Occurrence of hydrogen sulfide in wine and in fermentation: influence of yeast strain and supplementation of yeast available nitrogen

Hydrogen sulfide (H₂S) is a powerful aroma compound largely produced by yeast during fermentation. Its occurrence in wines and other fermented beverages has been associated with off-odors described as rotten egg and/or sewage. While the formation of hydrogen sulfide (H₂S) during fermentation has been extensively studied, it is the final H₂S content of wine that is actually linked to potential off-odors. Nevertheless, factors determining final H₂S content of wine have received little attention, and it is commonly assumed that high H₂S-forming fermentations will result in high final concentrations of H₂S. However, a clear relationship has never been established. In this report, we investigated the contribution of yeast strain and nitrogen addition to H₂S formation during fermentation and its consequent occurrence the resulting wines. Five commercial Saccharomyces cerevisiae wine yeast strains were used to ferment a Chardonnay juice containing 110 mg/l of YAN (yeast assimilable nitrogen), supplemented with di-ammonium phosphate (DAP) to increase YAN concentration to moderate (260 mg/l) and high (410 mg/l) levels. In contrast to the widely reported decrease in H₂S production in response to DAP addition, a non-linear relationship was found such that moderate DAP supplementation resulted in a remarkable increase in H₂S formation by each of the five wine yeasts. H₂S content of the finished wine was affected by yeast strain, YAN, and fermentation vigor. However, we did not observe a correlation between concentration of H₂S in the finished wines and H₂S produced during fermentation, with low-forming fermentations often having relatively high final H₂S and vice versa. Management of H₂S in wine through nitrogen supplementation requires knowledge of initial YAN and yeast H₂S characteristics.     

The production of hydrogen sulphide and other aroma compounds by wine strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in synthetic media with different nitrogen concentrations

The aim of this study was to evaluate the combined effect of initial nitrogen content on the production of hydrogen sulphide and other volatile compounds during alcoholic fermentation. For that propose, three commercial wine strains of Saccharomyces cerevisiae were used to ferment synthetic grape juice media under different nitrogen concentrations. H₂S was measured throughout fermentations and other aroma compounds were analyzed at the end of the experiments. The trigger levels at which an inverse relationship between the initial nitrogen present in media and total H₂S production varied among the three strains tested. For UCD522 and PYCC4072, the highest H₂S levels were produced in media with 267 mg N l⁻¹ of initial nitrogen, whereas the lowest levels were detected with nitrogen limitation/starvation conditions (66 mg N l⁻¹). Moreover, 21 other aroma compounds belonging to different chemical classes were identified and quantified by solid phase micro-extraction (SPME) coupled to gas chromatography–mass spectrometry (GC–MS). The initial nitrogen concentration more than yeast strain had a decisive effect on the final aroma composition, suggesting that modulation of nutrients emerges as a useful tool for producing desired flavour and odour compounds.     

Enhanced volatile phenols in wine fermented with Saccharomyces cerevisiae and spoiled with Pichia guilliermondii and Dekkera bruxellensis

Aims: To investigate whether the presence of Pichia guilliermondii impacts on the production of volatile phenols from mixed wine fermentations with Dekkera bruxellensis and Saccharomyces cerevisiae. Methods and Results: Four inoculation strategies were performed in small‐scale fermentations involving P. guilliermondii, D. bruxellensis and S. cerevisiae using Syrah grape juice supplemented with 100 mg l^?1 of p‐coumaric acid. High pressure liquid chromatography was used for the quantification or volatile phenols. Significant high levels of 4‐ethylphenol and 4‐ethylguaicol (720 and 545 μg l^?1, respectively), as well as the highest levels of 4‐vinylphenol (>4500 μg l^?1), were observed when P. guilliermondii species was inoculated from the beginning of the fermentation. Conclusions: The metabolic interaction occurring between the high vinylphenol producer species P. guilliermondii and D. bruxellensis exhibiting a high vinylphenol reductase activity resulted in an increased production of volatile phenols in wine. Significance and Impact of the Study: Pichia guilliermondii must be considered a very important spoilage yeast in the wine industry capable of producing large amounts of volatile phenols.     

MET2 affects production of hydrogen sulfide during wine fermentation

The production of hydrogen sulfide (H₂S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H₂S. A dominant R₃₁₀G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 _R310G allele confers reduced H₂S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H₂S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H₂S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H₂S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H₂S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H₂S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.     

Improvement of alcoholic fermentation by calcium ions under enological conditions involves the increment of plasma membrane H<Superscript>+</Superscript>-ATPase activity

The effect of Ca²⁺ on alcoholic fermentation and plasma membrane H⁺-ATPase activity of wine yeast under enological conditions were investigated in this study. The results showed that fermentation rate, cell growth and ethanol production were improved by 0.5 and 1.5 mM Ca²⁺ supplementation, which correlated well with the increment of ATPase activity and protein levels. Considering the important role of ATPase in the tolerance of yeast to ethanol, the improvement could be, at least partially, attributed to the increment of ATPase activity. No activation of ATPase by Ca²⁺ was observed in the early phase of fermentation and the increment of activity was only observed when ethanol concentration exceeded 6.5%. Therefore, the enhancement of ATPase activity by Ca²⁺ was ascribed to alleviating the inhibition of ATPase activity by ethanol through protection of membrane structure. Our results suggest that, besides maintenance of cell membrane structure, the increment of plasma membrane ATPase activity was also responsible for the improvement of alcoholic fermentation by Ca²⁺ supplementation.     

Effects of acetic acid, ethanol, and SO2 on the removal of volatile acidity from acidic wines by two Saccharomyces cerevisiae commercial strains

Herein, we report the influence of different combinations of initial concentration of acetic acid and ethanol on the removal of acetic acid from acidic wines by two commercial Saccharomyces cerevisiae strains S26 and S29. Both strains reduced the volatile acidity of an acidic wine (1.0 g l⁻¹ acetic acid and 11% (v/v) ethanol) by 78% and 48%, respectively. Acetic acid removal by strains S26 and S29 was associated with a decrease in ethanol concentration of 0.7 and 1.2% (v/v), respectively. Strain S26 revealed better removal efficiency due to its higher tolerance to stress factors imposed by acidic wines. Sulfur dioxide (SO₂) in the concentration range 95–170 mg l⁻¹ inhibits the ability of both strains to reduce the volatile acidity of the acidic wine used under our experimental conditions. Therefore, deacidification should be carried out either in wines stabilized by filtration or in wines with SO₂ concentrations up to 70 mg l⁻¹. Deacidification of wines with the better performing strain S26 was associated with changes in the concentration of volatile compounds. The most pronounced increase was observed for isoamyl acetate (banana) and ethyl hexanoate (apple, pineapple), with an 18- and 25-fold increment, respectively, to values above the detection threshold. The acetaldehyde concentration of the deacidified wine was 2.3 times higher, and may have a detrimental effect on the wine aroma. Moreover, deacidification led to increased fatty acids concentration, but still within the range of values described for spontaneous fermentations, and with apparently no negative impact on the organoleptical properties.     

Hydrogen sulphide production by Zymomonas and its elimination in a mutant strain

Strains of Zymomonas mobilis grown in media containing either glucose or sucrose were assessed for the production of hydrogen sulphide (H₂S). In a liquid medium with low glucose concentration (20 g l^?1) only a proportion of the strains tested formed H₂S, but in medium containing a higher glucose concentration (100 g l^?1) all the strains tested produced H₂S. Four Z. mobilis strains were assayed quantitatively for H₂S production and strain ZM4 was found to produce the most H₂S in glucose medium. The amount of yeast extract and glucose, and the type of sugar used in the medium affected the amount of H₂S formed by strain ZM4. A mutant, designated ZM4701, of strain ZM4 was isolated which did not produce any detectable H₂S in liquid medium containing yeast extract plus either glucose or sucrose. The nutritional requirements of ZM4701 were investigated.     

Evaluation of the acetaldehyde production and degradation potential of 26 enological <Emphasis Type="Italic">Saccharomyces</Emphasis> and non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeast strains in a resting cell model system

Acetaldehyde is relevant for wine aroma, wine color, and microbiological stability. Yeast are known to play a crucial role in production and utilization of acetaldehyde during fermentations but comparative quantitative data are scarce. This research evaluated the acetaldehyde metabolism of 26 yeast strains, including commercial Saccharomyces and non-Saccharomyces, in a reproducible resting cell model system. Acetaldehyde kinetics and peak values were highly genus, species, and strain dependent. Peak acetaldehyde values varied from 2.2 to 189.4 mg l⁻¹ and correlated well (r ² = 0.92) with the acetaldehyde production yield coefficients that ranged from 0.4 to 42 mg acetaldehyde per g of glucose in absence of SO₂. S. pombe showed the highest acetaldehyde production yield coefficients and peak values. All other non-Saccharomyces species produced significantly less acetaldehyde than the S. cerevisiae strains and were less affected by SO₂ additions. All yeast strains could degrade acetaldehyde as sole substrate, but the acetaldehyde degradation rates did not correlate with acetaldehyde peak values or acetaldehyde production yield coefficients in incubations with glucose as sole substrate.     

Acid hydrolysis of sugarcane bagasse for lactic acid production

In order to use sugarcane bagasse as a substrate for lactic acid production, optimum conditions for acid hydrolysis of the bagasse were investigated. After lignin extraction, the conditions were varied in terms of hydrochloric (HCl) or sulfuric (H₂SO₄) concentration (0.5–5%, v/v), reaction time (1–5 h) and incubation temperature (90–120 °C). The maximum catalytic efficiency (E) was 10.85 under the conditions of 0.5% of HCl at 100 °C for 5 h, which the main components (in g l⁻¹) in the hydrolysate were glucose, 1.50; xylose, 22.59; arabinose, 1.29; acetic acid, 0.15 and furfural, 1.19. To increase yield of lactic acid production from the hydrolysate by Lactococcus lactis IO-1, the hydrolysate was detoxified through amberlite and supplemented with 7 g l⁻¹ of xylose and 7 g l⁻¹ of yeast extract. The main products (in g l⁻¹) of the fermentation were lactic acid, 10.85; acetic acid, 7.87; formic acid, 6.04 and ethanol, 5.24.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Batch and continuous fermentative production of hydrogen with anaerobic sludge entrapped in a composite polymeric matrix

Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrose-based synthetic wastewater was converted to H₂ using batch and continuous cultures. A novel composite polymeric material comprising polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H₂ production. Using the PMMA immobilized cells, the favorable conditions for batch H₂ fermentation were 35 °C, pH 6.0, and an 20 g COD l⁻¹ of sucrose, giving a H₂ production rate of 238 ml h⁻¹ l⁻¹ and a H₂ yield of 2.25 mol H₂ mol sucrose⁻¹. Under these optimal conditions, continuous H₂ fermentation was conducted at a hydraulic retention time (HRT) of 4–8 h, giving the best H₂-producing rate of 1.8 l h⁻¹ l⁻¹ (over seven-fold of the best batch result) at a HRT of 6 h and a H₂ yield of 2.0 mol H₂ mol sucrose⁻¹. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H₂ and CO₂. The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMA-immobilized-cell system developed in this work seems to be a promising H₂-producing process due to the high stability in continuous operations and the capability of achieving a competitively high H₂ production rate under a relatively low organic loading rate.     

Utilization of dry distiller’s grain and solubles as nutrient supplement in the simultaneous saccharification and ethanol fermentation at high solids loading of corn stover

Dry distiller’s grain and solubles (DDGS) is a major by-product of corn-based ethanol production and is usually used as animal feed. Here, it was added to the simultaneous saccharification and ethanol fermentation (SSF) carried out at high solids loading of steam explosion pretreated corn stover using a mutant strain Saccharomyces cerevisiae DQ1. The performance of SSF process with DDGS was comparable to those using the expensive yeast extract supplementation. With 30% (w/w) solids plus the addition of cellulase and 1 g DDGS l⁻¹, the final ethanol reached 55 g l⁻¹ (7% v/v). The results indicated that the expensive supplement of yeast extract could be replaced by DDGS.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway

Aims: An evolution‐based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically, molybdate and chromate resistance was used as selectable phenotype to select sulfate permease–deficient variants that unable to produce sulfites and hydrogen sulfide (H₂S). Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the conjugation of gametes. Then, the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty‐four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfite production (SO₂ <10 mg l^?1) and with an impaired H₂S production in grape must without added sulfites were selected. Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly, resistance to toxic analogues of sulfate allowed us to detect strains that unable to assimilate sulfates. Significance and Impact of the Study: This strategy that combines the sexual recombination of spores and application of a specific selective pressure provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H₂S.     

Optimization of fermentation conditions for production of anti-TMV extracellular ribonuclease by Bacillus cereus using response surface methodology

Bacillus cereus ZH14 was previously found to produce a new type of antiviral ribonuclease, which was secreted into medium and active against tobacco mosaic virus. In order to enhance the ribonuclease production, in this study the optimization of culture conditions using response surface methodology was done. The fermentation variables including culture temperature, initial pH, inoculum size, sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃ were considered for selection of significant ones by using the Plackett–Burman design, and four significant variables (sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃) were further optimized by a 2⁴ factorial central composite design. The optimal combination of the medium constituents for maximum ribonuclease production was determined as 8.50 g/l sucrose, 9.30 g/l yeast extract, 2.00 g/l MgSO₄·7H₂O, and 0.62 g/l KNO₃. The enzyme activity was increased by 60%. This study will be helpful to the future commercial development of the new bacteria-based antiviral ribonuclease fermentation process.     

Studies on the kinetic parameters for alcoholic fermentation by flocculent Saccharomyces cerevisiae strains and non-hydrogen sulfide-producing strains

Summary The growing demand for high quality products and the immense export potential that cacha?a represents, demonstrated especially during the past few years, have clearly indicated the necessity of establishing well-defined standards of quality, as well as effective means of controlling the process of production of this beverage. The objective of this study was the selection of S. cerevisiae yeast strains and the investigation of their influence on the kinetic parameters of fermentation. Ninety strains of S. cerevisiae isolated from distilleries of the state of Minas Gerais were evaluated with respect to the following parameters: flocculation capacity, production of H₂S and kinetic parameters of fermentation. The UFMGA 905 strain was used as a reference because it presented desirable characteristics for the production of cacha?a. Five strains presented high specific sedimentation velocities (SSV), indicating a high flocculation capacity, and two did not produce H₂S. The strains presented significant statistical differences for fermentation parameters: yield of ethanol; efficiency of substrate conversion to ethanol; ratio of substrate conversion to ethanol (Y _p/s), to cells (Y _x/s), to organic acids (Y _ac/s), and to glycerol (Y _g/s); and productivity. In general, the strains presented a good fermentative potential, with ethanol yields varying from 74.7 to 82.1% and an efficiency of 76.1–84.4%. All strains presented high productivities (4.6–6.6 g l⁻¹ h⁻¹), indicating that this parameter can be used in the selection of strains for the production of cacha?a.     

Succinic acid production from sugarcane bagasse hemicellulose hydrolysate by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis>

Succinic acid, a four-carbon diacid, has been the focus of many research projects aimed at developing more economically viable methods of fermenting sugar-containing natural materials. Succinic acid fermentation processes also consume CO₂, thereby potentially contributing to reductions in CO₂ emissions. Succinic acid could also become a commodity used as an intermediate in the chemical synthesis and manufacture of synthetic resins and biodegradable polymers. Much attention has been given recently to the use of microorganisms to produce succinic acid as an alternative to chemical synthesis. We have attempted to maximize succinic acid production by Actinobacillus succinogenes using an experimental design methodology for optimizing the concentrations of the medium components. The first experiment consisted of a 2⁴⁻¹ fractional factorial design, and the second entailed a Central Composite Rotational Design so as to achieve optimal conditions. The optimal concentrations of nutrients predicted by the model were: NaHCO₃, 10.0 g l⁻¹; MgSO₄, 3.0 g l⁻¹; yeast extract, 2.0 g l⁻¹; KH₂PO₄. 5.0 g l⁻¹; these were experimentally validated. Under the best conversion conditions, as determined by statistical analysis, the production of succinic acid was carried out in an instrumented bioreactor using sugarcane bagasse hemicellulose hydrolysate, yielding a concentration of 22.5 g l⁻¹.