AU2014202663B2 - Enhanced biogas production - Google Patents
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- AU2014202663B2 AU2014202663B2 AU2014202663A AU2014202663A AU2014202663B2 AU 2014202663 B2 AU2014202663 B2 AU 2014202663B2 AU 2014202663 A AU2014202663 A AU 2014202663A AU 2014202663 A AU2014202663 A AU 2014202663A AU 2014202663 B2 AU2014202663 B2 AU 2014202663B2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
A methane producing aqueous environment including a source of reducing equivalents liberated from within the aqueous environment; substrates including hydrogen and a carbon containing 5 compound selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids, or a combination thereof, methanogenic organisms; and an electron transfer mediator to catalyse the conversion of the substrates by the methanogenic organisms to methane using the reducing equivalents. The invention also relates to the method of increasing the rate of production of methane in an aqueous environment.
Description
The invention also relates to the method of increasing the rate of production of methane in an aqueous environment.
ι
2014202663 15 May 2014
AUSTRALIA
Patents Act 1990
STANDARD COMPLETE SPECIFICATION
FOR THE INVENTION ENTITLED:
Enhanced biogas production
Applicant:
NewSouth Innovations Pty Ltd
The invention is described in the following statement:
1000636231 1.doc
Enhanced biogas production
2014202663 15 May 2014
Field of the invention
The invention is directed to a method of producing and/or improving production of a biogas that includes methane from a carbonaceous source material.
Background of the invention
Natural gas production is a matter of global political, social and environmental concern. Humanity faces the concurrent challenges of 1) meeting the energy demands of increasing populations with increasing energy consumption per capita and 2) reducing the environmental impacts of energy production, most notably the production of greenhouse gases but also habitat 10 degradation for all organisms including humans.
Biologically produced combustible gas (biogas) has a large role to play in the world’s future energy security and in meeting the energy needs of the human race whilst reducing greenhouse gas emissions. Natural gas (primarily methane) can be used for vehicle transport and for generating electricity with reduced greenhouse gas emissions compared to petroleum or coal.
This is exemplified in the USA, where they have recently reduced greenhouse gas emissions through the growth of the shale gas extraction industry and downstream shifts away from coal-fired power plants to gas powered electricity generation. Similar reductions in greenhouse gas emissions are anticipated in Australia as a result of the emergence of the coal seam gas and ultimately the shale gas industry. The production of biogas through anaerobic digestion of renewable sources such as organic feedstock or municipal and industrial waste is also set to become a significant statistic in energy markets with associated benefits of net neutral carbon emissions.
Biological methane production occurs through the anaerobic respiratory activity of methanogenic archaea. With 109 tons of methane produced annually, methanogenesis represents a major driver of organic matter turnover in anaerobic environments. Given this quantity, methanogenesis can also be appreciated for its potential as a major route to energy harvesting for human activity.
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Hydrogenotrophic and acetoclastic methanogenesis represent the two dominant methane producing processes in nature. The substrates for methanogenesis (acetate, hydrogen and carbon dioxide) are generated by fermentative bacteria through the anaerobic oxidation of complex organic molecules derived primarily from the biomass of photosynthetic organisms. The 5 fermentation of complex organic matter and subsequent production of methane are processes that are relatively well understood having received intense attention from scientific and engineering research communities globally for decades.
Hydrogenotrophic methanogenesis involves oxidation of hydrogen and reduction of carbon dioxide to methane (CO2 + 4H2 CH4 + 2H2O). Initially, carbon dioxide is fixed by the 10 Ci carrier methanofuran, which is reduced to formylmethanofuran. Subsequent transfer of the formyl group to another Ci carrier tetrahydromethanopterin precedes stepwise reduction of the formyl group to a methyl residue through activity of the coenzyme F42o-dependent dehydrogenase. The methyl group is then transferred to coenzyme M, which undergoes a final reduction through reaction with coenzyme B resulting in the release of methane and production 15 of the heterodisulfide C0B-S-S-C0M molecule.
In contrast acetoclastic methanogenesis carried out exclusively by members of the Methanosarcinales order involves activation of acetate to acetyl-CoA followed by cleavage of acetate and transfer of the methyl group to coenzyme-M. As in hydrogenotrophic methanogenesis subsequent reduction of the methyl group to methane by coenzyme-B results in production of C0B-S-S-C0M though the electrons are derived from oxidation of the carbonyl group originating from acetate cleavage to CO2.
In both hydrogenotrophic and acetoclastic methanogenesis the heterodisulfide CoB-S-SCoM serves as a terminal electron acceptor reduced by a heterodisulfide reductase linked to the generation of a proton motive force for subsequent ATP generation (see Figure 1). In many
Methanosarcinales species, the heterosulfide reductase is known to source reducing equivalents from the reduced form of methanophenazine (MP), the only biologically produced phenazine involved in energy metabolism. Methanophenazine itself is maintained in the reduced state as dihydromethanophenazine (DHMP) by the coenzyme F420-dependent dehydrogenase responsible for reduction of the formyl group described above.
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The biochemical processes for the synthesis of methane from methanogenic archaea are generally understood.
Efforts to improve methane yields from anaerobic digestion or fermentation of renewable or non-renewable resources have been carried out with an engineering focus on system design, 5 mixing, biomass immobilization, temperature, pH, short chain fatty acid concentrations, codigestion, pre-treatments (alkali, thermal, ultrasonic) and nutrient or metal additives. Significant scientific research effort has also been directed towards understanding the biochemistry and underlying genetics of fermentation and methanogenesis and currently towards linking key functions in biogassification processes with specific microbial phylotypes. However, there has 10 been little research into applying this knowledge on an industrial scale to improve the production of methane from carbonaceous sources by methanogenic organisms. There is a need to improve process efficiencies, rates of production, and yields of biogas from the decomposition of carbonaceous material, particularly on an industrial scale, particularly from non-renewable resources such as coal and renewable resources such as food waste, wastewater sludge and 15 landfill.
It is an object of the invention to provide a means for addressing at least some of the above aforementioned problems.
Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.
Summary of the invention
The invention relates to a method of increasing the rate of production of methane by methanogenic organisms from a reduced organic carbon source in an aqueous solution through the addition of an electron transfer mediator. The electron transfer mediator catalyses the production of methane performed by the methanogenic organisms. By increased rate it is meant that the rate of production of methane is increased as compared with the same system absent the electron transfer mediator.
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In one aspect of the invention there is provided a method of increasing the rate of production of methane in an aqueous environment, the aqueous environment including: a carbonaceous material, anaerobic hydrocarbon oxidising and fermentative microbes, and methanogenic organisms, wherein the anaerobic hydrocarbon oxidising and fermentative 5 microbes liberate a source of reducing equivalents through oxidising the carbonaceous material to substrates including hydrogen and a carbon containing compound selected from the group consisting of alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids or a combination thereof, the method including: the steps of mixing an electron transfer mediator into an aqueous environment and catalysing the conversion of the substrates to methane 10 performed by the methanogenic organisms, using the reducing equivalents liberated by the anaerobic hydrocarbon oxidising or fermenting microbes.
Preferably the carbon containing compound is selected from the group consisting of acetate, carbon dioxide, methanol, trimethylamine or a combination thereof.
The anaerobic hydrocarbon oxidising and fermentative microbes are capable of oxidising 15 or fermenting the carbonaceous material to substrates which include acetate, carbon dioxide, hydrogen, methanol, trimethylamine or a combination thereof. This oxidation process also provides a source of reducing equivalents. The methanogenic organisms are able to convert the substrates to methane. The electron transfer mediator provides the source of reducing equivalents to the methanogenic organism to enhance the rate of conversion of the substrates to methane through a series of reduction-oxidation (redox) reactions. The reducing equivalent interacts with the electron transfer mediator by reducing the electron transfer mediator. The reduced electron transfer mediator then provides the reducing equivalent to the methanogenic organism, enhancing the rate of methane production. Preferably, the transfer of the reducing equivalent from the electron transfer mediator to the methanogenic organism is conducted at a membrane of the methanogenic organism.
The term reducing equivalent refers to any of a number of chemical species which transfer the equivalent of one electron in redox reactions. Examples of reducing equivalents include: a lone electron, a hydrogen molecule, formate and a hydride ion. As discussed, the reducing equivalents are generated by the anaerobic hydrocarbon oxidising and fermentative microbes from the carbonaceous material in the aqueous environment and in the presence of the methanogenic organisms, which provides a source of reducing equivalents to the electron
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2014202663 15 May 2014 transfer mediator to catalyse the conversion of the substrates to methane performed by the methanogenic organisms. This is advantageous as the system does not require an additional external source of energy to supplement the conversion of the substrates to methane, for example in one or more preferred embodiments the system is able to generate methane at an increased 5 rate in the absence of an external supply of electricity. That is, in a preferred embodiment, there is no external supply of reducing equivalents provided to the electron transfer mediator to catalyse the conversion of the substrates to methane. Even more preferably the system does not include an external supply of electricity to generate methane at an increased rate.
In another aspect of the invention there is provided a method of increasing the rate of 10 production of methane in an aqueous environment, the aqueous environment including: a source of reducing equivalents being liberated from within the aqueous environment; substrates including hydrogen and a carbon containing compound selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids or a combination thereof; and methanogenic organisms; the method including the steps of: mixing an 15 electron transfer mediator into the aqueous environment and catalysing the conversion of the substrates to methane performed by the methanogenic organisms, using the reducing equivalents liberated within the aqueous environment.
Preferably the carbon containing compound is selected from the group consisting of acetate, carbon dioxide, methanol, trimethylamine or a combination thereof.
Advantageously, for both of the above described aspects, the generation and use of the reducing equivalents occurs within the same aqueous environment. This means that separate environments for the cathodic and anodic half reactions - which generate and consume the reducing equivalents - is not required. In one or more preferred embodiments, the method is able to produce methane at an increased rate in the absence of separate cathodic and anodic half cells.
Preferably the electron transfer mediator is added to the aqueous environment as an aqueous solution.
In an embodiment, the methanogenic organisms have a respiratory process that generates methane from the carbon containing compound (i.e. acetate or hydrogen and CO2 derived from the carbonaceous material), the respiratory process including the step of reducing a heterodisulfide compound to regenerate a coenzyme central to methane production, and wherein
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2014202663 15 May 2014 the electron transfer mediator catalyses the reduction of the heterodisulfide compound to enhance methane production.
In an embodiment, the heterodisulfide compound is CoM-S-S-CoB and includes component parts coenzyme M (CoM) and coenzyme B (CoB). The respiratory process reduces 5 the heterodisulfide compound to its component parts CoM and CoB. CoM then becomes available for reduction to methyl-CoM, the direct precursor for methane formation. The electron transfer mediator catalyses the reduction of the heterodisulfide compound via a membrane associated heterodisulfide reductase enzymes to generate the essential precursor (coenzyme M) for methane production.
In an embodiment the electron transfer mediator is a synthetic compound. Synthetic compound is intended to refer to a compound that is made via artificial means i.e. it is a manmade compound. Synthetic compounds may include compounds that are found in nature as well as compounds which do not occur naturally.
In an embodiment the electron transfer mediator is selected from the group consisting of:
a phenazine, a quinone, a flavin, or a viologen. Preferably the electron transfer mediator is a phenazine. More preferably the electron transfer mediator is a synthetic phenazine.
In an embodiment the electron transfer mediator has the structure:
wherein C1 is a carbon atom that forms a double bond with an adjacent carbon atom, or 20 with R2; R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of H, Ci-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or R5 and R6 together are a nitrosyl or an isocyano; X1 and X2 are independently selected from the group consisting of N or S; and when X1 or X2 is N, the N may be substituted, unsubstituted, or form a double bond
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2014202663 15 May 2014 with an adjacent carbon atom, and wherein when the N is substituted it is substituted with a substituent selected from the group consisting of H, C1-C3 alkanes, or C2-C3 alkenes.
Preferably X1 and X2 are N, and are substituted with -CH3. Preferably R1, R5, and R6 are -CH3. Preferably R3, R4, and R7 are H. Preferably R2 is selected from the group consisting of: an
O and C1 forms a double bond with R2, or H and C1 forms a double bond with an adjacent carbon atom. Preferably R8 is selected from the group consisting of amide or H. In an embodiment, R8 is CONH2.
In an embodiment the electron transfer mediator has the structure:
wherein: R1, R5, and R6 are independently selected from the group consisting of: H, CiC3 alkanes, or C2-C3 alkenes, halogen,carboxyl, amide, or amine; R2, R3, R4, R5, R6, R7, R8 are independently selected from the group consisting of: H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine.
Preferably R1, R5, and R6 are -CH3 It is also preferred that R2, R3, R4, R5, R6, or R7 are
H. Preferably R8 is selected from the group consisting of H or amide. In an embodiment R8 is CONH2.
In an embodiment, the electron transfer mediator is a substituted or unsubstituted molecule selected from the group consisting of:
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In an embodiment, the electron transfer mediator is a substituted or unsubstituted neutral 5 red or derivatives of neutral red. Preferably the electron transfer mediator is neutral red. It has been found that electron transfer agents having the structure of neutral red are particularly effective at improving the generation of methane. Without wishing to be bound by theory, the inventors believe that the observed increase in methane generation arises as a result of the neutral red mimicking methanophenazine by donating electrons to the heterodisulfide reductase enzyme in the methanogen respiratory chain.
In an embodiment the concentration of the electron transfer mediator in the aqueous environment is in the range of from about 50pm ol/L to about 1000pmol/L and more preferably from about 100pmol/L to about 500pmol/L. Preferably the lower limit in the concentration range is greater than 100pmol/L and may be about 200pmol/L. Preferably the upper limit in the concentration range is 400pmol/L and more preferably about 300pmol/L. The inventors have found that maintaining the concentration of the electron transfer mediator within this range favours the formation of methane.
In an embodiment, the method further includes the step of forming a precipitate of the electron transfer mediator on at least a portion of the surface of the carbonaceous material.
Without wishing to be bound by theory, the inventors believe that the electron transfer mediator
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2014202663 15 May 2014 affects the surface free energy and/or the electrochemical potential of the carbonaceous material allowing the carbonaceous material to be more readily converted into methane.
In one or more embodiments that precipitate formed on the surface of the carbonaceous material alters a surface property of the carbonaceous material. In certain embodiments the 5 surface property is hydrophobicity, and the precipitate increases the hydrophobicity of the surface of the carbonaceous material.
In one or more embodiments the precipitate exhibits a needle-like structure. Preferably the needle-like precipitate has a length of about 100-1500 pm and a width of about 1-5 pm.
In one or more embodiments the precipitate is crystalline. Preferably molecules of the 10 electron transfer mediator are arranged in the crystalline structure of the precipitate so that aromatic regions of adjacent molecules overlap to cause π stacking. More preferably each aromatic region of the adjacent molecules overlap.
In one or more embodiments hydrogen bonding retains the molecules of the electron transfer mediator in position in the crystalline structure of the precipitate. Preferably, the crystalline structure is formed from molecules of an electron transfer mediator and water.
Advantageously it has been found that the presence of crystallites of the electron transfer mediator on the surface of the carbonaceous material and in the presence of the methanogenic organisms further enhances the production of methane.
In an embodiment the electron transfer mediator is a semiconductor. Preferably, the 20 electron transfer mediator has a resistivity of from about 2xl0'9 Ohm.m to about 3xl0'9 Ohm.m. More preferably, the resistivity has a lower limit of 2.1xl0'9 Ohm.m, even more preferably 2.2xl0'9 Ohm.m. Alternatively, or in addition to, it is preferred that the resistivity has an upper limit of 2.5xl0'9 Ohm.m , more preferably 2.4xl0'9 Ohm.m.
In an embodiment, the electron transfer mediator has an electrochemical potential less 25 than -180mV. Preferably the electron transfer mediator has an electrochemical potential less than
-300mV. The electrochemical potential is referenced against a standard hydrogen electrode.
In an embodiment, the pH of the aqueous environment is from about pH 5.5 to about pH
8.5. If the pH is outside of this range, it is preferred that the method additionally includes
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2014202663 15 May 2014 adjusting the pH of the aqueous environment to fall within a pH range of from about pH 5.5 to about pH 8.5. Preferably, the lower value of the pH range is 6 and/or the upper value of the pH range is 8. More preferably, the pH of the aqueous environment is from about pH 6.5 to about pH 7.5. If the pH is outside of this range, it is preferred that the method additionally includes 5 adjusting the pH of the aqueous environment to fall within a pH range of from about pH 6.5 to about pH 7.5.
In an embodiment, the aqueous environment is an anaerobic aqueous environment.
In an embodiment, the anaerobic hydrocarbon oxidizing or fermenting microbes include: Rhizobium and relatives of Rhizobium sp., Phenylobacterium, Achromohacter, Alishewanella, 10 Aquahacteriitm, Desulfur omonas, Clostridium, Geosporobacter, Tessaracoccus, Unclassified Ruminococcaceae and Bacteroidetes, and mixtures thereof.
In an embodiment, the methanogenic organisms include: hydrogenotrophic, acetoclastic, methylotrophic methanogens, and mixtures thereof. Preferably the methanogenic organisms include: Methermicoccus, Methanosarcina, Methanosaeta and Methanobacterium, and mixtures thereof.
In an embodiment, the carbonaceous source is selected from the group consisting of: coal, oil shale, organic waste, organic feedstock, landfill, or biomass. The biomass may be raw organic feed or partially processed, such as in an anaerobic digester. Preferably the carbonaceous source surface area to aqueous environment volume ratio is no more than about 10 cm2 to 1 ml and no less than 0.1 cm2 to 1 ml.
In a further aspect of the invention there is provided a methane producing aqueous environment including: a carbonaceous material; anaerobic hydrocarbon oxidising or fermenting microbes to liberate a source of reducing equivalents within the aqueous environment through oxidising the carbonaceous material to substrates including hydrogen and a carbon containing material selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids or a combination thereof; methanogenic organisms; and an electron transfer mediator to catalyse the conversion of the substrates to methane performed by the methanogenic organisms using the reducing equivalents liberated by the anaerobic hydrocarbon oxidising or fermenting microbes.
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Preferably, the carbon containing material selected from the group consisting of: acetate, carbon dioxide, methanol, trimethylamine, or a combination thereof.
In yet another aspect of the invention there is provided a methane producing aqueous environment including: a source of reducing equivalents liberated from within the aqueous 5 environment; substrates including hydrogen and a carbon containing compound selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids or a combination thereof; methanogenic organisms; and an electron transfer mediator to catalyse the conversion of the substrates by the methanogenic organisms to methane using the reducing equivalents.
Preferably, the carbon containing material selected from the group consisting of: acetate, carbon dioxide, methanol, trimethylamine, or a combination thereof.
In one or more embodiments the methane producing aqueous environment is a hydrocarbon reservoir or landfill.
Various embodiments of these aspects are also described above in relation to the method 15 aspects.
Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.
Brief description of the drawings
Figure 1 is a schematic illustrating the roles of coenzyme F42o, methanophenazine (MP),
CoB-S-S-CoM, the heterosulfide reductase and the F42oH2 dehydrogenase in respiration by methanogens (based on Methanosarcina mazei).
Figure 2 shows related structures of the basic phenazine molecule, coenzyme F42o involved in dehydrogenase activity in methanogens, methanophenazine produced by
Methanosarcina species for energy generation, and the synthetic phenazine neutral red.
Figure 3 is a bright field microscopy image of neutral red crystals.
Figure 4 shows a crystal of neutral red, and the arrangement of neutral red in the crystal.
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Figure 5 is a Graph showing methane production in neutral red incubations (black bars = coal + LSCM3 + neutral red, white bars = LSCM3 + neutral red, lined bars = coal + neutral red). Methane analysis was carried out monthly over a period of 5 months. All incubations performed in triplicate.
Figure 6 is an illustration of a conceptual ‘circuit board’ model showing ‘rewiring’ of electron flow through an idealised microbial community with the use of neutral red.
Figure 7 shows epifluorescence images of 250 μΜ neutral red cultures. Neutral red + LSCM 3 + coal (A-F), neutral red + LSCM 3 (G-H), neutral red + coal (J-K). (Scale bars = 5 μΜ). Red crystals are formed from neutral red. Microbial cells are shown attaching to coal 10 (black) and neutral red crystals.
Figure 8 shows Melhanosarcinales-WVQ cluster formed in 250 μΜ neutral red cultures. On coal surface (A-C) and around/on neutral red crystals (D-H). (Scale bars = 5 μΜ).
Figure 9 shows Relative abundance of bacterial genera in neutral red treated coal fed methanogenic cultures at the start of incubation (TO), after 3 months incubation (Tl) and after 6 months incubation (T2).
Figure 10 shows Relative abundance of archaeal genera in neutral red treated coal fed methanogenic cultures at the start of incubation (TO), after 3 months incubation (Tl) and after 6 months incubation (T2).
Figure 11 is a microscopy image of neutral red crystals showing neutral red crystals 20 formed immediately after the addition (A). Cells were attached to the coal particles and neutral red crystals (B-D). (Scale bar = 5 μΜ).
Figure 12 is a microscopy image of neutral red crystals showing that after 2 months cell and neutral-red crystal densities were high (A-B). Cell clusters were formed around the neutral red crystals (C-D). (Scale bars = 5 μΜ).
Figure 13 is a graph showing methane concentration in 1000 L well headspace for wells amended with 250 μΜ neutral red, nutrients plus or minus acetate or left untreated.
Figure 14 is a graph showing cell concentrations in 475 L well water volume for wells amended with 250 μΜ neutral red, nutrients plus or minus acetate or left untreated.
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Figure 15 is a graph that shows the relationship between current and distance between electrodes along a single neutral red crystal with a potential of -5 volts.
Figure 16 shows two graphs that illustrate the relationship between voltage and current density (upper panel) and power density and current density (lower panel) in anodes with (upper 5 line) and without (central line) NR crystals (SP - synthetic phenazine). Anode chambers were inoculated with 10 mM acetate. An uninoculated anode chamber with NR crystals was included for comparison.
Detailed description of the embodiments
The invention is directed to a method of producing and/or improving production of a 10 biogas that includes methane from a carbonaceous source material. The inventors have discovered that the addition of certain chemical additives substantially increases methane production from carbon fed microorganism cultures.
Without wishing to be bound be theory, the leading hypothesis regarding the mechanism by which methane production is enhanced is that the electron transfer mediator is reduced cometabolically by a complex variety of reduced redox active elements and compounds (organic and inorganic) in anaerobic cultures and that the reduced electron transfer mediator delivers electrons directly and preferentially into the respiratory machinery of methanogenic organisms. Alternative mechanisms have also been proposed, such as the electron transfer mediator inhibiting the growth of microbes that compete with methanogens for reducing equivalents.
Specifically, as discussed in the background section, in the respiratory process of a methanogenic organism, there is a step wherein a methyl-carrier molecule is reduced to form a heterodisulfide compound, which is the final step in the production of methane. The inventors believe that certain electron transfer mediators interact with the respiratory process of the methanogenic organism reducing the heterodisulfide via heterodisulfide reductase thereby regenerating the methyl-carrier molecule (methyl coenzyme A) and hence produce methane at an enhanced rate (i.e. a rate which is greater than the methanogenic organism would naturally produce methane).
The use of these electron transfer mediators to rationally manipulate electron flow to improve methane yields from complex organic substrates is novel. This mechanism represents a
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2014202663 15 May 2014 new approach to maximising biogas yields in a variety of applications, for example from nonrenewable resources such as coal and renewable resources such as food waste, wastewater sludge, landfill and digester biomass.
A number of different types of chemical additives may be employed, for example: 5 phenazines, quinones, flavins, and viologens. Preferred phenazines include substituted or unsubstituted pyocyanin, phenazine, phenazine-1-carboxamide, neutral red, and chemical derivatives thereof. Preferred quinones include substituted or unsubstituted juglone, menaquinone, ubiquinone, and chemical derivatives thereof. Preferred flavins include substituted or unsubstituted riboflavin, FAD, FMN, and chemical derivatives thereof. Preferred viologens 10 include substituted or unsubstituted methyl viologen, benyl viologen, and chemical derivatives thereof.
The most preferred additive electron transfer mediators are phenazines. Phenazines are a large group of nitrogen containing heterocyclic compounds. Over 100 phenazines have been identified as natural products of a variety of microbes including Pseudomonas and Streptomyces species and over 6000 synthetic derivatives of phenazines have been generated.
The invention will now be described generally with reference to the electron transfer mediator being neutral red. Neutral red (2-amino-8-dimethylamino-3-methylphenazine) is a synthetic phenazine. Neutral red (Eo = -325 mV) has the redox potential to reduce methanophenazine (Eo = -165) and CoB-S-S-CoM (Eo = -143) directly. It is also possible that neutral red serves as a substrate for the F42o-dependent dehydrogenase or the heterodisulfide reductase, which is more likely given the structural similarities with methanophenazine, although neutral red shares some features of Coenzyme F42o (see Figure 2). Additionally neutral red has other interesting properties affected by pH and redox potential including polymer and colloid formation.
It is known that neutral red deprotonates at about neutral pH or above resulting in the formation of colloidal precipitate crystals. The inventors have observed this phenomenon under aerobic and anaerobic conditions (see Figure 3), and in ground water associated with a coal seam. The inventors have also observed that the formation of colloidal precipitate particles of neutral red is accelerated by organic matter. Without wishing to be bound by theory, it is thought that aggregates composed of coal and neutral red crystals represent an electrochemically active
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2014202663 15 May 2014 matrix upon which exchange of reducing equivalents between methanogens and other microbes could occur.
Figure 4 shows a crystal 400 of neutral red 402, and the arrangement of neutral red 402 in the crystal 400. The ladders 404 running up the structure are a hydrogen-bonded network of four 5 water molecules. The b-axis on the figure follows the long axis of the needles i.e. the long axis of needles would run up the page. This crystalline structure of deprotonated neutral red with hydrogen bonding with 4 molecules of water per molecule of neutral red enables neutral red molecules to be stacked with overlapping π orbitals. Stacking of π orbitals suggest that the crystals are conductive. The crystals are flexible given their dimensions of about 100-1500 pm in 10 length and about 1-5 pm in width.
Without wishing to be bound by theory, the inventors consider that the exposed tertiary amine group of the neutral red is the electron accepting and donating chemical moiety in neutral red interacting with heterodisulfide reductase.
The inventors have found that neutral red substantially increases methane production 15 from anaerobic coal fed enrichment cultures. The results shown in Figure 5 indicate that methane production from anaerobic coal fed enrichment cultures can be increased by 15-fold on addition of neutral red. Additionally, the results shown in Figure 5 indicate that methane production is negligible in the absence of coal or in the absence of microbes.
In addition to increased methane production an accumulation of acetate has been 20 observed, with concentrations of 209 and 380 pM acetate produced in response to application of and 250 pM neutral red respectively.
This data suggests that synthetic phenazines may improve biogas yields and CH4/CO2 ratios in commercial anaerobic digestion processes using renewable feed-stocks such as algal biomass, landfill or food waste. Electron transfer mediators (e.g. neutral red) have not previously been tested in this context. Neutral red and other electron transfer mediators have been used to catalyse reduction of chlorinated solvents and azo dyes and shown to modify electron flow in alcohol yielding fermentations, but there exists no mention in the scientific literature of application to methane generation during anaerobic digestion.
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Neutral red can be maintained in the reduced state through the activity of anaerobic microbial communities. This suggests that neutral red can be used to siphon reducing equivalents towards methanogenesis from the metabolism of microbes traditionally thought to compete with methanogens (e.g. homoacetogens and iron, sulphate and nitrate reducing bacteria). In effect, the 5 flow of electrons through the community can be ‘rewired’ to maximise methane production.
Figure 6 is an illustration of a conceptual ‘circuit board’ model showing ‘rewiring’ of electron flow through an idealised microbial community with the use of neutral red. In this case, the neutral red acts to ‘short circuit’ the microbial community resulting in decreased electron transfer for the reduction of nitrate, iron, and sulphate species, and instead provides increased 10 electron transfer to the methanogens for reduction of substrates to methane. Electrons are liberated from carbonaceous material by nitrate, iron, sulphate reducing and fermentative bacteria. The neutral red reacts with the electron to form a reduced neutral red. The reduced neutral red then interacts with membrane proteins of methanogens like heterodisulfide reductase, to transfer the electron into the respiratory process of the methanogens which enhances the 15 production of methane from the reduction of substrate compounds. No external source of electrons is required and no external source of electrons is introduced into the system for enhancing the production of methane. Electrons are liberated within the local environment from anaerobic digestion of carbonaceous material by the microbial community.
Examples
Example 1
An experiment was conducted to test the impact of neutral red on biogenic methane production from coal (complex organic matter). A defined anaerobic mineral salts medium containing sub-bituminous coal was inoculated (10% v/v) with a mixed species microbial community and treated with a range of neutral red concentrations (0 - 500 μΜ final concentration). Controls lacking coal or lacking the inoculum were also established. Cultures have been incubated anaerobically in the dark without agitation at 22 °C for 5 months.
Figure 5 shows that the coal fed mixed species microbial community responds to 250 μΜ neutral red with rapid methane production (approx. 15 fold increase in methane production rate). The monthly increase in methane concentration in this treatment is increasing (i.e. methane production rate is accelerating). Figure 5 also shows that the 500 μΜ neutral red treatment
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2014202663 15 May 2014 displays increased methane production, though a lag time is evident. Treatments with 50 or 100 μΜ neutral red are no different from the control lacking (0 μΜ) neutral red. Control cultures lacking coal or inoculum show limited or no response to neutral red indicating that the phenomenon is dependent on microbes and coal.
Epifluorescence microscopy analyses of the cultures revealed an increase in cell concentration along with increasing neutral red concentration joined by the formation of neutral red “needles” (Figures 7 and 8). The “needle” formation in the cultures due to neutral red were only observed in a concentration range from 100-500 μΜ neutral red, increasing in density with increased concentration. The presence of these needles appears to be important for enhancing 10 methane producti on.
Without wishing to be bound by theory, the inventor’s believe the observed activity occurs as neutral red mimics methanophenazine by donating electrons to the heterodisulfide reductase enzyme in the methanogen respiratory chain.
An experiment was conducted to test this hypothesis, involving reduction of 10 μΜ 15 neutral red with titanium (III) citrate and incubation with 12 μg of a membrane fraction of acetate-grown Methanosarcina mazei and 15 μΜ heterodisulfide in 40 mM potassium phosphate buffer (pH 7.0, 5 mM DTE). Absorbance at 530 nm was used to monitor neutral red oxidation. Results indicate that neutral red transfers electrons to the CoM-S-S-CoB heterodisulfide in the presence of methanogen membrane fractions with an activity of 700 mU/mg protein. Control reactions lacking membranes or the heterodisulfide showed that both components were required to observe neutral red oxidation. The main mode of action of neutral red therefore appears to be in heterodisulfide reduction; however, its unusual physical chemistry properties (needle formation) and interaction with coal also appear to play a role.
Example 2
Microbial community dynamics during enhanced biogas production.
Pyrosequencing of 16S rRNA gene amplicons was used to describe shifts in relative abundance of the most abundant Bacteria and Archaea in the coal fed methanogenic microbial communities treated with varying concentrations of neutral red. This data implicates specific
1000636231 1.doc
2014202663 15 May 2014 bacterial and archaeal genera involved in the process. Figure 9 shows bacterial abundances. Figure 10 shows archaeal abundances.
Bacterial sequences closely related to known hydrocarbon degrading bacteria were enriched in the neutral red cultures, predominantly, relatives of Rhizobium sp. accompanied by 5 Phenylobacterium, Achromobacter, Alishewanella, Aquabacterium, Desulfuromonas, Clostridium, Geosporobacter, Tessaracoccus, Unclassified Ruminococcaceae and Bacteroidetes. Bacteria belonging to these genera are likely to be involved in transferring reducing equivalents from coal to methanogens via neutral red crystals. In contrast, sulfate-reducing bacteria (Desulfovibrio, Desulfotomaculum) were inhibited by neutral red treatment.
The archaeal community consisted of hydrogenotrophic, acetoclastic and methylotrophic methanogens with a shift towards the enrichment of methylotrophic and hydrogenotrophic archaea through neutral red addition. However, Methanosarcina sp. was still abundant in the neutral red cultures and is characterized to carry out acetoclastic and/or hydrogenotrophic methane formation. Archaea belonging to the genera Methermicoccus, Methanosarcina,
Methanosaeta and Methanobacterium are likely responsible for methane production using reducing equivalents from neutral red.
Example 3
In situ field demonstration of enhanced biogas production with neutral red.
Neutral red was amended into a test well constructed 80 m below ground connecting with a 3 m thick seam of methane free subbituminous coal. The well contains 475 L of groundwater and a 1000 L headspace. Approximately 2 m2 of coal surface area was exposed to the treatable groundwater. The pH of the groundwater was approximately 8. The temperature was approximately 17 °C. The sulphate concentration in the groundwater was approximately 250 mg/L. The final neutral red concentration was 250 μΜ. Microscopy revealed the formation of neutral red crystals in situ (Fig. 11 and Fig. 12).
The sulphate concentration is of interest because in many cases methane production does not commence, or commences only very slowly while sulphate is being reduced to sulphide. The presence of sulphide is generally undesirable as it can be growth inhibitory to microbes and
1000636231 1.doc
2014202663 15 May 2014 cause corrosion of infrastructure. An advantageous feature of the neutral red amendments is that there is an inhibition of sulphate reducing bacteria and sulphate reduction.
Methane concentrations in the 1000 L well headspace and cell concentrations in the 475 L bulk aqueous phase were monitored in the neutral red treated well along with a nutrient 5 amended well, a nutrient plus acetate (20 mM) amended well and an untreated control well (Fig. 13 and Fig. 14). Neutral red addition resulted in a rapid increase in cell concentrations and methane accumulation in the headspace comparable to results achieved in the laboratory and superior to other treatments administered in the field. The neutral red treatment outperformed nutrient and acetate amendments alone or in combination. No methane or biomass production 10 was observed in the unamended control well. The neutral red treatment generated methane at a rate relative to coal surface area of approximately 5 mMoles/m2/day or 0.11 L/m2/day.
Example 4
Atomic force microscopy reveals NR crystals are semiconductors
Atomic force microscopy has been used to determine electrical properties of the neutral 15 red crystals. Figure 15 shows the relationship between current and distance between electrodes along a single neutral red crystal with a potential of -5 volts. From this data the resistivity of the crystal was calculated to be 2.386 x 10'9 Ohm m. This suggests the crystals are semiconductors, confirming the speculation that the pi orbital stacking of the neutral red molecules in the crystal structure may enable electron transfer along the length of the crystal.
Example 5
NR increases coal surface hydrophobicity enhancing bacterial attachment
The effect of neutral red to the coal surface properties was determined using contact angle measurement of subbituminous coal incubated in anaerobe media and water with the addition of neutral red (0, 50, 100, 250 and 500 μΜ) for a period of 5 days (Table 1). The coal incubated without neutral red revealed a highly hydrophilic surface (contact angle below 10°) possibly due to a rough coal surface containing small cracks. The surface of the coal particles in the neutral red incubations showed an increase towards hydrophobicity (contact angle ~ 30°) suggesting that the amendment of neutral red alters the coal surface enhancing the process of microbial attachment. In all coal containing neutral red cultures as well as in groundwater in the
1000636231 1.doc
2014202663 15 May 2014 field more cells were observed being attached to coal particles compared to the incubations lacking neutral red.
Table 1: Contact angle measurement of coal incubated with different concentrations of Neutral red. All measurements were carried out in triplicates.
| Coal + Neutral Red (pm) | Average contact angle |
| 0 | <10° |
| 50 | <10° |
| 100 | 5° |
| 250 | 28° |
| 500 | 32° |
Example 6
Bacteria can deliver electrons to NR crystals
Using microbial fuel cells with NR crystals entrained in a conductive carbon felt anode it has been demonstrated that reducing equivalents from acetate (10 mM) were diverted away from the carbon felt electrode in the presence of NR crystals. The presence of the NR crystals increased acetate consumption but decreased coulombic efficiency (Table 2) indicating that electrons sourced by bacteria from acetate were transferred to NR crystals at the expense of the electrode (Fig. 16).
Table 2: Bioelectrochemical data describing the impact of NR on electron fate
| Digestate + NR crystals | Digestate | NR Crystals | |
| Maximum power density | 61.41 | 191.21 | 9.06 |
| (mW/m3) |
1000636231 1.doc
2014202663 15 May 2014
Internal resistance (Ohms)
Acetate consumed (mM)
Coulombic efficiency
4400.5 1480.6 4400.9
8.58
6.39
-0.18
72.96 84.83 0.00 (%)
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
1002099254
2014202663 06 Mar 2018
Claims (10)
1. A method of increasing the rate of production of methane in an aqueous environment the aqueous environment including:
a carbonaceous material, anaerobic hydrocarbon oxidising or fermenting microbes, and 5 methanogenic organisms, wherein the anaerobic hydrocarbon oxidising or fermenting microbes liberate a source of reducing equivalents within the aqueous environment through oxidising the carbonaceous material to substrates including hydrogen and a carbon containing compound selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines,
0 organic acids, or a combination thereof;
the method including the steps of:
mixing an electron transfer mediator into the aqueous environment and catalysing the conversion of the substrates to methane performed by the methanogenic organisms using the reducing equivalents liberated by the anaerobic hydrocarbon oxidising or fermenting microbes;
5 wherein the electron transfer mediator is selected from the group consisting of:
wherein C1 is a carbon atom that forms a double bond with an adjacent carbon atom, or with R ;
R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group 20 consisting of H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or R5 and R6 together are a nitrosyl or an isocyano;
1 2
X and X are independently selected from the group consisting of N or S; and
1 2 when X or X is N, the N may be substituted, unsubstituted, or form a double bond with an adjacent carbon atom, and wherein when the N is substituted it is substituted with a substituent
25 selected from the group consisting of H, C1-C3 alkanes, or C2-C3 alkenes; or
1002099254
2014202663 06 Mar 2018 wherein Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8 are independently selected from the group consisting of: H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or
I i ch3 cr ch3
2. A method of increasing the rate of production of methane in an aqueous environment the aqueous environment including: a source of reducing equivalents being liberated from within the aqueous environment; substrates including hydrogen and a carbon containing compound
10 selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids, or a combination thereof; and methanogenic organisms;
the method including the steps of:
mixing an electron transfer mediator into the aqueous environment and catalysing the conversion of substrates to methane performed by the methanogenic organisms, using the
15 reducing equivalents liberated within the aqueous environment;
1002099254
2014202663 06 Mar 2018
wherein C1 is a carbon atom that forms a double bond with an adjacent carbon atom, or with R ;
5 R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or R5 and R6 together are a nitrosyl or an isocyano;
X and X are independently selected from the group consisting of N or S; and when X or X is N, the N may be substituted, unsubstituted, or form a double bond with an
0 adjacent carbon atom, and wherein when the N is substituted it is substituted with a substituent selected from the group consisting of H, C1-C3 alkanes, or C2-C3 alkenes; or wherein Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8 are independently selected from the group consisting of: H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or
O
1002099254
2014202663 06 Mar 2018
3. The method of claim 1 or 2, wherein the methanogenic organisms have a respiratory process that generates methane from the carbon containing compound, the respiratory process
5 including the step of reducing a heterodisulfide compound to regenerate a precursor coenzyme for methane production, and wherein the electron transfer mediator catalyses the reduction of the heterodisulfide compound to enhance methane production.
4. The method of claim 3, wherein the heterodisulfide compound is CoM-S-S-CoB and the electron transfer mediator catalyses the reduction of the CoM-S-S-CoB via membrane associated
0 heterodisulfide reductase enzymes to generate the precursor enzyme.
5. The method of claim 3, wherein the heterodisulfide compound is reduced by a heterodisulfide reductase enzyme, and wherein the electron transfer mediator catalyses the reduction of the heterodisulfide reductase.
6. The method of any one of the preceding claims, wherein the electron transfer mediator 15 is selected from the group consisting of: a phenazine, a quinone, a flavin, or a viologen; and when the electron transfer mediator is a phenazine, it is not methanophenazine.
7. The method of any one of the preceding claims wherein the electron transfer mediator is selected from the group consisting of:
1002099254
2014202663 06 Mar 2018
12 15
8. The method of claim 7 wherein X and X are N, and are substituted with -CH3; R , R ,
6 2 1 and R are -CH3; R is selected from the group consisting of: O and C forms a double bond with R , or H and C forms a double bond with an adjacent carbon atom; and R is selected from the group consisting of an amide or H.
5 9. The method of any one of any one of claims 1 to 6, wherein the electron transfer mediator is selected from the group consisting of:
15 6 8
10. The method of claim 9 wherein Y , Y , and Y are -CH3; and Y is selected from the group consisting of H or amide.
0 11. The method of any one of claims 1 to 6 wherein the electron transfer mediator is a substituted or unsubstituted molecule selected from the group consisting of:
1002099254
2014202663 06 Mar 2018
12. The method of any one of any one of the preceding claims, wherein electron transfer mediator is neutral red.
13. The method of any one of the preceding claims, wherein the concentration of the electron transfer mediator in the aqueous source is from about 100 pmol/L to about 500 pmol/L.
5 14. The method of claim 13, wherein the electron transfer mediator concentration is greater than 100 itmol/L.
15. The method of any one of the preceding claims, further including during the step of mixing, forming precipitate crystals of the electron transfer mediator.
16. The method of claim 15, wherein the precipitate exhibits a needle-like structure.
0 17. The method of any one of the preceding claims wherein the electron transfer mediator has an electrochemical potential lower than -180mV.
18. The method of claim 17, wherein the electron transfer mediator has an electrochemical potential of lower than -300mV.
19. The method of any one of the preceding claims, wherein the pH of the aqueous 5 environment is from about pH 6.5 to about pH 8.5.
20. The method of any one of the preceding claims, wherein the aqueous environment is an anaerobic aqueous environment.
21. The method of any one of the preceding claims, wherein the carbonaceous source is selected from the group consisting of: coal, oil shale, organic waste, organic feedstock, landfill,
20 or biomass.
22. A methane producing aqueous environment, including:
a carbonaceous material;
anaerobic hydrocarbon oxidising or fermenting microbes to liberate a source of reducing equivalents within the aqueous environment through oxidising the carbonaceous
25 material to substrates including hydrogen and a carbon containing material selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids, or a combination thereof;
1002145595 methanogenic organisms; and an electron transfer mediator to catalyse the conversion of the substrates to methane performed by the methanogenic organisms using the reducing equivalents liberated by the anaerobic hydrocarbon oxidising or fermenting microbes;
5 wherein the electron transfer mediator is selected from the group consisting of:
2014202663 19 Apr 2018 wherein C1 is a carbon atom that forms a double bond with an adjacent carbon atom, or with R2;
R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group 0 consisting of H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or R5 and R6 together are a nitrosyl or an isocyano;
X andX are independently selected from the group consisting of N or S; and when X or X is N, the N may be substituted, unsubstituted, or form a double bond with an adjacent carbon atom, and wherein when the N is substituted it is substituted with a substituent
5 selected from the group consisting of H, C1-C3 alkanes, or C2-C3 alkenes; or wherein Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8 are independently selected from the group consisting of: H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or
1002145595
2014202663 19 Apr 2018 ch3 ?
Cl Cl
CH,© 'γ CHa ?
OH O ch3 I (CH? — CH=C-CH2), o-H
23. A methane producing aqueous environment including:
5 a source of reducing equivalents liberated from within the aqueous environment;
substrates including hydrogen and a carbon containing compound selected from the group consisting of: alcohols, fatty acids, organic sulphides, carboxylates, organic amines, organic acids, or a combination thereof;
methanogenic organisms; and
10 an electron transfer mediator to catalyse the conversion of the substrates by the methanogenic organisms to methane using the reducing equivalents;
wherein the electron transfer mediator is selected from the group consisting of:
wherein C1 is a carbon atom that forms a double bond with an adjacent carbon
15 atom, or with R ;
1002145595
2014202663 19 Apr 2018
R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or R5 and R6 together are a nitrosyl or an isocyano;
X and X are independently selected from the group consisting of N or S; and 5 when X1 or X2 is N, the N may be substituted, unsubstituted, or form a double bond with an adjacent carbon atom, and wherein when the N is substituted it is substituted with a substituent selected from the group consisting of H, C1-C3 alkanes, or C2-C3 alkenes; or wherein Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8 are independently selected from the 0 group consisting of: H, C1-C3 alkanes, C2-C3 alkenes, halogen, carboxyl, amide, or amine; or
24. The methane producing aqueous environment of claim 22 or 23 wherein the electron 15 transfer mediator is added at a concentration of greater than 100 pmol/L to 500 pmol/L.
1002145595
3 19 Apr 2018
25. The method of any one of claims 1 to 21, wherein the carbon containing compound is selected from the group consisting of: acetate, carbon dioxide, methanol, trimethylamine, or a combination thereof.
26. The methane producing aqueous environment of any one of claims 22 to 24, wherein ) the carbon containing compound is selected from the group consisting of: acetate, carbon dioxide, methanol, trimethylamine, or a combination thereof.
Ό
Ό
CM o
CM o
CM
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2014202663 15 May 2014
1/10
FIGURES
Figure 1
Phenazine
A. -x x ,
Χίο
Coenzyme ^423 Ii x'X. xx^ x\ < w X-z
HO' x’X.X'X
NH X ,xks~
R
Methanophenazins ,0
T I! T ,z'XX\/
-- η
XX XX χχ
X.
j 3 Neutral Red .X z·^ .NHz H <C V % XX %
Χχ^ΧχχΧ^ 'CH.
XxXyl
Figure 2
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2014202663 15 May 2014
Figure 3
402
Figure 4
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2014202663 15 May 2014
Neutral Red
Figure 5
Reduced Organic Substrate (Coal, oil shale, organic waste, organic feedstock)
4'
Bmttelrbd ??????<
KsV’Xx'S^'Sx^SxxilCM,
Figure 6
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2014202663 15 May 2014
Figure 7
Figure 8
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2014202663 15 May 2014
Figure 9
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6/10
2014202663 15 May 2014 ω_ φ
η φ
ω η
ΓΗ
C “3 *<
Ο>
“3
Γ>
ω φ
Ο r+ ω
ΑΛΟ
Ό ο
r+
Φ
Ο>
φ
Ο f—t·
Ο>
αλω
Ζ5
Ο
CT ω
Ζ5
Ο
Ζ5 ω
φ’ Q. φ’ C ϋ S-L S-L 3 £ 6 $
Ζ3 Ζ3 Ο Ο σ ω ο
Φ φ Ζ3 ο
Ο ο
ο
C
Ο ο
Ο ο
ο ω
ο φ
ω φ
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28 ©
Figure 10
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A , 8
Figure 11
Figure 12
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Figure 13
Inucbation time [months]
Figure 14
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Distance (pm)
Figure 15
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Figure 16
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000003447A1 (en) * | 1998-07-09 | 2000-01-20 | Michigan State University | Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration |
| WO2011056183A1 (en) * | 2009-11-06 | 2011-05-12 | Sequesco | Biological and chemical process utilizing chemoautotrophic microorganisms for the chemosynthetic fixation of carbon dioxide and/or other inorganic carbon sources into organic compounds, and the generation of additional useful products |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000003447A1 (en) * | 1998-07-09 | 2000-01-20 | Michigan State University | Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration |
| WO2011056183A1 (en) * | 2009-11-06 | 2011-05-12 | Sequesco | Biological and chemical process utilizing chemoautotrophic microorganisms for the chemosynthetic fixation of carbon dioxide and/or other inorganic carbon sources into organic compounds, and the generation of additional useful products |
Non-Patent Citations (4)
| Title |
|---|
| PARK, D. H. ET AL: ‘Bioelectrosynthesis of Organic Chemicals and Fuels’, Communications- Faculty of Agricultural and Applied Biological Sciences (Ghent University), 2001, Vol. 66(3a), p. 129-131 * |
| PARK, D. H. ET AL: ‘Microbial Utilization of Electrically Reduced Neutral Red as the Sole Electron Donor for Growth and MetaboliteProduction’, Applied and Environmental Microbiology, 1999, Vol. 65, No. 7, p. 2912-2917 * |
| PICIOREANU, C. ET AL: ‘Model Based Evaluation of the Effect of pH and Electrode Geometry on Microbial Fuel Cell Performance’,Bioeletrochemistry, 2010, Vol. 78, No. 1, p. 8-24 * |
| STEINBUSCH, K. J. J. ET AL: ‘Bioeletrochemical Ethanol Production Through Mediated Acetate Reduction by Mixed Cultures’, Environmental Science & Technology, 2010, Vol. 44, No. 1, p. 513-517 * |
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