Contribution of the fermenting yeast strain to ethyl carbamate generation in stone fruit spirits

Fermented fruit and beverages frequently contain ethyl carbamate (EC), a potentially carcinogenic compound that can be formed by the reaction of urea with ethanol. Both are produced by the yeast Saccharomyces cerevisiae with ethanol as the major end product of hexose fermentation and urea as a by-product in arginine catabolism. In spirit production, EC can also be derived from cyanide introduced by stone fruit. To determine the relative contribution of yeast metabolism to EC production, we genetically engineered a diploid laboratory strain to reduce the arginase activity, thus blocking the pathway to urea production. For this purpose, strains with either a heterozygous CAR1/car1 deletion or a homozygous defect (car1/car1) were constructed. These strains were compared to the parental wild type and to an industrial yeast strain in cherry mash fermentations and spirit production. The strain with the homozygous car1 deletion showed a significant reduction of EC in the final spirits in comparison to the non-engineered controls. Nevertheless, using this strain for fermentation of stoneless cherry mashes did not completely impede EC formation. This indicates another, as yet unidentified, source for this compound.     

清香型白酒发酵过程中酵母群落结构及其与尿素代谢的关系

【目的】探索清香型白酒发酵过程中酵母群落结构及演变,分析潜在的关键尿素代谢酵母及其环境调控因素,为降低发酵过程中氨基甲酸乙酯的含量提供理论依据。【方法】通过相关性分析明确发酵过程中氨基甲酸乙酯主要前体物质及其代谢微生物类群,利用高通量测序技术解析酵母群落结构组成,并结合偏最小二乘回归分析寻找潜在的关键尿素代谢酵母。采用冗余分析评价发酵过程中环境因素对酵母群落结构的影响。【结果】尿素是清香型白酒发酵过程中氨基甲酸乙酯的主要前体物质,酵母是尿素代谢的主要微生物。高通量测序结果显示,在97%的相似度下进行操作分类单元聚类后,共鉴定出22个酵母种。其中,嗜高压有孢汉生酵母(Hanseniaspora osmophila)、发酵毕赤酵母(Pichia fermentans)和酿酒酵母(Saccharomyces cerevisiae)与尿素合成存在正相关性,库德毕赤酵母(Pichia kudriavzevii)与尿素降解存在正相关性。酒醅含水量、p H、乙醇和精氨酸是影响发酵过程中酵母群落演替的重要环境因素。【结论】环境因素影响潜在的关键尿素代谢酵母,为降低发酵过程中尿素与氨基甲酸乙酯含量提供理论依据。     

Dynamics of indigenous and inoculated yeast populations and their effect on the sensory character of Riesling and Chardonnay wines

To study the impact of yeast populations on wine flavour and to better understand yeast growth dynamics, wines were produced by the (i) indigenous microflora, (ii) vigorous yeast starter EC1118 and (iii) slowly fermenting yeast Assmannshausen. Sensory analysis revealed that wines differed depending on the fermentation type. However, these yeast-related differences did not exceed the varietal character. Both added starter cultures clearly dominated the Saccharomyces population from the middle of fermentation onwards. The starter cultures differed in their repression of indigenous non- Saccharomyces yeast. EC1118 limited growth of non- Saccharomyces yeasts more strongly than Assmannshausen. Sulphite addition further repressed growth of non- Saccharomyces yeasts. On completion, more than one Saccharomyces strain was present in each fermentation, with the largest variety in the non-inoculated and the smallest in the EC1118-inoculated fermentation. Results from the two genetic assays, karyotyping, and PCR using δ-primers were not fully equivalent, limiting the usefulness of δ-PCR in studies of native Saccharomyces yeasts.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

Mechanism of Fermentation Conversion from Polyalcohol Fermentation to Ethanol Fermentation by Pichia miso

A possible mechanism of fermentation conversion is described from polyalcohol fermentation to ethanol fermentation by Pichia miso. Little alcohol dehydrogenase activity was found in polyalcohol-producing cells, whereas higher enzyme activity was induced by ethanol-producing cells. The fermentation conversion may be caused by the different levels of alcohol dehydrogenase activity between polyalcohol- and ethanol-producing cells. It was also shown that yeast growth was inhibited and that yeast cells were lysed by ethanol (at 6g/100ml) that accumulated in 24 hr.     

Studies on the Flavors in Saké

A very small amount of vanillin was found in Saké, but the mechanism of its formation during Saké brewing has not yet been elucidated. Therefore, shaking culture of a Saké yeast (Kyokai No. 7 strain) was carried out in the Hayduck’s solution containing ferulic acid which was considered to be a precursor of vanillin. By the analysis of the fermentation products, formation of p-hydroxybenzoic acid and vanillic acid was elucidated. On the other hand, in the similar experiment using vanillin in place of ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid were identified.

On these results, it was suggested that vanillin might be formed as an intermediate of the degradation reaction of ferulic acid, and also, the demethoxylation of vanillin might be occurred in the fermentation of yeast.     

Enrichment of wheat bran byRhodotorula gracilis through solid-state fermentation

The potential oil-producing yeast Rhodotorula gracilis was found to produce higher yields of biomass (13.7 g/L) and lipids (20.3%) in a nitrogen-limited and economically cheaper medium (molasses without yeast extract) in a submerged fermentation system. But, when the yeast was grown on four different wheat bran media by solid-state fermentation technique, different media combinations affected the percent increase in biomass, protein, oil production, fatty acid profile and degree of saturation and unsaturation. The initial lipid content in the control medium was 3.5% while in a medium with wheat bran, molasses, and minerals it was 69.8%. The yeast did not produce alpha-amylase, amyloglucosidase and cellulolytic enzymes for the breakdown of wheat bran. The yeast produced red carotenoids, a precursor of vitamin B12 and some oligounsaturated fatty acids in the fermented product.     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

HTST-extrusion cooking in ethanol production from starchy materials

Starch syrup for ethanol fermentation is conventionally produced by acid or enzymatic hydrolysis. Recently, however, promising results have been obtained using HTST-extrusion cooking in starch liquefaction. The starchy material was pregelatinized and preliquefied in a Creusot-Loire BC45 twin-screw HTST-extrusion cooker before simultaneous saccharification by amyloglucosidase and fermentation by Saccharomyces cerevisiae or Zymomonas mobilis. With pretreatment of milled whole grain or starch by HTST-extrusion cooking a significantly shorter fermentation time could be achieved. Maximum ethanol yield was obtained in 45 h using conventional yeast and amyloglucosidase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) dosage, even without addition of Termamyl α-amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) during thermomechanical liquefaction. Immobilized yeast could also be used to produce ethanol both by a batch or continuous process. In this case, for a continuous process the DE-value of the syrup should be sufficiently high. A model for ethanol production as a function of dry matter, fermentation time, and yeast and Termamyl quantities has been developed.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Polyploid engineering by increasing mutant gene dosage in yeasts

The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best-understood biological systems. Diploid strains of S. cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole-genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth-based screening.     

Improvement of Fermentation Conditions for the Production of X-Prolyl-Dipeptidyl Aminopeptidase from Lactococcus Lactis

The quantitative effects of some fermentation conditions on the production of the enzyme X-prolyl-dipeptidyl aminopeptidase (PepXP)(EC 3.4.14.5) of Lactococcus lactis subsp. lactis and cremoris were studied. The PepXP activity was found both in the membrane and in the cytoplasm, suggesting the presence of multiple molecular forms. Both microorganisms showed higher PepXP activities when glucose (5 g/l) was used as the carbon source and the yeast extract in the culture medium was increased to 3.5 g/l. In these conditions, 226 mU/ml of PepXP activity were obtained with L. lactis subsp. lactis and 235 mU/ml with the subsp. cremoris after 6 h. The best fermentation temperature was in the 30–32 °C range. The enzyme activity remained stable even during the stationary phase.     

Synthetic Leaders with Potential BiP Binding Mediate High-Yield Secretion of Correctly Folded Insulin Precursors fromSaccharomyces cerevisiae

Secretion leaders are essential for expression of many heterologous proteins including insulin in yeast. The function of secretion leaders and their interaction with the secretory pathway is not clear. To determine what constitutes functional pre-pro-leader sequences inSaccharomyces cerevisiae,synthetic leader sequences for secretion of the insulin precursor were developed by a combination of rational design and stepwise systematic optimization. The synthetic leaders efficiently facilitate secretion of the insulin precursor fromS. cerevisiaewhen compared with the α-factor leader, leading to a high yield of correctly folded insulin precursor in the culture supernatant. The synthetic leaders feature two potential N-linked glycosylation sites which are efficiently glycosylated during secretion. Pulse–chase analysis indicates that the synthetic leaders/insulin precursor fusion protein have a prolonged residence in the endoplasmic reticulum compared to the α-factor leader/insulin precursor fusion protein. The longer transition time in the endoplasmic reticulum mediated by the synthetic leaders might provide additional time for correct folding of the insulin precursor and account for the increased fermentation yield.     

Fructo-oligosaccharides production by the Gluconacetobacter diazotrophicus levansucrase expressed in the methylotrophic yeast Pichia pastoris

Levansucrase (LsdA) (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus) yields high levels of fructo-oligosaccharides (FOS) from sucrose. A DNA fragment encoding the precursor LsdA lacking the first 57 amino acids was fused to the pho1 signal sequence under the control of the Pichia pastoris-alcohol oxidase 1 (AOX1) promoter. Methanol induction of a P. pastoris strain harboring a single copy of the lsdA expression cassette integrated in the genome resulted in the production of active levansucrase. After fermentation of the recombinant yeast, LsdA activity was detected in the periplasmic fraction (81%) and in the culture supernatant (18%) with an overall yield of 1% of total protein. The recombinant LsdA was glycosylated and displayed optimal pH and temperature for enzyme activity similar to those of the native enzyme, but thermal stability was increased. Neither fructosylpolymerase activity nor FOS production was affected. Incubation of recombinant LsdA in sucrose (500 g l(-1)) yielded 43% (w/w) of total sugar as 1-kestose, with a conversion efficiency about 70%. Intact recombinant yeast cells also converted sucrose to FOS although for a 30% efficiency.     

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.     

Identification and characterization of yeasts in sugarcane silages

Aims: To enumerate the micro‐organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. Method: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. Conclusions: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. Significance and Impact of the Study: Knowledge of the population of micro‐organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.     

Engineering Saccharomyces cerevisiae for direct conversion of raw,uncooked or granular starch to ethanol

The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.     

Eicosapentaenoic and docosahexaenoic acids production by and okara-utilizing potential of thraustochytrids

Nine thraustochytrid strains isolated from subtropical mangroves were screened for their eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production potential in a glucose yeast extract medium. Their ability to utilize okara (soymilk residue) for growth and EPA and DHA production was also evaluated. EPA yield was low in most strains, while DHA level was high on glucose yeast extract medium, producing 28.1–41.1% of total fatty acids, for all strains, with the exception of Ulkenia sp. KF13. The DHA yield of Schizochytrium mangrovei strains ranged from 747.7 to 2778.9 mg/l after 52 h of fermentation at 25°C. All strains utilized okara as a substrate for growth, but DHA yield was lower when compared with fermentation in a glucose yeast extract medium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 199–202. Received 11 December 2000/ Accepted in revised form 29 June 2001     

Lumping kinetics of ABE fermentation wastewater treatment by oleaginous yeast Trichosporon cutaneum

Lumping kinetics models were built for the biological treatment of acetone–butanol–ethanol (ABE) fermentation wastewater by oleaginous yeast Trichosporon cutaneum with different fermentation temperatures. Compared with high temperature (33°C, 306?K) and low temperature (23°C, 296?K), medium temperature (28°C, 301?K) was beneficial for the cell growth and chemical oxygen demand (COD) degradation during the early stage of fermentation but the final yeast biomass and COD removal were influenced little. By lumping method, the materials in the bioconversion network were divided into five lumps (COD, lipid, polysaccharide, other intracellular products, other extracellular products), and the nine rate constants (k₁–k₉) for the models can well explain the bioconversion laws. The Gibbs free energy (G) for this bioconversion was positive, showing that it cannot happen spontaneous, but the existence of yeast can after the chemical equilibrium and make the bioconversion to be possible. Overall, the possibility of using lumping kinetics for elucidating the laws of materials conversion in the biological treatment of ABE fermentation wastewater by T. cutaneum has been initially proved and this method has great potential for further application.