Considerazioni su Gelsi Ingialliti per Fame

Jerusalem artichoke (Helianthus tuberosus L.), an important crop, containing over 50% inulin in its tubers on a dry weight basis is an agricultural and industrial crop with a great potential for production of ethanol and industrial products. Inulin is a good substrate for bioethanol production. Saccharomyces cerevisiae 6525 can produce high concentrations of ethanol, but it cannot synthesize inulinase. In this study, a new integration vector carrying inuA1 gene encoding exoinulinase was constructed and transformed into 18SrDNA site of industrial strain S. cerevisiae 6525. The obtained transformant, BR8, produced 1.1 U mL^? 1 inulinase activity within 72 h and the dry cell weight reached 12.3 g L^? 1 within 48 h. In a small-scale fermentation, BR8 produced 9.5% (v/v) ethanol, with a productivity rate of 0.385 g ethanol per gram inulin, while wild-type S. cerevisiae 6525 produced only 3.3% (v/v) ethanol in the same conditions. In a 5-L fermentation, BR8 produced 14.0% (v/v) ethanol in fermentation medium containing inulin and 1% (w/v) (NH4)₂SO₄. The engineered S. cerevisiae 6525 carrying inuA1 converted pure nonhydrolyzed inulin directly into high concentrations of ethanol.     

Effect of Ethanol,Sulfur Dioxide and Glucose on the Growth of Wine Spoilage Yeasts Using Response Surface Methodology

Response surface methodology (RSM) was used to study the effect of three factors, sulfur dioxide, ethanol and glucose, on the growth of wine spoilage yeast species, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Seventeen central composite rotatable design (CCRD) trials were designed for each test yeast using realistic concentrations of the factors (variables) in premium red wine. Polynomial regression equations were fitted to experimental data points, and the growth inhibitory conditions of these three variables were determined. The overall results showed Sa. ludwigii as the most resistant species growing under high ethanol/free sulfur dioxide concentrations, i.e., 15% (v/v)/20 mg L^-1, 14% (v/v)/32 mg L^-1 and 12.5% (v/v)/40 mg L^-1, whereas other yeasts did not survive under the same levels of ethanol/free sulfur dioxide concentrations. The inhibitory effect of ethanol was primarily observed during longer incubation periods, compared with sulfur dioxide, which showed an immediate effect. In some CCRD trials, Sa. ludwigii and S. cerevisiae showed growth recovery after a short death period under the exposure of 20–32 mg L^-1 sulfur dioxide in the presence of 11% (v/v) or more ethanol. However, Sc. pombe and Z. bailii did not show such growth recovery under similar conditions. Up to 10 g L^-1 of glucose did not prevent cell death under the sulfur dioxide or ethanol stress. This observation demonstrates that the sugar levels commonly used in wine to sweeten the mouthfeel do not increase wine susceptibility to spoilage yeasts, contrary to the anecdotal evidence.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Bioethanol production from Gracilaria verrucosa using Saccharomyces cerevisiae adapted to NaCl or galactose

This study examined the pretreatment, enzymatic saccharification, and fermentation of the red macroalgae Gracilaria verrucosa using adapted saccharomyces cerevisiae to galactose or NaCl for the increase of bioethanol yield. Pretreatment with thermal acid hydrolysis to obtain galactose was carried out with 11.7% (w/v) seaweed slurry and 373 mM H₂SO₄ at 121 °C for 59 min. Glucose was obtained from enzymatic hydrolysis. Enzymatic saccharification was performed with a mixture of 16 U/mL Celluclast 1.5L and Viscozyme L at 45 °C for 48 h. Ethanol fermentation in 11.7% (w/v) seaweed hydrolysate was carried out using Saccharomyces cerevisiae KCTC 1126 adapted or non-adapted to high concentrations of galactose or NaCl. When non-adapted S. cerevisiae KCTC 1126 was used, the ethanol productivity was 0.09 g/(Lh) with an ethanol yield of 0.25. Ethanol productivity of 0.16 and 0.19 g/(Lh) with ethanol yields of 0.43 and 0.48 was obtained using S. cerevisiae KCTC 1126 adapted to high concentrations of galactose and NaCl, respectively. Adaptation of S. cerevisiae KCTC 1126 to galactose or NaCl increased the ethanol yield via adaptive evolution of the yeast.

     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Fermentation of Detoxified Acid-Hydrolyzed Pyrolytic Anhydrosugars into Bioethanol with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 2.399

Pyrolysate obtained from the pyrolysis of waste cotton is a source of fermentable sugars that could be fermented into bioethanol fuel and other chemicals via microbial fermentation. However, pyrolysate is a complex mixture of fermentable and non-fermentable substrates causing inhibition of the microbial growth. The aim of this study was to detoxify the hydrolysate and then ferment it into bio-ethanol fuel in shake flasks and fermenter applying yeast strain Saccharomyces cerevisiae 2.399. Pyrolysate was hydrolyzed to glucose with 0.2 M sulfuric acid, neutralized with Ba(OH)₂ followed by treatment with ethyl acetate and activated carbon to remove fermentation inhibitors. The effect of various fermentation parameters such as inoculum concentration, pH and hydrolysate glucose was evaluated in shake flasks for optimum ethanol fermentation. With respect to inoculum concentration, 20% v/v inoculum i.e. 8.0 × 10⁸–1.2 × 10⁹ cells/mL was the optimum level for producing 8.62 ± 0.33 g/L ethanol at 9 h of fermentation with a maximum yield of 0.46 g ethanol/g glucose. The optimum pH for hydrolysate glucose fermentation was found to be 6.0 that produced 8.57 ± 0.66 g/L ethanol. Maximum ethanol concentration, 14.78 g/L was obtained for 4% hydrolysate glucose concentration after 16 h of fermentation. Scale-up studies in stirred fermenter produced much higher productivity (1.32 g/L/h^–1) compared to shake flask fermentation (0.92 g/L/h^–1). The yield of ethanol reached a maximum of 91% and 89% of the theoretical yield of ethanol in shake flasks and fermenter, respectively. The complex of integrated models of development was applied, that has been successfully tested previously for the mathematical analysis of the fermentation processes.     

Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?

Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml^–1 of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5–5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l^–1 to 14.5 g l^–1 when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42×10^–7 g to 24.0×10^–7 g ethanol CFU^–1 h^–1), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml^–1) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

Optimization of process variables for minimization of byproduct formation during fermentation of blackstrap molasses to ethanol at industrial scale

Aims: To investigate the effect of molasses concentration, initial pH of molasses medium, and inoculum’s size to maximize ethanol and minimize methanol, fusel alcohols, acetic acid and aldehydes in the fermentation mash in industrial fermentors. Methods and Results: Initial studies to optimize temperature, nitrogen source, phosphorous source, sulfur supplement and minerals were performed. The essential nutrients were urea (2 kg in 60 m³), 0·5 l each of commercial phosphoric acid and sulfuric acid (for pH control) added at the inoculum preparation stage only. Yields of ethanol, methanol, fusel alcohols, total acids and aldehydes per 100‐l fermentation broth were monitored. Molasses at 29°Brix (degree of dissolved sugars in water), initial pH 4·5, inoculum size 30% (v/v) and anaerobic fermentation supported maximum ethanol (7·8%) with Y_P/S = 238 l ethanol per tonne molasses (96·5% yield) (8·2% increase in yield), and had significantly lower values of byproducts than those in control experiments. Conclusions: Optimization of process variables resulted in higher ethanol yield (8·2%) and reduced yield of methanol, fusel alcohols, acids and aldehydes. Significance and Impact of the Study: More than 5% substrate is converted into byproducts. Eliminating or reducing their formation can increase ethanol yield by Saccharomyces cerevisiae, decrease the overall cost of fermentation process and improve the quality of ethanol.     

Variations of two pools of glycogen and carbohydrate in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> grown with various ethanol concentrations

Glycogen, a major reservoir of energy in Saccharomyces cerevisiae, is found to be present as soluble and membrane-bound insoluble pools. Yeast cells can store excess glycogen when grown in media with higher concentration of sugar or when subjected to nutritional stress conditions. Saccharomyces cerevisiae NCIM-3300 was grown in media having ethanol concentrations up to 12% (v/v). The effects of externally added ethanol on glycogen and other carbohydrate content of yeast were studied by using alkali digestion process. Fermentative activities of cells grown in the presence of various ethanol concentrations (2–8% v/v) exhibited increase in values of glycogen and other carbohydrate, whereas cells grown with higher concentrations of ethanol (10–12% v/v) exhibited depletion in glycogen and carbohydrate content along with decrease in cell weight. Such inhibitory effect of ethanol was also exhibited in terms of reduction in total cell count of yeast grown in media with 2–16% (v/v) ethanol and 8% (w/v) sugar. These data suggest that, as the plasma membrane is a prime target for ethanol action, membrane-bound insoluble glycogen might play a protective role in combating ethanol stress. Elevated level of cell-surface α-glucans in yeast grown with ethanol, as measured by using amyloglucosidase treatment, confirms the correlation between ethanol and glycogen.     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey

The purpose of this study was to evaluate the important technological and fermentative properties of wine yeast strains previously isolated from different wine producing regions of Turkey. The determination of the following important properties was made: growth at high temperatures; fermentative capability in the presence of high sugar concentration; fermentation rate; hydrogen sulfide production; killer activity; resistance to high ethanol and sulfur dioxide; foam production; and enzymatic profiles. Ten local wine yeast strains belonging to Saccharomyces, and one commercial active dry yeast as a reference strain were evaluated. Fermentation characteristics were evaluated in terms of kinetic parameters, including ethanol yield (Y_P/S), biomass yield (Y_X/S), theoretical ethanol yield (%), specific ethanol production rate (q_p; g/gh), specific glucose uptake rate (q_s; g/gh), and the substrate conversion (%). All tested strains were able to grow at 37 °C and to start fermentation at 30° Brix, and were resistant to high concentrations of sulfur dioxide. 60 % of the strains were weak H₂S producers, while the others produced high levels. Foam production was high, and no strains had killer activity. Six of the tested strains had the ability to grow and ferment at concentrations of 14 % ethanol. Except for one strain, all fermented most of the media sugars at a high rate, producing 11.0–12.4 % (v/v) ethanol. Although all but one strain had suitable characteristics for wine production, they possessed poor activities of glycosidase, esterase and proteinase enzymes of oenological interest. Nine of the ten local yeast strains were selected for their good oenological properties and their suitability as a wine starter culture.     

Production of bioethanol by direct bioconversion of oil-palm industrial effluent in a stirred-tank bioreactor

The purpose of this study was to evaluate the feasibility of producing bioethanol from palm-oil mill effluent generated by the oil-palm industries through direct bioconversion process. The bioethanol production was carried out through the treatment of compatible mixed cultures such as Thrichoderma harzianum, Phanerochaete chrysosporium, Mucor hiemalis, and yeast, Saccharomyces cerevisiae. Simultaneous inoculation of T. harzianum and S. cerevisiae was found to be the mixed culture that yielded the highest ethanol production (4% v/v or 31.6 g/l). Statistical optimization was carried out to determine the operating conditions of the stirred-tank bioreactor for maximum bioethanol production by a two-level fractional factorial design with a single central point. The factors involved were oxygen saturation level (pO₂%), temperature, and pH. A polynomial regression model was developed using the experimental data including the linear, quadratic, and interaction effects. Statistical analysis showed that the maximum ethanol production of 4.6% (v/v) or 36.3 g/l was achieved at a temperature of 32°C, pH of 6, and pO₂ of 30%. The results of the model validation test under the developed optimum process conditions indicated that the maximum production was increased from 4.6% (v/v) to 6.5% (v/v) or 51.3 g/l with 89.1% chemical-oxygen-demand removal.     

Pure culture fermentation of green olives

The method previously developed by us for the pure-culture fermentation of brined cucumbers and other vegetables has been applied successfully to Manzanillo variety olives. Field-run grade fruit was processed first by conventional procedures to remove most of the bitterness. Then the relative abilities of Lactobacillus plantarum, L. brevis, Pediococcus cerevisiae, and Leuconostoc mesenteroides to become established and produce acid in both heat-shocked (74 C for 3 min) and unheated olives, brined at 4.7 to 5.9% NaCl (w/v basis), were evaluated. The heat-shock treatment not only proved effective in ridding the fruit of naturally occurring, interfering, and competitive microbial groups prior to brining and inoculation, but also made the olives highly fermentable with respect to growth and acid production by the introduced culture, particularly L. plantarum. Of the four species used as inocula, L. plantarum was by far the most vigorous in fermentation ability. It consistently produced the highest levels of brine acidity (1.0 to 1.2% calculated as lactic acid) and the lowest pH values (3.8 to 3.9) during the fermentation of heat-shocked olives. Also, L. plantarum completely dominated fermentations when used in two-species (with P. cerevisiae) and three-species (with P. cerevisiae and L. brevis) combinations as inocula. In contrast, when L. plantarum was inoculated into the brines of unheated olives it failed to become properly established; the same was true for the other species tested, but even to a more pronounced degree. L. brevis was the only species used that failed to develop in brines of both heat-shocked and unheated olives. Modification of the curing brine by the addition of lactic acid at the outset, either with or without dextrose, led to a much earlier onset of fermentation with accompanying acid development, as compared to treatments with dextrose alone or nonadditive controls. Reasons for the marked improvement of the fermentability of Manzanillo olives receiving the prebrining heat-shock treatment are discussed.     

Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2

When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl₂, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g⁻¹, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars.     

Ethanol formation by hybrid yeasts

Summary Fusion products between Saccharomyces cerevisiae and Saccharomyces mellis were examined for their fermentative capacity. While there was little difference in the fermentation rate between S. cerevisiae (industrial strain) and the hybrids at low sugar concentrations (4% glucose), at 35% the hybrid strains were far superior (13.6% w.w. vs. 9.0% w.w.). Data on the accumulation of intracellular ethanol seem to indicate that the high fermentation efficiency of the hybrid strains may be due, in addition to their osmotolerance, also to their improved ability to excrete ethanol from their cells.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001