Identifying the Main Drivers in Microbial Diversity for Cabernet Sauvignon Cultivars from Europe to South Africa: Evidence for a Cultivar-Specific Microbial Fingerprint.

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.

Antifungal activity of non-conventional yeasts against Botrytis cinerea and non-Botrytis grape bunch rot fungi.

Grapes harbour a plethora of non-conventional yeast species. Over the past two decades, several of the species have been extensively characterised and their contribution to wine quality is better understood. Beyond fermentation, some of the species have been investigated for their potential as alternative biological tools to reduce grape and wine spoilage. However, such studies remain limited to a few genera. This work aimed to evaluate the antagonistic activity of grape must-derived non-conventional yeasts against Botrytis cinerea and non-Botrytis bunch-rotting moulds and to further elucidate mechanisms conferring antifungal activity. A total of 31 yeast strains representing 21 species were screened on different agar media using a dual culture technique and liquid mixed cultures, respectively. Pichia kudriavzevii was the most potent with a minimum inhibitory concentration of 102 cells/mL against B. cinerea but it had a narrow activity spectrum. Twelve of the yeast strains displayed broad antagonistic activity, inhibiting three strains of B. cinerea (B05. 10, IWBT FF1 and IWBT FF2), a strain of Aspergillus niger and Alternaria alternata. Production of chitinases and glucanases in the presence of B. cinerea was a common feature in most of the antagonists. Volatile and non-volatile compounds produced by antagonistic yeast strains in the presence of B. cinerea were analysed and identified using gas and liquid chromatography mass spectrometry, respectively. The volatile compounds identified belonged mainly to higher alcohols, esters, organosulfur compounds and monoterpenes while the non-volatile compounds were cyclic peptides and diketopiperazine. To our knowledge, this is the first report to demonstrate inhibitory effect of the non-volatile compounds produced by various yeast species.

Towards a Better Understanding of the Potential Benefits of Seaweed Based Biostimulants in Vitis vinifera L. Cultivars.

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 (Ascophyllum spp.) improves plant growth and agricultural productivity, hormonal signalling, and an improved secondary plant metabolism. It also provides an alternative to soil supplementation, avoiding some of the negative effects of fertilizers through the leaching of nutrients into groundwater sources. This review aims to provide a summary of the use of seaweed extracts in grape production and their influence on grapevine physiology and stress adaptation mechanisms.

Phenotypic characterization of cell-to-cell interactions between two yeast species during alcoholic fermentation.

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.

Inoculation of grape musts with single strains of Saccharomyces cerevisiae yeast reduces the diversity of chemical profiles of wines.

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.

Ecological interactions are a primary driver of population dynamics in wine yeast microbiota during fermentation.

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.

Corrigendum: Investigating the Effect of Selected Non-Saccharomyces Species on Wine Ecosystem Function and Major Volatiles.

[This corrects the article DOI: 10.3389/fbioe.2018.00169.].

Lachancea yeast species: Origin, biochemical characteristics and oenological significance.

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.

Investigating the biochemical and fermentation attributes of Lachancea species and strains: Deciphering the potential contribution to wine chemical composition.

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 ß-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 ß-glucosidase but with different substrate specificities. Moreover, L. theromotolerans and L. fermentati also produced ß-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 ß-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).

Investigating the Effect of Selected Non-Saccharomyces Species on Wine Ecosystem Function and Major Volatiles.

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.

Evaluating fermentation characteristics of Kazachstania spp. and their potential influence on wine quality.

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.

Co-Flocculation of Yeast Species, a New Mechanism to Govern Population Dynamics in Microbial Ecosystems.

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.

Nesterenkonia suensis sp. nov., a haloalkaliphilic actinobacterium isolated from a salt pan.

A Gram-positive, non-motile, non-spore-forming actinobacterium designated strain Sua-BAC020(T) 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-BAC020(T) 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-BAC020(T) 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-C(15:0) and anteiso-C(17 : 0). The DNA G+C content of strain Sua-BAC020(T) was 64.8 mol%. Based on DNA-DNA hybridization, and physiological and biochemical tests, strain Sua-BAC020(T) 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-BAC020(T) (= DSM 22748(T)=NCCB 100309(T)).

The vineyard yeast microbiome, a mixed model microbial map.

Vineyards harbour a wide variety of microorganisms that play a pivotal role in pre- and post-harvest grape quality and will contribute significantly to the final aromatic properties of wine. The aim of the current study was to investigate the spatial distribution of microbial communities within and between individual vineyard management units. For the first time in such a study, we applied the Theory of Sampling (TOS) to sample gapes from adjacent and well established commercial vineyards within the same terroir unit and from several sampling points within each individual vineyard. Cultivation-based and molecular data sets were generated to capture the spatial heterogeneity in microbial populations within and between vineyards and analysed with novel mixed-model networks, which combine sample correlations and microbial community distribution probabilities. The data demonstrate that farming systems have a significant impact on fungal diversity but more importantly that there is significant species heterogeneity between samples in the same vineyard. Cultivation-based methods confirmed that while the same oxidative yeast species dominated in all vineyards, the least treated vineyard displayed significantly higher species richness, including many yeasts with biocontrol potential. The cultivatable yeast population was not fully representative of the more complex populations seen with molecular methods, and only the molecular data allowed discrimination amongst farming practices with multivariate and network analysis methods. Importantly, yeast species distribution is subject to significant intra-vineyard spatial fluctuations and the frequently reported heterogeneity of tank samples of grapes harvested from single vineyards at the same stage of ripeness might therefore, at least in part, be due to the differing microbiota in different sections of the vineyard.

Screening and identification of endomannanase-producing microfungi from hypersaline environments.

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.