The effect of pretreatment on the invertase activity of gel-entrapped yeast biomass

Summary Gel-entrapped, non-viable yeast biomass with specific invertase activity has been produced by two different pretreatment protocols: a short-time thermal treatment and a brief contact with concentrated ethanol solutions. Four yeast strains were most promising:K. fragilis L-293,C. utilis L-282,S. cerevisiae L-170 and L-209. Of these, the ethanol-tolerant L-282 and the ethanol-tolerant and heat-resistant L-170 gave the most active gel-entrapped biocatalysts: around 2 mg of reducing sugars produced per mg dry yeast per min.     

The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.     

Nucleic acid reduction in yeast

Summary A method for reduction of nucleic acid levels in preparations of the yeastsSaccharomyces cerevisiae andKluyromyces fragilis by means of alkaline treatment has been developed. Under similar conditions (4.5% NH₄OH, 65° C, 30 min) a low nucleic acid content (less than 2%) was obtained for both strains. Higher losses of proteins and biomass were obtained withK. fragilis than withS. cerevisiae.     

Comparison of various ways of extraction of nucleic acids and of preparation of yeast extract from saccharomyces cerevisiae and candida utilis

Six different variations of the extraction procedure applied to yeast cells of Saccharomyces cerevisiae and Candida utilis to optimize the production of yeast extract and isolation of nucleic acids were compared. The autolysis of C. utilis at 50 to 52°C without adding chemical agents was found to be the best for the production of yeast extract. The most suitable procedures used for the extraction of nucleic acids were those which were carried out from C. utilis at pH 7.5 (92°C) and the other with 0.4 M NH₄OH (40°C). Both these modifications yielded the highest amounts of polymer nucleic acids. Applying all procedures compared to S. cerevisiae an increased content of sterols (including Δ^5.7-sterols, predominantly ergosterol) was detected.     

Studies on Substances Which Increase RNA Content of Yeast Cells

Active substances which increased RNA content and RNA productivity in yeast culture without affecting the growth rate of yeast were investigated.

The remarkable effect of zinc ion on RNA accumulation was found in flask cultures of Candida utilis.

The active substance of culture of Saccharomyces cerevisiae was isolated from the culture filtrate of Streptomyces sp. S–22 and it was identified as anisomycin, an antiprotozoal and antifungal antibiotic. The effect of anisomycin on the enhancement of yeast RNA formation was shown only with the Saccharomyces genus, which was more sensitive to the antibiotic than other genus. This phenomenon was exhibited only in the case of anisomycin and cycloheximide, whose modes of action were similar among various antibiotics. The ratio of four nucleotides in RNA fraction was almost equal to that of ribosomal RNA.     

Functional Purification of the Monocarboxylate Transporter of the Yeast Candida utilis

Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-β-d-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

Improved inhibitor tolerance in xylose-fermenting yeast <Emphasis Type="BoldItalic">Spathaspora passalidarum</Emphasis> by mutagenesis and protoplast fusion

The xylose-fermenting yeast Spathaspora passalidarum showed excellent fermentation performance utilizing glucose and xylose under anaerobic conditions. But this yeast is highly sensitive to the inhibitors such as furfural present in the pretreated lignocellulosic biomass. In order to improve the inhibitor tolerance of this yeast, a combination of UV mutagenesis and protoplast fusion was used to construct strains with improved performance. Firstly, UV-induced mutants were screened and selected for improved tolerance towards furfural. The most promised mutant, S. passalidarum M7, produced 50% more final ethanol than the wild-type strain in a synthetic xylose medium containing 2 g/l furfural. However, this mutant was unable to grow in a medium containing 75% liquid fraction of pretreated wheat straw (WSLQ), in which furfural and many other inhibitors were present. Hybrid yeast strains, obtained from fusion of the protoplasts of S. passalidarum M7 and a robust yeast, Saccharomyces cerevisiae ATCC 96581, were able to grow in 75% WSLQ and produce around 0.4 g ethanol/g consumed xylose. Among the selected hybrid strains, the hybrid FS22 showed the best fermentation capacity in 75% WSLQ. Phenotypic and partial molecular analysis indicated that S. passalidarum M7 was the dominant parental contributor to the hybrid. In summary, the hybrids are characterized by desired phenotypes derived from both parents, namely the ability to ferment xylose from S. passalidarum and an increased tolerance to inhibitors from S. cerevisiae ATCC 96581.     

Alcohol production by selected yeast strains in lactase-hydrolyzed acid whey

Ethanol production by Kluyveromyces fragilis and Saccharomyces cerevisiae was studied using cottage cheese whey in which 80 to 90% of the lactose present had been prehydrolyzed to glucose and galactose. Complete fermentation of the sugar by K. fragilis required 120 hr at 30°C in lactase-hydrolyzed whey compared to 72 hr in nonhydrolyzed whey. This effect was due to a diauxic fermentation pattern in lactase-hydrolyzed whey with glucose being fermented before galactose. Ethanol yields of about 2% were obtained in both types of whey when K. fragilis was the organism used for fermentation. Saccharomyces cerevisiae produced alcohol from glucose more rapidly than K. fragilis, but galactose was fermented only when S. cerevisiae was pregrown on galactose. Slightly lower alcohol yields were obtained with S. cerevisiae, owing to the presence of some lactose in the whey which was not fermented by this organism. Although prehydrolysis of lactose in whey and whey fractions is advantageous in that microbial species unable to ferment lactose may be utilized, diauxie and galactose utilization problems must be considered.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

A quick screening method to identify β-glucosidase activity in native wine yeast strains: application of Esculin Glycerol Agar (EGA) medium

One hundred and fifty-four yeast strains were isolated from grapes and musts of Uruguayan vineyards and wineries. Only thirty strains showed β-glucosidase activity in Esculin Glycerol Agar (EGA) solid medium. Twenty-one were non-Saccharomyces and nine were Saccharomyces cerevisiae strains. The objective of this study was to evaluate the suitability of Esculin Glycerol Agar (EGA) solid medium for screening β-glucosidase activity in native yeasts strains. Halo sizes measured in the EGA solid medium were correlated to the Glycosyl-Glucose (GG) indexes measured after fermentation of grape musts with each strain. The two S. cerevisiae strains with the best performance were selected for further fermentations on a Muscat Miel grape must, rich in bound monoterpenes. The levels of free linalool, hodiol I and geraniol increased significantly as compared to fermentation with a commercial wine yeast strain. These results show the suitability of this simple and economic medium to identify S. cerevisiae glucosidase producers with a potential impact on real winemaking conditions. On the other hand, great variability was found for the non-Saccharomyces strains, and this would demand further studies for each species. In conclusion, the use of EGA solid medium shows that the screening method is suitable for exploring the glucosidase activity of native strains of S. cerevisiae and shows good correlation with its real impact on free aroma compounds in the final wine.     

Microbial population and biochemical changes during production of protein-enriched fufu

Summary Candida utilis strain BKT4 and Saccharomyces cerevisiae strain BKT7 isolated from burukutu (a local wine brewed from sorghum) were used to enrich fufu. During the fermentation process, there were changes in the microbiological and biochemical characteristics of the cassava. The total viable counts increased with increasing fermentation time while the counts of the lactics and fungi increased at the later stages of the fermentation due to the acidity of the medium. Various bacteria (Bacillus, Staphylococcus, Klebsiella, Escherichia, Streptococcus, Lactobacillus, Leuconostoc, Corynebacterium), moulds (Penicillium, Aspergillus, Fusarium, Mucor, Rhizopus) and yeasts (Candida, Saccharomyces, Hansenula, Rhodotorula) were found to be associated with the fermentation process. The pH of the fermenting cassava increased from 4.2 to 5.7 after 72 h while the cyanide level decreased from 2.2 mg/kg to 0.7 mg/kg over the same period of fermentation. Fufu (prepared by crushing and sieving fermenting cassava roots) enriched with 0.5 g of C. utilis strain BKT4, S. cerevisiae BKT7 and a mixed culture of the two organisms revealed a crude protein of 7.90, 6.34 and 10.0% respectively as compared to 2% protein content of the enriched fufu. There was a corresponding increase in protein content of the product as the quantity of the enrichment yeast was increased from 0.5 to 3.0 g. The aroma of the enriched fufu was preferred to that of the commercial fufu. Generally, good acceptability and organoleptic qualities (colour, taste, texture and aroma) of the protein enriched fufu was best achieved within 48 h of enrichment. The results of this study suggest that fufu can be made more nutritious with yeasts particularly Candida utilis strain BKT4 and Saccharomyces cerevisiae strain BKT7.     

Dates as a potential substrate for single cell protein production

The use of date juice as a substrate for single cell protein production was investigated. Four strains of Saccharomyces cerevisiae and two strains of Candida utilis were examined as possible production cultures. The criteria used for screening the organisms were total cell count, total protein and decrease in soluble solids. S. cerevisiae ATCC 4111 gave the highest protein and cell production. The optimum substrate concentration was 4 - 5% soluble solids. At this concentration, 55% of the sugars was utilized. Cell mass after 12 h fermentation was 4.86 g l⁻¹. The harvested and freeze-dried cells contained 8.6% nitrogen. The best combination of nutrient supplementation was found to be 0.25% (NH₄)₂HPO₄ and 0.1% FeNH₄(SO₄)₂; addition of MgSO₄ and (NH₄)₂SO₄ did not increase cell production.     

Enzymatic RNA reduction in disintegrated cells of Saccharomyces cerevisiae

Degradation of UNA by endogenous RNase in cell suspensions of Saccharomyces cerevisiae was found to be achieved by mechanical disintegration followed by incubation in the presence of NaCl. The incubation parameters pH, temperature, time, and concentration of NaCl were investigated. Protein concentrates with a low content of RNA were obtained by precipitation of the incubated suspensions and separation of the degradation products. On a pilot plant scale the incubation was performed at 50°C and pH 5.6 in the presence of 3% NaCl for 20 min. Kilogram quantities of protein concentrates containing 1.4% RNA and 8.2% nitrogen were obtained. The RNA reduction and the nitrogen yield was 85 and 60%, respectively. The yield of amino acids was about 75%. The process described can probably be applied for large-scale production.     

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Production of single-cell protein from enzymatic hydrolyzate of rice straw

Summary The components of rice straw, pretreated with sodium chlorite, cellulose and hemicellulose were solubilized with culture filtrate of Pellicularia filamentosa or Trichoderma reesei. The ratio of glucose to total sugar in the solution obtained from the cellulose component with the culture filtrate of Pellicularia filamentosa was approximately twice that of Trichoderma reesei.Ten yeast strains (Candida utilis, C. tropicalis, C. guilliermondii, C. parapsilosis, Torulopsis xylinus, Trichosporon cutaneum, Debaryomyces hansenii, Rhodotorula glutinis, Saccharomyces fragilis and Saccharomyces cerevisiae) were cultivated as test organisms for single-cell protein (SCP) production on sugar solutions obtained from the straw, cellulose and hemicellulose components, pretreated with the culture filtrate of Pellicularia filamentosa. Sugar consumption, in terms of total sugar and cell yield, of the culture with the sugar solution obtained from pretreated straw were; 70% and 6.8 g/l for Candida tropicalis, 56% and 6.4 g/l for Torulopsis xylinus, 76% and 10.1 g/l for Trichosporon cutaneum, and 74% and 7.6 g/l for Candida guilliermondii. In addition, the highest consumption with respect to total sugar (87%) and the best dry cell yield (15.6 g/l) were observed with the culture of Trichosporon cutaneum using the sugar solution obtained from the hemicellulose component.     

<Emphasis Type="Italic">MPK1</Emphasis> gene is required for filamentous growth induced by isoamyl alcohol in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from the alcoholic fermentation

The aim of this study was to evaluate the MPK1 (SLT2) gene deletion upon filamentous growth induced by isoamyl alcohol (IAA) in two haploid industrial strains of Saccharomyces cerevisiae using oligonucleotides especially designed for a laboratory S. cerevisiae strain. The gene deletion was performed by replacing part of the open reading frames from the target gene with the KanMX gene. The recombinant strains were selected by their resistance to G418, and after deletion confirmation by polymerase chain reaction, they were cultivated in a yeast extract peptone dextrose medium + 0.5% IAA to evaluate the filamentous growth in comparison to wild strains. Mpk1 derivatives were obtained for both industrial yeasts showing the feasibility of the oligonucleotides especially designed for a laboratory strain (Σ1278b) by Martinez-Anaya et al. (In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J Cell Sci 116:3423–3431, 2003). The filamentation rate in these derivatives was significantly lower for both strains, as induced by IAA. This drastic reduction in the filamentation ability in the deleted strains suggests that the gene MPK1 is required for IAA-induced filamentation response. The growth curves of wild and derivative strains did not differ substantially. It is not known yet whether the switch to filamentous growth affects the fermentative characteristics of the yeast or other physiological traits. A genetically modified strain for nonfilamentous growth would be useful for these studies, and the gene MPK1 could be a target gene. The feasibility of designed oligonucleotides for this deletion in industrial yeast strains is shown.     

Antistress cross-protection of UV-irradiated yeast cells with participation of extracellular peptide factors

Antistress effect of extracellular peptides on UV-irradiated yeast of different phylogenetic groups was studied. Yeast from different ecotopes and taxonomic groups exposed to UV radiation of a lethal intensity showed a protective effect and reactivating effect with participation of extracellular peptides. The highest protective activity was found in peptide reactivation factors (RFs) of bakery yeast—Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida utilis; the highest reactivating activity was exhibited by factors of the above-mentioned cultures and Debariomyces hansenii. Cross-protective and reactivating effects of RFs of yeast belonging to different taxonomic groups were demonstrated. Cross-protection increased two to three times after preexposure of reactivation factors to UV light (activation) in contrast to their reactivating effect.     

Isolation of total RNA from baker’s yeast

A simple method of production of total RNA from baker’s yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100°C for 40–60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.     

Biosynthesis regulation of the β-glucosidase produced by a yeast strain transformed by genetic engineering

The biosynthesis of the -glucosidase enzyme was studied in a transformed yeast obtained by cloning in Saccharomyces cerevisiae the structural gene coding for -glucosidase in Kluyveromyces fragilis. The enzyme biosynthesis was found to be non-adaptative, and repressed by glucose. These features are similar to those observed in K. fragilis. -Glucosidase activity in the transformed yeast was much higher than in K. fragilis. We attempted to ferment cellobiose with the transformed yeast: practically no cellobiose was consumed, growth and ethanol production were negligible. Warburg experiments showed that cellobiose fermentation did not occur when the respiratory chain was not functioning.