ADVANCED PROCESS CONTROL FOR FERMENTATION

Reference to Related Applications

[0001] This application claims priority to and the benefit of U.S. provisional patent application No. 61/792,482 filed 15 March 2013, the entirety of which is incorporated by reference herein.

Technical Field

[0002] 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.

Background

[0003] Ethyl alcohol or ethanol, as it is commonly known, 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. While glucose is the most common monosaccharide, sugars derived from cellulose also contain other sugars such as pentose and xylose depending on the source of cellulose. [0004] 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. 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.

Very-high-gravity ethanol fermentation

[0005] Very-high-gravity fermentation was extensively studied by Thomas et al., (1994) and Devantier et al., (2005). In VHG ethanol fermentation the high glucose concentrations can induce additional osmotic stress on the yeast cells during the initial growth phase (lag phase), while the very high final ethanol concentration can inhibit yeast survival towards the end of the process leading to loss of cell viability (Feng et al., 2012; Lin et al., 2010; Liu et al., 201 la, 201 lb; Piddocke et al., 2009). High inhibitory ethanol concentrations are observed under VHG conditions due to high feed glucose concentrations used in the process (Feng et al., 2012;

Ingledew and Lin, 201 1 ; Lin et al., 2010; Liu et al., 201 la, 201 lb). Ethanol concentrations over 40 g/L are believed to exert inhibition on yeast metabolism. In some cases, inhibition can turn to toxicity when ethanol concentrations in the fermentation broth exceed -90 g/L (Feng et al, 2012; Lin et al., 2010). Inhibitory stresses combined with loss of cell viability can result in shifting of metabolic flux away from ethanol production.

[0006] 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. Thus, microbial growth is a requirement for ethanol production. Ethanol production is not efficient in a completely anaerobic or aerobic environment. Preferably, O2 should be present for microbial growth and efficient ethanol production. This was demonstrated by Lin et al.

(2010) and Liu et al. (201 la, 201 lb). They concluded that sparging air through the fermentation broth during the course of fermentation improved yeast viability and consequently ethanol production. Therefore it has been stressed that despite the advantages of ethanol fermentation under VHG environments, inhibitions due to the lack of oxygen, high initial glucose concentrations and high final ethanol concentrations should be taken into consideration while designing efficient fermentation processes in general for the production of bio-ethanol (Hill, 2006; Ho and Shanahan, 1986; Jones and Greenfield, 1982; Kuhbeck et al., 2007; Kuriyama et al., 1993;).

Glycolysis in Saccharomyces cerevisiae

[0007] The production of ethanol from glucose in S. cerevisiae follows the glycolytic pathway. Glycolysis culminates in two distinct metabolisms; aerobic and anaerobic. Figure 1 represents a simplified representation of glycolysis in unicellular organisms. The acetaldehyde-ethanol shuttle is a part of anaerobic metabolism and is preceded by the conversion of pyruvate to acetaldehyde and carbon dioxide (CO ₂ ). Hence, ethanol production is accompanied by the simultaneous production of CO ₂ , a by-product of glycolysis. 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+. In the presence of oxygen, 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. 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).

[0009] It is believed that from the biochemical perspective of yeast growth, stress associated with either yeast growth or ethanol production is usually exhibited in the form of production of certain metabolites not produced otherwise. The production of such metabolites is accompanied by the production of ATP to meet the increased energy demands of the cell under stress.

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. In the context of VHG fermentation, 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).

[0010] 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.

[0011] In order to compensate for the loss of production of NAD ⁺ , theoretically, metabolic flux shifts from production of pyruvate to production of other metabolites that are accompanied by the oxidation of NADH to NAD ⁺ . Generation of glycerol from glyceraldehyde-6-phosphate is one such mechanism.

[0012] 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. In 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. [0013] The inhibitory effects of the metabolic products of glycolysis have been extensively studied by various authors (Brown et al., 1981 ; Feng et al., 2012; Ingledew and Lin, 201 1 ; Liu et al., 201 la; Maiorella et al., 1983; Pampulha and Loureiro-Dias, 1989; Piddocke et al., 2009). Inhibitory pressures tend to shift the pathway away from ethanol production. Maiorella et al. (1983) pointed to the inhibitory effect of non-volatile by-products like organic acids and aldehydes that are concentrated in the fermenter as a consequence of ethanol removal. They found that ethanol concentrations over 80 g/L resulted in 80% reduction of cell mass and termed this value as the inhibitory concentration. The findings of later authors regarding inhibition induced by high feed glucose concentrations (Feng et al., 2012; Ingledew and Lin, 201 1 ; Liu et al., 201 la) agreed with that of Maiorella et al. (1983). Maiorella et al. (1983) also hypothesized from their investigations that ethanol productivity (g ethanol/g cell) could be increased as a result of certain type of inhibitions. Inhibitions that usually affect the membrane transport

characteristics of the cells were focused upon for this study. Thus, the various forms of inhibition experienced during the course of fermentation tend to reduce the efficiency of conversion of glucose to ethanol in turn affecting fermentation performance.

Carbon dioxide in fermentation

[0014] 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.

C ₆ H ₁₂ 0 ₆ →2C ₂ H ₅ OH + 2C0 ₂ ^

[0015] Stoichiometry dictates that for every mole of glucose utilized by yeast, 2 moles each of ethanol and CO2 are produced during glycolysis (Daoud and Searle, 1990). Apart from CO2 produced by the decarboxylation of pyruvate to acetaldehyde, decarboxylation steps in the TCA cycle also result in CO2 production. However, for the production of certain metabolites in the carboxylation steps of the TCA cycle like succinic acid, CO2 also acts as a substrate (Ho and Shanahan, 1986; Lee et al., 1999; Song et al., 2007; Xi et al., 201 1).

Fermentation measurement using carbon dioxide

[0016] 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).

[0017] Measurements based on off-gas CO2 tend to be erroneous due to the dissolution of CO2 in the fermentation broth. Dahod, (1993) in his investigations concurred that the dissolved CO2 concentration was always higher than the equilibrium gas phase CO2 concentration. The variation between dissolved CO2 and the corresponding equilibrium gas phase concentration increased with increase in air flow rates. This variation and dependence were higher for broths of higher viscosity than for broths of lower viscosity. The observation was extrapolated and concluded that dissolved and off-gas CO2 do not exist in equilibrium in fermentation systems except under near plug flow conditions (Ho and Shanahan, 1986). Works of several authors (Montague et al., 1986; Pattison et al., 2000; Renger et al., 1992) in the area of C0 ₂ inhibition assumed near equilibrium conditions between off-gas and dissolved CO2. However, a few earlier authors (Dixon and Kell, 1989; Ho and Shanahan, 1986; Jones and Greenfield, 1982; Royce and Thornhill, 1991 ; Royce P. N., 1992; Zosel et al., 201 1) recommended against assuming such equilibrium environments except under specific conditions.

[0018] Typically 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. According to 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). The exact threshold of ethanol concentration was not mentioned though. This has been argued in terms of decrease in pH with increase in ethanol concentration as well. As described below, lower pH favors the presence of CO2 as dissolved CO2. Although dissolution of CO2 in the fermenter broth was accounted for in some models (e.g. El Haloui et al., 1988) by the use of constants, most off-gas measurements do not account for this discrepancy.

Carbon dioxide inhibition in ethanol fermentation

[0019] Despite its ability to represent fermentation progress, CO ₂ has been known to have an inhibitory effect on microorganisms typically used in fermentations. Various studies have been conducted on the effects of different parameters on fermentation processes using S. cervisiae such as pH (Dombek and Ingram, 1987), temperature (Saerens et al., 2008), pitching rate (Verbelen et al., 2009), oxygen requirement (Bonnefond et al., 2002; Verbelen et al., 2009;) and addition of certain flavor active solids (Kuhbeck et al., 2007). Although these apparently visible inhibitions have so far been taken into consideration in industrial VHG ethanol fermentation, the effect of CO ₂ produced during yeast metabolism has largely been neglected due to the fact that this effect is not as conspicuous as other forms of inhibition (El-Sabbagh et al., 2006; Jones and Greenfield, 1982; Kuriyama et al., 1993; Kruger et al., 1992; Mclntyre and McNeil, 1997; Mclntyre and McNeil, 1998; Mostafa and Gu, 2003; Shimoda et al., 2001). Primary work in the area of CO2 inhibition was pioneered by Jones and Greenfield (1982). Works performed in the last 3 decades have been built upon this work as a basis. Jones and Greenfield (1982) were of the opinion that CO ₂ not only played the role of an inhibitor in fermentations using S. cerevisiae, but also a role in improving yeast survival under conditions of stress in the fermentation broth. They put forward a theory that CO ₂ is involved in two different roles in the metabolism of S.

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.

Carbon dioxide behavior in aqueous systems

[0020] Theories pertaining to inhibition of microbial activity by CO ₂ have been proposed on the basis of the behavior of CO ₂ in aqueous environments. This description is necessary in order to justify the use of CO ₂ measurements as measures of microbial activity (Dixon and Kell, 1989; Royce, 1992; Yagi and Yoshida, 1977). CO ₂ in aqueous environments primarily can exist as three different species viz., CO2 (aq.) or dissolved CO2, HC0 ₃ ^~ ions and carbonate ions ( CO^ ).The pH of the aqueous system governs the equilibrium between the three species as described by Equations 2-5. The existence of CO2 as either dissolved CO2, HC0 ₃ or CO^ ions hence depends on the fermentation media in the present case, irrespective of the organism used for fermentation (Daoud and Searle, 1990; Frick and Junker, 1999; Golobic and Gjerkes, 1999; Zosel et al., 201 1). The general behavior of the three species was described through Figure 1 of Zosel et al. (201 1). According to Zosel et al. (201 1) and Frick and Junker (1999), an increase in dissolved CO2 is usually witnessed with a decrease in pH. Under the pH values of 4-6 that are prevalent in ethanol fermentation systems, CO2 exists primarily as dissolved CO2 with very minor concentrations of ions. 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).

CO2 (aqueous) -> H2CO ₃ [2]

Γ31

CO2 (gas phase) -> CO2 (aqueous) ^{1 J} C0 ₂ +H ₂ 0 ^ H ⁺ + HCO3 ^" iw = 5.35xlO ^"7 mol/L ^[4]

HCO _} -> H ⁺ + CO 3 - K ₂ = 6.12x 10 ^"11 mol/L [5]

[0021] 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.

Mechanism of carbon dioxide inhibition

[0022] 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.

[0023] Various theories proposed seem to agree on the fact that the CO2, whether in its native dissolved form or as HC0 ₃ ion, projects its effect on microorganisms on both intracellular as well as extracellular fronts starting with its absorption into the cell membrane. In contrast, literature also points out the importance of the presence of CO ₂ in the fermentation system as dissolved CO ₂ to aid in the growth and survival of yeast cells by playing a role in the construction of the cell membrane and facilitating transport through the membrane (in carboxylation reactions) (Dixon and Kell, 1989; Jones and Greenfield, 1982).

Effects of carbon dioxide inhibition on microorganisms

[0024] A few authors who have studied CO ₂ inhibition in S. cerevisiae and other

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.

[0025] Some authors have found that higher CO ₂ partial pressure (p _co ) can have a negative effect on yeast growth. Some authors have found that 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)).

[0026] Galazzo and Bailey (1990) postulated that the effects of CO ₂ inhibition on yeast and ethanol production are essentially separate and decoupled. They attributed this to feedback effects of products generated post glycolysis and postulated that CO ₂ inhibition only affects yeast growth and not ethanol production during fermentation.

[0027] In contrast to these studies, Kuriyama et al. (1993) suggested that higher CO ₂ concentrations actually increase ethanol productivity per cell. To support the claim, they suggested that higher CO ₂ concentrations decreased cell viability due to inhibition and this resulted in higher ethanol produced per cell. The reduction in the number of cells also agrees with the findings of Isenschmid et al. (1995). They observed that the presence of CO2 either in the off-gas or aqueous phase reduced the number of larger cells. They argued that decrease in cells larger than 5.6xl0 ^~n mL in volume could be a result of the solvent effect exerted by CO2 and could result in reduced fermentation rates. They also proposed a mechanism wherein stress alters activity of certain key enzymes in glycolysis. They attributed this alteration under high CO2 partial pressure to the shift in equilibrium during glycolysis. Since CO2 is a by-product of the pyruvate-dehydrogenase system, higher CO2 concentration tends to shift equilibrium to the pyruvate side thereby resulting in increased pyruvate production. This increase in pyruvate translates to increased acetaldehyde production followed by ethanol. This effect is similar to those proposed by Galazzo and Bailey (1990).

[0028] Thus, support in literature exists for both theories; the one where inhibitions and stress reduce growth as well as metabolite production and the other where inhibitions limit growth but enhance metabolite production, effectively decoupling growth and ethanol production.

Role of oxygen in ethanol fermentation

[0029] 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;

Ligthelm et al., 1988). The process of ethanol production through fermentation by S. cerevisiae is essentially an anaerobic process while the generation of biomass during this process is O2 dependent. Daoud and Searle (1990) proposed two separate equations, Equations 6 and 7, for anaerobic and aerobic fermentation of glucose by S. cerevisiae respectively. According to these equations, aerobic oxidation of glucose releases more energy in the form of ATP in comparison to anaerobic oxidation. The energy released in the process is used for cell growth and maintenance.

C ₆ H _n 0 ₆ → 2C ₂ H ₅ OH + 2C0 ₂ + 2 ATP [6]

C ₆ H _n 0 ₆ + 6<¾→ 6C0 ₂ + 6H ₂ 0 + 28 ATP

[7] [0030] Addition of O2 to the fermentation broth during sluggish fermentation may reduce fermentation time because of the effect of O2 on cell viability (Fornairon-Bonnefond et al., 2002; Verduyn et al., 1990). Optimized oxygenation during high-gravity fermentation may achieve improvements in fermentation efficiency and yeast cell viability (Verbelen et al., 2009).

[0031] On the other hand researchers have also pointed towards the existence of toxicity due to excess oxygen in the system (Belo et al., 2003; Fornairon-Bonnefond et al., 2002;). Belo et al. (2003) termed this as hyperoxia. Hyperoxia was witnessed when S. cerevisiae was cultivated under hyperbaric conditions of O2 partial pressure greater than 0.32 MPa.

Carbon dioxide removal in submerged fermentations

[0032] Several authors (D'Amore and Stewart, 1987; Kuhbeck et al., 2007; Reddy and Reddy, 2005; Shen et al., 2004) have shown that the addition of particulate materials improves fermentation. It has been postulated that addition of particulates provides higher nucleation sites for dissolved CO2 to form bubbles and desorb from the aqueous media. Kuhbecket al. (2007) postulated that addition of particulate matter induces a bubbling effect that results not only in the decrease of dissolved CO2 concentration due to CO2 desorption but also increases concentration of cells in suspension. The increase of cells in suspension improves the surface area of contact between the cells and the fermentation broth consequently improving cell metabolic rates and metabolite production due to improved mass transfer rates. VHG fermentation broths are more viscous than traditional fermentation broths that generally contain less than 12% (w/v) sugar. In terms of mass transfer required for desorption of CO2 from the aqueous to the gas phase, higher viscosity reduces the mass transfer coefficient. Redox potential based control of very-high-gravity fermentation

[0033] The combination of oxidation and reduction reactions mentioned above, otherwise known as redox reactions, results in the medium being electron rich or electron deficient. This addition or subtraction of charge results in a net potential difference in the fermentation media (Lin et al., 2010). This potential, otherwise known as redox potential or oxidation-reduction potential (ORP), is used as a measure of fermentation progress. Metabolic activity of a microorganism results in the release of electrons into the surrounding environment. When metabolic activity slows or ceases as a result of ethanol toxicity, the release of electrons is also slowed or stopped, and the resulting increase in the concentration of protons in the fermentation broth causes the oxidation-reduction potential activity to increase when metabolic activity in the fermenter is slowed or shut down.

[0034] Redox potential profiles for VHG ethanol fermentation under different glucose concentrations were reported by Lin et al. (2010). Lin et al. (2010) reported that the different regions depicted for the ORP profile correspond to different levels of yeast activity (Fig. 1 of Lin et al., 2010). The decreasing region or Region I corresponded to lag and exponential phases where reducing power outweighs the oxidizing power indicating increase in yeast activity.

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. Hence, 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.

[0035] 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. [0036] Liu et al. (201 la, 201 lb) utilized ORP as a control measure to improve fermentation efficiencies in VHG environments. They hypothesized ORP could be controlled by sparging air into the fermentation broth. The basis behind their hypothesis was that ORP being 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. [0037] Controlled fermentations were carried out in batch and continuous modes to assess their efficacies. It was concluded that maintaining ORP at specific levels (-50, -100 and -150 mV) had different effects on the ethanol productivity of the process. While it was easy to maintain control for a longer period at -50 mV, the duration of control reduced with decrease in the ORP values. The results also pointed to increased ethanol production at -150 mV level when compared to -50 mV for four different feed glucose concentrations (150, 200, 250 and 300 g/L). Although these results were promising, the major flaw with the control methodology utilizing ORP was the nature of the measurement made. Redox potential being a measure of electron activity is relative in nature. A relative measure changes with each process run depending on the initial conditions of the process.

[0038] The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. There remains a need for improved control techniques for microbial fermentation. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

Summary

[0039] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements. [0040] 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. In some embodiments, the substrate, product or by-product of fermentation that is monitored is a concentration of dissolved CO ₂ in the fermentation broth. In some embodiments, controlling the fermentation is done by detecting when a concentration of feed in the fermentation broth approaches or reaches zero. In some embodiments, 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. In some embodiments, 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.

[0041] In some embodiments, 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. When it has been determined that the rate of decrease in the concentration of the substrate, product or byproduct of fermentation broth is accelerating, a portion of the spent fermentation broth is removed and an approximately equivalent volume of fresh feed and media are added to the fermentation broth. In some embodiments, the substrate, product or by-product of fermentation is CO ₂ . In some embodiments, the microorganism is yeast. In some embodiments, the desired product is ethanol.

[0042] In some embodiments, 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. In some embodiments, the substrate, product or by-product that is controlled is dissolved CO ₂ . In some embodiments, a level of dissolved CO ₂ is controlled at approximately a predetermined target level during fermentation. In some embodiments, the concentration of dissolved CO ₂ present in the fermentation broth is controlled by sparging air through the fermentation broth. In some embodiments, a

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.

[0043] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.

Brief Description of the Drawings

[0044] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0045] 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.

[0046] Figure 2 shows an exemplary embodiment of a method for controlling a batch fermentation process by monitoring a control parameter.

[0047] Figure 3 shows an exemplary embodiment of a method for controlling a repeated-batch fermentation process by monitoring a control parameter.

[0048] Figure 4 shows an exemplary embodiment of a method for controlling a continuous fermentation process by monitoring a control parameter.

[0049] Figure 5 shows an exemplary embodiment of a method for controlling the concentration of dissolved CO2 while conducting fermentation using a batch process.

[0050] Figure 6 shows an exemplary embodiment of a method for controlling the concentration of dissolved CO2 while conducting fermentation using a repeated-batch process. [0051] Figure 7 shows an exemplary embodiment of a method for controlling the concentration of dissolved CO2 while conducting fermentation using a continuous process.

[0052] Figure 8 shows a line diagram of an experimental set-up used to perform batch ethanol fermentation using yeast in one example embodiment.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Figure 12 shows representative plots of ethanol concentration and cell viability profiles corresponding to profiles in Figures 9-1 1.

[0057] 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. [0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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. [0064] 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.

[0065] 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.

[0066] 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.

[0067] 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. In Figure 23(a) 1, 2 and 3 represent dissolved CO2 set points 500, 750 and 1000 mg/L respectively. In Figure 23(b) 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. [0068] 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. [0069] 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)

303.92±10.66 g glucose/L from duplicate experiments. 1, 2 and 3 in Figure 25(a) denote dissolved CO2 set points of 500, 750 and 1000 mg/L respectively; 1 and 2 in Figure 25(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. Conversion efficiency was calculated as

[Ethanol Produced] , _{nn n /}

xlOO %

[Glucose Consumed] x 0.511

where 0.511 represents the theoretical ethanol production based on the stoichiometric relationship between glucose utilization and ethanol production by weight. for

[0070] 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]—

Fermentation Duration

[0071] 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).

[0072] 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. [0073] Figure 29 shows a plot of the concentration of ethanol in the repeated-batch process of the example shown in Figure 28. Description

[0074] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0075] In some embodiments, measurement of a metabolic product or by-product produced during fermentation by a microorganism (i.e. a control parameter) 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. In some embodiments, the metabolic product or by-product (i.e. control parameter) that is measured is dissolved CO2 concentration. In some embodiments, 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). In such embodiments, the substrate so measured is a control parameter. In some embodiments, the substrate measured is dissolved CO2

concentration. In some embodiments, the concentration of a control parameter is controlled to improve fermentation.

[0076] 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.

[0077] As used herein, "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. In some embodiments in which the desired product is succinic acid, the microorganism is Actinobacillus succinogenes , Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, or Escherichia coli. In some embodiments, 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.

[0078] As used herein, "fermentation" means the conversion of carbohydrates to a desired product, e.g. other small molecules such as alcohols or organic acids, using a microorganism. In some embodiments, the fermentation process used is a very-high-gravity (VHG) fermentation process. In some embodiments, 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. [0079] The fermentation process is carried out under suitable conditions of temperature, atmosphere, agitation and the like to facilitate growth of the microorganism being used.

Selection and optimization of such conditions for any given species of microorganism and fermentation broth is within the expected ability of one skilled in the art. In some embodiments where the fermentation is a continuous or repeated-batch process, a portion of the fermentation broth that has been used to support fermentation (i.e. spent fermentation broth) is periodically removed and replaced with fresh media and feed (i.e. media and feed that have not yet been used to support fermentation by the microorganism). [0080] As used herein, "feed" means a suitable substrate provided to a microorganism to be converted to a desired product through fermentation. Examples of feeds that can be used in some embodiments of the invention include carbohydrates. In some embodiments, 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. Thus, 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. In some

embodiments, the media includes elements that enhance the growth of the microorganism but are not necessarily essential for microbial growth.

[0081] Examples of products that can be produced by fermentation using some embodiments include ethanol or succinic acid. In some embodiments, the product is a TCA cycle metabolite such as succinate, fumarate or malate.

[0082] In some embodiments, the feed used is a substrate that results in microbial metabolism using a metabolic pathway that results in the production of CO ₂ . In some embodiments, the feed used is a substrate that results in microbial metabolism involving glycolysis followed by the TCA cycle to produce CO ₂ . In some embodiments, the feed is glucose.

[0083] In some embodiments, the feed provided to the microorganisms yields a desired product that is produced by a metabolic pathway that uses CO ₂ as a substrate. For example, the production of succinic acid involves usage of CO2 as a substrate to enhance succinate production. Similarly, 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.

[0084] 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.

Rate of accumulation C0 ₂ desorption rate

CO ₂ evolution rate Rate of conversion of C0 ₂ in aqueous by physiochemical [8] by yeast of HCOj from C0 ₂ phase process

[0085] 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 (μ, 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. Thus, CER(t) is related to yeast growth with the assumption that μ (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.

CER(t) = MXY _{COi [9]}

[0086] During VHG batch ethanol fermentation S. cerevisiae follows four distinct growth phases, namely, lag, exponential, stationary and death phase (Lin et al., 2010; Liu et al., 201 la). While an increase in viable biomass is usually witnessed from the lag to the exponential phase, it remains nearly constant during the stationary phase and decreases abruptly in the death phase. Consequently, CER(t) being a function of viable biomass and CO ₂ being a metabolic by-product, the value of CER(t) increases with the viable cell concentration during the lag and exponential phases and begins decreasing during the stationary phase. As viable cell concentrations drop during the death phase, so does the associated CER(t) in Equation 9 due to the decline in X. This decrease in CO ₂ evolution can be attributed to the slowing down of yeast metabolism in the stationary phase and the absence of it in the death phase. Thus, Equation 9 relates CO ₂ produced during fermentation to yeast activity in the fermenter. In addition, 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.

[0087] 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.

[0088] The conversion of dissolved CO ₂ (DCO ₂ ) to HCO ₃ ^" ion is governed by the forward and backward reactions depicted by Equations 10-14, where k is the reaction rate constant for the forward reaction (1/h) and k_ is the reaction rate constant for the reverse reaction (L/mole-h). C0 ₂ + H ₂ 0^^→H ⁺ + HCO; [10]

HCO; + H ^{+ k} - ^b C0 ₂ + H ₂ 0 [1 1]

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.

d([HCO ^~ ])

- k _b [C0 ₂ ] - k_ _b [HCO-][ ⁺ ] [12] d _([ HCO- _]) _ k _{b [} ^ _] j K^ _[C0 ^ _ _{

dt K, [H ⁺ ] where K _x = -^- (mole/L) is the equilibrium constant for Equation 13. Under the steady state condition, d([HC0 ₃ ]) gq _uat j _on 1 3 j _{s e} q _ua j† _{0 zero} Hence, Equation 13 reduces to Equation dt

14.

[0089] The reaction rate constants k _t , and k- _b can be determined with the aid of the Arrhenius relation for reaction rate constants.

[HCO ] = -^-[C0 ₂ ] [14] Note that [CO2] in Equation 14 refers to the dissolved CO2 concentration that is symbolized as [DCO2] in Equation 17.

«WCO: ]) _ _[ H ^* ] \ _{[ 15]} dt K, [H ⁺

[0090] 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. This aspect of CO2 behavior is represented in the second term on the right hand side of Equation 8. In mass transfer, the rate of transfer of mass owing to a concentration gradient is governed by the volumetric mass transfer coefficient, a constant for any given system. In the conditions utilized in the examples presented

CO

below, the volumetric transfer coefficient (K _L ² (l/h)) remains constant throughout the fermentation process due to the maintenance of constant agitation rates and the absence of any air flow within the fermenter. Equation 16 clarifies this aspect of Equation 8.

C0 ₂ desorption rate = K^° ² a([DC0 ₂ ] - -¾-) [ 16]

[0092] 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). This is due to the very high solubility of CO2 in aqueous media in comparison to the solubility of O2 (typically, in fermentation broths the solubility of CO2 is ten times that of O2) (Spinnler et al., 1987). When the concentration of a gas dissolved in an aqueous solution is above the equilibrium concentration, the solution is termed to be supersaturated with the gas.

[0093] Substituting Equations 9, 15 and 16 in Equation 8 yields Equation 17. d ^{([D l ])} = CER{t) - K^ a{[DC0 ₂ ] -^ [ 17] For simplicity, the following notations are substituted for terms in Equation 17:

[DC0 ₂ ]; V _mt = K _L ^c ° ^> a ; C _g = j^ and ₇ = ^[H ⁺ ] ^- _i [DC0 ₂ ] - [HCO; ] dC

CER(t) - V _mt (C - C _g ) - _r [ 18] Equation 18 is further simplified for the purpose of discussion in the examples described below based on the processes they represent in Equation 8. The notations are as follows: a = CER(t) _a nd = V _mt (C- C _g ) . [0094] In some embodiments of the invention, 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.

[0095] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0096] In some embodiments, 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. In one embodiment, 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. wherein the magnitude of the slope of each adjacent measured interval is larger in the negative direction) for adjacent consecutive time intervals wherein 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.

[0097] For example only and not by way of limitation, if the slopes of dissolved CO2 concentration versus time measured at three different time points were Ti -50, T2 -100, and T3 - 200 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 consecutive adjacent time intervals is increasing (i.e. the absolute difference between T3- T2 of 100 ppm CO2 min ^"1 is larger in magnitude than the absolute difference between T ₂ - Ti of 50 ppm CO2 min ^"1 ).

Similarly, if 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 ). In contrast, if the slopes of dissolved CO2 concentration versus time measured at three different consecutive time points were Ti -50, T2 - 100, and T ₃ - 150 ppm CO2 min ^"1 , that would not indicate an abrupt drop in dissolved CO2 concentration. Although the slopes are getting progressively steeper in the negative direction, the magnitude of the difference between the slopes for adjacent consecutive time intervals is not increasing (i.e. the absolute difference between T ₃ -T2 of 50 ppm CO2 min ^"1 is not larger than the absolute difference between T ₂ -Ti of 50 ppm CO2 min ^"1 ).

[0098] 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. In one example embodiment utilizing Saccharomyces cerevisiae as the microorganism, the concentration of dissolved CO2 is measured at time intervals on the scale of minutes, e.g. between about 30 seconds and two minutes. In one such example embodiment, 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.

[0099] 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. In some embodiments, 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. In some

embodiments, 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). Comparison of a larger number of adjacent slopes may provide better accuracy in detecting the point at which there is an acceleration in the rate of decrease of the concentration of CO2, particularly in those embodiments in which the acceleration in the rate of reduction in the concentration of dissolved CO2 is lower (e.g. for higher glucose concentrations). [0100] In some embodiments, the fermentation process is a high-gravity fermentation process. In some embodiments, the fermentation process is a very-high-gravity (VHG) fermentation process. In some embodiments, the initial concentration of feed is between about 150 g/L and about 300 g/L. In some embodiments, the feed is glucose, and 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.

[0101] In some embodiments, 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. With reference to Figure 2, an example embodiment of a batch process 100 is illustrated. At 102, fermentation is initiated by combining the

microorganism and feed with media to provide a fermentation broth and allowing the microorganism to grow under appropriate conditions of temperature and atmosphere. At 104, the concentration of a control parameter is measured. At 106, when the control parameter indicates that the feed has been completely or nearly completely utilized, fermentation is stopped and the desired product is collected. In one example embodiment, 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.

[0102] In some embodiments, 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.

Further portions of the feed and fresh media can be added after each subsequent portion of the feed has been completely or nearly completely utilized. In some embodiments, 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. In some embodiments the fermentation process is a repeated-batch process and the concentration of dissolved CO2 is monitored. In some such embodiments, at approximately the time that an abrupt reduction in the concentration of dissolved CO2 is detected, a portion of the fermentation broth is withdrawn and an equal volume of fresh media with feed is added to replenish the fermentation broth. In some embodiments, 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.

[0103] In some embodiments in which the fermentation process is a repeated-batch process, the microorganism is maintained in its active growth phase or substantially in its active growth phase throughout the fermentation process. In some such embodiments, 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.

[0104] In some embodiments in which the fermentation process is a repeated-batch process, 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). In some embodiments in which 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. In some such embodiments, the accelerated fermentation rate increases the annual ethanol productivity. In some embodiments, 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. In some embodiments, 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.

[0105] Figure 3 shows an example embodiment of a repeated-batch process 110. At 112, 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. At 114, the concentration of a control parameter is measured. At 116, when 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. At 118, 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. [0106] In some embodiments, 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. 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. In some embodiments, 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.

[0107] In some embodiments in which the microorganism is yeast, the feed is glucose, and the desired product is ethanol, 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. [0108] In some embodiments, 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 . In a continuous process, substrate concentration can be maintained by the dilution rate (i.e. the volumetric feeding rate divided by the working volume of the fermenter). In some embodiments, 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 .

[0109] In some embodiments, 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. At approximately the time at which a peak of dissolved CO ₂ concentration is detected, 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.

[0110] Figure 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

microorganism is yeast. In some such embodiments, exponential growth is detected at 136 by detecting a peak in the concentration of dissolved CO ₂ . [0111] In some embodiments, 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. In some such embodiments, 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. During the late stage of fermentation, 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. In some embodiments, 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. In some embodiments, 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.

[0112] In some embodiments, 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. In some embodiments, improving feed utilization means increasing the efficiency of conversion of the feed to a desired product of fermentation. In some embodiments, improving feed utilization means that the microorganism utilizes a greater proportion of the feed over the course of the fermentation. In some embodiments, improving feed utilization means that the microorganism utilizes all or nearly all of the feed provided in the fermentation broth. In some embodiments, improving feed utilization means that the microorganism converts the feed to the desired product more rapidly, resulting in higher productivity.

[0113] In some embodiments, 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. In some embodiments, 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. In some embodiments, improvements in yield and/or productivity are achieved by increasing the biomass of the microorganism present in the fermentation broth.

[0114] In some embodiments, 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.

[0115] In some embodiments, 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. In some such embodiments, 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.

[0116] In some embodiments, 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.

[0117] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, it is determined that the predetermined level of feed utilization has been reached by detecting an abrupt decrease in the concentration of dissolved CO2 that indicates the first predetermined portion of the feed has been nearly or completely utilized.

[0118] In some embodiments, 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%. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. For example, 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. [0119] In some embodiments, 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.

[0120] The concentration of dissolved CO2 present in the fermentation broth can be controlled in any suitable manner. In some embodiments, the concentration of dissolved CO2 present in the fermentation broth is controlled by sparging air through the fermentation broth. In some such embodiments, 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. In some embodiments, 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.

[0121] In some embodiments, O2 is supplied to the fermentation broth in any suitable manner during fermentation. In some embodiments in which the 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.

[0122] 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. At 152, 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. At 154, the concentration of dissolved CO2 is measured. At 156, if the concentration of dissolved CO2 measured at block 154 is higher than a predetermined target concentration, 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. In some embodiments, including the illustrated embodiment, when 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. In one example embodiment, 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.

[0123] Figure 6 shows an example embodiment of a repeated-batch process 160

incorporating control of the concentration of dissolved CO2 and using the concentration of dissolved CO2 as a control parameter. At 162, 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. At 164, the concentration of dissolved CO2 is measured. At 166, if the concentration of dissolved CO2 measured is higher than a predetermined target concentration, 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.

[0124] When monitoring the concentration of dissolved CO2 164 indicates that the feed has been completely or nearly completely utilized, 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. At 172, 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. [0125] In some embodiments in which the microorganism is yeast, the feed is glucose, and the desired product is ethanol, 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. In some embodiments in which the microorganism is yeast, the feed is glucose, and the desired product is ethanol, 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.

[0126] Figure 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. At 186, exponential growth is detected by detecting a peak in the concentration of dissolved CO ₂ . Once exponential growth has been detected, 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. While fresh feed and media are being added at 188 and spent fermentation broth is being removed at 190, the concentration of dissolved CO ₂ is monitored at 192. At 194, if 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.

[0127] In some embodiments, both the concentration of dissolved CO ₂ in the fermentation broth and the oxidation-reduction potential of the fermentation broth may be controlled.

[0128] In one example embodiment, the desired product is succinic acid and the feed is glucose. In some embodiments in which the desired product is succinic acid, the microorganism is Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, or Escherichia coli. The production of succinic acid involves usage of CO2 as a substrate. In some embodiments, CO2 is also used as a control parameter to control the fermentation. In some embodiments, the concentration of CO2 in the fermentation broth is controlled to optimize the production of succinic acid.

Examples

[0129] Specific embodiments are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature. Example 1 - Materials and Methods

1.1 Strain and Growth Media

[0130] Ethanol Red™ 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.

[0131] 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. The concentration of 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.

Media Constituent Concentration (M)

Ammonium sulphate, NH4SO4 1

Calcium chloride dihydrate, CaCl ₂ .2H ₂ 0 0.082

Copper sulphate, CuS0 ₄ 0.0100

Ferric chloride, FeCi ₃ .6H ₂ 0 0.1000

Hydrogen borate, H3BO3 0.0240

Magnesium sulphate heptahydrate, MgS0 ₄ .7H ₂ 0 1

Manganese sulphate, MnS0 ₄ .H ₂ 0 0.0020

Potassium iodide, KI 0.0018

Potassium phosphate (monobasic), KH ₂ PO ₄ 0.7350

Sodium molybdenate, Na ₂ Mo0 ₄ 0.0015

Urea 1.6000

Zinc sulphate heptahydrate, ZnS0 ₄ .7H ₂ 0 1

1.1.1 Yeast Pre-culture

[0132] Prior to inoculation in the fermenter, 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.

1.2 Batch fermentation

1.2.1 Experimental set up

[0133] Production and concentrations of ethanol and CO ₂ by S. cerevisiae from glucose substrates under VHG conditions were investigated using a batch fermentation process. The fermentation apparatus used in the work were jar fermenters (Model: Omni Culture, New York, NY, USA) with a capacity of 2 L and a 1 L working volume (Figure 8). The ferm enter was covered with a detachable stainless steel (SS) lid screwed to the top of the jar to maintain sterility during the process. The cover was equipped with ports accessible for measuring temperature, agitation speed, dissolved CO2 concentration and redox potential. Agitation was achieved through a six-b laded impeller mounted to the agitator shaft that was fixed to the SS cover.

1.2.2 Measurement of dissolved carbon dioxide and redox potential

[0134] 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. It is to be noted that the measurement of dissolved CO2 by the InPro 5000 is based on the Severinghaus potentiometric principle (Janata, 2009; Kocmur et al., 1999), which has been previously used to develop and build custom dissolved CO2 sensors (Ho and Shanahan, 1986; Shoda and Ishikawa, 1981 ; Zosel et al., 201 1). Figure 8 shows a line diagram of the

experimental set-up used to perform batch ethanol fermentation.

[0135] Autoclavable ORP electrodes from Cole-Parmer Inc. (12 mm x 250 mm, Vernon Hills, IL, USA) were used to measure ORP values during batch fermentations. All measurements made through different sensors were acquired using a custom built data acquisition system (DAS) and LabView.

1.2.3 Control of dissolved carbon dioxide concentration

[0136] 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). Based on this premise three different dissolved CO2 control set points were chosen for glucose feeds of approximately 250 g/L while two different set points were chosen for glucose feeds of approximately 300 g/L. Control of dissolved CO2 was achieved using a PID control algorithm implemented through a LabView VI (Version 8.5, National Instrument, Austin, TX, USA). The concentration of dissolved CO2 was monitored using a dissolved CO2 sensor, available from Mettler Toledo.

1.2.3.1 Carbon dioxide control using calcium hydroxide

[0137] 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. Moreover, 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. However, 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.

[0138] For the purpose of controlling dissolved CO2 concentration in the broth using calcium hydroxide, 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.

1.2.3.2 Carbon dioxide control using air

[0139] Based on the acquired dissolved CO2 concentration signal, 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

[0140] The fermentation of glucose to ethanol was carried out aseptically in the steam sterilized fermenter at a working volume of 1 L. The variation in initial glucose concentrations among experiments in the absence as well as presence of control was expressed in terms of standard deviation values between the different initial glucose concentrations. It is to be noted that experiments were performed in triplicate for batches without control and in duplicate for batches under dissolved CO2 control. The media in the fermenter was inoculated with yeast from the pre-culture flask at 5 % of the working volume, and the pitching rate was adjusted to approximately 10 ⁷ viable yeast cells per mL for all experiments. The temperature of the broth was maintained at 33 °C by circulating water at 33 °C through the equipped heating/cooling coil of the fermenter. The agitation speed was maintained at 150 rpm for the duration of fermentation. Samples for analytical analysis were withdrawn as 5-mL fermentation broth aliquots every 4-6 hours.

1.2.5 Analytical analysis

[0141] 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 (Klett™ 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).

1.2.5.1 Analysis of carbohydrates and organic acids

[0142] 2 mL of the aliquots were centrifuged at 10,000 rpm for 10 minutes and the concentrations of sugars and organic acids in the culture supernatants were analyzed by HPLC (Series 1100, Agilent Technologies, Mississauga, ON) equipped with a refractive index (PJ) detector (HP 1047A, Hewlett Packard, Mississauga, ON). The metabolites were separated using an ion exclusion ION-300 column (Transgenomic, Inc., Omaha, NE, USA) with 8.5 mM H2SO4 at 0.4 mL/min as mobile phase. The column temperature was maintained at 65 °C and the RI detector temperature was maintained at 35 °C during the course of analysis. Each sample was injected into the column three times as 10 μΐ _^ injections each and subject to analysis. 1.3 Determination of ethanol toxic concentrations

[0143] 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. (201 la). A plot of biomass production rate (first-order derivative of biomass concentration against time) vs. ethanol concentration was created. The ethanol concentration corresponding to where the biomass production rate approaches to zero was considered to be the ethanol toxic concentration. This toxic ethanol concentration limit was determined to be 85 g/L.

Example 2 - Testing of Four Exemplary Glucose Feed Concentrations

[0144] The observed dissolved CO2 concentration profiles in the presence and absence of control for four different glucose concentrations of 150, 200.05±0.21, 250.32±0.12, and 300.24±0.28 g/L were determined. Feeds of -150 and -200 g glucose/L represent high-gravity fermentations. Very-high-gravity fermentations are represented by feeds of -250 and -300 g glucose/L. These profiles are representative of the absolute concentration of CO2 present in the form of dissolved CO2.

2.1 Dissolved carbon dioxide concentration and yeast growth

2.1.1 Dissolved carbon dioxide concentration and evolution

[0145] 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. In order to describe the combined physical, chemical and biological effects attributable to the changes of dissolved CO2 concentration in the fermentation broth during the course of VHG ethanol fermentation, 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

dC

equivalent to the term of Equation 18. Two distinct regions are evident in these figures: the dt

first region (Region I), where there is a peak in the accumulation rates, and the second region (Region II), where the accumulation rates remain at or below zero for all glucose feeds. A comparison of the accumulation profiles with their corresponding dissolved CO2 profiles (Figures 9-10) shows that transition from the first to the second region is not abrupt but rather spread over a 2-3 hour period of fermentation otherwise termed here as the transition period.

[0146] 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.

dt

'CER (¾)~0' denotes zero glucose concentration and the resulting abrupt decline in yeast metabolism. A corresponding abrupt drop in the concentration of dissolved CO2 is also observed. Experiments were performed in triplicate.

[0147] 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.

[0148] 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.

[0149] These regions can be interpreted in terms of Equation 18. During the lag and exponential phases of yeast growth where cell metabolism and hence CO2 evolution are at their dC

peaks, CER(t) is much higher resulting in α > (β + γ) in Equation 18 and a net (it is to be dt noted that CER(t) cannot be negative). This is seen in Region I of Figures 9-10. In Region I, CER(t) is much higher than the CO2 desorption rate, resulting in a net CO2 accumulation and a corresponding increase in dissolved CO2 concentration. Although, the exponential phase lasts as long as 20h, (refer to biomass concentrations in Figures 9-10) the dissolved CO2 concentration profiles do not directly correspond to the increase in biomass. Without being bound by theory, this may be due to the fact that the biomass profiles are representative of the total cell concentration rather than the viable cell concentration making this measure indifferent to actual metabolic activity. It is also possible that desorption of CO2 from the fermentation broth and other physiochemical processes affect measurement of dissolved CO2 concentration. The peak in the profiles in Figures 9 and 10 also explains two facts that were stated earlier; (1) the fermentation broth is supersaturated with CO2 as (2) the desorption of CO2 does not increase exponentially with increasing dissolved CO2 concentration. [0150] A negative accumulation of dissolved CO2, witnessed in Region II of Figures 9-10, is a physical outcome of CO2 evolution rate being less than the CO2 desorption rate resulting in a decreasing dissolved CO2 concentration (Region II of Figures 9-10). Correspondingly in dC

Equation 18 this would be represented as a < (β + γ) resulting in a negative , with y being dt

considered negligible relative to either a or β. Note that y value is one to two orders of magnitude dC

smaller than a and B. The decline in in Region II can be ascribed to the decrease in yeast dt

metabolic activity.

[0151] 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. Note that 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.

[0152] As illustrated in the dissolved CO2 profiles, the dissolved CO2 concentration remains dC

nearly constant in the transition regions resulting in being zero for this period (Figure 9 and dt

10). Speaking in terms of the dissolved CO2 mass balance described by Equation 18, this would mean a = β (assuming a negligible y similar to previous cases under current operating conditions). In terms of physical quantities this would mean that the rate of removal of dissolved CO2 from the fermentation broth by desorption is equal to rate of evolution of CO2 from microorganisms. This could be either due to the decrease in CER(t) (a) as a consequence of decrease in yeast activity or due to the increase in the CO2 desorption rate (β). 2.1.2 Effect of ethanol toxicity and osmosis on carbon dioxide evolution

[0153] Higher initial glucose concentrations tend to increase the osmotic stress, ultimately resulting in a longer lag phase (Liu et al., 201 la). Also the buildup of ethanol concentration may arrest yeast propagation, due to the toxic effect of the ethanol produced (Brown et al., 1981 ; Lin et al., 2010; Liu et al., 201 1a;). The ethanol toxic concentration for the yeast used in this study was determined to be -85 g/L. Without being bound by theory, rationalization of the differences among the CO2 accumulation rates and CER(t) observed between Region I and II of Figures 9 and 10 can be done based on the glucose concentration in the media and the toxic effect of ethanol produced by yeast. [0154] The ethanol concentrations in the fermenter measured at the end of Region I of Figures 9 and 10 for -150, -200, -250 and -300 g glucose/L were 18, 20, 30, and 28 g ethanol/L, respectively. These ethanol concentrations are lower than the ethanol toxic level, 85 g/L. Hence, without being bound by theory, it is postulated that the delay of yeast growth is mainly attributed to the high initial glucose concentration rather than ethanol toxicity. This can be noticed in Figure 12 illustrating the cell viabilities and corresponding ethanol concentrations for four glucose concentrations. In Figure 12 the cell viabilities decrease with increasing ethanol concentrations as well as progress of fermentation. The drop in viabilities is more drastic with increasing glucose concentrations. Accordingly, due to increase in substrate concentration, 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.

[0155] In Region II of Figures 9 and 10, the glucose concentrations after 10- 12h of fermentation are much lower than initial values, indicating a moderate reduction in osmotic stress for all four glucose feeds. At initial concentrations of -150 and -200 g glucose/L, final ethanol concentrations seen at the end of fermentation, when glucose is completely utilized, are 68 and 72 g/L respectively (Figure 1 1). Hence, yeast metabolism and the production of CO2 were hampered by glucose depletion rather than ethanol toxicity (Figures 1 1 and 12). At initial concentrations of -250 and -300 g glucose/L, although sufficient glucose (75 and 150 g/L) is present even following the peak in CO2 concentrations (32 and 34h respectively, where typically stationary phase begins; Lin et al., 2010; Liu et al., 201 1a), the corresponding ethanol concentration exceeds the toxic level of 85 g/L (Figures 11 and 12). As fermentation continues, the adverse effect of ethanol toxicity worsens. The decrease in dissolved CO2 concentration and evolution rates inferred from Region II of Figures 9 and 10 can be attributed to the death of yeast cells that cease to survive in ethanol concentrations exceeding the toxic level (Figure 2 in Lin et al. (2010) and Figure 12 of the present specification). Cessation of yeast growth and survival directly impacts the value of CER(t) and CO2 accumulation (see e.g. Figure 10 where once past dC the transition region the dissolved CO2 concentration starts decreasing and becomes dt increasingly negative).

[0156] From Equation 18 and Region II of Figures 9 and 10 it was further concluded that once accumulation of CO2 in the aqueous phase ceased, there was no change in the rate of dC

decrease of dissolved CO2 concentration and the value of remained fairly constant until dt ^y

CER(t) approached zero (denoted by CER(t)~0 for the case of -150 and -200 g glucose/L and illustrated in Figure 9). But for the case of -250 and -300 g glucose/L there was no abrupt decline in the dissolved CO2 concentration leading us to the observation that the point of CER(t)~0 was not evident from Figure 10. This observation led to the conclusion that the CO2 desorption rate remained almost constant irrespective of the changing CER(t), provided the assumption of a slowly decreasing CER(t) is valid. Thus from Equation 18 it can be inferred that the rate of CO2 desorption remains fairly constant during the entire fermentation process. This strengthens the observation of the fact that equilibrium between off-gas CO2 and dissolved CO2 does not appear to exist under the current experimental conditions. Furthermore, the decreasing gradient between dissolved and off-gas CO2 concentrations will result in a reduced driving force for CO2 desorption from the fermentation broth. Hence, it is postulated that the rate of desorption of CO2 will decrease with decrease in dissolved CO2 concentration in the fermentation broth.

2.2 Dissolved carbon dioxide control in very-high-gravity fermentation

[0157] Subsequently, control of dissolved CO2 concentration was incorporated into the VHG fermentation process. Earlier Liu et al. (201 1a) had concluded that incorporating an oxidation- reduction potential (ORP) based control strategy did not provide any benefit to fermentations processes operated under initial concentrations less than 200 g glucose/L. In addition, glucose was completely consumed in fermentations with initial substrate concentration less than 200 g glucose/L within a reasonable period of time (Figure 9). Aiming to improve ethanol fermentation for feeds greater than 200 g glucose/L, dissolved CO2 was controlled at three different levels of 500, 750 and 1000 mg/L for the case of -250 g glucose/L and at two levels of 750 and 1000 mg/L for -300 g glucose/L. The dissolved CO2 concentration profiles for each individual case have been presented in this section along with the corresponding biomass, glucose and ethanol concentration profiles.

[0158] 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. Hence, this caused cell viabilities to drop below 50 % after -6 h of fermentation when control of dissolved CO2 concentration was initiated by addition of Ca(OH)2. A drop in cell viabilities would reduce yeast growth as well as ethanol production rates. As control using Ca(OH)2 did not yield the desired results under the currently employed example conditions, the following discussions pertaining to dissolved CO2 concentration control are based on CO2 control achieved using sparged air. 2.2.1 Characteristics of dissolved carbon dioxide profiles in the presence of CO 2 control

[0159] In their basic form 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 low biomass concentrations in Region I stand witness to this fact (Figures 13 and 14). Longer lag phase is seen in the form of increase in fermentation duration for -300 g glucose/L in comparison to -250 _i A R _tt e _r aonae

g glucose/L (Table 2). Hence, Region I in Figures 13 and 14 was hypothesized to characterize the lag phase and the very early part of the exponential phase.

[0160] 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. In addition to the usual increase in metabolic rates witnessed in the mid- exponential phase, 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. Despite increased desorption, 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.

Table 2. Comparison of fermentation duration ^a in hours for fermentation under different dissolved CO2 control set points and aeration rates used for control under -250 and -300 g glucose/L initial concentration for batch ethanol fermentation.

Initial Concentration -250 g glucose/L -300 g 1 ¾lucose/L

DCO2 set point

500 750 1000 750 1000 (mg/L)

820 26.35±0.21 27.70±0.85 26.65±0.64 41.65±1.63 32.5±0.0 1300 26.50±0.71 27.50±0.56 27.45±1.91 36.80±1.13 36.30±0.71

Note: For uncontrolled batches under initial concentrations -250 and -300 g glucose/L presented in Section 2.1 glucose was not completely exhausted. Hence, the fermentation duration could not be accurately calculated and has been mentioned to be greater than 50 h.

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 in the fermentation broth, is almost zero in Region III.

dt

[0162] While the absence of net CO2 accumulation in Region III could be argued to be a result of increased stripping of CO2 from the broth, in the present case it is rather a consequence of a decrease in CO2 production due to drop in yeast metabolic activity. Thus in Equation 18, CER(t) reduces with no significant change to the rate of CO2 desorption (β) in comparison to Region II. The decrease in yeast metabolism was inferred from the biomass profiles pertinent to Region III in Figures 13 and 14. The smaller change in biomass points to growth slowing down post-exponential phase and entering the stationary phase. Despite the reduction in metabolism, yeast still thrives in the fermentation broth with slight drop in viabilities (Figure 19) not only due to presence of substrate but also the supply of oxygen through air sparging. This ensures yeast survival as well ethanol production even in the stationary phase represented by Region III.

[0163] In Region TV of Figures 13 and 14 the balance between CO2 production and stripping is offset. This departure from the balance that was present in Region III was attributed to complete substrate exhaustion (Figures 13(b), 14(b)). Substrate exhaustion results in complete cessation of yeast metabolism resulting in CER(t) in Equation 18 becoming zero. This resembles the condition similar to that which was illustrated in Section 2.1.1 where CER(t) is less than the dC

rate of CO2 desorption resulting in a net decline in dissolved CO2 ( <0). The net decline is dt

observed as the abrupt drop in the dissolved CO2 concentration depicted in Region IV of Figures 13 and 14. [0164] The features of the 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.

2.2.2 Comparison of profiles in the presence and absence of CO 2 control

[0165] While the dissolved CO2 profiles observed in the absence of control displayed only three distinct regions, namely, Regions I, II and a transition region (Figures 9 and 10), four distinct regions were exhibited when dissolved CO2 was controlled, namely, Regions I, II, III and IV (Figures 13 and 14). [0166] The primary distinction between the profiles was the absence of a transition region between Regions I and II in the presence of CO2 control. Without being bound by theory, the transition region observed in batch fermentations in the absence of dissolved CO2 control in Figures 9 and 10 was attributed to the possible extension of the osmotic effect, exerted by high initial glucose concentrations, beyond the lag phase and into the exponential growth phase of yeast. It was also postulated that the reduction in the period of the transition region with increase in glucose concentrations from 150 to 300 g/L was due to this osmotic effect.

[0167] In the case of profiles observed in the presence of dissolved CO2 control, the absence of such a transition region could be explained in terms of yeast metabolism observed under conditions of dissolved CO2 control. In the absence of dissolved CO2 control, no oxygen was supplied to the broth between lag and exponential phases. This essentially would mean a lower level of metabolic activity in batches without CO2 control relative to batches with CO2 control. The reduced level of metabolic activity could be interpreted as a lower CER(t) value in Equation 18. It was postulated above that either a decrease in the value of CER(t) or an increase in the value of β, as a result of increased CO2 desorption, would result in a condition where net accumulation of dissolved CO2 does not occur. This was seen during the transition region in Figures 9 and 10. In contrast, the supply of oxygen as a consequence of CO2 control results in increased yeast metabolism towards the end of Region I and beginning of Region II. The consequential increase in CER(t) as a result of increased metabolism thereby eliminates zero accumulation. This effect is observed despite the increase in CO2 desorption (β) brought about by air sparging. Without being bound by theory, it is believed this is why no transition region is observed between Regions I and II when dissolved CO2 is controlled through air sparging. Further differences between fermentation characteristics in the presence and absence of CO2 control could be made with the biomass concentrations and fermentation duration (Table 2). Biomass concentrations for fermentations without control are lower than that of fermentations where dissolved CO2 was controlled (Figure 24). The higher biomass for controlled fermentations could be attributed to air sparging. Higher biomass results in faster glucose consumption resulting in shorter duration for complete substrate utilization in controlled fermentations (Table 2). [0168] While distinctions between dissolved CO2 profiles in the presence and absence of CO2 control were focused on the transition region, it is observed that Region I in the dissolved CO2 profiles in both cases have a similar trend. Region I (for both cases) 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.

[0169] Similarities can also be observed based on the fact that dissolved CO2 profiles are capable of identifying complete glucose consumption during fermentation. While a non-zero residual glucose was observed for batches with initial concentrations of -250 and -300 g glucose/L in the absence of control (Figure 1 1), a zero residual glucose point was observed for similar initial glucose concentrations in the presence of CO2 control (Figures 13 and 14). The point of zero residual glucose was represented by an abrupt drop in dissolved CO2 concentration as observed in Region IV of the dissolved CO2 profiles similar to the drops observed for batches of -150 and -200 g glucose/L in the absence of CO2 control (Figure 9). This observation leads to the conclusion that, irrespective of the presence of a dissolved CO2 concentration control strategy, the dissolved CO2 profiles were able to accurately detect complete glucose exhaustion in VHG fermentation environments.

2.2.3 Effect of dissolved carbon dioxide set point on very-high-gravity fermentation

[0170] Controlling dissolved CO2 at different levels yielded distinct values for the various fermentation parameters like glucose consumption, ethanol production and conversion efficiency. As reported earlier higher initial glucose concentrations (>200 g/L) resulted in incomplete and sluggish fermentations with unspent residual glucose present at the end of batches without a CO2 control strategy. Incorporation of dissolved CO2 control into the process not only facilitated complete substrate consumption for initial glucose concentrations of -250 and -300 g/L, but also reduced fermentation times and improved ethanol productivities. Contrasts among these observations could be drawn on the basis of the level of dissolved CO2 control, the initial glucose concentrations and the aeration rate used to achieve control.

[0171] 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.

2.2.3.1 Dissolved carbon dioxide controlled at 500 mg/L

2.2.3.1.1 Effect of carbon dioxide removal

[0172] Jones and Greenfield (1982) mentioned that CO2 inhibited yeast growth through several physiological changes in yeast. Thus, choosing an appropriate set point to control dissolved CO2 is important. 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. In comparison dissolved CO2 profiles shown in Figures 18(b) and 21 obtained for dissolved CO2 set points under initial conditions of 750 mg/L, 308.49±12.87 g glucose/L and 1000 mg/L, 299.36±6.66 g glucose/L respectively have different characteristics from that shown in Figure 14.

[0173] It was observed from Figure 16 that in comparison to controlling dissolved CO2 at 750 (Figure 19) and 1000 mg/L (Figure 22), a control set point of 500 mg/L yielded lower final ethanol concentrations. Without being bound by theory, the difference in ethanol concentration could be explained on the basis that controlling dissolved CO2 at 500 mg/L resulted in excessive stripping of CO2 from the broth. Excessive stripping might deprive yeast cells of the required quantity of CO2 leading to a reduction in metabolism that is reflected in the lower ethanol concentration. Jones and Greenfield (1989) noted that CO2 is a requirement for various carboxylation reactions that are a part of yeast metabolism. 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. In contrast to the above two cases, controlling dissolved CO2 at 750 mg/L could have resulted in more optimal stripping of CO2 so as to not only diminish the effect of inhibition but also support cell maintenance at optimum levels for efficient ethanol production. Ethanol concentrations in the fermentation broth exceeding ~90 g/L (Figures 16, 19 and 22) also tend to exacerbate the aforementioned effects. Reduction in glucose consumption rates are also a possibility as a result. 2.2.3.1.2 Effect of oxygen supply

[0174] While the hypothesis stated earlier explains the differences among the set points based on dissolved CO2 concentration, it did not explain the effect that aeration would have on these dissolved CO2 levels. Two different aeration rates (820 and 1300 mL/min) were studied for the purpose of stripping dissolved CO2 from the fermentation broth to achieve CO2 control. Generally aeration rates were seen to be interrelated to the dissolved CO2 set points.

[0175] Dissolved oxygen concentrations in VHG broths are less in comparison to their low gravity counterparts because of the higher gravity and viscosity (Schumpe and Deckwer, 1979; Schumpe et al., 1982). The need for oxygen during fermentation is further exacerbated by the very nature of the process itself. Although ethanol production in yeast is essentially anaerobic, yeast growth and proliferation require oxygen. Furthermore, the production of a primary metabolite like ethanol is dependent on yeast growth. Aeration improves the dissolved O2 level in the broth leading to increase in cell viability (Fornairon-Bonnefond et al., 2002; Ligthelm et al., 1988; Verduyn et al., 1990). [0176] Yeast viability in the absence of air supply was observed to characteristically reduce with the progress of batch fermentation (Figure 12). This was not just due to progressive substrate depletion but also a concomitant increase in ethanol concentrations. Ethanol concentrations over 40 g/L are known to cause inhibitions for yeast growth while concentrations over 85 g/L result in cell death due to toxicity (Lin et al., 2010; Liu et al., 201 la, 201 lb). Hence, it is possible that supply of oxygen could have improved the ethanol tolerance of yeast resulting in higher cell viabilities. Figure 16 shows cell viabilities to be over 90% for the entire duration of fermentation when compared to those seen in Figure 12 for batches without control. Higher viabilities not only result in higher CO2 production (as a result of increase in CER(t)) but also result in faster consumption of glucose resulting in shorter fermentation durations in comparison to batches without control (Table 2).

[0177] However more oxygen does not necessarily result in a parallel increase in fermentation performance. Higher aeration in cases where dissolved CO2 was controlled at 500 mg/L could have also resulted in hyperoxia eventually facilitating a reduction in cell metabolism (Belo et al., 2003; Fornairon-Bonnefond et al., 2002) and hence a lower conversion efficiency and final ethanol concentration. An argument could also be made that excess oxygen supplied could have diverted the metabolic flux towards biomass generation rather than ethanol production resulting in reduced efficiencies but not significant reduction in productivities (Zeng and Deckwer, 1994). [0178] Based on the hypothesis that controlling dissolved CO2 at 500 mg/L not only resulted in excessive stripping of CO2 from the broth but also excessive oxygenation leading to hyperoxia, it was postulated that control of dissolved CO2 at 500 mg/L would have lower conversion efficiency than when dissolved CO2 was controlled at 750 mg/L. Hence, it was decided not to control dissolved CO2 at 500 mg/L for -300 g glucose/L. Under the tested conditions (see below), it was shown that controlling dissolved CO2 at 500 mg/L for -250 g glucose/L was less efficient despite the similarity in the duration of fermentation among the different CO2 control set points.

2.2.3.2 Dissolved carbon dioxide controlled at 750 mg/L

[0179] In VHG fermentation initial glucose concentration was seen to play an important role in determining the duration of fermentation. Using two different initial glucose concentrations for fermentation using the same dissolved CO2 control set point demonstrated the effects such a control has on various measurable parameters like the concentration profiles of dissolved CO2, glucose, ethanol and biomass. The dissolved CO2 profiles for CO2 control at 750 mg/L under different aeration rates and initial glucose concentrations are shown in Figures 17 and 18. Figure 19 illustrates the corresponding glucose, ethanol and biomass concentration and cell viability profiles.

2.2.3.2.1 Effect on dissolved carbon dioxide profiles

[0180] Although characteristics of dissolved CO2 profiles for -250 g glucose/L when dissolved CO2 was controlled at 750 mg/L (Figure 17), resembled that shown in Figure 13, it was observed that dissolved CO2 profiles differed from Figure 14 for the case of -300 g glucose/L (Figure 18(b)) depended on the set point as well as the aeration rate used to achieve control. It was observed that the difference between Figure 18(b) and Figure 14 was restricted to Region II of Figure 14 or Figure 18(a). In Section 2.2.1 it was established that the increase in dissolved CO2 concentration over the set point value in Region II of Figures 13 and 14, despite the presence of CO2 control, could be due to a combination of physiochemical as well as biological processes. While yeast metabolism was the only biological activity in the fermenter, discussions on physiochemical processes were restricted to CO2 desorption. Without being bound by theory, it is believed that the rise in dissolved CO2 concentration was a result of increased yeast activity and increased CO2 dissolution. Therefore, the loss of resemblance between Figures 18(a) and (b) in Region II was construed as a lesser increase in yeast activity as well as improved stripping of dissolved CO2. Without being bound by theory, these effects could arise due to various reasons.

[0181] A first potential reason could be the higher osmotic pressure in the case of -300 g glucose/L. In comparison to the -250 g glucose/L case, 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). On the other hand, 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.

[0182] It could be argued that certain physiochemical effects pertaining to the solubility of CO2 in the fermentation broth could have also affected the dissolved CO2 profiles. Chief among them are broth viscosity and pH. Viscosity of the fermentation broth increases with an increase in specific gravity of the broth or an increase in the initial glucose concentration. Increased broth viscosity not only lowers the solubility of oxygen but also improves the binding of dissolved CO2 to the protein molecules present in the broth (Dixon and Kell, 1989; Ho and Shanahan, 1986). Increased binding reduces the rate and quantity of CO2 desorption from the broth thereby enhancing dissolved CO2 accumulation and inhibition as well. As for pH, it is known to affect the equilibrium of dissolved CO2 mentioned in Equations 4-5. Lower pH favors the presence of CO2 as dissolved CO2. Fermentation broth pH is known to drop from 5.5 to 3.9 when left uncontrolled with the progress of fermentation thereby facilitating CO2 dissolution rather than desorption. Thus, without being bound by theory, it is believed that viscosity and pH do not play a significant role in the effects observed in Region II of the dissolved CO2 profiles for 300 g glucose/L in Figure 18.

[0183] 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).

2.2.3.2.2 Effect on aeration

[0184] It was noted above that the effect brought about by a change in aeration rate is interrelated with that brought about by change in dissolved CO2 set point for a given glucose concentration. Higher set points would mean less CO2 would have to be removed in comparison to its lower set point equivalent. This would mean that a lesser quantity of air is needed to remove the corresponding dissolved CO2 accumulated in the broth. This can be verified from Figure 23 illustrating the quantity of oxygen supplied for each case. Data shown in Figure 23 was estimated with the assumption that 21% of air was oxygen by volume under experimental conditions. Considering the fact that the duration of fermentation as well as control did not vary much between the two aeration rates for a given glucose concentration it is no surprise that more oxygen was bubbled through when higher aeration rates were used. It can also be noted from this figure that the quantity of oxygen bubbled through decreases progressively with increase in the dissolved CO2 set point. Hence, the quantity of oxygen bubbled through for control at 750 mg/L was lower than the case of 500 mg/L when initial glucose was at -250 g/L. But, the difference in the duration of fermentation is seen to have impacted the quantity of oxygen bubbled through for the case of -300 g glucose/L with dissolved CO2 controlled at 750 mg/L (Figure 23(b)). [0185] Here the trend in the quantity of oxygen supplied is reversed, i.e. a higher quantity of oxygen is supplied when control is achieved using an aeration rate of 820 mL/min than when an aeration rate of 1300 mL/min is used (Figure 23). This difference has been attributed to the longer duration of fermentation under aeration rates of 820 mL/min (Table 2) in comparison to aeration rates of 1300 mL/min for -300 g glucose/L. The longer duration also explains the general increase in the quantity of oxygen supplied for the case of -300 g glucose/L when compared to the case of -250 g glucose/L for both dissolved CO2 set points of 750 and 1000 mg/L.

[0186] It is highly probable that the higher quantity of oxygen supplied was in part also responsible for the distinction seen in Region II of Figure 18(b) from Figure 18(a). Higher aeration in conjunction with higher dissolved CO2 set points result in more efficient stripping of CO2 from the broth and prevents net accumulation of dissolved CO2. Reduction in metabolic activity also plays an important role as it results in reduced net CER(t).

2.2.3.2.3 Effect on metabolite consumption and production

[0187] Controlling dissolved C0 ₂ at 750 mg/L yielded the highest final ethanol concentration amongst the three set points for 250 g glucose/L (Figure 19). While rate of change of ethanol concentration did not vary much with aeration rates, they varied with initial glucose concentrations. Slower change in ethanol as well as biomass concentration was observed with increase in initial glucose concentration (Figure 19). Without being bound by theory, this was reasoned to be due to higher osmosis in higher glucose concentrations during the first 10-12 h of fermentation.

[0188] However, during the later stages of fermentation the above reasoning would not hold when glucose concentrations have reduced and osmosis is negligible (glucose concentrations after 12-15 h are lower than -150 g/L for 250 g glucose/L and -200 g/L for 300 g glucose/L). In comparison, the ethanol concentrations at this point are higher than their initial values and over the inhibitory concentration of 40 g/L (Figure 19). While this would explain the decrease in growth as well as associated glucose consumption and ethanol production in the case of -250 g glucose/L, it would also explain the reduced cell viabilities seen in the case of -300 g glucose/L despite oxygen supply. Although controlling dissolved CO2 at 750 mg/L improved fermentations for both initial glucose concentrations, fermentation was longer for -300 g glucose/L (Table 2) along with a need for increased air supply (Figure 23).

[0189] Glucose and ethanol aside, glycerol was one other metabolite whose concentrations followed a particular trend with variation in glucose concentrations (Figure 24). There were no appreciable differences in glycerol concentration with changes in aeration rate and dissolved CO2 set points. Despite the fact that supply of oxygen results in alleviating oxidative stresses associated with reduced dissolved oxygen concentration, glycerol concentrations increase with increasing initial glucose (Figure 24). While the increase in substrate alone could not result in an increase in glycerol, it stands to reason that other associated factors may also be responsible. These factors may include but are not limited to the higher osmotic pressure as well as ethanol inhibition and toxicity seen with increasing glucose concentrations.

2.2.3.2.4 Effect on yeast growth and viability

[0190] Changes in yeast growth and survival characteristics also tend to affect metabolite consumption and production patterns. As pointed out earlier controlling dissolved CO2 at 750 mg/L could be an optimum level of the tested conditions in relation to the quantity of CO2 stripped as well as oxygen supplied. This could have resulted in not only the increased cell viabilities explained earlier but consequential higher biomass concentrations relative to the VHG fermentation batches without control (Figure 24). However, differences in biomass concentration and cell viability profiles were observed between the two glucose concentrations (Figure 19).

[0191] Yeast growth is affected in the lag phase due to osmosis associated with high glucose concentrations. Osmosis not only leads to slower growth but also to slower ethanol production as noted in the previous section (Figure 19). But, this does not explain the reduction in the maximum biomass concentration with increase in glucose concentration (Figures 19 and 24). This observation has been made despite the higher viabilities seen in the case of -300 g glucose/L (Figures 19(c) and 19(d)). It is expected that the higher ethanol concentrations are responsible for this effect. Although in comparison to the 250 g glucose/L case, ethanol concentrations are at similar levels (-1 10 g/L) for 300 g glucose/L at 30 h of fermentation, it is likely that despite the increase in cell viability, cell tolerance to higher ethanol concentrations is reduced with increase in glucose concentration. This would not only explain the reduction in ethanol production with increase in broth ethanol concentration but also the concomitant reduction in cell viabilities with the progress of fermentation for -300 g glucose/L in comparison to -250 g glucose/L (Figures 19(a) and 19(b)). The associated reduction in cell metabolism and growth could also explain the lower biomass concentrations observed for the case of -300 g glucose/L especially during the final stages of fermentation. 2.2.3.3 Dissolved carbon dioxide controlled at 1000 mg/L

[0192] While controlling dissolved CO2 at 750 mg/L had several positive influences on fermentation performance, controlling dissolved CO2 at 1000 mg/L did not significantly change the interpretation of fermentation performance. Insignificant changes were observed due to change in the initial glucose concentration. Figures 20 and 21 show the dissolved CO2 profiles under -250 and -300 g glucose/L initial concentration for different aeration rates respectively. The corresponding glucose, ethanol and biomass concentration and cell viability profiles are plotted in Figure 22.

2.2.3.3.1 Effect on dissolved carbon dioxide profile

[0193] As explained earlier and illustrated in Figures 13, 14 and 18, differences in dissolved CO2 profiles between -250 and -300 g glucose/L were restricted to Region II. Region II in Figure 21 was similar to Region II of Figure 18(b).

2.2.3.3.2 Effect on aeration

[0194] Without being bound by theory, lower oxygen supply and a higher dissolved CO2 set point could potentially be blamed for an increase in CO2 inhibition in the broth. The observed cell viabilities (>90%) and biomass concentrations in both -250 and -300 g glucose/L (Figure 22) show that yeast metabolism is not inhibited at the macroscopic level. Lower oxygen supply for dissolved CO2 controlled at 1000 mg/L seen in Figure 23 could be justified in terms of the shorter fermentation duration seen for these cases in comparison to control at 750 mg/L (Table 2). Lower air supply might also economically justify the use of a higher set point and lower aeration rate for the case of -300 g glucose/L.

2.2.3.3.3 Effect on metabolite consumption and production

[0195] Apart from the effects mentioned in Section 2.2.3.2.2 no significant differences were observed as a result of the increase in dissolved CO2 set point from 750 to 1000 mg/L. Hence, in this example, differences in ethanol production were not contingent upon either the dissolved CO2 set point or aeration rate but only on the initial glucose concentration. 2.2.3.3.4 Effect on yeast growth and viability

[0196] While cell viabilities were maintained at very high levels (>90%) for the case of -250 g glucose/L for the entire process, an abrupt drop in viabilities were observed for the case of -300 g glucose/L towards the end of the process (Figure 22). While it is possible that the higher dissolved CO2 set point is responsible for this effect, it could be argued otherwise given that a similar trend was observed when dissolved CO2 was controlled at 750 mg/L (Figure 19). Hence, it is possible that other factors apart from dissolved CO2 inhibition may be involved. [0197] Without being bound by theory, one such factor could be deduced from the glycerol concentrations. 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). One could argue that the glycerol production pathway in yeast shown in Figure 1 could be robust and rigid to stresses and beyond control. In this regard it is of significance to note that glycerol concentrations did not vary significantly from batch counterparts without CO2 control for a given glucose concentration (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. Thus, it is highly possible that the shortfall in NAD ⁺ to maintain the NADH/NAD ⁺ balance is offset by directing flux to other pathways. At the same time, an equally valid argument would be that the inability of the cell to offset this shortfall results in loss of cell viability. These factors may be magnified in the presence of high toxic ethanol concentrations during the final stages of fermentation as well as high initial glucose concentrations.

2.2.4 Effect of dissolved carbon dioxide control on glucose conversion efficiency and ethanol productivity

[0198] Glucose conversion efficiencies and ethanol productivities under various fermentation conditions discussed previously are compared in Figures 25 and 26. The values of conversion efficiency show that controlling dissolved CO2 at 500 mg/L was the least efficient in the tested conditions. This conclusion is based on the low ethanol concentration (Figure 16) as well as the highest quantity of air supplied (Figure 23). The differences arising out of variation in dissolved CO2 set points was hypothesized to be a result of the compounded effect of CO2 removal as well as oxygen supply. These effects could explain the slightly lower conversions witnessed when dissolved CO2 was controlled at 1000 mg/L in comparison to when dissolved CO2 was controlled at 750 mg/L for -250 g glucose/L.

[0199] But, the hypothesis does not explain the uniformity in ethanol productivities for -250 g glucose/L initial concentration irrespective of the aeration rates or the very slight difference in conversion efficiencies between 750 and 1000 mg/L for either glucose concentration (Figure 26). It is possible that bubbling of air for dissolved CO2 control is responsible for this effect.

[0200] 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.

[0201] Increase in suspended cell concentration could also explain the variations seen in the biomass estimations of processes under CO2 control (Figures 19 and 22). Suspended cell concentration is typically higher when air is being bubbled through the broth. In the absence of any air bubbling, cells tend to precipitate from the fermentation broth. Since the final biomass estimated is usually in the absence of any air bubbling, biomass estimations tend to be significantly lower than a previous measurement. An increase in suspended cell concentration translates into an increase in number of active cells. Without being bound by theory, this combined with the higher viabilities and higher biomass (Figure 24) could potentially explain the higher fermentation rates and ethanol productivities as seen in the form of reduced fermentation times and complete glucose utilization even for glucose concentrations as high as 300 g/L. This physiochemical effect could also be responsible for the higher ethanol productivities seen when dissolved CO2 was controlled at 1000 mg/L rather than at 750 mg/L for -300 g glucose/L (Figure 26).

[0202] However, ethanol productivities and conversion efficiencies for -300 g glucose/L were lower than that of -250 g glucose/L by 13.77±9.60 % and 2.27±5.27 % respectively irrespective of the dissolved CO2 set points and aeration rates. The relatively lower biomass concentrations (Figure 24) may be responsible in part for the reduced conversion efficiencies and ethanol productivities seen in the -300 g glucose/L case. In addition, longer duration of fermentation (Table 2) in the case of -300 g glucose/L could be a factor for decrease in ethanol productivities. Longer durations in conjunction with higher osmosis and increasing ethanol concentrations as the fermentation progresses also tend to induce collective inhibitory pressure in this regard as explained earlier. In addition, physiochemical effects of lower pH and higher broth viscosity exacerbate the inhibitory effects of dissolved CO2 thereby affecting fermentation performance.

2.3 Comparison with redox potential measurement and profiles

2.3.1 Similarities between dissolved carbon dioxide and redox potential measurements

[0203] A relationship between dissolved CO2 and ORP profiles was established through their mutual association to yeast growth in terms of similarities and contrasts between the two measurement profiles. Figure 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. While osmotic effects increase the duration of the lag phase, ethanol inhibition and toxicity reduce cell viability resulting in sluggish fermentations and incomplete substrate utilization (Feng et al., 2012; Lin et al., 2010; Liu et al., 201 1a). Hence, similarity between the measurement of ORP and dissolved CO2 are restricted to Regions I and IV of both profiles. The increment in lag phase duration can be observed as a decrease in slope of the ORP and dissolved CO2 curves in Region I of the respective figures (Figure 27). Region IV is a result of decreasing yeast activity in both cases. In reference to Figure 27(b) (Region IV) where VHG fermentation was conducted under -300 g glucose/L condition, it can be seen that the concentration of ethanol is well beyond the toxic limit of 85 g/L, and glucose is nearly utilized. In comparison, 12.53±1 1.06 g glucose/L was unfermented when ORP control was implemented. Hence, it was hypothesized that although differences exist in Region IV in terms of the glucose concentration represented by the two profiles, both measurements represent the common element of cessation/reduction in yeast activity and consequential end of fermentation. ORP measurements are capable of representing loss of cell viability as a result of ethanol toxicity but not due to glucose exhaustion. In contrast, loss of cell activity or metabolism as a result of substrate exhaustion was much more accurately represented and conspicuously observed in fermentations with dissolved CO2 based control irrespective of the feed glucose concentrations.

2.3.2 Contrasts between dissolved carbon dioxide and redox potential measurements

[0204] Although there are similarities that exist between the two measurements in terms of physical and dynamic properties of the system they represent, these similarities do not extend beyond Regions I and IV. The contrast between the two profiles on the other hand can be drawn in terms of physio-chemical and biological factors that ultimately result in cessation of yeast metabolism and hence fermentation. These contrasts focus on Regions II and III of either profile. In Regions II and III although the profiles capture similar regions of yeast growth, the difference in quantities they measure is the reason for their dissimilar nature. In Region II of the dissolved CO2 curve, an increase in yeast activity is witnessed as a result of oxygen supplied to the system (Section 2.2.1); while no change in activity is seen in the case of ORP (referring to Region II). Even if there were a change in activity, it is not possible to observe the change through measurement of ORP. This is due to the very nature of ORP control that 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. In the case of dissolved CO2 based control, dissolved CO2 set point values are based on the solubility of CO2 for a given media under given conditions of temperature and pressure. Moreover, initial dissolved CO2 concentrations remain the same in the absence of any yeast activity prior to inoculation, i.e. an accurate estimate of yeast activity can be made for a given dissolved CO2 concentration based on the size of the inoculum, which is not possible in case of ORP measurements. Hence, dissolved CO2 measurements are absolute unlike their ORP counterparts.

[0205] 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.

[0206] Thus, despite the similarities and contrasts between ORP and dissolved CO2 measurement, when used in conjunction with each other they should be capable of representing cessation of yeast metabolism due to both glucose exhaustion as well as ethanol toxicity. Example 3.0 - Repeated-Batch Fermentation

[0207] 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. As an abrupt reduction of the concentration of dissolved CO2 was detected, approximately one-half of the fermentation broth was withdrawn, and an equal volume of fresh media was replenished (thereby removing approximately half the ethanol from the fermentation broth and increasing the feed concentration of glucose to approximately half its initial concentration). The self-cycling period was estimated by measuring time elapsed between peaks of two contiguous cycles. In this example, 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. [0208] In this example, an abrupt reduction in the concentration of dissolved CO2 in the fermentation broth indicating complete or nearly complete glucose utilization was detected by measuring the slope of a plot of the concentration of dissolved CO2 versus time, as measured at one-minute intervals. The slope of this plot was calculated by taking the difference between two consecutive measured dissolved CO2 concentrations with a one-minute time interval and dividing this by one minute. As the magnitude of the rate of decrease in the slope of the concentration of dissolved CO2 versus time becomes larger and larger, it is determined that there has been an abrupt reduction in the concentration of dissolved CO2. Depending on the initial glucose concentration, three to five consecutive slopes were compared. For this exemplary case of glucose at an initial concentration of 200 g/L, three consecutive slopes were compared. When a trend was observed that (a) each subsequent slope was smaller than the immediately preceding slope (i.e. the magnitude of each subsequent slope was greater in the negative direction) for three consecutive one -minute intervals and (b) the magnitude of the difference between each consecutive slope had increased over those three consecutive one-minute intervals, it was concluded that there has been an abrupt reduction in dissolved CO2 concentration.

[0209] 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. One skilled in the art would reasonably infer based on this example that 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.

Furthermore, 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. [0210] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub- combinations thereof. For example, it is not necessary that adjacent time intervals compared to determine that rate of decrease in the slope of a plot of the concentration of dissolved CO2 versus time is accelerating be immediately adjacent to each other; a gap in time could be present. It is therefore intended that the following appended aspects and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations thereof that are not mutually exclusive. The scope of the appended claims and claims hereafter introduced should not be limited by the exemplary embodiments and examples specifically set forth herein, but should be given the broadest interpretation consistent with the specification as a whole.

References

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