Automatic method for evaluating the activity of sourdough strains based on gas pressure measurements

A new method is proposed for the evaluation of the activity of sourdough strains, based on gas pressure measurements in closed air-tight reactors. Gas pressure and pH were monitored on-line during the cultivation of commercial yeasts and heterofermentative lactic acid bacteria on a semi-synthetic medium with glucose as the major carbon source. Relative gas pressure evolution was compared both to glucose consumption and to acidification and growth. It became obvious that gas pressure evolution is related to glucose consumption kinetics. For each strain, a correlation was made between maximum gas pressure variation and amount of glucose consumed. The mass balance of CO2 in both liquid and gas phase demonstrated that around 90% of CO2 was recovered. Concerning biomass production, a linear relationship was found between log colony-forming units/ml and log pressure for both yeasts and bacteria during the exponential phase; and for yeasts, relative gas pressure evolution also followed optical density variation.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

A study of sulfite-tolerant yeasts from comminuted lamb products

The sulfite tolerance of meat yeasts was shown to be determined by pH, sulfite concentration, substrate availability, and the composition of the preincubation medium. Acetaldehyde production by Candida norvegica was sulfite-induced and occurred during the exponential growth phase in sulfited (500 micrograms SO2 ml-1) lab lemco glucose broth cultures buffered at pH 5, 6, or 7. Growth at pH 4, however, was inhibited by sulfite. Acetaldehyde production occurred in sulfited medium containing fructose or ethanol but not lactate nor a range of other assimilable substrates. A non-acetaldehyde-producing yeast, Candida vini, grew in sulfited (500 micrograms SO2 ml-1) lab lemco broth containing glucose or lactate buffered at pH 6 or 7 but not at pH 4 or 5.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Evaluation of thermotolerant yeasts in controlled simultaneous saccharifications and fermentations of cellulose to ethanol

Simultaneous saccharification and fermentation (SSF) experiments were performed at selected temperatures (37, 41, and 43 degrees C) to obtain comprehensive material balance and performance data for several promising strains of thermotolerant yeast. Parameters measured were ethanol concentration, yeast cell density, and residual sugar and cellulose concentrations. The three yeasts Saccharomyces uvarum, Candida brassicae, and C. lusitaniae and two mixed cultures of Brettanomyces clausenii with S. cerevisiae (mixed culture I) and C. Iusitaniae with S. uvarum (mixed culture II) exhibited rapid rates of fermentation, high ethanol yields, strong viability, or high cellobiase activity. Overall, mixed culture II at 41 degrees C performed better than either component yeast by themselves because it combined a cellobiose fermenting capability with the high ethanol tolerance and rapid glucose fermentation of conventional industrial yeasts. Thus, the mixed cultures provide good initial rates by preventing buildup of cellobiose (a strong inhibitor of enzyme activity) while attaining high ultimate yields of ethanol for high cellulase concentrations. However, C. brassicae and S. uvarum gave similar results to mixed culture II at 37 degrees C.     

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.     

Fermentation of D-xylose by free and immobilized Saccharomyces cerevisiae

With D-xylose (50 g l ) as sole carbon substrate, aerobic cultures of S. cerevisiae consumed significant amounts of sugar (26.4 g l ), producing 4.0 g xylitol l but no ethanol. In the presence of a mixture of glucose (35 g l ) and xylose (15 g l ), yeasts consumed 1.6 g xylose l that was converted nearly stoichiometrically to xylitol. Anaerobic conditions lessened xylose consumption and its conversion into xylitol. Traces of ethanol (0.4 g l ) were produced when xylose was the only carbon source, however. Agar-entrapped yeasts behaved as anaerobically-grown cultures but with higher specific rates of xylose consumption and xylitol production.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Possible use of Biolog methodology for monitoring yeast presence in alcoholic fermentation for wine‐making

Aims: A research was undertaken to explore the possibility to use Biolog system of microbial metabolic characterization for the monitoring of yeast population evolution during alcoholic fermentation for wine production. Methods and Results: An application of Biolog system was employed for the characterization of yeasts of oenological interest, in pure cultures and mixed consortia, in various cell concentrations. The system’s capacity to discriminate among different cell concentrations of the same yeast strain was ascertained, along with the capacity to discriminate between mixed and pure populations. Conclusions: The tested application of Biolog system resulted suitable for a quick recognition (24 h) of the presence of starter cultures within mixed populations of autochthonous yeasts. Such discrimination was confirmed with the one resulting from molecular techniques. Significance and Impact of the Study: The study suggests the possibility to employ Biolog system for an early monitoring of yeast evolution in modern wine‐making fermentations, where specialized yeasts are more and more frequently used as starters and their ability to overcome autochthonous yeast populations is crucial.     

Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation

Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.     

Efficient production of L-lactic acid from xylose by Pichia stipitis

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to approximately 15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.     

Sugar and organic acid metabolism in mixed cultures of Pediococcus pentosaceus and Leuconostoc oenos isolated from wine

Pediococcus pentosaceus 12p and Leuconostoc oenos X₂L isolated from Argentinian wine were examined for growth and changes in the concentrations of glucose, fructose, sucrose and mannitol and malic, citric, acetic and lactic acids in pure and mixed cultures. In mixed cultures a mutualistic growth response and a change in the balance of end-products of sugar and organic acid metabolism were observed. The production of mannitol and acetic acid was lower while D(-) and L(+) lactic acids were detected in higher levels than in pure cultures. Malic and citric acids were metabolized simultaneously, but the amount of citric acid consumed was lower than in pure culture of Leuc. oenos.     

Factors Affecting Organic Acid Production by Sourdough (San Francisco) Bacteria

Previous workers from this laboratory observed considerable variation in the proportions of acetic and lactic acids produced in pure broth culture as compared to consistently high proportions of acetic acid produced in the sourdough and flour suspension systems. In the latter the proportion of acetic acid was always in the range of 20 to 35% of the total, whereas in pure broth culture frequently less than 5% acetic acid was produced. In the natural environment, the sourdough bacteria, tentatively identified as lactobacilli, coexist with a yeast, Saccharomyces exiguus, and this study was undertaken to determine whether this yeast or flour ingredients including glucose or other factors were involved in this variable production of acetic acid. The proportion of acetic acid produced in broth culture on maltose, the preferred carbohydrate source, was found to depend almost entirely on the degree of aeration. Essentially anaerobic conditions, as obtained by thorough evacuation and flushing with CO(2) or N(2), resulted in very low (5% or less) proportions of acetic acid. Aerobic conditions, achieved by continuous shaking in cotton-plugged flasks, yielded high levels (23 to 39% of the total) of acetic acid. Similar effects of aeration were observed with glucose as the substrate, although growth was considerably slower, or in nonsterile flour suspension systems. It is theorized that, under aerobic conditions, the reduced pyridine nucleotides generated in the dissimilation of carbohydrate are oxidized directly by molecular oxygen, thereby becoming unavailable for the reduction of the acetyl phosphate intermediate to ethyl alcohol, the usual product of anaerobic dissimilation of glucose by heterofermentative lactic acid bacteria. Comparative studies with known strains of homo- and heterofermentative lactobacilli showed similar effects of aeration only on the heterofermentative strains, lending additional support to the tentative grouping by previous workers from this laboratory of the sourdough bacteria with the heterofermentative lactobacilli.     

Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

The effect of acetic acid bacteria upon the growth and metabolism of yeasts during the fermentation of grape juice

The influence of species of Acetobacter and Gluconobacter upon growth of the wine yeasts Saccharomyces cerevisiae, Kloeckera apiculata and Candida stellata was examined during mixed culture in grape juice. Acetobacter pasteurianus, A. aceti and Gluconobacter oxydans grew in conjunction with yeasts during juice fermentation. As determined by viable counts, yeast growth was only slightly impaired by the presence of bacteria. However, as judged by the concentrations of glucose, fructose, ethanol, glycerol, acetaldehyde, ethyl acetate, iso -amyl alcohol and organic acids in the fermented juice, acetic acid bacteria significantly influenced the alcoholic fermentation by yeasts.     

A comparison of carbon/energy and complex nitrogen sources for bacterial sulphate-reduction: potential applications to bioprecipitation of toxic metals as sulphides

Detailed nutrient requirements were determined to maximise efficacy of a sulphate-reducing bacterial mixed culture for biotechnological removal of sulphate, acidity and toxic metals from waste waters. In batch culture, lactate produced the greatest biomass, while ethanol was more effective in stimulating sulphide production and acetate was less effective. The presence of additional bicarbonate and H₂ only marginally stimulated sulphide production. The sulphide output per unit of biomass was greatest using ethanol as substrate. In continuous culture, ethanol and lactate were used directly as efficient substrates for sulphate reduction while acetate yielded only slow growth. Glucose was utilised following fermentation to organic acids and therefore had a deleterious effect on pH. Ethanol was selected as the most efficient substrate due to cost and efficient yield of sulphide. On ethanol, the presence of additional carbon sources had no effect on growth or sulphate reduction in batch culture but the presence of complex nitrogen sources (yeast extract or cornsteep) stimulated both. Cornsteep showed the strongest effect and was also preferred on cost grounds. In continuous culture, cornsteep significantly improved the yield of sulphate reduced per unit of ethanol consumed. These results suggest that the most efficient nutrient regime for bioremediation using sulphate-reducing bacteria required both ethanol as carbon source and cornsteep as a complex nitrogen source.