Specific growth rates (mu) of two strains of Saccharomyces cerevisiae decreased exponentially (R2 > 0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30 degrees C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05-0.1% w/v and lactic acid at concentrations of 0.2-0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30 degrees C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P < or = 0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic.

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (⌬pH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.The yeast Saccharomyces cerevisiae under aerobic conditions can use short-chain organic acids, such as acetic acid and lactic acid, as carbon sources. The process involves induction of certain anabolic pathways, enzymes, and specific transport mechanisms (2,4,5,6,17). If glucose is available in the growth medium, these pathways and permeases are repressed. Glucose-repressed yeast cells are unable to take up the anions of these acids (6), but the undissociated acids diffuse freely into the cells. Once inside, these acids dissociate because of the higher intracellular pH and cause acidification of the cytoplasm. Generally, eucaryotic cells maintain their intracellular pH within a narrow range despite wide variations that may occur in the extracellular pH (7). Under fermentation conditions, the intracellular pH of S. cerevisiae is usually maintained between 5.5 and 5.75 when the external pH is 3.0 (9) or between 5.9 and 6.75 when the external pH is varied between 6.0 and 10.0 (8). To maintain the intracellular pH within a physiological range optimum for metabolism, the cells pump out protons at the expense of metabolic energy (ATP). Increased diversion of energy (ATP) to pump out protons results in decreased molar growth yield with respect to glucose (Y glucose ) (19,24). It has also been reported that the Y ATP (grams of biomass produced per mole of ATP generated) decreased from 14 to 4 when the concentration of acetic acid was increased from 0 to 170 mM (19). As the gap between the extracellular pH and the intracellular pH widens, greater stress i...

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20°C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.

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Normal-gravity (22 to 24°Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at ϳ10 5 , ϳ10 6 , ϳ10 7 , ϳ10 8 , and ϳ10 9 CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at ϳ10 7 CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at ϳ10 6 CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10 5 CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10 5 CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10 9 CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.

The antibiotic virginiamycin was investigated for its effects on growth and lactic acid production by seven strains of lactobacilli during the alcoholic fermentation of wheat mash by yeast. The lowest concentration of virginiamycin tested (0.5 mg Lactrol kg-1 mash), was effective against most of the lactic acid bacteria under study, but Lactobacillus plantarum was not significantly inhibited at this concentration. The use of virginiamycin prevented or reduced potential yield losses of up to 11% of the produced ethanol due to the growth and metabolism of lactobacilli. However, when the same concentration of virginiamycin was added to mash not inoculated with yeast, Lactobacillus rhamnosus and L. paracasei grew after an extensive lag of 48 h and L. plantarum grew after a similar lag even in the presence of 2 mg virginiamycin kg-1 mash. Results showed a variation in sensitivity to virginiamycin between the different strains tested and also a possible reduction in effectiveness of virginiamycin over prolonged incubation in wheat mash, especially in the absence of yeast.

Although the practice of cultivating mass fl owering crops (MFCs) is seen as a means of counteracting the widespread decline of insect pollinators, no study to-date has evaluated whether the increased pollinator abundance associated with MFCs infl uences pollinator visits to assemblages of adjacent native, non-crop plant species (pollinator ' spill-over ' ). In this study we quantifi ed bumblebee abundance along hedgerow transects in MFC (fi eld bean) vs non-MFC (wheat) margins. Surveys were conducted on east and west-facing margins twice daily (morning and afternoon) for three or four days during the main MFC fl owering period (June) over four years involving paired bean and wheat fi elds in Devon and Cornwall, southwest England. Although no single bumblebee species showed any consistent change in relative frequency, when taken across all years and bumblebee species combined, we observed twice as many bumblebees visiting fl owers adjacent to the MFC. However, when we compared bumblebee activity along hedgerow transects two weeks after bean fl owering, there was no diff erence between crops. We thus conclude that although there is evidence for pollinator spillover from the bean MFC to nearby semi-natural habitat, the eff ect is comparatively short lived and not specifi c to any single bumblebee species. We also suggest that while pollinator services to native plant species might be briefl y enhanced by MFC cultivation, the possible repercussions of bumblebee spill-over from MFCs to semi-natural habitats and the other pollinator groups they support should be evaluated before MFC cultivation is considered as a means of conserving declining bumblebee populations.

Aims: The aim of this study was to evaluate interactions between Saccharomyces cerevisiae and selected strains of lactobacilli regarding cell viabilities, and production of organic acids and ethanol during fermentation. Methods and Results: Corn mashes were inoculated with yeasts and selected strains of lactobacilli, and fermented in batch or semi-continuous (cascade) mode. Ethanolic fermentation rates and viabilities of yeast were not affected by lactobacilli unless the mash was pre-cultured with lactobacilli. Then, yeast growth was inhibited and the production of ethanol was reduced by as much as 22%. Conclusions: Yeasts inhibited the multiplication of lactobacilli and this resulted in reduced production of acetic and lactic acids. The self-regulating nature of the cascade system allowed the yeast to recover, even when the lactobacilli had a head start, and reduced the size of the population of the contaminating Lactobacillus to a level which had an insigni®cant effect on fermentation rate or ethanol yield. Signi®cance and Impact of the Study: Contamination during fermentation is normally taken care of by the large yeast inoculum, although yeast growth and fermentation rates could be adversely affected by the presence of high numbers of lactobacilli in incoming mash or in transfer lines.