energy source is determined.

Cultures, established with DM media (prepared as described above) containing varying concentrations of acetate as the sole carbon and energy source, are sub-cultured with the same media (in the absence of vanadium) or media supplemented with either 5 or 10 μM VCl₃. Methane production is measured after 48 hours of growth at 50° C.

Example 5 Stimulation of Methane Production by Vanadium in Cultures using Various Other Substrates

Hydrogen, butyrate, propionate and CO₂are found in coal formation water, are likely products of coal matrix degradation and can act a substrate for methanogenic microbes. Hydrogen, butyrate, propionate and CO₂are found in coal formation water, are likely products of coal matrix degradation and can act a substrate for methanogenic microbes.

The anaerobic reactor system described above in Example 1 contains methane-producing species in the enrichment culture. Methanogens in the culture were previously identified by 16sRNA sequencing.

A 10% vol./vol. inoculum is taken from the anaerobic reactor system described above in Example 1, and the effect of vanadium on methane production by methanogens utilizing hydrogen, butyrate, propionate and/or CO₂as a carbon and energy source is determined.

Cultures, established with DM media (prepared as described above) containing varying concentrations of hydrogen, butyrate, propionate or CO₂as the sole carbon and energy source, are sub-cultured with the same media (in the absence of vanadium) or media supplemented with either 5 or 10 μM VCl₃. Methane production is measured after 48 hours of growth at 50° C.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that elements of the embodiments described herein can be combined to make additional embodiments and various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and equivalents are within the scope of the invention as described and claimed herein.

Headings within the application are solely for the convenience of the reader, and do not limit in any way the scope of the invention or its embodiments.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method of stimulating production of a metabolic product with enhanced hydrogen content from a hydrocarbon material, comprising contacting said hydrocarbon material with a composition comprising sulfur, an oxyanion of sulfur or vanadium.

2. The method of claim 1, wherein said metabolic product with enhanced hydrogen content is methane.

3. The method of claim 1, wherein said hydrocarbon material comprises coal, oil, kerogen, peat, heavy oil, oil shale, tar sands, oil sands, bitumen or tar.

4. The method of claim 1, wherein said oxyanion of sulfur is thiosulfate, sulfuric acid, disulfuric acid, peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid, thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid or polythionic acid.

5. The method of claim 1, wherein said oxyanion of sulfur is thiosulfate.

6. The method of claim 1, wherein said vanadium is vanadium (III) chloride (VCl3), vanadium (II) chloride (VCl2), or vanadium (I) chloride (VCl).

7. A method of stimulating production of methane from a hydrocarbon material, comprising contacting said hydrocarbon material with a composition comprising sulfur, an oxyanion of sulfur or vanadium.

8. The method of claim 7, wherein said hydrocarbon material comprises coal, oil, kerogen, peat, heavy oil, oil shale, tar sands, oil sands, bitumen or tar.

9. The method of claim 7, wherein said oxyanion of sulfur is thiosulfate, sulfuric acid, disulfuric acid, peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid, thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid or polythionic acid.

10. The method of claim 7, wherein said oxyanion of sulfur is thiosulfate.

11. The method of claim 7, wherein said vanadium is vanadium (III) chloride (VCl3), vanadium (II) chloride (VCl2), or vanadium (I) chloride (VCl).

12. The method of claim 7, wherein contacting comprises injection of an aqueous composition comprising sulfur, an oxyanion of sulfur or vanadium into a subterranean formation containing said hydrocarbon material.

13. The method of claim 7, further comprising contacting said hydrocarbon material with a composition comprising one or more microorganisms, wherein at least one of said microorganisms is a methanogen.

14. The method of claim 13, wherein the one or more microorganisms comprise at least one species that is not indigenous to the hydrocarbon material.

15. The method of claim 7, wherein said sulfur, oxyanion of sulfur, or vanadium stimulates microorganisms to metabolize carbonaceous material into methane.

16. A method of stimulating methane production from coal, comprising contacting said coal with a composition comprising sulfur, an oxyanion of sulfur, or vanadium.

17. The method of claim 16, wherein contacting comprises injection of an aqueous composition comprising sulfur, an oxyanion of sulfur or vanadium into a subterranean coal formation.

18. The method of claim 16, further comprising contacting the coal with a composition comprising one or more microorganisms, wherein at least one of same microorganisms is a methanogen.

19. The method of claim 18, wherein the one or more microorganisms comprise at least one species that is not indigenous to the coal formation where the coal is present or extracted from.

20. The method of claim 16, wherein said sulfur, oxyanion of sulfur or vanadium stimulates microorganisms to metabolize carbonaceous material into methane.

21. The method of claim 16, wherein said oxyanion of sulfur is thiosulfate, sulfuric acid, disulfuric acid, peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid, thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid or polythionic acid.

22. The method of claim 16, wherein said oxyanion of sulfur is thiosulfate.

23. The method of claim 16, wherein said vanadium is vanadium (III) chloride (VCl3), vanadium (II) chloride (VCl2), or vanadium (I) chloride (VCl).