US20130217096A1

US20130217096A1 - Novel bacteria and methods of use thereof

Info

Publication number: US20130217096A1
Application number: US13/812,029
Authority: US
Inventors: Bjorn Daniel Heijstra; Evegenia Kern; Michael Koepke; Simon Segovia; Fung Min Liew
Original assignee: Lanzatech New Zealand Ltd
Current assignee: Lanzatech NZ Inc
Priority date: 2010-07-28
Filing date: 2011-07-28
Publication date: 2013-08-22
Also published as: WO2012015317A1; CN103415612A; CN103415612B; JP2013532481A; EA025778B1; BR112013003644A2; EP2598630A4; ZA201300612B; BR112013003644B1; US10494600B2; EP2598630A1; EA201390179A1; KR20130097174A; CA2786903A1; AU2011283282B2; AU2011283282C1; KR101375038B1; AU2011283282A1; JP5922657B2; EP2598630B1

Abstract

This invention relates generally to the field of microbial fermentation of gases. It more particularly relates to a novel strain of Clostridium autoethanogenum bacteria with improved efficiency in the production of ethanol by anaerobic fermentation of substrates containing carbon monoxide (CO).

Description

FIELD OF THE INVENTION

This invention relates generally to the field of microbial fermentation of gases. It more particularly relates to a novel class of bacteria with improved efficiency in the production of ethanol by anaerobic fermentation of substrates containing carbon monoxide (CO).

BACKGROUND OF THE INVENTION

Ethanol is rapidly becoming a major hydrogen-rich liquid transport fuel around the world. Worldwide consumption of ethanol in 2005 was an estimated 12.2 billion gallons. The global market for the fuel ethanol industry has also been predicted to grow sharply in future, due to an increased interest in ethanol in Europe, Japan, the USA, and several developing nations.
For example, in the USA, ethanol is used to produce E10, a 10% mixture of ethanol in gasoline. In E10 blends the ethanol component acts as an oxygenating agent, improving the efficiency of combustion and reducing the production of air pollutants. In Brazil, ethanol satisfies approximately 30% of the transport fuel demand, as both an oxygenating agent blended in gasoline, and as a pure fuel in its own right. Also, in Europe, environmental concerns surrounding the consequences of Green House Gas (GHG) emissions have been the stimulus for the European Union (EU) to set member nations a mandated target for the consumption of sustainable transport fuels such as biomass derived ethanol.
The vast majority of fuel ethanol is produced via traditional yeast-based fermentation processes that use crop derived carbohydrates, such as sucrose extracted from sugarcane 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 animal feed, while the cultivation of starch or sucrose-producing crops for ethanol production is not economically sustainable in all geographies. Therefore, it is of interest to develop technologies to convert lower cost and/or more abundant carbon resources into fuel ethanol.
CO is a major, free, energy-rich by-product of the incomplete combustion of organic materials such as coal or oil and oil derived products. For example, the steel industry in Australia is reported to produce and release into the atmosphere over 500,000 tonnes of CO annually.
Catalytic processes may be used to convert gases consisting primarily of CO and/or CO and hydrogen (H2) into a variety of fuels and chemicals. Micro-organisms may also be used to convert these gases into fuels and chemicals.
The ability of micro-organisms to grow on CO as a sole carbon source was first discovered in 1903. This was later determined to be a property of organisms that use the acetyl coenzyme A (acetyl CoA) biochemical pathway of autotrophic growth (also known as the Woods-Ljungdahl pathway and the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) pathway). A large number of anaerobic organisms including carboxydotrophic, photosynthetic, methanogenic and acetogenic organisms have been shown to metabolize CO to various end products, namely CO2, H2, methane, n-butanol, acetate and ethanol. While using CO as the sole carbon source, all such organisms produce at least two of these end products.
Anaerobic bacteria, such as those from the genus Clostridium, have been demonstrated to produce ethanol from CO, CO2 and H2 via the acetyl CoA biochemical pathway. For example, various strains of Clostridium ljungdahlii that produce ethanol from gases are described in WO 00/68407, EP 117309, U.S. Pat. Nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438. The bacterium Clostridium autoethanogenum sp is also known to produce ethanol from gases (Abrini et al., Archives of Microbiology 161, pp 345-351 (1994)).
However, ethanol production by micro-organisms by fermentation of gases is always associated with co-production of acetate and/or acetic acid. As some of the available carbon is converted into acetate/acetic acid rather than ethanol, the efficiency of production of ethanol using such fermentation processes may be less than desirable. Also, unless the acetate/acetic acid by-product can be used for some other purpose, it may pose a waste disposal problem. Acetate/acetic acid is converted to methane by micro-organisms and therefore has the potential to contribute to GHG emissions.
Microbial fermentation of CO in the presence of H2 can lead to substantially complete carbon transfer into an alcohol. However, in the absence of sufficient H2, some of the CO is converted into alcohol, while a significant portion is converted to CO2 as shown in the following equations:
6CO+3H₂O→C₂H₅OH+4CO₂
12H₂+4CO₂→2C₂H₅OH+6H₂O
The production of CO₂represents inefficiency in overall carbon capture and if released, also has the potential to contribute to Green House Gas emissions.
WO2007/117157 describes a process that produces alcohols, particularly ethanol, by anaerobic fermentation of gases containing carbon monoxide. Acetate produced as a by-product of the fermentation process is converted into hydrogen gas and carbon dioxide gas, either or both of which may be used in the anaerobic fermentation process.
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(es) in a first bioreactor can be used to produce products in a second bioreactor. Furthermore, by-products of the second fermentation stage can be recycled to the first bioreactor to produce products.
WO2009/064200 describes a novel class of bacteria which has improved efficiency in the production of ethanol by anaerobic fermentation of substrates containing carbon monoxide.
It would be beneficial to provide micro-organisms that are capable of fermentation of gases containing carbon monoxide to ethanol at increased efficiency, that is micro-organisms capable of improved uptake of carbon monoxide, of producing more ethanol, and/or a greater ratio of ethanol to acetate from the same substrate, than do micro-organisms of the prior art.
It is an object of the present invention to provide a new class of bacteria which overcomes one or more of the limitations of the prior art in the conversion of gaseous sources containing CO into ethanol, or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a biologically pure isolate of a bacterium wherein 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 about 2 g ethanol/L fermentation broth/gram of biomass/day.
In other embodiments, the bacterium is capable of producing ethanol at a specific productivity of at least about 3 g ethanol/L fermentation broth/gram of biomass/day, at least about 4 g ethanol/L fermentation broth/gram of biomass/day, at least about 5 g ethanol/L fermentation broth/gram of biomass/day, at least about 6 g ethanol/L fermentation broth/gram of biomass/day or at least about 7 g ethanol/L fermentation broth/gram of biomass/day.
In another aspect the invention provides a biologically pure isolate of a bacterium wherein 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 about 10 g ethanol/L of fermentation broth/day.
In other embodiments, the bacterium is capable of producing ethanol at a productivity of at least about 20 g ethanol/L of fermentation broth/day, at least about 30 g ethanol/L of fermentation broth/day, at least about 40 g ethanol/L of fermentation broth/day or at least about 50 g ethanol/L of fermentation broth/day, or at least about 60 g ethanol/L of fermentation broth/day, or at least about 70 g ethanol/L of fermentation broth/day.
In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of a specific uptake of CO of at least about 1.0 mMol CO/min/g of biomass.
In one embodiment the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass, at least about 1.4 mMol CO/min/g of biomass, at least about 1.6 mMol CO/min/g of biomass, at least about 1.8 mMol CO/min/g of biomass, or at least about 2.0 mMol CO/min/g of biomass. In one particular embodiment, the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass.
In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of a specific growth rate of at least about 0.8 day⁻¹.
In certain embodiments the bacterium is capable of a specific growth rate of at least about 1.0 day⁻¹, at least about 1.2 day⁻¹, at least about 1.4 day⁻¹, at least about 1.6 day^−1,at least about 1.8 day⁻¹or at least about 2.0 day⁻¹.
In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of producing ethanol at an ethanol to acetate ratio of at least about 2:1.
In certain embodiments the bacterium is capable of producing ethanol at an ethanol to acetate ratio of at least about 3:1, of at least about 4:1, of at least about 5:1, of at least about 7:1 or of at least about 10:1.
In one embodiment, the bacterium is capable of producing ethanol with substantially no acetate.
In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium is capable of tolerating alcohol of up to about 30 g/L of fermentation broth.
In certain embodiment the bacterium is capable of tolerating alcohol of up to about 40 g/L of fermentation broth, of up to about 50 g/L of fermentation broth, of up to about 60 g/L of fermentation broth, or of up to about 70 g/L of fermentation broth.
In another aspect the invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate comprising CO, and wherein the bacterium has two or more of the following characteristics:
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 about 2 g of ethanol/L of fermentation broth/gram of biomass/day;
is capable of producing ethanol at a concentration of at least about 10 g ethanol/L of fermentation broth/day;
is capable of a specific uptake of CO of at least about 1.0 mMol CO/min/g of biomass;
is capable of a growth rate of at least about 1.0 g/day;
is capable of producing ethanol at an ethanol to acetate ratio of at least about 2:1; and,
is capable of tolerating alcohol of up to about 30 g/L of broth. 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.
In a particular embodiment the bacterium has the defining characteristics of the Clostridium autoethanogenum strain deposited at DSMZ under the accession number DMS23693. In one embodiment the bacterium is the Clostridium autoethanogenum strain deposited at DSMZ under the accession number DMS23693.
In a further aspect the invention provides a biologically pure isolate of the Clostridium autoethanogenum strain deposited at DSMZ under the accession number DMS23693
In another aspect, the invention provides a method for the production of one or more alcohols comprising fermenting a substrate comprising CO using a bacterium as herein before described.
In one embodiment the method comprises the steps of:

- (a) providing a substrate comprising CO to a bioreactor containing a culture of a bacterium of the invention; and
- (b) anaerobically fermenting the culture in the bioreactor to produce one or more alcohols.

In a further aspect, the invention provides a method for reducing the total atmospheric carbon emissions from an industrial process, the method comprising:
(a) capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere; (b) the anaerobic fermentation of the CO-containing gas to produce one or more alcohols by a culture containing one or more bacterium of the invention.
In one embodiment of the method aspects, the fermentation is conducted at a temperature of about 34° C. to about 37° C. In one preferred embodiment, the fermentation is conducted at a temperature of about 34° C.
In certain embodiments of the method aspects, acetate is produced as a by-product of the fermentation. Preferably the one or more alcohols produced includes ethanol.
In particular embodiments of the method aspects, the bacterium is maintained in an aqueous culture medium.
In particular embodiments of the method aspects, the fermentation of the substrate takes place in a bioreactor.
In certain embodiments the substrate comprises at least about 25% CO by volume, at least about 30% CO by volume, at least about 40% CO by volume, at least about 50% CO by volume, at least about 65% CO by volume or at least about 70% CO by volume. In particular embodiments the substrate comprises at least about 75% CO by volume, at least about 80% CO by volume, at least about 85% CO by volume, at least about 90% CO by volume or at least about 95% CO by volume.
In one embodiment the substrate comprises about 30% or less H₂by volume. In another embodiments, the substrate comprises about 20% or less H₂by volume, about 15% or less H₂by volume, about 10% or less H₂by volume, about 5% or less H₂by volume, about 4% or less H₂by volume, about 3% or less H₂by volume, about 2% or less H₂by volume, about 1% or less H₂by volume, or substantially no H₂.
In one embodiment the substrate comprises less than or equal to about 20% CO₂by volume. In particular embodiments the substrate comprises less than or equal to about 15% CO₂by volume, less than or equal to about 10% CO₂by volume, less than or equal to about 5% CO₂by volume or substantially no CO₂.
In certain embodiments the substrate comprising CO is a gaseous substrate containing CO.
In certain embodiments, the gaseous substrate comprises a gas obtained as a by-product of an industrial process.
In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of biomass, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.
In one embodiment, the gaseous substrate may comprise a gas obtained from a steel mill.
In another embodiment, the gaseous substrate may comprise automobile exhaust fumes.
In certain embodiments of the method aspects the alcohol is recovered from the fermentation broth, the fermentation broth being the aqueous culture medium comprising bacterial cells and the alcohol.
In certain embodiments acetate is produced as a by-product of the fermentation.
In a further embodiment the alcohol and the acetate are recovered from the broth.
Although the invention is broadly as defined above, it is not limited thereto and also includes embodiments of which the following description provides examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in detail with reference to the accompanying Figures in which:

FIG. 1: Shows the CO consumption of DSM19630 and DSM23693

FIG. 2: Shows the metabolite production of DSM19630 (FIG. 2 a) and DSM23693 (FIG. 2 b)

FIG. 3: Shows the optimised biomass accumulation and metabolite production of DSM23693 as described in Example 2.

FIG. 4: Shows a genetic map of new C. autoethanogenum strain LZ1561 (DSM23693) showing the variations to strain LZ1560 (DSM19630)

FIG. 5 : Seq. ID. 1: Nucleotide sequence of DNA mismatch repair protein MutS gene in strain LZ1561

FIG. 6: Seq. ID. 2: Nucleotide sequence of DNA mismatch repair protein MutS gene in strain LZ1560

FIG. 7: Seq. ID. 3: Amino Acid sequence of DNA mismatch repair protein MutS gene in strain LZ1561

FIG. 8: Seq. ID. 4: Amino Acid sequence of DNA mismatch repair protein MutS gene in strain LZ1560

FIG. 9: Seq. ID. 5: Nucleotide sequence found to be re-arranged in strains LZ561 and LZ1560

FIG. 10: Seq. ID. 6: Nucleotide sequence of putative promoter region of F₁F_OATP synthase operon in strain LZ1561

FIG. 11: Seq. ID. 7: Nucleotide sequence of putative promoter region of F₁F_OATP synthase operon in strain LZ1560

FIG. 12: Seq. ID. 8: Nucleotide sequence of putative promoter region of Rnf complex operon in strain LZ1561

FIG. 13: Seq. ID. 9: Nucleotide sequence of putative promoter region of Rnf complex operon in strain LZ1560

FIG. 14: Seq. ID. 10: Nucleotide sequence of putative promoter region of carbon starvation protein in strain LZ1561

FIG. 15: Seq. ID. 11: Nucleotide sequence of putative promoter region of carbon starvation protein in strain LZ1560

FIG. 16: Seq. ID. 12: Nucleotide sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1561

FIG. 17: Seq. ID. 13: Nucleotide sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1560

FIG. 18: Seq. ID. 14: Amino Acid sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1561

FIG. 19: Seq. ID. 15: Amino Acid sequence of CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit gene in strain LZ1560

FIG. 20: Seq. ID. 16: Nucleotide sequence of 5,10-methylenetetrahydrofolate reductase gene in strain LZ1561

FIG. 21: Seq. ID. 17: Nucleotide sequence of 5,10-methylenetetrahydrofolate reductase gene in strain LZ1560

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed novel bacteria. The bacteria are characterised by having one or more of a number of unexpected properties (as outlined herein after), and in one preferred embodiment all of these properties. The use of these novel bacteria in anaerobic fermentation processes provides an unexpected benefit over existing strains of bacteria which may allow for an increase in the overall efficiency of a fermentation process for producing products such as ethanol and/or acetate.
Accordingly, in broad terms, in one aspect, the present invention relates to a novel 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.
In a further aspect, the invention relates to processes for producing an alcohol, preferably ethanol, by anaerobic fermentation of a CO-containing substrate by the bacteria of the invention.

Definitions

Unless otherwise defined, the following terms as used throughout this specification are defined as follows:
A “substrate containing CO”, a “substrate comprising CO” and like terms should be understood to 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 “substrate containing CO” is gaseous. Such substrates may be referred to herein as “gaseous substrates containing CO”, “gaseous substrates comprising CO” and the like.
In the description which follows, embodiments of the invention are described in terms of delivering and fermenting a “gaseous substrate containing CO”. However, it should be appreciated that the gaseous substrate may be provided in alternative forms. For example, the gaseous substrate containing CO may be provided dissolved in a liquid. Essentially, a liquid is saturated with a carbon monoxide containing gas and then that liquid is added to the bioreactor. This may be achieved using standard methodology. By way of 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) could be used. By way of further example, the gaseous substrate containing CO may be adsorbed onto a solid support. Such alternative methods are encompassed by use of the term “substrate containing CO”.
The terms “increasing the efficiency”, “increased efficiency” and the like, when used in relation to a fermentation process, include, but are not limited to, increasing one or more of: the rate of growth of micro-organisms catalysing the fermentation, the uptake or consumption of CO by the micro-organisms, the volume of desired product (such as alcohols) produced per volume of substrate (such as CO) consumed, the concentration of the desired product (such as alcohols) produced in the culture medium, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared with other by-products of the fermentation.
The term “acetate” includes both acetate salt alone and a mixture of molecular or free acetic acid and acetate salt, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as described herein. The ratio of molecular acetic acid to acetate in the fermentation broth is dependent upon the pH of the system.
The term “bioreactor” includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact.
The term “alcohol tolerance” as used herein 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, grow and/or to produce at least a level of the desired product.
Bacteria of the invention, or cultures or isolates thereof, may be described to be in an “isolated” or “biologically pure” form. These terms are intended to mean that the bacteria have been separated from an environment or one or more constituents, cellular or otherwise, which they may be associated with if found in nature or otherwise. The terms “isolated” or “biologically pure” should not be taken 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.
The invention provides a biologically pure isolate of a bacterium wherein the bacterium is capable of producing products including ethanol and optionally acetate, by anaerobic fermentation of a substrate containing CO and wherein the bacterium is capable of one or more of:
producing ethanol at a specific productivity of about 2 g ethanol/L fermentation broth/gram of biomass/day;
producing ethanol at a productivity of at least about 10 g /L of fermentation broth/day;
a specific uptake of CO of at least about 1.0 mMol CO/min/g of biomass; a specific growth rate of at least about 0.8 day⁻¹;
producing ethanol at an ethanol to acetate ratio of at least about 2:1; and,
tolerating alcohol of up to about 30 g/L of broth.
In a preferred embodiment, a bacterium of the invention is capable of two, three, four, or five of the above features.
In certain embodiments, the bacterium is capable of producing ethanol at a specific productivity of at least about 3 g ethanol/L fermentation broth/gram of biomass/day, at least about 4 g ethanol/L fermentation broth/gram of biomass/day, at least about 5 g ethanol/L fermentation broth/gram of biomass/day, at least about 6 g ethanol/L fermentation broth/gram of biomass/day or at least about 7 g ethanol/L fermentation broth/gram of biomass/day.
In certain embodiments, the bacterium is capable of producing ethanol at a productivity of at least about 20 g ethanol/L of fermentation broth/day, at least about 30 g ethanol/L of fermentation broth/day, at least about 40 g ethanol/L of fermentation broth/day or at least about 50 g ethanol/L of fermentation broth/day. The maximum value takes into account stoichiometry, CO uptake and ethanol stripping.
In certain embodiments the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass, at least about 1.4 mMol CO/min/g of biomass, at least about 1.6 mMol CO/min/g of biomass, at least about 1.8 mMol CO/min/g of biomass, or at least about 2.0 mMol CO/min/g of biomass. In one particular embodiment, the bacterium is capable of a specific uptake of CO of at least about 1.2 mMol CO/min/g biomass.
In certain embodiments the bacterium is capable of a specific growth rate of at least about 1.0 day^−1,at least about 1.2 day⁻¹, at least about 1.4 day⁻¹, at least about 1.6 day⁻¹, at least about 1.8 day⁻¹or at least about 2.0 day⁻¹.
In certain embodiments the bacterium is capable of producing ethanol at an ethanol to acetate ratio of at least about 3:1, of at least about 4:1, of at least about 5:1, of at least about 7:1 or of at least about 10:1. In one particular embodiment, there is no net production of acetate during fermentation.
In certain embodiments the bacterium is capable of tolerating alcohol of up to about 40 g/L of fermentation broth, of up to about 50 g/L of fermentation broth, or of up to about 60 g/L of fermentation broth. In one particular embodiment, the bacterium is capable of tolerating alcohol of up to about 70 g/L of fermentation broth.
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 Clostridium autoethanogenum strain DSM19630 (DSMZ, Germany) (described in WO2009/064200).
In a preferred embodiment, the bacterium of the invention is a strain of Clostridium autoethanogenum.
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.
In certain embodiments of the invention, the bacteria have the defining characteristics of Clostridium autoethanogenum strain DSM23693 deposited at DSMZ, Germany, in accordance with the Budapest Treaty, on 7 Jun. 2010. In a particular embodiment, the bacterium is Clostridium autoethanogenum strain DSM23693.
The invention also relates to bacteria derived from the bacteria of the invention.
The bacteria of certain embodiments of the invention are capable of an increased alcohol production rate, an increased growth rate, an increased CO consumption or update rate, a higher alcohol to acid production ratio, and/or an increased tolerance to alcohol. This provides a benefit over other strains of Clostridia sp including Clostridium autoethanogenum. Therefore, use of bacteria of the present invention may increase the overall efficiency of a fermentation process for producing products such as acetate and/or ethanol.
In certain embodiments the bacteria of the invention are capable of the productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance mentioned herein before at elevated levels of CO in the gaseous substrate. For example, the gaseous substrate may comprise at least about 50% CO by volume, at least about 65% CO by volume, or at least about 70% CO by volume. In certain embodiments the gaseous substrate comprises at least about 80% CO by volume, or at least about 85% CO by volume, or at least about 90% CO by volume or at least about 95% CO by volume.
Similarly the productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance herein before described are achievable in certain embodiments at low to non-existent levels of H₂in the gaseous substrate. The gaseous substrate may comprise about 30% or less H₂by volume. In particular embodiments the gaseous substrate comprises about 20% or less H₂by volume, about 15% or less H₂by volume, about 10% or less H₂by volume, about 5% or less H₂by volume, about 4% or less H₂by volume, about 3% or less H₂by volume, about 2% or less H₂by volume, about 1% or less H₂by volume, or substantially no H₂.
In certain embodiments the bacteria of the invention are also capable of the productivity, growth rates, alcohol to acid ratio, CO consumption and alcohol tolerance mentioned herein before when supplied with gaseous substrate comprising relatively little CO₂. In one embodiment the gaseous substrate comprises less than or equal to about 20% CO₂by volume. In certain embodiments the gaseous substrate comprises less than or equal to about 15% CO₂by volume, less than or equal to about 10% CO₂by volume, or less than or equal to about 5% CO₂by volume. In one particular embodiment, the gaseous substrate comprises substantially no CO₂.
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 media are known in the art and described for example in U.S. Pat. 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 herein after in the Examples section.
The invention also provides methods for the production of one or more alcohols from a gaseous substrate comprising CO, the methods comprising maintaining a culture of one or more bacterial isolate of the invention in the presence of the substrate, and the anaerobic fermentation of the substrate to one or more alcohols by the one or more bacterial isolate.
The invention also provides a method for reducing the total atmospheric carbon emissions from an industrial process, the method comprising:
(a) capturing CO-containing gas produced as a result of the industrial process, before the gas is released into the atmosphere;
(b) the 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.
In certain embodiments of the methods of the invention, acetate is produced as a by-product of the fermentation. The alcohol produced is ethanol.
In certain embodiments, the culture is maintained in a liquid nutrient medium.
The fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CTSR), a bubble column reactor (BCR) or a trickle bed reactor (TBR). Also, in some preferred embodiments of the invention, the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second, fermentation reactor, to which fermentation broth from the growth reactor is fed and in which most of the fermentation product (ethanol and acetate) is produced.
As described above, the carbon source for the fermentation reaction is a gaseous substrate containing CO. The gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing. In these embodiments, the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method. Depending on the composition of the gaseous CO-containing substrate, it may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known methods.
In addition, it is often desirable to increase the CO concentration of a substrate stream (or CO partial pressure in a gaseous substrate) and thus increase the efficiency of fermentation reactions where CO is a substrate. Increasing CO partial pressure in a gaseous substrate increases CO mass transfer into a fermentation media. The composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, O2 may reduce the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases in stages of a fermentation process before or after fermentation can increase the burden on such stages (e.g. where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in the fermentation). Accordingly, 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.
Substrate streams derived from an industrial source are typically variable in composition. Furthermore, substrate streams derived from industrial sources comprising high CO concentrations (such as, for example, at least 40% CO, at least 50% CO or at least 65% CO) often have a low H2 component (such as less than 20% or less than 10% or substantially 0%). As such, it is particularly desirable that micro-organisms are capable of producing products by anaerobic fermentation of substrates comprising a range of CO and H2 concentrations, particularly high CO concentrations and low H2 concentrations. The bacteria of the present invention have a surprisingly high growth rate and ethanol production rate while fermenting a substrate comprising CO (and no H2).
The presence of hydrogen in the substrate stream can lead to an improvement in efficiency of overall carbon capture and/or ethanol productivity. For example, WO02/08438 describes the production of ethanol using gas stream of various compositions. WO02/08438 reports a substrate stream comprising 63% H2, 32% CO and 5% CH4 being provided to a culture of C. ljungdahlii in a bioreactor to promote microbial growth and ethanol production. When the culture reached a steady 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 provide CO in a slight excess and promote ethanol production. This document also describes gas streams with higher and lower CO and H2 concentrations.
It will be appreciated that the processes of the present invention as described herein can be used to reduce the total atmospheric carbon emissions from industrial processes, by capturing CO-containing gases produced as a result of such processes and using them as substrates for the fermentation processes described herein.
Alternatively, in other embodiments of the invention, the CO-containing gaseous substrate may be sourced from the gasification of biomass. The process of gasification involves partial combustion of biomass in a restricted supply of air or oxygen. The resultant 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 sugar from sugarcane, or starch from maize or grains, or non-food biomass waste generated by the forestry industry may be gasified to produce a CO-containing gas suitable for use in the present invention.
It is generally preferred that the CO-containing gaseous substrate contains a major proportion of CO. In particular embodiments, the gaseous substrate comprises at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 65%, or at least about 70% to about 95% CO by volume. It is not necessary for the gaseous substrate to contain any hydrogen. The gaseous substrate also optionally contains some CO₂, such as about 1% to about 30% by volume, such as about 5% to about 10% CO₂.
It will be appreciated that for growth of the bacteria and CO-to-ethanol fermentation to occur, in addition to the CO-containing substrate gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. A nutrient medium will contain vitamins and minerals sufficient to permit 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 media are described in U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438 as well as other publications referred to herein before. In one embodiment of the invention the media is as described in the Examples section herein after.
The fermentation should desirably be carried out under appropriate conditions for the CO-to-ethanol fermentation to occur. Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.
The optimum reaction conditions will depend partly on the particular micro-organism of the invention used. However, in general, it is preferred that the fermentation be performed at pressure higher than ambient pressure. Operating at increased pressures allows a significant increase in the rate of CO transfer from the gas phase to the liquid phase where it can be taken up by the micro-organism as a carbon source for the production of ethanol. This in turn means that the retention time (defined as the liquid volume in the bioreactor divided by the input gas flow rate) can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure.
Also, since a given CO-to-ethanol conversion rate is in part a function of the substrate retention time, and achieving 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 bioreactor required, and consequently the capital cost of the fermentation equipment. According to examples given in U.S. Pat. No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.
The benefits of conducting a gas-to-ethanol fermentation at elevated pressures have also been described elsewhere. For example, WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively. However, example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.
It is also desirable that the rate of introduction of the CO-containing gaseous substrate is such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that the ethanol product is consumed by the culture.
In certain embodiments, a fermentation process according to the present invention described above will result in a fermentation broth comprising ethanol, as well as bacterial cells, in the aqueous culture medium. In preferred embodiments of the method the ethanol is recovered from the fermentation broth.
In certain embodiments, the recovering of ethanol comprises continuously removing a portion of broth and recovering the alcohol from the removed portion of the broth.
In particular embodiments the recovery of ethanol includes passing the removed portion of the broth containing ethanol through a separation unit to separate bacterial cells from the broth, to produce a cell-free alcohol-containing permeate, and returning the bacterial cells to the bioreactor.
In certain embodiments, the methods of the invention are continuous processes.
In particular embodiments, acetate is produced as a by-product of the fermentation.
In a further embodiment the ethanol and the acetate are recovered from the broth.
In certain embodiments, the recovering of ethanol and acetate comprises continuously removing a portion of the broth and recovering separately ethanol and acetate from the removed portion of the broth.
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 the ethanol and acetate, to produce a cell-free ethanol-and acetate-containing permeate, and returning the bacterial cells to the bioreactor.
In the above embodiments, the recovery of ethanol and acetate preferably includes first removing ethanol from the cell-free permeate followed by removing acetate from the cell-free permeate. Preferably the cell-free permeate is then returned to the bioreactor.
Ethanol is the preferred desired end product of the fermentation. The ethanol may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, and extractive fermentation. Distillation of ethanol from a fermentation broth yields an azeotropic mixture of ethanol and water (i.e. 95% ethanol and 5% water). Anhydrous ethanol can subsequently be obtained through the use of molecular sieve ethanol dehydration technology, which is also well known in the art. Extractive fermentation procedures involve the use of a water-miscible solvent that presents a low toxicity risk to the fermentation organism, to recover the ethanol from the dilute fermentation broth. For example, oleyl alcohol is a solvent that may be used in this type of extraction process. Oleyl alcohol is continuously introduced into a fermenter, whereupon this solvent rises forming a layer at the top of the fermenter which is continuously extracted and fed through a centrifuge. Water and cells are then readily separated from the oleyl alcohol and returned to the fermenter while the ethanol-laden solvent is fed into a flash vaporization unit. Most of the ethanol is vaporized and condensed while the oleyl alcohol is non volatile and is recovered for re-use in the fermentation.
Acetate may also be recovered from the fermentation broth using methods known in the art. Methods for the recovery of acetate are described in detail in WO2007/117157 and WO2008/115080.
In certain embodiments of the invention, ethanol and acetate are recovered from the fermentation broth by continuously removing a portion of the broth from the fermentation bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering first ethanol and then acetate from the broth. The ethanol may conveniently be recovered by distillation, and the acetate may 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 the ethanol and acetate have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may 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 enhance adsorption of acetic acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.

Reaction Stoichiometry

Without wishing to be bound by any theory, the chemical reactions for the fermentation of CO to ethanol (a) and acetic acid (b) in the process of the present invention are believed to be as follows:

(a) 6CO+3H₂O=>CH₃CH₂OH+4CO₂
(b) 4CO+2H₂O=>1CH₃COOH+2CO₂

The invention will now be described in more detail with reference to the following non-limiting examples.

EXAMPLES

Materials And Methods


Solution A

NH₄Ac	3.083	g
MgCl₂•6H₂O	0.4	g
CaCl₂•2H₂O	0.294	g
KCl	0.15	g
NaCl (optional)	0.12	g

Distilled Water

Up to 1 L

Solution B

Biotin	20.0	mg
Folic acid	20.0	mg
Pyridoxine•HCl	10.0	mg
Thiamine•HCl	50.0	mg
Riboflavin	50.0	mg
Nicotinic acid	50.0	mg
Calcium D-(*)-pantothenate	50.0	mg
Vitamin B12	50.0	mg
p-Aminobenzoic acid	50.0	mg
Thioctic acid	50.0	mg

	Distilled water	To 1 Litre

Solution C

	Component	mmol/L H2O

	FeCl₃	0.1
	CoCl₂	0.05
	NiCl₂	0.05
	H₃BO₃	0.01
	Na₂SeO₃	0.01
	Na₂MoO₄	0.01
	ZnCl₂	0.01
	MnCl2	0.01
	Na2WO3	0.01

Preparation of Cr (II) Solution

A 1 L three necked flask was fitted with a gas tight inlet and outlet to allow working under inert gas and subsequent transfer of the desired product into a suitable storage flask. The flask was charged with CrCl₃.6H₂O (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 flushing with N₂for one hour, the mixture was warmed to about 80° C. to initiate the reaction. Following two hours of stirring under a constant N₂flow, the mixture was cooled to room temperature and continuously stirred for another 48 hours by which time the reaction mixture had turned to a deep blue solution. The solution was transferred into N₂purged serum bottles and stored in the fridge for future use.

Bacteria

The two types of Clostridium autoethanogenum used were those deposited at the German Resource Centre for Biological Material (DSMZ) and allocated the accession numbers DSM 19630 and DSM 23693. DSM 23693 was developed from Clostridium autoethanogenum strain DSM19630 (DSMZ, Germany) via an iterative selection process.

Sampling And Analytical Procedures

Media samples were taken from the CSTR reactor at intervals over the course of each fermentation. Each time the media was sampled care was taken to ensure that no gas was allowed to enter into or escape from the reactor.

HPLC

HPLC System Agilent 1100 Series. Mobile Phase: 0.0025N Sulfuric Acid. Flow and pressure: 0.800 mL/min. Column: Alltech 10A; Catalog #9648, 150×6.5 mm, particle size 5 ρm. Temperature of column: 60° C. Detector: Refractive Index. Temperature of detector: 45° C.

Method For Sample Preparation

400 μL of sample and 50 μL of 0.15M ZnSO₄and 50 μL of 0.15M Ba(OH)₂are loaded into an Eppendorf tube. The tubes are centrifuged for 10 min. at 12,000 rpm, 4° C. 200 μL of the supernatant are transferred into an HPLC vial, and 5 μL are injected into the HPLC instrument.

Headspace Analysis

Measurements were carried out on a Varian CP-4900 micro GC with two installed channels. Channel 1 was a 10 m Mol-sieve column running at 70° C., 200 kPa argon and a backflush time of 4.2 s, while channel 2 was a 10 m PPQ column running at 90° C., 150 kPa helium and no backflush. The injector temperature for both channels was 70° C. Runtimes were set to 120 s, but all peaks of interest would usually elute before 100 s.

Cell Density

Cell density was determined by counting bacterial cells in a defined aliquot of fermentation broth. Alternatively, the absorbance of the samples was measured at 600 nm (spectrophotometer) and the dry mass determined via calculation according to published procedures.

Sequencing

Genome sequencing revealed several changes in genomes of C. autoethanogenum strain LZ1560 (DSM19630) and new strain LZ1561 (DSM23693), which are likely to contribute to the improved performance.
Both strains were grown anaerobically in PETC media to an optical density (OD_{600 nm}) of 1 and genomic DNA was isolated from 100 ml overnight cultures. Cells were harvested by centrifugation (6,000×g, 15 min, 4° C.), washed with potassium phosphate buffer (10 mM; pH 7.5) and suspended in 1.9 ml 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 of a SDS solution (10% (w/v); 10 min). RNA was digested by addition of 240 μl of an EDTA solution (0.5 M; pH 8), 20 μl Tris-HCl (1 M; pH 7.5), and 10 μl RNase A (50,000 U) for 1 hour. Proteolysis was performed by addition of 100 μl Proteinase K (0.5 U) for 1-3 h at 37° C. Finally, 600 μl of sodium perchlorate (5 M) were added, followed by a phenol-chloroform extraction and an isopropanol precipitation. Purity and quantity of DNA was verified using a NanoDrop® 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) and by gel electrophoresis.
Shotgun genome sequencing was performed using a 454 GS (Roche Applied Science, Indianapolis, Ind., USA). 191,368 single reads with a total length of 44,424,523 bases were created for LZ1560 (10× coverage), while 579,545 paired-end reads with a total length of 202,591,572 by were created for LZ1561 (47.5× coverage).The reads were assembled using the Newbler package (Roche Applied Science, Indianapolis, Ind., USA) and 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).
A total of 64 changes were found in assembled genome sequences of LZ1560 (DSM19630) and new strain LZ1561 (DSM23693) (FIG. 4). While most changes were single base variations, one 21 bp deletion (in gene encoding a putative DNA mismatch repair protein MutS; Seq. ID. 1-4) and a rearrangement event of a 15,408 by region (Seq. ID. 5) containing 11 genes (involved in nitrogen fixation, sugar metabolism, sugar transport and catabolite control) were found. From the 62 single base variations, 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 didn't result in a change of amino acid sequence, 14 resulted in a single amino acid change and 25 in a frameshift.
Most notably were changes in positions 212,530 (putative promoter region of F₁F_OATP synthase operon, Seq. ID. 6-7), 1,171,874 (putative promoter region of Rnf complex operon, Seq. ID. 8-9), 3,717,495 (putative promoter region of carbon starvation protein, Seq. ID. 10-11), and two variations in the Wood-Ljungdahl-gene cluster at positions 3,741,730 (CO dehydrogenase/CO-methylating acetyl-CoA synthase complex beta subunit, Seq. ID. 12-15) and 3,748,058 (5,10-methylenetetrahydrofolate reductase gene, Seq. ID. 16-17), which can be traced back directly to growth on CO/H₂and energy metabolism. Most other genes affected are uncharacterized genes.

Example 1

A: Batch Fermentation In CSTR

Approximately 1500 mL of solution A was transferred into a 1.5 L fermenter and sparged with nitrogen. Resazurin (1.5 mL of a 2 g/L solution) and H₃PO₄(85% solution, 2.25 mL) was added and the pH adjusted to 5.3 using concentrated NH₄OH (aq). Nitrilotriacetic acid (0.3 ml of a 0.15M solution) was added prior to 1.5 ml of solution C. This was followed by NiCl2 (0.75 ml of 0.1M solution) and Na₂WO₃(1.5 mL of a 0.01M solution). 15 ml of solution B was added and the solution sparged with N2 before switching to CO containing gas (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, Na2S solution (0.2M solution) was added at a rate of approx 0.3 ml/hour. Substrate supply was increased in response to the requirements of the microbial culture.
The bacterial culture did not proliferate in the experimental conditions used. The culture showed a 350 mM CO uptake after 48 hrs of growth (FIG. 1 a and Table 2) while the doubling time of the culture was 40.8 hrs (FIG. 2 a). This corresponds to a specific growth rate of 0.41 day⁻¹. The specific CO uptake increased during the experiment with a maximum value of 0.54 mM CO/min/g biomass. Day 1.0 specific uptake: 0.28 mM CO/min/g biomass (Table 1). Day 2.0 specific uptake: 0.54 mM CO/min/g biomass (Table 2).

B: Batch Fermentation In CSTR

Approximately 1500 mL of solution A was transferred into a 1.5 L fermenter and sparged with nitrogen. Resazurin (1.5 mL of a 2 g/L solution) and H₃PO₄(85% solution, 2.25 mL) was added and the pH adjusted to 5.3 using concentrated NH₄OH (aq). Nitrilotriacetic acid (0.3 ml of a 0.15M solution) was added prior to 1.5 ml of solution C. Na₂WO₃(1.5 mL of a 0.01M solution) was added. 15 ml of Solution B was added and the solution sparged with N2 before switching to CO containing gas (50% CO; 50% N2) at 60 mL/min. The fermenter was then inoculated with 180 ml of a Clostridium autoethanogenum 23693 culture. The fermenter was maintained at 37° C. and stirred at 300 rpm. During this experiment, Na2S solution (0.5M solution) was added at a rate of approx 0.12 ml/hour. Substrate supply was increased in response to the requirements of the microbial culture.
The bacterial culture proliferated in the experimental conditions used. The culture showed a 8400 mM CO uptake after 43 hrs of growth (FIG. 2 b) while the doubling time of the culture was 9.6 hrs (FIG. 2 b). This corresponds to a specific growth rate of 1.73 day⁻¹. The maximum specific CO uptake reached during the experiment was 1.17 mMol CO/min/g biomass. Day 1.0 specific uptake: 1.17 mM CO/min/g biomass (Table 1). Day 2.0 specific uptake: 1.03 mM CO/min/g biomass (Table 2). The fermentation conditions were identical or at least highly similar to the conditions used in Example 1A. The media preparation has identical components at similar concentrations while both gasses contained CO at least 50% (v/v). The similar fermentation conditions compared to the vast difference in CO uptake indicates the culture performance varied due to the improved efficiency of the developed Clostridium autoethanogenum 23693 culture compared to the parent strain Clostridium autoethanogenum 19630.

Results

TABLE 1

Day 1

Strain	DSM19630	DSM23693

CO consump-	113 mM	3700 mM
tion mM/L
Ethanol	0.48 g/L	7.98 g/L
Production g/L
Acetate	4.58 g/L	4.06 g/L
Production g/L
Biomass g/L	0.29 g/L	1.83 g/L
Specific uptake	0.28 CO/min/g biomass	1.17 CO/min/g biomass
Specific ethanol	2.5 g/L/g biomass/day	4.3 g/L/g biomass/day
production

TABLE 2

Day 2

Strain	DSM19630	DSM23693

CO consump-	350 mM	8150 mM
tion mM/L
Ethanol	1.84 g/L	26.14 g/L
Production g/L
Acetate	4.5 g/L	3.47 g/L
Production g/L
Biomass g/L	0.41 g/L	5.42 g/l
Specific uptake	0.54 CO/min/g biomass	1.03 CO/min/g biomass
Specific ethanol	3.0 g/L/g biomass/day	6.5 g/L/g biomass/day
production

Example 2

Approximately 1500 mL of solution A was transferred into a 1.5 L fermenter and sparged with nitrogen. Resazurin (1.5 mL of a 2 g/L solution) and H₃PO₄(85% solution, 0.56 mL) was added and the pH adjusted to 5.3 using concentrated NH₄OH(aq). Solution C (1.5 mL) was added after which Na₂WO₃(1.5 mL of a 0.01M solution) was added. 15 ml of Solution B was added and the solution sparged with N2 before switching to CO containing gas (50% CO; 50% N2) at 60 mL/min. The fermenter was then inoculated with 100 ml of a Clostridium autoethanogenum 23693 culture. The fermenter was maintained at 37° C. and stirred at 300 rpm. During this experiment, Na2S solution (0.5M solution) was added at a rate of approx 0.15 ml/hour. Substrate supply was increased in response to the requirements of the microbial culture.
The bacterial culture proliferated in the experimental conditions used. The fermentation conditions were identical or at least highly similar to the conditions used in Example 1A+B while both gasses contained CO at least 50% (v/v). The culture was grown to the stationary phase where maximum ethanol concentration was measured by HPLC (55.8 g/L).
The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the scope and spirit of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practised in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, the terms “comprising”, “including”, “containing” etc are to be read expansively and without limitation. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.
The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

1-29. (canceled)

30. A biologically pure isolate of a bacterium derived from Clostridium autoethanogenum wherein the selected bacterium ferments a substrate comprising CO to products comprising ethanol and wherein the bacterium has one or more improvements compared to a parent strain, the one or more improvements being selected from the group consisting of;

a. an increased production rate;

b. an increased growth rate;

c. an increased carbon monoxide uptake;

d. an increased tolerance to alcohol; and

e. a higher alcohol to acid production ratio.

31. The bacterium of claim 1 wherein the parent strain is Clostridium autoethanogenum strain deposited at the German Collection of Microorganisms and cell cultures (DSMZ) under the accession number DSM19630.

32. The selected bacterium of claim 1 wherein the bacterium is Clostridium autoethanogenum strain deposited at DSMZ under the accession number DSM23693.

33. A biologically pure isolate of Clostridium autoethanogenum having at least one DNA sequence selected from Seq. ID. 1 to Seq. ID. 17.

34. The bacterium of claim 30 wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day.

35. The bacterium of claim 31 wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day.

36. The bacterium of claim 32 wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day.

37. The bacterium of claim 33 wherein the bacterium ferments a substrate comprising CO to products comprising ethanol and wherein the production rate of the bacterium is at least 2 g ethanol/L fermentation broth/gram of biomass/day

38. The bacterium of claim 34 wherein the specific productivity is at least 7 g ethanol/L fermentation broth/gram of biomass/day.

39. The bacterium of claim 30 wherein the products comprise ethanol and acetate.

40. The bacterium of claims 30 wherein production rate of the bacterium is at least 10 g of ethanol/L of fermentation broth/day.

41. The bacterium of claim 40 wherein the production rate of the bacterium is at least 50 g of ethanol/L of fermentation broth/day.

42. The bacterium of claims 39 wherein the ethanol to acetate ratio is at least 2:1

43. The bacterium of any one of claim 30 wherein the bacterium has a specific uptake of CO of at least 1.0 mmol CO/min/g biomass.

44. The selected bacterium of claim 43 wherein the bacterium has a specific uptake of CO of at least 2.0 mmol CO/min/g biomass.

45. The bacterium of claim 30 wherein the bacterium has a specific growth rate of at least 0.8 day⁻¹.

46. The bacterium of claim 30 wherein the bacterium tolerates an alcohol concentration of up to 30 g/L of fermentation broth.

47. The bacterium of claim 46 wherein the bacterium tolerates an alcohol concentration of up to 70 g/L of fermentation broth.