Continuous ethanol fermentation coupled with recycling of yeast flocs / 生物工程学报

A continuous ethanol fermentation system composed of three-stage tanks in series coupled with two sedimentation tanks was established. A self-flocculating yeast strain developed by protoplast fusion from Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage enzymatic hydrolysate of corn powder containing 220g/L of reducing sugar, supplemented with 1.5g/L (NH4)2HPO4 and 2.5g/L KH2PO4, was used as the ethanol fermentation substrate and fed into the first fermentor at the dilution rate of 0.057h(-1). The yeast flocs separated by sedimentation were recycled into the first fermentor as two different models: activation-recycle and direct recycle. The quasi-steady states were obtained for both operation models after the fermentation systems experienced short periods of transitions. Activation process helped enhance the performance of ethanol fermentation at the high dilution rates. The broth containing more than 101g/L ethanol, 3.2g/L residual reducing sugar and 7.7g/L residual total sugar was produced. The ethanol productivity was calculated to be 5.77g/(L x h), which increased by more than 70% compared with that achieved in the same tank in series system without recycling of yeast cells.

Breeding of Yeast Fusant for Efficient Ethanol Fermentation from Xylose / 中国生物工程杂志

Yeast strains with improved ethanol yield are important for efficient bioconversion of lignocellulosic biomass for fuel ethanol.Candida shehatae CICC1766 was adapted to 4%(v/v)ethanol,and then subjected to UV mutagenesis.One respiration deficient mutant Rd-5 with improved xylose fermentation capability was selected.Protoplasts of Rd-5 were inactivated by UV treatment,followed by the PEG-mediated protoplast fusion with a Saccharomyces cerevisiae strain with good ethanol-fermenting capability.The xylose fermenting capability of the fusants was investigated,and the fusant F6 demonstrated good ethanol fermentation performance,producing 18.75g/L ethanol from 50g/L xylose with an ethanol yield of 0.375 or 73.4% of its theoretical value of 0.511.Comparing with its parent Candida shehatae strain,the ethanol yield of F6 was increased by 28%.

The Effects of Dilute Acid Hydrolysate By-products of Corn Stover on Ethanol Fermentation of Xylose-utilising Saccharomyces cerevisiae 6508-127 / 中国生物工程杂志

During the dilute acid pretreatment of lignocellulosic materials such as corn stover, hemicellulose is hydrolyzed into monosaccharides, and meanwhile, toxic by-products are simultaneously generated, which may influence ethanol fermentation thereafter. Studies on the inhibitory effects of the by-products on ethanol fermentation are of practical use for further improvement of ethanol yield from lignocellulosic materials. Five by-products, including acetic acid, formic acid, vanillin, furfural and 5-hydroxymethylfurfural, were identified to be the main components in the hydrolysate of dilute acid pretreatment of local corn stover, which were added into the medium at different concentrations to study their impacts on the growth and ethanol fermentation of a recombinant xylose-utilizing yeast strain, S. cerevisiae 6508-127. The ethanol production was inhibited by formic acid and acetic acid to a lesser extent than that to the growth, and formic acid was shown to be much more toxic than acetic acid, showing severe inhibitory effects at the concentration of 1g/L, half of the concentration for acetic acid which showed remarkably negative effects on ethanol fermentation. Vanillin caused a much longer lag-phase in growth when the concentration was 2g/L, and the lag-phase was not obvious at lower concentrations. At the concentration of 6g/L, vanillin completely inhibited the fermentation as well as the cell growth. 5-Hydroxymethylfurfural was showed to remarkably inhibit ethanol production, but the biomass yield was higher by exogenous addition of 5-Hydroxymethylfurfural than control. Furfural at 0.5~1.5g/L inhibited the cell growth, but the ethanol yield was higher than that of the control experiment. It was also found that vanillin, furfural and 5-hydroxymethylfurfural could be assimilated and metabolized by S. cerevisiae 6508-127 under the experimental conditions.

Optimized culture medium and fermentation conditions for lipid production by Rhodosporidium toruloides / 生物工程学报

Culture medium and fermentation conditions for lipid production by Rhodosporidium toruloides were optimized with single factor and uniform design experiment. The best medium recipe was found with 70 g/L glucose, 0.1 g/L (NH4)2SO4, 0.75 g/L yeast extract, 1.5 g/L MgSO4. 7H2O, 0.4g/L KH2PO4, sterilized at 121 degrees C for 15 min, and then supplemented with ZnSO4 1.91 x 10(-6) mmol/L, CaCl2 1.50 mmol/L, MnCl2 1.22 x 10(-4) mmol/L and CuSO4 1.00 x 10(-4) mmol/L. The optimal fermentation conditions were as follows: 50 mL of medium (pH 6.0) in 250 mL Erlenmeyer flask with 10% inoculum (28h) under orbital shaking at 200 r/min for 120h at 30 degrees C. Under these conditions, yeast biomass accumulated lipids up to 76.1%.

Protein amino acid composition of plasma membranes affects membrane fluidity and thereby ethanol tolerance in a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae / 生物工程学报

A combination of three amino acids including 1.0 g/L isoleucine, 0.5 g/L methionine and 2.0 g/L phenylalanine was found to enhance ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. When subjected to 20% (V/V) ethanol for 9 h at 30 degrees C, all cells died whereas 57% remained viable for the cells grown in the presence of the three amino acids. Based on the analysis of protein amino acid composition of plasma membranes and the determination of plasma membrane fluidity by measuring fluorescence anisotropy using diphenylhexatriene as a probe, it was found that the significantly increased ethanol tolerance of cells grown with the three amino acids was due to the incorporation of the supplementary amino acids into the plasma membranes, thus resulting in enhanced ability of the plasma membranes to efficiently counteract the fluidizing effect of ethanol when subjected to ethanol stress. This is the first time to report that plasma membrane fluidity can be influenced by protein amino acid composition of plasma membranes.

Continuous ethanol fermentation using self-flocculating yeast strain and bioreactor system composed of multi-stage tanks in series / 生物工程学报

A continuous ethanol fermentation system composed of four-stage tank fermentors in series and with a total working volume of 4000 mL was established. The first fermentor was designated as the seed fermentor and the others for ethanol fermentation. A self-flocculating yeast strain developed by protoplast fusion of Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage corn powder enzymatic hydrolyzate containing reducing sugar 100 g/L, together with 2.0 g/L (NH4)2HPO4 and KH2PO4, was used as yeast seed culture medium and fed into the seed fermentor at the dilution rate of 0.017h (-1). Meanwhile, the hydrolyzate containing reducing sugar 220 g/L, added with 1.5 g/L (NH4)2HPO4 and 2.5 g/L KH2PO4, was used as ethanol fermentation substrate and fed into the second fermentor at the dilution rates of 0.017, 0.025, 0.033, 0.040 and 0.050 h(-1) (based on the total working volume of the three fermentors), respectively. The chemostat states on which all of the monitoring parameters, including residual sugar, ethanol and yeast cell biomass concentrations, were maintained relatively constant were observed for seed cultivation and ethanol fermentations when the fermentation system was operated at the dilution rates of 0.017, 0.025, 0.033 and 0.050 h(-1). Yeast cells were observed being partly immobilized because significant yeast cell biomass concentration differences between the broth out of and inside the fermentors were detected. Moreover, the oscillations of residual sugar, ethanol and yeast cell biomass concentrations were observed when the fermentation system was operated at the dilution rate of 0.040 h(-1). The broth containing more than 12% (V/V) ethanol and less than 0.11% (W/V) residual reducing sugar and 0.35% (W/V) residual total sugar was produced when the dilution rate was controlled at no more than 0.033 h(-1). The ethanol productivity was calculated to be 3.32(g x L(-1) x h(-1)) for the dilution rate of 0.033 h(-1), which increased nearly 100% compared with that for conventional ethanol fermentation technologies using freely suspended yeast cells.

Effect of flocculence of a self-flocculating yeast on its tolerance to ethanol and the mechanism / 生物工程学报

Investigation was undertaken for the purpose of examining any possible correlation between flocculence of a self-flocculating fusant of Schizosaccharomyces pombe mutant and Saccharomyces cerevisiae mutant (called fusant SPSC for short) and the tolerance of this strain to ethanol. When exposed to 18% (V/V) ethanol for 7 h at 30 degrees C, 52%, 37% and 9% of viability levels remained for the cells of fusant SPSC and its two parental strains, Sch. pombe mutant and S. cerevisiae mutant respectively. Analysis of phospholipid fatty acid composition of plasma membrane showed that the content of palmitic acid of each flocculating yeast (fusant SPSC or Sch. pombe mutant) was around 2-fold higher than that of free S. cerevisiae mutant, with remarkably lower contents of palmitoleic and oleic acids than the latter. When 0.1 mol/L sodium citrate was initially included in the medium in which cells of each flocculating yeast were grown, free cells rather than aggregates were finally obtained. Furthermore, the content of palmitic acid in the phospholipid fatty acid composition of the plasma membranes of the free cells of each flocculating yeast was found to decrease significantly, with a marked increase in the contents of palmitoleic and oleic acids. As a result, the characteristics of the phospholipid fatty acid composition of the plasma membranes of the free cells of each flocculating yeast were similar to those of S. cerevisiae mutant. Meanwhile, the disappearance of flocculence of each flocculating yeast caused by the action of sodium citrate brought about a steeply decreased tolerance of the free cells to ethanol, thus being equivalent to that of S. cerevisiae mutant. These data suggest that the stronger ethanol tolerance of each flocculating yeast is related to the higher content of palmitic acid in the phospholipid fatty acid composition of the plasma membranes. Thus, the enhancement by flocculence on the tolerance of yeast cells to ethanol as well as its mechanism are first reported in this work.

Effects of dilution rates on the oscillatory behaviors of a very high gravity continuous ethanol fermentation system / 生物工程学报

Continuous ethanol fermentation using very high gravity medium containing 280 g/L glucose, 5 g/L yeast extract and 3 g/L peptone was run at the dilute rates of 0.006 h(-1), 0.012 h(-1), 0.017 h(-1), 0.024 h(-1) and 0.032 h(-1) (based on the total working volume) in a combined bioreactor system composed of a stirred tank and three-stage tubular bioreactors in series. Oscillations marked by big fluctuations of residual glucose, ethanol and biomass were observed at the dilution rate of 0.012 h(-1). The Hopf Bifurcation theory was used to analyze and predict the occurring of these oscillations and the dilution rates that incited oscillations. Theoretical analysis revealed that oscillations can occur at designated specific growth rates and was validated by experimental results. The benefits of oscillations for the fermentation system were also discussed by comparing the fermentation results with those without oscillations.

Continuous ethanol fermentation using self-flocculating yeast in multi-stage suspended bioreactors coupled with directly recycling of waste distillage / 生物工程学报

A fermentation system composed of four airlift suspended-bed bioreactors in series and with a total working volume of 4800 mL was established. Continuous ethanol fermentation using self-flocculating yeast SPSC01, a fusant from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two-stage enzymatic hydrolyte of dry milling corn powder, was continuously run for 120 days. All of the backset distillage collected after distilling the final beer was used to mix the corn powder and no any other wastes except the solid residue of corn powder was discharged from the fermentation system, which guaranteed the distillage to be recycled at its maximum. The experimental results revealed that both ethanol and residual sugar in the final beer could be maintained relatively stable with their average levels of 93.6 and 7.9 g/L, respectively when the fermentation system was operated at the dilution rate of 0.05 h(-1). Parameter oscillations reported previously were also observed for the first and second bioreactors, but were effectively attenuated thereafter, which indicated that high yeast cell concentrations resulted from the self-immobilization of this special self-flocculating strain contributed to damp these oscillations. The monitoring of residual nitrogen and phosphor indicated that the accumulations of these nutritional elements occurred and the amount of these inorganic salts supplemented in the substrate should be decreased properly.

Influence of phospholipid fatty acid composition of plasma membrane on sensitivity of plasma membrane ATPase of a self-flocculating yeast to in vivo ethanol activation and its relationship to ethanol tolerance / 生物工程学报

Although alterations in fatty acid composition of phospholipids in plasma membranes had no effect on activities of plasma membrane ATPases of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae cells grown in the absence of ethanol (basal enzymes), they significantly affected the susceptibilities of the enzymes to in vivo activation induced by ethanol: the maximal values for the activated enzymes in cells pregrown with 0.6 mmol/L palmitic, linoleic or linolenic acid respectively were 3.6, 1.5 and 1.2-fold higher than their respective basal levels (in cells grown without ethanol), whereas the corresponding value for cells pregrown in the absence of fatty acid was 2.3-fold, with the concentrations of ethanol for the above maximal in vivo activation of enzymes being 7%, 6%, 6% and 7% (V/V) respectively. The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the basal and the activated plasma membrane ATPases were essentially identical; however, the v(max) values of activated enzymes increased significantly. It was found that the characteristics of phospholipid fatty acid composition of plasma membrane leading to the enhanced ethanol tolerance of this strain, were also efficacious to increase the percentage of activation of plasma membrane ATPase per unit of ethanol. These data support a close correlation between the ethanol tolerance of this strain and the sensitivity of its plasma membrane ATPase to the in vivo ethanol-induced activation.

Enhancements in ethanol tolerance of a self-flocculating yeast by calcium ion through decrease in plasmalemma permeability / 生物工程学报

Ca2+ at 1.64 mmol/L markedly increased ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. After 9 h of exposure to 20% (V/V) ethanol at 30 degrees C , no viability remained for the control whereas 50.0% remained for the cells both grown and incubated with ethanol in Ca2+ -added medium. Furthermore, when subjected to 15% (V/V) ethanol at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient (P' ) of the cells both grown and incubated with ethanol in Ca2+ -added medium accounted for only 50.0% and 29.3% those of the control respectively, indicating that adding Ca2+ can markedly reduce plasma membrane permeability of yeast cells under ethanol stress as compared with the control. Meanwhile, high viability levels acquired by the addition of Ca2+ exactly corresponded to the striking decreases in extracellular nucleotide concentration and P' achieved with identical approach. Therefore, the enhancing effect of Ca2+ on ethanol tolerance of this strain is closely related to its ability to decrease plasma membrane permeability of yeast cells subjected to ethanol stress.