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.     

Continuous production ofL-isoleucine using immobilized growing Serratia marcescens cells

An immobilized growing cell system was applied to the continuous L -isoleucine production by Serratia marcescens. In the new immobilized-cell systems using the carrageenan gel method. S. marcescens cells in the gel required nutrients and oxygen for growth, and the numbers of living cells per milliliter of gel increased to the levels of that of free cells in the liquid medium. This immobilized growing cell system exhibited high and stable activity for isoleucine production under steady-state conditions. Continuous isoleucine production was carried out by feeding the nutrient medium under aeration into a fluidized bed reactor containing the immobilized cells. In the continuous operation, an efficient production was maintained by automatically controlling the pH of the reaction mixture at 7.5. The productivity of isoleucine increased using multibed reactors. In a two-bed reactor system, the effluent L -isoleucine concentration reached 4.5 mg/ml at a retention time of 10 hr, and a steady state was maintained for longer than 30 days.     

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 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.     

Production of ethanol and biomass from various carbohydrates byKluyveromyces fragilis

Summary Growth ofKluyveromyces fragilis NRC 2475 and the production of ethanol by the yeast were studied in the media containing one of the following sugars: glucose, lactose, galactose, or a glucose-galactose (50% 50%) mixture as a carbon source.The largest biomass yield and the lowest yield of ethanol were obtained in the medium containing glucose. The medium containing galactose gave the lowest yield of biomass and the largest yield of ethanol. When lactose was used for the growth and production of ethanol the obtained results for both biomass and ethanol were between those obtained with glucose and galactose.The ethanol productivities, expressed in terms of ethanol produced either per unit of cells, or per unit of cells and time, were the highest in the system with galactose and the lowest in that with glucose.     

Ethanol fermentation technologies from sugar and starch feedstocks

This article critically reviews some ethanol fermentation technologies from sugar and starch feedstocks, particularly those key aspects that have been neglected or misunderstood. Compared with Saccharomyces cerevisiae, the ethanol yield and productivity of Zymomonas mobilis are higher, because less biomass is produced and a higher metabolic rate of glucose is maintained through its special Entner-Doudoroff pathway. However, due to its specific substrate spectrum as well as the undesirability of its biomass to be used as animal feed, this species cannot readily replace S. cerevisiae in ethanol production. The steady state kinetic models developed for continuous ethanol fermentations show some discrepancies, making them unsuitable for predicting and optimizing the industrial processes. The dynamic behavior of the continuous ethanol fermentation under high gravity or very high gravity conditions has been neglected, which needs to be addressed in order to further increase the final ethanol concentration and save the energy consumption. Ethanol is a typical primary metabolite whose production is tightly coupled with the growth of yeast cells, indicating yeast must be produced as a co-product. Technically, the immobilization of yeast cells by supporting materials, particularly by gel entrapments, is not desirable for ethanol production, because not only is the growth of the yeast cells restrained, but also the slowly growing yeast cells are difficult to be removed from the systems. Moreover, the additional cost from the consumption of the supporting materials, the potential contamination of some supporting materials to the quality of the co-product animal feed, and the difficulty in the microbial contamination control all make the immobilized yeast cells economically unacceptable. In contrast, the self-immobilization of yeast cells through their flocculation can effectively overcome these drawbacks.     

A new immobilized cell system with protection against toxic solvents

A new immobilized cell system providing protection against toxic solvents was investigated so that normal fermentations could be carried out in a medium containing toxic solvents. The system consists of immobilized growing cells in Ca-alginate gel beads to which vegetable oils, which are inexpensive absorbents of solvents, had been added. The ethanol fermentation of Saccharomyces cerevisiae ATCC 26603 was used as a model fermentation to study the protection afforded by the system against solvent toxicities. The fermentation was inhibited by solvents such as 2-octanol, benzene, toluene, and phenol. Ethanol production of one batch was not finished even after 35 h using immobilized growing yeast cells in conventional Ca-alginate gel beads in an ethanol production medium (5% glucose) containing 0.1% 2-octanol, which is used as a solvent for liquid-liquid extraction and is one of the most toxic solvents in our experiments. With the new immobilized growing cell system using vegetable oils, however, four repeated batch fermentations were completed in 35 h. Castor oil provided even more protection than soy bean, olive, and tung oils, and it was possible to complete six repeated batches in 35 h. The immobilized cell system with vegetable oils also provided protection against other toxic solvents such as benzene and toluene. A possible mechanism for the protective function of the new immobilized cell system is discussed.     

Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.     

Control of packed column fouling in the continuous fermentation and stripping of ethanol

By recycling the contents of a 14 L fermentor through a stripping column to continuously remove ethanol and reduce product inhibition, continuous complete conversion of nutrient feed containing 600 g/L glucose was achieved in a small pilot plant. Ethanol was recovered from the carbon dioxide stripping gas in a refrigerated condenser, and the gas was reheated with steam and recycled by a blower. Productivity of ethanol in the fermentor as high as 15.8 g/L/h and condensate production of up to 10 L/day of almost 50% by volume ethanol were maintained for up to 60 days of continuous operation. Weekly washing of the column packing in situ was required to prevent loss of performance caused by attached growth of yeast cells, which restricts the gas flow rate through the stripping column. (c) 1996 John Wiley & Sons, Inc.     

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.     

The use of plant cell vesicles for immobilization of yeast cells producing ethanol

The preparation of immobilized living yeast cells adsorbed into or onto delipided specimens of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) is reported. These yeast cell-plant cell conjugates were used for the repeated batch production of ethanol from glucose (143 to 246 g/l) or saccharose (150 g/l). Up to 25 fermentation cycles at 30°C were performed. The cycle time for complete substrate conversion to ethanol was reduced 10-fold by a 5-fold increase of the yeast cell Wolffia conjugate concentration (ε = 0.08 to ε =0.4) ε = volume of cell conjugate/totnl reaction volume. The corresponding ethanol production was 11.5 to 13.5 vol% and 9 vol% respectively. The reported results on the discontinuous ethanol fermentation with Wolffia-immobilized yeast cells open the field for their application in continuous ethanol production processes.     

Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae.

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With such supplementation, the viability of the yeast cells was maintained above 80% throughout the fermentation, while it dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effective than proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% with glycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested that glycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine bases increased the sugar uptake and ethanol production but did not allow the population to maintain the high level of cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence of particulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar utilization to 68, 75, 81, and 82%, respectively.     

Ethanol Production by Saccharomyces cerevisiae Immobilized in Hollow-Fiber Membrane Bioreactors

Saccharomyces cerevisiae ATCC 4126 was grown within the macroporous matrix of asymmetric-walled polysulfone hollow-fiber membranes and on the exterior surfaces of isotropic-walled polypropylene hollow-fiber membranes. Nutrients were supplied and products were removed by single-pass perfusion of the fiber lumens. Growth of yeast cells within the macrovoids of the asymmetric-walled membranes attained densities of greater than 10¹⁰ cells per ml and in some regions accounted for nearly 100% of the available macrovoid volume, forming a tissue-like mass. A radial distribution of cell packing existed across the fiber wall, indicating an inadequate glucose supply to cells located beyond 100 μm from the lumen surface. By comparison, yeast cell growth on the exterior surfaces of the isotropic-walled membranes resulted in an average density of 3.5 × 10⁹ viable cells per ml. Ethanol production by reactors containing isotropic polypropylene fibers reached a maximum value of 26 g/liter-h based on the total reactor volume. Reactor performance depended on the fiber packing density and on the glucose medium flow rate and was limited by low nutrient and product transport rates. The inhibition of ethanol production and the reduction in fermentation efficiency arose primarily from the accumulation of CO₂ gas within the sealed reactor shell space.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Comparative study of ethanol production by an immobilized yeast in a tubular reactor and in a multistage reactor

Summary The productivity of continuous ethanol fermentation has been increased using fixed bed reactors where a high density of yeast cells was maintained on a packing of wood chips. Two different systems have been used: 1. A tubular reactor which produced alcohol solutions containing up to 13.5% (V/V) ethanol. High CO₂ retention and a poor mass transfer between bulk medium and immobilized biomass prevented production rates higher than 2.2 g/l·h. 2. A multistage reactor where a better utilisation of the reactor volume led to improved performances. Solutions containing 132 g/l of ethanol (16.5% V/V) were produced with a productivity increased up to 4.8 g/l·h. A better distribution of the active biomass and a lower gradient of alcohol concentration between support and bulk medium are possible reasons for this improvement.     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on alpha-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His(6)) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-alpha-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley beta-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and beta-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing beta-glucan as the sole carbon source and could directly ferment 45 g of beta-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.     

Regulation of beta-1, 4-Glucosidase Expression by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 expressed beta-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed beta-glucosidase expression (<0.3 U/ml); however, this yeast did produce beta-glucosidase when the initial glucose concentration was 相似文献