Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

The timing of diammonium phosphate supplementation of wine must affects subsequent H2S release during fermentation

Aims:  The aim of this study was to evaluate the impact of supplementation by diammonium phosphate (DAP) on hydrogen sulfide (H₂S) production, when DAP given either prior to fermentation or during the early stationary growth phase of yeast. Methods and Results:  Three contrasting Saccharomyces cerevisiae wine strains were used to ferment synthetic grape juice (GJ) containing 67 mg l^?1 of initial yeast assimilable nitrogen (YAN), supplied either as DAP or as mixture of amino acids. Sufficient DAP was added either prior to or 72 h after the initiation of fermentation to achieve a final YAN concentration of 267 mg l^?1. Supplementation prior to fermentation stimulated H₂S production. The results obtained in model solutions were validated using natural GJ. Conclusion:  The timing of DAP supplementation is critical for ensuring that fermentation proceeds without excessive release of H₂S. Significance and Impact of the Study:  This result has important implications for the wine‐making industry, because it highlights the value of determining the initial nitrogen level of a GJ. It raises awareness of the dependence of wine quality on the correct timing of DAP supplementation.     

The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

The growth kinetics and fermentation behaviour of some non-Saccharomyces yeasts associated with wine-making

Summary The growth kinetics and fermentation behaviour of five non-Saccharomyces yeast species associated with wine-making were evaluated.The results showed that the Candida stellata and Torulspora delbrueckii species are interesting for biotechnological applications in wine-making, whereas small-size apiculate yeasts could be profitably used in the production of wine for vinegar manufacture.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Effect of nitrogen limitation and surplus upon trehalose metabolism in wine yeast

Trehalose metabolism in yeast has been related to stress and could be used as a stress indicator. Winemaking conditions are stressful for yeast and understanding trehalose metabolism under these conditions could be useful for controlling alcoholic fermentation. In this study, we analysed trehalose metabolism of a commercial wine yeast strain during alcoholic fermentation by varying the nitrogen levels from low (below adequate) to high (excess). We determined trehalose, nitrogen, sugar consumption and expression of NTH1, NTH2 and TPS1. Our results show that trehalose metabolism is slightly affected by nitrogen availability and that the main consumption of nitrogen occurs in the first 24 h. After this period, nitrogen is hardly taken up by the yeast cells. Although nitrogen and sugar are still available, no further growth is observed in high concentrations of nitrogen. Increased expression of genes involved in trehalose metabolism occurs mainly at the end of the growth period. This could be related to an adaptive mechanism for fine tuning of glycolysis during alcoholic tumultuous fermentation, as both anabolic and catabolic pathways are affected by such expression.     

Genetic Basis of Variations in Nitrogen Source Utilization in Four Wine Commercial Yeast Strains

The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeast fermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogen compounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H₂S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.     

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.     

Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.     

Effect of Sugar Transport Inactivation in Saccharomyces cerevisiae on Sluggish and Stuck Enological Fermentations

Sluggish and stuck (i.e., very delayed or incomplete) fermentations have been often observed in wine making. Some of them appeared to be associated with insufficient levels of yeast nutrients such as assimilable nitrogen. In these conditions, sugar transport catabolite inactivation, which is triggered by the protein synthesis arrest, may account in part for the inhibition of fermentation. Moreover, this mechanism of inhibition may explain the failure of added ammoniacal nitrogen to nitrogen-limited musts to restore or elevate rate of fermentation after the early yeast growth phase.     

Monitoring stress-related genes during the process of biomass propagation of Saccharomyces cerevisiae strains used for wine making

Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.     

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.