Effect of C/N ratio and starch concentration on ethanol production from sago starch using recombinant yeast

Recombinant Saccharomyces cerevisiae YKU 131 (capable of expressing glucoamylase) was used to produce ethanol from sago starch. The optimum C/N ratio for ethanol production by the recombinant yeast was 7.9, where 4.7 and 10.1 g/l ethanol was produced from 20 and 40 g/l sago starch, respectively. At sago starch concentration higher than 40 g/l and C/N ratio higher than 10.4, glucoamylase production and rate of starch hydrolysis were reduced, which in turn, reduced ethanol production significantly. The theoretical yield of ethanol based on sago starch consumed in fermentation using 40 g/l was 72.6%. This yield was slightly lower than those obtained in fermentation using soluble starch such as potato and corn starch, which ranged from 80–90% as reported in the literature. However, S. cerevisiae YKU 131 could only utilize 62% of the total amount of starch added to a medium.     

Fermentation of starch by Klebsiella oxytoca p2, containing plasmids with alpha-amylase and pullulanase genes.

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 degrees C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12-24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower alpha-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

High-level ethanol production from starch by a flocculent <Emphasis Type="Italic">Saccharomyces cerevisiae </Emphasis>strain displaying cell-surface glucoamylase

A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from SaccharomYces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/alpha-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h(-1) l(-1)) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g l(-1). In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol.     

Direct alcohol fermentation of starch by a derepressed mutant ofSchwanniomyces castellii

Summary Production of ethanol by direct fermentation of soluble starch was carried out using a derepressed mutant M-6 ofSchwanniomyces castellii NRRL Y-2477. Under optimal fermentation conditions, 58.4g/l ethanol was produced utilizing 86% of 150g/l soluble starch. With a mixed culture of mutant M-6 andSaccharomyces cerevisiae STV 89, 64.3g/l of ethanol was produced utilizing 94% of 150g/l soluble starch.     

Production of ethanol and xylitol from corn cobs by yeasts

Saccharomyces cerevisiae and Candida tropicalis were used separately and as co-culture for simultaneous saccharification and fermentation (SSF) of 5-20% (w/v) dry corn cobs. A maximal ethanol concentration of 27, 23, 21 g/l (w/v) from 200 g/l (w/v) dry corn cobs was obtained by S. cerevisiae, C. tropicalis and the co-culture, respectively, after 96 h of fermentation. However, theoretical yields of 82%, 71% and 63% were observed from 50 g/l dry corn cobs for the above cultures, respectively. Maximal xylitol concentration of 21, 20 and 15 g/l from 200 g/l (w/v) dry corn cobs was obtained by C. tropicalis, co-culture, and S. cerevisiae, respectively. Maximum theoretical yields of 79.0%, 77.0% and 58% were observed from 50 g/l of corn cobs, respectively. The volumetric productivities for ethanol and xylitol increased with the increase in substrate concentration, whereas, yield decreased. Glycerol and acetic acid were formed as minor by-products. S. cerevisiae and C. tropicalis resulted in better product yields (0.42 and 0.36 g/g) for ethanol and (0.52 and 0.71 g/g) for xylitol, respectively, whereas, the co-culture showed moderate level of ethanol (0.32 g/g) and almost maximal levels of xylitol (0.69 g/g).     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes.

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08

Enterobacter cloacae IIT-BT08 was found to produce both !-amylase and hydrogen in a batch system using soluble starch as substrate. Incubation time, temperature, pH and substrate concentration for the maximum !-amylase activity (130 U/ml) were 8 h, 37 °C, 6.00 and 10 g/l of soluble potato starch respectively. However, the optimum temperature and pH for the crude !-amylase activity were 60 °C and 4 respectively. The maximum rate of hydrogen production was observed at 10th h of fermentation and corresponding hydrogen yield was 7.6 mmol H₂/g soluble potato starch.     

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Summary Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

Production of Thermostable alpha-Amylase, Pullulanase, and alpha-Glucosidase in Continuous Culture by a New Clostridium Isolate

The production of alpha-amylase, pullulanase, and alpha-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60 degrees C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of alpha-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.     

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 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.     

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.     

The fermentative production of acetone-butanol by Clostridium acetobutylicum.

Fourteen different media were used in the fermentative production of acetone-butanol. The highest total yields were achieved in medium I. Potato starch and soluble starch were suitable as carbon sources. The best concentrations of potato starch and soluble starch were 500.0 and 10.0 g/l, respectively. Peptone was the most favourable nitrogen source. The best concentration of peptone was 4.0 g/l. Calcium carbonate in 3.6 g/l acted as buffering agent in the fermentation process. The best initial pH value of the fermentation medium was 6.0. The optimum temperature was 32--33degreesC. The fermentation process required 120 h to obtain maximum yields of acetone-butanol.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH₄NO₃) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.