Comparative proteome analysis of robust <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> insights into industrial continuous and batch fermentation

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide identification of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genes required for tolerance to acetic acid

Background  

Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory.     

Proteomic insights into adaptive responses of Saccharomyces cerevisiae to the repeated vacuum fermentation

The responses and adaptation mechanisms of the industrial Saccharomyces cerevisiae to vacuum fermentation were explored using proteomic approach. After qualitative and quantitative analyses, a total of 106 spots corresponding to 68 different proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differentially expressed proteins were involved in amino acid and carbohydrate metabolisms, various signal pathways (Ras/MAPK, Ras–cyclic adenosine monophosphate, and HOG pathway), and heat shock and oxidative responses. Among them, alternations in levels of 17 proteins associated with carbohydrate metabolisms, in particular, the upregulations of proteins involved in glycolysis, trehalose biosynthesis, and the pentose phosphate pathway, suggested vacuum-induced redistribution of the metabolic fluxes. The upregulation of 17 heat stress and oxidative response proteins indicated that multifactors contributed to oxidative stresses by affecting cell redox homeostasis. Taken together with upregulation in 14-3-3 proteins levels, 22 proteins were detected in multispots, respectively, indicating that vacuum might have promoted posttranslational modifications of some proteins in S. cerevisiae. Further investigation revealed that the elevations of the differentially expressed proteins were mainly derived from vacuum stress rather than the absence of oxygen. These findings provide new molecular mechanisms for understanding of adaptation and tolerance of yeast to vacuum fermentation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell recycling during repeated very high gravity bio-ethanol fermentations using the industrial Saccharomyces cerevisiae strain PE-2

A very high gravity (VHG) repeated-batch fermentation system using an industrial strain of Saccharomyces cerevisiae PE-2 (isolated from sugarcane-to-ethanol distillery in Brazil) and mimicking industrially relevant conditions (high inoculation rates and low O₂ availability) was successfully operated during fifteen consecutive fermentation cycles, attaining ethanol at 17.1 ± 0.2% (v/v) with a batch productivity of 3.5 ± 0.04 g l⁻¹ h⁻¹. Moreover, this innovative operational strategy (biomass refreshing step) prevented critical decreases on yeast viability levels and promoted high accumulation of intracellular glycerol and trehalose, which can provide an adaptive advantage to yeast cells under harsh industrial environments. This study contributes to the improvement of VHG fermentation processes by exploring an innovative operational strategy that allows attaining very high ethanol titres without a critical decrease of the viability level thus minimizing the production costs due to energy savings during the distillation process.     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

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.     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Continuous fermentation systems for alcohol production

Many batch fermentation processes are operated throughout the world for the production of alcohol (ethanol) from sugar-based feedstocks but very few truly continuous processes are in operation. This review compares the two modes of operation and some advantages of the continuous processes are examined. Possible markets for continuous processes are identified and some possible explanations for the, as yet, lack of wide acceptance of continuous processes are offered. Seven different commercially available processes are reviewed.     

Metabolic flux and cell cycle analysis indicating new mechanism underlying process oscillation in continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions

Process oscillation characterized by long oscillation period and large oscillation amplitude was observed in continuous ethanol fermentation with Saccharomyces cerevisiae under very high gravity conditions. Metabolic flux analysis was applied to the fermentation system, and the results indicated that carbon flux distributions at the metabolic notes oscillated, correspondingly, and the root reason for the process oscillation was the intracellular metabolism of yeast cells. Cell cycle analysis with the flow cytometry showed that no cell-cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation, and thus different mechanism existed compared with the oscillation observed in the continuous culture of Saccharomyces cerevisiae and triggered by the synchronization of the daughter and mother cells under specific conditions. Furthermore, the overall metabolic activity of the yeast cells was examined, which was found not exactly out of phase but lag behind ethanol concentration that accumulated within the fermentation system and its inhibition on the yeast cells as well, which supported the mechanistic speculation for the process oscillation: the lag response of yeast cells to ethanol inhibition.     

Real‐time monitoring of high‐gravity corn mash fermentation using in situ raman spectroscopy

In situ Raman spectroscopy was employed for real‐time monitoring of simultaneous saccharification and fermentation (SSF) of corn mash by an industrial strain of Saccharomyces cerevisiae. An accurate univariate calibration model for ethanol was developed based on the very strong 883 cm^?1 C–C stretching band. Multivariate partial least squares (PLS) calibration models for total starch, dextrins, maltotriose, maltose, glucose, and ethanol were developed using data from eight batch fermentations and validated using predictions for a separate batch. The starch, ethanol, and dextrins models showed significant prediction improvement when the calibration data were divided into separate high‐ and low‐concentration sets. Collinearity between the ethanol and starch models was avoided by excluding regions containing strong ethanol peaks from the starch model and, conversely, excluding regions containing strong saccharide peaks from the ethanol model. The two‐set calibration models for starch (R² = 0.998, percent error = 2.5%) and ethanol (R² = 0.999, percent error = 2.1%) provide more accurate predictions than any previously published spectroscopic models. Glucose, maltose, and maltotriose are modeled to accuracy comparable to previous work on less complex fermentation processes. Our results demonstrate that Raman spectroscopy is capable of real time in situ monitoring of a complex industrial biomass fermentation. To our knowledge, this is the first PLS‐based chemometric modeling of corn mash fermentation under typical industrial conditions, and the first Raman‐based monitoring of a fermentation process with glucose, oligosaccharides and polysaccharides present. Biotechnol. Bioeng. 2013; 110: 1654–1662. © 2013 Wiley Periodicals, Inc.     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

Ethanol production by Saccharomyces cerevisiae in biofilm reactors

Biofilms are natural forms of cell immobilization in which microorganisms attach to solid supports. At ISU, we have developed plastic composite-supports (PCS) (agricultural material (soybean hulls or oat hulls), complex nutrients, and polypropylene) which stimulate biofilm formation and which supply nutrients to the attached microorganisms. Various PCS blends were initially evaluated in repeated-batch culture-tube fermentation with Saccharomyces cerevisiae (ATCC 24859) in low organic nitrogen medium. The selected PCS (40% soybean hull, 5% soybean flour, 5% yeast extract-salt and 50% polypropylene) was then used in continuous and repeated-batch fermentation in various media containing lowered nitrogen content with selected PCS. During continuous fermentation, S. cerevisiae demonstrated two to 10 times higher ethanol production in PCS bioreactors than polypropylene-alone support (PPS) control. S. cerevisiae produced 30 g L⁻¹ ethanol on PCS with ammonium sulfate medium in repeated batch fermentation, whereas PPS-control produced 5 g L⁻¹ ethanol. Overall, increased productivity in low cost medium can be achieved beyond conventional fermentations using this novel bioreactor design. Received 20 May 1997/ Accepted in revised form 29 August 1997     

Fermentation behaviour of controlled mixed and sequential cultures of Candida cantarellii and Saccharomyces cerevisiae wine yeasts

Behaviour of Candida cantarellii and Saccharomyces cerevisiae strains during the fermentation of Syrah grape must using pure, mixed and sequential yeast cultures was studied. Different kinds of inocula have been tested according to the type of culture. Inocula proportions used in mixed C. cantarellii and S. cerevisiae strains reflect the population levels in natural grape microbiota. Biomass evolution of both strains was analysed in relation to different byproduct levels. Saccharomyces cerevisiae overcame C. cantarellii in the different co-culture assays at 48 h of fermentation. The final concentration of ethanol was similar in mixed and both sequential tests and higher (from 7.8 to 10.6%) than in S. cerevisiae pure culture. In mixed and sequential cultures, the glycerol content of the final products was 44.3 to 52.8% higher than the one obtained with pure S. cerevisiae fermentation. Wine analytical profiles of experiments that involved S. cerevisiae and C. cantarellii strains differed from the pure ones mainly in acetoin, propanol and succinic acid contents. From an enological point of view, analysed byproducts are relevant. Considering this, mixed assay and the inoculation of S. cerevisiae after 3 days of pure C. cantarellii fermentation appear to be the more appropriate options to develop the particular characteristics of Syrah wines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Investigation of vinegar production using a novel shaken repeated batch culture system

Nowadays, bioprocesses are developed or optimized on small scale. Also, vinegar industry is motivated to reinvestigate the established repeated batch fermentation process. As yet, there is no small‐scale culture system for optimizing fermentation conditions for repeated batch bioprocesses. Thus, the aim of this study is to propose a new shaken culture system for parallel repeated batch vinegar fermentation. A new operation mode — the flushing repeated batch — was developed. Parallel repeated batch vinegar production could be established in shaken overflow vessels in a completely automated operation with only one pump per vessel. This flushing repeated batch was first theoretically investigated and then empirically tested. The ethanol concentration was online monitored during repeated batch fermentation by semiconductor gas sensors. It was shown that the switch from one ethanol substrate quality to different ethanol substrate qualities resulted in prolonged lag phases and durations of the first batches. In the subsequent batches the length of the fermentations decreased considerably. This decrease in the respective lag phases indicates an adaptation of the acetic acid bacteria mixed culture to the specific ethanol substrate quality. Consequently, flushing repeated batch fermentations on small scale are valuable for screening fermentation conditions and, thereby, improving industrial‐scale bioprocesses such as vinegar production in terms of process robustness, stability, and productivity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1158–1168, 2013     

Analysis of adaptation to high ethanol concentration in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using DNA microarray

In industrial process, yeast cells are exposed to ethanol stress that affects the cell growth and the productivity. Thus, investigating the intracellular state of yeast cells under high ethanol concentration is important. In this study, using DNA microarray analysis, we performed comprehensive expression profiling of two strains of Saccharomyces cerevisiae, i.e., the ethanol-adapted strain that shows active growth under the ethanol stress condition and its parental strain used as the control. By comparing the expression profiles of these two strains under the ethanol stress condition, we found that the genes related to ribosomal proteins were highly up-regulated in the ethanol-adapted strain. Further, genes related to ATP synthesis in mitochondria were suggested to be important for growth under ethanol stress. We expect that the results will provide a better understanding of ethanol tolerance of yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High solids fermentation of hydrolysates of wheat starch B in a continuous dynamic immobilized biocatalyst bioreactor

Summary A simple and efficient method of conversion of wheat starch B to ethanol was investigated. Employing a two-stage enzymatic saccharification process, 95% of the wheat starch was converted to fermentable sugars in 40 h. From 140 g/l total sugars in the feed solution, 63.6 g/l ethanol was produced continuously with a residence time of 3.3 h in a continuous dynamic immobilized biocatalyst bioreactor by immobilized cells ofSaccharomyces cerevisiae. The advantages and the application of this bioreactor to continuous alcoholic fermentation of industrial substrates are presented.     

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy‐demanding growth‐related processes. Studies using genome‐wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain