WO2009064201A2 - Use of carriers in microbial fermentation - Google Patents

Use of carriers in microbial fermentation Download PDF

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Publication number
WO2009064201A2
WO2009064201A2 PCT/NZ2008/000306 NZ2008000306W WO2009064201A2 WO 2009064201 A2 WO2009064201 A2 WO 2009064201A2 NZ 2008000306 W NZ2008000306 W NZ 2008000306W WO 2009064201 A2 WO2009064201 A2 WO 2009064201A2
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WO
WIPO (PCT)
Prior art keywords
fermentation
carrier
bioreactor
substrate
products
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PCT/NZ2008/000306
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French (fr)
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WO2009064201A3 (en
Inventor
Christophe Daniel Mihalcea
Sean Dennis Simpson
Nicholas Troy Moleta
Richard Llewellyn Sydney Forster
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Lanzatech New Zealand Limited
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Publication of WO2009064201A2 publication Critical patent/WO2009064201A2/en
Publication of WO2009064201A3 publication Critical patent/WO2009064201A3/en

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    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54—Acetic acid
    • 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 and/or productivity of .processes of making products by microbial fermentation. More particularly, the invention relates-to-the use of carriers for elements required -as-a substrate by a microbial fermentation, particularly carbon containing substrates. The invention particularly relates to processes of producing acids and or alcohols (particularly ethanol and/or butanol). BACKGROUND OF THE INVENTION
  • Ethanol is rapidly becoming a major liquid transport fuel around the world.
  • Worldwide consumption of ethanol in 2005 was an estimated 12.2 billion gallons.
  • the global market for the fuel ethanol industry has also been predicted to grow sharply in future, due to an increased - interest in ethanol in Europe, Japan, the USA and several developing nations.
  • ethanol is used to produce ElO, a 10% mixture of ethanol in gasoline.
  • ElO blends the ethanol component acts as an oxygenating agent, improving the efficiency of combustion and reducing the production of air pollutants.
  • ethanol satisfies approximately 30% of the transport fuel demand, as both an oxygenating agent blended in gasoline, or as a pure fuel in its own right.
  • GOG Green House Gas
  • EU European Union
  • CO is a major free energy-rich by-product of the incomplete combustion of organic materials such as coal or oil and oil derived products.
  • organic materials such as coal or oil and oil derived products.
  • the steel industry in Australia is . reported to produce and release into the atmosphere over 500,000 tonnes of CO annually.
  • catalytic processes may be used to convert gases consisting primarily of CO and/or CO2 and hydrogen (H2) into a variety of fuels and chemicals.
  • H2 hydrogen
  • ⁇ micco-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 -pver-catalytic- ⁇ rocesses, including higher specificity, higher yields, lower energy costs and greater resistance to poisoning.
  • Anaerobic bacteria such as those from the genus Clostridium, have been demonstrated to produce ethanol from-CO, CO2 and H2 via the acetyl CoA biochemical pathway.
  • 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)). -
  • CO 2 represents inefficiency in overall carbon capture and if released, also has the potential to contribute to Green House Gas emissions.
  • WO2007/117157 describes a process that produces alcohols, particularly ethanol/by anaerobic fermentation of gases containing carbon monoxide. Acetate produced as a by-product of the fermentation process isxonverted into hydrogen gas and carbon dioxide gas, either or both of which may be -used in the anaerobic fermentation process.
  • WO2008/115080 describes a process for, the production. of alcohol(s) in multiple fermentation stages.
  • By-products produced .as a result of anaerobic fermentation of gas(es) in a first bioreactor can be used to produce products -in a second bioreactor. Furthermore, by-products of the second fermentation stage can be recycled to the first bioreactor to produce products.
  • fermentation reactions have been fed essential -elements in a gaseous form ⁇ ;
  • gas streams containing CO and/ ⁇ r CO2 and/or 02 and/or H2 may be pumped into a bioreactor such "that they bubble through the fermentation broth and/or are provided in any headspace in the bioreactor. At least a portion of the gases " in the-streams becomes dissolved in the fermentation broth such that it is then usable by the microbes used in the particular reaction.
  • Sources of gaseous streams containing one or more of the above components may be in remote or generally isolated locations.
  • steel mills produce large quantities of CO.
  • Another source-of_CO and H2 is the gasification of biomass. It would be advantageous to have " methods of trapping/capturing substrate components of gaseous streams in order to effectively optionally transport and/or store and ultimately provide a substrate to a fermenter.
  • the invention provides a method of producing products by microbial fermentation of a substrate, the method including at least the steps of: capturing one or more element(s)jn and/or on one or more carriers; and treating the carrier(s), such that the elements are available as substrate for conversion into products by a microbial culture in a bioreactor.
  • the treating step incl ⁇ des ⁇ adding at least a portion of the carrier(s) and captured -element(s)-directly- to the bioreactor, such that the element(s) is available as substrate-for conversion into products. "
  • the treating step includes releasing at least a portion of the element(s) such that the released element(s) can be added to the bioreactor as substrate.
  • the carrier(s) is adapted to physically and/or chemically capture the element(s).
  • the carrier may be selected from any one or more of molecular sieve(s), zeolite(s), apolar liquid(s) and metal(s).
  • the- one or more elements include one or more of C, O and H. According to. some embodiments, the one or more elements- may be provided to and/or carried by and/or releasable in the form of one or more Of CO, CO 2 , O 2 and H 2 .
  • the carrier is a transition metal.
  • the transition metal carrier is Fe and/or Ni and captures the CO as Fe(CO) 5 and/or Ni(CO) 4 .
  • the carriers include one or more porphyrins, such as but not limited to-haemoglobin and/or myoglobin and/or chlorins, including chlorophyll. Other heterocycles such as corrins, bateriochlorophylls and corphins may additionally/alternatively be used.-
  • an apolar liquid is used as a carrier.
  • the ⁇ apolar-liq ⁇ id carries one or more of said element(s) and/or another carrier and is added directly to ⁇ he bioreactor.
  • the apolar liquid carrier may optionally form an emulsion with an aqueous fermentation media.
  • a surfactant may be added to the apolar liquid-to stabilise the emulsion ⁇ .
  • the apolar liquid is preferably selected from any one orjnore of carbon-based oils, silicon-based-oils, olefins, perfluorinated hydrocarbons and aromatic compounds.
  • the apolar liquid is removed from the bioreactor and used to capture more or replacement one or more said element(s), wherein the recharged apolar liquid is returned to the bioreactor.
  • the method may optionally include recovering one or more products from the apolar liquid.
  • the apolar liquid may be used to remove products of a fermentation and/or provide replacement substrate thereto.
  • the microbial fermentation reaction is anaerobic.
  • the reaction is used to produce acids (particularly butyric and/or acetic add) and/or alcohols (particularly ethanol and/or butanol and/or isopropa ⁇ ol).
  • the method comprises capturing and recovering one or more of the products produced by the fermentation.
  • the level or concentration of the one or more elements is kept within a desired range within the bioreactor.
  • the level or concentration of the one or more elements may be maintained within a desired range by adding a predetermined amount of the one or more carriers to the bioreactor at a predetermined time or after a predetermined time interval.
  • the method may comprise monitoring the level or concentration of one or more of the elements and/or " one or more of the carriers and determining whether to add any one or more thereof.
  • Elements may be aUded to the bioreactor by adding appropriate carriers therefor, the carriers having previously been provided with the element(s).
  • the step of determining preferably includes determining how much of the one or more elements and/or carriers to add. As required, desired quantities may be added to the bioreactor.
  • the one or more elements may be continuously fed to the broth, via the carriers.
  • the rate of flow of the feed is preferably controlled to maintain the levels or concentrations of one or more of the elements and/or carriers within a predetermined range (i.e., greater than a respective first threshold and/or lower than a respective second threshold).
  • the levels or concentrations of one or more of the elements and/or carriers may be monitored and the feed or flow rate adjusted based at least in part thereon.
  • o_ne or more-elements_iand/or carriers are preferably added with one or more diluents and/or, other ingredients required by the fermentation, such as nutrients and ingredients ..required to maintain pH levels according to processes known in -the art.
  • the element(s) may be . released from “the carrier(s) according to known mear ⁇ s " , ⁇ such as through the provision of light and/or an oxidfser.
  • the carrier(s) may be readily -removed from the generally aqueous fermentation broth. .Following said removal, the carriers may be "re-charged" with the element(s).
  • the CO is released and added to the bioreactor, wherein "the treating includes at least one or more of heating, irradiation and mixing with acid.
  • the treating includes at least one or more of heating, irradiation and mixing with acid.
  • one or more of the elements are obtained from gases.
  • - •they may be obtained from gases which are 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.
  • this carrier may be produced as a byproduct or waste- product -of a ferrous metal products manufacturing process. More particularly, the Fe(CO) 5 may be produced by passing CO over fine iron particles (preferably with the CO being a waste product as aformentioned).
  • iron or any of its oxides depending on whether and which oxidisers are usjd wijl-be produced.
  • - Elementary iron may be produced if light is used for the release of CO from the Fe(CO) 5j , Due to .its higher density than other components in the bioreactor, it may easjly be removed- therefrom and used in the same or a different ferrous metal products manufacturing processr " More particularly,-iron particles will-accumulate in the broth and any rust particles would precipitate, allowing for easy removal of the iron or iron salt.
  • Other oxides such as bromides: or-iodides- would be soluble and more- difficult to remove.
  • the metals left following the fermentation may similarly be removed.
  • the fermentation reaction is-carried out by one or more strains of carboxytrophic bacteria.
  • the carboxytrophic bacterium is selected from Clostridium f - ⁇ Moorella - and Carhoxy ⁇ thermus, such as Clostridium autoethanogenum, Clostridium Jjungdahlii and Morella thermoacetica.
  • the carboxytrophic bacterium is Clostridium autoethanogenum.
  • an apparatus for producing one or more carriers for use as a substrate in a, microbial fermentation reaction including a chamber containing fine iron particles, the chamber having gas inlet and outlet means.
  • the gas inlet means are configured to receive CO (preferably in the form of a waste gas from an industrial process).
  • the gas outlet means is ' configured to exhaust gases and/or vapours from the chamber. Note that, depending on how the process is conducted, as would be apparent to those skilled in the art, the products of the chamber may appear in liquid form directly.
  • the outlet means may therefore be adapted to remove said liquid, and/or a combination of liquid and gas, from the chamber.
  • the apparatus includes or is coupleable to (preferably via the gas outlet means) to a condenser.
  • a system adapted to produce products by anaerobic fermentation, wherein the system includes at least: a. - - a chamber for capturing a substrate in and/or on one or more carriers; b. an optional release chamber; c. a bioreactor containing a microbial culture and configured to receive the substrate; and d. transfer means adapted to pass the carrier(s) from (a) to: i. (b) to (c); or ii. (c); or iii. (b), wherein at least a portion of the substrate is then passed to (c).
  • carriers may be used to . provide all elements of, -or at least all of one or more particular elements, used as a substrate in .a fermentation reaction,
  • one or " more ⁇ of the elements / or at least a portion thereof, may be provided in- an alternative manner.
  • gases may -be fed: to a bioreactor in a conventional manner with the- same -or an ⁇ additio_naj inlet provided tp.add one or more carriers of the invention to the bioreactor.
  • -elements may be. added as required depending on the amount required to be added and the " availability of the different sources.
  • the invention ⁇ provides a method of maintaining or increasing r efficiency of a microbial fermentation reaction in which at least a portion of the feedstock has . a np-;or limited solubility in. media used for. the reaction, the method including: adding the at least a portion of the feedstock to a liquid in which the at least a portion is soluble or more soluble SQ;,as to produce a comnosite stream: and feedin ⁇ the fermentation reaction with the composite stream.
  • the_ feedstock includes a gas.
  • the feedstock includes at least one of CO, CO 2 ⁇ O 2 .and H 2 .
  • the feedstock includes at least CO. :
  • the gas or gases are obtained as a byproduct of an industrial process.
  • the microbial fermentation reaction is an anaerobic fermentation reaction.
  • the reaction is used to produce acids (particularly, butyric and/or acetic acid) and/or alcohols (particularly ethanol and/or butanol and/or isopropano)).
  • the liquid is apolar and/or, has improved (gas) solubility over the generally aqueous fermentation media.
  • the polarity may gradually change and that certain polarities may be better at dissolving certain gases than others.
  • Use of apolar liquids is preferred since these provide for improved solubility (over aqueous solutions) for portions of preferred feedstocks, particularly CO.
  • the liquid may be selected from any one or more of carbon-based oils, silicon-based oils, olefins, perfluorinated hydrocarbons and aromatic compounds. Other liquids may be apparent to those of skill in the art and are included within the scope of the invention.
  • the method includes adding a surfactant to the liquid and/or to the fermentation broth.
  • a surfactant to the liquid and/or to the fermentation broth.
  • the invention provides a system for maintaining or increasing efficiency .of-a : microbjaMermentatipn reaction in which at least a portion of the feedstock has no or ⁇ limited solubility jn.media used for the reaction; the system.including: means for adding the at _leastia_Bortron:-pftJie feedstock to a liquid in which the at least a portion is soluble or more .soluble so as:tj ⁇ .fpr.o_duce,a:cpjnposite stream; and means forieeding the. fermentation reaction with the composite stream.
  • -the feedstock includes a gas.
  • the ; feedstock includes at. least one or more of CO> CO 2 / O 2 and H 2 .
  • the feedstock includes at least CO.
  • the gas or gases are obtained as a byproduct of an. industrial process.
  • the system includes conduit or transport means for passing the gas from the industrial processing plant to the means for adding.
  • the microbial fermentation reactio ' ⁇ is an anaerobic fermentation reaction.
  • the reaction may be used to produce acids (particularly butyric and/or aceticacid) anjd/or alcohols (particularly ethanol and/or butanol and/or isopropanol).
  • the liquid is apolar.
  • apolar liquids Use of apolar liquids is preferred since these provide for improved .
  • the liquid may be selected from any one or more of carbon-based oils, silicon- based oils, olefins, perfluorinated hydrocarbons and aromatic compounds.
  • the system includes means for adding a surfactant to the liquid and/or to-the; fermentation -broth. While there is merit in adding a " surfactant, the addition thereof is -not essential to the invention. Moreover, according to particular embodiments, it may be preferable not to add a surfactant so as to improve ease of separation of the liquid from the fermentation broth.
  • the system includes means for mixing the liquid and/or the composite streanvwith an aqueous solution.
  • a surfactant is used, the surfactant is also preferably .mixed by the means for mixing.
  • the means for mixing may be provided inside of the bioreactor in. which the fermentation reaction takes place.
  • a separate chamber is used in such the mixed composite stream (including or not a surfactant) is introduced into the bioreactor.
  • the system includes means tor removing at least a portion of the liquid from the bioreactor.
  • a simple port in the bioreactor may be used to ,remove the liquid from therefrom.
  • conventional separation techniques and . means may be used for the removal, regardless of whether a surfactant is used.
  • means_are provided for adding a further at least a portion of the feedstock thereto (note that separate means may not be provided for this purpose).
  • the stored at least a portion of the feedstock becomes depleted as it is taken up by the micro-organisms, used in the fermentation reaction.
  • the liquid may then be "re-charged" so that it may be re-used in the fermentation reaction.
  • the system includes means for capturing and recovering one or more of the products produced by .the fermentation.
  • Figure 1 is a schematic representation of a system including a chamber for element capture according to certain embodiments of the invention.
  • Figure 2 is a schematic representation of a system including chambers for element capture and carrier dissociation according to certain embodiments of the invention.
  • Figure 3 is a schematic representation of a system including means for mixing and separating an apolar liquid according to certain embodiments of the invention.
  • Figure 4 is a light microscopy photo of a water droplet containing bacteria in an oily medium.
  • Figures 5 and 6 show acetate and ethanol productivity of C. Autoethanogenum in various mediums.
  • the carrier may be used to capture a substrate from a source, optionally transport and/or store the substrate in a more desirable form and subsequently provide.the_substrate to a fermentation reaction for conversion into products.
  • any carrier suitable for capturing and releasing a substrate may, be used in the methods of the invention.
  • the substrate may be captured by physical means; such as trapping within a matrix, or chemical means, such as chemical bonding or interaction with the carrier.
  • the substrate_ may be released prior to being provided to a fermentation reaction.
  • the substrate carrier may be added directly "to a fermentation reaction, where the substrate becomes available or more available for conversion to products by microbial fermentation.
  • the carrier is selected from metal carbonyls, molecular sieves (e.g. zeolites), apolar liquids and the like.
  • the invention provides for higher availability of elements of the feedstock for the reactions (note that, ⁇ for example, oils are able to hold more gas such as CO in solution than aqueous solutions), improved ease of removing products of the fermentation from the broth (such as through extractive fermentation processes) and further has the unexpected additional benefit of enabling "recharging" of the liquid with fresh feedstock at the same time as removing the products from the liquid, such as through gas stripping.
  • the invention has significant benefit in maintaining or increasing efficiency of fermentation processes and/or lowering process operating costs, particularly those configured for butanol production.
  • the invention provides methods for maintaining or increasing the efficiency of microbial fermentation processes. These methods involve utilising an apolar liquid such as an oil " or perfluorocarbon to provide at least a portion of the feedstock for a -fermentation reaction- and/or to remove " products " of ⁇ said reaction!
  • additional feedstock may be provided in a more conventional manner, such as through the provision of one or more gases directly to the fermentation broth.
  • an apolar liquid is provided to increase the solubility of CO in a fermentation reaction.
  • increasing the efficiency 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 CO) 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.
  • desired product such as alcohols
  • substrate such as CO
  • co-substrate refers to a substance that while not being the primary energy and material source for product synthesis, can be utilised for product synthesis when added in addition to 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 may be described herein.
  • the ratio of molecular acetic acid to acetate in the fermentation broth is dependent upon the pH of the system. Similar meanings may be attributed to “butyrate” and “butyric acid” as would be apparent to one of skill in the art.
  • biomass includes a fermentation device consisting of one or more vessels and/or towers or piping arrangements, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Membrane ReactoLSuch as Hollow Fibre Membrane Bioreactor (HFMBR), 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 or other vessel or other device suitable for gas-liquid contact.
  • the phrases "fermenting”, “fermentation process” or “fermentation reaction” and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of the process.
  • the bioreactor may comprfse a firsr growth reactorand a second fermentation reactor.
  • the addition of carriers or compositions to a fermentation reaction should be understood to include addition to either or both of these reactors.
  • element is to be interpreted broadly as meaning any element which may be used in -a substrate, regardless ofthfe form in which it is usable or may be used. Thus, it not only refers to, for example; C, O and H, but also, for example, CO, CO2, H2 or 02. Other “elements” mav be selected dependent on the particular fermentation reaction of concern.
  • apolar liquid includes carbon-based oils, silicon-based oils, olefins, perfluorinated hydrocarbons ahd aromatic compounds, and is intended to include any liquid that is generally immiscible with a substantially more polar aqueous fermentation media.
  • the invention provides methods for capturing one or more elements and providing said elements to a fermentation reaction.
  • the invention provides methods for providing a gaseous substrate comprising CO to a fermenter.
  • the method includes capturing at least a portion of CO or an alternative element from a gaseous source and providing the captured element to a fermenter.
  • the method includes capturing CO using a CO-carrier.
  • the CO-carrier can be provided to the fermenter directly, where the CO may be utilised in situ.
  • the CO may be released from the CO-carrier prior to-the CO being provided-to the fermentation reaction.
  • the nature of the CO-carrier may determine whether the CO needs to be released from the capture means (carrier) prior to providing the substrate to the fermentation reaction.
  • CO any suitable-carrier can be used to capture or trap CO.
  • preferred carriers provide reversible capture and enable the release of CO for provision to the fermentation reaction.
  • CO - may be captured physically and/or chemically or a combination thereof.
  • CO may- be captured -using- a variety of physical-means,-such ⁇ as ⁇ adsorption or-absorption into/onto a suitable carrier or trapping within a matrix.
  • Adsorption is the accumulation of gases, liquids or solutes on the surface of a solid or liquid.
  • Absorption is the process by which one substance, such as a solid or liquid, takes up another substance, such as a liquid or gas, through minute pores or spaces between its molecules. :
  • Relatively high surfa_Ge ⁇ area ⁇ iaterials can also be used to trap and/or adsorb gases " such " as CO.
  • a molecular sieve is a material containing tiny pores ' of a precise and uniform size that is used as arj-adsorbent for gases and liquids. Molecules that are small enough to pass through the pores are adsorbed while larger molecules are not.
  • a molecular-sieve is similar to ajommon filter b.ut operates on a molecular level.
  • Molecular sieves often consist of aluminpsjjicate minerals, clays, porous glasses, microporous charcoals, zeolites, active carbons, - or synthetic compounds that;have open structures through which small molecules, such as N2. and H2O, can diffuse. Jvlethods for regeneration of molecular sieves include pressure changing (e.g. in 02 concentrators) and heating and purging with a carrier gas.
  • Zeolites are a family of naturally occurring or synthetic microporous aluminosilicates. Zeolites are widely used as ion-exchange beds in domestic and commercial water purification. However, they also have applications in separating and trapping molecules based -on size. Zeolites-provide precise " and specific separation and capture of gases such as H2O, CO2 and -SO2 from natural gas streams. Additionally, zeolites can be used to separate and capture other molecules such as N2, 02 and CO.
  • Apolar liquids such as oijs and/or perfluorinated hydrocarbons can also be used to trap/absorb elements, such as gaseous elements, and make them available to a fermentation reaction.
  • CO is significantly- more soluble in apolar oils than it is in water.
  • an apolar oil may be charged with CO then directly added to a fermentation reaction, where the CO will be available for conversion into products by a microbial culture.
  • a surfactant may also be added to a fermentation reaction to promote the stability of an emulsion between the CO carrying oil and the aqueous fermentation media, thus improving CO availability.
  • an apolar liquid such as an oil or perfluorinated hydrocarbon and optionally one or more surfactants may be added directly to a fermentation reaction.
  • Gaseous substrates, such as CO, are more soluble in apolar liquids than water. Accordingly, when a substrate stream is provided using an apolar liquid, a higher concentration of substrate will be attained in the fermenter.
  • oils in the fermentation reaction may also have additional advantages where fermentation products are soluble in oil.
  • the oil may also act as a carrier to remove one or more products from the fermentati ⁇ rrreaction.
  • butanol is known to be toxic to_soj ⁇ e microbial cultures at elevated concentrations.
  • butanol is typically atjleast as soluble in.apolar liquids such as oils than it is in aqueous fermentation media. Accordingly, at least a portion " of butanol produced during fermentation may be removed by extraction with the apolar.liquid carrier,
  • CO can be trapped or captured chemically by reacting CO with a " reactant to produce a CO-carrier suitable for transport and/or storage.
  • the CO-carrier can be added " directly to the fermentation reaction or treated to release the CO for ⁇ provision to the fermentation.
  • Any compounds which may be used to contain (or carry)-and release a substrate such as CO under controllable conditions may be suitable for use in -the methods of the invention.
  • metal complexes such as metal, .carbonyls (e.g. Fe(CO) 5 and Ni(CO) 4 ) and porphyrins (e.g. haemoglobin, myoglobin, chlorophyll) may provide suitable reversible CO-carrying means.
  • the use of one or more carriers to capture CO from a source may have additional advantages where the CO source comprises further components that may be undesirable in a fermentation reaction.
  • CO derived from gaseous waste streams exhausted from industrial processes may contain components toxic to a microbial culture.
  • the CO-carrier may be used to selectively capture CO from a waste stream and enable separation of CO from unwanted components.
  • a non-gaseous carrier can be used to capture CO from a gaseous stream comprising CO and the non-gaseous CO-carrier simply separated using conventional means.
  • the CO-carrier can then be delivered directly to a fermentation reaction.
  • the CO can be liberated from the carrier and provided to the fermentation reaction for conversion into products.
  • the removal of unwanted components from a substrate stream can increase-the CO concentration (o/.CO.partial pressure in a gaseous substrate) and so increase the efficiency of fermentation reactions where CO is a substrate.
  • Increasing CO partial pressure in a gaseous substrate increases CO mass transfer into a fermentation media.
  • the composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, 02 may reduce the efficiency of an anaerobic fermentation process.
  • processing of unwanted or unnecessary gases in stages of a fermentation process before or after- fermentation can increase the burden on ⁇ such stages (e.g. where the gas-stream is compressed before entering a bioreactor, unnecessary energy may be used- to compress gases that are not needed in the fermentation).
  • the unwanted component(s) of a particular waste stream may be of " greater value if used elsewhere than in the fermentation reaction.
  • substantially pure CO may be provided to a fermentation reaction, where a CO-carrier has been used to capture CO from an industrial waste stream.
  • While-certain embodiments-of the invention namely those that include the production of ethanol by anaerobic fermentation using CO as the primary substrate, are readily recognized as being valuable improvements to technology of great interest today, it should be appreciated that the invention is applicable to production of alternative products such as other alcohols and the use of alternative substrates, particularly gaseous substrates, as will be known by persons of ordinary skill in the art to which the invention relates upon consideration of the - instant disclosure.
  • alternative substrates particularly carbon dioxide and hydrogen may be used in particular embodiments of the invention.
  • the invention may be applicable to fermentations to produce acetate, butyrate, propionate, caproate, ethanol, propanol, and butanol, and hydrogen.
  • these products may be produced by fermentation using microbes from the genus Moorella, Clostridia, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium, Oxobacter, Methanosarcina, Methanosarcina, and Desulfotomaculum,
  • the invention may further be applied to other fermentation reactions, as would be apparent to one of skill in the art.
  • the invention has particular applicability to supporting the production of ethanol and butanol from substrates, particularly those derived from high volume CO-containing industrial flue gases.
  • the invention is also applicable to reactions which produce alternative alcohols.
  • ⁇ Processes for the production of ethanol and ofheralcohols ⁇ frorh gaseous substrates " (such as " those described in the background section above) are known.
  • Exemplary processes include
  • a number of anaerobic bacteria are known to be capable of carrying out the fermentation of CO to alcohols, including n-butanohand ethanol, and acetic acid, and are suitable for use in _ processes of the present invention.
  • Examples of such bacteria include those of the genus Clostridium, such as strains of Clostridium ljungdahlii, including those described in WO £>0/68407, ⁇ P 117309, US patent No.'s 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438, and Clostridium autoethanogenum (Abrini et al, Archives of Microbiology 161: pp 345-351).
  • Suitable bacteria include those of the genus Moorella, including Moorella - ( sp_HU022'-l, (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). Further examples include Morella thermoacetica, Moorella, thermoautotrophica, Ruminococcus productus, Acetobacterium woodii, Eubacterium limosum, Butyribacterium methylotrophicum, Oxobacter pfennigii, Methanosarcina barken, .
  • Methanosarcina acetivorans Desulfotomaculum kuznetsovii (Simpa et. al. Critical Reviews in Biotechnology, 2006 Vol. 26. Pp41-65).
  • carboxydotrophic anaerobic bacteria can be used in the processes of the invention by a person of skill in the art. It will also be appreciated upon consideration of the instant disclosure that a mixed culture of two or more bacteria may be used in the process of the present invention.
  • 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.
  • those processes generally described in the following articles using gaseous .substrates ffpr fermentation may be utilised: (i) K. T. Klasson, et jal. (1991). Bioreactors for synthesis gas fermentations resources. Conservation and Recycling, 5; 145-165; (ii) K. T. Klasson, et al. (1991). Bioreactor design for synthesis gas fermentations. Fuel. 70. 605-614; (iii) K.
  • Clostridium ⁇ utoeth ⁇ nogenum is Clostridium ⁇ utoeth ⁇ nogenum.
  • the Clostridium ⁇ utoeth ⁇ nogenum is " a Clostridium ⁇ utoeth ⁇ nogenum 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 ⁇ utoeth ⁇ nogenum is a Clostridium ⁇ utoeth ⁇ nogenum - having the identifying characteristics of DSMZ deposit number DSMZ 10061.
  • Other microorganisms may be selected depending on the fermentation reaction to be performed.
  • fermentation is carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CTSR), a-bubble column reactor (BCR) a membrane reactor, such as a Hollow
  • HMBR Fibre Membrane Bioreactor
  • TBR trickle bed reactor
  • the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second fermentation reactor, to which fermentation'
  • broth from the-growth-reactor is fed and in which most of the fermentation product (ethanol and acetate and/or-butanol and butyrate) is produced.
  • the carbon source for the fermentation reaction is preferably a substrate — containing CO.
  • the substrate may be derived from a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as 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 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 substrate may be filtered or scrubbed using known methods.
  • the gases are typically provided to a bioreactor in a more conventional mariner (i.e., by feeding it * directly " into tlTe ⁇ fermentation bro ⁇ th ⁇ and7o7 ⁇ t ⁇ the headspace of the bioreactor).
  • at least a portion of the gases is bound to one or more carriers which are then- fed to the bioreactor.
  • at least a portion of the gases are released- from the carrier then fed to the bioreactor.
  • the CO-containing 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 02.
  • the resultant gas typically comprises mainly CO and H2, with minimal volumes of CO2, 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 nonfood 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 gas stream added to a carrier and/or a fermentation broth preferably contains a significant proportion of CO, preferably at least about 20% to about 100% CO by volume, more preferably from 40% to 95% CO by volume, from 60% to 90% CO by volume, or- from 70% to 90% CO by volume.
  • Gaseous substrates having lower concentrations of CO, such * as 6%, may also be appropriate, particularly when H2 and CO2 are also present.
  • An additional advantage of embodiments in which particular element(s) are captured is that lower concentration streams may be used to obtain higher concentration streams.
  • the gaseous substrate While it is not necessary for the gaseous substrate to contain any H2, the presence of H2 will generally not be detrimentaLto product formation in accordance with the methods of the invention. However, in certain embodiments of the invention, the gaseous substrate is substantially H2 free (less than 1%).
  • the gaseous substrate may also contain some CO2, such as about 1% to about 30% by volume, or such as about 5% to about 10% CO2.
  • a suitable liquid nutrient medium will need to be fed to the bioreactor.
  • a nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used.
  • Anaerobic media suitable for the fermentation of ethanol using CO as the sole carbon source are known in the art.
  • suitable media may be prepared in accordance with standard procedures known in the art as exemplified in JS patent No.s 5,173,429 and -5,593,886-and WO " 02/08438. " Prior to use, if required, " the " media- can - be made, anaerobic using standard procedures as -exemplified herein. The " 'Examples” herein provide other exemplary media.
  • he fermentation should desirably be carried out under appropriate conditions for the desired ermentation to occur (e.g. CO-to-alcohoI).
  • Reaction conditions that should be considered nclude pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox lotential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas) substrate concentrations to ensure that CO (for example) in the liquid phase does- not iecome limiting, and maximum product concentrations to avoid product inhibition.
  • he optimum reaction conditions will depend partly on the particular micro-organism used, lowever, in general, it may be preferable that the fermentation be performed at a pressure iigher than ambient 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.
  • theTate of introduction of the CO-containing substrate is such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that the ethanol product is consumed - by the culture. Limitations of other processes and for other substrates will be apparent to those skilled in the art and the rates of introduction may be controlled accordingly.
  • ethanol may be recovered from the fermentation broth by methods such as fractional distillation or evaporation, and extractive fermentation.
  • Extractive fermentation procedures involve the use of a water-immiscible 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 extra " ction ⁇ process.
  • oleyl alcohol is continuously introduced into a fermenter, whereupon this solvent rises forming a layer at the top of the fermenter which is continuously extracted and fed through a centrifuge. Water and cells are then readily separated from the oleyl alcohol and returned to the fermenter while the ethanol-laden solvent is fed into a flash vaporization unit. Most of the ethanol is vaporized and condensed while the oleyl alcohol is non volatile and is recovered for re-use in the fermentation.
  • Acetate which is produced as a by-product in the fermentation reaction, may also be recovered from the fermentation broth using methods known in the art.
  • an adsorption system involving an activated charcoal filter may ⁇ be ⁇ used.
  • In lKis ⁇ case7 microbial cells are- typically first removed from the fermentation broth using a suitable separation method. Numerous filtration-based methods of generating a cell free fermentation broth for product recovery are known in the art.
  • the cell free ethanol- and acetates-containing permeate is .then passed through a column containing activated charcoal to adsorb the a_cetate.
  • Acetate ]n the acid form (acetic acid) rather than the salt (acetate) form is more readily adsorbed by activated charcoal. It is therefore preferred that the pH of the fermentation broth is 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. In certain embodiments, ethanol produced by the fermentation process itself may be used to eiute the acetate. Because the boiling point of ethanol is 78.8 9 C and that of acetic acid is 107 9 C, ethanol and acetate can readily be separated from each other using a volatility-based method such as distillation.
  • the products of the fermentation reaction may be 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 one or more product from the broth simultaneously or sequentially.
  • Ethanol may be conveniently recovered by distillation, and acetate may be recovered by ⁇ adsorption on activated_charcoal, " Using " the methods " described ' above.
  • the ⁇ separated ⁇ microbial cells can be returned to the fermentation bioreactor.
  • the cell free permeate remaining after the ethanol and acetate have been removed is-also preferably returned to the fermentation bioreactor.
  • Additional nutrients may be added to the cell free permeate to-ceplenish-the nutrient medium before it is returned to the bioreactor.
  • additional nutrients such as B vitamins
  • the pH of the broth was adjusted as described above to enhance adsorption of acetjc acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
  • a method of capturing one or more elements, such as CO, from a gaseous source then providing the captured element(s) to a fermentation reaction.
  • the element(s) may be captured using any suitable physical or chemical carrier and then added , directly to a fermentation reaction.
  • the captured element(s) may be released from the carrier to provide a gaseous substrate that may be passed to a fermentation reaction for conversion into products.
  • one or more carriers charged with one or. more elements are added to a fermentation reaction, which typically takes place in a-, bioreactor, preferably at one or more time points.
  • the carrier(s) may be added to the fermentation reaction at any point at which one is concerned that they are depleted to the point that they may be limiting the rate of alcohol production by the micro-organisms or otherwise to ensure the concentration remains within a predetermined range.
  • the carrier(s) may be added at discrete time points or continuously fed to the bioreactor at a rate calculated to ensure that the concentration in the reaction broth is within the predetermined range. Diluents and/or other nutrients of use in the reaction may additionally be added. According to certain embodiments, the carrier(s) are mixed therewith prior to being added to the bioreactor, although the invention is not limited thereto.
  • the fermentatio ⁇ rprocess ⁇ by taking samples frbrrftne " bioreactor at particular time points to determine a status of the reaction e.g., by testing a ⁇ sample-qf one or more_products of-the fermentation reaction. (for example an alcohol and/or - acid) and/or determining- a cell density.
  • Known parameters of particular fermentation processes may _be_ monitored using known methodologies .and equipment-may be used in " determining whether additional feedstock is required.
  • Cell density may be measured using standard techniques known in the art. By way of example, . manual observation under a microscope " or preferably measurement of optical density using a spectrophotometer may be used. Measurement of optical density at 600nm is particularly useful.
  • the level .of one or more fermentation products can be measured using standard methodology known in " the art. By “ way of example, these may be identified and measured _using gas chromatography, high pressure liquid chromatography, enzyme-based assays or colourimetric or fluorometric assays.
  • The, carrier(s) may be added to the bioreactor by any known means.
  • solutions may be introduced into the reactor automatically through a dedicated pump or manually via a septum covered. port using a syringe.
  • known means may be provided for adding the carrier(s) to the bioreactor in the form of a gas.
  • element(s) such as CO
  • a carrier such that the element(s) can be provided to the bioreactor as a substrate for conversion to one or more products by a microbial culture.
  • the element(s) may be released from the carrier by any means known in the art and in certain embodiments, the carrier may be reused ⁇ to capture further element(s).
  • the element may be released from the carrier and provided to - a fermentation reaction at a sufficient rate suitable to maximise product biosynthesis.
  • the element can be released and stored in a reservoir. At least a portion of the stored substrate can be provided to a fermentation. reaction on an as required basis to maintain efficient product synthesis. Exemplary methods for storing a gaseous substrate and providing the substrate to a bioreactor are described in PCT/IMZ2008/275.
  • -sieves usually comprise . aluminosilicate minerals (zeolites), clays, porous glass, microporous
  • rrLoleculer_siev_es_usualLy_ dependss.on-the pore size and/or cavity size. For example, 3A sieves adsorbTNK3 and H2O molecules, while 4 A adsorb H2O, CO2, SO2, H2S and ethanol. Larger pore, sizes can selectively capture larger molecules. Typically captured molecules will be
  • elements may even form covalent bonds within the cavities of carriers such as zeolite(s).
  • Fermentation substrate molecules such as CO may be trapped by molecular sieves by simply passing a stream comprising such elements over the surface of the sieve.
  • the "charged" sieve . (carrier) may then- be transported and/or stored until the substrate is required for fermentation.-
  • the charged sieve can be added directly to a ' fermentation reaction-such that the element(s) will be available, or at least partially available for conversion into, ⁇ products by a microbial culture.
  • the element(s) may be released from the carrier by any known means prior to passing the substrate to the bioreactor.
  • Methods for releasing the substrate from molecular sieve carriers include pressure change, heating and purging with a carrier gas or heating under vacuum.
  • One exemplary_method-for releasing an element from a carrier is pressure swing adsorption (PSA).
  • PSA is an adiabatic process which may be used for the purification of gases to remove accompanying impurities by adsorption through suitable adsorbents in fixed beds contained in pressure vessels under high pressure. Regeneration of adsorbents is accomplished by countercurrent depressurization and by purging at low pressure with previously recovered - near product quality gas.
  • a gas stream such as a ⁇ waste/exhaust/biogas gas stream
  • adsorbents may readily be selected by one of skill in the art dependent on the type of impurity to be adsorbed and removed. Suitable adsorbents include zeolitic molecular sieves, activated carbon, silica gel oractivated alumina.” Combinations of adsorbentrbeds may be used ⁇ on " top " of on " e ⁇ another " thereby dividing the adsorber contents into a number of- distinct zones. PSA-involves a pendulating swing in -parameters such as pressure, temperature, flow ,and composition of gaseous and adsorbed phase.
  • PSA Purification or separation of gases " using PSA normally takes place at near ambient feed gas " temperatures, whereby the components to be removed are selectively adsorbed. Adsorption -should ideally be- sufficiently reversible to enable regeneration of adsorbents at similar ambient temperature.
  • PSA may be used for treatment, purification and potential capture of most common gases including CO, CO2 and H2. Examples of PSA techniques are described in detail in Ruthven, Douglas M. et al., 1993 Pressure Swing Adsorption, John Wiley and Sons.
  • Molecules such as metal carbonyls contain several moles of CO per mole of metal atom.
  • Metal carbonyls are coordination complexes of transition metals with CO. Most metal carbonyls are relatively stable and are prepared in several steps, however Fe(CO) 5 and Ni(CO) 4 can be f prepared directly by treating the pure metal with CO. Substances such as Fe(CO) 5 and Ni(CO) 4 are not very stable and tend to decompose at least when they are exposed to heat or light. The principal reaction usually involves the oxidation of the transition metal (if there is an oxidiser present) with the simultaneous release of CO. In the absence of any other chemicals, the molecule just decomposes to produce the elementary metal and CO.
  • iron pentacarbonyl is produced by reacting a stream comprising CO with elemental iron, such as iron filings.
  • elemental iron such as iron filings.
  • the resultant Fe(CO) 5 can be decomposed by any known means, however in certain embodiments, the metal carbonyl is reacted with strong acid (eg H 2 SO 4 ) to provide CO.
  • the dissociated metal eg Fe
  • the dissociated metal can be reused to capture further CO.
  • Elements such as CO which may form at least a portion of the feedstock for a fermentation reaction are typically more soluble in apolar liquids, such as oils or perfluorinated hydrocarbons, than the aqueous phase of most fermentation reactions. Accordingly, one or -more apolar liquids -carrbezused as a " carrier for elements IrTa " feedstock " to Increase the availability of the particular element in a fermentation reaction.
  • the feedstock preferably includes elements such as CO that are converted to-products by microbial fermentation;
  • a feedstock " comprises multiple elements,; particular elements will be more soluble in apolar liquids than others. Accordingly, the .-dissolved- portions of the feedstock become the substrate for the fermentation reaction.
  • apolar liquids are gen era Ny immiscible with aqueous solutions.
  • a surfactant such as lecithin
  • an emulsion may be formed so as to increase mass transfer of the at least a portion of the feedstock from the liquid to thejnicro-organisms used in the fermentation (note the at least a portion of the feedstock may become ⁇ dissolved in the fermentation media before it is taken up by the microorganisms). This is achieved by increasing the effective surface area of the liquid by dispersing ⁇ it throughout the fermentation broth (or at least throughout a larger portion thereof). Note -that the aforementioned liquids will tend to float on the surface of the broth unless such a' surfactant is added.
  • a surfactant While there is merit in adding a surfactant, the addition thereof is not essential to the invention. Moreover, according to particular embodiments, it may be preferable not to add a surfactant so as to improve ease of separation of the liquid from the fermentation broth.
  • the method includes mixing the liquid and/or the composite stream with an. aqueous solution.
  • the surfactant is also preferably mixed during this mixing step. Note that mixing may be performed when no surfactant is used so that the liquid or composite stream becomes dispersed in the broth (ideally in small droplets)neig but may be_periodically interrupted to allow components of the broth to separate (namely the liquid from the aqueous portion of the broth), so that the liquid and/or the aqueous portion of the broth may be drawn off as desired.
  • the .aqueous solution may, include at least a portion of the fermentation broth and/or supplementary media used for the reaction.
  • the step of adding and/or mixing may take place in the bioreactor(s) used to conduct the -fermentation-reaction.
  • the feedstock, or at least a -iportion thereof may be added to the liquid to form ⁇ the:compos ⁇ te streaTrf before being added uto the bioreactor,. although the invention is not limited thereto.
  • the -. composite-stream may.be formed and then mixed with an-aqueous solution outside- of -the- .ibjpreactor, suclias in a pre-treatment chamber or conduit used to feed the reaction inside the- further alternative, :the_atJeast a portion of the feedstock-may-be added
  • the at least a portiorrof the feedstock is preferably in gaseous form when added to the liquid; the invention is not limjted thereto. More particularly, carriers may be used and added to the liquid, the carriers being able to release quantities of the at least a portion of the .feedstock, preferably in a controllable manner.
  • the meino ⁇ mciu ⁇ es removing a ⁇ leas ⁇ a porxion OT the liquid from the bioreactor.
  • a further at least a ⁇ portion- otihsJeedstock is. preferably added thereto.
  • the stored at least a portion of the feedstock becomes depleted as it is taken up by /, the micro-organisms used jnjthe fermentation reaction.
  • the liquid may then be "re-charged" ' chorus so that it may be re-used.
  • butanol is typically comparably or more miscible in oil than aqueous solutions.
  • a proportion of butanol produced by a fermentation reaction will remain in the aqueous portion of the fermentation broth, a significant, portion will be captured within the apolar liquid.
  • This also has a further benefit in that excessive quantities of butanol- in the aqueous portion of the- fermentation broth are toxic to the micro-organisms used therefor.
  • the method includes removing one or " more products from the apolar liquid removed from the bioreactor. Since particular products may have higher miscibility.in partic ⁇ lariiquids " trran m ⁇ aq ⁇ e ⁇ s solutions, the productsl ⁇ ay be at " a " higher concentration therein providing for " a " ⁇ potentially more efficient product extraction process (i.e., the. concentration of alcohol will be larger in the liquid, e.g., oil), and it is
  • the liquids (e.g; JDiIs)- used will tend to have a . higher. boiling point than an aqueous solution, which is advantageous ; because the difference between the boiling point of the product to be.
  • liquids such as oils have a much lower heat capacity than water or aqueous solutions, meaning that less energy is needed for heating in the distillation process. Note that energy:; costs associated with heating for evaporation/distillation to
  • ⁇ capture/remove products from fermentation reactions are a sizeable proportion of the total cost of such processes.
  • At least one product such as butanol
  • Gas stripping is a technique which allows for the selective removal. of volatiles from a medium without the need for expensive membranes or chemicals. Gas. may be sparged into the medium to create bubbles which burst and cause the surrounding liquid to vibrate. These vibrations are sufficient to result in volatiles being removed from the medium.
  • the liquid is sparged with the at least a portion of--the feedstock such that "re-charging" of the liquid takes place as the products of the fermentation are stripped from the liquid.
  • oil such as pump oil or canola oil
  • CO gas so as to substantially concurrently charge the oil with CO and extract butanol.
  • the flow rate of the composite. stream into the bioreactor is controlled according to techniques known to those skilled in the art.
  • At least a portion of the substrate for a fermentation reaction is provided to a bioreactor after it has been dissolved in a liquid such as " an oil or a perfluorocarbon and/or such a liquid is used in the removal and/or separation of products of the reaction.
  • a liquid such as " an oil or a perfluorocarbon and/or such a liquid is used in the removal and/or separation of products of the reaction.
  • known parameters may be monitored using known methodologies and equipment so as to determine whether additional feedstock is required. Additional feedstock may be added at discrete time points. One could determine average rates of alcohol production and depletion of the feedstock used in the reaction and calculate points in the fermentation reaction at which it is most likely it will be necessary to supplement the feedstock. .Alternatively, one could actively monitor the fermentation process by taking samples from the bioreactor at particular time points to determine a status of the reaction e.g., by.testing a sample of one or more products of the fermentation reaction (for example an alcohol and/or acid) and/or determining a cell density.
  • a status of the reaction e.g., by.testing a sample of one or more products of the fermentation reaction (for example an alcohol and/or acid) and/or determining a cell density.
  • Cell density and/or the level of one or more fermentation products can be measured using standard methodology as discussed hereinbefore.
  • the feedstock maybe added to the bioreactor by any known means.
  • solutions may be introduced into the reactor automatically through a dedicated pump or manually via a-septum covered port using a syringe.
  • known means may be provided for adding the feedstock to the bioreactor in the form of a gas.
  • Desired products from microbial fermentation processes include alcoholsrketonesraldehydes and carboxylic acids. Usually these are waste products of the bacteriological metabolism and toxic to the bacteria themselves. The concentration of the desired products in the bacterial broth is . therefore .usually limited to the amounts the bacteria can maximally tolerate. For example, the.maxLmu.rn amount of butanol that can be tolerated by its producing microbe (e.g.
  • Clostridium such as C acetobutylicum, C. saccharobutylicum is of the order of only 2%.
  • the butanol has to be retrieved from this dilute solution using distillation or other techniques. Since this process, is very energy consuming it would be desirable to have a fermenter system that can tolerate higher amounts of products (so as to eliminate or more widely space apart interruptions in the process); and be able to use separation processes (such as distillation processes) to remove products of a fermentation from solutions containing larger amounts of these desired end products. .
  • separation processes such as distillation processes
  • Particular embodiments of the invention provide a leaching (cleaning) procedure of the ⁇ fermenter solution that may be applied in situ to remove these (toxic) products, thereby ⁇ allowing the microbial solution to be used for an extended time.
  • the invention is based on the solubility difference of the. desired end products between the aqueous bacterial phase and another suitable more apolar phase that is not toxic to the system (for example, canola oil or any other oils such as silicones and perfluorinated aliphatic and aromatic compounds). Both phases are preferably mixed together either by vigorous stirring or by applying surfactants to either of the phases with the aim of producing an emulsion and thereby increasing the contact area of the two phases under consideration.
  • the desired product i.e., the toxin for the bacteria
  • apolar phase which-can then be extracted by simply decanting it or by other means to separate the oily phase again.
  • Such embodiments readily provide for a continuous system or process of production where some oil is dispersed in the fermenter solution and the separated product rich phase (probably floating on the surface depending on the density of the oil used) taken out of the system for distillation or gas stripping etc.
  • emulsions In fermentations using gases, the use of emulsions as gas carriers could dramatically reduce the energy and operating costs associated with overcoming the gas to liquid mass transfer barrier in the fermentation system. This is particularly significant in fermentations using CO, as CO gas is sparingly soluble in water. Sterile emulsions could be "loaded” with gas separately in a dedicated unit before being introduced into a fermenter or reactor. A further advantage may be that emulsions could be continuously removed from a fermenter, separated from media and biomass and be "re-charged” with gas before reintroduction to the fermenter vessel as a continuous gas feeding process. This is made all the more possible since the " droplets that constitute the emulsion exist as a separate liquid phase within the media broth. This process could be applied to aerobic fermentations where the introduction of oxygen is required, and " anaerobic fermentations using CO containing gases.
  • the carrier is adapted to selectively carry desirable elements for optimal fermentation " efficiency.
  • the carrier can selectively capture CO from a waste stream from an industrial process and provide a substantially pure substrate stream comprising CO to the bioreactor.
  • further desirable elements such as H2,-can-also be captured by the same or different carrier(s) and provided to the bioreactor to enhance productivity.
  • CO and H2 are more soluble in apolar liquids than aqueous nutrient media. Accordingly, use of an apolar oil as a carrier will improve availability -of CO and H2 for conversion into products by microbial fermentation General
  • Embodiments of the invention are described by way of example. However, it should be appreciated that particular steps or stages necessary to perform the methods of one -embodiment may not be necessary in another. Conversely, steps or stages included in the description -of - a particular- embodiment can be optionally advantageously utilised in embodiments where they are not specifically mentioned. . While, the - invention-: — ⁇ s ⁇ broadly described with - reference to any - type of •feedstock/element/substrate- and/or carrier streams that -may be moved through or around the system(s) by- any known transfer means,., in certain embodiments, the substrate(s) are •gaseous. Those skilled in the art- wijj appreciate that particular stages may be coupled by .
  • suitable conduit means or the like configurable to receive or pass streams throughout a system.
  • a pump or compressor may be provided to facilitate delivery of the streams to particular stages.
  • a compressor can be used to increase the pressure of gas -provided to one or more stages, for example a capture chamber or the bioreactor.
  • the pressure of gases within a bioreactor can affect the efficiency of the fermentation reaction performed therein. The pressure can be adjusted to improve the efficiency of the fermentation. Suitable pressures for common reactions are known in the art.
  • the systems or processes of the invention may optionally include means for regulating and/or controlling -Other parameters to improve overall efficiency of the process.
  • particular embodiments may include determining means to monitor the composition of substrate and/or exhaust stream(s).
  • particular embodiments may include a means for controlling the delivery of substrate stream(s) to particular stages within a particular system if the determining means determines the stream has a composition suitable for a particular stage. For example, in instances where a gaseous substrate stream contains low levels of CO that may be detrimental to a fermentation reaction, additional CO may be liberated from a CO charged carrier and fed to a bioreactor.
  • the system includes means for monitoring and controlling the destination of a substrate stream and/or the flow rate, such that a stream with a desired or suitable composition can be delivered to a particular stage.
  • a carrier may be provided for a period such that the carrier is substantially fully charged with one or more elements before it is passed to a latter stage.
  • certain embodiments of the invention include means for monitoring the amount of element (e.g., CO) captured by a carrier.
  • the release rate may be ⁇ controlled such that a substrate stream is passed to a bioreactor at an optimised rate.
  • the system may include one or more pre/post treatment steps to improve the operation or efficiency of a particular stage.
  • a pre-treatment step may include means for removing particulate matter and/or long chain hydrocarbons or tars from a gaseous substrate stream.
  • Other pre- or post-operations that may be conducted include separation of desired product(s) from particular stages, such as, for example, the bioreactor production stage (e.g. removal of ethanol by distillation).
  • Figure 1 is a schematic representation of a system 100, according to an embodimeni configured to produce one or more CO-carriers and use the carrier(s) produced as a feedstock for -a fermentation reaction.
  • System 100 may be adapted to produce other carriers or ma ⁇ receive carriers that have been pre-manufactured. Additionally, the fermentation reaction may receive feedstock from more than one source.
  • System. 100 includes chamber 11, optional treatment unit 12 and bioreactor 13.
  • Chamber 11 contains CO capture means, such as fine particles of iron.
  • CO gas passes through chamber 11 towards treatment unit 12.
  • Treatment unit 12 performs any desired pre-treatment steps before the stream is fed to bioreactor 13.
  • treatment unit 12 may alternatively be placed upstream of chamber 11.
  • treatment units 12 may be placed upstream and downstream of chamber 11.
  • no treatment units 12 may be included in system 100.
  • Treatment unit 12 may be configured to remove unwanted components of the stream, such as by scrubbing. It may also include a condenser for condensing the Fe(CO) 5 vapours.
  • Bioreactor ⁇ 13 ⁇ contams " a " sliitable media inoculated with bacteria required ⁇ f ⁇ rthe ⁇ particular reaction. It receives the stream in its final form, as vapours and/or a liquid. Bioreactor 13 may -be provided with additional inlets and-outlets " as would be apparent to those skilled in the art to " enable the reaction to perform as desired and for the products thereof to be removed.
  • FIG. 2 is a schematic representation of a system 101 according to another embodiment "configured to capture-CO from a source using one or more CO-carriers, release the CO from the carrier(s) and provide the released CO to a fermenter.
  • System 101 further includes " dissociation " chamber 14r Chamber 11 contains a carrier, such as fine particles of iron or similar means for capturing and/or carrying CO. The CO-carrier then passes to dissociation chamber 14, where CO may be released from the carrier.
  • Chamber 11 and dissociation chamber 14 may be proximate to each other and joined by any suitable conduit means. Alternatively, Chamber 11 and dissociation chamber 14 may be remote and any known transfer means may be provided to transfer the CO-carrier therebetween, for example, a pipeline or vehicular transfer means.
  • CO may be captured from a waste stream exiting an industrial process using iron to form Fe(CO) 5 .
  • the capturing of the CO will take place at or near the exhaust of a particular process generating CO, for example, the waste stack of a steel mill.
  • the Fe(CO) 5 or other CO-carrier can be transported to a remote location through a pipeline.
  • liquid Fe(CO) 5 can be pumped through a pipeline over large distances.
  • the CO-carrier can be placed in transportable tanks or containers for delivery to optional treatment stage 12.
  • tanks or containers containing Fe(CO) 5 can be delivered to optional treatment 12 by train, ship or truck.
  • the CO-carrier is passed to dissociation chamber 14, where the -CO can be liberated by any known means and converted into a gaseous substrate.
  • CO can be liberated from Fe(CO) 5 by heating, irradiation or treatment with acid.
  • Fe(CO) 5 is treated with concentrated H 2 SO 4 to liberate the COr
  • the liberated CO can be passed directly to the fermentation reaction by any known means.
  • optional treatment stage 12 may be downstream of the dissociation chamber 14. As such, the treatment stage 12 would be adapted to treat or optimise the CO liberated in the chamber 14.
  • - Figure 3 is a schematic-representation of a system 102— according to a further embodiment, wherein-an apolar liquid is used-as a substrate carrier- A substrate stream (e.g. CO) 15is ⁇ nixed with water 16 and oil 17- in a hojmogenization chamber 18 to form an emulsion charged with - -CO.
  • the homogenisatjon chamber can include any mixing means known in the art for j)reparing an emulsion on a continuous basis.
  • Suitable mixing means include mechanical agitation and/or utilisation of some form of static mixer/homogeniser e.g. forcing the emulsion components through an orifice plate.
  • the CO charged carrier 19 emulsion comprising oil/water/CO
  • optional emujsion preparation tank 21 where optionally recycled microorganisms 20 and other components necessary for microbial fermentation, such as nutrients may be added.
  • the substrate carrier can be passed to bioreactor 22.
  • substrate stream 15 comprising CO may optionally be passed through the ferr ⁇ enter for conversion into products and any exhausted gas can be directed away from bioreactor 22 as indicated by arrow 23.
  • a trickle bed_reactor can be prepared by adding a packing to the column and trickling the emulsion down the column over the packing.
  • the gas phase of the reactor could be monitored and controlled.
  • the fermented emulsion 24 can then be passed to a processing unit 25.
  • a process unit may optionally be included to separate the emulsion components so that the ethanol containing aqueous component could be further processed and the oil rich component replenished with CO and returned to the reactor.
  • a standard -industrialjtubular bowl centrifuge or similar may be used to separate these components on a continuous basis.
  • the aqueous component 16 may be further treated before distillation and the oil component 17 is returned to the homogeniser 18 as the emulsion stream once bacteria have been optionally isolated using microbe separation means 27.
  • the microbe separation means 27 may be any known means used for separating bacteria from a liquid fermentation broth such as a cross -flow membrane or hollow fibre membranes. Isolated bacteria may optionally be redirected into emulsion preparation tank 21.
  • Clostridium autoethanogenum were obtained from the German Resource Centre for Biological
  • DSMZ The accession number given to the bacteria is DSMZ 10061.
  • Fe(CO) 5 was purchased from Sigma Aldrich (195731).
  • Lecithin was purchased in granulated form from a local supermarket Pluronic F-68 was purchased from Sigma Aldrich
  • Ethanol and butanol solutions were prepared and the concentrations measured using gas chromatography. The solutions were measured individually and also in a mix as depicted in Table 1 below. "An aliquot of the aqueous alcoholic phase (representing the fermenter solution) was taken and shaken with an equal amount of canola oil (representing the extraction, oily phase) in a beaker. After letting the beaker stand for a while to wait for the separation of the phases, the alcohol amounts in the aqueous phase was measured again.
  • the ethanol is not leached out of the water whereas about 40% the butanol is (more, particularly, 35%, 42% and 44%).
  • Theiajm ofthis experiment was to determine if emulsions of oils and surfactants could be used as carriers of CO in a nutrient media for bacteria that use CO as a source of carbon and energy.
  • Each portion of the emulsion was separately sparged with gas while cooling.
  • One half of the solution was sparged with N2 gas, while the other was sparged with a gas mix of 70% CO, 15% CO2,-14% N2, and 1% H2.-
  • Each solution was sparged with these gases until they reached room temperature.
  • a 2x concentrated solution of LM23 media was prepared lacking cysteine and B-vitamins. This solution.was boiled in the microwave to de-gas it and sparged with N2 gas as it cooled to room temperature. Once cool, the cysteine and b-vitamins were added and the pH of the media adjusted to 5.4 with 5M NaOH. Additionally dH 2 O was boiled and cooled under N 2 gas.
  • Six 250 ml serum.bottles were prepared in which to test the impact of emulsions in growth media on bacterial.growtruand their- utility as a CO carrier. All bottles were prepared under CO gas to ensure anaerobicity. The bottles were prepared as follows:
  • each bottle was immediately stoppered with a butyl rubber septum, and crimp- sealed with an aluminium cap to ensure anaerobicity. All 6 bottles were autoclaved at 121°C for 20 min and allowed to cool to room temperature. Once cooled each bottle was inoculated with 500 ⁇ l of a growing culture of bacteria (C. autoethanogenum). After inoculation, a 1 ml sample was removed from each serum bottle. The headspace gas was then pressurised to 35 psig with either N 2 gas or the 70% CO gas mix described above as shown in Table 2:
  • control treatment 3 25 ml of 2 X LM23 + 25 ml emulsion sparged with N2, with N 2 in the headspace
  • Control treatment 4 25 ml of 2 X LM23 + 25 ml emulsion sparged with N 2 , with 70% CO in the headspace
  • the emulsion did not inhibit the microbial population in the uptake of CO from the headspace.
  • Two emulsions were prepared by mixing canola oil with two different surfactants.
  • lecithin - was used and in the other case a tri-block copolymer (Pluronic F-68, trademark from BASF) i.e., a synthetically produced surfactant.
  • 24 g of surfactant was mixed with 80 g of canola oil and 300 ml of LM 23 media (prepared in the same fashion as for Example 2a) and magnetically stirred-until a homogeneous appearing emulsion resulted.
  • the pluronic emulsion was of white appearance, while the lecithin emulsion was yellowish in colour.
  • the emulsions were heated in the microwave and subsequently split into two fractions.
  • Each portion of the emulsion was separately sparged with gas while cooling.
  • One half of the solution was sparged with l ⁇ h gas, while the other was sparged with a gas mix of 70% CO, 15% CO 2 , 14% N 2 , and 1% H 2 .
  • Each solution was sparged with these gases until they reached room temperature.
  • Serum bottles were filled with 50 ml of the emulsions, sealed and subsequently autoclaved. Control bottles containing only LM 23 but no emulsions were prepared and treated otherwise in the same way. Bottles were evacuated and refilled with CO (70% in CO 2 ) twice and the final pressure adjusted to about 35psi. Control bottles were treated in the same way with the exception that N 2 was used instead of CO to saturate the oil. Once cooled, each bottle was inoculated with 1 ml of a growing culture of bacteria (C. autoethanogenum).
  • Bottles were placed on shaking tables inside an incubator and the products traced by taking 1 ml samples daily for GC analysis using techniques known in the art.
  • Figure 4 is a light microscopy photograph of a water droplet containing bacteria in an oily medium.
  • the sample was prepared by shaking (mixing) equal amounts of bacteria containing fermenter broth and a purified vegetable oil. After keeping the sample still for some time, the two phases almost completely separated and the oily phase was investigated for the presence of bacteria.
  • the bacteria avoid the oil phase entirely -there are bacteria (darker rod shapes) inside the water droplet (centre of image) but not outside (i.e., in the oily phase - identified by smaller droplets surrounding the larger water droplet).
  • This has a major positive implication with regard to the application of the use of oils in an industrial fermentation process.
  • gaseous feedstocks using the apolar liquids such as oils and deplete the solution of toxic solvent products like butanol. Doing this will not remove cells from the fermenter broth.
  • the large amount of dissolved gases in the oils can be used to cool the fermenter broth and vigorously mix the broth due to the large gas solubility gradient with temperature.
  • the oil may be saturated with, for example, a CO-containing gas at ambient temperature or cooler (noting that the solubility of gases in liquids is inversely proportional to temperature i.e., higher solubility at lower temperatures).
  • the oils may, for example, then be introduced into the fermenter via a small nozzle in a jet-like manner. Since the reactor is usually kept at higher temperature ( ⁇ 37°C), the gas would instantly go out of solution from the- oil to reach the new equilibrium solubility at that respective new temperature.
  • Example 4 Because of the speed the gas bubbles form and expand, a large cooling effect associated with this adiabatic expansion corresponds. Additionally, the feed gas replenishes the fermenter broth with carbon and energy in the form of CO gas. With an optimized fermenter layout, the rapid expansion of gas can also be used simultaneously to create turbulence in the fermenter so that no or minimal additional stirring/mixing techniques are needed.
  • Example 4
  • a preparation of LM23 fermentation , media (media recipe described above) at pH 5.5 was prepared as follows:
  • the serum bottle containing 180 ml NaOH was connected to a_series of Hungate " tubes filled with 10 ml pump oil; the remaining gas was pumped through these tubes.

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Abstract

This invention relates generally to methods for increasing the efficiency and/or productivity of processes of making products by microbial fermentation. More particularly, the invention relates to the use of carriers for elements required as a substrate by a microboial fermentation, particularly carbon containing substrates. In certain aspects, the invention provides means for capturing CO from an industrial waste stream using a carrier, and providing the CO to a bioreactor containing a microbial culture for conversion into products.

Description

USE OF CARRIERS IN MICROBIAL FERMENTATION FIELD OF THE INVENTION
This invention relates generally to methods for increasing the efficiency and/or productivity of .processes of making products by microbial fermentation. More particularly, the invention relates-to-the use of carriers for elements required -as-a substrate by a microbial fermentation, particularly carbon containing substrates. The invention particularly relates to processes of producing acids and or alcohols (particularly ethanol and/or butanol). BACKGROUND OF THE INVENTION
Ethanol is rapidly becoming a major liquid transport fuel around the world. Worldwide consumption of ethanol in 2005 was an estimated 12.2 billion gallons. The global market for the fuel ethanol industry has also been predicted to grow sharply in future, due to an increased - interest in ethanol in Europe, Japan, the USA and several developing nations.
For example, in the USA, ethanol is used to produce ElO, a 10% mixture of ethanol in gasoline. In ElO blends the ethanol component acts as an oxygenating agent, improving the efficiency of combustion and reducing the production of air pollutants. In Brazil, ethanol satisfies approximately 30% of the transport fuel demand, as both an oxygenating agent blended in gasoline, or as a pure fuel in its own right. Also, in Europe, environmental concerns surrounding the consequences of Green House Gas (GHG) emissions have been the stimulus for the European Union (EU) to set member nations a mandated target for the consumption of sustainable transport fuels such as biomass derived ethanol.
The vast majority of fuel ethanol is produced via traditional yeast-based fermentation processes that use crop derived carbohydrates, such as sucrose extracted from sugarcane or starch extracted from grain crops, as the main carbon source. However, the cost of these carbohydrate feed stocks is influenced by their value as human food or animal feed, while the cultivation of starch or sucrose-producing crops for ethanol production is not economically sustainable in all geographies. Therefore, it is of interest to develop technologies to convert lower cost and/or more abundant carbon resources into fuel ethanol.
CO is a major free energy-rich by-product of the incomplete combustion of organic materials such as coal or oil and oil derived products. For example, the steel industry in Australia is . reported to produce and release into the atmosphere over 500,000 tonnes of CO annually. It has long been recognised that catalytic processes may be used to convert gases consisting primarily of CO and/or CO2 and hydrogen (H2) into a variety of fuels and chemicals. However, ■ ;micco-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 -pver-catalytic-φrocesses, including higher specificity, higher yields, lower energy costs and greater resistance to poisoning.
The ability of micro-organisms to grow on CO as their 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 andr the carbon monoxide dehydrogenase / acetyl CoA synthase (CODH/ACS) pathway). -A large number of anaerobic organisms including carboxydotrophic, photosynthetic, methanogenic and acetogenic organisms have been shown to metabolize CO to various end products, namely CO2, Hl1 methane, n-butanol, acetate and ethanol. While using CO,- as the sole- carbon source all such organisms produce at least two of these end products.
Anaerobic bacteria, such as those from the genus Clostridium, have been demonstrated to produce ethanol from-CO, CO2 and H2 via the acetyl CoA biochemical pathway. For example, various strains of Clostridium ljungdahlii that produce ethanol from gases are described in WO 00/68407, EP 117309, 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)). -
Microbial fermentation of CO in the presence of H2 can lead to substantially complete carbon transfer into an.alcohol. However, in the absence of sufficient H2, some of the CO is converted into alcohol, while a significant portion is converted to CO2 as shown in the following equations:
6CO + 3H2O -^ C2H5OH + 4CO2 12H2 + 4CO2 -» 2C2H5OH + 6H2O
The production of. CO2 represents inefficiency in overall carbon capture and if released, also has the potential to contribute to Green House Gas emissions. WO2007/117157 describes a process that produces alcohols, particularly ethanol/by anaerobic fermentation of gases containing carbon monoxide. Acetate produced as a by-product of the fermentation process isxonverted into hydrogen gas and carbon dioxide gas, either or both of which may be -used in the anaerobic fermentation process. WO2008/115080 describes a process for, the production. of alcohol(s) in multiple fermentation stages. By-products produced .as a result of anaerobic fermentation of gas(es) in a first bioreactor can be used to produce products -in a second bioreactor. Furthermore, by-products of the second fermentation stage can be recycled to the first bioreactor to produce products.
According to particular-prior art arrangements, fermentation reactions have been fed essential -elements in a gaseous form~;For example/ gas streams containing CO and/όr CO2 and/or 02 and/or H2 may be pumped into a bioreactor such" that they bubble through the fermentation broth and/or are provided in any headspace in the bioreactor. At least a portion of the gases" in the-streams becomes dissolved in the fermentation broth such that it is then usable by the microbes used in the particular reaction. -
-The availabijity-or concentration of these" essential elements in the fermentation broth can have a significant impact on the productivity of fermentation processes. However, gases such -as CO and 02 have poor solubility in the generally aqueous broth contained within bioreactors, making it difficult and/or slow to transfer desired quantities of the gases to the broth and then to the micro-organisms used in the fermentation process.
Sources of gaseous streams containing one or more of the above components may be in remote or generally isolated locations. For example, steel mills produce large quantities of CO. Another source-of_CO and H2 is the gasification of biomass. It would be advantageous to have" methods of trapping/capturing substrate components of gaseous streams in order to effectively optionally transport and/or store and ultimately provide a substrate to a fermenter.
It is an object of the present to provide a process that goes at least some way towards improving the efficiency and/or productivity of microbial fermentations. Alternatively, it is an object to at least provide the public with a useful choice.
SUMMARY OF THE INVENTION fa. a jBrst broad aspect, the invention provides a method of producing products by microbial fermentation of a substrate, the method including at least the steps of: capturing one or more element(s)jn and/or on one or more carriers; and treating the carrier(s), such that the elements are available as substrate for conversion into products by a microbial culture in a bioreactor.
In some embodiments, the treating step inclϋdes~adding at least a portion of the carrier(s) and captured -element(s)-directly- to the bioreactor, such that the element(s) is available as substrate-for conversion into products. "Alternatively, or additionally the treating step includes releasing at least a portion of the element(s) such that the released element(s) can be added to the bioreactor as substrate.
In certain embodiments, the carrier(s) is adapted to physically and/or chemically capture the element(s). In particular, -the carrier may be selected from any one or more of molecular sieve(s), zeolite(s), apolar liquid(s) and metal(s).
In some embodiments, the- one or more elements include one or more of C, O and H. According to. some embodiments, the one or more elements- may be provided to and/or carried by and/or releasable in the form of one or more Of CO, CO2, O2 and H2.
In particular embodiments, the carrier is a transition metal. According to some embodiments, the transition metal carrier is Fe and/or Ni and captures the CO as Fe(CO)5 and/or Ni(CO)4. Additionally/alternatively, the carriers include one or more porphyrins, such as but not limited to-haemoglobin and/or myoglobin and/or chlorins, including chlorophyll. Other heterocycles such as corrins, bateriochlorophylls and corphins may additionally/alternatively be used.-
According to particular embodiments, an apolar liquid is used as a carrier. In certain embodiments, the~apolar-liqιιid carries one or more of said element(s) and/or another carrier and is added directly to^he bioreactor. The apolar liquid carrier may optionally form an emulsion with an aqueous fermentation media. Furthermore, a surfactant may be added to the apolar liquid-to stabilise the emulsionτ. The apolar liquid is preferably selected from any one orjnore of carbon-based oils, silicon-based-oils, olefins, perfluorinated hydrocarbons and aromatic compounds.
According to particular embodiments, at least a portion of the apolar liquid is removed from the bioreactor and used to capture more or replacement one or more said element(s), wherein the recharged apolar liquid is returned to the bioreactor. The method may optionally include recovering one or more products from the apolar liquid. Thus, the apolar liquid may be used to remove products of a fermentation and/or provide replacement substrate thereto. According to certain embodiments, the microbial fermentation reaction is anaerobic. In particular embodiments, the reaction is used to produce acids (particularly butyric and/or acetic add) and/or alcohols (particularly ethanol and/or butanol and/or isopropaøol).
In some embodiments, the method comprises capturing and recovering one or more of the products produced by the fermentation.
In particular embodiments, the level or concentration of the one or more elements is kept within a desired range within the bioreactor. The level or concentration of the one or more elements may be maintained within a desired range by adding a predetermined amount of the one or more carriers to the bioreactor at a predetermined time or after a predetermined time interval. Alternatively, the method may comprise monitoring the level or concentration of one or more of the elements and/or "one or more of the carriers and determining whether to add any one or more thereof. Elements may be aUded to the bioreactor by adding appropriate carriers therefor, the carriers having previously been provided with the element(s). The step of determining preferably includes determining how much of the one or more elements and/or carriers to add. As required, desired quantities may be added to the bioreactor.
As a further alternative, the one or more elements may be continuously fed to the broth, via the carriers. According to such embodiments, the rate of flow of the feed is preferably controlled to maintain the levels or concentrations of one or more of the elements and/or carriers within a predetermined range (i.e., greater than a respective first threshold and/or lower than a respective second threshold). Again, the levels or concentrations of one or more of the elements and/or carriers may be monitored and the feed or flow rate adjusted based at least in part thereon.
As would be apparent to one of skill in the art, in order for a fermentation reaction to continue" operating within desired performance parameters, the elements needed to feed that reaction must be provided in sufficient quantities. However, if these quantities become too high, the broth can .become toxic to the microbes used in the reaction, which at best lowers the efficiency and/or productivity of the reaction. Conversely, if the quantities become too low, the microbes may not be provided with sufficient feedstock to maintain the fermentation reaction above acceptable production levels. The:o_ne or more-elements_iand/or carriers are preferably added with one or more diluents and/or, other ingredients required by the fermentation, such as nutrients and ingredients ..required to maintain pH levels according to processes known in -the art.
The element(s) may be .released from "the carrier(s) according to known mearϊs",~such as through the provision of light and/or an oxidfser. Thus, embodiments of the invention provide for a level of control over the release of elements, such as CO, into the fermentation broth. As would be apparent to those skilled in the art, the apolar solution (and the carrier(s) therein) may be readily -removed from the generally aqueous fermentation broth. .Following said removal, the carriers may be "re-charged" with the element(s).
-According to particular embodiments, the CO is released and added to the bioreactor, wherein" the treating includes at least one or more of heating, irradiation and mixing with acid. In-certain embodiments, one or more of the elements are obtained from gases. In particular, - •they may be obtained from gases which are a by-product of an industrial process.
In certajn embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining -processes, gasification of biomass, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing; Preferably, the gaseous substrate comprises a gas obtained from a steel mill.
-According to embodiments involving use of Fe(CO)5, this carrier may be produced as a byproduct or waste- product -of a ferrous metal products manufacturing process. More particularly, the Fe(CO)5 may be produced by passing CO over fine iron particles (preferably with the CO being a waste product as aformentioned). Note that as a by product of the fermentation process, iron (or any of its oxides depending on whether and which oxidisers are usjd) wijl-be produced.- Elementary iron may be produced if light is used for the release of CO from the Fe(CO)5j, Due to .its higher density than other components in the bioreactor, it may easjly be removed- therefrom and used in the same or a different ferrous metal products manufacturing processr" More particularly,-iron particles will-accumulate in the broth and any rust particles would precipitate, allowing for easy removal of the iron or iron salt. Other oxides such as bromides: or-iodides- would be soluble and more- difficult to remove. Where other •metal carbonyls are used, the metals left following the fermentation may similarly be removed. In certain^ embodimentsy the fermentation reaction is-carried out by one or more strains of carboxytrophic bacteria. Preferably, the carboxytrophic bacterium is selected from Clostridium f-~ Moorella - and Carhoxyφthermus, such as Clostridium autoethanogenum, Clostridium Jjungdahlii and Morella thermoacetica. In a particular embodiment, the carboxytrophic bacterium is Clostridium autoethanogenum. In a second broad aspect, there is provided an apparatus for producing one or more carriers for use as a substrate in a, microbial fermentation reaction, the apparatus including a chamber containing fine iron particles, the chamber having gas inlet and outlet means.
In -certajn embodiments,-the gas inlet means are configured to receive CO (preferably in the form of a waste gas from an industrial process).
In particularembodiments, the gas outlet means is' configured to exhaust gases and/or vapours from the chamber. Note that, depending on how the process is conducted, as would be apparent to those skilled in the art, the products of the chamber may appear in liquid form directly. The outlet means may therefore be adapted to remove said liquid, and/or a combination of liquid and gas, from the chamber.
According to some embodiments, the apparatus includes or is coupleable to (preferably via the gas outlet means) to a condenser.
In a third broad aspect, there is provided a system adapted to produce products by anaerobic fermentation, wherein the system includes at least: a. - - a chamber for capturing a substrate in and/or on one or more carriers; b. an optional release chamber; c. a bioreactor containing a microbial culture and configured to receive the substrate; and d. transfer means adapted to pass the carrier(s) from (a) to: i. (b) to (c); or ii. (c); or iii. (b), wherein at least a portion of the substrate is then passed to (c).
According to the preceding aspects of the invention, carriers may be used to. provide all elements of, -or at least all of one or more particular elements, used as a substrate in .a fermentation reaction, However; according to an alternative embodiment, one or " more ■ of the elements/ or at least a portion thereof, may be provided in- an alternative manner. For [ example, gases, may -be fed: to a bioreactor in a conventional manner with the- same -or an ^additio_naj inlet provided tp.add one or more carriers of the invention to the bioreactor. Thus, -elements may be. added as required depending on the amount required to be added and the " availability of the different sources.
In .a fourth broad aspect, the invention^ provides a method of maintaining or increasingr efficiency of a microbial fermentation reaction in which at least a portion of the feedstock has .anp-;or limited solubility in. media used for. the reaction, the method including: adding the at least a portion of the feedstock to a liquid in which the at least a portion is soluble or more soluble SQ;,as to produce a comnosite stream: and feedinε the fermentation reaction with the composite stream.
In . certain , em.bod.im.ents,,.the_ feedstock includes a gas. Preferably, the feedstock includes at least one of CO, CO2^O2.and H2. In particular embodiments, the feedstock includes at least CO.:
According to- eertam embodiments of the invention, the gas or gases are obtained as a byproduct of an industrial process.
According to particular embodiments, the microbial fermentation reaction is an anaerobic fermentation reaction. Preferably, the reaction is used to produce acids (particularly, butyric and/or acetic acid) and/or alcohols (particularly ethanol and/or butanol and/or isopropano)).
In certain embodiments, the liquid is apolar and/or, has improved (gas) solubility over the generally aqueous fermentation media. It should be noted that the polarity may gradually change and that certain polarities may be better at dissolving certain gases than others. Use of apolar liquids is preferred since these provide for improved solubility (over aqueous solutions) for portions of preferred feedstocks, particularly CO.
The liquid may be selected from any one or more of carbon-based oils, silicon-based oils, olefins, perfluorinated hydrocarbons and aromatic compounds. Other liquids may be apparent to those of skill in the art and are included within the scope of the invention.
According to one embodiment, the method includes adding a surfactant to the liquid and/or to the fermentation broth. In a fifth broMfaspe_et, . the invention provides a system for maintaining or increasing efficiency .of-a: microbjaMermentatipn reaction in which at least a portion of the feedstock has no or ^limited solubility jn.media used for the reaction; the system.including: means for adding the at _leastia_Bortron:-pftJie feedstock to a liquid in which the at least a portion is soluble or more .soluble so as:tjθ.fpr.o_duce,a:cpjnposite stream; and means forieeding the. fermentation reaction with the composite stream.
!n particular embodiments, -the feedstock includes a gas. In certain embodiments, :the ; feedstock includes at. least one or more of CO> CO2/ O2 and H2. "In one embodiment, the feedstock includes at least CO.
According, to SQm.e ^embodiments of the invention, the gas or gases are obtained as a byproduct of an. industrial process. Thus, the system includes conduit or transport means for passing the gas from the industrial processing plant to the means for adding.
According to some , embodiments, the microbial fermentation reactio'ή is an anaerobic fermentation reaction.- The reaction may be used to produce acids (particularly butyric and/or aceticacid) anjd/or alcohols (particularly ethanol and/or butanol and/or isopropanol).
Jn particular.embodiments, the liquid is apolar. Use of apolar liquids is preferred since these provide for improved .solubility (over aqueous solutions) for portions of preferred feedstocks, - particularly CQ:: The liquid .may be selected from any one or more of carbon-based oils, silicon- based oils, olefins, perfluorinated hydrocarbons and aromatic compounds.
According to one embodiment, the system includes means for adding a surfactant to the liquid and/or to-the; fermentation -broth. While there is merit in adding a" surfactant, the addition thereof is -not essential to the invention. Moreover, according to particular embodiments, it may be preferable not to add a surfactant so as to improve ease of separation of the liquid from the fermentation broth.
In particular embodiments, the system includes means for mixing the liquid and/or the composite streanvwith an aqueous solution. Where a surfactant is used, the surfactant is also preferably .mixed by the means for mixing. The means for mixing may be provided inside of the bioreactor in. which the fermentation reaction takes place. However, according to certain embodiments, a separate chamber is used in such the mixed composite stream (including or not a surfactant) is introduced into the bioreactor.
According to particular embodiments, the system includes means tor removing at least a portion of the liquid from the bioreactor. Where a surfactant is not used, a simple port in the bioreactor may be used to ,remove the liquid from therefrom. In any event, conventional separation techniques and . means may be used for the removal, regardless of whether a surfactant is used.
In some embodiments, after the at least a portion of the feedstock is removed from the liquid, means_are provided for adding a further at least a portion of the feedstock thereto (note that separate means may not be provided for this purpose). During the time the liquid is in the bioreactor, the stored at least a portion of the feedstock becomes depleted as it is taken up by the micro-organisms, used in the fermentation reaction. The liquid may then be "re-charged" so that it may be re-used in the fermentation reaction.
In particular embodiments, the system includes means for capturing and recovering one or more of the products produced by .the fermentation.
Other aspects of the invention will become apparent from the detailed description of embodiments and examples of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be described in detail with reference to the accompanying Figures in which:
Figure 1: is a schematic representation of a system including a chamber for element capture according to certain embodiments of the invention.
Figure 2: is a schematic representation of a system including chambers for element capture and carrier dissociation according to certain embodiments of the invention.
Figure 3: is a schematic representation of a system including means for mixing and separating an apolar liquid according to certain embodiments of the invention.
Figure 4: is a light microscopy photo of a water droplet containing bacteria in an oily medium.
Figures 5 and 6: show acetate and ethanol productivity of C. Autoethanogenum in various mediums.
DETAILED DESCRIPTION OF THE INVENTION -•"Where -gaseous substrates-are used irra fermentation reaction^ the speed and --quantity of
* supply of essential 'elements forming a substrate for the reaction may be somewhat limited and -in accordance :with the~ methods of the invention, this speed and quantity may be rimproved by using a carrier for providing the elements. The invention has significant benefit in maintaining or -increasing efficiency of fermentation processes and/or lowering process operating costs. Furthermore, the carrier may be used to capture a substrate from a source, optionally transport and/or store the substrate in a more desirable form and subsequently provide.the_substrate to a fermentation reaction for conversion into products.
-As wilLbe clear to those skilled in the art, any carrier suitable for capturing and releasing a substrate may, be used in the methods of the invention. Furthermore, the substrate may be captured by physical means; such as trapping within a matrix, or chemical means, such as chemical bonding or interaction with the carrier. The substrate_may be released prior to being provided to a fermentation reaction. Alternatively the substrate carrier may be added directly "to a fermentation reaction, where the substrate becomes available or more available for conversion to products by microbial fermentation. In particular embodiments, the carrier is selected from metal carbonyls, molecular sieves (e.g. zeolites), apolar liquids and the like.
' There a number of unexpected benefits that may be derived through use of apolar liquid carriers, such as oils and perfluorocarbons, in fermentation reactions in which the media solution used therefor Js generally aqueous. More particularly, the invention provides for higher availability of elements of the feedstock for the reactions (note that,~for example, oils are able to hold more gas such as CO in solution than aqueous solutions), improved ease of removing products of the fermentation from the broth (such as through extractive fermentation processes) and further has the unexpected additional benefit of enabling "recharging" of the liquid with fresh feedstock at the same time as removing the products from the liquid, such as through gas stripping. The invention has significant benefit in maintaining or increasing efficiency of fermentation processes and/or lowering process operating costs, particularly those configured for butanol production.
In particular aspects, the invention provides methods for maintaining or increasing the efficiency of microbial fermentation processes. These methods involve utilising an apolar liquid such as an oil" or perfluorocarbon to provide at least a portion of the feedstock for a -fermentation reaction- and/or to remove "products" of~said reaction! Note that" additional feedstock may be provided in a more conventional manner, such as through the provision of one or more gases directly to the fermentation broth. In particular embodiments, an apolar liquid is provided to increase the solubility of CO in a fermentation reaction.
Definitions
Unless otherwise defined, the following terms as used throughout this specification are defined as follows:
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 CO) consumed, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared with other by-products of the fermentation.
The term "co-substrate" refers to a substance that while not being the primary energy and material source for product synthesis, can be utilised for product synthesis when added in addition to the primary substrate.
The term "acetate" includes both acetate salt alone and a mixture of molecular or free acetic acid and acetate salt, such as the mixture of acetate salt and free acetic acid present in a fermentation broth as may be described herein. The ratio of molecular acetic acid to acetate in the fermentation broth is dependent upon the pH of the system. Similar meanings may be attributed to "butyrate" and "butyric acid" as would be apparent to one of skill in the art.
The term "bioreactor" includes a fermentation device consisting of one or more vessels and/or towers or piping arrangements, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Membrane ReactoLSuch as Hollow Fibre Membrane Bioreactor (HFMBR), Static Mixer, or other vessel or other device suitable for gas-liquid contact.
Unless the context requires otherwise, the phrases "fermenting", "fermentation process" or "fermentation reaction" and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of the process. As will be described, in some embodiments the bioreactor may comprfse a firsr growth reactorand a second fermentation reactor. As such, the addition of carriers or compositions to a fermentation reaction should be understood to include addition to either or both of these reactors.
The term "element" is to be interpreted broadly as meaning any element which may be used in -a substrate, regardless ofthfe form in which it is usable or may be used. Thus, it not only refers to, for example; C, O and H, but also, for example, CO, CO2, H2 or 02. Other "elements" mav be selected dependent on the particular fermentation reaction of concern.
The term "apolar liquid" includes carbon-based oils, silicon-based oils, olefins, perfluorinated hydrocarbons ahd aromatic compounds, and is intended to include any liquid that is generally immiscible with a substantially more polar aqueous fermentation media.
The invention provides methods for capturing one or more elements and providing said elements to a fermentation reaction. In particular embodiments, the invention provides methods for providing a gaseous substrate comprising CO to a fermenter.
In certain embodiments, the method includes capturing at least a portion of CO or an alternative element from a gaseous source and providing the captured element to a fermenter. In particular, the method includes capturing CO using a CO-carrier. In some embodiments of the-Jnvention, the CO-carrier can be provided to the fermenter directly, where the CO may be utilised in situ. In alternative embodiments, the CO may be released from the CO-carrier prior to-the CO being provided-to the fermentation reaction. Those skilled in the art will appreciate the nature of the CO-carrier may determine whether the CO needs to be released from the capture means (carrier) prior to providing the substrate to the fermentation reaction.
Any suitable-carrier can be used to capture or trap CO. However, preferred carriers provide reversible capture and enable the release of CO for provision to the fermentation reaction. CO - may be captured physically and/or chemically or a combination thereof. By way of example, CO may- be captured -using- a variety of physical-means,-such~as~adsorption or-absorption into/onto a suitable carrier or trapping within a matrix. Adsorption is the accumulation of gases, liquids or solutes on the surface of a solid or liquid. Absorption is the process by which one substance, such as a solid or liquid, takes up another substance, such as a liquid or gas, through minute pores or spaces between its molecules. : Relatively high surfa_Ge~areaττiaterials can also be used to trap and/or adsorb gases" such" as CO.
.For example, a molecular sieve is a material containing tiny pores'of a precise and uniform size that is used as arj-adsorbent for gases and liquids. Molecules that are small enough to pass through the pores are adsorbed while larger molecules are not. A molecular-sieve is similar to ajommon filter b.ut operates on a molecular level. Molecular sieves often consist of aluminpsjjicate minerals, clays, porous glasses, microporous charcoals, zeolites, active carbons, - or synthetic compounds that;have open structures through which small molecules, such as N2. and H2O, can diffuse. Jvlethods for regeneration of molecular sieves include pressure changing (e.g. in 02 concentrators) and heating and purging with a carrier gas.
Zeolites are a family of naturally occurring or synthetic microporous aluminosilicates. Zeolites are widely used as ion-exchange beds in domestic and commercial water purification. However, they also have applications in separating and trapping molecules based -on size. Zeolites-provide precise" and specific separation and capture of gases such as H2O, CO2 and -SO2 from natural gas streams. Additionally, zeolites can be used to separate and capture other molecules such as N2, 02 and CO.
Apolar liquids such as oijs and/or perfluorinated hydrocarbons can also be used to trap/absorb elements, such as gaseous elements, and make them available to a fermentation reaction. For example CO is significantly- more soluble in apolar oils than it is in water. As such, an apolar oil may be charged with CO then directly added to a fermentation reaction, where the CO will be available for conversion into products by a microbial culture. In particular embodiments of the invention, a surfactant may also be added to a fermentation reaction to promote the stability of an emulsion between the CO carrying oil and the aqueous fermentation media, thus improving CO availability.
In an alternative embodiment, an apolar liquid, such as an oil or perfluorinated hydrocarbon and optionally one or more surfactants may be added directly to a fermentation reaction. Gaseous substrates, such as CO, are more soluble in apolar liquids than water. Accordingly, when a substrate stream is provided using an apolar liquid, a higher concentration of substrate will be attained in the fermenter.
The use of oils in the fermentation reaction may also have additional advantages where fermentation products are soluble in oil. In such embodiments, the oil may also act as a carrier to remove one or more products from the fermentatiσrrreaction. "For example, "butanol is known to be toxic to_sojηe microbial cultures at elevated concentrations. However, butanol is typically atjleast as soluble in.apolar liquids such as oils than it is in aqueous fermentation media. Accordingly, at least a portion "of butanol produced during fermentation may be removed by extraction with the apolar.liquid carrier,
Additionally or alternatively, CO can be trapped or captured chemically by reacting CO with a" reactant to produce a CO-carrier suitable for transport and/or storage. In particular embodiments the CO-carrier can be added "directly to the fermentation reaction or treated to release the CO for^ provision to the fermentation. Any compounds which may be used to contain (or carry)-and release a substrate such as CO under controllable conditions may be suitable for use in -the methods of the invention. However, by way of example, metal complexes such as metal, .carbonyls (e.g. Fe(CO)5 and Ni(CO)4) and porphyrins (e.g. haemoglobin, myoglobin, chlorophyll) may provide suitable reversible CO-carrying means. "
The use of one or more carriers to capture CO from a source may have additional advantages where the CO source comprises further components that may be undesirable in a fermentation reaction. For example^CO derived from gaseous waste streams exhausted from industrial processes may contain components toxic to a microbial culture. The CO-carrier may be used to selectively capture CO from a waste stream and enable separation of CO from unwanted components. For example, a non-gaseous carrier can be used to capture CO from a gaseous stream comprising CO and the non-gaseous CO-carrier simply separated using conventional means. The CO-carrier can then be delivered directly to a fermentation reaction. Alternatively, the CO can be liberated from the carrier and provided to the fermentation reaction for conversion into products.
Furthermore, the removal of unwanted components from a substrate stream can increase-the CO concentration (o/.CO.partial pressure in a gaseous substrate) and so increase the efficiency of fermentation reactions where CO is a substrate. Increasing CO partial pressure in a gaseous substrate increases CO mass transfer into a fermentation media. Furthermore, the composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, 02 may reduce the efficiency of an anaerobic fermentation process. In addition, processing of unwanted or unnecessary gases in stages of a fermentation process before or after- fermentation can increase the burden on~such stages (e.g. where the gas-stream is compressed before entering a bioreactor, unnecessary energy may be used- to compress gases that are not needed in the fermentation). Additionally or alternatively, the unwanted component(s) of a particular waste stream may be of "greater value if used elsewhere than in the fermentation reaction.
In certain embodiments; substantially pure CO may be provided to a fermentation reaction, where a CO-carrier has been used to capture CO from an industrial waste stream.
While-certain embodiments-of the invention, namely those that include the production of ethanol by anaerobic fermentation using CO as the primary substrate, are readily recognized as being valuable improvements to technology of great interest today, it should be appreciated that the invention is applicable to production of alternative products such as other alcohols and the use of alternative substrates, particularly gaseous substrates, as will be known by persons of ordinary skill in the art to which the invention relates upon consideration of the - instant disclosure. For example, gaseous substrates containing carbon dioxide and hydrogen may be used in particular embodiments of the invention. Further, the invention may be applicable to fermentations to produce acetate, butyrate, propionate, caproate, ethanol, propanol, and butanol, and hydrogen. By way of example, these products may be produced by fermentation using microbes from the genus Moorella, Clostridia, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium, Oxobacter, Methanosarcina, Methanosarcina, and Desulfotomaculum, The invention may further be applied to other fermentation reactions, as would be apparent to one of skill in the art.
Fermentation reaction
The invention-has particular applicability to supporting the production of ethanol and butanol from substrates, particularly those derived from high volume CO-containing industrial flue gases. Examples include gases produced during ferrous metal products manufacturing, non- ferrojus products manufacturing, petroleum refining processes, gasification of coal, gasification of biomass, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing. The invention is also applicable to reactions which produce alternative alcohols. ^Processes for the production of ethanol and ofheralcohols~frorh gaseous substrates" (such as" those described in the background section above) are known. Exemplary processes include
-.those described for example in WO 2007/117157, WO2008/115080 and US Patent Nos. 6,340,581, 6,136,577, 5,593,886, 5,807,722 and 5,821,111.
A number of anaerobic bacteria are known to be capable of carrying out the fermentation of CO to alcohols, including n-butanohand ethanol, and acetic acid, and are suitable for use in _ processes of the present invention. Examples of such bacteria include those of the genus Clostridium, such as strains of Clostridium ljungdahlii, including those described in WO £>0/68407,ΕP 117309, US patent No.'s 5,173,429, 5,593,886, and 6,368,819, WO 98/00558 and WO 02/08438, and Clostridium autoethanogenum (Abrini et al, Archives of Microbiology 161: pp 345-351). Other suitable bacteria include those of the genus Moorella, including Moorella -(sp_HU022'-l, (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). Further examples include Morella thermoacetica, Moorella, thermoautotrophica, Ruminococcus productus, Acetobacterium woodii, Eubacterium limosum, Butyribacterium methylotrophicum, Oxobacter pfennigii, Methanosarcina barken, . Methanosarcina acetivorans, Desulfotomaculum kuznetsovii (Simpa et. al. Critical Reviews in Biotechnology, 2006 Vol. 26. Pp41-65). In addition, other carboxydotrophic anaerobic bacteria can be used in the processes of the invention by a person of skill in the art. It will also be appreciated upon consideration of the instant disclosure that a mixed culture of two or more bacteria may be used in the process of the present invention.
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 jfurther example, those processes generally described in the following articles using gaseous .substrates ffpr fermentation may be utilised: (i) K. T. Klasson, et jal. (1991). Bioreactors for synthesis gas fermentations resources. Conservation and Recycling, 5; 145-165; (ii) K. T. Klasson, et al. (1991). Bioreactor design for synthesis gas fermentations. Fuel. 70. 605-614; (iii) K. T. Klasson, et al. (1992). Bioconversion of synthesis gas into liquid or gaseous fuels. Enzyme and Microbial Technology. 14; 602-608; (iv) J. L. Vega, et al. (1989). Study of Gaseous Substrate Fermentation: Carbon Monoxide Conversion to Acetate. 2. Continuous Culture. Biotech. ,_. _ Bioeng. 34. 6. 785-793; (Vi)-J^L Vega, et al. (-1989). Study of gaseous substrate ferrήenfations: Carbonjnonoxide. conversion to acetate. 1. Batch culture. Biotechnology and Bioengineering. 34. 6. 774-784; (vii) J. L -Vega, et al. -(1990).. Design of Bioreactors for Coal Synthesis Gas
. Fermentations. Resources, Conservation and Recycling. 3. 149-160.
One exemplary micro-organism suitable for use in the present invention is Clostridium αutoethαnogenum. In one embodiment; the Clostridium αutoethαnogenum is "a Clostridium αutoethαnogenum having the identifying characteristics of the strain deposited at the German Resource Centre for Biological Material (DSMZ) under the identifying deposit number 19630. In another embodiment, the Clostridium αutoethαnogenum is a Clostridium αutoethαnogenum - having the identifying characteristics of DSMZ deposit number DSMZ 10061. Other microorganisms may be selected depending on the fermentation reaction to be performed.
Typically, fermentation is carried out in any suitable bioreactor, such as a continuous stirred tank reactor (CTSR), a-bubble column reactor (BCR) a membrane reactor, such as a Hollow
Fibre Membrane Bioreactor (HFMBR), or a trickle bed reactor (TBR). Also, in some preferred
- embodiments of the invention, the bioreactor may comprise a first, growth reactor in which the micro-organisms are cultured, and a second fermentation reactor, to which fermentation'
, broth from the-growth-reactor is fed and in which most of the fermentation product (ethanol and acetate and/or-butanol and butyrate) is produced.
As described above, the carbon source for the fermentation reaction is preferably a substrate — containing CO. The substrate may be derived from a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production", carbon black production, ammonia production, methanol production and coke manufacturing. In these embodiments, the CO-containing gas may be captured from the industrial "process before it is emitted into the atmosphere, using any convenient method. Depending on the composition of the gases, it- may be desirable to treat them to remove any undesired impurities, such as dust particles. For example, the gaseous substrate may be filtered or scrubbed using known methods. The gases are typically provided to a bioreactor in a more conventional mariner (i.e., by feeding it* directly" into tlTe~fermentation bro~th~and7o7ϊπtό the headspace of the bioreactor). However, according to certain embodiments of the invention, at least a portion of the gases is bound to one or more carriers which are then- fed to the bioreactor. In other embodiments of the invention, at least a portion of the gases are released- from the carrier then fed to the bioreactor.
The CO-containing 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 02. The resultant gas typically comprises mainly CO and H2, with minimal volumes of CO2, methane, ethylene and ethane. For example, biomass by-products obtained during the extraction -and processing of-foodstuffs such as sugar from sugarcane, or starch from maize or grains, or nonfood 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 gas stream added to a carrier and/or a fermentation broth preferably contains a significant proportion of CO, preferably at least about 20% to about 100% CO by volume, more preferably from 40% to 95% CO by volume, from 60% to 90% CO by volume, or- from 70% to 90% CO by volume. Gaseous substrates having lower concentrations of CO, such* as 6%, may also be appropriate, particularly when H2 and CO2 are also present. An additional advantage of embodiments in which particular element(s) are captured is that lower concentration streams may be used to obtain higher concentration streams.
While it is not necessary for the gaseous substrate to contain any H2, the presence of H2 will generally not be detrimentaLto product formation in accordance with the methods of the invention. However, in certain embodiments of the invention, the gaseous substrate is substantially H2 free (less than 1%). The gaseous substrate may also contain some CO2, such as about 1% to about 30% by volume, or such as about 5% to about 10% CO2.
It will be appreciated that for growth of the bacteria and CO-to-ethanol fermentation to occur, in addition to the CO-containing substrate gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. A nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used. Anaerobic media suitable for the fermentation of ethanol using CO as the sole carbon source are known in the art. For example, suitable media may be prepared in accordance with standard procedures known in the art as exemplified in JS patent No.s 5,173,429 and -5,593,886-and WO" 02/08438." Prior to use, if required," the " media- can - be made, anaerobic using standard procedures as -exemplified herein. The" 'Examples" herein provide other exemplary media.
"he fermentation should desirably be carried out under appropriate conditions for the desired ermentation to occur (e.g. CO-to-alcohoI). Reaction conditions that should be considered nclude pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox lotential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas) substrate concentrations to ensure that CO (for example) in the liquid phase does- not iecome limiting, and maximum product concentrations to avoid product inhibition. he optimum reaction conditions will depend partly on the particular micro-organism used, lowever, in general, it may be preferable that the fermentation be performed at a pressure iigher than ambient pressure. Operating at increased pressures allows a significant increase in he rate of CO transfer from the gas phase to the liquid phase where it can be taken up by the nicro-organism as a carbon source for the production of ethanol. This in turn means that the'f etention time (defined as the liquid volume in the bioreactor divided by the input gas flow ate) can- be reduced when bioreactors are maintained at elevated pressure rather than tmospheric pressure.
Jso, because a given CO-to-ethanol conversion rate is in part a function of the substrate stention time, and achieving a desired retention time in turn dictates the required volume of bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor squired, and consequently the capital cost of the fermentation equipment. According to examples given in US patent No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.
The benefits of conducting a gas-to-ethanol fermentation at elevated pressures have been described elsewhere. For example, WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively. However, example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day. "ΑS"Will be~appareτit tothose of skill in the art, embodiments of theinvention in which" gaseous - substrates are solely provided to a bioreactor after having already been dissolved, or are carried in a form other than a gas, the need to elevate the pressure within the bioreactor will be removed or at least diminished.
It is" also desirable that theTate of introduction of the CO-containing substrate is such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that the ethanol product is consumed - by the culture. Limitations of other processes and for other substrates will be apparent to those skilled in the art and the rates of introduction may be controlled accordingly.
Product recovery
The products of the fermentation reaction can be recovered using known methods. Exemplary "methods Include" those'described in WO2007/117157, WO2008/115080, US 6,340,581, US 6,136,577, US 5,593,886, US 5,807,722 and US 5,821,111. However, briefly and by way of example only, ethanol may be recovered from the fermentation broth by methods such as fractional distillation or evaporation, and extractive fermentation.
Distillation of ethanol from a fermentation broth yields an azeotropic mixture of ethanol and water (ie 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-immiscible solvent that presents a low toxicity risk to the fermentation organism, to recover the ethanol from the dilute fermentation broth. For example, oleyl alcohol is a solvent that may be used in this type of extra"ction~process. In this process, oleyl alcohol is continuously introduced into a fermenter, whereupon this solvent rises forming a layer at the top of the fermenter which is continuously extracted and fed through a centrifuge. Water and cells are then readily separated from the oleyl alcohol and returned to the fermenter while the ethanol-laden solvent is fed into a flash vaporization unit. Most of the ethanol is vaporized and condensed while the oleyl alcohol is non volatile and is recovered for re-use in the fermentation.
Acetate, which is produced as a by-product in the fermentation reaction, may also be recovered from the fermentation broth using methods known in the art. For example, an adsorption system involving an activated charcoal filtermay~be~used. "In lKis~case7 microbial cells are- typically first removed from the fermentation broth using a suitable separation method. Numerous filtration-based methods of generating a cell free fermentation broth for product recovery are known in the art. The cell free ethanol- and acetates-containing permeate is .then passed through a column containing activated charcoal to adsorb the a_cetate. Acetate ]n the acid form (acetic acid) rather than the salt (acetate) form is more readily adsorbed by activated charcoal. It is therefore preferred that the pH of the fermentation broth is 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. In certain embodiments, ethanol produced by the fermentation process itself may be used to eiute the acetate. Because the boiling point of ethanol is 78.8 9C and that of acetic acid is 107 9C, ethanol and acetate can readily be separated from each other using a volatility-based method such as distillation.
Other methods for recovering acetate from a fermentation broth are also known in the art and may be used in the processes of the present invention. For example, US patent No.'s 6,368,819 and 6,753,170 describe a solvent and cosolvent system that can be used for extraction of acetic_acid from fermentation broths. As with the example of the oleyl alcohol- based system described for the extractive fermentation of ethanol, the systems described in US patent nos. 6,368,819 and 6,753,170 describe a water immiscible solvent/co-solvent that can be mixed with the fermentation broth in either the presence or absence of the fermented micro-organisms in order to extract the acetic acid product. The solvent/co-solvent containing the acetic acid product is.then separated from the broth by distillation. A second distillation step may then be used to purify the acetic acid from the solvent/co-solvent system.
The products of the fermentation reaction (for example ethanol and acetate) may be 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 one or more product from the broth simultaneously or sequentially. Ethanol may be conveniently recovered by distillation, and acetate may be recovered by ■adsorption on activated_charcoal," Using" the methods" described' above. The~separated~ microbial cells can be returned to the fermentation bioreactor. The cell free permeate remaining after the ethanol and acetate have been removed is-also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to-ceplenish-the nutrient medium before it is returned to the bioreactor. Also, if the pH of the broth was adjusted as described above to enhance adsorption of acetjc acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
Element Capture Using Carriers
In accordance with particular aspects of the invention, there is provided a method of capturing one or more elements, such as CO, from a gaseous source, then providing the captured element(s) to a fermentation reaction. In particular embodiments of the invention, the element(s) may be captured using any suitable physical or chemical carrier and then added , directly to a fermentation reaction. In alternative embodiments, the captured element(s) may be released from the carrier to provide a gaseous substrate that may be passed to a fermentation reaction for conversion into products.
In accordance with certain aspects of the invention, one or more carriers charged with one or. more elements are added to a fermentation reaction, which typically takes place in a-, bioreactor, preferably at one or more time points. The carrier(s) may be added to the fermentation reaction at any point at which one is concerned that they are depleted to the point that they may be limiting the rate of alcohol production by the micro-organisms or otherwise to ensure the concentration remains within a predetermined range.
The carrier(s) may be added at discrete time points or continuously fed to the bioreactor at a rate calculated to ensure that the concentration in the reaction broth is within the predetermined range. Diluents and/or other nutrients of use in the reaction may additionally be added. According to certain embodiments, the carrier(s) are mixed therewith prior to being added to the bioreactor, although the invention is not limited thereto.
As to addition at discrete time points, one could determine average rates of alcohol production and depletion of the feedstock used in the reaction and calculate points in the fermentation reaction at which it is most likely it will be necessary to supplement the feedstock.
93 - -Alternatively,
Figure imgf000026_0001
the fermentatioτrprocess~by taking samples frbrrftne" bioreactor at particular time points to determine a status of the reaction e.g., by testing a ^sample-qf one or more_products of-the fermentation reaction. (for example an alcohol and/or - acid) and/or determining- a cell density. Known parameters of particular fermentation processes may _be_ monitored using known methodologies .and equipment-may be used in " determining whether additional feedstock is required.
Cell density may be measured using standard techniques known in the art. By way of example, . manual observation under a microscope" or preferably measurement of optical density using a spectrophotometer may be used. Measurement of optical density at 600nm is particularly useful.
The level .of one or more fermentation products (alcohols and/or acids) can be measured using standard methodology known in "the art. By "way of example, these may be identified and measured _using gas chromatography, high pressure liquid chromatography, enzyme-based assays or colourimetric or fluorometric assays.
- The, carrier(s) may be added to the bioreactor by any known means. By way of example, solutions may be introduced into the reactor automatically through a dedicated pump or manually via a septum covered. port using a syringe. Alternative, known means may be provided for adding the carrier(s) to the bioreactor in the form of a gas.
In accordance with another aspect of the invention, element(s) such as CO, is released from a carrier such that the element(s) can be provided to the bioreactor as a substrate for conversion to one or more products by a microbial culture. The element(s) may be released from the carrier by any means known in the art and in certain embodiments, the carrier may be reused ■ to capture further element(s). The element may be released from the carrier and provided to - a fermentation reaction at a sufficient rate suitable to maximise product biosynthesis. Additionally or alternatively the element can be released and stored in a reservoir. At least a portion of the stored substrate can be provided to a fermentation. reaction on an as required basis to maintain efficient product synthesis. Exemplary methods for storing a gaseous substrate and providing the substrate to a bioreactor are described in PCT/IMZ2008/275.
Carriers "As noted-previσιrsly,-elementsrsuch as COrcan be captOredTising molecular^sievesr Molecular "
-sieves usually comprise . aluminosilicate minerals (zeolites), clays, porous glass, microporous
-charcoals, active carbons or.syjnthetic -compounds that- have open structures through which-^
-selected smalhmolecules such as CO may pass. The adsorption selectivity and efficiency of rrLoleculer_siev_es_usualLy_:depends.on-the pore size and/or cavity size. For example, 3A sieves adsorbTNK3 and H2O molecules, while 4 A adsorb H2O, CO2, SO2, H2S and ethanol. Larger pore, sizes can selectively capture larger molecules. Typically captured molecules will be
. additionally held within^a cavity by intermolecular interactions such as_ Van der Waals or dipolar forces. In-some examples, elements may even form covalent bonds within the cavities of carriers such as zeolite(s).
Fermentation substrate molecules, such as CO, may be trapped by molecular sieves by simply passing a stream comprising such elements over the surface of the sieve. The "charged" sieve . (carrier) may then- be transported and/or stored until the substrate is required for fermentation.- In particular embodiments, the charged sieve can be added directly to a ' fermentation reaction-such that the element(s) will be available, or at least partially available for conversion into,^products by a microbial culture. In alternative embodiments, the element(s) may be released from the carrier by any known means prior to passing the substrate to the bioreactor. Methods for releasing the substrate from molecular sieve carriers" include pressure change, heating and purging with a carrier gas or heating under vacuum. One exemplary_method-for releasing an element from a carrier is pressure swing adsorption (PSA).
PSA is an adiabatic process which may be used for the purification of gases to remove accompanying impurities by adsorption through suitable adsorbents in fixed beds contained in pressure vessels under high pressure. Regeneration of adsorbents is accomplished by countercurrent depressurization and by purging at low pressure with previously recovered - near product quality gas. To- obtain a continuous flow of product, preferably at least two adsorbers are provided, such that at least one adsorber is receiving a gas stream (such as a ~waste/exhaust/biogas gas stream) and actually produces a product of desired purity. Simultaneously, the subsequent steps of depressurization, purging and repressurization back to the adsorption pressure are executed by the other adsorber(s). Common adsorbents may readily be selected by one of skill in the art dependent on the type of impurity to be adsorbed and removed. Suitable adsorbents include zeolitic molecular sieves, activated carbon, silica gel oractivated alumina." Combinations of adsorbentrbeds may be used~on" top "of on"e~another" thereby dividing the adsorber contents into a number of- distinct zones. PSA-involves a pendulating swing in -parameters such as pressure, temperature, flow ,and composition of gaseous and adsorbed phase.
Purification or separation of gases" using PSA normally takes place at near ambient feed gas " temperatures, whereby the components to be removed are selectively adsorbed. Adsorption -should ideally be- sufficiently reversible to enable regeneration of adsorbents at similar ambient temperature. PSA may be used for treatment, purification and potential capture of most common gases including CO, CO2 and H2. Examples of PSA techniques are described in detail in Ruthven, Douglas M. et al., 1993 Pressure Swing Adsorption, John Wiley and Sons.
Molecules such as metal carbonyls contain several moles of CO per mole of metal atom. Metal carbonyls are coordination complexes of transition metals with CO. Most metal carbonyls are relatively stable and are prepared in several steps, however Fe(CO)5 and Ni(CO)4 can be f prepared directly by treating the pure metal with CO. Substances such as Fe(CO)5 and Ni(CO)4 are not very stable and tend to decompose at least when they are exposed to heat or light. The principal reaction usually involves the oxidation of the transition metal (if there is an oxidiser present) with the simultaneous release of CO. In the absence of any other chemicals, the molecule just decomposes to produce the elementary metal and CO. However, where the substance decomposes through oxidation, a salt of the metal is produced and not the .elemental metak In almost any case, it is possible to decompose the molecules purposely to produce (release) CO (and/or another desired gas). Given the high density of the element, such as CO, provided by metal carbonyls, the (CO) load in the broth can be increased.
In particular embodiments of the invention, iron pentacarbonyl is produced by reacting a stream comprising CO with elemental iron, such as iron filings. The resultant Fe(CO)5 can be decomposed by any known means, however in certain embodiments, the metal carbonyl is reacted with strong acid (eg H2SO4) to provide CO. In a particular embodiment of the invention the dissociated metal (eg Fe) can be reused to capture further CO.
Elements such as CO which may form at least a portion of the feedstock for a fermentation reaction are typically more soluble in apolar liquids, such as oils or perfluorinated hydrocarbons, than the aqueous phase of most fermentation reactions. Accordingly, one or -more apolar liquids -carrbezused as a "carrier for elements IrTa" feedstock" to Increase the availability of the particular element in a fermentation reaction. -In such embodiments, the feedstock preferably includes elements such as CO that are converted to-products by microbial fermentation; Those skilled in the art will appreciate that where a feedstock" comprises multiple elements,; particular elements will be more soluble in apolar liquids than others. Accordingly, the .-dissolved- portions of the feedstock become the substrate for the fermentation reaction.
As will be apparent to those skilled in the art, the abovementioned apolar liquids are gen era Ny immiscible with aqueous solutions. Through use of a surfactant such as lecithin, an emulsion may be formed so as to increase mass transfer of the at least a portion of the feedstock from the liquid to thejnicro-organisms used in the fermentation (note the at least a portion of the feedstock may become^dissolved in the fermentation media before it is taken up by the microorganisms). This is achieved by increasing the effective surface area of the liquid by dispersing^ it throughout the fermentation broth (or at least throughout a larger portion thereof). Note -that the aforementioned liquids will tend to float on the surface of the broth unless such a' surfactant is added.
While there is merit in adding a surfactant, the addition thereof is not essential to the invention. Moreover, according to particular embodiments, it may be preferable not to add a surfactant so as to improve ease of separation of the liquid from the fermentation broth.
In certain embodiments; the method includes mixing the liquid and/or the composite stream with an. aqueous solution. Where a surfactant is used, the surfactant is also preferably mixed during this mixing step. Note that mixing may be performed when no surfactant is used so that the liquid or composite stream becomes dispersed in the broth (ideally in small droplets) „ but may be_periodically interrupted to allow components of the broth to separate (namely the liquid from the aqueous portion of the broth), so that the liquid and/or the aqueous portion of the broth may be drawn off as desired.
The .aqueous solution may, include at least a portion of the fermentation broth and/or supplementary media used for the reaction.
The step of adding and/or mixing may take place in the bioreactor(s) used to conduct the -fermentation-reaction. According to an alternative embodiment, the feedstock, or at least a -iportion thereof, may be added to the liquid to form~the:composιte streaTrf before being added uto the bioreactor,. although the invention is not limited thereto. As a further alternative, the -. composite-stream may.be formed and then mixed with an-aqueous solution outside- of -the- .ibjpreactor, suclias in a pre-treatment chamber or conduit used to feed the reaction inside the-
Figure imgf000030_0001
further alternative, :the_atJeast a portion of the feedstock-may-be added
"to the liquid after it has been added to or mixed with the aforementioned liquid- and/or a surfactant.
While the at least a portiorrof the feedstock is preferably in gaseous form when added to the liquid; the invention is not limjted thereto. More particularly, carriers may be used and added to the liquid, the carriers being able to release quantities of the at least a portion of the .feedstock, preferably in a controllable manner.
Ir+ particular embodiments of the invention, the meinoα mciuαes removing aτ leasτ a porxion OT the liquid from the bioreactor. After the at least a portion is removed, a further at least a ~ portion- otihsJeedstock is. preferably added thereto. During the time the liquid is in the . bioreactor, the stored at least a portion of the feedstock becomes depleted as it is taken up by /, the micro-organisms used jnjthe fermentation reaction. The liquid may then be "re-charged" '„ so that it may be re-used.
For example, butanol is typically comparably or more miscible in oil than aqueous solutions. Thus, according to embodiments of the invention, while a proportion of butanol produced by a fermentation reaction will remain in the aqueous portion of the fermentation broth, a significant, portion will be captured within the apolar liquid. As described hereinabove, it is relatively straightforward to isolate and remove the apolar liquid from the fermentation broth, improving the ease of capturing and recovering the products of the fermentation. This also has a further benefit in that excessive quantities of butanol- in the aqueous portion of the- fermentation broth are toxic to the micro-organisms used therefor. The use of oil serves to -isolate at least a portion of the butanol produced from the aqueous portion of the solution (which contains the micro-organisms) and holds it in the apolar liquid. Thus, it is possible to run a fermentation reaction for a longer period than previously because it will be longer before the concentration of butanol in the aqueous portion of the broth will reach an unacceptable level (around 2%). Through suitable control of the circulation of the apolar liquid through a : bioreactor> it may be possible to remove sufficient butaήol to enable the fermentatiόϊi reaction to operate continuously or semi-continuously.-
Ih particular embodiments of the invention, the method includes removing one or "more products from the apolar liquid removed from the bioreactor. Since particular products may have higher miscibility.in particύlariiquids"trran m~aqϋeδϋs solutions, the productslήay be at"a" higher concentration therein providing for " a"~ potentially more efficient product extraction process (i.e., the. concentration of alcohol will be larger in the liquid, e.g., oil), and it is
-therefore more economical to remove it than as a weaker solution from the generally aqueous fermentation media. For example, where evaporation/distillation is used to separate different
-portions of the product stream (i.e., at least the apolar liquid from one product or vice versa), there;will be. a smaller mass to heat to enable such evaporation to take place. Furthermore, ,the liquids (e.g; JDiIs)- used will tend to have a . higher. boiling point than an aqueous solution, which is advantageous ; because the difference between the boiling point of the product to be.
,. removed (e.g. alcohol) and the liquid (e.g. oil) is much higher than the difference between the boiling point of the product to be removed and the watery fermentation broth which makes,
-distillation, easier, . Furthermpre, liquids such as oils have a much lower heat capacity than water or aqueous solutions, meaning that less energy is needed for heating in the distillation process. Note that energy:; costs associated with heating for evaporation/distillation to
^capture/remove products from fermentation reactions are a sizeable proportion of the total cost of such processes.
According to a certain embodiment, at least one product, such as butanol, may be removed from the liquid using. a gas stripping process. Gas stripping is a technique which allows for the selective removal. of volatiles from a medium without the need for expensive membranes or chemicals. Gas. may be sparged into the medium to create bubbles which burst and cause the surrounding liquid to vibrate. These vibrations are sufficient to result in volatiles being removed from the medium.
According to a particular embodiment involving gas stripping, the liquid is sparged with the at least a portion of--the feedstock such that "re-charging" of the liquid takes place as the products of the fermentation are stripped from the liquid. Thus, according to a particular embodiment, oil (such as pump oil or canola oil) obtained'from a bioreactor is sparged with at least CO gas so as to substantially concurrently charge the oil with CO and extract butanol.
As_would be apparent to one of skill in trfe'art; in order for a fermentation reaction to continue operating within desired performance parameters, elements of the required feedstock need to be fed to the reaction in sufficient quantities or sufficient rates. However, if these quantities become too high, the broth can become toxic to the microbes used in the reaction, which at best lowers the efficiency and/or productivity of the reaction. Conversely, if the quantities become too low, the microbes may not be provided with sufficient feedstock to maintain the fermentation reaction above acceptable production levels. Thus, in certain embodiments, the flow rate of the composite. stream into the bioreactor is controlled according to techniques known to those skilled in the art.
In broad terms, according to the invention, at least a portion of the substrate for a fermentation reaction is provided to a bioreactor after it has been dissolved in a liquid such as "an oil or a perfluorocarbon and/or such a liquid is used in the removal and/or separation of products of the reaction.
During fermentation processes, known parameters may be monitored using known methodologies and equipment so as to determine whether additional feedstock is required. Additional feedstock may be added at discrete time points. One could determine average rates of alcohol production and depletion of the feedstock used in the reaction and calculate points in the fermentation reaction at which it is most likely it will be necessary to supplement the feedstock. .Alternatively, one could actively monitor the fermentation process by taking samples from the bioreactor at particular time points to determine a status of the reaction e.g., by.testing a sample of one or more products of the fermentation reaction (for example an alcohol and/or acid) and/or determining a cell density.
Cell density and/or the level of one or more fermentation products (alcohols and/or acids) can be measured using standard methodology as discussed hereinbefore.
The feedstock maybe added to the bioreactor by any known means. By way of example, solutions may be introduced into the reactor automatically through a dedicated pump or manually via a-septum covered port using a syringe. Additionally, known means may be provided for adding the feedstock to the bioreactor in the form of a gas. Desired products from microbial fermentation processes include alcoholsrketonesraldehydes and carboxylic acids. Usually these are waste products of the bacteriological metabolism and toxic to the bacteria themselves. The concentration of the desired products in the bacterial broth is. therefore .usually limited to the amounts the bacteria can maximally tolerate. For example, the.maxLmu.rn amount of butanol that can be tolerated by its producing microbe (e.g. Clostridium such as C acetobutylicum, C. saccharobutylicum) is of the order of only 2%. Thus the butanol has to be retrieved from this dilute solution using distillation or other techniques. Since this process, is very energy consuming it would be desirable to have a fermenter system that can tolerate higher amounts of products (so as to eliminate or more widely space apart interruptions in the process); and be able to use separation processes (such as distillation processes) to remove products of a fermentation from solutions containing larger amounts of these desired end products. . Such objects are achieved according to embodiments of the invention by choosing an apolar solvent having a higher boiling point but lower heat capacity compared to the aqueous fermenter broth.
Particular embodiments of the invention provide a leaching (cleaning) procedure of theτ fermenter solution that may be applied in situ to remove these (toxic) products, thereby< allowing the microbial solution to be used for an extended time. The invention is based on the solubility difference of the. desired end products between the aqueous bacterial phase and another suitable more apolar phase that is not toxic to the system (for example, canola oil or any other oils such as silicones and perfluorinated aliphatic and aromatic compounds). Both phases are preferably mixed together either by vigorous stirring or by applying surfactants to either of the phases with the aim of producing an emulsion and thereby increasing the contact area of the two phases under consideration.
The desired product, i.e., the toxin for the bacteria, preferably dissolves in the apolar phase which-can then be extracted by simply decanting it or by other means to separate the oily phase again. Such embodiments readily provide for a continuous system or process of production where some oil is dispersed in the fermenter solution and the separated product rich phase (probably floating on the surface depending on the density of the oil used) taken out of the system for distillation or gas stripping etc. Note that removal of any portion (not necessarily* a major portion) of the" pMSϋct of the fermentation may be beneficial in -enabling the reaction to continue for longer and" thus; generally more .efficiently and with-a higher productivity.- Thus a higher proportion of the product of the fermentation may remain in the generally aqueous fermentation media than the apolar liquid (e.g., oil/perfluorocarbon).
In fermentations using gases, the use of emulsions as gas carriers could dramatically reduce the energy and operating costs associated with overcoming the gas to liquid mass transfer barrier in the fermentation system. This is particularly significant in fermentations using CO, as CO gas is sparingly soluble in water. Sterile emulsions could be "loaded" with gas separately in a dedicated unit before being introduced into a fermenter or reactor. A further advantage may be that emulsions could be continuously removed from a fermenter, separated from media and biomass and be "re-charged" with gas before reintroduction to the fermenter vessel as a continuous gas feeding process. This is made all the more possible since the" droplets that constitute the emulsion exist as a separate liquid phase within the media broth. This process could be applied to aerobic fermentations where the introduction of oxygen is required, and " anaerobic fermentations using CO containing gases.
In particular embodiments, the carrier is adapted to selectively carry desirable elements for optimal fermentation "efficiency. For example, the carrier can selectively capture CO from a waste stream from an industrial process and provide a substantially pure substrate stream comprising CO to the bioreactor. Additionally or alternatively, further desirable elements, such as H2,-can-also be captured by the same or different carrier(s) and provided to the bioreactor to enhance productivity. For example, CO and H2 are more soluble in apolar liquids than aqueous nutrient media. Accordingly, use of an apolar oil as a carrier will improve availability -of CO and H2 for conversion into products by microbial fermentation General
Embodiments of the invention are described by way of example. However, it should be appreciated that particular steps or stages necessary to perform the methods of one -embodiment may not be necessary in another. Conversely, steps or stages included in the description -of - a particular- embodiment can be optionally advantageously utilised in embodiments where they are not specifically mentioned. .While, the - invention-: — \s~ broadly described with - reference to any - type of •feedstock/element/substrate- and/or carrier streams that -may be moved through or around the system(s) by- any known transfer means,., in certain embodiments, the substrate(s) are •gaseous. Those skilled in the art- wijj appreciate that particular stages may be coupled by. suitable conduit means or the like, configurable to receive or pass streams throughout a system. A pump or compressor may be provided to facilitate delivery of the streams to particular stages. Furthermore, a compressor can be used to increase the pressure of gas -provided to one or more stages, for example a capture chamber or the bioreactor. As discussed hereinabove, the pressure of gases within a bioreactor can affect the efficiency of the fermentation reaction performed therein. The pressure can be adjusted to improve the efficiency of the fermentation. Suitable pressures for common reactions are known in the art.
In addition, the systems or processes of the invention may optionally include means for regulating and/or controlling -Other parameters to improve overall efficiency of the process. For example particular embodiments may include determining means to monitor the composition of substrate and/or exhaust stream(s). In addition, particular embodiments may include a means for controlling the delivery of substrate stream(s) to particular stages within a particular system if the determining means determines the stream has a composition suitable for a particular stage. For example, in instances where a gaseous substrate stream contains low levels of CO that may be detrimental to a fermentation reaction, additional CO may be liberated from a CO charged carrier and fed to a bioreactor.
In particular embodiments of the invention, the system includes means for monitoring and controlling the destination of a substrate stream and/or the flow rate, such that a stream with a desired or suitable composition can be delivered to a particular stage. For example, where a feedstock source is intermittent in nature, a carrier may be provided for a period such that the carrier is substantially fully charged with one or more elements before it is passed to a latter stage. As such, certain embodiments of the invention include means for monitoring the amount of element (e.g., CO) captured by a carrier. Additionally or alternatively, in embodiments where element(s) are released from the carrier, the release rate may be ■ controlled such that a substrate stream is passed to a bioreactor at an optimised rate. Those -skilled in the~art will appreciate suitable monitoring means and controlling means in order to promote efficient fermentation. In addition, it may be necessary to heat or cool particular system" components or substrate -stream(s) prior- to or during one or more stages in the process. In such instances, known heating or cooling means may be usedr
Furthermore, the system may include one or more pre/post treatment steps to improve the operation or efficiency of a particular stage. For example, a pre-treatment step may include means for removing particulate matter and/or long chain hydrocarbons or tars from a gaseous substrate stream. Other pre- or post-operations that may be conducted include separation of desired product(s) from particular stages, such as, for example, the bioreactor production stage (e.g. removal of ethanol by distillation).
.Various embodiments of the systems of the invention are described in the accompanying Figures. The alternative embodiments described in Figures 1 and 2 comprise features in common with one another and the same reference numbers have been used to denote the same or similar features in the various figures. Only the new features (relative to Figure 1) of Figure 2 are described, and so Figure 2 should be considered in conjunction with the description of Figure 1.
Figure 1 is a schematic representation of a system 100, according to an embodimeni configured to produce one or more CO-carriers and use the carrier(s) produced as a feedstock for -a fermentation reaction. System 100 may be adapted to produce other carriers or ma\ receive carriers that have been pre-manufactured. Additionally, the fermentation reaction may receive feedstock from more than one source.
System. 100 includes chamber 11, optional treatment unit 12 and bioreactor 13. Chamber 11 contains CO capture means, such as fine particles of iron. CO gas passes through chamber 11 towards treatment unit 12. As the CO passes through chamber 11, it comes into contact with the iron particles and forms Fe(CO)5 vapours which pass to treatment unit 12. Treatment unit 12 performs any desired pre-treatment steps before the stream is fed to bioreactor 13. Note that treatment unit 12 may alternatively be placed upstream of chamber 11. Alternatively, treatment units 12 may be placed upstream and downstream of chamber 11. As a further alternative, no treatment units 12 may be included in system 100. Treatment unit 12 may be configured to remove unwanted components of the stream, such as by scrubbing. It may also include a condenser for condensing the Fe(CO)5 vapours. Bioreactor~13~contams"a"sliitable media inoculated with bacteria required~fσrthe^particular reaction. It receives the stream in its final form, as vapours and/or a liquid. Bioreactor 13 may -be provided with additional inlets and-outlets "as would be apparent to those skilled in the art to "enable the reaction to perform as desired and for the products thereof to be removed.
Figure 2 is a schematic representation of a system 101 according to another embodiment "configured to capture-CO from a source using one or more CO-carriers, release the CO from the carrier(s) and provide the released CO to a fermenter. System 101 further includes "dissociation" chamber 14r Chamber 11 contains a carrier, such as fine particles of iron or similar means for capturing and/or carrying CO. The CO-carrier then passes to dissociation chamber 14, where CO may be released from the carrier. Chamber 11 and dissociation chamber 14 may be proximate to each other and joined by any suitable conduit means. Alternatively, Chamber 11 and dissociation chamber 14 may be remote and any known transfer means may be provided to transfer the CO-carrier therebetween, for example, a pipeline or vehicular transfer means. For example CO may be captured from a waste stream exiting an industrial process using iron to form Fe(CO)5. In certain embodiments, the capturing of the CO will take place at or near the exhaust of a particular process generating CO, for example, the waste stack of a steel mill. The Fe(CO)5 or other CO-carrier can be transported to a remote location through a pipeline. For example, liquid Fe(CO)5 can be pumped through a pipeline over large distances. Alternatively, the CO-carrier can be placed in transportable tanks or containers for delivery to optional treatment stage 12. For example, tanks or containers containing Fe(CO)5 can be delivered to optional treatment 12 by train, ship or truck.
Following optional treatment, the CO-carrier is passed to dissociation chamber 14, where the -CO can be liberated by any known means and converted into a gaseous substrate. In certain embodiments, CO can be liberated from Fe(CO)5 by heating, irradiation or treatment with acid. In a particular embodiment, Fe(CO)5 is treated with concentrated H2SO4 to liberate the COr The liberated CO can be passed directly to the fermentation reaction by any known means.
In an alternative arrangement, optional treatment stage 12 may be downstream of the dissociation chamber 14. As such, the treatment stage 12 would be adapted to treat or optimise the CO liberated in the chamber 14. -Figure 3 is a schematic-representation of a system 102— according to a further embodiment, wherein-an apolar liquid is used-as a substrate carrier- A substrate stream (e.g. CO) 15isτnixed with water 16 and oil 17- in a hojmogenization chamber 18 to form an emulsion charged with - -CO. The homogenisatjon chamber can include any mixing means known in the art for j)reparing an emulsion on a continuous basis. Suitable mixing means include mechanical agitation and/or utilisation of some form of static mixer/homogeniser e.g. forcing the emulsion components through an orifice plate. The CO charged carrier 19 (emulsion comprising oil/water/CO) is passed_to optional emujsion preparation tank 21, where optionally recycled microorganisms 20 and other components necessary for microbial fermentation, such as nutrients may be added. At desired time points or continuously the substrate carrier can be passed to bioreactor 22. Additionally substrate stream 15 comprising CO may optionally be passed through the ferrηenter for conversion into products and any exhausted gas can be directed away from bioreactor 22 as indicated by arrow 23.
Any conventional bioreactor may be used for the fermentation, including a bubble column reactor. In one embodiment a trickle bed_reactor can be prepared by adding a packing to the column and trickling the emulsion down the column over the packing. The gas phase of the reactor could be monitored and controlled. The fermented emulsion 24 can then be passed to a processing unit 25. A process unit may optionally be included to separate the emulsion components so that the ethanol containing aqueous component could be further processed and the oil rich component replenished with CO and returned to the reactor. A standard -industrialjtubular bowl centrifuge or similar may be used to separate these components on a continuous basis. The aqueous component 16 may be further treated before distillation and the oil component 17 is returned to the homogeniser 18 as the emulsion stream once bacteria have been optionally isolated using microbe separation means 27. The microbe separation means 27 may be any known means used for separating bacteria from a liquid fermentation broth such as a cross -flow membrane or hollow fibre membranes. Isolated bacteria may optionally be redirected into emulsion preparation tank 21.
EXAMPLES
The invention will now be further described in more detail with reference to the following non- limiting examples.. /-The media and solutions used in the fermentations described in these examples contain the following components, unless otherwise noted. Media:
Fermentation media: LM23
Figure imgf000039_0001
Figure imgf000040_0001
-HCI were mixed in 400ml distilled water. This solution was made anaerobic by heating to boiling and allowing it to cool to r-oom temperature under a constant flow of 95% CO, 5% CO2 gas. Once cool, the Cysteine-HGI was added and the pH of the solution adjusted to 5.5 before making the volume up to 1000ml; anaerobicity was maintained throughout the experiments.
Bacteria:
Clostridium autoethanogenum were obtained from the German Resource Centre for Biological
Material (DSMZ). The accession number given to the bacteria is DSMZ 10061.
Sampling and analytical procedures: Cell Density:
To determine the cell density in these experiments, the absorbance of the samples was τmeasured -at 600nm (spectrophotometer) and the dry mass determined via calculation :ording to "published procedures. The level of metabolites" was characterized using High rformance Liquid Chromatography (HPLC)
1C:
1LC System-Agilent 1100 Series. Mobile Phase: 0.0025N Sulphuric Acid. Flow and pressure: 500 mL/min. Column: Alltech 1OA; Catalog # 9648,- 150 x 6.5 mm, particle size 5 μm. mperature of column: 600C. Detector: Refractive Index. Temperature of detector: 45°C.
... Ξthodfor sample preparation:
400 μL of sample and 50 μL of 0.15M ZnSO4 and 50 μL of 0.15M Ba(OH)2 are loaded fnto an Eppendorf tube. The tubes are centrifuged for 10 min. at 12,000rpm, 4°C. 200 μL of the supernatant are transferred into an HPLC vial, and 5μL are injected into the HPLC instrument.
Reagents
Fe(CO)5 was purchased from Sigma Aldrich (195731).
Lecithin was purchased in granulated form from a local supermarket Pluronic F-68 was purchased from Sigma Aldrich
Example 1
Ethanol and butanol solutions were prepared and the concentrations measured using gas chromatography. The solutions were measured individually and also in a mix as depicted in Table 1 below. "An aliquot of the aqueous alcoholic phase (representing the fermenter solution) Was taken and shaken with an equal amount of canola oil (representing the extraction, oily phase) in a beaker. After letting the beaker stand for a while to wait for the separation of the phases, the alcohol amounts in the aqueous phase was measured again.
As can be seen from Table 1, the ethanol is not leached out of the water whereas about 40% the butanol is (more, particularly, 35%, 42% and 44%).
Figure imgf000041_0001
Figure imgf000042_0001
Table 1 Example 2A
Theiajm ofthis experiment was to determine if emulsions of oils and surfactants could be used as carriers of CO in a nutrient media for bacteria that use CO as a source of carbon and energy.
Procedure:
An emulsion of canola oil and lecithin was autoclaved at 121°C for 20 minutes. Once the solution had-cooled to less than 1000C, the emulsion solution was split into two equal portions, approximately 100 ml per portion.
Each portion of the emulsion was separately sparged with gas while cooling. One half of the solution was sparged with N2 gas, while the other was sparged with a gas mix of 70% CO, 15% CO2,-14% N2, and 1% H2.- Each solution was sparged with these gases until they reached room temperature.
A 2x concentrated solution of LM23 media was prepared lacking cysteine and B-vitamins. This solution.was boiled in the microwave to de-gas it and sparged with N2 gas as it cooled to room temperature. Once cool, the cysteine and b-vitamins were added and the pH of the media adjusted to 5.4 with 5M NaOH. Additionally dH2O was boiled and cooled under N2 gas. Six 250 ml serum.bottles were prepared in which to test the impact of emulsions in growth media on bacterial.growtruand their- utility as a CO carrier. All bottles were prepared under CO gas to ensure anaerobicity. The bottles were prepared as follows:
Treatment:
1. 25 ml of 2 X LM23 + 25 ml dH2O
2. 25 ml of 2 X LM23 + 25 ml dH2O
3. 25 ml of 2 X LM23 + 25 ml emulsion sparged with N2
4. 25 ml of 2 X LM23 + 25 ml emulsion sparged with N2
5. 25 ml of 2 X LM23 + 25 ml emulsion sparged with 70%.CO
6. 25 ml of 2 X LM23 + 25 ml emulsion sparged with 70% CO
Once prepared each bottle was immediately stoppered with a butyl rubber septum, and crimp- sealed with an aluminium cap to ensure anaerobicity. All 6 bottles were autoclaved at 121°C for 20 min and allowed to cool to room temperature. Once cooled each bottle was inoculated with 500 μl of a growing culture of bacteria (C. autoethanogenum). After inoculation, a 1 ml sample was removed from each serum bottle. The headspace gas was then pressurised to 35 psig with either N2 gas or the 70% CO gas mix described above as shown in Table 2:
Figure imgf000043_0001
Table 2
Once pressurised with gas each bottle was placed on a shaking incubator at 37°C. The pH of the samples taken was measured to be 5.4 for each sample. Results:
After 4 days-of incubation, 1 ml samples were taken -from each of the-six serum bottles. The pH of each sample was measured and__used as an indicator of bacterial growth, the rationale and our experience having shown that as the bacterial population grows acetic acid is produced resulting in-a-_reduction in the pH of the growth media. The results are provided in Table 3:
Figure imgf000044_0001
Table 3
The results for treatment 6 (25 ml of 2 X LM23 + 25 ml emulsion sparged with 70% CO, with N2 in the headspace) indicate that the emulsion acted as a carrier for CO gas, enabling microbial growth and associated acetic acid production. In this bottle there was no CO in the headspace, therefore the only source of carbon and energy was the CO carried by the emulsion.
The~control treatment 3 (25 ml of 2 X LM23 + 25 ml emulsion sparged with N2, with N2 in the headspace) showed no drop in pH, demonstrating that the bacterial population were unable to use the carbon and energy associated with the oil and surfactants in the emulsion. Control treatment 4 (25 ml of 2 X LM23 + 25 ml emulsion sparged with N2, with 70% CO in the headspace) demonstrated that the emulsion did not inhibit the microbial population in the uptake of CO from the headspace. "
Example 2B
Two emulsions were prepared by mixing canola oil with two different surfactants. In one case lecithin -was used and in the other case a tri-block copolymer (Pluronic F-68, trademark from BASF) i.e., a synthetically produced surfactant. 24 g of surfactant was mixed with 80 g of canola oil and 300 ml of LM 23 media (prepared in the same fashion as for Example 2a) and magnetically stirred-until a homogeneous appearing emulsion resulted. The pluronic emulsion was of white appearance, while the lecithin emulsion was yellowish in colour. The emulsions " were heated in the microwave and subsequently split into two fractions. Each portion of the emulsion was separately sparged with gas while cooling. One half of the solution was sparged with l\h gas, while the other was sparged with a gas mix of 70% CO, 15% CO2, 14% N2, and 1% H2. Each solution was sparged with these gases until they reached room temperature.
After, cooling, cysteine was added and the pH adjusted to 5.5. Serum bottles were filled with 50 ml of the emulsions, sealed and subsequently autoclaved. Control bottles containing only LM 23 but no emulsions were prepared and treated otherwise in the same way. Bottles were evacuated and refilled with CO (70% in CO2) twice and the final pressure adjusted to about 35psi. Control bottles were treated in the same way with the exception that N2 was used instead of CO to saturate the oil. Once cooled, each bottle was inoculated with 1 ml of a growing culture of bacteria (C. autoethanogenum).
Bottles were placed on shaking tables inside an incubator and the products traced by taking 1 ml samples daily for GC analysis using techniques known in the art.
In detail the samples prepared were:
• Control CO; LM 23 only saturated and gassed with CO (Fig 5: Acetate = open triangle; Ethanol = solid triangle)
• Pluronic-N2; Emulsion with Pluronic saturated with N2 (Fig 5: Acetate = open square; Ethanol = solid square)
• Pluronic CO; Emulsion with Pluronic saturated with CO (Fig 6: Acetate = open diamond; Ethanol = solid diamond)
• Lecithin CO; Emulsions with Lecithin saturated with CO (Fig 6: Acetate = open circle; Ethanol = solid circle)
The results for Ethanol/Acetate concentrations as a function of time are given in Figures 5 and 6. As can be seen in Figures 5 and 6, the N2 gassed samples showed little or no production as -was~to be expectecL-The LM 23 control bottle without surfactants showed~theTTsuaTproduct .accumulation pattern. Both types of emulsions produced more ethanol than the control.
Example 3
Figure 4 is a light microscopy photograph of a water droplet containing bacteria in an oily medium. The sample was prepared by shaking (mixing) equal amounts of bacteria containing fermenter broth and a purified vegetable oil. After keeping the sample still for some time, the two phases almost completely separated and the oily phase was investigated for the presence of bacteria.
As can be seen in-Figure 4, the bacteria avoid the oil phase entirely -there are bacteria (darker rod shapes) inside the water droplet (centre of image) but not outside (i.e., in the oily phase - identified by smaller droplets surrounding the larger water droplet). This has a major positive implication with regard to the application of the use of oils in an industrial fermentation process. Specifically, it is possible to deliver, in particular, gaseous feedstocks using the apolar liquids such as oils and deplete the solution of toxic solvent products like butanol. Doing this will not remove cells from the fermenter broth. Hence, there seems to be no need to apply any filtering techniques etc that seek to retain the cells in the fermentation vessel.
It has also been found that the large amount of dissolved gases in the oils can be used to cool the fermenter broth and vigorously mix the broth due to the large gas solubility gradient with temperature. More particularly, the oil may be saturated with, for example, a CO-containing gas at ambient temperature or cooler (noting that the solubility of gases in liquids is inversely proportional to temperature i.e., higher solubility at lower temperatures). The oils may, for example, then be introduced into the fermenter via a small nozzle in a jet-like manner. Since the reactor is usually kept at higher temperature (~37°C), the gas would instantly go out of solution from the- oil to reach the new equilibrium solubility at that respective new temperature. Because of the speed the gas bubbles form and expand, a large cooling effect associated with this adiabatic expansion corresponds. Additionally, the feed gas replenishes the fermenter broth with carbon and energy in the form of CO gas. With an optimized fermenter layout, the rapid expansion of gas can also be used simultaneously to create turbulence in the fermenter so that no or minimal additional stirring/mixing techniques are needed. Example 4
Procedure .
A preparation of LM23 fermentation, media (media recipe described above) at pH 5.5 was prepared as follows:
1. All -ingredients _with the exception of cysteine HCL were mixed in 800ml of distilled -water. Th is -solution was made anaerobic by heating to boiling and allowing it to cool to room temperature under a constant flow of 95%CO and 5%CO2 gas. Once cool, the cysteine HCL was added and the pH of the solution adjusted to 5.5 before making the volume up to 1000ml. Anaerobicity was maintained throughout.
2. Under a continuous gas flow of 95%CO and 5%CO2, 50ml of LM23 media was dispensed into 250 ml serum bottles. All bottles were stopped with gas impermeable butyl rubber septa and crimp sealed before autoclaving at 121°C for 20 minutes.
3. Five serum bottles were inoculated with 5ml of dense sample from a continuous culture of Clostridium autoethanogenum. ll.βpsi of CO was pumped into these serum bottles as a result of the CO release procedure described below.
4. CO2 was added in order to pressurise the serum bottles to 20psig.
5. Samples were then taken to observe growth over time. CO release Procedure:
1. ImI of liquid Fe(CO)5 was added to a 250 ml serum bottle with 180ml H2SO4. This was connected to a 250ml serum bottle containing 180ml NaOH (30%wt) by tubing to pump through the resulting gas.
2. The serum bottle containing 180 ml NaOH was connected to a_series of Hungate" tubes filled with 10 ml pump oil; the remaining gas was pumped through these tubes.
3. Once filtered through the pump oil, the gas was then passed through a series of 3 Hungate tubes in ice.
4. The resulting gas was filtered once more through 10ml of pump oil and then pumped into the 250ml serum bottles. The CO generated from the Fe(COJs was at ll.δpsia
Results: -Five serum bottles were-sampled and growth analyzed-and~the results are presented-ϊn Table , 4. The bacteria showed no signs of growth until day~3. Rapid growth was observed between" days 3 and 5 after which growth stopped.
Results
Figure imgf000048_0001
Table'4
These results showjthat Clostridium autoethanogenum converts CO released from Fe(CO)5 into products.
The invention has been described with reference to certain embodiments, in order to enable the reader to practice the invention without undue experimentation. Those skilled in the art will appreciate the invention is susceptible to variations and modifications other than those specifically described. It is to be understood the invention includes all such variations and modifications. Furthermore, titles, heading, or the like are provided to enhance the reader's comprehension and should not be read as limiting the scope of the invention.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world. The entire disclosures of all applications, patents and publications cited herein are incorporated by reference.
Throughout this specification and claims, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive-sense, that is to say, in the sense of "including, but not limited to".

Claims

-CLAIMS -
1. A method of producing products bv microbial fermentation of a substrate, the method including at least the steps of:
_a._ jcapjuring one or more element(s) in and/or on one or more carriers; ^nd
b. treating-the carrier(s), such that the elements are available as substrate for conversion into products by a microbial culture in a bioreactor.
2. A method according to claim I1 wherein the treating step includes adding at least a portion of . the carrier(s) and captured element(s) directly to the bioreactor, such that the element(s) is available as substrate for conversion into products.
3. A method according to claim 1 or 2, wherein the treating step includes releasing at least a portion of the element(s) such that the released element(s) can be added to the bioreactor as substrate.
4. A method according to any one of claims 1 to 3, wherein the carrier(s) is adapted to physically and/or chemically capture the element(s).
5. A method according to any one of claims 1 to 4, wherein the carrier is selected from:
a. molecular sieve(s)
b. zeolite(s)
c. apolar liquid(s)
d. metal(s).
6. A method according to any one of claims 1 to 5, wherein the element is CO.
7. A method according to claim 6, wherein the carrier is a transition metal.
8. A method according to claim 7, wherein the transition metal carrier is Fe and/or Ni and captures the CO as Fe(CO)5 and/or Ni(CO)4. -9. -A method according to claim-8 when -dependant on claim 3, wherein the CO is released and added to the bioreactor, wherein the treating includes at least one or more of:
a. heating
b. irradiation
c. mixing with acid.
10. A method according to any one of claims 1 to 9, wherein an apolar liquid is used as a carrier.
11. A method according to claim 10, wherein the apolar liquid carries one or more of said element(s) and or another carrier and is added directly to the bioreactor.
12. A method according to claims 10 or 11, wherein the apolar liquid carrier forms an emulsion with an aqueous fermentation media.
13. A method according to_ claim 12, wherein a surfactant is added to the apolar liquid to stabilise the emulsion.
14. A method according to any one of claims 10 to 13, wherein the apolar liquid is selected from carbon-based oils, silicon-based oils, olefins, perfluorinated hydrocarbons and aromatic compounds.
15. A method according to any one of claims 10 to 14, wherein at least a portion of the apolar liquid is removed from the bioreactor and used to capture more or replacement one or more said element(s), wherein the recharged apolar liquid is returned to the bioreactor.
16. A method according to claim 15, including recovering one or more products from the apolar liquid.
17. A method according to any one of claims 1 to 16, wherein the microbial fermentation is an anaerobic fermentation.
18. A method according to claim 17, wherein the element(s) is derived as a by-product of an industrial process. ~19^-A-method accordjng-to any-one of claims 1 to 18, wherein the microbial culture-comprises at least one strain of carboxydotrophic bacterium,
20. A method according to claim 19, wherein the bacterium is Clostridium autoethanogenum and the products are acetate and/or ethanol.
21. -A system adapted to produce products by anaerobic fermentation, wherein the system includes at least:
a. a chamber for capturing a substrate in and/or on one or more carriers;
b. an optional release chamber;
c. - a- bioreactor containing a microbial culture and configured to receive the substrate; and
d. transfer means adapted to pass the carrier(s) from (a) to:
i. (b) to (c); or
ii. (c); or
iii. (b), wherein at least a portion of the substrate is then passed to (c).
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