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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
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  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
  • C12M1/38—Temperature-responsive control
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/02—Separating microorganisms from their culture media
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  • 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
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  • 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
• • 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
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  • 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
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
  • C01B3/00—Hydrogen; Gaseous mixtures containing hydrogen; Separation of hydrogen from mixtures containing it; Purification of hydrogen
  • C01B3/02—Production of hydrogen or of gaseous mixtures containing a substantial proportion of hydrogen
• • 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
  • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
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  • 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
  • C12P7/16—Butanols
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/145—Clostridium
• • 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
• • 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
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/842—Clostridium

Definitions

• the present invention is directed to improvements in microbial fermentation methods for the production of alcohol from a gaseous substrate containing at least one reducing gas containing at least one acetogenic microorganism.
• Clostridium autoethanogenum (Abrini et al., 1994); Clostridium ljungdahlii (Arora et al, 1995; Barik et al., 1988; Barik et al. 1990; and Tanner et al., 1993).
• Clostridium ljungdahlii and Clostridium autoethanogenum are known to convert CO to ethanol.
• U.S. Pat. No. 5,173,429 to Gaddy et al. discloses Clostridium ljungdahlii ATCC
• No. 49587 an anaerobic microorganism that produces ethanol and acetate from CO and H. sub.20 and/or CO. sub.2 and H.sub.2 in synthesis gas.
• U.S. Pat. No. 5,192,673 to Jain et al. discloses a mutant strain of Clostridium acetobytylicum and a process for making butanol with the strain.
• U.S. Pat. No. 5,593,886 to Gaddy et al. discloses Clostridium ljungdahlii ATCC
• This microorganism can anaerobically produce acetate and ethanol using waste gas (e.g. carbon black waste gas) as a substrate.
• waste gas e.g. carbon black waste gas
• U.S. Pat. No. 5,807,722 to Gaddy et al. discloses a method and apparatus for converting waste gases into useful products such as organic acids and alcohols using anaerobic bacteria, such as Clostridium ljungdahlii ATCC No. 55380.
• U.S. Pat. No. 6,136,577 to Gaddy et al. discloses a method and apparatus for converting waste gases into useful products such as organic acids and alcohols (particularly ethanol) using anaerobic bacteria, such as Clostridium ljungdahlii ATCC Nos. 55988 and 55989.
• U.S. Pat. No. 6,136,577 to Gaddy et al. discloses a method and apparatus for converting waste gases into useful products such as organic acids and alcohols (particularly acetic acid) using anaerobic strains of Clostridium ljungdahlii.
• U.S. Pat. No. 6,753,170 to Gaddy et al. discloses an anaerobic microbial fermentation process for the production of acetic acid.
• U.S. Pat. No. 7,285,402 to Gaddy et al. discloses an anaerobic microbial fermentation process for the production of alcohol.
• strains of aceotgens have also been described for use in the production of liquid fuels from synthesis gas, e.g.: Butyribacterium methylotrophicum (Grethlein et al., 1990, Appl. Biochem. Biotech. 24/24:875-884); and Clostridium autoethanogenum (Abrini et al., 1994, Arch. Microbiol. 161:345-351).
• the present invention relates to methods for sustaining microorganism culture in a syngas fermentation reactor in decreased concentration or absence of various substrates comprising: adding carbon dioxide and optionally alcohol; maintaining free acetic acid concentrations; and performing the above mentioned steps within specified time.
• the present invention further contemplates a method for preventing rapid loss of microorganism culture in a syngas fermentation reactor in decreased concentration or absence of various substrates comprising: adding carbon dioxide and optionally alcohol; decreasing temperature from the operating temperature; maintaining free acetic acid concentrations; and performing the above mentioned steps within specified time.
• the present invention further provides a method for sustaining microorganism culture in a syngas fermentation reactor due to decreased concentration or absence of various substrates in feed gas supply comprising: adding carbon dioxide and optionally alcohol; decreasing temperature from operating temperature; maintaining free acetic acid concentrations; and performing the above mentioned steps within specified time.
• alcohol can be utilized as a substrate. Although several alternative growth substrates were tried, none performed as well as alcohol and none are as readily available as the alcohol. When synthesis gas supply is restored the microorganism culture readily returns to utilizing the syngas. Additionally, as an embodiment, solely utilizing the acetate / alcohol pathway does not provide the opportunity for other competing bacteria to grow that may be present in the culture broth or process piping. Whereas a growth substrate such as glucose would be readily available to any organisms present for their growth.
• Prior art would include adjustments to the culture broth to maintain a low free acetic acid concentration. These would include raising the pH and increasing the liquid flow to wash out the acetyl. As an embodiment, temperature reduction to reduce culture activity and using the product ethanol and carbon dioxide to provide energy back to the culture to maintain viability. Additionally, the concept of novel alternative substrate.
• Another application of the present invention comprises transporting innoculum from one site to another.
• the culture may not not have a syngas supply, therefore an alternative substrate would be required. Having the capability to maintain viability for
• FIG. 1 is a schematic diagram illustrating an embodiment of overall process flow contemplated during normal operations of the present invention. Although ethanol is indicated in the diagram, other alcohols are also contemplated by the present invention.
• FIG. 2 is a schematic diagram illustrating embodiments of the present invention showing trends with carbon dioxide addition, alcohol consumption, and culture recovery.
• FIG. 3 is a schematic diagram illustrating comparisons of the present invention demonstrating lack of alcohol consumption and lack of culture recovery.
• any amount refers to the variation in that amount encountered in real world conditions of sustaining microorganism culture, e.g., in the lab, pilot plant, or production facility.
• an amount of an ingredient employed in a mixture when modified by “about” includes the variation and degree of care typically employed in measuring in an experimental condition in production plant or lab.
• the amount of a component of a product when modified by “about” includes the variation between batches in an multiple experiments in the plant or lab and the variation inherent in the analytical method. Whether or not modified by “about,” the amounts include equivalents to those amounts. Any quantity stated herein and modified by “about” can also be employed in the present invention as the amount not modified by "about.”
• acetate is used to describe the mixture of molecular or free acetic acid and acetate salt present in the fermentation broth.
• the ratio of molecular acetic acid to acetate is dependent upon the pH of the system, i.e., at a constant "acetate" concentration, the lower the pH, the higher the molecular acetic acid concentration relative to acetate salt.
• acetogen or "acetogenic” refers to a bacterium that generates acetate as a product of anaerobic respiration. This process is different from acetate fermentation, although both occur in the absence of oxygen and produce acetate. These organisms are also referred to as acetogenic bacteria, because all known acetogens are bacteria. Acetogens are found in a variety of habitats, generally those that are anaerobic (lack oxygen). Acetogens can use a variety of compounds as sources of energy and carbon; the best studied form of acetogenic metabolism involves the use of carbon dioxide as a carbon source and hydrogen as an energy source.
• bioreactor include a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas lift Fermenter, Static Mixer, or other device suitable for gas-liquid contact.
• the fermentation bioreactor comprises a growth reactor which feeds the fermentation broth to a second fermentation bioreactor, in which most of the product, ethanol, is produced.
• Cell concentration in this specification is based on dry weight of bacteria per liter of sample. Cell concentration is measured directly or by calibration to a correlation with optical density.
• continuous method refers to a fermentation method which includes continuous nutrient feed, substrate feed, cell production in the bioreactor, cell removal (or purge) from the bioreactor, and product removal. This continuous feeds, removals or cell production may occur in the same or in different streams.
• a continuous process results in the achievement of a steady state within the bioreactor.
• steady state is meant that all of these measurable variables (i.e., feed rates, substrate and nutrient concentrations maintained in the bioreactor, cell concentration in the bioreactor and cell removal from the bioreactor, product removal from the bioreactor, as well as conditional variables such as temperatures and pressures) are constant over time.
• Ethanol productivity is the volumetric productivity of ethanol, calculated as the ratio of the steady state ethanol concentration and the liquid retention time (LRT) in continuous systems, or the ratio of the ethanol concentration and the time required to produce that concentration in batch systems.
• LRT liquid retention time
• high ethanol productivity describes a volumetric ethanol productivity of greater than 10 g/Lday.
• Excess H.sub.2 is available for ethanol production when the ratio of the moles of H.sub.2 in the feed gas to the sum of two times the moles of CO converted and three times the moles of CO.sub.2 converted is greater than 1.0. If this ratio is less than 1.0, excess H.sub.2 is not available and ethanol can only be produced through a different controlling mechanism.
• fermentation means fermentation of CO to alcohols and acetate.
• Anaerobic bacteria are known to be capable of carrying out the fermentation of CO to alcohols, including butanol and ethanol, and acetic acid, and are suitable for use in the process of the present invention.
• Clostridium such as strains of Clostridium lungdahlii, including those 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, and Clostridium autoethanogenum (Aribini et al, Archives of Microbiology 161: pp 345-351).
• 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). The disclosures of each of these publications are fully incorporated herein by reference.
• other acetogenic anaerobic 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.
• One micro-organism suitable for use in the present invention is Clostridium autoethanogenum that is available commercially from DSMZ and having the identifying characteristics of DSMZ deposit number DSMZ 10061.
• the fermentation may be carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CTSR), a bubble column reactor (BCR) or a trickle bed reactor (TBR).
• CTSR continuous stirred tank reactor
• BCR bubble column reactor
• TBR trickle bed reactor
• the bioreactof may comprise a first, growth reactor in which the micro-organisms are cultured, and a second, fermentation reactor, to which fermentation broth from the growth reactor is fed and in which most of the fermentation product (ethanol and acetate) is produced.
• gaseous substrates as used herein means CO alone, CO and H.sub.2, CO.sub.2 and H.sub.2, or CO, CO.sub.2 and H.sub.2, optionally mixed with other elements or compounds, including nitrogen and methane in a gaseous state.
• gaseous substrates include gases or streams, which are typically released or exhausted to the atmosphere either directly or through combustion.
• the gaseous substrate comprises CO.
• the gaseous substrate comprises CO.sub.2 and H.sub.2.
• the gaseous substrate comprises CO and H.sub.2.
• the gaseous substrate comprises CO, CO.sub.2 and H.sub.2.
• substrates of the invention may include those components mentioned above and at least one gas of nitrogen, CO.sub.2, ethane and methane.
• gases include what is conventionally referred to as “syngas” or synthesis gas from the gasification of carbon products (including methane), as well as waste gases from a variety of industrial methods.
• high concentration of ethanol means greater than about 10 g/L, preferably greater than 15 g/L ethanol in fermentation broth or a product ratio of ethanol to acetate of 5: 1 or more.
• limiting substrate or “limiting nutrient” define a substance in the nutrient medium or gaseous substrate which, during bacterial culture growth in the bioreactor, is depleted by the culture to a level which no longer supports steady state or stable bacterial growth in the bioreactor. All other substances in the nutrient medium or gas substrate are thus present in excess, and are "non-limiting".
• the evidence for limitation is that an increase in the rate of addition of the limiting substrate, i.e. in the nutrient feed rate or gas feed rate, to the culture causes a corresponding increase in the rate of gas uptake (mmol/min of gas) due to increase in cell density.
• microorganism includes bacteria, fungi, archaea, and protists; microscopic plants (called green algae); and animals such as plankton, the planarian and the amoeba. Some also include viruses, but others consider these as non-living. Microorganisms live in all parts of the biosphere where there is liquid water, including soil, hot springs, on the ocean floor, high in the atmosphere and deep inside rocks within the Earth's crust. Microorganisms are critical to nutrient recycling in ecosystems as they act as decomposers. Microbes are also exploited by people in biotechnology, both in traditional food and beverage preparation, and in modern technologies based on genetic engineering.
• mixed strain microorganisms that may or may not contain strains of various microorganisms, will be utilized in the present invention.
• recombinant DNA technology can create microorganisms using select strains of existing microorganisms.
• several exemplary strains of C. ljungdahlii include strain PETC (U.S. Pat. No. 5,173,429); strain ERI2 (U.S. Pat. No. 5,593,886) and strains C-Ol and O-52 (U.S. Pat. No. 6,136,577). These strains are each deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va.
• Each of the strains of C. ljungdahlii is an anaerobic, gram-positive bacterium with a guanine and cytosine (G+C) nucleotide content of about 22 mole %. These bacteria use a variety of substrates for growth, but not methanol or lactate. These strains differ in their CO tolerance, specific gas uptake rates and specific productivities. In the "wild" strains found in nature, very little ethanol production is noted. Strains of C. ljungdahlii operate ideally at 37.degree.
• ljungdahlii strains have been used to produce ethanol and acetyl with a product ratio of 1 : 1 (equal parts ethanol and acetyl), but the ethanol concentration is less than 10 g/L, a level that results in low productivity, below 10 g/Lday.
• culture stability is an issue, primarily due to the relatively high (8-10 g/L) concentration of acetyl (2.5-3 g/L molecular acetic acid) in combination with the presence of ethanol.
• the gas rate is increased in an effort to produce more ethanol, the culture is inhibited, first by molecular acetic acid and then by CO. As a result, the culture becomes unstable and fails to uptake gas and produce additional product.
• mixed strains it is meant a mixed culture of two or more of the microorganism.
• Such “mixed strains” of the microorganism enumerated hereinabove are utilized in the methods of this invention.
• natural state describes any compound, element, or pathway having no additional electrons or protons that are normally present.
• reduction state describes any compound, element, or pathway having an excess of one or more electrons.
• the “reduction state” is achieved by adding one or more electrons to the “natural state”, i.e. by lowering the redox potential of the fermentation broth.
• Nutrient medium is used generally to describe conventional bacterial growth media which contain vitamins and minerals sufficient to permit growth of a selected subject bacteria. Sugars are not included in these media. Components of a variety of nutrient media suitable to the use of this invention are known and reported in prior publications, including those of the inventors. See, e.g. the nutrient media formulae described in International Patent Application No. WO08/00558; U.S. Pat. No. 5,807,722; U.S. Pat. No. 5,593,886, and U.S. Pat. No. 5,821,111, as well as in the publications identified above.
• a typical laboratory nutrient medium for acetate production from CO, CO.sub.2, and H.sub.2 contains 0.9 mg/L calcium pantothenate.
• a typical laboratory nutrient medium for ethanol production from CO, CO.sub.2, and H.sub.2 contains 0.02 mg/L calcium pantothenate.
• reducing gas means either or both CO or H.sub.2.
• an amount of reducing gas greater than that required for growth of the bacteria' is mean that amount of reducing gas that exceeds the amount that the bacteria can use for growth or metabolism, given the nutrient medium ingredients. This amount can be achieved by increasing the net amount of reducing gas, or by reducing key nutrient ingredients, so that the excess amount of gas is achieved without increasing the gas, or by increasing the rate of gas delivery to the bacteria.
• the bacteria respond by increasing the producing of ethanol.
• Subject bacteria are acetogenic anaerobic (or facultative) bacteria, which are able to convert CO and water or H.sub.2 and CO.sub.2 into ethanol and acetic acid products.
• Useful bacteria according to this invention include, without limitation, Acetogenium kivui, Acetobacterium woodii, Acetoanaerobium noterae, Clostridium aceticum, Butyribacterium methylotrophicum, C. acetobutylicum, C. thermoaceticum, Eubacterium limosum, C. ljungdahlii PETC, C. ljungdahlii ERI2, C. ljungdahlii C-Ol, C. ljungdahlii 0-52, and Peptostreptococcus productus.
• Other acetogenic anaerobic bacteria are selected for use in these methods by one of skill in the art.
• syngas means synthesis gas which is the name given to a gas mixture that contains varying amounts of carbon monoxide and hydrogen.
• Examples of production methods include steam reforming of natural gas or liquid hydrocarbons to produce hydrogen, the gasification of coal and in some types of waste-to-energy gasification facilities. The name comes from their use as intermediates in creating synthetic natural gas (SNG) and for producing ammonia or methanol.
• Syngas is also used as an intermediate in producing synthetic petroleum for use as a fuel or lubricant via Fischer-Tropsch synthesis and previously the Mobil methanol to gasoline process.
• Syngas consists primarily of hydrogen, carbon monoxide, and very often some carbon dioxide, and has less than half the energy density of natural gas. Syngas is combustible and often used as a fuel source or as an intermediate for the production of other chemicals
• the present invention relates to methods for sustaining microorganism culture in a syngas fermentation reactor in decreased concentration or absence of various substrates comprising: adding carbon dioxide and optionally alcohol; maintaining free acetic acid concentration to less than 5 g/L free acetic acid; and performing the above mentioned steps within 0-30 minutes, within 0-15 minutes, within 15 -30 minutes.
• the present invention further contemplates a method for preventing rapid loss of microorganism culture in a syngas fermentation reactor in decreased concentration or absence of various substrates comprising: adding carbon dioxide and optionally alcohol; decreasing temperature from the operating temperature to between 0-25 degrees C while maintaining the temperature between 0-25 C; maintaining free acetic acid concentration to less than 5 g/L free acetic acid; and performing the above mentioned steps within 0-30 minutes, within 0-15 minutes, within 15 -30 minutes.
• the present invention further provides a method for sustaining microorganism culture in a syngas fermentation reactor due to decreased concentration or absence of various substrates in feed gas supply comprising: adding carbon dioxide and optionally alcohol; decreasing temperature from operating temperature to between 0-25 degrees C while maintaining the temperature between 0-25 C; maintaining free acetic acid concentration to less than 5 g/L free acetic acid; and performing the above mentioned steps within 0-30 minutes, within 0-15 minutes, within 15 -30 minutes.
• said sustaining microorganism culture comprises duration of about 0-30 hours.
• pH which can be maintained in the range of about 3.5-5.6.
• a bicarbonate solution is added to control pH.
• Bicarbonate solution can comprise: ammonium bicarbonate, sodium bicarbonate, and/or potassium bicarbonate.
• An embodiment of the present invention provides a method wherein optionally removing said carbon dioxide into the said reactor.
• optionally adding nutrients to said reactor is provided.
• the present invention provides optionally adding nutrients to said reactor.
• Further embodiments of the present invention provide alcohol comprising one or more of the following: ethanol, butanol, ethanol and butanol.
• the temperature can be decreased from the operating temperature to between 0-25 degrees C while maintaining the temperature between 0-25 C; optionally water can be added to said reactor.
• This water can comprise fresh water, make-up water, recycle water, distilled water, deionized water or their combinations.
• the present invention contemplates a method wherein said microorganism culture contains at least one acetogenic bacteria.
• the microorganism culture can comprise one or more strains selected from Clostridium, Moorella, and Carboxydothermus or their genetic modifications.
• the microorganism can comprise Clostridium ljungdahlii selected from the strains consisting of PETC, ERI-2, 0-52 and C-Ol or their combinations.
• the present invention also provides a method wherein microorganism culture is returned to pre suspension conditions comprising addition of syngas.
• the present invention can provide for: removing permeate; purging said reactor with inert gas; or maintaining low agitation to keep solids in suspension.
• Acetogenic autotrophic bacteria that utilize carbon monoxide and/or hydrogen and carbon dioxide (synthesis gas) to produce alcohol require a constant supply of the gas to produce alcohol.
• An interim product in the production of ethanol is acetic acid, which may be intercellular and extracellular. Without a sufficient synthesis gas supply, limited alcohol is produced in favor of acetic acid.
• the culture can convert alcohol back to acetic acid in the presence of carbon dioxide.
• Ethanol is already present in the culture broth and is readily available in the event of limited or no synthesis gas. Additional alcohol could also be supplied as needed.
• Carbon dioxide can be added by bubbling the CO2 gas into the culture or it can be formed in the culture broth by the addition of bicarbonate. Sodium bicarbonate can be used in the fermentation to maintain the desired pH and is therefore readily available. In the acidic culture broth the bicarbonate buffer reacts to form carbon dioxide. The formed carbon dioxide is then available to the bacterium to shift alcohol back to acetic acid.
• the equilibrium of acetyl to acetic acid is controlled in part by pH. Raising the pH during synthesis gas supply interruption permits the total acetyl concentration, acetyl plus acetic acid, to be higher while maintaining a lower free acid concentration.
• a third method with the potential for controlling the free acid concentration is an increased liquid flow through the system. As the free acid concentration increases, increasing the flow of a liquid stream into the system with an increase in the permeate purge will wash more free acid out of the culture while preventing unwanted cell washout.
• the additional liquid into the system may be an additional water stream or an increase in the flow of the nutrient supply stream.
• the present invention involves methods for the anaerobic fermentation of gaseous substrates containing at least one reducing gas, particularly the gaseous components of industrial waste and synthesis gases (e.g., CO, CO.sub.2 and H.sub.2) to ethanol. These methods yield ethanol productivities greater than 10 g/Lday by manipulating the biological pathways of the subject bacteria.
• One method of the invention causes an abundance of NAD(P)H over NAD(P).
• the oxidation of NAD(P)H to NAD(P) causes acetic acid produced by the culture to be reduced to ethanol.
• the inventors theorize that the methods for increasing the anaerobic production of ethanol from the methods described herein are based upon the biological pathways involving the conversion OfNAD(P)H to NAD(P) in the basic pathway cycles of the acetogenic pathway for autotrophic growth.
• the invention involves manipulating those pathways to enable continuous production and maintenance of high concentrations of ethanol with low acetate concentrations under stable operating conditions, thereby providing commercially useful methods for ethanol production from industrial gases.
• the essential involvement of NAD(P)H to NAD(P) in the biological pathways is described as follows: The production of ethanol from gaseous components, such as CO, CO.sub.2, and H.sub.2 occurs in a three step biological method. In the first step, the substrates CO and H.sub.2 are oxidized and, in doing so, release NAD(P)H: NAD(P).fwdarw.NAD(P)H
• step 1 The products of step 1 are then converted to acetic acid, a step that requires NAD(P)H: NAD(P)H.fwdarw.NAD(P) CO+CO.sub.2+6 H.sup.+.fwdarw.CH.sub.3COOH+H.sub.2O Finally, if excess NAD(P)H is available because the reaction of step 1 proceeds at a faster rate than the reaction of step 2, acetic acid is reduced to ethanol. NAD(P)H.
• the sequence for the generation of ethanol and acetic acid therefrom is illustrated in J. R. Phillips et al, 1994 Applied Biochemistry and Biotechnology, 45/46:145.
• the Acetyl-CoA cycle has an inner cycle, referred to herein as the CO cycle.
• the CO cycle normally reacts clockwise, ferredoxin is reduced.
• Ferredoxin can also be reduced by H.sub.2 as it is oxidized on the enzyme hydrogenase.
• the Acetyl-CoA cycle also reacts clockwise, and ferredoxin is oxidized.
• ferredoxin is in a redox-state equilibrium. If however, these two cycles do not occur at the same rate, i.e., the CO cycle reacts at a faster rate than the Acetyl-CoA cycle, reduced ferredoxin is built up. Also with excess H.sub.2, reduced ferredoxin can also be produced in excess. This excess reduced ferredoxin causes the NAD(P) to be regenerated (reduced) to NAD(P)H, which builds an excess that must be relieved to equilibrium and in doing so, reduces acetic acid to ethanol.
• the THF cycle functions for cell growth and is necessary for a continuous culture; therefore it cannot be completely stopped. Reducing the THF cycle rate also serves to cause a higher NAD(P)H to NAD(P) ratio.
• NAD(P)H is oxidized in two places. By limiting this oxidation, which would keep the total cellular NAD(P)H to NAD(P) ratio in balance, the NAD(P)H is used to reduce acetic acid to ethanol.
• a second basic method of causing acetic acid to be reduced to ethanol is by directly lowering the redox potential of the fermentation broth. A reduction state sufficiently lower than the natural state of the culture causes NAD(P)H to be in abundance and promote the reduction of acetic acid to ethanol.
• a continuous fermentation method with product recovery is described by reference to FIG. 1.
• a continuous flow of gaseous substrate 1 comprising at least one reducing gas, e.g., CO or H.sub.2, is supplied at a selected gas feed rate and a continuous flow of liquid phase nutrient medium 2 at a selected nutrient feed rate are supplied to a fermentation bioreactor 3 containing a subject bacteria.
• the medium and gaseous substrate are fermented by the bacteria to produce ethanol and acetate acid.
• the components of the continuous system are manipulated to reduce the redox potential, or increase the NAD(P)H to NAD(P) ratio, in the fermentation broth, while keeping the free acetic acid concentration in the bioreactor less than 5 g/L.
• the methods of this invention are designed to permit and maintain production of ethanol and acetate in the fermentation broth such that the ethanol productivity is greater than 10 g/Lday at an ethanol to acetate ratio of between 1:1 and 20:1. In one embodiment, that ratio is greater than 3:1. In another embodiment, that ratio is greater than 5:1. In still another embodiment, that ratio is greater than 10:1. In still another embodiment that ratio is greater than 15:1.
• the method of this invention is alternatively effective in enhancing stable continuous (steady state) production of high ethanol concentrations (15-35 g/L ethanol) and low acetate concentrations (0-5 g/L acetate), i.e., ethanol to acetate product ratio of 3:1 or more, from CO, CO.sub.2, and H.sub.2 with good method stability.
• samples of the broth are removed to determine the ratio by a conventional assay method.
• the cells are separated from the sample, e.g., by centrifugation and the cell-free sample is then subject to an assay method, such as the preferred method of gas chromatography.
• an assay method such as the preferred method of gas chromatography.
• other conventional assay methods are selected by one of skill in the art.
• the additional optional steps of the method are added to achieve and/or maintain the ratio.
• Steps used to manipulate the system components and maintain and/or achieve the desired ethanol productivity or the ethanol to acetate ratio include at least one, and desirably, combinations of the following steps: altering nutrient medium contents, nutrient feed rate, aqueous feed rate, operating pressure, operating pH, gaseous substrate contents, gas feed rate, fermentation broth agitation rate, avoiding product inhibition step, decreasing cell density in the bioreactor, or preventing substrate inhibition.
• Some preferred manipulations include supplying the bioreactor with liquid phase nutrient (pantothenate or cobalt) limitation, a slight excess of CO and H.sub.2 in the feed gas, minimizing acetate concentration, avoiding culture acclimation to low liquid phase nutrient concentrations, bringing the culture to a suitable cell concentration at a relatively fast rate, raising the pH of the culture above 4.5, purging bacterial cells from the bioreactor to a cell concentration less than the stable steady state concentration that utilizes all reducing gas or nutrient substrates in the bioreactor and increasing the aqueous feed rate when the free acetic acid portion of the acetate present in the fermentation bioreactor broth exceeds 2 g/L, thereby inhibiting any unwanted increase in the concentration of free acetic acid. All of these steps are described in detail below.
• Exhaust gas 4 containing gases other than CO, CO. sub.2 and H.sub.2 and unconverted CO, CO.sub.2 and H.sub.2 from the reactor are vented from the reactor and are used for their fuel value. If excess H.sub.2 as a controlling mechanism is employed, the H.sub.2 partial pressure in the outlet gas and ratio of H.sub.2 partial pressure to CO.sub.2 partial pressure in the exit gas are used to identify the control of the ethanol to acetate ratio by that step. Cell recycle is used (but is not required) to increase the concentration of cells inside the bioreactor, and thus provide more biocatalyst for CO,
• the cells 7 are sent back to the bioreactor and the permeate 8 is sent to product recovery.
• Cell separation is accomplished by using a continuous centrifuge, hollow fiber or spiral wound filtration system, ceramic filter system or other solid/liquid separator. Ethanol can be recovered from the permeate (or alternatively the effluent from the reactor 5 if cell separation is not employed) by a variety of techniques including distillation and adsorption.
• Permeate 8 is separated in a distillation column to produce 95% ethanol overhead 10, and water 11 for recycle back to the reactor 3.
• the recycle water 11 contains excess nutrients not used in the fermentation, but any excess vitamins from fermentation or cell lysis are destroyed by thermal distillation.
• the 95% ethanol overhead 10 is sent to a molecular sieve 12 where anhydrous ethanol 13, the desired final product, is separated from dilute ethanol 14 which is sent back to the distillation column 9.
• the continuous combination of growth, death and cell purge maintains a constant cell concentration, such that a continuous method used in producing ethanol (and small amounts of acetic acid) can operate for many months by being fed CO, CO.sub.2 and H.sub.2 along with nutrients without additional culture supplementation.
• the methods of this invention maintain and control conditions for continuous ethanol and acetic acid production and prevent or correct rapidly for method upsets.
• the methods of this invention also help prevent culture acclimation to low nutrient concentration, which can be detrimental to culture performance.
• the pressure used is 1 atmosphere and the temperature used is between 36-41. degree. C. Desirable temperatures and pressures may be determined by one of skill in the art, depending on the microorganism selected for use in the bioreactor.
• liquid phase nutrient limitation pantothenate or cobalt
• excess H.sub.2 or CO are the method steps of the invention, described in detail below, used to achieve and maintain the desired ethanol productivity and permit production of stable concentrations and ratios of ethanol to acetate in the fermentation broth.
• the ethanol to acetate product ratio produced in the fermentation broth is greater than 10:1 and the ethanol concentration is greater than 15 g/L.
• the method for manipulating the biological pathways to favor ethanol production and limit acetic acid production involves limiting the amount of calcium pantothenate in the nutrient medium to an amount which is less than required to maintain the bacteria at a stable, steady state concentration that would fully utilize the calcium pantothenate provided.
• Pantothenate is a component of Acetyl-CoA and therefore, by limiting calcium pantothenate in the nutrient medium, the Acetyl-CoA cycle rate is reduced relative to the CO cycle rate. This causes a build-up of reduced ferredoxin and the reduction of NAD(P) to NAD(P)H, and thereby increases the production of ethanol as the final product.
• Pantothenate limitation is observed when the micrograms (.mu.g) of calcium pantothenate fed to the reactor per gram (g) of cells (dry weight) produced in the reactor is in the range of 0.5 to 100.
• a more desirable pantothenate limitation is in the range of 2 to 75 .mu.g of calcium pantothenate per gram (g) of dry cells produced in the reactor.
• Still a preferred pantothenate limitation is in the range of 0.5 to 50 .mu.g of calcium pantothenate per gram (g) of cells produced in the reactor.
• Another embodiment of this limitation is at about 1-25 .mu.g of calcium pantothenate per grain (g) of cells produced in the reactor.
• Another embodiment of this limitation is at about 10-30 .mu.g of calcium pantothenate per gram (g) of cells produced in the reactor. This amount of the nutrient maintains ethanol production in preference to acetate production.
• the acclimation of the bacteria in the fermentation bioreactor to low limiting calcium pantothenate concentration is avoided by regulating or adjusting the fermentation parameters, so that a constant calcium pantothenate concentration is maintained, while at least one, and sometimes more than one, parameter of gas feed rate, liquid feed rate, agitation rate, or H.sub.2 partial pressure is adjusted.
• Major changes in nutrients are avoided, but a relatively constant nutrient feed concentration is maintained.
• the biological pathway is controlled to favor ethanol production and limit acetic acid production by first supplying excess H.sub.2 in the feed gas to the bioreactor, and then limiting calcium pantothenate in the nutrient medium as described above.
• the method for manipulating the biological pathways to favor ethanol production and limit acetic acid production involves limiting the amount of cobalt in the nutrient medium to an amount which is less than required to maintain the bacteria at a stable steady state concentration that would fully utilize the cobalt provided.
• Cobalt limitation is observed when the micrograms (.mu.g) of cobalt fed to the reactor per gram (g) of cells (dry weight) produced in the bioreactor is in the range of 5 to 100.
• a cobalt limitation involves providing between about 20 to 50 .mu.g of cobalt to the reactor per gram of cells produced in the reactor. This amount of cobalt maintains ethanol production in preference to acetate in the process.
• Limiting cobalt in the fermentation broth may also reduce the Acetyl-CoA cycle rate. Because cobalt is used to transfer a methyl group from the THF cycle to the Acetyl-
• CoA cycle limiting the amount of cobalt in the fermentation broth also reduces the THF cycle function by not permitting the transfer.
• Cobalt limitation reduces the THF cycle rate, which also causes a higher NAD(P)H to NAD(P) ratio, thereby producing ethanol.
• the method is further manipulated by preventing acclimation to low limiting cobalt concentration.
• acclimation to low pantothenate concentrations In much the same manner as acclimation to low pantothenate concentrations is avoided, a constant cobalt concentration is maintained while adjusting one or more of the fermentation parameters (gas rate, liquid rate, agitation rate, CO.sub.2 content, and H.sub.2 gas partial pressure). Major changes in nutrients is avoided, but instead a relatively constant nutrient feed concentration is maintained.
• the biological pathway is controlled to favor ethanol production and limit acetic acid production by first feeding excess H.sub.2 to the reactor and then limiting cobalt in the nutrient medium as described above.
• the limiting liquid phase nutrient cobalt is kept in excess to avoid acclimation to low nutrients concentration, a condition that can result in very poor culture performance and the loss of the culture's ability to produce product ratios greater than 1:1.
• the method for manipulating the biological pathways to favor ethanol production and limit acetic acid production involves feeding excess
• the H.sub.2 reducing gas is in excess relative to CO, and the excess H.sub.2 causes the bacteria to produce a high ethanol to acetate ratio in the fermentation broth. If the ratio of the H.sub.2 (moles of gas fed) to the sum of two times the CO (in moles of gas) converted and three times the CO.sub.2 (in moles of gas) converted is greater than 1, the fermenter is carbon limited.
• the H.sub.2 partial present in the exit gas is preferably greater than 0.4 atm. Finally the ratio of
• H.sub.2 partial pressure to CO.sub.2 partial pressure must be greater than 3.0 to assure that sufficient H.sub.2 is available to use all the CO.sub.2. If the CO.sub.2 partial pressure is greater than 0.1 atm, it is likely that growth has been otherwise limited.
• the bioreactor is suppled with gaseous substrate comprising CO where the amount of CO present in the bioreactor is greater than the amount required to maintain the bacteria at a stable, steady state concentration that would fully utilized the CO provided.
• CO oversupply as a method of favoring ethanol production over acetic acid production when the specific rate of CO uptake (millimoles of CO per gram of cells (dry weight) in the reactor per minute, or mmol/g cellmin) is greater than 0.3.
• this step involves a specific rate of CO uptake of greater than 0.5.
• each cell on the average is utilizing CO in its metabolism at a rate of at least 0.3 mmol/gmin., or more ideally at a rate of at least 0.5 mmol/gmin.
• the CO is provided at a rate at which the CO uptake is from 0.3 to 2 mmol CO/gram cell (dry weight) of bacteria/minute.
• the CO is provided at a rate of from 0.5 to 1.5 mmol CO/gram cell (dry weight) of bacteria/minute.
• the CO is provided at a rate of about 1 mmol CO/gram cell (dry weight) of bacteria/minute.
• This rate of CO uptake maintains ethanol production in preference to acetate production. If CO is supplied such that the dissolved CO in the fermentation broth is significant by gas pressure or extremely good mass transfer, the fermentation broth becomes more reduced. Oversupply of CO has two additional benefits. Excess CO may cause the CO cycle to operate at a faster rate, and if the Acetyl-CoA cycle is otherwise limited and cannot keep up with the CO cycle, reduced ferredoxin builds-up. CO may also slow down step 2 (production of the intermediate acetic acid) in the overall three- step method through substrate inhibition. This decreased rate of step 2 in relation to step 1 causes an excess of NAD(P)H, which leads to ethanol production in favor of acetic acid.
• One such additional embodiment involves ensuring that the mass transfer of the CO or H.sub.2 from the gas feed to the liquid fermentation broth is faster than the ability of the bacteria to utilize the dissolved gases. For example, if a bioreactor containing C. ljungdahlii is fed CO, CO. sub.2 and H.sub.2 and is operated without limitation on nutrients (such as pantothenate or cobalt) or the presence of excess H.sub.2, cell growth is limited by the amount of gas transferred into the liquid phase and the system produces acetic acid as the product.
• the culture is fed a slight amount of CO or H.sub.2 in excess of that required for culture growth, it produces ethanol.
• too much gas is transferred into the liquid phase for the culture to use, substrate inhibition occurs, which can lead to culture upset and cell death.
• the CO cycle or the reduction of ferredoxin through hydrogenase must occur faster than the Acetyl-CoA cycle.
• the methods described herein limit the rate at which the organisms can utilize the dissolved gases by restricting the rate at which essential nutrients e.g., calcium pantothenate or cobalt, or other substrates, such as CO.sub.2 gas, are available to the bacteria, or by providing excess substrate, H.sub.2 or CO to the culture.
• essential nutrients e.g., calcium pantothenate or cobalt, or other substrates, such as CO.sub.2 gas
• stability in the high ethanol concentration/limited acetic acid production is achieved in the methods which limit cobalt or calcium pantothenate, or provide an abundance of H.sub.2 or CO.
• CO and H.sub.2 are supplied in slight excess.
• a slight excess of CO and H.sub.2 is achieved by attaining steady operation and then gradually increasing the gas feed rate and/or agitation rate (10% or less increments) until the CO and H.sub.2 conversions just start to decline.
• Steps to avoid mass transfer limitation include an increase in the agitation rate or gas rate to transfer more CO and H.sub.2 into the liquid phase, and thus return to the presence of a slight excess CO and H.sub.2. If product inhibition occurs as a result of mass transfer limitation, it is necessary to increase the liquid feed rate to clear the acetic acid inhibition, by diluting to a lower resulting acetate concentration. Since increasing the medium feed rate would increase the .mu.g pantothenate or cobalt/g-cell produced, this must be done only briefly or the excess pantothenate or cobalt must be eliminated by adjusting the medium concentration or increasing the water feed rate.
• acetic acid product inhibition can occur if too much molecular acetic acid, i.e., >2 g/L, accumulates in the bioreactor to allow cell growth and further ethanol production. Another manipulating step is used to avoid culture failure.
• One modification involves briefly increasing the liquid or aqueous feed rate to reduce the liquid phase concentration of inhibiting acetic acid to lower than 2 g/L. 4. Water Recycle Step
• Still another optional method step for maintaining a stable culture which produces ethanol as the only product with no net acetic acid production in the methods of this invention involves adding water recycle from distillation back to the fermentation reactor.
• water (containing up to 5 g/L acetate) recycle has the benefit of recycling the produced acetate back to the reactor so that no net acetic acid is produced.
• An equilibrium is thus established between the ethanol and acetate in the reactor.
• all CO, CO.sub.2 and H.sub.2 fed to the reactor and converted to products results in ethanol production, except for that used for culture maintenance. 5.
• Still another manipulating step useful in the method is to initiate periodic or continuous purging of bacterial cells from the bioreactor to reduce the cell concentration in the bioreactor.
• This manipulation serves to reduce the cell concentration to less than a stable, steady state cell concentration that utilizes all reducing gas or nutrient substrates in the bioreactor. By thus, altering the cell density, the production of ethanol is favored over the production of acetate in the bioreactor. 6.
• This modification involves operating a two-stage CSTR, in which a growth reactor (Stage 1) to feed a production reactor (Stage 2) where the bulk of the ethanol production occurs.
• the growth reactor is not operated with the nutrient limitation steps described above, so the culture is not as susceptible to acclimation to a limited condition.
• the Growth Stage is operated at a liquid retention time (LRT) of about 24 hours.
• the Growth Stage CSTR 1 is fed enough pantothenate or cobalt in the medium 2 to yield a healthy culture (and may produce some acetic acid as well). Thus, excess acetic acid is produced in the reactor, but with increased stability. This pantothenate or cobalt concentration is in excess of what would normally be fed to a single CSTR used to produce ethanol.
• the gas feed to this reactor is unconverted gas 3 from the Production Stage 4 and the liquid feed is fresh medium 2.
• the Growth Stage CSTR is operated without cell recycle. The purpose of this Growth Stage reactor is to provide a healthy culture for later ethanol production that does not acclimate to low pantothenate concentrations.
• the Production stage reactor 4 is operated at a nominal LRT of less than 20 hours.
• This CSTR with cell recycle is fed a fresh gas feed 5, and may have low conversions. It is fed fresh medium feed 6 as well as culture feed 7 from the Growth Stage. Minimal pantothenate or cobalt is fed to this reactor since the excess from the Growth Stage is available.
• Cell recycle 8 is used in this reactor in order to get the most production out of the cells sent back to the reactor 9.
• the exit ethanol concentration in the liquid product 10 should be greater than 20 g/L.
• the features of the two-stage CSTR system include little change for acclimation to low pantothenate or cobalt concentrations; an overall LRT of less than or equal to 30 hours; an expected greater ethanol productivity and higher ethanol concentration than from a single CSTR of the same size. 7.
• Start-up Modifications Still other method steps, which are preferably utilized in the practice of this invention, involve cell production in the initial start-up of the fermentation culture.
• the start-up of a bioreactor fed CO, CO.sub.2 and H.sub.2 to produce ethanol and acetic acid is accomplished by batch inoculation from stock culture or by employing a continuous inoculum from an existing reactor as culture feed.
• the culture is most desirably brought up to a high cell concentration prior to limiting nutrients, but supplying excess H.sub.2 to the culture.
• This rapid start-up avoids culture acclimation and yields good product ratios (high ethanol and low acetate concentrations). If the rapid start-up is not employed, poor product ratios can occur and the culture can acclimate to low liquid phase nutrient concentrations and require reactor reinoculation.
• the reactor is started with a batch liquid phase (liquid medium is not initially fed continuously to the reactor), at low agitation rates (perhaps 400-600 rpm in a laboratory New Brunswick Scientific Bioflo.RTM.
• the liquid phase in the reactor thus consists of a batch of nutrient medium containing vitamins and salts, with a nominal concentration of limiting nutrient, either calcium pantothenate or cobalt (20 .mu.g/L pantothenate or 75 ppb cobalt). If continuous inoculum from an existing reactor is employed, batch liquid phase operation likely is not necessary. In this case, gas is fed continuously to the reactor during initial start-up at a slow rate. Ideally, the gas phase at start-up would be CO.sub.2-free, H.sub.2-abundant and the gas rate and agitation rate would be kept at low levels to avoid CO substrate inhibition.
• An exemplary general start-up protocol for producing and sustaining commercially viable ethanol concentrations from CO, CO.sub.2 and H.sub.2 consists of three distinct phases: (a) initial start-up, where cell production is critical; (b) start-up where production rate becomes critical; and (c) steady state operation.
• initial start-up is characterized by inoculation of a batch liquid, with a nominal limiting nutrient, selected from cobalt (75 ppb) or calcium pantothenate (20 .mu.g/L) at a desired pH (typically 4.5-5.5).
• the gas feed rate and agitation rate are preferentially kept low, while H.sub.2 is fed in excess.
• the culture is allowed to steady to confirm H.sub.2 uptake.
• the start-up shifts to a mode in which production rate becomes important. It is desirable to have CO conversions exceeding 80% and a high H.sub.2 partial pressure in the exit gas (at least 0.55 atm) to assure ethanol production while limiting acetate and the free molecular acetic acid concentration.
• the liquid medium feed rate is then turned on (for systems having batch inoculation from stock culture) to initiate continuous liquid feed and the gas rate is increased in 10% increments toward the target flow rate.
• H.sub.2 remains in excess to avoid excess acetic acid production.
• the liquid phase nutrients are limited (calcium pantothenate or cobalt), and the effect of such limitation is a small drop in H.sub.2 conversion, at the target production.
• the above described methods of continuously producing and maintaining high concentrations of ethanol with low by-product acetate concentrations under stable operating conditions enhance the use of the subject bacteria on a commercial scale for ethanol production.
• the steps outlined in the methods above overcome the limitations of utilizing the subject bacteria for commercial ethanol production from CO, CO.sub.2 and H.sub.2.
• the method employs a continuous bioreactor, although batch and fed- batch fermentation methods are also used, but are not likely to be economically viable for large-scale ethanol production.
• the following examples will serve to illustrate certain specific embodiments of the inventions herein disclosed. These Examples should not, however, be construed as limiting the scope of the novel invention as there are many variations which may be made thereon without departing from the spirit of the disclosed invention, as those of skill in the art will recognize.
• the measured ethanol and total acetyl concentrations had changed as was expected.
• the ethanol level decreased from 24.0 to 12.8g/L while the total acetyl level increased from 4.2 to lOAg/L.
• the temperature set point was returned to 38°C.
• the agitation was increased to the same level used prior to the experiment; the carbon dioxide was replaced with synthesis gas flow at 50 percent the flow rate used prior to the experiment.
• the permeate purge was stopped.
• the culture was maintained in this condition for 14 hours.
• the carbon monoxide uptake remained stable and the hydrogen uptake steadily improved.
• the gas flowrate was stepwise increased to reach the pre-experimental flowrate.
• the synthesis gas flow rate had returned to pre-experimental rates.
• the total acetyl concentration was decreasing and the ethanol concentration increased.
• the total acetyl concentration was back down to pre- experimental levels within 32 hours.
• the ethanol concentration reached near pre- experimental levels within 70.5 hours.
• the carbon dioxide was provided by a continuous flow of a 7.7% sodium bicarbonate solution into the culture.
• the temperature was reduced to 25°C.
• Free acetic acid concentration was controlled by lowering the temperature to 25°C, increasing pH and by increasing the liquid flow through the culture.
• the synthesis gas was turned off and replaced with a continuous flow of 7.7% sodium bicarbonate.
• sodium bicarbonate degrades into a sodium ion, water and carbon dioxide thus providing the necessary carbon dioxide for the conversion of ethanol to free acid. Agitation was decreased to a low level that provided just enough mixing to distribute heat and liquid additions into the reactor.
• the pH set point was not controlled, but as the bicarbonate was added to the culture, the pH slowly increased throughout the experiment which helped control the concentration of free acid.
• the reactor had a cell recycle loop using a hollow fiber membrane, which permitted a permeate purge to be used in order to prevent cell loss during the experiment.
• the permeate flow was equal to the flow of medium plus the additional flow of sodium bicarbonate into the system.
• the extra bicarbonate flow reduced the liquid retention time from 29 to 21 hours.
• the ethanol concentration decreased as the total acetyl concentration rose steadily. Within 5.5 hours the ethanol concentration had decreased from 21.0 to 14.1 g/L while the total acetyl level had increased from 4.4 to 9.1g/L.
• the measured pH had also increased from 4.48 to 4.84.
• the nutrient stream flow rate was increased from 1.33mL/min to 2.81 mL/min., 5.6 hours after the start of the experiment.
• the permeate purge was also increased from 1.86 to 3.48mL/min to prevent unwanted cell washout.
• These changes decreased the liquid retention time from 21 to 12 hours. This had the desired effect of holding the acid concentration down.
• Two hours after the changes in liquid flows the total acetyl concentration had increased to only 9.4g/L.
• the ethanol concentration dropped at a faster rate from 14.1 to 10. 7g/L. After 8 hours of no synthesis gas supply, the measured ethanol and total acetyl concentrations changed as expected.
• the ethanol level decreased from 21.0 to 10.7g/L while the total acetyl level increased from 4.4 to 9.4g/L.
• the temperature set point was raised back to 38°C.
• the agitation was increased to the same level used prior to the experiment; the sodium bicarbonate addition was stopped, synthesis gas flow was started at 50 percent the flow rate used prior to the experiment; and the permeate purge was stopped.
• the culture was maintained in that condition for only 50 minutes.
• the gas flow rate was stepwise increased to reach the prior flow rate. Within 29.2 hours the synthesis gas flow rate had returned to pre-experimental rates.
• the total acetyl concentration decreased and the ethanol concentration increased to pre- experimental concentrations within 43.2 hours.
• ethanol that is already in the fermenter can thus be used along with carbon dioxide to maintain culture viability during synthesis gas interruption.
• the purpose of the microorganism experimentation was to determine a method of sustaining culture in the event of a feed gas loss for an extended (>30 minute) period of time.
• the focus was on CO2 addition for the conversion of ethanol to free acid as a way for the culture to gain energy during the loss of synthesis gas. It has been known for quite some time that certain acetogenic microorganisms can convert ethanol back to acetic acid using CO2, but no testing had been done to determine if this process could be used to sustain the culture for long periods of time when there was no synthesis gas available.
• An embodiment of the present invention provides a solution for surviving a loss of feed gas since ethanol and CO2 (in the form of sodium bicarbonate) are readily available for use due to normal bioreactor operations.
• the culture temperature was decreased during some of the experiments as a way to slow culture activity. A slower cell activity should reduce the amount of energy needed, the amount of CO2 and ethanol required, and the amount of acid produced.
• the bioreactor was run as a straight through CSTR with both a cell recycle and culture cooling coil loop.
• a permeate purge was used during the experiments to prevent unwanted cell loss, but the purge stream was diverted to waste and was not recycled back into the bioreactor.
• the culture temperature was kept 38°C; agitation was 400rpm; the approximate culture volume was about 2.4L; and the culture pH set point was 4.5.
• a solution of 7.7% NaHCO3 was used for pH control.
• the feed gas was synthesis gas containing 15%H2, 45%N2, 30%CO and 10% CO2.
• the syngas feed rate was about 475mL/min.
• Medium was fed into the reactor at about 1.30-1.35mL/min, or about 1870-1940mL/day. Liquid and cell retention times averaged 25-30 hours.
• the medium used was the Ix EtOH medium regularly used for the C-Ol culture. Medium components and their concentrations are listed in Table 1 below.
• the cells can advantageously gain electrons for survival from the oxidation of the alcohol to the carboxylic acid form. If feed gas is interrupted, the culture typically gains electrons, while not gaining carbon thus decreasing cell growth. It is therefore believed that this process provides a means of culture survival, though not optimized production. Cell washout, or the removal of any cells from the system, should be avoided in order to maintain the cell density during the feed gas loss. This process leads to a buildup of acid. Steps must be taken to insure that the free acetic acid levels are maintained to concentration levels below inhibiting concentrations ( ⁇ 5g/L).
• culture temperature plays vital role in this process as a way to control the metabolic rate of the cells.
• the cell's metabolic rate slows. That, in turn, slows the production of acid and the use of ethanol and CO2.
• Lowering the temperature when the reactor is without feed gas extends the length of time the culture can survive.
• the temperature is kept at 38°C the acid production rate is at its highest and careful monitoring of the acid level and ethanol level is required to keep the culture healthy.
• Experiments have lowered the culture temperature to about 25°C, successfully maintaining the cell viability for about 30 hours without feed gas.
• An embodiment of the present invention provides a delivery method of a controlled amount of CO2 comprising NaHCO3 addition.
• a delivery method of a controlled amount of CO2 comprising NaHCO3 addition When sodium bicarbonate is introduced into an acid environment like the fermentation broth CO2 is produced as shown below in Equation 2. It is believed that this method of CO2 addition to the system is advantageous over sparging CO2 into the culture because the sodium bicarbonate not only adds CO2 but also increases the culture pH to about 5.1, helping to compensate for the system production of acid by balancing the free acid levels.
• NaHCO3 + H+ O Na+ + H2CO3 ⁇ > Na+ + H2O + CO2(g) Equation 2
• the conversion of ethanol to acid starts to take place almost immediately, or within seconds, after the loss of the feed gas. It is possible to prevent a quick and large buildup of acid at the start of the feed gas loss by stripping the dissolved CO2 present in the culture using a high N2 flow of about 400-450mL/min. Nitrogen should be sparged through the culture for approximately 0-15 minutes as soon as possible after the feed gas flow is lost, the faster this is done the more advantageous it is for the present invention. Nitrogen flow within the first 5 minutes is an embodiment of the present invention. Once the inventory of dissolved CO2 has been removed, the NaHCO3 addition can be started using a controlled feed rate.
• Using NaHCO3 addition to provide CO2 can increase the culture pH. If the cells remain active utilizing all of the sodium bicarbonate available, the pH should increase to about 5.1 then remain there. This is a desired secondary effect and should not be prevented. The slow and steady increase in pH will help to counter the rising acid production by keeping the free acid level in check. If however, the cell activity is compromised, the pH will increase beyond about 5.3 giving an indication that the culture may be denatured or otherwise functionally decreased.
• the sodium bicarbonate addition should be stopped and the mass transfer of the feed gas should be increased as quickly as possible, but taking care not to overwhelm the cells.
• the agitation should be increased back to the same setting used prior to the feed gas loss and the feed gas flow rate increased back to about 50% of the original feed rate. Because of the availability of substrate and the high level of total acetyl the culture will steadily convert the acid back into ethanol. This will be reflected in an increase in pH and is expected. Changes to the fermentor's feed gas flow rate during this time should be made based on gas conversions as in any normal reactor operations.
• the feed gas flow rate should be able to reach a normal operating setting within about 20-26 hours.
• the ethanol and acid concentrations can take longer to reach normal operating levels, about 26-72 hours.
• a minimum agitation is required to maintain the temperature distribution throughout the bioreactor and to maintain the culture pH.
• Minimum agitation would be defined as just enough mixing to keep the liquid distributed. This can be about 50rpm, or 40-60rpm, as compared to high rates, such as 400rpm, used during normal operations. This agitation in effect also keeps the cells suspended. It is believed that the cells should be suspended in order to provide constant contact with CO2 and ethanol to perform the needed reactions.
• Acetogenic microorganisms require CO or H2 and CO2 in order to gain the necessary electrons and carbon for cell growth. During the periods of time without feed gas neither CO nor H2 are available for the cell growth process. It is believed that cell growth is suspended during those times of feed gas loss. It may be reasoned that a lower supply of feed gas could be used for culture survival during times in which the feed gas supply may be limited. Diminished amount of feed gas supplied provides the culture reversion to acid production mode. When the substrate feed rate is decreased, the culture will automatically stop the conversion of acid to ethanol causing an increased drop in the ethanol to acid ratio.
• Na+ concentration is from NaCl only. It does not include Na+ from the other components such as Na2WO4 • 2H2O.
• Ca+2 concentration does not include calcium from pantothenic acid, calcium salt.
• Table 2 details the culture parameters before and after the experiment such as pH, redox, ethanol and acetic acid. Generally, when the culture uses ethanol and CO2 for survival, the ethanol level decreases as the acid concentration and culture pH increases.
• Table 2 also lists the high measured level of free acid during those experiments as well as the number of hours after the end of the experiment to recover to the original gas feed rate. It should be remembered that a key component in culture survival during the feed gas loss is the maintenance of a free acid concentration ⁇ 5.0g/L. As acid is being produced, a higher culture pH and a faster acid removal rate is required to prevent acid inhibition. As acid is being produced, a higher culture pH or a faster acid removal rate is required to prevent acid inhibition.
• Table 3 details the CO2 addition to the culture in mmol/min per gram of cells in the culture. The calculations were based on the feed rate of sodium bicarbonate and the total number of cells in the bioreactor. At 25°C a CO2 feed rate of 0.014mmol/min g was sufficient to sustain the culture for 12 hours without feed gas. When the experimental length of time was increased to 24 hours, an average CO2 feed rate of
• the culture cell density was 3.7g/L; pH was 4.44; redox was -44OmV; CO and H2 uptake was 5.0 and 1.2mmol/min respectively; CO and H2 conversions were 86 and 40% respectively; ethanol was 23.5g/L; and acid was 3.9g/L.
• the feed gas flow rate was decreased from 474mL/min to 53mL/min.
• the agitation was dropped from about 400 to about 50rpm, and the temperature set point on the reactor was decreased from 38 to 25°C within about 12 minutes.
• 38.5g/L sodium bicarbonate as started at 0.57mL/min providing a 0.030mmol/min per gram of cells CO2 feed rate; the feed gas flow was stopped; a permeate purge was started at 1.95mL/min, and the medium flow was kept at 1.37mL/min. Nitrogen was slowly added to the reactor headspace to prevent a vacuum from forming in the reactor. The culture was left in that condition for 16.9 hours.
• the temperature set point on the reactor was increased back to about 38°C; the feed gas was restarted at about 53mL/min; Medium B and permeate purge were stopped; and the N2 flow into the reactor headspace was stopped.
• the feed gas flow rate was increased to 143mL/min.
• the feed gas flow was increased again to 236mL/min, or 50% or the original gas flow rate.
• the agitation was increased to 200rpm.
• the agitation was increased to about 400rpm.
• the culture cell density was about 3.2g/L; pH was about 4.50; redox was about -425mV; CO and H2 uptake was 4.7 and 1.5mmol/min respectively; ethanol was about 17.7g/L; and acid was about 2.93g/L.
• the feed gas flow rate was decreased from about 474mL/min to 53mL/min.
• the agitation was dropped from about 400 to about 50rpm, and the temperature set point on the reactor was decreased from about 38 to about 25°C in about 14 minutes.
• the sodium bicarbonate addition was started using a about 38.5g/L NaHCO3 flow of O.58mL/min providing a CO2 feed rate of
• the culture condition provided: cell density of about 2.9g/L; pH of about 5.04; EtOH of about 4.10g/l; and acid was about 8.68g/L.
• the cell morphology showed about 10-15% of the cells had turned grainy or almost grainy.
• the temperature set point on the reactor was increased back to about 38°C; the feed gas was restarted at about 53ml/min; Medium B and permeate purge were stopped; and the N2 flow into the reactor headspace was stopped. Medium was changed to a normal Ix EtOH medium with no ethanol added.
• the feed gas and agitation were increased at regular intervals as the temperature increased stepwise. When the temperature reached about 28.0 0 C, the feed gas flow rate was increased to about 179mL/min. At about 30.0°C the feed gas flow was increased again to about 248ml/min, or about 50% or the original gas flow rate. At about 32.0°C, the agitation was increased to about 200rpm. At about 34°C the agitation was increased to about 400rpm.
• initial conversions about 35 minutes after the increase in gas, agitation and temperature were at 60%H2 and 84%CO. As an embodiment, approximately 15min later the conversions provided: 62%H2 and 91%CO. Gas flow rate increases were introduced immediately. In this case, it took about 19.5 hours to reach the maximum gas flow used prior to the start of the experiment.
• the 24 hour gas loss experiment showed that the culture can survive very well for about 24 hours without feed gas while providing 0.035mmol/min CO2 addition per gram of cells.
• the medium and sodium bicarbonate flows into the reactor during the experiment required about 2.6L of permeate to be removed to prevent cell loss due to washout. That is slightly more than the 2.4L of culture volume.
• the waste water amount must be monitored and, if needed, decreased.
• all parameters were kept the same as the previous, experiments except the medium flow rate was reduced by half to reduce the amount of permeate purge that is required. There have been some indications in past experiment that suggest that a reduction in the cysteine feed rate may interfere with the experiment, so during this experiment the cysteine concentration in medium was doubled to retain the cysteine feed rate.
• the culture cell density was about 2.5g/L; pH was about 4.50; redox was about -44OmV; CO and H2 uptake was about 4.8 and about 1.2mmol/min respectively; ethanol was about 21.3g/L; and acid was about 2.96g/L.
• the feed gas flow rate was decreased from 474mL/min to 53mL/min.
• the agitation was dropped from about 400 to about 50rpm, and the temperature set point on the reactor was decreased from 38 to 25 0 C within 13 minutes.
• the sodium bicarbonate addition was started using a 38.5g/L NaHCO3 solution at 0.57ml/min; the feed gas flow was stopped; a permeate purge was started at 1.20ml/min, and the medium flow was reduced to 0.68ml/min. Nitrogen was slowly added to the reactor headspace to prevent a vacuum from forming in the reactor.
• the cysteine concentration was increased to 5g/L in medium A.
• the CO2 was provided at 0.039mmol/min per gram of cells. The culture was maintained in that condition for 24 hours.
• This experiment evaluates the minimum CO2 addition rate needed to sustain the culture for 12 hours at 25°.
• NaHCO3 solution used as Medium B was decreased in concentration while keeping all other experimental parameters the same.
• the NaHCO3 concentration was dropped to about 19.3g/L providing a CO2 feed rate of about 0.014mmol/min CO2 per gram of cells in the reactor. This is a low feed rate with culture survival.
• the culture cell density was about 4.0g/L; pH was about 4.43; redox was about -43OmV; CO and H2 uptake was about 4.9 and about 1.3mmol/min respectively; CO and H2 conversions were about 86 and about 44% respectively; ethanol was about 18.9g/L; and acid was about 3.8g/L.
• t 9580.7 hours, the feed gas flow rate was decreased from about
• the temperature set point on the reactor was increased back to about 38°C; the feed gas was restarted at about 53mL/min; Medium B and permeate purge were stopped; and the N2 flow into the reactor headspace was stopped. While the culture warmed over the next 15 minutes, the feed gas flow rate and agitation were increased stepwise. When the temperature reached about 28.0 0 C, the feed gas flow rate was increased to about 178mL/min. The pH of the culture was slowing dropping indicating culture activity. At about 30.0 0 C the feed gas flow was increased again to 236mL/min, or 50% or the original gas flow rate. At about 32.0 0 C, the agitation was increased to about 200rpm. At about 34°C the agitation was increased to about 400rpm.
• the culture cell density was about 2.76g/L; pH was about 4.60; redox was about -44OmV; CO and H2 uptake was about 4.5 and about 1.2mmol/min respectively; ethanol was about 19.0g/L; and acid was about 2.43g/L.
• the feed gas flow rate was decreased from about 475ml/min to about 53ml/min then turned off.
• a high N2 flow was started through the feed gas sparger while the agitation was still at about 400rpm for about 3 minutes to strip the CO2 from the culture. While stripping the CO2, the pH control was turned off to prevent any sodium bicarbonate addition.
• the N2 flow was dropped and the N2 inlet was changed to the headspace rather than the sparger.
• the agitation was dropped from about 400 to about 50rpm.
• CO2 addition was started at about 0.82mL/min using about 77g/L NaHCO3 to provide about 0.114mmol/min CO2 per gram of cells in the reactor.
• the medium flow rate was left at about 1.34mL/min, and a permeate purge flow of 2.25ml/min was started.
• a synthesis or waste gas containing CO and/or carbon dioxide/gaseous hydrogen is continuously introduced into a stirred tank bioreactor containing a strain of C. ljungdahlii, along with a conventional liquid medium containing vitamins, trace metals and salts.
• the reactor is operated with a batch liquid phase, where the liquid medium is not fed continuously to the reactor.
• the liquid phase in the reactor thus consists of a batch of nutrient medium with a nominal concentration of limiting nutrient, either calcium pantothenate or cobalt.
• a rich medium containing yeast extract, trypticase or other complex nutrients can also be employed.
• the gas phase at start-up is CO2 free and contains excess H.sub.2.
• the gas rate and agitation rate are kept at low levels (less than 500 rpm in a New Brunswick Scientific Bioflo.RTM. fermentation bioreactor) to yield CO and H.sub.2 in slight excess, but at the same time, avoiding CO substrate inhibition.
• the gas rate and agitation rate are kept at low levels (less than 500 rpm in a New Brunswick Scientific Bioflo.RTM. fermentation bioreactor) to yield CO and H.sub.2 in slight excess, but at the same time, avoiding CO substrate inhibition.
• New Brunswick Scientific Bioflo.RTM. fermentation bioreactor as an example, where the feed gas composition is 63% H.sub.2, 32% CO and 5% CH.sub.4, the agitation rate to initiate start-up is 400 rpm and the gas rate is 20 ml/min.
• the cause of ethanol production during start-up is excess H.sub.2; limitation on nutrients occurs later.
• excess liquid nutrients pantothenate, cobalt are actually present during start-up to avoid unwanted culture acclimation to low nutrients.
• CO2 is produced from the conversion of CO, and H.sub.2 is consumed along with the CO.sub.2, which is a signal to nominally increase the agitation rate to avoid gas mass transfer limitation.
• the exit gas is 25% CO, 67% H.sub.2, 2% CO.sub.2, and 6% CH.sub.4. If the agitation rate is increased too fast, CO substrate inhibition occurs, as evidenced by a decrease in methane concentration after an increase in agitation. Thus the agitation rate might typically be increased by 200 rpm in 24 hours.
• a typical target agitation rate in the New Brunswick Scientific Bioflo.RTM. fermentation bioreactor is 900 rpm.
• cell production instead of product formation is of utmost importance.
• cell concentrations of about 1.5 g/L are attained, while typical product concentrations are 10 g/L ethanol and 2 g/L acetate from the batch culture.
• the system is allowed to grow to maximum H.sub.2 uptake. It is desirable to have very high H.sub.2 exit concentrations (typically >60%) to assure ethanol production while limiting acetic acid production.
• the liquid medium feed is then turned on (for systems having batch inoculation from stock culture) to initiate continuous liquid feed and the gas feed rate is increased toward the target flow rate.
• the liquid feed rate is typically 0.5 mL/min, while the gas flow rate is increased by 10 to 15% every 24 hours toward a target rate of 125 mL/min.
• the production method and reactor system are then maintained at a steady state producing 15 to 35 g/L ethanol and 0 to 5 g/L acetate as products, with only occasional small adjustments in limiting nutrients, liquid rates and gas rate.
• Typical steady state conditions in the laboratory New Brunswick Scientific Bioflo.RTM. fermentation bioreactor without cell recycle are a gas retention time (gas flow rate/reactor liquid volume) of 20 minutes, a liquid retention time (liquid flow rate/reactor liquid volume) of 30 hours and an agitation rate of 900 rpm, yielding CO conversions of 92% and H.sub.2 conversions of 60% with pantothenate limitation.
• acetic acid product inhibition is a more severe problem. If excess molecular acetic acid is produced by the culture as a result of long term substrate inhibition, excess nutrient addition, CO.sub.2 accumulation or mechanical problems of many types, the problem that led to the excess acetic acid must first be corrected. The excess acetic acid, which quickly leads to product inhibition, is then cleared from the system by increasing the liquid rate to wash the acetic acid (and unfortunately ethanol) from the system. Once the acetate level is below 3-5 g/L, the liquid rate is reset and the reactor is placed back under either excess H.sub.2 feed, or vitamin or cobalt limitation (with or without cell recycle). Bringing the reactor back involves reducing the gas rate to avoid substrate inhibition and/or agitation rate before cell washout and lysis takes place. The agitation rate or gas rate is then increased.
• a CSTR with cell recycle containing C. ljungdahlii, strain C-Ol that was producing ethanol and acetic acid from CO, CO.sub.2 and H.sub.2 began producing acetic acid in response to a mechanical problem.
• the 2100 ml reactor was fed gas containing 62% H.sub.2, 31% CO and 7% C.sub.2H.sub.6 at a gas retention time of 15 minutes, and was operating with an agitation rate of 600 rpm and a pH of 4.86.
• the liquid retention time was 23 hours and the cell retention time was 68 hours.
• B- vitamin solution (an aqueous mixture of 50.5 mg/1 calcium pantothenate, 20.6 mg/L d- biotin and 50.6 mg/L thiamine HCl) was present in the liquid nutrient medium containing salts and vitamins at a concentration of 0.4 ml vitamin solution per liter of medium (see Table T).
• the ethanol concentration fell to 7 g/L, while the acetate concentration rose to 7 g/L, conditions that are neither stable for operating the reactor nor economical for ethanol production.
• the cell concentration was 2.4 g/L, the CO conversion was 85% and the H.sub.2 conversion was 25%.
• the strategy used in recovering the reactor consisted of first dramatically reducing the gas feed rate to the reactor, followed by gradual recovery of the reactor in the presence of excess H.sub.2.
• the liquid rate to the reactor was not reduced to clear product inhibition in this example because the acetate concentration was not exceedingly " high. Instead, the acetate concentration was allowed to more gradually drop to non- inhibiting levels with the reduction in gas flow rate and subsequent operation in the presence of excess H.sub.2.
• the specific procedure in recovering the reactor is discussed below.

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