Flow-cytometric investigation of sterol content and profliferation activity of yeast

To understand and control the dynamics of microbial growth and metabolism, a theoretical background based on segregated models is necessary, and therefore flow cytometry is the suitable measuring method. It is shown that additional information to the usual mean-value information, and to the well-known cytometric methods for determining DNA content and size/shape measurement by scattered light, like the distribution of membrane sterol content of baker's yeast, leads to a more detailed knowledge of growth dynamics. We have found, that a low content of membrane sterols is a characteristic of cells with suppressed proliferation activity, but necessary for high fermentation activity. Also, a high content of membrane sterols seems to enhance the viability and survival capability of harvested and dryed baker's yeast cells. Therefore, we recommend harvesting cells at a point of medium content of membrane sterols.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

Anaerobic treatment of baker's yeast wastewater: I. Start-up and sodium molybdate addition

The anaerobic treatment of baker's yeast wastewater was studied using an anaerobic biological contact reactor (AnRBC) and a fixed-film reactor. The AnRBC had an active biomass developed within the reactor before this study commenced; however, the fixed-film reactor was started without attached biomass in a support structure. The gas production rates obtained for the AnRBC were between 0·55 and 0·61 litre methane per litre reactor per day. However, a gas production rate of only 0·46 litre methane per litre reactor per day was achieved after a four-month operating period for the fixed-film reactor. Higher chemical oxygen demand reduction was also found in the AnRBC. The results indicated that the presence of high sulfate concentration in baker's yeast wastewater affected teh start-up process. The reactor with fully developed active biomass was less susceptible to sulfate inhibition and showed improved anaerobic digestion. Results indicate that the reactor should be innoculated by feeding nutrient-balanced substrate before it was subjected to the digestion of baker's yeast wastewater. The fixed-film reactor was also fed with the substrate contianing sodium molybdate, an inhibitor of sulfate-reducing bacteria. The results indicated that both methanogenic and sulfate-reducing bacteria were inhibited.     

A Multivariate Re-Examination of Experimental Condition Effects on Acyloin-Type Condensation Mediated by Saccharomyces Cerevisiae

Statistical methods of optimization were applied to the stereoselective synthesis of (2S,3R)-5-phenylpent-4-en-2,3-diol mediated by baker's yeast. The quantitative effects of seven variables, i.e. pH, temperature, concentration of cinnamaldehyde, yeast and glucose, addition of pyruvate and acetaldehyde were investigated using a fractional factorial design. This approach gave informations about the chemical behaviour of the yeast. Response surface methodology was employed to describe the variability of the yield in the experimental domain.     

自絮凝酵母SPSC01悬浮液的流变行为

用旋转黏度计测定了自絮凝颗粒酵母悬浮液的流变特性,并考察了其流变特性的影响因素,如菌体生物量、葡萄糖质量浓度、温度等。结果表明：自絮凝颗粒酵母悬浮液呈假塑性非牛顿流体,其流变特性服从幂律指数模型,随着菌体浓度的增大,稠度系数增大,流动行为指数减小;絮凝悬浮液的表观黏度随着糖浓度的增加有所增加,同一生物量下稠度系数k随着糖浓度的增加而增加,流动行为指数n随着糖浓度的增加变化很小,悬浮液的表观黏度随着温度的升高而降低;相同生物量下的流变指数随温度的升高而升高,而稠度系数随温度升高有所下降。     

Kinetics of ethanol production by baker's yeast in an integrated process of fermentation and microfiltration

The productivity of a fermentation is proportional to the biomass concentration. The productivity can therefore be increased by retention of the cells in the fermentor. In this study microfiltration was used for cell retention in a fermentation of glucose to ethanol by baker's yeast. Compared to a system without cell retention the productivity could be increased 12-fold to 55 kg/m³ h at a biomass concentration of 135 kg/m³. Maximal ethanol concentrations of 76 kg/m³ were obtained at conditions of growth. At zero growth conditions in the integrated system the ethanol concentration could be increased to about 115 kg/m³, and could be produced for at least 10 hours. The fermentation results in the integrated system could be described reasonably well with a mathematical model based on a different linear inhibition kinetics for growth and substrate consumption.     

Estimation of the disruption in freeze-pressed Saccharomyces cerevisiae by an electronic particle counter

An electronic particle counter has been used to estimate the disintegration by freeze-pressing baker's yeast. A counter threshold level which just yielded the maximum count for intact cells was selected. The conductivity of the suspending medium was chosen such that maximum counts were obtained. Under these conditions, the electronic counts agreed well with the visual counts. At a certain threshold level the maximum count was obtained at a lower resistivity (higher conductivity) in the suspending solution with the freeze-pressed suspension than with untreated cells, indicating that damage to the permeability barrier may occur without disruntion of the cell envelope. Fresh baker's yeast cells do not behave as nonconducting particles. This has to be taken into account when volume determinations with electronic particle counters are performed.     

Mathematical modelling of the dehydrogenase catalyzed hexanol oxidation with coenzyme regeneration by NADH oxidase

The hexanol oxidation catalyzed by alcohol dehydrogenase from baker's yeast (YADH) has been investigated with two different forms of the biocatalyst: the isolated YADH as well as the YADH in the permeabilized whole cells. It was found that in this reaction, equilibrium is shifted to the reduction side. Hence, to increase the conversion it was necessary to regenerate NAD⁺. For that purpose, enzyme NADH oxidase isolated from Lactobacillus brevis was used. All biocatalysts were kinetically characterized. The overall reaction rate was described by the mathematical model which consisted of kinetics and balance equations. Due to the deactivation of NADH oxidase, only 50–58% hexanol was converted to hexanal in the batch reactor where the hexanol oxidation was catalyzed by isolated YADH. In the case of permeabilized baker's yeast cells, no enzyme deactivation occurred and 100% hexanol conversion in the hexanoic acid was detected.     

An extreme pressure pump for continuous cell disintegration

The use of an air operated extreme pressure hydraulic pump for continuous cell disintegration is described, together with figures obtained for soluble protein released from suspensions of commercially obtained baker's yeast (Saccharomyces cerevisiae).     

A milliliter-scale yeast-based fuel cell with high performance

Microbial fuel cells are attracting attention as one of the systems for producing electrical energy from organic compounds. We used commercial baker's yeast (Saccharomyces cerevisiae) for a glucose fuel cell because the yeast is a safe organism and relatively high power can be generated in the system. In the present study, a milliliter (mL)-scale dual-chamber fuel cell was constructed for evaluating the power generated by a variety of yeasts and their mutants, and the optimum conditions for high performance were investigated. When carbon fiber bundles were used as an electrode in the fuel cell, high volumetric power density was obtained. The maximum power produced per volume of anode solution was 850 W/m³ under optimum conditions. Furthermore, the power was examined using seven kinds of yeast. In Kluyveromyces marxianus, not only the power but also the power per consumed glucose was high. Moreover, it was suggested that xylose is available as fuel for the fuel cell. The fuel cell powered by K. marxianus may prove to be helpful for the effective utilization of woody biomass.     

Bioreduction of a carbon–nitrogen double bond using immobilized baker's yeast’a first report

Immobilized baker's yeast entrapped in calcium alginate beads efficiently reduces N-benzylidinemethylamine to N-methylbenzylamine in hexane at 37°C and tetrahydrofuran (THF) at 30°C in the presence of 18-crown-6, while in the presence of water as cosolvent and glucose as an additive N-benzylidinemethylamine undergoes decomposition. Benzaldoxime in a hexane–water (1:9) solvent system containing glucose as an additive is reduced to N-benzylhydroxylamine. On using an ethanol–water (1:1) solvent system, benzaldoxime is converted to benzyl alcohol and in hexane, benzene, THF, hexane–water (1:1) or acetonitrile–water (1:1) solvent systems, or using dried baker's yeast in different solvent systems, transformation of benzaldoxime does not occur.     

Baker's yeast mediated bioreduction: Practical procedure using EtOH as energy source

Baker's yeast mediated bloreduction of prochiral ketones was investigated with respect to its energy source. The rate of glucose consumption was five times that of the reduction of ethyl acetoacetate (EA), and the reduction of EA proceeded under aerobic condition after glucose had been consumed. From these results, reduction by baker's yeast based on the regeneration system for NAD (P) H through the oxidative pathway of ethyl alenhol (EtOH) was developed. Under anaerobic conditions, reduction by the new procedure did not occur. The optimum conditions of EtOH concentration and temperature were about 200 mM and 30°C, respectively. At higher than 400 mM EtOH concentration, the reduction soon leveled off. The rate of EtOH consumption was less than twice that of EA reduction, and no by-products except for carbon dioxide were detected, so the new procedure is a practical one.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Use of immobilized cell biocatalysts in baking

Baking using baker's yeast immobilized in a starch–gluten–milk matrix (traditional fermented cereal food trahanas), containing viable lactic acid bacteria (LAB), and kefir (natural co-culture of yeasts and LAB) immobilized on orange peel, were investigated. The use of immobilized cells increased shelf life, delayed staling, and improved overall the quality of bread, compared with the traditional baker's yeast bread. These improvements were attributed to the reduction of pH, the lower moisture loss rates, and the presence of LAB, which are known to exhibit antimould properties. Better results were obtained using the sourdough method compared to the straight dough bread-making method. Headspace SPME GC–MS analysis showed that the use of immobilized cells increased the number of bread aroma volatiles, especially esters. The best results, including shelf life and overall bread quality, were obtained in the case of baker's yeast immobilized on trahanas, although kefir immobilized on orange peel seems to be a more cost effective biocatalyst.     

Glutamine Production by Coupling with Energy Transfer: Employing Yeast Cells and Gluconobacter Glutamine Synthetase

A glutamine production process was established by combining alcoholic fermentation of baker's yeast cells with glutamine synthetase from the bacterium Gluconobacter suboxydans. The maximum amount of glutamine formed under optimum conditions was about 20 mM in 3 hr with 80% yield based on glutamate, substrate. The fermentation proceeded in two steps: the accumulation of energy in a form of fructose 1,6-diphosphate (FDP) by yeast fermentation of sugar based on the Harden-Young effect and the fermentation of FDP coupled with glutamine synthetase reaction (an endergonic reaction) through an ATP-ADP system. The following factors were found to be important: (a) the ratio of the activities of yeast fermentation of sugar and glutamine synthetase, (b) effect of contaminating enzyme(s) in glutamine synthetase preparation, and (c) enzymatic properties of glutamine synthetase.     

FTIR and UV spectroscopy in real-time monitoring of S. cerevisiae cell culture

A combination of FTIR and UV spectroscopy is proposed as a novel technique for integrated real-time monitoring of metabolic activity and growth rates of cell cultures, required for systematic studies of cellular low-frequency (LF) electric and magnetic field (EMF) effects. As an example, we investigated simultaneous influence of periodic LF 3D EMFs on a culture of Saccharomyces cerevisiae (baker's yeast) cells. Amplitudes, frequencies and phases of the field components were the variable parameters. Electromagnetic fields were found to efficiently control the activity of the yeast cells, with the resulting CO₂ production rates, as monitored by FTIR spectroscopy, varying by at least one order of magnitude due to the field action. Additionally, population dynamics of the yeast cells was monitored by UV absorption of the yeast culture at λ_prob = 320 nm, and compared to the CO₂ production rates. The detected physiologically active frequencies are all below 1 kHz, namely, 800 Hz excitation was effective in reducing the metabolic rates and arresting cell proliferation, whereas 200 Hz excitation was active in accelerating both cell proliferation and overall metabolic rates. The proposed methods produce objective, reliable and quantitative real-time results within minutes and may be used in various tasks that could benefit from a rapid feedback they provide in the form of metabolic and growth rates. Amplitude and frequency dependences of the LF EMF effects from individual field components with different polarizations were recorded and qualitatively interpreted based on a simple model, describing ion diffusion through a membrane channel.     

Regulation of sterol synthesis by glucose in baker's yeast

The ability of ten baker's yeast strains to synthesize sterols was checked. Ergosterol/24(28)-dehydroergosterol (E/D) ratio had the value of 0.8 to 1 in most strains grown in a medium containing molasses as the carbon source. The ratio values were significantly increased in the cultures grown in a glucose medium. Both a decrease in the content of 24(28)-dehydroergosterol and a slight increase of the ergosterol content were found to be responsible for the high values of E/D in the glucose-grown cells. The strains examined could be divided into three groups on the basis of their behaviour towards glucose. The effect of the type of cultivation on sterol accumulation is demonstrated by comparing the sterol content of the representatives of the three groups of baker's yeasts grown in a fermenter or in shaken flasks.     

一种改进型体液表观粘度函数测量仪

本文介绍一种性能较好的体液表观粘度函数快测系统,它是在较严密的流变学理论基础上通过较精细的硬、软件设计而研究成功的.其前身为L-1型粘度计,但作了重要改进.它的最大特点是能在一次测量过程完后得到不同切变率下的表观粘度;同时能在通常认为困难却十分重要的“低剪切”段令人满意地工作.     

‘Yeast Mail’: A Novel Saccharomyces Application (NSA) to Encrypt Messages

The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)‐based, one‐step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages.