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Microbial diversity in vineyards and in grapes has generated significant scientific interest. From a biotechnological perspective, vineyard and grape biodiversity has been shown to impact soil, vine, and grape health and to determine the fermentation microbiome and the final character of wine. Thus, an understanding of the drivers that are responsible for the differences in vineyard and grape microbiota is required. The impact of soil and climate, as well as of viticultural practices in geographically delimited areas, have been reported. However, the limited scale makes the identification of generally applicable drivers of microbial biodiversity and of specific microbial fingerprints challenging. The comparison and meta-analysis of different datasets is furthermore complicated by differences in sampling and in methodology. Here we present data from a wide-ranging coordinated approach, using standardized sampling and data generation and analysis, involving four countries with different climates and viticultural traditions. The data confirm the existence of a grape core microbial consortium, but also provide evidence for country-specific microbiota and suggest the existence of a cultivar-specific microbial fingerprint for Cabernet Sauvignon grape. This study puts in evidence new insight of the grape microbial community in two continents and the importance of both location and cultivar for the definition of the grape microbiome.

The general interest in microbial ecology has skyrocketed over the past decade, driven by technical advances and by the rapidly increasing appreciation of the fundamental services that these ecosystems provide. In biotechnology, ecosystems have many more functionalities than single species, and, if properly understood and harnessed, will be able to deliver better outcomes for almost all imaginable applications. However, the complexity of microbial ecosystems and of the interactions between species has limited their applicability. In research, next generation sequencing allows accurate mapping of the microbiomes that characterise ecosystems of biotechnological and/or medical relevance. But the gap between mapping and understanding, to be filled by "functional microbiomics", requires the collection and integration of many different layers of complex data sets, from molecular multi-omics to spatial imaging technologies to online ecosystem monitoring tools. Holistically, studying the complexity of most microbial ecosystems, consisting of hundreds of species in specific spatial arrangements, is beyond our current technical capabilities, and simpler model systems with fewer species and reduced spatial complexity are required to establish the fundamental rules of ecosystem functioning. One such ecosystem, the ecosystem responsible for natural alcoholic fermentation, can provide an excellent tool to study evolutionarily relevant interactions between multiple species within a relatively easily controlled environment. This review will critically evaluate the approaches that are currently implemented to dissect the cellular and molecular networks that govern this ecosystem. KEY POINTS: • Evolutionarily isolated fermentation ecosystem can be used as an ecological model. • Experimental toolbox is gearing towards mechanistic understanding of this ecosystem. • Integration of multidisciplinary datasets is key to predictive understanding.

Globally, 7.4 million hectares of arable land is planted with grapevine with a farm gate value of $68.3 billion. The production of grapes faces growing pressure associated with challenges such as climate change, diminishing resources as well as the overuse of chemical fertilizers and synthetic pesticides, which have an impact on sustainability. Consequently, viticulture has over the years embraced and implemented various practices such integrated pest management, organic and biodynamic farming to curb the high chemical inputs typically used in conventional farming. Biostimulants and biofertilizers are considered environmentally friendly and cost-effective alternatives to synthetic fertilizers and plant growth regulators. Seaweed is of particular interest because of its availability globally. It was reported that brown seaweed (

Shotgun sequencing was employed to explore the community structure (phylotyping of rRNA genes) andfunctional potential of Cabernet Sauvignon grape must microbiome. A metagenomic library, representing92.6 Mb of genetic information, was generated from DNA obtained from Cabernet Sauvignon grapemust.. Fungi were identified as the dominant domain (59.5%) followed by Streptophyta (39%). Amongthe 84 fungal species, 22 were yeasts of various genera. Additionally, grapevine endophytes such asDavidiella sp., Botryotinia fuckeliana, Alternaria sp., and Cladosporium sp. were identified. An unusuallyhigh prevalence of Mucor spp. was evidenced. Functional annotation revealed sequences of genesinvolved in metabolism (35.6%), followed by poorly characterized categories (28.3%), cellular processesand signalling (18.4%), and finally information storage (17.8%). Among the former, glycosidases wereabundant followed by glycogen debranching enzyme, 6-phosphofructokinase and trehalose-6-phosphatesynthase. Furthermore, the taxonomic analysis of the functional sequence data exhibited the eukaryoticgene pool that predominantly contains sequences derived from Streptophyta (mainly Vitis vinifera) 60% >Ascomycota (32%) > Basidiomycota (5%) > Bacteria (2.5%). Finally, sequences of a variety of hydrolyticenzymes of potential oenological relevance were retrieved, thereby confirming that grape juice is a richreservoir for valuable biocatalysts that should be explored further.

Anecdotal evidence suggests that spontaneous alcoholic fermentation of grape juice is becoming a more popular option in global wine production. Wines produced from the same grape juice by inoculation or spontaneous fermentation usually present distinct chemical and sensorial profiles. Inoculation has been associated with more similar end-products, a loss of typicity, and lower aroma complexity, and it has been suggested that this may be linked to suppression of the local or regional wine microbial ecosystems responsible for spontaneous fermentations. However, whether inoculated fermentations of different juices from different regions really end up with a narrower, less diverse chemical profile than those of spontaneously fermented juices has never been properly investigated. To address this question, we used grape juice from three different varieties, Grüner Veltliner (white), Zweigelt (red), and Pinot noir (red), originating from different regions in Austria to compare spontaneous and single active dry yeast strains inoculated fermentations of the same grape samples. The chemical analysis covered primary metabolites such as glycerol, ethanol and organic acids, and volatile secondary metabolites, including more than 40 major and minor esters, as well as higher alcohols and volatile fatty acids, allowing an in depth statistical evaluation of differences between fermentation strategies. The fungal (mainly yeast) communities throughout fermentations were monitored using automated ribosomal intergenic spacer analysis. The data provide evidence that inoculation with single active dry yeast strains limits the diversity of the chemical fingerprints. The fungal community profiles clearly show that inoculation had an effect on fermentation dynamics and resulted in chemically less diverse wines.

Microbial multispecies ecosystems are responsible for many biotechnological processes and are particularly important in food production. In wine fermentations, in addition to the natural microbiota, several commercially relevant yeast species may be co-inoculated to achieve specific outcomes. However, such multispecies fermentations remain largely unpredictable because of multilevel interactions between naturally present and/or co-inoculated species. Understanding the nature of such interactions has therefore become essential for successful implementation of such strategies. Here we investigate interactions between strains of Saccharomyces cerevisiae and Lachancea thermotolerans. Co-fermentations with both species sharing the same bioreactor (physical contact) were compared to co-fermentations with physical separation between the species in a membrane bioreactor ensuring free exchange of metabolites. Yeast culturability, viability and the production of core metabolites were monitored. The previously reported negative interaction between these two yeast species was confirmed. Physical contact greatly reduced the culturability and viability of L. thermotolerans and led to earlier cell death, compared to when these yeasts were co-fermenting without cell-cell contact. In turn, in the absence of cell-cell contact, L. thermotolerans metabolic activity led to an earlier decline in culturability in S. cerevisiae. Cell-cell contact did not result in significant differences in the major fermentation metabolites ethanol, acetic acid and lactic acid, but impacted on the production of some volatile compounds.

Background In wine fermentation starter cultures, the blending of non-Saccharomyces yeast with Saccharomyces cerevisiae to improve the complexity of wine has become common practice, but data regarding the impact of co-cultivation on yeast physiology and on genetic and metabolic regulation remain limited. Here we describe a transcriptomic analysis of mixed fermentations of Saccharomyces cerevisiae and Lachancea thermotolerans. The fermentations were carried out in carefully controlled environmental conditions in a bioreactor to reduce transcriptomic responses that would be due to factors other than the presence of the second species. Results The transcriptomic data revealed that both yeast species showed a clear response to the presence of the other. Affected genes primarily belonged to two groups: genes whose expression can be linked to the competition for certain trace elements such as copper and iron, as well as genes required for cell wall structure and integrity. Furthermore, the data revealed divergent transcriptional responses with regard to carbon metabolism in response to anoxic conditions. Conclusions The results suggest that the mixed fermentation created a more competitive and stressful environment for the two species than single strain fermentations independently from total biomass, i.e. competition between cells of the same species is less stressful, or may present a different set of challenges, than interspecies competition. The changes in cell wall and adhesion properties encoding genes suggest that the adjustment of physical contact between cells may play a direct role in the response to the presence of competing species. Electronic supplementary material The online version of this article (10.1186/s12864-019-5511-x) contains supplementary material, which is available to authorized users.

The yeast Lachancea thermotolerans is of significant biotechnological interest, and selected strains of this species have become commonly used starter cultures in wine fermentation. However, the impact of this species on wine is frequently limited by the rapid dominance of Saccharomyces cerevisiae strains which are better adapted to wine alcoholic fermentation conditions. Previous studies have shown that the major limiting factor for L. thermotolerans competitive performance in the wine ecosystem is oxygen availability, and not ethanol levels as had been previously suggested. Here we investigated the transcriptional response of L. thermotolerans to anaerobiosis in wine fermentation conditions. The data show that L. thermotolerans broadly redirects gene expression towards genes involved in central carbon metabolism, lipid metabolism, remodeling of the cell wall as well as autophagy. Furthermore, the induction of genes that are likely involved in the generation of lactate indicates a redirection of metabolic flux towards this metabolite. The data provide the first insight into the oxygen-dependent response of L. thermotolerans and suggest potential genetic targets to improve lactate production and/or anaerobic fermentation performance of this yeast.

Spontaneous wine fermentation is characterized by yeast population evolution, modulated by complex physical and metabolic interactions amongst various species. The contribution of any given species to the final wine character and aroma will depend on its numerical persistence during the fermentation process. Studies have primarily evaluated the effect of physical and chemical factors such as osmotic pressure, pH, temperature and nutrient availability on mono- or mixed-cultures comprising 2–3 species, but information about how interspecies ecological interactions in the wine fermentation ecosystem contribute to population dynamics remains scant. Therefore, in the current study, the effect of temperature and sulphur dioxide (SO2) on the dynamics of a multi-species yeast consortium was evaluated in three different matrices including synthetic grape juice, Chenin blanc and Grechetto bianco. The population dynamics were affected by temperature and SO2, reflecting differences in stress resistance and habitat preferences of the different species and influencing the production of most volatile aroma compounds. Evidently at 15 °C and in the absence of SO2 non-Saccharomyces species were dominant, whereas at 25 °C and when 30 mg/L SO2 was added S. cerevisiae dominated. Population growth followed similar patterns in the three matrices independently of the conditions. The data show that fermentation stresses lead to an individual response of each species, but that this response is strongly influenced by the interactions between species within the ecosystem. Thus, our data suggest that ecological interactions, and not only physico-chemical conditions, are a dominant factor in determining the contribution of individual species to the outcome of the fermentation.

Oxygen is sometimes deliberately introduced in winemaking at various stages to enhance yeast biomass formation and prevent stuck fermentation. However, there is limited information on how such interventions affect the dynamics of yeast populations. Our previous study in synthetic grape juice showed that oxygen supply enhances the persistence of Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia pulcherrima. The three non-Saccharomyces yeasts showed differences in growth as a function of oxygen. The present study focused on evaluating the influence of short oxygen pulses on population dynamics and the aroma profile of Chardonnay wine inoculated with L. thermotolerans and Saccharomyces cerevisiae. The results confirmed a positive effect of oxygen on the relative performance of L. thermotolerans. The mixed culture fermentation with L. thermotolerans with S. cerevisiae developed a distinct aroma profile when compared to monoculture S. cerevisiae. Specifically, a high concentration of esters, medium chain fatty acids and higher alcohols was detected in the mixed culture fermentation. The data also showed that the longer persistence of L. thermotolerans due to addition of oxygen pulses influenced the formation of major volatile compounds such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, propanol, isobutanol, butanol, isoamyl alcohol, hexanol, isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, octanoic acid, and decanoic acid.

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.

AIMS The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2 ) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B. bruxellensis cells in red grape juice. METHODS AND RESULTS Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B. bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of β-glucan, suggesting a compensatory response of the sensitive cells. CONCLUSIONS The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.

Natural alcoholic fermentation is initiated by a diverse population of several non-Saccharomyces yeast species. However, most of the species progressively die off, leaving only a few strongly fermentative species, mainly Saccharomyces cerevisiae. The relative performance of each yeast species is dependent on its fermentation capacity, initial cell density, ecological interactions as well as tolerance to environmental factors. However, the fundamental rules underlying the working of the wine ecosystem are not fully understood. Here we use variation in cell density as a tool to evaluate the impact of individual non-Saccharomyces wine yeast species on fermentation kinetics and population dynamics of a multi-species yeast consortium in synthetic grape juice fermentation. Furthermore, the impact of individual species on aromatic properties of wine was investigated, using Gas Chromatography-Flame Ionization Detector. Fermentation kinetics was affected by the inoculation treatment. The results show that some non-Saccharomyces species support or inhibit the growth of other non-Saccharomyces species in the multi-species consortium. Overall, the fermentation inoculated with a high cell density of Starmerella bacillaris displayed the fastest fermentation kinetics while fermentation inoculated with Hanseniaspora vineae showed the slowest kinetics. The production of major volatiles was strongly affected by the treatments, and the aromatic signature could in some cases be linked to specific non-Saccharomyces species. In particular, Wickerhamomyces anomalus at high cell density contributed to elevated levels of 2-Phenylethan-1-ol whereas Starm. bacillaris at high cell density resulted in the high production of 2-methylpropanoic acid and 3-Hydroxybutanone. The data revealed possible direct and indirect influences of individual non-Saccharomyces species within a complex consortium, on wine chemical composition.

Vitis vinifera is one of the most widely planted crops and holds important economic value in South Africa and Austria. Grapes obtained from this plant harbour a complex fungal community which plays a crucial role in the wine fermentation process and influences wine flavour and aroma. For many years the contributions of the natural yeasts has been eclipsed by the use of active dry yeast (ADY) inoculant, mainly of the species Saccharomyces cerevisiae. However, recent studies show a growing interest in deciphering the natural microbial diversity and in promoting its persistence during fermentation in order to enhance wine typicity. The current preliminary study aims to provide a first broad assessment of the fungal community fingerprint of different grape varietals from different wine producing areas in Austria and South Africa through Automated Ribosomal Intergenic Spacer Analysis (ARISA). The ARISA profiles separated the samples according to country of origin, and suggested some regional and varietal separation within each country. Future work will evaluate the contribution of these fungal communities to wine chemical composition and sensorial distinctness.

The genus Lachancea, first proposed in 2003, currently comprises 12 valid species, all found to have eight chromosomes. Lachancea spp. occupy a myriad of natural and anthropic habitats, and their geographic as well as ecological origin have been identified as key drivers in the genetic variations amongst strains of several of the species. Lachancea thermotolerans is the type species of the genus and also the most widely explored, especially for its role in fermentation environments. Indeed, L. thermotolerans is desired for its ability to acidify beer and wine through the production of lactic acid, and to enhance aroma and flavor through increased production of various compounds. Similarly, L. fermentati has been characterized for its potential contribution to the chemical composition of these beverages, albeit to a lesser extent, while other species have received little attention. Overall, members of the genus Lachancea form part of the microbiomes in many fermentation ecosystems and contribute directly or indirectly to the modulation of aroma and flavor of different products. The current review provides an overview of this genus, including the latest reports on the genetic and biochemical characteristics of member species, as well as their biotechnological potential.

Yeasts of various genera are increasingly used alongside Saccharomyces cerevisiae to drive wine fermentations owing to their positive contribution to the organoleptic profile of the resulting wines. One such yeast species is Lachancea thermotolerans. Other species of the genus Lachancea, namely, L. fermentati and L. lanzarotensis have also been isolated from the fermentation environment, but have not received the same degree of attention as L. thermotolerans. The aim of this study was to investigate the oenological potential of these three Lachancea species, regarding their expression of oenologically relevant enzymes, their fermentation attributes and the expression and location of β-glucosidase during fermentation of synthetic and real grape must (Muscat of Alexandria). In the current study we evaluated three species viz. L. thermotolerans (14 strains), L. fermentati (1 strain) and L. lanzarotensis (2 strains). Our data show that all the species and strains produced β-glucosidase but with different substrate specificities. Moreover, L. theromotolerans and L. fermentati also produced β-xylosidase. H2S production, SO2 and ethanol tolerance was variable between species and strains, with the L. lanzarotensis and L. fermentati displaying considerably high H2S production while L. thermotolerans and L. fermentati displayed higher ethanol tolerance. Furthermore, L. fermentati showed higher SO2 tolerance and could proliferate at 20 mg/L total SO2. Interestingly, an increase in β-glucosidase activity during fermentation did not result in a significant increase in monoterpene concentrations. However, mixed-fermentations with L. fermentati and L. thermotolerans Concerto enhanced geraniol levels. The data show that this activity was mostly cell-associated and constitutively expressed. Sequential fermentations with the Lachancea spp. and S. cerevisiae resulted in wines with significantly altered chemical compositions compared to that obtained from S. cerevisiae inoculated alone. Wines produced from L. thermotolerans and L. lanzarotensis mixed culture fermentations exhibited similar volatile compound composition. Conversely, L. fermentati produced chemically distinct wines consistently associated with high isobutanol and isobutyric acid, and higher monoterpenes. In particular, linalool and geraniol had potential to make perceivable aroma contribution (OAV ≥ 1).

This study investigated the microbial diversity present in grape juice and in the early stage of alcoholic fermentation. The grapes were obtained from a South African biodynamic vineyard in three consecutive vintages (2012, 2013, and 2014). Culture-dependent and culture-independent approaches were used to investigate yeast and bacterial diversity. For the culture-independent approach, Automated Ribosomal Intergenic Spacer Analysis (ARISA) was employed. Using basic microbiological analyses, 4, 12, and 15 yeast species were obtained in 2012, 2013, and 2014, respectively. In contrast, ARISA profiles revealed 13 fungal peaks for the year 2013 and 14 for 2012 and 2014. Out of 14 peaks in 2012, two peaks were identified as probable corresponding fungal Operational Taxonomical Units (OTUs) and six peaks were identified for the years 2013 and 2014. The bacterial ARISA revealed ten, seven, and 12 peaks for these three years, respectively. Furthermore, the same technique was used to assess the evolution of the fungal community in the first three days of fermentation of the 2013 grape must. The data show that the yeast population diversity declined rapidly and revealed 12, 10, and 6 peaks for days 1, 2, and 3, respectively. The study demonstrated the suitability of ARISA for studying microbial diversity and dynamics in grape must and during wine fermentation.

Natural, also referred to as spontaneous wine fermentations, are carried out by the native microbiota of the grape juice, without inoculation of selected, industrially produced yeast or bacterial strains. Such fermentations are commonly initiated by non-Saccharomyces yeast species that numerically dominate the must. Community composition and numerical dominance of species vary significantly between individual musts, but Saccharomyces cerevisiae will in most cases dominate the late stages of the fermentation and complete the process. Nevertheless, non-Saccharomyces species contribute significantly, positively or negatively, to the character and quality of the final product. The contribution is species and strain dependent and will depend on each species or strain's absolute and relative contribution to total metabolically active biomass, and will therefore, be a function of its relative fitness within the microbial ecosystem. However, the population dynamics of multispecies fermentations are not well understood. Consequently, the oenological potential of the microbiome in any given grape must, can currently not be evaluated or predicted. To better characterize the rules that govern the complex wine microbial ecosystem, a model yeast consortium comprising eight species commonly encountered in South African grape musts and an ARISA based method to monitor their dynamics were developed and validated. The dynamics of these species were evaluated in synthetic must in the presence or absence of S. cerevisiae using direct viable counts and ARISA. The data show that S. cerevisiae specifically suppresses certain species while appearing to favor the persistence of other species. Growth dynamics in Chenin blanc grape must fermentation was monitored only through viable counts. The interactions observed in the synthetic must, were upheld in the natural must fermentations, suggesting the broad applicability of the observed ecosystem dynamics. Importantly, the presence of indigenous yeast populations did not appear to affect the broad interaction patterns between the consortium species. The data show that the wine ecosystem is characterized by both mutually supportive and inhibitory species. The current study presents a first step in the development of a model to predict the oenological potential of any given wine mycobiome.

From the time when microbial activity in wine fermentation was first demonstrated, the microbial ecology of the vineyard, grape, and wine has been extensively investigated using culture-based methods. However, the last 2 decades have been characterized by an important change in the approaches used for microbial examination, due to the introduction of DNA-based community fingerprinting methods such as DGGE, SSCP, T-RFLP, and ARISA. These approaches allowed for the exploration of microbial community structures without the need to cultivate, and have been extensively applied to decipher the microbial populations associated with the grapevine as well as the microbial dynamics throughout grape berry ripening and wine fermentation. These techniques are well-established for the rapid more sensitive profiling of microbial communities; however, they often do not provide direct taxonomic information and possess limited ability to detect the presence of rare taxa and taxa with low abundance. Consequently, the past 5 years have seen an upsurge in the application of high-throughput sequencing methods for the in-depth assessment of the grapevine and wine microbiome. Although a relatively new approach in wine sciences, these methods reveal a considerably greater diversity than previously reported, and identified several species that had not yet been reported. The aim of the current review is to highlight the contribution of high-throughput next generation sequencing and metagenomics approaches to vineyard microbial ecology especially unraveling the influence of vineyard management practices on microbial diversity.

The current study is the first one to demonstrate the wine fermentation potential of members of several species of the genus Kazachstania including strains derived from grape must. The fermentation characteristics were evaluated in synthetic grape juice medium and in Sauvignon blanc. Our data show that none of the species evaluated could ferment to dryness in monoculture fermentations. However, at least 75% of the sugar was consumed before the fermentations got stuck. In mixed-culture fermentations with Saccharomyces cerevisiae diverse aroma profiles were evident especially in Sauvignon blanc fermentations. Four distinct potential aroma associations were identified: (i) Kazachstania solicola-vinegar and solvent-like, (ii) Kazachstania hellenica-spirituous, cheesy, (iii) Kazachstania aerobia CBS-fruity, floral (iv) K. aerobia IWBT, Kazachstania unispora and Kazachstania servazii-rancid, harsh. Furthermore, strain variation was apparent as the two K. aerobia strains displayed distinct karyotypes and aroma potential. Our data show that although members of the genus Kazachstania are typically encountered at low frequency in grape must, some of the species have positive aroma attributes that should be explored further.

Wine spoilage associated with Brettanomyces bruxellensis is a major concern for winemakers. An effective and reliable method to control the proliferation of this yeast is therefore of utmost importance. To achieve this purpose, sulphur dioxide (SO2) is commonly employed but the efficiency of this chemical compound is subject to wine composition and it can elicit allergic reactions in some consumers. Biological alternatives are therefore actively sought. The current study focused on identifying and characterizing killer toxins which are antimicrobial compounds that show potential in inhibiting B. bruxellensis in wine. Two killer toxins, CpKT1 and CpKT2, from the wine isolated yeast Candida pyralidae were identified and partially characterized. The two proteins had a molecular mass above 50kDa and exhibited killer activity against several B. bruxellensis strains especially in grape juice. They were active and stable at pH3.5-4.5, and temperatures between 15 and 25°C which are compatible with winemaking conditions. Furthermore, the activity of these killer toxins was not affected by the ethanol and sugar concentrations typically found in grape juice and wine. In addition, these killer toxins inhibited neither the Saccharomyces cerevisiae nor the lactic acid bacteria strains tested. These preliminary results indicated that the application of these toxins will have no effect on the main microbial agents that drive alcoholic and malolactic fermentations and further highlight the potential of using these toxins as agents to control the development of B. bruxellensis in grape juice or wine.

The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis ( TPS1 , encoding trehalose-6-phosphate synthase), central glycolysis ( TDH3 , encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway ( ZWF1 , encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle ( ACO1 and ACO2 , encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type ( tps1 Δ and tdh3 Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines.

The sequential or co-inoculation of grape must with non-Saccharomyces yeast species and Saccharomyces cerevisiae wine yeast strains has recently become a common practice in winemaking. The procedure intends to enhance unique aroma and flavor profiles of wine. The extent of the impact of non-Saccharomyces strains depends on their ability to produce biomass and to remain metabolically active for a sufficiently long period. However, mixed-culture wine fermentations tend to become rapidly dominated by S. cerevisiae, reducing or eliminating the non-Saccharomyces yeast contribution. For an efficient application of these yeasts, it is therefore essential to understand the environmental factors that modulate the population dynamics of such ecosystems. Several environmental parameters have been shown to influence population dynamics, but their specific effect remains largely uncharacterized. In this study, the population dynamics in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species: Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, was investigated as a function of oxygen availability. In all cases, oxygen availability strongly influenced population dynamics, but clear species-dependent differences were observed. Our data show that L. thermotolerans required the least oxygen, followed by T. delbrueckii and M. pulcherrima. Distinct species-specific chemical volatile profiles correlated in all cases with increased persistence of non-Saccharomyces yeasts, in particular increases in some higher alcohols and medium chain fatty acids. The results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and suggests that more stringent aeration strategies would be necessary to support the persistence of non-Saccharomyces yeasts in real must fermentations.

Halophilic and haloterant eubacteria have been isolated from different marine and hypersaline environments. Halophilic eubacteria also occur in environments typified by more than one soda lakes which are both hypersaline and extremely alkaline. These organisms have been shown to produce a wide array of hydrolytic enzymes including proteases, amylases, xylanases, cellulases as well as lipases and DNases. These enzymes are commonly applied in the production of fermented food and food supplements, in animal feed, laundry detergents and textile industries. Several studies have shown that enzymes derived from halophilic and halotolerant eubacteria are not only halostable but may also be thermostable and alkalistable. This extremophilicity make the enzymes suitable candidates in various fields of biotechnology and may even open up new application opportunities.

Red wine spoiled by the yeast Brettanomyces bruxellensis is characterised by off-odours commonly described as horse sweat, phenolic, varnish and band-aid. The growth of this yeast in wine is traditionally controlled by the use of sulphur dioxide (SO2). However, the concentration of SO2, the pH of the wine, the presence of SO2-binding chemical compounds in the wine, as well as the strain of B. bruxellensis, determine the effectiveness of SO2. Other chemical preservatives have been tested, but are not much more efficient than SO2, and methods used to clean barrels are only partially effective. Filtration of wine and the use of electric currents/fields are also reported to alter the physical and sensory properties of wine. In this context, alternative methods are currently sought to achieve full control of this yeast in wine. Killer toxins have recently been proposed to fulfil this purpose. They are antimicrobial compounds secreted by Saccharomyces and non-Saccharomyces yeasts, displaying killer activity against other yeasts and filamentous fungi. They are believed to play a role in yeast population dynamics, and this killer phenotype potentially could be exploited to inhibit the growth of undesired microorganisms within a microbial ecosystem such as that occurring in wine. In this review, non-Saccharomyces killer toxins are described and their potential application in inhibiting B. bruxellensis in wine is discussed in comparison to other tried methods and techniques.

Flocculation has primarily been studied as an important technological property of Saccharomyces cerevisiae yeast strains in fermentation processes such as brewing and winemaking. These studies have led to the identification of a group of closely related genes, referred to as the FLO gene family, which controls the flocculation phenotype. All naturally occurring S. cerevisiae strains assessed thus far possess at least four independent copies of structurally similar FLO genes, namely FLO1, FLO5, FLO9 and FLO10. The genes appear to differ primarily by the degree of flocculation induced by their expression. However, the reason for the existence of a large family of very similar genes, all involved in the same phenotype, has remained unclear. In natural ecosystems, and in wine production, S. cerevisiae growth together and competes with a large number of other Saccharomyces and many more non-Saccharomyces yeast species. Our data show that many strains of such wine-related non-Saccharomyces species, some of which have recently attracted significant biotechnological interest as they contribute positively to fermentation and wine character, were able to flocculate efficiently. The data also show that both flocculent and non-flocculent S. cerevisiae strains formed mixed species flocs (a process hereafter referred to as co-flocculation) with some of these non-Saccharomyces yeasts. This ability of yeast strains to impact flocculation behaviour of other species in mixed inocula has not been described previously. Further investigation into the genetic regulation of co-flocculation revealed that different FLO genes impact differently on such adhesion phenotypes, favouring adhesion with some species while excluding other species from such mixed flocs. The data therefore strongly suggest that FLO genes govern the selective association of S. cerevisiae with specific species of non-Saccharomyces yeasts, and may therefore be drivers of ecosystem organisational patterns. Our data provide, for the first time, insights into the role of the FLO gene family beyond intraspecies cellular association, and suggest a wider evolutionary role for the FLO genes. Such a role would explain the evolutionary persistence of a large multigene family of genes with apparently similar function.

The current study evaluated the diversity of yeast species in Cabernet Sauvignon grape must derived from three neighbouring vineyards from a similar terroir but on which significantly different management practices are employed. The fermentation kinetics and yeast population dynamics were monitored from the beginning to the end of spontaneous fermentation. The grape musts were characterised by distinct yeast populations comprising oxidative, weakly fermentative and strongly fermentative yeasts. Different combinations of dominant non-Saccharomyces yeasts were observed in each must, with significantly different assortments of dominant species, including Starmerella bacillaris (synonym Candida zemplinina), Lachancea thermotolerans, Hanseniaspora uvarum, Candida parapsilosis and Wickerhamomyces anomalus. None of these yeast consortia appeared to affect the growth of Saccharomyces cerevisiae or inhibit the overall progress of fermentation. However, the percentage of fermentative yeasts was positively correlated with the fermentation rate. Glucose and fructose consumption rates suggested active participation of both glucophilic and fructophilic yeasts from the onset of fermentation. The data highlight two parameters, viz. initial cell concentration and yeast community composition, as important fermentation drivers and open the possibility to predict fermentation behaviour based on the initial composition of the yeast community.

There was an error in the last author's name. The correct name is: Florian Franz Bauer. The correct citation is: Setati ME, Jacobson D, Andong U-C, Bauer FF (2012) The Vineyard Yeast Microbiome, a Mixed Model Microbial Map. PLoS ONE 7(12): e52609. doi:10.1371/journal.pone.0052609 The correct Author Contributions are: Conceived and designed the experiments: MES DJ FFB. Performed the experiments: MES U-CA DJ. Analyzed the data: MES U-CA DJ. Contributed reagents/materials/analysis tools: MES DJ FFB. Wrote the paper: MES DJ FFB.

A Gram-positive, non-motile, non-spore-forming actinobacterium designated strain Sua-BAC020T was isolated from brine from Sua salt pan in Botswana. The strain was alkaliphilic and moderately halophilic, displaying optimal growth at 35–37 °C, pH 9 and 2.5 % (w/v) NaCl. Comparative 16S rRNA gene sequence analysis showed that strain Sua-BAC020T belonged to the genus Nesterenkonia , sharing 96.2–99.0 % sequence similarity with the type strains of recognized species within this genus. DNA–DNA hybridization with the type strains of species that showed the closest phylogenetic affiliation, Nesterenkonia xinjiangensis (16S rRNA gene sequence similarity, 98.9 %), Nesterenkonia aethiopica (99.0 %), Nesterenkonia halophila (97.5 %), Nesterenkonia flava (97.4 %) and Nesterenkonia halobia (97.2 %), gave relatedness values of 10–45 %. The peptidoglycan type of strain Sua-BAC020T was A4α, l-Lys–Gly–d-Asp. Cells of the isolate contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and unidentified glycolipids as major polar lipids, MK-8, MK-9 and MK-7 were the predominant menaquinones, and the major fatty acids (>10 %) were anteiso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of strain Sua-BAC020T was 64.8 mol%. Based on DNA–DNA hybridization, and physiological and biochemical tests, strain Sua-BAC020T is distinct from all recognized Nesterenkonia species, suggesting that this strain represents a novel species, for which the name Nesterenkonia suensis sp. nov. is proposed. The type strain is Sua-BAC020T ( = DSM 22748T = NCCB 100309T).

A culture-dependent enrichment technique was used to isolate endo-1,4-beta-mannanase-producing fungi from a hypersaline environment. Galactomannan was used as carbon source and resulted in isolation of strains of Scopulariopsis brevicaulis, S. candida, and Verticillium dahliae. The Scopulariopsis isolates were found to be more dominant and could be isolated from consecutive evaporation ponds, whereas Verticillium was only isolated from one pond. The Scopulariopsis strains exhibited only endomannanase activity, whereas Verticillium displayed broad-activity spectrum by secreting endoxylanases and cellulases in addition to endomannanases. S. candida LMK004 and LMK008 produced 7,420 and 14,750 nkat g(-1) biomass, respectively. Endomannanase production in these strains increased with an increase in NaCl concentration up to 10% (w/v), after which both growth and enzyme production was decreased. V. dahliae LMK006 grew and produced up to 5,000 nkat g(-1) biomass endomannanase in the absence of NaCl. Increased NaCl concentration had a negative effect on this strain. S. brevicaulis LMK002 showed poor endomannanase production but a similar growth trend as the other Scopulariopsis strains. In general, the Scopulariopsis strains exhibited better halotolerance than V. dahliae and could grow in the presence of 20% NaCl on solid medium.