Subsequent Fermentation Performance Research Articles

Strain-Specific Responses by Saccharomyces cerevisiae to Competition by Non-Saccharomyces Yeasts

The use of non-Saccharomyces yeast species generally involves sequential or co-inoculation of a Saccharomyces cerevisiae strain to complete fermentation. While most studies have focused on characterising the impact that S. cerevisiae has on the growth and metabolic activity of these non-Saccharomyces species, microbial interactions work reciprocally. Antagonism or competition of non-Saccharomyces species against S. cerevisiae has been shown to impact subsequent fermentation performance. To date, it remains unclear whether these negative interactions are strain specific. Hence, characterisation of strain-specific responses to co-inoculation would enable the identification of specific S. cerevisiae strain/non-Saccharomyces combinations that minimise the negative impacts of sequential fermentation on fermentation performance. The competitive fitness response of 93 S. cerevisiae strains to several non-Saccharomyces species was simultaneously investigated using a barcoded library to address this knowledge gap. Strain-specific fitness differences were observed across non-Saccharomyces treatments. Results obtained from experiments using selected S. cerevisiae strains sequentially inoculated after Metschnikowia pulcherrima and Torulaspora delbrueckii were consistent with the competitive barcoded library observations. The results presented in this study indicate that strain selection will influence fermentation performance when using non-Saccharomyces species, therefore, appropriate strain/yeast combinations are required to optimise fermentation.

Precise pretreatment of lignocellulose: relating substrate modification with subsequent hydrolysis and fermentation to products and by-products

BackgroundPretreatment is a crucial step for valorization of lignocellulosic biomass into valuable products such as H2, ethanol, acids, and methane. As pretreatment can change several decisive factors concurrently, it is difficult to predict its effectiveness. Furthermore, the effectiveness of pretreatments is usually assessed by enzymatic digestibility or merely according to the yield of the target fermentation products. The present study proposed the concept of “precise pretreatment,” distinguished the major decisive factors of lignocellulosic materials by precise pretreatment, and evaluated the complete profile of all fermentation products and by-products. In brief, hemicellulose and lignin were selectively removed from dewaxed rice straw, and the cellulose was further modified to alter the crystalline allomorphs. The subsequent fermentation performance of the selectively pretreated lignocellulose was assessed using the cellulolytic, ethanologenic, and hydrogenetic Clostridium thermocellum through a holistic characterization of the liquid, solid, and gaseous products and residues.ResultsThe transformation of crystalline cellulose forms from I to II and from Iα to Iβ improved the production of H2 and ethanol by 65 and 29%, respectively. At the same time, the hydrolysis efficiency was merely improved by 10%, revealing that the crystalline forms not only influenced the accessibility of cellulose but also affected the metabolic preferences and flux of the system. The fermentation efficiency was independent of the specific surface area and degree of polymerization. Furthermore, the pretreatments resulted in 43–45% of the carbon in the liquid hydrolysates unexplainable by forming ethanol and acetate products. A tandem pretreatment with peracetic acid and alkali improved ethanol production by 45.5%, but also increased the production of non-ethanolic low-value by-products by 136%, resulting in a huge burden on wastewater treatment requirements.ConclusionCellulose allomorphs significantly affected fermentation metabolic pathway, except for hydrolysis efficiency. Furthermore, with the increasing effectiveness of the pretreatment for ethanol production, more non-ethanolic low-value by-products or contaminants were produced, intensifying environmental burden. Therefore, the effectiveness of the pretreatment should not only be determined on the basis of energy auditing and inhibitors generated, but should also be assessed in terms of the environmental benefits of the whole integrated system from a holistic view.

The effect of preconditioning cells under osmotic stress on high alcohol production

This paper focuses on the research into the influence of salt on physiology of the yeast, Saccharomyces cerevisiae. Specifically, the work focused on how NaCl affected the growth, viability and fermentation performance of this yeast in laboratory-scale experiments. One of the main findings of the research presented involved the influ?ence of salt ?preconditioning? of yeasts which represents a method of pre-culturing of cells in the presence of salt in an attempt to improve subsequent fermentation performance. Such an approach resulted in preconditioned yeasts having an improved capability to ferment high-sugar containing media (up to 60% w/v of glucose) with increased cell viability and with increased levels of produced ethanol (higher than 20% in vol.). Salt-preconditioning was most likely influencing the stress-tolerance of yeasts by inducing the synthesis of key metabolites such as trehalose and glycerol which act to improve cells? ability to withstand osmostress and ethanol toxicity. The industrial-scale trials using salt-preconditioned yeasts verified the benefit of the physiological engineering approach to practical fermentations. Overall, this research has demonstrated that a relatively simple method designed to adapt yeast cells physiologically - by salt-preconditioning - can have distinct advantages for al?cohol fermentation processes.

Effects of rehydration nutrients on H2S metabolism and formation of volatile sulfur compounds by the wine yeast VL3.

In winemaking, nutrient supplementation is a common practice for optimising fermentation and producing quality wine. Nutritionally suboptimal grape juices are often enriched with nutrients in order to manipulate the production of yeast aroma compounds. Nutrients are also added to active dry yeast (ADY) rehydration media to enhance subsequent fermentation performance. In this study we demonstrate that nutrient supplementation at rehydration also has a significant effect on the formation of volatile sulfur compounds during wine fermentations. The concentration of the 'fruity' aroma compounds, the polyfunctional thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), was increased while the concentration of the 'rotten egg' aroma compound, hydrogen sulfide (H2S), was decreased. Nutrient supplementation of the rehydration media also changed the kinetics of H2S production during fermentation by advancing onset of H2S production. Microarray analysis revealed that this was not due to expression changes within the sulfate assimilation pathway, which is known to be a major contributor to H2S production. To gain insight into possible mechanisms responsible for this effect, a component of the rehydration nutrient mix, the tri-peptide glutathione (GSH) was added at rehydration and studied for its subsequent effects on H2S formation. GSH was found to be taken up during rehydration and to act as a source for H2S during the following fermentation. These findings represent a potential approach for managing sulfur aroma production through the use of rehydration nutrients.

Physiological analysis of yeast cells by flow cytometry during serial-repitching of low-malt beer fermentation

At the end of beer brewing fermentation, yeast cells are collected and repitched for economical reasons. Although it is generally accepted that the physiological state of inoculated yeast cells affects their subsequent fermentation performance, the effect of serial-repitching on the physiological state of such yeast cells has not been well clarified. In this study, the fermentation performance of yeast cells during serial-repitching was investigated. After multiple repitchings, the specific growth rate and maximum optical density (OD(660)) decreased, and increases in isoamyl alcohol, which causes an undesirable flavor, and residual free amino acid nitrogen (FAN) concentrations were observed. The physiological state of individual cells before inoculation was characterized by flow cytometry using the fluorescent dyes dehydrorhodamine 123 (DHR) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). The fluorescence intensities of DHR, an indicator of reactive oxygen species (ROSs), and OXN, which indicates membrane potential, gradually increased as the number of serial-repitching cycles increased. Fluorescence intensity correlated strongly with cell growth. The subsequent fermentation performance can be predicted from this correlation.

Detection of Proteinases inSaccharomyces cerevisiaeby Flow Cytometry

The physiological state of a yeast population used for inoculation determines how rapidly the cells adapt to new environmental conditions, begin proliferating and utilising extract. The decision as to whether a yeast culture is suitable for re-pitching should not be based only on viability determinations since this can be misleading. Increased proteolytic activity in a yeast population indicates the onset of senescence. A flow cytometric method has been developed for measuring a wide variety of proteinases in Saccharomyces cerevisiae employing a commercially available casein-dye conjugate. The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population. This knowledge will assist the industry to optimize the selection of yeast and its subsequent fermentation performance. Yeast cell autolysis with all its negative consequences for beer quality and stability will thus be minimised.

Linoleic Acid Supplementation of a Cropped Brewing Lager Strain: Effects on Subsequent Fermentation Performance with Serial Repitching

Most breweries collect yeast from a previous fermentation cycle for further use in a subsequent cycle. However, the cropped cells are deficient in membrane sterols and unsaturated fatty acids (UFAs) which are required for good fermentation performance in the next cycle. Consequently, the cellular levels of these compounds must be restored to obtain an optimal fermentation performance. There are currently three possibilities to satisfy this requirement. The common practice is aeration of the wort before pitching, thus providing oxygen needed for lipid synthesis during the first stages of fermentation. Oxygenation (aeration) of cropped yeast slurries is a second alternative. Finally, the addition of the required lipids to wort is sometimes suggested as an alternative to aeration. We examined a fourth possibility, namely the supplementation with UFA of cropped cells. Previously, we reported that the supplementation of stationary phase cropped brewer's yeast with linoleic acid is a good alternative to wort aeration. This conclusion resulted from results obtained with a well-defined stirred synthetic fermentation medium. We also showed that cells cropped from non-stirred tall-tube fermented malt wort incorporated linoleic acid into different cellular lipid fractions, when suspended and supplemented in fermented wort. Now, we report that such yeast, pitched in malt wort in non-stirred tall-tubes, showed growth and attenuation profiles comparable to unsupplemented yeast in pre-aerated wort. Moreover, the synthesis of acetate esters, which is known to be affected when UFAs are added directly to the wort, was not significantly affected. We hypothesize that the active uptake of linoleic acid during fermentation and its activation by coenzyme A (CoA) and phospholipid synthesis are responsible for the effects on ester synthesis, through repression of the alcohol acetyltransferase-encoding gene, ATF1. In supplemented cropped yeast, these reactions occur prior to fermentation, thus avoiding interferences with acetate ester synthesis. In serial repitching experiments with repeated linoleic acid supplementation of the cropped yeast, the fermentation performances of the yeast remained comparable to those of non-supplemented yeast in pre-aerated wort. However, due to a progressive increase of cellular UFA, negative effects on acetate ester synthesis appeared. Nevertheless, the supplementation of cropped yeast with UFAs can be considered as an interesting alternative to wort oxygenation to restore optimal membrane functions.

The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis

For beer wort fermentation the addition of unsaturated fatty acids has sometimes been suggested as an alternative to wort oxygenation. This can however negatively affect the synthesis of acetate esters and consequently beer flavour. This work investigates the effect of supplementing a cropped yeast with an unsaturated fatty acid on the fermentation performance of the pitching yeast. Cropped yeast is in a different physiological state to yeast pitched in unfermented wort. Using a synthetic medium for the fermentations, it was found that the incubation of cropped yeast with linoleic acid resulted in two important changes in the yeasts composition: (1) the ratio of unsaturated fatty acids to total fatty acids increased from 0.53 to 0.66 and (2) the ratio of trehalose to glycogen increased from 0.17 to 0.49. The performance of this yeast in subsequent fermentations was compared to unsupplemented yeast under three conditions: medium pre-aeration, de-aerated medium and de-aerated medium with newly added unsaturated fatty acid. It was found that the supplemented pitching yeast showed growth, attenuation and ethanol formation profiles similar to those obtained with unsupplemented yeast in pre-aerated medium, which simulated the normal brewing practice. Compared to fermentations with unsaturated fatty acids added to the medium, the supplemented cropped yeast did not induce a reduction in acetate ester synthesis. Results indicated that the supplementation of cropped yeast with unsaturated fatty acids could be an interesting alternative to wort oxygenation to restore the optimal membrane fluidity of the yeast.

A NOVEL AND PRACTICAL YEAST VITALITY METHOD BASED ON MAGNESIUM ION RELEASE

Using a commercial lager brewing yeast, the immediate release of magnesium, potassium and phosphate ions by cells when inoculated into wort was evaluated to be directly related to its subsequent fermentation performance. Yeast which released appreciable amounts of these ions immediately after inoculation mediated improved fermentations as evidenced by better growth, higher ethanol concentrations and lower diacetyl levels at the end of fermentation. Conversely, yeast that absorbed these ions or released them at very low concentrations performed poorly throughout fermentation producing beers with lower ethanol concentrations and higher diacetyl levels. These observations led to the identification and development of a rapid, practical and highly sensitive method to measure Mg++ released or absorbed by yeast as an indicator of its vitality and a predictor of its subsequent fermentative performance. Full method details of the Magnesium Release Test (MRT) are given.

Effect of Starvation on the Flocculation of Ale and Lager Brewing Yeasts

Starvation has been shown to influence the technological behavior of brewing yeast. Starved yeast were observed to be less flocculant in beer, therefore, the effect of starvation on the flocculation of lager and ale brewing strains was examined. The characterization of the yeast cell surface involved the use of techniques such as dye retention, solvent partitioning, latex microsphere attachment, biochemical analysis, and electron microscopy. Starved lager yeast cells exhibited significantly less negative surface charge than did those that were non-starved, although surface hydrophobicity was largely unaffected. Starved ale yeast exhibited an increased surface charge and a reduced surface hydrophobicity compared with those that were non-starved. The effect of starvation on the subsequent fermentation performance of the lager yeast was examined in wort. Starvation-induced modifications in flocculation were retained by subsequent generations. It was observed that starvation resulted in a reduction in surface phosphate concentration, and, although the thickness of the cell wall decreased after starvation, the relative proportions of other cell wall components were not observed.

Effect of Physiological Stress on the Surface Properties of Brewing Yeasts

Brewer's yeast may be stored before inoculation into a fermentation vessel, and during these periods of storage, starvation may occur. The effect of starvation on yeast inoculum condition and its subsequent fermentation performance in brewing is important but not well understood. Although the physical properties of brewing yeast cell surfaces influence flocculation, they have not previously been used to ascertain the physiological state of brewing yeast. Starvation has been shown to influence the technological behavior of yeast. Indeed, starved yeast were observed to be less flocculant in beer. The characterization of the yeast cell surfaces involved the use of techniques such as dye retention, electrophoretic mobility, solvent partition, whole cell low-pressure chromatography, and electron microscopy. Starved cells were significantly less negatively charged compared with those that were non-starved, although surface hydrophobicity was largely unaffected. Modifications in cell surface topography and colony morphology also were observed. It is suggested that the cell surface characteristics reflect some aspect of the physiological condition of brewing yeast.

The effect of high electric fields on the subsequent fermentation performance of cells of Saccharomyces cerevisiae

Suspensions of cells of Saccharomyces cerevisiae were electrostatically sprayed through a charged nozzle into an inert heavy hydrocarbon solvent, collected and used to conduct fermentation of malt extract media. Cell growth and ethanol production were unaffected by voltages up to 25kV.

Changes in Brewer's Yeast during Storage and the Effect of These Changes on Subsequent Fermentation Performance

A slurry of brewer's yeast in beer was stored in two, 200-bbl cylindroconical vessels at 1.6° C. One was maintained full, while 34% of the yeast was removed from the other vessel after 24 hr. The yeast was stored in both vessels for a total of 80 hr. Samples were removed at intervals and analyzed for glycogen, trehalose, and viability. Aliquots of each yeast sample were pitched into 2 L of wort. Yeast glycogen and trehalose declined only slightly in the full vessel, but in the other vessel, glycogen declined significantly after removal of a portion of the yeast. Viability of the yeast stored by either method remained between 90 and 96%. Fermentations of the yeast stored by either method showed no relationship between yeast fermentation performance and glycogen or trehalose content. The fermentation performance of yeast stored under these industrial conditions was independent of glycogen or trehalose content and several reasons for this are discussed.

Changes in Brewer�s Yeast During Storage and the Effect of These Changes on Subsequent Fermentation Performance

Changes in Brewer�s Yeast During Storage and the Effect of These Changes on Subsequent Fermentation Performance

Selective solvent delignification for fermentation enhancement

Cellulose and hemicellulose in renewable biomass resources such as cornstover and wheat straw have been examined as substrates for the production of ethanol. A mixed culture of selected strains of Clostridium thermocellum and Clostridium thermosaccharolyticum are used to accomplish both the hydrolysis and fermentation of these carbohydrates in a single step. However, lignin and related phenolic materials are shown to diminish the rate, extent, and yield at which these carbohydrates can be utilized for ethanol production. In order to overcome this problem, a selective solvent pretreatment with alkaline-ethanol-water mixtures was examined for the delignification of cellulosic biomass under conditions where very little loss of fermentable carbohyrates results. Under optimal conditions, up to 67% of the initial lignin in cornstover can be extracted while 95% of the alpha-cellulose and pentosan carbohydrates remain insoluble. Subsequent mixed culture fermentation of the treated material has shown a 400% increase in the rate of degradation and greater than 85% utilization of the substrate. The effects of various extraction parameters on delignification kinetics and subsequent fermentation performance are discussed.