Note: Descriptions are shown in the official language in which they were submitted.
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INCREASED MICROBIAL PRODUCTION OF METHANE GAS FROM
SUBSURFACE HYDROCARBON CONTAINING FORMATIONS
[0001] This
application claims priority to U.S. Provisional Patent Application
Serial No. 60/800,857, filed May 17, 2006 and U.S. Provisional Patent
Application
Serial No. 60/808,110, filed May 25, 2006,
FIELD
[0002] The present
invention relates to increased microbial release and
production of methane gas from subsurface hydrocarbon formations.
BACKGROUND
[0003] Methane gas
is used as an energy source throughout the world.
Compared to other conventional hydrocarbon fuels, methane gas is clean burning
and results in low levels of carbon dioxide and toxin emissions. Currently, a
majority of methane gas is obtained from conventional methane gas reservoirs.
In
recent years, however., these reservoirs have become increasingly depleted.
There is therefore a need to obtain methane gas from other sources, such as
hydrocarbon containing formations and coal containing formations.
[0004] Furthermore,
it is projected that the demand for methane gas will
continue to increase in the future as the world's fuel needs increase and as
the
demand for clean burning fuels and for domestic fuels increase. There Is
therefore a need for new methods to increase the production of methane gas
from
hydrocarbon containing formations.
[0005] In addition
to the need to increase production, there is also a need
for new methods which are cost effective and which do not adversely effect the
environment. Conventional
methods for producing methane gas from
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hydrocarbon formations can involve the release into the environment of
substantial amounts of water and can also affect the status of underground
aquifers. Accordingly, there is a need for methods for obtaining methane gas
from
formations that have already been commercially exploited and are now
abandoned. Obtaining methane gas from such sources provides a commercial
benefit because the value of these abandoned formations has already been
depreciated. Obtaining methane from such sources provides an environmental
benefit because many of the environmental issues regarding these abandoned
formations have already been addressed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] Figure 1 is a graph showing mean methane production, in millimoles
(mmol) over time in the S24C160 culture given different concentrations of
amino
acids. Cultures were also amended with 0.5 grams (g) of coal.
[0007] Figure 2 is a graph showing mean methane production (in mmol) in
the S24C160 culture given either 0.5 g coal and 0.003 g/milliliter (m1) amino
acids,
0.5 g coal, 0.003 g/m1 amino acids, or nothing.
[0008] Figure 3 is a graph showing methane production (in mmol) in
S24C160 cultures amended with different concentrations of amino acids and
dosed with amino acids during the course of the incubation period. AA is an
abbreviation for amino acids.
[0009] Figure 4 is a graph showing mean methane production (in mmol) in
different methanogenic cultures grown with 0.5 g coal and with and without
0.006
g/m1 amino acids. ARC S1 is an abbreviation for ARC Sample 1. Obed MS is an
abbreviation for Obed Mine Sludge.
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[0010] Figure 5 is a graph showing mean methane production in the
S24C160 culture incubated with different ranked coals and with and without
0.006
g/ml of amino acids.
[0011] Figure 6 is a graph showing changes in culture pH with time in
S24C160 culture incubated with coals of different rank and with and without
amino
acids. The percent CH4 values represent the day 125 measurement.
[0012] Figure 7 is a graph showing mean methane production in mmol over
99 days in coal methanogenic culture with and without amino acids and
initially
adjusted to a pH range of 5.0 to 9Ø
[0013] Figure 8 is a graph showing mean methane production (in mmol) in
coal methanogenic cultures adjusted to different salinities with NaCI and with
and
without amino acids.
[0014] Figure 9 is a graph showing mean methane production (in mmol) in
coal S24C160 culture, with and without amino acids and adjusted to salinities
of
4.0, 8.0, 12.0, and 15.0 mg/ml NaCI.
[0015] Figure 10 is a graph showing mean methane production (in mmol) in
S24C160 cultures grown with different nutrient broths and with and without
crushed Obed coal. BHI is an abbreviation for the nutrient Brain Heart
Infusion;
YE is an abbreviation for the nutrient yeast extract.
[0016] Figure 11 is a graph showing methane production (in mmol) in high-
pressure vessels containing crushed coal and mineral salts medium and
inoculated with the S24C160 culture. A is an abbreviation for vessel A. B is
an
abbreviation for vessel B.
[0017] Figure 12 is a graph showing stable isotope values of CH4 and CO2
produced in Vessel A and B over time. dC1 is an abbreviation for 513CcH4. dCO2
is abbreviation for 813Cco2.
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[0018] Figure 13 is a graph showing methane production (in mmol) in
vessels A-D containing coal cores at elevated pressures.
[0019] Figure 14 is a graph showing 513CcH4 and 813Cco2 values generated
over time in Vessel A (core only). dC1 is an abbreviation for 513CcH4. dCO2 is
an
abbreviation for 813Cco2.
[0020] Figure 15 is a graph showing 813CcH4 and 813Cco2 values generated
over time in Vessel B (core + inoculum). dC1 is an abbreviation for 4513CcH4.
dCO2 is an abbreviation for 813Cc02-
[0021] Figure 16 is a graph showing 4513CcH4 and .513Cco2 values
generated
over time in Vessel C (core + inoculum + amino acids).
[0022] Figure 17 is a graph showing 813CcH4 and 813Cco2 values generated
over time in Vessel D (core + amino acids).
SUMMARY
[0023] The invention provides a method of increasing methane gas
released and produced from a subsurface coal containing formation, comprising
increasing methane gas released and produced from the formation by contacting
amino acids with the formation, where the contacting occurs in situ in the
formation, the formation contains methanogenic microorganisms, the amino acids
are in an amount effective to increase the release or production of methane
gas
from the formation, the amount of amino acids is less than 30 kilograms of
amino
acids per metric tonne of coal contained in a predetermined location of the
formation and the amino acids are obtained from a source outside of the
formation.
[0024] The invention further provides that the methanogenic
microorganisms are naturally occurring in the formation and have not been
removed from the formation.
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[0025] Moreover, the invention provides a method of increasing methane
gas
released or produced from a subsurface coal formation, comprising increasing
methane gas
release from the formation by lowering an amount of amino acids contacting the
formation in
situ, wherein the amino acids of the lowered amount are obtained from a source
outside of
said formation.
[0026] The invention also provides increasing the methane release from a
formation
by further decreasing the amount of amino acids.
[0027] The invention further provides a subsurface coat containing
formation,
wherein an amount of amino acids less than 30 kilograms per metric tonne of
coal contained
in a predetermined location of said formation is in contact with said coal,
wherein said
amount is effective to increase the release of methane gas from said coal and
wherein said
amino acids are obtained outside of said formation.
[0028] The invention further provides methane gas that is obtained by the
methods
provided herein.
[0028a] According to an aspect, there is provided a method of increasing
methane
gas produced or released from a subsurface coal containing formation,
comprising:
increasing methane gas produced or released from said formation by contacting
amino acids with said formation, wherein:
said contacting occurs in situ;
said formation contains methanogenic microorganisms;
said amino acids are in an amount effective to increase the release or
production of
methane gas from said formation;
said amount is greater than 60 grams and less than 30 kilograms of amino acids
per
metric tonne of coal contained in a predetermined location of said formation;
said formation is substantially saturated with said amount of amino acids; and
said amino acids are obtained from a source outside of said formation.
[0028b] According to an aspect, there is provided a method of increasing
methane
gas released or produced from a subsurface hydrocarbon containing formation,
comprising:
increasing methane gas released or produced from said formation by contacting
amino acids with said formation, wherein:
said hydrocarbon is peat, coal, shale, tar sand, heavy oil or mixtures
thereof;
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said contacting occurs in situ;
said formation contains methanogenic microorganisms;
said amino acids are in an amount effective to increase the release or
production of
methane gas from said formation;
said amount is greater than 60 grams and less than 30 kilograms of amino acids
per
metric tonne of coal contained in a predetermined location of said formation;
said formation is substantially saturated with said amount of amino acids; and
said amino acids are obtained from a source outside of said formation.
[0028c] According to an aspect, there is provided a subsurface coal
containing
formation, wherein an amount of amino acids greater than 60 grams and less
than 30
kilograms per metric tonne of coal contained in a predetermined location of
said formation is
in contact with said coal, said formation is substantially saturated with said
amount of amino
acids, wherein said amount is effective to increase the release or production
of methane gas
from said coal and wherein said amino acids are obtained outside of said
formation.
[0028d] According to another aspect, there is provided a use of an amount
of amino
acids effective to increase the release or production of methane gas from coal
in a
subsurface coal containing formation, wherein an amount of said amino acids
greater than
60 grams and less than 30 kilograms per metric tonne of said coal contained in
a
predetermined location of said formation is in contact with said coal, said
formation is
substantially saturated with said amount of amino acids, and wherein said
amino acids are
obtained outside of said formation.
DETAILED DESCRIPTION
[0029] Methods are provided for increasing methane gas release or
production from
subsurface coal containing formations. Subsurface coal containing formations
are geological
formations containing coal that are found below the surface of the ground.
Such formations
are found throughout the world and are located at varying depths. Because of
changes to
the earth's crust over time, subsurface coal containing formations may also be
found near or
contiguous to the surface and may also be found under water. Examples of
subsurface coal
containing formations are coal fields, coal reservoirs, coal basins, coalbeds,
coal seams,
coal horizons or coal mines.
[0030] Coal can be classified by rank or grade. A coal's grade refers to
its purity.
Coals of various grades are included in the invention. With regard to rank,
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a coal's rank refers to the degree of coalification. Coalification refers to
the
chemical composition of coal which depends on the amount of pressure and heat
that, in nature, has been applied to form the coal. The major ranks of coal,
listed
from the lowest rank to the highest rank are lignite, sub-bituminous,
bituminous,
semiantharacite and anthracite. The higher rank of a coal signifies that a
greater
amount of heat and pressure formed the coal compared to a lower ranked coal.
Generally, higher rank coals contain more carbon, but less oxygen and less
water
or moisture content. In one aspect of the invention, the subsurface coal is
lignite,
sub-bituminous, bituminous or mixtures thereof.
[0031] Methane gas, as a result of, for example, naturally occurring
processes, is trapped in many subsurface coal containing formations. Methane
gas is found trapped in coal containing formations, for example, in three
states:
as free gas, as gas dissolved in water in contact with the coal (for examples,
water
found in coal seam fractures, also known as cleats) or as gas adhering to the
coal
itself or contained in micropores in the coal. The gas may also be found
trapped
in cleats or in interbeds of non-coal. The methane gas is, for example, held
in
place in the formation by pressure.
[0032] In an aspect of the invention, the methane gas is modified. For
example, the methane gas molecules are altered or combined with other atoms or
molecules. Also, the form of the methane gas may be modified, for example by
liquification. As a further example, reagents or inert ingredients may be
added to
the methane gas. Such modifications may be made, for example, to improve the
methane gas' production, release, collection, measurement, storage, transport
or
commercial use. In the context of this invention, methane gas includes the
above
modifications and any similar modifications.
[0033] Commercially, methane gas is typically obtained from subsurface
coal containing formations by drilling a well into the formation or by
fracturing the
formation with, by, for example, horizontal drilling. Obtaining methane gas
from a
subsurface formation often involves pumping water out of the formation. As a
result of such drilling, fracturing or water extraction, the pressure causing
the
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methane gas to be trapped in the formation is reduced permitting the methane
gas
to be released. Furthermore, in an aspect of the invention, the released
methane
gas is measured, compared to prior amounts of released methane gas or
collected. In a further aspect of the invention, the methane gas is
transported
away from the formation.
[0034] In an aspect of the invention, methods are provided for increasing
the release or production of methane gas from the formation. In a further
aspect
of the invention, the increase is relative to a prior amount of methane gas
released
or produced from the formation. In a further aspect of the invention, the
increase
is relative to an equivalent time period of methane gas release or production
and
an equivalent location of methane gas release or production from the
formation.
In this aspect, the equivalent location and time period may be estimated or
extrapolated. For example, the equivalent location can be estimated based on
coal samples or based on the estimated amount, grade or rank of coal from
which
methane gas is released or produced.
[0035] In an aspect of the invention, the increased release or production
of
methane gas from the formation results from the metabolic production of
methane
gas by methanogenic microorganisms as discussed herein. In another aspect of
the invention, the release of methane gas results from the release of methane
gas
that is trapped in the formation, for example, prior to addition of amino
acids and is
released because of coal degradation, microfractures, or other aspects created
by
a consortium of microorganisms stimulated by the amino acids as described
herein.
[0036] Additionally, the methods of the present invention further provide
increasing the release or production of methane gas from the formation by
contacting the formation with amino acids. In an aspect of the invention, the
contacting is made via a well or fracture in the formation using water or a
liquid to
disperse the amino acids in the formation. However, other methods can be used
to contact the amino acids with the subsurface coal containing formation, such
as
dispersing the amino acids as dry matter.
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[0037] As discussed below, amino acids contacted with a subsurface coal
containing formation are metabolized by a consortium of microorganisms. These
metabolic processes include the production of methane by methanogenic
microorganisms, included, for example, in the consortium. Therefore, in an
aspect
of the invention, the purpose of contacting the formation with the amino acids
is to
provide the amino acids as a substrate for the microorganisms located in the
formation in order to increase methane production and methane release from the
formation. In an aspect of the invention, contacting the amino acids with the
formation refers to locating the amino acids in the immediate proximity of the
formation so that a consortium of microorganisms in the formation have access
to
the amino acids as substrates.
[0038] In an aspect of the invention, the amino acids contacting occurs
in
situ in the subsurface coal containing formation. That is, the contacting
occurs in
the formation itself in contrast to coal being extracted from the formation.
However, the invention includes the option that, in addition to such
contacting
occurring in situ, a portion of the coal or the formation may also be
extracted and
contacted with amino acids outside of the formation, for testing or for other
purposes.
[0039] In an aspect of the invention, the subsurface coal containing
formation contains methanogenic microorganisms. In the context of this
invention,
methanogenic microorganisms are microorganisms that produce methane from
substrates located in (naturally occurring or introduced into) a subsurface
coal or
hydrocarbon containing formation. Common
substrates for methanogenic
microorganisms of the invention are acetic acids and carbon dioxide.
[0040] In an aspect of the invention, the methanogenic microorganisms are
obligate anaerobes. In another aspect of the invention, the methanogenic
microorganisms are facultative anaerobes. The methanogenic microorganisms
included in the present invention are commonly archaebacteria but also include
other microorganisms capable of producing methane in subsurface coal or
hydrocarbon containing formations. In an aspect of the invention, the
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methanogenic microorganisms are naturally occurring in the subsurface coal
containing formations, for example, on the coal, interbed non-coal or in water
of
the formation. In
another aspect of the invention, the methanogenic
microorganisms are introduced into subsurface coal containing formations, for
example, after being genetically modified, nutrient stressed or subject to
other
processes.
[0041] In an
aspect of the invention, the methanogenic microorganisms
produce methane in a symbiotic or syntropic relationship with a consortium of
other microorganisms. A syntropic relationship in this context refers to a
relationship between two or more different species or strains of
microorganisms
where the different microorganisms provide each other with nutrients. Such
consortium include, for example, hydrolitic microorganisms, fermentative
microorganisms and acetogenic microorganisms.
[0042] In an
aspect of the invention, methane production from coal results
from a series of biochemical reactions under anaerobic or substantially
anaerobic
conditions. That is, a consortium of microorganisms, degrade coal in a
stepwise
fashion such that the products of some microorganisms serve as substrates for
other microorganisms of the consortium.
[0043] For
example, proteins, polypeptides and small peptides are
degraded by hydrolytic microorganisms and fermentative anaerobic
microorganisms producing monomeric compounds. The monomeric compounds
produced include amino acids, carbon dioxide, 'acetate and hydrogen gas. These
monomeric compounds serve as substrates, for example, for acetogenic
microorganisms which produce, for example, carbon dioxide and acetate.
Methanogenic microorganisms produce methane from, for example, the carbon
dioxide and acetate products of the acetogenic microorganisms.
[0044] A common
methanogenic microorganism pathway uses CO2-type
substrates in a carbonate reduction pathway to produce methane:
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CO2 + 4112 -4 CH4 + 21120
[0045] Methanogenic microorganisms also cleave acetate to CO2 plus CH4
in what is called the acetoclastic or fermentative pathway:
CH3C00- + H20 ---> CH + HCO3-
In the above reaction, the CO2 is shown as bicarbonate (HCO3-) because carbon
dioxide is predominately bicarbonate in neutral or slightly alkaline water.
[0046] In another aspect of the invention, the coal is depolymerized,
either
aerobically or anaerobically, as part of the process leading to the production
of
methane from the methanogenic microorganisms. In an aspect of the invention,
depolymerization is achieved by microorganisms and in another aspect by other
means known to the skilled artisan.
[0047] In the context of the invention, amino acids include one or more
types of amino acids. Regarding size, amino acids include free monomer amino
acids, small peptide chains, polypeptide chains and proteins. In an aspect of
the
invention, the amino acids are contained in a composition containing other
ingredients, such as lipids (e.g., fatty acids), vitamins, non-protein
nitrogen and
other non-amino acids ingredients. In another aspect of the invention, the
amino
acids are contained in a fish enzyme hydrolysate composition.
[0048] The amino acids that are contacted in the formation according to
an
aspect of the invention are obtained from a source outside of the formation.
It is
understood, however, that such externally obtained amino acids, when put in
contact with the formation, are metabolized or otherwise broken down into
smaller
amino acids (or other products) in situ in the formation, which in turn are
used in
the metabolic processes resulting in the production of methane. Also, it is
understood that in this aspect of the invention, the externally obtained amino
acids
may be combined with amino acids that are produced in situ in the formation.
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[0049] In an aspect of the invention, the amino acids are obtained from
fish.
In a further aspect of the invention, the amino acids are fish amino acids
that are
obtained by enzymatic hydrolysis of fish material. In another aspect of the
invention, the fish amino acids are obtained from fish material that is the
waste
product of commercial fish manufacturing for human consumption.
[0050] In an aspect of the invention, fish amino acids are used having
the
following distribution shown in the below Table 1:
Amino acids Abbreviation Percentage of Amino acids to
Total Amino acids
Alanine Ala 7.0
Arginine Arg 6.0
Aspartic Acid Asp 6.5
Cystein/Cystine Cys 0.6
Glutamic Acid Glu 11.5
Glycine Gly 13.0
,
Histidine His 2.4
Isoleucine Ile 2.5
Leucine Leu 4.9
Lysine Lys 5.2
Methionine Met 2.0
Phenylalanine Phe 2.7
Proline Pro 6.0
Serine Ser 3.9
Threonine Thr 3.5
Tryptophan Trp 0.5
Tyrosine Tyr 1.5
_
Valine Val 3.0
OH-proline 0Hpro 3.4
Taurine Tau 3.4
[0051] In an aspect of the invention, amino acids are used which are
under
60,000 daltons in size. In a further aspect of the invention, amino acids are
used,
wherein 90 percent or more of amino acids used are 10,000 daltons or less in
size, wherein 70 percent or more of amino acids used are 5,000 daltons or less
in
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size and wherein 50 percent or more of amino acids used are 1,000 daltons or
less in size.
[0052] In other aspects of the invention, the amino acids are obtained by
enzymatic hydrolysis. Methods for enzymatic hydrolysis are known in the art.
Furthermore, descriptions of enzymatic hydrolysis and fish amino acids
obtained
by enzymatic hydrolysis are found, for example, in U.S. published patent
application, publication number: US 2005/0037109 Al to Soerensen at al.
[0053] The invention further includes contacting the subsurface coal
containing formation with an amount of amino acids that is effective to
increase
the release or production of methane gas from the formation. This amount can
be
determined by measuring the amount of release or production of methane gas,
resulting from contacting with amino acids, from the formation itself, for
example
by measuring release or production of methane gas at the location of
contacting
or at more remote locations in the formation. This amount can also be
calculated,
for example, by measuring the amount of methane produced at the location of
contacting or by measuring the amount of methane produced from formation
samples, contacted with an amount of amino acids, that have been extracted
from
the in situ formation.
[0054] In an aspect of the invention, it has been discovered that amino
acids above a certain amount result in a decrease of methane production from
coal by methanogenic microorganisms. As a corollary, it has also been
discovered that lowering the amount of amino acids b eiow a certain amount
results in an increase in methane production from methanogenic microorganisms.
Without being bound by a specific theory, it is predicted that this decrease
in
methane production results from inhibitory compounds that are produced when
the amount of amino acids exceeds a certain level.
[0055j. Based on this discovery, aspects of the invention irxiude using the
following amounts of amino acids per metric tonne of coal: less than 30
kilograms
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of amino acids; less than 30 kilograms but greater than 60 grams; less than 15
kilograms, but greater than 600 grams; and less than 6 kilograms, but greater
than
1.2 kilograms.
[0056] Also contemplated are further ranges containing all integers
between the foregoing ranges down to 0 grams and up to 30 kilograms. Also
included are the above ranges where the gram and kilograms shown above are
less than or equal to or are preceded by about.
[0057] Furthermore, grams of amino acids per tonne of coal is not
intended
to be a limiting. The aspect of the invention regarding the amount of amino
acids
per amount of coal may be calculated in several alternative ratios of units
such as
weight/volume (w/v), volume/weight (v/w), volume/volume (v/v) or weight/weight
(w/w). As used herein, but without being limiting, grams of amino acids per
tonne
of coal is expressed in w/w units. The w/w units also correspond to v/w units
provided in the Examples herein, such as ml or m3 of 60% amino acids
hydrolysate per tonne of coal. For instance, 3 kilograms of amino acids
hydrolysate corresponds to 0.005 m3 of 60% amino acids hydrolysate at the same
final volume of 0.5 m3.
[0058] With regard to calculating metric tonnes of coal, this amount may
include coal volume, coal density or a combination of the two. A metric tonne
of
coal can also correlate to a determination of gas resource, as measured by,
for
example, a billion cubic feet (bcf).
[0059] The invention further provides that the amount of amino acids is
determined based on the metric tonnes of coal in a predetermined location of
the
formation. This predetermined location may include the entire formation or a
portion or portions thereof. The purpose of predetermining a location is to
identify
the area or areas in the formation where it is desired to put the amino acids
in
contact with the formation. For instance, depending on the attributes of the
formation (such as cleats, interbeds and amount of water), the skilled artisan
will
make a determination of a location of the formation to disperse the amino
acids.
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[0060] The invention further provides methods for increasing methane gas
released or produced from a subsurface coal formation
by lowering the amount of amino acids contacting the formation. In this aspect
the
amount of amino acids is lower relative to a prior amount of amino acids that
has
been added to the ,formation and the increase in methane gas release or
production is relative to the amount released or produced with regard to the
prior
amount of amino acids.
[0061] Furthermore, the invention includes applying the methods herein to
inactive or abandoned formations and formations where methane gas has already
been released from the formation or methane gas is no longer being collected
from the formation.
[0062] In addition to coal, the present invention also includes
subsurface
formations including the following hydrocarbon containing materials: peat,
shale,
tar sand, heavy oil or mixtures thereof.
[0063] As used in the context of the invention, words such as "or" or
"and"
refer to each element described individually or one or more of the elements in
combination. As used in the context of the invention, the word "including"
means
including without limitation. As used in the context of the invention,
singular terms
such as "a" do not exclude the presence of two or more elements. For example,
the phrase "a consortium of microorganisms" used herein includes two or more
consortia of microorganisms.
EXAMPLES
STAGE 1 OF THE EXAMPLES
[0064] The objective of Stage 1 was to verify an amino acids mixture has
(or does not have) an effect on enhancing and/or increasing biogenic methane
production from coal. Growth Examples with crushed coal were conducted at
atmospheric pressures in sealed glass bottles. A variety of Examples were
done,
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including looking at dosing, pH, salinity and coal rank effects and how the
presence of the amino acids mixture influenced rnethanogenesis under these
different conditions.
[0065] Different methanogenic cultures enriched from coal cores and from
other coal-related environments were used in the Examples. Table 2 briefly
outlines the culture characteristics. CBM is an abbreviation for coalbed
methane.
Table 2. Methanogenic cultures used in research project.
Culture Name Characteristic
S24C 160 Enriched from a coal core taken from a CBM well, grown at
30 C. '
S22C150 Enriched from a coal core taken from a CBM well, grown at
30 C.
S26C162 Enriched from a coal core taken from a CBM well, grown at
30 C.
S32C169 Enriched from a coal core taken from a CBM well, grown at
30 C.
ARC Sample 1 Enriched from a coal core taken from a CBM well, grown at
30 C.
ARC Therm Enriched from a coal core taken from a CBM well, grown at
50 C.
Obed Mine sludge Enriched from coaly sludge taken from a coal mine, grown at
30 C.
[0066] For many of the Examples herein, the S24C160 culture was used as
the inoculum since it showed, compared to the other cultures, the greatest
methanogenic activity.
[0067] The growth medium for growing anaerobic consortia and for
culturing core samples consisted of a mineral salts medium (MSM). The medium
was boiled for 2 minutes and cooled while 02-free 100%. N2 was bubbled through
the liquid. The medium was transferred to serum bottles sparged with 02-free
100% N2. The bottles were sealed with butyl rubber stoppers and crimped down
with aluminum seals. Just prior to inoculation, the culture bottles were
reduced to
-571 E0' (mV) by 0.1 ml sodium sulfide (25 g/l stock solution). Cultures were
prepared in triplicate to account for any variation in microbial activity and
culture
preparation.
[0068] The amino acids were received as both a liquid fish hydrolysate
(60% dry matter) and as powder. The 60% (w/v) hydrolysate was used for the
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Examples and dilutions were made and kept frozen until needed. As well,
aliquots
of the original, un-diluted hydrolysate were kept frozen.
[0069] For the majority of Examples, a sub-bituminous coal was used as
the coal source. This coal came from Obed Mine (Luscar Coal Ltd., Alberta) and
was surface collected. The coal was subsequently ground using a mortar and
pestle to a mesh size between 24-32 (a mesh opening size of 0.50-0.71 mm).
The coal was added after sterilization (and prior to medium reduction and
inoculation) to a concentration of 0.05-0.10% (w/v). Other coals that were
used in
the project (see Example 5) were also crushed to a mesh size between 24-32.
[0070] For subsequent culturing and transferring of the cultures into
fresh
media, a 20% (v/v) inoculum size was used. The cultures were incubated at
different temperatures ranging from 30 to 50 C, in the dark. The cultures were
kept stationary.
EXAMPLE 1
[0071] To initiate the research project the methanogenic cultures
S24C160,
S22C150, S32C169, and Arc Therm (Table 2) were grown with 0.5 g crushed coal
and amended with 0.03 g/ml of the amino acids mixture. Methane yields were
very low and the CO2 yields were very high in all of the cultures tested.
[0072] In order to determine whether the amino acids were inhibiting
methanogenesis at the concentration of 0.03 g/ml, the S24C160 culture was
grown with coal and the amino acids at the following reduced amino acids
concentrations: 0.006, 0.003, and 0.0003 g/ml. Lowering the amino acids
concentration did reverse the inhibition effect (Figure 1) as methane
production
was detected in those cultures given a 5-fold and 10-fold diluted amino acids
solution (0.006 and 0.003 g/ml, respectively). There was an enhancement in
methane production of 18.4-fold when the amino acids mixture concentration was
lowered from 0.03 to 0.006 g/ml (0.022 mmol compared to 10404 mmol
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methane/day, respectively). The methane yield decreased to 0.260 mmol at the
amino acids concentration of 0.003 g/ml. This yield is still 11.8-fold higher
than
with the 0.03 g/ml amino acids-amended cultures. Cultures amended with 0.0003
g/ml amino acids (a 100-fold dilution of the original concentration of 0.03
g/ml) had
a slightly higher methane production than those cultures given 0.03 g/ml amino
acids solution (0.042 mmol compared to 0.022 mmol methane at day 74,
respectively).
[0073] CO2 production decreased in the cultures given the diluted amino
acids mixture. CO2 yields on day 74 of the Example varied from 0.392 mmol in
the 0.03 g/ml amino acids-amended cultures to 0.188 mmol and 0.119 mmol CO2
for the 0.006 and 0.003 g/ml amino acids-amended cultures, respectively (Table
3). The cultures given 0.0003 g/ml amino acids solution had the lowest CO2
yield
of 0.042 mmol. The methanogenesis rates are also summarized in Table 3,
clearly showing that the culture amended with 0.006 g/ml amino acids had the
highest methane production rate amongst the cultures (0.00712 mmol
methane/day compared to 0.0046 mmol methane/day with 0.003 g/ml amino
acids).
Table 3. Methanogenesis rates and yields in S24C160 coal cultures amended with
different
concentrations of the amino acids mixture. Cultures were also amended with 0.5
g coal.
- Culture Amendment Methanogenesis rate r Yield (mmol)
,
(mmol C114/day) Methane CO2 i
0.03 g/ml amino acids 0.000366 0.022 0.392 '
i
0.006 g/ml amino acids 0.00712 0.404 0.188
0.003 g/ml amino acids 0.0046 0.260 0.119
0.0003 g/ml amino acids 0.00083 0.042 0.042
I
'Yield on day 74.
[0074] The efficacy of the amino acids mixture to enhance methanogenesis
was correlated to its concentration in the consortia. At a concentration of
0.03
g/ml, very little methane was produced (0.0037 mmol/day), but with a five-fold
dilution of this concentration (to give 0.006 g/ml), enhanced methane
production
occurred (0.00712 mmol/day). With further dilution of the amino acids mixture,
the
rate of methane production correspondingly decreased. At 0.003 g/ml the rate
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was 0.0046 mmol methane /day and at 0.0003 g/ml the rate was only 0.00083
mmol methane /day.
EXAMPLE 2
[0075] Example 2, outlined in Table 4, was then done in order to gain a
better understanding of how the amino acids affect methane production. In
particular, the question of whether the culture, when amended with the amino
acids, preferentially uses the amino acids as substrates for growth over the
coal
was to be addressed by this Example.
Table 4. Example design of cultures to determine effect of 0.003 g/ml amino
acids mixture on
methane production. Inoculum was the S24C160 culture and crushed Obed coal
served as the coal
source (0.5 g/culture).
Culture Amendments Purpose/Comment
Coal Amino acids
Mixture
Coal and amino Measures effect of amino acids addition on
methane
acids (0.003 production from coal.
g/ml)
Coal Measures methane production from coal
metabolism.
Amino acids Measures methane production from amino acids
(0.003 g/ml) metabolism.
No additions Measures methane production from media
components and carry-over of amino acids and coal
from inoculum bottle.
[0076] The cultures given coal and the amino acids mixture had a
significantly higher methane yield and production rate than those given only
amino
acids or only coal (Figure 2). When the culture was only given coal, 0.005
mmol
methane was generated after 74 days (Table 5). Cultures given only the amino
acids mixture had a 16-fold increase in methane yield on day 74 over the coal-
only cultures. When the culture was given both coal and amino acids, the
methane yield increased by 3.2-fold over those cultures with only the amino
acids
mixture and 52-fold over those cultures with only coal. This culture also had
the
greatest methane production rate at 0.0049 mmol methane/day; 4.0-fold higher
than the production rate of the amino acids-only culture. The culture with
only
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coal had a methane production rate 51 times slower than the culture amended
with coal and amino acids. The culture bottles with no additions had
negligible
methane production indicating carry over of amino acids and coal from the
inoculum bottles did not result in any significant methane production.
Table 5: Methane production rates and yields in the S24C160 culture amended
with either 0.5 g
coal and 0.003 g/m1 amino acids, 0.5 g coal, 0.003 g/ml amino acids, or
nothing.
Culture Methanogenesis rates I Methane Yield'
(mmol/day) (mmol)
, Coal and amino acids 0.0049 0.258
(0.003 g/ml)
! Coal 0.000096 0.005
Amino acids 0.001 0.0802
(0.003 g/ml)
No additions . 0.0000112 I 0.00089
'Yield on day 74 of the incubation period.
[0077] Under
methanogenic conditions, amino acids are fermented to
volatile fatty acids (VFA) and finally to methane and CO2 by trophically
different
microorganisms (Tang et al., J. Bioscience Bioengineering 99(2):150-164
(2005)).
Acetic, propionic and butyric acid are the major VFA formed during anaerobic
biodegradation. Dhaked at al., Bioresources Technol. 87:299-303 (2003)
reported
that these main substrates in the terminal step of methanogenesis are
inhibitory to
the process at higher concentrations with propionate more toxic than the
others.
[0078] . It is also possible that the Stickland reaction (microbial
fermentation
of amino acids whereby the.amino acids act as either electron donor or
electron
acceptor) dominates in environments rich in amino acids (Tang at al., 2005).
In
environments with low amino acids and high methanogenic activity,
methanogenesis may dominate over the Stickland reaction as the reducing
equivalents (H2) would be scavenged quickly by the methanogenic
microorganisms.
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[0079] At a concentration of 0.03 g/m1 in the coal cultures, the amino
acids
could have been preferentially degraded by the Stickland reaction or possibly
the
resulting VFA concentration proved too toxic for methanogenesis. In the
cultures
given 0.03 g/ml, a large amount of CO2 was produced. CO2 is one of the
products
of the Stickland reaction. All the cultures tested at the highest amino acids
concentration (0.03 g/m1) did not produce methane.
EXAMPLE 3
[0080] In Examples 1 & 2, the addition of amino acids provided an
increased production of methane from methanogenesis in the presence of coal.
The methane production rates in the cultures began. to slow down and plateau
after 50 to 60 days of incubation. Example 3 investigated the effect of dosing
the
amino acids-amended coal cultures with additional amounts of the amino acids
mixture at time points when the methanogenesis rates appeared to be slowing
down. The cultures selected for this Example were the S24C160 cultures given
coal and 0.03, 0.006, 0.003, and 0.0003 g/m1 amino acids. Two of the
triplicate
bottles from each culture condition were given the amino acids doses while the
third bottle remained un-dosed and served as the control (it had received
amino
acids at the start of the Example). The dosing regimen is shown in Table 6.
Table 6. Amino acids dosing regimen of S24C160 coal cultures grown with
different
concentrations of amino acids mixture over a 264-day period.
Culture Original Amino acids Dose First Dose Second Dose
(Time Zero) (Day 88) (Day 196)
0.03 + coal 0.03 g/m1 0.03 g/m1 0.006 g/m1
0.006 + coal 0.006 g/m1 0.006 g/m1 0.006 g/m1
0.003 + coal 0.003 g/m1 0.003 g/m1 0.006 g/m1
0.0003 + coal 0.0003 g/m1 0.0003 giml 0.006 g/m1
[0081] For the cultures originally given 0.006, 0.003, and 0.0003 g/ml of
the
amino acids mixture, the addition of an equivalent concentration of amino
acids on
day 88 of the incubation period resulted in a modest increase in methane
production rate (Figure 3) from 1.2-fold to 3.1-fold increase.
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[0082] The cultures given 0.003 g/ml amino acids showed the greatest
increase in methane production rates; 3-fold over its un-dosed equivalent from
0.00307 to 0.00964 mmol methane/day (Table 7). For the second dose on day
196, 0.006 g/ml amino acids were given to all the cultures and the methane
production rates continued to rise, 3-fold in the 0.006 g/ml culture and by 30-
fold
in the 0,0003 g/ml culture compared to their previous growth phase (first
dosage,
days 88-196). The one culture bottle from each concentration series that did
not
receive any amino acids dose continued to produce methane during the entire
incubation period though the rates were slower from days 88-264 than they were
from the start of the Example, time zero, to day 88. Not surprisingly, the
culture
given the highest amino acids concentration of 0.03 g/ml, did not produce any
significant amounts of methane during the entire incubation period with and
without dosing with amino acids.
Table 7. Methane production rates and yields of the S24C160 cultures amended
with different
amino acids concentrations before and after dosing with the amino acids
mixture.
Methane Production Rates (mmol CH4/day)and Yield (mmol CH4)
Culture Original Amino First Dosage Second Dosage (Days No
Dose
(mg/ml acids dosage (Days 88-196) 196-264)
(Days 88-264)
amino (Time Zero-Day 88)
acid) Rate Yield Rate Yield Rate Yield Rate
Yield
(day 88) (day 196) (day 264)
(day
264)
0.03 + coal 0.000103 0.0259 0.000099 0.0326
0.000064 0.0340 0.000143 0.0525
(0.0454)a
0.006+ 0.0059 0.523 0.0073 1.30 0.020 1.80 0.0028
1.101
coal (0.866)
0.003 + 0.00307 0.288 0.00964 1.004 n.a.b nab
0.00243c 0.473
coal (0.259)
0.0003 + 0.000556 0.0515 0.000835 0.144
0.017 1.303 0.000298 0.1657
coal I (0.0837)
'Values in brackets are the methane yield for the culture bottle that was not
given the amino acids
dosage during the 88-196-day period.
bThe 0.003 g/ml culture series were accidentally destroyed on day 160,
therefore rate is from days
88-160.
'Time period from Day 88-160.
[0083] The CO2 production in the 0.03 g/ml amino acids-amended cultures
greatly increased upon addition of the amino acids doses, from 30% to 53% of
the
headspace gas (0,48 to 1.69 mmol methane). This increase in CO2 production
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occurred within the first week of the amino acids addition and did not
increase with
further incubation. The cultures dosed with the lower concentrations of the
amino
acids solution also produced CO2 but at correspondingly lower amounts. With
each dose of the amino acids, CO2 production increased as compared to the un-
dosed cultures. The following table (Table 8) compares the production rates of
CO2 in the cultures before and after amino acids additions.
Table 8. CO2 production rates and yields of the S24C160 cultures amended with
different amino
acids concentrations before and after dosing with the amino acids solution.
CO2 Production Rates (mmol CO2klay)and YieldImmol CO2)
Culture Original Amino First Dosage Second Dosage
(Days No Dose
(mg/ml acids dosage (Days 88-196) 196-264) (Days 88-264)
amino (Time Zero-Day 88)
acid) Rate Yield Rate Yield Rate Yield Rate
Yield
(day 88) (day 196) (day 264) (day
I
264) ,
0.03 + 0.00221 0.480 0.0112 1.69 0.0031 1.90 0.0030
1.001
coal (0.786)a _
0.006+ 0.00217 0.227 0.0037 0.626 0.0087 1.20 0.0019 0.534
_ coal (0.336)
0.003 + 0.00223 0.197 0.00413 0.443 n.a.b n.a.b
0.0016' 0.259
coal (0.259)
0.0003+ 0.000655 0.0575 0.00119 0.186 0.0081 0.734 -
0.00068 0.175
coal (0.116)
'Values in brackets are the CO2 yield for the culture bottle that was not
given the amino acids
dosage during the 88-196-day period.
bThe 0.003 g/m1 culture series were accidentally destroyed on day 160,
therefore rate is from days
88-160.
'Time period from Day 88-160.
[0084] At the completion of Example 3, the culture fluid was analyzed for
acetic acid and ammonia concentrations (Table 9). The highest amounts of
acetic
acid and ammonia were observed in the cultures with 0.03 g/ml of the amino
acids
mixture. The amounts of acetic acid and ammonia decreased with decreasing
concentration of the amino acids mixture.
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Table 9. Comparison of acetic acid and ammonia levels in dosed and un-dosed
S24C160 coal
cultures amended with different amino acids concentrations on day 264.
Culture Acetic acid (g/1) Ammonia (g/1)
(mg/ml amino
acid) Dosed Un-dosed Dosed Un-dosed
0.03 + coal 16.41 8.15 7A 0 5.01
0.006 + coal 3.16 4.42 2.88 3.22
0.0003 + coal 0.89 0.00 0.95 0.02
[0085] After approximately 50 days of incubation, methane production
began to level off and the cultures entered a stationary phase due to either
the
depletion of an essential nutrient or the build up of an inhibitory waste
product.
The largest response, in terms of increased methane production rates and
yields,
upon the addition of a dose of amino acids occurred in the culture with the
0.003
g/ml amino acids mixture. There was an increase of 3.14-fold in production
rate
and a 3.5-fold increase in yield after the culture was dosed with 0.003 g/ml
of the
amino acids mixture. In contrast, an average of 1.37-fold increase in methane
production rates and 2.7-fold increase in methane yield occurred in the 0.006
g/ml- and 0.0003 g/ml-amino acids cultures after dosing with an equivalent
amount of amino acids as the starting amino acids concentration. Overall,
dosing
the culture with the amino acids mixture did result in increased methane
yields
and enhanced rates of production when compared to the un-dosed culture in each
culture series. An increase in 1.63-fold and 7.86-fold in methane yields in
the
dosed 0.006 g/ml and 0.003 g/ml amino acids cultures, respectively, over the
un-
dosed cultures was observed (the 0.0003 g/ml amino acids culture was dosed
with 0.006 g/ml amino acids at the second dosing event). There was a 7.14-fold
and 57.04-fold increase in methane production rates in the dosed 0.006 g/ml
and
0.0003 g/m1 amino acids cultures over the un-dosed cultures, respectively.
EXAMPLE 4
[0086] In order to verify that the amino acids mixture produces a similar
enhancement in methane production in other methanogenic cultures as it does in
the S24C160 culture, Example 4 was conducted whereby four different
methanogenic cultures were grown with and without the amino acids mixture at a
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concentration of 0.006 g/ml. Crushed coal was present in all of the cultures.
The
cultures were ARC Sample 1, Obed Mine sludge, S26C162, and S32C169 (see
Stage 1 for details on the cultures). Only one of these cultures, S32C169, was
used in the original amino acids-amendment Example 1. It was decided to use
the three new cultures as the inoculum bottles for these cultures were very
active
in methane production. The three new cultures represented a more diverse
selection of methanogenic cultures than the ones used in Example 1, as the
cultures were enriched from different environments and coal cores.
[0087] The presence of amino acids had a significant effect on enhancing
methanogenesis over those cultures not given the amino acids mixture (Figure
4).
The cultures, however, were not equal in their response to the amino acids
amendments as the methanogenesis rates in Table 10 show. The S32C169
culture amended with amino acids had the highest rate of all the cultures
tested at
0.0050 mmol/day. The lowest rate of the cultures amended with amino acids was
generated by the ARC Sample 1 culture at 0.0025 mmol/day.
Table 10. Methane production rates and yields of methanogenic cultures grown
with coal and
with and without 0.006 g/ml amino acids mixture.
Methanogenesis Rate Yielda
Culture (mmol 014/day) (mmol CH4)
ARC Sample 1 0.000080 0.00156
ARC Sample 1 + amino acids 0.0025 0.075
Obed Mine Sludge 0.000047 0.00084
Obed Mine Sludge + amino acids 0.0043 0.133
S26C162 0.000111 0.00195
S26C162 + amino acids 0.0034 0.119
S32C169 0.000112 0.00209
S32C169 + amino acids 0.0050 0.174
'Yield on day 36.
EXAMPLE 5
[0088] A series of tests were conducted to determine the effect of
physical
parameters of coal and culture medium on methanogenesis and how this affected
the efficacy of the amino acids mixture. Coals of different rank and that can
be
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found in the provinces of Alberta and Saskatchewan, Canada, were used. Typical
coals in Alberta that are targeted for coalbed methane production range from
sub-
bituminous to high-volatile bituminous coal. The
effect of coal rank on
methanogenesis and how the addition of the amino acids mixture may alter or
interact with the coal and microorganisms was investigated by growing the
S24C160 culture with coals of different ranks in the presence or absence of
the
amino acids.
[0089] Five
different coals were used to test the effect of coal rank on
methanogenesis. The coals are listed in Table 11. The coals were crushed and
0.5 g added to each culture bottle (0.05% w/v).
Table 11. Characteristics of coals used in the coal rank methanogenesis.
Volatile Fixed
Coal Source Rank matter CYO Carbon (%)
Lignite Poplar River mine, Lignite 56-64 naa
Saskatchewan
Wabamum Whitewood Mine, Sub-bituminous C 50-52 70
Luscar Mines Ltd.,
Alberta
Obed Obed Mine, Luscar Sub-bituminous A 44-46 70
Mines Ltd., Alberta.
Coal Coal Valley Mine, High-volatile bituminous 42-44 70
Valley Luscar Mines Ltd.,
Alberta
Cardinal Cardinal River Mine, Medium-volatile 24-28
80
River Luscar Mines Ltd., bituminous
Alberta
'Information not available
[0090] In all of
the cultures, the addition of the amino acids significantly
enhanced the methane production rate (6-to 22-fold increase) over their
parallel
cultures with no amino acids additions (Figure 5). Only the Wabamum coal
cultures with no amino acids did not show any methanogenic activity.
[0091] There
were significant differences in methane yield and production
rates between cultures Containing coals of different ranks. Lignite and
Wabamum
coal cultures amended with amino acids had the highest activity; on average
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mmol methane after 123 days of incubation (Table 12). Obed and Coal Valley
coal cultures had lower methane yields of 0.56 mmol after 123 days. Cardinal
River coal cultures with amino acids had a significantly lower methane yield
than
the other amino acids-amended cultures; 0.183 mmol methane for Cardinal *River
coal compared to between 0.56 and 0.66 mmol methane for the other cultures
after 123 days of incubation. The cultures with the lignite coal had the
fastest
methane production rate of 0.0118 mmol methane/day, followed closely by the
Wabamum coal cultures (0.00932 mmol methane/day). In summary, the cultures
can be ordered by decreasing methane production rates and increasing coal
ranks:
Lignite > Wabamum > Obed > Coal Valley > Cardinal River
(Lignite > Sub-bituminous C> Sub-bituminous A> High-volatile bituminous C>
Medium-volatile
bituminous)
Table 12. Methane production rates and yields of the S24C160 culture incubated
with coals of
different ranks and with and without 0.006 giml amino acids mixture.
Culture Metbanogenesis rate Yield'
(mmol CH4/day) (mmol CH4)
_ _
Obed 0.000475 0.052
Obed + amino acids 0.00556 0.56
Lignite 0.000531 0.058
Lignite + amino acids 0.0118 0.66
Wabamum 0.000012 0.0014
Wabamum + amino acids 0.00938 0.64
Cardinal River 0.000614 0.069
Cardinal River + amino 0.000684 0.183
acids
Coal Valley 0.00070 0.085
Coal Valley + amino acids 0.00415 0.56 I
aYield on day 123.
[0092] The pH
of the culture fluid was measured at three different time
points (start, day 0; middle, day 49; and end, day 125) during the incubation
of the
cultures in order to determine whether the different coals, due to their
chemistry,
changed the culture pH and negatively affected methanogenesis. In all amino
acids-amended cultures except Cardinal River coal, the pH went up in value
with
time of nearly one full pH unit (e.g. 6.40 to 7.40 in Lignite cultures with
amino
acids over 125 days) (Figure 6). In the cultures with no coal, the pH values
were
generally lower than in the amino acids-amended cultures. In those cultures,
such
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as Wabamum coal and Obed coal-amended cultures, the pH of the culture
medium was approximately 6.00 at the end of the incubation period. These
cultures had low methane yields of between 0.11 and 1.5% or 0.0014 and 0.0522
mmol methane. Cardinal River coal culture behaved differently; these cultures
exhibited high pH values approaching 7.60 and they had the highest methane
yield amongst the coal-only cultures. The Cardinal River coal with amino acids
cultures had a slightly lower pH, but slightly higher methane yields. But,
this yield
was significantly lower than the other amino acids-amended cultures.
[0093] Results from the consortia studies demonstrated greatest methane
production occurred with the lower rank coals. Although the exact chemical
structure of coal is unknown, structure models have been deduced. The so-
called
coal structure changes with rank of coal. Coal ranks are based on fixed carbon
content, volatile matter content, and calorific value. The four ranks of coal
mined
today and ordered by increasing carbon content are: lignite, sub-bituminous,
bituminous, and anthracite. With increasing carbon content, there is a
corresponding decrease in the oxygen content in the coal. This makes coal
resistant to biogasification because microbial activity generally decreases
with
decreasing oxygen content. Lignites and/or sub-bituminous coals are used as
substrates in nearly all coal biogasification experiments because their
structure is
more amenable to biodegradation than the higher rank coals. Very little coal
biodegradation research, therefore, has been performed in bituminous and
anthracite coals.
[0094] The pH data from the coal rank study showed how the pH was
elevated in the cultures given the amino acids mixture compared to those
cultures
with only the coal. With the exception of the cultures with Cardinal River
coal
(highest coal rank of the Example, medium-volatile bituminous), there seemed
to
be a correlation between relatively high culture pH (from 7.20 to 7.40) and
the
presence of amino acids. The coal cultures with no amino acids all had final
culture fluid pH values of less than 7.00 (exception was the Cardinal River
coal
cultures) and with a corresponding low methane yield. The amino acids mixture
seemed to have caused an increase in pH over time. This may be due to an
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increase in ammonia over time as the amino acids were degraded. The VFAs
produced from the degradation of the amino acids would either be consumed or
buffer the effect of the ammonia so that the pH did not become excessively
alkaline. The slightly alkaline conditions may have then increased the
solubilisation of coal humates as described above.
EXAMPLE 6
[0095] Example 5 indicated that the addition of the amino acids as well
as
the presence of the coal affected the pH of the culture medium and that the pH
had an effect on methanogenesis. Example 6 was then done to examine the
effect of culture pH on methanogenesis and how the addition of the amino acids
mixture can modify the pH effect. The mineral salts medium was prepared and
adjusted to give 5 different pH ranges: pH 5.0, 6.0, 7.0, 8.0, and 9Ø Two
culture
series were prepared, one series received only Obed coal, and the other
received
Obed coal and 0.006 g/m1 of the amino acids mixture. S24C160 served as the
source of the methanogenic culture.
[0096] Those coal cultures that received the amino acids mixture had
significantly higher methane yields than those coal cultures without the amino
acids mixture (Figure 7). The cultures at a pH of 9.0 with the amino acids
mixture
had the highest methane yield, 0.33 mmol compared to 0.17 to 0.26 mmol for the
pH 5.0-8.0 culture with amino acids.
[0097] The pH of the culture medium was measured twice during the
course of the incubation period on day 52 and 100. The pH of the cultures with
amino acids and initially adjusted to pH 5.0-8.0 were similar to each other on
day
52 and were between pH 7.11 and 7.16 (Table 13). The pH of the culture medium
of pH 9.0 and amino acids was slightly higher on day 52 at pH 7.37. The
cultures
without the amino acids addition all had lower pH values than those with the
amino acids, between 6.70 and 6.92. The pH did not vary much within each
culture bottle over the course of Example 6 as, on average, the pH of the
culture
medium only varied by 0.043 units between days 52 and 100. An un-inoculated
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control bottle of just coal and the mineral salts medium and adjusted to pH
7.0
measured pH 7.0 at the end of the Example 6 time period.
Table 13. pH of culture fluid in coal-cultures with and without the amino
acids mixture at days 52
and 100 of incubation (the cultures were adjusted to various pH values at
start of incubation).
Culture pH on day 52 pH on day 100
pH 5.0, coal only 6.79 6.68
pH 5.0, coal with amino acids 7.12 7.12
pH 6.0, coal only 6.70 6.67
pH 6.0, coal with amino acids 7.16 7.16
pH 7.0, coal only 6.93 6.85
pH 7.0, coal with amino acids 7.12 7.19
pH 8.0, coal only 6.83 6.84
pH 8.0, coal with amino acids 7.11 7.16
pH 9.0, coal only 6.92 6.85
pH 9.0, coal with amino acids 7.37 7.36
[0098] Example
6 may substantiate the effect of alkaline conditions on
increased methane production from coal. The cultures amended with the amino
acids all had culture fluid pH values of 7.12 to 7.36, whereas the cultures
without
the amino acids had pH values below 7Ø Despite being adjusted initially to
the
different pH values, the pH of the culture fluid did not remain at this pH
despite the
presence of a phosphate buffer. The change in pH was most likely due to the
actions of the microbial culture and the presence of the amino acids, as an un-
inoculated control bottle containing just the coal and mineral salts medium
remained at its originally adjusted pH of 7.00 throughout the course of
Example 6.
The culture with the highest methane yield and production rate was the one
adjusted to pH 9.00 initially and that at the end had a culture fluid pH of
7.36 (the
highest of all the cultures). The very alkaline conditions of the pH 9.0
culture at
the beginning of Example 6 may have resulted in increased solubilization of
the
coal humates. Combined then with the effect of the amino acids, this resulted
in
enhanced methane production over the other cultures at lower pH values.
EXAMPLE 7
[0099] Example
7 was done to determine the effect of increasing salinity on
methanogenesis and how the presence of the amino acids mixture affects culture
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activity at the different salinities. The anaerobic culture medium was
prepared
and aliquoted into equal portions. Each portion was then amended with varying
amounts of sodium chloride (NaCI) in order to achieve a salinity range of 0.5,
1.0,
1.5, 2.0, 4.0, 8.0, 12.0, and 15.0 mg/ml NaCI. One portion did not receive any
NaCI and these culture bottles served as the control (0.05 mg/ml NaCI is in
the
original culture medium). The culture bottles all received 0.5 g of the
crushed
Obed coal. Half of the culture bottles were then given the amino acids mixture
at
a final concentration of 0.006 mg/ml. The bottles were inoculated with an
S24C160 culture.
[00100] Example 7 was done in two stages. The first stage compared
methane production rates of the culture at the lower salinities, 0.5 - 4.0
mg/ml
NaCI, to the control. As evident in Figure 8, those cultures given the amino
acids
mixture had a 5- to 22-fold increase in methane production over their
corresponding cultures with no amino acids. The cultures given amino acids
grew
well at all salinities.
[00101] Table 14 summarizes the methane production rates and yields of all
the cultures. Amongst the amino acids-amended cultures, the one at 4.0 mg/ml
NaCI had the highest yield (0.574 mmol methane by 126 days of incubation) and
rate (0.0048 mmol/day). In contrast, amongst the cultures without the amino
acids
solution, the 4.0 mg/ml NaCI culture had a significantly higher yield (0.091
mmol at
day 126) and rate (0.0014 mmol/day) compared to the other coal-only cultures.
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Table 14. Methane production rates and yields of the S24C160 culture at
different salinities and
incubated with coal and with and without the amino acids mixture.
Culture Amendment Methanogenesis Rate Yield'
(mmol CH4/day) (mmol)
..
Control 0.00034 0.050
Control + amino acids 0.00390 0.452
0.5 mg/ml NaCI 0.00026 0.036
0.5 mg/ml NaCI + amino acids 0.00360 0.462
1.0 mg/ml NaCI 0.00021 0.027
1.0 mg/ml NaCI + amino acids 0.00400 0.477
1.5 mg/ml NaCI 0.00027 0.036
1.5 mg/ml NaCI + amino acids 0.00400 0.490
2.0 mg/ml NaC1 0.00030 0.036
2.0 mg/ml NaCI + amino acids 0.00460 0.560
4.0 mg/ml NaC1 0.00140 0.091
4.0 mg/ml NaC1+ amino acids 0.00480 0.574
-a-Yield on day 126 of Example.
[00102] The second phase of Example 7 compared methane production
rates and yields of the culture at the higher salinities of 4.0 to 15.0 mg/ml
NaCI.
The objective was to determine the upper limit of salt tolerance in the
culture and
the effect of amino acids addition on methanogenesis. These cultures were
incubated less than half the time the lower salinity cultures were grown, but
definite trends in methane production can be seen in Figure 9. Methane
production was observed at all salinities. However, those cultures amended
with
the amino acids had a 6- to 15-fold increase in their methane production rates
over their corresponding cultures with no amino acids mixture. The highest
rate
was with the 4.0 mg/ml NaCI culture with 0.0096 mmol CH4/day generated (Table
15). The methane production rates decreased with increasing salinity.
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Table 15. Methane production rates and yields of the S24C160 culture incubated
with coal and
with and without the amino acids mixture and adjusted to salinities of 4.0,
8.0, 12.0, and 15.0
mg/ml NaCl.
Culture Amendment Methanogenesis Rate Yield'
(mmol C114/day) (mmoll
4.0 mg/ml NaCI 0.00070 0.0186
4.0 mg/m1NaC1+ amino acids 0.00960 0.263
8.0 mg/nil NaC1 0.00054 0.0176
8.0 mg/ml NaC1+ amino acids 0.00830 0.229
12.0 mg/ml NaCI 0.00058 0.0168
12.0 mg/ml NaCI + amino acids 0.00610 0.221
15.0 mg/ml NaCI 0.00061 0.0151
15.0 mg/ml NaC1+ amino acids 0.00370 0.142
aYield on day 42 of Example.
[00103] Overall,
the highest rate of methane production for the amino acids-
amended cultures was observed in the culture given 4.0 mg/ml NaCI, followed by
the cultures given 8.0 and 12.0 mg/ml NaCI. The remaining cultures had
essentially the same rates. For the cultures not given the amino acids
solution,
the highest rate occurred again with the 4.0 mg/ml NaCI culture. As mentioned
previously, there was no significant difference in methane production rates
among
the rest of the cultures. For practical applications, the addition of the
amino acids
mixture to coal beds With different salinities would not have a negative
effect on
methanogenesis.
EXAMPLE 8
[00104] Example
8 was conducted to compare the effectiveness of the
amino acids mixture against other microbiological nutrient solutions.
Concentrated stock solutions of different nutrient broths were prepared and
added
to the bottles to give a final concentration range of 0.0046-0.006 g/m1 as per
Table
16. Culture bottles were divided in half; one half received Obed coal and the
different nutrient broths, the other half received the nutrient broths only.
The
cultures were inoculated with the S24C160 culture.
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Table 16. The concentration of nutrients used in the methane production
comparison Example 8.
Nutrient/Growth Medium Final Concentration in Culture Bottles
Brain Heart Infusion (BHI) 0.0046 g/m1
Yeast Extract 0.005 g/m1
Soytone 0.005 g/m1
_
Tryptone 0.005 g/m1
Amino acids mixture including Fish 0.006 g/ml
Hydrolysate
[00105] The other Nutrient/Growth Medium included Brain-Heart Infusion
(dehydrated infusion of beef or porcine brains and hearts), yeast extract
(water
soluble portion of autolyzed yeast containing a vitamin B complex), soytone
(enzymatic digests of plant protein), and tryptone (enzymatic digest of
casein, the
main protein of milk). All of these complex nitrogen sources serve as an
excellent
source of amino acids, vitamins and act as stimulators of bacterial growth.
[00106] Methane production was detected in all of the cultures, regardless
of
which nutrient broth it was given (Figure 10). However, methane production was
enhanced 1.73- (soytone) to 31.7- (amino acids) fold when the cultures were
amended with coal. The amino acids mixture on its own produced the lowest
methane production rate (0.000144 mmol/day). When coal was present, the
amino acids mixture had the highest methane production rate (0.005 mmol/day)
of
all the cultures (Table 17). The culture with amino acids and coal had a
methane
production rate statistically higher than the other cultures including when
the rates
were compared based on the amount of nutrient given to each culture. The amino
acids-amended culture had a rate of 0.0842 mmol methane/day/g nutrient
whereas the tryptone-amended culture had a rate of 0.0786 mmol methane/day/g
nutrient.
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Table 17. Methane production rates and yields of the S24C160 culture incubated
with the
different nutrients and with and without coal.
Culture Methanogenesis Rate Methanogenesis Rate Yield'
Amendment (mmol C114/day) (mmol CH4/day/g nutrient)
(mmol)
Amino acids 0.000144 0.0024 0.0107
solution
Amino acids 0.00505 0.0842 0.339
solution + coal
Brain Heart Infusion 0.000463 0.01 0.0296
(BHI)
Bill + coal 0.00260 0.0565 0.167
Yeast Extract 0.000541 0.011 0.0360
Yeast Extract + coal 0.00314 0.0628 0.201
Soytone 0.00164 0.033 0.108
Soytone + coal 0.00267 0.0534 0.187
Tryptone 0.000684 0.014 0.044
Tryptone + coal 0.00393 0.0786 0.253
-aYield on day 68 of Example 8.
[00107] A lower concentration of amino
acids could be used to stimulate coal
seam microorganisms, and methane production could be increased to high levels
with periodic dosing or feeding with the amino acids mixture at the same lower
concentration than the highest concentration observed to produce the greatest
enhancement in methanogenesis rates. Using a lower concentration of amino
acids would be more economical than a higher concentration. Another advantage
of using a lower concentration of amino acids in dosing the coal seam is the
generation of lower amounts of acetic acid. As
discussed above, the
accumulation of large amounts of VFA such as acetic acid may inhibit
methanogenesis.
[00108] The
Examples demonstrated how the addition of the amino acids
greatly enhanced methane production. The amino acids were acting as a source
of nitrogen for the cultures. This indicates the coal-only cultures were
lacking a
nitrogen source and the presence of the amino acids increased the carbon to
nitrogen ratio to acceptable levels.
[00109] As the
growth Example demonstrated, the addition of these complex
nitrogen sources to the coal cultures all stimulated and enhanced
methanogenesis
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over the culture growing with only the complex nitrogen sources (without
coal).
Some methane was generated in the nutrient-only cultures, but significantly
higher
methane was produced in cultures given the coal as the carbon source. The
greatest methane production amongst the nutrient and coal cultures occurred
with
the cultures given the amino acids mixture (although the amino acids mixture
was
added at a slightly higher concentration than the other nutrients (0.006 g/ml
compared to 0.005 and 0.0045, respectively).
[00110] Stage 1 was done in small glass bottles at atmospheric pressure
with small amounts of crushed coal (for increased surface area). However, in
coal
containing formations, the amount of coal is large and solid and has reduced
surface area as compared to the crushed coal. Furthermore, coalbeds are often
located from 500 to 1000 meters below the surface and thus elevated pressures
are often present.
STAGE 2 OF THE EXAMPLES
[00111] In Stage 2, batch Examples were done to simulate in-situ
conditions
and to evaluate the performance of the amino acids mixture under these
conditions. A ramped experimental process was used whereby growth studies
proceeded from glass bottles and crushed coal at atmospheric pressure to
stainless steel vessels and crushed coal at elevated pressures to finally
larger
stainless steel vessels and coal cores at elevated pressures. To carry out
these
Examples cultures were grown in specially-designed growth vessels that can be
pressurized to a maximum of 3000 psi (207 bars). Both crushed and consolidated
coal cores saturated with a mineral salts medium were tested. The effects of
adding the amino acids mixture on methane production were determined by
monitoring pressure and gas composition changes.
[00112] Elevated pressure growth Examples were conducted using stainless
steel pressure vessels (available by Parr Instrument Company, Illinois), with
either
a capacity of 300 ml or 1000 ml and a maximum pressure rating of 3000 psi (207
bar).
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[00113] All the
vessels were operated in batch mode and incubated in a
water bath set at 30 C. The volume of gas sample removed from each vessel for
analysis was recorded and taken into account in the yield calculations.
Pressure
transducers monitored pressure changes within the vessels.
[00114] If
vessels were to be amended with the amino acids solution, the
vessels were depressurized to atmospheric pressures. This allowed the addition
of the nutrient through a port without addressing high pressures. The vessels
were then re-pressurized to the selected pressure.
[00115] To detect
and quantify the components of the culture headspace
gas, a Hewlett Packard QUADH Micro Gas Chromatograph (CG) was used. This
GC contained two columns; a molecular sieve column and a Poraplot U column.
Helium was the carrier gas and a 30 second sampling time was used. The
columns were able to detect H2, 02, N2, CH4, CO2 and H2S.
[00116] Carbon
isotope ratios were obtained with a Finnigan-MAT 252 GC-C
CF IRMS CONFLO II system. The gas chromatograph was equipped with a
PLOT fused silica capillary column (27.5 m X o.45 mm, 0.32 ID. Carbon isotope
compositions are reported as 513C values in ppt (%0) relative to the PDB
international standard.
Reproducibility of the 813C values was 0.2 %o for
methane and CO2. Acetic acid and ammonia were measured using analysis kits
manufactured by Meg azyme (www.megazyme.corn).
EXAMPLE 9
[00117] Example 9
compared methane production rates in two 300 ml
vessels, A and B, which each contained 10 g of crushed coal, 100 ml of MSM
growth medium, and was inoculated with the same culture, S24C160. Vessel A
received the amino acids mixture (final concentration of 0.006 g/m1), whereas
vessel B did not. The vessels were initially pressurized to 24 psi with 100%
oxygen-free nitrogen. After a week of incubation, the pressure was increased
to
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50 psi and, after another week of incubation, to a final pressure of 100 psi.
This
period signified the first growth period and lasted 55 days.
[00118] On day 56, Vessel A was depressurized and "fed" 10 ml of the
amino acids mixture so that a final concentration of 0.006 g/ml "fresh" amino
acids
was obtained in the vessel. Vessel 6 was also depressurized at the same time
as
Vessel A, but was not given the amino acids, instead it was given an equal
volume of the mineral salts medium. The vessels were then pressurized with
nitrogen to 150 psi. This signified the second growth phase, from days 56 to
146
for Vessel A and days 56 to 138 for Vessel B. During the de-pressurization,
the
headspace gas was slowly vented. There was still some residual methane in the
vessel after the de-pressurization was complete and the vessel re-pressurized.
That is why on the methane production graph (Figure 11) the methane line did
not
start at 0 mmol during the second growth phase.
[00119] On day 139, Vessel B was fed 11 ml of the amino acids mixture for
a
final concentration of 0.006 g/ml. On day 146, Vessel A received its second
dose
of the amino acids solution (final concentration of 0.006 g/ml). Both vessels
were
pressurized to 150 psi after dosing and incubated for a total time of 208 days
(Figure 11).
[00120] The presence of the amino acids greatly increased methane
production over the culture with no amino acids amendment. There was a 180-
fold increase in methane production in the amino acids-amended vessel A than
in
the un-amended vessel B during the first growth phase; 0.266 compared to
0.00148 mmol/day, respectively (Table 18). The methane production rate in the
amino acids amended-vessel (Vessel A) tapered off after 50 days of incubation.
Upon the first dose of the amino acids to Vessel A (the second growth phase),
the
methane production rate decreased by slightly less than half (from 0.266 to
0.147
mmol/day) and tapered off after 56 days from time of dosage. The methane yield
also dropped slightly from 8.80 in the first phase to 8.22 mmol in the second
phase. After the second dose of amino acids to Vessel A on day 147, the
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methane production rate increased from 0.147 (second phase) to 0.153
nn m o lid a y
,[00121] The methane production rate in the un-amended vessel (Vessel B)
remained very low during the first two growth phases, although there was a
slight
increase in the rates (from 0.00148 to 0.00186 mmol methane/day) after the
addition of the mineral salts medium solution on day 56. The overall methane
yield decreased, however, during the second growth phase. Upon the addition of
the amino acids solution to Vessel B at the start of the third growth phase,
the
methane production rate increased, approximately 200-fold, from 0.00186 to
0.403 mmol/day. The highest methane yield (11.5 mmol methane on day 208)
was recorded in this vessel during the entire course of Example 9.
Table 18. Methane production rates and yields of a methanogenic culture
(S24C160) grown at
elevated pressures with crushed coal and amino acids mixture (Vessel A) and
with coal only
(Vessel B).
¨ ¨ - ¨ - -
First phase:¨ - ¨Second Phase: Third Phase:
Days 0-55 Days 56-146 (A) and Days 147-
208 (A) and
100psi 56-138 (B) 139-208 (B)
150 psi, Vessel A dosed 150 psi, Vessels A,B dosed
Rate Yield' Rate Yielda Rate Yields
Vessel (mmol/day) (mmol) (mmol/day) (mmol) (mmol/day)
A 0.266 8.80 0.147 8.22 0.153 8.09
0.00148 0.18 0.00186 0.127 0.403 11.5
'Methane yield at end of each phase.
[00122] Culture fluid was analyzed at the end of Example 9 for acetic acid
and ammonia concentrations and pH (Table 19). Vessel A, which had three
additions of amino acids compared to a single dose to Vessel B, had the
highest
amount of acetic acid (0.125 g/L) and ammonia levels (2.783 g/L) in the
culture
fluid at the end of the Example. Vessel B had 0.013 g/L of acetic acid and
1.034
g/L ammonia. The pH of the culture fluid in Vessel A was also higher than in
Vessel B (7.68 compared to 7.36, respectively).
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Table 19. Comparison of acetic acid and ammonia levels and pH of culture fluid
in Vessels A and
B after 230 days of incubation (inoculated crushed coal at elevated
pressures).
Vessel Acetic Acid (g/L) Ammonia (g,/L) PH
A 0.125 2.783 7.68
0.013 1.034 7.36
[00123] Periodic gas samples were taken from the vessels and the isotopic
composition of the methane and CO2 were analyzed. Figure 12 shows the results
of the isotopic analysis. The 813CcH4 in Vessel A stabilized at approximately -
34.00 to -35.00 %o during the first growth phase. With each subsequent amino
acids dosage in phases 2 and 3, the 513CcH4 became more negative from -52.30
to -66.90 %o, respectively. The 813Cc02 values became more positive after each
amino acids dose. Isotopic analysis of the methane produced in Vessel B varied
considerably over the first growth phase, from -48.97 to -16.60 %o and finally
to -
42.17 on day 55. During the second growth phase, the 813CcH4 was more
negative than in the first phase and remained fairly stable from -53.17 to -
55.03
%o. Upon the addition of the amino acids in the third phase, the 513CcH4
shifted
from -54.99 to -30.59 %o with a final 4313CcH4 value of -37.54 %o on day 208.
This
shift in 513CcH4 values mirrored what occurred in Vessel A during the first
growth
phase. The 813Cc02 in Vessel B became slightly positive during each growth
phase, though the values were not as positive as in the 813Cco2 values in
Vessel
A.
[00124] In Example 9, two vessels were used to grow the S24C160 culture
at elevated pressures. Both vessels were identical in terms of growth medium,
inoculum, crushed coal, headspace gas and pressure, except one vessel was
given the amino acids mixture (Vessel A), the other was not (Vessel B). The
presence of the amino acids caused a methanogenesis enhancement ratio of 180
over the non amino acids-amended vessel when both vessels were at 150 psi.
[00125] Vessel A was dosed with the amino acids mixture during the course
of Example 9 to re-stimulate the microbial culture. In Vessel B, the culture
was
able to produce some methane but was essentially non-active. However microbial
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activity and methane production was activated with little lag time (7-10 days)
upon
the addition of the amino acids mixture. This indicates that microbial cells
can
remain inactive or dormant for lengthy periods of time and then become quickly
active when environmental conditions change that allow opportunities for
growth.
[00126] Stable
isotope analysis of the headspace gas during the course of
Example 9 can give an indication of methanogenesis pathways. The different
isotopic forms of methane exhibit virtually identical chemical behaviour but
have
different masses. Therefore, measurements of the ratios of 13C to 12c, 14c to
12c
and deuterium (D) to 1H in the individual atoms of methane can be used to
reveal
clues as to the sources of methane. The 813C of methane in the deep subsurface
is commonly measured to determine whether the gas is biogenic or
thermocatalytic in origin. Thermogenic methane is generally, but not
exclusively,
enriched in 13C compared with bacterial methane. These isotopic ratios vary
because kinetic processes such as bacterial reactions preferentially use the
lighter
isotope of an element due to a lower activation energy for bond breaking and
because isotopic exchange occurs between different chemical substances,
different phases, or individual molecules as chemical processes move toward
'isotopic equilibrium. It is
also possible to distinguish between acetate
fermentation and bacterial carbonate reduction. In bacterial carbonate
reduction
the methane is generally more depleted in 13C and enriched in D.
[00127] The
repeated addition of the amino acids to Vessel A (originally
given amino acids at the start of the Example) caused a shift in the 813CcH4
towards more negative values and towards a typical 513CcH4 value observed in
cultures using the fermentative pathway for methanogenesis. Since the culture
vessel was operated in a static or batch mode, the repeated additions of the
amino acids led to a build-up of amino acids breakdown products (there was
0.125 g/L acetic acid in Vessel A at the end of the Example compared to only
0.013 g/L acetic acid in Vessel B) and this may have been the cause in the
shift in
methanogenesis from a predominately carbonate reduction to a fermentative
pathway.
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EXAMPLE 10
[00128] Example 10 involved using four 1000-ml vessels. Each vessel
received a coal core. These Alberta cores came from an ARC-led project on CO2
storage in coal beds. The cores were approximately 4 inches in height and 3.0
inches in diameter. Table 20 gives a description of each of the cores. Enough
MSM growth medium (340 ml) was added to submerge the core. The headspace
gas was N2.
Table 20. Description of the cores used in the high-pressure growth Examples.
Vessel Description of coal core
A Shiny, 3.5 inches height, 3 inches diameter, broken on top
B Shiny, 5 inches height, 1.75 inches in width. Slabbed core, mud on
surface was
washed off with distilled water. Core was divided into two and placed side by
side in
vessel.
C Shiny, 4 inches height, 3 inches diameter. Removed part of top of
core in order for it
to fit in vessel better.
D Dull, 3.5 inches in height, 3 inches in diameter. Clay/mud present.
[00129] The next step in the progression towards simulating in-situ
conditions was to use coal cores instead of crushed coal under elevated
pressures. Therefore, the effect of amino acids addition on methanogenesis by
both indigenous and introduced methanogenic consortia growing in the presence
of large, solid pieces of coal, i.e. coal cores, was investigated using four
1000-ml
high-pressure vessels. The Example design is summarized in Table 21.
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Table 21: Example design of the high-pressure growth study using coal cores.
The inoculum used
was the S24C160 culture.
11
Vessel Culture Media Components Coal Purpose of Vessel
Weight (g) ,
A Mineral salts medium 432.0 Measures background methane
levels present in coal (i.e. degassing
and some biogenic production).
B Mineral salts medium, inoculum 385.2 Measures biogenic methane
production from coal after
inoculation with methanogenic
culture
C Mineral salts medium, 508.4 Measures the effect of amino
acids
inoculum, amino acids mixture on biogenic methane production 1
from coal by introduced
methanogenic culture.
D Mineral salts medium, amino 762 Measures whether possible
acids mixture indigenous microbes can be
stimulated into methane production ,
by the addition of amino acids. (
[00130] As in Example 9 using crushed coal, Example 10 was also divided
into several growth phases (Figure 13). During the first growth phase, the
vessels
were all pressurized to 100 psi with nitrogen (the vessels were initially at
20 psi
only during the first week of incubation). After 51 days of incubation, the
pressure
inside the four vessels was increased to 200 psi. Vessels A and B remained at
this pressure, growth phase 2, for the duration of the Example (132 days).
Vessels C and D, on the other hand, remained at 200 psi until day 79 (growth
phase 2a) when they were de-pressurized to allow the addition of the amino
acids
mixture to a final concentration of 0.006 g/ml. Vessels C and D were then
pressurized up to 200 psi and incubated until a total time of 132 days had
elapsed
(growth phase 3).
[00131] Methane production was detected in all of the vessels as indicated
in
Figure 13. During the first growth phase, the lowest methane production rates
and yields (0.014 mmol/d and 0.688 mmol after 51 days, respectively) was
observed in Vessel A, which consisted of only the core in the mineral salts
medium (Table 22). Methane continued to be generated in Vessel A when it was
pressurized to 200 psi, but the production rate declined with time at this
higher
pressure (from 0.014 to 0.006 mmol/day). The inoculated vessel, B, had a 2.4 -
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fold higher methane production rates than the un-inoculated vessel A. The rate
decreased as well when the pressure was increased to 200 psi from 0.033 to
0.11
mmol/day, respectively. The methane yields in Vessel B were 4- and 3-fold
higher
than in A at 100 and 200 psi, respectively.
[00132] Vessel C, which was inoculated and given the amino acids mixture,
had a 16-fold increase in methane production rate and 6.5-fold increase in
methane yield over Vessel B which had the inoculum but no amino acids
addition.
When Vessel C was pressurized up to 200 psi, methane continued to be
generated, though at a slower rate (0.214 mmol/d, Table 22) than during the
first
growth phase at 100 psi (0.550 mmol/d). The methane production rate increased
slightly from 0.214 to 0.304 mmol/d when Vessel C was fed the amino acids
solution on day 79. The methane that was accumulated in the vessel during the
first two phases was vented to allow the de-pressurization of the vessel and
subsequent feeding with the amino acids solution. The highest yields were
obtained in Vessel C, between 17.94 and 19.50 mmol methane after growth
phase 1 and 2a, respectively.
[00133] The highest methane production rate was observed during the first
growth phase in Vessel D which was amended with only the amino acids mixture.
The rate was 0.763 mmol/day compared to 0.550 mmol/day in Vessel C. After 23
days of incubation it appeared that methane production in Vessel D stopped as
indicated by the plateauing curve of Vessel D in Figure 13. At the higher
pressure
of 200 psi, methane production appeared to be proceeding at a very slow rate
(0.063 mmol/day). The methane yield did not increase during this time. Methane
production began again when Vessel D was fed the amino acids mixture on day
79. The rate increased from 0.063 to 12.38 mmol/day and the yield after 53
days
of incubation from the feeding (day 112) was 16.35 mmol, surpassing that
obtained during the first growth phase (13.11 mmol after 33 days). The methane
production rate in Vessel D was greater than the rate in Vessel C during the
third
growth phase.
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[00134] It should . be noted that for both Vessels C and D, methane
production tapered off and stopped after only 14 days of incubation from when
the
pressure inside the two vessels was increased to 200 psi. At the lower
pressure
of 100 psi, it took 51 and 23 days for the methane production rates to slow
down
or stop in Vessel C and D, respectively.
Table 22. Methanogenesis rates and yields of methanogenic cultures growing in
the presence of
coal cores with and without the amino acids mixture at elevated pressures.
First Phase, Second Phase, Second Phase (2a),
Third Phase, !
100 psi 200 psi 200 psi 200 psi
Days 0-51 Days 51-112 Days 51-79 Days 79-132
Rate Yield' Rate Yield' Rate Yield' Rate
Yield
(mmol/ (mmol) (mmol/ (mmol) (mmol/ (mmol) (mmol/ (mmol)
Vessel day) day) day) day)
A 0.014 0.688 0.006 0.988
0.033 2.73 0.011 3.08
0.550 17.94 0.214 19.50 0.304
16.06
0.763 13.11 0.063 12.38 0.481
16.35 ,
'Methane yield at end of each phase.
[00135] The culture fluid was examined at the end of the Example for pH
values and for amounts of acetic acid and ammonia (Table 23). As was observed
with the vessels containing the crushed coal, those vessels that had the amino
acids additions (C and D) had the highest concentrations of acetic acid and
ammonia. Vessel C had 6.7-fold higher amounts of acetic acid than Vessel D
(0.128 compared to 0.019 g/L, respectively). Likewise, ammonia levels were
also
higher in Vessel C than in D (6.7-fold difference). The highest pH (8.03) was
recorded in Vessel A which contained only the core, the lowest pH (7.40) was
recorded in Vessel D.
Table 23. Comparison of acetic acid and ammonia levels and pH of culture fluid
in Vessels A-D
after 139 days of incubation (coal cores at elevated pressures).
Vessel Acetic Acid (g/L) Ammonia (g/L) pH
A 0.002 0.047 8.03
0.006 0.125 7.81
C 0.128 1.183 7.48
D 0.019 0.177 7.40
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[00136] The headspace gas from the vessels was also analyzed for the
stable isotope composition of methane and CO2. A "time zero" isotope analysis
was done on the headspace gas at start-up of the Example. There is always
some carry-over of methane and CO2 from the inoculum, so the resulting isotope
data reflects "old" or residual methane/CO2 and should be considered as
background levels and not a true representation of new methane/CO2 isotope
data.
[00137] The 813CcH4 and 813Cco2 varied quite a bit from each vessel and
over time within each vessel. In vessel A (no amendments) the 813CcH4 started
at
-32.88 %0 and then decreased to -55.92 % by day 20 (Figure 14). As the Example
progressed the 513CcH4 value stabilized to around -38.00 %0. The 513Cco2
remained fairly constant at -20.00 % over the course of the Example.
[00138] In vessel B, the 513CcH4 showed an opposite trend to that in
vessel
A. The 813CcH4 started off at a low value of -42.32 % and increased to -24.96
%
by 15 days (Figure 15). Upon further incubation and elevation of vessel
pressure
to 200 psi, the 513CcH4 stabilized to approximately -38.00 %G. The 813Cco2
changed quite dramatically when compared to vessel A. During the first growth
phase at 100 psi, the 813Cco2 became more positive changing from -16.26 to -
2.98
%0 over the 50 days. The pattern of high 813Cco2 and low o13CcH4 shown in
vessel
B is indicative of a typical fermentation pathway for methanogenesis. At the
higher pressure of 200 psi, the 813Cco2 values became more negative. The
increased solubility of CO2 at higher pressures may have accounted for this
change in 813Cco2. Since there was more CO2 generated in vessel B than A
(0.230% vs. 0.040% CO2 at day 112, respectively), there was more of an
isotopic
effect/solubility effect in B than in A.
[00139] The two vessels given amino acids, vessels C and D, showed
different isotope fractionation results from each other. Vessel C, which was
inoculated with the S24C160 culture, had residual 813CcH4 of -36.02 %. (time
zero)
which within 10 days decreased to -51.71 % and, with time, became more
positive
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and settled around -37.00 Ifoc; (Figure 16). Upon the addition of the amino
acids
mixture on day 79, the 813CcH4 became more negative, settling down to around -
47.00 %o by the end of the Example. The 813Cc02 in Vessel C became more
positive with time during the first growth phase, from -15.20 % to -9.87 %o.
The
addition of the amino acids mixture on day 79 caused the 813Cco2 value to
become more negative (-19.48 %o), but with time this value became more
positive.
There developed a big difference in the 813CcH4 to .513Cco2 values in Vessel
C.
[00140] In contrast, there was less of a spread in the E=13CcH4 and
813Cco2
values in Vessel D (Figure 17). Vessel D was given the amino acids solution
only,
and presumably stimulated a microbial community present on the coal core. The
813CcH4 formation follows the same pattern as that observed in Vessel A (no
amendments). The 813CcH4 changed from -50.75 %o at the start of the Example
(day 7) and increased to -24.94 %o by day 20. The 513C0H4 remained fairly
stable
at -26.00 Too, including when the vessel was pressurized to 200 psi. This
roughly
corresponded to the period within the vessel when methane production had
ceased (Figure 13). Upon the addition of the amino acids on day 79, the
813CoH4
decreased to -37.13 % and then by the end of the Example had increased to -
28.28 %0. In contrast, the 813Cco2 remained fairly constant after stabilizing
within
the first 20 days of incubation to approximately -14.00 /00.
[00141] The methane observed in Vessel A which was not amended with the
amino acids or inoculated with the culture most likely arose from residual gas
still
desorbing off the coal. Even though the 813CcH4 seemed to vary a bit during
the
first growth phase, it stabilized at approximately -38.00 %o by day 50 and
remained
at this value for the duration of the Example. The .513Cc02 remained quite
negative
(approximately -20.00 %o). A 8"Cco2 value that becomes positive with time is
indicative of biogenic processes since 12C is preferentially used by microbes
over
13c.
[00142] Inoculating the core with the methanogenic culture, S24C160,
resulted in an increase in methane production over the un-inoculated core (2.4-
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fold increase). Compared, however, to those vessels (C and D) given amino
acids, the methane production rate and yield in Vessel B was low. As in
Examples 1-9, in Example 10 the addition of the amino acids mixture
significantly
increased methane production rates and yields. The core in Vessel D harboured
a methanogenic consortium that was stimulated by the amino acids mixture. It
is
very likely that all the cores had microorganisms associated with the coal but
they
were not very active or low in numbers. The amino acids stimulated microbial
growth of the hydrolytic bacteria resulting in the creation of substrates for
methanogenesis. There were two different cultures in Vessels C and D as the
stable isotope data indicated. Different 613CcH4 and 513Cco2 values were
generated
over time in each vessel. If the same types of bacteria were in each vessel,
then
the isotope values would be similar. The methanogenic consortium, S24C160,
used to inoculate Vessel C was slightly more active than the one that was
stimulated in Vessel D. S24C160 was enriched from coal and through continuous
culturing in the laboratory was very active and adapted to growing with coals
and
the amino acids mixture.
[00143] Overall,
stage 2 demonstrated that the cultures could grow and
produce methane at elevated pressures as would be encountered in the deep
subsurface. The addition of the amino acids mixture greatly stimulated
microbial
activity.
[00144] The
methane production rates and yields observed in the high-
pressure growth vessels using coal cores may translate into economical and
commercially viable rates and yields in the field. Factors
such as coal
permeability, groundwater effects, transport, etc. are assumed to have
negligible
effect on methanogenesis. Table 24 summarizes the extrapolated methane rates
of production and yields from the high-pressure vessels containing cores (See
Example 10 Vessels A-D). Methane yields are given in standard cubic feet
(scf),
the unit used by the CBM industry. Mscf is an abbreviation for a thousand
standard cubic feet; MMscf is an abbreviation for a million standard cubic
feet.
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Table 24. Extrapolated methane yields and rates of production from the high-
pressure growth
vessels containing coal cores.
scf/tonne scf/CBIVI SCF/tonne Mscf/ CBM MMscf/ CBM
Vessel' nib coalb reservoir' coal/day Reservoir/day' Reservoir/year'
A 7.79e 1.64 0.80e6 0.012 5.90 2.20
4
2.43e 5.11 2.50er 0.039 19.30 7.00
3
1.27e" 26.7 13.20e6 0.20 98.74 36.00
2
1.29e 27.1 13.40e6 0.21 103.70 39.00
2
'Vessel A, core only; Vessel B, core + inoculum; Vessel C, core, inoculum, and
amino acids
mixture; Vessel D, core + amino acids mixture.
bAfter 132 days of incubation, 200 psi
'Assume a 10 m thick coal covering 10 acres with a coal tonnage of 493,714
tonnes
[00145] Using
Vessel D's extrapolated methane production rate, 39
MMscf/day methane could be produced from a large volume of coal. The data
from the coal core Examples at elevated pressures can also be used to
determine
how much of the amino acids mixture could be used for a field application. A
total
volume of 340 ml of the mineral salts medium (which contained 0.006 g/m1 of
the
amino acids mixture) was used to submerge and saturated an average mass of
coal of 744 g (an average of the Vessel C and D's cores). The volume of
liquid,
based on the above ratio of 340 ml per 744 g coal, to use to saturate one
tonne of
coal is approximately 0.50 m3. If a concentrated stock of 60% (w/v) of the
amino
acids hydrolysate is used to make up a 0.006 g/m1 solution (for a total volume
of
0.50 m3), then 5.0 litres of the hydrolysate is used. For a coalbed containing
10
meters of coal, covering an area of 10 acres for a total coal tonnage of
493,714
tonnes, the volume of liquid used to saturate the coal would be 2.5e5 m3. To
make an amino acids solution of 0.006 g/ml, 2,269 m3 of a 60% (w/v) amino
acids
hydrolysate is used.
[00146] The data
from the coal core Examples can also be used to
determine how many grams or kilograms of amino acids mixture would be useful
for a field application. A total volume of liquid useful to saturate one tonne
of coal
could be as low as about 0.20 m3 or as high as about 1 m3. As shown in Table
25, if a concentrated stock of 60% (w/v) of the amino acids hydrolysate is
used to
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make up a 0.006 g/ml solution, a total volume of 0.2 m3 would require 1.2
kilograms of amino acids. Similarly, assuming a 1.0 m3 volume to saturate a
metric tonne of coal, 30 kilograms of amino acids hydrolysate is used for a
final
concentration of 0.03 g/ml.
[00147] For a
range of grams of amino acids hydrolysate, such as from
about 600 grams to about 15 kilograms, the final volume to saturate a tonne of
coal may include the range of about 0.2 m3 to about 1.0 m3, then th e final
concentration of amino acids hydrolysate may include the range of about 0.003
g/m1 to about 0.015g/ml. For a range of grams of amino acids hydrolysate, such
as from about 1.2 kilograms to about 6 kilograms, again the final volume to
saturate a tonne of coal may include the range of about 0.2 m3 to about 1.0
m3,
therefore the final concentration of amino acids hydrOlysate may include about
0.006 g/mi.
Table 25. Weight of Amino acids per Metric Tonne of Coal.
Final Grams / Kilograms of Amino acids per Metric Tonne of Coal
Concentration
of Amino
acids 0.2 m3 Final Volume 0.5 m3 Final Volume 1.0 m3 Final
Hydrolysate Volume
0.03 g/m1 6.0 kg 15 kg 30 kg
0.015 g/m1 3.0 kg 7.5 kg 15 kg
0.006 g/m1 1.2 kg 3.0 kg 6.0 kg
0.003 g/m1 600 g 1.5 kg 3.0 kg
0.0003 g/m1 60 g 150g 300g
[00148] A tonne
refers to a metric tonne which is a measurement of mass
equal to 1,000 kilograms. With an average mass of 744 g coal core, a
multiplier of
1292 extrapolates the average coal core mass to a tonne. Using the same 1292
multiplier, 440,000 ml could be used to saturate a tonne of extrapolated coal
core
since 340 ml can be used to saturate the average mass of 744 g coal core.
[00149] The
foregoing are examples of aspects of the invention and the
invention should not be interpreted as limited to the specific examples
provided
herein.
49
