Fermentation — Open Access Journal

Volatility of the petroleum market has renewed research into butanol as an alternate fuel. In order to increase the selectivity for butanol during glycerol fermentation with Clostridium pasteurianum, butyric acid can be added to the medium. In this manuscript, different methods of extracellular butyric acid addition are explored, as well as self-generation of butyric acid fermented from sugars in a co-substrate strategy. Molasses was used as an inexpensive sugar substrate, and the optimal molasses to glycerol ratio was found to allow the butyric acid to be taken back up into the cells and increase the productivity of butanol from all carbon sources. When butyric acid is added directly into the media, there was no significant difference between chemically pure butyric acid, or butyric acid rich supernatant from a separate fermentation. When low concentrations of butyric acid (1 or 2 g/L) are added to the initial media, an inhibitory effect is observed, with no influence on the butanol selectivity. However, when added later to the fermentation, over 1 g/L butyric acid is taken into the cells and increased the relative carbon yield from 0.449 to 0.519 mols carbon in product/mols carbon in substrate. An optimized dual substrate fermentation strategy in a pH-controlled reactor resulted in the relative carbon yield rising from 0.439 when grown on solely glycerol, to 0.480 mols C product/mols C substrate with the dual substrate strategy. An additional benefit is the utilization of a novel source of sugars to produce butanol from C. pasteurianum. The addition of butyric acid, regardless of how it is generated, under the proper conditions can allow for increased selectivity for butanol from all substrates. Full article

Hanseniaspora vineae is an apiculate yeast that plays a significant role at the beginning of fermentation, and it has been studied for its application in the improvement of the aromatic profile of commercial wines. This work evaluates the use of H. vineae in alcoholic fermentation compared to Saccharomyces cerevisiae and in ageing on the lees process (AOL) compared to Saccharomyces and non-Saccharomyces yeasts. The results indicated that there were not significant differences in basic oenological parameters. H. vineae completed the fermentation until 11.9% v/v of ethanol and with a residual sugars content of less than 2 g/L. Different aroma profiles were obtained in the wines, with esters concentration around 90 mg/L in H. vineae wines. Regarding the AOL assay, the hydroalcoholic solutions aged with H. vineae lees showed significantly higher absorbance values at 260 (nucleic acids) and 280 nm (proteins) compared to the other strains. However, non-significant differences were found in the polysaccharide content at the end of the ageing process were found compared to the other yeast species, with the exception of Schizosaccharomyces pombe that released around 23.5 mg/L of polysaccharides in hydroalcoholic solution. The use of H. vineae by the wineries may be a viable method in fermentation and AOL to improve the quality of white wines. Full article

Wine spoilage yeasts are one of the main issues in the winemaking industry, and the control of the Brettanomyces and Pichia genus is an important goal to reduce economic loses from undesired aromatic profiles. Previous studies have demonstrated that Candida intermedia LAMAP1790 produces antimicrobial peptides of molecular mass under 10 kDa with fungicide activity against Brettanomyces bruxellensis, without affecting the yeast Saccharomyces cerevisiae. So far, it has not been determined whether these peptides show biocontroller effect in this yeast or other spoilage yeasts, such as Pichia guilliermondii. In this work, we determined that the exposure of B. bruxellensis to the low-mass peptides contained in the culture supernatant of C. intermedia LAMAP1790 produces a continuous rise of reactive oxygen species (ROS) in this yeast, without presenting a significant effect on membrane damage. These observations can give an approach to the antifungal mechanism. In addition, we described a fungicide activity of these peptides fraction against two strains of P. guilliermondii in a laboratory medium. However, carrying out assays on synthetic must, peptides must show an effect on the growth of B. bruxellensis. Moreover, these results can be considered as a start to develop new strategies for the biocontrol of spoilage yeast. Full article

Lactic acid bacteria (LAB) are fastidious microorganisms that have specific nutritional requirements. The de Man, Rogosa, and Sharpe (MRS) is an expensive standard growth medium for LAB to produce lactic acid, and the industry is always looking for an alternative low-cost medium. The date palm (Phoenix dactylifera L.) is naturally full of essential nutrients that lead to stimulate or promote the growth of Lactobacillus spp. The date fruit industries generate a large amount of unwanted date by-product. Thus, the objective of this study was to examine the effect of different nitrogen sources on the growth of Lactobacillus reuteri grown in a date base medium. In this study, date palm fruit was pressed, and the fiber was blended with distilled water, centrifuged, and the supernatant was autoclaved to obtain date palm extract (DPE). The date palm medium (DPM) was formed by mixing the DPE with buffer solution. The DPM was then supplemented with different concentrations of different nitrogen sources. Lactobacilli MRS was used as a standard growth medium. Three different L. reuteri strains were individually inoculated into batches of MRS and DPMs at an initial inoculum 2.5 Log CFU/mL, and then incubated at 37 °C for 18 h. Bacterial growth was monitored by measuring the optical density readings (O.D 610 nm) for up to 18 h. At the end of the incubation period, final populations of each individual strain were verified by enumeration of the MRS agar. Our results showed that the bacterial population in DPM (control; without nitrogen), reached 3.55 ± 0.5 Log CFU/mL. However, the bacterial populations that reached 7.03 ± 0.1 Log CFU/mL in the DPM medium were supplemented with 0.8% phytone peptone, compared to the MRS 7.90 ± 0.24 Log CFU/mL. Our findings thus suggest that date by-products could be used as a low-cost alternative for the LAB growth medium. Full article

The production of wines with peculiar analytical and sensorial profiles, together with the microbiological control of the winemaking process, has always been one of the main objectives of the wine industry. In this perspective, the use of oenological starters containing non-Saccharomyces yeasts can represent a valid tool for achieving these objectives. Here we present the results of seven pilot scale fermentations, each of which was inoculated with a different non-Saccharomyces yeast strain and after three days with a commercial Saccharomyces cerevisiae starter. The fermentations were carried out in double on 70 L of Sangiovese grape must, the most widely planted red grape variety in Italy and particularly in Tuscany, where it is utilized for the production of more than 80% of red wines. Fermentations were monitored by assessing both the development of the microbial population and the consumption of sugars at the different sampling times. The impact of the different starters was assessed after stabilization through the evaluation of the standard analytical composition of the resulting wines, also taking into account polysaccharides and volatile compounds. Moreover, quantitative descriptive sensory analyses were carried out. Compared to the control wines obtained by inoculating the S. cerevisiae starter strain, those inoculated with non-Saccharomyces/Saccharomyces mixed starters presented a significant differentiation in the chemical-analytical composition. Moreover, sensory analysis revealed differences among wines mainly for intensity of color, astringency, and dryness mouthfeel perception. Full article

Uninoculated wine fermentations are conducted by a consortium of wine yeast and bacteria that establish themselves either from the grape surface or from the winery environment. Of the additives that are commonly used by winemakers, sulphur dioxide (SO₂) represents the main antimicrobial preservative and its use can have drastic effects on the microbial composition of the fermentation. To investigate the effect of SO₂ on the resident yeast community of uninoculated ferments, Chardonnay grape juice from 2018 and 2019 was treated with a variety of SO₂ concentrations ranging up to 100 mg/L and was then allowed to undergo fermentation, with the yeast community structure being assessed via high-throughput meta-barcoding (phylotyping). While the addition of SO₂ was shown to select against the presence of many species of non-Saccharomyces yeasts, there was a clear and increasing selection for the species Hanseniaspora osmophila as concentrations of SO₂ rose above 40 mg/L in fermentations from both vintages. Chemical analysis of the wines resulting from these treatments showed significant increases in acetate esters, and specifically the desirable aroma compound 2-phenylethyl acetate, that accompanied the increase in abundance of H. osmophila. The ability to modulate the yeast community structure of an uninoculated ferment and the resulting chemical composition of the final wine, as demonstrated in this study, represents an important tool for winemakers to begin to be able to influence the organoleptic profile of uninoculated wines. Full article

The fermentation of synthesis gas, or syngas, by acetogenic bacteria can help in transitioning from a fossil-fuel-based to a renewable bioeconomy. The main fermentation products of Clostridium ljungdahlii, one of such microorganisms, are acetate and ethanol. A sensitive, robust and reproducible system was established for C. ljungdahlii syngas fermentation, and several process parameters and medium components (pH, gas flow, cysteine and yeast extract) were investigated to assess its impact on the fermentation outcomes, as well as real time gas consumption. Moreover, a closed carbon balance could be achieved with the data obtained. This system is a valuable tool to detect changes in the behavior of the culture. It can be applied for the screening of strains, gas compositions or media components, for a better understanding of the physiology and metabolic regulation of acetogenic bacteria. Here, it was shown that neither yeast extract nor cysteine was a limiting factor for cell growth since their supplementation did not have a noticeable impact on product formation or overall gas consumption. By combining the lowering of both the pH and the gas flow after 24 h, the highest ethanol to acetate ratio was achieved, but with the caveat of lower productivity. Full article

Over the last decades, the average alcohol content of wine has increased due to climate change and consumer preferences for particular wine styles that resulted in increased grape sugar levels at harvest. Therefore, alcohol reduction is a current challenge in the winemaking industry. Among several strategies under study, the use of non-conventional yeasts in combination with Saccharomyces cerevisiae plays an important role for lowering ethanol production in wines nowadays. In the present work, 33 native non-Saccharomyces strains were assayed in sequential culture with a S. cerevisiae wine strain to determine their potential for reducing the alcohol content in Malvar white wines. Four of the non-Saccharomyces strains (Wickerhamomyces anomalus 21A-5C, Meyerozyma guilliermondii CLI 1217, and two Metschnikowia pulcherrima (CLI 68 and CLI 460)) studied in sequential combination with S. cerevisiae CLI 889 were best able to produce dry wines with decreased alcohol proportion in comparison with one that was inoculated only with S. cerevisiae. These sequential fermentations produced wines with between 0.8% (v/v) and 1.3% (v/v) lower ethanol concentrations in Malvar wines, showing significant differences compared with the control. In addition, these combinations provided favorable oenological characteristics to wines such as high glycerol proportion, volatile higher alcohols, and esters with fruity and sweet character. Full article

Pectinases are the group of enzymes that catalyze the degradation of pectic substances. It has wide applications in food industries for the production and clarification of wines and juices. The aim of this study was to isolate, screen and characterize pectinase from fungi isolated from various soil samples and evaluate its application in juice clarification. Fungal strains were isolated and screened primarily using 1% citruspectin incorporated potato dextrose agar (PDA) and secondarily using pectinase screening agar medium (PSAM) for pectinolytic organisms. The enzyme was produced by submerged state fermentation and assayed using the dinitro salicylic acid (DNS) method. From 20 different soil samples, 55 fungal isolates were screened primarily and, among them, only 14 isolates were subjected for secondary screening. Out of 14, only four strains showed the highest pectinolytic activity. Among four strains, Aspergillus spp. Gm showed the highest enzyme production at a 48-h incubation period, 1% substrate concentration, and 30 °C temperature. The thermal stability assessment resulted that the activity of pectinase enzyme declines by 50% within 10 min of heating at 60 °C. The optimum temperature, pH, and substrate concentration for the activity of enzyme was 30 °C (75.4 U/mL), 5.8 (72.3 U/mL), and 0.5% (112.0 U/mL), respectively. Furthermore, the yield of the orange juice, the total soluble solid (TSS), and clarity (% transmittance) was increased as the concentration of the pectinase increased, indicating its potential use in juice processing. Overall, the strain Aspergillus spp. Gm was identified as a potent strain for pectinase production in commercial scale. Full article

Worldwide, there are various feedstocks such as straws, corn stover, sugarcane bagasse, sweet sorghum bagasse (SSB), grasses, leaves, whey permeate, household organic waste, and food waste (FW) that can be converted to valuable biofuels such as butanol. For the present studies, an economic analysis was performed to compare butanol production from three feedstocks (SSB; FW; and yellow top presscake, YTP or YT) using a standard process and an advanced integrated process design. The total plant capacity was set at 170,000–171,000 metric tons of total acetone butanol ethanol (ABE) per year (99,300 tons of just butanol per year). Butanol production from SSB typically requires pretreatment, separate hydrolysis, fermentation, and product recovery (SHFR). An advanced process was developed in which the last three steps were combined into a single unit operation for simultaneous saccharification, fermentation, and recovery (SSFR). For the SHFR and SSFR plants, the total capital investments were estimated as $213.72 × 10⁶ and $198.16 × 10⁶, respectively. It was further estimated that the minimum butanol selling price (using SSB as a feedstock) for the two processes were $1.14/kg and $1.05/kg. Therefore, SSFR lowered the production cost markedly compared to that of the base case. Butanol made using FW had an estimated minimum selling price of only $0.42/kg. This low selling price is because the FW to butanol process does not require pretreatment, hydrolysis, and cellulolytic enzymes. For this plant, the total capital investment was projected to be $107.26 × 10⁶. The butanol selling price using YTP as a feedstock was at $0.73/kg and $0.79/kg with total capital investments for SSFR and SHFR of $122.58 × 10⁶ and $132.21 × 10⁶, respectively. In the Results and Discussion section, the availability of different feedstocks in various countries such as Brazil, the European Union, New Zealand, Denmark, and the United States are discussed. Additionally, the use of various microbial strains and product recovery technologies are also discussed. Full article

The fermentation industry is known to be very conservative, relying on traditional yeast management. Yet, in the modern fast-paced world, change comes about in facets such as climate change altering the quality and quantity of harvests, changes due to government regulations e.g., the use of pesticides or SO₂, the need to become more sustainable, and of course by changes in consumer preferences. As a silent companion of the fermentation industry, the wine yeast Saccharomyces cerevisiae has followed mankind through millennia, changing from a Kulturfolger, into a domesticated species for the production of bread, beer, and wine and further on into a platform strain for the production of biofuels, enzymes, flavors, or pharmaceuticals. This success story is based on the ‘awesome power of yeast genetics’. Central to this is the very efficient homologous recombination (HR) machinery of S. cerevisiae that allows highly-specific genome edits. This microsurgery tool is so reliable that yeast has put a generally recognized as safe (GRAS) label onto itself and entrusted to itself the life-changing decision of mating type-switching. Later, yeast became its own genome editor, interpreted as domestication events, to adapt to harsh fermentation conditions. In biotechnology, yeast HR has been used with tremendous success over the last 40 years. Here we discuss several types of yeast genome edits then focus on HR and its inherent potential for evolving novel wine yeast strains and styles relevant for changing markets. Full article

Increasing beer quality demands from consumers have put pressure on brewers to target specific steps within the beer-making process to modify beer styles and quality traits. However, this demands more robust methodologies to assess the final aroma profiles and physicochemical characteristics of beers. This research shows the construction of artificial intelligence (AI) models based on aroma profiles, chemometrics, and chemical fingerprinting using near-infrared spectroscopy (NIR) obtained from 20 commercial beers used as targets. Results showed that machine learning models obtained using NIR from beers as inputs were accurate and robust in the prediction of six important aromas for beer (Model 1; R = 0.91; b = 0.87) and chemometrics (Model 2; R = 0.93; b = 0.90). Additionally, two more accurate models were obtained from robotics (RoboBEER) to obtain the same aroma profiles (Model 3; R = 0.99; b = 1.00) and chemometrics (Model 4; R = 0.98; b = 1.00). Low-cost robotics and sensors coupled with computer vision and machine learning modeling could help brewers in the decision-making process to target specific consumer preferences and to secure higher consumer demands. Full article

The management of the alcoholic fermentation (AF) in wine is crucial to shaping product quality. Numerous variables (e.g., grape varieties, yeast species/strains, technological parameters) can affect the performances of this fermentative bioprocess. The fact that these variables are often interdependent, with a high degree of interaction, leads to a huge ‘oenological space’ associated with AF that scientists and professionals have explored to obtain the desired quality standards in wine and to promote innovation. This challenge explains the high interest in approaches tested to monitor this bioprocess including those using volatile organic compounds (VOCs) as target molecules. Among direct injection mass spectrometry approaches, no study has proposed an untargeted online investigation of the diversity of volatiles associated with the wine headspace. This communication proposed the first application of proton-transfer reaction-mass spectrometry coupled to a time-of-flight mass analyzer (PTR-ToF-MS) to follow the progress of AF and evaluate the impact of the different variables of wine quality. As a case study, the assessment of VOC variability associated with different combinations of Saccharomyces/non-Saccharomyces was selected. The different combinations of microbial resources in wine are among the main factors susceptible to influencing the content of VOCs associated with the wine headspaces. In particular, this investigation explored the effect of multiple combinations of two Saccharomyces strains and two non-Saccharomyces strains (belonging to the species Metschnikowia pulcherrima and Torulaspora delbrueckii) on the content of VOCs in wine, inoculated both in commercial grape juice and fresh grape must. The results demonstrated the possible exploitation of non-invasive PTR-ToF-MS monitoring to explore, using VOCs as biomarkers, (i) the huge number of variables influencing AF in wine, and (ii) applications of single/mixed starter cultures in wine. Reported preliminary findings underlined the presence of different behaviors on grape juice and on must, respectively, and confirmed differences among the single yeast strains ‘volatomes’. It was one of the first studies to include the simultaneous inoculation on two non-Saccharomyces species together with a S. cerevisiae strain in terms of VOC contribution. Among the other outcomes, evidence suggests that the addition of M. pulcherrima to the coupled S. cerevisiae/T. delbrueckii can modify the global release of volatiles as a function of the characteristics of the fermented matrix. Full article

A relevant trend in winemaking is to reduce the use of chemical compounds in both the vineyard and winery. In organic productions, synthetic chemical fertilizers, pesticides, and genetically modified organisms must be avoided, aiming to achieve the production of a “safer wine”. Safety represents a big threat all over the world, being one of the most important goals to be achieved in both Western society and developing countries. An occurrence in wine safety results in the recovery of a broad variety of harmful compounds for human health such as amines, carbamate, and mycotoxins. The perceived increase in sensory complexity and superiority of successful uninoculated wine fermentations, as well as a thrust from consumers looking for a more “natural” or “organic” wine, produced with fewer additives, and perceived health attributes has led to more investigations into the use of non-Saccharomyces yeasts in winemaking, namely in organic wines. However, the use of copper and sulfur-based molecules as an alternative to chemical pesticides, in organic vineyards, seems to affect the composition of grape microbiota; high copper residues can be present in grape must and wine. This review aims to provide an overview of organic wine safety, when using indigenous and/or non-Saccharomyces yeasts to perform fermentation, with a special focus on some metabolites of microbial origin, namely, ochratoxin A (OTA) and other mycotoxins, biogenic amines (BAs), and ethyl carbamate (EC). These health hazards present an increased awareness of the effects on health and well-being by wine consumers, who also enjoy wines where terroir is perceived and is a characteristic of a given geographical area. In this regard, vineyard yeast biota, namely non-Saccharomyces wine-yeasts, can strongly contribute to the uniqueness of the wines derived from each specific region. Full article

Celiac disease (CD) is an immune-mediated gluten-sensitive enteropathy. Currently, it affects around 1% of world population, but it is constantly growing. Celiac patients have to follow a strict gluten-free (GF) diet. Beer is one of the most consumed beverages worldwide, but it is not safe for people with CD. It has a gluten content usually above the safe threshold (20 ppm), determined by the official method for hydrolyzed foods (R5-competitive-ELISA). The demand on the market for GF beers is increasingly growing. This review aims to provide a comprehensive overview of different strategies to produce GF beer, highlighting strengths and weaknesses of each approach and taking into account technological and sensory issues. GF cereals or pseudocereals have poor brewing attitudes (if used as main raw material) and give the beer unusual flavour. Instead, enzymatic treatments allow traditional brewing process followed by gluten content reduction. A survey on 185 GF-producing breweries (both industrial and craft) from all over the world have been considered to assess which approach is most used. Beers brewed with GF cereals and pseudocereals (used in well-balanced proportions) are more common than gluten-removed (GR) beers, obtained by enzymatic treatment. Full article

This study evaluated the feasibility of concomitant nutrient removal, cleaner water recovery, and improved ethanol production via glucose fermentation in the liquid fraction of anaerobic digestate (ADE) by Saccharomyces cerevisiae. The 25%, 50%, and 100% (v/v) ADE supported the growth of S. cerevisiae, glucose utilization (~100 g/L) and ethanol production (up to 50.4 ± 6.4 g/L). After a 144 h fermentation in the 50% ADE, the concentrations of ammonia, total nitrogen, phosphate, and total phosphorus decreased 1000-, 104.43-, 1.94-, and 2.20-fold, respectively. Notably, only 0.40 ± 0.61 mg/L ammonia was detected in the 50% ADE post-fermentation. Similarly, the concentrations of aluminum, copper, magnesium, manganese, molybdenum, potassium, sodium, iron, sulfur, zinc, chloride, and sulfate decreased significantly in the ADE. Further analysis suggests that the nitrogen (ammonia and protein), phosphate, and the metal contents of the digestate work in tandem to promote growth and ethanol production. Among these, ammonia and protein appear to exert considerable effects on S. cerevisiae. These results represent a significant first step towards repurposing ADE as a resource in bio-production of fuels and chemicals, whilst generating effluent that is economically treatable by conventional wastewater treatment technologies. Full article

Khadi is a popular traditional alcoholic beverage in rural households in Botswana. The product is produced by fermentation of ripened sun-dried Grewia flava (Malvaceae) fruits supplemented with brown table sugar. Despite its popularity, its growing consumer acceptance, its potential nutritional value, and its contribution to the socio-economic lifestyle of Botswana, the production process remains non-standardized. Non-standardized production processes lead to discrepancies in product quality and safety as well as varying shelf life. Identification of unknown fermentative microorganisms of khadi is an important step towards standardization of its brewing process for entrance into commercial markets. The aim of this study was to isolate and identify bacteria and yeasts responsible for fermentation of khadi. Yeasts and bacteria harbored in 18 khadi samples from 18 brewers in central and northern Botswana were investigated using classic culture-dependent techniques and DNA sequencing methods. Additionally, we used the same techniques to investigate the presence of bacteria and yeasts on six batches of ripened-dried G. flava fruits used for production of the sampled brews. Our results revealed that Saccharomyces cerevisiae closely related to a commercial baker’s yeast strain sold locally was the most predominant yeast species in khadi suggesting a possible non-spontaneous brewing process. However, we also detected diverse non-Saccharomyces yeasts, which are not available commercially in retail shops in Botswana. This suggests that spontaneous fermentation is partially responsible for fermentation of khadi. This study, presenting the first microbiological characterization of a prominent traditional alcoholic beverage in Botswana, is vital for development of starter cultures for the production of a consistent product towards the commercialization of khadi. Full article

Traditionally, Saccharomyces species are those used to conduct industrial alcoholic fermentations. Recently, an increasing interest has arisen with respect to the potential of so-called non-conventional yeasts to improve wine and beer aroma profiles, keeping the particular terroir of each region or for the development of craft beers. In this study, the potential of diverse non-conventional yeasts to improve aroma in winemaking and brewing was investigated, testing several pure and mixed culture combinations. In addition, a comparison between microscale and labscale was carried out in order to assess the value of microwine and microbeer as screening tools. The results indicated that non-Saccharomyces yeasts were good candidates to enhance or diversify aroma profiles in alcoholic beverages, especially regarding acetate ester yield and fruity aromas. However, mixed cultures with Saccharomyces spp. are normally required to achieve a successful fermentation. The adjustment of pithing ratios is crucial for this purpose. Microscale is presented as an effective and efficient screening tool to compare different culture combinations, although scaling-up will always be necessary in order to get results closer to real winemaking or brewing processes. Full article

Recently, there has been a growing interest in the role of non-Saccharomyces yeast (NSY) as a coculturing partner with Saccharomyces cerevisiae during grape must fermentation. We investigated three new strains, namely Nakazawaea ishiwadae, Candida railenensis and Debaryomyces hansenii, for their oenological potential in mixed-culture micro-vinifications with S. cerevisiae Vin13 using Muscaris grape must. None of the NSY strains impeded the fermentation performance as all the mixed-culture experiments finished at the same time. Coculturing with N. ishiwadae yielded significantly higher concentrations of ethyl and acetate esters in the final wine product. Apart from higher acetic acid levels, wines produced with C. railenensis and D. hansenii yielded much lower esters concentrations. The concentrations of certain terpenes and norisoprenoids were also significantly modulated in the mixed-culture fermentations. This study reveals the rarely reported species of N. ishiwadae as a promising coculturing partner for increasing aroma-active compounds in a wine. Full article