US20100252255A1

US20100252255A1 - Increased microbial production of methane gas from subsurface hydrocarbon containing formations

Info

Publication number: US20100252255A1
Application number: US11/798,937
Authority: US
Inventors: Karen Budwill; Twyla Malcolm
Original assignee: GREEN EARTH INDUSTRIES LLC; Alberta Research Council
Current assignee: GREEN EARTH INDUSTRIES LLC; Alberta Research Council
Priority date: 2006-05-17
Filing date: 2007-05-17
Publication date: 2010-10-07
Also published as: CA2652144A1; WO2007136716A3; AU2007254192B2; WO2007136716A2; AU2007254192A1; AU2007254192B9; CA2652144C

Abstract

Methods and compositions are provided for increasing methane gas produced or released from a subsurface hydrocarbon containing formation containing methanogenic microorganisms. The methods and compositions provide using an amount of amino acids effective to increase methane gas released from the formation.

Description

This application claims priority to U.S. Provisional Patent Application Ser. No. 60/800,857, filed May 17, 2006 and U.S. Provisional Patent Application Ser. No. 60/808,110, filed May 25, 2006, the disclosures of which are expressly incorporated by reference herein in their entireties.

FIELD

The present invention relates to increased microbial release and production of methane gas from subsurface hydrocarbon formations.

BACKGROUND

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.
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.
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 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

FIG. 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.

FIG. 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 (ml) amino acids, 0.5 g coal, 0.003 g/ml amino acids, or nothing.

FIG. 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.

FIG. 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/ml amino acids. ARC S1 is an abbreviation for ARC Sample 1. Obed MS is an abbreviation for Obed Mine Sludge.

FIG. 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.

FIG. 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 CH₄values represent the day 125 measurement.

FIG. 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 9.0.

FIG. 8 is a graph showing mean methane production (in mmol) in coal methanogenic cultures adjusted to different salinities with NaCl and with and without amino acids.

FIG. 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 NaCl.

FIG. 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.

FIG. 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.

FIG. 12 is a graph showing stable isotope values of CH₄and CO₂produced in Vessel A and B over time. dC1 is an abbreviation for δ¹³C_CH4. dCO2 is abbreviation for δ¹³C_CO2.

FIG. 13 is a graph showing methane production (in mmol) in vessels A-D containing coal cores at elevated pressures.

FIG. 14 is a graph showing δ¹³C_CH4and δ¹³C_CO2values generated over time in Vessel A (core only). dC1 is an abbreviation for δ¹³C_CH4. dCO2 is an abbreviation for δ¹³C_CO2.

FIG. 15 is a graph showing δ¹³C_CH4and δ¹³C_CO2values generated over time in Vessel B (core+inoculum). dC1 is an abbreviation for δ¹³C_CH4. dCO2 is an abbreviation for δ¹³C_CO2.

FIG. 16 is a graph showing δ¹³C_CH4and δ¹³C_CO2values generated over time in Vessel C (core+inoculum+amino acids).

FIG. 17 is a graph showing δ¹³C_CH4and δ¹³C_CO2values generated over time in Vessel D (core+amino acids).

SUMMARY

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.
The invention further provides that the methanogenic microorganisms are naturally occurring in the formation and have not been removed from the formation.
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.
The invention also provides increasing the methane release from a formation by further decreasing the amount of amino acids.
The invention further provides a subsurface coal 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.
The invention further provides methane gas that is obtained by the methods provided herein.

DETAILED DESCRIPTION

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.
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, 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.
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.
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.
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 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.
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.
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.
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.
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.
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.
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.
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 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.
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.
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.
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.
A common methanogenic microorganism pathway uses CO₂-type substrates in a carbonate reduction pathway to produce methane:
CO₂₊₄H₂→CH₄₊₂H₂O
Methanogenic microorganisms also cleave acetate to CO₂plus CH₄in what is called the acetoclastic or fermentative pathway:
CH₃COO⁻+H₂O→CH₄+HCO₃ ⁻
In the above reaction, the CO₂is shown as bicarbonate (HCO₃ ⁻) because carbon dioxide is predominately bicarbonate in neutral or slightly alkaline water.
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.
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.
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.
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.
In an aspect of the invention, fish amino acids are used having the following distribution shown in the below Table 1:


		Percentage of Amino acids to
Amino acids	Abbreviation	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	OHpro	3.4
Taurine	Tau	3.4

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 size and wherein 50 percent or more of amino acids used are 1,000 daltons or less in size.
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 A1 to Soerensen et al., the contents of which are expressly incorporated herein by reference.
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.
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 elow 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.
Based on this discovery, aspects of the invention include using the following amounts of amino acids per metric tonne of coal: less than 30 kilograms 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.
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.
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 m³of 60% amino acids hydrolysate per tonne of coal. For instance, 3 kilograms of amino acids hydrolysate corresponds to 0.005 m³of 60% amino acids hydrolysate at the same final volume of 0.5 m³.
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).
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.
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.
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.
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.
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

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, including looking at dosing, pH, salinity and coal rank effects and how the presence of the amino acids mixture influenced methanogenesis under these different conditions.
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

S24C160	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.

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.
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 O₂-free 100% N₂was bubbled through the liquid. The medium was transferred to serum bottles sparged with O₂-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.
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 Examples and dilutions were made and kept frozen until needed. As well, aliquots of the original, un-diluted hydrolysate were kept frozen.
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.
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

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 CO₂yields were very high in all of the cultures tested.
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 (FIG. 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 0.404 mmol 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).
CO₂production decreased in the cultures given the diluted amino acids mixture. CO₂yields 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 CO₂for 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 CO₂yield 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.

Methanogenesis rate

Yield (mmol)^a

Culture Amendment	(mmol CH₄/day)	Methane	CO₂

0.03 g/ml amino acids	0.000366	0.022	0.392
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

^aYield on day 74.

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 was 0.0046 mmol methane/day and at 0.0003 g/ml the rate was only 0.00083 mmol methane/day.

Example 2

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).

Amendments

		Amino acids
Culture	Coal	Mixture	Purpose/Comment

Coal and amino	+	+	Measures effect of amino acids addition on methane
acids (0.003 g/ml)			production from coal.
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.

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 (FIG. 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 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/ml amino acids, 0.5 g coal, 0.003
g/ml amino acids, or nothing.

	Methanogenesis rates	Methane Yield^a
Culture	(mmol/day)	(mmol)

Coal and amino	0.0049	0.258
acids (0.003 g/ml)
Coal	0.000096	0.005
Amino acids	0.001	0.0802
(0.003 g/ml)
No additions	0.0000112	0.00089

^aYield on day 74 of the incubation period.

Under methanogenic conditions, amino acids are fermented to volatile fatty acids (VFA) and finally to methane and CO₂by 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 et 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.
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 et 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.
At a concentration of 0.03 g/ml 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 CO₂was produced. CO₂is one of the products of the Stickland reaction. All the cultures tested at the highest amino acids concentration (0.03 g/ml) did not produce methane.

Example 3

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/ml 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.

	Original Amino acids Dose	First Dose	Second Dose
Culture	(Time Zero)	(Day 88)	(Day 196)

0.03 + coal	0.03 g/ml	0.03 g/ml	0.006 g/ml
0.006 + coal	0.006 g/ml	0.006 g/ml	0.006 g/ml
0.003 + coal	0.003 g/ml	0.003 g/ml	0.006 g/ml
0.0003 + coal	0.0003 g/ml	0.0003 g/ml	0.006 g/ml

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 (FIG. 3) from 1.2-fold to 3.1-fold increase.
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)

	Original Amino
Culture	acids dosage	First Dosage	Second Dosage (Days	No Dose
(mg/ml	(Time Zero-Day 88)	(Days 88-196)	196-264)	(Days 88-264)

amino		Yield		Yield		Yield		Yield
acid)	Rate	(day 88)	Rate	(day 196)	Rate	(day 264)	Rate	(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	n.a.^b	0.00243^c	0.473^c
coal				(0.259)
0.0003 +	0.000556	0.0515	0.000835	0.144	0.017	1.303	0.000298	0.1657
coal				(0.0837)

^aValues 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.
^cTime period from Day 88-160.

The CO₂production 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 CO₂production 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 CO₂but at correspondingly lower amounts. With each dose of the amino acids, CO₂production increased as compared to the un-dosed cultures. The following table (Table 8) compares the production rates of CO₂in the cultures before and after amino acids additions.

TABLE 8

CO₂production rates and yields of the S24C160 cultures amended with different amino
acids concentrations before and after dosing with the amino acids solution.

CO₂Production Rates (mmol CO₂/day)and Yield (mmol CO₂)

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^c	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)

^aValues in brackets are the CO₂yield 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.
^cTime period from Day 88-160.

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.

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
(mg/ml amino	Acetic acid (g/l)		Ammonia (g/l)

acid)	Dosed	Un-dosed	Dosed	Un-dosed

0.03 + coal	16.41	8.15	7.10	5.01
0.006 + coal	3.16	4.42	2.88	3.22
0.0003 + coal	0.89	0.00	0.95	0.02

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/ml amino acids cultures over the un-dosed cultures, respectively.

Example 4

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 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.
The presence of amino acids had a significant effect on enhancing methanogenesis over those cultures not given the amino acids mixture (FIG. 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	Yield^a
Culture	(mmol CH₄/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

^aYield on day 36.

Example 5

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 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.
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
			matter	Carbon
Coal	Source	Rank	(%)	(%)

Lignite	Poplar River mine,	Lignite	56-64	na^a
	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	42-44	70
Valley	Luscar Mines Ltd.,	bituminous C
	Alberta
Cardinal	Cardinal River Mine,	Medium-volatile	24-28	80
River	Luscar Mines Ltd.,	bituminous
	Alberta

^aInformation not available

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 (FIG. 5). Only the Wabamum coal cultures with no amino acids did not show any methanogenic activity.
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 0.65 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 g/ml amino acids mixture.

	Methanogenesis rate	Yield^a
Culture	(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

^aYield on day 123.

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) (FIG. 6). In the cultures with no coal, the pH values were generally lower than in the amino acids-amended cultures. In those cultures, such 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.
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.
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 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

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 9.0. Two culture series were prepared, one series received only Obed coal, and the other received Obed coal and 0.006 g/ml of the amino acids mixture. S24C160 served as the source of the methanogenic culture.
Those coal cultures that received the amino acids mixture had significantly higher methane yields than those coal cultures without the amino acids mixture (FIG. 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.
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 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

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 7.0. 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

Example 7 was done to determine the effect of increasing salinity on methanogenesis and how the presence of the amino acids mixture affects culture 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 (NaCl) 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 NaCl. One portion did not receive any NaCl and these culture bottles served as the control (0.05 mg/ml NaCl 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.
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 NaCl, to the control. As evident in FIG. 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.
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 NaCl 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 NaCl 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.

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.

	Methanogenesis Rate	Yield^a
Culture Amendment	(mmol CH4/day)	(mmol)

Control	0.00034	0.050
Control + amino acids	0.00390	0.452
0.5 mg/ml NaCl	0.00026	0.036
0.5 mg/ml NaCl + amino acids	0.00360	0.462
1.0 mg/ml NaCl	0.00021	0.027
1.0 mg/ml NaCl + amino acids	0.00400	0.477
1.5 mg/ml NaCl	0.00027	0.036
1.5 mg/ml NaCl + amino acids	0.00400	0.490
2.0 mg/ml NaCl	0.00030	0.036
2.0 mg/ml NaCl + amino acids	0.00460	0.560
4.0 mg/ml NaCl	0.00140	0.091
4.0 mg/ml NaCl + amino acids	0.00480	0.574

^aYield on day 126 of Example.

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 NaCl. 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 FIG. 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 NaCl culture with 0.0096 mmol CH₄/day generated (Table 15). The methane production rates decreased with increasing salinity.

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.

	Methanogenesis Rate	Yield^a
Culture Amendment	(mmol CH₄/day)	(mmol)

4.0 mg/ml NaCl	0.00070	0.0186
4.0 mg/ml NaCl + amino acids	0.00960	0.263
8.0 mg/ml NaCl	0.00054	0.0176
8.0 mg/ml NaCl + amino acids	0.00830	0.229
12.0 mg/ml NaCl	0.00058	0.0168
12.0 mg/ml NaCl + amino acids	0.00610	0.221
15.0 mg/ml NaCl	0.00061	0.0151
15.0 mg/ml NaCl + amino acids	0.00370	0.142

^aYield on day 42 of Example.

Overall, the highest rate of methane production for the amino acids-amended cultures was observed in the culture given 4.0 mg/ml NaCl, followed by the cultures given 8.0 and 12.0 mg/ml NaCl. 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 NaCl 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

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/ml 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.

TABLE 16

The concentration of nutrients used in the methane
production comparison Example 8.

		Final Concentration
	Nutrient/Growth Medium	in Culture Bottles

	Brain Heart Infusion (BHI)	0.0046 g/ml
	Yeast Extract	0.005 g/ml
	Soytone	0.005 g/ml
	Tryptone	0.005 g/ml
	Amino acids mixture including	0.006 g/ml
	Fish Hydrolysate

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.
Methane production was detected in all of the cultures, regardless of which nutrient broth it was given (FIG. 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.

TABLE 17

Methane production rates and yields of the S24C160 culture
incubated with the different nutrients and with and without coal.

	Methanogenesis	Methanogenesis Rate
Culture	Rate	(mmol CH₄/day/g	Yield^a
Amendment	(mmol CH₄/day)	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)
BHI + 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.

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.
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.
As the growth Example demonstrated, the addition of these complex nitrogen sources to the coal cultures all stimulated and enhanced methanogenesis 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).
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

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.
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).
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.
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.
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, O₂, N₂, CH₄, CO₂and H₂S.
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×0.45 mm, 0.32 ID. Carbon isotope compositions are reported as 8¹³C values in ppt (‰) relative to the PDB international standard. Reproducibility of the δ¹³C values was ±0.2‰ for methane and CO₂. Acetic acid and ammonia were measured using analysis kits manufactured by Megazyme (www.megazyme.com).

Example 9

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/ml), 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 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.
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 B 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 (FIG. 11) the methane line did not start at 0 mmol during the second growth phase.
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 (FIG. 11).
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 methane production rate increased from 0.147 (second phase) to 0.153 mmol/day.
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).

	Second Phase:	Third Phase:
	Days 56-146 (A) and	Days 147-208 (A) and
First phase:	56-138 (B)	139-208 (B)
Days 0-55	150 psi, Vessel A	150 psi, Vessels
100 psi	dosed	A, B dosed

	Rate	Yield^a	Rate	Yield^a	Rate
Vessel	(mmol/day)	(mmol)	(mmol/day)	(mmol)	(mmol/day)	Yield^a(mmol)

A	0.266	8.80	0.147	8.22	0.153	8.09
B	0.00148	0.18	0.00186	0.127	0.403	11.5

^aMethane yield at end of each phase.

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).

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
B	0.013	1.034	7.36

Periodic gas samples were taken from the vessels and the isotopic composition of the methane and CO₂were analyzed. FIG. 12 shows the results of the isotopic analysis. The δ¹³C_CH4in Vessel A stabilized at approximately −34.00 to −35.00‰ during the first growth phase. With each subsequent amino acids dosage in phases 2 and 3, the δ¹³C_CH4became more negative from −52.30 to −66.90‰, respectively. The δ¹³C_CO2values 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‰ and finally to −42.17 on day 55. During the second growth phase, the δ¹³C_CH4was more negative than in the first phase and remained fairly stable from −53.17 to −55.03‰. Upon the addition of the amino acids in the third phase, the δ¹³C_CH4shifted from −54.99 to −30.59‰ with a final δ¹³C_CH4value of −37.54‰ on day 208. This shift in δ¹³C_CH4values mirrored what occurred in Vessel A during the first growth phase. The δ¹³C_CO2in Vessel B became slightly positive during each growth phase, though the values were not as positive as in the δ¹³C_CO2values in Vessel A.
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.
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 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.
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 δ¹³C 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.
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 δ¹³C_CH4towards more negative values and towards a typical δ¹³C_CH4value 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.

Example 10

Example 10 involved using four 1000-ml vessels. Each vessel received a coal core. These Alberta cores came from an ARC-led project on CO₂storage 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.

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.

TABLE 21

Example design of the high-pressure growth study using coal cores.
The inoculum used was the S24C160 culture.

		Coal
Vessel	Culture Media Components	Weight (g)	Purpose of Vessel

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
			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.

As in Example 9 using crushed coal, Example 10 was also divided into several growth phases (FIG. 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).
Methane production was detected in all of the vessels as indicated in FIG. 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-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.
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.
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 FIG. 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.
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		Rate		Rate		Rate
	(mmol/	Yield^a	(mmol/	Yield^a	(mmol/	Yield^a	(mmol/	Yield^a
Vessel	day)	(mmol)	day)	(mmol)	day)	(mmol)	day)	(mmol)

A	0.014	0.688	0.006	0.988	—	—	—	—
B	0.033	2.73	0.011	3.08	—	—	—	—
C	0.550	17.94	—	—	0.214	19.50	0.304	16.06
D	0.763	13.11	—	—	0.063	12.38	0.481	16.35

^aMethane yield at end of each phase.

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
B	0.006	0.125	7.81
C	0.128	1.183	7.48
D	0.019	0.177	7.40

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 CO₂from the inoculum, so the resulting isotope data reflects “old” or residual methane/CO₂and should be considered as background levels and not a true representation of new methane/CO₂isotope data.
The δ¹³C_CH4and δ¹³C_CO2varied quite a bit from each vessel and over time within each vessel. In vessel A (no amendments) the δ¹³C_CH4started at −32.88‰ and then decreased to −55.92‰ by day 20 (FIG. 14). As the Example progressed the δ¹³C_CH4value stabilized to around −38.00‰. The δ¹³C_CO2remained fairly constant at −20.00‰ over the course of the Example.
In vessel B, the δ¹³C_CH4showed an opposite trend to that in vessel A. The δ¹³C_CH4started off at a low value of −42.32‰ and increased to −24.96‰ by 15 days (FIG. 15). Upon further incubation and elevation of vessel pressure to 200 psi, the δ¹³C_CH4stabilized to approximately −38.00‰. The δ¹³C_CO2changed quite dramatically when compared to vessel A. During the first growth phase at 100 psi, the δ¹³C_CO2became more positive changing from −16.26 to −2.98‰ over the 50 days. The pattern of high δ¹³C_CO2and low δ¹³C_CH4shown in vessel B is indicative of a typical fermentation pathway for methanogenesis. At the higher pressure of 200 psi, the δ¹³C_CO2values became more negative. The increased solubility of CO₂at higher pressures may have accounted for this change in δ¹³C_CO2. Since there was more CO₂generated in vessel B than A (0.230% vs. 0.040% CO₂at day 112, respectively), there was more of an isotopic effect/solubility effect in B than in A.
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 δ¹³C_CH4of −36.02‰ (time zero) which within 10 days decreased to −51.71‰ and, with time, became more positive and settled around −37.00‰ (FIG. 16). Upon the addition of the amino acids mixture on day 79, the δ¹³C_CH4became more negative, settling down to around −47.00‰ by the end of the Example. The δ¹³C_CO2in Vessel C became more positive with time during the first growth phase, from −15.20‰ to −9.87‰. The addition of the amino acids mixture on day 79 caused the δ¹³C_CO2value to become more negative (−19.48‰), but with time this value became more positive. There developed a big difference in the δ¹³C_CH4to δ¹³C_CO2values in Vessel C.
In contrast, there was less of a spread in the δ¹³C_CH4and δ¹³C_CO2values in Vessel D (FIG. 17). Vessel D was given the amino acids solution only, and presumably stimulated a microbial community present on the coal core. The δ¹³C_CH4formation follows the same pattern as that observed in Vessel A (no amendments). The δ¹³C_CH4changed from −50.75‰ at the start of the Example (day 7) and increased to −24.94‰ by day 20. The δ¹³C_CH4remained fairly stable at −26.00‰, including when the vessel was pressurized to 200 psi. This roughly corresponded to the period within the vessel when methane production had ceased (FIG. 13). Upon the addition of the amino acids on day 79, the δ¹³C_CH4decreased to −37.13‰ and then by the end of the Example had increased to −28.28‰. In contrast, the δ¹³C_CO2remained fairly constant after stabilizing within the first 20 days of incubation to approximately −14.00‰.
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 δ¹³C_CH4seemed to vary a bit during the first growth phase, it stabilized at approximately −38.00‰ by day 50 and remained at this value for the duration of the Example. The δ¹³C_CO2remained quite negative (approximately −20.00‰). A δ¹³C_CO2value that becomes positive with time is indicative of biogenic processes since ¹²C is preferentially used by microbes over ¹³C.
Inoculating the core with the methanogenic culture, S24C160, resulted in an increase in methane production over the un-inoculated core (2.4-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 δ¹³C_CH4and δ¹³C_CO2values 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.
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.
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.

TABLE 24

Extrapolated methane yields and rates of production from the high-pressure growth
vessels containing coal cores.

		scf/tonne	scf/CBM	SCF/tonne	Mscf/CBM	MMscf/CBM
Vessel^a	scf^b	coal^b	reservoir^c	coal/day	Reservoir/day^c	Reservoir/year^c

A	7.79e⁻⁴	1.64	0.80e⁶	0.012	5.90	2.20
B	2.43e⁻³	5.11	2.50e⁶	0.039	19.30	7.00
C	1.27e⁻²	26.7	13.20e⁶	0.20	98.74	36.00
D	1.29e⁻²	27.1	13.40e⁶	0.21	103.70	39.00

^aVessel 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
^cAssume a 10 m thick coal covering 10 acres with a coal tonnage of 493,714 tonnes

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/ml 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/ml 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 2.5e⁵m³. To make an amino acids solution of 0.006 g/ml, 2,269 m³of a 60% (w/v) amino acids hydrolysate is used.
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 m³or 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 make up a 0.006 g/ml solution, a total volume of 0.2 m³would require 1.2 kilograms of amino acids. Similarly, assuming a 1.0 m³volume to saturate a metric tonne of coal, 30 kilograms of amino acids hydrolysate is used for a final concentration of 0.03 g/ml.
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 m³to about 1.0 m³, then the final concentration of amino acids hydrolysate may include the range of about 0.003 g/ml to about 0.015 g/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 m³to about 1.0 m³, therefore the final concentration of amino acids hydrolysate may include about 0.006 g/ml.

TABLE 25

Weight of Amino acids per Metric Tonne of Coal.

Final
Concentration	Grams/Kilograms of
of Amino	Amino acids per Metric Tonne of Coal

acids	0.2 m³		1.0 m³Final
Hydrolysate	Final Volume	0.5 m³Final Volume	Volume

0.03 g/ml	6.0	kg	15	kg	30	kg
0.015 g/ml	3.0	kg	7.5	kg	15	kg
0.006 g/ml	1.2	kg	3.0	kg	6.0	kg
0.003 g/ml	600	g	1.5	kg	3.0	kg
0.0003 g/ml	60	g	150	g	300	g

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.
The foregoing are examples of aspects of the invention and the invention should not be interpreted as limited to the specific examples provided herein.

Claims

1. 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 less than 30 kilograms of amino acids per metric tonne of coal contained in a predetermined location of said formation; and

said amino acids are obtained from a source outside of said formation.

2. The method of claim 1, further comprising increasing the methane gas released or produced from said formation by further decreasing said amount of amino acids.

3. The method of claim 1, wherein said amino acids are contained in a composition comprising said amino acids and wherein said amino acids are enzymatically hydrolyzed fish amino acids.

4. The method of claim 3, wherein said composition comprises fatty acids and vitamins.

5. The method of claim 1, wherein said coal is lignite, sub-bituminous, bituminous or mixtures thereof.

6. The method of claim 1, wherein said formation is a coalbed.

7. The method of claim 1, wherein collection of methane gas has been abandoned from said formation.

8. The method of claim 1, wherein one or more of said methanogenic microorganisms are naturally occurring in situ in said formation and have not been removed from said formation.

9. The method of claim 1, wherein said amount is greater than 60 grams of amino acids per said metric tonne.

10. The method of claim 1, wherein said amount is greater than 600 grams and less than 15 kilograms of amino acids per said metric tonne.

11. The method of claim 1, wherein said amount is greater than 1.2 kilograms and less than 6 kilograms of amino acids per said metric tonne.

12. A method of increasing methane gas released or produced from a subsurface coal formation, comprising:

increasing methane gas released or produced from said formation by lowering an amount of amino acids contacting said formation in situ, wherein said amino acids of said lowered amount are obtained from a source outside of said formation.

13. The method of claim 12, further comprising increasing methane gas released from said formation by further lowering said amount of amino acids.

14. The method of claim 12, wherein said subsurface coal formation is a coalbed.

15. 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;

said contacting occurs in situ;

said formation contains methanogenic microorganisms;

said amount is less than about 30 kilograms of amino acids per metric tonne of coal contained in a predetermined location of said formation; and

said amino acids are obtained from a source outside of said formation.

16. A subsurface coal 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 or production of methane gas from said coal and wherein said amino acids are obtained outside of said formation.

17. The formation of claim 16, wherein said formation is a coalbed.

18. The formation of claim 16, wherein collection of methane gas has been abandoned from said formation.

19. The method of claim 12, wherein said released methane gas is measured and collected.