Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
  • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
  • C12P5/023—Methane
• • 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
  • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
• • 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
  • C12P33/00—Preparation of steroids
• • 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
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
  • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
• • 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
• • 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
  • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

• Method to produce a synthesis product e.g. methane utilizing methanogenic microorganisms in a microbial electrolysis cell (MEC) by applying a separated nutrient feeding strategy
• the present invention refers to a method to produce methane during the methane production phase or at least another synthesis product by methanogenic microorganisms from at least one inorganic carbon source in a microbial electrolysis cell (MEC) by involving an improved separated nutrient feeding strategy.
• MEC microbial electrolysis cell
• Methane has the highest energy density per carbon atom among fossil fuels and its potential for energy conversion is far greater than any other natural gas, obtained directly by combustion in presence of oxygen or using fuel cells to produce electricity. Methane’s potential for energy generation has become increasingly relevant in the global market.
• methane constitutes a sustainable and renewable energy source and already today increasingly substitutes coal and other fossil fuels.
• the current invention is based on microbial electrochemical technology (MET) devoted to bio-electromethanation.
• MET microbial electrochemical technology
• This process is realized in a microbial electrolysis cell (MEC), which is a unique system capable of converting chemical energy into electrical energy (and vice-versa) while employing microbes as catalysts.
• MEC microbial electrolysis cell
• the system achieves the combination of electrolysis and methane production in one single reactor, the MEC.
• the methanogenic microorganisms reside e.g. at the cathode (“bio-cathode”).
• the reactor may comprise a single compartment, or the cathodic compartment, or chamber, may be separated from the anodic compartment, or chamber, e.g. via a semipermeable membrane.
• methanogenesis by the methanogenic microorganisms takes place directly in the (bio)cathode compartment, while the electron flow required for the cathodic reduction of classical CO 2 to methane is formed in the anode compartment by water oxidation (cf. Figure 1).
• electrical power is used to enhance the potential difference between the anode and the cathode of MEC to enable the bio-electromethanation reaction.
• a culture of methanogenic microorganisms e.g. using hydrogen may catalyse the methanation reaction as follows:
• the water produced by this methanation process is also called “metabolic water” or “free water”.
• a problem associated with the production of metabolic water is the dilution factor of the medium components within the culture medium (catholyte part of electrolytes) that must be specifically addressed.
• This dilution factor is modified by the liquid migration processes that take place between the anodic and the cathodic chamber which may be separated classically by a proton exchange membrane.
• the nutrient requirements of the methanogenic organisms are typically supplied as culture medium or as concentrated medium stock solutions in the prior art, which have to be added continuously in continuous or fed batch modes to guarantee a normal methanation rate during operation. This continuous addition of fresh medium stock is a significant detrimental part of the operational costs of the process.
• this state of the art feeding strategy does not specifically meet the actual microbial nutrient requirements that the microorganisms face in MEC as the bio-electromethanation process at the cathode imposes specific nutrient requirements.
• special strains of methanogenic microorgansims also have special and different needs with regards to nutrient usage.
• One way to face this problem in the state of the art is to supply cell culture media compositions differently formulated with respect to nutrients and nutrients amounts to fulfil the needs of specific strains.
• a method is provided to produce methane during the methane production phase and/or at least one other synthesis product by methanogenic microorganisms in a microbial electrolysis cell (MEC), the method comprising the steps: i. providing a MEC, comprising an anode, a cathode and a culture of methanogenic microorganisms in a suitable liquid aqueous electrolytic culture medium; ii. culturing the methanogenic microorganisms in a continuous process; iii. supplying electrons from the anode to the cathode of the MEC and contacting the methanogenic microorganisms with said electrons; iv.
• MEC microbial electrolysis cell
• contacting the methanogenic microorganisms with a at least one inorganic carbon source v. contacting the methanogenic microorganisms with a nitrogen source and/or a sulfur source by separately supplying the nitrogen source and/or the sulfur source in a discrete or a continuous manner into the culture medium; vi. collecting methane, a methane enriched gas composition and/or at least one other synthesis product from the MEC.
• the method provides improved methane production during a continuous operating process during a methane production phase, after a cell growth phase.
• the continuous operating process may comprise supply of the nitrogen source and/or the sulfur source in a discrete manner or in a continuous manner.
• a method is provided to produce and collect a synthesis product other than methane.
• a method is provided to produce methane and at least one other synthesis product and then to separately collect methane, a methane enriched gas composition and the at least other synthesis product from the MEC.
• the nitrogen source and the sulfur source may each supplied in a discrete manner timewise separated of each other for example, pulsed within 24 hours of each other.
• the nitrogen source and the sulfur source may alternatively each supplied in a discrete manner at same time points or overlappingtime points, i.e. in a simultaneous manner.
• Supplyingthe nitrogen source and the sulfur source in a simultaneous manner can be performed by providing each the sulfur source and the nitrogen source as separate stock solutions and supplying both, e.g. at least at some points by separate supplying means to the culture medium.
• the sulfur source and the nitrogen source may be already premixed together as at least one to multiple different stock mix solutions with a certain individual stock concentration of the nitrogen source and the sulfur source each. Such a specific stock mix solution may then be supplied as needed by a supplying mean to the culture medium.
• the remaining separated electrolytic culture medium comprising vitamins, non-toxic salt ions and other nutrients necessary for cell growth, may be supplied in a discrete manner or in a continuous manner, preferably in a continuous manner, as long as it is supplied separately from the nitrogen source and/or the sulfur source.
• the inorganic carbon source is also preferably supplied separately but may be supplied as part of the separated culture medium.
• the inventors of the present invention have advantageously and surprisingly found by running a MEC under such separated supply regime (nitrogen source, sulfur source, inorganic carbon source) that this increases the overall efficiency of the system.
• the overall efficiency of the system was observed to be 30% or higher or preferably to be 50% or higher than in comparable experiments where a standard whole culture medium supply strategy was applied. Processes, which may be included in the calculation of this efficacy are the reduction of costs, saving of nutrients while increasing the overall methanation rate.
• each of the at least one inorganic carbon source, the nitrogen source and/or the sulfur source are supplied separately into the culture medium.
• These feeding strategies disclosed herein provide beneficial flexibility in terms of process operation with the key goal of maximizing methanogenesis, i.e. energy production in form of methane. With respect of energy recovery in an MFC, high coulombic efficiency is to be targeted. According to the present invention the “coulombic efficiency” expresses the number of electrons that ends ideally in the desired methane produced by electro-methanation.
• a "phase" in the sense of the invention describes a condition or state of the methanogenic microorganisms in the bioreactor of the invention, which is characterized by specific fermentation conditions, which are applied to the methanogenic microorganism, e.g., the ratio of the partial pressures of hydrogen and carbon dioxide or a specific value or range of at least one nutrient, which is applied, e.g. ammonium and/or the settings of the bioreactorto keep cells in the reactor (cell retention) or not.
• a “cell growth phase” according to the present invention is a phase mainly characterized by an increase of the biomass of the methanogenic microorganisms by cell division and cell growth.
• a “methane production phase” according to the present invention is a phase mainly characterized by methane production rather than cell division and cell growth.
• the cells may also or may not produce methane and during any methane production phase, the overall biomass may also increase.
• the methanogenic microorganisms When the microorganisms are in an operating state, the methanogenic microorganisms may be in one of a variety of metabolic phases, which differ with regard to the methanation rate and the division rate and growth rate of the microorganisms, the latter, which can be expressed, as doubling time of microorganism number (division rate) or cell mass (growth rate).
• the phases typically observed include a lag phase, an active growth phase (also known as exponential or logarithmic phase when microorganisms multiply rapidly), a stationary phase, and a death phase (exponential or logarithmic decline in cell numbers).
• the microorganisms of the disclosure are in a lag phase, an active growth phase, a stationary phase, or a nearly stationary phase.
• the stationary phase is generally the main methane production phase.
• the method of the present invention does comprise a step of culturing methanogenic archaea, which is based on typical culture conditions for archaea, which have been previously described and which are known to the practitioner. Such conditions are influenced and controlled - according to the skills of a practitioner by common parameters affecting the culture including temperature, pressure, volume, salt ion content, conductivity, carbon content, nitrogen content, vitamin content, amino acid content, mineral content, or any combination thereof may be varied and are encompassed by the method of the present invention.
• the present invention can be performed under so called “cell retention conditions” as described in the international application PCT /EP2020/060979 to avoid - as this widely happens in classical culturing methods of the prior art - that substantial numbers of cells are continuously washed out of the reaction vessel. These washed out cells have to be replaced by further cycles of cell division and cell growth therefore by utilization of CO 2 and H 2 for the generation and growth of cells rather than for the generation of the aimed methane output. This is unfavourable for the efficiency of the system. Alternatively, and/or additionally there may be the option to supply a sufficient amount of new methanogenic microorganisms to compensate the amount of cells washed out if the MEC is running under no cell-retention conditions (see PCT/EP2020/060979).
• the inventors of the present invention have advantageously and surprisingly found out that a separate feeding strategy of either a nitrogen source or a sulfur source or of both improves methane production efficiency and each separately supplied advantageously meet the physiological needs of a respective methanogenic microorganism on a strain dependent manner.
• feeding in a discrete manner is meant that the nitrogen source and/or the sulfur source supply will be done discontinuously at certain time points e.g. as a single pulse or a multiplicity of pulses and may be performed according to the concrete and specific demands of the methanogenic microorganism strain.
• FIG. 2 A possible system for the separate feedingstrategy of the nitrogen source and/or the sulfur source in a discrete or continuous manner is depicted in Figure 2 and represented with the letter A-B and C.
• the Figure also represents the inorganic carbon source applied to the cathode chamber, the pump driving the recirculation of the anolyte and the catholyte, the potentiostat-power source to establish a potential difference between anode and cathode, and the Micro-GC for quality measurements of the out-product gas.
• methanation or “methanogenesis” or “bio-(electro)methanation”, is understood as the production of methane or a methane enriched gas composition as carried out by methanogenic microorganisms, such as those included in a list of methanogenic microorganisms suitable to carry out the present invention as described below.
• methanogenic microorganisms are cultured in a microbial electrolysis cell (MEC) in order to produce one or more synthesis products, preferably biomethane.
• MEC microbial electrolysis cell
• steps are regularly disclosed concerning the production of methane without always explicitly adding that they are regularly also concerning the production of the at least one synthesis product different from methane.
• MEC microorganisms
• a “MEC” stands for a bioreactor, and is either a bioreaction vessel, or a bioreaction enclosure, or a bioreaction tank, and/or at least a bioreaction chamber, and/or a cell, or a combination thereof, as also intended in the state of the art.
• the MEC may comprise a single compartment, or the cathodic compartment, or chamber, may be separated from the anodic compartment, or chamber, e.g. via a semipermeable membrane.
• the MEC has to be able to withstand variations of e.g. temperature and/or pressure, among others, and/or able to maintain whichever imparted values of e.g. temperature, and/or pressure are assigned or have to be maintained, before, after or during the reaction process, and wherein the intended reactions relevant for carrying out the invention may take place.
• Such reactions are understood as bioreactions as they pertain to the domain of reactions wherein microorganisms are involved, and herein referring to their normal physiology - such as e.g. metabolic fermentation, or aerobic or anaerobic digestion - and that, as such, require suitable environments, suitable cultures of microorganisms, suitable culture mediums and suitable reactants to occur.
• a MEC in the meaning of the invention performs reliably within the tolerance values of each variable in order to enable the method as disclosed, and it is expected to allow the listed steps to be carried out reliably over time.
• a MEC may comprise one or more sensors or components that measure and/or regulate values of, for example, (a) temperature, (b) pressure and/or (c) electrical potential difference, within a pre-set range.
• the values may be measured and/or regulated before, after or during the reaction process (e.g. methane production).
• Cultured methanogenic microorganisms according to the present invention, or autotrophic methanogenic microorganisms may be anaerobic archaea or even recently classified aerotolerant archaea, either in pure strains, or in consortia with a plurality of, i.e. two or more, strains, or in mixed cultures wherein methanation may be also encouraged bysyntrophic exchange across different species.
• methanogenic refers to microorganisms that produce methane as a metabolic byproduct.
• culture refers to a population of living microorganisms in or on culture medium.
• the culture medium When part of the MEC, the culture medium also serves as the electrolytic medium facilitating electrical conduction within the MEC.
• the method herein disclosed is concerned with the culturing of methanogenic microorganisms in a “continuous process”, wherein such continuity is understood as continuity in the production of methane or at least another synthesis product by the methanogenic microorganisms (continuous operating process) and continuity in the culture, wherein no step of separating inactive terminal biomass from active members of the colony is required. It is instead encouraged that dead biomaterial is kept in the reactor together with the active members across several stages of growth, as it is found advantageous that said biomass or biomaterial provide further substrate for the active culture, intensifying nutrition availability.
• the methanogenic microorganisms may be but not necessarily cultured with dead biomaterial inside the bioreactor for a certain period of time, at least 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, or more.
• suitable reactants other than the separate supply of the nitrogen source and/or the sulfur source, are supplied to the MEC in a continuous or fed batch manner, allowing the methanogenic microorganisms to carry out methane production without significant alteration of the measured amount of produced methane (i.e. yield of methane) obtained from any cycle of methanogenic activity across the culture and within the operational phases of the reactor.
• the nitrogen source or the sulfur source or both are supplied by feeding in a discrete manner instead of a continuous manner, while the other reactants are supplied in a continuous manner.
• methane is produced by methanogenic archaea from single strains or in mixed cultures, wherein a mixed culture is either a culture where a plurality of, therefore two or more, strains may also be employed, or a culture where a plurality of additional species interact with methanogenic archaea, or any combination thereof.
• a “refreshing of the culture medium” can be realized by exchanging the cell culture medium at least partly or by adding at least one nutrient, which triggers cell division and cell growth.
• Nutrients, which trigger cell growth and cell division are well known by an artisan and include the addition orthe increase of a nitrogen source, a sulfur source, phosphorous and cell growth factors.
• a combination of the described options for refreshing of the culture medium is also a possible option according to the present invention.
• Such a “refreshing of the culture medium” may be but not necessarily be applied every month, every half year for at least one day or at least one day to five days or at least one day to four days at least one day to three days.
• a conductive conduit connected to a power source, may connect the anode and the cathode such that the power source provides an electrical potential difference between the anode and the cathode.
• the MEC comprises a sensor to measure the electrical potential difference between the anode and the cathode, or the oxidative reduction potential.
• the MEC further comprises a sensorthat measures current density, temperature, or pressure.
• the MEC may also comprise one or more sensors that measure each the input of separately supplied culture medium, nitrogen source and/or sulfur source.
• step v. further comprises: continuously controlling and regulating the concentration of the nitrogen source in the culture medium to maintain the nitrogen source concentration in the culture medium to be at a given amount of 0.005 to 0.2 M or of 0.02 to 0.2 M, preferably between 0.01 to 0.02 M.
• the nitrogen source is preferably supplied in a discrete manner.
• the methanogenic microorganism culture is continuously controlled and regulated, i.e. stabilized to be kept cultured at a nitrogen source concentration at a given amount.
• the nitrogen source is NH 3 and the method of the present invention step v. further comprises: continuously controlling the nitrogen source in the culture medium and regulating said concentration of the nitrogen source, when the nitrogen source concentration in the culture medium is lower than 0.2 M, lower than 0.02 M, lower than 0.01 M or lower than 0.005 M to maintain the nitrogen source concentration in the culture medium to be at a given amount of 0.2 to 0.005 M or of 0.2 to 0.02 M, preferably between 0.02 to 0.01 M.
• the nitrogen source is preferably supplied in a discrete manner.
• the nitrogen source is supplied at the lowest minimum levels, for example, in a discrete manner or continuous manner.
• the method may comprise regulating the nitrogen source concentration in the cathode chamber of the MEC to regulate the concentration of nitrogen within a specified range, e.g. a level lower than 0.8 M, or 0.07 M, or 0.007 M.
• Methanogenic microorganisms generally need a nitrogen source and accordingly all published prior art documents teach the supply of nitrogen in one or the other way.
• the influence how a nitrogen source pulse or a sulfur source pulse (each discrete supply) influences parameters of the methanation process - without being bound by the theory - as salinity of the culture medium (cathode side), the pH, the current, the CH 4 /CO 2 conversion, the hydrogen production and the optical density is depicted in Figure 7.
• step v. further comprises: continuously controlling and regulating the sulfur source concentration in the culture medium to maintain the sulfur source concentration in the culture medium to be at a given amount of 0.1 to 100 mM or of 10 to 80 mM, preferably between 15 to 30 mM and preferably discretely supply the sulfur source.
• the methanogenic microorganism culture is continuously controlled and regulated, i.e. stabilized to be kept cultured at a sulfur source concentration at a given amount.
• the sulfur source is supplied at the lowest minimum levels necessary, for example, in a discrete manner or continuous manner.
• the method may comprise regulating the sulfur source concentration in the cathode chamber of the MEC to regulate the concentration of sulfur within a specified range, e.g. a level lower than 100 mM, or lowerthan 50 mM, or lowerthan 20 mM or lowerthan 10 mM.
• the method comprises regulating the sulfur source in dependance to the oxidation-reduction potential (ORP).
• ORP oxidation-reduction potential
• the sulfur source concentration in the culture medium further comprises: continuously controlling the sulfur source concentration in the culture medium and regulating the sulfur source concentration in the culture medium to maintain the sulfur source concentration in the culture medium to be at a given amount of 0.1 to 100 mM or of 10 to 80 mM, preferably between 15 to 30 mM and discretely supply the sulfur source, when the oxidation-reduction potential (ORP) in the culture medium is lower than -200, lower than -350, lower than -400 or lower than -450.
• ORP oxidation-reduction potential
• Sulfur may act as a reducing agent to maintain the low oxidation-reduction potential (ORP) in the growth medium that is regarded as important to a productive operating of the methanogenic microorganisms, e.g. for methanogenesis.
• ORP oxidation-reduction potential
• methanogens is a key element of enzymes catalysing the reactions involved in the CO 2 reduction to methane with H 2 , e.g., as part of the prosthetic groups in the [4Fe 4 S]-cluster of the F 420 -reducing hydrogenases that catalyse the reversible reaction of coenzyme F 420 with H 2 .
• step v. further comprises: continuously controlling and regulating the concentration of the nitrogen source in the culture medium to maintain the nitrogen source concentration in the culture medium to be at a given amount of 0.005 to 0.2 M or of 0.02 to 0.2 M, preferably between 0.01 to 0.02 M.
• the nitrogen source is preferably supplied in a discrete manner. continuously controlling and regulating the sulfur source concentration in the culture medium to maintain the sulfur source concentration in the culture medium to be at a given amount of 0.1 to 100 mM or of 10 to 80 mM, preferably between 15 to 30 mM and d iscretely su p p ly the su Ifu r sou rce.
• step v. further comprises: continuously controlling the nitrogen source in the culture medium and regulating said concentration of the nitrogen source, when the nitrogen source concentration in the culture medium is lower than 0.2 M, lower than 0.02 M, lower than 0.01 M or lower than 0.005 M to maintain the nitrogen source concentration in the culture medium to be at a given amount of 0.2 to 0.005 M or of 0.2 to 0.02 M, preferably between 0.02 to 0.01 M.
• the nitrogen source is preferably supplied in a discrete manner.
• the nitrogen source and the sulfur source are preferably supplied in a discrete manner.
• the oxidation-reduction potential (ORP) or redox potential is a measure of the tendency of an aqueous solution to either gain or lose electrons when it is subjected to change by introduction of a new chemical species.
• Methanogens can be generally sensitive towards O 2 and can be inhibited or even killed by certain amounts or even traces of oxygen (strain dependently). Therefore, a low ORP, which varies linearly with the logarithm of O 2 concentration, helps to maintain their metabolic activity.
• the ORP is e.g. determined by measuringthe potential of a chemically-inert (platinum) electrode which is immersed in the solution.
• the sensing electrode potential is read relative to the reference electrode of the pH probe and the value is presented in millivolts (mV).
• mV millivolts
• the potential difference between these electrodes or ORP (mV) is measured by using an ORPmeter.
• ORP Normally the ORP during a methane production phase according to the present invention is in a range of -450 to -200 mV.
• the inventors of the present invention have surprisingly and advantageously found byways of initial comparative experimentations that providing of the nitrogen source and the sulfur source (e.g. each by means of a pulse (discrete supply)) together but timewise separated from each other, e.g.
• common culture or growth mediums to be provided to the culture of methanogenic organisms may include common inorganic elements, in their elemental forms or in any suitable non-toxic salt ions thereof, e.g. sodium, potassium, magnesium, calcium, iron, chloride, sources of sulfur, e.g. hydrogen sulfide or elemental sulfur, phosphorus sources, e.g. phosphate, nitrogen sources, e.g. ammonium, nitrate or nitrogen gas.
• the culture medium according to the present invention is supplied separately from the nitrogen source and sulfur source and optionally also separately from the at least one inorganic carbon source. It may comprise other nutrients necessary for cell growth, including vitamins, and non-toxic salt ions, and optionally comprise minimal amounts of sulfur and nitrogen, but preferably omit sulfur and nitrogen.
• Typical supplied salts utilized for culturing methanogenic organisms according to the present invention are NaCI, KH 2 PO 4 , FeCI 2 -4H 2 O, Na 2 SeO 3 , Na 2 S, NH 4 0H and MgCI 2 .
• the present invention is besides others characterized by a step of controllingthe external supply of the nitrogen source and/or the (resulted) concentration of the nitrogen source (e.g. ammonia) within the cell culture medium.
• the present invention is also characterized by a step of controlling the external supply of the sulfur source and/or the (resulted) concentration of the sulfur source (e.g. Na 2 S) within the cell culture medium.
• controlling is understood in the general common meaning of keeping under constant monitoring the parameters related to the culture and essentially measuring said parameters or status indicators, using common methodologies and measuring instrumentation known in the art. Since it might not be sufficient to keep under constant monitoring and therefore only control this parameter of the culture; therefore, a further embodiment of the present invention comprises in particular regulating the nitrogen source concentration and/or the sulfur source concentration within the cell culture medium continuously.
• regulating is intended as actively maintaining a “given value” or a given value span for a parameter, e.g. the nitrogen sou rce/sulfur source concentration of the culture, by using appropriate means to do so.
• a parameter e.g. the nitrogen sou rce/sulfur source concentration of the culture
• a “given value” according to the invention may be a defined value with given tolerances, tolerances within the measurements system or tolerances due to the variability within the culture or due to the culture diversity, wherein said value is suitable for enabling methanation; or a given value may be a range of suitable values, which achieve the same effect on methanation as a given value.
• the inventors of the present invention have surprisingly found that if the nitrogen source and the sulfur source are both controlled and separately supplied in a need-related manner (by discrete or continuous supply) that this feeding regime in particular allows for increased flexibility in terms of needed process operations with the key goal of maximizing methanogenesis, i.e., e.g. energy production in form of methane. Moreover, with a situation- and need-dependent supply of the nitrogen source and the sulfur source the more chemicals and connected cost to maintain the methanation process are saved and associated waste amounts of chemicals not needed in the process are beneficially more reduced as when supplying only one eitherthe nitrogen source or the sulfur source alone.
• Na 2 S in the culture medium can advantageously act situation-dependent on several MEC performance variables, e.g., pH, ORP, cell proliferation, etc. that are at the same time interrelated each other as depicted in Fig. 3.
• MEC performance variables e.g., pH, ORP, cell proliferation, etc. that are at the same time interrelated each other as depicted in Fig. 3.
• Some of these variables responses are directly connected with the nitrogen source (e.g. pH) and the sulfur source (e.g. ORP) and their change concurrently triggers the variation in a battery of indirect variables, e.g., methanation rate.
• the rest of the media components e.g.
• vitamins, minerals, salt ions etc. can be supplied together compensating the catholyte dilution factor, i.e., the water formation produced because of the methanation reaction driven by the methanogenic microorganisms (metabolic water) and/or the dilution, which occurs due to the migration of the catholyte and anolyte part of the electrolytes through the membrane.
• the catholyte dilution factor i.e., the water formation produced because of the methanation reaction driven by the methanogenic microorganisms (metabolic water) and/or the dilution, which occurs due to the migration of the catholyte and anolyte part of the electrolytes through the membrane.
• the method comprises regulating the sulfur source proportionate to the current density or to the projected electrode area.
• the sulfur source is supplied to maintain a ratio of a projected electrode area (m 2 ) to sulfur source concentration in the culture medium (mol/L) in the range of 1:0.1 to 1:10, or 1:1.5 to 1:3, preferably in the range of 1:2 to 1:3 or 1:2 to 1:2.5.
• the amount of sulfur supplied is reduced and the ratio is correspondingly reduced, e.g. to ranges of 1:0.001 to 1: 0.1.
• the projected electrode area is the surface area of an electrode, i.e. the geometrical area. It can be measured by methods of the state of the art as geometric methods well known by the skilled person. These methods use simple mathematical formulas to calculate areas of regular geometrical figures, such as triangles, trapeziums*, or areas bounded by an irregular curve.
• H 2 O is the primary net electron donor for the methanogenic organisms.
• the electrolyte pH and on the strain optical density (OP) can act as a pH regulator balancing acidity created by the dissolution of the inorganic carbon source, e.g. CO 2 (as NH 4 0H) or alkalinity by the OH' generation (by means of H 2 O reduction) at the cathode (as NH 4 + ).
• the buffering role of the nitrogen source may be combined with an extra buffer, e.g. a phosphate buffer.
• the nitrogen requirement for pH balancing may be lower than for cell growth, wherein the nitrogen source, e.g. NH 3 acts as a key nutrient for protein synthesis in archaea.
• NH 3 may be supplied at the cathodic chamber as far as needed to balance pH and maximize methanation, both variable in combination with the lowest OD.
• the sulfur source e.g. in form of Na 2 S may establish a low ORP, a fundamental requirement to provide the adequate thermodynamic conditions for the methanation reaction.
• ORP a fundamental requirement to provide the adequate thermodynamic conditions for the methanation reaction.
• concentration of such a compound will be only increased if needed for further synthesis stimulation of the necessary enzymatic components required for methanation, e.g., [4Fe 4 S]-cluster of the F 420 -reducing hydrogenases that catalyse the reversible reaction of coenzyme F 420 with H 2 .
• the inventors of the present invention found that the necessary compounds needed to act on pH, OD, ORP and methanation rates may be also regulated to adapt to new potential methanation scenarios in terms of the concentration of the inorganic carbon source, e.g. CO 2 concentration. These new CO 2 scenarios depend on the CO 2 influx flow that at the same time depends on the electron flow from the anodic toward the cathodic chamber.
• Stepwise variation of the sulfur source supply to adapt to the different system evolution phases, i.e., lag, exponential and stationary phase in terms of strain proliferation and methanation rate.
• This variation must be determined on the flow of the inorganic carbon source, e.g. in form of CO 2 .
• ORP oxidation-reduction potential
• Asuboptimal value of above -250 mV, caused by e.g. an oxygen diffusion to the catholyte may be an event where Na 2 S may be supplied.
• Na 2 S may function to reverse the inadequate ORP conditions due to deficient membrane performance (oxygen diffusion through the cathode) by acting as a scavenger agent for oxygen and thereby decreasing the ORP.
• Provisional oxygen contamination may also occur through other mechanisms as e.g.
• Adjustment of the electron flow in the MEC i.e., the electron donor at the bio-electromethanation cathodic reaction may react to different variable, e.g., salinity, MEC voltage, temperature, electrolyte composition, etc..
• the oxidation of the electron donor (e.g. H 2 O) and the resulting electron flow from the anode to the cathode determine the potential flow of the inorganic carbon source(e.g. CO 2 ) that may be converted to methane due to the 8 electrons required for its formation.
• the variation in this carbon flow supply influences pH, ORP, cell growth, methanation rate, etc., see e.g. in the following potential exemplary scenarios.
• the methods provided herein also provide steps to respond and counteract to variations from the desired ranges by controlling and regulating the inorganic carbon source, the nitrogen source and the sulfur source as will be described in the following: Scenario A: Increased current -> Counter action: Increasing inorganic carbon source flow (e.g. CO 2 flow)
• the counter action is to increase the inorganic carbon source flow (e.g. CO 2 flow).
• the pH and ORP may vary due to the increase of the CO 2 flow.
• the pH may decrease due to more CO 2 in solution (as carbonic acid).
• the ORP may increase due to more acidic condition that trigger the conversion of present Na 2 S to H 2 S (the latter, which will leave the system as a gas).
• Extra buffer/nitrogen source e.g. ammonia
• Extra buffer/nitrogen source should be spiked when the ORP is e.g. under -450 mV (as targeted) but the pH is detrimental still under a threshold, e.g. under pH value 6.5.
• Scenario B Decreased current -> Counter action: Lowering inorganic carbon source flow (e.g. CO 2 flow)
• the counter action is to decrease the inorganic carbon sourceflow (e.g. CO 2 flow).
• the pH value may increase due to less CO 2 in solution (as carbonic acid).
• Scenario C Decreased conversion of the inorganic carbon source (e.g. CO 2 ) to methane i) If this scenario is associated with an increased OD 6 IO during the last operation hours; Potential counter action: supply growth media (e.g. strain-specific), optional supply nickel and/or the sulfur source (e.g. Na 2 S) to maximize the synthesis of the enzymatic metabolic components of the strain that may maximize the CO 2 to methane conversion, e.g., [4Fe 4 S]-cluster of the F 420 -reducing hydrogenases that catalyse the reversible reaction of coenzyme F 420 with H 2 . ii) If this scenario is associated with a decreased OD 6 IO during the last operation hours;
• growth media e.g. strain-specific
• nickel and/or the sulfur source e.g. Na 2 S
• the separated supply of the nitrogen source and/or the sulfur source e.g. NH 4 0H/CI, Na 2 S
• the classical separated supply of the inorganic carbon source e.g. CO 2
• Adapting to new electron donor and acceptor flows scenarios according to the present invention beneficially reduces the times of adaptation of a certain experimental strain culture to these new flows in terms of cell proliferation and growth, pH and ORP stability and methanation rates.
• the inventors of the present invention surprisingly found out that this separated supply of necessary nutrients has the advantage of acting independently on different variables of the methanation process of a methanogenic microorganism culture to allow for a situation-dependent and strain-dependent optimized methanation rate.
• this feeding regime further provides flexibility in terms of operation during the MEC methanation process.
• step v. further comprises to continuously control and regulate the concentration of at least one other source of inorganic elements in the culture medium in their elemental form or in form of any suitable non-toxic salt ions thereof, e.g. selected from the group consisting of sodium, potassium, magnesium, calcium, iron, chloride or phosphate by a separate supply in a continuous or discrete manner.
• any suitable non-toxic salt ions thereof e.g. selected from the group consisting of sodium, potassium, magnesium, calcium, iron, chloride or phosphate by a separate supply in a continuous or discrete manner.
• the concentration of said at least one other source of inorganic elements in the culture medium may be continuously controlled and regulated to be maintained, i.e. stabilized at a certain given value or range in relation to the methanation rate and I or to the metabolic water production rate. It is done to address / counteract the rate of consumption of the at least one other source of inorganic elements in the culture medium driven by the methanation process and/or to counteract the progressive dilution due to the production of metabolic water.
• at least one or all said inorganic elements may be already premixed together as at least one to multiple different stock mix solutions with a certain individual stock concentration of each of the individual different inorganic elements.
• Such a specific stock mix solution may then be supplied as needed by a supplying mean to the culture medium and optionally in relation to the methanation rate and/or metabolic water production rate orto the rate and amount of removal of diluted cell culture medium from the overall cell culture medium volume to remove excess metabolic water from the system.
• the inventors of the present invention believe - without being bound by that theory - that the more individual nutrients are separately supplied in a continuous or discrete manner if needed the more increased is the flexibility in terms of process operations with respect to methanogenesis.
• the step of culturing the methanogenic organisms further comprise keeping the temperatures in a range between 32°C and 85°C; preferably 50-70°C or 62-67°C.
• the step of culturing the methanogenic organisms additionally comprise recirculating the culture, wherein the recirculating of the culture can be carried out regularly, in intervals, continuously, or keeping the soluble culture at least in a certain slow and constant movement.
• temperatures may vary according to the presence of selected microorganism species within the culture, each of which betterthrive within set ranges of temperatures, for most of the methanogenic microorganisms increased temperatures are not detrimental, and they may even assist in optimizing cellular metabolism and thus metabolic turnover or even methanation.
• a temperature must be controlled by energetic regulation; in this regard it is to be considered a valuable feature to reduce energy expenditure by enabling temperature control.
• the method of the present invention was found to be most efficient in a temperature range between 32°C and 85°C, or alternatively 50 to 70°C or further alternatively 62-67°C at atmospheric pressure. If accordingto some embodiments one or more steps of the method accordingto the invention are carried out in a pressurized atmosphere, then the pressure is chosen to be preferably up to 16 bar, alternatively up to 20 bar, alternatively up to 50 bar, alternatively up to 68 bar, alternatively up to 110 bar or even up to 420 bar.
• the present invention also refers to a culturing process at pressures equal or between the range of I to 10 bar.
• High pressure e.g. 16 bar, 20 bar, 35 bar, 40 bar or 60 bar and correspondingly, higher temperatures, which would allow the same hydrogen solubility as at a temperature range between 32°C and 85°C, or alternatively 50 to 70°C or further alternatively 62-67°C at atmospheric pressure are also encompassed.
• Methanogenic microorganisms in general, may live and grow also in a plurality of other and even extreme temperature ranges up to and well above 100°C, e.g. 140°C; accordingly, the above temperature range is an indication of a preferred range, but it is not to be understood as limiting the scope of the invention.
• the nitrogen source is but not limited to diatomic nitrogen (N 2 ), ammonia (NH 3 ), nitrate or nitrite salt ions, ammonium (NH 4 + ) compounds, preferably in the form of NH 4 0H or NH 4 CI or combinations of the aforementioned.
• the nitrogen source is ammonia.
• the nitrogen source is an ammonium compound, preferably in the form of NH 4 0H.
• the concentration of living cells in the culture medium is in some embodiments maintained above 0.01 g dry weight/L. In certain embodiments, the density may be 50 g dry weight/L or higher.
• the OD 6 IO optical density at 610 nm
• the optical density (OD) of the culture according to the present invention is measured utilizing common methods and standards known in the art.
• Optical density, or, rather, turbidity measurements as a form of cell counting are performed using a spectrophotometer, is typically operated around or at 600 nm, but accordingly other wavelengths may be suitable.
• the culture of the methanogenic microorganisms can be guided or led into a high density culture with an OD 6 IO of at least 14, but preferably above 20, further also above 30, further above 40 and even up to 120 or 200 by supplying sufficient nutrient to the culture and simultaneously removing free or metabolic water from the culture.
• the method of the present invention can thus be suitably performed in culture of one or more strains of methanogenic microorganism, having throughout the various developmental stages a measurable OD 6 IO between 60 - 200; further an OD 6 IO between 14 - 120; further an OD 6 IO between 20 - 120; further an OD610 between 30 - 120; further an OD 6 IO between 40 - 120; further an OD 6 IO between 50 - 120; further an OD 6 IO between 50 - 100; further an OD 6 IO between 14 - 80; further an OD 6 IO between 20 - 80; further an OD 6 IO between 30 - 80; further an OD 6 IO between 40 - 80; further an OD 6 IO between 20 - 80; further an OD 6 IO between 30 - 40; further an OD 6 IO between 40 - 60; further an OD 6 IO between 20 - 40.
• a high optical density corresponding to a high number of cells is obtained into the growth phase and maintained by keeping the members of the culture in the bioreactor across the entire stages of their lives to their terminal stage, so that the remains of the inactive cellular bodies may provide nutrients to the active members of the culture.
• the culture of the methanogenic microorganisms can be guided or led into a density culture with an OD 6 IO of at least 0.04 or at least 0.1, but preferably above 0.3, further also above 0.4, further above 0.5 and even up to 0.6, 0.7, 0.8, 1.0, 1.5, 2.0 or 2.5 by supplying sufficient nutrient to the culture and simultaneously removing free or metabolic water from the culture.
• the method of the present invention can thus be suitably performed in culture of one or more strains of methanogenic microorganism, having throughout the various developmental stages a measurable OD 6 IO between 0.1 - 2.5; further an OD 6 IO between 0.3 - 2.5; further an OD 6 IO between 0.4 - 2.5; further an OD 6 IO between 0.5 - 2.5; further an OD 6 IO between 0.6 - 2.5; further an OD 6 IO between 0.1 - 2.0; further an OD 6 IO between 0.1 - 1.5; further an OD 6 IO between 0.1 - 0.8, further an OD 6 IO between 0.1 - 0.7; further an OD 6 IO between 0.1 - 0.6.
• the sulfur source is selected from the group consisting of hydrogen sulfide, Na 2 S, L-cysteine, elemental sulfur or sulphate or combinations of the aforementioned.
• the method further comprises a source of a reductive element in the culture medium selected from the group consisting of hydrogen, hydrogen sulfide, the sulfur source, formic acid, carbon monoxide, reduced metals, sugars, acetate, cathodic electrodes or combinations of the aforementioned.
• a source of a reductive element in the culture medium selected from the group consisting of hydrogen, hydrogen sulfide, the sulfur source, formic acid, carbon monoxide, reduced metals, sugars, acetate, cathodic electrodes or combinations of the aforementioned.
• the sulfur source can also act as a reductive element. Na 2 S will be applied when the ORP a suboptimal value of above -250 mV, caused by e.g. an oxygen diffusion.
• the at least one inorganic carbon source comprises electron equivalents and is selected from the group consisting of CO 2 gas, sodium carbonate, potassium carbonate and ammonium carbonate or combinations of the aforementioned.
• an organic carbon source may be used instead of or additionally to the at least one inorganic carbon source as disclosed above.
• the at least one organic carbon source may be selected from the group consisting of formate, acetate, methanol, methylamines and sugars or combinations of the aforementioned.
• the at least one inorganic carbon source can be applied in a need-dependent and situation-dependent manner to face the particular needs of a culture of a certain microorganism strain specifically.
• the inorganic carbon source classically in the form of CO 2 gas is already given separately.
• the inorganic carbon source e.g. in form of CO 2 may be applied as pure gas or alternatively delivered using the supply of industrial gases.
• industrial gases depending on their source may comprise very different gas compositions. They have primarily in common that they contain a relatively high amount of CO 2 in comparison to air. They may contain a normal (air-like) partial amount of oxygen and/or nitrogen, however, depending on their origin they may also be oxygen free.
• they may contain substantial amounts of at least one of the following, particularly carbon monoxide, hydrogen and hydrogen sulfide, other sulphur compounds (sulfides, disulfides, thiols), siloxanes (organic silicon compounds), halogenated compounds, ammonia, and organochlorines, i.e. pesticides and other synthetic organic compounds with chlorinated aromatic molecules.
• the inorganic carbon source is supplied or regulated proportionate to the electron flow, wherein the inorganic carbon source comprising electron equivalents is supplied in at an electron equivalent to electron ratio.
• the method further comprises the step of continuously controlling and regulating the flow of the inorganic carbon source comprising electron equivalents in dependence of the electron flow in an electron equivalent to electron ratio in a range of 1 : 20 to 1 : 1, or at 1 : 20, 1 : 18, 1 : 15, 1 : 12, 1 :10, 1 : 9, 1 : 8, 1 : 7, 1 : 6, 1 : 5, 1 : 4, 1 : 3, 1 : 2 or 1 : 1, preferably in a range of 1 : 10 to 1 : 6, more preferably at 1 : 8 and the flow of the inorganic carbon source, e.g. of CO 2 into the MEC may be measured.
• the flow of the inorganic carbon source e.g. of CO 2 into the MEC may be measured.
• the at least one inorganic carbon source is CO 2 gas and the method further comprises the step of continuously controlling and regulating the CO 2 flow to receive a CO 2 : electron ratio in a range of 1 :20 to 1:1, or at 1 : 20, 1 : 18, 1 : 15, 1 : 12, 1 :10, 1 : 9, 1 : 8, 1 : 7, 1 : 6, 1 : 5, 1 : 4, 1 : 3, 1 : 2 or 1 : 1, preferably in a range of 1 : 10 to 1 : 6 more preferably at 1 : 8.
• a current of 100 mA is equal to a CO 2 : electron ratio of 1 : 4 with a CO 2 flow of 0.2 mL/min. Techniques and means how to measure the current are well known by the artisan, e.g., by mean of potentiostatic control in a 3-electrode system or using a power source with a 2-electrode system.
• the inventors of the present invention have found that the controlling and regulating of the flow of the inorganic carbon source (e.g. the CO 2 flow) by a separate supply regime in dependence of the electron flow under such ratios will provide beneficial flexibility in terms of process operation with respect to maximizing of the methanogenesis.
• the inorganic carbon source e.g. the CO 2 flow
• the at least one inorganic carbon source is CO 2 and methane or a methane enriched gas composition is collected.
• the at least one synthesis product different from methane or different from the methane enriched gas composition is selected from the group consisting of geraniol, vitamin A, cholesterol, carotenoids, and natural rubber.
• the method further comprises the step of setting an initial pH value to be at a given value of pH 6 to 11, of pH 7 to 10 or at pH 8 and subsequent continuously controlling and regulating, i.e. stabilizing the pH value.
• the separated nutrient supply system (nitrogen source and/or sulfur source); (nitrogen source, sulfur source, inorganic carbon source) may be applied to regulate an initial set pH value to be at a given value of pH 6.0 to 7.5 to promote cell division and cell growth or to regulate an initial set pH value to be at a given value of pH 7.5 to 9.0 to promote CO 2 capturing and thus CH 4 /CO 2 conversion.
• the separated feeding supply beneficially also allows for a situation-dependent shift between such pH values of above, thus promoting cell growth and CH 4 /CO 2 conversion as needed.
• the step of controlling and regulating the pH value continuously to be kept at a given different value is done by dosing suitable amounts of a base and/or an acid, e.g. NaOH/HCI or NH 4 0H/HCI to the culture.
• a base and/or an acid e.g. NaOH/HCI or NH 4 0H/HCI
• a responsive step comprises optionally (a) decreasing the supply of inorganic carbon source (e.g. CO 2 flow) or (b) pulsing additional fresh culture medium (e.g. separated culture medium) into the cathode chamber to increase salinity and therefore current production.
• inorganic carbon source e.g. CO 2 flow
• additional fresh culture medium e.g. separated culture medium
• the sulfur source supply when delivered as a buffer form, may be temporarily increased in response.
• acid may be added as a responsive step.
• the sulfur source supply may be temporarily increased in response, although the sulfur source may also increase pH.
• a responsive step comprises increasing the supply of culture media, or nickel and/or the sulfur source (e.g. Na 2 S); or alternatively increasing the supply of the nitrogen source.
• the sulfur source e.g. Na 2 S
• the inventors have found that in general there is no need to supply externally H 2 to maintain overall methanation efficiency as e.g. the hydrolysis of water generates sufficient H 2 for the metabolism and maintenance of the culture of methanogenic microorganisms. However, if needed, e.g. when there is a MEC misfunction, the external supply of H 2 in a discrete manner as an emergency treatment or just for assistance purposes to recover the culture could be appropriate.
• the method further comprises the step of contacting the methanogenic microorganisms with at least one feeding gas comprising H 2 .
• the used culture of methanogenic microorganisms resides floating within the culture medium or is at least partially bound to the cathode, e.g. as biofilm.
• the organic inorganic carbon source is absent in the culture medium.
• the methanogenic microorganisms are hydrogenotrophic and are selected from at least one of the group of Archaea or archaebacteria comprising of Methanobacterium, Methanobrevibacter, Methanothermobacter, Methanococcus, Methanosarcina, Methanopyrus or mixtures thereof.
• hydrogenotrophic refers to a microorganism capable of converting hydrogen to another compound as part of its metabolism.
• Classical hydrogenotrophic methanogenic microorganisms are capable of utilizing hydrogen (H 2 ) and an inorganic carbon source as CO 2 in the production of methane.
• classical hydrogenotrophic methanogenic microorganisms according to the definitions as given above may be modified, e.g. by way of genetic modification to produce additionally other synthesis products as methane from H 2 and a carbon source, e.g. geraniol as described in Lyu etal., 2016.
• the cathode accordingto the invention may be of a high surface to volume electrically conductive material.
• the cathode may be made of an electrically conductive material, e.g. graphite.
• the cathode may be porous or non-porous at least at its surface.
• the cathode may be made from a reticulated vitreous carbon foam.
• the pores of the cathode may be large enough (e.g., greater than 1-2 micrometers in minimum dimension) to accommodate living methanogenic microorganisms within the pores.
• the electrical conductivity of the cathode matrix is preferably at least two orders of magnitude greaterthan the ion conductivity of the aqueous electrolytic medium contained within its pores.
• the role of the cathode is to supply electrons to the microorganisms while minimizing side-reactions and minimizing energy loss.
• Fig.l MEC scheme for different possible ways of methane production in the process of bio-electromethanation.
• Anode indicated by A
• bio-cathode indicated by C
• PEM ionic exchange membrane
• Flow of electrons is indicated by “e-” and associated arrows indicate the direction of the flow.
• NHE Normal Hydrogen Electrode.
• D indicates a methanogen
• E and G indicate indirect electron transfer mechanisms
• F indicates direct electron transfer mechanism
• H and I indicate the chemical reactions for methane production under indirect and direct electron transfer mechanisms respectively.
• L indicates the chemical reaction for water oxidation at the anode.
• J and K indicate redox mediators.
• Fig.2 General overview on the MEC bio-electromethanogenesis process using a separate media supply system.
• the strain media requirements are supplied through a separate feeding system (indicated by three separated compartments) to allow for separated and independent nutrient supply.
• This system provides the versatility to act specifically on the potential different process scenarios.
• Fig. 3 Scheme on relations of variables that interact bi-directionally with the sulfur source and the nitrogen source (theory of the inventors).
• A indicates the nitrogen source and B the sulfur source.
• Fig.4 Discrete supply of a punctual sulfur source pulse (Na 2 S; 1 ml, 100 g/L) results in a fast increase in the conversion of CO 2 to CH 4 .
• X axis indicates time (hours) after biocatalyst inoculation;
• Y axis indicates CH 4 /CO 2 conversion in %;
• A indicates reactor inoculation.
• Fig.5 Increase of the initial concentration of the sulfur source (Na 2 S, “sulfide feeding”) results in a fast and concentration dependent increase of the conversion of CO 2 to CH 4 (short term experiment).
• X axis indicates sulfurfeeding at 4 different concentrations (1X-2X-3X and 4X);
• Y axis indicates CH 4 /CO 2 conversion in %.
• Fig.6 Doublingthe initial concentration of the sulfursource (Na 2 S) results in an increase in the conversion of CO 2 to CH 4 over time (long term experiment).
• the initial concentration of Na 2 S is supplied at time 0, i.e., just right before the strain inoculation.
• Periodic Na 2 S (each 4 hours) was applied in both experiments maintaining the relation in the Na 2 S concentration applied, i.e., doubling it in one of the experiments regarding the other.
• X axis indicates time (hours) after biocatalyst inoculation in hours after sulfur feeding at 2 different concentrations (1X-2X); Y axis indicates CH 4 /CO 2 conversion in %.
• Fig.7 Flow chart showing how a nitrogen and/or sulfur pulse (discrete supply) in the culture medium of a MEC influences parameters of the methanation process of an inoculated methanogen: Legend: 1- Pulse of nitrogen and/or sulfur source; 2- Increased conductivity at the cathode chamber; 3-Effect on pH and ORP; 4-lncreased current production; 5-lncreased hydrogen production; 6-lncreased CO 2 inflow (under operation control); 7-Effect on CH 4 /CO 2 conversion rate (increases); 8-lncreased volumetric methane production; 9-Effect on cell density (increased OD).
• the CO 2 flow is adapted according to the current in order to establish the desired CO 2 : electron ratio.
• Fig. 8 The supply of two subsequent sulfur source pulses (Na 2 S; indicated by arrow heads) results in a fast increase in the conversion of CO 2 to CH 4 , while physical-chemical parameters do not change notably.
• X axis indicates time (hours) after inoculation;
• Y left axis indicates CH 4 /CO 2 conversion in %;
• Y right axis indicates pH, ORP, and conductivity (o (mS/cm)) respectively.
• Fig. 9 The supply of two subsequent sulfur source pulses (Na 2 S; indicated by arrow heads) results in a fast increase in the utilization of H 2 to reduce CO 2 to CH 4 (cf. columns), while the current production stays stable.
• X axis indicates time (hours) after biocatalyst inoculation
• Y left axis indicates CH 4 /H 2 conversion in %
• Y right axis indicates current production in amperes (/ (A); cf. squares).
• Fig. 10 A punctual supply of ammonia (nitrogen source) results in a fast increase in the conversion of CO 2 to CH 4 .
• the increase of CO 2 conversion to CH 4 is limited for the H 2 availability at the cathode chamber.
• X axis indicates time (hours) before (negative) and after (positive) the ammonia pulse;
• Y left axis indicates CH 4 /CO 2 conversion in % (cf. columns);
• Y right axis indicates conductivity (o (mS/cm), cf. triangle) and pH (cf. squares).
• Fig. 11 The supply of ammonia (nitrogen source) results in a fast decrease of H 2 in the system while the current production stays stable.
• X axis indicates time (hours) before (negative) and after (positive) the ammonia pulse;
• Y left axis indicates H 2 at the product gas in % (cf. columns);
• Y right axis indicates current production in amperes (cf. squares);
• A indicates two outgas composition measurements where H 2 is totally depleted.
• Fig. 12 The simultaneous supply of a sulfur source (Na 2 S) and nitrogen source (NH 3 ) pulse results in a fast increase in the utilization of H 2 and CO 2 to produce methane (CH 4 ), increasing over 50% the conversion percentage of these 2 reactants to CH 4 in 24 hours.
• X axis indicates time (hours) before (negative) and after (positive) the simultaneous supply of ammonia and sulfur;
• Y left axis indicates H 2 , CO 2 and CH 4 at the product gas in %;
• Y right axis indicates CH 4 /CO 2 and CH 4 /H 2 conversion in %.
• MEC start-up The procedure of the general experimental set-up (MEC start-up) was as follows: • Add distill water at the anode and cathode circuit until the anode and cathode reservoir are fill up totally. Preferably, add 250 ml_ of distill water at the anode and cathode reservoir, each.
• Heating system on heat applied directly to the anode and cathode reservoirs (independently)
• the output gas i.e. gas that leaves the system should be harvested during a fixed time.
• the gas volume and the electrical charge per unit of time must be registered.
• CE measurements should be a key parameter to evaluate the quality of our ratio CO 2 to electrons;
• Example 1 A punctual pulse of the sulfur source results in a fast percentage increase of the CO 2 / CH 4 conversion
• Example 2 Increasing the initial sulfur source concentration (Na 2 S) led to a sulfur source concentration dependent increase of the CO 2 to CH 4 conversion in the first 24 hours (short term experimentation)
• Methanothermobacter thermautotrophicus strain UC 120910 EHlOO or ECH0100
• methanogenic microorganisms content of 10 mL culture bottle, OD 6 I 0 5, preferably final ODeiol.8
• CO 2 flow regulated comprising electron equivalents in dependence of the electron flow in an electron equivalent to electron ratio in a range of 1 : 20 to 1 : 1, more preferably at 1 : 10.
• setting initial CO 2 flow at 1 mL / min
• Pulse parameters Resulting molarity in the cell Measured ratio of the culture medium afterthe projected electrode area pulse [m 2 ] : sulfur source concentration [mol/L] in the culture medium
• CO 2 flow regulated comprising electron equivalents in dependence of the electron flow in an electron equivalent to electron ratio in a range of 1 : 20 to 1 : 1, more preferably at 1 : 10.
• Example 4 A punctual supply of the sulfur source results in a fast percentage increase of the CO 2 and H2 conversion to CH 4
• flow regulated comprising electron equivalents in dependence of the electron flow in an electron equivalent to electron ratio in a range of 1 : 20 to 1 : 1, more preferably at 1 : 10.
• CO2 flow regulated comprising electron equivalents in dependence of the electron flow in an electron equivalent to electron ratio in a range of 1 : 20 to 1 : 1, more preferably at 1 : 10.
• Example 6 A simultaneous pulse of nitrogen and sulfur sources results in a fast percentage increase of the CO 2 and H 2 conversion to CH 4
• the experimental procedure was as follows: • Inoculating the MEC with Methanothermobacter thermautotrophicus strain UC 120910 (ECH100 or ECH0100) (final concentration OD 6 IO 1.8)
• CO 2 flow regulated comprising electron equivalents in dependence of the electron flow in an electron equivalent to electron ratio in a range of 1 : 20 to 1 : 1, more preferably at 1 : 10. • No regular feeding applied

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