Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids
  • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/52—Propionic acid; Butyric acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• biofuels such as biodiesel and bioethanol
• biodiesel and bioethanol generally produced by transesterif ⁇ cation using vegetable oils or animal fats and an alcohol.
• a byproduct of this reaction is glycerol, and there is currently a surplus of waste glycerol produced in biofuel plants.
• Glycerol is a viscous liquid that is difficult to process, and it is generally disposed of by incineration or chemical processing.
• incineration of glycerol may release pollutants into the air. Rather than disposing of the glycerol, it would be better to process the glycerol into another chemically useful product.
• Glycerol can be used as a feedstock in biological conversion, a process in which a microorganism enzymatically converts glycerol into specific chemical products, such as ethanol or lactic acid. Conversion of glycerol to ethanol or other products is advantageous because they have value as fuel or as feedstocks for other processes.
• Anaerobic fermentation and aerobic respiration have been used for the industrial production of chemicals, and are the two methods applied to date for glycerol-based cultures.
• Oxygen rich respiration offers very efficient cell growth (growth rate and yield) and converts a 31175413-032 high percentage of the carbon source into carbon dioxide and cell mass (see table below).
• Anaerobic fermentation results in poor cell growth, but the synthesis of several fermentation products at high yields (e.g. lactate, formate, ethanol, propionate, succinate, etc.).
• anaerobic methods are usually preferred where possible, and it is typical to grow cells to a large number aerobically, and then switch the cells to anaerobic culture for the production of desired molecules.
• Another anaerobic method has been developed to biologically ferment glycerol into ethanol and other chemicals under anaerobic conditions. This method is described in co-pending WO2007115228, incorporated herein by reference in its entirety for all purposes. While an advantage of anaerobic fermentation of glycerol is the increased percentage yield of the desired end product, a disadvantage is slower bacterial growth associated with the lack of oxygen in the system. Thus, again what is needed in the art is a better system of culturing bacteria, using glycerol as a feedstock to produce high yields.
• HOUDMS/260652.1 31175413-032 but with a small amount of oxygen or other electron acceptor such as nitrate or nitrite, so that ultimately the redox balance for the conversion of the substrate into the product in a stoichiometrically defined pathway is equal to or greater than zero and a fraction of the substrate is used to generate cell mass.
• microaerobic what is meant is 1-20 mg O 2 /L/h or its equivalent.
• O 2-15 O 2 /L/h is used, and more preferably 3-10 or 5 mg O 2 /L/h.
• microaerobic conditions can also be produced by decreasing the stir rate (thus decreasing the oxygenation of a large culture), or by adding more feedstock to increase the cell density (and hence higher oxygen demand), or combinations thereof.
• ⁇ deletion or “disruption”
• Genes can be partially or completely deleted, or interrupted with stop codons, or mutated so as to yield an inactive protein product. Proteins can also be inactivated with inhibitors or by suppression of expression or translation, and the like.
• overexpression what is meant is that a gene or protein is changed so as to result in at least 150% activity as compared with an appropriate control strain. Preferably as much as 2, 5 or 10 fold increases in activity are obtained. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or upregulating the endogenous gene, and the like.
• a method for producing a chemical product in a microorganism that includes providing conditions to produce the product, wherein the degree of reduction balance for the conversion of the substrate into the product in a stoichiometrically defined pathway is equal to or greater than zero, the degree of reduction balance for the conversion of the substrate into cell mass is greater than zero, and a fraction of the substrate is used to generate cell mass. In addition, the degree of reduction balance for the conversion of the substrate into cell mass is greater than zero.
• the chemical product so produced can be, but is not limited to, lactate, ethanol, and succinate.
• FIG. 1 illustrates the production of reducing equivalents generated by the metabolism of glucose, xylose, or glycerol to produce succinate, ethanol, lactate, and acetate in accordance with the present techniques
• FIG. 2 illustrates the relationship between glycerol, chemical products produced from glycerol, and cell mass; and the net production of reducing equivalents in accordance with the present techniques
• FIG. 3 illustrates the pathway for producing succinate from glycerol in accordance with the present techniques
• FIG. 4 illustrates the pathway for producing ethanol from glycerol in accordance with the present techniques
• FIG. 5 illustrates the pathway for producing lactate from glycerol in accordance with the present techniques
• FIG. 6A is a graph comparing the fermentation of glycerol in a wild-type bacterial strain under microaerobic conditions and anaerobic conditions;
• FIG. 6B is a graph showing a characteristic profile of dissolved oxygen, OTR
• volumetric oxygen transfer rate and cell growth (OD 550 ) in a wild-type bacterial strain under microaerobic conditions
• FIG. 7 is a bar graph comparing the product and biomass yields of the fermentation of glycerol (solid bars), glucose (clear bars) and xylose (gray bars) in a wild-type bacterial strain under microaerobic conditions. Data is also shown for "oxygen yield", which represents the amount of oxygen consumed in the metabolism of the specific substrate;
• FIG. 8A is a graph comparing the production of ethanol, formate, and organic acids from glycerol under microaerobic conditions in a strain SY04 [pZSKLMgldA], which was engineered for the co-production of ethanol and formate (i.e. ⁇ pta, AfrdA, /SfdhF, gldA+, dhaKLM+);
• FIG. 8B is a graph comparing the production of ethanol, formate, and succinate from glycerol under microaerobic conditions in a mutagenic strain EH05 (pZSKLMgldA), which
• HOUDMS/260652.1 31175413-032 was engineered for the co-production of ethanol and hydrogen (i.e. Apta, AfrdA, AldhA, gldA+, dhaKLM+);
• FIG. 9A is a bar graph comparing the product yields of the fermentation of glycerol in wild-type MG1655 and strain LAOl (pZSglpKglpD) engineered for the production of D-lactate under microaerobic conditions (i.e. ApflB, AfrdA, glpK+, glpD+);
• FIG. 9B is a bar graph comparing the concentration of products of the fermentation of glycerol in wild-type MGl 655 and strain LA20 (pZSglpKglpD) engineered for the production of L-lactate under microaerobic conditions (i.e. AfrdA Apta AadhE AmgsA ⁇ t ⁇ /z ⁇ (ldhbovis), glpK+, glpD+);
• FIG. 9C is a bar graph comparing the concentration of products of the fermentation of glycerol in wild-type MG 1655 and strain SA24 (pZSpyc) engineered for the production of succinate under microaerobic conditions (i.e. AadhE Apta ApoxB Appc, pyc+);
• FIG. 10 illustrates the pathways engineered for the production of succinate from glycerol in exemplary mutagenic microorganisms in accordance with the present techniques
• FIG. 11 illustrates the pathways engineered for the production of ethanol from glycerol in exemplary mutagenic microorganisms in accordance with the present techniques.
• FIG. 12 illustrates the pathway engineered for the production of lactate from glycerol in exemplary mutagenic microorganisms in accordance with the present techniques.
• the synthesis of microbial cell mass from certain carbon sources may be harnessed under the appropriate conditions to produce a useful chemical product.
• the microbial synthesis of cell mass from carbon sources that are more reduced than biomass, such as glycerol, is a metabolic process that results in the net generation of reducing equivalents.
• reaction conditions in which there are substantially no electron acceptors available e.g. under micro- respiratory or micro-aerobic conditions
• conversion of some of the feedstock into a reduced product allows the reaction to maintain redox balance.
• the activity of this conversion to a reduced product facilitates attaining redox balance by consuming the excess reducing equivalents generated in the synthesis of cell mass from the carbon source.
• a redox- balanced system allows for increased microbial growth, which in turn allows the reaction to produce increased amounts of the desired chemical product.
• the present techniques may release redox constraints imposed by the incorporation of carbon sources into cellular biomass, a metabolic process that results in the generation of reducing equivalents when the carbon sources are more reduced than the biomass.
• the low levels of oxygen available under microaerobic conditions may serve as a sink for the excess reducing equivalents, thus improving the redox balance, allowing the incorporation of a larger fraction of glycerol into cell mass, and generating additional ATP.
• biomass or “cell mass” relate to the amount of a given cell culture, i.e. any organism or living cell growing in a medium, that is actively growing.
• the cell mass yield of a cell culture may be measured as gram cell mass obtained per liter of culture medium.
• the present techniques may be useful for the production of any suitable product wherein the degree of reduction balance for the conversion of the carbon source into the chemical product is equal to or greater than zero, i.e., the conversion of glycerol into the chemical product does not consume more reducing equivalents than it produces.
• Such products may include alcohols, alcohol derivatives, sugar alcohols ⁇ e.g. ethanol, propanol, butanol, butanediol, arabitol, xylitol), organic acids (e.g. formate, succinate, propionate, fumarate, malate, lactate, propionate, pyruvate,), amino acids (e.g. alanine, asparagine, aspartic acid,), or lactones (e.g. gamma-butyrolactone, caprolactone/lactone).
• alcohols alcohol derivatives
• sugar alcohols ⁇ e.g. ethanol, propanol, butanol, butanediol, arabi
• Table 1 Analysis of redox balance for the conversion of glycerol into cell mass and selected products.
• eCell mass formula is the average of reported for different microorganisms, including bacteria, yeast, and fungi (Nielsen et al., 2003). Conversion of glycerol into cell mass neglects carbon losses as 1-C metabolites. In consequence, the degree of reduction balance in this case represents the minimum amount of redox units generated.
• Table 2 Analysis of redox balance for the conversion of glycerol into cell mass and selected fermentation products.
• Table 3 Analysis off redox balance for the conversion of sugars into cell mass and selected fermentation products.
• the synthesis of cell mass results in the net consumption of reducing equivalents (i.e., ⁇ K ⁇ 0).
• Redox balance in such cases can be achieved in the absence of electron acceptors.
• the present techniques address disadvantages associated with anaerobic fermentation of reduced carbon sources such as glycerol and the by-products created during the production of chemical products from glycerol.
• the conversion of glycerol into cell mass results in the net gain of reducing equivalents (Table 1).
• the conversion of glycerol into succinate or lactate for example, does not consume the excess reducing equivalents generated by the conversion of glycerol into cell mass; in the case of converting glycerol into lactate, the redox balance is further perturbed by a net gain of two reducing equivalents. This redox potential imbalance can be ameliorated through the co-production of a chemical like 1,3-
• glycerol may be used to produce ethanol, lactic acid, and succinic acid by wild-type E. coli.
• much larger concentrations of succinate were produced under microaerobic conditions (FIG. 6) with improved product yields (FIG. 7) as compared to the succinate production under anaerobic conditions.
• mutant bacterial strains were evaluated for their ability to produce products whose synthesis results in no net generation or consumption of reducing equivalents.
• production of ethanol (along with co-products formate or hydrogen) is one of the products in this category. Evaluation
• the ethanol yield was 0.96 mole/mole of glycerol utilized and volumetric productivities for glycerol utilization and ethanol synthesis were 4.65 mole/L/h and 4.61 mole/L/h, respectively (FIG. 8B).
• the high productivities realized were the result of both higher cell density and higher specific rates under microaerobic conditions, compared to those obtained with the use of anaerobic conditions.
• the microaerobic conditions used were characterized by a volumetric oxygen transfer rate of 5 mg O 2 /L/h.
• FIG 9A, 9B, and 9C show increased production of lactic acid and succinic acid by a combination of appropriate microaerobic conditions and specific genetic manipulations.
• the microaerobic conditions used in these experiments were characterized by a volumetric oxygen transfer rate of 20 mg O 2 /L/L
• any appropriate microbial strain may be used with the present techniques, including members of the genera Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Dunaliella, Debaryomyces, Mucor, Torylopsis, Bacillus, Paenibacillus and Escherichia.
• a suitable microbial strain for the microbial synthesis process may be any wild type strain capable of producing the reduced chemical compound of interest.
• a suitable microbial strain may be obtained and/or improved by subjecting a parent strain of interest to a classical mutagenic treatment or to recombinant DNA techniques.
• strains may then be subjected to sequential selection processes of metabolic evolution in which progeny strains are evaluated for improved performance under the desired reaction conditions.
• progeny strains may be selected on the basis of high-performing biocatalysts.
• Markers of improved performance of a progeny strain may be increased production or yield of the desired chemical product from the glycerol feedstock.
• metabolic evolution is conducted in the presence of decreasing amounts of oxygen, nitrate, nitrite or other electron acceptor.
• Genes that will positively affect the production of the desired chemical product may be expressed in suitable hosts. For example it may be highly desirable to over-express certain enzymes in the glycerol to dihydroxyacetone pathway and/or other pathways at levels far higher than currently found in wild type cells. As shown in FIGs. 8A and B, and FIGs. 9A, 9B, and 9C the overexpression of gldA in a suitable microbial host in combination with the inhibition of enzymes involved in competing pathways may increase the production of succinate, ethanol, or lactic acid.
• Recombinant organisms that have been genetically engineered to express one or more exogenous polynucleotide coding sequences, i.e., a gene sequence that is not native, or endogenous, to the host organism can also be used in the invention.
• Overexpression of desired genes may be accomplished by the selective cloning of the genes encoding those enzymes into multicopy plasmids or placing those genes under a strong inducible or constitutive promoter. Methods for overexpressing desired proteins are common and well known in the art of molecular biology. It is envisioned that any of the genes provided in Table 4 may be overexpressed in a microbial strain.
• Fumarate reductase frdABCD E. coli
• phosphoenolpyruvate carboxylase ppc E. coli
• the present method will be able to make use of cells having single or multiple mutations specifically designed to enhance the production of the desired end product.
• Cells that normally divert a carbon feed stock into nonproductive pathways, or that exhibit significant catabolite repression could be mutated to avoid these phenotypic deficiencies.
• many wild type cells are subject to catabolite repression from glucose and by-products in the media and it is contemplated that mutant strains of these wild type organisms would be particularly useful in certain embodiments of the present
• any of the genes in Table 5 may be mutated, either alone or in combination with other genes, in a microbial organism for use in the present techniques.
• microorganisms that lack an indicated gene are considered to be knock outs of that gene.
• FIG. 10 depicts a schematic pathway for the production of succinate or propionate from glycerol in accordance with the present techniques.
• a suitable microorganism may include a mutation of the ⁇ dhE gene.
• Additional mutations may involve the mutation of the pt ⁇ , ⁇ ckA, or poxB genes involved in acetate formation; the ppc gene involved in the conversion of phosphoenolpyruvate into oxaloacetate; the glpABC operon, glpD, or glpK, involved in the conversion of glycerol to glycerol-3-P to dihydroxyacetone phosphate; or dh ⁇ KLM
• HOUDMS/260652.1 31175413-032 involved in the conversion of dihydroxyacetone to dihydroxyacetone phosphate.
• mutations or combinations thereof may be combined with the overexpression of either endogenous or heterologous genes, including but not limited to: gldA from E. coli and comparable genes from other organisms; dhaKLcff ⁇ om Citrobacter freundii; pepck from Actinobacillus succinogenes; or pyc from Rhizobium etli.
• ethanol production from glycerol may be increased through the inhibition of competing pathways that produce acetate and succinate byproducts.
• These mutations may involve the mutation of the pta, ackA, or poxB genes involved in acetate formation; the frdABCD operon involved in succinate formation; the glpABC operon, glpD or glpK, involved in the conversion of glycerol to dihydroxyacetone phosphate.
• These mutations or combinations thereof may be combined with the overexpression of either endogenous or heterologous genes, including but not limited to: gldA from E. coli and comparable genes from other organisms or dhaKLcff ⁇ om Citrobacter freundii.
• lactate production from glycerol may be increased through the inhibition of competing pathways that produce acetate, succinate, and ethanol by-products.
• These mutations may involve the mutation of the pta, ackA, or poxB genes involved in acetate formation; the frdABCD operon involved in succinate formation; the adhE gene involved in ethanol formation.
• Further genetic alterations may involve overexpression of the glpABC operon, glpD or glpK, involved in the conversion of glycerol to dihydroxyacetone phosphate.
• mutants having the desired phenotype may be selected by a variety of methods. Random screening is most common where the mutagenized cells are selected for the ability to produce the desired product. Alternatively, selective isolation of mutants can be performed by growing a mutagenized population on selective media where only resistant colonies can develop. In one embodiment, cells may be subjected to mutagenesis prior to or during selection for metabolic evolution. Targeted mutagenesis through recombinant techniques can also be employed separately, or in combination with random mutagenic techniques.
• Preferred growth media may include minimal media or minimal salt media.
• suitable media may include 4-morpholinepropanesulfonic acid media (MOPS).
• MOPS 4-morpholinepropanesulfonic acid media
• the phosphate salts in the media may be replaced with sodium salts.
• Glycerol feedstock may be provided in any suitable amount.
• glycerol may be provided at 10g/L.
• Suitable pH ranges for the fermentation may be between pH 5.0 to pH 9.0. It is envisioned that alkaline pH may be suitable for improved yield of certain products while acidic pH may be suitable for improved yield of other products. For example, acidic pH may promote production of ethanol and hydrogen while alkaline pH may promote the production of ethanol and formic acid.
• Fermentations may be conducted in any suitable microaerobic or microrespiratory fermentation system. It is envisioned that room air, oxygen, and/or nitrogen may be provided to the system in suitable amounts and at a suitable rate such that, as the growth rate of the microbes approaches log phase, there are substantially no detectable electron acceptors in the culture.
• the present process may employ a batch method of fermentation.
• a batch method of fermentation As used herein,
• “fermentation” may refer to microbial growth under microaerobic or microrespiratory conditions suitable for the production of reduced chemical products from glycerol.
• a classical batch fermentation may be a closed system where the composition of the media is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the media is inoculated with the desired organism or organisms and fermentation is permitted to occur adding nothing to the system.
• a "batch" fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration.
• the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted.
• Sequential fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses.
• a fed-batch strategy for example, may be advantageous.
• a fed-batch fermentation is an open system where a defined fermentation medium is added at the beginning of
• HOUDMS/260652 1 31175413-032 the fermentation and is inoculated with the desired organism or organisms and the fermentation is permitted to occur.
• a fed-batch fermentation involves the periodic or continuous addition of the carbon source and any additional limiting nutrients, with little or no removal of culture medium from the fermentation reaction during the fermentation process.
• Continuous fermentation is an open system where a defined fermentation media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing.
• Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
• Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration.
• the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable.
• cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production of reduced products from glycerol.

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