Fermentation, Volume 4, Issue 3 (September 2018) – 32 articles

Flocculation is one of the most important characteristics of brewing yeast as it allows for the easy and cheap removal of cells after fermentation. The genes responsible for both the Flo1 and NewFlo flocculation phenotypes are well characterized. However, the relationship between Flo protein abundance and flocculation efficiency is poorly understood. In this present study, we used mass spectrometry proteomics to compare the cell wall and whole cell proteomes of commercial yeast strains with diverse flocculation behaviors. We found that the relative abundance of Flo1/5 or Flo10 in the cell wall was correlated with the ability of these yeast strains to flocculate. Analysis of whole cell proteomes identified differences in the proteomes of yeast strains and identified the potential for high metabolic diversity. Characterization of the cell wall and whole cell proteomes during fermentation showed high levels of Flo10 in cells that settled early during fermentation. Our data reveal the diversity of the cell wall and global proteomes of brewing yeast, highlighting the potential biochemical diversity present in yeast that can be utilized in the production of fermented beverages. Full article

At specific growth rates above a particular critical value, Crabtree-positive microorganisms exceed their respiratory capacity and enter diauxic growth metabolism. Excess substrate is converted reductively to an overflow metabolite, resulting in decreased biomass yield and productivity. To prevent this scenario, the cells can be cultivated in a fed-batch mode at a growth rate maintained below the critical value, µ_crit. This approach entails two major challenges: accurately estimating the current specific growth rate and controlling it successfully over the course of the fermentation. In this work, the specific growth rate of S. cerevisiae and E. coli was estimated from enhanced on-line biomass concentration measurements obtained with dielectric spectroscopy and turbidity. A feedforward-feedback control scheme was implemented to maintain the specific growth rate at a setpoint below µ_crit, while on-line FTIR measurements provided the early detection of the overflow metabolites. The proposed approach is in line with the principles of Bioprocess Analytical Technology (BioPAT), and provides a means to increase the productivity of Crabtree-positive microorganisms. Full article

This special issue collected recent developments on the microbiota of fermented beverages, from raw materials to the finished product, as well as the use of specific starter cultures. In particular, several studies investigated the occurrence and use of conventional and non-conventional yeasts in distilled alcoholic beverages, wine, and beer production, while other papers investigated probiotic and health-promoting compounds. Results indicated that the management of microbiota greatly improves the analytical, sensorial, and healthy characteristics of fermented beverages. Full article

This project aims to characterize and define an autochthonous yeast, Saccharomyces bayanus CN1, for wine production from partially dehydrated grapes. The yeast was identified via PCR and Basic Local Alignment Search Tool (BLAST) analysis as Saccharomyces bayanus, and then subsequently used in fermentations using partially dehydrated or control grapes. Wine grapes were dried to 28.0°Brix from the control grapes at a regular harvest of 23.0°Brix. Both the partially dehydrated and control grapes were then vinified with each of two yeast strains, S. bayanus CN1 and S. cerevisiae EC1118, which is a common yeast used for making wine from partially dehydrated grapes. Chemical analysis gas chromatography-flame ionization detector (GC-FID) and enzymatic) of wines at each starting sugar level showed that CN1 produced comparable ethanol levels to EC1118, while producing higher levels of glycerol, but lower levels of oxidative compounds (acetic acid, ethyl acetate, and acetaldehyde) compared to EC1118. Yeast choice impacted the wine hue; the degree of red pigment coloration and total red pigment concentration differed between yeasts. A sensory triangle test (n = 40) showed that wines made from different starting sugar concentrations and yeast strains both differed significantly. This newly identified S. bayanus strain appears to be well-suited for this style of wine production from partially dehydrated grapes by reducing the oxidative compounds in the wine, with potential commercial application for cool climate wine regions. Full article

Apiculate yeasts of the genus Hanseniaspora/Kloeckera are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine color and flavor. Ten species of the genus Hanseniaspora have been recovered from grapes and are associated in two groups: H. valbyensis, H. guilliermondii, H. uvarum, H. opuntiae, H. thailandica, H. meyeri, and H. clermontiae; and H. vineae, H. osmophila, and H. occidentalis. This review focuses on the application of some strains belonging to this genus in co-fermentation with Saccharomyces cerevisiae that demonstrates their positive contribution to winemaking. Some consistent results have shown more intense flavors and complex, full-bodied wines, compared with wines produced by the use of S. cerevisiae alone. Recent genetic and physiologic studies have improved the knowledge of the Hanseniaspora/Kloeckera species. Significant increases in acetyl esters, benzenoids, and sesquiterpene flavor compounds, and relative decreases in alcohols and acids have been reported, due to different fermentation pathways compared to conventional wine yeasts. Full article

Glycerol carbonate (GC) is a value-added product originating from the valorization of widely available glycerol (Gly), a side stream from the production of biodiesel. Here we approach the production of this chemical comparing two reactions based on the transesterification of Gly with dimethyl carbonate (DMC) and ethylene carbonate (EC). When using DMC, it was observed that the free enzyme CALB (lipase B from Candida antarctica) gave the best results, whereas Eversa Transform (a genetic modification of Thermomyces lanuginosus lipase) performed better than the rest if EC was the reagent. With the selected catalysts, their immobilized analogous enzymes Novozym 435 and Lypozyme TL IM, respectively, were also tested. Observing that the yields for the reaction with EC were significantly faster, other operating variables were evaluated, resulting the best performance using a closed system, tert-butanol as solvent, a concentration of enzyme Eversa Transform of 3% w/w, a molar excess of EC:Gly of 9:1 and a temperature of 60 °C. Finally, several runs were conducted at different temperatures and molar ratios of EC:Gly, fitting a kinetic model to all experimental data for the reaction catalyzed with Eversa Transform. This model included the bimolecular transesterification reaction of Gly and EC catalyzed by the lipase and a reversible ring-opening polymerization of EC. Full article

Candida stellata is an imperfect yeast of the genus Candida that belongs to the order Saccharomycetales, while phylum Ascomycota. C. stellata was isolated originally from a must overripe in Germany but is widespread in natural and artificial habitats. C. stellata is a yeast with a taxonomic history characterized by numerous changes; it is either a heterogeneous species or easily confused with other yeast species that colonize the same substrates. The strain DBVPG 3827, frequently used to investigate the oenological properties of C. stellata, was recently renamed as Starmerella bombicola, which can be easily confused with C. zemplinina or related species like C. lactis-condensi. Strains of C. stellata have been used in the processing of foods and feeds for thousands of years. This species, which is commonly isolated from grape must, has been found to be competitive and persistent in fermentation in both white and red wine in various wine regions of the world and tolerates a concentration of at least 9% (v/v) ethanol. Although these yeasts can produce spoilage, several studies have been conducted to characterize C. stellata for their ability to produce desirable metabolites for wine flavor, such as acetate esters, or for the presence of enzymatic activities that enhance wine aroma, such as β-glucosidase. This microorganism could also possess many interesting technological properties that could be applied in food processing. Exo and endoglucosidases and polygalactosidase of C. stellata are important in the degradation of β-glucans produced by Botrytis cinerea. In traditional balsamic vinegar production, C. stellata shapes the aromatic profile of traditional vinegar, producing ethanol from fructose and high concentrations of glycerol, succinic acid, ethyl acetate, and acetoin. Chemical characterization of exocellular polysaccharides produced by non-Saccharomyces yeasts revealed them to essentially be mannoproteins with high mannose contents, ranging from 73–74% for Starmerella bombicola. Numerous studies have clearly proven that these macromolecules make multiple positive contributions to wine quality. Recent studies on C. stellata strains in wines made by co-fermentation with Saccharomyces cerevisiae have found that the aroma attributes of the individual strains were apparent when the inoculation protocol permitted the growth and activity of both yeasts. The exploitation of the diversity of biochemical and sensory properties of non-Saccharomyces yeast could be of interest for obtaining new products. Full article

The interest in non-Saccharomyces yeast for use in sparkling wine production has increased in recent years. Studies have reported differences in amino acids and ammonia, volatile aroma compounds (VOCs), glycerol, organic acids, proteins and polysaccharides. The aim of this review is to report on our current knowledge concerning the influence of non-Saccharomyces yeast on sparkling wine chemical composition and sensory profiles. Further information regarding the nutritional requirements of each of these yeasts and nutrient supplementation products specifically for non-Saccharomyces yeasts are likely to be produced in the future. Further studies that focus on the long-term aging ability of sparkling wines made from non-Saccharomyces yeast and mixed inoculations including their foam ability and persistence, organic acid levels and mouthfeel properties are recommended as future research topics. Full article

Two correlations were developed to calculate the composition of binary ethanol-water solutions from experimental temperature and density inputs. The first correlation is based on a Redlich-Kister (R-K) expansion and computes mixture composition within an average accuracy of ±0.45 wt.%. The R-K model is a non-linear function of composition and therefore requires the use of an iterative solving tool. A polynomial correlation was additionally developed which utilizes a direct solving method, and computes ethanol composition over a range of 0–100 wt.% [283.15–313.15 K] with an accuracy better than ±0.37 wt.%. The polynomial model is particularly advantageous as it can be tailored to specific composition ranges for increased accuracy. Both correlations are intended to provide a method for monitoring ethanol concentration within a chemical process in real time without off-line sample analysis, allowing for precise in-situ system control and optimization. Full article

Non-Saccharomyces yeasts are becoming important because most of them are considered as spoilage species in winemaking processes, among them the species Saccharomycodes ludwigii. This species is frequently isolated at the end of the fermentation process and/or during storage of the wine, i.e., it can to grow in the presence of high levels of ethanol. Besides, this species is adaptable to unfavorable conditions such as high concentrations of SO₂ and is characterized by its capacity to produce high amounts of undesirable metabolites as acetoin, ethyl acetate or acetic acid. To the present, physical (gamma irradiation and continuous pulsed electric fields), chemical (inhibitory compounds such as chitosan and dimethyl dicarbonate) and biological (antagonistic biocontrol by killer yeasts) treatments have been developed in order to control the growth of this spoilage yeast in wines and other fruit derivatives. Therefore, this review is focused on the most relevant studies conducted to control contamination by S. ludwigii. Moreover, potential applications of S. ludwigii in alternative winemaking techniques, for example for ageing-on-lees and stabilization of red wines, and improvement of aromatic profile are also examined. Full article

There are numerous yeast species related to wine making, particularly non-Saccharomyces, that deserve special attention due to the great potential they have when it comes to making certain changes in the composition of the wine. Among them, Schizosaccharomyces pombe stands out for its particular metabolism that gives it certain abilities such as regulating the acidity of wine through maloalcoholic fermentation. In addition, this species is characterized by favouring the formation of stable pigments in wine and releasing large quantities of polysaccharides during ageing on lees. Moreover, its urease activity and its competition for malic acid with lactic acid bacteria make it a safety tool by limiting the formation of ethyl carbamate and biogenic amines in wine. However, it also has certain disadvantages such as its low fermentation speed or the development of undesirable flavours and aromas. In this chapter, the main oenological uses of Schizosaccharomyces pombe that have been proposed in recent years will be reviewed and discussed. Full article

The genus Zygosaccharomyces is generally associated to wine spoilage in the winemaking industry, since a contamination with strains of this species may produce re-fermentation and CO₂ production in sweet wines. At the same time, this capacity might be useful for sparkling wines production, since this species may grow under restrictive conditions, such as high ethanol, low oxygen, and harsh osmotic conditions. The spoilage activity of this genus is also found in fruit juices, soft drinks, salad dressings, and other food products, producing besides package expansion due to gas production, non-desired compounds such as ethanol and esters. Despite these drawbacks, Zygosaccharomyces spp. produces high ethanol and acetoin content in wines and may play an important role as non-Saccharomyces yeasts in differentiated wine products. Control strategies, such as the use of antimicrobial peptides like Lactoferricin B (Lfcin B), the use of dimethyl dicarbonate (DMDC) or non-thermal sterilization techniques may control this spoilage genus in the food industry. Full article

Nowadays it is widely accepted that non-Saccharomyces yeasts, which prevail during the early stages of alcoholic fermentation, contribute significantly to the character and quality of the final wine. Among these yeasts, Wickerhamomyces anomalus (formerly Pichia anomala, Hansenula anomala, Candida pelliculosa) has gained considerable importance for the wine industry since it exhibits interesting and potentially exploitable physiological and metabolic characteristics, although its growth along fermentation can still be seen as an uncontrollable risk. This species is widespread in nature and has been isolated from different environments including grapes and wines. Its use together with Saccharomyces cerevisiae in mixed culture fermentations has been proposed to increase wine particular characteristics. Here, we review the ability of W. anomalus to produce enzymes and metabolites of oenological relevance and we discuss its potential as a biocontrol agent in winemaking. Finally, biotechnological applications of W. anomalus beyond wine fermentation are briefly described. Full article

Each year, winemakers can face sluggish or stuck fermentations during wine making, especially when a spontaneous fermentation is performed, even if strains of the classical wine yeast Saccharomyces cerevisiae are applied. Problems are inevitable when low ammonium concentrations (<160 mg L⁻¹ grape must) or an excess of fructose compared to glucose are observed during grape must fermentation. S. cerevisiae strains cannot use all kinds of amino acids as the sole nitrogen source but usually need free ammonium (optimal concentration: 600 mg L⁻¹ grape must). It preferably consumes glucose, leading often to an excess of fructose in the fermenting must, which contains glucose and fructose in an equal ratio at the beginning of fermentation. Yeast hybrids have been isolated from wines several times and different strains are already commercially available. The united properties of the parent strains can provide advantages under sophisticated fermentation conditions. However, the involvement of a hybrid yeast for the rectification of fermentation disorders in spontaneous fermentations has only been described recently in the literature. Recent investigations have provided convincing evidence that fermentation problems can be overcome when must fermentations are successively performed with Saccharomyces bayanus strain HL 77 and the triple hybrid S. cerevisiae × Saccharomyces kudriavzevii × S. bayanus strain HL 78. The triple hybrid strain HL 78 uses amino acids as a nitrogen source in the absence of ammonium and it also exhibits a fructophilic character with an enhanced uptake of fructose in comparison to glucose. The application of genetically modified yeast strains is not allowed for starter cultures in wine making, but the usage of yeast mixtures and hybrid strains could be a promising tool for winemakers to solve fermentation problems during spontaneous fermentation or for the creation of novel wine types with desired sensory characteristics under more challenging conditions, especially when the composition of the must components is not optimal because of, e.g., critical climatic or soil conditions. Full article

Alcohol-free beer (AFB) is no longer just a niche product in the beer market. For brewers, this product category offers economic benefits in the form of a growing market and often a lower tax burden and enables brewers to extend their product portfolio and promote responsible drinking. Non-Saccharomyces yeasts are known for their flavor-enhancing properties in food fermentations, and their prevailing inability to ferment maltose and maltotriose sets a natural fermentation limit and can introduce a promising approach in the production of AFB (≤0.5% v/v). Five strains isolated from kombucha, Hanseniaspora valbyensis, Hanseniaspora vineae, Torulaspora delbrueckii, Zygosaccharomyces bailii and Zygosaccharomyces kombuchaensis were compared to a commercially applied AFB strain Saccharomycodes ludwigii and a Saccharomyces cerevisiae brewer’s yeast. The strains were characterized for their sugar utilization, phenolic off-flavors, hop sensitivity and flocculation. Trial fermentations were analyzed for extract reduction, ethanol formation, pH drop and final beers were analyzed for amino acids utilization and fermentation by-products. The performance of non-Saccharomyces strains and the commercial AFB strain were comparable during fermentation and production of fermentation by-products. An experienced sensory panel could not discriminate between the non-Saccharomyces AFB and the one produced with the commercial AFB strain, therefore indicating their suitability in AFB brewing. Full article

Fusicocca-2,10(14)-diene (FCdiene) is a tricyclic diterpene which has many pharmaceutical applications, for example, it is a precursor for different anticancer drugs, including fusicoccin A. Chemical synthesis of this diterpene is not economical as it requires 14 steps with several stereospecific reactions. FCdiene is naturally produced at low titers in phytopathogenic filamentous fungi. However, production of FCdiene can be achieved via expression of fusicoccadiene synthase in yeast. The objective of this study is to increase FCdiene production by optimizing the yeast fermentation process. Our preliminary fermentations showed influences of carbon sources, buffer agents, and oxygen supply on FCdiene production. Buffer agents as well as oxygen supply were investigated in detail at 0.2 and 1.8 L cultivation volumes. Using glucose as the carbon source, FCdiene concentrations were increased to 240 mg_FCdiene/L by optimizing pH and oxygen conditions. In situ extraction and adsorption techniques were examined at the 0.2 L scale to determine if these techniques could improve FCdiene yields. Different adsorbents and solvents were tested with in situ product recovery and 4-fold increases in FCdiene productivity could be shown. The results generated in this work provide a proof-of-concept for the fermentative production of FCdiene from S. cerevisiae as a practical alternative to chemical synthesis. Full article

Distillers dried grains with solubles (DDGS) is a coproduct of corn-based ethanol production that can be a valuable source of energy, digestible amino acids, and available phosphorus in poultry feeds. Dietary incorporation of DDGS reduces the amount of primary ingredients such as corn and soybean meal needed to formulate poultry diets, improving the sustainability of both biofuel and poultry production. The nutritional value of DDGS has been extensively evaluated since it became increasingly available to feed producers in the early 2000s, but evolving methods of ethanol production and coproduct fractionation necessitate its continued characterization. Attempts to relate nutrient utilization of DDGS to its chemical composition have revealed that fiber content is a primary determinant of dietary energy value of DDGS for poultry. Distillers corn oil, which is extracted from thin stillage during production of distillers grains, can also be supplemented into poultry diets as an energy-dense lipid source in place of animal fats or other vegetable-based oils. Poultry feeding practices in the United States are also evolving, including increased adoption of all vegetable-based diets and reduced use of in-feed antimicrobials. Therefore, further characterization of both the nutritional value of DDGS and its impact on gastrointestinal health will support its continued use in poultry diets. Full article

Cupriavidus necator, a facultative hydrogen-oxidizing bacterium, was grown on carbon dioxide, hydrogen, and oxygen for value-added products. High cell density and productivity were the goal of gas fermentation, but limited by gas substrates because of their low solubility in the aqueous medium solution. Enhancement of gas fermentation was investigated by (i) adding n-hexadecane as a gas vector to increase the volumetric mass transfer coefficient (k_La) and gas solubility, (ii) growing C. necator under a raised gas pressure, and (iii) using cell mass hydrolysates as the nutrients of chemolithotrophic growth. In contrast to previous studies, little positive but negative effects of the gas vector were observed on gas mass transfer and cell growth. The gas fermentation could be significantly enhanced under a raised pressure, resulting in a higher growth rate (0.12 h⁻¹), cell density (18 g L⁻¹), and gas uptake rate (200 mmole L⁻¹ h⁻¹) than a fermentation under atmospheric pressure. The gain, however, was not proportional to the pressure increase as predicted by Henry’s law. The hydrolysates of cell mass were found a good source of nutrients and the organic nitrogen was equivalent to or better than ammonium nitrogen for chemolithotrophic growth of C. necator on carbon dioxide. Full article

Reductive sulfurous off-odors are still one of the main reasons for rejecting wines by consumers. In 2008 at the International Wine Challenge in London, approximately 6% of the more than 10,000 wines presented were described as faulty. Twenty-eight percent were described as faulty because they presented “reduced characters” similar to those presented by “cork taint” and in nearly the same portion. Reductive off-odors are caused by low volatile sulfurous compounds. Their origin may be traced back to the metabolism of the microorganisms (yeasts and lactic acid bacteria) involved in the fermentation steps during wine making, often followed by chemical conversions. The main source of volatile sulfur compounds (VSCs) are precursors from the sulfate assimilation pathway (SAP, sometimes named as the “sulfate reduction pathway” SRP), used by yeast to assimilate sulfur from the environment and incorporate it into the essential sulfur-containing amino acids methionine and cysteine. Reductive off-odors became of increasing interest within the last few years, and the method to remove them by treatment with copper (II) salts (sulfate or citrate) is more and more questioned: The effectiveness is doubted, and after prolonged bottle storage, they reappear quite often. Numerous reports within the last few years and an ongoing flood of publications dealing with this matter reflect the importance of this problem. In a recent detailed review, almost all relevant aspects were discussed on a scientific data basis, and a “decision tree” was formulated to support winemakers handling this problem. Since we are dealing with a very complicated matter with a multitude of black spots still remaining, these advices can only be realized using specific equipment and special chemicals, not necessarily found in small wineries. The main problem in dealing with sulfurous compounds arises from the high variability of their reactivities. Sulfur is a metalloid with a large valence span across eight electron transformations from S (−II) up to S (+VI). This allows it to participate in an array of oxidation, reduction and disproportionation reactions, both abiotic and linked to microbial metabolism. In addition, sulfur is the element with the most allotropes and a high tendency to form chains and rings, with different stabilities of defined species and a high interconvertibility among each other. We suppose, there is simply a lack of knowledge of what is transferred during filling into bottles after fermentation and fining procedures. The treatment with copper (II) salts to remove sulfurous off-odors before filling rather increases instead of solving the problem. This paper picks up the abundant knowledge from recent literature and tries to add some aspects and observations, based on the assumption that the formation of polythionates, hitherto not taken into consideration, may explain some of the mystery of the re-appearance of reductive off-odors. Full article

The main hypothesis of this work is that facultative and obligate heterofermentative Lactobacillus species can differently impact the final characteristics of pizza. The objective was to evaluate separately the behaviors of the obligate heterofermentative species (OHS), such as Lactobacillus sanfranciscensis, Lactobacillus brevis, and Lactobacillus rossiae, and the facultative heterofermentative species (FHS), including Lactobacillus plantarum, and Lactobacillus curvatus, in the sourdoughs to be used for pizza production. The production of the experimental pizzas was carried out with semolina (Triticum turgidum L. ssp. durum). The acidification process—which was followed by pH, total titratable acidity (TTA), and lactic acid bacteria (LAB) development—indicated for all of the experimental trials that the kinetics is comparable to those of the controls. The fermentation quotient of the FHS trial was particularly higher than that of the other trials, including the control production performed with a sourdough inoculum used in an artisanal bakery. The dominance of the added strains indicated the clear persistence of L. sanfranciscensis PON100336, L. brevis 200571, and L. plantarum PON100148 in the obligate–facultative heterofermentative species (OFHS) trial. The pizzas were baked without seasoning in order to investigate weight loss, color, morphology, and a generation of volatile organic compounds (VOCs). The data showed the differences among trials regarding the inocula. Eight classes of VOCs were detected in the pizza samples with aldehydes, esters, alcohols, and acids as major compounds. The sensory attributes were significantly different for the judges and the pizzas. The multivariate statistical approach found marked differences among the trials. The results indicated that the application of mixed cultures of the facultative heterofermentative species of Lactobacillus determined high quality pizzas. Full article

A two-year study was performed to evaluate the effects of the timing of cluster thinning on Pinot noir grapes and wines in the central coast of California. Vines were thinned to one cluster per shoot at three selected time-points during the growing season, and fruit was harvested and made into wine. No consistent effect of cluster thinning was found in wine phenolic profile or color across a cool (2016) and a warm (2017) growing season. The growing season had a more significant effect than the cluster thinning treatment for most parameters measured. There was no detectable overall sensory difference between the non-thinned control wines and any of the thinned treatment wines. Based on current results, Pinot noir vineyards on the central coast of California can support crop loads that result in Ravaz Index values from 3 to 6 without concern for impacting ripening potential or negatively affecting fruit composition. Full article

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L⁻¹ glucose and 95 g L⁻¹ xylose generated 120.6 g L⁻¹ ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g⁻¹, and ethanol productivity of 5.26 g L⁻¹·h⁻¹. Full article

The effectiveness of polyhydroxyalkanoates (PHAs) production process from a waste stream is determined by the selection of a suitable mixed microbial culture (MMC). In this work, a feedstock from the paper industry, hardwood spent sulfite liquor (HSSL), supplemented with short-chain organic acids (SCOAs) to simulate a fermented effluent, was used as substrate to enrich a MMC in PHA-storing microorganisms. A stable culture was quickly established, and during the accumulation step the selected MMC reached a maximum PHA content of 34.6% (3HB:3HV-76:24). The bacterial community was analyzed through FISH analysis. Bacteria belonging to the four main classes were identified: Betaproteobacteria (44.7 ± 2.7%), Alphaproteobacteria (13.6 ± 1.3%) and Gammaproteobacteria (2.40 ± 1.1%) and Bacteroidetes (9.20 ± 3.8%). Inside the Betaproteobacteria class, Acidovorax (71%) was the dominant genus. Full article

The objectives of this paper were to summarize the status of the livestock (pig and cattle) industry and its waste management in Taiwan. The Water Pollution Control Act authorized the reuse of liquor and digestate from anaerobic digestion (AD) as fertilizers for agricultural lands on 24 November 2015. A large number of official databases and literature have been surveyed and analyzed to address the characterization of the biogas (AD-based) digestate and the potential benefits of biogas-to-power in Taiwan. On the promulgation of the Act, the central ministries have jointly managed the applications of livestock farms for reusing the AD-based liquor and digestate as fertilizers for farmlands. The survey findings revealed that the biogas digestate from a pig farm in Taiwan contained significant amounts of nitrogen, phosphorus, and other soil nutrients such as calcium and magnesium. However, it is necessary to control zinc present in the biogas digestate from pig-raising farms. A preliminary analysis based on 123 large-scale pig farms with a total of 1,223,674 heads showed the annual benefits of methane reduction of 6.1 Gg, electricity generation of 3.7 × 10⁷ kW-h, equivalent electricity charge saving of 4.0 × 10⁶ US$, and equivalent carbon dioxide mitigation of 152.5 thousand tons (Gg). Obviously, the integration of AD and biogas-to-power for treating animal manure is a win-win option for livestock farms to gain environmental, energy, and economic benefits. Full article

To improve the quality of fermented drinks, or more specifically, wine, some strains of yeast have been isolated, tested and studied, such as Saccharomyces and non-Saccharomyces. Some non-conventional yeasts present good fermentative capacities and are able to ferment in quite undesirable conditions, such as the case of must, or wines that have a high concentration of acetic acid. One of those yeasts is Lachancea thermotolerants (L. thermotolerans), which has been studied for its use in wine due to its ability to decrease pH through L-lactic acid production, giving the wines a pleasant acidity. This review focuses on the recent discovery of an interesting feature of L. thermotolerans—namely, its ability to decrease wines’ volatile acidity. Full article

Clostridium acetobutylicum has traditionally been used for production of acetone, butanol, and ethanol (ABE). Butanol is a commodity chemical due in part to its suitability as a biofuel; however, the current yield of this product from biological systems is not economically feasible as an alternative fuel source. Understanding solvent phase physiology, solvent tolerance, and their genetic underpinning is key for future strain optimization of the bacterium. This study shows the importance of a [NiFe]-hydrogenase in solvent phase physiology. C. acetobutylicum genes ca_c0810 and ca_c0811, annotated as a HypF and HypD maturation factor, were found to be required for [NiFe]-hydrogenase activity. They were shown to be part of a polycistronic operon with other hyp genes. Hydrogenase activity assays of the ΔhypF/hypD mutant showed an almost complete inactivation of the [NiFe]-hydrogenase. Metabolic studies comparing ΔhypF/hypD and wild type (WT) strains in planktonic and sessile conditions indicated the hydrogenase was important for solvent phase metabolism. For the mutant, reabsorption of acetate and butyrate was inhibited during solventogenesis in planktonic cultures, and less ABE was produced. During sessile growth, the ΔhypF/hypD mutant had higher initial acetone: butanol ratios, which is consistent with the inability to obtain reduced cofactors via H₂ uptake. In sessile conditions, the ΔhypF/hypD mutant was inhibited in early solventogenesis, but it appeared to remodel its metabolism and produced mainly butanol in late solventogenesis without the uptake of acids. Energy filtered transmission electron microscopy (EFTEM) mapped Pd(II) reduction via [NiFe]-hydrogenase induced H₂ oxidation at the extracelluar side of the membrane on WT cells. A decrease of Pd(0) deposits on ΔhypF/hypD comparatively to WT indicates that the [NiFe]-hydrogenase contributed to the Pd(II) reduction. Calculations of reaction potentials during acidogenesis and solventogenesis predict the [NiFe]-hydrogenase can couple NAD⁺ reduction with membrane transport of electrons. Extracellular oxidation of H₂ combined with the potential for electron transport across the membrane indicate that the [NiFe}-hydrogenase contributes to proton motive force maintenance via hydrogen cycling. Full article

Yeast—especially Saccharomyces cerevisiae—have long been a preferred workhorse for the production of numerous recombinant proteins and other metabolites. S. cerevisiae is a noteworthy aroma compound producer and has also been exploited to produce foreign bioflavour compounds. In the past few years, important strides have been made in unlocking the key elements in the biochemical pathways involved in the production of many aroma compounds. The expression of these biochemical pathways in yeast often involves the manipulation of the host strain to direct the flux towards certain precursors needed for the production of the given aroma compound. This review highlights recent advances in the bioengineering of yeast—including S. cerevisiae—to produce aroma compounds and bioflavours. To capitalise on recent advances in synthetic yeast genomics, this review presents yeast as a significant producer of bioflavours in a fresh context and proposes new directions for combining engineering and biology principles to improve the yield of targeted aroma compounds. Full article

Lachancea (kluyveromyces) thermotolerans is a ubiquitous yeast that can be naturally found in grapes but also in other habitats as soil, insects and plants, extensively distributed around the world. In a 3-day culture, it shows spherical to ellipsoidal morphology appearing in single, paired cells or short clusters. It is a teleomorph yeast with 1–4 spherical ascospores and it is characterized by a low production of volatile acidity that helps to control global acetic acid levels in mixed or sequential inoculations with either S. cerevisiae or other non-Saccharomyces species. It has a medium fermentative power, so it must be used in sequential or mixed inoculations with S. cerevisiae to get dry wines. It shows a high production of lactic acid able to affect strongly wine pH, sometimes decreasing wine pH by 0.5 units or more during fermentation. Most of the acidification is produced at the beginning of fermentation facilitating the effect in sequential fermentations because it is more competitive at low alcoholic degree. This application is especially useful in warm areas affected by climatic change. pH reduction is produced in a natural way during fermentation and prevents the addition of tartaric acid, that produces tartrate precipitations, or the use of cation exchangers resins highly efficient reducing pH but with undesirable effects on wine quality. Production of lactic acid is done from sugars thus reducing slightly the alcoholic degree, especially in strains with high production of lactic acid. Also, an improvement in the production of 2-phenylethanol and glycerol has been described. Full article

Enzymes are used in modern wine technology for various biotransformation reactions from prefermentation through fermentation, post-fermentation and wine aging. Industrial enzymes offer quantitative benefits (increased juice yields), qualitative benefits (improved color extraction and flavor enhancement) and processing advantages (shorter maceration, settling and filtration time). This study gives an overview about key enzymes used in winemaking and the effects of commercial enzyme preparations on process engineering and the quality of the final product. In addition, we highlight on the presence and perspectives of beneficial enzymes in wine-related yeasts and lactic acid bacteria. Full article

Biological acidification is a common and beneficial way for breweries to adjust the pH value of their mash or wort to improve enzymatic activity during mashing, raise yeast activity during fermentation, and increase the flavor stability of the finished beer. The reactors are mostly run for many years without re-inoculating a fresh culture, creating the possibility of changes in the culture, genetic drifts, or the survival of different strains. In this study, a biological acidification culture that had been continuously run for 20 years was analyzed by GTG5 PCR and IGS2-314 rDNA PCR fingerprinting, as well as 16S and 26S rDNA sequencing, and real-time PCR was applied to differentiate the bacterial and yeast strains and species. The applied real-time PCR primers for Lactobacillus amylolyticus and Lactobacillus amylovorus have not been published yet. It was shown that only strains of the species L. amylolyticus were present, with low contamination of yeast strains from the species Saccharomyces cerevisiae. As the original starter culture was Lactobacillus amylolyticus, the acidification plant ran for 20 years, and no Lactobacillus sp. cross-contamination could be analyzed using culture-dependent methods after 20 years. The microflora composition is a decisive factor for the final beer flavor. Full article