U.S. patent application number 13/153120 was filed with the patent office on 2012-06-07 for methods to stimulate biogenic methane production from hydrocarbon-bearing formations.
This patent application is currently assigned to SYNTHETIC GENOMICS, INC.. Invention is credited to Brian G. Clement, James G. Ferry, Stuart Underwood.
Application Number | 20120138290 13/153120 |
Document ID | / |
Family ID | 45067319 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120138290 |
Kind Code |
A1 |
Clement; Brian G. ; et
al. |
June 7, 2012 |
METHODS TO STIMULATE BIOGENIC METHANE PRODUCTION FROM
HYDROCARBON-BEARING FORMATIONS
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.
Inventors: |
Clement; Brian G.; (Houston,
TX) ; Ferry; James G.; (State College, PA) ;
Underwood; Stuart; (Roanoke, VA) |
Assignee: |
SYNTHETIC GENOMICS, INC.
La Jolla
CA
|
Family ID: |
45067319 |
Appl. No.: |
13/153120 |
Filed: |
June 3, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61351709 |
Jun 4, 2010 |
|
|
|
Current U.S.
Class: |
166/246 ;
166/279 |
Current CPC
Class: |
C12N 1/20 20130101; Y02E
50/343 20130101; Y02E 50/30 20130101; C09K 8/582 20130101; C09K
8/70 20130101; C12N 1/26 20130101; C12P 5/023 20130101 |
Class at
Publication: |
166/246 ;
166/279 |
International
Class: |
E21B 43/22 20060101
E21B043/22 |
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 (VCl.sub.3), vanadium (II) chloride (VCl.sub.2), 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 (VCl.sub.3), vanadium (II) chloride (VCl.sub.2), 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 (VCl.sub.3), vanadium (II) chloride (VCl.sub.2), or
vanadium (I) chloride (VCl).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] 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.
[0002] 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
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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
CO.sub.2 (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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] In various embodiments, the stimulant involved in the
conversion of hydrocarbon to methane is yeast extract, sulfur, an
oxyanion of sulfur (e.g., thiosulfate (S.sub.2O.sub.3), sodium
thiosulfate (Na.sub.2S.sub.2O.sub.3), potassium thiosulfate
(K.sub.2S.sub.2O.sub.3), sulfuric acid, disulfuric acid,
peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid,
thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid
or polythionic acid), NH.sub.4Cl, KCl, vanadium, VCl.sub.3,
VCl.sub.2, VCl, Na.sub.2SO.sub.3, MnCl.sub.2, Na.sub.2MoO.sub.4,
FeCl.sub.3 or Na.sub.2SO.sub.4. In a preferred embodiment, the
stimulant is vanadium, VCl.sub.3, VCl.sub.2, VCl, sulfur,
thiosulfate or sodium thiosulfate.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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
[0020] 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:
[0021] FIGS. 1A and 1B illustrate methane production from coal
after stimulation of a functional microbial subcommunity with a
composition comprising vanadium (III) chloride (VCl.sub.3) and a
composition comprising sodium thiosulfate (NaS.sub.2O.sub.3),
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.
[0022] FIGS. 2A and 2B illustrate methane production from coal
after stimulation of a functional microbial subcommunity with a
composition comprising ammonium chloride (NH.sub.4Cl) and a
composition comprising sodium sulfite (Na.sub.2SO.sub.3),
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.
[0023] FIGS. 3A and 3B illustrate methane production from coal
after stimulation of a functional microbial subcommunity with a
composition comprising manganese chloride (MnCl.sub.2) and a
composition comprising sodium molybdic (Na.sub.2MoO.sub.4),
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.
[0024] 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 (FeCl.sub.3), 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.
[0025] FIG. 5 illustrates methane production from coal after
stimulation of a functional microbial subcommunity with a
composition comprising sodium sulfate (Na.sub.2SO.sub.4). 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.
[0026] 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 VCl.sub.3.
[0027] 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
NaS.sub.2O.sub.3.
[0028] FIG. 8 illustrates a variety of potential enzymatic pathways
in the conversion of coal to methane.
[0029] 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
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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
CO.sub.2 to CH.sub.4 by adding one hydrogen atom at a time
(CO.sub.2-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).
[0051] 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).
[0052] 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.
[0053] 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
[0054] 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.
[0055] 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.
[0056] 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
CO.sub.2 reducing and acetoclastic), and any combinations thereof
to achieve conversion of a hydrocarbon to methane.
[0057] Examples of stimulants include, for example, yeast extract,
sulfur, an oxyanion of sulfur (e.g., thiosulfate (S.sub.2O.sub.3),
sodium thiosulfate (Na.sub.2S.sub.2O.sub.3), potassium thiosulfate
(K.sub.2S.sub.2O.sub.3), sulfuric acid, disulfuric acid,
peroxymonosulfuric acid, peroxydisulfuric acid, dithionic acid,
thiosulfuric acid, disulfurous acid, sulfurous acid, dithionus acid
or polythionic acid), NH.sub.4Cl, KCl, vanadium, VCl.sub.3,
VCl.sub.2, VCl, Na.sub.2SO.sub.3, MnCl.sub.2, Na.sub.2MoO.sub.4,
FeCl.sub.3 or Na.sub.2SO.sub.4. In a preferred embodiment, the
stimulant is vanadium or thiosulfate.
Incorporation of Stimulants to Increase Methane Production
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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 10.sup.7 cells 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, milli.OMEGA. 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).
[0064] 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.
[0065] 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)
[0066] An inoculum containing a functional methane producing
community enriched from formation water was collected from the San
Juan Basin.
[0067] 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 .mu.m 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
N.sub.2 using 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.
[0068] 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 .mu.g sodium resazurin. A
1 L volume of this solution was sparged with N.sub.2 gas 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.
[0069] 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 N.sub.2 gas, capped with a
butyl rubber stopper, sealed with an aluminum crimp seal and
autoclaved at 121.degree. C. for 20 minutes.
[0070] 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
N.sub.2 gas, capped with a butyl rubber stopper, sealed with an
aluminum crimp seal and autoclaved at 121.degree. C. for 20
minutes.
[0071] 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 N.sub.2
sparged syringes and needles and inoculated with 1 ml of anoxic
formation water. The primary enrichment was incubated at 50.degree.
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 N.sub.2 sparged 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.
[0072] 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.degree. 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% N.sub.2/20%
CO.sub.2). The reactor was started DMY-media with a hydraulic
retention time (HRT) of 48 h.
Example 2
Stimulation of Methane Production
[0073] 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
[0074] Below is the detailed description of growth media
composition, coal sample used and microbial inoculum for the batch
experiments.
TABLE-US-00001 Inoculum 2L-245 (10% vol./vol.) Growth Medium DM
Incubation Temp 50.degree. C. Head-space atmosphere 20%
CO.sub.2/Bal. N.sub.2 Coal Sterility Autoclave 30' Liquid
Additives/Stimulants:
[0075] The experiments were conducted at three different levels of
additives (0.5.times., 1.times. and 2.times.): additives
(stimulants) were added in the following three concentrations.
TABLE-US-00002 Concentration (.mu.M) Additive/Stimulant High Medium
Low KCl 8000 4000 2000 FeCl.sub.3 74 37 18.5 Na.sub.2MoO.sub.4 10 5
2.5 MnCl.sub.2 10 5 2.5 VCl.sub.3 10 5 2.5 Na.sub.2SO.sub.4 500 250
125 Na.sub.2S.sub.2O.sub.3 200 100 50 NH.sub.4Cl 21 10 5
Na.sub.2SO.sub.3 500 250 125
Media Preparations
[0076] DM media: the media composition in milli-Q water contains
1.2 g/L NaCl, 0.4 g/L MgCl.sub.2.times.6 H.sub.2O, 0.2 g/L
CaCl.sub.2.times.2 H.sub.2O, 0.3 g/L NH.sub.4Cl, 0.3 g/L KCl, 2.4
g/L NaHCO.sub.3, 0.25 g/L Na.sub.2S, and 0.2 g/L K.sub.2HPO.sub.4
(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 N.sub.2/CO.sub.2 mixture (80%/20%) to make the
solution anaerobic.
[0077] DMY media: DM media composition in milli-Q water is made as
described above with the addition of 0.5 g/L yeast extract.
[0078] 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.
[0079] 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)
TABLE-US-00003 [0080] Wolfe's Trace elements (100X) EDTA 0.5 g/L
MgSO.sub.4.cndot.7H.sub.2O 3 g/L MnSO.sub.4.cndot.H.sub.2O 0.5 g/L
NaCl 1 g/L FeSO.sub.4.cndot.7H.sub.2O 0.1 g/L
Co(NO.sub.3).sub.2.cndot.6H.sub.2O 0.1 g/L CaCl.sub.2(anhydrous)
0.1 g/L ZnSO.sub.4.cndot.7H.sub.2O 0.1 g/L
CuSO.sub.4.cndot.5H.sub.2O 0.01 g/L
TABLE-US-00004 Wolfe's Trace elements (100X)
AlK(SO.sub.4).sub.2(anhydrous) 0.01 g/L H.sub.3BO.sub.3 0.01 g/L
Na.sub.2MoO.sub.4.cndot.2H.sub.2O 0.01 g/L
Na.sub.2SeO.sub.3(anhydrous) 0.001 g/L
Na.sub.2WO.sub.4.cndot.2H.sub.2O 0.01 g/L
NiCl.sub.2.cndot.6H.sub.2O 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%
N.sub.2/20% CO.sub.2 and added to samples anaerobically.
TABLE-US-00005 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
[0081] 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% N.sub.2). Coal
used in the experiments described herein had a particle size of 150
.mu.m to 250 .mu.m.
Methods
[0082] 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.times. (low), 1.times. (medium), and 2.times. (high)) as
described above. Each sample type was conducted in 4 replicates.
Samples were incubated anaerobically for 5 weeks at 50.degree. 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.
[0083] At each time point, headspace measurements were taken using
gas chromatography (microGC 3000A, Agilent Technologies, Inc.).
Tubes were sampled for H.sub.2, CO.sub.2 and CH.sub.4 at 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
[0084] 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.
[0085] Methane production from each additive experiment is provided
in the Tables below.
TABLE-US-00006 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-US-00007 TABLE 2 VCl.sub.3 Week RepA RepB RepC Rep D Average
DMY 1 0.22 0.20 0.21 0.21 0.21 2.5 .mu.M VCl.sub.3 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 .mu.M VCl.sub.3 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 .mu.M VCl.sub.3 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 .mu.M VCl.sub.3 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 .mu.M
VCl.sub.3 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 .mu.M VCl.sub.3 3 1.15 1.04
1.04 1.01 1.06 5 1.49 1.31 1.30 1.33 1.36
TABLE-US-00008 TABLE 3 Na.sub.2SO.sub.4 Week RepA RepB RepC Rep D
Average DMY 1 0.24 0.21 0.20 0.19 0.21 125 .mu.M Na.sub.2SO.sub.4 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 .mu.M Na.sub.2SO.sub.4 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
.mu.M Na.sub.2SO.sub.4 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 .mu.M
Na.sub.2SO.sub.4 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 .mu.M Na.sub.2SO.sub.4 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 .mu.M Na.sub.2SO.sub.4 3 1.11 1.08 1.02
1.38 1.15 5 1.39 1.28 1.29 1.32 1.32
TABLE-US-00009 TABLE 4 Na.sub.2S.sub.2O.sub.3 Week RepA RepB RepC
Rep D Average DMY 1 0.24 0.24 0.22 0.23 0.23 50 .mu.M
Na.sub.2S.sub.2O.sub.3 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 .mu.M
Na.sub.2S.sub.2O.sub.3 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 .mu.M
Na.sub.2S.sub.2O.sub.3 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 .mu.M
Na.sub.2S.sub.2O.sub.3 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 .mu.M
Na.sub.2S.sub.2O.sub.3 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 .mu.M
Na.sub.2S.sub.2O.sub.3 3 1.18 0.85 0.82 0.93 0.95 5 1.14 1.13 1.11
1.27 1.16
TABLE-US-00010 TABLE 5 NH.sub.4Cl Week RepA RepB RepC Rep D Average
DMY 1 0.26 0.27 0.27 0.27 0.26 5 mM NH.sub.4Cl 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 NH.sub.4Cl 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 NH.sub.4Cl 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 NH.sub.4Cl 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 NH.sub.4Cl
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 NH.sub.4Cl 3 1.30 20 1.08 1.15 1.18 5
1.63 1.48 1.37 1.36 1.46
TABLE-US-00011 TABLE 6 Na.sub.2SO.sub.3 Week RepA RepB RepC Rep D
Average DMY 1 0.20 0.20 0.21 0.22 0.21 125 .mu.M Na.sub.2SO.sub.3 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 .mu.M Na.sub.2SO.sub.3 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 .mu.M Na.sub.2SO.sub.3 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 .mu.M
Na.sub.2SO.sub.3 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 .mu.M Na.sub.2SO.sub.3 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 .mu.M Na.sub.2SO.sub.3 3 1.01 0.98 1.07
1.04 1.04 5 1.34 1.26 1.37 1.41 1.35
TABLE-US-00012 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-US-00013 TABLE 8 MnCl.sub.2 Week RepA RepB RepC Rep D Average
DMY 1 0.40 0.32 0.35 0.36 0.36 2.5 .mu.M MnCl.sub.2 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 .mu.M MnCl.sub.2 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 .mu.M
MnCl.sub.2 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 .mu.M MnCl.sub.2 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 .mu.M MnCl.sub.2 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 .mu.M MnCl.sub.2
3 1.45 1.49 1.43 1.48 1.46 5 1.63 1.82 1.71 1.70 1.72
TABLE-US-00014 TABLE 9 Na.sub.2MoO.sub.4 Week RepA RepB RepC Rep D
Average DMY 1 0.38 0.36 0.38 0.35 0.37 2.5 .mu.M Na.sub.2MoO.sub.4
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 .mu.M Na.sub.2MoO.sub.4 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 .mu.M Na.sub.2MoO.sub.4 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 .mu.M
Na.sub.2MoO.sub.4 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 .mu.M Na.sub.2MoO.sub.4 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 .mu.M Na.sub.2MoO.sub.4 3 1.03 1.15 1.14
1.07 1.10 5 1.11 1.14 1.15 1.13 1.13
TABLE-US-00015 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-US-00016 TABLE 11 FeCl.sub.3 Week RepA RepB RepC Rep D
Average DMY 1 0.42 0.43 0.38 0.42 0.41 18.5 .mu.M FeCl.sub.3 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 .mu.M FeCl.sub.3 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 .mu.M
FeCl.sub.3 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 .mu.M FeCl.sub.3 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 .mu.M FeCl.sub.3 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 .mu.M
FeCl.sub.3 3 1.31 0.05 1.30 1.94 1.15 5 1.51 0.06 1.48 2.26
1.33
[0086] VCl.sub.3: A statistically significant positive effect on
the production of methane was observed at the intermediate
concentration of VCl.sub.3 (P=0.0084 at 5 .mu.M VCl.sub.3, P=0.0771
at 2.5 .mu.M VCl.sub.3); methane production was only observed in
the presence of coal (FIG. 1A).
[0087] 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).
[0088] NH.sub.4Cl: A statistically significant positive effect on
the production of methane was observed at the intermediate
concentration of NH.sub.4Cl (P=0.0007); methane production was only
observed in presence of coal (FIG. 2A).
[0089] Molybdate (Na.sub.2MoO.sub.4): There was a negative
correlation in methane production when molybdate was added to the
cultures (R.sup.2=0.69). Effects with or without coal were not
statistically significantly different (P=0.063) (FIG. 3B).
[0090] 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).
[0091] 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).
[0092] 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
[0093] Formate is a likely product of coal matrix degradation and
acts a substrate for methanogenic microbes.
[0094] 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.
[0095] 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.
[0096] 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 .mu.M
VCl.sub.3. Methane production was measured after 48 hours of growth
at 50.degree. C.
TABLE-US-00017 Replicate Cultures Methane (% (.mu.M VCl.sub.3 added
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
[0097] The VCl.sub.3-supplemented cultures showed an average of
approximately 2-fold greater methane formation than cultures
without VCl.sub.3. The increase in methane production was observed
when VCl.sub.3 was 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
[0098] Acetate is found in coal formation water, is a likely
product of coal matrix degradation and acts a substrate for
methanogenic microbes.
[0099] 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.
[0100] 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.
[0101] 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
.mu.M VCl.sub.3. Methane production is measured after 48 hours of
growth at 50.degree. C.
Example 5
Stimulation of Methane Production by Vanadium in Cultures using
Various Other Substrates
[0102] Hydrogen, butyrate, propionate and CO.sub.2 are found in
coal formation water, are likely products of coal matrix
degradation and can act a substrate for methanogenic microbes.
Hydrogen, butyrate, propionate and CO.sub.2 are found in coal
formation water, are likely products of coal matrix degradation and
can act a substrate for methanogenic microbes.
[0103] 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.
[0104] 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 CO.sub.2 as a carbon and energy source is
determined.
[0105] Cultures, established with DM media (prepared as described
above) containing varying concentrations of hydrogen, butyrate,
propionate or CO.sub.2 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 .mu.M VCl.sub.3. Methane
production is measured after 48 hours of growth at 50.degree.
C.
[0106] 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.
[0107] 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.
[0108] 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.
* * * * *