BRPI1008162B1 - METHOD FOR IMPROVING THE EFFICIENCY OF MICROBIAL FERMENTATION OF A SUBSTRATE COMPRISING CARBON MONOXIDE - Google Patents

Abstract

METHODS FOR IMPROVING THE EFFICIENCY OF MICROBIAL FERMENTATION OF A SUBSTRATE COMPRISING CO AND OF THE PRODUCTION OF ONE OR MORE ACIDS AND/OR ALCOHOLS BY SUCH FERMENTATION, AS WELL AS A SYSTEM FOR SUCH FERMENTATION. The present invention relates to the production of products such as alcohols and acids by microbial fermentation, particularly microbial fermentation of substrates comprising CO. More particularly it relates to methods and systems for improving the efficiency of products by microbial fermentation. In particular embodiments, the invention provides a method for optimizing the production of desired products including the step of checking the proportion of CO converted to CO2.

Description

FIELD OF INVENTION

The present invention generally relates to methods for increasing the efficiency of microbial growth and product production by microbial fermentation on gaseous substrates. More particularly the invention relates to processes for producing alcohols, particularly ethanol, by microbial fermentation of gases containing carbon monoxide. In particular embodiments, the invention relates to methods for determining the total net conversion of CO to products during microbial fermentation.

BACKGROUND OF THE INVENTION

Ethanol is rapidly becoming an important hydrogen-rich liquid transportation fuel worldwide. Global ethanol consumption in 2005 was an estimated 12.2 billion liters. The global market for the fuel ethanol industry is also predicted to continue growing sharply in the future, due to increased interest in ethanol in Europe, Japan, the United States, and several developing nations.

For example, in the US, ethanol is used to produce E10, a 10% blend of ethanol in gasoline. In E10 blends, the ethanol component acts as an oxygenating agent, improving combustion efficiency and reducing the production of air pollutants. In Brazil, ethanol meets approximately 30% of transportation fuel demand, both as an oxygenating agent blended into gasoline and as a pure fuel on its own. Furthermore, in Europe, environmental concerns surrounding the consequences of greenhouse gas (GHG) emissions have prompted the European Union (EU) to set a mandatory target for member countries to consume sustainable transportation fuels such as biomass-derived ethanol.

The vast majority of fuel ethanol is produced through traditional yeast-based fermentation processes that use crop-derived carbohydrates, such as sucrose extracted from sugarcane or starch extracted from grain crops, as the primary carbon source. However, the cost of these carbohydrate feedstocks is influenced by their value as human food or animal feed, and growing sucrose- or starch-producing crops for ethanol production is not economically sustainable in all geographies. Therefore, it is of interest to develop technologies to convert lower-cost and/or more abundant carbon resources into fuel ethanol.

CO is a free, energy-rich byproduct of the incomplete combustion of organic materials such as coal or oil or petroleum 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 can be used to convert gases consisting primarily of CO and/or CO and hydrogen (H2) into a variety of fuels and chemicals. Microorganisms can also be used to convert these gases into fuels and chemicals. These biological processes, although generally slower than chemical reactions, have several advantages over catalytic processes, including greater specificity, higher product yields, lower energy costs, and greater resistance to poisoning.

The ability of microorganisms 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 biochemical acetyl coenzyme A (acetyl CoA) reaction series of autotrophic growth (also known as the Woods-Ljungdahl reaction series and the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) reaction series). A large number of anaerobic organisms including carboxydotrophic, photosynthetic, methanogenic, and acetogenic organisms have been shown to metabolize CO to various end products, i.e., CO2, H2, methane, n-butanol, acetate, and ethanol. While utilizing CO as the sole carbon source, all such organisms produce at least two of these end products.

Anaerobic bacteria, such as those of the genus Clostridium, have been shown to produce ethanol from CO, CO2, and H2 through a series of biochemical reactions involving acetyl CoA. For example, several strains of Clostridium Ijungdahlii that produce ethanol from gases are described in WO 00/68407, EP 117309, U.S. Patent Nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558, and WO 02/08438. The bacterium Clostridium autoethanogenum sp is also known to produce ethanol from gases (Abrini et al., Archives of Microbiology 161, pp. 345-351 (1994)).

However, ethanol production by microorganisms through gas fermentation is typically associated with the co-production of acetate and/or acetic acid. When some of the available carbon is converted to acetate/acetic acid instead of ethanol, the efficiency of ethanol production using such fermentation processes may be lower than desirable. Furthermore, the acetate/acetic acid by-product may be used for some other purpose, which may pose a waste disposal problem. Acetate/acetic acid is converted to methane by microorganisms and therefore has the potential to contribute to GHG emissions.

Several enzymes known to be associated with the ability of microorganisms to utilize carbon monoxide as their sole source of carbon and energy are known to require metal cofactors for their activity. Examples of major enzymes requiring metal cofactor binding for activity include carbon monoxide dehydrogenase (CODH), and acetyl-CoA synthase (ACS). W02007/117157, W02008/115080, W02009/022925, W02009/058028, W02009/064200, W02009/064201, and W02009/113878, the description of which is incorporated herein by reference, describe processes that produce alcohols, particularly ethanol, by anaerobic fermentation of gases containing carbon monoxide. Acetate produced as a byproduct of the fermentation process described in WO2007/117157 is converted to hydrogen gas and carbon dioxide gas, either or both of which may be utilized in the anaerobic fermentation process. WO2009/022925 describes the effect of pH and ORP on the conversion of substrates comprising CO to products such as acids and alcohols by fermentation. WO2009/058028 describes the use of industrial waste gases for the production of products, such as alcohol, by fermentation. WO2009/064201 describes carriers for CO and the use of CO in fermentation. WO2009/113878 describes the conversion of acid(s) to alcohol(s) during the fermentation of a substrate comprising CO.

Microbes capable of growing on Co-containing gases are known to do so at a slower rate than is traditionally associated with microbes grown on sugars. From a commercial perspective, in a fermentation process the time required for a microbial population to grow to a sufficiently high cell density to allow an economically viable level of product to be synthesized is a major operational cost affecting the viability of the process. Technologies that act to enhance culture productivities and/or growth rates and therefore reduce the time required to achieve desired cell densities and/or desired product levels may serve to improve the commercial viability of the entire process.

In fermentation processes dedicated to the production of alcohols from gaseous feedstocks, ensuring appropriate conditions for microbial growth and/or alcohol production can be critical to maintaining optimal microbial growth and/or alcohol productivities. For example, during the initial start-up of a fermentation, the primary objective may be microbial growth. However, once the desired microbial density is achieved, the primary objective may be alcohol production. Understanding how the product profile changes during the course of a fermentation, when changes occur, particularly in response to changes in operating conditions, can enable an operator to optimize productivity. Providing a substrate comprising CO and optionally H2 at an optimal level, or in an optimal range for particular requirements, such as rapid growth and/or alcohol production, can also be challenging. For example, too much CO can lead to CO inhibition as described in US 7,285,402, which is fully incorporated herein by reference. Furthermore, too little CO and metabolic rates including microbial growth and alcohol production may decrease.

It is an object of the present invention to provide a process which will at least somewhat overcome the above disadvantages, or at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

The invention generally relates to a method for producing products including acids and/or alcohols by microbial fermentation of a substrate comprising CO, wherein at least a portion of a microbial culture converts: at least a portion of the substrate comprising CO to microbial biomass; and/or at least a portion of the substrate comprising CO to acid(s); and/or at least a portion of the substrate comprising CO to alcohol(s); and/or acid(s) and at least a portion of the substrate comprising CO to alcohol(s).

In one embodiment, the microbial culture converts: at least a portion of the substrate comprising CO to acid(s); and at least a portion of the substrate comprising CO to alcohol(s).

In another embodiment, the microbial culture converts: at least a portion of the substrate comprising CO to acid(s); and at least a portion of the substrate comprising CO to alcohol(s); and acid(s) and at least a portion of the substrate comprising CO to alcohol(s).

In particular embodiments of the invention, the substrate comprises CO and H2. However, according to the invention, conversion proceeds with insufficient H2 for total carbon fixation in products and/or cellular material. In particular embodiments, H2 is provided such that less than 2:1 H2:CO is converted by the culture, such as approximately 1:1; or approximately 1:2; or approximately 1:3; or approximately 1:4; or approximately 1:5; or approximately 1:10. In particular embodiments, H2 is not provided.

In a first aspect of the invention, in which aspect there is provided a method for improving the efficiency of microbial fermentation of a substrate comprising CO and optionally H2, the method including supplying the substrate to a microbial culture such that a first proportion of CO is fixed as one or more desired products including acid(s) and/or alcohol(s) and a second proportion of CO is converted to CO2. In particular embodiments, determining the proportion of CO converted to CO2 is used to determine a substrate supply rate for the production of the one or more desired products.

In particular embodiments, the substrate supply rate is either: i. increased if the ratio of CO converted to CO2 is determined to be below an ideal range or value; or ii. decreased if the ratio of CO converted to CO2 is determined to be above an ideal range or value; or iii. maintained if the ratio of CO converted to CO2 is determined to be substantially within an ideal range or value.

In particular embodiments, the substrate supply rate is automatically adjusted such that the ratio of converted CO to CO2 is maintained substantially within an ideal range or value.

In particular embodiments, the optimal range or value can be experimentally verified based on the desired fermentation products. In particular embodiments, where alcohol is the desired product, the substrate can be provided such that at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the fixed carbon is fixed as alcohol. Additionally or alternatively, where the desired product is acetate, the substrate can be provided such that at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the fixed carbon is fixed as acetate.

In particular embodiments, a proportion of fixed carbon as a desired product can be maintained substantially constant. In particular embodiments, where the proportion of fixed carbon as a desired product deviates from a predetermined range, the supply of the substrate is controlled such that the proportion is returned to the predetermined range. In particular embodiments, the predetermined range is about ±1%, or about ±2%, or about ±3%, or about ±4%, or about ±5%.

In particular embodiments, the substrate supply is automatically adjusted in response to deviations from the predetermined range.

In a second aspect of the invention, in that aspect there is provided a method for improving the efficiency of production of one or more acids and/or alcohols by microbial fermentation of a substrate comprising CO and optionally H2, wherein at least a portion of a microbial culture is transitioned from converting: at least a portion of the substrate comprising CO to acid(s); or at least a portion of the substrate comprising CO to alcohol(s); or acid(s) and at least a portion of the substrate comprising CO to alcohol(s); to converting: at least a portion of the substrate comprising CO to acid(s); or at least a portion of the substrate comprising CO to alcohol(s); or acid(s) and at least a portion of the substrate comprising CO to alcohol(s).

In particular embodiments of the invention, at least a portion of the microbial culture can be transitioned by preparing an adjustment to the microbial culture and/or the substrate stream. In certain embodiments, the anaerobic fermentation is carried out in a bioreactor, where the microbial culture is at least partially suspended in a fermentation broth comprising a liquid nutrient medium. In particular embodiments, at least a portion of the microbial culture can be transitioned by preparing an adjustment to the fermentation broth and/or liquid nutrient medium.

In certain embodiments, the adjustment includes one or more of: changing the pH of the fermentation broth; changing the redox potential of the fermentation broth; changing the CO concentration of the fermentation broth; changing the composition of the substrate stream; changing the pressure of the substrate stream; changing the agitation rate of the fermentation broth; removing product; changing the acid and/or alcohol concentration of the fermentation broth; changing one or more nutrients in the liquid nutrient medium; changing the rate of supply of one or more nutrients.

In particular embodiments of the invention, the substrate comprising CO also comprises H2. In some embodiments, the adjustment may include altering the H2 concentration of the fermentation broth and/or altering the ratio of CO:H2 in the fermentation broth. In certain embodiments, the substrate comprising CO and optionally H2 is gaseous. In some embodiments, the adjustment may include altering the partial pressure of CO and/or H2 in the bioreactor.

In particular embodiments of the invention, the method includes determining the ratio of oxidized CO to CO2, such that a net conversion by the microbial culture can be determined. In particular embodiments, the complete net conversion of the microbial fermentation is: at least a portion of the substrate comprising CO to acid(s); or at least a portion of the substrate comprising CO to alcohol(s); or acid(s) and at least a portion of the substrate comprising CO to alcohol(s).

In a third aspect, in this aspect there is provided a method for improving the efficiency of production of one or more acids and/or alcohols by microbial fermentation of a substrate comprising CO under predetermined operating parameters, the method including determining a proportion of carbon targeted for one or more products and depending on the determination, either: i. making an adjustment to one or more operating parameters such that the proportion of carbon fixed as a desired product increases; or ii. maintaining the operating parameters such that the proportion of carbon fixed as a desired product remains substantially constant.

In certain embodiments, adjusting includes changing one or more of the following operating parameters: changing the pH of the fermentation broth; changing the redox potential of the fermentation broth; changing the CO concentration of the fermentation broth; changing the substrate supply rate; changing the composition of the substrate stream; changing the pressure of the substrate stream; changing the agitation rate of the fermentation broth; removing product; changing the acid and/or alcohol concentration of the fermentation broth; changing one or more nutrients in the liquid nutrient medium; changing the rate of supply of one or more nutrients.

In particular embodiments of the invention, the substrate comprising CO also comprises H2. In some embodiments, the adjustment may include altering the H2 concentration of the fermentation broth and/or altering the ratio of CO:H2 in the fermentation broth. In certain embodiments, the substrate comprising CO and optionally H2 is gaseous. In some embodiments, the adjustment may include altering the partial pressure of CO and/or H2 in the bioreactor.

In particular embodiments of the first, second and third aspects, the method of improving efficiency includes improving a rate at which one or more products, such as alcohol, in particular ethanol, are produced.

In a fourth aspect of the invention, in that aspect there is provided a method for determining a total net conversion in a microbial fermentation of a substrate comprising CO, the method including determining the proportion of fixed carbon as a particular product by a microbial culture.

In particular embodiments, the proportion of fixed carbon as a particular product can be established by determining the ratio of oxidized CO to CO2.

In particular embodiments, the total net conversion in microbial fermentation is: at least a portion of the substrate comprising CO to acid(s); or at least a portion of the substrate comprising CO to alcohol(s); or acid(s) and at least a portion of the substrate comprising CO to alcohol(s);

In particular embodiments, the ratio of CO converted to CO2 is determined by measuring CO and optionally H2 consumed by the microbial culture and CO2 produced by the microbial culture.

In certain embodiments of the first, second, third or fourth aspects, the CO, H2 and/or CO2 entering and/or leaving the bioreactor can be monitored substantially continuously or at discrete time points, such as before and/or after an adjustment has been made. In some embodiments, the amounts of CO, CO2 and/or H2 entering and/or leaving the bioreactor can be determined using gas chromatography. In particular embodiments, gas chromatography is used to determine a ratio of converted CO to CO2. In one embodiment, gas chromatography is introduced using a micro GC.

It is recognized that portions of the microbial culture may be involved with alternative conversions, however the total net conversion by the total culture may be determined. In particular embodiments of the invention where H2 is substantially limited, such as embodiments where a substrate stream comprises less than 5% H2; or less than 4% H2; or less than 3% H2; or less than 2% H2; or less than 1% H2; a CO2 produced/CO2 consumed ratio of 0.5 indicates a conversion of a substrate comprising CO to acid(s) and optionally microbial cells. A CO2 produced/CO2 consumed ratio of 0.667 indicates a net conversion of a substrate comprising CO to alcohol(s). A CO2 produced/CO2 consumed of 0.5-0.667 indicates a net conversion of a substrate comprising CO to acid(s) and alcohol(s) and optionally microbial cells. A CO2produced/COconsumed ratio above 0.667 indicates a net conversion of a substrate comprising CO and acid(s) to alcohol(s).

It is recognized that in various embodiments of the above aspects, at least a portion of the microbial culture can convert alcohol(s) to acid(s) and carbon monoxide. However, in particular embodiments, the anaerobic fermentation results in a net total conversion of the substrate comprising CO to the products. In other embodiments, where CO2produced/COConsumed θ is less than 0.5, the net conversion is alcohol(s) to acid(s) and reduction of CO2 and/or H2O, which may be undesirable.

In a fifth aspect of the invention, in which aspect there is provided a system for the microbial fermentation of a substrate stream comprising CO to products such as acid(s) and/or alcohol(s) comprising a bioreactor containing a microbial culture; measuring means adapted to determine a proportion of fixed carbon as a particular product and at least one adjustment means adapted to make one or more adjustments to the microbial culture and/or the substrate stream.

In particular embodiments, the measuring means includes at least one means adapted to determine the composition of an exhaust stream exiting the bioreactor and optionally the substrate stream entering the bioreactor. The measuring means may optionally be coupled to a processing means such that a proportion of fixed carbon as a desired product may be determined. In one embodiment, the measuring means is a gas chromatograph.

In certain embodiments, the adjustment means is configured to make one or more adjustments if the determination means determines a fixed carbon ratio when a desired product has deviated from a predetermined range or value. In particular embodiments, the adjustment means is configured to make adjustments by: changing the pH of the fermentation broth; changing the redox potential of the fermentation broth; changing the CO concentration of the fermentation broth; changing the H2 concentration of the fermentation broth; changing the composition of the substrate stream; changing the pressure of the substrate stream; agitation rate of the fermentation broth; removal of product; changing the acid and/or alcohol concentration of the fermentation broth; changing one or more nutrients in the liquid nutrient medium; changing the rate of supply of one or more nutrients.

In particular embodiments, the system includes processing means adapted to control one or more adjustment means, such that one or more adjustment means, such that one or more adjustment(s) may be made to the microbial culture and/or substrate stream if it is determined that a proportion of fixed carbon as a desired product has deviated from a predetermined range or value. In other embodiments, the system may include visual and/or aural feedback to an operator, such that the operator may manually control the adjustment means.

In particular embodiments of the various aspects, the substrate comprises a gas obtained as a byproduct of an industrial process. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal product manufacturing, nonferrous product manufacturing, petroleum refining processes, biomass gasification, coal gasification, electric power production, carbon black production, ammonia production, methanol production, and coke manufacturing. In a particular embodiment, the gaseous substrate comprises a gas obtained from a steel mill.

In certain embodiments the substrate comprises from 20% CO to 100% CO by volume, such as from 40% to 95% CO by volume, such as from 60% to 90% CO by volume, or such as from 70% to 90% CO by volume. In particular embodiments, the substrate comprises 25%, or 30%, or 35%, or 40%, or 45%, or 50% CO by volume. While it is not necessary for the substrate to contain any hydrogen, the presence of H2 should not be detrimental to product formation according to the methods of the invention. In particular embodiments, the presence of hydrogen results in an improved overall alcohol production efficiency. The gaseous substrate may also contain some CO2, for example, such as from about 1% to about 80% CO2 by volume, or from 1% to about 30% CO2 by volume.

In particular embodiments of the various aspects, the substrate comprising CO is gaseous.

In particular embodiments, the alcohol produced by the fermentation process is ethanol. The fermentation reaction can also produce acetate.

In particular embodiments, the fermentation reaction is carried out by one of a majority of carboxydotrophic bacterial strains. Preferably, the carboxydotrophic bacteria is selected from Clostridium, Moorella and Carboxydothermus, such as Clostridium autoethanogenum, Clostridium Ijungdahlii, Clostridium ragsdalei, Clostridium carboxydivorans and Moorella thermoacetica. In one embodiment, the carboxydotrophic bacteria is Clostridium autoethanogenum.

Although the invention is broadly as defined above, it is not limited thereto and also includes embodiments of which the following description provides examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in detail with reference to the accompanying figures in which: Figure 1 is a graph showing changes in acetate and alcohol production and CO2 produced/CO2 consumed in a batch fermentation of a substrate comprising CO to produce products including alcohol. Figure 2 is a graph showing changes in acetate and alcohol production and CO2 produced/CO2 consumed in a batch fermentation of a substrate comprising CO to produce products including alcohol. Figure 3 is a schematic representation of a system including means for determining the ratio of CO2 produced/CO2 consumed in accordance with certain embodiments of the invention. Figure 4: is a graph showing the amount of CO and H2 consumed by a microbial culture of example 3. Figure 5: is a graph showing the metabolite production and growth of a microbial culture of example 3. Figure 6: is a graph showing the amount of CO and consumed by a microbial culture of example 4. Figure 7: is a graph showing the metabolite production and growth of a microbial culture of example 4. Figure 8: is a graph showing the metabolite production in a continuous fermentation of example 5.

DETAILED DESCRIPTION OF THE INVENTION

Carboxydotrophic bacteria such as Clostridium autoethanogenum unexpectedly produce products such as acid(s) and alcohol(s) by anaerobic fermentation of a substrate comprising CO and optionally H2, by several different mechanisms simultaneously. It has surprisingly been recognized that production of acid(s) and alcohol(s) by carboxydotrophic microorganisms can occur without concomitant water production. In previously reported fermentation of substrates comprising CO and H2, products such as alcohols and/or acids are considered to be produced together with water. However, it has surprisingly been recognized that when insufficient H2 is available for complete carbon fixation in products and cellular materials, such as alcohols and/or acids, fermentation proceeds without concomitant water production. In particular embodiments, insufficient H2 is available for complete carbon fixation when H2 and CO are consumed by a microbial culture in a H2:CO ratio of less than 2:1; such as approximately 1:1; or approximately 1:2; or approximately 1:3; or approximately 1:4; or approximately 1:5; or approximately 1:10. In particular embodiments, H2 is substantially unavailable to the microbial culture. Without wishing to be bound by theory, the products such as acetate and ethanol are produced by at least one, or at least two, or at least three, or all of the following mechanisms simultaneously: 1. Fixation of carbon monoxide to acetic acid 2CO + 2H2 → CH3COOH 2. Fixation of carbon monoxide to ethanol 3CO + 3H2 → CH3CH2OH + CO2 3. Reduction of acetic acid to ethanol CH3COOH + H2 → CH3CH2OH + CO2 4. Oxidation of ethanol to acetic acid CH3CH2OH + H2O → CH3COOH + 2H2

Microbial cell mass accumulation or anabolism typically occurs concomitantly with at least mechanism 1. However, it is considered that only a small proportion of carbon is directed to anabolism compared to other metabolites.

In addition, microorganisms can efficiently produce their own H2 through water gas shift reaction (CO + H2O → CO2 + H2). In this way metabolites including acetate and ethanol are also produced according to A-C: A) 4CO + 2H2O → CH3COOH + 2CO2 B) 6CO + 3H2O → CH3CH2OH + 4CO2 C) CH3COOH +2CO + H2O → CH3CH2OH + 2CO2

It is recognized that microbial culture is dynamic and without wishing to be bound by theory, it is assumed that microbial culture converts at least a portion of a substrate comprising CO and optionally H2, to product according to one or more of 1-3 and A-C simultaneously. Thus, in dynamic microbial culture, several different mechanisms may be occurring in the system to produce a total net conversion of CO to alcohols and/or acids. To determine how CO is metabolized, the influence of H2 and the weighting of equations 1-3 relative to A-C need to be determined. Once determined, the individual relative influence of equations 1-3 and A-C can be established by determining CO2 produced, and H2 and CO consumed.

According to one aspect of the invention, in that aspect there is provided a method for producing products including acids and/or alcohols by microbial fermentation of a substrate comprising CO, wherein at least a portion of a microbial culture converts: at least a portion of the substrate comprising CO to acid(s) and microbial cells; and/or at least a portion of the substrate comprising CO to acid(s); and/or at least a portion of the substrate comprising CO to alcohol(s); and/or acid(s) and at least a portion of the substrate comprising CO to alcohol(s).

Surprisingly, it has been found that by determining the ratio of CO converted to CO2, a modeling system can be developed to predict the production profile of products, such as alcohol and/or acids, for a CO metabolizing bacterium. Because the degree of oxidation in the products differs depending on whether the bacteria are synthesizing organic acids or alcohols, the proportion of carbon in the bacteria that is devoted to solventogenesis (such as alcohol production) can be predicted based on the stoichiometry of the underlying chemical processes.

Understanding how the product profile of a system is changing allows adjustments or changes to be made to the operating conditions of a system to promote a desirable outcome, such as increased alcohol production. Furthermore, in a particular embodiment, the invention provides a method for improving the fermentation efficiency of a substrate comprising CO to produce products including alcohol(s) and/or acid(s), the method including providing a substrate at an optimal level or in an optimal range.

In particular embodiments where CO is supplied in the absence of H2 or with limited amounts of H2, the influence of 1-3 will be minimal. Limited amounts of H2 are considered to be available when a proportion of H2 in a substrate stream is less than 5%; such as less than 4%; such as less than 3%; such as less than 2%; such as less than 1%. As such, the relative influence of equations A-C can be established by calculating a ratio of CO2produced/COCOconsumption according to y = 2/3x + 0.5. In such embodiments, equation A will determine a ratio of 0.5, equation B will determine a ratio of 0.667, equation C will determine a value of >0.667, and equation 4 will determine a value of <0.5. From the calculated value, the relative influence of each equation can be determined.

The invention also provides a method for providing efficient production of one or more acids and/or alcohols by microbial fermentation of a substrate comprising CO, wherein at least a portion of a microbial culture is transitioned from converting: at least a portion of the substrate comprising CO to acid(s) and microbial cells; or at least a portion of the substrate comprising CO to acid(s); or at least a portion of the substrate comprising CO to alcohol(s); or acid(s) and at least a portion of the substrate comprising CO to alcohol(s); to converting: at least a portion of the substrate comprising CO to acid(s) and microbial cells; or at least a portion of the substrate comprising CO to acid(s); or at least a portion of the substrate comprising CO to alcohol(s); or acid(s) and at least a portion of the substrate comprising CO to alcohol(s).

Definitions:

Unless otherwise defined, the following terms as used throughout this specification are defined as follows:

The terms "increasing 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 microorganisms catalyzing the fermentation, the volume of desired product (such as alcohols) produced per volume of substrate (such as 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 to other by-products of the fermentation.

The term "substrate comprising carbon monoxide" and other terms should be understood to include any substrate on which carbon monoxide is available to one or more strains of bacteria for growth and/or fermentation, for example.

"Gaseous substrate comprising carbon monoxide" includes any gas that contains carbon monoxide. The gaseous substrate will typically contain a significant proportion of CO, preferably at least about 5% to about 100% CO by volume.

In the context of fermentation products, the term "acid" as used herein includes both the carboxylic acid and the associated carboxylate anion, such as the mixture of free acetic acid and acetate present in a fermentation broth as described herein. The ratio of molecular acid to carboxylate in the fermentation broth is dependent on the pH of the system. The term "acetate" includes both the acetate salt alone and a mixture of molecular or free acetic acid and acetate salt, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as described herein. The ratio of molecular acetic acid to acetate in the fermentation broth is dependent on the pH of the system.

The term "bioreactor" includes a fermentation device consisting of one or more vessels and/or towers or piping arrangements, which includes Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Flowing Bed Reactor (TBR), Bubble Column, Gas Support Fermenter, Membrane Reactor such as Hollow Fiber Membrane Bioreactor (HFMBR), Static Mixer, or other vessel or other device suitable for gas-liquid contact.

Unless the context otherwise requires, the phrases "fermentation", "fermentation process" or "fermentation reaction" and the like, as used herein, are intended to encompass both the growth phase and the product biosynthesis phase of the process. As will also be described herein, in some embodiments the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, the addition of metals or compositions to a fermentation reaction should be understood to include addition to one or both of these reactors.

The term "total net conversion" and the like, as used herein, is intended to describe the conversion of substrates, such as CO, to products including acid(s) and/or alcohol(s) by a microbial culture at a particular point in time. It is recognized that portions of a microbial culture may be devoted to different functions at a particular point in time and multiple products may be produced. Furthermore, one or more of the products present in the fermentation broth may be converted to other products. Consequently, total net conversion includes all products produced by the microbial culture at any particular point in time.

While the following description focuses on particular embodiments of the invention, i.e. the production of ethanol and/or acetate using CO as the primary substrate, it should be appreciated that the invention may be applicable to the production of alternative alcohols and/or acids and the use of alternative substrates as will be known to persons of ordinary skill in the art to which the invention relates. For example, gaseous substrates containing carbon dioxide and hydrogen may be used.

Furthermore, the invention may be applicable to fermentation to produce butyrate, propionate, caproate, ethanol, propanol, and butanol. The methods may also be of use in the production of hydrogen. By way of example, these products may be produced by fermentation using microbes of the genera Moorella, Clostridia, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium, Oxobacterium, Methanosarcina, Methanosarcina, and Desulfotomaculum.

Certain embodiments of the invention are adapted to the use of gas streams produced by one or more industrial processes. Such processes include steelmaking processes, particularly processes that produce a gas stream having an elevated CO content or a CO content above a predetermined level (i.e., 5%). According to such embodiments, acetogenic bacteria are preferably used to produce acids and/or alcohols, particularly ethanol or butanol, in one or more bioreactors. Those skilled in the art will be aware from the present description that the invention may be applied to various industries or waste gas streams, including those from vehicles with an internal combustion engine. Furthermore, those skilled in the art will be aware from the present description that the invention may be applied to other fermentation reactions including those utilizing the same or different microorganisms. It is therefore intended that the scope of the invention be not limited to the particular embodiments and/or applications described, but is rather to be understood in a broader sense; for example, the source of the gas stream is not limiting except that at least one component thereof is usable to fuel a fermentation reaction. The invention has particular applicability to improving total carbon capture and/or production of ethanol and other alcohols from gaseous substrates such as automobile exhaust gases and industrial fuel gases containing high volume CO.

Fermentation

Processes for producing ethanol and other alcohols from gaseous substrates are known. Exemplary processes include those described, for example, in WO2007/117157, WO2008/115080, US 6,340,581, US 6,136,577, US 5,593,886, US 5,807,722 and US 5,821,111, each of which is incorporated herein by reference.

Various 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 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 Ijungdahlii, including those described in WO 00/68407, EP 117309, United States Patent Nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438, Clostridium carboxydivorans (Liou et al., International Journal of Systematic and Evolutionary Microbiology 33: pp 2085- 2091), Clostridium ragsdalei (WO/2008/028055) and Clostridium autoethanogenum (Abrini et al., Archives of Microbiology 161: pp 345-351). Other suitable bacteria include those of the genus Moorella, including Moorella sp HUC22-1, (Sakai et al., Biotechnology 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). Other examples include Moorella thermoacetica, Moorella thermoautotrophica, Ruminococcus productus, Acetobacterium woodii, Eubacterium limosum, Butyribacterium methylotrophicum, Oxobacter pfennigii, Methanosarcina barken, Methanosarcina acetivorans, Desulfotomaculum kuznetsovii (Simpa et al., Critical Reviews in Biotechnology, 2006 Vol. 26. Pp41-65). Furthermore, it should be understood that other anaerobic acetogenic bacteria may be applicable to the present invention as would be understood by one of skill in the art. It will also be appreciated that the invention may be applied to a mixed culture of two or more bacteria.

An exemplary microorganism suitable for use in the present invention is Clostridium autoethanogenum. In one embodiment, the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identification characteristics in the strain deposited at the German Resource Center for Biological Material (DSMZ) under the identification deposit number 19630. In another embodiment, the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identification characteristics of DSMZ deposit number DSMZ 10061.

Cultivation of the bacteria used in the methods of the invention may be conducted using any number of methods known in the art for cultivating and fermenting substrates using anaerobic bacteria. Exemplary techniques are provided in the "Examples" section below. By way of further examples, those methods generally described in the following articles using gaseous substrates for fermentation may be used: (i) K. T. Klasson, et al. (1991). Bioreactors for synthesis gas fermentation resources. Conservation and Recycling, 5; 145-165; (ii) K. T. Klasson, et al. (1991). Bioreactor design for synthesis gas fermentation. Fuel. 70. 605-614; (iii) K. T. Klasson, et al. (1992). Bioconversion of synthesis gas into liquid or gaseous fuels. Enzyme and Microbial Technology. 14; 602-608; (iv) J. L. Vega, et al. (1989). Study of Gaseous Substrate Fermentation: Carbon Monoxide Conversion to Acetate. 2. Continuous Culture. Biotech. Bioeng. 34. 6. 785-793; (v) J. L Vega, et al. (1989). Study of Gaseous Substrate fermentations: Carbon Monoxide conversion to acetate. 1. Batch culture. Biotechnology and Bioengineering. 34. 6. 774-784; (vi) J. L Vega, et al. (1990). Design of Bioreactors for Coal Synthesis Gas Fermentations. Resources, Conservation and Recycling. 3. 149-160; all of which are incorporated herein by reference.

The fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CSTR), an immobilized cell reactor, a gas-sustained reactor, a bubble column reactor (BCR), a membrane reactor such as a Hollow Fiber Membrane Bioreactor (HFMBR), or a flow-through bed reactor (TBR). Furthermore, in some embodiments of the invention, the bioreactor may comprise a first, growth factor in which the microorganisms are grown, and a second, fermentation reactor, to which the fermentation broth from the growth reactor is fed and in which the majority of the fermentation product (e.g., ethanol and acetate) is produced.

According to various embodiments of the invention, the carbon source for the fermentation reaction is a gaseous substrate containing CO. The substrate may be a CO-containing waste gas obtained as a byproduct of an industrial process, or from some other source such as automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal product manufacturing, such as steel milling, non-ferrous product manufacturing, petroleum refining processes, coal gasification, electric power production, carbon black production, ammonia production, methanol production, and coke manufacturing. In these embodiments, the CO-containing substrate may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method. Depending on the composition of the CO-containing substrate, it may also be desirable to treat it to remove any unwanted impurities, such as dust particles, before introducing it into the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known methods.

Alternatively, CO-containing substrates may originate from biomass gasification. The gasification process involves partial combustion of biomass in a restricted supply of air or oxygen. The resulting gas typically comprises primarily CO and H2, with minimal amounts of CO2, methane, ethylene, and ethane. For example, biomass byproducts obtained during the extraction and processing of foodstuffs such as sugar from sugarcane, or cornstarch and grains, or non-food biomass residues generated by the forestry industry may be gasified to produce a CO-containing gas suitable for use in the present invention.

The CO-containing substrate will typically contain a major proportion of CO, such as at least about 20% to about 100% CO by volume, from 40% to 95% CO by volume, from 60% to 90% CO by volume, and from 70% to 90% CO by volume. In particular embodiments, the substrate comprises 25%, or 30%, or 35%, or 40%, or 45%, or 50% CO by volume. Substrates having lower CO concentrations, such as 6%, may also be appropriate, particularly when H2 and CO2 are also present.

While it is not necessary for the substrate to contain any hydrogen, the presence of H2 should not be detrimental to product formation according to the methods of the invention. In particular embodiments, the presence of hydrogen results in an improved overall efficiency of alcohol production. For example, in particular embodiments, the substrate may comprise up to a 2:1, or 1:1, or 1:2 ratio of H2:CO. In other embodiments, the substrate stream comprises low concentrations of H2, e.g., less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially free of hydrogen. The substrate may also contain some CO2, e.g., such as about 1% to about 80% CO2 by volume, or 1% to about 30% CO2 by volume.

Typically, carbon monoxide will be added to the fermentation reaction in a gaseous state. However, the methods of the invention are not limited to adding the substrate in this state. For example, the carbon monoxide may be supplied in a liquid. For example, a liquid may be saturated with a gas containing carbon monoxide and that liquid added to the bioreactor. This may be accomplished using standard technology. For example, a microbubble dispersion generator (Hensirisak et al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3 / October, 2002) may be used for this purpose.

It will be appreciated that for bacterial growth and CO fermentation to occur to alcohol, in addition to the substrate containing CO gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. A nutrient medium contains sufficient vitamins and minerals to allow growth of the microorganism utilized. Suitable anaerobic media for ethanol fermentation using CO as the sole carbon source are known in the art. For example, suitable media are described in U.S. Patent Nos. 5,173,429 and 5,593,886 and WO 02/08438, WO2007/115157 and WO2008/115080 referred to above. The present invention provides a novel medium that has increased effectiveness in supporting the growth of microorganisms and/or alcohol production in the fermentation process. This medium will be described in greater detail below.

Fermentation should desirably be carried out under conditions appropriate for the desired fermentation to occur (e.g., CO to ethanol). Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, medium pH, medium redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO in the liquid phase is not limiting, and maximum product concentrations to avoid product inhibition. Suitable conditions are described in WO02/08438, WO07/117157, and WO08/115080.

The optimum reaction conditions will depend partly on the particular microorganism used. However, in general, it is preferred that fermentation be carried out at a pressure greater than ambient pressure. Operating at increased pressures allows a significant increase in the rate of transfer of CO from the gas phase to the liquid phase where it can be taken up by the microorganism as a carbon source for ethanol production. This in turn means that the retention time (defined as the liquid volume in the bioreactor divided by the inflow gas flow rate) can be reduced when bioreactors are maintained at elevated pressures other than atmospheric pressure.

Furthermore, since the rate of conversion of CO to ethanol is in part a function of the retention time of the substrate, and achieving a desired retention time ultimately dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the required bioreactor volume, and consequently the capital cost of the fermentation equipment. According to the examples given in U.S. Patent No. 5,593,886, the reactor volume can be reduced in linear proportion to the increase 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 gas-to-ethanol fermentation at elevated pressures have also been described elsewhere. For example, WO 02/08438 describes gas-to-ethanol fermentations carried out at pressures of 206.85 kPa (30 psig) and 517.11 kPa (75 psig), reporting ethanol productivities of 150 g/L/day and 369 g/L/day respectively. However, exemplary fermentations carried out using similar media and inflow gas compositions at atmospheric pressure have been found to produce between 10 and 20 times less ethanol per liter per day.

It is also desirable that the rate of introduction of the substrate containing gaseous CO be such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that the ethanol product is consumed by the crop.

Product Recovery

The products of the fermentation reaction can be recovered using known methods. Exemplary methods include those described in WO07/117157, WO08/115080, US 6,340,581, US 6,136,577, US 5,593,886, US 5,807,722 and US 5,821,111. However, briefly and by way of example only ethanol can be recovered from the fermentation broth by methods such as evaporation or fractional distillation, and extractive fermentation.

Distillation of ethanol from a fermentation broth produces an azeotropic mixture of ethanol and water (i.e., 95% ethanol and 5% water). Anhydrous ethanol can subsequently be obtained through the use of molecular sieve ethanol dehydration technology, which is also well known in the art.

Extractive fermentation procedures involve the use of a water-miscible solvent that poses a low risk of toxicity to the fermenting organism to recover ethanol from the diluted fermentation broth. For example, oleyl alcohol is a solvent that can be used in this type of extraction process. The oleyl alcohol is continuously fed into a fermenter, where this solvent rinses to form a layer on top of the fermenter that is continuously extracted and fed through a centrifuge. The water and cells are then easily separated from the oleyl alcohol and returned to the fermenter while the ethanol-laden solvent is fed to a flash evaporator unit. Most of the ethanol is vaporized and condensed while the oleyl alcohol is non-volatile and recovered for reuse in the fermentation.

Acetate, which is produced as a byproduct in the fermentation reaction, can also be recovered from the fermentation broth using methods known in the art. For example, an absorption system involving an activated carbon filter can be used. In this case, it is preferred that the microbial cells are first removed from the fermentation broth using a suitable separation unit. Numerous filtration-based methods of generating a cell-free fermentation broth for product recovery are known in the art. The permeate containing ethanol and cell-free acetate is then passed through a column containing activated carbon to absorb the acetate. Acetate in the acid form (acetic acid) rather than the salt form (acetate) is more readily absorbed by activated carbon. It is therefore preferred that the pH of the fermentation broth be reduced to less than about 3 before being passed through the activated carbon column, to convert most of the acetate to the acetic acid form.

Acetic acid absorbed by activated carbon can be recovered by elution using methods known in the art. For example, ethanol can be used to elute bound acetate. In certain embodiments, ethanol produced by the fermentation process itself can be used to elute acetate. Because the boiling point of ethanol is 78.8 °C and that of acetic acid is 107 °C, ethanol and acetate can easily be separated from each other using a volatility-based method such as distillation.

Other methods for recovering acetate from a fermentation broth are also known in the art and can be utilized in the processes of the present invention. For example, U.S. Patent Nos. 6,368,819 and 6,753,170 describe a solvent and cosolvent system that can be utilized for extracting acetic acid from fermentation broths. As with the example of the oleyl alcohol-based system described for the extractive fermentation of ethanol, the systems described in U.S. Patent Nos. 6,368,819 and 6,753,170 describe a water-immiscible solvent/cosolvent that can be mixed with the fermentation broth either in the presence or absence of the fermenting microorganisms to extract the acetic acid product. The solvent/cosolvent containing the acetic acid product is then separated from the broth by distillation. A second distillation step can then be used to purify the acetic acid from the solvent/cosolvent system.

Fermentation reaction products (e.g., ethanol and acetate) can be recovered from the fermentation broth by continuously removing a portion of the broth from the fermentation bioreactor, separating the microbial cells from the broth (conveniently by filtration), and recovering one or more products from the broth simultaneously or sequentially. In the case of ethanol, it can be conveniently recovered by distillation, and acetate can be recovered by adsorption onto activated carbon, using the methods described above. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell-free permeate remaining after the ethanol and acetate have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) can be added to the cell-free permeate to replace the nutrient medium before it is returned to the bioreactor. Additionally, if the pH of the broth has been adjusted as described above to enhance acetic acid uptake by activated carbon, the pH should be readjusted to a pH similar to that of the broth in the fermentation bioreactor before being returned to the bioreactor.

Determination of Carbon Fixation in Fermentation

By determining a ratio of CO converted to CO2, a modeling system was devised to predict the product production profile for a CO2-metabolizing bacterium. Since the degree of oxidation in the products differs depending on whether the bacteria are synthesizing alcohols or organic acids, the proportion of carbon in the bacteria that are engaged in solventogenesis can be predicted based on the stoichiometry of the underlying chemical processes. Analysis and/or quantification of the degree of oxidized byproducts (CO2) effectively provides a real-time indication of the total product conversion by a microbial culture:

The system models the reactor state as being a composite of one or more "ideal" states with calculated underlying stoichiometry. The modeling system assigns a specific gas sample to a "best fit" compromise between two primary states and a modification condition that generates two secondary hybrid reactions depending on the hydrogen available, and two tertiary states. The primary states are: Carbon monoxide fixation to acetic acid 2CO + 2H2 → CH3COOH (a CO2/CO ratio of O) Carbon monoxide fixation to ethanol 3CO + 3H2 → CH3CH2OH + CO2 (a CO2/CO ratio of 0.3333)

In the absence of free hydrogen gas, both of these primary reactions are supplemented by a water-gas shift reaction; CO + H2O → H2+ CO2

It can be assumed that this water-gas shift occurs simultaneously with carbon fixation when carbon fixation is carried out in the absence of free hydrogen.

The combination of the water-gas shift with the two primary reactions produces a pair of secondary hybrid reactions that occur in the absence of free hydrogen gas. Carbon monoxide fixation to acetic acid in the absence or free hydrogen 4CO + 2H2O → CH3COOH + 2CO2 (a CO2/CO ratio of 0.5) Carbon monoxide fixation to ethanol in the absence of free hydrogen 6CO + 3H2O → CH3CH2OH + 4CO2 (a CO2/CO ratio of 0.6667).

Additionally there were two potential tertiary states; Reduction of acetic acid to ethanol CH3COOH + 2CO + H2O → CH3CH2OH + 2CO2 (a CO2/CO ratio of 1) Oxidation of ethanol to acetic acid CH3CH2OH + H2O → CH3COOH + 2H2 (a CO2/CO ratio of 0) By observing the ratio of CO2(produced)/CO(consumed), the state of the culture can be deduced and its product yield calculated. In a culture with 100% hydrogen consuming character, the ratio will vary between 0 and 1/3. This can be graphed as a linear function with the equation y = 1/3x. In a culture with 100% water-gas character deviation, the ratio will vary between 1/2 and 2/3, which can probably be graphed as a linear function with the equation y = 2/3x + 0.5.

When hydrogen consumption is negligible, the first linear function can effectively be ignored for modeling purposes and by solving only the second equation, using the observed CO2/CO value, the calculated x value will be a proportion of carbon directed to ethanol production. Subtracting the total CO2 released from the total carbon absorbed will determine the carbon available for fixation, and multiplying this by the previously calculated ratio determines the predicted fixed carbon as ethanol. Since two carbon atoms are fixed in one ethanol molecule, this value must be halved to convert the Mol of carbon input to Mol of ethanol output.

When hydrogen consumption is not negligible but is insufficient to complete carbon fixation in products and/or cellular material, neither the CO2/CO ratio nor the amount of hydrogen can be used to directly infer crop status. Hydrogen ethanol production occupies a continuum between 3CO + 3H2 → CH3CH2OH + CO2 (a CO2/CO ratio of 0.3333) and 2CO + 2H2 → CH3COOH (a CO2/CO ratio of 0).

Both utilize CO and H2 in a 1:1 ratio. Without knowing the position on this continuum occupied by the hydrogen-consuming portion of the microbial culture, the relative CO2 input by the hydrogen-consuming microorganisms is unknown. An accurate total CO2/CO ratio cannot be calculated without knowing the CO2 produced by the underlying water-gas diversion using the population, and which figure cannot be calculated without first knowing precisely the CO2/CO ratio of the hydrogen consumers and the relative amount of CO2 they are producing. However, this can be circumvented by considering that in a hydrogen-consuming state, H2 consumption equals CO consumption, and the second equation of state can also be a CO2/H2 ratio, represented as a z value except for an x value; y = 2/3x + 0.5 and y = 1/3z

However, without a third equation to connect x and z, the simultaneous equation cannot be solved. Because the state of the culture can change, this third equation; y = ax + bz is indeed variable, as is to be expected given that the rate at which a culture produces acetate or ethanol during a fermentation process changes with conditions.

In the circumstance that a culture was fully hydrogen-consuming, both the CO2/CO and CO2/H2 ratios would be equal, since CO consumption would be 1:1 with hydrogen consumption. From this it can be inferred that a line drawn between a point calculated with the CO2/CO ratio and the CO2/H2 ratio will tend to be horizontal as the hydrogen-consuming character of the microbial population increases, and that the z-intercept of the axis would be directly proportional to the proportion of carbon being fixed in ethanol when the line became fully horizontal, as in a hydrogen-fed state, CO2 is produced on a 1:1 basis with ethanol.

From this information, an approximation can be added to allow the third "hybrid" equation to be calculated.

The gradient of a line between the CO2/CO and CO2/H2 ratios is used to designate a weight for the z-intercept of this hybrid line; if this line were horizontal, the gradient ([CO2/CO]/[CO2/H2]) would be 1, indicating a pure hydrogen consumption culture in which all ethanol production came from hydrogen consumers, meaning that the only equation to consider is the hydrogen equation.

As this line moves away from the horizontal, its gradient ([CO2/CO]/[CO2/H2]) will tend to zero. Using this as a multiplicative weighting value to multiply by the z-intercept, the inferred amount of CO2 (and ethanol) produced by hydrogen consumers will likely tend to zero as the lines between the CO2/CO and CO2/H2 ratios move further and further from the horizontal.

The z-intercept multiplied with the gradient-derived weighting factor yields an approximation of the total CO2 produced by the hydrogen-consuming microorganisms, and the CO consumed by the hydrogen consumers will be 1:1 with the hydrogen consumption, and this value can then be substituted into to solve the equation y = 1/3x. The CO consumed and CO2 produced by the hydrogen-consuming microorganisms can be subtracted from the total production and consumption to yield the remaining CO and CO2 that the water-gas diversion population is responsible for, and the ratio of this remainder can be substituted into to solve the equation y = 2/3x + 0.5. In this way the proportion of fixed carbon in a particular product such as acid(s) and/or alcohol(s) can be determined.

Those skilled in the art will appreciate that an amount of CO and optionally H2 consumed and CO2 produced can be monitored continuously or at discrete time points as desired. Any means known in the art can be used to determine an amount of CO2, CO and H2; however, in one embodiment of the invention, gas chromatography (GC) is used to measure the amount of CO2, CO and H2 present in an exhaust stream exiting a bioreactor. The proportion of fixed carbon as alcohol and/or acid can be calculated if the composition of the substrate stream entering the bioreactor is known. If the composition of the substrate stream is unknown, another gas chromatography can be used to determine the composition. Other means for determining the amount of CO2 produced and substrate consumed include mass spectroscopy (MS), GCMS and internal sensors.

Thus, in accordance with the invention, the proportion of fixed carbon as a particular product such as acetate and/or ethanol can be determined by measuring the CO2 produced, CO consumed and optionally H2 consumed. It is recognized that the rate at which substrate (e.g., CO and optionally H2) becomes available to a microbial culture can affect the relative proportion of products as well as the rate at which they are produced. For example, increasing the supply of substrate to an acetate-producing culture can increase the proportion of carbon directed to alcohol production.

A substrate comprising CO and optionally H2 is typically supplied in gaseous form and the availability of CO and H2 to a microbial culture will be dependent on the mass transfer properties of the fermentation system. For example, the availability of CO and/or H2 to a microbial culture suspended in a fermentation broth is dependent on factors known to those skilled in the art including temperature, broth composition, gas supply rate, gas composition, CO vapor pressure, H2 vapor pressure, mixing. Thus, increasing the availability of CO and/or H2 to a microbial fermentation requires improving the mass transfer properties of the system, such as increasing the substrate supply rate and/or increasing the agitation of a mechanically stirred bioreactor.

In accordance with the methods of the invention, the efficiency of fermentation can be improved by supplying the substrate comprising CO and optionally H2 in or at an optimum range or level. An optimum level can be determined based on the desired fermentation product. For example, if alcohol and microbial growth are desired, the substrate comprising CO and optionally H2 can be supplied such that the carbon is predominantly fixed as alcohol, while a portion is available for microbial growth. For example, a substrate comprising CO can be supplied to a microbial culture such that microbial growth and alcohol production occur.

The conditions, particularly the substrate supply rate and/or relative CO and H2 concentrations, can be varied until microbial growth and alcohol production are optimized to the operator's satisfaction. Once the influence of each series of carbon fixation reactions on products can be determined, the substrate supply can be adjusted to trap and/or maintain desirable conditions during fermentation. For example, it is recognized that a substrate stream may comprise fluctuating CO and/or H2 components. However, using the methods of the invention, the net production of products can be maintained at a substantially constant ratio by adjusting the substrate supply.

Additionally or alternatively, as a microbial culture grows, or microbial density fluctuates, the substrate supply can be altered according to the microbial culture requirements based on the determination of CO2 produced and CO and H2 consumed.

In this regard, the proportion of carbon directed to a particular product can be maintained substantially constant regardless of changes to the substrate supply and/or microbial density. In particular embodiments of the invention, a proportion of carbon directed to a particular product can be selected by an operator and conditions adjusted to maintain the proportion substantially constant. For example, if an operator requires 90% of the fixed carbon to be directed to ethanol production, the substrate can be supplied such that the proportion does not deviate outside of a predetermined range, such as ±1%, or +2%, or ±3%, or ±4%, or ±5%. In particular embodiments, the substrate supply can be controlled in response to determining a proportion of carbon directed to a particular product. In particular embodiments, the substrate supply is automatically adjusted in response to changes in a proportion of carbon directed to a particular product.

In particular embodiments, where CO is supplied in the absence of appreciable amounts of H2, the ratio of CO2 produced/CO2 consumed can be determined. In particular embodiments of the invention, microbial growth and alcohol production can be optimized when acetate is concomitantly produced. As such a ratio of CO2 produced/CO2 consumed of <0.667, such as approximately 0.66, or approximately 0.65, or approximately 0.64, or approximately 0.63, or approximately 0.62, or approximately 0.61, or approximately 0.60, or less would be expected. Alternatively, microbial growth and alcohol production can be optimal when acetate is consumed. As such, a ratio of CO2 produced/CO2 consumed of >0.667, such as approximately 0.67, or approximately 0.68, or approximately 0.69, or approximately 0.70, or greater would be expected.

Once an optimum range or level has been determined, the fermentation, or future fermentations, may be operated under similar conditions where the substrate is supplied such that the experimentally determined fixed carbon and/or CO2 produced/CO2 consumed ratio is substantially maintained. An optimum fermentation ratio of a substrate comprising CO and H2 may be similarly determined and applied.

In a further or alternative embodiment, the method can be used to indicate when and/or how a microbial culture can or should be transitioned from one total net conversion to another. For example, as noted previously, if growth is the primary objective, then the microbial culture can desirably be maintained such that the majority of the carbon is directed toward acetate production. For example, with the substrate stream comprising CO and minimal or no H2, the ratio of CO2 produced/CO2 consumed is maintained at about 0.5. If the ratio of CO2 produced/CO2 consumed deviates beyond a predetermined range or threshold, such as about 0.45-0.55, or about 0.48-0.52, an adjustment can be made to the culture and/or the substrate stream to transition at least a portion of the microbial culture such that the total net conversion by the entire culture is as desired. For example, transition the culture such that the ratio of CO2/CO is about 0.5. In the presence of H2, equivalent adjustments can similarly be made, such that carbon fixation remains substantially constant.

In particular embodiments of the invention, at least a portion of the microbial culture can be transitioned by making an adjustment to the microbial culture and/or the substrate stream. In certain embodiments, the anaerobic fermentation is carried out in a bioreactor, where the microbial culture is at least partially suspended in a fermentation broth comprising a liquid nutrient medium. In particular embodiments, at least a portion of the microbial culture can be transitioned by making an adjustment to the fermentation broth and/or liquid nutrient medium.

In certain embodiments, adjusting includes one or more of: changing the pH of the fermentation broth; changing the redox potential of the fermentation broth; changing the CO concentration of the fermentation broth; changing the H2 concentration of the fermentation broth; changing the composition of the substrate stream; changing the pressure of the substrate stream; changing the agitation rate of the fermentation broth; removing product; changing the acid and/or alcohol concentration of the fermentation broth; changing one or more nutrients in the liquid nutrient medium; changing the rate of supply of one or more nutrients.

Additionally or alternatively, if alcohol production is the primary objective then the substrate may be supplied such that substantially all of the carbon is fixed as ethanol. In particular embodiments where no H2 is available, the CO2 produced/CO2 consumed ratio may desirably be maintained at approximately 0.667. If the CO2 produced/CO2 consumed ratio deviates beyond a predetermined range or threshold, such as 0.58-0.73 or 0.63-0.7, an adjustment may be made to the culture and/or substrate stream to transition at least a portion of the microbial culture such that the total net conversion by the total culture is as desired, e.g., returned to a CO2 produced/CO2 consumed ratio of approximately 0.667.

In a further or alternative embodiment, an alcohol-producing crop maintained at a CO2 produced/CO2 consumed ratio of approximately 0.667 may have significant amounts of undesirable acetate, e.g., residual acetate left over from a previous growth period. The acetate may be converted to alcohol by carrying over at least a portion of the acetate reduction to alcohol (equation 3). As such, the crop may be adjusted until the CO2 produced/CO2 consumed ratio increases above 0.667 until the desired conversion is complete.

The ratio of oxidized CO to CO2 can be used to determine the total net conversion of a microbial culture. The amount of CO consumed by the culture also provides an indication of the viability of the culture (specific uptake: CO uptake rate/cell density). Accordingly, the methods of the invention can be used in combination with specific uptake monitoring. For example, if a proportion of fixed carbon as a particular product and/or specific uptake deviates from predetermined ranges or thresholds, one or more adjustments can be made to the culture such that the desired viability and conversion are maintained. In particular embodiments, where H2 is limited or substantially unavailable, specific CO uptake is expected to be at least 0.5 mmol/gram microbial cells dry weight/minute (mmol/g/min), such as approximately 0.6 mmol/g/min, such as approximately 0.7 mmol/g/min, such as approximately 0.8 mmol/g/min, such as approximately 0.9 mmol/g/min, such as approximately 1.0 mmol/g/min.

In this regard, a proportion of fixed carbon as a particular product in combination with specific CO uptake can be used to determine the rate at which particular desired metabolites, such as acids and/or alcohols, are produced. In particular embodiments, the method can be used to improve the efficiency of microbial fermentation by optimizing (i.e., improving) the rate at which one or more products (such as alcohols) are produced. For example, ethanol production can be improved by making one or more adjustments to the microbial culture that increase specific CO uptake while maintaining a CO2 produced/CO2 consumed ratio of approximately 0.667. Additionally or alternatively, one or more adjustments can be made to increase CO uptake and CO2 produced/CO2 consumed ratio from (e.g.) 0.5 to (e.g.) 0.667 to improve the rate of alcohol production.

In particular embodiments of the invention, continuous fermentation of substrates comprising CO and optionally H2 can be achieved for extended periods of at least 2 days, such as at least 3 days, or at least 5 days, or at least 1 week, or at least 1 month. Continuous fermentation includes supplying fresh media to a fermentation broth and removing the fermentation broth containing the products and microbial cells to maintain a substantially constant fermentation broth volume. In particular embodiments, the concentrations of products, including alcohol(s) and optionally acid(s), and microbial cells are maintained substantially constant in the continuous process. In particular embodiments of the invention, to maintain a continuous fermentation for an extended period, the microbial culture fixes at least a portion of carbon as acid, such as acetate. Acetate can be produced at a concentration of less than 5 g/L. However, according to the methods of the invention, the majority of the carbon is fixed as alcohol, such as ethanol, in excess of 10 g/L, or 15 g/L. Thus, to maintain continuous operation, the substrate needs to be supplied such that carbon is fixed as acetate, ethanol and biomass.

In a particular embodiment, the fermentation producing ethanol and small amounts of acetate continuously over an extended period is maintained in a CO2 produced/CO2 consumed ratio range of approximately 0.61-0.65; such as between 0.62-0.64. Such a CO2 produced/CO2 consumed ratio ensures that a majority of the fixed carbon is directed to alcohol production, while a smaller amount is directed to acetate and cellular material to maintain microbial growth, thereby prolonging a continuous culture. In particular embodiments, the substrate is provided such that the specific CO2 uptake is maintained at least 0.8 mmol/g/min, such as approximately 1.0 mmol/gram dry cell mass/minute.

According to another embodiment, non-continuous (batch) fermentation may be conducted such that alcohol is produced without concomitant acid production. In such embodiments, the substrate is supplied such that alcohol and optionally cell material (biomass) are produced. According to the invention, the substrate is supplied such that a CO2/CO ratio of approximately 0.667 is maintained. It is recognized that as a microbial culture grows, the amount of CO (and optionally H2) required increases. However, according to the invention, an optimal amount of CO can be supplied while maintaining a CO2/CO ratio as the substrate supply rate increases. Figure 1 is a schematic representation of a system 100 according to an embodiment of the invention. The substrate stream 1 enters the bioreactor 2 through a suitable conduit 3. The substrate stream 1 comprises CO and optionally CO2 and/or H2 and in certain embodiments, the substrate stream is a waste gas stream from an industrial process, such as the decarburization of steel. The substrate stream 1 may be a constant stream in the sense that it is constantly supplied, but the content of the stream may vary over time. The composition of the substrate stream, particularly the concentration of CO and CO2 may be known, or may alternatively be determined by optional determination means (not shown).

Bioreactor 2 is configured to perform the desired fermentation reaction to produce the products. According to certain embodiments, bioreactor 2 is configured to convert CO into products including one or more acids and/or alcohols. Bioreactor 2 may comprise more than one tank, each tank configured to perform the same reaction and/or different stages in a particular fermentation process and/or different reactions, including different reactions for different fermentations that may include one or more common stages.

The products produced in bioreactor 2, such as acids and/or alcohols, can be recovered by any recovery process known in the art.

The components of the substrate stream that are not consumed in the fermentation reaction and any byproduct of the fermentation reaction, such as CO2, exit bioreactor 2 through exhaust outlet 4. In particular embodiments of the invention, measuring means 5 are adapted to determine the concentration of CO, CO2, and optionally H2 in the exhaust stream exiting bioreactor 2 through exhaust outlet 4. In particular embodiments, the proportion of carbon directed to acid(s) and/or alcohol(s) may be determined from an amount of CO, CO2, and H2 supplied to and the amount exiting bioreactor 2. Accordingly, an operator may optionally make adjustments to the microbial culture in bioreactor 2 and/or to the substrate stream 1 using adjustment means 6 to maintain the microbial culture in, or transition the culture to, a desired state of production. Adjustments to maintain or transition the culture include one or more of: changing the pH of the fermentation broth; changing the redox potential of the fermentation broth; changing the CO concentration of the fermentation broth; changing the H2 concentration of the fermentation broth; changing the composition of the substrate stream; changing the pressure of the substrate stream; agitation rate of the fermentation broth; product removal; changing the acid and/or alcohol concentration of the fermentation broth; changing one or more nutrients in the liquid nutrient medium; changing the rate of supply of one or more nutrients. Additionally or alternatively, the system 100 includes optional processing means 7 adapted to determine a ratio of direct carbon to the particular products and control adjustment means 6 such that the culture can be maintained in or transitioned to a desired state.

In particular embodiments, the CO2, H2, and CO entering and/or leaving the bioreactor 2 may be monitored continuously or at a discrete time point and carbon fixation determined. In addition, the adjustment means 6 may be configured to make continuous adjustments or adjustments at discrete time points if necessary.

Any means for determining the ratio of CO2 produced/CO2 consumed may be utilized, however in particular embodiments, one or more gas chromatography is utilized to determine the CO2 and CO concentrations of the stream exiting bioreactor 2 and optionally substrate stream 1. In one embodiment, the means for determining the CO and CO2 concentrations in the stream exiting bioreactor 2 is a Varian micro GC CP-4900. EXAMPLES Materials and Methods (Examples 1 and 2):

Preparation of Media (Example 1 and 2):

The medium was prepared as follows: 85% H3PO4 (20 mmoles) was added to a 1 L solution of solution A. The pH of the medium was adjusted to 5.3 by the addition of a 5 M NaOH solution. Metal salts were then optionally added according to solution(s) B.

The vehicle solution was sterilized by autoclaving for 30 minutes at 121 °C, or by filter sterilization before use. Resazurin was added as a redox indicator and 10 ml of Vitamin B solution (solution C) was added.

Preparation of Na2Sx

A 500-ml flask was charged with Na2S (93.7 g, 0.39 mol) and 200 ml of H2O. The solution was stirred until the salt dissolved and sulfur (25 g, 0.1 mol) was added under a constant flow of N2. After 2 hours of stirring at room temperature, the "Na2Sx" solution (approximately 4 M with respect to [Na] and approximately 5 M with respect to [S]), now a clear reddish-brown liquid, was transferred into N2-purged serum bottles wrapped in aluminum foil.

Materials and Methods Examples 3, 4 and 5:

Preparation of Cr(II) solution

A 1 L three-necked flask was equipped with a narrow gas inlet and outlet to permit operation under inert gas and subsequent transfer of the desired product to a suitable storage bottle. The flask was charged with CrCl3.6H20 (40 g, 0.15 mol), zinc granules [20 mesh] (18.3 g, 0.28 mol), mercury (13.55 g, 1 mL, 0.0676 mol), and 500 mL of distilled water. Following stimulation with N2 for 1 h, the mixture was heated to approximately 80°C to initiate the reaction. Following 2 h of stirring under a constant N2 flow, the mixture was cooled to room temperature and continuously stirred for another 48 h, during which time the reaction mixture turned into a deep blue solution. The solution was transferred to N2-purged saline bottles and stored in the refrigerator for future use. Bacteria: Clostridium autoethanogenum used is the one deposited at the German Resource Center for Biological Material (DSMZ) and allocated the accession number DSMZ 19630.

Sampling and Analytical Procedures

Vehicle samples were taken from the CSTR reactor at intervals over periods of up to 20 days. Each time the vehicle was sampled care was taken to ensure that no gases were allowed to enter or escape from the reactor.

HPLC:

Agilent 1100 Series HPLC System. Mobile Phase: 0.0025 N Sulfuric Acid. Flow and Pressure: 0.800 mL/min. Column: Alltech 1OA; Catalog No. 9648, 150 x 6.5 mm, 5 μm particle size. Column Temperature: 60°C. Detector: Refractive Index. Detector Temperature: 45°C.

Method for sample preparation:

400 μL of the sample and 50 μL of 0.15M ZnSO4 and 50 μL of 0.15M Ba(OH)2 are loaded into an Eppendorf tube. The tubes are centrifuged for 10 min at 12,000 rpm, 4°C. 200 μL of the supernatant is transferred into an HPLC vial, and 5μL is injected into the HPLC instrument.

Free Top Analysis:

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

Example 1: Batch fermentation in CSTR

1.5 liters of the vehicle solution containing solution(s) B was aseptically and anaerobically transferred into a 2 L CSTR vessel, and continuously sparged with N2. Once transferred to the fermentation vessel, the reduction state and pH of the transferred vehicle could be measured directly through the probes. The vehicle was heated to 37°C and stirred at 300 rpm. The vehicle was then further reduced to -130 mV by the addition of 0.3 M Cr(II)chloride solution.

Polysulfide solution (0.1% v/v, 1.5 mL) was added to the solution, and a black precipitate was observed on the vehicle. An initial drop in potential to -300 mV was also observed, which settled to -150 mV over several hours. N2 was continuously sparged through the solution following the addition of the polysulfide solution.

Prior to inoculation, the gas was switched to a premixed mixture of 70% CO, 1% H2, 15% CO2, and 14% N2, which was continuously sprayed into the fermentation broth throughout the experiment. An actively growing culture of Clostridium autoethanogenum was inoculated into the CSTR at a level of approximately 7.5% (v/v). During this experiment, the pH was maintained at approximately 5.5.

Results:

A comparison between metabolite production and the ratio of CO2Produced/COconsumed can be seen in Figure 1. After a long period during which the culture consumed no significant amounts of gas, gas consumption and metabolite production started after about 5 days.

During the first three days the ratio of CO2produced/COCOnsumed remained between 0.55 and 0.62, a value at which the model implies ethanol will be produced co-currently with acetate; an increase in both ethanol and acetate is observed by HPLC analysis at the same time.

On day nine, CO2 produced/CO2 consumed was 0.66, a value projected by the model to be indicative of nearly all carbon capture going into ethanol production with little or no acetate production. HPLC analysis from that same day shows continued ethanol production by the crop, but acetate levels have leveled off. A slightly higher ratio would indicate acetate consumption, an event observed to have occurred during the overnight period between days 11 and 12 of HPLC analysis when head gas analysis was not performed.

On days 12 and 13, the ratio moved closer to 0.6, indicating continued ethanol production with some acetate production; again, an observation matched by HPLC measurements. From days 14 through 16, the ratio increased to over 0.667, indicating ethanol production from acetate consumption. HPLC data from these days showed continued ethanol accumulation, but acetate levels fluctuated, with minor decreases and increases. By day 19 the ratio dropped sharply to less than 0.5, suggesting ethanol consumption, with HPLC analysis during this period showing a reduction in ethanol concentration. After day 19, reactor gas consumption was close to zero and the culture was predicted to be dormant.

Example 2: Batch fermentation in CSTR

1.5 liters of the vehicle solution without solution(s) B were aseptically and anaerobically transferred into a 2 L CSTR vessel and continuously sparged with N2. Once transferred to the fermentation vessel, the reduction state and pH of the transferred vehicle could be measured directly through the probes. The vehicle was heated to 37°C and stirred at 300 rpm. The vehicle was then reduced further to -130 mV by the addition of 0.3 M Cr(II)chloride solution.

Polysulfide solution (3M solution, 1.0 mL) was added to the solution. An initial drop in potential to -220 mV was also observed, which settled to -100 mV over several hours. Following 12 h of continuous N2 sparging, solution(s) D was added to the solution and the ORP adjusted to approximately -200 mV by addition of Cr(II).

Prior to inoculation, the gas was switched to a premixed mixture of 70% CO, 1% H2, 15% CO2, and 14% N2, which was continuously sprayed into the fermentation broth throughout the experiment. An actively growing culture of Clostridium autoethanogenum was inoculated into the CSTR at a level of approximately 7.5% (v/v). During this experiment, the pH was not externally controlled.

Results:

A comparison of metabolite production and the ratio of CO2 produced/CO2 consumed can be seen in Figure 2. After a long period during which the culture consumed no significant amounts of gas, gas consumption and metabolite production began around day 3. Initially the CO2 produced/CO2 consumed is observed to be very high, in fact above 1:1. Calculated values above 1:1 are treated by the model as being equal to 1:1. Such a ratio suggests high acetate consumption and ethanol production. HPLC analysis over this time period showed a corresponding decrease in acetate levels and an increase in ethanol. Between days 5 and 9, the ratio drops to between 0.6 and 0.5, indicating that ethanol production is co-occurring with acetate production, according to HPLC analysis. Between days 8 and 11 the ratio of CO2 produced/CO2 consumed drops to below 0.5, indicating ethanol consumption. On days 9 and 10 HPLC analysis shows a drop in ethanol concentration. After day 11 the ratio increases to above 0.667, indicating that ethanol production is due to acetate consumption, in agreement with the observed decrease in acetate from HPLC analysis. This consumption becomes more pronounced on days 13 and 14, with a corresponding jump in the ratio above 1:1.

During the period from day 15 to day 18, no free gas top was realized, although HPLC analysis showed a gradual decrease in ethanol levels and a slight increase in acetate.

On day 19 when free top analysis was resumed, the ratio was above 0.667, indicating ethanol production and acetate consumption. HPLC analysis showed that ethanol levels increased sharply during the period when no sampling was performed, but acetate levels remained more or less static. No further data collection was performed on the culture after day 20.

Example 3: Batch fermentation in CSTR

The vehicle was prepared as follows: 85% H3PO4 (45 mmol) was added to a 1.5 L solution of solution A. The pH of the vehicle was adjusted to 5.3 by the addition of a 5 M NaOH solution. The vehicle solution was sterilized by autoclaving for 30 min at 121 °C, or by filter sterilization before use. Resazurin was added as a redox indicator. The vehicle solution was aseptically and anaerobically transferred into a 1.5 L CSTR vessel, and continuously sparged with N2. Once transferred to the fermentation vessel, the reduction state and pH of the transferred vehicle could be measured directly by probes. The vehicle was heated to 37 °C and stirred at 300 rpm.

Sodium sulfide solution (3.75 mL of a 0.2 M solution) was added, followed by nitriloacetic acid (1.5 mL of a 0.1 M solution), trace metal solution B (1.5 mL) Na2WO4 (1.5 mL of a 0.01 M solution) then vitamin B solution C (15 mL). The ORP of the solution was adjusted to approximately -200 mV using the Cr(II) solution.

Prior to inoculation, the gas was switched to a mixture of 33% H2, 23% N2, 31% CO, 13% CO2.

An actively growing culture of Clostridium autoethanogenum was inoculated into the CSTR at a level of approximately 10% (v/v). During this experiment, the pH was maintained at approximately 5.3 and Na2S solution was added at a rate of approximately 0.16mMol/day.

Agitation and gas supply were increased during the fermentation time course in response to changes in the gas stream exiting the bioreactor. In accordance with the methods of the invention, the proportion of carbon directed to alcohol was maintained at a high level by maintaining a breakpoint greater than 70% but less than 100%. The CO, H2 uptake and breakpoint are shown in Figure 4, while microbial growth and metabolite production are shown in Figure 5. By maintaining the substrate supply at a level to promote alcohol production, no acetate was produced, while ethanol and biomass rapidly accumulated.

Example 4: Batch fermentation in CSTR

The vehicle was prepared as follows: 85% H3PO4 (45 mmol) was added to a 1.5 L solution of solution A. The pH of the vehicle was adjusted to 5.3 by the addition of a 5 M NaOH solution. The vehicle solution was sterilized by autoclaving for 30 min at 121 °C, or by filter sterilization before use. Resazurin was added as a redox indicator. The vehicle solution was aseptically and anaerobically transferred into a 1.5 L CSTR vessel, and continuously sparged with N2. Once transferred to the fermentation vessel, the reduction state and pH of the transferred vehicle could be measured directly through the probes. The vehicle was heated to 37 °C and stirred at 300 rpm.

Sodium sulfide solution (3.75 mL of a 0.2 M solution) was added, followed by nitriloacetic acid (1.5 mL of a 0.1 M solution), trace metal B solution (1.5 mL), Na2WO4 (1.5 mL of a 0.01 M solution) and then Vitamin B solution C (15 mL). The ORP of the solution was adjusted to approximately -200 mV using the Cr(II) solution.

Prior to inoculation, the gas was switched to a mixture of 50% CO and 50% N2, which was continuously sprayed into the fermentation broth throughout the experiment. An actively growing culture of Clostridium autoethanogenum was inoculated into the CSTR at a level of approximately 10% (v/v). During fermentation, the pH was maintained at approximately 5.3 and Na2S solution was added at a rate of approximately 0.16mMol/day.

Agitation and gas supply were increased during the fermentation time course in response to changes in the gas stream exiting the bioreactor. In accordance with the methods of the invention, the ratio of CO2 produced/CO2 consumed was maintained at approximately 0.667 such that substantially all of the carbon was directed toward alcohol production. The CO uptake and ratio of CO2 produced/CO2 consumed are shown in Figure 6, while microbial growth and metabolite production are shown in Figure 7. By maintaining the substrate supply at a level to promote alcohol production, no acetate was produced, while ethanol and biomass rapidly accumulated.

Example 5: Continuous Fermentation in CSTR

A 2 L CSTR was set up under the following conditions: Vehicle was prepared as follows: 85% H3PO4 (30 mM) was added to 1.5 µL solution A. The pH of the vehicle was adjusted to 5.3 by the addition of NH4OH. The vehicle solution was sterilized by autoclaving for 30 min at 121 °C, or by filter sterilization before use. Resazurin was added as a redox initiator. The vehicle solution was aseptically and anaerobically transferred into a 1.5 L CSTR vessel, and continuously sparged with N2. Once transferred to the fermentation vessel, the reduction state and pH of the transferred vehicle could be directly measured through probes. The vehicle was heated to 37°C and stirred at 300 rpm, then trace metal solution B including 0.3 mol/l_ nitriloacetic acid (1.5 mL), then Na2WO4 (1.5 mL_ of a 0.01 M solution) and then Solution C (15 mL) were added. Prior to inoculation, the gas was switched to 2% H2, 28% N2, 48% CO, and 22% CO2. An actively growing culture of Clostridium autoethanogenum was inoculated into the CSTR at a level of approximately 10% (v/v). During this experiment, Na2S solution (0.2 M) was added at a rate of approximately 0.3 mL/hr.

The microbial culture was allowed to grow in batch mode for approximately 1 day. On day 1, fermentation was switched to continuous operation where fresh vehicle was supplied to obtain a dilution ratio of approximately 1 to 1.8. Substrate supply was increased in response to the requirements of the microbial culture.

The fermentation results are shown in Figure 8. The substrate supply rate and agitation rate were increased or decreased during the time course of the fermentation in response to changes in the ratio of CO converted to CO2. In accordance with the invention, sustainable continuous operation was achieved by maintaining a CO2/CO ratio of approximately 0.62-0.64. Sustainable continuous operation resulted in a stable biomass of approximately 3 g/L, substantially stable acetate concentration of approximately 5 g/L and substantially stable ethanol concentration of at least 10 g/L. Specific CO uptake by the microbial culture was maintained at approximately 1.0 mmol/g/min.

Example 6: Prophetic

The fermentation of a substrate comprising CO by a carboxydotrophic microbial culture comprised in a liquid nutrient medium, where conditions (such as those well known in the art) promote rapid growth of the culture with concomitant production of acetate. Under such conditions, the ratio of CO2 produced/CO2 consumed will ideally be maintained at approximately 0.5. However, at a stage when an operator wishes to switch the culture from acetate production to alcohol production, one or more adjustments may be made. In particular embodiments, the pH of the liquid nutrient medium may be lowered such that at least a portion of the microbial culture is transitioned to a state where alcohol is produced. When the desired transition is made the ratio of CO2 produced/CO2 consumed will increase to approximately 0.667. In another embodiment, rather than manually altering the pH, the adjustment may be accomplished by ceasing pH control so that the microbial culture alone could regulate the pH.

Example 7: Prophetic

The fermentation of a substrate comprising CO by a carboxydotrophic microbial culture in liquid nutrient medium, where conditions (such as those well known in the art) promote the production of alcohol. Under such conditions, the ratio of CO2 produced/CO2 consumed will ideally be maintained at approximately 0.667. However, if the ratio of CO2 produced/CO2 consumed deviates from this value, for example falling to approximately 0.5, one or more adjustments may be made to increase the ratio back to the ideal value. For example, the hydrogen component of the gas stream may be increased such that alcohol production is promoted.

The invention has been described herein with reference to certain preferred embodiments, to enable the reader to practice the invention without undue experimentation. Those skilled in the art will appreciate that the invention is susceptible to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications. In addition, titles, headings, or the like are provided to enhance the reader's understanding of this document, and should not be construed as limiting the scope of the present invention. The full description of all applications, patents, and publications cited above and below, if any, is incorporated herein by reference.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavor in any country in the world.

Throughout this specification and any claims that follow, unless the context otherwise requires, the words "comprises," "comprising," and the like, are to be taken in an inclusive sense as opposed to an exclusive sense, that is, in the sense of "including but not limited to."

Claims (9)

1. A method for improving the efficiency of microbial fermentation of a substrate comprising CO, comprising: a. providing the substrate to a microbial culture to produce products selected from the group consisting of acids, alcohols, and mixtures thereof, and CO2 as a byproduct; b. measuring the total amount of CO consumed by the microbial culture and the amount of CO2 produced by the microbial culture and calculating a ratio of CO2 produced/CO consumed; c. adjusting the substrate supply rate to maintain the ratio of CO2 produced/CO consumed within a predetermined range; and d. repeating steps b. and c. to maintain the ratio of CO2 produced/CO consumed within the predetermined range. 2. The method of claim 1, wherein the substrate supply rate is: i. increased if the ratio of CO converted to CO2 is determined to be below the predetermined range; or ii. decreased if the ratio of CO converted to CO2 is determined to be above the predetermined range; or iii. maintained if the ratio of CO converted to CO2 is determined to be within the predetermined range. 3. Method according to claim 2, characterized in that the substrate supply rate is automatically adjusted such that the ratio of converted CO to CO2 is maintained in the predetermined range. 4. The method of any one of claims 1 to 3, wherein the substrate comprising CO comprises less than 5% by volume of H2. 5. Method according to any one of claims 1 to 4, characterized in that the alcohol is ethanol. 6. Method according to any one of claims 1 to 5, characterized in that the microbial culture comprises a microorganism selected from the group consisting of Clostridia, Moorella, Pyrococcus, Eubacterium, Desulfobacterium, Carboxydothermus, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium and Peptostreptococcus. 7. Method according to claim 6, characterized in that the Clostridia microorganism is Clostridium autoethanogenum. 8. Method according to claim 1, characterized in that the CO2 produced/CO consumed ratio is 0.5 to optimize acid production. 9. Method according to claim 1, characterized in that the CO2 produced/CO consumed ratio is 0.667 to optimize alcohol production.