Enhancing hydrogen and carbon dioxide use in acetogenic monocultures and cocultures

Abstract

A method for enhancing hydrogen uptake by cells of an acetogenic bacterium in a culture is provided. The method comprises incubating the cells in a liquid medium supplemented with an external gas mixture, wherein the external gas mixture comprises H2 and CO2; overexpressing an uptake hydrogenase and an associated electron transfer protein in the cells; increasing uptake of the H2 by the cells; and enhancing uptake of the CO2 by the cells. The culture may be a coculture with additional cells of a non-acetogenic bacterium, which produces natively H2 from catabolism of an organic molecule.

Description

[0001] ENHANCING HYDROGEN AND CARBON DIOXIDE USE IN ACETOGENIC

[0002] MONOCULTURES AND COCULTURES

[0003] CROSS-REFERENCE TO RELATED APPLICATION This application claims priority7to U.S. Provisional Application No. 63 / 553,918 filed

[0004] February715, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes.

[0005] REFERENCE TO U.S. GOVERNMENT SUPPORT This invention was made with government support under contract awarded DE-

[0006] AR0001505 - UNIVERSITY OF DELAWARE by ARPA-E US Department of Energy. The United States has certain rights in the invention.

[0007] FIELD OF THE INVENTION The invention relates to cultures of acetogenic bacteria and their uptake of hydrogen and carbon dioxide.

[0008] BACKGROUND OF THE INVENTION

[0009] Acetogenic bacteria, also referred to as acetogens, are bacteria species capable of converting hydrogen (H2), carbon dioxide (CO2) and carbon monoxide (CO) to commodity chemicals, including acetic acid and ethanol, via the Wood-Ljungdahl Pathway (WLP). Due to their ability to convert carbon dioxide, a greenhouse gas, into useful products, certain acetogenic bacterial species have been proposed as promising platform strains for renewable, carbon negative production of chemicals / metabolites when acetogens are grown with various organic molecules, for example, simple carbohydrates such as methanol and complex carbohydrates, together with gaseous substrates, for example, hydrogen (H2), carbon dioxide (CO2) and / or carbon monoxide (CO). Such a mixture of the acetogens w ith the organic molecules and gaseous substrates is referred to as acetogenic mixotrophy. When all the carbon from the said organic-molecule substrates, which are the substrates other than the gaseous components (notably H2, and CO2, w ith or without CO in the gas mixture), is incorporated into metabolite chemicals, which are also referred to as products, this process is fermentation or culturing a carbon-neutral fermentation. If more carbon (for example, derived from CO2 and possibly CO) is recovered into metabolites, this process is fermentation or culture carbon-negative. Acetogens can be grown on their own in monocultures or together with other microbes in cocultures that can be set up to achieve a syntrophic coculture whereby one microbe’s metabolism benefits the other. The one-way beneficial relationship is known as commensalism. The mutually beneficial syntrophy is known as mutualism. Unfortunately, low ATP yield of carbon dioxide consumption limits the growth, activity, and chemical production available to acetogenic bacteria when utilizing carbon dioxide as the primary carbon source. This problem has been addressed by others by using carbon monoxide, which has a much higher ATP yield per carbon mole fixed, as the primary carbon and electron source. However, growth on carbon monoxide decreases or eliminates CO2 fixation, and can even results in a net CO2 emission due to the oxidation of CO to CO2 in order for the organism to extract more electrons for growth and metabolite production. This is not desirable because net fixation of the greenhouse gas CO2 is one of the main attractions for using acetogenic bacteria in biological manufacturing.

[0010] There remains a need for improving growth of acetogens in a culture with external H2 and CO2, either alone or with other organisms that produce H2 and CO2.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides a method for enhancing hydrogen uptake by acetogenic bacteria. The invention is based on the inventors' disco very of acetogenic bacteria overexpressing uptake hydrogenase and an associated electron transfer protein. The acetogenic bacteria may be in a monoculture and in a co-culture with one or more additional bacteria where the additional bacteria may produce H2 and CO2 via fermentation of biomass derived and related carbohydrates. A method for enhancing hydrogen uptake by cells of an acetogenic bacterium in a culture is provided. The method comprises: (a) incubating the cells in a liquid medium supplemented with an external gas mixture, wherein the external gas mixture comprises H2 and CO2; (b) overexpressing an uptake hydrogenase and / or an associated electron transfer protein in the cells; (c) increasing uptake of the H2 by the cells; and (d) enhancing uptake of the CO2 by the cells.

[0013] The method may further comprise producing a metabolite by the cells with electrons from the H2 of step (c) and carbon from the CO2 of step (d). The external gas mixture may further comprise CO.

[0014] The external gas mixture may consist of H2 and CO2.

[0015] The associated electron transfer protein may be selected from the group consisting of formate dehydrogenase subunit, molybdopterin molybdotransferase, molybdopterin- guanine dinucleotide biosynthesis adapter protein, formate dehydrogenase subunit, NADH dehydrogenase I, NADH dehydrogenase I, NADH dehydrogenase related protein, 4Fe-4S ferredoxin, and electron transfer protein.

[0016] The method may further comprise increasing growth rate of the cells. The method may further comprise increasing density of the cells.

[0017] The method may further comprise supplying an organic molecule. The organic molecule may be selected from the group consisting of non-glucose sugars, tricarboxylic acid (TCA) cycle intermediates, methanol, formate, and pentose phosphate pathway (PPP) intermediates. The non-glucose sugar may be fructose.

[0018] The method may further comprise reducing production of a metabolite by the culture, wherein the metabolite is selected from the group consisting of acetone, nitrate, carboxylic acid, ketone, and aldehyde. The acetogenic bacterium may be selected from the group consisting of Clostridium ljungdahlii (Clj), Acetobacterium carbinolicum. Acetobacterium wieringae. Acetobacterium malicum, Acetobacterium woodii, Clostridium aceticum, Clostridium autoethanogenum, Clostridium drakei, Clostridium luticellarii, Clostridium carboxidivorans, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa acidovorans, Sporomusa aerivorans, Sporomusa malonica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Thermoanaerobacter kivui, Thermacetogenium phaeum, and Eubacterium limosum.

[0019] The culture may be a monoculture. The culture may be a coculture with additional cells of a non-acetogenic bacterium, wherein the additional cells produce H2 from catabolism of an organic molecule. The non- acetogenic bacterium may be Clostridium. The Clostridium may be selected from the group consisting of Clostridium acetobutylicum (Cac), Clostridium beijerinckii, Clostridium sacdiarolylicum. Clostridium butyricum, Clostridium cellulovorans , and Clostridium cellulolyticum. The Clostridium may be a butyric-acid Clostridium. The method may further comprise increasing hydrogen production by the additional cells. The method may further comprise overexpressing by the additional cells a gene encoding a protein selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenases, ferredoxins, and allogeneic NADH kinase. The method may further comprise overexpressing by the additional cells a gene encoding a production hydrogenase.

[0020] The method may further comprise increasing the overall interfacial transport coefficient (kta) in the culture. The culture may be in a bioreactor selected from the group consisting of stirred tank bioreactor, bubble column bioreactor, airlift bioreactor, falling film bioreactor, trickle-bed bioreactor, and fixed bed bioreactor.

[0021] Where the liquid medium comprises iron silicate or magnesium iron silicate ore particles, the method may further comprise increasing hydrogen production at the immediate vicinity of the acetogenic bacteria.

[0022] The culture may produce metabolic products in a sugar-carbon neutral manner.

[0023] The culture may produce metabolic products in a sugar-carbon negative manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figs. 1A-1C show overexpression of hydA for enhanced H2 production capability. (A) A scheme of alcohol and H2 production related to hydrogenase. (B) Comparison of H2 and CO2 yields from the hydA overexpressing strain vs. WT Cac. (C) Ethanol, acetone, and butanol yields of the hydA overexpressing strain vs. WT Cac.

[0025] Figs. 2A-2E show effects of engineered electron management system of Cac for acetone and hydrogen production. (A) A glycolytic metabolic map of Cac and cofactor dependency. (B) Genetic architecture of p95ace06a. (C) Glucose consumption by Cac harboring either hydA-only (HydA) or p95ace06a (ACE06) plasmid. (D) Full metabolite profiles from mono- and co-culture of Cac 824 / p95ace06a with Clj. (E) Calculated hydrogen yield of engineered Cac strains.

[0026] Fig. 3 shows change in CO2 incorporation and fixation rate for varied fructose (Fruc.) feed rates and cell densities. 0Dx2 and 0Dx3 means twice or thrice as high starting cell densities.

[0027] Figs. 4A-4D show complete sugar-electron utilization, exogenous H2 utilization, & strong CO2 fixation in IPA producing CACas9(-)hbd / p95ace02atoB-C / / cocultures (vs Chc4C- monoculture) under atmosphere of: (A, B) N2 (zero H2 formation; complete sugar electron use); (C) H2 (strong exogenous H2 uptake, enhanced CO2 uptake); & (D) 80% H2+20% CO2 (additional CO2 uptake). 160 mL serum bottles.

[0028] Figs. 5A-5B show profiles of metabolites from high cell density fermentation with sugar co-feeding. (A) Profile of liquid metabolites (B) H2 and CO2 profiles.

[0029] Figs. 6A-6D show repeated batch fermentation with high cell density. (A) Profile of biomass. (B) Profile of glucose consumption. (C) Profile of isopropanol and acetone production. (D) Profile of acetate and ethanol production. Figs 7 shows ethanol, acetone and H2 production yields by CACas9 (-)hbd strains overexpressing the electron flux / management related genes. The WT indicates the plasmid free control. Figs. 8A-8C show carbon negative mixotrophic fermentation with fructose cofeeding . (A) Carbon recovery at each timepoint of the coculture (B) Headspace composition of the coculture assessed by gas chromatography. The vertical lines represent manual additions of exogenous gas. (C) Time dependent profiles of soluble metabolites assessed by HPLC.

[0030] Figs. 9A-9B show engineering electron flow / flux for higher IPA selectivity in coculture. (A) Schematic of carbon and electron flow in Cac. (B) Metabolite yields from the cocultures of different Cac strains with Clj. Figs. 10A-10D show coculture of CACas9 (-)hbd / p95ace02atoB and

[0031] C7 / 7p l OOptaHalo under different gas transfer conditions. (A) A picture of roller bottle culture system. (B) A picture of rotary shaker bottle culture system. (C) A kinetic metabolite profile under the roller bottle culture system. (D) A kinetic metabolite profile under the rotary shaker bottle culture system.

[0032] Figs. 11 A-l IB show coculture of Cac 824 / p95ace02a and C / 7pl OOptaHalo under different gas transfer conditions. (A) A kinetic metabolite profile under the roller bottle culture system. (B) A kinetic metabolite profile under the rotary shaker bottle culture system.

[0033] Figs. 12A-12B show Clj monoculture under roller bottle culture system and rotary shaker bottle culture system. (A) The amount of acetate formation and CO2 fixation. (B) Maximum OD. Figs. 13A-13C show data that identify H2-solubility as the bottleneck for CO2 fixation by Clj. (A) Clj biomass accumulation and (B) headspace concentrations of hydrogen and carbon dioxide for the experiment with 30 psig starting pressure of 80 / 20 H2 / CO2 gas mix. (C) Clj biomass accumulation for the experiment with 30 psig starting pressure of 80 / 20 H2 / CO2 gas mix and periodic re-pressurization to 20 psig.

[0034] Figs. 14A-14B show A) Hydrogen consumption kinetics and B) growth curves for Clj cultures grown under 5, 15, and 30 psig 80% / 20% H2:CO2 gas mixture in roller bottle system. Fig. 15 shows ore particles to generate hydrogen when used in cultures containing acetogenic bacteria.

[0035] DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for enhancing hydrogen uptake by an acetogenic bacterium. The inventors have surprisingly discovered that overexpression of uptake hydrogenase complex proteins by acetogenic bacterium cells incubated in a liquid medium supplemented with external gas mixture of H2 and CO2 enhanced uptake of H2 and CO2. The inventors have disclosed a strategy of co-feeding CO2 with an organic-molecule substrate, such as a sugar, amino acid, or an intermediate of the energy generation pathways (e.g., glycolysis, the TCA cycle or the pentose phosphate pathway (PPP)). This supplements the energetic requirements of the acetogen without possibly suppressing CO2 utilization. This was surprising because while in continuous culture (without cell retention) of the thermophilic acetogen Moorella thermoacetica, slow glucose feeding enables good CO2 utilization, at high concentrations, glucose inhibited CO2 utilization via the Wood- Ljungdahl Pathway (WLP). Moorella thermoacetica is a thermophilic acetogen that uses glucose, which is the most well studied sugar inducing carbon catabolite repression (CCR), a mechanism through which glucose inhibits the use of other substrates. Unlike Moorella thermoacetica, Clostridium ljungdahlii (Clj) is a mesophile and cannot use glucose as a substrate. Instead, it uses fructose and some other 6 and 5-carbon sugars (e.g., xylose, through the PPP). Working with Clj, the inventors have disclosed that feeding of fructose in batch and fed- batch cultures of Clj enables good CO2 co-utilization. The inventors have also disclosed that xylose can also be used in this context as well as other non-glucose sugars, TCA and PPP intermediates. Fructose does not inhibit CO2 utilization in Clj, and also possibly in other mesophilic acetogens that do not use glucose. It is unlikely that other non-glucose sugars. TCA and PPP intermediates would inhibit CO2 utilization in Clj and in other mesophilic acetogens that do not use glucose. Another potential strategy developed by the inventors is coculture of acetogenic bacteria in syntrophic consortia with other bacteria to enable tight exchange of carbon and electrons, leading to an overall higher carbon recovery in the system. The inventors have demonstrated genetic engineering and bioreactor design strategies for boosting the growth, activity, and chemical production of acetogenic bacteria grown with carbon dioxide as the primary7carbon source both in coculture with one or more carbohydrate-fermenting partner bacteria or in monoculture.

[0036] Transfer of insoluble gaseous substrates (such as Oxygen [O2]. Hydrogen [H2], carbon dioxide [CO2], methane, etc.) in bioreactor systems where one grows various kinds of cells such as in this case Clj and Clostridium acetobutylicum (Cac), involves several steps from the gas phase (frequently in the form of gas bubbles) to the cell surface. This is well studied in aerobic systems for O2 transfer. For transfer of H2, the H2 transfer rate (HTR) is calculated as follows:

[0037] HTR = (H2 rate, mmol H2 / cm3 / h) = Kra (Cm- CLH) = k a (CH*- CLH)

[0038] Here, a = (total gas surface area) / (liquid volume), KL is the real interfacial transport coefficient on the liquid side of the gas bubble, to account for the resistance to H2 transfer from the gas phase to the liquid phase by the stagnant thin liquid film surrounding each bubble. Cm is the concentration of H2 at the gas-bubble surface, and CLH is the concentration of H2 away from the gas bubble, that is, in the bulk liquid. HTR can also have units of mg H2 / cm3 / h. cm3refers to the culture volume. Because we cannot measure easily Cm, one typically uses (CH*- CLH) as the driving force for interfacial transport of the insoluble gas (here H2). CH* is the H2 concentration in the liquid which would be in equilibrium with gas phase H2 concentration or partial pressure in the absence of a film resistance on the liquid side due to the stagnant thin liquid film surrounding each bubble. CH* is easily calculated from the known thermodynamic relationship (known as Henry's law in its simplest form) for H2 equilibrium betw een the gas phase and the liquid phase, which here is the growth medium of the culture. When using (CH*- CLH) as the driving force, one must use the modified coefficient kr and thus kta is the overall interfacial transport coefficient, also known as volumetric mass transfer coefficient, for the transfer of hydrogen from the gas phase to the liquid medium, ki.a depends on the mixing intensity in the fermentation broth in a bioreactor, which is a device vessel where cells are grown. The kra also depends on the composition of the culture medium (for example the concentration of salts in the medium), the presence of cells and components of lysed cells, the temperature, the osmotic pressure of the medium, and the presence of surfactants in the medium, among other factors that are well known, kt can be increased by changing the composition of the medium and the salt (electrolyte) composition of the culture medium. “a” can be increased by increasing the interfacial are between the gas and the liquid. Several ways can be used to increase a: increase the gas sparging rate, decrease the bubble size, and use packed columns, whereby the packing forces an increased mass transfer of hydrogen between the gas and the liquid phases. Like in the transport of other insoluble gases such as O2, there are nine steps for hydrogen (H2) transfer from the gas phase (bubble) to cells: 1, from the bulk of gas to the gas-liquid interface; 2, interfacial equilibration between the partial pressure of H2 at the gas side and the liquid leading to the interfacial concentration of dissolved H2; 3, transfer of dissolved H2 from the gas liquid interface to the bulk liquid through the stagnant thin film (boundary layer for H2 transfer) around the gas phase-bubble; 4, transfer of dissolved H2 through the well bulk liquid; 5, transfer of dissolved H2 from through the thin film (a different than step 3 boundary layer for H2 transfer) around the cell aggregate or single cells; 6. transfer of dissolved H2 across the liquid-aggregate interface; 7. transfer of dissolved H2 through the cell aggregate; 8, transfer of dissolved H2 from the cell surface through the cell membrane; 9, utilization of dissolved H2 in chemical reaction(s) in the cell or right on the cell membrane. Step 7 is not relevant if all cells exist as single cells and not in aggregates. In that case, steps 6 and 8 are the same as the cell aggregate is replaced by a single cell. Step 3 is the rate limiting step for H2 transfer rate (HTR).

[0039] Our data support the disclosed concept that the transfer of H2 from the bulk gas to the cells is limiting Clj grow th when grown on H2 and CO2 only, whether in monoculture or co-culture. While overall O2 transfer is limited by equivalent step 3 above, here we disclose that H2 is limited by steps 8 and 9, in addition to step 3. We disclose two strategies to enhance overall H2 transfer to the Clj cells in monoculture or cocultures.

[0040] The present invention relates to enhancing carbon recovery' to products and rate of carbon fixation of cocultures which include acetogenic bacterial species alongside carbohydrate-fermenting bacteria species as well as monocultures including only acetogenic bacterial species. This invention addresses 2 major technical challenges: 1) Overcoming energetic and mass transfer limitations inherent in ty pical gas fermentations with acetogenic bacteria; and 2) improving growth rate, biomass accumulation, CO2 fixation rate, and metabolite production rate of acetogenic bacteria when grown with CO2 as their primary carbon source or mixotrophically together with organic molecules as additional carbon and energy sources, both in monocultures of acetogens and in cocultures of acetogens with other microbes. According to the present invention, Clostridium acetobutylicum (Cac), an anaerobic, carbohydrate fermenting, solventogenic bacterial species, was genetically engineered to increase production of carbon dioxide and hydrogen and then co-cultured with Clostridium ljungdahlii (Clj). Cac was transformed with plasmids causing it to overexpress its endogenous hydrogenase (hydA, CA_C0028), its endogenous glyceraldehyde-3-phosphate dehydrogenase (gapN, CA_C3657), or a Cac codon-optimized heterologous NADH kinase (pos5. YPL188W) from Saccharomyces cerevisiae. The hydA overexpression strain produced 42% more H2 and 9% more CO2 in monoculture compared to the wild type parent strain. Coculturing of the hydA overexpression Cac strain with Clj produced a 311% higher acetate yield and 44% higher isopropanol (IP A) yield compared to coculture with the wild type strain, both demonstrating the enhanced Clj activity in the coculture. The gapN overexpression strain produced 50% more H2 and 13% more CO2 in monoculture compared to the wild type strain. The pos5 overexpression strain produced 15% more H2 in monoculture compared to the parent Cac strain. Cac was also engineered to overexpress the binary combinations of each of these genes (hydA-gapN, gapN-pos5, and pos5-hydA), as well as the hydrogenase combined with key genes of the acetone pathway (acetoacetate decarboxylase, adc. CA_P0165; acetate CoA / acetoacetate CoA-transferase, ctfAB, CA_P0163 and 0164) from the endogenous pSOLl megaplasmid. All these Cac strains produced as much or more hydrogen and carbon dioxide in monoculture than their corresponding single gene strains. Coculture of each of these Cac strains with Clj also led to increased acetate yield and carbon recovery compared to coculture of Clj with wild type Cac.

[0041] An additional component of the invention relates to the engineering of Clj to facilitate enhanced hydrogen uptake to improve rate of CO2 fixation and growth of Clj. The hydrogenase used to produce hydrogen which we overexpressed in Cac is highly homologous to the Clj hydrogenase (hydA2, CLJU_c07070) used to uptake hydrogen. RNAseq data show that this Clj hydrogenase is downregulated by coculture with Cac. Overexpressing hydA2 in Clj facilitated faster uptake of hydrogen from the media by Clj. Additional components of the hydrogenase complex when overexpressed in Clj enhanced hydrogen uptake. In coculture, faster uptake of hydrogen by Clj depletes the liquid medium of hydrogen, encouraging faster production of hydrogen by Cac according to the principle of reaction enhanced mass transfer. In both monoculture and coculture faster uptake of hydrogen by Clj also encouraged faster diffusion of exogenous hydrogen from the headspace according to the same principle of reaction-enhanced and accelerated mass (here H2) transfer. An additional component of the invention relates to culturing Clj in a system, such as a roller bottle system or as described in examples in falling-film or trickle-bed bioreactors, such that the effective surface area to volume ratio, and consequently gas-liquid mass transfer is massively increased. Employing this strategy led to 80% greater CO2 fixation, 70% greater acetate production, and 48% higher biomass accumulation in Clj monoculture relative to a typical lab scale shaker fermentation inside a serum bottle on a rotary shaker. Clj performance in the coculture was similarly enhanced by this strategy.

[0042] This component can be combined with the Clj engineering component in monoculture for superior monoculture growth on only CO2 and H2 or combined with the Cac and Clj engineering components for superior coculture performance on a mixotrophic combination of fermentable carbohydrates and exogenous CO2 and H2.

[0043] An additional component of the invention relates to the feeding of fructose to Clj at a such that fructose is metabolized by the cells.. This approach supports CO2 fixation by partially fulfilling the maintenance energy requirements of the acetogenic bacteria, enabling the accumulation of a higher cell density of the acetogen, leading to more overall CO2 fixation in the culture. This approach is directly applicable to acetogenic monocultures. It can also be used in coculture with Cac. This strategy can also be used in coculture with a Cac strain that has been genetically modified to delete the genes of the fructose utilization operon (CA_C0231, CA_C0232, CA_C0233, CA_C0234), such that Cac loses the ability to metabolize fructose. The same strategy' of feeding of an organic molecule (other non- glucose sugars, TCA and PPP intermediates) can be also utilized to enable sustainable CO2 utilization by Clj and other mesophilic acetogens that do not use glucose.

[0044] Monocultures of Clj on CO2 and H2 in the absence of CO may be sustained to reach high cell densities and fast grow th by two disclosed approaches: a. Increased hydrogen uptake by Clj by overexpressing components of the uptake hydrogenase complex and related proteins. The higher gene / protein dosage increases hydrogen uptake / utilization by the cells thus reducing the concentration of hydrogen on the surface of the cells. This then increases the driving force (CLH- CH step 8 for cell aggregates or 6 for single cells, as described above). CH is the concentration of hy drogen), thus overcoming the limitation of hydrogen supply to Clj cells (this is a reaction-driven, enhanced hydrogen mass transfer rate leading to enhanced hydrogen supply to Clj cells). The limitation of hydrogen supply to Clj cells is identified and disclosed here as limiting the growth rate and high cell densities of Clj when cultured on CO2 and H2 alone without CO in the gas mixture. This also applies when fructose and / or other organic molecules are used as carbon co-substrates. b. Increased ki a for H2 transfer where k a is the overall interfacial transport coefficient.

[0045] The inventors have identified hydrogen as the growth limiting substrate (and not CO2) for acetogens grown on CO2 and H2 in monoculture, for two primary reasons. First, the stoichiometry of the WLP requires two moles of hydrogen for every one mole of CO2 utilized to form one mole of acetate.

[0046] WL Pathway : CO2 + 2H2= (1 / 2) Acetyl-CoA + H2O + ((n-l) / 2) ATP where n is membrane-generation coefficient.

[0047] Therefore, on a molar basis, two times more H2 is required than CO2 for the growth of acetogenic bacteria. Second, at 37-40 degrees C, the molar solubility of H2 is only about 0.7 mM (similar to O2, 0.9 mM), whereas the solubility of CO2 is 34 mM, more than forty times higher. In coculture, Cac produces both CO2 and H2 in situ, partially overcoming gas liquid mass transfer limitations. However, Cac produces more CO2 than H2 on a molar basis, so, according to the aforementioned stoichiometry of the WLP, additional exogenous H2 transfer is always required to utilize all of the CO2 produced by Cac. Thus, energetic growth limitations imposed by inherent difficulties in hydrogen transport and uptake is the key limiting factor for the growth of acetogenic bacteria, both in monoculture and coculture, that our invention addresses and discloses.

[0048] Metabolic energy limitations prevent acetogenic bacteria from robust growth or production of products other than acetic acid with conventional gas fermentation using primarily CO2 and H2. These metabolic energy limitations can be overcome by replacing or supplementing CO2 and H2 with carbon monoxide, enabling stronger growth and a broader product portfolio. Unfortunately, carbon monoxide addition decreases, eliminates, or even reverses carbon dioxide fixation, one of the key selling points of gas fermentation platforms. Coculture with a second, carbohydrate-fermenting bacterial species (such as Cac and other clostridia species described in examples below) enables upgrading of the acetate produced from CO2 fixation into a more diverse, valuable range of products and facilitates tight exchange of carbon and electrons via direct (such as various electron carriers like NADH and reduced ferredoxin via exchange of cytoplasmic material) or indirect exchange through production and uptake of hydrogen.

[0049] The present invention enhances the exchange of carbon and electrons between the two bacteria in coculture systems, increasing the carbon recovery' and expanding the potential product portfolio of the coculture to include less reduced final products without CO2 emission. The present invention makes possible to produce metabolite chemicals in a carbon-neutral and carbon-negative fermentation process.

[0050] An important technical problem the present invention addresses is overcoming mass transport limitations inherent to gas fermentation. These mass transport limitations are important even in carbon monoxide fermentation, but they become especially critical when carbon dioxide is the primary carbon source because gas transfer limitation can cause the acetogenic bacteria to become energy limited due to the lower ATP yield of carbon dioxide from the WLP. The invention addresses these limitations by increasing in situ generation of CO2 and H2 in the liquid medium, increasing transport of exogenous CO2 and H2 into the liquid media, and by increasing uptake of CO2 and H2 by the acetogenic bacteria, driving further production and diffusion of CO2 and H2 via reaction enhanced mass transfer.

[0051] The present invention provides a method for enhancing hydrogen uptake by cells of an acetogenic bacterium in a culture. The method comprises (a) incubating the cells in a liquid medium. The liquid medium is supplemented with an external gas mixture. The external gas mixture comprises H2 and CO2. The method also comprises (b) overexpressing one or more uptake hydrogenase complex proteins, for example, an uptake hydrogenase and / or an associated electron transfer protein in the cells. The method further comprises (c) increasing uptake of the H2 by the cells, and (d) enhancing uptake of the CO2 by the cells.

[0052] The method may further comprise producing a metabolite by the cells w ith electrons from the H2 of step (c) and carbon from the CO2 of step (d). The external gas mixture may further comprise CO. The external gas mixture may further comprise nitrogen (N2). The external gas mixture may further comprise traces of air at, for example, less than about 3%, 5% or 7% air. The external gas mixture may consist of H2 and CO2. The uptake hydrogenase may be a Fe-Fe hydrogenase. Examples of the Fe-Fe hydrogenase include HydA2, and CLJU_cO7O7O.

[0053] The associated electron transfer protein may be selected from the group consisting of formate dehydrogenase subunit, molybdopterin molybdotransferase, molybdopterin- guanine dinucleotide biosynthesis adapter protein, formate dehydrogenase subunit, NADH dehydrogenase I, NADH dehydrogenase I, NADH dehydrogenase related protein, 4Fe-4S ferredoxin, and electron transfer protein. The formate dehydrogenase subunit may be alpha subunit, or CLJU_c06990. The molybdopterin molybdotransferase may be MoeA, or CLJU_c07000. The molybdopterin- guanine dinucleotide biosynthesis adapter protein may be CLJU_c07010. The formate dehydrogenase subunit may be FdhD or CLJU_c07020. The NADH dehydrogenase may be E subunit. CLJU_c07030. The NADH dehydrogenase I may be F subunit, or CLJU_c07040.

[0054] The NADH dehydrogenase related protein may be CLJU_c07050. The 4Fe-4S ferredoxin may be CLJU_c07060. The electron transfer protein may be CLJU_c07080.

[0055] The method may further comprise increasing growth rate of the cells. The growth rate may be increased by at least about 5%. 10%, 20%, 50%, 80%, 100%, or 200%. or about 5-50%, 5-100%, 5-200%, 50-100%, 50-200%, or 100-200%.

[0056] The method may further comprise increasing density of the cells. The density maybe increased by at least about 5%, 10%, 20%, 50%, 80%, 100%, or 200%, or about 5-50%, 5-100%, 5-200%, 50-100%, 50-200%, or 100-200%.

[0057] The method may further comprise supplying an organic molecule. The organic molecule in the liquid medium in an amount without suppressing CO2 utilization by the cells. For example, the liquid medium may comprise fructose maintained at a concentration of less than 3 mM, 5 rnM, 8 mM. For other organic molecules, the concentration may be maintained at a concentration of less than about 100 mM, or about 0 mM - 100 mM. The organic molecule may be selected from the group consisting of non-glucose sugars, tricarboxylic acid (TCA) cycle intermediates, methanol, formate, amino acids such as histidine or arginine, and pentose phosphate pathway (PPP) intermediates. The non-glucose sugar may be fructose, xylose, arabinose, ribose, or erythrose.

[0058] The method may further comprise the chemical reduction of a metabolite by the culture. The metabolite may be selected from the group consisting of acetone, nitrate, carboxylic acid, ketone, and aldehyde. The acetogenic bacterium may be selected from the group consisting of Clostridium ljungdahlii (Clj Acetobacterium carbinolicum, Acelobaclerium wieringcie.

[0059] Acetobacterium malicum. Acetobacterium woodu. Clostridium aceticum, Clostridium autoethanogenum, Clostridium drakei, Clostridium luticellarii, Clostridium carboxidivorans, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa acidovorans, Sporomusa aerivorans, Sporomusa malonica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Thermoanaerobacter kivui, Thermacetogenium phaeum, and Eubacterium limosum.

[0060] The culture may be a monoculture.

[0061] The culture may be a coculture with additional cells of a non-acetogenic bacterium.

[0062] The additional cells may produce H2 natively from catabolism of an organic molecule. The H2 production by the additional cells may be enhanced by expression a heterologous gene and / or overexpression of a native gene. The organic molecule may be used as a carbon and / or energy substrate by the additional cells.

[0063] The non-acetogenic bacterium may be Clostridium. The Clostridium may be selected from the group consisting of Clostridium acetobutylicum (Cac). Clostridium beijerinckii, Clostridium saccharolyticum, Clostridium butyricum, Clostridium cellulovorcms , and Clostridium cellulolyticum. The Clostridium may be a butyric-acid Clostridium.

[0064] Where the culture is a co-culture, the method may further comprise increasing hydrogen production by the additional cells. The method may further comprise overexpressing by the additional cells a gene encoding a protein. The protein may be selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenases, ferredoxins, and allogeneic NADH kinase. Examples of the glyceraldehyde-3-phosphate dehydrogenases include autologous gapN, and CA_C3657. Examples of the ferredoxins include autologous ferredoxin, CA C0303, and CA C3527. Examples of the allogeneic NADH kinase include allogeneic pos5, and YPL188W. The method may further comprise overexpressing by the additional cells a gene encoding a production hydrogenase. Examples of the production hydrogenase include autologous hydA, and CA_C0028.

[0065] The method may further comprise increasing the overall interfacial transport coefficient (k a) in the culture. The ki.a may be increased by at least about 5%, 10%, 20%, 50%, 80%, 100%, or 200%, or about 5-50%, 5-100%, 5-200%, 50-100%, 50-200%, or 100- 200%. The culture may be in a bioreactor. The bioreactor may be selected from the group consisting of stirred tank bioreactor, bubble column bioreactor, airlift bioreactor, falling film bioreactor, trickle-bed bioreactor, and fixed bed bioreactor.

[0066] The liquid medium may comprise iron silicate or magnesium iron silicate ore particles. The method may further comprise increasing hydrogen production at the immediate vicinity of the acetogenic bacteria.

[0067] According to the method of the present invention, the culture may produce metabolic products in a sugar-carbon neutral manner or in a sugar-carbon negative manner. The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%. more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate. Example 1. Overexpression of the Cac hydA gene in Cac drives electrons towards

[0068] H2 and Acetone / IPA production.

[0069] Hydrogenase (HydA) is the major enzyme that drives conversion of electrons produced via glycolysis into H2 gas. Increasing Cads H2 production accomplishes the dual project objectives of producing more H2 for Clj to use as an electron source as well as directing Cads metabolism away from production of more reduced products, such as ethanol and butanol, towards acetone (Fig. 1). A recent publication reported that overexpression of HydA increased H2 production from glucose by 40% and decreased production of other reduced byproducts. To test the hypothesis, the native Cac hydA gene (CA_C0028) was cloned under control of our two strongest Cac promoters, the Clj pta promoter (Ppta_clj) and an engineered thiolase promoter (Pthlsup) and transformed into Cac. Both strains were tested in monoculture to determine which promoter would be most effective in driving H2 and acetone production. A quick characterization in bottle cultures without pH control showed that Pthlsupwas superior to Ppta_clj for hydA overexpression. Especially, hydA expression under the Pthlsupincreased 40% more H2 than the WT strain (Fig. IB) and 2.1 -fold higher acetone yield compared to the WT control (Fig. 1C). Next, the Pthlsupbased hydA overexpressing strain was cultured in our pH controlled mini-bioreactors both as a monoculture and in coculture with Clj. In the coculture, butanol was decreased by 61% relative to the monoculture experiment, and acetoin, ethanol, and lactate production were eliminated in coculture. These results support the conclusion from the bottle cultures that overexpression of HydA helps direct electrons and carbon towards H2 and acetone. The 2.8 mol IPA / mol BuOH achieved in this experiment is the highest IPA to butanol ratio observed in a Cac-Clj coculture. Therefore, the hydA overexpression strategy will be pursued in engineered strains including the hbd deleted M5 strain for enh an ced el ectron man agement.

[0070] Example 2. Investigating effects of electron management related genes on hydrogen and acetone production.

[0071] Our experiment results highlighted the importance of electron management by Cac for efficient acetone production as well as isopropanol in Cac-Clj coculture. Therefore, we investigated two metabolic pathways - hydrogenase (hydA) and NADPH dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (gapN) that are closely related to the electron management of Cac (Fig. 2A). We had demonstrated that overexpression of hydrogenase (hydA) gene can increase selectivity for desired products acetone and hydrogen by 100% and 40%, respectively. We also showed that coculture of this strain with Clj produced the highest isopropanol to butanol ratio (2.8 mol IPA / mol BuOH) ever observed by our group. Based on these results, we constructed a new plasmid, p95ace06a (Fig. 2B), that overexpresses both the hydA and the acetone pathw ay genes and transformed it into Cac. We tested this strain in both monoculture and coculture with Clj. Previous testing with the hydA-ovAy plasmid showed increased hydrogen production, but the strain grew slowly and did not consume all the available sugar. As we hypothesized, overexpressing the hydrogenase and acetone pathway together restored robust cell growth and sugar consumption in our pH-controlled reactors (Figs. 2C, 2D). This is a potent strategy to improve acetone production applicable to the 4C pathway knockouts and / or knockdown strains. Next, we tested whether improving NADPH availability from glycolysis will improve hydrogen production (Fig. 2A). Overexpressing hydA converts electrons into H2. but only if those electrons can first be converted to the form usable by the hydrogenase: reduced ferredoxin. Reduced ferredoxin is naturally generated during the pyruvate decarboxylation to acetyl-CoA, but additional reducing equivalents are produced in the form of NADH during glycolysis. Ideally, these additional reducing equivalents would be converted to reduced ferredoxin and then hydrogen. A recent paper isolated and characterized the enzyme responsible for this reaction in Cac, showing that the Bcd-etfAB complex which catalyzes the final step of butyrate formation in Cac is also an NADH- ferredoxin reductase. However, our crotonate feeding experiments with the hbd deleted Cac M5 strain strongly suggested that crotonyl-CoA reduction and conversion of NADH to ferredoxin are directly coupled, meaning that knocking out butyrate and butanol production in Cac also eliminates its ability to directly convert NADH to reduced ferredoxin.

[0072] Conversely, NADPH conversion to ferredoxin appears to happen readily without a coupled reaction. If NADPH could be produced instead of NADH during glycolysis, NADPH could be used to reduce ferredoxin and then produce H2. Cac possesses a NADPH dependent non- phosphorylating glyceraldehyde-3-phosphate dehydrogenase (gapN), which produces NADPH instead of NADH from glycolysis, but flux through this alternate reaction is generally low. We cloned and overexpressed gapN in Cac under the control of our strongest pta clj promoter to investigate if overexpression of gapN would increase hydrogen production by substituting NADH with NADPH. which can more easily be converted to hydrogen. The results supported this hypothesis, showing that our gapN overexpressing strain generated more hydrogen per glucose molecule utilized compared to the WT Cac strain. Interestingly, overexpression of gapN was even more effective in increasing hydrogen selectivity than overexpressing hydA. and it was nearly as effective as the combined overexpression of hydA and the acetone pathway (Fig. 2E).

[0073] Overall, we demonstrated that the engineering electron management system of Cac drives hydrogen and acetone production. We are pursuing a combinatorial approach to combine and optimize these strategies.

[0074] Example 3. Improving CO2 fixation of Clj through fructose co-feeding.

[0075] Acetogens utilizing the Wood-Ljungdahl Pathway (WLP) cannot grow to high cell densities with CO2 as the sole carbon source (and H2 as an electron source) due to energetic and regulato y- limitations. To solve this problem, we investigated a low fructose feeding strategy for cocultures at a rate to support the estimated Clj biomass maintenance. We hypothesized that we could feed fructose at a rate high enough to provide extra energy for maintaining high cell densities, but low enough such that the cells would still need to fix large amounts of CO2 to fulfill all their energetic requirements. Using constant low feed rates, as opposed to intermittent batch additions, fructose was consumed immediately and did not accumulate in the medium. We have conducted tests of this strategy with Clj monocultures in serum bottles pressurized with CO2 and H2 and fed small amounts of fructose through a syringe pump. These results showed that CO2 fixation by Clj improves when fructose availability decreases, and that the percent incorporation of exogenous CO2 into acetate can be tuned from 4% to 63% simply by varying the specific fructose feed rate (Fig. 3). Critically, these results show that the specific rate of CO2 fixation remains similar even as cell inoculum density is increased, demonstrating that overall CO2 fixation can be scaled by increasing cell density. Finally, even at our lowest specific feed rate, biomass still increased by 30-84% over the course of the experiment. This suggests that, so far. we have fed fructose in excess of what the cells require to maintain their cell mass. We hypothesize that even lower fructose feed rates will substantially decrease CCR and increase CO2 utilization once we achieve a high cell density in the bioreactor. We contrast these findings from the findings whereby, in continuous culture (without cell retention) of a thermophilic acetogen Moorella thermoacetica, they reported that slow glucose feeding enables good CO2 utilization. At high concentrations, they found that glucose inhibits CO2 utilization via the Wood-Ljungdahl Pathway (WLP). Clostridium ljungdahlii (Clj) is a mesophile that does not use glucose and significantly its WLP and thus CO2 utilization is not inhibited by fructose and other sugars that is capable of metabolizing, unlike the thermophilic acetogen Moorella thermoacetica.

[0076] Example 4. Complete substrate electron utilization plus exogenous H2 utilization towards a CCh-negative Cac-Clj coculture fermentation.

[0077] An important goal is achieving sugar carbon neutral and sugar-carbon negative metabolite formation. Carbon neutral means to recover all the C atoms from the sugar into metabolites. Carbon negative means that the number of C atoms in metabolites is higher than the C atoms in the sugars used by the cells. These extra C atoms derive by the fed CO2 utilized together with the sugars. In other words, carbon neutral or carbon negative metabolite formation is the ability to use all available electrons in a substrate such as a sugar and derive additional electrons for additional carbon utilization (including the use of exogenous CO2) from H2. We demonstrated this core capability here using sealed serum bottles that permit gas composition measurements (Fig. 4). The coculture of C ACas9(-) / ty<7 / p95ace02atoB (atoB) with Clj under a N2 headspace showed no accumulation of H2 (Fig. 4B) as opposed to the monoculture (Fig. 4A), demonstrating complete utilization of the available electrons in the glucose substrate. Significantly, the absence of H2 in the gas phase demonstrates that there is direct transfer of electrons in the form of reduced electron carriers from Cac to Clj. This shows the importance of heterologous cell-fusion and associated exchange of electrons and cellular material for achieving the goal of complete substrate electron utilization towards sugar-carbon negative product formation. Moreover, we have demonstrated that the coculture can utilize exogenous H2 to fix additional, exogenous CO2 (Figs. 4C and 4D). The next step is to demonstrate complete utilization of all the carbons in glucose, as well as exogenous CO2 added to the system. Since Clj is the coculture partner responsible for CO2 fixation, we disclose that we can improve CO2 fixation rates by increasing the cell density of metabolically active Clj in our coculture fermentations.

[0078] Example 5. High cell density fermentation with sugar co-feeding.

[0079] Typically, we start our cocultures around an optical density of 1 with a 10:1 ratio of Clj to Cac. Since interaction between the two species seems to favor improved IPA selectivity, we investigated whether higher density inoculum can increase interspecies proximity from the beginning of coculture. We hypothesized that this approach would increase both IPA selectivity and productivity. First, we conducted experiments in serum bottles in which ODeoo of Cac and Clj at 1 and 4. respectively. We also connected these bottles with a syringe pump so that they could receive a continuous low fructose feed, to support Clj, and a continuous moderate glucose feed, to support Cac. These cultures accumulated roughly twice the typical amount of biomass (ODeoo of 20) and demonstrated rapid glucose utilization, H2 utilization, and IPA production (Fig. 5 A). They also produced our highest observed IPA titer thus far of 250 mM. fixed 1.4 mM exogenous H2 per mM glucose utilized (Fig. 5B), and recovered 78% of the carbon from glucose.

[0080] Next, we conducted a repeated batch fermentation with high cell density7in 15 rnL stationary7centrifuge tubes in an anaerobic chamber. We started them at the same 5x starting cell density as the serum bottle experiment, but instead of feeding additional glucose and fructose via syringe pump, we spun down the cells and resuspended them in fresh media every 12 hours. The maximum ODeoo of the cultures was approximately 27 (Fig. 6 A). One of these cultures achieved glucose utilization rate as high as 22 mM / hr (Fig. 6B) resulting in our highest demonstrated IPA productivity of 11.4 mM / hr, with an additional 5.1 mM / hr of acetone (Fig. 6C). As the two byproducts, acetate and ethanol were produced at rates of 7.9 mM / hr and 22.3 mM / hr, respectively (Fig. 6D). Another culture demonstrated similar biomass accumulation and glucose utilization, but it made proportionally more ethanol, as high as 44 mM / hr. The results suggest that cell retention will significantly improve glucose consumption and IPA productivity.

[0081] Example 6. Electron flux engineering for improving acetone selectivity. Another reason for the strong ethanol production is the insufficient rate of ferredoxin reduction (e‘ + Fdox= Fdred) and H2 production (Fdred = H2). We have previously shown that individual overexpression of Cac ’s primary hydrogenase (hydA), glyceraldehyde-3- phosphate dehydrogenase (gapN), and a yeast NADEI kinase (pos5) gene can improve FI2 production by Cac. hydA overexpression improves H2 production rate by increasing conversion of reduced ferredoxin to H2, and gapN and pos5 overexpression improve H2 production by increasing production of NADPH which can be converted to reduced ferredoxin (and then H2) by Cac 's NADPH-Fd reductase. Here, we tested combinatorial expression of these three H2-production enhancing genes, hypothesizing that they would work together synergistically to direct electrons towards H2 and away from ethanol. We constructed and tested three plasmid constructs for overexpression in Cac: gapN-pos5, gapN-hydA, pos5-hydA. All the genes were expressed from monocistronic operons under the control of the two strongest known promoters (Ppta clj and Psthl). To evaluate the performance of each new strain, we performed a simple monoculture fermentation in serum bottles, measured the soluble metabolites, normalized the metabolite production by the amount of glucose consumed, and compared the results to previously obtained results for the baseline CACas9(-) / ? ?J strain as well as the C ACas9(-) / ?6t / strain transformed with our best acetone plasmid obtained thus far, p95ace02atoB (ace02_atoB) (Fig. 7). These results show that all three new constructs improved H2 production compared to the plasmid free control (WT). The best strain was determined to be gapN-hydA, which resulted in 2.4- and 1.4-fold higher H2 yields compared to the WT and ace02_atoB, respectively. Interestingly, gapN-hydA also outperformed ace02_atoB in terms of acetone production, improving acetone yields by 24%. The results proved our hypothesis that manipulating electron flow will be important for controlling acetone production. This strategy will be applied to genomic integration since this collection of acetone production pathway genes and the electron flux engineering genes is too large (~11 kb) to express from a single plasmid.

[0082] Another interesting result was that the gapN-pos5 overexpressing strain produced 58% more ethanol than the baseline strain with no plasmid, contrary7to our hypothesis. We expected overproduction of NADPH via gapN and pos5 to decrease ethanol production since the adhEl and adhE2 genes from the pSOLl megaplasmid cannot utilize the NADPH for ethanol production. The results can be explained by the fact that the three chromosomal NADPH-dependent butanol dehydrogenases (DHs) bdhA. bdhB. and bdhC are capable of taking over ethanol production using NADPH. Therefore, we disclose that multiple alcohol DHs including adhEl, adhE2, and bdhABC be deleted to decrease ethanol production through genetic modification. Particularly, BdhA / B / C are responsible for over 95% of NADPH-dependent alcohol dehydrogenase activity in Cac. Therefore, to achieve our goal, we disclose that knocking-out both bdhA and bdhB (and also bdhC if necessary), while simultaneously knocking in the synthetic acetone production operons as well as hydA and gapN.

[0083] Example 7. Sugar co-feeding enables carbon-negative mixotrophic fermentation. We had demonstrated that continuous feeding of fructose throughout the coculture allows Clj to obtain enough energy to maintain its biomass while still fixing large amount of CO2. Here , our experiments (Fig. 8) demonstrate 112% carbon recover}', representing a carbon negative mixotrophic coculture of Cac and Clj in a simpler, low cell density coculture system.

[0084] Through the literature and a series of experiments with monocultures, we determined that a feed rate of 0.060 mM fructose per hour per OD of Clj was sufficient to maintain active Clj cell mass while maximizing CO2 fixation. We chose to start a coculture with a Clj starting OD of 4 and a starting OD of 1 of Cac (4: 1 ratio of Clj to Cac). In this experiment, we used C7 / / pl00ptaHalo strain and CACas9 dHBD / p95ace02atoB strain grown under a headspace with 80% H2 and 20% CO2 at the beginning of the experiment and at every' timepoint. The coculture with starting condition combined with a fructose feeding rate of 2.4 mM fructose per hour (the rate described above scaled with the starting Clj biomass) led to a coculture that maintained carbon negative conditions throughout the fermentation and finished with a 112% carbon yield relative to sugars consumed, showing that extra CO2 was incorporated from the headspace in addition to all CO2 produced by Cac. These cocultures accumulated by far the largest amount of acetate we’ve observed, over 500 mM, suggesting that CO2 fixation by Clj was faster than acetate assimilation by Cac. Accordingly, glucose consumption was only 109 mM, leading to production of 87 mM of ethanol and 52 mM of isopropanol, suggesting that Cac performance was suboptimal. When we deleted the 4C pathway from Cac, we observed that Clj appeared to inhibit Cat's growth in coculture. We hypothesized that this phenomenon is due to redox stress caused by direct electron transfer from Cac to Clj faciliated by cell-to-cell contact. We ameliorated this issue by overexpression of the redox -insensitive thiolase atoB from E. coll. How ever, now7that we have substantially increased Clj 's ability to fix carbon and electrons via supporting a higher active Clj cell fraction with fructose feeding, we appear to be running into this issue again. The issue can be addressed by the high-cell density coculture with cell retention as demonstrated above. We disclose that this experiment with the same starting amount of Clj but a higher amount of Cac (i.e., 1: 1 Clj-.Cac ratio) maintains a high enough active fraction of Cac to fully utilize the available glucose in the presence of a high density, highly active Clj cell fraction that maintains carbon negative conditions throughout the fermentation.

[0085] Example 8. Combinatorial expression of H2-production enhancing proteins (genes) in coculture.

[0086] We demonstrated above that combinatorial expression of genes related to H2 production and cofactor interconversion could significantly improve H2 and acetone production in CACas9 dHBD monoculture. The two plasmids that gave the best H2 and acetone yields combined overexpression of Cac ’s glyceraldehyde-3-phosphate dehydrogenase (gapN) with either a yeast NADH kinase (pos5) or Cac ’s primary hydrogenase hydA) (Fig. 9A). We tested these two strains, CACas9 dHBD / gapN-pos5 (blue bars in Fig. 9B) and CACas9 dHBD / gapN-hydA (orange bars in Fig. 9B), in coculture with Clj.

[0087] Coculture of both strains show ed very similar results, substantially decreasing the yield of EtOH per glucose to as low7as 15% for the coculture with gapN-pos5. This EtOH yield is nearly 50% lower than a typical fermentation with our C ACas9 dHBD p95ace02atoB strain (grey bars in Fig. 9B). Because the gapN-pos5 and gapN-hydA strains did not overexpress the acetone pathway genes (the plasmid backbone is too large to cany7both the acetone pathway genes and H2 genes simultaneously), the carbon w as redirected away from EtOH towards acetate instead of acetone or IP A. Based on the glucose consumption and carbon balance, 35-40% of Cac ’s total carbon products were acetate (not counting the additional acetate produced by Clj via CO2 fixation). Normally, when Cac constitutively overexpressing the acetone pathw ay genes, Clj is the sole producer of acetate and Cac imports acetate to synthesize additional acetone. Based on these results, we hypothesize that combined overexpression of these H2 genes with the acetone pathway (via knock-in of one set of genes combined with plasmid expression or knock-in of both sets) will result in a Cac strain that, when cocultured, maintains the low EtOH yield but produces acetone (which is converted to isopropanol via Clj) instead of acetate as the primary or sole byproduct.

[0088] Example 9. Increasing Eh-gas transfer improves CO2 fixation but inhibits glucose consumption by CACas9 (-)hbd.

[0089] We had demonstrated that Clj biomass accumulation and activity can be improved by feeding low7levels of fructose to provide enough energy to maintain biomass w ithout inhibiting autotrophic gas fixation, enabling carbon neutral or carbon negative fermentation. Here, we tested strategies for increasing exogenous-H2 transfer from the headspace, based on the hypothesis that this is an additional limiting factor for Clj activity7and exogenous CO2 fixation.

[0090] To test this hypothesis, we compared coculture in a typical shaker bottle setup, 20 mL of liquid culture in 250 mL serum bottles shaken at 90 RPM on a rotary shaker with the same bottles mixed with a roller bottle system in which the bottles are turned on their side and rotated rapidly (Figs. 10A, 10B). The roller bottle system provides a huge increase in gaseous mass transfer because the rolling motion of the bottle causes a thin film of culture to form around the entire bottle. This thin film has a much larger surface area and much less depth, drastically increasing the effective surface area to volume ratio, maximizing gas transfer from the headspace to the liquid medium (Fig. 10A). The roller bottle system is used for culture of slow growing mammalian cells that need to form a monolayer, and not for its higher gas transfer rates. The roller bottle system has not been used for microbial cultures and certainly not for culturing acetogens, in any scale, industrial, pilot or laboratory scale. Thus, the use of the roller bottle system has not been considered despite a well-known need for higher gas transfer rates. The use of the roller bottle system and its scale up equivalents are discussed in Example 17.

[0091] In addition to utilizing the roller bottles, we also increased the driving force by reflushing all controls with H2 / CO2 gas mixture (80 / 20) at every timepoint as opposed to charging the headspace with the gas mix at the beginning. Since Clj requires tw o moles of H2 for every mole of CO2 fixed, and because our CACas9 (-)hbd strain produces approximately twice as much CO2 as H2, the requirement for exogenous H2 transfer into the liquid medium is far greater than the requirement for exogenous CO2 transfer. By reflushing the headspace with the H2 / CO2 gas mixture at every timepoint, we maximized the partial pressure of H2, and therefore the mass transfer driving force, throughout the experiment. Data showing results from both systems is the average of two biological replicates (Figs. IOC, 10D).

[0092] Interestingly, coculture in the roller-bottle culture system resulted in much higher acetate production, acetate yields, and carbon recovery, all indicative of strong Clj activity, compared to the shaker system. However, we did observe less glucose utilization in the roller-bottle cultures, suggesting that the increased Clj activity or higher level of acetate due to the increased gas fixation may be limiting or inhibiting Cac cell growth. Example 10. Increasing H2- gas transfer enhances Clj metabolism that causes inhibition of glucose consumption by Cac 824 / p95ace02a.

[0093] We have consistently shown that the presence of Clj shifts the product spectrum of Cac towards more oxidized products, such as acetone. Using a fluorescent protein under the control of a redox sensitive promoter, we have shown that coculture with Clj directly leads to a more oxidized cytoplasm in Cac cells. Since the four carbon reaction in wild type Cac (ATCC 824) is important for balancing the cellular redox state, we tested whether the behavior of the Cac 824 / p95ace02a in coculture is also affected by enhanced H2-gas transfer. Our core hypothesis was that the use of the 824 / p95ace02a strain would impart more robust electron management and therefore increased glucose utilization in the presence of highly active Clj cultures.

[0094] Similar to the experiment we performed with our -4C strain (CACas9 (-)7?M / p95ace02atoB), we performed the cultivation using the roller-bottle culture system or the rotatory shaker culture system. Interestingly, the coculture under the roller bottle culture system exhibited much different behavior with severe inhibitions on glucose consumption (Fig. 11 A). Accordingly, a high carbon recovery (120%) and acetate titers (>500 mM) were achieved. However, contrary to our expectations, the use of the +4C strain resulted in even lower glucose utilization than the similar experiment with the -4C strain described earlier in this report. The carbon recovery and acetate titers indicate an exceptionally robust Clj activity. In contrast, the coculture under the rotary shaker culture system behaved similar with previously reported coculture experiments (Fig. 11B). One potential explanation for the decreased glucose utilization is that the Cac with 4C pathway harboring the original version of our acetone plasmid with redox sensitive thiolase (824 / p95ace02a) casued the Cac cell growth cessation after the cellular redox states becomes more oxidized. One solution is to replace the Cac thiolase with the redox-insensitive E. coll thiolase atoB (p95ace02atoB).

[0095] Overall, we have unequivocally proved that controlling H2 / CO2 gas transfer can activate the Clj activities in Cac-Clj coculture, potentially beneficial for population control and optimization for the selective IPA production.

[0096] Example 11. Eh-gas transfer is the limiting factor for Clj cell growth and CO2 fixation under auxotrophic conditions.

[0097] Our data show that energy’ availability was the limiting factor for Clj growth and activity in our cocultures. Here, we tested if Clj growth and activity could be improved by increasing the energy available via autotrophic gas fixation.. This is a well-recognized and significant problem: the inability to sustain growth and reach high cell densities of Clj (and other acetogens) growing on H2 / CO2 alone (in contrast, growth on CO is sustainable and enables high cell densities, as in the Lanzatech technology). This problem limits the use of acetogens for using H2 / CO2 without significant amounts of CO in the gas mixture.

[0098] To improve the gas transfer to Clj in the liquid medium, we used the roller bottle culture system described above. We did not completely reflush our bottles at every’ timepoint but recharged the headspace up to 20 psig of our H2 / CO2 gas mixture at every timepoint. To test the effect of improved gas transfer to the liquid medium, we compared the Clj monocultures in the roller bottle culture system to those in shaker bottles. We claim that increased H2-gas transfer w ould lead to improved Clj cell growth, higher cell densities and CO2 fixation in the roller bottle culture system. To test combinatorial effects of fructose concentration with the gas transfer, w e used either low level (1 g / L) or a high level (5 g / L) fructose concentration (Fig. 12).

[0099] Regarding the effects of gas transfer, we observe relatively similar growth, acetate production, and CO2 fixation, with a slight advantage to the roller in all categories. The slight improvement in the roller condition is to be expected since Clj will begin to utilize the headspace gases as the fructose level in the media is exhausted, giving an advantage to the roller system. Under the low fructose condition, Clj performed much better in the roller bottle culture system in terms of biomass accumulation (48% higher), acetate production (70% higher), and CO2 fixation (80% higher) compared to the rotary shaker bottle culture system (Fig. 12). Based on these findings, we disclose that fib-gas transfer, and therefore energy production, is limiting Clj cell growth and activity in our system. The high fructose condition accumulated 15% more biomass compared to the low fructose condition. However, the low7fructose condition made 29% more acetate and fixed 88% more CO2.

[0100] Example 12. High growth rates and cell densities are achieved by enhancing the interfacial gas transfer thus demonstrating that gas transger limits Clj (and likely all acetogen) growth on H2 / CO2 as sole substrates.

[0101] Above, we presented data comparing Clj monoculture performance in our roller bottle system compared to typical serum bottle fermentation conditions. Our data suggested that gas transfer rate from the headspace is limiting autotrophic growth of Clj on CO2 and H2 in monoculture (and likely also in coculture with Cac). However, in these Clj monoculture experiments, we added either 1 g / L or 5 g / L fructose to all conditions so we could increase the cellular growth rate.We also w anted to explore the potential of purely autotrophic Clj growth using the roller bottle system in order to identify factors which are rate-limiting CO2 fixation by Clj in the Cac-Clj coculture. Studies quantifying the growth rate and performance of acetogens, including Clj, on pure CO2 and H2 have been conducted, but we suspected that these studies may have been performed under gas-transfer limited conditions, masking the true potential growth rate of Clj on H2 / CO2.

[0102] First, we performed a simple growth curve of Clj in our roller bottle system (Fig. 10) using TCGM medium without glucose or fructose. To start, we pressurized the bottle with 30 psig of our 80 / 20 H2 / CO2 mixture. We also performed a N2-headspace control to ensure that Clj could not grow to any meaningful level on the yeast extract and other nutrients present in the medium (as expected, this control did not show meaningful growth) (Fig. 13A). The results show that, in the roller bottle system, Clj was capable of strong, robust growth up to an OD600 of 1.95 in under 40 hours, exhibiting a doubling time of under 7 hours during the exponential growth phase.

[0103] These results are in stark contrast to several other sources in the literature which (using Clostridium autoethanogenum, which is virtually identical with Clj) either fail to achieve an OD600 over 1.0 under pure growth on H2 / CO2, even with 90% cell retention, or required 100 hours in a pH-controlled bioreactor to reach OD 1.0. In addition, our results represent the fastest recorded doubling time for Clj (or C. autoethanogenum) on only H2 and CO2, and it is comparable to the maximum doubling time of 7.3 hours observed for C. autoethanogenum on syngas (50% CO with additional CO2 and H2) Moreover, in our experiment, the headspace gas analysis showed that the cells ran out of carbon dioxide at 38 hours (Fig. 13B), which coincided with the end of biomass accumulation. This shows that, in the presence of more CO2, the cells may have accumulated to even greater densities.

[0104] We repeated the experiment to confirm our results. This time, we took more samples during the exponential growth phase for detailed kinetics and re-pressurized the headspace with our 80 / 20 (H2 / CO2) gas mixture whenever the pressure dropped below 20 psig in order to maximize CO2 and H2 partial pressure throughout the experiment and prevent the cells from running out of carbon dioxide. In this case, the performance was even more impressive (Fig. 13C). In under 50 hours, the cells accumulated to an even higher OD600 of 2.58, and the doubling time was approximately 5 hours during the fastest part of the exponential grow th phase, comparable to the growth rates for Clj we typically observe on mixotrophic growth with H2. CO2, and 5 g / L fructose. These results show that the gas transfer from the headspace to the liquid phase is the limiting factor for the growth rate, biomass accumulation, and CO2 fixation for autotrophic growth on H2 / CO2 in Clj (and likely similar acetogens such as C. autoethanogenum and other acetohens discussed below ).

[0105] Example 13: Calculating the potential of acetogenic CO2 fixation (growing on CO2 + H2 without CO) in monoculture and coculture based on maintenance energy requirements and the volumetric mass transfer coefficient

[0106] We had demonstrated that CO2 fixation by Clj, both in monoculture and in coculture with Cac. is limited by H2 transport. Here, we performed calculations designed to estimate the potential of acetogenic CO2 fixation at scale based on this key insight. The meaning of all variables used in this section is described in Table 1.

[0107] Table 1. Definition of variables used in this example

[0108] First, we perform a steady-state cellular ATP balance on Clj (Eq. 1), applicable for a Clj or other acetogen monoculture in continuous fermentation. Second, we assume that the cells will be growing very' little and operating primarily in cellular maintenance mode, as expected in the continuous cell retention bioreactors in which we will perform our fermentations. This assumption allows us to set the growth rate term to zero. Next, the key insight of H2 transport limitation enables us to assume that the soluble H2 concentration will be approximately zero. Solving the resulting equation for steady state Clj biomass enables us to write Eq. 2.

[0109] Here, we now see that assuming CO2 and H2 as the sole carbon and energy’ sources, full cell retention, and continuous operating conditions, the Clj biomass cell density depends only on the volumetric mass transfer coefficient for H2 transport and H2 partial pressure; all of the other quantities in Eq. 2 are known constants. Next, if w e multiply the Clj biomass density by the per cell specific rate of H2 uptake by Clj, we can set this equal to the H2 uptake rate (Eq. 3). Because of the stoichiometry of the Wood-Ljundahl Pathway (2 mol H2 needed per mol CO2 fixed), we know- that the CO2 uptake rate (under conditions of H2 limitation) will simply be one half of the H2 uptake rate. Finally, because of the insight of H2 limitation, we can once again eliminate the soluble H2 concentration term and combine equations 2 and 3, yielding equation 4. Based on the key insight of H2 transport limitation, this equation shows that, in monoculture, Clj CO2 fixation will depend only on the bioreactor H2 partial pressure, volumetric mass transfer coefficient for H2 (i.e. bioreactor mixing), and the maximum specific H2 fixation rate by an acetogen such as Clj. The H2 partial pressure and reactor mass transfer coefficient are easily manipulated in well -characterized reactor systems. The maximum specific uptake rate of gaseous substrates by a given organism can be easily measured with a well-established method in simple batch serum bottle cultures, and experiments are currently underway in our lab to directly measure the maximum specific uptake rate of H2 by Clj according to this method.

[0110] To test the validity of the monoculture equations shown above, we compared the predictions of these equations to results from one paper in the literature which tested growth of Clostridium autoethanogenum (which is virtually identical to Clj) on CO2 and H2 under continuous conditions with 90% cell retention. This is the only study we were able to find which tested either Clj or C. autoethanogenum growing on only CO2 and H2 in a continuous system with a high cell retention rate. Under the conditions descnbed in this study, the steady state cell concentration of C. autoethanogenum reached an ODeoo of 0.69. We estimated the volumetric mass transfer coefficient of their reactor system to be approximately 10-50 h'1. Plugging in these estimated ki.a values into eq. 4 gives a range of 0.20- 1.0 for ODeoo, a range which includes the experimental value.

[0111] Equation 4 assumes that H2 transport from the headspace is the only cellular electron (and thus energy) source (eq. 1), as would be the case in acetogenic monoculture. However, equation 1 can be easily modified to include an additional energy term representing the soluble hydrogen Cac generates in the liquid phase when it metabolizes glucose (eq. 5). Performing the same analysis as before results in Equation 6. Here, by comparison with Equation 4, we clearly see that, for fixed H2 partial pressure and mixing conditions, cocultured Clj will always have a higher CO2 fixation rate than a monoculture due to the increased H2 generated by Cac from glucose in the liquid phase.

[0112] This analysis was performed assuming that Clj makes only acetate using only CO2 and H2. However, the energy yield coefficients, gaseous solubility constants, and Cac H2 yield coefficient applied in this analysis can easily be modified to estimate the likely performance of other hypothetical acetogenic monoculture or coculture systems using different gaseous (such as CO) or carbohydrate (such as xylose or fructose) substrates to make different target products (such as acetone, ethanol, butanol, hexanol). We also disclose that the HTR (hydrogen transfer rate) to acetogens can be also effectively increased by promoting the attachment of acetogens to the bubbles of the hydrogen containing bubbles when the bioreactor (any bioreactor, see Example 17) uses forced gas aeration resulting in bubbles to gases. Microbes attach to gas bubbles because the gas surface is attracting and collecting microbes and all cells. As a result, microbes have direct access to the hydrogen arriving at the gas-bubble surface and prior to being transported into the bulk liquid of the culture medium. One can use additives (electrolytes, such as simple inorganic salts) or remove additives (such as proteins and other micromolecules) that increase the ability of the gas bubble surface area to attract microbes such as acetogens. This mechanism and approach has never been disclosed or used for culturing acetogens to increase the rate of hydrogen transfer to and use by the cells. Furthermore, this has never been discussed or used for cultures of acetogens, monocultures or cocultures.

[0113] Example 14. Estimation of ki a in different bioreactor configurations to enable retrospective insight and future experimental design and reactor performance scaling.

[0114] We have shown that growth on CO2 and H2 and the rate of CO2 fixation by Clj is hydrogen mass-transfer limited, both in monoculture and in coculture with Cac. Based on this finding, we increased exogenous gas transfer in our coculture using our roller bottles (batch) and the packed column of the SuperBrewed Foods reactor system (continuous with cell retention), enabling us to achieve carbon-negative mixotrophic production of isopropanol in both systems. We also developed a set of equations that allow us to predict the required ki.a and Clj biomass required to achieve a given CO2 fixation rate, in monoculture or coculture, across different scales.

[0115] Motivated by the success of these approaches, we evaluated the approximate ki .a values of our various reactor configurations, enabling the retrospective analysis of past results and the planning of future experiments. Many of our earliest coculture fermentations were performed using miniature bioreactor system. The vessels for this reactor system are fashioned from spinner flasks, and mixing is achieved with stir bars. According to correlations developed from a detailed study of the mass-transfer characteristics of extremely similar flasks the ki.a value in our MBR platform is likely between 2-7 hr'1. This is likely a similar k a value to what we typically achieve in our standard, simple serum bottle fermentations performed on a rotary shaker. In contrast, we used headspace gas profdes (assessed by gas chromatography) and cellular growth data (Figs. 14 A, 14B) from several Clj fermentations to measure the kta in our roller bottle system at 50 hr'1. Based on our CO2 fixation rate and kta scaling equations, this 10-fold difference in rate of gaseous mass transfer readily explains the stark difference in carbon recovery between experiments conducted using the two different fermentation platforms.

[0116] Example 1 . Acetogens utilizing hydrogenases to uptake H2 as an electron source.

[0117] There are many acetogens, but they all have the common characteristic of utilizing WLP for CO2 fixation using H2 as an electron donor. Various acetogens have different H2 uptake characteristics, suggesting that their H2 utilization and CO2 fixation will be significantly improved by implementing the examples described above. Those acetogens expected to be relevant to the described technologies include, but not limited to, Acetobacterium carbinolicum.. Acetobacterium wieringae, Acetobacterium malicum,

[0118] Acetobacterium woodii, Clostridium aceticum, Clostridium autoethanogenum, Clostridium drakei, Clostridium luticellarii, Clostridium carboxidivorans, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa acidovorans, Sporomusa aerivorans, Sporomusa malonica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Thermoanaerobacter ki vui, Thermacetogenium phaeum, Eubacterium limosum. Successful expression or overexpression of uptake hydrogenases from an acetogen into another acetogen would improve hydrogen uptake by the latter. The hy drogen uptake sy stems in acetogens include several proteins working together, including, but not limited to formate dehydrogenase subunits, endogenous molybdopterin molybdotransferase, molybdopterin-guanine dinucleotide biosynthesis adapter protein, formate dehydrogenase subunit, NADH dehydrogenase I, NADH dehydrogenase related protein, 4Fe-4S ferredoxin, Fe-Fe hydrogenase, and endogenous electron transfer protein. There are additional proteins that would be beneficial for engineering the hydrogen uptake capabilities of acetogens. The genes for such additional proteins can be identified by screening genomic libraries from one or more acetogens. Screening genomic libraries in organism to identify desirable traits, here enhanced hydrogen uptake, is a well-known process in the art.

[0119] Example 16. Engineering the hydrogen uptake and fixation machinery for improved uptake of hydrogen in acetogens, such as Clostridium ljungdahlii, leading to improved uptake of CO2 by acetogens.

[0120] In the examples above, we have shown that the rate-limiting step for utilization of CO2 by acetogens is the consumption of hydrogen which provides the reducing equivalents required by the Wood-Ljungdahl pathway (WLP) for metabolizing CO2. Because acetogens can uptake hydrogen very effectively, engineering their hydrogen uptake machinery has been viewed as counter-intuitive and not necessary. Directly engineering the enzymes and enzyme complexes associated with hydrogen uptake will significantly improve the rate at which the engineered acetogen can consume CO2. Using Clostridium ljungdahlii as an example of an acetogenic bacterium, we specifically claim the overexpression of modification of the following enzymes as a method to increase hydrogen (and by extension CO2) fixation: endogenous formate dehydrogenase subunit (alpha subunit, CLJU_c06990), endogenous molybdopterin molybdotransferase (MoeA, CL JU c07000), endogenous molybdopterin-guanine dinucleotide biosynthesis adapter protein (CLJU_c07010), endogenous formate dehydrogenase subunit (FdhD, CLJU_c07020), endogenous NADH dehydrogenase I (E subunit, CLJU_c07030), endogenous NADH dehydrogenase I (F subunit, CLJU_c07040), endogenous NADH dehydrogenase related protein (CLJU_c07050), endogenous 4Fe-4S ferredoxin (CLJU_c07060), endogenous Fe-Fe hydrogenase (HydA2, CLJU_cO7O7O), and endogenous electron transfer protein (CLJU_c07080). We also claim the extension of this principle to other acetogens which contain the homologous proteins in well-conserved organization on their genomes.

[0121] Example 17. Use of falling film, trickle bed and packed bed columns and bioreactors.

[0122] The demonstrated technology can be also achieved by applying various types of bioreactor designed to increase gas to liquid mass transfer to emulate and surpass the increased hydrogen transfer rate (HTR) demonstrated using the roller bottles of Fig. 10. Those bioreactors include, but not limited to: stirred tank bioreactors, bubble column bioreactor, airlift bioreactor, fixed bed bioreactor, and significantly falling film and trickle- bed reactors. Stirred tank bioreactors, bubble column bioreactors, airlift bioreactors, and fixed bed bioreactors have been used extensively to carry7out industrial fermentations and are well known. Falling film reactors are used in chemical processing, notably the manufacture of sulfuric acid, but they have not been used in industrial fermentations because the oxygen transfer needs (high enough ki.a values) of industrial fermentations can be met by stirred tank bioreactors, bubble column bioreactors, airlift bioreactors, and to a lessed extent fixed bed bioreactors. Here we need higher ki.a values to increase the HTR. The concept of the roller bottle (Fig. 10) that has never been used in microbial fermentations can be scaled up using the falling film reactor, where the liquid falls down from the top a large surface area (flat or cylindrical) in a tower configuration while the gas is moving up from the bottom in counter-current configuration. This emulates and surpasses the gas transfer intensity of the roller bottle system (Fig. 10). It achieves much higher kta values than can be achieved in stirred tank bioreactors, bubble column bioreactors, airlift bioreactors. Instead of a flat or cylindrial surface, one can use a tower packed with packing particles of large surface so that the overall surface area for the contact of the liquid with gas is dramatically increased. The liguid is flowing from the top down while the gas flows up in a counter-current configuration. This is what is known as a trickle bed reactor.

[0123] Example 18. Microbes that produce hydrogen (from catabolizing various organic substrates) as coculture partners of acetogen.

[0124] There are a large number of microbes that use organic chemicals (including simple and complex carbohydrates, and proteins) and generate H2 and CO2. Such microorganisms are suitable for use in cocultures with acetogens like C. acetobutylicum in the examples above. All that is needed is the ability to grow in a medium that acetogens can grow- so that the hydrogen generated by these hydrogen producing organisms becomes available to feed the acetogen through close contact of the two types of microbes. The close contact and the generation of dissolved hydrogen in the immediate microenvironment of the acetogen makes hydrogen immediately available to the acetogen without the need for hydrogen to be transferred from a gas to the liquid for use by the acetogen. These other microorganisms are selected from a group of non-acetogenic bacteria that includes Clostridium beijerinckii,

[0125] Clostridium saccharolyticum, Clostridium butyricum, Clostridium cellulovorans , and Clostridium cellulolyticum, and other butyric acid bacteria, that is bacteria that produce buty ric acid and its derivatives when using the said organic molecules as substrates and while they produce hydrogen and carbon dioxide. Example 19. Feeding of other organic molecule substrates to support Clj and other acetogens for autotrophic metabolism.

[0126] We also claim the extension of feeding non-glucose sugars (arabinose, ribose. xylose, erythrose, and threose) which Clostridium ljungdahlii and other acetogens have the genes to utilize. We also claim the extension of this principle to the co-feeding with CO2 of alternative Cl substrates, such as formate and methanol; amino acids which can be broken down to generate energy by acetogens, such as histidine and arginine; and TCA intermediates, such as citrate, cis-aconitate, isocitrate, 2-oxo-glutarate, pyruvate, phosphoenol-pyruvate, oxaloacetate, malate, fumarate, and succinate. All these strategies rest on the same underlying protected principle of supplementing the energetic requirements of the cell with additional electron or energy sources to enable them to devote more cellular energy and machinery to consuming CO2.

[0127] Example 20. Combined engineering of hydrogenase and electron interconversion enzymes for increasing hydrogen production by solventogenic Clostridia, such as Clostridium acetobutylicum, as well as other microbes producing hydrogen as a method for increasing feeding of hydrogen to acetogens in cocultures.

[0128] We have shown that combining the overexpression of electron interconversion enzymes, such as glyceraldehyde-3-phosphate dehydrogenases (for example the endogenous gapN, CA_C3657), and / or exogenous NADH kinase (for example the heterologous pos5, YPL188W), alone or in combination with endogenous or heterologous production hydrogenases (for example the endogenous hydA, CA_C0028), can significantly increase the production of H2 by Clostridium acetobutylicum and other clostridia such as those described in Example 18. We also claim the overexpression of the electron-carrier protein ferredoxin (for example the endogenous ferredoxin, CA_C0303) alone or in combination with one or more of the aforementioned genes. These strategies work through the general principle of maximizing the electrons converted to and sequestered in the form of reduced ferredoxin, which can then be converted to hydrogen by a hydrogenase. Overexpression of electron interconversion enzymes works because, by increasing the redirection of NADH (which is produced in glycolysis) to NADPH (which is crucial for many growth associated biological reactions), electrons in the form of reduced ferredoxin that otherwise would have gone to NADPH production can instead be used for H2 generation. Overexpression of ferredoxin works by increasing the total amount of ferredoxin protein in the cell, which increases the amount of electrons that can be stored in the cell as reduced ferredoxin at any one time. Overexpressing a hydrogenase in combination with one or both of these two strategies works by increasing the rate of conversion of reduced ferredoxin to hydrogen. We claim the principle that, when combined in coculture with an acetogen such as Clostridium ljungdahlii, additional H2 production by a solventogenic clostridia, such as Clostridium acetobutylicum, due to the overexpression of one or more of these electron interconversion enzymes (with or without the additional overexpression of a hydrogenase) can stimulate the grow th and CO2 consumption rates of the acetogen due to direct exchange of H2 in the liquid phase of the coculture.

[0129] Example 21. Conversion of chemicals by Clostridium ljungdalii (Clj) and other acetogens in monoculture and coculture. Clj and other acetogens have extensive capabilities to reduce a broad variety of chemicals when added to its own cultures (monocultures) or when those chemicals are produced by partner microbes in cocultures. The reduction energy derives from sources of electrons such as hydrogen or the catabolism of other substrates by the acetogen. For partner microbes that produce H2 and CO2. Clj forms very close cell-to-cell contacts with those partner microbes including membrane and cell fusions and exchange of cellular material, and in the process alters the metabolism of those partner cells to make them produce more metabolites, such as acetone, carboxylic acids, ketones and aldehydes. These said, close cell-to-cell contacts are essential in imparting these capabilities to acetogens such as Clj and have remained unknown and unexplored for practical applications in biocatalysis until recently by work done in the laboratory of one of the inventors of this presently disclosed technology7. Clj and other acetogens can reduce those compounds to their corresponding chemicals: acetone to isopropanol, carboxylic acids to their corresponding aldehydes and alcohols, ketones to the corresponding alcohols, and aldehydes to the corresponding alcohols. Clj and other acetogens can also reduce nitrate salts (nitrates) to ammonium salts (frequently referred to as ammonia source). These capabilities enable acetogens to carry out a broad biocatalytic chemical reductions to produce a large array of chemicals. To do so. hydrogen is a most important, although not the only, source of electrons.

[0130] Example 22. Use of ore particles in cultures of acetogenic bacteria to generate hydrogen in the immediate vicinity of the acetogenic bacteria. Iron Silicate [(Fe)2SiO4] or magnesium iron silicate [(Mg,Fe)2SiC>4] are natural inexpensive minerals used as a blasting abrasive, in iron production and in concrete manufacturing and construction. Iron Silicate is known as fayalite Fe2SiO4 exists in nature as a component of the crystalline solid solution known as olivine (magnesium iron silicate).

[0131] Iron Silicate and magnesium iron silicate ore particles (referred to here and below as “ore particles”) catalyze the production of hydrogen in aqueous solutions. Although hydrogen production is relatively slow at the temperatures where cells grow (20-75 degrees centigrade), we found that when acetogens are grown with such ore particles, they accelerate the production of hydrogen and thus the use of CO2 by the acetogens. This is because the microbes, by attaching to the surface of the ore particles, accelerate the formation of hydrogen from water as catalyzed by the ore particles (Fig. 16). Here we claim the use of iron-silicate and magnesium-iron silicate ore particles to enhance the availability of molecular hydrogen in the immediate molecular vicinity of the microbes thus enhancing their growth and their ability' to use more CO2. The immediate use of H2 produced on the surface of the ore particles by the microbes in the same ore-surface microenvironment reduces the local partial pressure of H2 to zero and accelerates hydrogen production by the ore particles. This is called mass-transfer driven enhanced catalytic production of hydrogen, as catalyzed by the ore particles.

[0132] These ore particles that lead to hydrogen production in the micro-vicinity' of the acetogen microbes can be used in suspension cultures, whereby the small ore particles (size can vary from 0.5 to 800 microns) are suspended freely in the medium where the microbes grow under the mixing power from mixing devices such as impellers, other mixing devices, forced gas streams in combination. Packed-bed columns packed with iron silicate and magnesium iron silicate ore particles would enhance the kta coefficient of H2 transfer from externally supplied H2 or gas mixtures containing H2 such as H2 / CO2 / CO / N2 gas mixtures (Fig. 15), while also generating additional soluble H2 due to chemical catalysis by the column packing particles. The ore particle size in such packed bed columns can vary from 0.5 microns to several centimeters depending in the cross sectional area of the packed bed column. The ore particle size should be a fraction of the largest dimension of the cross section of the packed bed column. One can also use flat surfaces coated with iron Silicate and magnesium iron silicates such as monoliths, similar in geometric configuration to the monoliths used in catalytic converters of gas-powered cars. The combination of H2 evolution from the ore particles and the enhanced kta coefficient will significantly enhance H2 utilization and thus CO2 use leading to higher cell densities and higher production of metabolic products by the acetogens, growing alone or in coculture with other microbes such as in the examples above.

[0133] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED:

1. A method for enhancing hydrogen uptake by cells of an acetogenic bacterium in a culture, comprising: (a) incubating the cells in a liquid medium supplemented with an external gas mixture, wherein the external gas mixture comprises H2 and CO2;(b) overexpressing an uptake hydrogenase and / or an associated electron transfer protein in the cells; (c) increasing uptake of the H2 by the cells; and(d) enhancing uptake of the CO2 by the cells.

2. The method of claim 1, further comprising producing a metabolite by the cells with electrons from the H2 of step (c) and carbon from the CO2 of step (d).

3. The method of claim 1 or 2. wherein the external gas mixture further comprises CO.

4. The method of claim 1 or 2, wherein the external gas mixture consists of H2 and CO2.

5. The method of any one of claims 1-4. wherein the associated electron transfer protein is selected from the group consisting of formate dehydrogenase subunit, molybdopterin molybdotransferase, molybdopterin-guanine dinucleotide biosynthesis adapter protein, formate dehydrogenase subunit, NADH dehydrogenase I, NADH dehydrogenase I, NADH dehydrogenase related protein, 4Fe-4S ferredoxin, and electron transfer protein.

6. The method of any one of claims 1-5, further comprising increasing growth rate of the cells.

7. The method of any one of claims 1-6, further comprising increasing density of the cells.

8. The method of any one of claims 1 -7, further comprising supplying an organic molecule.

9. The method of claim 8, wherein the organic molecule is selected from the group consisting of non-glucose sugars, tricarboxylic acid (TC A) cycle intermediates, methanol, formate, and pentose phosphate pathway (PPP) intermediates.

10. The method of claim 9, wherein the non-glucose sugar is fructose.

11. The method of any one of claims 1-10, further comprising reducing production of a metabolite by the culture, wherein the metabolite is selected from the group consisting of acetone, nitrate, carboxylic acid, ketone, and aldehyde.

12. The method of any one of claims 1-11 , wherein the acetogenic bacterium is selected from the group consisting of Clostridium ljungdahlii (Clf), Acetobacterium carbinolicum, Acetobacterium v.'ieringae. Acetobacterium malicum, Acetobacterium woodii, Clostridium aceticum, Clostridium autoethanogenum, Clostridium drakei, Clostridium luticellarii , Clostridium carboxidivorans, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa acidovorans, Sporomusa aerivorans, Sporomusa malonica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Thermoanaerobacter kivui, Tltermacetogenium phaeum, and Eubacterium limosum.

13. The method of any one of claims 1-12, wherein the culture is a monoculture.

14. The method of any one of claims 1-12, wherein the culture is a coculture with additional cells of a non-acetogenic bacterium, wherein the additional cells produce H2 from catabolism of an organic molecule.

15. The method of claim 14, wherein the non-acetogenic bacterium is Clostridium.

16. The method of claim 15, wherein the Clostridium is selected from the group consisting of Clostridium acetobutylicum (Cac). Clostridium beijerinckii, Clostridium saccharolyticum. Clostridium butyricum, Clostridium cellulovorans. and Clostridium cellulolyticum.

17. The method of claim 15, wherein the Clostridium is a butyric-acid Clostridium.

18. The method of any one of claims 14-17, further comprising increasing hydrogen production by the additional cells.

19. The method of claim 18, further comprising overexpressing by the additional cells a gene encoding a protein selected from the group consisting of glyceraldehyde-3- phosphate dehydrogenases, ferredoxins, and allogeneic NADH kinase.

20. The method of claim 18, further comprising overexpressing by the additional cells a gene encoding a production hydrogenase.

21. The method of any one of claims 1-20, further comprising increasing the overall interfacial transport coefficient (kr.a) in the culture.

22. The method of claim 21, wherein the culture is in a bioreactor selected from the group consisting of stirred tank bioreactor, bubble column bioreactor, airlift bioreactor, falling film bioreactor, trickle-bed bioreactor, and fixed bed bioreactor.

23. The method of any one of claims 1-22, wherein the liquid medium comprises iron silicate or magnesium iron silicate ore particles, further comprising increasing hydrogen production at the immediate vicinity of the acetogenic bacteria.

24. The method of any one of claims 1-23, wherein the culture produces metabolic products in a sugar-carbon neutral manner.

25. The method of any one of claims 1-23, wherein the culture produces metabolic products in a sugar-carbon negative manner.

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