NZ583586A - Processes of producing alcohols by microbial fermentation of carbon monoxide - Google Patents

Abstract

Disclosed is a method for increasing the efficiency of capturing carbon and producing alcohol by microbial fermentation, the method comprising: a.) Culturing in a bioreactor, an isolate of the bacterium Clostridium autoethanogenum in a liquid nutrient medium; b.) Anaerobically fermenting a substrate comprising CO in the bioreactor of step (a) to produce one or more alchohols; c.) Monitoring a pH of the liquid nutrient medium to detect changes in the pH of the liquid nutrient medium; d.) If a change in the pH is detected, altering the pH of the liquid nutrient medium such that the pH is maintained within an optimum range for alcohol production, wherein the optimum range for alcohol production comprises substantially pH 4.5 to 6.5.

Description

*10058806880* 8 3 5 8 6 PATENTS FORM NO. 5 Our ref: TIS508231NZPR Divisional of NZ 556615/560756 Antedating requested to 15 August 2008 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION PROCESSES OF PRODUCING ALCOHOLS We, LANZATECH NEW ZEALAND LIMITED of 24 Balfour Road, Parnell, Auckland 1052, New Zealand, hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: 301396453:TIS:glw 301400726:508231 NZ FIELD OF THE INVENTION This invention relates generally to methods for increasing the efficiency of processes of producing products by microbial fermentation of gases and/or dissolved 5 gases and/or carbohydrates, particularly but not exclusively, to processes of producing ethanol by microbial fermentation of gaseous substrates comprising carbon monoxide.

BACKGROUND OF THE INVENTION Ethanol is rapidly becoming a major hydrogen-rich liquid transport fuel around the 10 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 continue 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 15 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, or as a pure fuel in its own right. Also, in Europe, environmental concerns surrounding the consequences of Green House Gas (GHG) 20 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 25 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, and 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 30 resources into fuel ethanol. 301400726:508231 NZ 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. These biological processes, although generally slower than chemical reactions, have several advantages over catalytic processes, including higher specificity, higher yields, lower energy costs and greater 10 resistance to poisoning.

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 15 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 H2via 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, US patent nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438. The bacterium Clostridium 25 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 30 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 301400726:508231 NZ 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.

W02007/117157 describes a process that produces alcohols, particularly ethanol, 5 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. US 7,078,201 and WO 02/08438 also describe fermentation processes for producing ethanol which vary conditions (e.g. pH and redox potential) of the liquid nutrient medium 10 in which the fermentation is performed.

There may be considered to be a need to provide improved means to control or regulate conditions in fermentation reactions which result in production of alcohols.

It is an object of the present invention to meet such a need and/or provide improved methods for producing alcohols by microbial fermentation, or at least to 15 provide the public with a useful choice.

SUMMARY OF THE INVENTION In a first aspect, the invention provides a process for producing one or more alcohols by anaerobic bacterial fermentation of a substrate comprising CO, the process 20 comprising: (a) culturing in a bioreactor, one or more strains of bacteria in a liquid nutrient medium and supplying the substrate to the bioreactor; (b) monitoring a redox potential of the liquid nutrient medium; and (c) controlling the redox potential such that it is substantially maintained within an 25 optimum range for alcohol production.

In certain embodiments, the process further comprises: (a) monitoring pH of the liquid nutrient medium; and (b) controlling the pH such that it is substantially maintained above an optimum growth pH. 301927972:508231 NZ Received at IPONZ on 16.08.2011 In a second aspect, the invention provides a process for producing one or more alcohols by anaerobic bacterial fermentation of a substrate comprising CO, the process comprising: (a) culturing in a bioreactor, one or more strains of bacteria in a liquid nutrient 5 medium at an optimum growth pH and supplying the substrate to the bioreactor such that microbial growth is promoted; (b) either, i) at a desired time point, adjusting pH of the liquid nutrient medium to a level or a range above the optimum growth pH, or ii) at a desired time point, adjusting the redox potential of the liquid nutrient 10 medium to an optimum level or range; (c) monitoring the pH and/or redox potential of the liquid nutrient medium; and, (d) controlling the pH such that it is substantially maintained above an optimum growth pH and/or controlling the redox potential such that it is substantially maintained at an optimum level or range.

In a third aspect, the invention provides a process of producing one or more alcohols by anaerobic bacterial fermentation of acetate and a substrate comprising CO, wherein the process comprises: (a) in a first bioreactor, fermenting a substrate containing a carbon and energy source 20 to produce acetate, (b) in a second bioreactor, culturing in a bioreactor one or more strains of anaerobic bacteria in a liquid nutrient medium and supplying acetate produced from the fermentation (a) and the substrate to the bioreactor, wherein the pH and/or redox potential of the liquid nutrient medium are maintained at levels that allow fermentation of acetate and the substrate comprising CO by the bacteria to produce one or more alcohols.

In a further aspect, the invention provides a method of capturing carbon by microbial fermentation, the method comprising; 302014135:508231 NZ Received at IPONZ on 27.01.2012 -5A- (a) culturing in a bioreactor, an isolate of the bacterium Clostridium autoethanogenum in a liquid nutrient medium; (b) anaerobically fermenting a substrate comprising CO in the bioreactor of step (a) to produce one or more alcohols; (c) monitoring a pH of the liquid nutrient medium to detect changes in the pH of the liquid nutrient medium; (d) if a change in the pH is detected, altering the pH of the liquid nutrient medium such that the pH is maintained within an optimum range for alcohol production, wherein the optimum range for alcohol production 10 comprises substantially pH 4.5 to substantially pH 6.5.

In embodiments of each of the various aspects, the optimum redox potential range is about -450 to -550 mV.

In embodiments of some aspects, the pH is maintained at about 5.7 to about 7.0. 301400726:508231 NZ In embodiments of each of the various aspects the redox potential is controlled by one or more of the following: (a) adding one or more oxidising agents, (b) adding one or more reducing agents, (c) adjusting pH.

In embodiments of each of the various aspects, the reducing agent is methyl viologen or cysteine.

In embodiments of each of the various aspects, the pH is controlled or adjusted by adding acid(s) and/or base(s).

In a fourth aspect, the invention provides a method for increasing the efficiency of a fermentation process, the method comprising: (a) detecting a change in the intracellular redox potential of micro-organisms used in the fermentation process; and (b) controlling at least one parameter of the fermentation process based on 15 whether a change is detected in step (a).

In particular embodiments, the step (a) of detecting a change comprises removing a sample of the micro-organisms from a bioreactor, wherein the fermentation process is, at least in part, performed in the bioreactor. In certain embodiments, the step (a) of detecting a change is repeated one or more times to obtain a plurality of measurements, 20 preferably, after predetermined intervals. Note that the intervals may or may not be the same for a particular process and may change from process to process as would be apparent to one of skill in the art in view of this disclosure.

The parameters controlled may comprise any one or more of pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential/agitation 25 rate, inoculum level, maximum gas substrate concentrations, maximum product concentrations and the commencement of product recovery.

Any method known in the art may be used to detect a change in the intracellular redox potential. However, in certain embodiments, a flow cytometry approach is provided, whereby a reagent, which is an indicator of bacterial reductase activity, is 30 added to the sample and then penetrates the bacteria. Following reduction, the reagent 301400726:508231 NZ produces a fluorescent signal which varies depending on the intracellular redox potential, and may be analysed using a flow cytometer.

In particular embodiments, the controlling of step (b) comprises determining, based on whether a change in intracellular redox potential is detected at step (a), 5 whether to alter at least one parameter of the fermentation process and/or maintain at least one parameter at its present value and/or to take no action to change a parameter based on the result of step (a).

Note that while no action may be taken based on the result of step (a), the skilled artisan would be aware in view of the present disclosure that conventional forms of 10 control and adjustment may still be carried out, such as maintenance of pH within a predetermined range.

In particular embodiments, the fermentation process produces acetate and/or one or more alcohols by anaerobic fermentation of a substrate comprising CO by one or more strains of acetogenic bacteria.

In certain embodiments, step (a) comprises culturing in a bioreactor one or more strains of anaerobic bacteria in a liquid nutrient medium and supplying a gaseous substrate comprising CO to the bioreactor, and maintaining the pH and redox potential of the liquid nutrient medium at levels which allow the bacteria to grow and produce acetate.

In some embodiments, the step of determining is based on whether there is a detected rise in intracellular redox potential. Additionally or alternatively, the step of determining may be based on whether there is a detected drop in intracellular redox potential. In the case of a detected drop in intracellular redox potential, the controlling of step (b) may comprise ceasing the fermentation process and/or adding micro-organisms 25 to the contents of the fermentation tank and/or removing a portion of the contents of the fermentation tank and/or changing one or more parameters (see the above list) of the fermentation process.

In a particular embodiment, the method of the invention is adapted for use in a process of producing alcohols, more particularly ethanol and/or butanol and/or 30 isopropanol, by anaerobic fermentation of gases, particularly gases containing carbon 302191566_1, DO C :TIS508231N ZPR RECEIVED at IPONZ on 18 May 2012 -8- monoxide. However, the invention is not limited thereto and may be applied to other uses, including aerobic fermentations, and to fermentations of substrates not including CO or only including a small proportion thereof (e.g. 6% CO), particularly where the substrate contains C02 and/or H2. Embodiments of the invention include, for example and without limitation, 5 fermentations of carbohydrates and, additionally or alternatively, the production of acids (and/or salts thereof) and/or H2.

According to a fifth aspect, the invention provides a fermentation system for carrying out a fermentation process, the system comprising: (a) means for detecting a change in the intracellular redox potential of micro- organisms used in the fermentation process; and (b) means for controlling at least one parameter of the fermentation process based on whether a change is detected by the means for detecting.

In a further aspect, the invention provides an anaerobic system for carrying out a method as defined in any one of the aspects hereinbefore, the system comprising: (a) A bioreactor containing an isolate of the bacterium Clostridium autoethanogenum in a liquid nutrient medium; (b) means for detecting a change in the pH of the liquid nutrient medium; and (c) means for controlling the pH of the fermentation process based on whether a change is detected in the pH by the means for detecting.

The system may be configured to carry out anaerobic and/or aerobic fermentation processes. In one embodiment, the invention relates to anaerobic fermentations.

Embodiments of each of the various aspects find particular application in the fermentation of a substrate comprising CO to produce acids and/or alcohols. In particular embodiments, the substrate comprising CO is gaseous. The gaseous substrate may comprise 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.

Preferably, the gaseous substrate comprises a gas obtained from a steel mill. 301927972:508231 NZ Received at IPONZ on 16.08.2011 -8A- ln certain embodiments of each of the various aspects the gaseous substrate comprises from 15% CO to 100% CO by volume, such as from 20% to 95% by volume, such as from 75% CO to 95% CO by volume, such as from 80% to 90% CO by volume. Lower CO levels, such as 6%, may be envisaged where the substrate also contains C02 and H2.

In embodiments of each of the various aspects, the alcohol produced by the fermentation process is ethanol. The fermentation reaction may also produce acetate and/or butanol and/or isopropanol. 301400726:508231 NZ ln particular embodiments of each of the various aspects, the fermentation reaction is carried out by one of more strains of acetogenic bacteria.

In embodiments of each of the various aspects, the anaerobic, acetogenic bacteria are selected from the group consisting of Clostridium, Moorella, Carboxydothermus, 5 Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium and Peptostreptococcus. In particular embodiments, the acetogenic bacterium is Clostridium autoethanogenum or Clostridium carboxydivorans.

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 more detail and with reference to the accompanying figures, in which: Figure 1 is a chart showing the typical life cycle of Clostridium autoethanogenum; Figure 2 is a graph showing the variation of RS positive cell proportion in a culture treated with methyl viologen compared to an untreated culture over against time; Figure 3 is a graph showing acetate and ethanol production by Clostridium autoethanogenum over time at optimum growth pH and then elevated pH.

DETAILED DESCRIPTION OF THE INVENTION The following is a description of the present invention, including various embodiments thereof, given in general terms. The invention is further exemplified in the disclosure given under the heading "Examples" herein below, which provides experimental data relating to making, using, and practicing the invention, specific examples of aspects and embodiments of the invention, and illustrative means of performing the invention.

Definitions Unless otherwise defined, the following terms as used throughout this specification are defined as follows: 301400726:508231 NZ 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 volume of desired product (such as alcohols) produced per volume of substrate (such as 5 carbon monoxide) consumed, 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 "substrates comprising carbon monoxide" include any solid, liquid or gaseous material containing CO that may be present or introduced into a bioreactor for 10 fermentation.

The term "co-substrate" refers to a substance that, while not necessarily being the primary energy and material source for product synthesis, can be utilised for product synthesis when added in addition to another, such as the primary, substrate.

The term "acetate" includes both acetate salt alone and a mixture of molecular or 15 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 any fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank 20 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 "optimum growth pH" and the like include the pH level or pH range of the liquid nutrient medium which promotes and/or supports growth in bacteria. It will be 25 appreciated that the optimum growth pH may vary depending on the micro-organism used and/or other fermentation conditions.

The term "optimum redox potential" or "optimum ORP" and the like include the ORP level or ORP range of the liquid nutrient medium which promotes and/or supports alcohol production. It will be appreciated that the optimum ORP may vary depending on 30 the micro-organism used and/or other fermentation conditions. 301400726:508231 NZ The term "limiting concentration" means an initial concentration of a given component in a microbial fermentation medium that is sufficiently low to ensure that it will be depleted at some stage in the fermentation.

The term "carbon capture" as used herein refers to the sequestration of carbon 5 compounds including CO2 and/or CO from a stream comprising CO2 and/or CO and either: (a) converting the CO2 and/or CO into products; or (b) converting the C02 and/or CO into substances suitable for long term storage; or (c) trapping the C02 and/or CO in substances suitable for long term storage; or a combination of these processes.

Unless the context requires otherwise, the phrases "fermenting", "fermentation process" or "fermentation reaction" and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of a process involving the growth/and or biosynthesis of a product by a micro-organism.

The present invention generally relates to processes of producing alcohols by 15 fermentation of gaseous substrates containing carbon monoxide (CO). The methods of the present invention also generally relate to improvements in carbon capture, wherein CO and optionally C02 are converted into useful products, namely alcohols.

Processes of producing alcohols by anaerobic bacterial fermentation of gaseous substrates containing CO as the carbon and energy source are known in the art. These 20 fermentation processes, in addition to producing the desired alcohol products, may also produce acetate as a by-product. Thus, the efficiency of conversion of the available carbon from the CO to the desired alcohol product is less than optimal.

Without wishing to be bound by theory, in fermentation processes, there are often two metabolic phases, the acidogenic phase in which the bacteria grows and 25 produces an acid and the solventogenic phase in which solvents such as alcohols are produced. By way of example, in fermentation processes using Clostridium species for the production of solvents, two metabolic phases are known and are shown in Figure 1. The acetogenic phase is associated with bacterial growth during which Clostridium species produce, for example, H2, C02, and/or organic acids such as butyrate and acetate. While 30 solvents such as alcohols may optionally be produced during the acetogenic phase, they are generally produced in insignificant amounts. 301400726:508231 NZ Th e acetogenic phase is followed by the solventogenic phase during which the metabolism of the cells undergoes a shift to favour solvent production. Typically, there is a reduction in, or cessation of, organic acid production and microbial growth in this phase. Different environmental parameters (e.g. pH, redox potential, etc of the media) 5 are preferable for the bacteria depending on which phase they are in.

The inventors have surprisingly shown that by elevating the pH of a culture of anaerobic acetogenic bacteria to above the optimum growth pH, while maintaining the redox potential of the culture at a low level (about -450 mV or below), the bacteria produce alcohol and optionally acetate. Under such conditions, alcohol and optionally 10 acetate are produced in a molar ratio of at least about 1:1, preferably in a molar ratio favouring alcohol. In one particular embodiment, the molar ratio of alcohol to acetate is about 1.4 to 1. Furthermore, the bacteria convert at least a portion of the acetate produced as a by-product of the fermentation to ethanol, at a much higher rate than under lower pH conditions, which would normally be used in such fermentation 15 processes.

Furthermore, it has been surprisingly shown that there is a notable decrease in the intracellular redox potential of micro-organisms, particularly Clostridium autoethanogenum, in a fermentation process when they switch from the acetogenic phase to the solventogenic phase. Consequently, in broad terms, certain aspects of the 20 invention relate to detection of a status or change in status of micro-organisms in a fermentation process using one or more indicators of the intracellular redox potential of the micro-organisms.

While the following description focuses on certain embodiments of the invention, namely the production of ethanol using CO as the primary substrate, it should be 25 appreciated that the invention may be applicable to production of other alcohols and the use of other substrates as will be known by persons of ordinary skill in the art to which the invention relates upon consideration of the present disclosure. Also, while particular mention is made to fermentations carried out using acetogenic bacteria, for example, Clostridium autoethanogenum and Clostridium corboxydivorans, the invention is also 30 applicable to other micro-organisms which may be used in the same or different processes, including other species of Clostridia which may be used to produce useful 301400726:508231 NZ products, including but not limited to butanol. Accordingly, in one aspect, the invention relates to processes of producing alcohols, such as butanol, for example, n-butanol, by anaerobic bacterial fermentation of gaseous substrates comprising CO as the carbon and energy source.

Processes of the invention will typically involve culturing, in a bioreactor containing a liquid nutrient medium, one or more strains of anaerobic bacteria that are capable of producing alcohols and optionally acetate from a CO-containing gaseous substrate, and supplying the gaseous substrate to the bioreactor. This fermentation produces one or more desired alcohols, and generally also acetate.

In one particular embodiment the process includes controlling the redox potential of the liquid nutrient medium such that it is maintained at an optimum level or range, in particular about -450 mV or lower, for example -450mV to about -550 mV. In a particular embodiment the process also includes the step of controlling the pH of the liquid nutrient medium such that it is maintained at an elevated level (i.e. increased above the optimum 15 growth pH), in particular from about 5.7 to about 7.0, or from about 5.8 to about 6.5. In a particular embodiment, the liquid nutrient medium is maintained under the relevant redox conditions, or under the relevant pH and redox conditions, for at least a period of time sufficient to allow the bacteria to produce one or more alcohols and optionally acetate in a molar ratio of at least 1:1, or at a higher ratio favouring alcohol. The 20 conditions further promote fermentation of any acetate present in the liquid nutrient medium to the desired alcohol, for example ethanol or n-butanol. This acetate to alcohol conversion increases the yield of ethanol from the fermentation process, thus further improving the ethanol to acetate ratio.

The redox potential of the liquid nutrient medium can be adjusted by adding one 25 or more suitable reducing agents or oxidising agents, as required. Reducing agents suitable for lowering ORP will be known to those skilled in the art. However, by way of example, sulphites, bisulphites, sulphur dioxide, ammonia, hydrazine or reducing gases such as hydrogen or even CO, may be used to reduce the ORP. In particular embodiments of the invention reducing agents suitable for use in the processes of the invention include 30 methyl viologen and cysteine. Oxidising agents suitable for increasing the ORP will be known to those skilled in the art, but by way of example may include hydrogen peroxide, 301400726:508231 NZ -14 ozone, dihalide (chlorine, bromine or iodine), chlorine dioxide, potassium permanganate, air or oxygen. Those skilled in the art will also appreciate that the ORP may also be decreased or increased by raising or lowering the pH of the liquid nutrient medium respectively. For example, by increasing the pH of a theoretical aqueous system by one 5 pH unit, the ORP will decrease by 59mV.

The pH of the liquid nutrient medium may be adjusted as required by adding one or more suitable pH-adjusting agents or buffers. For example, bases such as NaOH and acids such as sulphuric acid may be used to increase or decrease, respectively, the pH, as required. Those skilled in the art will be aware of acids and/or bases available for 10 adjusting the pH of the liquid nutrient medium.

Those of skill in the art will also appreciate that the methods of the invention therefore typically involve one or more steps of measuring the pH and/or redox potential of the fermentation medium.

In particular embodiments of the invention, the bioreactor includes means for 15 continuously or intermittently determining liquid nutrient media pH and/or ORP. Those skilled in the art will appreciate suitable means or sensors for monitoring these parameters, however by way of example, ORP may be continuously or intermittently monitored by immersion of an ORP probe into the liquid nutrient media. Similarly, pH can be monitored by immersion of a pH probe in the liquid nutrient media. 20 The feedback provided by the pH and/or ORP determining means allow monitoring of the conditions in the bioreactor. The conditions within the reactor can be controlled as a result of the feedback or otherwise, by altering particular parameters or adding particular reagents.

ORP can be controlled by adding reducing or oxidising reagents to the liquid 25 nutrient medium. For example, if the ORP increases or decreases above or below the optimum ORP, reducing agents or oxidising agents may be added to the liquid nutrient media to maintain the media within the optimum ORP range. Alternatively, the ORP may be controlled by introduction of electrodes into the liquid nutrient medium to maintain or achieve a desired ORP.

Similarly, pH can also be controlled. For example, if the pH increases above or decreases below the desired pH range, acids or bases may be added to the liquid nutrient media to maintain the media within a desired pH range.

Those skilled in the art will appreciate that the oxidising or reducing agents, or acids or bases may be added manually in response to deviation from optimum conditions. For example, one or more alarms may be activated in order to alert an operator that a particular reagent needs to be added. However, in certain embodiments, the pH and/or ORP are monitored continuously or intermittently and suitable reagents are added automatically in response to the deviation from optimum conditions. For example, pH and/or ORP probes provide feedback to a central processor that monitors the conditions with the bioreactor. If the ORP increases above a predetermined threshold, the processor will initiate the introduction of a reducing agent until the desired ORP is maintained. Alternatively, or in addition, if the pH decreases below a predetermined threshold, the processor can initiate the introduction of a base until the desired pH is maintained.

In one embodiment of the invention, continuous monitoring and subsequent maintenance of pH and ORP in desired ranges has led to continuous production of ethanol over a 9 day period. It is contemplated that monitoring and maintaining liquid nutrient media conditions in this manner will result in continuous alcohol production for several weeks or months.

In another aspect, the invention relates to detection of a status or a change in status of micro-organisms, in a fermentation process using one or more indicators of the intracellular redox potential of the micro-organisms. The intracellular redox potential if largely independent of the ORP of the liquid nutrient medium and may be used as an indirect measure of the viability and/or status of a microbial population in a bioreactor. Based on the determined status, the conditions within the bioreactor housing the microorganisms may be controlled so that they are optimised for the determined status, thereby increasing the efficiency of the process. For example, in the case of acetogenic micro-organisms, if it is determined that the micro-organisms are in the growth or acetogenic phase, parameters may be controlled so as to provide for optimal growth. Alternatively, if micro-organisms are in the alcohol or solventogenic producing phase, conditions may be optimised for the production of alcohol. For example, in the alcohol 301400726:508231 NZ production phase, the pH and/or ORP can be maintained within optimum ranges to promote alcohol production.

Optimisation of bioreactor conditions is highly desirable in such processes. In particular, more rapid accumulation of bacterial cell biomass, such as C. autoethanogenum, is highly desirable in the context of a commercial process, such as ethanol production from CO-containing gases. This is because increases in bacterial biomass accumulation allow a reduction in the time required for the bacterial concentration in a fermentation process to reach a bacterial cell density that allows maximal process productivity per volume of input gas, per volume of reactor media. This 10 reduction in time is associated with a concomitant reduction in operating costs and nonproductive down time.

Alternatively, or in addition to the above, the optimisation of bioreactor conditions comprises determining, based on whether a change in intracellular redox potential is detected, whether to alter at least one parameter of the fermentation 15 process and/or maintain at least one parameter at its present value and/or to take no action to change a parameter based on the result of step (a).

The parameters controlled may comprise any one or more of pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate, inoculum level, maximum gas substrate concentrations, maximum product 20 concentrations and the commencement or cessation of product recovery.

Thus, according to some embodiments of the invention, samples of microorganisms are taken from a bioreactor and a determination is made as to whether there is a change in the intracellular redox potential thereof. Any known method may be used to determine the intracellular redox potential of the micro-organisms, including those 25 involving the use of spectrophotometers. The samples are preferably obtained at regular intervals although the interval may be varied depending on known characteristics of particular reactions. For example, if it is known that a particular micro-organism has a minimum growth period, there may be an initial period when fewer or no samples are taken followed by a period when more regular samples are taken until a change is 30 detected. Where there is a significant change in the intracellular redox potential, it is 301400726:508231 NZ possible to then come to a conclusion on the status thereof, and use this information to optimise conditions for the micro-organisms.

Intracellular redox states can be assayed by measuring the concentrations of redox couples such as NAD+/NADH, NADP+/NADPH, and cystine/cysteine, and the 5 oxidised and reduced forms of glutathione and metalloenzymes. Tools utilised to achieve such measurements include the use of fluorescence (flow cytometry, microscopy and fluorometric assays), colourmetric assays, radiolabeling and magnetic resonance imaging.

In a particular embodiment of the invention, flow cytometry is used to determine the reductase activity in bacterial samples taken from the bioreactor. Any known 10 indicator of bacterial reductase activity may be used. However, by way of example, the RedoxSensor™ Green reagent (RS) is a suitable indicator of bacterial reductase activity used in some embodiments of the invention. Reductase activity is, in turn, a reliable marker for changes in electron transport chain function. Following reduction, RS will produce a stable green-fluorescent signal in 10 minutes. Stain intensity is altered when 15 cells are treated with reagents that disrupt electron transport, such as sodium azide, or carbonyl cyanide 3-chlorophenylhydrazone.

Fermentation The invention has particular applicability to improving the production of ethanol 20 and/or other alcohols such as butanol and/or isopropanol from substrates comprising CO. In particular embodiments, the substrate comprising CO is gaseous. The gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process. Aspects of the invention are also applicable to reactions which produce acids and/or alcohols of a wide variety of types. For example, butyrate, acetate, butanol, 25 acetone, ethanol or isopropanol production by species of Clostridia such as C. acetobutylicum, C. bejerenkii and C. saccarolyticum, including fermentation on cellulose, cellulose hydrolysate, starch, starch hydrolysate, glucose, sucrose, fructose xylose, arabinose, glycerol and lactose.

Processes for the production of ethanol and other alcohols from gaseous 30 substrates (such as those described above) are known. Exemplary processes include 301400726:508231 NZ -18 those described for example in W02007/117157 and US 6,340,581, US 6,136,577, US 5,593,886, US 5,807,722 and US 5,821,111.

A number of anaerobic bacteria are known to be capable of carrying out the fermentation of CO to alcohols, including n-butanol and ethanol, and acetic acid, and are 5 suitable for use in the process of the present invention.

Examples of such bacteria that are suitable for use in the invention include those of the genus Clostridium, such as strains of Clostridium ljungdahlii, including those described in WO 00/68407, EP 117309, US patent no's 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438, Clostridium autoethanogenum (Abrini et al, 10 Archives of Microbiology 161: pp 345-351), Clostridium aceticum, Clostridium acetobutylicum and Clostridium thermoaceticum. Other useful bacteria include Acetogenium kivui, Acetobacterium woodii, Acetoanaerobium noterae, Butyribacterium methylotrophicum and Peptostreptococcus productus. Other suitable bacteria include those of the genus Moorella, including Moorellasp HUC22-1, (Sakai et al, Biotechnology 15 Letters 29: pp 1607-1612), and those of the genus Carboxydothermus (Svetlichny, V.A., Sokolova, T.G. et al (1991), Systematic and Applied Microbiology 14: 254-260). In addition, other anaerobic bacteria, particularly acetogenic bacteria, may be selected for use in the process of the invention by a person of skill in the art. It will also be appreciated that a mixed culture of two or more bacteria may be used in the process of 20 the present invention.

One exemplary micro-organism suitable for use in the present invention is Clostridium autoethanogenum. In one embodiment, the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identifying characteristics of the strain deposited at the German Resource Centre for Biological Material (DSMZ) under the 25 identifying deposit number 19630. In another embodiment, the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identifying characteristics of DSMZ deposit number DSMZ 10061. Another suitable micro-organism is Clostridium carboxydivorans, available commercially from DSMZ and having the identifying characteristics of 15243.

Culturing of the bacteria used in a method of the invention may be conducted using any number of processes known in the art for culturing and fermenting substrates 301400726:508231 NZ using anaerobic bacteria. Exemplary techniques are provided in the "Examples" section below. By way of further example, those processes generally described in the following articles using gaseous substrates for fermentation may be utilised: K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1991). Bioreactors for synthesis gas 5 fermentations resources. Conservation and Recycling, 5; 145-165; K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1991). Bioreactor design for synthesis gas fermentations. Fuel. 70.605-614; K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1992). Bioconversion of synthesis gas into liquid or gaseous fuels. Enzyme and Microbial Technology. 14; 602-608; J. L. Vega, G. M. Antorrena, E. C. Clausen and J. L. 10 Gaddy (1989). Study of Gaseous Substrate Fermentation: Carbon Monoxide Conversion to Acetate. 2. Continuous Culture. Biotech. Bioeng. 34. 6. 785-793; J. L. Vega, E. C. Clausen and J. L. Gaddy (1989). Study of gaseous substrate fermentations: Carbon monoxide conversion to acetate. 1. Batch culture. Biotechnology and Bioengineering. 34. 6. 774-784; and, J. L. Vega, E. C. Clausen and J. L. Gaddy (1990). Design of Bioreactors for Coal 15 Synthesis Gas Fermentations. Resources, Conservation and Recycling. 3.149-160. The fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CSTR), an immobilised cell reactor, a gas-lift reactor, a bubble column reactor (BCR) or a trickle bed reactor (TBR).

As described above, the carbon source for the fermentation reaction is a substrate 20 containing CO. In particular embodiments, the substrate comprising CO is gaseous. 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 25 manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production, methanol cracking 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 30 containing substrate, it may also be desirable to treat it to remove any undesired 301400726:508231 NZ impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known methods.

Alternatively, the CO-containing gaseous substrate may be sourced from the gasification of biomass. The process of gasification involves partial combustion of 5 biomass in a restricted supply of air or oxygen. The resultant gas typically comprises mainly CO and H2, with minimal volumes of C02, 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-10 containing gas suitable for use in the present invention.

The CO-containing gaseous substrate will ideally contain a significant proportion of CO, such as at least about 70% to about 95% CO by volume. However, the proportion of CO may be from about 15% to about 100% CO by volume, from about 20% to about 95% CO by volume, from about 40% to about 95% CO by volume, from about 60% to about 15 90% CO by volume and from 70% to about 90% CO by volume. In one embodiment, the gaseous substrate comprises approximately 80% CO by volume. It may be lower than 15%, such as 6%, if H2 and C02 are also present. For example/applications of the invention involving the use of biomass syngas may contain around 20% or less CO.

While it is not necessary for the gaseous substrate to contain any hydrogen, the presence 20 of hydrogen should not generally be detrimental to product formation in accordance with the methods of the invention. However, in certain embodiments of the invention, the gaseous substrate is substantially hydrogen free (less than 1%). The gaseous substrate may also contain some C02, such as about 1% to about 30% by volume, such as about 5% to about 10% C02.

In some embodiments of the invention, the substrate comprising CO is derived from carbon containing waste, for example, industrial waste gases or from the gasification of other wastes. As such, the methods of the invention represent effective processes for capturing carbon that would otherwise be exhausted into the environment. In certain embodiments, the methods provide improved processes for capturing CO and 30 optionally C02 by conversion into useful products such as alcohol. 301400726:508231 NZ Typically, the carbon monoxide will be added to the fermentation reaction in a gaseous state. However, the methods of the invention are not limited to addition of the substrate in this state. For example, the carbon monoxide can be provided in a liquid. For example, a liquid may be saturated with a carbon monoxide containing gas and that 5 liquid 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 for this purpose. It will be appreciated from consideration of this disclosure, that for growth of the bacteria 10 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 contains vitamins and minerals sufficient to permit growth of the microorganism 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 US 15 patent IMos. 5,173,429 and 5,593,886 and WO 02/08438 referred to above.

The fermentation should be carried out under appropriate conditions for the CO-to-ethanol fermentation to occur. In addition to maintaining the pH and redox potential within the ranges described above for a sufficient time to produce alcohol and/or allow conversion of acetate by-product to ethanol to occur, other reaction conditions that 20 should be considered and may be monitored and controlled include pressure, temperature, gas flow rate, liquid flow rate, agitation rate (if using a continuous stirred tank reactor), and inoculum level, and maximum product concentration (for example, to avoid product inhibition).

The optimum reaction conditions will depend partly on the particular micro-25 organism used. However, in general, it may be beneficial to perform the fermentation 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 30 bioreactor divided by the input gas flow rate) can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure. 301400726:508231 NZ Also, because 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 5 equipment. According to examples given in US patent 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 10 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. 15 It is also desirable that the rate of introduction of the CO-containing substrate is such as to ensure that the concentration of CO in the liquid medium does not become limiting. This is because a consequence of CO-limited conditions may be that the alcohol product is consumed by the culture.

Alcohol recovery In certain embodiments, a fermentation process according to the present invention described above will result in a fermentation broth comprising one or more alcohols, for example, ethanol, as well as bacterial cells, in the liquid nutrient medium.

In one embodiment of the invention, ethanol is the preferred desired end product of the 25 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 30 be obtained through the use of molecular sieve ethanol dehydration technology, which is also well known in the art. 301400726:508231 NZ 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 5 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 10 fermentation.

Modulation of media conditions In particular embodiments, the process of the present invention is carried out in a single bioreactor, and the pH and/or redox potential of the liquid nutrient medium are 15 modulated to allow the metabolic activity of the bacterial culture that uses CO-containing gas to be transitioned between various states. As noted previously, continuous or intermittent monitoring of and maintenance of pH and/or ORP in desired ranges results in improved alcohol production. Therefore, in certain embodiments, the process involves transitioning the bacterial culture between a growth phase where microbial growth 20 and/or acetate production are promoted and an alcohol production phase, where alcohol is produced from acetate and/or a substrate comprising CO. In a particular embodiment, the culture is transitioned between the following states: (!) Growth and acetate production. The pH of the liquid nutrient medium is, in one embodiment, maintained in the range of about 5.0 to about 5.7 and the redox 25 potential at about -450 mV or higher, for example, about -400 mV to about -250 mV. (2) Ethanol production from acetate and/or CO-containing gas. The pH of the liquid nutrient medium is, in one embodiment, maintained in the range of about 5.8 to about 6.5, and the redox potential at about -450 mV or lower. The medium is 30 maintained under these conditions for sufficient time to allow the bacteria to produce ethanol and optionally acetate in at least about a 1:1 ratio favouring 301400726:508231 NZ ethanol, and/or to convert acetate produced in the first state (1) to be converted to ethanol.

As described above, the pH and/or redox potential of the liquid nutrient medium can be adjusted as required using pH-adjusting agents, oxidising agents and reducing agents, 5 to transition the metabolic activity of the bacterial culture between the above states.

Two-stage fermentation In another aspect, the present invention relates to a process of producing one or more alcohols, for example, ethanol, by anaerobic bacterial fermentation of acetate and a 10 gaseous substrate comprising CO. The process involves culturing in a bioreactor one or more strains of anaerobic bacteria in a liquid nutrient medium and supplying acetate from an external source, and the gaseous substrate, to the bioreactor.

The pH and redox potential of the liquid nutrient medium are maintained at levels that allow fermentation of both the added acetate and the gaseous substrate by the 15 bacteria to produce one or more alcohols. In various embodiments, the pH and redox potential are maintained in a range of about 5.7 to about 7.0 and equal to or below about -450 mV, respectively, to allow the bacteria to convert the added acetate (as well as any acetate produced in the CO to alcohol fermentation) to alcohol.

This process may conveniently involve two fermentations carried out in separate 20 bioreactors. In such embodiments, the process will include a first-stage fermentation dedicated to acetate production from a suitable carbon and energy source. In various embodiments, the first-stage fermentation involves conversion of a CO-containing gaseous substrate to acetate, using anaerobic acetogenic bacteria selected from, for example, those described above as being suitable for the CO to alcohol fermentation. The 25 media conditions for the first stage fermentation should be optimised for bacterial growth and acetate production. In this embodiment, the pH of the liquid nutrient medium is maintained in the range of about 5.0 to about 5.7 and the redox potential at about -450 mV or higher, for example, from about -400 mV to about -250 mV.

The acetate product is recovered from the first stage fermentation and introduced, 30 together with a CO-containing gas, to a second bioreactor, under conditions optimised for ethanol production from acetate and/or CO-containing gas by one or more microbes 301400726:508231 NZ suitable for converting acetate to alcohol. The pH of the liquid in the second fermenter is maintained above the optimum growth pH and the ORP is maintained at less than about -450mV. In one particular embodiment, the pH of the liquid nutrient medium is maintained in the range of from about 5.8 to about 6.5 and the redox potential at about -5 450 mV or lower.

Acetate recovery Acetate may be continuously recovered from the first stage fermentation bioreactor using methods known in the art. These methods include but are not restricted 10 to distillation, the use of permeable acid and/or acetate selective membranes, and/or the use of adsorption columns.

For example, an adsorption system involving an activated charcoal filter may be used. In this embodiment of the invention, the microbial cells are first removed from the fermentation broth using a suitable separation unit. Numerous filtration-based methods 15 of generating a cell free fermentation broth for product recovery are known in the art. The cell free acetate-containing permeate is then passed through a column containing activated charcoal to adsorb the acetate. Acetate in the acid form (acetic acid) rather than the salt (acetate) form is more readily adsorbed by activated charcoal. The pH of the fermentation broth (medium) is therefore typically reduced to less than about 3 before it 20 is passed through the activated charcoal column, to convert the majority of the acetate to the acetic acid form.

Acetic acid adsorbed to the activated charcoal may be recovered by elution using methods known in the art. For example, ethanol may be used to elute the bound acetate. Because the boiling point of ethanol is 78.8 9C and that of acetic acid is 107 9C, ethanol 25 and acetate can readily be separated from each other using a volatility-based method such as distillation.

In certain embodiments of the invention, acetate is 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 30 filtration), and recovering acetate from the broth. The acetate may be recovered by adsorption on activated charcoal, using the methods described above. The separated 301400726:508231 NZ microbial cells can then be returned to the fermentation bioreactor. The cell free permeate remaining after the acetate has been removed is also typically 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 5 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 is re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor. The invention will now be described in more detail with reference to the following non-limiting experimental section. 0 EXAMPLES Materials and Methods Media: * Combine NaH2P04 (13.2g) and Na2HP02.7H20 (l.lg) in H20 (1L).

Media Component Concentration per 1.0L of Media (LM23) Concentration per 1.0L of | Media (LM33) | | MgCI2.6H20 0.5 g | 0.5g 1 1 NaCI 0.2g 0-2g I CaCI2 0.2 g 0.2g 1 lOOmM sodium I phosphate buffer (pH 6.°)* 160 ml j j NaH2P04 - 2.04g 1 NH4CI 0.6 g 2.5g | 85% H3PO4 0.05 ml I 1 KCI 0.15g 0.15g f | Composite trace metal 1 solution (LS06) mL j i 10m L I Composite B vitamin Solution (LS03) mL 10m L | | Resazurin (1000 mg/L stock) 1 mL 2mL | i FeCls 0.0025g l °-°ie J Cysteine HCI monohydrate 0.75g 0.5g Agarose (optional) 15g 15g Distilled water | To 1 litre To 1 litre 301400726:508231 NZ Composite B vitamin | Solution (LS03) | per L of Stock Composite trace metal ] solution (LS06) [ per L of 1 stock. 1 Biotin ( .0 mg Nitrilotriacetic Acid l-5g I Folic acid | .0 mg MgS04.7H20 3.0g | Pyridoxine hydrochloride .0 mg MnS04.H20 0.5g | Thiamine. HCI 50.0 mg NaCl l.Og | Riboflavin 50.0 mg FeS04.7H20 O.lg Nicotinic acid 50.0 mg Fe(S04)2(NH4)2.6H20 j O 00 era jMWg Calcium D-(*)-pantothenate 50.0 mg CoCl2. 6H20 0.2g Vitamin B12 50.0 mg ZnS04.7H20 0.2g | p-Aminobenzoic acid 50.0 mg CuCI2. 2H20 JM*. : Thioctic acid 50.0 mg AIK(S04)2.12H20 0.02g Distilled water To 1 Litre H3BO3 0.30g NaMo04.2H20 0.03g \ Na2Se03 0.02g 1 NiCl2.6H20 1 0.02g Na2W04.6H20 ~~| 0 02g Distilled water To 1 1 Litre > Media was prepared at pH 5.5 as follows. All ingredients with the exception of Cysteine-HCI were mixed in either 400ml or 800ml distilled water. This solution was 5 made anaerobic by heating to boiling and allowing it to cool to room temperature under a constant flow of 95% CO, 5% C02 gas. Once cool, the Cysteine-HCI was added and the pH of the solution adjusted to 5.5 before making the volume up to 1000ml; anaerobicity was maintained throughout the experiments.

Bacteria: Clostridium autoethanogenum were obtained from the German Resource Centre for Biological Material (DSMZ). The accession number given to the bacteria is DSMZ 10061. Alternatively, the Clostridium autoethanogenum used is that deposited at the German Resource Centre for Biological Material (DSMZ) and allocated the accession 301400726:508231 NZ number 19630. Clostridium carboxydivorans with the accession number 15243 was also obtained from DSMZ.

Fermentation in CSTR batch reactor: The media for CSTR reactor experiments was prepared as described above. The media solutions were introduced into the fermenters and optionally sparged with the respective CO containing gases from the start of the experiment, or after a predetermined interval. During these experiments, the pH was adjusted and/or maintained by a controller through the automated addition of buffers (0.5 M NaOH or 2N 10 H2SO4). An actively growing Clostridium autoethanogenum culture was inoculated into the reactor at a level of 5 % or 7.5% (v/v). The temperature of the reactor was maintained at 37°C and agitation rate was 400 rpm.

Sampling and analytical procedures: Media samples were taken from the CSTR reactor at intervals over periods up to days. Each time the media was sampled care was taken to ensure that no gas was allowed to enter into or escape from the reactor.

Cell Density: To determine the cell density in these experiments, the absorbance of the samples was measured at 600nm (spectrophotometer) and the dry mass determined via calculation according to published procedures. The level of metabolites was characterized using High Performance Liquid Chromatography (HPLC) and in some cases Gas Chromatography (GC).

HPLC: HPLC System Agilent 1100 Series. Mobile Phase: 0.0025N Sulphuric Acid. Flow and pressure: 0.800 mL/min. Column: Alltech IOA; Catalog# 9648,150x 6.5 mm, particle size 5 nm. Temperature of column: 60°C. Detector: Refractive Index. Temperature of 30 detector: 45°C. 301400726:508231 NZ Method for sample preparation: 400 [iL of sample and 50 |iL of 0.15M ZnS04 and 50 nL of 0.15M Ba(OH)2 are loaded into an Eppendorf tube. The tubes are centrifuged for 10 min. at 12,000rpm, 4°C. 200 nL of the supernatant are transferred into an HPLC vial, and 5^L are injected into the 5 HPLC instrument.

Gas Chromatography: Gas Chromatograph HP 5890 series II utilizing a Flame Ionization Detector.

Capillary GC Column: EC1000- Alltech EC1000 30m x 0.25mm x 0.25um. The Gas 10 Chromatograph was operated in Split mode with a total flow of hydrogen of 50 mL/min with 5 mL purge flow (1:10 split), a column head pressure of 10 PIS resulting in a linear velocity of 45 cm/sec. The temperature program was initiated at 60°C, held for 1 minute then ramped to 215°C at 30°C per minute, then held for 2 minutes. Injector temperature was 210°C and the detector temperature was 225°C.

Method for sample preparation: 500 nL sample is centrifuged for 10 min at 12,000rpm, 4°C. 100 nL of the supernatant is transferred into an GC vial containing 200 \il water and 100 |iL of internal standard spiking solution (10 g/L propan-1-ol, 5 g/L iso-butyric acid, 135 mM hydrochloric 20 acid). 1 piL of the solution is injected into the GC instrument.

Intracellular redox potential of Bacteria: RedoxSensor green was used to measure intracellular reductase activity, providing a measure of intracellular redox potential. The BacLight™ RedoxSensor™ Green Vitality 25 Kit was used (Invitrogen, catalogue number 349554) in accordance with the manufacturers instructions.

Redox staining procedure: 1. Cell counts were taken and samples diluted in medium to approx. 1 x 106 cells/mL, 30 with 500 |iL placed in each tube. 301400726:508231 NZ - 2. 0.5 piL of each stain (RedoxSensor™ Green and PI (Propidium Iodide)) was added to each tube, mixed and incubated for 10 min. at room temperature in darkness, before analysis on a flow cytometer.

Flow Cytometry: Flow cytometric analysis of RedoxSensor green and PI stained bacterial suspensions was performed using a Coulter Epics Elite flow cytometer (Epics Division of Coulter Corporation, Hialeah, FL, USA), fitted with a Uniphase 2201-20SLE air cooled 588 nm Argon laser. RedoxSensor green fluorescence was measured on PMT2 (525 nm band pass filter) and PI fluorescence was measured on PMT3 (610 nm band pass filter). 10 Compensation settings comprised of 22% of the PMT2 signal subtracted from the PMT3 signal and 2% of the PMT3 signal subtracted from the PMT2 signal. Expo32 vl.2B Cytometer software (Applied Cytometry Systems, Sacramento, CA, USA) was used to acquire list mode files generated from samples run on the flow cytometer. Analysis of list mode files was achieved using FlowJo v8.5.1 (Macintosh OSX) software (Tree Star 15 Incorporated, Ashland, OR, USA).

Example 1: Effect ofpH and oxidation and reduction potential (ORP) levels on ethanol production using CO containing gases: This example describes testing to demonstrate the kinetics of ethanol production 20 by Clostridium autoethanogenum culture in a fermenter at different pH values and ORP levels with a continuous feed of CO as the sole source of carbon and energy.

Procedure 1. 2 L media of anaerobic LM23 fermentation media were inoculated with an actively 25 growing Clostridium autoethanogenum culture (DSMZ 10061) at a level of 7.5% (v/v). A continuous flow of 95% CO and 5% CO2 gas was introduced into the bottom of the fermenter vessel through a diffusing sparger at a volumetric flow rate of 5 ml/minute. The initial pH of the fermenter was set to 5.5. 2. The culture was allowed to grow to a bacterial cell density of 0.1 g of dry cell mass 30 per litre of media, at which point the pH and ORP of the culture were adjusted and ethanol production monitored. 301400726:508231 NZ 3. To maintain the ORP of the media at a consistently low level, methyl viologen was added to the fermentation media to a final concentration of lOmg/L. 4. The level of ethanol and acetate in the media were monitored over time as the fermentation media was maintained at three different pH values (6.5, 5.8 and 4.5) and an ORP level at or below -450mV.

. Each pH value was maintained and monitored for at least a 72 hour period.

Results Results for ethanol production rates achieved by the culture at each pH value are presented in the table below. At pH values of 6.5 and 5.8, ethanol production was found to coincide with acetate degradation at the rates described. Little or no acetate degradation was observed during ethanol production at pH 4.5.

PH Specific ethanol production rate (g/g/day) Specific acetate 1 degradation rate (g/g/day) 6.5 16.0 21.3 .8 18.0 .5 \ 4.5 j .0 0.0 Other tests by the inventors have shown that the optimum pH for microbial growth and acetate production is 5-5.7. The above results reveal that a C. autoethanogenum culture using CO containing gas as the sole source of carbon and energy is able to produce ethanol at elevated pH (pH 5.8 to 6.5) in a media containing acetate with an ORP less than -450 mV. By increasing the pH, the bacteria produced ethanol in association with acetate degradation at a specific ethanol production rate of ~15 g/g/day. Acetate was simultaneously degraded at a specific rate of ~20 g/g /day.

However, at a low pH (pH 4.5) and in a culture containing acetate, the bacteria produced ethanol without the associated acetate degradation observed at elevated pH levels. Under these conditions, the specific ethanol production rate was ~5 g/g/day. 301400726:508231 NZ Example 2: Monitoring intracellular redox potential of a microbial culture Example 2A: Monitoring intracellular redox potential and cell vitality in a batch microbial culture.

Procedure 1. A preparation of LM23 fermentation media at pH 5.5 was prepared as previously described. 2. Under a continuous gas flow of 95%CO and 5%C02; 50ml of LM23 media was dispensed into 250 ml serum bottles. All bottles were stopped with gas impermeable butyl rubber septa and crimp sealed before autoclaving at 121°C for 20 minutes. 3. Once cool, serum bottles were divided into two groups. In one group, methyl viologen was introduced into the media to a final concentration of 40|ig/ml. The pH of all bottles was checked and adjusted to 5.5 where necessary. All bottles were inoculated with 1 ml of a Clostridium autoethanogenum culture that was actively growing on 95%CO and 5%C02- Headspace gas was pressurised to 35 psig with 95%CO and 5%C02. An initial media sample was taken aseptically from each bottle. Bottles were placed on a shaking incubator at 37°C. 4. Media samples were taken at regular intervals over a 15 day period. Each time the media was sampled, the headspace of each bottle was flushed three times with 95%C0 and 5%C02 gas before pressurising with this gas to 35 psig.

. Samples were prepared from each serum bottle and assayed for: Optical density (150 piL at 620 nm), Ethanol levels, Acetate levels, Redox staining for flow cytometry, and Live/dead staining for flow cytometry.

Clostridium autoethanogenum (DSMZ 10061) was grown in serum bottles - control vs. methyl viologen (40 jig/mL). Samples were taken every day, stained with RS and analysed via flow cytometry. Cells were categorised based on being RS positive (green staining, high reductase activity), RS negative (red staining, low reductase activity) or neither (unknown reductase activity). A measure was then taken of the proportion of cells with a high redox potential to those with a low redox potential. 301400728:508231 NZ Results Referring to Figure 2, the RS positive cell proportion increased as the culture progressed, reaching a peak between day 4 and 8. From day 9, the proportion of RS positive cells declined, and by day 14, very few RS positive cells remained. In the methyl 5 viologen treated culture, a peak in the proportion of RS positive cells was reached at day 4, and declined sooner than in the control.

These results show that an increase in reductase activity is associated with an actively growing C. autoethanogenum culture, with a decrease in reductase activity observed in ageing cultures undergoing solventogenesis. Thus, for the data shown in 10 Figure 2, it is possible to determine that environmental parameters for the media should preferably be switched from those suitable for the acetogenic phase to those suitable for the solventogenic phase sometime around or after day 4. After day 8, the percentage of RS positive cells began to drop, indicating cells were dying. This indication, in an industrial process, can be used as a cue to take action to revert to conditions suitable for 15 the acetogenic phase and/or to add further cells and/or to add further nutrients to the fermentation tank in accordance with the methods of the invention. This action may coincide with a portion of the contents being removed. Alternatively, this indication of cell death (or other indicator of cell death) may be used in accordance with the methods of the invention as a cue for replacing the contents of the fermentation tank, which is 20 particularly applicable to batch processing. When cells are added, they may or may not be of the same species of micro-organism as those previously existing inside the fermentation tank without departing from the scope of the invention. For example, in a two-stage process performed inside a single fermentation tank, it may be desirable in many embodiments for the cell lines to be different.

The RS green reagent provides a measure of the intracellular redox potential of in a microbial population and thus provides an indication of the status or viability of a microbial population within a bioreactor. When a change in the intracellular redox potential is detected, fermentation parameters or conditions within the bioreactor can be altered to improve alcohol producing conditions. 301400726:508231 NZ 34 Example 2B: Monitoring the intracellular redox potential and population vitality of microbes taken from a continuous culture configured for stable growth and acetate production.

Procedure 1. Continuous cultures of C. autoethanogenum (DSMZ 10061) were initiated by inoculation of 5 L of anaerobic LM33 medium (pH of 5.5 and temperature of 37°C) from serum bottle cultures, which in turn were inoculated from glycerol storage stocks. A gas blend of 70% CO, 15% C02,1% H2 and 14% N2 gas was sparged into the base of the culture at a rate of 64 mL/min. 2. Cultures were then allowed to grow and increase in biomass for 5 days as batch cultures, before initiation of a continuous culture regime. 3. Fresh anaerobic LM33 medium was then continuously introduced to the reactor at a rate of 3 mL/min, with the culture intermittently removed based on a level probe set to a volume of 5 L.

Results Once stabilized, the continuous cultures of C. autoethanogenum maintained a dry weight biomass ranging from 0.6-0.8 g/L, with resident acetate levels ranging from 6-8 g/L over the course of 15 days. Alcohol production was either not observed, or led to only very low levels. The proportion of viable cells to total cells in the continuous cultures was 20 consistently in the range of 80-90% (assayed using flow cytometric analysis of RedoxSensor green and propidium iodide dye fluorescence).

During active growth, as seen in the continuous cultures, C. autoethanogenum produced high levels of acetic acid, coupled to very low levels of ethanol. Biomass production does not seem to be compatible with the stimulation of ethanol production. 25 Flow cytometric observation of C. autoethanogenum cells stained with RedoxSensor green and propidium iodide give useful insights into the population dynamics of the continuous cultures. In addition to measuring the proportion of viable (RedoxSensor green positive, propidium iodide negative) versus non-viable (RedoxSensor green negative, propidium iodide positive) cells, an intermediate population is also evident. This 30 dual positively stained cell population is minimal in continuous cultures (ranging from 3- 301400726:508231 NZ 9% of the total culture), but makes up a more substantial proportion in cultures undergoing solventogenesis (e.g., see example 4).

During this active growth phase, fermentation parameters and/or bioreactor conditions can be altered or maintained to promote microbial growth and/or acetate 5 production.

Example 2C: Monitoring intracellular redox potential and cell vitality in a batch microbial culture at pH 5.5.

Procedure 1. A batch culture of C. autoethanogenum (DSMZ 10061) was initiated by inoculation of 1 L of anaerobic LM33 medium (pH of 5.5 and temperature of 37°C) from an actively growing continuous culture. A gas blend of 70% CO, 15% C02,1% H2 and 14% N2 gas was sparged into the base of the culture at a rate of 64 mL/min. 2. The culture was then allowed to grow and increase in biomass, with periodic 15 sampling, but no further nutrient addition.

The ethanol to acetic acid production ratio over the course of a 24 h period in a batch culture of C. autoethanogenum (grown at pH 5.5 on a gas blend of 70% CO, 15% CO2,1% H2 and 14% N2 gas) was approximately 1:6. During this production window, the redox potential of the culture ranged between -446 and -430 mV. The internal redox potential 20 of the cells tracked over time using the RedoxSensor green reagent, revealed viability of between 90% (day 3) and 77% (day 7) of the culture. This proportion decreased sharply to only 18% by day 10, suggesting an environment not suitable for continued culture growth due to the closed nature of this batch culture.

Again, during this active growth phase, fermentation parameters and/or bioreactor 25 conditions can be altered or maintained to promote microbial growth and/or acetate production.

Example 3: Effect of oxidation and reduction potential (ORP) levels on ethanol production using CO containing gases This example describes ethanol production at different ORP levels and controlled pH (pH at 6.2). 301400726:508231 NZ Procedure 1. 1 L media of anaerobic LM33 fermentation media in a 1 litre CSTR was inoculated with an actively growing Clostridium autoethanogenum culture (DSMZ 19630) at a level of 5% (v/v). A continuous flow of 95%CO and 5%C02 gas was introduced at the bottom of the fermenter vessel through a diffusing sparger at a volumetric flow rate of 19ml/minutes. The initial pH of the fermenter was adjusted to 5.5. 2. The culture was allowed to grow to a bacterial cell density of 0.35 g of dry cell mass per litre of media, at which point the pH of the culture were adjusted to 6.2. 3. Another culture was set up as above. The culture was allowed to grow to a bacterial cell density of 0.3 g of dry cell mass per litre of media, at which point the pH of the culture was adjusted to 6.2. 4. To achieve the required ORP level in each culture, 0.5 g/L L-cysteine.H20 was added to the first culture and lOmg/L methyl viologen was added to the second culture.

. The viability of cultures was monitored by bacterial oxidation-reduction activity. Baclight redoxsensor green viability for flow cytometry kit purchased from Invitrogen was used to indicate intracellular redox potential and hence cell viability.

Results The addition of L-cysteine.HCl monohydrate to the first culture maintained the ORP of the fermentation medium at -500 mV. The addition of methyl viologen to the second culture maintained the ORP of the fermentation medium at -600 mV. Results for acetate degradation and ethanol production rates achieved by each culture are presented in the table below.

ORP (mV) Acetate degradation (mM/day) Ethanol production (mM/day) -500 16.7 34 -600 0 0 301400726:508231 NZ Prior to the addition of the cysteine or methyl viologen supplements, the microbial cell viability was approximately 72%. On addition of cysteine, the ORP reduced to approximately -500mV, and the viability of culture decreased to approximately 32%. The proportion of intermediate cells increased from approximately 6% to 16% on addition 5 of cysteine.

After adding methyl viologen to the culture, approximately 5% of population was considered viable.

The results reveal the Clostridium autoethanogenum culture using CO gas as the sole source of carbon and energy became substantially inactive at the level of -600mV. At 10 the level of-500 mV and pH 6.2, acetate was consumed, while ethanol levels increased, indicating conversion of at least a portion of acetate into ethanol.

Determination of the intracellular redox potential showed there was a significant drop in the proportion of viable cells and intermediate cells when the ORP was reduced to -500mV and ethanol was produced. Thus, in accordance with the methods of the 15 invention, the change in viable and/or intermediate cells indicates the overall status of the culture with respect to whether ethanol is produced and/or acetate is consumed.

Example 4: Effect ofpH on ethanol production using CO containing gas This example describes ethanol production at different pH values in the absence 20 of added reducing agents. Figure 3 provides a summary of the concentrations of acetate, ethanol and biomass over a 2 week period. Example 4A outlines the first 4 days of the fermentation where the pH was maintained at 5.6, then increased to pH 5.9 for 1 day. Example 4B outlines the next 9 days, wherein the pH was maintained at 6-6.1.

Procedure 1. 1 L media of anaerobic LM33 fermentation media in a 1 Litre CSTR was inoculated with an actively growing Clostridium autoethanogenum culture (DSMZ 19630) at a level of 5% (v/v). A continuous flow of 70%CO and 15 %C021% H214% N2 gas was introduced at the bottom of the fermenter vessel through a diffusing sparger at a 30 volumetric flow rate of 19ml/minutes. The initial pH of the fermenter was set to .5 301400726:508231 NZ 38 2. For the majority of the experiment, the acetic acid concentration of the culture was maintained below 4 g/L by a cell recycle and media exchange system. The cells were passed through a cross flow membrane Viva 200, the filtrate was collected and the cells were returned to the reactor vessel. The filtrate was replaced with fresh media to ensure the medium volume inside the reactor remained constant. 3. The culture was operated continuously for at least 14 days. The cell recycle system removed 1-1.5L of liquid nutrient media every 1-2 days without removing bacteria from the bioreactor. The removed media was replaced with fresh media, to maintain constant volume.

Example 4A The pH was maintained at approximately 5.6 for 3 days, during which the ORP fluctuated between -400 and -430mV. After 3 days, the pH was adjusted to approximately 5.9, and the ORP decreased to approximately -470mV. pH Acetate (mM/day) Ethanol (mM/day) Acetate:Ethanol ratio J .6 (3 days) .3 9.8 1.56 :1 | .9 (1 day) r\. <- .A,-., -5.17 I j 31.3 tm 1 1 These results show that, at pH 5.6, acetate and ethanol are produced simultaneously, with an excess of acetate. Referring to Figure 3, this stage of the fermentation is also associated with a period of microbial growth. When the pH is increased and the ORP reduced to approximately -470mV, the rate of alcohol production increases, while some acetate is consumed.

Example 4B The same culture was adjusted to and maintained at pH 6 to 6.1 by adding buffers (base and acid). At the pH 6 to 6.1, the culture maintained ORP from -480 to -487 mV. 25 Results for acetate and ethanol production rates achieved by the culture at different pH values are shown in the table below. 301400726:508231 NZ 39- PH Acetate (mM/day) Ethanol (mM/day) Acetate:Ethanol ratio 6-6.1 (9 days) 19.75 28.9 | 1:1.46 wot These results show that the molar ethanol to acetate ratio can be maintained at higher than 1:1 (favouring ethanol) for an extended period at elevated pH (6-6.1) and ORP (-480 to -487mV). While there is an overall increase in acetate, the rate of ethanol 5 production is significantly higher, indicating that at least a portion of acetate formed in the fermentation is converted to ethanol. In this method of contiguous pH and ORP monitoring and maintenance within a desired range, the alcohol product is produced in excess of the less desirable acid by-product.

The intracellular redox potential was also monitored. At pH 5.6 (see example 4A), 10 the viable population was 79%, and the intermediate population was 10.6%. However, when the pH was increased, the viable population was 44% (average) and the intermediate population was 20% (average). Under these conditions alcohol is the major product and fermentation parameters and/or bioreactor conditions can be altered or maintained to promote alcohol production for an extended period.

Example 5: Effect ofpH on ethanol production using CO containing gas in batch culture This example describes ethanol production at different pH values without adding reducing agents in batch culture.

Procedure 1. 10 L media of anaerobic LM33 fermentation media (media recipe described above) in a 1 Litre CSTR was inoculated with an actively growing Clostridium autoethanogenum culture at a level of 5% (v/v). The temperature of fermenter was set at 37°C and agitation rate was 400 rpm. A continuous flow of 95%CO and 5 %C02 gas was introduced at the bottom of the fermenter vessel through a 25 diffusing sparger at a volumetric flow rate of 19ml/minutes. The initial pH of the fermenter was set to 5.5 2. Then the pH of media was adjusted to 7 and then decreased to 6.5, 6 and 5.7 every two days thereafter by adding buffers. 301400726:508231 NZ Results When the pH of media was adjusted from 5.5 to 7, the culture became inactive with respect to ethanol production, and the ORP ranged from -550 to -600 mV. When the pH was decreased from 7 to 6.5, ethanol was produced at 26 mmol per gram of cell dry 5 weight per day and acetate degradation rate was 13 mmol per gram of cell dry weight per day. The ethanol production continued at the same rate when pH was dropped from 6.5 to 6. ORP was between -510 to -550 mV.

A combination of low ORP (-550mV to -600mV) and a relatively high pH (pH 7) appeared to inhibit ethanol production in the culture substantially. However, when the 10 pH was reduced (to between pH 6 to 6.5), the ORP increased (-510mV to -550mV), and acetic acid is converted to ethanol, leading to increased ethanol production.

Example 6: Effect of increasing pH on ethanol production using CO containing gas with different microorganism Materials 1. Actively growing culture of Clostridium carboxidivorans cultivated and maintained using gases as its sole source of carbon and energy (70% CO 1% H2 15 %C02 14% N2 gas stream) Procedure 1. 1.5 L media of anaerobic LM33 fermentation media in a 2 litre CSTR was inoculated with an actively growing Clostridium carboxidivorans culture at a level of 5% (v/v). The temperature of fermenter was set at 37°C and agitation rate was 400 rpm. A continuous flow of 70%CO and 15 %C02 1% H2 14% N2 gas was introduced at the bottom of the fermenter vessel through a diffusing sparger at a 25 volumetric flow rate of 19ml/minutes. The initial pH of the fermenter was adjusted to 6. 2. The culture was allowed to grow to an optical density of 0.9 at 600 nm, and then the pH was adjusted to 6.5 and 7. 3. The samples were analysed with HPLC and Gas Chromatography. 301400726:508231 NZ Results Results of acetic acid and ethanol production of culture are presented in the table below. • '-s Jti. fi*. ,»iAs l » m- ■% >*. -f t .-%• ,* „ - * > Jiv t pH ORP (mV) Acetic acid production (mM/day) Ethanol Production j (mM/day) 1 6 -480 12.5 0 t 6.5 -528 16.7 4 7 -573. -1 | The optimum pH for growth of a Clostridium carboxidivorans culture and associated acetate production is generally 6-6.5. The above results show that, at pH 6, acetate is produced without the concomitant production of alcohol. At pH 6.5, acetic acid and ethanol were produced. At pH 7, acetate was consumed while ethanol was produced. This indicates at elevated pH (pH 7), at least a portion of the acetate in a fermentation 10 broth is converted into ethanol. Clostridium carboxidivorans is known to produce acetic acid, butyric acid, ethanol and butanol.

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 15 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 20 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" and the like are to be read expansively and without limitation.

Titles, headings, or the like are provided to enhance the reader's comprehension 25 of this document, and should not be read as limiting the scope of the present invention. :508231 NZ 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 5 suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims (16)

301927972:508231 NZ Received at IPONZ on 27.01.2012 -43- What we claim is:

1. A method of capturing carbon by microbial fermentation, the method comprising; (a) culturing in a bioreactor, an isolate of the bacterium Clostridium autoethanogenum in a liquid nutrient medium; 5 (b) anaerobically fermenting a substrate comprising CO in the bioreactor of step (a) to produce one or more alcohols; (c) monitoring a pH of the liquid nutrient medium to detect changes in the pH of the liquid nutrient medium; (d) if a change in the pH is detected, altering the pH of the liquid nutrient medium such 10 that the pH is maintained within an optimum range for alcohol production, wherein the optimum range for alcohol production comprises substantially pH 4.5 to substantially pH 6.5.

2. A method according to claim 1, wherein the method further comprises; (a) monitoring a redox potential of the liquid nutrient medium to detect changes in the 15 redox potential of the liquid nutrient medium; and (b) if a change in the redox potential is detected, altering the redox potential of the liquid nutrient medium such that the redox potential is maintained within an optimum range for alcohol production.

3. A method according to any one of claims 1 or 2, wherein the bacterium is Clostridium 20 autoethanogenum strain deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM 10061.

4. A method according to any one of claims 1 to 3 wherein the pH is maintained at a range of about 5.7 to 6.5.

5. A method according to any one of claims 2 to 4 wherein the optimum range for redox 25 potential is about -450 to about -550mV.

6. A method according to anyone of claims 1 to 4 wherein the pH is controlled or adjusted by adding acid(s) and/or base(s).

7. A method according to any one of claims 2 to 6 wherein the redox potential is controlled by one or more of the following; 30 (a) adding one or more oxidising agents; (b) adding one or more reducing agents; (c) adjusting pH. TIS5O0231NZPR 302455503 RECEIVED at IPONZ on 22 June 2012 44-

8. A method according to claim 7, wherein the reducing agent is selected from the group consisting of methyl viologen or cysteine.

9. A method according to claim 2 wherein the step of monitoring the redox potential of the liquid nutrient medium comprises removing a sample of the bacterium from the 5 bioreactor, wherein the fermentation process, is at least in part, performed in the bioreactor.

10. A method according to any one of claims 1 to 9 wherein the substrate comprising CO is a gaseous substrate.

11. A method according to claim 10 wherein the substrate comprising CO, comprises at least 10 about 15% to about 100% CO by volume.

12. A method according to claim 10 or 11 wherein the substrate comprising CO comprises at least about 70% CO to about 95% CO.

13. A method according to any one of claims 10 to 12 wherein the substrate comprises a gas obtained from a steel mill. 15

14. A method according to any one of claims 1 to 10 wherein the alcohol is ethanol.

15. An anaerobic fermentation system when used for carrying out the method of any one of the preceding claims, the system comprising: (a) A bioreactor containing an isolate of the bacterium Clostridium autoethanogenum in a liquid nutrient medium; 20 (b) means for detecting a change in the pH of the liquid nutrient medium; and (c) means for controlling the pH of the fermentation process based on whether a change is detected in the pH by the means for detecting.

16. A fermentation system according to claim 15, further comprising: (d) means for detecting a change in the intracellular redox potential of a microorganism 25 used in the fermentation process; and (e) means for controlling at least one parameter of the fermentation process based on whether a change is detected in the intracellular redox potential by the means for detecting.