METHOD

Abstract

The present invention provides a process for the microbiological production of hydrogen from a hydrocarbon-rich deposit, said process comprising the step of modifying the composition of the deposit by the introduction into the deposit of at least one non-native hydrogen producing microorganism selected positively to diversify the microbiological abundance of hydrogen-producing microorganisms in the deposit and for the preferential production of hydrogen over methane.

Claims

1. A process for the microbiological production of hydrogen from a hydrocarbon-rich deposit, said process comprising the step of modifying the composition of the deposit by the introduction into the deposit of at least one non-native hydrogen producing microorganism selected positively to diversify the microbiological abundance of hydrogen-producing microorganisms in the deposit and for the preferential production of hydrogen over methane.

2. The process according to claim 1, wherein the non-native hydrogen producing microorganism is: a. a microorganism not naturally present in the hydrocarbon-rich deposit; and/or b. of a strain of microorganisms not naturally present in the hydrocarbon-rich deposit; and/or c. of a species of microorganisms not naturally present in the hydrocarbon-rich deposit; and/or d. of a genus of microorganisms not naturally present in the hydrocarbon-rich deposit; and/or e. a microorganism naturally present in the hydrocarbon-rich deposit but genetically modified to increase (relative to the naturally present microorganism) its propensity for hydrogen production by the metabolization by that microorganism of one or more hydrocarbons contained within the deposit.

3. The process according to claim 1, wherein the at least one non-native hydrogen producing microorganism is one of a plurality of different non-native hydrogen producing microorganisms, strains of microorganisms, species of microorganisms, genera of microorganisms and/or naturally occurring but genetically modified organisms introduced into the deposit.

4. The process according to claim 3, wherein the plurality is greater than two, greater than three, greater then four and/or greater than five.

5. The process according to claim 1, wherein the non-native hydrogen producing microorganism has a propensity to metabolize one or more hydrocarbons contained within the deposit to molecular hydrogen in preference to methane such that the yield of production of molecular hydrogen from the metabolization is higher than the yield of production of methane by at least 1%, by at least 10%, by at least 100% and/or by at least 1000%.

6. The process according to claim 1, wherein the non-native hydrogen producing microorganism is introduced into the deposit and accompanied during, after or upon its introduction by at least one nutrient selected to promote the growth of said microorganism and introduced into the deposit for that purpose.

7. The process according to claim 6, wherein the at least one nutrient is selected preferentially to promote the growth of the said microorganism in preference to at least one, to at least some or to all of any native microorganisms in the deposit.

8. The process according to claim 6, wherein the nutrient comprises one or more of: a. one or more salts selected from: i. phosphates; and/or ii. halides; and/or iii. nitrates/ammonium salts/nitrogenous salts b. one or more carbohydrates selected from: i. sugars; and/or ii. starches; and/or c. one or more vitamins; d. complex nutrients, optionally selected from yeast extracts, corn steep liquor, biomass, bacterial and/or algal biomass.

9. The process according to claim 1, wherein the hydrogen producing microorganism is introduced into the deposit and accompanied during, after or upon its introduction by at least one pH regulator selected to regulate the pH environment in which the microorganism resides in the deposit and introduced into the deposit for that purpose.

10. The process according to claim 9, where in the pH regulator is selected to regulate the pH of the hydrogen producing microorganism environment in the deposit to a pH within the range of from about 5 to about 9, from about 6 to about 8 and/or from about 6 to about 7.

11. The process according to claim 1, wherein the hydrocarbon-rich deposit is a liquid hydrocarbon-rich deposit.

12. The process according to claim 1, wherein the at least one non-native hydrogen producing microorganism has a genus of Syntrophobacter, Syntrophus, Syntrophomonas, Thermoanaerobacter, Thermotoga, Pseudothermotoga, Thermoanaerobacterium, Fervidobacterium, Thermosipho, Haloanaerobium, Acetoanaerobium, Anaerobaculum, Geotoga, Petrotoga, Thermococcus, Pyrococcus, Clostridium, Enterobacter, Klebsiella, Ethanoligenens, Pantoea, Escherichia, Bacillus, Caldicellulosiruptor, Pelobacter, Caldanaerobacter, Marinitoga, Oceanotoga, Defluviitoga, Kosmotoga, or a combination or mixture thereof.

13. The process according to claim 12, wherein the non-native hydrogen producing microorganism or the recombinant microorganism expresses at least one protein selected from hydrogenases, dehydrogenases, hydroxylases, carboxylases, esterases, hydratases and acetyltransferases having an amino acid sequence at least 95% identical to a sequence expressed by an upregulated or downregulated gene selected from mth (EC 1.12.98.2), mrt, hycA (ID: 45797123), fdhF (ID: 66346687), fhlA (ID: 947181), ldhA (ID: 946315), nuoB (ID: 65303631), hybO (ID: 945902), fdhl, narP, ppk or Pepc by expressing a non-native protein expressing nucleotide sequence, wherein an amount of hydrogen produced or protein produced by the non-native hydrogen producing microorganism or the recombinant microorganism is greater than that produced relative to a control microorganism lacking the non-native protein expressing nucleotide sequence.

14. The process according to claim 1, wherein the environment of the hydrocarbon-rich deposit and the introduced hydrogen producing microorganism constitutes an enclosed bioreactor, being a bioreactor subterranean formation, a bioreactor landfill enclosure, or a combination thereof.

15. The process according to claim 14 comprising: a. providing a baseline reaction mixture in the enclosed bioreactor, wherein the baseline reaction mixture includes a hydrocarbon having up to 120 carbon atoms, water, and a baseline amount of at least one microorganism; producing baseline microorganism data on an identity and a baseline percentage of the at least one microorganism, relative to a baseline total percentage of microorganisms in the baseline reaction mixture, by performing DNA and/or RNA sequencing of a baseline microorganism sample from the baseline reaction mixture; measuring a baseline amount of hydrogen in a baseline gas sample of gasses collected from the enclosed bioreactor; increasing hydrogen production from the enclosed bioreactor by forming a synthetic reaction mixture, and harvesting the hydrogen from the enclosed bioreactor at a hydrogen harvesting rate by separating the hydrogen from other gasses and transferring the hydrogen into a hydrogen storage container; and/or b. providing at least one anode and at least one cathode connected to an interior of the enclosed bioreactor, wherein the enclosed bioreactor is a subterranean formation, an enclosed landfill, or a combination thereof, and the at least one anode and the at least one cathode are connected through the enclosed bioreactor by at least one bioreactor liquid pathway; providing a baseline reaction mixture in the enclosed bioreactor, wherein the baseline reaction mixture includes an organic substrate, water, and a baseline amount of at least one microorganism; measuring a baseline amount of hydrogen in a baseline gas sample of gasses collected from the enclosed bioreactor; increasing hydrogen production from the enclosed bioreactor from the baseline amount of hydrogen to a production amount of hydrogen by applying a potential between the at least one anode and the at least one cathode; and harvesting the hydrogen from the enclosed bioreactor at a hydrogen harvesting rate by separating the hydrogen from other gasses and transferring the hydrogen into a hydrogen storage container, wherein the production amount of hydrogen is at least 20% greater than the baseline amount of hydrogen; and/or c. providing a baseline reaction mixture in the enclosed bioreactor, wherein the baseline reaction mixture includes a substrate, water, and a baseline amount of at least one microorganism, wherein the substrate includes a nitrogen source, an unsaturated hydrocarbon having from 2 to 120 carbon atoms, methane, hydrogen, or a combination thereof, wherein the hydrogen-containing liquid includes ammonia, ammonium, methanol, a saturated hydrocarbon having from 2 to 120 carbon atoms, or a combination thereof; producing baseline microorganism data on an identity and a baseline percentage of the at least one microorganism, relative to a baseline total percentage of microorganisms in the baseline reaction mixture, by performing DNA and/or RNA sequencing of a baseline microorganism sample from the baseline reaction mixture; measuring a baseline amount of hydrogen-containing liquid in a baseline sample collected from the enclosed bioreactor; increasing production of the hydrogen-containing liquid from the enclosed bioreactor by forming a synthetic reaction mixture, and harvesting the hydrogen-containing liquid from the enclosed bioreactor at a hydrogen-containing liquid harvesting rate by separating the hydrogen-containing liquid from solids and other liquids by transferring the hydrogen-containing liquid into a hydrogen-containing liquid storage container; and/or d. providing hydrocarbon wastewater from a hydrocarbon producing site; forming a baseline reaction mixture by transferring the hydrocarbon wastewater into an enclosed bioreactor, wherein the baseline reaction mixture includes the hydrocarbon wastewater and a baseline amount of at least one microorganism; producing baseline microorganism data on an identity and a baseline percentage of the at least one microorganism, relative to a baseline total percentage of microorganisms in the baseline reaction mixture, by performing DNA and/or RNA sequencing of a baseline microorganism sample from the baseline reaction mixture; measuring a baseline amount of hydrogen in a baseline gas sample of gasses collected from the enclosed bioreactor; measuring a baseline amount of hydrocarbons in a baseline liquid sample of a liquid collected from the enclosed bioreactor; producing hydrogen and forming purified water from the hydrocarbon wastewater by forming a synthetic reaction mixture in the enclosed bioreactor, harvesting the hydrogen from the enclosed bioreactor at a hydrogen harvesting rate by separating the hydrogen from other gasses and transferring the hydrogen into a hydrogen storage container, and gathering the purified water from the enclosed bioreactor by transferring the purified water from the enclosed bioreactor to a purified water liquid path at a purified water rate, optionally of from about 10 L/hr to about 10,000 L/hr; and/or

16. The process according to claim 14 for increasing hydrogen production from the enclosed bioreactor comprising: a. providing a baseline reaction mixture in the enclosed bioreactor, wherein the baseline reaction mixture includes a hydrocarbon having up to 120 carbon atoms, water, and a baseline amount of at least one microorganism; b. producing baseline microorganism data on an identity and a baseline percentage of the at least one microorganism, relative to a baseline total percentage of microorganisms in the baseline reaction mixture, by performing DNA and/or RNA sequencing of a baseline microorganism sample from the baseline reaction mixture; c. measuring a baseline amount of hydrogen in a baseline gas sample of gasses collected from the enclosed bioreactor; d. increasing hydrogen production from the enclosed bioreactor by forming a synthetic reaction mixture, and e. harvesting the hydrogen from the enclosed bioreactor at a hydrogen harvesting rate by separating the hydrogen from other gasses and transferring the hydrogen into a hydrogen storage container; f. forming the synthetic reaction mixture by: i. adding at least one non-native hydrogen producing microorganism until a percentage of the non-native hydrogen producing microorganism in the synthetic reaction mixture is at least 20% of a total amount of microorganisms in the synthetic reaction mixture; or ii. adding at least one hydrogen production enhancer to the baseline reaction mixture until a post-baseline amount of hydrogen in a post-baseline gas sample of gasses collected from the enclosed bioreactor is at least 10% higher than the baseline amount of hydrogen; or iii. adding at least one recombinant microorganism to the baseline reaction mixture until a percentage of the at least one recombinant microorganism in the synthetic reaction mixture is at least 20% of a total amount of microorganisms in the reaction mixture, or iv. a combination thereof.

17. The process according to claim 16 wherein: a. the hydrogen production rate of the enclosed bioreactor is from about 0.1 L/hr to about 10.sup.6 L/hr; and/or b. the enclosed bioreactor is a subterranean formation comprising a natural formation, non-natural formation, a hydrocarbon-bearing formation, a natural gas-bearing formation, a methane-bearing formation, a depleted hydrocarbon formation, a depleted natural gas-bearing formation, a wellbore, or a combination thereof; and/or c. the enclosed bioreactor is a landfill enclosure comprising a landfill that is enclosed by a building material, wherein the building material includes at least one of a brick, a cement, a plastic, a non-natural rubber, a geomembrane of any kind, concrete, steel, a glass, or a combination thereof; and/or d. the hydrogen production enhancer is a biocidal inhibitor (optionally glutaraldehyde, a quaternary ammonium compound, formaldehyde, a formaldehyde releaser such as 3,3′-methylenebis[5-methyloxazolidine], dibromonitrilopropionamide, tetrakis hydroxymethyl phosphonium sulfate, chlorine dioxide, peracetic acid, tributyl tetradecyl phosphonium chloride, methylisothiazolinone, chloromethylisothiazolinone, sodium hypochlorite, dazomet, dimethyloxazolidine, trimethyloxazolidine, N-Bromosuccinimide, Bronopol, or 2-propenal, or a mixture thereof), a methanogenesis inhibitor (optionally bromethane sulfonic acid, an Aminobenzoic acid, 2-bromoethanesulfonate, 2-chloroethanesulfonate, 2-mercaptoethanesulfonate, lumazine, a fluoroacetate, nitroethane, or 2-nitropropanol, or a mixture thereof), a sulfate reduction inhibitor (optionally a molybdate salt, a nitrate salt, a nitrite salt, a chlorate salt, or a perchlorate salt or a mixture thereof), a nitrate reduction inhibitor (optionally sodium chlorate, a chlorate salt, or a perchlorate salt, or a mixture thereof), an iron reduction inhibitor, or a combination thereof.

18. The process according to claim 16, further comprising: a. producing carbon dioxide from the enclosed bioreactor at a carbon dioxide producing rate, b. separating the carbon dioxide from other gasses by filtering the carbon dioxide through a carbon dioxide-selective membrane filter; and i. pumping the carbon dioxide into the enclosed bioreactor at a replenishment rate or to a different enclosed bioreactor at an injection rate; and/or ii. forming an algal biomass by reacting the carbon dioxide with an algae reaction mixture in an algal bioreactor, and pumping the algal biomass into the reaction mixture of the enclosed bioreactor or a different enclosed bioreactor.

19. The process according to claim 16, wherein forming the synthetic reaction mixture includes: a. adding at least one non-native hydrogen producing microorganism until a percentage of the non-native hydrogen producing microorganism in the synthetic reaction mixture is at least 20% of a total amount of microorganisms in the synthetic reaction mixture; and/or b. adding at least one hydrogen production enhancer to the baseline reaction mixture until a post-baseline amount of hydrogen in a post-baseline gas sample of gasses collected from the enclosed bioreactor is at least 10% higher than the baseline amount of hydrogen; and/or c. adding at least one recombinant microorganism to the baseline reaction mixture until a percentage of the at least one recombinant microorganism in the synthetic reaction mixture is at least 20% of a total amount of microorganisms in the reaction mixture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0126] The invention will now be more particularly described with reference to the following examples and figures, in which;

[0127] FIG. 1 is a schematic illustration of a system for increasing hydrogen production from an enclosed bioreactor according to some embodiments herein.

[0128] FIG. 2 is a schematic illustration of a system for increasing hydrogen production from an enclosed bioreactor according to some embodiments herein.

[0129] FIG. 3 is a flow chart depicting an embodiment of a method of increasing hydrogen production from an enclosed bioreactor according to some embodiments herein.

[0130] FIG. 4 is a schematic illustration of a system for increasing hydrogen production from an enclosed bioreactor according to some embodiments herein.

[0131] FIG. 5 is a schematic illustration of a system for increasing hydrogen production from an enclosed bioreactor according to some embodiments herein.

[0132] FIG. 6 is a schematic illustration of a system for increasing production of a hydrogen containing liquid from an enclosed bioreactor according to some embodiments herein.

[0133] FIG. 7 is a schematic illustration of a system for increasing production of a hydrogen containing liquid from an enclosed bioreactor according to some embodiments herein.

[0134] FIG. 8 is a schematic illustration of a system for increasing hydrogen production and hydrocarbon wastewater purification according to some embodiments herein.

[0135] FIG. 9 is a schematic illustration of a system for the microbiological production of hydrogen from a hydrocarbon-rich deposit in accordance with the first aspect of the invention described above, and as exemplified in Example 10 below. It will be apparent to the skilled addressee that recovery of hydrogen from the subterranean deposit may be effected by various means, and that the schematically depicted H.sub.2 separator membrane is merely illustrative.

[0136] The foregoing summary, as well as the following detailed description of the embodiments, will be better understood when read in conjunction with the attached drawings. For the purpose of illustration, there are shown in the drawings some embodiments, which may be preferable. It should be understood that the embodiments depicted are not limited to the precise details shown. Unless otherwise noted, the drawings are not to scale.

DETAILED DESCRIPTION

[0137] Unless otherwise noted, all measurements are in standard metric units.

[0138] Unless otherwise noted, all instances of the words “a,” “an,” or “the” can refer to one or more than one of the word that they modify.

[0139] Unless otherwise noted, the phrase “at least one of” means one or more than one of an object. For example, “at least one of a single walled carbon nanotube, a double walled carbon nanotube, and a triple walled carbon nanotube” means a single walled carbon nanotube, a double walled carbon nanotube, or a triple walled carbon nanotube, or any combination thereof.

[0140] Unless otherwise noted, the term “about” refers to ±10% of the non-percentage number that is described, rounded to the nearest whole integer. For example, about 100 mm, would include 90 to 110 mm. Unless otherwise noted, the term “about” refers to ±5% of a percentage number. For example, about 20% would include 15 to 25%. When the term “about” is discussed in terms of a range, then the term refers to the appropriate amount less than the lower limit and more than the upper limit. For example, from about 100 to about 200 mm would include from 90 to 220 mm.

[0141] Unless otherwise noted, properties (height, width, length, ratio etc.) as described herein are understood to be averaged measurements.

[0142] Unless otherwise noted, the terms “provide”, “provided” or “providing” refer to the supply, production, purchase, manufacture, assembly, formation, selection, configuration, conversion, introduction, addition, or incorporation of any element, amount, component, reagent, quantity, measurement, or analysis of any method or system of any embodiment herein.

[0143] Unless otherwise noted, the term “non-native” refers to a microorganism that is not naturally occurring in a particular location, such as a particular subterranean formation.

[0144] Unless otherwise noted, the term “recombinant microorganism” refers to a microorganism that does not occur in nature and is the synthetic product of recombinant DNA engineering.

[0145] Unless otherwise noted, the term “hydrocarbon” refers to a compound that contains only contains hydrogen and carbon atoms.

[0146] Unless otherwise noted, the term “gas path” is interchangeable with the term “gas flow path.” Unless otherwise noted, the term “gas path” refers to an enclosed solid structure or channel that a gas can move or be pumped through. For example, in various embodiments of the systems and methods disclosed herein, a gas path includes one or more pipes and/or tubes connected to or connected through one or more valves or pumps, so long as gas can flow or be pumped continuously through the structure of the gas path.

[0147] Unless otherwise noted, the term “liquid path” is interchangeable with the term “liquid flow path.” Unless otherwise noted, the term “liquid path” refers to an enclosed solid structure or channel that a gas can move or be pumped through. For example, in various embodiments of the systems and methods disclosed herein, a gas path includes one or more pipes and/or tubes connected to or connected through one or more valves or pumps, so long as gas can be made to flow or be pumped continuously through the structure of the gas path.

[0148] Unless otherwise noted, the term “electrically connected” refers to connecting two or more objects such they can conduct electricity.

[0149] Unless otherwise noted, the term “biomass” refers to a product which can contain one or more microorganisms, such as alga, living or dead, colonies of those organisms, and/or the contents of one or more microorganisms, such as enzymes, cytoplasm, nutrients, and the like. An example of a “biomass” can include alga that have been mechanically disrupted.

[0150] Unless otherwise noted, the term “enclosed” or “enclosure” refers to a structure that is sealable or resealable, such that when the structure is sealed, the contents of the structure are not free to mix with the open air.

[0151] Unless otherwise noted, the term “enclosed bioreactor” refers to a subterranean formation or a landfill enclosure in which a microorganism can be introduced.

[0152] Unless otherwise noted, the term “hydrogen-containing liquid” refers to a molecule that contains hydrogen atoms and from 80% to 100% weight of the compound, relative to the total weight of the compound, is a liquid or liquid slurry at standard temperature and pressure.

EXAMPLES

Example 1: Initial Set-Up for a Depleted Oil Well

[0153] Purchasing or leasing land having a depleted oilwell with a wellbore and a well casing already in place such that the wellbore and well casing extend into a subterranean formation that has been substantially depleted of hydrocarbons. Attaching a valve assembly to the head of the wellbore such that the valves of the valve assembly can control what enters and leaves the wellbore. A suitable valve assembly can be purchased from oil field service companies such as Mogas, Suez Water Technologies, and Halliburton, among others.

[0154] Using a bulldozer to dig a pool into the surface within about 100 to 200 meters out of the valve assembly of the depleted oil well. Digging the pool to a depth up about 5 feet any length and width of about 100 meters. The pool would be filled with water and alga of the genera Chlorella or Scenedesmus which can be purchased from UTEX Culture Collection of Algae at the University of Texas at Austin. A series of rods would be extended over the length and width of the pool to form a support structure, and a transparent polyethylene cover would be used to seal the top of the pool, making it substantially airtight. The covered pool would serve as an algal bioreactor.

[0155] A free-standing structure would be connected by one or more gas pipes to the algal reactor to form a hydrogen separation building. The hydrogen separation building would be connected to the subterranean formation either directly by drilling a wellbore into the subterranean formation or by one or more pipes connecting to the valve assembly. The freestanding structure would contain a T-junction connecting a gas path from the subterranean formation to a hydrogen selective membrane, where in on one side of the hydrogen selective membrane (the filtered hydrogen side) the hydrogen separator is connected to a hydrogen storage tank by a gas pipe. A suitable hydrogen selective membrane can be hollow microfiber membranes, which can be purchased from Generon located in California, among other suppliers. Alternatively, palladium-based membranes, such as those available from HySep, can be used for hydrogen separation. The other side of the membrane (the carbon dioxide side) would be connected to the algal bioreactor by a gas pipe.

[0156] The valve assembly would be connected to two containers, one serving is a microorganism container and one serving as a hydrogen production enhancer container. The valve assembly would further connect to a DNA testing facility wherein the DNA testing facility includes a DNA sequencer and would further connect the valve assembly to the DNA sequencer, such that sequencing could be controlled buy a computer or remotely. A suitable DNA sequencer would include the MinIon nanopore sequencer, which can be commercially purchased from Oxford Nanopore Technologies located in the United Kingdom.

[0157] The algal bioreactor would further be connected to the subterranean formation either directly by a well bore and liquid tube or indirectly by connecting the algal bioreactor to the valve assembly.

Example 2: Initial Set-Up for a Landfill

[0158] Purchasing or leasing land having a commercial landfill. Drilling wellbores into the landfill using a commercial drilling rig. Forming liquid distributors by placing one or more pipes over the landfill and drilling holes into the pipes at regular intervals to allow for liquid and slurries to be distributed onto the landfill. Constructing a dome over the landfill to and sealed around the liquid additive pathways, forming a gastight structure that is sealed around the liquid additive pathways. A suitable material for the dome can include polyvinyl chloride, which can be purchased commercially from Membrane Systems Europe located in The Netherlands.

[0159] Using a bulldozer to dig a pool into the surface within about 100 to 200 meters out of the valve assembly of the depleted oil well. Digging the pool to a depth up about 5 feet any length and width of about 100 meters. The pool would be filled with water and alga of the genus Chlorella or Scenedesmus which can be purchased from UTEX Culture Collection of Algae at the University of Texas at Austin. A series of rods would be extended over the length and width of the pool to form a support structure, and a transparent polyethylene cover would be used to seal the top of the pool, making it substantially airtight. The covered pool would serve as an algal bioreactor.

[0160] A free-standing structure would be connected by one or more gas pipes to the algal reactor to form a hydrogen separation building. The hydrogen separation building would be connected to the landfill by a one gas pipe or tube connected to the liquid additive pathway. The freestanding structure would contain a T-junction connecting a gas path from the landfill dome to a hydrogen selective membrane, where in on one side of the hydrogen selective membrane (the filtered hydrogen side) the hydrogen separator is connected to a hydrogen storage tank by a gas pipe. A suitable hydrogen selective membrane can be hollow microfiber membranes, which can be purchased from Generon located in California, among other suppliers. Alternatively, palladium-based membranes, such as those available from HySep, can be used for hydrogen separation. The other side of the membrane (the carbon dioxide side) would be connected to the algal bioreactor by a gas pipe.

[0161] The liquid additive pathway or sprinkler system could be connected to two containers, one serving is a microorganism container and one serving as a hydrogen production enhancer container. The liquid additive pathway or sprinkler system could be separate from or connect to a DNA testing facility. The DNA testing facility would contain a DNA sequencer. A suitable DNA sequencer would include the MinIon nanopore sequencer, which can be commercially purchased from Oxford Nanopore Technologies located in the United Kingdom.

[0162] The algal bioreactor would further be connected to the landfill dome by the liquid additive pathway to the algal bioreactor. The liquid additive pathway or sprinkler system could include more than one set of pipes for distributing liquids and slurries. For example, one set of pipes over the landfill might carry a biomass liquid slurry. Another set of pipes could be connected to the microorganism container to distribute the microorganisms over the landfill.

Example 3: Increasing Hydrogen Production from a Subterranean Formation Having a Low Amount of Hydrogen Producing Microorganisms

[0163] Providing the set up according to Example 1 above, with the following changes.

[0164] Taking a gas sample of the hydrogen produced by the subterranean information and analyzing the amount of hydrogen in the gas sample using a gas chromatograph with PDHID (Pulse Discharge Helium Ionization Detection) can be purchased from Custom Solutions Group which is located in Houston, Tex. Determining that the hydrogen output is too low.

[0165] Taking a liquid sample from the subterranean formation. Performing a bulk DNA extraction by performing the steps detailed in the DNeasy PowerSoil Pro Kit from Qiagen (Hilden, Germany). Quantifying the amount of DNA using real-time PCR and primers that target the 16S rRNA gene. Sequencing the DNA from the samples using a commercially available kit such as the 16S sequencing kit, which is commercially available from Oxford Nanopore Technologies.

[0166] Further testing the liquid sample to determine pH, temperature, and level of nutrients present in the liquid sample.

[0167] Analyzing the data from the microorganism population and determining that there are microorganisms present in the subterranean formation, but that less than 1% of the microorganisms present produce hydrogen. Adding 1-50 barrels of ˜10E8 cells/mL of a nonnative hydrogen producing microorganism, such as Clostridium spp., which is known to be a hydrogen producing organism and compatible with a pH of 5-8 and temperature of 77-95 F, until the amount is projected to be over 20% of the total microorganisms present. Suitable Clostridium can be purchased from ATCC, which is located in Manassas, Va.

[0168] Harvesting an amount of hydrogen by pumping the gasses from the subterranean formation through the hydrogen selective filter into a hydrogen storage tank at a rate of about 0.3 tons/hr to 30 tons/hr, wherein the percentage of hydrogen in the gas sample is increased by at least 10%. Pumping they non-hydrogen gases such as carbon dioxide into the algal bioreactor.

[0169] Pumping water, nutrients, and alga from the algal reactor as needed into the subterranean formation to feed the reaction mixture.

[0170] Using DNA sequencing to monitor liquid samples about once a month to ensure that the amount of hydrogen producing microbes does not fall below 20% of the total amount of microbes present.

Example 4: Increasing Hydrogen Production from a Subterranean Formation Having a High Amount of Hydrogen Consuming Microorganisms

[0171] Performing the same steps as in Example 3, except the DNA analysis indicates that there are hydrogen producing microorganisms present in an amount of at least 20% of the total amount of microorganisms present, but there is a high amount or percentage of microorganisms such as sulfate reducing microbes or nitrate reducing microbes, which are known to be a microorganism that consumes hydrogen. This hydrogen consumer is decreasing the amount of hydrogen which can be harvested from the subterranean formation. Therefore, instead of adding a native hydrogen producing microorganism, an inhibitor such as sodium nitrate, which is known to inhibit sulfate reducing microbes is pumped into the subterranean formation at about 50 mM concentration.

[0172] Taking gas samples from the subterranean formation and adding the inhibitor until the increase in hydrogen percentage relative to the total amount of gases is increased by at least 10%.

Example 5: Increasing Hydrogen Production from a Subterranean Formation Having a High Temperature and Low pH

[0173] Providing this setup according to Example 1 and performing the method according to Example 3 above, except that the DNA analysis of the microbial population and the water testing step indicate that the subterranean formation would be unlikely to support a sustainable population of naturally occurring hydrogen producing microorganisms.

[0174] Creating recombinant microorganism by inserting DNA having a sequence, which is known to code for a hydrogen producing protein, into a microorganism, which is known to thrive in environments having the high temperature as well as the low pH. Adding amounts of the recombinant microorganism to the subterranean formation until the total amount of percentage in the population increases above 20% relative to the total population of microorganisms.

[0175] Using DNA sequencing to monitor liquid samples from the subterranean formation about once a month to ensure that the amount of recombinant microorganisms does not fall below 20% of the total amount of microbes present.

Example 6—Applying Potential to Increase Hydrogen Production Using Hydrocarbons in Place as Substrate in the Subterranean Formation

[0176] Providing the setup according to Example 1 above.

[0177] Electric current would be applied to the reservoir by electrodes placed in water injection wells and production wells. Salt water (recycled produced water) would be injected simultaneously with application of electric current. To reduce the flow of electricity to overlying beds, casing above the electrode would be electrically isolated. Both water and electric current might be transmitted in the well through electrically conductive tubing, so that both the tubing and injected salt water would be utilized as electric conductors. The tubing could be externally insulated, or it could be equipped with non-conductive centralizers and installed with an insulating fluid in the casing-tubing annulus.

[0178] Providing the setup according to Example 3, 4, or 5 above.

Example 7: Applying Potential to Increase Hydrogen Production Using Alternative Organic Mass as Substrate in the Subterranean Formation

[0179] Providing the setup according to Example 1 and Example 6 above except there is not enough recalcitrant hydrocarbons left in situ to produce hydrogen to the desired degree.

[0180] A biomass consisting of biodegradable waste, paper waste, plant waste, pulp waste, or a combination thereof is pumped into the subterranean formation.

[0181] Providing the setup according to Example 3, 4, or 5 above.

Example 8: Producing Hydrogen Carriers in the Subterranean Formation

[0182] Providing the setup according to Example 1 above.

[0183] Providing the setup according to Example 3 above except that the DNA analysis is used to determine presence of hydrogen carrier producing microorganisms is less than 1%.

[0184] Adding 1-50 barrels of ˜10E8 cells/mL of a non-native hydrogen carrier producing microorganism, such as a recombinant Methanothermobacter which are known to be hydrogen carrier (methanol) producing organisms until the amount is projected to be over 20% of the total microorganisms present. Suitable anaerobic methanotrophs can be isolated from landfills or anaerobic digesters.

[0185] Harvesting an amount of hydrogen carriers by pumping the liquids from the subterranean formation into a hydrogen carrier storage tank at a rate of about 0.3 tons/hr to 30 tons/hr, wherein the percentage of hydrogen carrier in the liquid sample is increased by at least 10%. Pumping they non-hydrogen gases such as carbon dioxide into the algal bioreactor. Pumping water, nutrients, and alga from the algal reactor as needed into the subterranean formation to feed the reaction mixture.

[0186] Using DNA sequencing to monitor liquid samples about once a month to ensure that the amount of hydrogen producing microbes does not fall below 20% of the total amount of microbes present.

Example 9: Producing Hydrogen from Oil and Gas Wastewater Treatment Process

[0187] Providing the setup according to Example 1 above.

[0188] Produced water that has been separated from the total fluids production would be placed in an enclosed bioreactor. Hydrogen would be produced from the enclosed setup providing the setup according to Example 3 above.

Example 10: Field Well Trial

[0189] Schematically illustrated (for a single well) in FIG. 9, two low producing oil wells were stimulated in a huff-n-puff application to increase microbial hydrogen production. An oil sample from each well was taken and its indigenous microbiological content determined, with the results set out in Tables 1 and 2 below:

TABLE-US-00001 TABLE 1 Well 1 - Indigenous microbial population Halanaerobium praevalens DSM 2228 18.1% Acinetobacter johnsonii 13.4% Desulfohalobium retbaense DSM 5692 12.1% Halanaerobium hydrogeniformans 7.4% Methanohalophilus halophilus 6.0% Methanohalophilus mahii DSM 5219 4.7% Escherichia coli 2.0% Halobacteroides halobius DSM 5150 2.0% Azospirillum thiophilum 1.3% Keratinibaculum paraultunense 1.3%

TABLE-US-00002 TABLE 2 Well 2 - Indigenous microbial population Methanohalophilus halophilus 13.2% Methanohalophilus mahii DSM 5219 11.0% Halanaerobium praevalens DSM 2228 7.3% Desulfohalobium retbaense DSM 5692 6.8% Halanaerobium hydrogeniformans 3.7% Acinetobacter johnsonii 3.2% Petrotoga mobilis SJ95 3.2% Halothermothrix orenii H 168 2.3% Flexistipes sinusarabici DSM 4947 2.3% Pelobacter acetylenicus 1.8% Methanotorris igneus Kol 5 1.4% Bacillus mycoides 1.4%

[0190] In the first well, nutrients were blended as described below in Table 3 into 500 bbls of produced water in a frac tank.

TABLE-US-00003 TABLE 3 Nutrient package mixed into the 500 bbls: Reagent [g/L] K.sub.2HPO.sub.4 1.044 NH.sub.4Cl 1.5 Sucrose 1.41 Yeast extract 1.5 Tween 80 0.081

[0191] The nutrient mix was injected down the annulus of the well and an additional 500 bbls of produced water was pumped down the annulus on top of the nutrient mixture. In the second well, the same process occurred with the exception that a consortium of microbes capable of producing hydrogen from hydrocarbon fermentation was added to the first 500 bbls of produced water along with the nutrient package.

[0192] The consortium was prepared by combining non-native hydrogen producing microorganisms selected to be different from the indigenous microbial populations, and for their capability to digest hydrocarbons to yield hydrogen in preference to methane, in the proportions identified in Table 4:

TABLE-US-00004 TABLE 4 Well 2 - Exogenous microbial population Pseudothermotoga elfii ~20% Pseudothermotoga hypogea ~20% Thermotoga petrophila ~20% Petrotoga mobilis ~20% Caldanaerobacter tengcongensis ~20%

[0193] The exogenous microbes were maintained in anaerobic liquid culture and nurtured for 2 months under nitrogen (100% N2) at 150 F (65.56 degC), with fresh media inoculated every 3-4 days to provide 100 L kegs for field deployment. The selected media was an ATCC 2114 medium modified for preferential culturing of extremophiles.

[0194] Approximately 400 L of microbial culture consisting of approximately 10.sup.8 cells/mL was added to the 500 bbls.

[0195] Following addition of the nutrient package (Well 1) and the nutrient/microbial consortium package (Well 2), the two wells were shut-in for 4 days. After the four-day shut-in period the wells were opened and samples were collected off the gas flow line for analysis with respect to H.sub.2 content on a gas chromatograph, with the results presented in Table 5 below:

TABLE-US-00005 TABLE 5 Gas Chromatography characterization of samples: Baseline H2 After shut-in H2 Well (ppm) (ppm) 1 (nutrients only) <112 (LOD) 1761 2 (nutrients and microbes) <112 (LOD) 13251

[0196] The gas chromatography was carried out using a standard protocol as follows: 10 milliliter gas samples were extracted from culture bottles using 10 milliliter plastic luer lock syringes. Field gas samples were collected in multi-layer foil gas sampling bags connected via tygon tubing to a sampling valve directly off the of wellhead flow line. Gas samples were injected immediately into the inlet port of an SRI 8610C Gas Chromatograph. The sample was analyzed using a Flame Photometric Detector (FPD), a Flame Ionization Detector (FID), an FID with a large methanizer (FIDM), and a Thermal Conductivity Detector (TCD).

[0197] The samples were passed through an 18-inch HayeSep D Packed Column, a 3-foot Molecular Sieve 5A Packed Column, and then into the TCD and FIDM detectors following relay G injection. When relay F was turned on the samples were run through a 6-foot HayeSep D Column and a 60-meter MXT-1 Capillary Column before being analyzed using the FID and FPD. The G relay was turned on at time 0.020 minutes and was turned off at 1.000 minutes, while the F relay was turned on after 4.500 minutes. The initial temperature was set for 50° C. and held for 6 minutes before ramping to 270° C. at a rate of 30° C. per minute. The temperature was held at 270° C. for 6.500 minutes to remove excess sample from the columns.

[0198] Any peak areas produced were converted into ppm values using the trend lines of calibration curves derived from standards of various concentrations.

[0199] It will be seen from the results in Table 5 that modifying the composition of the well by the introduction into the well of a nutrient package and of consortium of non-native hydrogen producing microorganisms selected positively to diversify the microbiological abundance of hydrogen-producing microorganisms in the well and for the preferential production of hydrogen over methane increased hydrogen production from the well by two orders of magnitude with respect to baseline H.sub.2 production, and by an order of magnitude with respect to introduction of the nutrient package alone.

Example 11: Microbe Laboratory Data

[0200] The consortium of microbes described in Example 10 and capable of producing hydrogen from hydrocarbon fermentation was used to inoculate 6 different synthetic seawater blends in triplicate as described below in Table 6.

TABLE-US-00006 TABLE 6 Synthetic seawater blends: Brine Description A Synthetic seawater B Synthetic seawater with oil C Synthetic seawater with nutrients D Synthetic seawater with nutrients and oil E Synthetic seawater with enhanced nutrients F Synthetic seawater with enhanced nutrients and oil G Synthetic seawater with algae biomass and oil

[0201] Synthetic seawater is a simple reproducible representative of produced water brines. It was produced using NeoMarine aquarium salts by Brightwell Aquatics. The oil used in this example was a sweet west Texas crude blend was used (API 25-35). 4 mL of the oil was used in 100 mL synthetic seawater sample. The nutrient packages employed were as follows in Tables 7, 8 and 9:

TABLE-US-00007 TABLE 7 Synthetic seawater with nutrients: Reagent [g/L] Aquarium Salts 35.40290621 K.sub.2HPO.sub.4 0.348 KH.sub.2PO.sub.4 0.227 NH.sub.4Cl 0.5 Wolfes Vitamin solution  10 mL Reducing agent 1 Resazurin solution  ~1 mL dH.sub.2O 989 mL Combine, pH to desired 6.5 +− 0.5), filter sterilize

TABLE-US-00008 TABLE 8 Synthetic seawater with enhanced nutrient package: Reagent [g/L] Aquarium Salts 35.40290621 K.sub.2HPO.sub.4 0.348 NH.sub.4Cl 0.5 Glucose 0.47 Yeast extract 0.5 Tween 80 0.027 Reducing agent 1 Resazurin solution  ~1 mL dH.sub.2O 999 mL Combine, pH to desired 6.5 +− 0.5), filter sterilize

TABLE-US-00009 TABLE 9 Synthetic seawater with algae biomass nutrient package: Reagent [g/L] Aquarium Salts 35.40290621 K.sub.2HPO.sub.4 0 NH.sub.4Cl 0 Glucose 0 Yeast extract 0 Chlorella algae powder 0.5 Tween 80 0.027 Reducing agent 1 Resazurin solution  ~1 mL dH.sub.2O 999 mL

[0202] A 100 mL sample of each brine A-E was prepared anaerobically in glass bottles and sealed. Following inoculation, the bottles were incubated at 65 C for 48 hours along with abiotic controls for each brine.

[0203] At 48 hours, samples were taken for ATP analysis (microbial enumeration) and gas analysis, the results of which are shown in Table 10.

TABLE-US-00010 TABLE 10 ATP Analysis: Abiotic Control Inoculated H2 Microbial H2 Microbial Concentration enumeration Concentration enumeration Brine Description (ppm) (cells/mL) (ppm) (cells/mL) A Synthetic seawater 0 6.92E+03 0 1.51E+06 B Synthetic seawater 0 2.00E+04 82.33 7.54E+06 with oil C Synthetic seawater 0 1.40E+03 119 7.55E+06 with nutrients D Synthetic seawater 0 7.67E+03 1406.7 2.58E+07 with nutrients and oil E Synthetic seawater 0 9.80E+03 62.33 3.04E+07 with enhanced nutrients F Synthetic seawater 0 2.95E+04 2735.33 2.07E+07 with enhanced nutrients and oil G Synthetic sea water 0 1.18E+07 2365.5 1.01E+08 with algae biomass and oil

[0204] In sample E, the enhanced nutrient package used causes rapid microbial growth at 24 hours and all of the carbon source is consumed which leads to a lower reading at 48 hours when no oil is present to maintain microbial activity, which rationalizes the lower H.sub.2 concentration observed for this sample relative to comparative sample C.

[0205] The test kit used for the determination of ATP was the Luminultra QGO-M which is compliant with ASTM Standard E2694 for the measurement of ATP in Metalworking Fluids and D7687 for the measurement of ATP in fuels, fuel/water mixtures and fuel-associated water.