Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.     

Inferring ethanol tolerance of Saccharomyces and non-Saccharomyces yeasts by progressive inactivation

The kinetics of cell inactivation in the presence of ethanol at 20, 22.5% and 25% (v/v), was measured by progressive sampling and viable counting, and used as an inference of the ethanol resistance status of five non-Saccharomyces strains and one strain of Saccharomyces cerevisiae. The capacity of standard inocula of the same strains to establish growth at increasing initial ethanol concentrations was employed as a comparison. The effect of various different pre-culture conditions on the ethanol resistance of the 6 strains was analysed by the cell inactivation method and by the cell growth method. Exposing cells to 25% (v/v) ethanol for 4 min enabled the differentiation of the yeasts in terms of their resistance to ethanol. The results suggest that the two methods are generally concordant and that the cell inactivation method can, thus, be used to infer ethanol resistance of yeast strains.     

Study of a single and mixed culture for the direct bio-conversion of sorghum carbohydrates to ethanol

Fusaium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae 2541 fermented soluble and insoluble carbohydrates of sweet sorghum stalk directly to ethanol. Both microorganisms were first grown aerobically and fermented sorghum stalk to ethanol thereafter. During fermentation, insoluble carbohydrates were hydrolysed to soluble sugars by the celluloytic system of F. oxysporum. Ethanol yields as high as 24.4 and 33.5 g/100 g dry stalks were obtained by F. oxysporum and the mixed culture respectively, representing a theoretical yield enhancement of 11.6% and 53.6% respectively. The corresponding ethanol concentrations in the fermentation medium were 4.6% and 6.4% (w/v). These results clearly demonstrated that a large portion of insoluble carbohydrate from sorghum was converted by simultaneous saccharification and fermentation to ethanol, making the process promising for bioethanol production.     

The plasma membrane ATPase of Kloeckera apiculata: purification,characterization and effect of ethanol on activity

Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35°C, a K _m of 3.6 mm ATP and a V _max of 11 mol P_i/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol.     

Purification of glycyrrhizin from Glycyrrhiza uralensis Fisch with ethanol/phosphate aqueous two phase system

Phase diagrams of alcohol (ethanol or 2-propanol)/salt (phosphate or sulfate) aqueous two-phase systems were made. The system consisting of 60% (v/v) ethanol and 15% (w/v) phosphate was then used to separate glycyrrhizin from an extract of Glycyrrhiza uralensis Fisch and gave a 92% recovery of glycyrrhizin with 2.6-fold purification.     

Dietary ethanol and lipid synthesis in Drosophila melanogaster

When cultured on a defined diet, ethanol was an efficient substrate for lipid synthesis in wild-type Drosophila melanogaster larvae. At certain dietary levels both ethanol and sucrose could displace the other as a lipid substrate. In wild-type larvae more than 90% of the flux from ethanol to lipid was metabolized via the alcohol dehydrogenase (ADH) system. The ADH and aldehyde dehydrogenase activities of ADH were modulated in tandem by dietary ethanol, suggesting that ADH provided substrate for lipogenesis by degrading ethanol to acetaldehyde and then to acetic acid. The tissue activity of catalase was suppressed by dietary ethanol, implying that catalase was not a major factor in ethanol metabolism in larvae. The activities of lipogenic enzymes, sn-glycerol-3-phosphate dehydrogenase, fatty acid synthetase (FAS), and ADH, together with the triacylglycerol (TG) content of wild-type larvae increased in proportion to the dietary ethanol concentration to 4.5% (v/v). Dietary ethanol inhibited FAS and repressed the accumulation of TG in ADH-deficient larvae, suggesting that the levels of these factors may be subject to a complex feedback control.This research was supported by National Institutes of Health Grant GM-28779 to B.W.G. and a Monash University Research Grant to S.W.M.     

Mobile phase compositions for ceramide III by normal phase high performance liquid chromatography

Ceramide III was prepared by the cultivation ofSaccharomyces cerevisiae. Ceramide III was partitioned from the cell extracts by solvent extraction and analyzed by Normal Phase High Performance Liquid Chromatography (NP-HPLC) using Evaporative Light Scattering Detector (ELSD). We experimentally determined the mobile phase composition to separate ceramide III with NP-HPLC. Three binary mobile phases of n-hexane/ethanol,n-hexane/Isoprophyl Alcohol (IPA) andn-hexane/n-butanol and one ternary mobile phase ofn-hexane/IPA/methanol were demonstrated. For the binary mobile phase ofn-hexane/ethanol, the first mobile phase composition, 95/5 (v/v), was step-increased to 72/23 (v/v) at 3 min. In the binary mobile phase, the retention time of ceramide III was 7.87 min, while it was 4.11 min respectively in the ternary system, where the mobile phase composition ofn-hexane/IPA/methanol, 85/7/8 (v/v/v), was step-increased to 75/10/15 (v/v/v) at 3 min. However, in the ternary mobile phase, the more peak area of ceramide III was observed.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.     

Growth and Fermentation Characteristics of a Strain of the Wine Yeast Kluyveromyces thermotolerans Isolated in Greece

Summary During the single culture fermentation of grape must K. thermotolerans, strain TH941, isolated in a wine-producing region in northern Greece, reached a very high cell concentration of 8.4 log (c.f.u ml⁻¹), followed by a rapid decline of the viable cells. The yeast produced 9.6 g L-lactic acid l⁻¹ during the growth phase, 7.58% v/v of ethanol and showed a limited degradation of L-malic acid as well as a low production of volatile acidity. In the presence of 3% v/v and 6% v/v of ethanol the K. thermotolerans isolate was able to grow. At 9% v/v of ethanol it could not grow but showed no loss of viability for 10 days.     

Variations of two pools of glycogen and carbohydrate in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> grown with various ethanol concentrations

Glycogen, a major reservoir of energy in Saccharomyces cerevisiae, is found to be present as soluble and membrane-bound insoluble pools. Yeast cells can store excess glycogen when grown in media with higher concentration of sugar or when subjected to nutritional stress conditions. Saccharomyces cerevisiae NCIM-3300 was grown in media having ethanol concentrations up to 12% (v/v). The effects of externally added ethanol on glycogen and other carbohydrate content of yeast were studied by using alkali digestion process. Fermentative activities of cells grown in the presence of various ethanol concentrations (2–8% v/v) exhibited increase in values of glycogen and other carbohydrate, whereas cells grown with higher concentrations of ethanol (10–12% v/v) exhibited depletion in glycogen and carbohydrate content along with decrease in cell weight. Such inhibitory effect of ethanol was also exhibited in terms of reduction in total cell count of yeast grown in media with 2–16% (v/v) ethanol and 8% (w/v) sugar. These data suggest that, as the plasma membrane is a prime target for ethanol action, membrane-bound insoluble glycogen might play a protective role in combating ethanol stress. Elevated level of cell-surface α-glucans in yeast grown with ethanol, as measured by using amyloglucosidase treatment, confirms the correlation between ethanol and glycogen.     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

Ethanol tolerance and carbohydrate metabolism in lactobacilli

Summary This paper describes the ethanol tolerance and metabolism of 31 strains ofLactobacillus on glucose, xylose, lactose, cellobiose and starch. The purpose of this work was to determine the suitability of the 31 strains as potential host for the ethanol producing genes, pyruvate decarboxylase and aldehyde dehydrogenase, fromZymomonas mobilis. The 31 strains were screened for their ability to grow in 0 to 8% v/v ethanol on all five carbohydrates. Those strains that were able to grow to an OD of 1.0 in 8% ethanol were evaluated at ethanol concentrations up to 16%. v/v. The fermentative products from the five carbohydrates were analyzed to determine the ratios of lactic acid, ethanol, and acetic acid.Published as Paper No. 9786, Journal Series Nebraska Agricultural Experiment Station, Lincoln, NE 68583-0704.     

Improved adaptation to heat,cold, and solvent tolerance in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40°C) and cold shock (12°C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Characteristics of Egyptian boza and a fermentable yeast strain isolated from the wheat bread

A sample of indigenous beer, boza was collected at Cairo, Egypt and analysed. Boza was an off-white porridge-like slurry containing 3.8% (v/v) ethanol. Volatile esters and higher alcohols such as ethyl acetate and isoamyl alcohol were detected in the boza by gas chromatography. The pH of the boza was 3.7. Organoleptically, this alcoholic beverage had an estery flavour and a sour taste. A fermentable yeast strain EG1 was isolated from the material wheat bread and identified, and was considered to resemble Candida krusei. The rice sake made with the yeast strain C. krusei EG1 at 30 °C contained 11.7% ethanol, 74.1 mg/l ethyl acetate and its pH value was 4.2.     

Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

Mapping ethanol-induced deletions

Summary Chromosomal rearrangements, uniformly represented by very large deletions, were stimulated upon transiently exposing Escherichia coli cells with a defective lambda prophage to about 18% (v/v) ethanol. It was shown that the ethanol treatment induced deletion formation rather than enriching for ethanol-tolerant cells. The deletions in 435 mutants were mapped to 26 groups. Ethanol treatment changed the spectrum of deletions relative to those arising spontaneously, and stimulated the formation of deletions with endpoints in E. coli DNA flanking the lambda fragment. The promotion of deletion formation by ethanol involves the joining of distant, nonhomologous linear DNA segments, which can be considered an illegitimate recombination event; however, activity of the E. coli recA gene product was also required. Although spontaneous deletions arose in comparable cells defective for recA, the incidence of deletion formation in recA cells was not altered by ethanol. It is proposed that ethanol stimulates chromosomal rearrangements involving two oppositely oriented replication forks, since the localized deletions commonly removed or inactivated the chromosomal segment including the bidirectional lambda origin of replication. The results imply a novel mutagenic process induced by an agent that does not act directly on DNA.     

Improvement of ethanol yield from raw corn flour by <Emphasis Type="Italic">Rhizopus</Emphasis> sp.

A novel raw-starch-digesting glucoamylase producer, Rhizopus sp. W-08, and Saccharomyces cerevisiae Z-06 were used in a fed batch process for simultaneous saccharification and fermentation of raw corn flour. Ethanol concentration of 21% (v/v) was obtained after 48 h. The conversion efficiency of raw corn flour to ethanol was 94.5% of the theoretical ethanol yield.     

Extraction of fucoxanthin and polyphenol from <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> using supercritical carbon dioxide with co-solvent

Extraction of brown seaweed (Undaria pinnatifida) oil was carried out by using supercritical carbon dioxide (SCO₂) and ethanol as co-solvent. The flow rate of ethanol was 3.0% (v/v) as compared to that of SCO₂. Experiments were performed in a semi-batch flow apparatus on dried samples at temperatures from 303 to 333 K and pressures from 80 to 300 bar. Fucoxanthin and polyphenol were quantitatively analyzed by using HPLC and UV-spectrometer. The highest yields of fucoxanthin and polyphenol were shown at 200 bar, 323 K and 250 bar, 333 K, respectively. The solubility of fucoxanthin in SCO₂ agreed well with the Chrastil model.