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    • 专利摘要:
    BACTERIA AND METHODS OF USE OF THE SAME. The present invention generally relates to the field of microbial fermentation of gases. More particularly, it refers to a new class of bacteria with improved efficiency in ethanol production by anaerobic fermentation of substrates containing carbon monoxide (CO).
    公开号:BR112013003644B1
    申请号:R112013003644-3
    申请日:2011-07-28
    公开日:2020-11-17
    发明作者:Bjorn Daniel Heijstra；Evgenia Kern；Michael Koepke；Simon Segovia；Fungmin Liew
    申请人:Lanzatech New Zealand Limited；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [001] The present invention generally relates to the microbial gas fermentation field. More particularly it refers to a new class of bacteria with improved efficiency in ethanol production by anaerobic fermentation of substrates containing carbon monoxide (CO). BACKGROUND OF THE INVENTION
    [002] Ethanol is fast becoming a major hydrogen-rich liquid transport fuel around the world. World ethanol consumption in 2005 was approximately 12.2 billion gallons. The global market for the fuel ethanol industry was also predicted to grow sharply in the future, due to an increased interest in ethanol in Europe, Japan, the USA and several developing countries.
    [003] For example, in the USA, ethanol is used to produce E10, a mixture of 10% ethanol in gasoline. In the E10 mixture, the ethanol component acts as an oxygenation agent, improving combustion efficiency and reducing the production of air pollutants. In Brazil, ethanol satisfies approximately 30% of the transport fuel requirement, as much as an oxygenation agent mixed with gasoline, as a pure fuel in itself. Also, in Europe, environmental issues surrounding the consequences of greenhouse gas (GHG) emissions have been the stimulus of the European Union (EU) to establish a mandate target for the consumption of sustainable transport fuels, such as as ethanol derived from biomass.
    [004] The vast majority of fuel ethanol is produced through traditional yeast-based fermentation processes using crop-derived carbohydrates, such as sucrose extracted from sugar cane or starch extracted from grain crops, as the main carbon source . However, the cost of these carbohydrate feed stocks is influenced by their value as human food or fodder, while the cultivation of starch or sucrose-producing crops for ethanol production is not economically sustainable in all geographic areas. For this reason, it is of interest to develop technologies to convert lower-cost and / or more abundant carbon resources into fuel ethanol.
    [005] CO is a major, free by-product, rich in energy from the incomplete combustion of organic materials, such as coal or oil and petroleum products. For example, the steel industry in Australia is reported to produce and release more than 500,000 tonnes of CO into the atmosphere annually.
    [006] Catalytic processes can be used to convert gases that consist mainly of CO and / or CO and hydrogen (H2) into a variety of fuels and chemicals. Microorganisms can also be used to convert these gases into fuels and chemicals.
    [007] The ability of microorganisms to thrive in CO as a single carbon source was first discovered in 1903. This was later determined to be a property of organisms that use the biochemical pathway of acetyl coenzyme A (acetyl CoA) for growth autotrophic (also known as Woods-Ljungdahl pathway and carbon monoxide dehydrogenase / acetyl CoA synthase pathway (CODH / ACS)). It has been shown that a large number of anaerobic organisms including carboxidotrophic, photosynthetic, methanogenic and acetogenic organisms metabolize CO to various end products, that is, CO2, Ft, methane, n-butanol, acetate and ethanol. Using CO as the sole source of carbon, all such organisms produce at least two of these end products.
    [008] Anaerobic bacteria, such as those of the genus Clostridium, have been shown to produce ethanol from CO, CO2 and H2 through the biochemical pathway of acetyl CoA. For example, several strains of Clostridium Ijungdahlii that produce ethanol from gases are described in WO 00/68407, EP 117309, US patent nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438. It is also known that the bacterium Clostridium autoethanogenum sp produces ethanol from gases (Abrini et al., Archives of Microbiology 161, pp 345-351 (1994)).
    [009] However, the production of ethanol by microorganisms by the fermentation of gases is always associated with the co-production of acetate and / or acetic acid. As a part of the available carbon is converted to acetate / acetic acid instead of ethanol, the efficiency of ethanol production using such fermentation processes may be less than desirable. Also, unless the acetate by-product / acetic acid by-product can be used for some other purpose, it can represent a waste disposal problem. Acetate / acetic acid is converted to methane by microorganisms and therefore has the potential to contribute to GHG emissions.
    [0010] Microbial fermentation of CO in the presence of H2 can lead to substantially complete carbon transfer in an alcohol. However, in the absence of sufficient H2, some of the CO is converted to alcohol, while a significant portion is converted to CO2 as shown in the following equations: 6CO + 3H2O -► C2H5OH + 4CO2 12H2 + 4CO2 -> 2C2H5OH + 6H2O
    [0011] CO2 production represents a lack of efficiency in capturing total carbon and if released, it also has the potential to contribute to greenhouse gas emissions.
    [0012] WO2007 / 117157 describes a process that produces alcohols, particularly ethanol, by anaerobic fermentation of gases containing carbon monoxide. The acetate produced as a by-product of the fermentation process is converted to hydrogen gas and carbon dioxide gas, or both of which can be used in the anaerobic fermentation process.
    [0013] WO2008 / 115080 describes a process for the production of alcohol (s) in multiple fermentation stages. By-products produced as a result of anaerobic fermentation of gas (s) in a first bioreactor can be used to produce products in a second bioreactor. In addition, by-products from the second fermentation stage can be recycled to the first bioreactor to produce products.
    [0014] WO2009 / 064200 describes a new class of bacteria that has improved efficiency in ethanol production by anaerobic fermentation of substrates containing carbon monoxide.
    [0015] It would be beneficial to provide microorganisms that are capable of fermenting gases containing carbon monoxide to ethanol with increased efficiency, which are microorganisms capable of improved absorption of carbon monoxide, the production of more ethanol, and / or a higher proportion of ethanol to acetate of the same substrate, than the microorganisms of the previous technique.
    [0016] It is an object of the present invention to provide a new class of bacteria that overcomes one or more of the limitations of the prior art in converting gaseous sources containing CO into ethanol, or at least providing the public with a useful choice. SUMMARY OF THE INVENTION
    [0017] In a first aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, at a specific productivity of at least approximately 2 g of ethanol / L of fermentation broth / gram of biomass / day.
    [0018] In other modalities, the bacterium is capable of producing ethanol at a specific productivity of at least approximately 3 g of ethanol / L of fermentation broth / gram of biomass / day, at least approximately 4 g of ethanol / L of broth fermentation / gram of biomass / day, at least approximately 5 g of ethanol / L of fermentation broth / gram of biomass / day, at least approximately 6 g of ethanol / L of fermentation broth / gram of biomass / day or at least minus approximately 7 g of ethanol / L of fermentation broth / gram of biomass / day.
    [0019] In another aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, at a productivity of at least approximately 10 g of ethanol / L of fermentation broth / day.
    [0020] In other modalities, the bacterium is capable of producing ethanol at a productivity of at least approximately 20 g of ethanol / L of fermentation broth / day, at least approximately 30 g of ethanol / L of fermentation broth / day, at least approximately 40 g of ethanol / L of fermentation broth / day or at least approximately 50 g of ethanol / L of fermentation broth / day, or at least approximately 60 g of ethanol / L of fermentation broth / day, or at least approximately 70 g of ethanol / L of fermentation broth / day.
    [0021] In another aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and in which the bacterium is capable of specific absorption of at least approximately CO 1.0 mMol CO / min / g of biomass.
    [0022] In one embodiment, the bacterium is capable of a specific CO absorption of at least approximately 1.2 mMol of CO / min / g of biomass, at least approximately 1.4 mMol of CO / min / g of biomass, at least approximately 1.6 mMol of CO / min / g of biomass, at least approximately 1.8 mMol of CO / min / g of biomass, or at least approximately 2.0 mMol of CO / min / g of biomass. In a particular embodiment, the bacterium is capable of a specific CO input of at least approximately 1.2 mMol of CO / min / g of biomass.
    [0023] In another aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and in which the bacterium is capable of specific growth rate of at least approximately 0.8 day-1.
    [0024] In certain embodiments, the bacterium is capable of a specific growth rate of at least approximately 1.0 day1, at least approximately 1.2 day1, at least approximately 1.4 day1, at least approximately 1.6 day1, at least at least approximately 1.8 days1 or at least approximately 2.0 days1.
    [0025] In another aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and in which the bacterium is capable of producing ethanol in an ethanol to acetate ratio of at least approximately 2: 1.
    [0026] In certain embodiments, the bacterium is capable of producing ethanol in an ethanol to acetate ratio of at least approximately 3: 1, at least approximately 4: 1, at least approximately 5: 1, at least approximately 7 : 1 or at least approximately 10: 1. In one embodiment, the bacterium is capable of producing ethanol with substantially no acetate.
    [0027] In another aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and in which the bacterium is capable of tolerating alcohol of up to approximately 30g / L of fermentation broth.
    [0028] In a certain modality, the bacteria is capable of tolerating alcohol of up to approximately 40g / L of fermentation broth, of up to approximately 50g / L of fermentation broth, of up to approximately 60g / L of fermentation broth, or up to approximately 70g / L of fermentation broth.
    [0029] In another aspect, the invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and in which the bacterium has two or more of the following characteristics: it is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, in a specific productivity of at least approximately 2 g of ethanol / L of fermentation broth / gram of biomass / day; it is capable of producing ethanol in a concentration of at least approximately 10 g of ethanol / L of fermentation broth / day; it is capable of a specific CO absorption of at least approximately 1.0 mMol of CO / min / g of the biomass; it is capable of a growth rate of at least approximately 1.0 g / day; it is capable of producing ethanol in an ethanol to acetate ratio of at least approximately 2: 1; and, it is capable of tolerating alcohol of up to approximately 30g / L of the broth.
    [0030] In one embodiment, the bacteria of the invention are derived from Clostridium autoethanogenum. In a preferred embodiment, the bacterium of the invention is a strain of Clostridium autoethanogenum.
    [0031] In a particular modality, the bacterium has the defining characteristics of the Clostridium autoethanogenum strain deposited in the DSMZ under accession number DMS23693. In one embodiment, the bacterium is the strain of Clostridium autoethanogenum deposited in the DSMZ under accession number DMS23693.
    [0032] In a further aspect, the invention provides a biologically pure isolate of the strain of Clostridium autoethanogenum deposited in the DSMZ under accession number DMS23693.
    [0033] In another aspect, the invention provides a method of producing one or more alcohols comprising fermenting a substrate comprising CO using a bacterium as described earlier in this application.
    [0034] In one embodiment, the method comprises the steps of: (a) supplying a substrate comprising CO to a bioreactor containing a culture of a bacterium of the invention; and (b) anaerobic fermentation of the culture in the bioreactor to produce one or more alcohols.
    [0035] In an additional aspect, the invention provides a method to reduce the total atmospheric carbon emissions of an industrial process, the method comprising: (a) capturing gas containing CO produced as a result of the industrial process, before the gas is released In the atmosphere; (b) anaerobic fermentation of CO containing gas to produce one or more alcohols by a culture containing one or more bacteria of the invention.
    [0036] In an embodiment of the method aspects, fermentation is conducted at a temperature of approximately 34 ° C to approximately 37 ° C. In a preferred embodiment, fermentation is conducted at a temperature of approximately 34 ° C.
    [0037] In certain modalities of method aspects, acetate is produced as a by-product of fermentation. Preferably one or more alcohols produced include ethanol.
    [0038] In particular modalities of aspects of the method, the bacterium is kept in an aqueous culture medium.
    [0039] In particular modalities of aspects of the method, substrate fermentation is carried out in a bioreactor.
    [0040] In certain embodiments, the substrate comprises at least approximately 25% by volume of CO, at least approximately 30% by volume of CO, at least approximately 40% by volume of CO, at least approximately 50% by volume of CO, at least approximately 65% by volume of CO or at least approximately 70% by volume of CO. In particular embodiments, the substrate comprises at least approximately 75% by volume of CO, at least approximately 80% by volume of CO, at least approximately 85% by volume of CO, at least approximately 90% by volume of CO or at least approximately 95% by volume of CO.
    [0041] In one embodiment, the substrate comprises approximately 30% or less of H2 by volume. In other embodiments, the substrate comprises approximately 20% or less H2 by volume, approximately 15% or less H2 by volume, approximately 10% or less H2 by volume, approximately 5% or less H2 by volume, approximately 4% or less H2 in volume, approximately 3% or less H2 by volume, approximately 2% or less H2 by volume, approximately 1% or less H2 by volume, or substantially without H2.
    [0042] In one embodiment, the substrate comprises less than or equal to approximately 20% by volume CO2. In particular embodiments the substrate comprises less than or equal to approximately 15% by volume CO2, less than or equal to approximately 10% by volume CO2, less than or equal to approximately 5% by volume CO2 or substantially without CO2.
    [0043] In certain embodiments, the substrate comprising CO is a gaseous substrate containing CO.
    [0044] In certain embodiments, the gaseous substrate comprises a gas obtained as a by-product of an industrial process.
    [0045] In certain modalities, the industrial process is selected from the group consisting of the manufacture of ferrous metal products, the manufacture of non-ferrous products, petroleum refining processes, biomass gasification, coal gasification, electricity production, production carbon black, ammonia production, methanol production and coke production.
    [0046] In one embodiment, the gaseous substrate may comprise a gas obtained from a steel mill.
    [0047] In another embodiment, the gaseous substrate may comprise automobile exhaust fumes.
    [0048] In certain modalities of aspects of the method, alcohol is recovered from the fermentation broth, the fermentation broth which is the aqueous culture medium comprising bacterial cells and alcohol.
    [0049] In certain embodiments, acetate is produced as a by-product of fermentation.
    [0050] In an additional modality, alcohol and acetate are recovered from the broth.
    [0051] Although the invention is broadly as defined above, it is not limited to it and also includes embodiments of which the following description provides examples. BRIEF DESCRIPTION OF THE DRAWINGS
    [0052] The invention will now be described in detail with reference to the accompanying Figures in which: Figure 1: Shows the consumption of CO of DSM19630 and DSM23693 Figure 2: Shows the metabolite production of DSM19630 (Figure 2a) and DSM23693 (Figure 2b) Figure 3: Shows the optimized biomass accumulation and metabolite production of DSM23693 as described in Example 2.
    [0053] Figure 4: Shows that a genetic map of the new strain of C. autoethanogenumLZ1561 (DSM23693) showing the variations for the strain LZ1560 (DSM19630) Figure 5: Seq. ID.l: Nucleotide sequence of the MutS gene of the DNA incompatibility repair protein in the LZ1561 strain Figure 6: Seq. ID.2: Nucleotide sequence of the MutS gene of the DNA incompatibility repair protein in the LZ1560 strain Figure 7: Seq. ID.3: Amino acid sequence of the MutS gene of the DNA incompatibility repair protein in the LZ1561 strain Figure 8: Seq. ID.4: Amino acid sequence of the MutS gene of the DNA incompatibility repair protein in the LZ1560 strain Figure 9: Seq. ID.5: Nucleotide sequence found to be rearranged in strains LZ561 and LZ1560 Figure 10: Seq. ID.6: Nucleotide sequence of the putative promoter region of the FiPo operon of ATP synthase in strain LZ1561 Figure 11: Seq. ID.7: Nucleotide sequence of the putative promoter region of the FiPo operon of ATP synthase in strain LZ1560 Figure 12: Seq. ID.8: Nucleotide sequence of the putative promoter region of the Rnf complex operon in the LZ1561 strain Figure 13: Seq. ID.9: Nucleotide sequence of the putative promoter region of the Rnf complex operon in the LZ1560 strain Figure 14: Seq. ID. 10: Nucleotide sequence of the putative promoter region of carbon deficiency protein in the LZ1561 strain Figure 15: Seq. ID.ll: Nucleotide sequence of the putative promoter protein region of carbon deficiency in the LZ1560 strain Figure 16: Seq. ID. 12: Nucleotide sequence of the beta subunit gene of the CO dehydrogenase / acetyl-CoA synthase CO-methylase complex in the LZ1561 strain Figure 17: Seq. ID. 13: Nucleotide sequence of the beta subunit gene of the CO dehydrogenase / acetyl-CoA synthase CO-methylase complex in the LZ1560 strain Figure 18: Seq. ID. 14: Amino acid sequence of the beta subunit gene of the CO dehydrogenase / acetyl-CoA synthase CO-methylase complex in the LZ1561 strain Figure 19: Seq. ID. 15: Amino acid sequence of the beta subunit gene of the CO dehydrogenase / acetyl-CoA synthase CO-methylase complex in the LZ1560 strain Figure 20: Seq. ID. 16: Nucleotide sequence of the 5,10-methylenetetrahydrofolate reductase gene in the LZ1561 strain Figure 21: Seq. ID. 17: Nucleotide sequence of the 5,10-methylenetetrahydrofolate reductase gene in strain LZ1560 DETAILED DESCRIPTION OF THE INVENTION
    [0054] The inventors developed new bacteria. Bacteria are characterized by having one or more of several unexpected properties (as outlined below), and in a preferred embodiment all of these properties. The use of these new bacteria in anaerobic fermentation processes provides an unexpected benefit over existing strains of bacteria that can allow an increase in the total efficiency of a fermentation process for producing products, such as ethanol and / or acetate.
    [0055] Consequently, in broad terms, in one aspect, the present invention relates to a new bacterium and a biologically pure isolate of a bacterium with increased efficiency in an anaerobic fermentation process. In one aspect, the bacterium is capable of producing an alcohol, preferably ethanol, from a substrate comprising CO.
    [0056] In a further aspect, the invention relates to processes for producing an alcohol, preferably ethanol, by anaerobic fermentation of a substrate containing CO by the bacteria of the invention. Definitions
    [0057] Unless otherwise defined, the following terms as used throughout this specification are defined as follows:
    [0058] A "substrate containing CO", it is to be understood that a "substrate comprising CO" and as terms include any substrate in which carbon monoxide is available to bacteria for growth and / or fermentation, for example. In particular embodiments of the invention, the "CO containing substrate" is gaseous. Such substrates may be referred to in this application as "gaseous substrates containing CO", "gaseous substrates comprising CO" and the like.
    [0059] In the description that follows, the modalities of the invention are described in terms of delivery and fermentation of a "gaseous substrate containing CO". However, it must be appreciated that the gaseous substrate can be supplied in alternative forms. For example, the gaseous substrate containing CO can be supplied dissolved in a liquid. Essentially, a liquid is saturated with a gas containing carbon monoxide and then that liquid is added to the bioreactor. This can be achieved using standard methodology. For example, a microbubble dispersion generator (Hensirisak et al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3 / October 2002 can be used. gaseous substrate containing CO can be adsorbed on a solid support, such alternative methods are encompassed by the use of the term "substrate containing CO".
    [0060] The terms "increased efficiency", "increased efficiency" and the like, when used in connection with a fermentation process, include, but are not limited to, increase in one or more of: microorganism growth rate that catalyze fermentation, absorption or consumption of CO by microorganisms, the volume of the desired product (such as alcohols) produced by the volume of the substrate (such as CO) consumed, the concentration of the desired product (such as alcohols) produced in culture medium, the production rate or production level of the desired product, and the relative proportion of the desired product produced compared to other fermentation by-products.
    [0061] The term "acetate" includes both the acetate salt alone and a mixture of acetate salt and free or molecular acetic acid, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as described in this application. The proportion of molecular acetic acid to acetate in the fermentation broth depends on the pH of the system.
    [0062] The term "bioreactor" includes a fermentation device consisting of one or more vessels and / or towers or arrangement of tubes, which includes the Continuous Agitated Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Bed Reactor Drip (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for contacting gas and liquid.
    [0063] The term "alcohol tolerance", as used in this application, should be taken to refer to the level of alcohol, preferably ethanol, that a bacterium or population of bacteria will tolerate while continuing to survive, cultivate and / or produce at least one level of the desired product.
    [0064] The bacteria of the invention, or cultures or isolates from them, can be described to be in an "isolated" or "biologically pure" form. These terms are meant to mean that bacteria have been separated from an environment or one or more constituents, cellular or otherwise, with which they can be associated if found in nature or otherwise. The terms "isolated" or "biologically pure" should not be used to indicate the extent to which the bacteria have been purified. However, in one embodiment, the isolates or cultures of the bacteria contain a predominance of the bacteria of the invention.
    [0065] The invention provides a biologically pure isolate of a bacterium in which the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate containing CO and in which the bacterium is capable of one or more of: produce ethanol at a specific productivity of approximately 2 g of ethanol / L of fermentation broth / gram of biomass / day; produce ethanol at a productivity of at least approximately 10 g / L of broth / day of fermentation; a specific CO absorption of at least approximately 1.0 mMol of CO / min / g of biomass; a specific growth rate of at least approximately 0.8 day 1 produces ethanol in an ethanol to acetate ratio of at least approximately 2: 1; and, tolerate alcohol of up to approximately 30 g / L of broth.
    [0066] In a preferred embodiment, a bacterium of the invention is capable of two, three, four, or five of the above attributes.
    [0067] In certain embodiments, the bacterium is capable of producing ethanol at a specific productivity of at least approximately 3 g of ethanol / L of fermentation broth / gram of biomass / day, at least approximately 4 g of ethanol / L of broth fermentation / gram of biomass / day, at least approximately 5 g of ethanol / L of fermentation broth / gram of biomass / day, at least approximately 6 g of ethanol / L of fermentation broth / gram of biomass / day or at least minus approximately 7 g of ethanol / L of fermentation broth / gram of biomass / day.
    [0068] In certain embodiments, the bacterium is capable of producing ethanol at a productivity of at least approximately 20 g of ethanol / L of fermentation broth / day, at least approximately 30 g of ethanol / L of fermentation broth / day, at least approximately 40 g of ethanol / L of fermentation broth / day or at least approximately 50 g of ethanol / L of fermentation broth / day. The maximum value takes into account stoichiometry, CO absorption and ethanol removal.
    [0069] In certain embodiments, the bacterium is capable of a specific CO absorption of at least approximately 1.2 mMol of CO / min / g of biomass, at least approximately 1.4 mMol of CO / min / g of biomass, at least approximately 1.6 mMol of CO / min / g of biomass, at least approximately 1.8 mMol of CO / min / g of biomass, or at least approximately 2.0 mMol of CO / min / g of biomass. In a particular embodiment, the bacterium is capable of a specific CO absorption of at least approximately 1.2 mMol of CO / min / g of biomass.
    [0070] In certain embodiments, the bacterium is capable of a specific growth rate of at least approximately 1.0 day1, at least approximately 1.2 day1, at least approximately 1.4 day1, at least approximately 1.6 day1, at least approximately 1.8 days1 or at least approximately 2.0 days1.
    [0071] In certain embodiments, the bacterium is capable of producing ethanol in an ethanol to acetate ratio of at least approximately 3: 1, at least approximately 4: 1, at least approximately 5: 1, at least approximately 7 : 1 or at least approximately 10: 1. In a particular embodiment, there is no liquid production of acetate during fermentation.
    [0072] In certain modalities, the bacterium is able to tolerate alcohol up to approximately 40 g / L of fermentation broth, up to approximately 50 g / L of fermentation broth, or up to approximately 60 g / L of fermentation broth. In a particular embodiment, the bacteria is able to tolerate alcohol up to approximately 70 g / L of fermentation broth.
    [0073] In a preferred embodiment, the bacteria of the invention are derived from Clostridium autoethanogenum. In a more preferred embodiment of the invention, the bacteria are derived from the strain DSM19630 of Clostridium autoethanogenum (DSMZ, Germany) (described in W02009 / 064200).
    [0074] In a preferred embodiment, the bacterium of the invention is a strain of Clostridium autoethanogenum.
    [0075] Clostridium autoethanogenum is described, for example, in Abrini et al; Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide, Arch Microbiol (1994) 161: 345-351.
    [0076] In certain embodiments of the invention, bacteria have the defining characteristics of the strain of Clostridium autoethanogenum DSM23693 deposited in DSMZ, Germany, according to the Treaty of Budapest, on June 7, 2010. In a particular embodiment, the bacterium is the strain DSM23693 from Clostridium autoethanogenum.
    [0077] The invention also relates to bacteria derived from the bacteria of the invention.
    [0078] Bacteria of certain embodiments of the invention are capable of an increased alcohol production rate, an increased growth rate, an increased rate of CO consumption or renewal, a higher proportion of alcohol production in relation to acid production and / or an increased tolerance to alcohol. This provides a benefit over other strains of Clostridiasp including Clostridium autoethanogenum. Therefore, the use of bacteria of the present invention can increase the total efficiency of a fermentation process for producing products, such as acetate and / or ethanol.
    [0079] In certain embodiments, the bacteria of the invention are capable of productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance mentioned earlier in this application to high levels of CO in the gaseous substrate. For example, the gaseous substrate may comprise at least approximately 50% by volume of CO, at least approximately 65% by volume of CO, or at least approximately 70% by volume of CO. In certain embodiments, the gaseous substrate comprises at least approximately 80% by volume of CO, or at least approximately 85% by volume of CO, or at least approximately 90% by volume of CO or at least approximately 95% by volume of CO.
    [0080] Similarly, productivity, growth rates, alcohol-to-acid ratio, CO consumption and alcohol tolerance in this application described above are achievable in certain modalities at low to nonexistent levels of H2 in the gaseous substrate. The gaseous substrate can comprise approximately 30% or less by volume of H2. In particular embodiments, the gaseous substrate comprises approximately 20% or less by volume of H2, approximately 15% or less by volume of H2, approximately 10% or less by volume of H2, approximately 5% or less by volume of H2, approximately 4 % or less by volume of H2, approximately 3% or less by volume of H2, approximately 2% or less by volume of H2, approximately 1% or less by volume of H2, or substantially without H2.
    [0081] In certain embodiments, the bacteria of the invention are also capable of productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance mentioned in this application before when supplied with a gaseous substrate comprising relatively little CO2. In one embodiment, the gaseous substrate comprises less than or equal to approximately 20% CO2 by volume. In certain embodiments, the gaseous substrate comprises less than or equal to approximately 15% CO2 by volume, less than or equal to approximately 10% CO2 by volume, or less than or equal to approximately 5% CO2 by volume. In a particular embodiment, the gaseous substrate comprises substantially no CO2.
    [0082] In certain embodiments, a culture of a bacterium of the invention is maintained in an aqueous culture medium. Preferably, the aqueous culture medium is a minimal anaerobic microbial growth medium. Suitable means are known in the art and described, for example, in US Patent Nos. 5,173,429 and 5,593,886 and WO 02/08438, and in Klasson et al [(1992). Bioconversion of Synthesis Gas into Liquid or Gaseous Fuels. Enz. Microb. Technol. 14: 602-608.], Najafpour and Younesi [(2006). Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii. Enzyme and Microbial Technology, Volume 38, Issues 1-2, p. 223-228] and Lewis et al [(2002). Making the connection-conversion of biomass-generated producer gas to ethanol. Abst. Bioenergy, p. 2091-2094]. In particular embodiments of the invention, the minimal anaerobic microbial growth medium is as described below in the Examples section.
    [0083] The invention also provides methods for the production of one or more alcohols from a gaseous substrate comprising CO, methods comprising maintaining a culture of one or more bacterial isolates of the invention in the presence of the substrate, and the anaerobic fermentation of the substrate to one or more alcohols by one or more bacterial isolates.
    [0084] The invention also provides a method for reducing the total atmospheric carbon emissions of an industrial process, the method comprising: (a) capturing gas containing CO produced as a result of the industrial process, before the gas is released into the atmosphere; (b) anaerobic fermentation of the CO-containing gas to produce one or more alcohols by a culture containing one or more bacterial isolates of the invention.
    [0085] In certain embodiments of the methods of the invention, acetate is produced as a by-product of fermentation. The alcohol produced is ethanol.
    [0086] In certain modalities, the culture is maintained in a liquid nutritious medium.
    [0087] Fermentation can be carried out in any suitable bioreactor, such as a continuous agitated tank reactor (CTSR), a bubble column reactor (BCR) or a drip bed reactor (TBR). Also, in some preferred embodiments of the invention, the bioreactor may comprise a first growth reactor in which microorganisms are grown, and a second fermentation reactor, to which the growth reactor fermentation broth is fed and in which the most of the fermentation product (ethanol and acetate) is produced.
    [0088] As described above, the carbon source of the fermentation reaction is a gaseous substrate containing CO. The gaseous substrate may be a gas containing residual CO obtained as a by-product of an industrial process, or from some other source such as automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of the manufacture of ferrous metal products, such as a steel mill, the manufacture of non-ferrous products, oil refining processes, coal gasification, electricity production, production carbon black, ammonia production, methanol production and coke production. In these modalities, the gas containing CO can be captured from the industrial process before it is emitted into the atmosphere, using any suitable method. Depending on the composition of the gaseous substrate containing CO, it may also be desirable to treat it to remove any unwanted impurities, such as dust particles, before introducing it to fermentation. For example, the gaseous substrate can be filtered or washed using known methods.
    [0089] In addition, it is often desirable to increase the concentration of CO in a substrate stream (or partial pressure of CO in a gaseous substrate) and thereby increase the efficiency of fermentation reactions where CO is a substrate. Increasing the partial pressure of CO on a gaseous substrate increases the mass transfer of CO in the fermentation media. The composition of the gas streams used to power a fermentation reaction can have a significant impact on the efficiency and / or costs of that reaction. For example, 02 can reduce the efficiency of an anaerobic fermentation process. Processing unwanted or unnecessary gases at stages of a fermentation process before or after fermentation can increase the load at such stages (for example, where the gas stream is compressed before entering a bioreactor, unnecessary energy can be used to compress gases that are not needed in fermentation). Consequently, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove unwanted components and increase the concentration of desirable components.
    [0090] Substrate currents derived from an industrial source are typically variable in composition. In addition, substrate streams derived from industrial sources comprising high concentrations of CO (such as, for example, at least 40% CO, at least 50% CO or at least 65% CO) often have a low Fh component (such as less than 20% or less than 10% or substantially 0%). As such, it is particularly desirable for microorganisms to be able to produce products by anaerobic fermentation of substrates comprising a range of concentrations of CO and H2, particularly high concentrations of CO and low concentrations of H2. The bacteria of the present invention have a surprisingly high growth rate and production rate of ethanol by fermenting a substrate comprising CO (and without H2).
    [0091] The presence of hydrogen in the substrate stream can lead to an improvement in total carbon capture efficiency and / or ethanol productivity. For example, W002 / 08438 describes the production of ethanol using gas streams of various compositions. W002 / 08438 reports that a substrate stream comprising 63% H2, 32% CO and 5% CH4 that is supplied to a C. Ijungdahlii culture in a bioreactor promotes microbial growth and ethanol production. When the culture reached a permanent state and microbial growth was no longer the main objective, the substrate stream was switched to 15.8% H2, 36.5% CO, 38.4% N2 and 9.3% CO2 in order to supply CO in a slight excess and promote ethanol production. This document also describes gas streams with higher CO and lower H2 concentrations.
    [0092] It will be appreciated that the processes of the present invention as described in this application can be used to reduce the total atmospheric carbon emissions of industrial processes, capturing gases containing CO produced as a result of such processes and using them as substrates for fermentation processes described in this application.
    [0093] Alternatively, in other embodiments of the invention, the substrate containing CO gas can be obtained from biomass gasification. The gasification process involves the partial combustion of biomass with a restricted supply of air or oxygen. The resulting gas typically comprises mainly CO and H2, with minimal volumes of CO2, methane, ethylene and ethane. For example, biomass by-products obtained during the extraction and processing of foodstuffs, such as sugarcane sugar, or corn or grain starch, or non-food biomass residues generated by the forestry industry can be aerated to produce a CO containing gas suitable for use in the present invention.
    [0094] It is generally preferred that the substrate containing CO gas contains a major proportion of CO. In particular embodiments, the gaseous substrate comprises at least approximately 25%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 65%, or at least approximately 70% to approximately 95% by volume of CO. It is not necessary for the gaseous substrate to contain some hydrogen. The gaseous substrate also optionally contains some CO2, such as approximately 1% to approximately 30% by volume, such as approximately 5% to approximately 10% CO2.
    [0095] It will be appreciated that for bacterial growth and fermentation of CO to ethanol to occur, in addition to the gas containing CO in the substrate, an appropriate liquid nutrient medium will need to be fed to the bioreactor. A nutrient medium will contain enough vitamins and minerals to allow the growth of the micro-organism used. Anaerobic media suitable for the fermentation of ethanol using CO as the sole carbon source are known in the art. For example, suitable means are described in US patents 5,173,429 and 5,593,886 and WO 02/08438 as well as other publications referred to earlier in this application. In one embodiment of the invention, the means are as described in the Examples section below.
    [0096] Fermentation should desirably be carried out under conditions suitable for CO fermentation to ethanol. The reaction conditions that must be considered include pressure, temperature, gas flow rate, liquid flow rate, pH of the media, redox potential of the media, agitation rate (using a continuous agitated tank reactor), inoculum level, concentrations maximum gas substrate to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.
    [0097] The optimal reaction conditions will depend in part on the particular microorganism of the invention used. However, in general, it is preferable that the fermentation is carried out at a higher pressure than the ambient pressure. Operating at increased pressures allows for a significant increase in the transfer rate of CO from the gas phase to the liquid phase where it can be absorbed by the micro-organism as a carbon source for ethanol production. This in turn means that the retention time (defined as the net volume in the bioreactor divided by the flow rate of the inlet gas) can be reduced when the bioreactors are maintained at high pressure instead of atmospheric pressure.
    [0098] Furthermore, since a given conversion rate from CO to ethanol is partly a function of the substrate retention time, and the achievement of a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the necessary bioreactor, and consequently the capital cost of the fermentation equipment. According to the examples given in US Patent no. 5,593,886, the reactor volume can be reduced in linear proportion to increases in the reactor operating pressure, that is, bioreactors operated in 10 atmospheres of pressure need only have one tenth of the volume of those operated in 1 atmosphere of pressure.
    [0099] The benefits of conducting a gas to ethanol fermentation at high pressures have also been described in another application. For example, WO 02/08438 describes gas to ethanol fermentations carried out under pressures of 30 psig and 75 psig, providing ethanol yields of 150 g / l / day and 369 g / l / day respectively. However, it was found that example fermentations carried out using similar media and atmospheric pressure inlet gas compositions produced between 10 and 20 times less ethanol per liter per day.
    [00100] It is also desirable that the rate of introduction of the substrate containing CO gas is to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of the CO limiting conditions may be that the ethanol product is consumed by the culture.
    [00101] In certain embodiments, a fermentation process in accordance with the present invention described above will result in a fermentation broth comprising ethanol, as well as bacterial cells, in aqueous culture medium. In preferred methods of the method, ethanol is recovered from the fermentation broth.
    [00102] In certain embodiments, ethanol recovery involves continuously removing a portion of juice and recovering alcohol from the portion removed from the juice.
    [00103] In particular embodiments, ethanol recovery includes passing the removed portion of the broth containing ethanol through a separation unit to separate bacterial cells from the broth, to produce a permeate containing cellless alcohol, and restoring the bacterial cells to the bioreactor.
    [00104] In certain embodiments, the methods of the invention are continuous processes.
    [00105] In particular modalities, acetate is produced as a by-product of fermentation.
    [00106] In an additional modality, ethanol and acetate are recovered from the broth.
    [00107] In certain embodiments, the recovery of ethanol and acetate comprises continuously removing a portion of the broth and separately recovering ethanol and acetate from the portion removed from the broth.
    [00108] In some embodiments, the recovery of ethanol and acetate includes passing the removed portion of the broth containing ethanol and acetate through a separation unit to separate bacterial cells from ethanol and acetate, to produce a permeate containing ethanol and acetate without cells , and restitution of bacterial cells to the bioreactor.
    [00109] In the above embodiments, the recovery of ethanol and acetate preferably includes first removal of the cellless ethanol permeate followed by removal of the cellless acetate permeate. Preferably, cell-free permeates are then returned to the bioreactor.
    [00110] Ethanol is the preferred end product of fermentation. Ethanol can be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, and extractive fermentation. Distilling ethanol from a fermentation broth produces an azeotropic mixture of ethanol and water (ie 95% ethanol and 5% water). Anhydrous ethanol can later be obtained by using ethanol dehydration technology by molecular sieve, which is also well known in the art. Extractive fermentation procedures involve the use of a water-miscible solvent that presents a risk of low toxicity to the fermentation organism, to recover ethanol from the diluted fermentation broth. For example, oleyl alcohol is a solvent that can be used in this type of extraction process. Oleyl alcohol is continuously introduced into a fermenter, after which this solvent rises to form a layer above the fermenter that is continuously extracted and fed through a centrifuge. Water and cells are then promptly separated from the oleyl alcohol and returned to the fermenter while the solvent loaded with ethanol is fed to a rapid spray unit. Most of the ethanol is vaporized and condensed while oleyl alcohol is not volatile and is recovered for reuse in fermentation.
    [00111] Acetate can also be recovered from the fermentation broth using methods known in the art. Methods for the recovery of acetate are described in detail in W02007 / 117157 and W02008 / 115080.
    [00112] In certain embodiments of the invention, ethanol and acetate are recovered from the fermentation broth by continuously removing a portion of the fermentation bioreactor broth, separating microbial cells from the broth (conveniently by filtration), and first recovering ethanol and then acetate from of the broth. Ethanol can be conveniently recovered by distillation, and acetate can be recovered by adsorption on activated charcoal, using the methods described above. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell-free permeate remaining after ethanol and acetate are removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) can be added to the cell-free permeate to replenish the nutrient medium before it is returned to the bioreactor. Also, if the pH of the broth was adjusted as described above to increase the adsorption of acetic acid on the activated charcoal, the pH must be readjusted to a pH similar to that of the broth in the fermentation bioreactor, before being returned to the bioreactor. Reaction Stoichiometry
    [00113] Without wishing to be bound by any theory, it is believed that the chemical reactions of CO fermentation for ethanol (a) and acetic acid (b) in the process of the present invention are as follows: (a) 6CO + 3H2O = > CH3CH2OH + 4CO2 (b) 4CO + 2H2O => ICH3COOH + 2CO2
    [00114] The invention will now be described in more detail with reference to the following non-limiting examples. EXAMPLES Materials and Methods:
    Preparation of Cr (II) solution
    [00115] A 1 L flask with three necks was fitted with a firm gas inlet and outlet to allow working under inert gas and subsequent transfer of the desired product in a suitable storage flask. The flask was loaded with CrCh.ólUO (40 g, 0.15 mol), zinc granules [20 mesh] (18.3 g, 0.28 mol), mercury (13.55 g, 1 mL, 0.0676 mol) and 500 mL of distilled water. Following washing with N2 for one hour, the mixture was heated to approximately 80 ° C to initiate the reaction. The next two hours of stirring under a constant flow of N2, the mixture was cooled to room temperature and continuously mixed for another 48 hours at which point the reaction mixture turned into an intense blue solution. The solution was transferred into serum bottles purged with N2 and stored in the refrigerator for future use. Bacteria:
    [00116] The two types of Clostridium autoethanogenum used were those deposited at the German Resource Center for Biological Material (DSMZ) and allocated the access numbers DSM 19630 and DSM 23693. DSM 23693 was developed from DSM19630 of the strain of Clostridium autoethanogenum (DSMZ, Germany) through an iterative selection process. Sampling and analytical procedures
    [00117] Samples of media were taken from the CSTR reactor from time to time over the course of each fermentation. Each time the media were sampled, care was taken to ensure that no gas entered or escaped the reactor. HPLC:
    [00118] Agilent 1100 Series HPLC system. Mobile Phase: Sulfuric Acid 0.0025N. Flow and pressure: 0.800 mL / min. Column: Alltech IOA; Catalog # 9648, 150 x 6.5 mm, particle size 5 pm. Column temperature: 60 ° C. Detector: Refractive index. Detector temperature: 45 ° C.
    [00119] Sample preparation method:
    [00120] 400 pL of sample and 50 pL of 0.15M ZnSC> 4 and 50 pL of 0.15M Ba (OH) 2 are loaded into an Eppendorf tube. The tubes are centrifuged for 10 min. at 12,000 rpm, 4 ° C. 200 pL of the supernatant is transferred into an HPLC flask, and 5 pL is injected into the HPLC instrument. Head Space Analysis:
    [00121] The measurements were performed on a Varian CP-4900 micro GC with two channels installed. Channel 1 was a 10 m molecular sieve column running at 70 ° C, 200 kPa argon and a flow reversal time of 4.2 s, while channel 2 was a 10 m PPQ column running at 90 ° C, 150 kPa of helium and no flow reversal. The injector temperature of both channels was 70 ° C. Run times were set at 120 s, but all peaks of interest would normally elute before 100 s. Cell Density:
    [00122] Cell density was determined by counting bacterial cells in a defined aliquot of the fermentation broth. Alternatively, the absorbance of the samples was measured at 600 nm (spectrophotometer) and the dry mass determined by calculation according to published procedures. Sequencing:
    [00123] Genome sequencing revealed that several modifications in genomes of the C. autoethanogenum strain LZ1560 (DSM19630) and the new strain LZ1561 (DSM23693), which are likely to contribute to improved performance.
    [00124] Both strains were cultured anaerobically in PETC media at an optical density (ODeoonm) of 1 and genomic DNA was isolated from 100 ml of night cultures. The cells were collected by centrifugation (6,000 xg, 15 min, 4 ° C), washed with potassium phosphate buffer (10 mM; pH 7.5) and suspended in 1.9 ml of STE buffer (50 mM Tris-HCl) , 1 mM EDTA, 200 mM sucrose; pH 8.0). This suspension was treated with 300 µl lysozyme (-100,000 U; 30 min, 37 ° C) and 280 µl SDS solution (10% (w / v); 10 min). RNA was digested by adding 240 pl of an EDTA solution (0.5 M; pH 8), 20 pl of Tris-HCl (1 M; pH 7.5), and 10 pl of RNase A (50,000 U) per 1 hour. Proteolysis was performed by adding 100 µl Proteinase K (0.5 U) for 1 to 3 h at 37 ° C. Finally, 600 µl of sodium perchlorate (5 M) was added, followed by an extraction with phenol-chloroform and a precipitation with isopropanol. The purity and quantity of DNA were verified using a NanoDrop® 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by gel electrophoresis.
    [00125] Shotgun genome sequencing was performed using a 454 GS (Roche Applied Science, Indianapolis, IN, USA). 191,368 unique readings with a total length of 44,424,523 bases were created for LZ1560 (10x coverage), while 579,545 paired edge readings with a total length of 202,591,572 bp were created for LZ1561 (47,5x coverage). The readings were assembled using the Newbler package (Roche Applied Science, Indianapolis, IN, USA) and the sequences compared using Geneious (Biomatters Ltd., Auckland, NZ) with the MAUVE package (Darling et al., 2004, Genome Res. 14 : 1394-1403) and by Artemis Comparison Tool (Carver et al., 2008, Bioinformatics 24: 2672-6).
    [00126] A total of 64 modifications were found in genome sequences assembled from LZ1560 (DSM19630) and the new strain LZ1561 (DSM23693) (Fig. 4). While most of the modifications were single base variations, a 21 bp deletion (in the gene encoding a putative MutS DNA incompatibility repair protein; Seq. ID. 1-4) and a rearrangement event of a 15,408 region bp (Seq. ID.5) containing 11 genes (involved in nitrogen fixation, sugar metabolism, sugar transport and catabolite control) were found. Of 62 variations of single bases, 22 were point mutations, and 40 insertions / deletions. 18 of these variations were found in intergenic regions and 44 in coding regions. While 5 of the variations in the coding region were silent and did not result in a change in the amino acid sequence, 14 resulted in a single amino acid change and 25 in a reading frame.
    [00127] Most notably were changes in positions 212,530 (putative promoter region of the ATP synthase FiFo operon, Seq. ID.6-7), 1,171,874 (putative promoter region of the Rnf complex operon, Seq. ID.8 -9), 3,717,495 (putative promoter region of the carbon deficiency protein, Seq. ID. 10-11), and two variations in the Wood-Ljungdahl gene cluster at positions 3,741,730 (beta subunit of the CO dehydrogenase complex / acetyl-CoA synthase CO-methylating, Seq. ID.12-15) and 3,748,058 (5,10-methylenetetrahydrofolate reductase gene, Seq. LD.16-17), which can be traced back directly to develop in CO / H2 and energy metabolism. Most of the other affected genes are uncharacterized genes. Example 1; A: Batch batch fermentation in CSTR
    [00128] Approximately 1500 mL of solution A was transferred in a 1.5 L fermentor and dispersed with nitrogen. Resazurin (1.5 mL of a 2 g / L solution) and H3PO4 (85% solution, 2.25 mL) were added and the pH adjusted to 5.3 using concentrated NH4OH (aq). Nitriloacetic acid (0.3 ml of a 0.15 M solution) was added before 1.5 ml of solution C. This was followed by NiCl2 (0.75 ml of 0.1 M solution) and Na2WO3 (1.5 mL of a 0.01 M solution). 15 ml of solution B was added and the solution dispersed with N2 before switching to the gas containing CO (50% CO; 28% N2, 2% H2, 20% CO2) at 70 ml / min. The fermenter was then inoculated with 200 ml of a Clostridium autoethanogenum 19630 culture. The fermenter was maintained at 37 ° C and stirred at 300 rpm. During this experiment, the Na2S solution (0.2 M solution) was added at a rate of approximately 0.3 ml / hour. The substrate supply was increased in response to the requirements of microbial culture.
    [00129] The bacterial culture did not proliferate under the experimental conditions used. The culture showed an absorption of 350 mM CO after 48 hours of growth (Figure 1 a and Table 2) while the culture doubling time was 40.8 hours (Figure 2a). This corresponds to a specific growth rate of 0.41 day -1. The absorption of specific CO increased during the experiment with a maximum value of 0.54 mM CO / min / g of biomass. Day 1.0 specific absorption: 0.28 mM CO / min / g of biomass (Table 1). Day 2.0 specific absorption: 0.54 mM CO / min / g of biomass (Table 2). B: Batch fermentation in CSTR
    [00130] Approximately 1500 mL of solution A was transferred in a 1.5 L fermentor and dispersed with nitrogen. Resazurin (1.5 mL of a 2g / L solution) and H3PO4 (85% solution, 2.25 mL) were added and the pH adjusted to 5.3 using concentrated NH4OH (aq). Nitriloacetic acid (0.3 ml of a 0.15 M solution) was added before 1.5 ml of solution C. Na2WO3 (1.5 ml of a 0.01 M solution) was added. 15 ml of Solution B was added and the solution dispersed with N2 before switching to the gas containing CO (50% CO; 50% N2) at 60 ml / min. The fermenter was then inoculated with 180 ml of a culture of Clostridium autoethanogenum 23693. The fermenter was maintained at 37 ° C and stirred at 300 rpm. During this experiment, the Na2S solution (0.5 M solution) was added at a rate of approximately 0.12 ml / hour. The substrate supply was increased in response to the requirements of microbial culture.
    [00131] Bacterial culture proliferated under the experimental conditions used. The culture showed an absorption of 8400 mM CO after 43 hours of growth (Figure 2b) while the culture doubling time was 9.6 hours (Figure 2b). This corresponds to a specific growth rate of 1.73 days1. The maximum specific CO absorption achieved during the experiment was 1.17 mMol of CO / min / g of biomass. Day 1.0 specific absorption: 1.17 mM CO / min / g of biomass (Table 1). Day 2.0 specific absorption: 1.03 mM CO / min / g of biomass (Table 2). The fermentation conditions were identical or at least highly similar to the conditions used in Example IA. The media preparation has identical components in similar concentrations while both gases contained at least 50% (v / v) CO. Similar fermentation conditions compared to the vast difference in CO intake indicate varied culture performance due to the improved efficiency of the developed Clostridium autoethanogenum 23693 culture compared to the 19630 Clostridium autoethanogenum parent strain. Results: Table 1: Day 1
    Table 2: Day 2
    Example 2
    [00132] Approximately 1500 mL of solution A was transferred in a 1.5 L fermentor and dispersed with nitrogen. Resazurin (1.5 mL of a 2 g / L solution) and H3PO4 (85% solution, 0.56 mL) were added and the pH adjusted to 5.3 using concentrated NH4OH (aq). Solution C (1.5 mL) was added after which Na2WOβ (1.5 mL of a 0.01 M solution) was added. 15 ml of Solution B was added and the solution dispersed with N2 before switching to the gas containing CO (50% CO; 50% N2) at 60 ml / min. The fermenter was then inoculated with 100 ml of a culture of Clostridium autoethanogenum 23693. The fermenter was maintained at 37 ° C and stirred at 300 rpm. During this experiment, the Na2S solution (0.5 M solution) was added at a rate of approximately 0.15 ml / hour. The substrate supply was increased in response to the requirements of microbial culture.
    [00133] Bacterial culture proliferated under the experimental conditions used. The fermentation conditions were identical or at least highly similar to the conditions used in Example IA + B while both gases contained at least 50% (v / v) CO. The culture was grown at the stationary phase where the maximum ethanol concentration was measured by HPLC (55.8 g / L).
    [00134] The specific methods and compositions described in this application are representative of preferred modalities and are exemplary and not desired as limitations on the scope of the invention. Other objects, aspects and modalities will occur to those skilled in the art in consideration of this specification, and are included within the scope and spirit of the invention. It will be readily apparent to one skilled in the art that various substitutions and modifications can be made to the invention described in this application without departing from the scope and spirit of the invention. The invention described illustratively in this application can appropriately be practiced in the absence of any element or elements, or limitations or limitations, which are not specifically described in this application as essential. Thus, for example, in each example in this application, in embodiments or examples of the present invention, the terms "comprising", "including", "containing" etc. should be read expansively and without limitation. In addition, titles, headings or the like are provided to increase the reader's understanding of this document, and should not be read as a limitation on the scope of the present invention.
    [00135] The full disclosures of all patent applications, patents and publications, mentioned above and below, if any, are hereby incorporated by reference. However, reference to any patent applications, patents and publications in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that constitutes valid prior technique or is part of the common general knowledge in any country in the world.
    权利要求:
    Claims (12)
    [0001]
    1. Biologically pure isolate of a bacterium Clostridium autoethanogenum, characterized by the fact that the strain of Clostridium autoethanogenum is the strain of Clostridium autoethanogenum deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM23693 and in which the bacteria ferment a substrate comprising CO in products comprising ethanol.
    [0002]
    2. Biologically pure isolate of Clostridium autoethanogenum according to claim 1, characterized by the fact that the bacteria ferment a substrate comprising CO in products comprising ethanol in which the specific productivity of the bacterium is at least 2 g of ethanol / L fermentation broth / gram of biomass / day.
    [0003]
    3. Isolated according to claim 2, characterized by the fact that the specific productivity is at least 7g of ethanol / L of fermentation broth / gram of biomass / day.
    [0004]
    4. Isolated according to claim 1, characterized by the fact that the products comprise ethanol and acetate.
    [0005]
    An isolate according to any one of claims 1 to 4, characterized by the fact that the bacteria ferment a substrate comprising CO to produce ethanol at a productivity of at least 10g of ethanol / L of fermentation broth / day.
    [0006]
    6. Isolated according to claim 5, characterized by the fact that the productivity of the bacterium is at least approximately 50 g of ethanol / L of fermentation broth / day.
    [0007]
    An isolate according to any one of claims 1 to 6, characterized by the fact that the bacterium produces ethanol and acetate, whereby the ratio of ethanol to acetate is at least 2: 1.
    [0008]
    An isolate according to any one of claims 1 to 7, characterized by the fact that the bacterium has a specific CO absorption of at least 1.0 mMol of CO / min / g of biomass.
    [0009]
    9. Isolated according to claim 8, characterized by the fact that the bacteria has a specific CO absorption of at least 2.0 mMol of CO / min / g of biomass.
    [0010]
    An isolate according to any one of claims 1 to 9, characterized by the fact that the bacterium has a specific growth rate of at least 0.8 days 1
    [0011]
    11. Isolated according to any one of claims 1 to 10, characterized by the fact that the bacteria tolerates an alcohol concentration of up to 30 g / L of fermentation broth.
    [0012]
    12. Isolated according to claim 11, characterized by the fact that the bacteria tolerates an alcohol concentration of up to 70 g / L of fermentation broth.
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    BR112013003644A2|2018-07-31|
    US20130217096A1|2013-08-22|
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    AU2011283282A1|2013-02-21|
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    WO2012015317A1|2012-02-02|
    US20160017276A1|2016-01-21|
    AU2011283282B2|2013-10-03|
    US10494600B2|2019-12-03|
    EA201390179A1|2013-08-30|
    EP2598630A4|2013-10-23|
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    法律状态:
    2018-08-14| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-05-21| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2019-07-30| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-04-22| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-08-18| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-11-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/07/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    2022-01-25| B25G| Requested change of headquarter approved|Owner name: LANZATECH NEW ZEALAND LIMITED (NZ) |
    2022-02-08| B25G| Requested change of headquarter approved|Owner name: LANZATECH NEW ZEALAND LIMITED (NZ) |
    2022-02-22| B25G| Requested change of headquarter approved|Owner name: LANZATECH NEW ZEALAND LIMITED (NZ) |
    2022-03-08| B25G| Requested change of headquarter approved|Owner name: LANZATECH NEW ZEALAND LIMITED (US) |
    优先权:
    申请号 | 申请日 | 专利标题
    US36848610P| true| 2010-07-28|2010-07-28|
    US61/368,486|2010-07-28|
    PCT/NZ2011/000144|WO2012015317A1|2010-07-28|2011-07-28|Novel bacteria and methods of use thereof|
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