Ethanol production from whey with immobilized living yeast

Summary Living Kluyveromyces fragilis yeast cells were succesfully entrapped in calcium alginate gel beads at cell loadings of 4 to 16 g yeast (0.8 to 3.2 g d.m.) per 1 g of sodium alginate. In batch systems, about 90 % conversion in 48 h was obtained both with free and immobilized yeast using demineralized whey of 5 to 10 % lactose content as substrate. In continuous packed-bed column operation nearly a constant 2 % product ethanol concentration could be maintained at 5 % substrate lactose level for at least one month.     

Ethanol tolerance of immobilized brewers' yeast cells

A method based on the survival of yeast cells subjected to an ethanol or heat shock was utilized to compare the stress resistance of free and carrageenan-immobilized yeast cells. Results demonstrated a significant increase of yeast survival against ethanol for immobilized cells as compared to free cells, while no marked difference in heat resistance was observed. When entrapped cells were released by mechanical disruption of the gel beads and submitted to the same ethanol stress, they exhibited a lower survival rate than entrapped cells, but a similar or slightly higher survival rate than free cells. The incidence of ethanol- or heat-induced respiratory-deficient mutants of entrapped cells was equivalent to that of control or non-stressed cells (1.3 ± 0.5%) whereas ethanol- and heat-shocked free and released cells exhibited between 4.4% and 10.9% average incidence of respiration-deficient mutants. It was concluded that the carrageenan gel matrix provided a protection against ethanol, and that entrapped cells returned to normal physiological behaviour as soon as they were released. The cell growth rate was a significant factor in the resistance of yeast to high ethanol concentrations. The optimum conditions to obtain reliable and reproducible results involved the use of slow-growing cells after exhaustion of the sugar substrate.     

Continuous production of ethanol in high concentration using immobilized growing yeast cells

Summary Application of an immobilized growing yeast cell system to continuous production of ethanol in high concentration (10%) was investigated using Saccharomyces cerevisiae IFO 2363. When a medium containing 25% glucose was fed, the growth of yeast cells in gel was inhibited. The inhibitory effect was found to be reduced by a stepwise increase in concentration of glucose in the feed medium. The stepwise operation resulted in constant growth of cells in the gel even in the medium containing 25% glucose. By this stepwise feeding system, continuous production of ethanol of 114 mg/ml was maintained at a retention time of 2.6 h for over 2 months and a conversion rate of glucose to ethanol of over 95% of theoretical, was achieved.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Continuous ethanol production by yeast cells immobilized in open pore gelatin matrix

Summary Open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of Ca alginate from the composite matrix followed by crosslinking with glutaraldehyde. Saccharomyces uvarum cells immobilized in the porous carrier showed high ethanol productivities with a maximum value of 25 g/l.h when monitored in packed bed reactors at 35°C with continuous cane molasses feedstock containing 10% fermentable sugars.     

Production of ethanol by immobilized Saccharomyces bayanus in an extractive fermentation system

An extractive fermentation system using immobilized yeast cells was developed to study the ethanol production at high sugar concentrations. Organic acids were used as extracting solvents of ethanol and their toxicity was tested in free and k-carrageenan entrapped cell preparations. Immobilization seems to protect cells against solvent toxicity, when long-chain organic acids, e.g., oleic acid, were used, probably due to steric and diffusional limitations, the free cells not being viable at high oleic acid concentrations. The entrapped cells also present a higher metabolic activity than their free counterparts at high glucose concentrations. A solution of 300 g/L of glucose was totally fermented by the immobilized yeast cells, which when free cannot normally convert more than 200 g/L. In situ recovery of ethanol by oleic acid in a batch immobilized cell system led to higher ethanol productivities and to the fermentation of 400 g/L, when an oleic acid/medium ratio of 5 was used.     

Immobilized <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.     

Continuous production of ethanol using immobilized growing yeast cells

Summary Immobilized growing yeast cells were prepared in kappa-carra-geenan gel. Gel beads containing a small number of cells were incubated in a complete medium. The cells grew very well in the gel and the number of living cells per ml of gel increased to over 10 times that of free cells per ml of culture medium. After growing in the gel, the cells formed a dense layer of cells near the gel surface and produced large amounts of ethanol. The conditions for continuous production of ethanol using immobilized growing yeast cells were investigated. The supply of appropriate nutrients for growth was essential for the continuous production. The living cells in the gel were maintained at the high level of 10⁹ per ml of gel and continuous production of ethanol using the complete medium containing 10% glucose was carried out with a retention time of 1 h. In this operation, a stable steady state was maintained for longer than 3 months. The ethanol concentration was 50 mg/ml and the conversion of glucose utilized to ethanol produced was almost 100% of the theoretical yield.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae

The effect of γ-ray irradiation on alcohol-dehydrogenase activity of yeast was investigated. The results suggested that low doses of γ-ray (10 and 20 Gy) significantly increased the enzyme activity. This work also describes the impact of irradiation on immobilization efficiency of biocatalyst entrapped on to alginate gel beads. When yeast irradiated to a dose of 20 Gy was immobilized, ADH stability was improved up to 1.4 times at 45 °C compared to the immobilized non-irradiated cells. Also, the irradiated biocatalyst, when immobilized, can be reused more than eight times in oxidation reaction of ethanol. This preparation also permitted to reach high yields of immobilization (79%) and activity (88%).     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

Continuous production of L-arginine using immobilized growing Serratia marcescens cells: Effectiveness of supply of oxygen gas

Summary The immobilized growing cell system using Serratia marcescens was applied to continuous L-arginine production. From the determination of oxygen uptake rate, it was shown that the cells entrapped in carrageenan gel were in an oxygen-limited state due to the diffusion barrier to oxygen transport created by the gel layer. This limited state in gel was relieved by supply of oxygen-enriched gas instead of air into the medium. The maximum population of immobilized cells increased to five times that of free cells with the supply of pure oxygen gas. The L-arginine-producing activity of the immobilized growing cells was proportional to the concentration of oxygen gas supplied and was 6 mg/h per millilitre in gel supplied with pure oxyges gas. The continuous L-arginine containing production was constantly maintained by controlling the medium penicillin G at pH 6.5 and more than 10 mg/ml of L-arginine were obtained at 10h of residence time for at least 12 days.     

Cell immobilization in kappa-carrageenan for ethanol production

A combination of extended Monod kinetics and the diffusional equation was used for evaluating the effectiveness factor of entrapped immobilized cells. Based on the kinetics of Zymomonas mobilis reported in the literature, the numerical results have revealed that the problem of mass transfer diffusional restrictions can be neglected by using small beads (1 mm in diameter) with a corresponding cell loading up to 276 g/L gel. On the basis of the numerical results obtained, the application of immobilized cells for continuous ethanol production was investigated. The kappa-carrageenan method was utilized to entrap Z. mobilis CP4, a potential ethanol producer. A two stage fermentation process has also been developed for ethanol production by the Z. mobilis carrageenan-bound cells. About 90 g/L ethanol was produced by immobilized cells at a total residence time of 1.56 h. The ethanol yield was estimated to be 93% of theoretical. The results obtained in this study also indicated that the control of optimum pH in an immobilized cell column is necessary to enhance the rate of ethanol production.     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

Alumina-doped alginate gel as a cell carrier for ethanol production in a packed-bed bioreactor

Alumina-doped alginate gel (AEC) was developed as a new type of cell carrier to be used in ethanol fermentation. The presence of the alumina particles in alginate gel not only improved the porous structure of the carrier, but also provided many advantageous characteristics including good mechanical strength, high stability, and high immobilization yield. The attachment of alumina particles and yeast cells by electrostatic attraction was shown to promote cell growth and increase ethanol productivity. The AEC carrier was found to be more effective for the immobilization of Saccharomyces cerevisiae M30 than the conventional Ca-alginate bead. Ethanol productivities of 1.4 and 7.9 ∼ 12.6 g/(L/h) were obtained using the AEC cultures in batch and continuous modes of operation, respectively, with an ethanol yield of 43.9 ∼ 46.7% and an immobilized yield of 81.4 ∼ 84.5%. Ethanol fermentation in a continuous packed-bed reactor using the AEC carrier was stable for > 30 days.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Alcoholic fermentation of D-xylose by immobilizedPichia stipitis yeast

Summary Pichia stipitis NRRL Y-7124 yeast cells were for the first time immobilized both in agar gel beads and on fine nylon net for ethanol fermentation on D-xylose, in order to investigate the possibility of using the biocatalyst for improved utilization of the biomass pentose fraction. With free cells the initial xylose level affected little ethanol production, with a maximum of 22 g/l ethanol obtained in 5 days on 5% and of 40 g/l in 8 days on 10% xylose, and an average volumetric productivity of about 0.22 g/lh. The maximum ethanol concentration of 19.5% on 5% xylose with the nylon net attached cells in a continuous packed-bed column reactor was obtained with 35 h residence time. The volumetric productivities of 0.56 g/lh at 19.5 g/l ethanol and 1.0 g/lh at 15.0 g/l ethanol were markedly higher than those obtained with free cells. The stability of the immobilized biocatalyst was excellent. The same reactor could be used for at least 80 days without significant activity loss.     

Continuous ethanol production by immobilized yeast reactor

Summary Saccharomyces cerevisiae yeast immobilized in calcium alginate gel beads was employed in packed-bed column reactors for continuous ethanol production from glucose or cane molasses, and for beer fermentation from barley malt wort. With properly balanced nutrient content or periodical regeneration of cells by nutrient addition and aeration, ethanol production could be maintained for several months. About 7 percent (w/v) ethanol content could be easily maintained with cane molasses diluted to about 17.5 percent (w/v) of total reducing sugars at about 4 to 5 h residence time. Beer of up to 4.5 percent (wv) of ethanol could be produced from barley wort at about 2 h residence time without any addition of nutrients.     

Scanning electron microscopy of immobilizedHumicola lutea during semicontinuous production of acid proteinases

The morphology of the fungusHumicola lutea (strain 120–5), immobilized in polyacrylamide and polyhydroxyethylmethacrylate and used for the semicontinuous production of acid proteinases, was examined by scanning electron microscopy. The fungus developed a dense mycelium below the bead surface as well as in the bead interior after precultivation of entrapped spores. During maximal semicontinuous enzyme biosynthesis, formation of numerous large bulbous cells with a different shape was observed. Lysis of the cells was observed mainly in the centre of the gel beads after 13 successive fermentations with polyacrylamide-immobilized cells or after 21 re-uses of polyhydroxyethylmethacrylate-immobilized mycelia, respectively. Growth and changes in the cellular morphology of immobilizedH. lutea, accompanying biosynthesis of acid proteinases, were comparable in both gel matrices but mycelia immobilized in polyhydroxyethylmethacrylate maintained their productivity twice as long.     

Metabolic flux and cell cycle analysis indicating new mechanism underlying process oscillation in continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions

Process oscillation characterized by long oscillation period and large oscillation amplitude was observed in continuous ethanol fermentation with Saccharomyces cerevisiae under very high gravity conditions. Metabolic flux analysis was applied to the fermentation system, and the results indicated that carbon flux distributions at the metabolic notes oscillated, correspondingly, and the root reason for the process oscillation was the intracellular metabolism of yeast cells. Cell cycle analysis with the flow cytometry showed that no cell-cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation, and thus different mechanism existed compared with the oscillation observed in the continuous culture of Saccharomyces cerevisiae and triggered by the synchronization of the daughter and mother cells under specific conditions. Furthermore, the overall metabolic activity of the yeast cells was examined, which was found not exactly out of phase but lag behind ethanol concentration that accumulated within the fermentation system and its inhibition on the yeast cells as well, which supported the mechanistic speculation for the process oscillation: the lag response of yeast cells to ethanol inhibition.