Simultaneous saccharification of inulin and ethanol fermentation by recombinantSaccharomyces cerevisiae secreting inulinase

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.     

Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.)

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.     

Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1

A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl2 and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30 degrees C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm2 for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.     

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.     

Role of Tween 80 and Monoolein in a Lipid-Sterol-Protein Complex Which Enhances Ethanol Tolerance of Sake Yeasts

An exogenous ternary complex composed of Tween 80, ergosterol, and albumin increased the final ethanol concentration of fermentation by sake yeasts from 17.2 to 19.0% (vol/vol) and reduced the fermentation time from 30 to 25 days. Likewise, a complex of monoolein, albumin, and either ergosterol or ergosteryl oleate increased the final ethanol concentration of fermentation to 19.7 or 19.8% (vol/vol), respectively, and reduced the fermentation time to 25 days. Both Tween 80 and monoolein promoted the fermentative activity (Q_CO2) of cells, and the effect was enhanced by the presence of ergosterol.     

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations

By using 7 x 10(8) cells of Saccharomyces cerevisiae per ml with which 25 degrees Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 x 10(7) to 7.8 x 10(8) cells per ml. At the lowest level of inoculum, 1.1 x 10(7) cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar.     

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40?°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7?%, respectively. In the range of 30 to 40?°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40 degrees C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

Acid-hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol/l sulfuric acid using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol after 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol after 18 h. The results showed that acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by it in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.     

Selection and study of potent lactose-fermenting yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen most potent strains isolated from milk products fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11-12, and 10-12%, respectively (4 degrees C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30 degrees C for 2-3 days, ethanol concentration was 4-5%.     

Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity

Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. (c) 1997 John Wiley & Sons, Inc.     

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

Biotechnological potential of inulin for bioprocesses

Inulin consists of linear chains of β-2,1-linked d-fructofuranose molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end. In this review article, inulin and its applications in bioprocesses are overviewed. The tubers of many plants, such as Jerusalem artichoke, chicory, dahlia, and yacon contain a large amount of inulin. Inulin can be actively hydrolyzed by microbial inulinases to produce fructose, glucose and inulooligosaccharides (IOS). The fructose and glucose formed can be further transformed into ethanol, single-cell protein, single cell oil and other useful products by different microorganisms. IOS formed have many functions. Therefore, inulin can be widely used in food, feed, pharmaceutical, chemical and biofuels industries.     

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes.

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

Isolation of thermotolerant ethanologenic yeasts and use of selected strains in industrial scale fermentation in an Egyptian distillery

An enrichment and isolation program for new ethanol-producing thermotolerant yeasts as well as a screening program of some known thermotolerant strains resulted in the selection of several strains capable of growth at 40-43 degrees C. Among these strains four grew and fermented sugar cane molasses at 43 degrees C under batch conditions with sugar-conversion efficiencies >94% and ethanol concentrations 6.8-8.0% (w/v). The two best-performing strains, a Saccharomyces cerevisiae F111 and a Kluyveromyces marxianus WR12 were used in eight 87.5 m(3) fermentation runs (four using each strain) for industrial ethanol production in an Egyptian distillery using sugar cane molasses. Mean ethanol production was 7.7% and 7.4% (w/v), respectively, with an added advantage of cooling elimination during fermentation and higher ethanol yields compared to the distillery's S. cerevisiae SIIC (ATCC 24855) strain in use. The isolate S. cerevisiae F111 was subsequently adopted by the distillery for regular production with significant economical gains and water conservation.     

Characterization of cellobiose fermentations to ethanol by yeasts

Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test cultures. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When higher carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of beta-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, beta-glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the beta-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When empolying these organisms in a simultaneous saccharification-fermentation of avicel, using Trichoderma reesei cellulase as the saccharifying agent, 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae.