+

WO2009022925A1 - Procédés de production d'alcools - Google Patents

Procédés de production d'alcools Download PDF

Info

Publication number
WO2009022925A1
WO2009022925A1 PCT/NZ2008/000213 NZ2008000213W WO2009022925A1 WO 2009022925 A1 WO2009022925 A1 WO 2009022925A1 NZ 2008000213 W NZ2008000213 W NZ 2008000213W WO 2009022925 A1 WO2009022925 A1 WO 2009022925A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation
substrate
acetate
bioreactor
redox potential
Prior art date
Application number
PCT/NZ2008/000213
Other languages
English (en)
Inventor
Sean Dennis Simpson
Richard Llewellyn Sydney Forster
Phuong Loan Tran
Joshua Jeremy Conolly
Matthew James Rowe
Original Assignee
Lanzatech New Zealand Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lanzatech New Zealand Limited filed Critical Lanzatech New Zealand Limited
Priority to CN200880111599.9A priority Critical patent/CN102016052B/zh
Publication of WO2009022925A1 publication Critical patent/WO2009022925A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • This invention relates generally to methods for increasing the efficiency of processes of producing products by microbial fermentation of gases and/or dissolved gases and/or carbohydrates, particularly but not exclusively, to processes of producing ethanol by microbial fermentation of gaseous substrates comprising carbon monoxide.
  • Catalytic processes may be used to convert gases consisting primarily of CO and/or CO and hydrogen (H 2 ) into a variety of fuels and chemicals. Micro-organisms may also be used to convert these gases into fuels and chemicals. These biological processes, although generally slower than chemical reactions, have several advantages over catalytic processes, including higher specificity, higher yields, lower energy costs and greater resistance to poisoning.
  • micro-organisms to grow on CO as a sole carbon source was first discovered in 1903. This was later determined to be a property of organisms that use the acetyl coenzyme A (acetyl CoA) biochemical pathway of autotrophic growth (also known as the Woods-Ljungdahl pathway and the carbon monoxide dehydrogenase / acetyl CoA synthase (CODH/ACS) pathway).
  • CODH/ACS carbon monoxide dehydrogenase / acetyl CoA synthase
  • a large number of anaerobic organisms including carboxydotrophic, photosynthetic, methanogenic and acetogenic organisms have been shown to metabolize CO to various end products, namely CO 2 , H 2 , methane, n-butanol, . acetate and ethanol. While using CO as the sole carbon source all such organisms produce at least two of these end products.
  • Anaerobic bacteria such as those from the genus Clostridium, have been demonstrated to produce ethanol from CO, CO 2 and H 2 via the acetyl CoA biochemical pathway.
  • various strains of Clostridium ljungdahlii that produce ethanol from gases are described in WO 00/68407, EP 117309, US patent nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438.
  • the bacterium Clostridium autoethanogenum sp is also known to produce ethanol from gases (Abrini et al, Archives of Microbiology 161, pp 345-351 (1994)).
  • ethanol production by micro-organisms by fermentation of gases is always associated with co-production of acetate and/or acetic acid.
  • the efficiency of production of ethanol using such fermentation processes may be less than desirable.
  • the acetate/acetic acid by-product can be used for some other purpose, it may pose a waste disposal problem.
  • Acetate/acetic acid is converted to methane by micro-organisms and therefore has the potential to contribute to GHG emissions.
  • WO2007/117157 describes a process that produces alcohols, particularly ethanol, by anaerobic fermentation of gases containing carbon monoxide.
  • Acetate produced as a by-product of the fermentation process is converted into hydrogen gas and carbon dioxide gas, either or both of which may be used in the anaerobic fermentation process.
  • US 7,078,201 and WO 02/08438 also describe fermentation processes for producing ethanol which vary conditions (e.g. pH and redox potential) of the liquid nutrient medium in which the fermentation is performed.
  • the invention provides a process for producing one or more alcohols by anaerobic bacterial fermentation of a substrate comprising CO, the process comprising:
  • the process further comprises:
  • the invention provides a process for producing one or more alcohols by anaerobic bacterial fermentation of a substrate comprising CO, the process comprising:
  • the invention provides a process of producing one or more alcohols by anaerobic bacterial fermentation of acetate and a substrate comprising CO, wherein the process comprises:
  • the optimum redox potential range is about -450 to -550 mV.
  • the pH is maintained at about 5.7 to about 7.0.
  • the redox potential is controlled by one or more of the following:
  • the reducing agent is methyl viologen or cysteine.
  • the pH is controlled or adjusted by adding acid(s) and/or base(s).
  • the invention provides a method for increasing the efficiency of a fermentation process, the method comprising:
  • step (b) controlling at least one parameter of the fermentation process based on whether a change is detected in step (a).
  • the step (a) of detecting a change comprises removing a sample of the micro-organisms from a bioreactor, wherein the fermentation process is, at least in part, performed in the bioreactor.
  • the step (a) of detecting a change is repeated one or more times to obtain a plurality of measurements, preferably, after predetermined intervals. Note that the intervals may or may not be the same for a particular process and may change from process to process as would be apparent to one of skill in the art in view of this disclosure.
  • the parameters controlled may comprise any one or more of pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate, inoculum level, maximum gas substrate concentrations, maximum product concentrations and the commencement of product recovery.
  • a flow cytometry approach whereby a reagent, which is an indicator of bacterial reductase activity, is added to the sample and then penetrates the bacteria. Following reduction, the reagent produces a fluorescent signal which varies depending on the intracellular redox potential, and may be analysed using a flow cytometer.
  • step (b) comprises determining, based on whether a change in intracellular redox potential is detected at step (a), whether to alter at least one parameter of the fermentation process and/or maintain at least one parameter at its present value and/or to take no action to change a parameter based on the result of step (a).
  • step (a) While no action may be taken based on the result of step (a), the skilled artisan would be aware in view of the present disclosure that conventional forms of control and adjustment may still be carried out, such as maintenance of pH within a predetermined range.
  • step (a) comprises culturing in a bioreactor one or more strains of anaerobic bacteria in a liquid nutrient medium and supplying a gaseous substrate comprising CO to the bioreactor, and maintaining the pH and redox potential of the liquid nutrient medium at levels which allow the bacteria to grow and produce acetate.
  • step of determining is based on whether there is a detected rise in intracellular redox potential.
  • the step of determining may be based on whether there is a detected drop in intracellular redox potential.
  • the controlling of step (b) may comprise ceasing the fermentation process and/or adding micro-organisms to the contents of the fermentation tank and/or removing a portion of the contents of the fermentation tank and/or changing one or more parameters (see the above list) of the fermentation process.
  • the method of the invention is adapted for use in a process of producing alcohols, more particularly ethanol and/or butanol and/or isopropanol, by anaerobic fermentation of gases, particularly gases containing carbon monoxide.
  • gases particularly gases containing carbon monoxide.
  • the invention is not limited thereto and may be applied to other uses, including aerobic fermentations, and to fermentations of substrates not including CO or only including a small proportion thereof (e.g. 6% CO), particularly where the substrate contains CO 2 and/or H 2 .
  • Embodiments of the invention include, for example and without limitation, fermentations of carbohydrates and, additionally or alternatively, the production of acids (and/or salts thereof) and/or H 2 .
  • the invention provides a fermentation system for carrying out a fermentation process, the system comprising:
  • the system may be configured to carry out anaerobic and/or aerobic fermentation processes.
  • the invention relates to anaerobic fermentations.
  • Embodiments of each of the various aspects find particular application in the fermentation of a substrate comprising CO to produce acids and/or alcohols.
  • the substrate comprising CO is gaseous.
  • the gaseous substrate may comprise a gas obtained as a by-product of an industrial process.
  • the industrial process is selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of biomass, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing.
  • the gaseous substrate comprises a gas obtained from a steel mill.
  • the gaseous substrate comprises from 15% CO to 100% CO by volume, such as from 20% to 95% by volume, such as from 75% CO to 95% CO by volume, such as from 80% to 90% CO by volume. Lower CO levels, such as 6%, may be envisaged where the substrate also contains CO 2 and H 2 .
  • the alcohol produced by the fermentation process is ethanol.
  • the fermentation reaction may also produce acetate and/or butanol and/or isopropanol.
  • the fermentation reaction is carried out by one of more strains of acetogenic bacteria.
  • the anaerobic, acetogenic bacteria are selected from the group consisting of Clostridium, Moorella, Carboxydothermus, Acetogenium, Acetobacterium, Acetoanaerobium, Butyribacterium and
  • the acetogenic bacterium is Clostridium autoethanogenum or Clostridium carboxydivorans.
  • Figure 1 is a chart showing the typical life cycle of Clostridium autoethanogenum
  • Figure 2 is a graph showing the variation of RS positive cell proportion in a culture treated with methyl viologen compared to an untreated culture over against time;
  • Figure 3 is a graph showing acetate and ethanol production by Clostridium autoethanogenum over time at optimum growth pH and then elevated pH.
  • the terms “increasing the efficiency”, “increased efficiency” and the like, when used in relation to a fermentation process, include, but are not limited to, increasing one or more of: the rate of growth of micro-organisms catalysing the fermentation, the volume of desired product (such as alcohols) produced per volume of substrate (such as carbon monoxide) consumed, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared with other by-products of the fermentation.
  • substrates comprising carbon monoxide include any solid, liquid or gaseous material containing CO that may be present or introduced into a biore'actor for fermentation.
  • co-substrate refers to a substance that, while not necessarily being the primary energy and material source for product synthesis, can be utilised for product synthesis when added in addition to another, such as the primary, substrate.
  • acetate includes both acetate salt alone and a mixture of molecular or free acetic acid and acetate salt, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as described herein.
  • the ratio of molecular acetic acid to acetate in the fermentation broth is dependent upon the pH of the system.
  • biomass includes any fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact.
  • CSTR Continuous Stirred Tank Reactor
  • ICR Immobilized Cell Reactor
  • TBR Trickle Bed Reactor
  • Bubble Column Gas Lift Fermenter
  • Static Mixer Static Mixer
  • optimum growth pH and the like include the pH level or pH range of the liquid nutrient medium which promotes and/or supports growth in bacteria. It will be appreciated that the optimum growth pH may vary depending on the micro-organism used and/or other fermentation conditions.
  • optimum redox potential or "optimum ORP” and the like include the ORP level or ORP range of the liquid nutrient medium which promotes and/or supports alcohol production. It will be appreciated that the optimum ORP may vary depending on the micro-organism used and/or other fermentation conditions. Th e term “limiting concentration” means an initial concentration of a given component in a microbial fermentation medium that is sufficiently low to ensure that it will be depleted at some stage in the fermentation.
  • carbon capture refers to the sequestration of carbon compounds including CO 2 and/or CO from a stream comprising CO 2 and/or CO and either:
  • the present invention generally relates to processes of producing alcohols by fermentation of gaseous substrates containing carbon monoxide (CO).
  • the methods of the present invention also generally relate to improvements in carbon capture, wherein CO and optionally CO 2 are converted into useful products, namely alcohols.
  • Processes of producing alcohols by anaerobic bacterial fermentation of gaseous . substrates containing CO as the carbon and energy source are known in the art. These fermentation processes, in addition to producing the desired alcohol products, may also produce acetate as a by-product. Thus, the efficiency of conversion of the available carbon from the CO to the desired alcohol product is less than optimal.
  • two metabolic phases are known and are shown in Figure 1.
  • the acetogenic phase is associated with bacterial growth during which Clostridium species produce, for example, H 2 , CO 2 , and/or organic acids such as butyrate and acetate. While solvents such as alcohols may optionally be produced during the acetogenic phase, they are generally produced in insignificant amounts.
  • the acetogenic phase is followed by the solventogenic phase during which the metabolism of the cells undergoes a shift to favour solvent production.
  • Different environmental parameters e.g. pH, redox potential, etc of the media
  • Different environmental parameters are preferable for the bacteria depending on which phase they, are in.
  • the inventors have surprisingly shown that by elevating the pH of a culture of anaerobic acetogenic bacteria to above the optimum growth pH, while maintaining the redox potential of the culture at a low level (about -450 mV or below), the bacteria produce alcohol and optionally acetate. Under such conditions, alcohol and optionally acetate are produced in a molar ratio of at least about 1:1, preferably in a molar ratio favouring alcohol. In one particular embodiment, the molar ratio of alcohol to acetate is about 1.4 to 1. Furthermore, the bacteria convert at least a portion of the acetate produced as a by-product of the fermentation to ethanol, at a much higher rate than under lower pH conditions, which would normally be used in such fermentation processes.
  • certain aspects of the invention relate to detection of a status or change in status of micro-organisms in a fermentation process using one or more indicators of the intracellular redox potential of the micro-organisms.
  • the invention relates to processes of producing alcohols, such as butanol, for example, n-butanol, by anaerobic bacterial fermentation of gaseous substrates comprising CO as the carbon and energy source.
  • Processes of the invention will typically involve culturing, in a bioreactor containing a liquid nutrient medium, one or more strains of anaerobic bacteria that are capable of producing alcohols and optionally acetate from a CO-containing gaseous substrate, and supplying the gaseous substrate to the bioreactor. This fermentation produces one or more desired alcohols, and generally also acetate.
  • the process includes controlling the redox potential of the liquid nutrient medium such that it is maintained at an optimum level or range, in particular about -450 mV or lower, for example -45OmV to about -550 mV.
  • the process also includes the step of controlling the pH of the liquid nutrient medium such that it is maintained at an elevated level (i.e. increased above the optimum growth pH), in particular from about 5.7 to about 7.0, or from about 5.8 to about 6.5.
  • the liquid nutrient medium is maintained under the relevant redox conditions, or under the relevant pH and redox conditions, for at least a period of time sufficient to allow the bacteria to produce one or more alcohols and optionally acetate in a molar ratio of at least 1:1, or at a higher ratio favouring alcohol.
  • the conditions further promote fermentation of any acetate present in the liquid nutrient medium to the desired alcohol, for example ethanol or n-butanol. This acetate to alcohol conversion increases the yield of ethanol from the fermentation process, thus further improving the ethanol to acetate ratio.
  • the redox potential of the liquid nutrient medium can be adjusted by adding one or more suitable reducing agents or oxidising agents, as required.
  • Reducing agents suitable for lowering ORP will be known to those skilled in the art.
  • sulphites, bisulphites, sulphur dioxide, ammonia, hydrazine or reducing gases such as hydrogen or even CO may be ' used to reduce the ORP.
  • reducing agents suitable for use in the processes of the invention include methyl viologen and cysteine.
  • Oxidising agents suitable for increasing the ORP will be known to those skilled in the art, but by way of example may include hydrogen peroxide, ozone, dihalide (chlorine, bromine or iodine), chlorine dioxide, potassium permanganate, air or oxygen.
  • the ORP may also be decreased or increased by raising or lowering the pH of the liquid nutrient medium respectively. For example, by increasing the pH of a theoretical aqueous system by one pH unit, the ORP will decrease by 59mV.
  • the pH of the liquid nutrient medium may be adjusted as required by adding one or more suitable pH-adjusting agents or buffers.
  • bases such as NaOH and acids such as sulphuric acid may be used to increase or decrease, respectively, the pH, as required.
  • acids and/or bases available for adjusting the pH of the liquid nutrient medium.
  • the methods of the invention therefore typically involve one or more steps of measuring the pH and/or redox potential of the fermentation medium.
  • the bioreactor includes means for continuously or intermittently determining liquid nutrient media pH and/or ORP.
  • ORP may be continuously or intermittently monitored by immersion of an ORP probe into the liquid nutrient media.
  • pH can be monitored by immersion of a pH probe in the liquid nutrient media.
  • the feedback provided by the pH and/or ORP determining means allow monitoring of the conditions in the bioreactor.
  • the conditions within the reactor can be controlled as a result of the feedback or otherwise, by altering particular parameters or adding particular reagents.
  • ORP can be controlled by adding reducing or oxidising reagents to the liquid nutrient medium. For example, if the ORP increases or decreases above or below the optimum ORP, reducing agents or oxidising agents may be added to the liquid nutrient media to maintain the media within the optimum ORP range. Alternatively, the ORP may be controlled by introduction of electrodes into the liquid nutrient medium to maintain or achieve a desired ORP. Similarly, pH can also be controlled. For example, if the pH increases above or decreases below the desired pH range, acids or bases may be added to the liquid nutrient media to maintain the media within a desired pH range.
  • the oxidising or reducing agents, or acids or bases may be added manually in response to deviation from optimum conditions.
  • one or more alarms may be activated in order to alert an operator that a particular reagent needs to be added.
  • the pH and/or ORP are monitored continuously or intermittently and suitable reagents are added automatically in response to the deviation from optimum conditions.
  • pH and/or ORP probes provide feedback to a central processor that monitors the conditions with the bioreactor. If the ORP increases above a predetermined threshold, the processor will initiate the introduction of a reducing agent until the desired ORP is maintained.
  • the processor can initiate the introduction of a base until the desired pH is maintained.
  • continuous monitoring and subsequent maintenance of pH and ORP in desired ranges has led to continuous production of ethanol over a 9 day period. It is contemplated that monitoring and maintaining liquid nutrient media conditions in this manner will result in continuous alcohol production for several weeks or months.
  • the invention relates to detection of a status or a change in status of micro-organisms, in a fermentation process using one or more indicators of the intracellular redox potential of the micro-organisms.
  • the intracellular redox potential if largely independent of the ORP of the liquid nutrient medium and may be used as an indirect measure of the viability and/or status of a microbial population in a bioreactor.
  • the conditions within the bioreactor housing the microorganisms may be controlled so that they are optimised for the determined status, thereby increasing the efficiency of the process.
  • parameters may be controlled so as to provide for optimal growth.
  • conditions may be optimised for the production of alcohol.
  • the pH and/or ORP can be maintained within optimum ranges to promote alcohol production.
  • Optimisation of bioreactor conditions is highly desirable in such processes.
  • more rapid accumulation of bacterial cell biomass such as C. autoethanogenum
  • C. autoethanogenum is highly desirable in the context of a commercial process, such as ethanol production from CO-containing gases.
  • increases in bacterial biomass accumulation allow a reduction in the time required for the bacterial concentration in a fermentation process to reach a bacterial cell density that allows maximal process productivity per volume of input gas, per volume of reactor media. This reduction in time is associated with a concomitant reduction in operating costs and nonproductive down time.
  • the optimisation of bioreactor conditions comprises determining, based on whether a change in intracellular redox potential is detected, whether to alter at least one parameter of the fermentation process and/or maintain at least one parameter at its present value and/or to take no action to change a parameter based on the result of step (a).
  • the parameters controlled may comprise any one or more of pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate, inoculum level, maximum gas substrate concentrations, maximum product concentrations and the commencement or cessation of product recovery.
  • samples of microorganisms are taken from a bioreactor and a determination is made as to whether there is a change in the intracellular redox potential thereof.
  • Any known method may be used to determine the intracellular redox potential of the micro-organisms, including those involving the use of spectrophotometers.
  • the samples are preferably obtained at regular intervals although the interval may be varied depending on known characteristics of particular reactions. For example, if it is known that a particular micro-organism has a minimum growth period, there may be an initial period when fewer or no samples are taken followed by a period when more regular samples are taken until a change is detected. Where there is a significant change in the intracellular redox potential, it is possible to then come to a conclusion on the status thereof, and use this information to optimise conditions for the micro-organisms.
  • Intracellular redox states can be assayed by measuring the concentrations of redox couples such as NAD+/NADH, NADP+/NADPH, and cystine/cysteine, and the oxidised and reduced forms of glutathione and metalloenzymes.
  • Tools utilised to achieve such measurements include the use of fluorescence (flow cytometry, microscopy and fluorometric assays), colourmetric assays, radiolabeling and magnetic resonance imaging.
  • flow cytometry is used to determine the reductase activity in bacterial samples taken from the bioreactor. Any known indicator of bacterial reductase activity may be used.
  • the RedoxSensorTM Green reagent is a suitable indicator of bacterial reductase activity used in some embodiments of the invention.
  • Reductase activity is, in turn, a reliable marker for changes in electron transport chain function.
  • RS will produce a stable green-fluorescent signal in 10 minutes. Stain intensity is altered when cells are treated with reagents that disrupt electron transport, such as sodium azide, or carbonyl cyanide 3-chlorophenyIhydrazone.
  • the invention has particular applicability to improving the production of ethanol and/or other alcohols such as butanol and/or isopropanol from substrates comprising CO.
  • the substrate comprising CO is gaseous.
  • the gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process.
  • aspects of the invention are also applicable to reactions which produce acids and/or alcohols of a wide variety of types. For example, butyrate, acetate, butanol, acetone, ethanol or isopropanol production by species of Clostridia such as C. acetobutylicum, C. bejerenkii and C. saccarolyticum, including fermentation on cellulose, cellulose hydrolysate, starch, starch hydrolysate, glucose, sucrose, fructose xylose, arabinose, glycerol and lactose.
  • Clostridia such as C. acetobutylicum, C. bejerenkii and
  • Processes for the production of ethanol and other alcohols from gaseous substrates are known. Exemplary processes include those described for example in WO2007/117157 and US 6,340,581, US 6,136,577, US 5,593,886, US 5,807,722 and US 5,821,111.
  • a number of anaerobic bacteria are known to be capable of carrying out the fermentation of CO to alcohols, including n-butanol and ethanol, and acetic acid, and are suitable for use in the process of the present invention.
  • Clostridium such as strains of Clostridium ljungdahlii, including those described in WO 00/68407, EP 117309, US patent no's 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438, Clostridium autoethanogenum (Abri
  • Other useful bacteria include Acetogenium kivui, Acetobacterium woodii, Acetoanaerobium noterae, Butyribacterium methylotrophicum and Peptostreptococcus productus.
  • Other suitable bacteria include those of the genus Moorella, including 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).
  • anaerobic bacteria particularly acetogenic bacteria
  • acetogenic bacteria may be selected for use in the process of the invention by a person of skill in the art. It will also be appreciated that a mixed culture of two or more bacteria may be used in the process of the present invention.
  • Clostridium autoethanogenum is Clostridium autoethanogenum.
  • the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identifying characteristics of the strain deposited at the German Resource Centre for Biological Material (DSMZ) under the identifying deposit number 19630.
  • the Clostridium autoethanogenum is a Clostridium autoethanogenum having the identifying characteristics of DSMZ deposit number DSMZ 10061.
  • Another suitable micro-organism is Clostridium carboxydivorans, available commercially from DSMZ and having the identifying characteristics of 15243.
  • Culturing of the bacteria used in a method of the invention may be conducted using any number of processes known in the art for culturing and fermenting substrates using anaerobic bacteria. Exemplary techniques are provided in the "Examples" section below. By way of further example, those processes generally described in the following articles using gaseous substrates for fermentation may be utilised: K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L Gaddy (1991). Bioreactors for synthesis gas fermentations resources. Conservation and Recycling, 5; 145-165; K. T. Klasson, M. D. Ackerson, E. C. Clausen and J. L Gaddy (1991). Bioreactor design for synthesis gas fermentations. Fuel. 70.
  • the fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CSTR), an immobilised cell reactor, a gas-lift reactor, a bubble column reactor (BCR) or a trickle bed reactor (TBR).
  • CSTR continuous stirred tank reactor
  • immobilised cell reactor such as a cell-lift reactor, a bubble column reactor (BCR) or a trickle bed reactor (TBR).
  • BCR bubble column reactor
  • TBR trickle bed reactor
  • the carbon source for the fermentation reaction is a substrate containing CO.
  • the substrate comprising CO is gaseous.
  • the gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes.
  • the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production, methanol cracking and coke manufacturing.
  • the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method.
  • the gaseous CO containing substrate may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation.
  • the gaseous substrate may be filtered or scrubbed using known methods.
  • the CO-containing gaseous substrate may be sourced from the gasification of biomass.
  • the process of gasification involves partial combustion of biomass in a restricted supply of air or oxygen.
  • the resultant gas typically comprises mainly CO and H 2 , with minimal volumes of CO 2 , methane, ethylene and ethane.
  • biomass by-products obtained during the extraction and processing of foodstuffs such as sugar from sugarcane, or starch from maize or grains, or non-food biomass waste generated by the forestry industry may be gasified to produce a CO- containing gas suitable for use in the present invention.
  • the CO-containing gaseous substrate will ideally contain a significant proportion of CO, such as at least about 70% to about 95% CO by volume.
  • the proportion of CO may be from about 15% to about 100% CO by volume, from about 20% to about 95% CO by volume, from about 40% to about 95% CO by volume, from about 60% to about 90% CO by volume and from 70% to about 90% CO by volume.
  • the gaseous substrate comprises approximately 80% CO by volume. It may be lower than 15%, such as 6%, if H 2 and CO 2 are also present.
  • applications of the invention involving the use of biomass syngas may contain around 20% or less CO.
  • the gaseous substrate is substantially hydrogen free (less than 1%).
  • the gaseous substrate may also contain some CO 2 , such as about 1% to about 30% by volume, such as about 5% to about 10% CO 2 .
  • the substrate comprising CO is derived from carbon containing waste, for example, industrial waste gases or from the gasification of other wastes.
  • the methods of the invention represent effective processes for capturing carbon that would otherwise be exhausted into the environment.
  • the methods provide improved processes for capturing CO and optionally CO 2 by conversion into useful products such as alcohol.
  • the carbon monoxide will be added to the fermentation reaction in a gaseous state.
  • the methods of the invention are not limited to addition of the substrate in this state.
  • the carbon monoxide can be provided in a liquid.
  • a liquid may be saturated with a carbon monoxide containing gas and that liquid added to the bioreactor. This may be achieved using standard methodology.
  • a microbubble dispersion generator Heensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and BiotechnoloRv Volume 101, Number 3 / October, 2002) could be used for this purpose.
  • a suitable liquid nutrient medium will need to be fed to the bioreactor.
  • a nutrient medium contains vitamins and minerals sufficient to permit growth of the micro- organism used.
  • Anaerobic media suitable for the fermentation of ethanol using CO as the sole carbon source are known in the art. For example, suitable media are described in US patent Nos. 5,173,429 and 5,593,886 and WO 02/08438 referred to above.
  • the fermentation should be carried out under appropriate conditions for the CO- to-ethanol fermentation to occur.
  • other reaction conditions that should be considered and may be monitored and controlled include pressure, temperature, gas flow rate, liquid flow rate, agitation rate (if using a continuous stirred tank reactor), and inoculum level, and maximum product concentration (for example, to avoid product inhibition).
  • the optimum reaction conditions will depend partly on the particular micro- organism used. However, in general, it may be beneficial to perform the fermentation at pressure higher than ambient pressure. Operating at increased pressures allows a significant increase in the rate of CO transfer from the gas phase to the liquid phase where it can be taken up by the micro-organism as a carbon source for the production of ethanol. This in turn means that the retention time (defined as the liquid volume in the bioreactor divided by the input gas flow rate) can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure.
  • reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.
  • WO 02/08438 describes gas-to- ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively.
  • example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.
  • the rate of introduction of the CO-containing substrate is such as to ensure that the concentration of CO in the liquid medium does not become limiting. This is because a consequence of CO-limited conditions may be that the alcohol product is consumed by the culture.
  • a fermentation process according to the present invention described above will result in a fermentation broth comprising one or more alcohols, for example, ethanol, as well as bacterial cells, in the liquid nutrient medium.
  • alcohols for example, ethanol
  • bacterial cells in the liquid nutrient medium.
  • ethanol is the preferred desired end product of the fermentation.
  • the ethanol may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, and extractive fermentation.
  • Distillation of ethanol from a fermentation broth yields an azeotropic mixture of ethanol and water (i.e. 95% ethanol and 5% water).
  • Anhydrous ethanol can subsequently be obtained through the use of molecular sieve ethanol dehydration technology, which is also well known in the art.
  • Extractive fermentation procedures involve the use of a water-miscible solvent that presents a low toxicity risk to the fermentation organism; to recover the ethanol from the dilute fermentation broth.
  • oleyl alcohol is a solvent that may be used in this type of extraction process. Oleyl alcohol is continuously introduced into a fermenter, whereupon this solvent rises forming a layer at the. top of the fermenter which is continuously extracted and fed through a centrifuge.
  • Water and cells are then readily separated from the oleyl alcohol and returned to the fermenter while the ethanol-laden solvent is fed into a flash vaporization unit. Most of the ethanol is vaporized and condensed while the oleyl alcohol is non volatile and is recovered for re-use in ' the fermentation.
  • the process of the present invention is carried out in a single bioreactor, and the pH and/or redox potential of the liquid nutrient medium are modulated to allow the metabolic activity of the bacterial culture that uses CO-containing gas to be transitioned between various states.
  • the process involves transitioning the bacterial culture between a growth phase where microbial growth and/or acetate production are promoted and an alcohol production phase, where alcohol is produced from acetate and/or a substrate comprising CO.
  • the culture is transitioned between the following states:
  • the pH of the liquid nutrient medium is, in one embodiment, maintained in the range of about 5.0 to about 5.7 and the redox potential at about -450 mV or higher, for example, about -400 mV to about -250 mV.
  • the pH of the liquid nutrient medium is, in one embodiment, maintained in the range of about 5.8 to about 6.5, and the redox potential at about -450 mV or lower.
  • the medium is maintained under these conditions for sufficient time to allow the bacteria to produce ethanol and optionally acetate in at least about a 1:1 ratio favouring ethanol, and/or to convert acetate produced in the first state (1) to be converted to ethanol.
  • the pH and/or redox potential of the liquid nutrient medium can ' be adjusted as required using pH-adjusting agents, oxidising agents and reducing agents, to transition the metabolic activity of the bacterial culture between the above states.
  • the present invention relates to a process of producing one or more alcohols, for example, ethanol, by anaerobic bacterial fermentation of acetate and a gaseous substrate comprising CO.
  • the process involves culturing in a bioreactor one or more strains of anaerobic bacteria in a liquid nutrient medium and supplying acetate from an external source, and the gaseous substrate, to the bioreactor.
  • the pH and redox potential of the liquid nutrient medium are maintained at levels that allow fermentation of both the added acetate and the gaseous substrate by the bacteria to produce one or more alcohols.
  • the pH and redox potential are maintained in a range of about 5.7 to about 7.0 and equal to or below about -450 mV, respectively, to allow the bacteria to convert the added acetate (as well as any acetate produced in the CO to alcohol fermentation) to alcohol.
  • the process may conveniently involve two fermentations carried out in separate bioreactors.
  • the process will include a first-stage fermentation dedicated to acetate production from a suitable carbon and energy source.
  • the first-stage fermentation involves conversion of a CO-containing gaseous substrate to acetate, using anaerobic acetogenic bacteria selected from, for example, those described above as being suitable for the CO to alcohol fermentation.
  • the media conditions for the first stage fermentation should be optimised for bacterial growth and acetate production.
  • the pH of the liquid nutrient medium is maintained in the range of about 5.0 to about 5.7 and the redox potential at about -450 mV or higher, for example, from about -400 mV to about -250 mV.
  • the acetate product is recovered from the first stage fermentation and introduced, together with a CO-containing gas, to a second bioreactor, under conditions optimised for ethanol production from acetate and/or CO-containing gas by one or more microbes suitable for converting acetate to alcohol.
  • the pH of the liquid in the second fermenter is maintained above the optimum growth pH and the ORP is maintained at less than about - 45OmV.
  • the pH of the liquid nutrient medium is maintained in the range of from about 5.8 to about 6.5 and the redox potential at about - 450 mV or lower.
  • Acetate may be continuously recovered from the first stage fermentation bioreactor using methods known in the art. These methods include but are not restricted to distillation, the use of permeable acid and/or acetate selective membranes, and/or the use of adsorption columns.
  • an adsorption system involving an activated charcoal filter may be used.
  • the microbial cells are first removed from the fermentation broth using a suitable separation unit.
  • a suitable separation unit Numerous filtration-based methods of generating a cell free fermentation broth for product recovery are known in the art.
  • the cell free acetate-containing permeate is then passed through a column containing activated charcoal to adsorb the acetate.
  • Acetate in the acid form (acetic acid) rather than the salt (acetate) form is more readily adsorbed by activated charcoal.
  • the pH of the fermentation broth (medium) is therefore typically reduced to less than about 3 before it is passed through the activated charcoal column, to convert the majority of the acetate to the acetic acid form.
  • Acetic acid adsorbed to the activated charcoal may be recovered by elution using methods known in the art. For example, ethanol may be used to elute the bound acetate.
  • acetate is recovered from the fermentation broth by continuously removing a portion of the broth from the fermentation bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering acetate from the broth.
  • the acetate may be recovered by adsorption on activated charcoal, using the methods described above.
  • the separated microbial cells can then be returned to the fermentation bioreactor.
  • the cell free permeate remaining after the acetate has been removed is also typically returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor.
  • the pH of the broth was adjusted as described above to enhance adsorption of acetic acid to the activated charcoal, the pH is re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
  • Nicotinic acid 50.0 mg Fe(SO 4 ) 2 (NH 4 ) 2 . 6H 2 O 0.8g
  • Vitamin B12 50.0 mg I ZnSO 4 JH 2 O 0.2g
  • p-Aminobenzoic acid 50.0 mg CuCI 2 . 2H 2 O 0.02g
  • Media was prepared at pH 5.5 as follows. All ingredients with the exception of Cysteine-HCI were mixed in either 400ml or 800ml distilled water. This solution was made anaerobic by heating to boiling and allowing it to cool to room temperature under a constant flow of 95% CO, 5% CO2 gas. Once cool, the Cysteine-HCI was added and the pH of the solution adjusted to 5.5 before making the volume up to 1000ml; anaerobicity was maintained throughout the experiments.
  • Clostridium autoethanogenum were obtained from the German Resource Centre for Biological Material (DSMZ).
  • the accession number given to the bacteria is DSMZ 10061.
  • the Clostridium autoethanogenum used is that deposited at the German Resource Centre for Biological Material (DSMZ) and allocated the accession number 19630.
  • Clostridium carboxydivorans with the accession number 15243 was also obtained from DSMZ.
  • Fermentation in CSTR batch reactor The media for CSTR reactor experiments was prepared as described above. The media solutions were introduced into the fermenters and optionally sparged with the respective CO containing gases from the start of the experiment, or after a predetermined interval. During these experiments, the pH was adjusted and/or maintained by a controller through the automated addition of buffers (0.5 M NaOH or 2N H 2 SO 4 ). An actively growing Clostridium autoethanogenum culture was inoculated into the reactor at a level of 5 % or 7.5% (v/v). The temperature of the reactor was maintained at 37 0 C and agitation rate was 400 rpm.
  • 500 ⁇ L sample is centrifuged for 10 min at 12,000rpm, 4°C.
  • 100 ⁇ L of the supernatant is transferred into an GC vial containing 200 ⁇ L water and 100 ⁇ L of internal standard spiking solution (10 g/L propan-1-ol, 5 g/L iso-butyric acid, 135 mM hydrochloric acid).
  • 1 ⁇ L of the solution is injected into the GC instrument.
  • RedoxSensor green was used to measure intracellular reductase activity, providing a measure of intracellular redox potential.
  • the Bac ⁇ ghtTM RedoxSensorTM Green Vitality Kit was used (Invitrogen, catalogue number 349554) in accordance with the manufacturers instructions.
  • Flow Cytometry Flow cytometric analysis of RedoxSensor green and Pl stained bacterial suspensions was performed using a Coulter Epics Elite flow cytometer (Epics Division of Coulter Corporation, Hialeah, FL, USA), fitted with a Uniphase 2201-20SLE air cooled 588 nm Argon laser.
  • RedoxSensor green fluorescence was measured on PMT2 (525 nm band pass filter) and Pl fluorescence was measured on PMT3 (610 nm band pass filter). Compensation settings comprised of 22% of the PMT2 signal subtracted from the PMT3 signal and 2% of the PMT3 signal subtracted from the PMT2 signal.
  • Expo32 vl.2B Cytometer software (Applied Cytometry Systems, Sacramento, CA, USA) was used to acquire list mode files generated from samples run on the flow cytometer. Analysis of list mode files was achieved using FlowJo v8.5.1 (Macintosh OSX) software (Tree Star Incorporated, Ashland, OR, USA).
  • Example 1 Effect ofpH and oxidation and reduction potential (ORP) levels on ethanol production using CO containing gases:
  • This example describes testing to demonstrate the kinetics of ethanol production by Clostridium autoethanogenum culture in a fermenter at different pH values and ORP levels with a continuous feed of CO as the sole source of carbon and energy.
  • the culture was allowed to grow to a bacterial cell density of 0.1 g of dry cell mass per litre of media, at which point the pH and ORP of the culture were adjusted and ethanol production monitored. 3. To maintain the ORP of the media at a consistently low level, methyl viologen was added to the fermentation media to a final concentration of lOmg/L
  • the level of ethanol and acetate in the media were monitored over time as the fermentation media was maintained at three different pH values (6.5, 5.8 and 4.5) and an ORP level at or below -45OmV.
  • Example 2 Monitoring intracellular redox potential of a microbial culture
  • Example 2A Monitoring intracellular redox potential and cell vitality in a batch microbial culture.
  • Procedure 1 A preparation of LM23 fermentation media at pH 5.5 was prepared as previously described.
  • Samples were prepared from each serum bottle and assayed for: Optical density (150 ⁇ L at 620 nm), Ethanol levels, Acetate levels, Redox staining for flow cytometry, and Live/dead staining for flow cytometry.
  • Clostridium autoethanogenum (DSMZ 10061) was grown in serum bottles - control vs. methyl viologen (40 ⁇ g/mL). Samples were taken every day, stained with RS and analysed via flow cytometry. Cells were categorised based on being RS positive (green staining, high reductase activity), RS negative (red staining, low reductase activity) or neither (unknown reductase activity). A measure was then taken of the proportion of cells with a high redox potential to those with a low redox potential. Results
  • the RS positive cell proportion increased as the culture progressed, reaching a peak between day 4 and 8. From day 9, the proportion of RS positive cells declined, and by day 14, very few RS positive cells remained. In the methyl viologen treated culture, a peak in the proportion of RS positive cells was reached at day 4, and declined sooner than in the control.
  • This action may coincide with a portion of the contents being removed.
  • this indication of cell death (or other indicator of cell death) may be used in accordance with the methods of the invention as a cue for replacing the contents of the fermentation tank, which is particularly applicable to batch processing.
  • cells When cells are added, they may or may not be of the same species of micro-organism as those previously existing inside the fermentation tank without departing from the scope of the invention.
  • the RS green reagent provides a measure of the intracellular redox potential of in a microbial population and thus provides an indication of the status or viability of a microbial population within a bioreactor.
  • Example 2B Monitoring the intracellular redox potential and population vitality of microbes taken from a continuous culture configured for stable growth and acetate production.
  • Procedure 1 Continuous cultures of C. autoethanogenum (DSMZ 10061) were initiated by inoculation of 5 L of anaerobic LM33 medium (pH of 5.5 and temperature of 37°C) from serum bottle cultures, which in turn were inoculated from glycerol storage stocks. A gas blend of 70% CO, 15% CO 2 , 1% H 2 and 14% N 2 gas was sparged into the base of the culture at a rate of 64 mL/min. 2. Cultures were then allowed to grow and increase in biomass for 5 days as batch cultures, before initiation of a continuous culture regime.
  • Fresh anaerobic LM33 medium was then continuously introduced to the reactor at a rate of 3 mL/min, with the culture intermittently removed based on a level probe set to a volume of 5 L.
  • the continuous cultures of C. autoethanogenum maintained a dry weight biomass ranging from 0.6-0.8 g/L, with resident acetate levels ranging from 6-8 g/L over the course of 15 days. Alcohol production was either not observed, or led to only very low levels.
  • the proportion of viable cells to total cells in the continuous cultures was consistently in the range of 80-90% (assayed using flow cytometric analysis of RedoxSensor green and propidium iodide dye fluorescence).
  • C. autoethanogenum produced high levels of acetic acid, coupled to very low levels of ethanol. Biomass production does not seem to be compatible with the stimulation of ethanol production.
  • RedoxSensor green and propidium iodide give useful insights into the population dynamics of the continuous cultures. In addition to measuring the proportion of viable (RedoxSensor green positive, propidium iodide negative) versus non-viable (RedoxSensor green negative, propidium iodide positive) cells, an intermediate population is also evident. This dual positively stained cell population is minimal in continuous cultures (ranging from 3- 9% of the total culture), but makes up a more substantial proportion in cultures undergoing solventogenesis (e.g., see example 4).
  • fermentation parameters and/or bioreactor conditions can be altered or maintained to promote microbial growth and/or acetate production.
  • Example 2C Monitoring intracellular redox potential and cell vitality in a batch microbial culture at pH 5.5.
  • Procedure 1 A batch culture of C. autoethanogenum (DSMZ 10061) was initiated by inoculation of 1 L of anaerobic LM33 medium (pH of 5.5 and temperature of 37°C) from an actively growing continuous culture. A gas blend of 70% CO, 15% CO 2 , 1% H 2 and 14% N 2 gas was sparged into the base of the culture at a rate of 64 mL/min. 2. The culture was then allowed to grow and increase in biomass, with periodic sampling, but no further nutrient addition.
  • the ethanol to acetic acid production ratio over the course of a 24 h period in a batch culture of C. autoethanogenum was approximately 1:6.
  • the redox . potential of the culture ranged between -446 and -430 mV.
  • the internal redox potential of the cells tracked over time using the RedoxSensor green reagent, revealed viability of between 90% (day 3) and 77% (day 7) of the culture. This proportion decreased sharply to only 18% by day 10, suggesting an environment not suitable for continued culture growth due to the closed nature of this batch culture.
  • fermentation parameters and/or bioreactor conditions can be altered or maintained to promote microbial growth and/or acetate production.
  • Example 3 Effect of oxidation and reduction potential (ORP) levels on ethanol production using CO containing gases This example describes ethanol production at different ORP levels and controlled pH (pH at 6.2). Procedure
  • the culture was allowed to grow to a bacterial cell density of 0.35 g of dry cell mass per litre of media, at which point the pH of the culture were adjusted to 6.2. 3.
  • Another culture was set up as above. The culture was allowed to grow to a bacterial cell density of 0.3 g of dry cell mass per litre of media, at which point the pH of the culture was adjusted to 6.2.
  • the microbial cell viability was approximately 72%.
  • the ORP reduced to approximately -50OmV, and the viability of culture decreased to approximately 32%.
  • the proportion of intermediate cells increased from approximately 6% to 16% on addition of cysteine.
  • the change in viable and/or intermediate cells indicates the overall status of the culture with respect to whether ethanol is produced and/or acetate is consumed.
  • Example 4 Effect ⁇ fpH on ethanol production using CO containing gas
  • Example 4A outlines the first 4 days of the fermentation where the pH was maintained at 5.6, then increased to pH 5.9 for 1 day.
  • Example 4B outlines the next 9 days, wherein the pH was maintained at 6-6.1.
  • the acetic acid concentration of the culture was maintained below 4 g/L by a cell recycle and media exchange system.
  • the cells were passed through a cross flow membrane Viva 200, the filtrate was collected and the cells were returned to the reactor vessel.
  • the filtrate was replaced with fresh media to ensure the medium volume inside the reactor remained constant.
  • the culture was operated continuously for at least.14 days.
  • the cell recycle system removed 1-1.5L of liquid nutrient media every 1-2 days without removing bacteria from the bioreactor.
  • the removed media was replaced with fresh media, to maintain constant volume.
  • the pH was maintained at approximately 5.6 for 3 days, during which the ORP fluctuated between -400 and -43OmV. After 3 days, the pH was adjusted to approximately 5.9, and the ORP decreased to approximately -47OmV.
  • the same culture was adjusted to and maintained at pH 6 to 6.1 by adding buffers (base and acid). At the pH 6 to 6.1, the culture maintained ORP from -480 to -487 mV. Results for acetate and ethanol production rates achieved by the culture at different pH values are shown in the table below.
  • the intracellular redox potential was also monitored. At pH 5.6 (see example 4A), the viable population was 79%, and the intermediate population was 10.6%. However, when the pH was increased, the viable population was 44% (average) and the intermediate population was 20% (average). Under these conditions alcohol is the major product and fermentation parameters and/or bioreactor conditions can be altered or maintained to promote alcohol production for an extended period.
  • Example 5 Effect ofpH on ethanol production using CO containing gas in batch culture This example describes ethanol production at different pH values without adding reducing agents in batch culture.
  • Procedure 1 10 L media of anaerobic LM33 fermentation media (media recipe described above) in a 1 Litre CSTR was inoculated with an actively growing Clostridium autoethanogenum culture at a level of 5% (v/v). The temperature of fermenter was set at 37 0 C and agitation rate was 400 rpm. A continuous flow of 95%CO and 5 %CO 2 gas was introduced at the bottom of the fermenter vessel through a diffusing sparger at a volumetric flow rate of 19ml/minutes. The initial pH of the fermenter was set to 5.5
  • Example 6 Effect of increasing pH on ethanol production using CO containing gas with different microorganism Materials
  • Procedure 1 1.5 L media of anaerobic LM33 fermentation media in a 2 litre CSTR was inoculated with an actively growing Clostridium carboxidivorans culture at a level of 5% (v/v). The temperature of fermenter was set at 37°C and agitation rate was 400 rpm. A continuous flow of 70%CO and 15 %CO 2 1% H 2 14% N 2 gas was introduced at the bottom of the fermenter vessel through a diffusing sparger at a volumetric flow rate of 19ml/minutes. The initial pH of the fermenter was adjusted to 6.
  • the culture was allowed to grow to an optical density of 0.9 at 600 nm, and then the pH was adjusted to 6.5 and 7.
  • the optimum pH for growth of a Clostridium carboxidivorans culture and associated acetate production is generally 6-6.5.
  • the above results show that, at pH 6, acetate is produced without the concomitant production of alcohol.
  • acetic acid and ethanol were produced.
  • pH 1 acetate was consumed while ethanol was produced. This indicates at elevated pH (pH 7), at least a portion of the acetate in a fermentation broth is converted into ethanol.
  • Clostridium carboxidivorans is known to produce acetic acid, butyric acid, ethanol and butanol.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des procédés pour augmenter l'efficacité des procédés de fabrication de produits par fermentation microbienne, comprenant des procédés dans lesquels le pH et/ou le potentiel redox sont mesurés et régulés.
PCT/NZ2008/000213 2007-08-15 2008-08-15 Procédés de production d'alcools WO2009022925A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN200880111599.9A CN102016052B (zh) 2007-08-15 2008-08-15 生产醇的工艺

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
NZ556615 2007-08-15
NZ55661507 2007-08-15
NZ56075607 2007-08-17
NZ560756 2007-08-17

Publications (1)

Publication Number Publication Date
WO2009022925A1 true WO2009022925A1 (fr) 2009-02-19

Family

ID=40350881

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NZ2008/000213 WO2009022925A1 (fr) 2007-08-15 2008-08-15 Procédés de production d'alcools

Country Status (2)

Country Link
CN (1) CN102016052B (fr)
WO (1) WO2009022925A1 (fr)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009064200A3 (fr) * 2007-11-13 2009-08-06 Lanzatech New Zealand Ltd Nouvelle bactérie et méthodes d'utilisation
WO2009113878A1 (fr) * 2008-03-12 2009-09-17 Lanzatech New Zealand Limited Procédé de production d’alcool
WO2009151342A1 (fr) 2008-06-09 2009-12-17 Lanzatech New Zealand Limited Production de butanediol par fermentation microbienne anaérobie
WO2010064932A1 (fr) 2008-12-01 2010-06-10 Lanzatech New Zealand Limited Milieu de fermentation optimisé
WO2010098679A1 (fr) 2009-02-26 2010-09-02 Lanzatech New Zealand Limited Procédés pour maintenir durablement la viabilité de culture
WO2011002318A1 (fr) 2009-07-02 2011-01-06 Lanzatech New Zealand Limited Procédé de production d'alcool
WO2011028137A1 (fr) 2009-09-06 2011-03-10 Lanzatech New Zealand Limited Procédé amélioré de fermentation de substrats gazeux
WO2011087380A1 (fr) 2010-01-14 2011-07-21 Lanzatech New Zealand Limited Procédé de production d'un alcool
US8119844B2 (en) 2008-05-01 2012-02-21 Lanzatech New Zealand Limited Alcohol production process
WO2012054798A2 (fr) 2010-10-22 2012-04-26 Lanzatech New Zealand Limited Procédés et systèmes pour produire des produits hydrocarbonés
WO2012058508A2 (fr) 2010-10-29 2012-05-03 Lanzatech New Zealand Limited Procédés et systèmes de production de produits de type hydrocarbure
WO2013147621A1 (fr) 2012-03-30 2013-10-03 Lanzatech New Zealand Limited Procédé de fermentation
US8580152B2 (en) 2010-04-13 2013-11-12 Ineos Usa Llc Methods for gasification of carbonaceous materials
US8585789B2 (en) 2010-04-13 2013-11-19 Ineos Usa Llc Methods for gasification of carbonaceous materials
US8592191B2 (en) 2011-06-30 2013-11-26 Ineos Bio Sa Process for fermentation of syngas
WO2013177466A1 (fr) 2012-05-23 2013-11-28 Lanzatech New Zealand Limited Procédé de fermentation à lit mobile simulé
US8663949B2 (en) 2010-12-20 2014-03-04 Lanzatech New Zealand Limited Fermentation method
WO2014075013A1 (fr) 2012-11-12 2014-05-15 Lanzatech New Zealand Limited Liquéfaction de biomasse par fermentation de gaz
WO2014088427A1 (fr) 2012-12-05 2014-06-12 Lanzatech New Zealand Limited Processus de fermentation
WO2014151158A1 (fr) 2013-03-15 2014-09-25 Lanzatech New Zealand Limitied Système et procédé pour réguler la production de métabolites lors d'une fermentation microbienne
WO2015042550A1 (fr) 2013-09-22 2015-03-26 Lanzatech New Zealand Limited Procédé de fermentation
US8999021B2 (en) 2010-04-13 2015-04-07 Ineos Usa Llc Methods for gasification of carbonaceous materials
US9193947B2 (en) 2012-05-22 2015-11-24 Ineos Bio Sa Process for culturing microorganisms on a selected substrate
WO2016065217A1 (fr) 2014-10-22 2016-04-28 Lanzatech New Zealand Limited Procédés en bioréacteur à plusieurs étages
WO2016065085A1 (fr) 2014-10-22 2016-04-28 Lanzatech New Zealand Limited Unité d'essai de gaz et procédé associé
US9617566B2 (en) 2014-07-11 2017-04-11 Lanzatech New Zealand Limited Control of bioreactor processes
US9624512B2 (en) 2009-01-29 2017-04-18 Lanzatech New Zealand Limited Alcohol production process
RU2639525C2 (ru) * 2012-05-22 2017-12-21 Инеос Био Са Способ и среда для ферментации синтез-газа
US10010807B2 (en) 2016-02-04 2018-07-03 Lanzatech New Zealand Limited Low pressure separator having an internal divider and uses therefor
US10100338B2 (en) 2012-05-22 2018-10-16 Ineos Bio S.A. Method of operation of a syngas fermentation process
EP3384005A4 (fr) * 2015-12-03 2019-05-08 Lanzatech New Zealand Limited Supplémentation en arginine pour améliorer l'efficacité des acétogènes de fermentation gazeuse
US10968465B2 (en) 2017-06-13 2021-04-06 Lanzatech, Inc. Biological conversion and product recovery processes
FR3133196A1 (fr) 2022-03-07 2023-09-08 Totalenergies One Tech Procede de fabrication d’un carbureacteur a partir de charges d’origine renouvelable
WO2023214014A1 (fr) 2022-05-06 2023-11-09 Totalenergies Onetech Procédé de fabrication d'un carburéacteur comprenant une étape de conversion d'un flux d'alcool dans un lit fluidisé, carburéacteur et installation associés
WO2023214001A1 (fr) 2022-05-06 2023-11-09 Totalenergies Onetech Procédé d'obtention d'hydrocarbures, et installation associée
WO2023214015A1 (fr) 2022-05-06 2023-11-09 Totalenergies Onetech Procédé de fabrication d'un carburéacteur, carburéacteur et installation associés
WO2023233098A1 (fr) 2022-05-30 2023-12-07 Totalenergies Onetech Procede de fabrication de fluides hydrocarbones a partir de charges d'origine renouvelable

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105177058B (zh) * 2015-08-27 2020-03-17 中国科学院天津工业生物技术研究所 一种调节厌氧食气微生物发酵产物中酸醇比例的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173429A (en) * 1990-11-09 1992-12-22 The Board Of Trustees Of The University Of Arkansas Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism
US20030211585A1 (en) * 2000-07-25 2003-11-13 Gaddy James L. Methods for increasing the production of ethanol from microbial fermentation
US20060115884A1 (en) * 2004-12-01 2006-06-01 Burmaster Brian M Ethanol fermentation using oxidation reduction potential
WO2007117157A1 (fr) * 2006-04-07 2007-10-18 Lanzatech New Zealand Limited Fermentation microbienne de substrats gazeux pour produire des alcools
US20070275447A1 (en) * 2006-05-25 2007-11-29 Lewis Randy S Indirect or direct fermentation of biomass to fuel alcohol

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5173429A (en) * 1990-11-09 1992-12-22 The Board Of Trustees Of The University Of Arkansas Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism
US20030211585A1 (en) * 2000-07-25 2003-11-13 Gaddy James L. Methods for increasing the production of ethanol from microbial fermentation
US20060115884A1 (en) * 2004-12-01 2006-06-01 Burmaster Brian M Ethanol fermentation using oxidation reduction potential
WO2007117157A1 (fr) * 2006-04-07 2007-10-18 Lanzatech New Zealand Limited Fermentation microbienne de substrats gazeux pour produire des alcools
US20070275447A1 (en) * 2006-05-25 2007-11-29 Lewis Randy S Indirect or direct fermentation of biomass to fuel alcohol

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABRINI E TAL.: "Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide", ARCHIVES OF MICROBIOLOGY, vol. 161, no. 4, 1994, pages 345 - 351, XP008024869 *
JAIN M.K. ET AL.: "Bioconversion of coal derived synthesis gas to liquid fuels", FINAL TECHNICAL REPORT, DOE/PC/90176-T53, 1991, pages 1 - 31, Retrieved from the Internet <URL:http://www.osti.gov/bridge/product.biblio.jsp?osti_id=10105716> *
YOUNESI ET AL.: "Liquid fuel production from synthesis gas via fermentation process in a continuous tank bioreactor", IRANIAN JOURNAL OF BIOTECHNOLOGY, vol. 4, no. 1, 2006, pages 45 - 53 *

Cited By (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101918538B (zh) * 2007-11-13 2013-01-30 兰扎泰克新西兰有限公司 一种厌氧菌及其用途
WO2009064200A3 (fr) * 2007-11-13 2009-08-06 Lanzatech New Zealand Ltd Nouvelle bactérie et méthodes d'utilisation
EA022710B1 (ru) * 2007-11-13 2016-02-29 Ланзатек Нью Зиленд Лимитед Штамм бактерии clostridium autoethanogenum, способный продуцировать этанол и ацетат путем анаэробной ферментации субстрата, содержащего co
US8119378B2 (en) 2008-03-12 2012-02-21 Lanzatech New Zealand Limited Microbial alcohol production process
WO2009113878A1 (fr) * 2008-03-12 2009-09-17 Lanzatech New Zealand Limited Procédé de production d’alcool
AU2009224112B9 (en) * 2008-03-12 2013-01-31 Lanzatech Nz, Inc. Microbial alcohol production process
AU2009224112B2 (en) * 2008-03-12 2012-08-16 Lanzatech Nz, Inc. Microbial alcohol production process
EA019266B1 (ru) * 2008-03-12 2014-02-28 Ланзатек Нью Зиленд Лимитед Микробиологический способ производства спирта
US8119844B2 (en) 2008-05-01 2012-02-21 Lanzatech New Zealand Limited Alcohol production process
WO2009151342A1 (fr) 2008-06-09 2009-12-17 Lanzatech New Zealand Limited Production de butanediol par fermentation microbienne anaérobie
US8354269B2 (en) 2008-12-01 2013-01-15 Lanzatech New Zealand Limited Optimised media containing nickel for fermentation of carbonmonoxide
WO2010064932A1 (fr) 2008-12-01 2010-06-10 Lanzatech New Zealand Limited Milieu de fermentation optimisé
US9624512B2 (en) 2009-01-29 2017-04-18 Lanzatech New Zealand Limited Alcohol production process
EP3399019A1 (fr) 2009-02-26 2018-11-07 LanzaTech New Zealand Limited Procédé de soutien de la viabilité de cultures
US8658415B2 (en) 2009-02-26 2014-02-25 Lanza Tech New Zealand Limited Methods of sustaining culture viability
WO2010098679A1 (fr) 2009-02-26 2010-09-02 Lanzatech New Zealand Limited Procédés pour maintenir durablement la viabilité de culture
WO2011002318A1 (fr) 2009-07-02 2011-01-06 Lanzatech New Zealand Limited Procédé de production d'alcool
CN107858381A (zh) * 2009-07-02 2018-03-30 朗泽科技新西兰有限公司 醇生产方法
EP3739056A1 (fr) 2009-07-02 2020-11-18 LanzaTech New Zealand Limited Procédé de production d'alcool
US8906655B2 (en) 2009-07-02 2014-12-09 Lanzatech New Zealand Limited Alcohol production process
WO2011028137A1 (fr) 2009-09-06 2011-03-10 Lanzatech New Zealand Limited Procédé amélioré de fermentation de substrats gazeux
EP3070170A1 (fr) 2010-01-14 2016-09-21 Lanzatech New Zealand Limited Fermentation du co2 en utilisant un potentiel électrique
WO2011087380A1 (fr) 2010-01-14 2011-07-21 Lanzatech New Zealand Limited Procédé de production d'un alcool
US8377665B2 (en) 2010-01-14 2013-02-19 Lanzatech New Zealand Limited Alcohol production process
US8999021B2 (en) 2010-04-13 2015-04-07 Ineos Usa Llc Methods for gasification of carbonaceous materials
US8585789B2 (en) 2010-04-13 2013-11-19 Ineos Usa Llc Methods for gasification of carbonaceous materials
US8580152B2 (en) 2010-04-13 2013-11-12 Ineos Usa Llc Methods for gasification of carbonaceous materials
WO2012054798A2 (fr) 2010-10-22 2012-04-26 Lanzatech New Zealand Limited Procédés et systèmes pour produire des produits hydrocarbonés
WO2012058508A2 (fr) 2010-10-29 2012-05-03 Lanzatech New Zealand Limited Procédés et systèmes de production de produits de type hydrocarbure
US8663949B2 (en) 2010-12-20 2014-03-04 Lanzatech New Zealand Limited Fermentation method
US8592191B2 (en) 2011-06-30 2013-11-26 Ineos Bio Sa Process for fermentation of syngas
US9976158B2 (en) 2011-06-30 2018-05-22 Peter Simpson Bell Method and apparatus for syngas fermentation with high CO mass transfer coefficient
WO2013147621A1 (fr) 2012-03-30 2013-10-03 Lanzatech New Zealand Limited Procédé de fermentation
US8735115B2 (en) 2012-03-30 2014-05-27 Lanzatech New Zealand Limited Method for controlling the sulphur concentration in a fermentation method
US10100338B2 (en) 2012-05-22 2018-10-16 Ineos Bio S.A. Method of operation of a syngas fermentation process
US9193947B2 (en) 2012-05-22 2015-11-24 Ineos Bio Sa Process for culturing microorganisms on a selected substrate
RU2639525C2 (ru) * 2012-05-22 2017-12-21 Инеос Био Са Способ и среда для ферментации синтез-газа
US10131872B2 (en) 2012-05-22 2018-11-20 Ineos Bio S.A. Process for fermenting co-containing gaseous substrates
US10100336B2 (en) 2012-05-22 2018-10-16 Ineos Bio S.A. Syngas fermentation process and medium
WO2013177466A1 (fr) 2012-05-23 2013-11-28 Lanzatech New Zealand Limited Procédé de fermentation à lit mobile simulé
US8980596B2 (en) 2012-05-23 2015-03-17 Lanzatech New Zealand Limited Fermentation and simulated moving bed process
WO2014075013A1 (fr) 2012-11-12 2014-05-15 Lanzatech New Zealand Limited Liquéfaction de biomasse par fermentation de gaz
US10724059B2 (en) 2012-12-05 2020-07-28 Lanzatech New Zealand Limited Fermentation process
WO2014088427A1 (fr) 2012-12-05 2014-06-12 Lanzatech New Zealand Limited Processus de fermentation
EP4424833A2 (fr) 2013-03-15 2024-09-04 LanzaTech NZ, Inc. Système et procédé de régulation de la production de métabolites dans une fermentation microbienne
WO2014151158A1 (fr) 2013-03-15 2014-09-25 Lanzatech New Zealand Limitied Système et procédé pour réguler la production de métabolites lors d'une fermentation microbienne
CN105051178A (zh) * 2013-03-15 2015-11-11 朗泽科技新西兰有限公司 用于在微生物发酵中控制代谢物产生的系统和方法
EP4166673A1 (fr) 2013-09-22 2023-04-19 LanzaTech NZ, Inc. Procédé de fermentation
WO2015042550A1 (fr) 2013-09-22 2015-03-26 Lanzatech New Zealand Limited Procédé de fermentation
US9771603B2 (en) 2013-09-22 2017-09-26 Lanzatech New Zealand Limited Fermentation process
EP3167068A4 (fr) * 2014-07-11 2018-03-21 Lanzatech New Zealand Limited Régulation de procédés en bioréacteur
EP3766983A1 (fr) 2014-07-11 2021-01-20 LanzaTech New Zealand Limited Commande de processus de bioréacteur
US9617566B2 (en) 2014-07-11 2017-04-11 Lanzatech New Zealand Limited Control of bioreactor processes
WO2016065217A1 (fr) 2014-10-22 2016-04-28 Lanzatech New Zealand Limited Procédés en bioréacteur à plusieurs étages
WO2016065085A1 (fr) 2014-10-22 2016-04-28 Lanzatech New Zealand Limited Unité d'essai de gaz et procédé associé
US10113194B2 (en) 2014-10-22 2018-10-30 Lanzatech New Zealand Limited Gas testing unit and method
US9834792B2 (en) 2014-10-22 2017-12-05 Lanzatech New Zealand Limited Multi-stage bioreactor processes
EP4180806A1 (fr) 2014-10-22 2023-05-17 LanzaTech NZ, Inc. Bioréacteur et unité et procédé de test de gaz
EP4198118A1 (fr) 2014-10-22 2023-06-21 LanzaTech NZ, Inc. Procédés de bioréacteur à plusieurs étages
US10995347B2 (en) 2015-12-03 2021-05-04 Lanzatech New Zealand Limited Arginine supplementation to improve efficiency in gas fermenting acetogens
EP3981869A1 (fr) * 2015-12-03 2022-04-13 LanzaTech NZ, Inc. Arginine en tant que source d'azote unique pour micro-organisme de fixation c1
EP3384005A4 (fr) * 2015-12-03 2019-05-08 Lanzatech New Zealand Limited Supplémentation en arginine pour améliorer l'efficacité des acétogènes de fermentation gazeuse
US10252183B2 (en) 2016-02-04 2019-04-09 Lanzatech New Zealand Limited Product management in biological conversion processes
US10010807B2 (en) 2016-02-04 2018-07-03 Lanzatech New Zealand Limited Low pressure separator having an internal divider and uses therefor
US10968465B2 (en) 2017-06-13 2021-04-06 Lanzatech, Inc. Biological conversion and product recovery processes
FR3133196A1 (fr) 2022-03-07 2023-09-08 Totalenergies One Tech Procede de fabrication d’un carbureacteur a partir de charges d’origine renouvelable
WO2023170360A1 (fr) 2022-03-07 2023-09-14 Totalenergies Onetech Procede de fabrication d'un carbureacteur a partir de charges d'origine renouvelable
WO2023214014A1 (fr) 2022-05-06 2023-11-09 Totalenergies Onetech Procédé de fabrication d'un carburéacteur comprenant une étape de conversion d'un flux d'alcool dans un lit fluidisé, carburéacteur et installation associés
WO2023214001A1 (fr) 2022-05-06 2023-11-09 Totalenergies Onetech Procédé d'obtention d'hydrocarbures, et installation associée
WO2023214015A1 (fr) 2022-05-06 2023-11-09 Totalenergies Onetech Procédé de fabrication d'un carburéacteur, carburéacteur et installation associés
FR3135264A1 (fr) 2022-05-06 2023-11-10 Totalenergies Onetech Procédé de fabrication d’un carburéacteur, carburéacteur et installation associés
FR3135263A1 (fr) 2022-05-06 2023-11-10 Totalenergies Onetech Procédé de fabrication d’un carburéacteur comprenant une étape de conversion d’un flux d’alcool dans un lit fluidisé, carburéacteur et installation associés
FR3135265A1 (fr) 2022-05-06 2023-11-10 Totalenergies Onetech Procédé d’obtention d’hydrocarbures, et installation associée
WO2023233098A1 (fr) 2022-05-30 2023-12-07 Totalenergies Onetech Procede de fabrication de fluides hydrocarbones a partir de charges d'origine renouvelable

Also Published As

Publication number Publication date
CN102016052A (zh) 2011-04-13
CN102016052B (zh) 2015-04-29

Similar Documents

Publication Publication Date Title
WO2009022925A1 (fr) Procédés de production d&#39;alcools
US8354269B2 (en) Optimised media containing nickel for fermentation of carbonmonoxide
EP2449121B1 (fr) Procédé de production d&#39;alcool
US8119378B2 (en) Microbial alcohol production process
US8377665B2 (en) Alcohol production process
EP2391724B1 (fr) Procédé pour améliorer l&#39;efficacité de la fermantation microbienne
US20110300593A1 (en) Optimised fermentation media
EP3399019A1 (fr) Procédé de soutien de la viabilité de cultures
US20110250629A1 (en) Alcohol production process
NZ583586A (en) Processes of producing alcohols by microbial fermentation of carbon monoxide

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200880111599.9

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08827215

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 698/DELNP/2010

Country of ref document: IN

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08827215

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载