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WO2023183931A2 - Bioréacteur à hydrogène - Google Patents

Bioréacteur à hydrogène Download PDF

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Publication number
WO2023183931A2
WO2023183931A2 PCT/US2023/064946 US2023064946W WO2023183931A2 WO 2023183931 A2 WO2023183931 A2 WO 2023183931A2 US 2023064946 W US2023064946 W US 2023064946W WO 2023183931 A2 WO2023183931 A2 WO 2023183931A2
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Prior art keywords
chamber
bioreactor
culture
hydrogen
sugar
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WO2023183931A3 (fr
Inventor
Daniel Christopher Ducat
Matthew Shaker
Biswarup MUKHOPADHYAY
Daniel Fleck
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Virginia Tech Intellectual Properties Inc
Michigan State University MSU
Altrix Medical Inc
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Virginia Tech Intellectual Properties Inc
Michigan State University MSU
Altrix Medical Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/02—Photobioreactors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/04—Bioreactors or fermenters specially adapted for specific uses for producing gas, e.g. biogas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M43/00—Combinations of bioreactors or fermenters with other apparatus
    • C12M43/08—Bioreactors or fermenters combined with devices or plants for production of electricity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/18—Gas cleaning, e.g. scrubbers; Separation of different gases

Definitions

  • Hydrogen is a clean fuel that produces water when consumed in a fuel cell. With a production of 55 million tones every year, hydrogen is an important eco- friendly industrial feedstock due to its high energy density. Hydrogen is produced by natural gas reforming (a thermal process) and electrolysis of water. Other methods include solar-driven and biological processes.
  • biohydrogen The biological production of hydrogen (“biohydrogen”) by organisms including algae, bacteria, and archaea involves cultivating the organisms with energy sources such as solar energy or fixed carbon substrates.
  • energy sources such as solar energy or fixed carbon substrates.
  • current strategies for biohydrogen production suffer from difficulties in process engineering, low productivity of organisms, oxygen sensitivity of hydrogen producing enzymes, operational costs, and inadequate insights on strain capacity. These difficulties hinder the commercialization of biohydrogen production.
  • bioreactors for efficiently producing clean renewable biologically produced hydrogen.
  • Apparatus and methods described herein provide the integration of two or more microbes, combined into a bioreactor comprising a syntrophic closed loop system wherein one or more microbes produces a feedstock of sugars that can be provided to other microbe(s) that produce hydrogen through fermentation processes in the absence of oxygen that can reduce productivity.
  • the fermentative microbes can produce a feedstock of acetate that can be provided to other microbes that produce hydrogen through photofermentative processes.
  • the bioreactor can comprise light sources, including solar light, and atmospheric carbon dioxide to drive these metabolic processes.
  • the bioreactors described herein can further comprise a fuel cell for production of electricity from the hydrogen. Moreover, the bioreactors described herein can be scaled for different hydrogen production needs including hydrogen fueling stations, hydrogen fueled vehicles for public transit, or hydrogen fueled vehicles for air travel.
  • Figures 1A-B illustrate a two-chamber bioreactor.
  • Figure 1 A illustrates a bioreactor with a first chamber comprising a sugar-producing microbe and a second chamber comprising an anaerobic fermentative microbe connected to a hydrogen storage container.
  • Figure IB illustrates a bioreactor with a first chamber comprising a sugar-producing microbe and a second chamber comprising an anaerobic fermentative microbe connected to a hydrogen-consuming fuel cell.
  • Figures 2A-B illustrate a three-chamber bioreactor.
  • Figure 2A illustrates a bioreactor with a first chamber comprising a sugar-producing microbe, a second chamber comprising an anaerobic fermentative microbe, and a third chamber comprising a phototrophic bacterium, wherein the second bioreactor and the third bioreactor are connected to a hydrogen storage container.
  • Figure 2B illustrates a bioreactor with a first chamber comprising a sugar-producing microbe, a second chamber comprising an anaerobic fermentative microbe, and a third chamber comprising a phototrophic bacterium, wherein the second bioreactor and the third bioreactor are connected to a hydrogen-consuming fuel cell.
  • Figure 3 illustrates photoproduction of enzymes and metabolites from cyanobacteria.
  • Figure 4 is an image of a cyanobacterial strain encapsuled in barium-alginate beads.
  • Figures 5A-B illustrate characteristics of S. elongatus CscB planktonic culture compared to alginate-encapsulated culture.
  • Figure 5A illustrates .S'. elongatus CscB planktonic (solid lines) and encapsulated A elongatus CscB 5 (dashed lines) sucrose accumulation after 66 hours incubation in Ml medium with salt (140 rnM NaCl or 160 mM NaCI).
  • Figure 5B graphically illustrates sucrose accumulation per Chia (w/w) after 66 hours of incubation in Ml medium with (140 mM NaCl or 160 mM NaCl) 10 SEM).
  • Figure 6 illustrates the accumulation of exported sucrose upon activation of a sucrose export pathway.
  • Figure 7 illustrates the effect of adding salt to the nutritional media of cyanobacteria on sucrose production.
  • bioreactors comprising sugar-producing microbes that can supply sucrose as a feedstock to anaerobic fermentative microorganisms that metabolize the sucrose to carbon dioxide, hydrogen, and acetate.
  • the acetate can be moved to another bioreactor comprising a phototrophic bacterium that can produce additional hydrogen from the acetate.
  • the bioreactor can include a hydrogen storage container for storing the hydrogen and/or a hydrogen consuming fuel cell for using the hydrogen to generate electricity.
  • a bioreactor 100 can comprise a first chamber 105 comprising a first culture 110 comprising a first nutritional medium 115, wherein the first culture 110 includes at least one species of a sugar-producing microbe that releases sugar into the first nutritional medium 115 (FIG. 1 A).
  • the sugar-producing microbe can be mixed with the first nutritional medium 115 or the sugar- producing microbe can be encapsulated such that the encapsulated sugar-producing microbe occupies a bottom portion of the first chamber 105.
  • a portion of the first nutritional medium 115 above the encapsulated sugar-producing microbe may contain the secreted sugar and be substantially free of the encapsulated sugar-producing microbe.
  • the bioreactor 100 can further comprise a second chamber 180 comprising a second culture 120 comprising a second nutritional medium mixed with the second culture 120 (FIG. 1A).
  • a first separator 125 can be disposed between and separate the first chamber 105 and the second chamber 180, wherein the first separator 125 allows a portion of the first nutritional medium 115 comprising the sugar to travel into the second chamber 180.
  • the first separator 125 can be any suitable device for providing a barrier between the first chamber 105 and the second chamber 180 and also allow for transfer of a portion of the first nutritional medium 115 comprising the sugar to travel into the second culture 120.
  • the first separator 125 can be a conduit with a valve or a membrane.
  • the first separator 125 can provide a controlled rate of transfer of the first nutritional medium 115 which can regulate the rate at which the first nutritional medium 115 leaves the first chamber 105. This can regulate and make predictable the timing and amount of the first nutritional medium 115 to be replaced in the first chamber 105.
  • the first nutritional medium 1 15 can be recycled from the second chamber 180 back into the first chamber 105, such as when both nutritional media are compatible for maintenance of both the sugar-producing microbe and the fermentative bacteria.
  • the second culture 120 can comprise at least one species of an anaerobic fermentative microbe that converts the sugar to a first product mixture comprising hydrogen and acetate.
  • the anaerobic fermentative microbe can be an obligate anaerobe or a facultative anaerobe.
  • the hydrogen produced by the anaerobic fermentative microbe collects in a headspace 130 of the second chamber 180.
  • the hydrogen can be transferred from the headspace 130 into a hydrogen storage container 175 (FIG 1A).
  • the hydrogen can be transferred from the headspace 130 into a fuel cell 165 (discussed below) (FIG. IB).
  • the second culture 120 can comprise a co-culture of the anaerobic fermentative microbe and a population of Geobacter sulfurreducens ( G. sulfurreducens).
  • G. sulfurreducens can convert the acetate in the first product mixture produced by the anaerobic fermentative microbe into hydrogen.
  • the hydrogen produced by the G. sulfurreducens can also collect in the headspace 130 of the second chamber 180 and be transferred into a hydrogen storage container 175.
  • the phototrophic bacteria can be cultured separately from the anaerobic fermentative microbe.
  • the bioreactor 100 as described above further comprises a third chamber 140 that is separated from the second chamber 180 by a second separator 155 (FIGS 2A-B).
  • the second separator 155 can be disposed between and separate the second chamber 180 from the third chamber 145.
  • the second separator 155 can be any suitable device for providing a barrier between the second chamber 155 and the third chamber 140 and also allow for at least a portion of the second nutritional medium mixed with the second culture 120 comprising the acetate to travel into the third culture 145.
  • the second separator 155 can be a conduit with a valve or a membrane.
  • the third culture 145 comprises at least one species of the phototrophic bacteria that converts the acetate in the first product mixture produced by the anaerobic fermentative microbe to a second product mixture comprising hydrogen.
  • the second separator 155 can keep the second nutritional medium in residence in the second chamber 180 until the fermentative bacteria consumes most or all of the sucrose, and transferring the second nutritional medium comprising acetate to the third culture 145.
  • the hydrogen produced by the phototrophic bacteria can collect in the headspace 150 of the third chamber 140.
  • the hydrogen can be transferred from the headspace 130 and a headspace 150 into the hydrogen storage container 175 (FIG 2A).
  • the hydrogen can be transferred from the headspace 130 and a headspace 150 into the fuel cell 165 (discussed below) (FIG. 2B).
  • the bioreactors described herein can comprise a variety of accessories that perform various functions including aeration, oxygen reduction, agitation, pressure regulation, and light.
  • the sugar-producing microbe may be an oxygenic phototrophic microbe, such as cyanobacteria, and may need light for photosynthesis to sustain the viability of the culture.
  • the first chamber 100 may include light source 155 (FIGS. 1 and 2).
  • the third chamber 140 can also include a light source 155 to provide light to the phototrophic bacteria in the third culture 145.
  • the light source 155 can include any suitable light source for providing sufficient light penetration into the first culture 110, the third culture 145, or both in a closed bioreactor.
  • the light source includes photosynthetically active radiation (PAR).
  • PAR can be provided in the form of unseparated wavelengths of light (such as sunlight), or selected wavelengths of light.
  • Light can be natural, such as by at least a portion of the bioreactor comprising a translucent wall to allow solar radiation to penetrate to an interior of the bioreactor.
  • the light can supplied by an artificial light source, such as light emitting diodes (LEDs).
  • the bioreactors can also comprise a gas delivery system 160 for delivering a gas to the first chamber 105, the second chamber 180, the third chamber 140, the first separator 125, or a combination thereof.
  • the gas delivery system 160 can provide functions including sparging of oxygen from the second chamber 180 and the first separator 125 and efficient mixing of the nutritional medium in the first chamber 105, the second chamber 180, the third chamber 140 (collectively the “bioreactor chambers”) or a combination thereof (FIGS. 1A-B and 2A-B).
  • the gas delivery system 160 can also be placed in the first separator 125 to provide degassing by bubbling an anaerobic gas such as carbon dioxide or nitrogen through the first medium 115 before it travels to the second chamber 180.
  • This function of degassing can also be called “scrubbing.”
  • Degassing of the first medium 115 prior to entry into the second chamber 180 can allow the anaerobic fermentative bacteria to more efficiently convert the sucrose into acetate and hydrogen.
  • the facultative anaerobic fermentative bacteria Escherichia coli E. coli
  • E. coli can be used to convert sucrose to acetate and hydrogen.
  • E. coli will convert the oxygen to water until the oxygen is consumed. Once the oxygen is consumed, E. coli will start converting the sugar to acetate and hydrogen. If oxygen is present in the first medium 115, there can be a lag time until E. coli completes oxygen consumption and starts anaerobically producing acetate and hydrogen.
  • the gas delivery system 160 can include any suitable mechanism for delivering a gas into one or more bioreactor chamber(s).
  • the gas delivery system 160 can be a tube with aeration holes for the gas to escape from the tube and enter the bioreactor chamber(s).
  • the gas delivery system 160 can be placed strategically along a bottom surface of the bioreactor chamber(s) to bubble gas through the first culture 110, the second culture 120, the third culture 145, or a combination thereof. Bubbling the gas through these cultures can provide agitation to keep cells and solids in suspension and to provide improved mixing of the nutritional medium. Mixing rate of the nutritional medium can be controlled by the gas delivery system 160 alone, or in combination with other agitation mechanisms, such as stir bars (not shown).
  • the gas provided through the gas delivery system 160 can be selected based on the individual needs of each of the bioreactor chambers.
  • the gas provided through the gas delivery system 160 in the first chamber 105 can be carbon dioxide (CO2) needed by the sugar-producing microbe cyanobacteria for carbon fixation reactions.
  • the gas provided through the gas delivery system 160 in the second chamber 180 can be carbon dioxide ( CO2(g)) or an inert gas such as nitrogen (N2(g)). Bubbling CO2(g) or N2(g) into the second culture 120 can maintain an oxygen depleted environment to support the viability of the anaerobic fermentative bacteria.
  • the oxygen from the second chamber 180 or the first separator 135 can be sparged out through the sparging ports 135 (FIGS. 1A-B and 2A-B).
  • the bioreactors can also comprise one or more pumps for injection of microbes, water, nutrients, pH stabilizers, trace elements, and/or other components into the bioreactor chambers (pump not shown).
  • the pump(s) can propel the first nutritional medium 115 through the first separator 125 and/or the second nutritional medium mixed with the second culture 145 through the second separator 155.
  • the bioreactors described herein can be provided with a fuel cell 165 (FIGS 1 and 2).
  • the fuel cell 165 can be any suitable hydrogen-consuming fuel cell connected to a bioreactor 100 or a bioreactor 200.
  • the fuel cell 165 can utilize and store the hydrogen produced in the second chamber 180, the third chamber 140, or both.
  • the fuel cell 165 can include an anode, a cathode, and an electrically-insulating ionconducting electrolyte (e.g., a membrane, such as a proton exchange membrane, or PEM) separating the anode and cathode, wherein at least one of the anode or cathode undergoes a chemical reaction that consumes hydrogen and generates an electrical potential across the electrodes.
  • the cathode of the fuel cell consumes hydrogen gas and generates electrons and hydrogen ions.
  • the hydrogen ions can travel across the electrolyte to the cathode, while the electrons can travel to the cathode via an electrical circuit connecting the anode to the cathode.
  • the hydrogen ions can react with oxygen gas and the electrons produced by the anode to form water.
  • the anode and/or the cathode of the fuel cell can be electrically coupled to an electricity storage device 170 configured to store the electricity.
  • the electricity storage device can comprise a battery or a capacitor.
  • the electricity storage device 170 whether a battery or capacitor, can provide power to the bioreactor, such as through an electrical conduit 185 (FIGS. IB and 2B) to run any processes in the bioreactor that can use electricity, such as the pump, the light source, a stirring mechanism.
  • Electrical conduit 185 can be connected to the bioreactor 100 or 200 in any suitable manner, such as to one or more chambers, for providing power to the bioreactors and the connections illustrated in FIGS. IB and 2B are nonlimiting example connections.
  • the bioreactors can be configured to (1) store hydrogen and use hydrogen to create electricity to power itself; (2) draw power from an external power source, such as an electrical outlet, and store hydrogen; (3) draw power from an external power source, use hydrogen to create electricity to power itself, and store hydrogen; or (4) store hydrogen and use hydrogen to create electricity to power itself, wherein the electricity is provided for external uses.
  • the sugar-producing microbes can be one or more strains of wild-type cyanobacteria, genetically modified cyanobacteria, or a combination thereof.
  • Cyanobacteria also known as bluegreen algae, blue-green bacteria, or Cyanophyta, is a phylum of bacteria that obtain their energy through photosynthesis. Cyanobacteria can produce metabolites, such as carbohydrates, proteins, lipids and nucleic acids, from CO2, water, inorganic salts and light. Any cyanobacteria may be used according to the present disclosure.
  • Cyanobacteria include both unicellular and colonial species. Colonies may form filaments, sheets or even hollow balls. Some filamentous colonies have the ability to differentiate into several different cell types, such as vegetative cells, the normal, photosynthetic cells that are formed under favorable growing conditions; akinetes, the climate-resistant spores that may form when environmental conditions become harsh; and thick-walled heterocysts, which contain the enzyme nitrogenase, vital for nitrogen fixation.
  • Heterocysts may also form under the appropriate environmental conditions (e.g., anoxic) whenever nitrogen is necessary. Heterocyst-forming species are specialized for nitrogen fixation and are able to fix nitrogen gas, which cannot be used by plants, into ammonia (NH3), nitrites (NO2-), or nitrates (NO3-), which can be absorbed by plants and converted to protein and nucleic acids.
  • anoxic e.g., anoxic
  • cyanobacteria also form motile filaments, called hormogonia, which travel away from the main biomass to bud and form new colonies elsewhere.
  • the cells in a hormogonium are often thinner than in the vegetative state, and the cells on either end of the motile chain may be tapered.
  • a hormogonium In order to break away from the parent colony, a hormogonium often must tear apart a weaker cell in a filament, called a necridium.
  • Each individual cyanobacterial cell typically has a thick, gelatinous cell wall.
  • Cyanobacteria differ from other gram-negative bacteria in that the quorum sensing molecules autoinducer-2 and acyl-homoserine lactones are absent. They lack flagella, but hormogonia and some unicellular species may move about by gliding along surfaces. In water columns, some cyanobacteria float by forming gas vesicles, like in archaea.
  • Cyanobacteria have an elaborate and highly organized system of internal membranes that function in photosynthesis. Photosynthesis in cyanobacteria generally uses water as an electron donor and produces oxygen as a by-product, though some cyanobacteria may also use hydrogen sulfide, similar to other photosynthetic bacteria. Carbon dioxide is reduced to form carbohydrates via the Calvin cycle (FIG. 3). In most forms, the photosynthetic machinery is embedded into folds of the cell membrane, called thylakoids.
  • cyanobacteria Due to their ability to fix nitrogen in aerobic conditions, cyanobacteria are often found as symbionts with a number of other groups of microorganisms such as fungi (e.g., lichens), corals, pteridophytes (e.g., Azolla), and angiosperms (e.g., Gunnera), among others.
  • fungi e.g., lichens
  • corals e.g., corals
  • pteridophytes e.g., Azolla
  • angiosperms e.g., Gunnera
  • Cyanobacteria are the only group of microorganisms that are able to reduce nitrogen and carbon in aerobic conditions.
  • the water-oxidizing photosynthesis is accomplished by coupling the activity of photosystem (PS) II and I (Z-scheme).
  • PS photosystem
  • cyanobacteria are also able to use only PS I (i.e., cyclic photophosphorylation) with electron donors other than water (e.g., hydrogen sulfide, thiosulphate, or molecular hydrogen), similar to purple photosynthetic bacteria.
  • cyanobacteria share an archaeal property; the ability to reduce elemental sulfur by anaerobic respiration in the dark.
  • the cyanobacterial photosynthetic electron transport system shares the same compartment as the components of respiratory electron transport.
  • the plasma membrane contains only components of the respiratory chain, while the thylakoid membrane hosts both respiratory and photosynthetic electron transport.
  • Cyanobacteria of the present disclosure may be from any genera or species of cyanobacteria that is genetically manipulable, i.e., permissible to the introduction and expression of exogenous genetic material.
  • Examples of cyanobacteria that can be employed include, but are not limited to, the genus Synechocystis, Synechococcus, Thermosynechococcus, Nostoc, Prochlorococcu, Microcystis, Anabaena, Spirulina, and Gloeobacter.
  • the cyanobacteria are Synechococcus elongatus.
  • the cyanobacteria can include Synechococcus elongatus PCC 7942 which can be obtained from the American Type Culture Collection as ATCC 33912 (see webpages for atcc.org/products/33912).
  • S. elongatus UTEX # 2973 (see webpage utex.org/products/utex-2973) can be used.
  • the Synechococcus UTEX 2973 strain (Yu, et al., 2015) is closely related to .S’. elongatus PCC 7942, but has faster growth rates and higher light tolerance.
  • FIG. 1 A schematic of a sucrose production pathway from a cyanobacterial strain is shown in FIG.
  • Strains of S. elongatus can be engineered to over-express a sucrose transporter gene chromosomally-encoded sucrose utilizing gene B (CscB) of Escherichia coll.
  • the CscB gene encodes the sucrose permease enzyme that actively transports sugars across the cell membrane.
  • the CscB permease is one member of the large major facilitator superfamily (MFS) of transporters that share conserved amino acid sequence motifs, exhibit similar predicted two-dimensional structures within the membrane, and are thus predicted to have a common mechanism for solute transport across the membrane.
  • MFS major facilitator superfamily
  • CscB has been grouped into the smaller family, called the oligosaccharide :H+ symporters family (OHS, family 5), consisting of several homologous secondary active sugar transporters.
  • .S' elongatus engineered to over-express CscB utilizes light and CO2 inputs to export sucrose that contribute to photosynthetic carbon fixation reactions.
  • PCC 6803 allows the sucrose production pathway to be induced in the absence of the salt stress conditions normally required for sucrose accumulation while CscB gene overexpression permits the export of cytosolic sucrose.
  • the cyanobacteria can be engineered to express sucrose phosphate synthase
  • SPS SPS catalyzes the rate limiting step of sucrose biosynthesis from UDP-glucose and fructose- 6-phosphate.
  • SPS catalyzes the transfer of a glycosyl group from an activated donor sugar, such as uridine diphosphate glucose (UDP-Glc), to a saccharide acceptor d-fructose 6- phosphate (F6P), resulting in the formation of UDP and d-sucrose-6'-phosphate (S6P).
  • an activated donor sugar such as uridine diphosphate glucose (UDP-Glc)
  • F6P saccharide acceptor d-fructose 6- phosphate
  • sucrose phosphate synthase SPS
  • sucrose permease CscB
  • fermentative microbe can be a wild type or modified strain of E. coli. Because E. coli is a facultative anaerobe, it can function under both aerobic and anaerobic conditions. Thus, E. coli can consume any remaining oxygen, then switch to anaerobic mode and produce hydrogen.
  • An alternative implementation of this invention can include use a method of consuming O2 without sparging with nitrogen or carbon dioxide, using E. coli only.
  • E. coli can produce hydrogen and acetate from the sucrose present in the first nutritional medium. Hydrogen can be collected and stored or collected in a fuel cell. The acetate-containing solution can be transferred to a third chamber containing a phototrophic bacterium.
  • the E. coli strain can include heterologous genes such as the scrK, scrY, scrA, scrB and scrR genes (Penfold & Macaskie, Production of hydrogen from sucrose by Escherichia coli strains carrying the pUR400 plasmid, which encodes invertase activity.
  • the scrA and scrY genes can be used in conjunction with native genes, to provide an engineered E. coli that can actively transport sucrose as sucrose-6-phosphate into the cell.
  • sucrose-6-phosphate can be hydrolyzed to glucose-6-phosphate and fructose using a scrB-encoded hydrolase, and the fructose can be converted to fructose-6-phosphate using a fructokinase (scrK).
  • the system can be controlled by a negative regulator, a product of the scrR gene.
  • Such an active transport system using scrA and scrY provides a modified E. coli that can utilize most of the sucrose made available to it.
  • An alternate system can also be used that employs a passive, and therefore non-energy dependent, transport system.
  • a passive, and therefore non-energy dependent, transport system can involve an E. coli W strain that include cscA, cscB, cscK and cscR genes inserted into the genome (Archer, Kim, & Jeong, 2011) (Sabri, Nielsen, & Vickers, 2013).
  • Other strains with similar capabilities can also be used (Carruthers, Saleski, Scholz, & Lin, 2020).
  • E. coli W lines and similar strains can be supported in co-culture with S.
  • E. coli W strain is available from the ATCC No. as 9637 (see webpages atcc.org/products/9637).
  • E. coli MC4100 available from ATCC No. 35695, see webpage atcc.org/products/35695 can also be used as a parent strain for genetic modification.
  • sucrose can enter the cell through a permease encoded by the cscB gene and be hydrolyzed to glucose and fructose by an invertase (cscA gene), which can then be converted into glucose-6-phosphate by the action of a native enzyme and to fructose-6- phosphate with the assistance of the cloned cscK gene, respectively (Robeis, et al., 2002) (Archer, Kim, & Jeong, 2011) (Sabri, Nielsen, & Vickers, 2013).
  • the system can be controlled by a negative regulator, such as a product of the cscR gene.
  • glucose-6-phosphate and fructose-6-phosphate can be generated from sucrose and fermented to hydrogen and acetate.
  • the strains described herein can efficiently produce hydrogen, especially when one or more genetic modifications are included.
  • one modification can include deletion of hycA that encodes a repressor of the formate hydrogen lyase system, which is a key route for hydrogen production in E. coli (Sauter, Bohm, & Bock, 1992) (Penfold, Forster, & Macaskie, Increased hydrogen production by Escherichia coli strain HD701 in comparison with the wild-type parent strain MC4100, 2003).
  • Another modification can include overexpressing one of the subunits of a hydrogen evolving [Fe]- hydrogenase (HydA) from Enterobacter cloacae 11T-BT 08 (Mishra, Khurana, Kumar, Ghosh, & Das, 2004) (Chittibabu, Nath, & Das, 2006).
  • HydA hydrogen evolving [Fe]- hydrogenase
  • E. coli strain ATCC 9637 CscB protein is provided above as the amino acid sequence of SEQ ID NO: 1.
  • SEQ ID NO: 18 An example of a Salmonella typhimurium ScrA protein is provided below as SEQ ID NO: 18.
  • genes and proteins employed can have at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence described herein.
  • enzymes can have conservative changes such as one or more deletions, insertions, replacements, or substitutions that have no significant effect on the activities of the sucrose transporter.
  • enzymes can have conservative changes such as one or more deletions, insertions, replacements, or substitutions that have no significant effect on the activities of the enzymes. Examples of conservative substitutions are provided in the table below.
  • the phototrophic bacteria can include any suitable phototrophic bacterium that can contain convert acetate to hydrogen.
  • the phototrophic bacterium can be Rhodobacter sphaeroides, such as strain O.U.OOl.
  • Rhodobacter sphaeroides is a phototrophic purple non-sulfur bacterium converting the acetate to hydrogen with assistance from sunlight (Nath, Kumar, & Das, 2005) (Chittibabu, Nath, & Das, 2006).
  • the Rhodobacter sphaeroides strain O.U.OOl is available as ATCC No. as 49419 (see webpage atcc.org/products/49419).
  • Rhodobacter sphaeroides can be grown in the third chamber in the second nutritional medium transferred from second chamber.
  • the third chamber can comprise the light source and/or a tubular reactor made of glass and illuminated from outside and inside and supplemented with malic acid and glutamic acid at the concentrations of 1 and 1.5 gram/liter concentrations and adjusted to pH 6.8 with sodium hydroxide (Nath, Kumar, & Das, 2005).
  • the third chamber can include Geobacter sulfurreducens, an exoelectrogenic bacterium, forming a Microbial Electrolysis Cell (MEC). With an input of power, this bacterium will yield hydrogen.
  • MEC Microbial Electrolysis Cell
  • the MEC can utilize the second nutritional medium from the second chamber, and an anode and cathode made of Nonporous flat-surface graphite, as described in Geelhoed, et al, 2011).
  • the hydrogen generated in the third chamber can collect in the headspace of the third chamber or be transferred to a fuel cell and/or a hydrogen storage container.
  • expression vectors or transgenes that encode one or more of the enzyme(s) described herein.
  • expression systems can be present in the cyanobacteria or the fermentative bacteria.
  • Cells containing such expression systems are further described herein.
  • the cells containing such expression systems can be used to manufacture the enzymes (e.g., for in vitro use) or products produced by the enzymes. Methods of using the enzymes or cells containing expression cassettes encoding such enzymes to make products, degrade products, and combinations thereof are also described herein.
  • Nucleic acids encoding the enzymes can have sequence modifications.
  • nucleic acid sequences described herein can be modified to express enzymes that have modifications.
  • Most amino acids can be encoded by more than one codon.
  • the codons are referred to as degenerate codons.
  • a listing of degenerate codons is provided in the table below. [0107] Table 2: Degenerate Amino Acid Codons
  • nucleic acid segment can be designed to optimize the efficiency of expression of an enzyme by using codons that are preferred by an organism of interest.
  • nucleotide coding regions of the enzymes described herein can be codon optimized for expression in various cyanobacterial or heterotroph species.
  • An optimized nucleic acid can have less than 98%, less than 97%, less than 95%, or less than 94%, or less than 93%, or less than 92%, or less than 91%, or less than 90%, or less than 89%, or less than 88%, or less than 85%, or less than 83%, or less than 80%, or less than 75% nucleic acid sequence identity to a corresponding nonoptimized (e.g., a non-optimized parental or wild type enzyme nucleic acid) sequence.
  • a corresponding nonoptimized e.g., a non-optimized parental or wild type enzyme nucleic acid
  • the enzymes described herein can be expressed from an expression cassette and/or an expression vector.
  • Such an expression cassette can include a nucleic acid segment that encodes an enzyme operably linked to a promoter to drive expression of the enzyme.
  • Convenient vectors, or expression systems can be used to express such enzymes.
  • the nucleic acid segment encoding an enzyme is operably linked to a promoter and/or a transcription termination sequence.
  • the promoter and/or the termination sequence can be heterologous to the nucleic acid segment that encodes an enzyme.
  • Expression cassettes can have a promoter operably linked to a heterologous open reading frame encoding an enzyme. The invention therefore provides expression cassettes or vectors useful for expressing one or more enzyme(s).
  • Constructs e.g., expression cassettes
  • vectors comprising the isolated nucleic acid molecule, e.g., with optimized nucleic acid sequence, as well as kits comprising the isolated nucleic acid molecule, construct or vector are also provided.
  • Nucleic acids encoding one or more enzyme(s) can have one or more nucleotide deletions, insertions, replacements, or substitutions.
  • the nucleic acids encoding one or more enzyme(s) can, for example, have less than 95%, or less than 30 94.8%, or less than 94.5%, or less than 94%, or less than 93.8%, or less than 94.50% nucleic acid sequence identity to a corresponding parental or wild-type sequence.
  • the nucleic acids encoding one or more enzyme(s) can have, for example, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at 90% sequence identity to a corresponding parental or wild-type sequence.
  • parental or wild type nucleic acid sequences for unmodified enzyme(s) with amino acid sequences SEQ ID NOs: 1, 3, 5-18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, and nucleic acid sequences SEQ ID NOs: 2, 4, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.
  • nuclei acid or amino acid sequences can, for example, encode or have sequences with less than 99.5%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94.8%, less than 94.5%, less than 94%, less than 93.8%, less than 93.5%, less than 93%, less than 92%, less than 91%, or less than 90% sequence identity to a corresponding parental or wild-type sequence.
  • promoters can be included in the transgenes, expression cassettes and/or expression vectors. Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells.
  • a promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences can also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression.
  • Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA.
  • Promoters can be strong or weak, or inducible.
  • a strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression.
  • An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.
  • a bacterial promoter such as the Pt ac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation.
  • a strong promoter for heterologous DNAs can be advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
  • the promoter within such expression cassettes / vectors can be functional during plant development or growth.
  • the promoter can be a promoter functional in a cyanobacteria such as a bacterial promoter, yeast promoter, viral promoter, or a mammalian promoter.
  • the promoter can be a heterologous promoter.
  • heterologous when used in reference to a gene or nucleic acid refers to a gene, nucleic acid, or enzyme that has been manipulated in some way.
  • a heterologous promoter is a promoter that contains sequences that are not naturally linked to an associated coding region.
  • a heterologous promoter is not the same one as the natural promoter that drives expression of an operably linked coding region.
  • promoters examples include, but are not limited to, the T7 promoter
  • the CaMV 35S promoter (Odell et al., Nature. 313:810- 812 (1985)), the CaMV 19S promoter (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nos promoter (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), Adhl promoter (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. USA.
  • .-tubulin promoter ubiquitin promoter
  • actin promoter Wang et al., Mol. Cell. Biol. 12:3399 (1992)
  • cab Sullivan et al., Mol. Gen. Genet. 215:431 (1989)
  • PEPCase promoter Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)
  • the CCR promoter cinnamoyl CoA:NADP oxidoreductase, EC 1.2.1.4434 isolated from Lollium perenne, (or a perennial ryegrass) and/or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)).
  • promoters can be used with or without associated enhancer elements. Examples include a baculovirus derived promoter, the plO promoter. Although some heterotrophs are bacteria or yeast, cyanobacteria and heterotrophs may also employ plant promoters.
  • Expression cassettes that include a promoter operably linked to a nucleic acid segment encoding a polypeptide or peptide can include other elements such as a segment encoding 3' nontranslated regulatory sequences, and restriction sites for insertion, removal and manipulation of segments of the expression cassettes.
  • the 3’ nontranslated regulatory DNA sequences can act as a signal to terminate transcription and in some cases can allow for the polyadenylation of the resultant mRNA.
  • the 3' nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains prokaryotic or eukaryotic transcriptional and translational termination sequences. Various 3' can be employed.
  • 3' nontranslated regulatory sequences can be obtained as described in An (Methods in Enzymology. 153:292 (1987)). Many such 3' nontranslated regulatory sequences are also present in plasmids available from commercial sources such as Clontech, Palo Alto, California.
  • the cyanobacteria can be encapsulated within a hydrogel matrix.
  • a hydrogel matrix that includes the cyanobacteria and one or more types of hydrogels.
  • encapsulation of cyanobacteria greatly enhances stability and productivity of cyanobacteria cultures while also enabling selective recovery and transfer of nutritional media comprising exported sugar.
  • Encapsulation of cyanobacteria minimizes cell escape while still maintaining the viability and photosynthetic activity of trapped cells.
  • encapsulation provides other advantages, including: 1) physical separation of cyanobacteria!
  • This method also allows for customizable alginate physical support structures (e.g., sheets, tubes) designed for maximal surface area across the light delivery mechanism.
  • additional genetic modifications can also improve sucrose production, including elimination of competing metabolic pathways (Ducat, Avelar-Rivas, Way, & Silvera, 2012) (Qiao, et al., 2018) and upregulation of rate-limiting steps for sucrose synthesis (e.g., sucrose phosphate synthase) (Abramson, Kachel, Kramer, & Ducat, 2016) (Santos-Merino, et al., 2021).
  • hydrogel materials examples include alginate, latex, silica, and combinations thereof. As illustrated herein, alginate forms an excellent encapsulation material that provides the benefits described in the foregoing paragraph. Latex is also a good encapsulation material. Latex is an easy-to-use, and inexpensive resin that has been used to embed bacteria. For example, one type of latex that can be used is Rhoplex SF012. Similarly, silica materials have superior properties for industrial use (e.g., thermostability). One example of a silica material that can be used is polyol silanes93.
  • the encapsulation procedure can include generation of beads or sheets of a diameter that allow diffusion of gases, nutrients, and other molecules to foster the stability and productivity of the cyanobacteria.
  • the size and highly cross-linked network of encapsulation polymers may present a physical constraint on the diffusion of small molecules and can present a barrier to large molecules, such as proteins.
  • the most salient molecules to consider are the permeability of CO2 into the matrix, and escape of O2 and sucrose.
  • the cell density, percent alginate slurry, crosslinking efficiency, photosynthetic rates, and mixing rates are all variables that can influence the diffusion of substrates and products within the bead, so calculation of an optimal encapsulation diameter is non-trivial.
  • Optimal encapsulation diameters can vary and can include encapsulation 20 diameters of about 0.05 mm to about 4 mm, or of about 0.075 mm to about 3.5 mm, or of about 0.1 mm to about 3 mm, or of about 0.15 mm to about 3 mm, or of about 0.15 mm to about 2.75 mm, or of about 0.25 mm to about 2.75 mm, or of about 0.2 mm to about 2 mm.
  • titers of encapsulated cyanobacteria can be determined and compared to a control.
  • the control can be nonencapsulated cyanobacteria or cyanobacteria encapsulated to a specified or desired diameter.
  • the specific productivity of secreted sucrose content of encapsulated cyanobacteria beads can be measured over time (see FIG. 5A) as a proxy for photosynthetic activity and cell viability. More precise measurements of photosynthetic parameters can be evaluated through monitoring chlorophyll a fluorescence dynamics embedded cells. These nondestructive methods can analyze the quantum efficiency of Photosystem II (PSII), electron transfer rates, and Photosystem I (PSI) activity.
  • One method for encapsulation of cyanobacteria is to drip a mixture of at least one hydrogel with cyanobacteria cells into a curing solution.
  • a cyanobacteria cell/alginate suspension can be dripped into a barium chloride (BaCl) curing solution.
  • BaCl barium chloride
  • the encapsulation methods can include use of a Nisco Encapsulation Unit VAR VI, which uses electrostatically assisted spraying for rapid generation of large, encapsulated bead volumes of user-specified sizes, for example, over the range of about 15 0.2 mm to 2 mm.
  • VAR VI Nisco Encapsulation Unit
  • the present technology provides a method of using a bioreactor.
  • the method can include maintaining a first culture comprising at least one sugar-producing microbe in a first nutritional medium that releases sugar into the first nutritional medium.
  • the sugar-producing microbe can be a wild-type or genetically modified cyanobacteria that can be maintained as a viable culture within the first nutritional medium, a light source, and carbon dioxide (CO2(g)).
  • the cyanobacteria can be encapsulated to provide separation of the cyanobacteria from the first nutritional medium to allow a portion of the first nutritional medium to be transferred into a second culture that is separated from the first culture.
  • the second culture can comprise a second culture medium and at least one anaerobic fermentative microbe that converts the sugar in the first nutritional medium into acetate and hydrogen gas.
  • the second culture can be maintained in anaerobic conditions, such as by sparging the second culture with CO2(g).
  • the method can include using a phototrophic bacterium to convert the acetate from the second culture medium into hydrogen.
  • a portion of the second culture medium comprising the acetate can be transferred into a third culture comprising the phototrophic bacterium.
  • the method can include providing the hydrogen formed by the anaerobic fermentative microbe and the phototrophic bacterium (if present), to a suitable hydrogen fuel cell (e.g., a hydrogen-consuming fuel cell), such as to a hydrogen-consuming electrode of the hydrogen fuel cell (e.g., an anode electrode or a cathode electrode, wherein the anode electrode and a cathode electrode are electronically connected to each other).
  • a suitable hydrogen fuel cell e.g., a hydrogen-consuming fuel cell
  • a hydrogen-consuming electrode of the hydrogen fuel cell e.g., an anode electrode or a cathode electrode, wherein the anode electrode and a cathode electrode are electronically connected to each other.
  • the hydrogen can contact the anode electrode and electrons in the second culture can be transferred to the anode electrode to produce electricity.
  • the hydrogen gas can be provided to the electrode in any suitable way, such as via a conduit, pipe, or other fluid connection between the hydrogen and the electrode.
  • Cyanobacteria can be cultured in a variety of simple media in the presence of light. For example, cyanobacteria can be grown in sea water. In some cases, cyanobacteria can be cultured in a medium that contains a nitrate salt, a phosphate salt, a magnesium salt, a calcium salt, a carbonate salt, a chelator, citric acid, ferric ammonium, and combinations thereof. Cyanobacteria are often cultured at about neutral pH or in slightly alkaline culture media.
  • a cyanobacteria medium can contain BG-11 medium (Sigma- Aldrich, C3061) buffered with 1 g L-l HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)) (pH 8.3 with NaOH).
  • BG-11 medium Sigma- Aldrich, C3061
  • HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
  • GRO-LUX Infors-Multitron photobioreactor incubator with ⁇ 125 pmol photons m-2 s-1 fluorescent (GRO-LUX) lighting supplemented with 2% CO2 was used at 32 °C with orbital shaking at 150 rpm. Cultures were typically maintained with daily back dilution to OD750 ⁇ 0.3.
  • 3% sodium alginate (Sigma-Aldrich) was created by slow mixing over several hours, followed by vacuum degassing and autoclave sterilization.
  • this solution was then added dropwise to a >20-fold larger volume of 20 mM BaCh using a vertically-oriented syringe pump (KD Scientific, Holliston, MA), 5 mL syringes (BD Biosciences), and 30G needles.
  • KD Scientific Holliston, MA
  • 5 mL syringes BD Biosciences
  • 30G needles The drops traveled ⁇ 35 cm from needle to the slowly stirred BaCh solution and cured at least 15 min in BaCh before being twice rinsed with deionized water and further incubated in deionized water for 5 min.
  • beads were transferred into >2 L of BG11 medium with ImM IPTG and indicated NaCl concentrations; the medium was exchanged at least 2x after >30 min incubations and the removal of excess residual barium was evaluated by precipitate formation after adding 1 M Na2SO4 to withdrawn supernatant. Equilibration was considered successful when residual barium no longer precipitated. Finally, beads were transferred into a baffled 4 L Fernbach flask for 12 h, with the intended final medium and salt under constant light. Beads intended for use in M2 medium experiments were transferred into BG11 containing no nitrate (BG11-N) at a volume appropriate to diluting the 17.6 mM nitrate contained within the BG11 infused beads to the desired 2 mM. The completed beads were then apportioned into experimental flasks with fresh medium to begin experiments.
  • BG11-N no nitrate
  • Sucrose was measured using a Sucrose/D-Glucose Assay Kit (Megazyme, Bray, Ireland).
  • Example 2 Sucrose production of alginate-encapsulated 5. elongatus CscB
  • cyanobacteria are encapsulated, they could facilitate selective harvesting of nutritional media comprising exported sucrose from the first chamber of the bioreactor for transfer into the second chamber comprising the anaerobic fermentative microbe.
  • sodium alginate- suspended 5. elongatus CscB were dripped into a barium chloride gelling solution, generating spherical, barium-alginate hydrogel beads of about 2.6 mm in diameter that contained entrapped cyanobacterial cells (FIG. 4).
  • sucrose productivity of encapsulated S. elongatus CscB was examined relative to planktonic (i.e., “free floating”) suspensions by culturing equal numbers of free and encapsulated cyanobacteria into separate cultures. As shown in FIGS. 5-7, the specific productivity of sucrose from encapsulated S. elongatus CscB was sustained, and likely enhanced, relative to planktonic cells. Similar amounts of sucrose were measured in the supernatant of both encapsulated and planktonic cultures over 66 hours, with encapsulated cells exhibiting somewhat higher sucrose productivity (FIG. 5A).
  • IPTG isopropyl P-D-l- thiogalactopyranoside
  • Example 3 Methods of maintaining an E. coli culture
  • Axenic cultures containing E. coli were grown in a BG-11 CO medium, a modified BG-11 including 20 g L-l sucrose, and 4 mM NH4CI. Cultures were grown for up to 72 h at 32 0 C with shaking at 200 rpm. E. coli cultures were grown to OD600 ⁇ 0.1 prior to induction with IPTG.
  • Example 4 Media for use with both cyanobacteria and anaerobic fermentative bacteria
  • Both cyanobacteria and anaerobic fermentative bacteria such as 5. elongatus and E. coli, respectively, can be maintained in a BG-11 co medium, a modified BG-11 including 20 g L-l sucrose, and 4 mM NH4CI.
  • the pH of the media for the cyanbacteria can be approximately pH 8 - 8.3 and the pH for the media for the anaerobic fermentative bacteria can be adjusted to approximately 7.2.
  • the media for the anaerobic fermentative bacteria can be degassed in the first separator and the second chamber.
  • a bioreactor comprising : a first chamber comprising a first culture comprising a first nutritional medium and at least one species of a sugar-producing microbe that releases sugar into the first nutritional medium; a second chamber comprising a second culture comprising a second nutritional medium and at least one species of an anaerobic fermentative microbe that converts the sugar to a first product mixture comprising hydrogen gas and acetate; and a first separator disposed between and separating the first chamber and the second chamber, wherein the first separator allows a portion of the first nutritional medium comprising the sugar to transfer into the second culture; wherein the hydrogen gas produced by the anaerobic fermentative microbe collects in a headspace of the second chamber.
  • a third culture comprising at least one species of a phototrophic bacteria that converts the acetate from the first product mixture to a second product mixture comprising hydrogen gas, wherein the third culture is in a third chamber that is separated from the second chamber by a second separator, wherein the hydrogen gas produced by the phototrophic bacterium collects in a headspace of the third chamber.
  • the sugar-producing microbe is a cyanobacteria.
  • cyanobacteria are Synechocystis, Synechococcus, Tltermosynechococcus, Nostoc, Prochlorococcu, Microcystis, Anabaena, Spirulina, Gloeobacter cyanobacteria, or a combination thereof.
  • the cyanobacteria comprise an expression system comprising at least one expression vector having a heterologous promotor operably linked to a nucleic acid segment encoding a sucrose phosphatase synthase, a sucrose permease, or a combination thereof.
  • nucleic acid segment encoding the sucrose phosphatase synthase encodes an amino acid sequence with at least 95% sequence identity to SEQ. ID NOS. 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, and the nucleic acid segment encoding the sucrose permease encodes an amino acid sequence with at least 95% sequence identity to SEQ. ID NO. 1.
  • the first chamber comprises a first nutritional media comprising approximately 140mM salt to approximately 160mM salt for increasing the sucrose production of the cyanobacteria compared to growing the cyanobacteria with less than approximately 140mM salt.
  • the at least one anaerobic bacterium is Escherichia coli, Geobacter sulfiirredticens, Celltdomonas uda, Clostridium lentocellum, Acetivibrio celluloyticus, Clostridium cellobioparum, Shewanella sp., or a combination thereof.
  • the Escherichia coli comprises an expression system comprising at least one expression vector having a heterologous promotor operably linked to a nucleic acid segment that encodes an amino acid sequence with at least 95% sequence identity to SEQ. ID Nos: 1, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • the second culture further comprises Geobacter sulfurreducens as a co-culture with Escherichia coli.
  • the phototrophic bacterium is Rhodobacter sphaeroides.
  • the separator comprises a scrubber and a sparging port for removing oxygen from the first culture medium before the first culture medium transfers into the second culture.
  • the second chamber further comprises a sparging port configured to release oxygen produced by the fermentative microbe to reduce an amount of oxygen in the second culture.
  • gas delivery system is configured to introduce anaerobic gases into the second chamber and the first separator to sparge oxygen from the first nutritional medium and the second nutritional medium through the sparging port.
  • a bioreactor comprising: a first chamber comprising a first culture comprising a first nutritional medium and at least one sugar-producing microbe that releases sugar into the first nutritional medium; a second chamber separated from the first chamber by a first separator configured to allow the sugar from the first nutritional medium to enter the second chamber, wherein the second chamber comprises a second nutritional medium and at least one anaerobic fermentative microbe that converts the sugar to a first product mixture comprising hydrogen gas and acetate; and a third chamber separated from the second chamber by a second separator configured to allow the acetate from the second chamber to enter the third chamber, wherein the third chamber comprises a phototrophic bacterium that converts the acetate to hydrogen gas; wherein the hydrogen gas collects in a headspace of the second chamber and the third chamber.
  • the second chamber further comprises a sparging port configured to release oxygen to reduce an amount of oxygen in the second culture.
  • a method of producing hydrogen gas comprising: maintaining a first culture comprising at least one sugar-producing microbe in a first nutritional medium that releases sugar into the first nutritional medium; transferring a portion of the first nutritional medium comprising the sugar into a second culture that is separated from the first culture, wherein the second culture comprises a second culture medium and at least one anaerobic fermentative microbe that converts the sugar into acetate and hydrogen gas; and collecting the hydrogen gas in a hydrogen storage container or a hydrogen consuming fuel cell for producing electricity.

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Abstract

L'invention concerne des procédés et un appareil pour un bioréacteur comprenant une première chambre comprenant une première culture comprenant un premier milieu nutritionnel et au moins une espèce d'un microbe produisant du sucre qui libère du sucre dans le premier milieu nutritionnel. Le bioréacteur comprend également une seconde chambre comprenant une seconde culture comprenant un second milieu nutritionnel et au moins une espèce d'un microbe de fermentation anaérobie qui convertit le sucre en un premier mélange de produits comprenant de l'hydrogène gazeux et de l'acétate. Un premier séparateur peut être disposé entre la première chambre et la seconde chambre et séparer la première chambre et la seconde chambre pour permettre à une partie du premier milieu nutritionnel comprenant le sucre de se déplacer dans la seconde culture. L'hydrogène gazeux produit par le microbe de fermentation anaérobie s'accumule dans un espace libre de la seconde chambre.
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