WO2014140703A1 - Régulation avancée de processus de fermentation - Google Patents

Régulation avancée de processus de fermentation Download PDF

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WO2014140703A1
WO2014140703A1 PCT/IB2013/056087 IB2013056087W WO2014140703A1 WO 2014140703 A1 WO2014140703 A1 WO 2014140703A1 IB 2013056087 W IB2013056087 W IB 2013056087W WO 2014140703 A1 WO2014140703 A1 WO 2014140703A1
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concentration
fermentation
dissolved
fermentation broth
glucose
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Yen-Han LIN
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University Of Saskatchewan
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    • 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
    • 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
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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

  • the present specification discloses methods for producing molecules by fermentation using microorganisms. Some embodiments of the present invention relate to methods for optimizing the yield of ethanol produced by fermentation by yeast.
  • Ethyl alcohol or ethanol is a major alcohol in today's energy intensive economy. Ethanol is used as biodiesel and is increasingly being used as a substitute for petroleum based fuels in blended forms. The current decline in fossil fuel production and increase in their cost has made alternative fuels like bio-ethanol and bio-diesel more attractive.
  • the main source of ethanol for both the food and energy industries are cereals and grains like corn and maize that are fermented to produce ethanol.
  • Currently, work on using cellulose to produce ethanol through biochemical and physiochemical processes has gained more importance as an alternative to using food grains and cereals.
  • the very essence of bio-ethanol production is the conversion of monosaccharides or sugars present in these sources to ethanol through oxidation.
  • Saccharomyces cerevisiae otherwise known as baker's yeast in general, is the most common species of yeast used in fermentation of glucose to ethanol. Ethanol obtained through traditional fermentation processes, used for the production of edible alcohol, tends to be of very low concentrations (10-12% v/v), thereby increasing downstream processing costs. The requirement for high ethanol concentration to reduce downstream processing costs has been satisfied by very-high-gravity (VHG) ethanol fermentation where typical feed sugar concentrations used for ethanol production are over 250 g/L.
  • VHG very-high-gravity
  • VHG fermentation processes Apart from producing higher ethanol concentrations and being an energy efficient process, VHG fermentation processes also increase the annual ethanol productivity (Devantier et al., 2005; Feng et al., 2012; Ho and Shanahan, 1986; Ingledew and Lin, 201 1; Piddocke et al., 2009; Saerens et al., 2008; Wang et al., 2007). All these advantages accrue together to lower per batch operating costs given that the majority of current VHG fermentation processes are batch processes.
  • Ethanol is a primary metabolite produced during the growth phases of yeast, i.e., lag and exponential phases, rather than the stationary phase where secondary metabolites are typically produced.
  • microbial growth is a requirement for ethanol production.
  • Ethanol production is not efficient in a completely anaerobic or aerobic environment.
  • O2 should be present for microbial growth and efficient ethanol production. This was demonstrated by Lin et al.
  • the CO 2 produced can exert a certain degree of inhibitory pressure on growth and survival of microorganisms (Jones and Greenfield, 1982; Lacoursiere et al., 1986; Mclntyre and McNeil, 1997; Mclntyre and McNeil, 1998; Mostafa and Gu, 2003; Pattison et al., 2000; Saucedo-Castaneda and Trejo-Hernandez, 1994). [0008] Acetaldehyde is reduced to ethanol with concomitant oxidation of NADH to NAD+.
  • the aerobic route leads to the production of acetyl-CoA (Ac-CoA) from pyruvate culminating in the tricarboxylic acid (TCA) cycle and production of ATP.
  • Ac-CoA acetyl-CoA
  • TCA tricarboxylic acid
  • Metabolites like acetic, succinic and pyruvic acid are produced when metabolic flux is present in this direction.
  • the aerobic route can also result in prioritizing production of biomass (from ATP) over production of ethanol (Daoud and Searle, 1990; Zeng and Deckwer, 1994).
  • Glycerol and trehalose are two such compounds. It has been shown that these compounds are produced through a branch in glycolysis, with intermediates produced during glycolysis acting as precursors for their production. These compounds remain within the cell membrane and protect the cells against stress while ATP is oxidized for cell maintenance. Once stress is relieved, production stops and these metabolites are excreted through the cell membrane.
  • glycerol is usually produced as a response to oxidative stress while trehalose is produced in response to the high osmotic pressure experienced by cells in VHG environments (Belo et al., 2003; Brown et al., 1981 ; Devantier et al., 2005; Wang et al., 2001).
  • Glycerol is believed to be produced as a means to produce NAD + . Accordingly a higher concentration of glycerol suggests a requirement for NAD + and consequent oxidation of NADH (Belo et al., 2003; Devantier et al., 2005). NAD + oxidizes glucose to pyruvate as it gets reduced to NADH. The reduction of acetaldehyde to ethanol is also accompanied by NADH oxidation to NAD + . Therefore during glycolysis, NADH and NAD + are continuously recycled (Figure 1). Stress induced by the high ethanol concentrations can reduce the production of ethanol and the associated NAD + as a result.
  • Glycerol is also believed to play the role of a redox sink. Consequently, a higher concentration of glycerol in the broth can also suggest oxidative stress in the fermentation broth.
  • VHG broths the very high sugar concentrations apart from resulting in higher osmotic pressures on yeast also increase the viscosity of the medium. This reduces the O2 solubility and as a result O2 concentration in the broth (Devantier et al., 2005; Gros et al., 1999; Ho and Shanahan, 1986; Schumpe and Deckwer, 1979; Schumpe et al., 1982; Verbelen et al., 2009).
  • Trehalose is produced to compensate for the higher osmotic stress witnessed during the initial stages under VHG conditions. Osmosis affects the intercellular transport characteristics of the cell membrane. Trehalose is believed to act as an osmo-protectant and maintain intercellular nutrient transport through the cell membrane under high osmotic pressures. Although these metabolites improve yeast survival under stress, they drain carbon from the intended product, i.e. ethanol.
  • CO2 is a major by-product of ethanol production. The evolution of CO2 from S.
  • cerevisiae can be stoichiometrically related to glucose consumption and ethanol production by Equation 1.
  • CO2 evolved Due to the stoichiometric relationship between glucose utilization, ethanol production, and yeast growth, the CO2 evolved is a direct measure of yeast activity in the fermentation broth. Hence, monitoring CO2 concentration should enable monitoring of the fermentation process in an inexpensive manner (Chen et al., 2008; Dahod, 1993; Daoud and Searle, 1990; El Haloui et al., 1988; Royce and Thornhill, 1991).
  • Carbon dioxide has been widely used as a measure of fermentation progress by several investigators (Golobic and Gjerkes, 1999; Manginot et al., 1997; Montague et al., 1986; Saucedo-Castaneda and Trejo-Hernandez, 1994; Taherzadeh et al., 1999).
  • the solubility of CO2 is about 10 times greater than that of oxygen in aqueous media (Kawase et al., 1992; Schumpe and Deckwer, 1979; Schumpe et al., 1982).
  • the protein rich fermentation broths tend to further decrease CO2 desorption as a result of increased binding of CO2 to the organic molecules on the cell membrane and in the broth.
  • Kruger et al. (1992), Kuriyama et al. (1993) and Kuhbeck et al. (2007) the presence of ethanol during VHG fermentation also enhances CO2 solubility in the broth.
  • Carbon dioxide is 4.5 times more soluble in aqueous solutions containing ethanol (Isenschmid et al., 1995).
  • cerevisiae one that of a product of decarboxylation reactions (conversion of pyruvate to acetaldehyde) or that of a substrate in carboxylation reactions (production of succinic acid from acetyl-CoA in the TCA cycle). They opined that presence of excessive CO 2 in the fermenter would result in growth inhibition while excessive stripping of CO2 would also stymie yeast metabolism during fermentation.
  • the concentration of CO ⁇ ions under this pH range is close to zero and between a pH of 6 and 7, is less than 0.3% (Dixon and Kell, 1989; Frahm, et al., 2002; Zosel et al., 201 1).
  • Equation 4 Due to the instantaneous nature of conversion of dissolved CO2 to carbonic acid (H 2 C0 3 ) the reactions illustrated by Equations 2 and 3 are combined and generally written Equation 4.
  • Carbon dioxide inhibition of microorganism growth can be classified into two distinct categories; inhibition due to HC0 3 ions and inhibition due to dissolved CO2. Despite the fact that two different species are involved, the underlying mechanism of inhibition remains the same. Effects exerted by bicarbonate ions or dissolved CO2 molecules are at the extracellular as well as intracellular level.
  • microorganisms (Dixon and Kell, 1989; Mclntyre and McNeil, 1997, 1998; Shang et al., 2003; Lacoursiere et al., 1986) have determined CO 2 based inhibition to have varied effects on different microorganisms. These microbes come from a variety of genus and species like Aspergillus niger, Escherichia coli, Penicillium chrysogenum, Ralstonia eutropha, and filamentous fungi.
  • CO 2 partial pressure p co
  • increased CO 2 partial pressures reduced production of undesired products, e.g. esters and fusel alcohols or flavor active volatiles, which could be attributed to growth inhibition caused by CO 2 .
  • Some authors have found that dissolved CO 2 can effect a change in cell morphology along with growth inhibition, including in studies of A. Niger and a few filamentous fungi, Z. mobilis, and Streptococcus mutants (Mclntyre and McNeil (1997, 1998); Dixon and Kell (1989)).
  • Oxygen (O2) has been considered as an important constituent in aerobic fermentation processes. In certain cases of aerobic fermentation the depletion of O2 in the fermentation system has been cited as a reason for decreased biomass as well as product yield (Belo et al., 2003;
  • VHG fermentation broths are more viscous than traditional fermentation broths that generally contain less than 12% (w/v) sugar.
  • higher viscosity reduces the mass transfer coefficient.
  • Metabolic activity is at its highest in the middle of the exponential phase. Increased activity is usually accompanied by net NADH production and hence increased presence of electrons in the system. This is observed as a steeply decreasing ORP.
  • the flatness of Region II was attributed to the balance between oxidizing and reducing powers in the system owing to the air supplied to the fermentation broth as a consequence of ORP control. This balance is due to the oxidizing nature of oxygen in air that acts as an electron acceptor. Towards the end, equilibrium shifts towards a highly oxidizing environment as a result of ethanol toxification, substrate exhaustion and the resulting reduction in metabolic activity.
  • An increase in ORP is usually a sign of reducing concentration of electrons in the system.
  • the increasing ORP values in Regions III and IV represent the late stationary and death phases. This is the case when yeast activity decreases accompanied by the consumption of NADH.
  • Redox potential has been used in several instances as a measure of fermentation progress in VHG ethanol fermentations. Feng et al. (2012) is a classic example of such work. Redox potential here was used to determine the point at which fresh feed was to be delivered for a fed- batch ethanol fermentation process. But, the inability to completely consume glucose in the case of 250 g/L feed glucose concentration resulted in the failure of this process being used for glucose concentrations higher than 200 g/L.
  • One of the interesting conclusions obtained through this study was the ability of yeast to adapt to ethanol concentrations higher than 85 g/L resulting in cell viabilities over 90% even towards the end of a given cycle when substrate concentrations were close to zero.
  • ORP was a measure of the number of electrons/protons in the system, the oxygen supplied into the fermenter would act as an electron acceptor and maintain the balance of electrons between source and sink.
  • a higher ORP indicated an abundant supply of electron acceptors while a lower ORP indicated a dearth in electron acceptors to compensate for the electrons released into the system by yeast.
  • Some embodiments provide methods of using a microorganism to produce a desired product from a feed by fermentation in a fermentation broth.
  • the concentration of a substrate, product or by-product of fermentation can be monitored in the fermentation broth and used as a basis for controlling fermentation.
  • the substrate, product or by-product of fermentation that is monitored is a concentration of dissolved CO 2 in the fermentation broth.
  • controlling the fermentation is done by detecting when a concentration of feed in the fermentation broth approaches or reaches zero.
  • the time at which the concentration of the feed in the fermentation broth approaches or reaches zero is detected by detecting a time at which there is an abrupt decrease in the concentration of dissolved CO 2 in the fermentation broth.
  • an abrupt decrease in the concentration of dissolved CO 2 in the fermentation broth is detected by detecting a time at which a rate of decrease in the concentration of dissolved CO 2 in the fermentation broth is accelerating.
  • the fermentation is a repeated-batch process.
  • a concentration of a substrate, product or by-product of fermentation in the fermentation broth is monitored.
  • a rate of decrease in the concentration of the substrate, product or by-product is determined.
  • the substrate, product or by-product of fermentation is CO 2 .
  • the microorganism is yeast.
  • the desired product is ethanol.
  • a microorganism is used to produce a desired product from a feed by fermentation, and a concentration of a substrate, product or by-product in the fermentation broth is controlled to enhance the fermentation process.
  • the substrate, product or by-product that is controlled is dissolved CO 2 .
  • a level of dissolved CO 2 is controlled at approximately a predetermined target level during fermentation.
  • the concentration of dissolved CO 2 present in the fermentation broth is controlled by sparging air through the fermentation broth.
  • a microorganism is used to produce a desired product from a feed by fermentation, and a concentration of a substrate, product or by-product in the fermentation broth is controlled to enhance the fermentation process.
  • the substrate, product or by-product that is controlled is dissolved CO 2 .
  • a level of dissolved CO 2 is controlled at approximately a predetermined target level during fermentation.
  • the concentration of dissolved CO 2 present in the fermentation broth is controlled by sparging air through the fermentation broth.
  • a microorganism is used to produce a desired product from a feed by fermentation
  • concentration of a substrate, product or by-product in the fermentation broth is controlled to enhance the fermentation process, and that substrate, product or by-product is used as a control parameter to control the fermentation process.
  • Figure 1 shows glycolysis in unicellular microorganisms for the utilization of glucose. Adopted from Biochemical pathways, 3rd edition, Part- 1 , Michal Gerhard (ed.), Roche Molecular Biochemicals.
  • Figure 2 shows an exemplary embodiment of a method for controlling a batch fermentation process by monitoring a control parameter.
  • Figure 3 shows an exemplary embodiment of a method for controlling a repeated-batch fermentation process by monitoring a control parameter.
  • Figure 4 shows an exemplary embodiment of a method for controlling a continuous fermentation process by monitoring a control parameter.
  • Figure 5 shows an exemplary embodiment of a method for controlling the concentration of dissolved CO2 while conducting fermentation using a batch process.
  • Figure 6 shows an exemplary embodiment of a method for controlling the concentration of dissolved CO2 while conducting fermentation using a repeated-batch process.
  • Figure 7 shows an exemplary embodiment of a method for controlling the concentration of dissolved CO2 while conducting fermentation using a continuous process.
  • Figure 8 shows a line diagram of an experimental set-up used to perform batch ethanol fermentation using yeast in one example embodiment.
  • Figure 9 shows representative profiles of biomass and dissolved CO2 concentration and dissolved CO2 accumulation observed during batch ethanol fermentation in the absence of CO2 control in example embodiments for a) 150 and b) 200.05 ⁇ 0.21 g glucose/L initial concentration from triplicate experiments.
  • Figure 10 shows representative profiles of biomass and dissolved CO2 concentration and dissolved CO2 accumulation observed during batch ethanol fermentation in the absence of CO2 control in example embodiments for a) 250.32 ⁇ 0.12 and b) 300.24 ⁇ 0.28 g glucose/L initial concentration from triplicate experiments.
  • Figure 1 1 shows representative concentration profiles of glucose and ethanol for example embodiments a) 150, b) 200.05 ⁇ 0.21, c) 250.32 ⁇ 0.12 and d) 300.24 ⁇ 0.28 g glucose/L initial concentration in batch ethanol fermentation from triplicate experiments without CO2 control. Initial concentration greater than 200 g glucose/L results in residual glucose even after -50 h of fermentation.
  • Figure 12 shows representative plots of ethanol concentration and cell viability profiles corresponding to profiles in Figures 9-1 1.
  • Figure 13 shows concentration profiles of a) dissolved CO2 and biomass and b) glucose and ethanol representing the four regions (I, II, III and IV) of the dissolved CO2 concentration profile observed in the presence of CO2 control for an example embodiment with an initial concentration of 259.72 ⁇ 7.96 g glucose/L. Dissolved CO2 was controlled at 750 mg/L using an aeration rate of 1300 mL/min.
  • Figure 14 shows concentration profiles of a) dissolved CO2 and biomass and b) glucose and ethanol representing the four regions of the dissolved CO2 concentration profile observed in the presence of control for initial concentration of 303.92 ⁇ 10.66 g glucose/L. Dissolved CO2 was controlled at 750 mg/L using an aeration rate of 820 mL/min.
  • Figure 15 shows representative profiles of example embodiments with dissolved CO2 concentration controlled at 500 mg/L under aeration rates of a) 820 and b) 1300 mL/min for initial concentration of 263.76 ⁇ 5.55 g glucose/L. Observed values were obtained from duplicate experiments.
  • Figure 16 shows profiles of glucose and ethanol concentration and cell viability representing duplicate experiments in example embodiments with dissolved CO2 controlled at 500 mg/L under aeration rates of a) 820 and b) 1300 mL/min for initial concentration of 263.76 ⁇ 5.55 g glucose/L. A final ethanol concentration of 103.31 ⁇ 2.19 g/L was obtained irrespective of the aeration rates.
  • Figure 17 shows representative profiles of example embodiments with dissolved CO2 concentration controlled at 750 mg/L under aeration rates of a) 820 and b) 1300 mL/min for initial concentration of 259.85 ⁇ 9.02 g glucose/L. Observed values were obtained from duplicate experiments.
  • Figure 18 shows representative profiles of example embodiments with dissolved CO2 concentration controlled at 750 mg/L under aeration rates of a) 820 and b) 1300 mL/min for initial concentration of 308.49 ⁇ 12.87 g glucose/L. Observed values were obtained from duplicate experiments.
  • Figure 19 shows profiles of glucose, ethanol and biomass concentration and cell viability representing duplicate experiments in example embodiments with dissolved CO2 controlled at 750 mg/L under initial concentrations and aeration rates of a) 259.85 ⁇ 9.02 and 820, b) 259.85 ⁇ 9.02 and 1300, c) 308.49 ⁇ 12.87 and 820 and d) 308.49 ⁇ 12.87 g glucose/L and 1300 mL/min respectively.
  • Figure 20 shows representative profiles of example embodiments with dissolved CO2 concentration controlled at 1000 mg/L under aeration rates of a) 820 and b) 1300 mL/min for initial concentration of 255.55 ⁇ 8.65 g glucose/L. Observed values were obtained from duplicate experiments.
  • Figure 21 shows representative profiles of example embodiments with dissolved CO2 concentration controlled at 1000 mg/L under aeration rates of a) 820 and b) 1300 mL/min for initial concentration of 299.36 ⁇ 6.66 g glucose/L. Observed values were obtained from duplicate experiments.
  • Figure 22 shows profiles of glucose, ethanol and biomass concentration and cell viability representing duplicate experiments in example embodiments with dissolved CO2 controlled at 1000 mg/L under initial concentrations and aeration rates of a) 255.55 ⁇ 8.65 and 820, b) 255.55 ⁇ 8.65 and 1300, c) 299.36 ⁇ 6.66 and 800 and d) 299.36 ⁇ 6.66 g glucose/L and 1300 mL/min respectively.
  • Figure 23 shows the quantity of oxygen bubbled through the fermentation broth representing the quantity of air bubbled through for example embodiments with initial concentrations of a) 259.72 ⁇ 7.96 and b) 303.92 ⁇ 10.66 under different set points and aeration rates.
  • 1 and 3 represent dissolved CO2 set points 500, 750 and 1000 mg/L respectively.
  • 1 and 2 represent dissolved CO2 set points 750 and 1000 mg/L;
  • a and B represent aeration rates 820 and 1300 mL/min respectively.
  • Figure 24 shows the maximum biomass and glycerol concentrations measured during the course of batch ethanol fermentation for example embodiments with different dissolved CO2 set points and aeration rates for initial concentrations of a) 259.72 ⁇ 7.96 and b) 303.92 ⁇ 10.66 g glucose/L in duplicate experiments for each case listed.
  • 1, 2 and 3 in Figure 24(a) denote dissolved CO2 set points of 500, 750 and 1000 mg/L respectively;
  • 1 and 2 in Figure 24(b) denote dissolved CO2 set points of 750 and 1000 mg/L while A and B refer to aeration rates of 820 and 1300 mL/min respectively.
  • Figure 25 shows the glucose conversion efficiencies obtained in the presence of CO2 control for example embodiments with initial concentration of a) 259.72 ⁇ 7.96 and b)
  • Figure 26 shows the ethanol productivities obtained in the presence of CO2 control example embodiments with an initial concentration of a) 259.72 ⁇ 7.96 and b) 303.92 ⁇ 10.66 g glucose/L from duplicate experiments.
  • 1, 2 and 3 in Figure 26(a) denote dissolved CO2 set points of 500, 750 and 1000 mg/L respectively;
  • 1 and 2 in Figure 26(b) denote dissolved CO2 set points of 750 and 1000 mg/L while A and B refer to aeration rates of 820 and 1300 mL/min respectively.
  • Ethanol productivity was calculated as [Ethanol Produced]—
  • Figure 27 shows a comparison of the a) O P and b) dissolved CO2 profiles observed under control of the respective quantities for an example embodiment with -300 g glucose/L initial concentration showing the four distinct regions of each profile.
  • Figure 27(a) was adapted from Lin et al. (2010) and Figure 27(b) is same as Figure 14 and Figure 18(a).
  • Figure 28 shows a plot of the concentration of glucose and the concentration of dissolved CO2 in a repeated-batch process conducted according to one example embodiment conducted at a glucose concentration of 200 g/L.
  • Figure 29 shows a plot of the concentration of ethanol in the repeated-batch process of the example shown in Figure 28. Description
  • measurement of a metabolic product or by-product produced during fermentation by a microorganism is used to provide information about the growth of the microorganism and/or the progress of the fermentation process (for example, the amount of substrate consumed and/or the amount of desired metabolic product produced), to allow the fermentation process to be controlled.
  • the metabolic product or by-product (i.e. control parameter) that is measured is dissolved CO2 concentration.
  • measurement of the concentration of a material utilized for fermentation by a microorganism i.e.
  • a substrate) to produce a desired product is used to provide information about the growth of the microorganism and/or the progress of the fermentation process (for example, the amount of substrate consumed and/or the amount of desired metabolic product produced).
  • the substrate so measured is a control parameter.
  • the substrate measured is dissolved CO2
  • the concentration of a control parameter is controlled to improve fermentation.
  • Some embodiments of the present invention pertain to monitoring the level of dissolved CO2 present in a medium in which fermentation is being conducted, i.e. in a fermentation broth. In some embodiments, such monitoring is used to monitor events such as the extent of feed utilization in the fermentation broth or the accumulation of toxic levels of products or by-products in the fermentation broth. Some embodiments of the present invention pertain to controlling fermentation by a microorganism by monitoring the level of dissolved CO2 present in a fermentation broth. The level of dissolved CO 2 present in the fermentation broth can indicate when various steps in the fermentation process should be taken, e.g. the addition of fresh feed in a continuous or repeat-batch process, or the halting of fermentation or collection of the desired product in any fermentation process including a batch process. Some embodiments of the present invention pertain to methods of controlling fermentation by a microorganism to optimize production of a desired product by controlling the level of dissolved CO 2 or other control parameter present in a fermentation broth.
  • microorganism means an organism that can be used to produce a small molecule through fermentation, and includes prokaryotes and fungi.
  • exemplary microorganisms include yeast and bacteria.
  • Exemplary species of microorganisms that can be used in some embodiments of the present invention include Saccharomyces cerevisiae, flocculating yeast, and other species of yeast, Escherichia coli or other species of bacteria, or any other microorganism that produces CO 2 as a byproduct of fermentation.
  • the microorganism is Actinobacillus succinogenes , Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, or Escherichia coli.
  • any organism or microorganism that generates CO 2 as a byproduct of fermentation can be used. For example, most organisms that utilize carbohydrates as a food source will generate CO 2 since CO 2 is a byproduct of the TCA cycle, and can be used to conduct fermentation in embodiments of the present invention.
  • the fermentation process used is a very-high-gravity (VHG) fermentation process.
  • VHG very-high-gravity
  • the fermentation process used is a batch process, repeated-batch process, or continuous process.
  • the fermentation process is conducted in a fermentation broth, which provides nutrients and an environment suitable for growth of the microorganisms.
  • the fermentation broth can comprise any appropriate media to support growth of the microorganism being used, together with an appropriate feed for the microorganism. Selection of an appropriate fermentation broth based on the microorganism is within the ability of one skilled in the art.
  • the fermentation process is carried out under suitable conditions of temperature, atmosphere, agitation and the like to facilitate growth of the microorganism being used.
  • feed means a suitable substrate provided to a microorganism to be converted to a desired product through fermentation.
  • feeds include carbohydrates.
  • the feed is a carbohydrate such as starch, cellulose, glucose, pentose, xylose or the like.
  • the type of feed used depends on the microorganism being used for fermentation. Not all microorganisms can ferment all types of carbohydrates. For example, some strains of S. cerevisiae may be unable to ferment pentose or xylose sugars, and thus pentose or xylose could not be used as feed for such strains because such sugars are not suitable substrates for that microorganism. Similarly, some strains of yeast can be engineered to utilize cellulose as a substrate and some bacteria can utilize cellulose as a substrate.
  • cellulose could provide a feed material for such strains of yeast or bacteria, but not for other strains of yeast or bacteria that cannot utilize cellulose as a substrate.
  • the selection of a suitable substrate to provide a feed to yield a desired product using a selected microorganism is within the ability of one skilled in the art.
  • Media includes all elements besides the feed that are required to support growth of the microorganism.
  • the media includes elements that enhance the growth of the microorganism but are not necessarily essential for microbial growth.
  • Examples of products that can be produced by fermentation using some embodiments include ethanol or succinic acid.
  • the product is a TCA cycle metabolite such as succinate, fumarate or malate.
  • the feed used is a substrate that results in microbial metabolism using a metabolic pathway that results in the production of CO 2 .
  • the feed used is a substrate that results in microbial metabolism involving glycolysis followed by the TCA cycle to produce CO 2 .
  • the feed is glucose.
  • the feed provided to the microorganisms yields a desired product that is produced by a metabolic pathway that uses CO 2 as a substrate.
  • a desired product that is produced by a metabolic pathway that uses CO 2 as a substrate.
  • the production of succinic acid involves usage of CO2 as a substrate to enhance succinate production.
  • other intermediates in the TCA cycle such as fumarate or malate also use CO 2 as a substrate and can be the desired product in some embodiments.
  • Equation 8 A mass balance equation based on the concentration of dissolved CO 2 as the control parameter was developed for use in some embodiments of the present invention.
  • the mass balance equation was developed with reference to the exemplary use of yeast to produce ethanol by fermentation as used in the examples described below, but could be applied to other types of microorganisms, substrates and/or fermentation products or by-products based on the exemplary results set forth herein. This mass balance is shown in Equation 8.
  • the left hand side (LHS) of Equation 8 relates the accumulation of CO 2 as dissolved CO 2 in the fermentation broth as a result of the deficit between generation and removal of dissolved CO 2 from the fermentation broth owing to the solubility of CO 2 represented on the right hand side ( HS) of Equation 8.
  • the first term on the RHS of Equation 8 refers to the rate of generation or production of CO 2 , otherwise known as CO 2 evolution rate ⁇ CER(t), mol/L-h), by yeast as a result of metabolic activity.
  • Metabolic activity being a function of yeast growth can be represented in terms of yeast specific growth rate ( ⁇ , 1/h) and instantaneous biomass concentration (in terms of viable cells) (X, g viable cell dry weight) (Equation 9).
  • a constant (Yco 2 , the biomass yield coefficient with respect to CO 2 ; g C(Vg viable cell dry weight) is used to represent the quantity of CO 2 generated per unit concentration of biomass.
  • CER(t) is related to yeast growth with the assumption that ⁇ (specific growth rate, 1/h) is representable of ft (maximum specific growth rate, 1/h) and Y co ⁇ is regarded as constant over the period of fermentation.
  • Equation 9 relates CO 2 produced during fermentation to yeast activity in the fermenter.
  • this equation can also be utilized to relate ethanol production to yeast activity during fermentation. Ethanol being a primary metabolite is produced only in the presence of active growth. Hence, a decrease in CER(t) not only indicates a reduction in viable cells, but also points to reduction in ethanol production during fermentation.
  • Equation 15 To mathematically express the conversion of dissolved CO 2 to HCO ⁇ ions as a result of pH fluctuations affecting the equilibrium between the different forms of existence of CO 2 in aqueous media, Equations 4-5 are considered. Based on these equations, the term corresponding to the third term on the RHS of Equation 8 was derived as follows in Equations 10 to 14. The derived term is given in Equation 15.
  • Equation 12 The rate of conversion of DCO2 to HCO 3 " in aqueous media is governed by the rate equation illustrated by Equation 12. Note that due to the absence of a limiting effect on the equilibrium of these ions, the concentration of water is left out of Equation 12.
  • Equation 13 Equation 13 reduces to Equation dt
  • reaction rate constants k t , and k- b can be determined with the aid of the Arrhenius relation for reaction rate constants.
  • Equation 15 considers CO2 present only in the form of HCO ⁇ ion and not as C0 3 2 ⁇ ion due to the pH of the system. Under pH of 4-6 witnessed in the examples described below, it was reported that less than 0.3% of CO2 exists as C0 3 2 ⁇ , about 3% exists as HCO ⁇ and the rest exists as dissolved CO2. Carbon dioxide present as dissolved CO2 over and above the equilibrium concentration, in theory, is desorbed into the fermenter headspace (Frahm, et al., 2002; Fig. 1 of Zosel et al., 2011). [0091] Desorption of CO2 from the aqueous to the gas phase is a physical process contingent upon the concentration gradient of CO2 between these two phases.
  • Equation 16 clarifies this aspect of Equation 8.
  • the rate of desorption is a function of the CO2 concentration gradient between the gas and liquid phase.
  • the gas phase equilibrium concentration of CO2 is expressed in terms of partial pressure as governed by Henry's Law in Equation 16 (where H c ° 2 is Henry's Law constant for CO2, L-atm/mole). Fermentation broths, under lab scale experimental conditions, are believed to be supersaturated with dissolved CO2 while microorganisms are metabolically active (Frahm, et al., 2002; Ho and Shanahan, 1986; Kuriyama et al., 1993; Song et al., 2007; Zosel et al., 201 1).
  • the concentration of dissolved CO2 in a fermentation broth is used as a control parameter and is monitored to control fermentation by a microorganism. In some embodiments, the concentration of dissolved CO2 in the fermentation broth is used to determine the point at which the feed in the fermentation broth has been completely or nearly completely used, i.e. the point at which the feed in the fermentation broth has been completely utilized or nearly completely utilized.
  • complete or nearly complete utilization of feed in a fermentation broth is detected by detecting an abrupt drop in dissolved CO2 concentration in the fermentation broth.
  • an abrupt drop in dissolved CO2 concentration in the fermentation broth is detected as a drop in dissolved CO2 concentration of more than about 200 mg/L in a time span of about 2 to 4 hours.
  • complete or nearly complete utilization of feed in a fermentation broth is detected by detecting the time at which the production rate of CO2 by the microorganism (CER(t) in Equation 18) approaches zero.
  • the fermentation process is stopped and/or the desired product of fermentation is collected after the time at which complete utilization of feed in the fermentation broth is detected.
  • an abrupt drop in dissolved CO2 concentration in the fermentation broth indicating that the feed has been completely or nearly completely utilized is detected as an accelerating rate of decrease in the concentration of dissolved CO2 in the fermentation broth.
  • the accelerating rate of decrease in the concentration of dissolved CO2 can be detected in any suitable manner.
  • the rate of decrease of the concentration of dissolved CO2 is determined by measuring the concentration of dissolved CO2 at periodic intervals and calculating the slope of a plot of dissolved CO2 concentration versus time across a time interval. A trend of progressively smaller slopes (i.e.
  • the magnitude of the slope of each adjacent measured interval is larger in the negative direction
  • the magnitude of difference between the slopes for adjacent consecutive time intervals is increasing (i.e. wherein the change in slope between each adjacent time interval is increasing in the negative direction, or in other words there is an acceleration in the rate of decrease in the concentration of dissolved CO2 in the fermentation broth) indicates that there has been an abrupt reduction in the concentration of dissolved CO2 in the fermentation broth.
  • the slopes of dissolved CO2 concentration versus time measured at three different time points were Ti -50, T2 -80, and T 3 -120 ppm CO2 min "1 , that would indicate an abrupt drop in dissolved CO2 concentration.
  • the slopes are getting progressively steeper in the negative direction (i.e. T2 is less than Ti, and T 3 is less than T 2 ), and the magnitude of the difference between the slopes for adjacent consecutive time intervals is increasing (i.e. the absolute difference between T 3 - T2 of 40 ppm CO2 min "1 is larger in magnitude than the absolute difference between T 2 - Ti of 30 ppm CO2 min "1 ).
  • the concentration of dissolved CO2 can be measured across any convenient time interval, depending on the rate of growth and metabolism of the microorganism in the fermentation broth.
  • the concentration of dissolved CO2 is measured at time intervals on the scale of minutes, e.g. between about 30 seconds and two minutes.
  • the concentration of dissolved CO2 is measured at time intervals of about one minute.
  • Use of electronic equipment to monitor the concentration of dissolved CO2 means this concentration can be sampled at any desired frequency (e.g. on a millisecond scale), but the choice of time interval between time points to be used to determine that the rate of decrease of dissolved CO2 concentration is increasing can be selected based on the timescale relevant to the rate of metabolism by the microorganism.
  • the number of measurements at adjacent consecutive time intervals of the rate of change of dissolved CO2 concentration versus time that should be compared to determine when there is an acceleration in the rate of decrease of the concentration of dissolved CO2 can vary.
  • the number of time points compared should be sufficient to ensure that environmental noise is not treated as an abrupt reduction in CO2 concentration.
  • comparison of the trend observed in three consecutive slopes of the rate of change of dissolved CO2 concentration versus time measured over adjacent intervals is used to determine when there is an acceleration in the rate of decrease of the concentration of dissolved CO2.
  • three, four, five, or more adjacent consecutive slopes are compared.
  • the selection of sampling frequency and the number of measurements to compare to detect an acceleration in the rate of decrease in the concentration of dissolved CO2 can be done by one skilled in the art based on the particular conditions of any given fermentation. For example, in embodiments in which the microorganism is Saccharomyces cerevisiae and the feed is glucose, the acceleration in the rate of reduction in the concentration of dissolved CO2 will be greater for lower glucose concentrations (e.g. in the range of about 200 g/L) than for higher glucose concentrations (e.g. in the range of about 250 or 300 g/L).
  • the fermentation process is a high-gravity fermentation process.
  • the fermentation process is a very-high-gravity (VHG) fermentation process.
  • the initial concentration of feed is between about 150 g/L and about 300 g/L.
  • the feed is glucose
  • the initial concentration of glucose in the fermentation broth is between about 150 g/L and 300 g/L, including any value therebetween, e.g. 175 g/L, 200 g/L, 225 g/L, 250 g/L or 275 g/L.
  • the fermentation process is a batch process and the process is stopped and/or the desired product collected when the feed in the fermentation broth has been completely or nearly completely utilized.
  • a batch process 100 is illustrated.
  • fermentation is initiated by combining the
  • the concentration of a control parameter is measured.
  • the control parameter indicates that the feed has been completely or nearly completely utilized, fermentation is stopped and the desired product is collected.
  • the control parameter is the concentration of dissolved CO2, and this parameter is monitored at 104. When the concentration of dissolved CO2 decreases abruptly, this indicates that the feed has been completely or nearly completely utilized and the fermentation is stopped and the desired product is collected at 106.
  • the fermentation process is a repeated-batch process in which a first portion of the feed is added to the fermentation broth, and a second portion of the feed is added to the fermentation broth after it has been determined that the feed in the first portion has been completely or nearly completely utilized based on the control parameter.
  • the repeated- batch process is a self-cycling fermentation process that monitors the concentration of dissolved CO2 as the feedback parameter (i.e. control parameter) used to control the process.
  • the fermentation process is a repeated-batch process and the concentration of dissolved CO2 is monitored.
  • a portion of the fermentation broth is withdrawn and an equal volume of fresh media with feed is added to replenish the fermentation broth.
  • the portion of the fermentation broth that is withdrawn is approximately one-half of the fermentation broth, e.g. between about 40% and about 60% or any portion therebetween of the fermentation broth.
  • the microorganism is maintained in its active growth phase or substantially in its active growth phase throughout the fermentation process.
  • maintenance of the microorganism in its active growth phase maximizes production of the desired product. It has been reported that growing yeast produces ethanol 33 times faster than stationary phase yeast (Snyder and Ingledew, Biofuels Business, May 2009, pp. 54-56, which is incorporated by reference). Thus, maintaining yeast in the active growth phase by conducting a repeated-batch process can enhance the production of ethanol by the yeast.
  • the microorganism adapts to the presence of inhibitory compounds in the fermentation broth, resulting in an accelerated fermentation rate (observable as a decreased cycle time between the complete utilization of the feed in the fermentation broth).
  • the microorganism is yeast and the desired product is ethanol
  • the yeast adapts to the presence of ethanol in the fermentation broth and/or to other conditions of fermentation during a repeated- batch process to accelerate the fermentation rate.
  • the accelerated fermentation rate increases the annual ethanol productivity.
  • the fermentation rate is increased after the completion of two or more cycles in the repeated-batch process, e.g. after two cycles, after three cycles, after four cycles, or the like.
  • this increased fermentation rate is observable as a decrease in the cycle time of the repeated-batch process.
  • the combination of accelerated fermentation rate and higher levels of ethanol production that can be achieved by keeping the microorganism in or substantially in its active growth phase during the repeated-batch process can increase the yield of ethanol and/or the annual productivity that can be obtained in some embodiments.
  • FIG. 3 shows an example embodiment of a repeated-batch process 110.
  • fermentation is initiated by combining the microorganism and feed in a fermentation broth and allowing the microorganism to grow under appropriate conditions of temperature and atmosphere.
  • concentration of a control parameter is measured.
  • the control parameter indicates the feed has been completely or nearly completely utilized, a portion of the spent fermentation broth including the desired product is removed and the desired product can be recovered from the spent fermentation broth at 120.
  • a portion of fresh media and feed is added to replace the removed spent fermentation broth.
  • the steps of monitoring the concentration of the control parameter 114, removing a portion of the spent fermentation broth including the desired product when the feed has been completely or nearly completely utilized 116, and adding a portion of fresh media including feed 118 are repeated any desired number of times. In some embodiments, these steps are repeated at least three times, at least four times, at least five times, at least six times, or more, e.g. more than ten times or between two and twenty times. In some embodiments, the volume of fermentation broth removed at 116 is approximately one-half the volume of the fermentation broth, e.g. between about 40% and about 60% of the volume.
  • the microorganism becomes acclimated to the fermentation conditions through repetition of steps 114, 116 and 118 so that the cycle time required to completely or nearly completely utilize the feed decreases after a few cycles, e.g. after two or three cycles in some embodiments.
  • the microorganism population is maintained approximately in an active growth phase throughout the repeated-batch process by repetition of steps 114, 116 and 118.
  • steps 114, 116 and 118 are repeated at least three times, at least four times, at least five times, at least six times, at least seven times, or more.
  • the control parameter measured at 114 is the concentration of dissolved CO2.
  • the concentration of dissolved CO2 indicates that the glucose feed has been completely or nearly completely utilized at 116 when the concentration of dissolved CO2 decreases abruptly, and ethanol is recovered at 120.
  • the fermentation process is a continuous process in which the concentration of dissolved CO2 in the fermentation broth is monitored and the rate of addition of feed is controlled based on the measured concentration of dissolved CO 2 .
  • substrate concentration can be maintained by the dilution rate (i.e. the volumetric feeding rate divided by the working volume of the fermenter).
  • the fermentation process is a continuous process in which the concentration of dissolved CO 2 in the fermentation broth is monitored and the rate and/or amount of air or other oxygen source introduced into the fermentation broth is regulated based on the concentration of dissolved CO 2 .
  • the fermentation process is a continuous process.
  • the continuous process is initiated in a manner analogous to a batch process, and the microorganisms are permitted to grow until the microorganisms reach the exponential growth phase.
  • the exponential growth phase can be detected by monitoring the concentration of dissolved CO 2 and detecting the time at which a peak of dissolved CO 2 concentration is reached.
  • feed and fresh media can be pumped in at a constant dilution rate, with spent broth being extracted from the fermenter simultaneously.
  • the concentration of dissolved CO 2 can then be monitored and/or the concentration of dissolved CO2 in the fermentation broth can be controlled to monitor the progress of and/or control the fermentation process.
  • FIG. 4 shows an example embodiment of a continuous fermentation process 130. Fermentation is initiated at 132 by combining the microorganism and feed in a fermentation broth and allowing the microorganism to grow under appropriate conditions of temperature and atmosphere. The microorganism is grown to exponential phase while the control parameter is monitored at 134. At 136, exponential growth is detected based on the control parameter. Once exponential growth has been detected, fresh feed and media is added to the fermentation broth at 138, and an approximately equivalent volume of spent fermentation broth is removed at 140. In some embodiments, fresh fermentation broth (i.e. fresh feed and media) is added (and spent fermentation broth is removed) at a constant or nearly constant dilution rate. In some embodiments, the control parameter is the concentration of dissolved CO 2 and the
  • the microorganism is yeast.
  • exponential growth is detected at 136 by detecting a peak in the concentration of dissolved CO 2 .
  • the fermentation process can be controlled by monitoring both the concentration of dissolved CO2 in the fermentation broth and the oxidation-reduction potential of the fermentation broth.
  • the concentration of dissolved CO2 is monitored to determine the point of complete feed utilization, while the oxidation- reduction potential of the fermentation broth is monitored to determine the point at which a concentration of the desired product in the fermentation broth reaches a toxic concentration.
  • an increase of oxidation-reduction potential value coincides with an abrupt reduction in yeast viability, and this is the point at which product toxicity becomes detrimental.
  • the fermentation process may be stopped after either complete or nearly complete feed utilization is detected based on dissolved CO2 concentration, or a toxic product concentration is achieved as detected by monitoring oxidation-reduction potential.
  • the fermentation process used is a repeated-batch process in which a first portion of the feed is added to the fermentation broth, and a second portion of the feed is added to the fermentation broth either after it has been determined that the first portion of feed has been completely or nearly completely utilized, as detected based on a substantial drop in dissolved CO2 concentration, or after a toxic product concentration is achieved as detected by monitoring oxidation-reduction potential.
  • it is determined that the first portion of feed has been completely or nearly completely utilized when the dissolved CO2 concentration decreases abruptly. Further portions of the feed are added after each subsequent portion of the feed has been completely or nearly completely utilized, or after a toxic product concentration is achieved, as detected by monitoring dissolved CO2 concentration and oxidation-reduction potential.
  • a concentration of dissolved CO2 or other control parameter in a fermentation broth is controlled to improve feed utilization by a microorganism in a fermentation process.
  • improving feed utilization means increasing the efficiency of conversion of the feed to a desired product of fermentation.
  • improving feed utilization means that the microorganism utilizes a greater proportion of the feed over the course of the fermentation.
  • improving feed utilization means that the microorganism utilizes all or nearly all of the feed provided in the fermentation broth.
  • improving feed utilization means that the microorganism converts the feed to the desired product more rapidly, resulting in higher productivity.
  • a concentration of dissolved CO 2 or other control parameter in a fermentation broth is controlled to improve the yield of a desired product produced by fermentation by a microorganism, i.e. to increase the overall amount of the desired product produced by the fermentation process.
  • a concentration of dissolved CO 2 or other control parameter in a fermentation broth is controlled to increase productivity by a microorganism, i.e. to increase the rate of production of a desired product in the fermentation process.
  • improvements in yield and/or productivity are achieved by increasing the biomass of the microorganism present in the fermentation broth.
  • a concentration of dissolved CO 2 or other control parameter in a fermentation broth is controlled to decrease the duration of a fermentation process used to produce a desired product using a microorganism.
  • a concentration of dissolved CO2 or other control parameter in a fermentation broth is controlled to increase the biomass of a microorganism in a fermentation process.
  • the increase in biomass of the microorganism decreases the duration of the fermentation process and/or increases the yield of the desired fermentation product and/or increases the productivity of the microorganism.
  • O 2 is provided to a fermentation broth to increase the metabolic activity of a microorganism during a fermentation process. In some embodiments, O 2 is provided to the fermentation broth by sparging air through the fermentation broth.
  • the fermentation process is a continuous process, i.e. a portion of the feed is being supplied to the fermentation process on a reasonably consistent basis throughout the course of fermentation while a corresponding amount of the fermentation broth is removed to maintain approximately the same volume.
  • the fermentation process is a batch fermentation process, i.e. the fermentation broth, the entire amount of feed to be used in the fermentation process, and the microorganism are combined and the fermentation process is permitted to continue until it is stopped and the desired fermentation product is collected.
  • the fermentation process is a repeated-batch process, i.e.
  • the fermentation broth, a first predetermined portion of the feed, and the microorganism are combined and the fermentation process is permitted to continue until a predetermined level of feed utilization is reached, at which time a second predetermined portion of the feed and fresh media is added to replenish the fermentation broth (after removal of a corresponding amount of the spent fermentation broth).
  • the time at which the predetermined level of feed utilization has been reached is determined by monitoring the concentration of dissolved CO2 in the fermentation broth.
  • the concentration of dissolved CO2 in the fermentation broth is controlled to a target level in the range of about 25% to about 70% of the maximum CO2 solubility in the fermentation broth, or any value therebetween, e.g. 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%.
  • the concentration of dissolved CO2 in the fermentation broth is controlled to a target level in the range of about 45% to about 60% of maximum CO2 solubility in the fermentation broth.
  • the concentration of dissolved CO2 in the fermentation broth is controlled to a target level of about 30%, about 45% or about 60% of the maximum CO2 solubility in the fermentation broth.
  • the solubility of CO2 in the fermentation broth is determined prior to selecting a target level for the concentration of dissolved CO2 in the fermentation broth.
  • an optimal target level of CO2 in the fermentation broth is determined empirically for the microorganism, feed and fermentation broth being used, and the concentration of dissolved CO2 in the fermentation broth is maintained at approximately the target level so determined, or within about ⁇ 5% of that level.
  • an optimal target level of CO2 in the fermentation broth may be determined empirically by conducting test fermentations at a plurality of different dissolved CO2 concentrations and selecting the dissolved CO2 concentration that results in the highest yield of ethanol, highest productivity, shortest fermentation time, or otherwise optimizes any other desired quality and using that concentration of dissolved CO2 as the target level of CO2.
  • the concentration of dissolved CO2 is controlled to a target level in the range of 500 mg/L to 1000 mg/L or any value therebetween, e.g. 550 mg/L, 600 mg/L, 650 mg/L, 700 mg/L, 750 mg/L, 800 mg/L, 850 mg/L, 900 mg/L or 950 mg/L. In some embodiments, the concentration of dissolved CO2 is controlled to a target level in the range of about 700 mg/L to about 800 mg/L.
  • the concentration of dissolved CO2 present in the fermentation broth can be controlled in any suitable manner.
  • the concentration of dissolved CO2 present in the fermentation broth is controlled by sparging air through the fermentation broth.
  • air is sparged through the fermentation broth at a rate in the range of about 700 mL/min to about 1400 mL/min or any value therebetween, e.g. 800 mL/min, 900 mL/min, 1000 mL/min, 1 100 mL/min, 1200 mL/min or 1300 mL/min.
  • the rate at which air is sparged through the fermentation broth is selected by balancing the cost of implementing a particular rate of air addition on an industrial scale with the efficiency and productivity associated with that particular rate of air addition.
  • O2 is supplied to the fermentation broth in any suitable manner during fermentation.
  • concentration of dissolved CO2 present in the fermentation broth is controlled by sparging air through the fermentation broth
  • O2 is provided to the fermentation broth in the sparged air.
  • Figure 5 shows an example embodiment of a batch process 150 incorporating control of the concentration of dissolved CO2 and using the concentration of dissolved C02 as a control parameter.
  • fermentation is initiated by combining the microorganism and feed with media in a fermentation broth and allowing the microorganism to grow under appropriate conditions of temperature and atmosphere.
  • the concentration of dissolved CO2 is measured.
  • air is sparged through the fermentation broth. The steps of measuring the concentration of dissolved CO2 154 and sparging air through the fermentation broth 156 are repeated to try to maintain the concentration of dissolved CO2 at the predetermined target concentration.
  • the step of monitoring the concentration of dissolved CO2 154 indicates that the feed has been completely or nearly completely utilized, fermentation is stopped and the desired product is collected at 158.
  • monitoring the concentration of dissolved CO2 at 154 indicates that the feed has been completely or nearly completely utilized when the concentration of dissolved CO2 decreases abruptly.
  • Figure 6 shows an example embodiment of a repeated-batch process 160
  • fermentation is initiated by combining the microorganism and feed in a fermentation broth and allowing the microorganism to grow under appropriate conditions of temperature and atmosphere.
  • concentration of dissolved CO2 is measured.
  • air is sparged through the fermentation broth. The steps of measuring the concentration of dissolved CO2 164 and sparging air through the fermentation broth 166 are repeated to try to maintain the concentration of dissolved CO2 at the predetermined target concentration.
  • a portion of the spent fermentation broth including the desired product is removed at 168 and the desired product can be recovered from the portion so removed at 170.
  • a portion of fresh media and feed is added to replace the removed spent fermentation broth.
  • the steps of monitoring the concentration of dissolved CO2 164, sparging air 166, removing a portion of the spent fermentation broth including the desired product 168, and adding a portion of fresh media including feed 172 are repeated any desired number of times. In some embodiments, these steps are repeated at least two times, at least three times, at least four times, at least five times, or more, e.g. more than ten times, or between ten and twenty times.
  • the concentration of dissolved CO2 indicates that the glucose feed has been completely or nearly completely utilized at block 164 when the concentration of dissolved CO 2 decreases abruptly.
  • the concentration of dissolved CO 2 indicates that the glucose feed has been completely or nearly completely utilized at block 164 when an acceleration in the rate of decrease in the concentration of dissolved CO 2 is detected, and ethanol is recovered at 170.
  • FIG. 7 shows an example embodiment of a continuous fermentation process 180 incorporating control of the concentration of dissolved CO 2 and using the concentration of dissolved CO 2 as a control parameter.
  • Fermentation is initiated at 182 by combining the microorganism and feed in a fermentation broth and allowing the microorganism to grow under suitable conditions of temperature and atmosphere.
  • the microorganism is grown to exponential phase while the concentration of dissolved CO 2 is monitored at 184.
  • exponential growth is detected by detecting a peak in the concentration of dissolved CO 2 .
  • fresh feed and media are added to the fermentation broth at a constant dilution rate at 188, and an approximately equivalent volume of spent fermentation broth is removed at 190.
  • the concentration of dissolved CO 2 is monitored at 192.
  • the concentration of dissolved CO 2 measured is higher than a predetermined target concentration, air is sparged through the fermentation broth.
  • the steps of measuring the concentration of dissolved CO 2 192 and sparging air through the fermentation broth 194 are repeated while steps 188 and 190 are being carried out to try to maintain the concentration of dissolved CO 2 at the predetermined target concentration.
  • the desired product is recovered from the spent fermentation broth removed at 190.
  • both the concentration of dissolved CO 2 in the fermentation broth and the oxidation-reduction potential of the fermentation broth may be controlled.
  • the desired product is succinic acid and the feed is glucose.
  • the microorganism is Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, or Escherichia coli.
  • the production of succinic acid involves usage of CO2 as a substrate.
  • CO2 is also used as a control parameter to control the fermentation.
  • the concentration of CO2 in the fermentation broth is controlled to optimize the production of succinic acid.
  • Ethanol RedTM strain of Saccharomyces cerevisiae obtained as dry yeast from Lesaffre Yeast Corp. (Milwaukee, MI, USA) was used during the course of this investigation. Prior to utilizing them batch fermentations, the dry yeast was rehydrated with 50 mL sterilized water, and cultured in YPD agar (10 g/L yeast extract, 10 g/L peptone, 20 g/L dextrose, and 20 g/L agar). Two sub-culture steps were performed to purify yeast strains and were stored in YPD agar coated petri dishes at 4 °C for later use.
  • the fermentation media was divided into three portions; part A: the required glucose concentration in 600 mL of reverse osmosis (RO) water; part B: 1% (w/v) of yeast extract, 0.2% (v/v) of MgS0 4 and 1% (v/v) of Urea in 100 mL of RO water; part C: 0.1% (w/v) of L-(+)- Sodium Glutamate Monohydrate, 0.5% (v/v) of KH 2 P0 4 , 0.1% (v/v) of (NH 4 ) 2 S0 4 and 0.1% (v/v) each of H 3 BO 3 , Na 2 Mo0 4 , MnS0 4 -H 2 0, CuS0 4 , KI, FeCl 3 -6H 2 0, CaCl 2 -2H 2 0 and ZnS0 4 -7H 2 0 in 100 mL of RO water.
  • part A the required glucose concentration in 600 mL of reverse osmosis (RO) water
  • part B 1% (w/v
  • each stock solution used in the media is given in Table 1. These portions were steam sterilized at 121 °C for 15 min as such and mixed aseptically in the fermenter/bioreactor after they cooled down to room temperature prior to fermentation. The fermentation media was made-up to the working volume by adding sterilized RO water to the mixture. Yeast extract was obtained from HiMedia Laboratories (Mumbai, India). All other chemicals were of reagent grade or higher purity. Table 1. Concentration of media constituents used as their stock solutions.
  • yeast grown on agar was pre-cultured until the mid-exponential phase in shake-flasks with a working volume of 100 mL for 18 hours at 32.3 °C in an incubator-shaker at 120 rpm.
  • the media for shake-flask cultures was separated into two parts; part A: the required glucose concentration (0.15, 0.20, 0.25 or 0.30% (w/v)) in 90 mL of RO water; part B: 1% (w/v) of yeast extract, 0.2% (v/v) of MgS0 4 and 0.5% (v/v) of Urea in 10 mL of RO water. All media constituents used from stock solutions follow the concentrations given in Table 2.1.
  • the media was steam sterilized in an autoclave at 121 °C for 15 min and allowed to cool down to room temperature prior to mixing. Yeast inoculation from agar plates was done aseptically after mixing.
  • Measurement of dissolved CO2 was done using a commercial autoclavable dissolved CO2 sensor (InPro 5000, Mettler-Toledo, Bedford, MA, USA). The measurement was done using an M400 controller (Mettler-Toledo, Bedford, MA, USA) and acquired using LabView (Version 8.5, National Instrument, Austin, TX, USA). The calibration of the sensor was performed using 1 -point and 2-point procedures provided by the manufacturer using pH buffers of 7.0 and 9.21 at 25 °C.
  • Dissolved CO2 concentrations during fermentations were controlled using two different techniques. Control was achieved by either using a mixture of calcium hydroxide (Ca(OH)2) and fermentation media or air. Dissolved CO2 control set points were set based on the maximum solubility of CO2 in aqueous fermentation media. The maximum solubility of CO2 in fermentation media is in the range of 1.5-1.8 g/L (Spinnler et al., 1987). The solubility of CO2 is influenced by the presence of organic and inorganic salts in the fermentation media (Royce and Thornhill, 1991; Royce P. N., 1992).
  • Calcium hydroxide (Ca(OH)2) was tested as a mechanism for controlling dissolved CO2 levels.
  • Calcium carbonate is an inert solid as far as ethanol fermentation is concerned. Calcium carbonate does not ionize and hence has negligible to no effect on yeast growth and survival and hence ethanol production.
  • the removal of the CaC0 3 formed as a result of CO2 absorption can be achieved through simple decantation of the spent fermentation broth. This is possible since CaC0 3 is insoluble in aqueous media.
  • calcium hydroxide was not found to sustain cell viabilities at acceptable levels under the conditions tested, and so only dissolved CO2 control with sparged air was further examined in these examples.
  • the fermenter was made accessible to three peristallic pump heads (Model 7013-20 and Model 7014-20, Cole-Parmer Canada Inc., QC, Canada).
  • the smaller head was connected to the nutrient and control solution reservoir, while the bigger head was used to discharge spent fermentation broth from the fermenter.
  • the working volume was kept constant by maintaining the harvesting tube at a fixed position.
  • Level maintenance was achieved through simultaneous addition of nutrient and control solution and removal of spent broth to and from the fermenter respectively through the PID controller.
  • the PID controller was used to actuate an air pump. Air from the pump was passed through a polytetrafluoroethylene (PTFE) membrane filter (PN 4251, Pall Corporation, Ann Arbor, MI, USA) prior to being bubbled through the broth by a sparger. Air was selected as the CO2 control agent based on its ability to not only remove dissolved CO2 but also supply O2 to the broth. 1.2.4 Fermentation conditions
  • the withdrawn aliquots were analyzed for biomass and cell viability.
  • the biomass was estimated semi-empirically in terms of optical density (OD) measurements made using a colorimeter (KlettTM Colorimeter, Belart, NJ, USA) at 600nm.
  • the OD values were calibrated for different values against cell dry weight (CDW) of yeast in order to obtain biomass concentrations for different OD values.
  • Cell viabilities were estimated using the methylene violet staining technique (Smart et al., 1999). In this technique yeast cells were stained with methylene violet stain and observed and counted under a microscope on a hemacytometer (Hausser Scientific, Horsham, PA, USA).
  • Toxic ethanol concentration limits were determined with the help of shake-flask experiments. Shake-flasks with media composition same as that described in Section 1.1.1 were used for this purpose. In addition to the growth medium, ethanol in concentrations varying from 4 % (v/v) through 12 % (v/v) in 2 % increments were added to the shake-flasks. The flasks were inoculated with yeast and cultured at 32.8 °C at 120 rpm for 24 hours. Measurements of biomass, cell viability and analysis of carbohydrates, organic acids and ethanol were made every two hours during this culture period. The biomass and ethanol concentrations were smoothed by a three-parameter logistic growth model as described in Section 2 of Liu et al.
  • Figures 9 and 10 show the dissolved CO2 concentration profiles as observed for 150, 200.05 ⁇ 0.21 and 250.32 ⁇ 0.12, 300.24 ⁇ 0.28 g glucose/L respectively in batch fermentations in the absence of any form of control of the concentration of dissolved CO2.
  • the corresponding glucose and ethanol concentration profiles are illustrated in Figure 1 1.
  • the first order derivative was applied to the observed dissolved CO2 concentration profiles shown in Figures 9 and 10.
  • the derived profiles as illustrated at the bottom of Figures 9 and 10 are
  • Figure 9 shows representative profiles of biomass and dissolved CO2 concentration and dissolved CO2 accumulation observed during batch ethanol fermentation in the absence of CO2 control for a) 150 and b) 200.05 ⁇ 0.21 g glucose/L initial concentration from triplicate experiments. Distinct regions observed during batch ethanol fermentation in the absence of any form of CO2 control are demarcated by dotted lines in Figure 9 as well as Figure 10.
  • the CO2 accumulation rate ( d([DC0 2 ]) ⁇ j s ⁇ Q fj rs t- 0 rder derivative of the CO2 concentration shown.
  • Figure 10 shows representative profiles of biomass and dissolved CO2 concentration and dissolved CO2 accumulation observed during batch ethanol fermentation in the absence of CO2 control for a) 250.32 ⁇ 0.12 and b) 300.24 ⁇ 0.28 g glucose/L initial concentration from triplicate experiments. Note the absence of the characteristic drop in dissolved CO2 concentration as seen in Figure 9.
  • Figure 1 1 shows representative concentration profiles of glucose and ethanol for a) 150, b) 200.05 ⁇ 0.21, c) 250.32 ⁇ 0.12 and d) 300.24 ⁇ 0.28 g glucose/L initial concentration in batch ethanol fermentation from triplicate experiments without control. Initial concentration greater than 200 g glucose/L results in residual glucose even after ⁇ 50 h of fermentation.
  • Equation 18 this would be represented as a ⁇ ( ⁇ + ⁇ ) resulting in a negative , with y being dt
  • Regions between Region I and Region II in Figures 9 and 10 are marked as transition regions. From the dissolved CO2 profiles for four glucose concentrations it can be observed that the duration of this region decreases significantly from -150 g/L to -300 g/L, with ⁇ 150g/L having the longest transition period. It is postulated that the change in duration of the transition period from lower to higher glucose concentration is affected by the presence of high glucose concentrations, i.e. lower glucose concentrations have a longer transition period due to lower osmosis.
  • the residual glucose concentration at the end of the transition periods for 150, 200.05 ⁇ 0.21, 250.32 ⁇ 0.12, and 300.24 ⁇ 0.28 g glucose/L were -130, -165, -210 and -270 g glucose/L respectively; the higher the initial glucose concentration, the higher the residual glucose concentration.
  • Inhibitory effects of ethanol on yeast activity were ruled out as the concentration of ethanol at the end of these transition regions were lower than the inhibitory concentration of 40g/L (18, 20, 30, and 28 g ethanol/L for -150, -200, -250, and -300 g glucose/L respectively; Figure 12). It is also possible that the osmotic effect due to higher glucose concentrations extends far beyond the lag phase into the exponential phase. Without being bound by theory, the drop in cell viability in high glucose concentration broths may be precipitated further by higher ethanol concentrations.
  • yeast growth is enhanced albeit with a delay in the lag phase resulting in an increase of CER(t) in Equation 18.
  • the absence of exponential increase in CO2 desorption rate with increase in CO2 dC evolution is reflected in the corresponding increase in dissolved CO2 concentration and as dt shown in Figures 9 and 10.
  • Control was achieved either using Ca(OH)2 or air.
  • Control with Ca(OH)2 was capable of absorbing dissolved CO2 and maintaining dissolved CO2 levels as dictated by the set points despite its very low solubility; however, addition of Ca(OH)2 to the fermentation broth reduced the ethanol concentration in the broth. Reduction of ethanol concentration defeated the purpose of VHG ethanol fermentation which is to produce high concentrations of ethanol.
  • Addition of Ca(OH)2 also increased the pH of the fermentation broth beyond 6. It is believed this resulted in an environment non-conducive for yeast growth. Yeast ceases to function at or close to neutral pH.
  • the dissolved CO2 profiles for initial concentrations of -250 g glucose/L can be split into four distinct regions irrespective of the level of CO2 control and aeration rate used.
  • Figures 13 and 14 illustrate differentiation of the dissolved CO2 profile into four distinct regions for the case of -250 and -300 g glucose/L, respectively, with dissolved CO2 controlled at 750 mg/L. These four regions have been hypothesized to represent the different levels of yeast activity and growth phases during fermentation. Initially yeast metabolism is known to be slow during the lag and early exponential phases of growth resulting in slower production of CO2. The osmotic effects induced by high initial glucose concentrations are known to compound this effect resulting in an increased duration for the lag phase.
  • the lag phase paves the way for the exponential phase towards the end of Region I.
  • Region II of Figures 13 and 14 characterize yeast growth during the mid and late exponential phases.
  • the increase in metabolic activity in this phase results in increased CO2 production and accumulation.
  • the supply of oxygen during transition from Region I to Region II, as a consequence of control is known to further enhance yeast vitality and consequently ethanol production (with reference to Equation 18, CER(t) is at its highest in this region).
  • the sparged air in addition to supplying oxygen also aids in the stripping of dissolved CO2 from the fermentation broth.
  • dissolved CO2 profiles depict an increase in the dissolved CO2 concentration over and above the set point values in Region II of Figures 13 and 14. This increase in concentration that can be construed as a net dissolved CO2 accumulation, has been postulated to represent the additional increase in CER(t) owing to oxygen supply as well as CO2 removal.
  • aFermentation duration was calculated on the basis of glucose concentration in the fermentation broth. A zero glucose concentration as pointed out by the dissolved C0 2 profile was considered as the end of fermentation. [0161] Further corroboration can be obtained from the higher rate of change of biomass seen in Region II when compared to other regions. While the mid exponential phase is the pinnacle of yeast activity, the late exponential phase signifies the transition to stationary phase growth and the associated reduction in metabolic activity. The complete transition from exponential to stationary phase is postulated to be represented in Region III of Figures 13 and 14 where the absence of a net dissolved CO2 accumulation is observed as an unchanging flat plateau in the dissolved CO2 concentration profiles. In reference to Equation 18, the net accumulation of dC
  • dissolved CO2 profiles observed in the presence of a dissolved CO2 based control strategy for VHG fermentation were characteristic for every batch for both initial concentrations of -250 and -300 g glucose/L. Except for certain specific cases (for 300 g glucose/L dissolved CO2 controlled at 1000 mg/L under aeration rates of 820 and 1300 mL/min as well as dissolved CO2 controlled at 750 mg/L under 1300 mL/min aeration) dissolved CO2 profiles depicted a marked increase in dissolved CO2 concentrations over and above the set point values in Region II for 250 as well as 300 g glucose/L.
  • Region I represents the lag phase growth of yeast during fermentation. In the lag phase or Region I, it is believed that control of dissolved CO2 does not play a role in altering fermentation characteristics in terms of yeast metabolism as is the case for the subsequent regions of the profile.
  • Incorporating the present CO2 control strategy in VHG fermentation processes achieves two objectives: 1) Removal of dissolved CO2 from the fermentation broth and 2) Oxygenation of the broth through aeration. For control to improve fermentation, both the aforementioned factors can be manipulated at optimum levels. To choose the optimum set point, dissolved CO2 was controlled at three distinct levels of 500, 750 and 1000 mg/L that represented 30, 45 and 60% of the maximum CO2 solubility in fermentation media respectively. While this was the case for -250 g/L initial glucose, only two set points of 750 and 1000 mg/L were used for -300 g glucose/L.
  • Dissolved CO2 profiles observed when dissolved CO2 was controlled for the case of -250 and -300 g glucose/L using different aeration rates are shown in Figures 15, 17, 18, 20 and 21.
  • Figures 16, 19 and 22 illustrate the corresponding glucose and ethanol concentration and cell viability profiles.
  • Figures 15, 17, 18, 20 and 21 display profiles for dissolved CO2 controlled at 500, 750 and 1000 mg/L under different initial glucose concentrations and aeration rates respectively. Dissolved CO2 profiles in Figure 15 (500 mg/L), 17 (750 mg/L) 18(a) (750 mg/L) and 20 (1000 mg/L) were obtained under 263.76 ⁇ 5.55, 259.85 ⁇ 9.02 308.49 ⁇ 12.87 and 255.55 ⁇ 8.65 g glucose/L initial condition and have attributes similar to those shown in Figures 13 and 14 and discussed in detail earlier in Section 2.2.1.
  • Carboxylation reactions play an important role in maintaining the rigidity and fluidity of the cell membrane (Jones and Greenfield, 1989). Excess stripping of CO2 might lead to lack of CO2 for maintenance of membrane fluidity that in turn affects inter-cellular transport characteristics of the cells as well as making them susceptible to ethanol inhibition and toxicity. Based on the same premise it could be postulated that controlling dissolved CO2 at 1000 mg/L resulted in insufficient stripping of CO2 from the broth causing an inhibitory effect. This would result in reduced conversion efficiencies for both 500 and 1000 mg/L set points.
  • a first potential reason could be the higher osmotic pressure in the case of -300 g glucose/L.
  • yeast under initial concentrations of -300 g glucose/L experience higher osmotic stress. This could be witnessed in the slow rate of decrease of glucose concentration as well as slow rate of increase in biomass concentration in Figures 19(c) and 19(d) when compared to that seen in Figures 19(a) and 19(b).
  • Osmotic effects could also explain the longer duration of fermentation required for -300 g glucose/L to attain zero residual glucose (Table 2).
  • the higher quantity of substrate supplied to the fermenter may in itself be responsible for the longer duration required to completely exhaust glucose to mark the end of fermentation in the case of -300 g glucose/L.
  • a second potential reason for a lack of resemblance in Region II could be the aeration rate.
  • a lower CER(t) as a result of osmotic effects combined with a higher aeration rate may result in removing excess dissolved CO2 thereby preventing net accumulation in Region II of Figure 18(b) as opposed to that seen in Figures 13-14 or Figure 18(a).
  • Glycerol concentrations were uniform irrespective of the dissolved CO2 set point as well as aeration rate but increased with increase in initial glucose (Figure 24).
  • Glycerol is produced as one of the several by-products of NAD + generation, just like ethanol. Given that ethanol production is strongly affected by changes in fermentation environment, ethanol production is neither a reliable nor a robust source for NAD + .
  • Glycerol production on the other hand being reliable as well as robust, contributes to a fixed flux for NAD + regeneration.
  • NAD + the shortfall in NAD + to maintain the NADH/NAD + balance is offset by directing flux to other pathways.
  • an equally valid argument would be that the inability of the cell to offset this shortfall results in loss of cell viability.
  • Air bubbling is also known to have a positive influence on the physiochemical aspects of fermentation. Air bubbling not only strips dissolved CO2 from the broth but also increases the concentration of cells that are in suspension. Increase in the suspended cell concentration improves mass transfer. Without being bound by theory, the summation of these effects could explain the very small variation in conversion efficiencies among the 750 and 1000 mg/L dissolved CO2 set points for different aeration rates under a given initial glucose concentration.
  • FIG. 27 compares profiles of dissolved CO2 and ORP for VHG ethanol fermentations in the presence of dissolved CO2 and ORP based control methodologies under similar initial glucose of -300 g/L. Visually, the two bath tub-shaped profiles are seen to be generally mirror images of each other. Due to the nature of ORP measurement the scrutiny of this discussion is restricted to the two major drawbacks of VHG ethanol fermentation; osmotic effects due to high initial glucose feeds and inhibition and toxicity due to high final ethanol concentrations.
  • ORP control is based on maintaining a redox balance in the system.
  • Control action in case of ORP measurement is initiated in response to imbalance of redox powers.
  • Redox potential measurements are thus relative in nature, constrained upon the initial electron/proton/redox activity in the fermentation broth. A low initial redox potential would result in more air being pumped into the system to maintain the redox balance during ORP control.
  • dissolved CO2 set point values are based on the solubility of CO2 for a given media under given conditions of temperature and pressure.
  • Region III in the dissolved CO2 curve is very similar in appearance to Region II of the ORP curve. While the zero slope regions in both the curves cannot be explained in terms of either the glucose or ethanol concentrations, it can be seen that alteration in yeast activity if any, due to the supply of air cannot be deciphered from the ORP curve. This is regarded as one of the major drawbacks of using a relativistic measure to control VHG ethanol fermentations. These similar regions (Region II in Figure 27(a) and Region III in Figure 27(b)) also represent regions of perfect control in their respective cases; i.e., a balance between reduction and oxidation is reached in case of ORP, and a balance between production and consumption of CO2 is reached in case of dissolved CO2.
  • Figures 28 and 29 show the results of the determination of the self-cycling period of yeast in an example repeated-batch fermentation process at 200 g glucose/L to produce ethanol.
  • the self-cycling period was estimated by measuring time elapsed between peaks of two contiguous cycles.
  • the batch fermentation time was reduced significantly from approximately 22 hours in the second and third cycle to an average of 14.67 ⁇ 2.7 hours for the last six cycles.
  • the glucose in the fermenter was completely or nearly completely utilized between cycles.
  • This example demonstrates that monitoring the concentration of dissolved CO2, one of the products of fermentation, can be used to control a repeated-batch process by detecting the point at which complete or nearly complete substrate utilization in the fermentation broth has been reached. This example also demonstrates that the abrupt reduction in dissolved CO2 concentration measured corresponds to complete glucose utilization.
  • monitoring the concentration of dissolved CO2 in the fermentation broth could be used to control a repeated-batch process using other microorganisms, feeds and substrates that utilize or result in the production of CO2.
  • this example demonstrates that the fermentation rate is accelerated as the yeast adapts to the fermentation environment, and that yeast can be maintained in its active growth phase to maximize ethanol production by monitoring the concentration of dissolved CO2 in a repeated-batch process.
  • yeast can be maintained in its active growth phase to maximize ethanol production by monitoring the concentration of dissolved CO2 in a repeated-batch process.

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Abstract

La présente invention concerne la surveillance d'une concentration d'un paramètre de régulation dans un bouillon de fermentation pour surveiller et/ou réguler la fermentation microbienne. Dans certains modes de réalisation, un taux d'un paramètre de régulation présent dans le bouillon de fermentation est régulé pour améliorer la fermentation microbienne. Dans certains modes de réalisation, le paramètre de régulation est la concentration en dioxyde de carbone dissous. Dans certains modes de réalisation, une chute brutale de la concentration en dioxyde de carbone dissous indique que l'alimentation en bouillon de fermentation a été complètement ou presque complètement utilisée.
PCT/IB2013/056087 2013-03-15 2013-07-24 Régulation avancée de processus de fermentation WO2014140703A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020152017A1 (fr) 2019-01-26 2020-07-30 Kazda Marian Procédé de surveillance automatique de réacteurs à biogaz
CN111836897A (zh) * 2018-03-30 2020-10-27 英威达纺织（英国）有限公司 用于在好氧生物合成期间控制氧浓度的方法
CN112326625A (zh) * 2020-11-06 2021-02-05 四川省丹丹郫县豆瓣集团股份有限公司 用于提升食品安全防控等级的成品检测方法
CN113462631A (zh) * 2021-06-30 2021-10-01 金华职业技术学院 一种有利于提高菌种密度的菌种发酵工艺
CN114752647A (zh) * 2022-04-08 2022-07-15 青岛啤酒股份有限公司 利用群体异质性评价酵母菌株抗逆性的方法

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Publication number Priority date Publication date Assignee Title
DE3939064A1 (de) * 1989-11-25 1991-05-29 Manfred Prof Dr Rer Grossmann Verfahren zur herstellung alkoholarmer bis alkoholfreier getraenke
US20020020667A1 (en) * 2000-06-13 2002-02-21 Brown David Geoffrey Method and apparatus for reducing foaming during fermentation
WO2007032265A1 (fr) * 2005-09-15 2007-03-22 New Century Fermentation Research Ltd. Appareil de culture continue pour bactérie productrice d’alcool et procédé de culture de la bactérie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3939064A1 (de) * 1989-11-25 1991-05-29 Manfred Prof Dr Rer Grossmann Verfahren zur herstellung alkoholarmer bis alkoholfreier getraenke
US20020020667A1 (en) * 2000-06-13 2002-02-21 Brown David Geoffrey Method and apparatus for reducing foaming during fermentation
WO2007032265A1 (fr) * 2005-09-15 2007-03-22 New Century Fermentation Research Ltd. Appareil de culture continue pour bactérie productrice d’alcool et procédé de culture de la bactérie

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111836897A (zh) * 2018-03-30 2020-10-27 英威达纺织（英国）有限公司 用于在好氧生物合成期间控制氧浓度的方法
CN111836897B (zh) * 2018-03-30 2023-10-17 英威达纺织（英国）有限公司 用于在好氧生物合成期间控制氧浓度的方法
WO2020152017A1 (fr) 2019-01-26 2020-07-30 Kazda Marian Procédé de surveillance automatique de réacteurs à biogaz
CN112326625A (zh) * 2020-11-06 2021-02-05 四川省丹丹郫县豆瓣集团股份有限公司 用于提升食品安全防控等级的成品检测方法
CN112326625B (zh) * 2020-11-06 2023-08-22 四川省丹丹郫县豆瓣集团股份有限公司 用于提升食品安全防控等级的成品检测方法
CN113462631A (zh) * 2021-06-30 2021-10-01 金华职业技术学院 一种有利于提高菌种密度的菌种发酵工艺
CN114752647A (zh) * 2022-04-08 2022-07-15 青岛啤酒股份有限公司 利用群体异质性评价酵母菌株抗逆性的方法

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