Comparative Performance of Zymomonas mobilis and Saccharomyces cerevisiae in Alcoholic Fermentation of Saccharified Wheat Starch B in a Continuous Dynamic Immobilized Biocatalyst Bioreactor

Glucose derived from an industrial, enzymatically saccharified wheat starch fraction (B starch) was investigated for ethanol production using free and immobilized cells of Saccharomyces cerevisiae and Zymomonas mobilis. Batch cultures of these organisms could successfully ferment this substrate within 24 h giving 7% ethanol by volume and over 96% sugar conversion. Fermentation with S cerevisiae and Z. mobilis immobilized on rotating fiber discs in a continuous dynamic biocatalyst bioreactor (CDIBB) resulted in very rapid and efficient fermentation and concurrent high ethanol productivity. Z. mobilis loaded at 30g/I cell density (based on bioreactor volume, 31) fermented saccharified wheat starch B slurry at 15% solids and having 128g/I glucose, in 0.9 h with 92% sugar utilization; and volumetric productivity based on liquid hold up was 65.5 g/I-h, being threefold higher than that obtained with immobilized yeast cells. Z. mobilis appears to be the preferred organism for the rapid fermentation of glucose rich solutions in the CDIBB. Advantages and the possible application of this bioreactor system to alcoholic fermentation of industrial derived substrates associated with high suspended solids are discussed. Die aus einer industriellen, enzymatisch verzuckerten Weizen-B-Stärke gewonnene Glucose wurde unter Verwendung von freien und immobilisierten Zellen von Saccharomyces cerevisiae und Zymomonas mobilis hinsichtlich der Ethanolgewinnung untersucht. Chargenweise Kulturen dieser Organismen vergoren dieses Substrat erfolgreich 24h; sie ergaben 7 Vol.% Ethanol bei 96% iger Zuckerumwandlung. Die Vergärung mit auf rotierenden Faserplatten immobilisierten S. cerevisiae und Z. mobilis in einem kontinuierlichen, dynamischen Biokatalysator-Bioreaktor (CDIBB) führte zu sehr schneller und wirksamer Gärung und vergleichbar hoher Ethanolproduktivität. Mit einer Zelldichte von 30g/1 (bez. auf das Bioreaktorvolumen, 31) gepackte Z. mobilis vergor eine Weizen-B-Stärkesuspension mit 15% Feststoff und 128g Glucose/1 in 0,9h bei 92% Zuckerverwertung. Die Volumenproduktivität, bezogen auf die Flüssigkeitsrückhaltung, betrug 65,5 g/1-h und war damit dreimal so hoch wie bei immobilisierten Hefezellen. Z. mobilis dürfte der bevorzugte Organismus für die rasche Vergärung von glucosereichen Lösungen im CDIBB sein. Die Vorteile und die mögliche Anwendung dieses Bioreaktorsystems für die alkoholische Vergärung von industriellen Substraten in Verbindung mit hoch suspendierten Feststoffen werden diskutiert.     

Enzymatic digestion of corncobs pretreated with low strength of sulfuric acid for bioethanol production

In this study, the effect and the optimum pretreatment condition of corncobs using low strength of H₂SO₄ were investigated, in which H₂SO₄ was used to improve the enzymatic digestibility of corncobs for saccharification without degradation of sugars released. The optimum pretreatment condition was found to be the addition of 0.5% (vol./vol.) H₂SO₄ and autoclaving at 122 °C for 20 min. Under this condition, the structural integrity of corncob was altered to make cellulose microfibrils more accessible for cellulase enzymes, and the enzymatic digestion of corncobs could be significantly enhanced. A high yield of sugar, 80% (wt./wt.), could be obtained at a low enzyme dosage of 0.024 g enzymes/g cobs, when pretreated. As a result, the ethanol production was obviously improved by the pretreatment, i.e., the ethanol yield of 77% (wt./wt.) was obtained within 36 h in the SSF fermentation using Saccharomyces cerevisiae NBRC2114.     

Comparative Production of Sugarcane Vinegar by Different Immobilization Techniques

Sugarcane juice was converted to ethanol by Saccharomyces cerevisiae producing 8% (v/v) ethanol. This ethanol was used for vinegar production using adsorbed (bagasse, corn cobs and wood shavings) and entrapped (calcium alginate) cells of Acetobacter aceti NRRL 746. All three adsorbed carrier materials were statistically similar for acetic acid production and produced acidity from 5.9 to 6.7% after 28 days of submerged fermentation. By recycling bagasse adsorbed cells, the time of acetic acid fermentation was reduced to 13 days. Semi‐continuous fermentation of bagasse adsorbed cells using a packed bed column further reduced the fermentation time to 80 h.     

Ethanol production from carob pods by Saccharomyces cerevisiae

The production of ethanol from carob pods extract by Saccharomyces cerevisiae in static and shake flask fermentation was investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The external addition of nutrients into the carob pods extract did not improve the production of ethanol. The maximum concentration of ethanol (75 g/l) was obtained at an inoculum amount of 0.3%, a pH of 4.5, 30°C and an initial sugar concentration of 200 g/1. Under the same fermentation conditions both sterilized and non‐sterilized carob pods extract gave the same final ethanol concentration.     

Production of a high‐protein meal and fermentable sugars from defatted soybean meal,a co‐product of the soybean oil industry

The goal of this research was to produce a high‐protein meal by treating defatted soybean meal, a by‐product of soybean oil production, with dilute acid. Treatments were a mild hydrolysis at 80 °C with sulphuric acid at concentrations ranging from 0.5% to 2.0% (w/v) and times varying from 1 to 16 h that were arranged according to a central composite rotatable experimental design. The end products were an enhanced‐protein meal and a carbohydrate concentrate of fermentable and nonfermentable sugars. The highest protein content rise, from 48% to 58%, was for treatments with concentrations of acid ranging between 1.2% and 1.7% and times between 1.0 and 2.6 h. The maximum yield of fermentable sugar was 21.0% d.b. at 2.0% H₂SO₄ and treatments of at least 6 h. The conditions that provide a highest protein and sugar contents were the treatments with concentrations of sulphuric acid ranging from 0.9 to 1.9% H₂SO₄ for 1–4 h.     

Scale‐up batch fermentation of bioethanol production from the dry powder of Jerusalem artichoke (Helianthus tuberosus L.) tubers by recombinant Saccharomyces cerevisiae

This study is a first exploration of industrial bioethanol production from the dry powder of Jerusalem artichoke (Helianthus tuberosus L.) tubers by recombinant S. cerevisiae 6525. Scale‐up fermentation of bioethanol production from the dry powder of Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae 6525 was carried out at the scale of 50 and 500 L agitating fermentors. For the 50 L fermentor, 85.67 g/L of ethanol was obtained within 72 h of fermentation, and the ethanol yield was 90.1%. For the 500 L fermentor, the highest ethanol concentration of 77.00 g/L was achieved at 84 h, and the ethanol yield was 81.01%. These results indicated that a relatively high yield of ethanol could also be obtained from a scaled‐up fermentation of Jerusalem artichoke powder. Thus, it may be feasible to use a Jerusalem artichoke tuber feedstock to carry out ethanol fermentations using the recombinant S. cerevisiae 6525 for industrial production. Copyright © 2016 The Institute of Brewing & Distilling     

Fermented sugarcane and pineapple beverage produced using Saccharomyces cerevisiae and non‐Saccharomyces yeast

The potential of Saccharomyces cerevisiae (strains UFLA CA11 and UFLA FW15) and Pichia caribbica (UFLA CAF733) to produce a fermented sugarcane and pineapple drink was evaluated. Co‐ and pure cultures using different proportions of sugarcane juice and pineapple pulp (80:20, 70:30 and 60:40) were prepared. The sugar concentration of the must was adjusted to 16° Brix and was inoculated with approximately 7 log CFU/mL. After a preliminary test and based on higher concentrations of desirable volatile components, low production of acetic acid, high production of ethanol, and kinetic parameters, P. caribbica was chosen to perform the fermentation in 5 L batches. The fermentation performed with P. caribbica in the proportion of 60:40 showed a yield in ethanol of 0.45 g/g, an ethanol productivity of 1.32 g/L/h and a fermentation efficiency of 88.22%. The maximum ethanol concentration was 79.78 g/L and P. caribbica increased concentrations of desirable volatile compounds, such as 2‐phenyethanol, 2‐methyl‐1‐propanol, 3‐methyl‐1‐butanol, ethyl acetate and phenylethyl acetate. Copyright © 2015 The Institute of Brewing & Distilling     

Production of Ethanol From Maize by Zymomonas mobilis

Maize (Zea mays Ganga-5, hybrid variety) was used as a substrate for ethanol production by batch fermentation using Zymomonas mobilis. 20% solids concentration, 72 h of duration, pH 5.5 and 30°C were found most effective in yielding maximum alcohol and proved optimum for highest fermentation efficiency.     

Factors Affecting Zymomonas mobilis subsp. francensis Growth and Acetaldehyde Production

An experimental plan was designed to determine the incidence of factors encountered during cider production on Zymomonas mobilis subsp. francensis growth and acetaldehyde production. Different factor combinations of pH (3.50 to 4.10), SO₂ addition (0, 50, 100 and 200 mg/L), nitrogen source concentrations (0.5 and 5.0 g/L), polyphenol cider marc extract supplementation (0.25 and 1.00 g/L), temperature (12°C, 18.5°C and 25°C) and inoculation level (10² and 10⁵ CFU/mL) were tested over a 30‐day period at regular time intervals in synthetic medium with ethanol. Viable cell counts and acetaldehyde production were correlated. Individually, and in decreasing significance, the following factors influenced acetaldehyde production: nitrogen source, SO₂ addition, inoculation level, temperature and pH. On the other hand, presence of polyphenol cider marc extract was not significant. A model was determined based on factor interactions. At high contamination levels (10⁵ CFU/mL), conditions leading to a high risk for spoilage were observed at pH values ranging from 3.75 to 4.10, at 0 to 50 mg/L total SO₂ and in the presence of 5 g/L nitrogen source (yeast extract) while temperature did not in fact appear to play a key role. At lower contamination levels (10² CFU/mL), the risk was drastically reduced. The only conditions leading to increased acetaldehyde levels were a high pH 4.1, no added SO₂ and high nitrogen source concentration (5 g/L yeast extract).     

Effect of added sulphur dioxide levels on the fermentation characteristics of strawberry wine

The aim of this study to evaluate the effects of sulphur dioxide (SO₂) on strawberry wine fermentation and on the quality of the final wine product. Major aroma compounds, reducing sugars, ethanol, titratable acid and microflora were analyzed during the fermentation of strawberry wine supplemented with 0–100 mg/L SO₂. As the amount of added SO₂ increased, the consumption of reducing sugars and soluble solids and ethanol production decreased during early fermentation, but increased during late fermentation. During the fermentation process, the concentrations of 2‐phenethanol, isobutanol and isopentanol significantly increased and those of n‐propanol, isoamyl acetate and ethyl lactate decreased with increasing amount of added SO₂. The production of n‐butanol, ethyl acetate and ethyl butyrate was slightly dependent on the amount of added SO₂. Yeast cells were the dominant microbe in the fermenting strawberry pulp, and indigenous bacteria and fungi populations decreased rapidly or disappeared because of their sensitivity to SO₂. It was concluded that 60–80 mg/L SO₂ should be added during strawberry wine fermentations to improve wine quality. Copyright © 2016 The Institute of Brewing & Distilling     

Optimization of fermentation conditions for Chinese bayberry wine by response surface methodology and its qualities

Colour and flavour are both important challenges for bayberry wine production because of a shortage of selected strains. The yeast Saccharomyces cerevisiae strain YF152, separated from a natural Chinese bayberry fermentation mash [alcohol tolerance capacity (18.0%), SO₂ (200 mg/L), sugar content (40%) and pH (2.50)] was employed to brew Chinese bayberry wine. Response surface methodology was used to simultaneously analyse the effects of the fermentation conditions on Chinese bayberry wine. The optimum conditions were found to be a temperature of 26.5 °C, an initial sugar content of 22.0°Brix, an inoculum size of 4.0% and an initial pH of 2.90. Under these conditions, the final alcohol content of the bayberry wine was 13.4%, the anthocyanin content was 77 mg/L and the residual sugar content was 1 g/L. These numbers agreed well with the predicted values. The major characteristic flavour components of the wine were 3‐methyl‐1‐butanol, 2‐phenylethanol, ethyl acetate, 2‐methylbutyl acetate and acetic acid. Copyright © 2016 The Institute of Brewing & Distilling     

Inoculated fermentation of orange juice (Citrus sinensis L.) for production of a citric fruit spirit

The yeast Saccharomyces cerevisiae UFLA CA1174 was evaluated for its potential to produce an orange spirit, as a possible alternative to reduce waste and increase income to citrus farmers. The sugar concentration of the orange juice was adjusted to 16ºBrix and the pH to 4.5. The orange juice was inoculated at approximately 7 log CFU/mL, and the fermentation was performed at room temperature until ºBrix stabilization. The yeast used showed high values for the conversion factors of the substrates into ethanol (Y_p/s 0.50 g/g), the volumetric productivity of ethanol (Q_p 1.78 g/L/h), the biomass productivity (P_x 58.47 g/g) and the fermentation efficiency (E_f 97.83%). The sugars were converted quickly, and a high ethanol concentration (58.13 g/L) was achieved after 24 h of fermentation. The orange wine was distilled in a copper alembic, and the head, heart and tail fractions were collected. The orange spirit produced (heart fraction) had high concentrations of acetaldehyde, ethyl acetate, isoamyl alcohol and 2‐phenylethanol. The results showed that orange juice could be a good substrate for fermentation and distillation, and the sensory analysis performed revealed that the produced beverage had good acceptance by the tasters. Copyright © 2013 The Institute of Brewing & Distilling     

Characterization of glycolytic metabolism and ion transport of Candida albicans

The main energetic pathways, fermentation and respiration, and the general ion transport properties of Candida albicans were studied. Compared to Saccharomyces cerevisiae, we found that in C. albicans: (a) the cell mass yield when grown in YPD was significantly larger; (b) it required longer times to be starved of endogenous substrates; (c) ethanol production was lower but significant; (d) respiration was also lower; (e) it showed a small activity of an alternative oxidase; (f) fermentation and oxidative phosphorylation seemed to compete for both ADP and NADH; and (g) NADH levels were lower. Regarding ion transport and compared to S. cerevisiae: (a) the general mechanism was similar, with a plasma membrane H⁺‐ATPase that generates both a plasma membrane ΔpH and a ΔΨ, the latter being responsible for driving K⁺ inside; (b) its acidification capacity is slightly smaller and less sensitive to activation by high pH; and (c) the presence of K⁺ results in a large activation of both respiration and fermentation, most probably due to the energy required in the process. ADP produced by H⁺‐ATPase stimulation by high pH or the addition of K⁺ at low pH results in the increase of both respiration and fermentation. Copyright © 2012 John Wiley & Sons, Ltd.     

Optimization of Calvatia gigantea mycelia production from distillery wastewater

The focus of wastewater management has evolved from treatment technology into resource recovery, which enables one to minimize contaminants and to generate value‐added products. Calvatia gigantea is used not only as a source of food, but has also been used as a traditional Chinese medicine for thousands of years. In this study, the mycelial production of C. gigantea was studied under submerged fermentation conditions using non‐pretreated distilled wastewater from Chinese liquor production. The fermentation medium composition was optimized using response surface methodology involving a Box–Behnken design. Fermentation conditions were optimized using an orthogonal experimental design. The optimized medium composition was the non‐pretreated distilled wastewater of Chinese liquor supplemented with cornflour at 2.35 g/100 mL, (NH₄)₂SO₄ at 1.11 g/100 mL and CuSO₄ at 0.12 g/100 mL. The optimized fermentation conditions were a rotation speed of 150 rpm, an inoculum size of 10% (v/v), a fermentation temperature of 26 °C and a fermentation time 4.5 days. A maximum mycelial biomass yield of 2.75 g/100 mL was achieved using the optimized medium under the optimized conditions. Results from this study suggest that this is a feasible technology for the mycelial production of C. gigantea using the non‐pretreated distilled wastewater from Chinese liquor production. Copyright © 2015 The Institute of Brewing & Distilling     

Multi‐Cereal Beverage Fermented by Lactobacillus Helveticus and Saccharomyces Cerevisiae

A novel multi‐cereal‐based fermented beverage with suitable aroma, flavor, and pH fermented by lactic acid bacteria and Saccharomyces cerevisiae was developed. Twenty‐seven lactobacilli strains were screened for acid production (pH and titratable acidity) in a mixture of malt, rice, and maize substrates. It was found that Lactobacillus helveticus KLDS1.9204 had the greatest acid production among 27 lactobacilli tested. The fermentation performance of L. helveticus KLDS1.9204 was also assayed and the fermentation parameters were optimized using Plackett–Burman design and steepest ascent method. L. helveticus KLDS1.9204 showed good proteolytic capability, however, the strain could not utilize starch. The optimum substrate consisted of 50% malt (25 g/100 mL), 25% rice (20 g/100 mL), and 25% maize (30 g/100 mL). The inoculum was 5% with a ratio of S. cerevisiae to L. helveticus KLDS1.9204 of 2.5:1. The optimum temperature was 37 °C and the time was 22 h. Lastly, the quality of the multi‐cereal‐based fermented beverage was evaluated. This beverage was light yellow, transparent, and it tasted well with a pleasant acid and a unique flavor of cereals. The beverage was rich in free amino acids and organic acids. The pH and titratable acidity of the beverage were 3.5 and 29.86 °T, respectively. The soluble solids content of the beverage was 6.5 °Brix, and the alcohol content was 0.67%.     

The influence of pre‐fermentative practices on the dominance of inoculated yeast starter under industrial conditions

The influence of pre‐fermentative practices on the growth dynamics of a ‘natural’ starter culture with specific phenotype (H₂S^?) concurrently with wild yeast populations was evaluated under winery conditions. Different clarification procedures and added SO₂ strongly influenced species and cell numbers isolable at the pre‐fermentation stage. Independent treatments of must with sulphite addition or vacuum‐filtering clarification caused a 30‐fold reduction in viable cells. Clarification procedures, enhanced by the selective effect of SO₂ addition, induced the appearance of Saccharomyces cerevisiae ‘wild’ yeasts. Correct application of the inoculum generally guarantees the dominance of fermentation by starter cultures. However, inoculated fermentations using unclarified white and red musts exhibited a consistent presence and persistence of non‐Saccharomyces and/or Saccharomyces ‘wild’ yeasts during fermentation. The extent and composition of the initial wild microflora at the start of fermentation may affect the presence and persistence of wild Saccharomyces and non‐Saccharomyces yeasts during guided fermentations under commercial conditions. The above findings confirm the results of previous works carried out at laboratory‐ or pilot‐scale level. Furthermore, they suggest a clear correlation between the modality of pre‐fermentative practices and the presence and persistence of ‘wild’ yeasts during fermentation. © 2002 Society of Chemical Industry     

Improvement of flavour compound synthesis in a mixed culture system at high temperature by solid‐state fermentation

In order to enhance the synthesis of flavour compounds in solid‐state fermentation (SSF) at a high temperature, Bacillus subtilis XJ‐013 and Saccharomyces cerevisiae Z‐06 were used as a mixed culture with Monascus HQ‐3. The culture temperature was enhanced from 37°C to 56°C by the synergetic effect, and the synthesis of esterase was enhanced from 85.43 U/g to 129.65 U/g in the mixed culture system (over 50% higher than that of the culture using a single strain). This resulted in the synthesis of favourable flavour compounds in the solid‐state fermentation. These results signified that a mixed culture of Monascus and S. cerevisiae was favourable for enzyme production. The mixed culture of Monascus and B. subtilis resulted in a high culture temperature that promoted flavour compound synthesis in the solid‐state fermentation system dramatically. These results present a model to explain the synergetic effects between the fungus and the Bacillus in the solid‐state fermentation. Copyright © 2015 The Institute of Brewing & Distilling     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

INTERACTIVE EFFECTS OF SELECTED NUTRIENTS AND FERMENTATION TEMPERATURE ON H2S PRODUCTION BY WINE STRAINS OF SACCHAROMYCES

ABSTRACT

Metabolic interactions between yeast assimilable nitrogen (YAN), biotin, pantothenic acid, and fermentation temperature that affect H₂S production by wine yeast during alcoholic fermentation were examined. Strains of Saccharomyces cerevisiae (UCD 522 and EC1118) were inoculated into a synthetic grape juice medium with H₂S evolution monitored under fermentative conditions. While a number of interactions affected the evolution of H₂S, YAN as a factor by itself was found to be not significant (P > 0.05) for both yeasts examined. Maximal cumulative H₂S production for strain UCD 522 occurred in media fermented at 30C with 60 mg/L YAN, 10 µg/L biotin, and 50 µg/L pantothenic acid while minimum production was observed with 250 mg/L YAN and 250 µg/L pantothenate. Similarly, strain EC1118 produced the most H₂S at 30C, but with 250 mg/L YAN, 0.5 µg/L biotin, and 50 µg/L pantothenic acid and the least in media that contained 250 mg/L YAN and 250 µg/L pantothenic acid.

PRACTICAL APPLICATIONS

“Reduced” off‐odors of wines, primarily associated with sulfur‐containing molecules such as H₂S, continue to be a difficulty facing winemakers worldwide. One strategy for wineries to limit these problems is to add yeast nutrients prior to fermentation, most commonly, nitrogen‐containing compounds such as diammonium phosphate. However, nitrogen deficiency is not always the sole cause for these problems. Rather, the current research suggests the need to consider factors other than nitrogen including availability of biotin and pantothenic acid as well as fermentation temperature in order to minimize these off‐odors.     

Determination of biotin in low‐temperature fish‐meal processed from different species by means of a microbiological method using Lactobacillus plantarum as test organism

Determination of biotin content in 10 fish‐meals produced from different species was performed by a microbiological assay using Lactobacillus plantarum as test organism. The contents of biotin in the fish‐meal samples ranged between 0.36 and 0.80 mg kg⁻¹. Most of the fish‐meals contained approximately 0.4 mg biotin kg⁻¹. The highest value was found in meal processed from sand eel (Ammodytes marinus). The precision of the assay showed a 2.7% relative standard deviation. Recovery using addition of biotin to the fish‐meal ranged between 93 and 95%. Testing of extraction procedure suggested that hydrolysis in 1.5 M H₂SO₄ at 121 °C for 3 h is sufficient for biotin to be liberated from proteins and amino acids. © 1999 Society of Chemical Industry