Application of gravitational sedimentation to efficient cellular recycling in continuous alcoholic fermentation

A mathematical model for the sedimentation velocity in an inclined parallel plate sedimenter is proposed. The parameters of the alcoholic fermentation broth (cell density of Saccharomyces cerevisiae, density of the fermentation medium, viscosity of the broth at various alcohol and biomass contents) were determined experimentally. The sedimentation velocities were predicted under the various operational conditions and parameters, both of the broth (the alcohol concentration and cell content) and the sedimenter prototype (length, distance between the plates, and slope). The proposed model for the sedimentation velocity presented a good correlation with the experimental results of continuous sedimentation. These sedimenter prototypes were assembled and tested for efficiency of separation of yeast cell under conditions considered for interest for continuous alcoholic fermentation. A selective filter for the overflow composed of calcium alginate gel improved operation. A high operational stability, high separation efficiency (over 98%), and adequate settler residence times (about 20 min) were attained. The operational results permitted the operation of continuous alcoholic fermentation with cellular recycling effected exclusively by gravitational sedimentation, this characterizing a process of enormous industrial interest because of the operational simplicity and low operational and maintenance costs. (c) 1993 John Wiley & Sons, Inc.     

Trehalose metabolism in Saccharomyces cerevisiae during alcoholic fermentation

Strains of Saccharomyces cerevisiae accumulated intracellular trehalose up to 105 mg/g cell dry wt with 90% survival. Viability could be correlated to trehalose levels during ethanol fermentation albeit the disaccharide did not seem to contribute to fermentation yields. Trehalose-6-phosphate synthase showed high activity (up to 279 mu/mg protein) even at high residual sucrose concentration (115 g/l) in the wort suggesting to be a response of yeast cells to the osmotic stress conditions.     

Mechanism of proanthocyanidins-induced alcoholic fermentation enhancement in Saccharomyces cerevisiae

Our previous work revealed proanthocyanidins (PAs) could pose significant enhancement on the activity of H⁺-ATPase and fermentation efficiency after a transient initial inhibition (Li et al in Am J Enol Vitic 62(4):512–518, 2011). The aim of the present work was to understand the possible mechanism for this regulation. At Day 0.5 the gene expression level of PMA1 in AWRI R₂ strain supplemented with 1.0 mg/mL PAs was decreased by around 54 % with a 50 % and a 56.5 % increase in the concentration of intracellular ATP and NADH/NAD⁺ ratio, respectively, compared to that of control. After the transient adaptation, the gene expression levels of PMA1 and HXT7 in PAs-treated cells were enhanced significantly accompanied by the decrease of ATP contents and NADH/NAD⁺ ratio, which resulted in the high level of the activities of rate-limiting enzymes. PAs could pose significant effects on the fermentation via glucose transport, the energy and redox homeostasis as well as the activities of rate-limiting enzymes in glycolysis.     

Intensification of alcoholic fermentation upon dehydration-rehydration of the yeast Saccharomyces cerevisiae

Summary In comparison with intact yeast, dehydrated-rehydrated cells of Saccharomyces cerevisiae show significantly higher ethanol production from exogenous substrate under both anaerobic and aerobic conditions, particularly when low concentration (0.1%) of glucose are used. For populations with a higher percentage of viable rehydrated cells (above 70%) a more notable decrease in the Pasteur effect (the difference between the quantity of ethanol formed under anaerobic and aerobic conditions) is observed.     

Relationship between the inhibition of alcoholic fermentation by Saccharomyces cerevisiae and the activities of hexokinase and alcohol dehydrogenase

Summary The cessation of fermentation by Saccharamyces cerevisiae in a medium rich in sugar is not due to an inhibition of the activities of hexokinase and alcohol dehydrogenase. The activity of alcohol dehydrogenase was significantly increased in a medium of low sugar concentration supplemented with toxic substances, ethanol and fatty acids.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Effect of anisothermal conditions on the kinetics of alcoholic fermentation by Saccharomyces cerevisiae

A comparison between isothermal and anisothermal alcoholic fermentation is made in this paper. Important differences were observed: in some anisothermal operations maximum rates of CO₂ production were reached towards the end of fermentation. Cultures with different initial nitrogen or biotin concentrations showed the importance of thermal conditions for the completion of the reaction. They indicated that the notion of the limiting nutrient does not have the same technological significance with respect to the mode of temperature processing. Thus, some studies at the laboratory scale should not be carried out under isothermal conditions, especially not within temperature ranges which may be critical for cell viability. This is, for example, the case with enological studies about red wine processing.     

Gaseous environments modify physiology in the brewing yeast Saccharomyces cerevisiae during batch alcoholic fermentation

Aims:  To investigate the impact of different gaseous atmospheres on different physiological parameters in the brewing yeast Saccharomyces cerevisiae BRAS291 during batch fermentation.
Methods and Results:  Yeasts were cultivated on a defined medium with a continuous sparging of hydrogen, helium and oxygen or without gas, permitting to obtain three values of external redox. High differences were observed concerning viable cell number, size and metabolites produced during the cultures. The ethanol yields were diminished whereas glycerol, succinate, acetoin, acetate and acetaldehyde yields were enhanced significantly. Moreover, we observed major changes in the intracellular NADH/NAD⁺ and GSH/GSSG ratio.
Conclusions:  The use of gas led to drastic changes in the cell size, primary energy metabolism and internal redox balance and E _h . These changes were different depending on the gas applied throughout the culture.
Significance and Impact of the Study:  For the first time, our study describes the influence of various gases on the physiology of the brewing yeast S. cerevisiae . These influences concern mainly yeast growth, cell structure, carbon and redox metabolisms. This work may have important implications in alcohol-related industries, where different strategies are currently developed to control better the production of metabolites with a particular attention to glycerol and ethanol.     

Improvement on laboratory fermentation equipment to study Saccharomyces cerevisiae metabolism

Beside being an ordinary fermenter, the present equipment was conceived to sample the medium, to store the samples and to record photographs of the yeasts. Ten sensors were used to measure gas exchanges. During the growth of ScM1 (a Saccharomyces cerevisiae strain) on glucose, we could observe two different linear decreases of CO₂ production rates (18.17±0.12 mmol CO₂ h^–2 (g biomass)^–1 and 8.67±0.12 mmol CO₂ h^–2 (g biomass)^–1), together with a sudden variation of slope during the respiro-fermentative phase. Nomenclature F_in InletairFlowl h ^–1 F_out OutletgasFlowl h ^–1 _in Inletairtemperature°C_out Outletgastemperature°CP _atm AtmosphericPressuremmHgP _in InletairOverPressuremmHgP _out OutletgasOverPressuremmHgDODissolvedO ₂ mg l^–1 pO₂ PartialPressureO ₂ in Outlet gas % (v/v) pCO₂ PartialPressureCO ₂ in Outlet gas % (v/v) Int(t) Whole number of hours     

Kinetics of volatile metabolites during alcoholic fermentation of cane molasses by Saccharomyces cerevisiae

Summary The kinetics of ethanol, acetaldehyde, ethyl acetate and fusel alcohols during alcoholic fermentations on cane molasses by Saccharomyces cerevisiae have been obtained via an in-situ gas membrane sensor connected to a gas chromatograph. Various operation parameters have been investigated such as inoculum rate, molasses concentration, operation mode (batch, fed-batch). The modification of fusel alcohols kinetics in response to addition of amino acids has been studied as well as the assimilation of two intermediary aldehydes (isovaleraldehyde and isobutyraldehyde) in the fusel alcohol synthesis pathway. Offprint requests to: M.-N. Pons     

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P₅₀)]. P₅₀ was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.     

Application of metabolically engineered Saccharomyces cerevisiae to extractive lactic acid fermentation

In this study, Saccharomyces cerevisiae OC-2T T165R, metabolically engineered to produce optically pure L(+)-lactic acid, was used to develop a high performance extractive fermentation process. Since the transgenic yeast could produce lactic acid efficiently even at lower than pH 3.5, high extractive efficiency was achieved when tri-n-decylamine (TDA), a tertiary amine, was used as the extractant. Separation of microorganisms by means of a hollow fiber module could not only improve the total amount of lactic acid produced but also increase the lactic acid concentration in the solvent. Moreover, pH had a significant effect on extractive fermentation. The highest rate of recovery of lactic acid could be obtained on pH-uncontrolled fermentation (pH 2.5); however, the lowest amount of lactic acid was produced. Taking into account the trade-off between the fermentation and extraction efficiencies, the optimum pH value was considered to be 3.5, with which the largest amount of lactic acid was produced and the highest lactic acid concentration in the solvent was obtained. The results show promise for the use of the transgenic yeast for extractive fermentation.     

Behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel and performing alcoholic fermentation

Summary The behaviour of Saccharomyces cerevisiae cells entrapped in a polyacrylamide gel was studied during their continuous function in an ethanol-producing reactor. Polymerization destroys 40% to 80% of the cells, depending on their physiological state. A three day adaptation phase is required before ethanol production stabilizes and this phase corresponds to an increase in cell concentration in the gels and to protein synthesis. The amounts of DNA, glucan, glycogen and trehalose are different in entrapped and free cells. Microscopic observation shows that 75% to 85% of the cells lose their integrity and that the remainder appear to multiply normally. Within a gel particle, both viability and fermentation activity are heterogeneous. A high percentage of cells have low viability and low fermentation activity. A proportion of cells remains capable of forming colonies and these cells have higher fermentation activity and are preferentially localized at the surface of gel particles.     

Industrial PE-2 strain of Saccharomyces cerevisiae: from alcoholic fermentation to the production of recombinant proteins

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