WO2009070022A1 - Production of a product in a microbial fuel cell - Google Patents

Production of a product in a microbial fuel cell Download PDF

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Publication number
WO2009070022A1
WO2009070022A1 PCT/NL2008/050754 NL2008050754W WO2009070022A1 WO 2009070022 A1 WO2009070022 A1 WO 2009070022A1 NL 2008050754 W NL2008050754 W NL 2008050754W WO 2009070022 A1 WO2009070022 A1 WO 2009070022A1
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microorganism
fuel cell
microbial fuel
product
electrode
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PCT/NL2008/050754
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French (fr)
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Ruud Alexander Weusthuis
Hubertus Victor Marie Hamelers
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Stichting Dienst Landbouwkundig Onderzoek
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Publication of WO2009070022A1 publication Critical patent/WO2009070022A1/en

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/90—Selection of catalytic material
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/10—Fuel cells with solid electrolytes
    • H01M2008/1095—Fuel cells with polymeric electrolytes
    • 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
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
    • Y02P70/50—Manufacturing or production processes characterised by the final manufactured product

Definitions

  • the invention relates to a fermentation process, wherein use is made of a microbial fuel cell for fermenting a substrate into a product, wherein said product is oxidised compared to the substrate present in the microbial fuel cell and wherein electrical energy is produced.
  • Fermentation is an important technology for the production of several types of products: bio transport fuels (such as ethanol, butanol or hydrogen) or food and feed ingredients (such as glutamate, lysine, citrate, or lactic acid) or chemical building blocks for products such as nylon.
  • bio transport fuels such as ethanol, butanol or hydrogen
  • food and feed ingredients such as glutamate, lysine, citrate, or lactic acid
  • chemical building blocks for products such as nylon.
  • the efficiency of fermentation processes is rather low due to among other the formation of undesired side products, the production of biomass and/or the low rate of oxygen transfer.
  • heat is produced during the conversion of oxygen into water. Therefore, expensive cooling equipments are needed in order to maintain the temperature of the fermentation to a suitable temperature for the microorganism used.
  • To counteract the formation of undesired side product one may genetically modify the microorganism used. However, this strategy is not always possible.
  • To improve the rate of oxygen transfer one may increase the inflow of the air into the fermentor, increase the pressure of the fermentor, lower the viscosity of the fermentation broth and/or increase the gas exchange area.
  • the energetic efficiency of a fermentation processes is to a large extent determined by the difference in the so-called degree of reduction of the fermentation products compared to the degree of reduction of the substrate.
  • This reduction degree equals the number of available electrons for a reaction.
  • the difference of the reduction degree between the substrate and the product partly determines the yield and the energetic efficiency of the process.
  • the invention in a first aspect, relates to a use of a microbial fuel cell for fermenting a substrate into a product, wherein said product is oxidised compared to the substrate present in the microbial fuel cell and wherein electrical energy is produced.
  • a microbial fuel cell is an electrochemical device (see for example figure 1 ) in which energy derived from chemical reactions is converted to electrical energy by means of the catalytic activity of microorganisms and/or their enzymes.
  • Microbial fuel cells generally use as substrate a complex molecule to generate at the anode the hydrogen ions required to reduce oxygen to water, while generating free electrons for use in electrical applications. At the cathode side the electrons and protons from the anode are used to reduce oxygen to water.
  • a microbial fuel cell comprises a cathode and anode separated by some sort of barrier or salt bridge, such as for example a polymer electrolyte membrane.
  • barrier or salt bridge such as for example a polymer electrolyte membrane.
  • microbial fuel cells rely on microorganisms and/or biological molecules such as enzymes to carry out the reaction. Examples of microbial fuel cells have been disclosed in US 2005/0095466 or in WO 2005/005981 or in US 3,284,239 or in WO 07/006107 or in WO 04/004036. Microbial fuel cells are known to the skilled person in mainly two types of applications which are the production of electricity and water purification treatment.
  • Microbial fuel cells used in water purification treatment focus on the optimization of the degradation of a waste organic substrate into carbon dioxide and of the production of electricity.
  • Microbial fuel cells are attractive for carrying out an (industrial) fermentation process since at least some of the drawbacks found in classical fermentation processes are not present in the microbial fuel cell.
  • the most important improvement relates to the fact that the fermentation reaction is carried out in two separate compartments: an oxidation reactor wherein an oxidation step is carried out and a reduction reactor wherein a reduction step is carried out.
  • the microorganisms may be present either in the oxidation and/or in the reduction reactor. Distinct microorganisms may be present in each reactor. In a preferred embodiment, the microorganisms are present in the oxidation reactor. The microorganisms present in the oxidation reactor are preferably not able to completely use the electrical energy to convert it into heat and biomass. Therefore, less biomass and less heat will be produced, which will improve the efficiency of the fermentation process. We expect that the substrate will be more efficiently used and converted into a product. The fact that the microbial fuel cell comprises two separate compartments gives the opportunity to optimize each reaction (oxidation and reduction) separately.
  • the rate of oxygen transfer may for example be optimised. Conditions may even be chosen for the reduction reactor, which are not optimal for the microorganism present in the oxidation reactor. In addition, the efficiency of electron transfer from an oxidation to a reduction reactor may also be further optimized.
  • the microbial fuel cell comprises two compartments: a. an oxidation reactor comprising a substrate and a microorganism , b. a reduction reactor which comprises oxygen.
  • a microorganism converts a substrate into a product, which results in the production of electrons and protons. A least part of these electrons (at least part of the electrical energy hence formed) and protons produced are used in a reduction reactor to convert oxygen into water.
  • a microbial fuel cell comprises two compartments that are separated by a membrane. Part of this electrical energy (moving electrons) produced in the oxidation reactor is used by the microorganism. The rest of said electrical energy is preferably transported with the electrons via the anode and cathode to a reduction reactor being the other compartment. These electrons are preferably used to produce electric energy. Protons preferably diffuse via the membrane to a reduction reactor.
  • any microorganism may be present in the microbial fuel cell (i.e. oxidation reactor) as long as at least part of the dissipated chemical energy of the fermentation reaction is transferred to an electrode (per definition said electrode is an anode).
  • the transfer of the electrons is preferably measured by recording the current as measured by a multimeter.
  • "at least part of the dissipated chemical energy of the fermentation reaction” means at least approximately 5% of the total formed electrical energy produced. More preferably at least approximately 7% thereof, even more preferably at least approximately 10% thereof.
  • the total formed energy produced by the microbial fuel cell may be determined by measuring the voltage and current of a microbial fuel cell, by a multimeter. The product of voltage and current is the produced energy at that point in time.
  • the microorganism may be any microorganism which is known to be used in a fermentation process.
  • the microorganism may be selected from a fungus, a bacteria, an archea or an algae.
  • the microorganism may belong to a species which is selected from the following list: Acetobacter aceti, A. hansenii, A. liquefaciens, A. mesoxydans, A. pasteurianus, A. suboxydans, A. xylinum, Achromobacter agile, A. lactium, Acinetobacter baumanii, A. calcoaceticus, A. genospecies, A. genospesis, A.
  • A.junii Acinetobacter sp. Actinomycete sp., Actinoplane missouriensis
  • Aerobacter aerogenes A. cloacae, Aeromonas culicicola, A. formicans, Aeromonas sp., Agrobacterium radiobacter, A. rhizogenes, A. tumefaciens, Alcaligenes faecalis, Alcaligenes sp., A. tolerans, A. viscolactis, Amylolatopsis mediterranei, Anabaena ambigua, A.
  • vinelandii Azotomonas insolita, Bacillus aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aporrheous, B. brevis, B. cereus, B. cereus subsp. mycoids, B. circulans, B. coagulans, B. firmus, B. freudenreichii, B. globigii, B. laevolacticus, B. laterosporus, B. lentus, B. licheniformis, B. licheninformis, B. macerans, B. macquariensis, B. marcescens, B. megaterium, B. mesentericus, B. pantothenticus, B.
  • Nematospora coryli Nitrobacter sp., Nitrosomonas sp., Nocardia asteroids, N. calcaria, N. cellulans, N. hydrocarbonoxydans, N. mediterranei, N. rugosa, Nocardia sp., Nocardiopsis rougevillei, Nostoc elipsosporum, N. entrophytum, N. muscorum, N. punctriforme, Oerskovia xanthineolytica, Oosporidium margaritiferum, Pachysolen tannophilus, Pachytichospora transvaalensis, Pediococcus acidilactici, P. cerevisiae,
  • Rhodosporidium torreloides Rhodotorula aurantiaca, R. glutinis, R. graminis, R. marina, R. minuta, R. rubra, Rhodotorula sp., Saccharomyces capsulraries, S. cerevisiae, Saccharomycodes ludwigii, Saccharomycopsis fibuligera, Salmonella abony, S. typhimurium, Sarcina lutea, Sarcina sp., S. subflava, Scenedesmus abundans, Schizosaccharomyces octosporus, S. pombe, S. slooffii, Schwanniomyces occidentalis,
  • Serratia marcescens S. marinorubra, S. plymuthiea, Spirulina sp., Sporobolomyces holsaticus, S. roseus, S. salmonicolor, Sreptomyces diastaticus, S. olivaceous, S. rimosus, Sreptomyces sp., S. venezuelae, Staphylococcus afermentans, S. albus, S. aureus, S. epidermidis, Streptococcus agalactiae, S. cremoris, S. diacetilactis, S. faecalis, S.faecium, S. lactis, S.
  • nitrosporeus S. niveus, S. noursei, S. olivaceous, S. olivaceus, S. phacochromogenes, S. pseudogriseolus, Streptomyces sp., S. thermonitrificans, S. venezualae, S. vinaceus, S. viridefaciens, Streptosporangium sp., Streptoverticillium cinnamoneum, S. mobaraense, Streptoverticillium sp., Thermospora sp., Thiobacillus acidophilus, T. ferrooxidans, T. novellus, T.
  • Absidia corymbifera Acremonium chrysogenum, Actinomucor sp., Agaricus bitorquis, Alternaria alternata, A. brassicicola, Alternaria sp., A. terreus, Artrhobotrys conoides, A. oligospora, A. gossypii, Aspergillus awamori, A. candidus, A. clavtus, A. fischeri, A. flavipes,A. flavus A. foetidus A. funiculosus A. luchuensis A. nidulans A. niger A. oryzae A. oryzae var. viridis A.
  • proliferans A. sojae Aspergillus sp, A. terreus, A. terreus var. aureus, A. ustus, A. versicolor, A. wentii, Aureobasidium mausonii, A. pullulans.
  • Chaetomella raphigera Chaetomium globosum, Cladosporium herbarum, Cladosporium sp., Claviceps paspali, C. purpurea, Cokeromyces recurvatus, Coriolus versicolor, Cunninghamella blakesleeana, C. echinulata, C. elegans, C. sp., Curvularia brachyspora, C. cymbopogonis, C.fallax, C.
  • Pencillium ochrochloron Pencillium sp., P. argillaceum, P. asperosporum, P. chrysogenum, P. citrinum, P.frequentans, P. funiculosum, P.janthinellum, P. lignorum, P. notatum, P. ochrochloron, P. pinophillum, P. purpurogenum, P. roqueforti, P. variabile, Phaenerochaete chrysosporium, Phialophora bubakii, P. calciformis, P. fastigiata, P. lagerbergii, P.
  • a substrate may be any oxidisable compound.
  • a substrate may be any undesired waste or left over compound obtained in any industry.
  • a substrate consists of or comprises at least one compound selected from the following group of substrates: carbohydrates in general such as cellulose, hemicellulose, starch, pectine, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, xylose, arabinose; alcohols in general such as methanol, ethanol, propanol, isobutanol, butanol, isopropanol, allyl alcohols, aryl alcohols; polyols in general like glycerol, propanediol, mannitol, xylitol; sugar acids in general like glucuronate and gluconate or polymer thereof such as pectin, organic acids in general like lactate, malate, citrate, fumarate, succinaat, formate, itaconate, acetate, isocitrate and pyruvate; lipids and fatty acids; aminoacids etc.
  • the substrate comprises or
  • a product that could be produced by a classical fermentation process in a fermentor could also be produced using a microbial fuel cell of the invention: bio transport fuel, food and feed ingredients, basic compounds for chemical industry.
  • the product is not carbon dioxide.
  • a product will be formed by fermentation, wherein said product is oxidised compared to the substrate present in the microbial fuel cell.
  • a formed product may be any organic compound.
  • an organic compound is a known fermentation product. More preferably, a known fermentation product is an organic acid that may be produced by fermentation.
  • a formed product is selected from: citrate, acetate, glutamate, lysine, gluconate, itaconate, succinate, lactic acid, ethanol, butanol, glycerol and 1,3 propanediol, butyrate.
  • the fermenting process in the microbial fuel cell may be anaerobic or aerobic.
  • “anaerobic” preferably means that the fermenting process herein defined is carried out in the absence of oxygen or wherein substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol h "1 gDW "1 , more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors.
  • the process is aerobic in a reduction reactor (cathode side). More preferably within this embodiment, the process is aerobic or anaerobic in an oxidation reactor (anode side).
  • the process is aerobic in a reduction reactor and anaerobic in an oxidation reactor.
  • a component is added to the fermentation broth which replaces oxygen as a nutrient.
  • Preferred components include ergosterol and/or oleic acid and/or molecules derived therefrom.
  • oxygen is present in a reduction reactor (aerobic process). Even more preferably, especially when the process in a reduction reactor is aerobic, the reduction reactor is adapted for optimizing oxygen transfer.
  • Suitable cathodes for microbial fuel cell have already been extensively described (see for example Electricity Generation Using an Air-Cathode Single Chamber Microbial Fuel Cell in the Presence and Absence of a Proton Exchange Membrane. Hong Liu and Bruce E. Logan, Environ. ScL Technol, 38 (14), 4040 -4046).
  • an oxidation reactor may be adapted for optimizing the electrons transfer to an electrode.
  • a shuttling agent is selected from the group consisting of: Thionine, brilliant cresyl blue, methyleen blue, benzyl viologen, methyl viologen, Fe(III) EDTA, phenoxazine, bipyridilium, neutral red, saphranine O, Azure A, 2-hydroxy-l,4-naphthoquinone, ubiquinones, dyes and metal complexes and disulphonated thionines.
  • Phenoxazine may be phenothiazine, phenazine or indophenol.
  • shuttling agents are known to have the capacity to enhance the electrons transfer to an electrode (Shukla A.K.P. et al, (2004), Curr. Science, 87:455-468, Bullen R.A.. et al, (2006), Biosens. Bioelectron. 21:2015-2045). They are believed to act as a shuttle for the electrons between a microorganism and an electrode.
  • a shuttling agent Alternatively or in combination with the addition of a shuttling agent, another preferred embodiment, is to use a microorganism that is able to produce a shuttling like-agent (Hernandez M.E., et al, (2004), Appl. Environ. Microbiol, 70:921-928 and Rabaey K. N., et al, (2004), Appl. Environ. Microbiol, 70: 5373-5382).
  • An example of such microorganisms is: Pseudomonas chlororaphis .
  • another preferred embodiment is to facilitate a direct contact of a microorganism with an electrode (i.e. an anode).
  • an electrode i.e. an anode
  • This is preferably realized by increasing the surface of an electrode. It is clear for the skilled person that increasing the surface of the electrode is preferably done in such a way that the current density (i.e. the current per surface unit area) reaches an acceptable value. This acceptable value is determined by the anode potential that is needed to sustain the current density. A too high anode potential reduces the electrical power output of the cell.
  • the anode potential is comprised between approximately 0.5 V (vs the Standard Hydrogen Electrode (SHE)) and approximately -0.6 V, more preferably between approximately 0.4V and approximately -0.5 V, even more preferably between approximately 0.3V and approximately -0.5 V, even more preferably between approximately 0.2V and approximately -0.5 V, even more preferably between approximately 0.1V and approximately -0.5 V, even more preferably between approximately 0.0V and approximately -0.5 V, even more preferably between approximately -0.1V and approximately -0.5 V, even more preferably between approximately -0.2V and approximately -0.5 V, even more preferably between approximately -0.3V and approximately -0.5 V.
  • a microorganism is in direct contact with an electrode. Even more preferably the surface of the electrode is increased and a microorganism is in direct contact with said electrode.
  • a shuttling agent e.g., a styrene-maleic anhydride
  • a microorganism which is able to produce a shuttling like-agent, and/or with facilitating a direct contact of a microorganism with an electrode
  • another preferred embodiment is to use a microorganism, which is able to produce a nanowire that electron transfer to an electrode (Reguera G., (2005), Nature, 435:1098-1101). ).
  • An example of such microorganisms is a Geobacter species.
  • a microorganism used in the present invention may already be known as transferring electron to an electrode or as producing a shuttling-like agent and/or a nano wire.
  • the invention relates to a method for screening a microorganism having a capacity to transfer electron to an electrode by culturing the microorganism for at least months in a microbial fuel cell as earlier defined herein and screening for microorganisms that are improved in their capacity of electron transfer to an electrode.
  • a microorganism is improved in its capacity of electron transfer to an electrode when its capacity of electron transfer to an electrode is at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, at least 200% or more compared to the capacity of electron transfer to an electrode of the microorganism it derives from.
  • the capacity of electron transfer of the improved microorganism is compared to the capacity of electron transfer of a Geobacter sulfurreducens which is taken as control.
  • the capacity of electron transfer is preferably assessed as earlier herein defined.
  • the invention provides a method for producing a product by fermentation, wherein the method comprises the following steps: a. providing a microorganism which is preferably able to transfer at least part of the electrons formed in a reaction to an electrode, b. culturing the microorganism in an oxidation reactor of a microbial fuel cell in the presence of a substrate, c. producing electrical energy by transferring at least part of the electrons formed in the oxidation reactor, d. producing a product in the oxidation reactor.
  • Figure 1 Scheme of a microbial bio fuel cell. It consists of two compartments separated by a membrane. On the left side, in the oxidation reactor sugar is converted into an oxidised product. As a result, free electrons and protons are generated. The electrons go through the anode and cathode to the compartment of the right side, which is the reduction reactor, wherein they give their energy away to the lamp (on this example). The protons diffuse throught the membrane to the compartment of the right side. In this compartment, electrons and protons are used to reduce oxygen into water.
  • Candida oleophila CBS 8108 used in these experiments was obtained from Centraalbureau voor Schimmelcultures (CBS, Delft, The Netherlands). It was cultivated on Yeast Malt Extract agar plates at 30 0 C for 2 days and stored at 4 0 C.
  • Inoculums for fermentation experiments were prepared by transferring a single colony aseptically from the YME plate to an Erlenmeyer with 50 ml mineral medium according to Anastassiadis et al (2002) with a total working volume 500 ml.
  • the growth medium contained sterilized mineral medium, trace elements, vitamins and glucose solution with a final concentration of glucose 120 g/1.
  • the medium contained 1.5 g/1 NH 4 Cl.
  • CaCO 3 Separately sterilized CaCO 3 (3 g/1) was added to cultures.
  • the medium was supplemented with ergosterol and Tween 80 as described by Andreasen & Stier, 1953.
  • Antibiotics were added to prevent contamination (2.5 ml/1 Penstrep, Sigma- Aldrich).
  • the yeast was grown aerobically, agitated at 200 rpm in a rotary water bath shaker and incubated at 30 0 C for 20-24 hours. The inoculum was used after 2 days of growth.
  • a two compartment (anode and cathode) microbial fuel cell was used as a fuel cell system for microbial electricity production.
  • the total volumes of each compartment were 900 ml and 800 ml, respectively.
  • the electrodes consisted of graphite felt sized 19cm x 19cm (thickness 3mm, FMI composites Ltd., Galashiels, Scotland). As defined by Min et al..(2005) this MFC has an effective channel surface of 290 cm 2 , which is 80% of the total surface area.
  • a gold wire pressed on the electrode had the function of current collector.
  • the electrodes in the anode and cathode compartment were connected to a fixed resistor with a range of 0-2000 ⁇ via a gold wire.
  • the reference electrodes were Ag/AgCl; 3M KCl electrodes (+205mV ns NHE, Prosense Qis, Oosterhout, The Netherlands).
  • a cation-selective membrane (Fumasep FKB, Fumatech, St. Ingbert, Germany ) was placed between the anode and cathode compartment in order to allow proton exchange during fermentation.
  • the anode compartment was connected with a partly filled bottle equipped with a dissolved oxygen electrode and a pH electrode.
  • the fermentation medium was circulated through anode compartment and expansion bottle using a pump.
  • the MFC was operated in a temperature controlled compartment at 30 0 C.
  • the pH was maintained at 5.0 by addition of 45% NaOH.
  • the cathode compartment was filled with potassium ferrycyanide solution in the concentration according to the theoretical equimolar of electrons produced from the amount of initial glucose concentration. In this case it was approximately 0.30M.
  • the cathode compartment was aerobic throughout the fermentation process.
  • the anode compartment was filled with fermentation medium through the expansion reservoir using a circulation pump. After the medium has been circulated for 10 minutes, it was inoculated with Candida oleophila CBS 8108 (10 %, v/v). The yeast cells were circulated in the MFC for 48 hour to promote initial growth, with addition of air (0.5 wm) before 5 mM Methylene blue was added as mediator and air addition was stopped. The mediator was injected into expansion reservoir using a syringe. Samples were taken after 48, 72, 96, 144 and 196 hours.
  • Citric acid was measured with an integrated HPLC system (Waters Chromatography B. V., The Netherlands). 1 ml sample was taken and put into a 1.5 ml eppendorf tube and then centrifuged at 13000 rpm for 3 minutes. 400 ⁇ l of supernatant was taken and mixed with 1 M H2SO4 solution (added with MES) at a ratio of 1 : 1. After 5 minutes, the mixture was injected into an individual HPLC vial through a 20 ⁇ m filter.
  • Electricity production was measured by connecting the circuit in the MFC to an online data acquisition system system.
  • the measured data shown were: (1) fixed external resistance used, (2) anode potential, (3) cathode potential, (4) cell potentials and (5) membrane potential.
  • the anode and cathode potential were measured against Ag/ AgCl reference electrodes.
  • the cell potential was obtained by the voltage difference between the cathode and anode reference electrode.
  • Power density P (W/m 2 ) and current density j (A/m 2 ) were calculated from the cell potential E cell (volts), circuit load resistance R ( ⁇ ) and anode electrode surface area A(m 2 ) according to equation 1 and 2.
  • the transferred charge Q can be calculated from the current obtained from equation 2. Then the number of mol electrons transferred in time N e can be calculated from equation 3. Where the Faraday's constant (F) is 96485 C/mol.
  • the open circuit voltage (OCV) was measured by disconnecting the electrodes in order to stop electron transfer within the system.
  • a low current production can be a result of high internal resistance.
  • This resistance in MFCs can be affected by the anolyte and catholyte composition and pH, electrode material, and microorganism resistive nature.
  • R INT Internal resistance within the system was calculated according to Eq.5. Where I r is the current produced under a certain resistance load (R EXT ) and E O c was the potential at open circuit.
  • Citric acid production by C. oleophila from glucose in three MFC devices was run under aerobic conditions with open circuit without addition of methyleen blue, device B was switched to anaerobic conditions after 48 hours, with addition of methyleen blue, with closed circuit and device C was run as device B but with closed circuit
  • citric acid production continued as before, reaching a final concentration of approximately 50 g/1.
  • citric acid production completely stopped after 48 hours.
  • citric acid production continued though at a lower rate as before, reaching a final concentration of approximately 20 g/1 after 196 hours.
  • the current output in device C was measured during the experiment. Using the equations 2,3 and 4, the output was calculated as 101, 174, 244 and 289 mmol electrons after 72, 96, 144 and 196 hours of cultivation respectively.

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Abstract

The invention relates to a specific use of a microbial fuel cell for fermenting a substrate into a product, wherein said product is oxidised compared to the substrate present in the microbial fuel cell and wherein electrical energy i s produced.

Description

Production of a product in a microbial fuel cell
Field of the invention The invention relates to a fermentation process, wherein use is made of a microbial fuel cell for fermenting a substrate into a product, wherein said product is oxidised compared to the substrate present in the microbial fuel cell and wherein electrical energy is produced.
Background of the invention
Fermentation is an important technology for the production of several types of products: bio transport fuels (such as ethanol, butanol or hydrogen) or food and feed ingredients (such as glutamate, lysine, citrate, or lactic acid) or chemical building blocks for products such as nylon. However, the efficiency of fermentation processes is rather low due to among other the formation of undesired side products, the production of biomass and/or the low rate of oxygen transfer. Furthermore, heat is produced during the conversion of oxygen into water. Therefore, expensive cooling equipments are needed in order to maintain the temperature of the fermentation to a suitable temperature for the microorganism used. To counteract the formation of undesired side product, one may genetically modify the microorganism used. However, this strategy is not always possible. One needs first to know which gene(s) has(have) to be inactivated. Furthermore, this is a long process and has to be applied again for each type of microorganism used and for each product to be produced. To improve the rate of oxygen transfer, one may increase the inflow of the air into the fermentor, increase the pressure of the fermentor, lower the viscosity of the fermentation broth and/or increase the gas exchange area.
The energetic efficiency of a fermentation processes is to a large extent determined by the difference in the so-called degree of reduction of the fermentation products compared to the degree of reduction of the substrate. This reduction degree equals the number of available electrons for a reaction. The difference of the reduction degree between the substrate and the product partly determines the yield and the energetic efficiency of the process. When the product is more reduced than the substrate, electrons should be added. When the substrate is more reduced than the product, electrons should be transported elsewhere.
Due to the low efficiency of fermentation processes as described above even applying some of the proposed improvements as described above , there is still a need for improved fermentation processes which do not have all of the drawbacks of conventional fermentation processes.
Description of the invention In a first aspect, the invention relates to a use of a microbial fuel cell for fermenting a substrate into a product, wherein said product is oxidised compared to the substrate present in the microbial fuel cell and wherein electrical energy is produced. A microbial fuel cell is an electrochemical device (see for example figure 1 ) in which energy derived from chemical reactions is converted to electrical energy by means of the catalytic activity of microorganisms and/or their enzymes. Microbial fuel cells generally use as substrate a complex molecule to generate at the anode the hydrogen ions required to reduce oxygen to water, while generating free electrons for use in electrical applications. At the cathode side the electrons and protons from the anode are used to reduce oxygen to water. This may be done directly or indirectly using an intermediate like hexacyanoferrate. A microbial fuel cell comprises a cathode and anode separated by some sort of barrier or salt bridge, such as for example a polymer electrolyte membrane. Rather than using precious metals as catalysts, microbial fuel cells rely on microorganisms and/or biological molecules such as enzymes to carry out the reaction. Examples of microbial fuel cells have been disclosed in US 2005/0095466 or in WO 2005/005981 or in US 3,284,239 or in WO 07/006107 or in WO 04/004036. Microbial fuel cells are known to the skilled person in mainly two types of applications which are the production of electricity and water purification treatment. Microbial fuel cells used in water purification treatment focus on the optimization of the degradation of a waste organic substrate into carbon dioxide and of the production of electricity. The inventors found out that a microbial fuel cell is an effective system for producing an attractive (industrial) product (instead of degrading a waste substrate), while still producing electricity. Microbial fuel cells are attractive for carrying out an (industrial) fermentation process since at least some of the drawbacks found in classical fermentation processes are not present in the microbial fuel cell. The most important improvement relates to the fact that the fermentation reaction is carried out in two separate compartments: an oxidation reactor wherein an oxidation step is carried out and a reduction reactor wherein a reduction step is carried out. As a consequence, part of the electrons produced in the oxidation reactor will be transported to the reduction reactor, which will produce electrical energy. The microorganisms may be present either in the oxidation and/or in the reduction reactor. Distinct microorganisms may be present in each reactor. In a preferred embodiment, the microorganisms are present in the oxidation reactor. The microorganisms present in the oxidation reactor are preferably not able to completely use the electrical energy to convert it into heat and biomass. Therefore, less biomass and less heat will be produced, which will improve the efficiency of the fermentation process. We expect that the substrate will be more efficiently used and converted into a product. The fact that the microbial fuel cell comprises two separate compartments gives the opportunity to optimize each reaction (oxidation and reduction) separately. The rate of oxygen transfer may for example be optimised. Conditions may even be chosen for the reduction reactor, which are not optimal for the microorganism present in the oxidation reactor. In addition, the efficiency of electron transfer from an oxidation to a reduction reactor may also be further optimized.
In a preferred embodiment, the microbial fuel cell comprises two compartments: a. an oxidation reactor comprising a substrate and a microorganism , b. a reduction reactor which comprises oxygen.
In this preferred embodiment, a microorganism converts a substrate into a product, which results in the production of electrons and protons. A least part of these electrons (at least part of the electrical energy hence formed) and protons produced are used in a reduction reactor to convert oxygen into water. In a more preferred embodiment, a microbial fuel cell comprises two compartments that are separated by a membrane. Part of this electrical energy (moving electrons) produced in the oxidation reactor is used by the microorganism. The rest of said electrical energy is preferably transported with the electrons via the anode and cathode to a reduction reactor being the other compartment. These electrons are preferably used to produce electric energy. Protons preferably diffuse via the membrane to a reduction reactor. In the context of the invention, any microorganism may be present in the microbial fuel cell (i.e. oxidation reactor) as long as at least part of the dissipated chemical energy of the fermentation reaction is transferred to an electrode (per definition said electrode is an anode). The transfer of the electrons is preferably measured by recording the current as measured by a multimeter. In a preferred embodiment, "at least part of the dissipated chemical energy of the fermentation reaction" means at least approximately 5% of the total formed electrical energy produced. More preferably at least approximately 7% thereof, even more preferably at least approximately 10% thereof. The total formed energy produced by the microbial fuel cell may be determined by measuring the voltage and current of a microbial fuel cell, by a multimeter. The product of voltage and current is the produced energy at that point in time.
The microorganism may be any microorganism which is known to be used in a fermentation process. The microorganism may be selected from a fungus, a bacteria, an archea or an algae. The microorganism may belong to a species which is selected from the following list: Acetobacter aceti, A. hansenii, A. liquefaciens, A. mesoxydans, A. pasteurianus, A. suboxydans, A. xylinum, Achromobacter agile, A. lactium, Acinetobacter baumanii, A. calcoaceticus, A. genospecies, A. genospesis, A. haemolyticus, A.junii, Acinetobacter sp. Actinomycete sp., Actinoplane missouriensis, Aerobacter aerogenes, A. cloacae, Aeromonas culicicola, A. formicans, Aeromonas sp., Agrobacterium radiobacter, A. rhizogenes, A. tumefaciens, Alcaligenes faecalis, Alcaligenes sp., A. tolerans, A. viscolactis, Amylolatopsis mediterranei, Anabaena ambigua, A. subcylindria, Aquaspirillium intersonii, Arthroascus javanensis, Arthrobacter albidus, A. citreus, A. luteus, A. nicotinae, A. polychromogenes, A. simplex, Arthrobacter sp., A. ureafaecalis, A. viscosus, Azomonas macrocytogenes, Azospirillum brasilense, A. lipoferum, Azotobacter chroococcum, A. agilis, A. chroococcum, A. macrocytogenes, Azotobacter sp., A. vinelandii, Azotomonas insolita, Bacillus aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aporrheous, B. brevis, B. cereus, B. cereus subsp. mycoids, B. circulans, B. coagulans, B. firmus, B. freudenreichii, B. globigii, B. laevolacticus, B. laterosporus, B. lentus, B. licheniformis, B. licheninformis, B. macerans, B. macquariensis, B. marcescens, B. megaterium, B. mesentericus, B. pantothenticus, B. pasteurii, B. polymyxa, B. pumilus, Bacillus sp., B. sphaericus; B. stearothermophilus, B. subtilis, B. thuringiensis, B. zopfii, B. subtilis, Beijerinckia indica, B. lactiogenes, Bordetella bronchiseptica, Brettanomyces intermedins, Brevibacterium ammoniagene, B. diverticatum, B. immariophilum, B. imperiale, B. linens, B. liquifaciens, B. luteum, B. roseum, B. saccharolyticum, B. vitarumen, Candida albicans, C. bombii, C. brumptii, C. catenulata, C. colliculosa, C. deformans, C. epicola, C. etchellsii, C.famata, C. freyschussii, C. glabrata, C. gropengiesseri, C. guilliermondii, C. krusei, C. lambica, C. lusitaniae, C. magnoliae, C. mannitofaciens, C. melibiosica, C. mucifera, C. parapsilosis, C. pseudotropicalis, C. rugosa, C. rugosa, C. tropicalis, C. utilis, C. versatilis, C. wickerhamii, C. sake, C. shehatae Candida Sp., C stellata, Cellulomonas bibula, C. bizotea, C. cartae, Cfimi, C. flavigena, C. gelida, C. uda, Chainia sp., Chlorella pyrenoidosa, Chromatium sp., Citeromyces matritensis, Citrobacter fruendii,
C. acetobutylicum, C. felsineum, C. pasteurianum, C. perfringens, C. roseum, C. sporogenes, C. tetanomorphum, Corynebacterium rubrum, Corynebacterium sp., Cryptococcus laurentii, C leteolus, C neoformans, C neoformans, Crytococcus sp., Cytophaga hutchinsonii, Debaryomyces castellii, D. fibuligera, D. hansenii, D. marama, D. polymorphus, D. vanriji, Dekerra anomala, D. claussenii, D. bruxellensis,
D. intermedia, D. naardensis, Desulfotomaculum nigrificans, Desulfovibrio desulfuricans, Enterobacter aerogenes, E. clocae, Erwinia cherysanthemi, Escherichia coli, E. intermedia, E. irregular, Euglena gracilis, Filobasidium capsuligenum, F. uniguttulatum, Flavobacterium dehydrogenans, F. devorans, F. odoratum, Flavobacterium sp., Geotrichum sp., Gluconobacter melanogenes, G. melanogenus, G. oxydans, G. roseus, Guilliermondella selenospora, Hafnia alvei, Halobacterium cutirubrum, H. halobium, H. salinarium, H. trapinium, Haneseniaspora vineae, Hansenula beckii, H. beijerinckii, H. canadensis, H. capsulata, H. ciferrii, H. polymorpha, H, valbiensis, Hormoascus ambrosiae, Issatchenkia orientalis, Janthinobacter lividum, Jensinia canicruria, Klebsiella aerogenes, K. pneumoniae, K. terrigena, Klockera corticis, K. javancia, Kluveromyces marxianus, Kluyvera citrophila, K. lodderi, K. marxianus, K. marxianus var. lactis, Lactobacillus acidophilus, L. brevis, L. buchneri, L. bulgaricus, L. casei, L. casei var. rhamnosus, L. delbrueckii, L. fermentum, L. helveticus, L. jugurti, L. lactis, L. leichmannii, L. pentosus, L. plantarum, Lactobacillus sp., L. sporogenes, L.viridescens, Leuconostoc mes enter oides, L. oenos, Leuconostoc sp., Leucosporidium frigidium, Lineola longa, Lipomyces lipofera, L. starkeyi, Metschnikowia pulcherrima, M. reukaufii, Micrococcus sp., Micrococcus flavus, M. glutamicus, M. luteus, Microcyclus aquaticus, M. flavus, Morexella sp., Mycobacterium phlei, M. smegmatis, Mycobacterium sp.,
Mycoplana bullata, M. dimorpha, Mycrocyclus aquaticus, Nadsonia elongata,
Nematospora coryli, Nitrobacter sp., Nitrosomonas sp., Nocardia asteroids, N. calcaria, N. cellulans, N. hydrocarbonoxydans, N. mediterranei, N. rugosa, Nocardia sp., Nocardiopsis dassonvillei, Nostoc elipsosporum, N. entrophytum, N. muscorum, N. punctriforme, Oerskovia xanthineolytica, Oosporidium margaritiferum, Pachysolen tannophilus, Pachytichospora transvaalensis, Pediococcus acidilactici, P. cerevisiae,
P. pentosaceous, Pichia anomala, P. carsonii, P. farinosa, P. fermentans, P. fluxuum,
P. guilliermondii, P. haplophila, P. ohmeri, P. pastoris, P. pijperi, P. rhodanensis, P. toletana, P. trihalophila, P. stipitis, Propionibacteriumfreudenreichii, P. shermanii, P. thoenii, P. zeae, Protaminobacter alboflavus, Proteus mirabilis, P. morganii, P. morganii, P. vulgaris, Prototheca moriformis, Providencia styartii, Pseudomonas aeruginosa, P. acidovorans, P. aeruginosa, P. aureofaciens, P. auruginosa, P. azotogensis, P. caryophylli, P. cepacia, P. convexa, P. cruciviae, P. denitrificans, P. desmolytica, P. desmolyticum, P. diminuta, P. fluorescens, P.fragi, P. glutaris, P. hydrophila, P. lemonnieri, P. maltophilia, P. mildenbergi, P. oleovorans, P. ovalis, P. pictorum, P. pisi, P. pseudoalcaligenes, P. pseudoflava, P. putida, P. reptilivora, P. resinivorans, P. solanacerum, Pseudomonas sp., P. stutzeri, P. syringae, P. testosteroni, P. viridiflava, Rhizobium indigofera, R. japonicum, R. leguminosarum, R. lupini, R. meliloti, R. phaseoli, Rhizobium sp, R. trifoli, Rhodococcus sp., R. terrae,
Rhodosporidium torreloides, Rhodotorula aurantiaca, R. glutinis, R. graminis, R. marina, R. minuta, R. rubra, Rhodotorula sp., Saccharomyces capsulraries, S. cerevisiae, Saccharomycodes ludwigii, Saccharomycopsis fibuligera, Salmonella abony, S. typhimurium, Sarcina lutea, Sarcina sp., S. subflava, Scenedesmus abundans, Schizosaccharomyces octosporus, S. pombe, S. slooffii, Schwanniomyces occidentalis,
Serratia marcescens, S. marinorubra, S. plymuthiea, Spirulina sp., Sporobolomyces holsaticus, S. roseus, S. salmonicolor, Sreptomyces diastaticus, S. olivaceous, S. rimosus, Sreptomyces sp., S. venezuelae, Staphylococcus afermentans, S. albus, S. aureus, S. epidermidis, Streptococcus agalactiae, S. cremoris, S. diacetilactis, S. faecalis, S.faecium, S. lactis, S. pyogenes, S. salivaris, Streptococcus sp., S. thermophilus, S. zymogenes, S. peuceticus, S. albogriseolus, S. albus, S. antibioticus, S. atrofaciens, S. aureofaciens, S.caelastis, S. diastaticus, S. erythraeus, S. fluorescens, S. fradiae, S. griseoflavus, S. griseus, S. hawaiiensis, S. hygroscopicus, S. kanamyceticus, S. lavendulae, S. lividans, S. nataliensis, S. nitrosporeus, S. niveus, S. noursei, S. olivaceous, S. olivaceus, S. phacochromogenes, S. pseudogriseolus, Streptomyces sp., S. thermonitrificans, S. venezualae, S. vinaceus, S. viridefaciens, Streptosporangium sp., Streptoverticillium cinnamoneum, S. mobaraense, Streptoverticillium sp., Thermospora sp., Thiobacillus acidophilus, T. ferrooxidans, T. novellus, T. thiooxidans, Torulaspora delbrueckii, Torulopsis ethanolitolerans, T. glabrata, Torulopsis sp., Tremella mesenterica, Trichosporon beigelii, T. capitatum, T. pullulans, Trichosporon sp, Trigonopsis variabilis, Williopsis californica, W. saturnus, Xanthomonas campestris, X. malvacearum, Yarrowia lipolytica, Zygosaccharomyces bisporus, Z. rouxii, Z. bisporus, Z. priorionus, Zygosporium aromyces, Z. priorionus, Zymomonas anaerobia, Z. mobilis .
Absidia corymbifera, Acremonium chrysogenum, Actinomucor sp., Agaricus bitorquis, Alternaria alternata, A. brassicicola, Alternaria sp., A. terreus, Artrhobotrys conoides, A. oligospora, A. gossypii, Aspergillus awamori, A. candidus, A. clavtus, A. fischeri, A. flavipes,A. flavus A. foetidus A. funiculosus A. luchuensis A. nidulans A. niger A. oryzae A. oryzae var. viridis A. proliferans A. sojae Aspergillus sp, A. terreus, A. terreus var. aureus, A. ustus, A. versicolor, A. wentii, Aureobasidium mausonii, A. pullulans. Auricularia polytricha Basidiobolus haptosporus, Beauveria bassiana, Benjaminella multispora, B. poitrasii, Botryodiplodia theobromae, Botryotrichum piluliferum, Botrytis allii, Cephaliophora irregularis Cephalosporium sp. Chaetomella raphigera, Chaetomium globosum, Cladosporium herbarum, Cladosporium sp., Claviceps paspali, C. purpurea, Cokeromyces recurvatus, Coriolus versicolor, Cunninghamella blakesleeana, C. echinulata, C. elegans, C. sp., Curvularia brachyspora, C. cymbopogonis, C.fallax, C. lunata, Daedalea flavida, Datronia mollis, Dipodascus uninucleatus, Flammulina velutipes, Fusarium moniliforme, F. oxysporum, F. proliferatum, Fusarium sp., F. tricinctum, Ganoderma lucidum, Georichum candidum, Gibber ella fujikuroi, G. saubinetti, Gliocladium roseum, Gongronella butleri, Helminthosporium sp., Humicola grisea, Hymenochaete rubigonosa, Laetiporus sulphur eus, Lenzites striata, Lepiota rhacodes, Monilinia fructicola, Mucor hiemails, M. plumbeus, Mucor sp., Mycotypha africana, M. microspora, Myrothecium roridum, M. verrucaria, Neurospora crassa, N. sitophila, Paecilomyces sp., P. varioti, Pencillium ochrochloron, Pencillium sp., P. argillaceum, P. asperosporum, P. chrysogenum, P. citrinum, P.frequentans, P. funiculosum, P.janthinellum, P. lignorum, P. notatum, P. ochrochloron, P. pinophillum, P. purpurogenum, P. roqueforti, P. variabile, Phaenerochaete chrysosporium, Phialophora bubakii, P. calciformis, P. fastigiata, P. lagerbergii, P. richardsiae, Phialophora sp., Phoma exigua, Phycomyces blakesleeanus, Pleurotus flabellatus, P.florida, P.floridanus, P. ostreatus, P. sajor-caju, Polyporus meliae, Porta placenta, Ptychogaster sp.,
Pycnoporus cinnabarinus, P. sanguineus, Rhizopus oryzae, R. stolonifer, Sclerotium rolfsii, Scopulariopsis brevicaulis, Sporothecium sp., Sporotrichum sp., Stachybotrys chartarum, Stemphylium sarcinaeforme, Stemphylium sp., Tolypocladium inflatum, Trametes cubensis, T. hirsuta, T. lactinea, T. serialis, T. versicolor, T. inaequalis, T. harzianum, T. reesei, Trichoderma sp.,T. viride, Trichosporon sp., Trichothecium roseum, Ustilago maydis, Volvariella diplasia, Volvariella sp., V. volvacea Most preferred are Saccharomyces cerevisiae, Aspergillus niger, Aspergillus terreus, Bacillus species, Pseudomonas species, Escherichia coli, Gluconobacter species, Acetobaceter species and Corynebacterium glutamicum. In the context of the invention, a substrate may be any oxidisable compound. A substrate may be any undesired waste or left over compound obtained in any industry. A substrate consists of or comprises at least one compound selected from the following group of substrates: carbohydrates in general such as cellulose, hemicellulose, starch, pectine, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, xylose, arabinose; alcohols in general such as methanol, ethanol, propanol, isobutanol, butanol, isopropanol, allyl alcohols, aryl alcohols; polyols in general like glycerol, propanediol, mannitol, xylitol; sugar acids in general like glucuronate and gluconate or polymer thereof such as pectin, organic acids in general like lactate, malate, citrate, fumarate, succinaat, formate, itaconate, acetate, isocitrate and pyruvate; lipids and fatty acids; aminoacids etc. In a preferred embodiment, the substrate comprises or consists of carbohydrates, in a more preferred embodiment the substrate comprises or consists of at least one of glucose, fructose, sucrose, maltose and starch.
We expect that any product that could be produced by a classical fermentation process in a fermentor could also be produced using a microbial fuel cell of the invention: bio transport fuel, food and feed ingredients, basic compounds for chemical industry. In a preferred embodiment, the product is not carbon dioxide. Depending on the identity of a substrate and of a microorganism chosen, a product will be formed by fermentation, wherein said product is oxidised compared to the substrate present in the microbial fuel cell. A formed product may be any organic compound. Preferably, an organic compound is a known fermentation product. More preferably, a known fermentation product is an organic acid that may be produced by fermentation. Even more preferably, a formed product is selected from: citrate, acetate, glutamate, lysine, gluconate, itaconate, succinate, lactic acid, ethanol, butanol, glycerol and 1,3 propanediol, butyrate.
The fermenting process in the microbial fuel cell may be anaerobic or aerobic. In the context of the invention, "anaerobic" preferably means that the fermenting process herein defined is carried out in the absence of oxygen or wherein substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol h"1 gDW"1, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors. In a preferred embodiment, the process is aerobic in a reduction reactor (cathode side). More preferably within this embodiment, the process is aerobic or anaerobic in an oxidation reactor (anode side). Even more preferably, the process is aerobic in a reduction reactor and anaerobic in an oxidation reactor. Even more preferably, when a microorganism used is only able to grow under aerobic conditions outside of the microbial fuel cell, a component is added to the fermentation broth which replaces oxygen as a nutrient. Preferred components include ergosterol and/or oleic acid and/or molecules derived therefrom. An example of a molecule derived from oleic acid in Tween 80. In another preferred embodiment, oxygen is present in a reduction reactor (aerobic process). Even more preferably, especially when the process in a reduction reactor is aerobic, the reduction reactor is adapted for optimizing oxygen transfer. Suitable cathodes for microbial fuel cell have already been extensively described (see for example Electricity Generation Using an Air-Cathode Single Chamber Microbial Fuel Cell in the Presence and Absence of a Proton Exchange Membrane. Hong Liu and Bruce E. Logan, Environ. ScL Technol, 38 (14), 4040 -4046).
Alternatively or in combination with earlier preferred embodiment directed to among other improvements of a reduction reactor, an oxidation reactor may be adapted for optimizing the electrons transfer to an electrode.
This is preferably realized by adding a redoxmediator also named as a shuttling agent to the medium of the oxidation reactor. More preferably, a shuttling agent is selected from the group consisting of: Thionine, brilliant cresyl blue, methyleen blue, benzyl viologen, methyl viologen, Fe(III) EDTA, phenoxazine, bipyridilium, neutral red, saphranine O, Azure A, 2-hydroxy-l,4-naphthoquinone, ubiquinones, dyes and metal complexes and disulphonated thionines. Phenoxazine may be phenothiazine, phenazine or indophenol. These shuttling agents are known to have the capacity to enhance the electrons transfer to an electrode (Shukla A.K.P. et al, (2004), Curr. Science, 87:455-468, Bullen R.A.. et al, (2006), Biosens. Bioelectron. 21:2015-2045). They are believed to act as a shuttle for the electrons between a microorganism and an electrode.
Alternatively or in combination with the addition of a shuttling agent, another preferred embodiment, is to use a microorganism that is able to produce a shuttling like-agent (Hernandez M.E., et al, (2004), Appl. Environ. Microbiol, 70:921-928 and Rabaey K. N., et al, (2004), Appl. Environ. Microbiol, 70: 5373-5382). An example of such microorganisms is: Pseudomonas chlororaphis .
Alternatively or in combination with the addition of a shuttling agent and/or with the use of a microorganism that is able to produce a shuttling like-agent, another preferred embodiment is to facilitate a direct contact of a microorganism with an electrode (i.e. an anode). This is preferably realized by increasing the surface of an electrode. It is clear for the skilled person that increasing the surface of the electrode is preferably done in such a way that the current density (i.e. the current per surface unit area) reaches an acceptable value. This acceptable value is determined by the anode potential that is needed to sustain the current density. A too high anode potential reduces the electrical power output of the cell. Therefore, in a preferred embodiment, the anode potential is comprised between approximately 0.5 V (vs the Standard Hydrogen Electrode (SHE)) and approximately -0.6 V, more preferably between approximately 0.4V and approximately -0.5 V, even more preferably between approximately 0.3V and approximately -0.5 V, even more preferably between approximately 0.2V and approximately -0.5 V, even more preferably between approximately 0.1V and approximately -0.5 V, even more preferably between approximately 0.0V and approximately -0.5 V, even more preferably between approximately -0.1V and approximately -0.5 V, even more preferably between approximately - 0.2V and approximately -0.5 V, even more preferably between approximately -0.3V and approximately -0.5 V. More preferably, a microorganism is in direct contact with an electrode. Even more preferably the surface of the electrode is increased and a microorganism is in direct contact with said electrode.
Alternatively or in combination with the addition of a shuttling agent and/or with the use of a microorganism that is able to produce a shuttling like-agent, and/or with facilitating a direct contact of a microorganism with an electrode, another preferred embodiment is to use a microorganism, which is able to produce a nanowire that electron transfer to an electrode (Reguera G., (2005), Nature, 435:1098-1101). ). An example of such microorganisms is a Geobacter species. A microorganism used in the present invention may already be known as transferring electron to an electrode or as producing a shuttling-like agent and/or a nano wire. Alternatively, a microorganism which does originally exhibit none of these characteristics, or only at least one of these could be modified for acquiring one of these or all. Alternatively, a microoorganism which exhibit at least one of these characteristics may be further improved. Accordingly, in a further aspect, the invention relates to a method for screening a microorganism having a capacity to transfer electron to an electrode by culturing the microorganism for at least months in a microbial fuel cell as earlier defined herein and screening for microorganisms that are improved in their capacity of electron transfer to an electrode. A microorganism is improved in its capacity of electron transfer to an electrode when its capacity of electron transfer to an electrode is at least 2%, at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, at least 200% or more compared to the capacity of electron transfer to an electrode of the microorganism it derives from. Preferably, the capacity of electron transfer of the improved microorganism is compared to the capacity of electron transfer of a Geobacter sulfurreducens which is taken as control. The capacity of electron transfer is preferably assessed as earlier herein defined.
In a further aspect, the invention provides a method for producing a product by fermentation, wherein the method comprises the following steps: a. providing a microorganism which is preferably able to transfer at least part of the electrons formed in a reaction to an electrode, b. culturing the microorganism in an oxidation reactor of a microbial fuel cell in the presence of a substrate, c. producing electrical energy by transferring at least part of the electrons formed in the oxidation reactor, d. producing a product in the oxidation reactor.
All the elements of this method have already been defined earlier herein.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Description of the figures
Figure 1. Scheme of a microbial bio fuel cell. It consists of two compartments separated by a membrane. On the left side, in the oxidation reactor sugar is converted into an oxidised product. As a result, free electrons and protons are generated. The electrons go through the anode and cathode to the compartment of the right side, which is the reduction reactor, wherein they give their energy away to the lamp (on this example). The protons diffuse throught the membrane to the compartment of the right side. In this compartment, electrons and protons are used to reduce oxygen into water.
Examples Materials and methods
Pre-culture and inoculum preparation Candida oleophila CBS 8108 used in these experiments was obtained from Centraalbureau voor Schimmelcultures (CBS, Delft, The Netherlands). It was cultivated on Yeast Malt Extract agar plates at 30 0C for 2 days and stored at 4 0C. Inoculums for fermentation experiments were prepared by transferring a single colony aseptically from the YME plate to an Erlenmeyer with 50 ml mineral medium according to Anastassiadis et al (2002) with a total working volume 500 ml. The growth medium contained sterilized mineral medium, trace elements, vitamins and glucose solution with a final concentration of glucose 120 g/1. The medium contained 1.5 g/1 NH4Cl. Separately sterilized CaCO3 (3 g/1) was added to cultures. The medium was supplemented with ergosterol and Tween 80 as described by Andreasen & Stier, 1953. Antibiotics were added to prevent contamination (2.5 ml/1 Penstrep, Sigma- Aldrich). The yeast was grown aerobically, agitated at 200 rpm in a rotary water bath shaker and incubated at 300C for 20-24 hours. The inoculum was used after 2 days of growth.
Microbial fuel cell system
A two compartment (anode and cathode) microbial fuel cell was used as a fuel cell system for microbial electricity production. The total volumes of each compartment were 900 ml and 800 ml, respectively. The electrodes consisted of graphite felt sized 19cm x 19cm (thickness 3mm, FMI composites Ltd., Galashiels, Scotland). As defined by Min et al..(2005) this MFC has an effective channel surface of 290 cm2, which is 80% of the total surface area. A gold wire pressed on the electrode had the function of current collector. The electrodes in the anode and cathode compartment were connected to a fixed resistor with a range of 0-2000 Ω via a gold wire. The reference electrodes were Ag/AgCl; 3M KCl electrodes (+205mV ns NHE, Prosense Qis, Oosterhout, The Netherlands). A cation-selective membrane (Fumasep FKB, Fumatech, St. Ingbert, Germany ) was placed between the anode and cathode compartment in order to allow proton exchange during fermentation.
The anode compartment was connected with a partly filled bottle equipped with a dissolved oxygen electrode and a pH electrode. The fermentation medium was circulated through anode compartment and expansion bottle using a pump.
Microbial fuel cell experiment
The MFC was operated in a temperature controlled compartment at 300C. The pH was maintained at 5.0 by addition of 45% NaOH. Before the batch fermentation process in the MFC began, the cathode compartment was filled with potassium ferrycyanide solution in the concentration according to the theoretical equimolar of electrons produced from the amount of initial glucose concentration. In this case it was approximately 0.30M. The cathode compartment was aerobic throughout the fermentation process.
The anode compartment was filled with fermentation medium through the expansion reservoir using a circulation pump. After the medium has been circulated for 10 minutes, it was inoculated with Candida oleophila CBS 8108 (10 %, v/v). The yeast cells were circulated in the MFC for 48 hour to promote initial growth, with addition of air (0.5 wm) before 5 mM Methylene blue was added as mediator and air addition was stopped. The mediator was injected into expansion reservoir using a syringe. Samples were taken after 48, 72, 96, 144 and 196 hours.
Metabolite analysis Citric acid was measured with an integrated HPLC system (Waters Chromatography B. V., The Netherlands). 1 ml sample was taken and put into a 1.5 ml eppendorf tube and then centrifuged at 13000 rpm for 3 minutes. 400 μl of supernatant was taken and mixed with 1 M H2SO4 solution (added with MES) at a ratio of 1 : 1. After 5 minutes, the mixture was injected into an individual HPLC vial through a 20μm filter.
Electricity production in MFC
Electricity production was measured by connecting the circuit in the MFC to an online data acquisition system system. The measured data shown were: (1) fixed external resistance used, (2) anode potential, (3) cathode potential, (4) cell potentials and (5) membrane potential. In these MFC experiments, the anode and cathode potential were measured against Ag/ AgCl reference electrodes. The cell potential was obtained by the voltage difference between the cathode and anode reference electrode. The cell potential should have the same value as Ecell = Ecathode - Eanode - Elosses
Power density P (W/m2) and current density j (A/m2) were calculated from the cell potential Ecell (volts), circuit load resistance R (Ω) and anode electrode surface area A(m2) according to equation 1 and 2. The external load value used for the experiments was in the range of 0-200 Ω. Power density was calculated as: P = Ecell 2/(R.A) (1)
and current density as
j=E/(R.A) (2)
The transferred charge Q can be calculated from the current obtained from equation 2. Then the number of mol electrons transferred in time Ne can be calculated from equation 3. Where the Faraday's constant (F) is 96485 C/mol.
Q = L t (3) and
Figure imgf000017_0001
The open circuit voltage (OCV) was measured by disconnecting the electrodes in order to stop electron transfer within the system.
According to Ieropoulos et al.. (2005), a low current production can be a result of high internal resistance. This resistance in MFCs can be affected by the anolyte and catholyte composition and pH, electrode material, and microorganism resistive nature.
Internal resistance (RINT) within the system was calculated according to Eq.5. Where Ir is the current produced under a certain resistance load (REXT) and EOc was the potential at open circuit.
Figure imgf000017_0002
Results
At t=0 h three MFC devices (A, B and C), aerated at 0.5 wm, were inoculated with 10 % v/v C. oleophila. Between 30-35 hours the ammonium was completely consumed and citric acid production started. At t=48 h the citric acid concentration was between 8 and 10 g/1. Aeration was stopped in MFC devices B and C and 5 mM methylene blue was added. This resulted in the decrease of the dissolved oxygen tension to zero. Device B was operated with closed circuit, device C with open circuit. Table 1 shows citric acid production in time for the three devices.
Table 1. Citric acid production by C. oleophila from glucose in three MFC devices. Device A was run under aerobic conditions with open circuit without addition of methyleen blue, device B was switched to anaerobic conditions after 48 hours, with addition of methyleen blue, with closed circuit and device C was run as device B but with closed circuit
Figure imgf000018_0001
In device A citric acid production continued as before, reaching a final concentration of approximately 50 g/1. In device B citric acid production completely stopped after 48 hours. In device C citric acid production continued though at a lower rate as before, reaching a final concentration of approximately 20 g/1 after 196 hours.
The current output in device C was measured during the experiment. Using the equations 2,3 and 4, the output was calculated as 101, 174, 244 and 289 mmol electrons after 72, 96, 144 and 196 hours of cultivation respectively.
References
Anastassiadis, S., Aivasidis, A. and Wandrey, C. (2002). Citric acid production by Candida strains under intracellular nitrogen limitation. Appl. Microbiol. Biotechnol. 60:81-87
Andreasen, A.A. and Stier, T.J.B. (1953). Anaerobic nutrition of Saccharomyces cerevisiae, unsaturated fatty acid requirement for growth in a defined medium. Journal of Celullar and Comparative Physiology. Min, J.R. Kim, S.E. Oh, J.M. Regan and B.E. Logan, (2005). Electricity generation from swine wastewater using microbial fuel cells. Water Res. 39 (2005), pp. 4961- 4968.

Claims

Claims
1.Use of a microbial fuel cell for fermenting a substrate into a product, wherein said product is oxidised compared to the substrate present in the microbial fuel cell and wherein electrical energy is produced.
2. The use according to claim 1, wherein the microbial fuel cell comprises two compartments: a. an oxidation reactor comprising a substrate and a microorganism, b. a reduction reactor which comprises oxygen.
3. The use according to claim 2, wherein the microorganism present in the oxidation reactor is able to transfer at least part of the formed electrical energy transferred to an electrode.
4. The use according to claim 2 or 3, wherein the microorganism is selected from a fungus, a bacteria, an archea or an algae.
5. The use according to any one of claims 1 to 4, wherein the substrate comprises or consists of at least one of glucose, fructose, sucrose, maltose and starch.
6. The use according to any one of claims 1 to 5, wherein the product is selected from: citrate, acetate, glutamate, lysine, gluconate, itaconate, succinate, lactic acid, ethanol, butanol, glycerol and 1,3 propanediol, butyrate.
7. The use according to any one of claims 2 to 6, wherein the reduction reactor is adapted for optimizing oxygen transfer.
8. The use according to any one of claims 2 to 7, wherein the oxidation reactor is adapted for optimizing the electrons transfer to an electrode.
9. Method for screening a microorganism having a capacity to transfer electron to an electrode by culturing the microorganism for at least months in a microbial fuel cell as defined in any one of claims 2 to 8 and screening for microorganisms that are improved in their capacity of electron transfer to an electrode.
10. Method for producing a product by fermentation, wherein the method comprises the following steps: a. providing a microorganism which is preferably able to transfer at least part of the electrons formed in a reaction to an electrode, b. culturing the microorganism in an oxidation reactor of a microbial fuel cell in the presence of a substrate, c. producing electrical energy by transferring at least part of the electrons formed in the oxidation reactor, d. producing a product in the oxidation reactor.
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EP2895594A4 (en) * 2012-09-17 2016-10-05 Musc Found For Res Dev Microbial electrosynthetic cells
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