High-Throughput Sequencing Approach to Analyze the Effect of Aging Time and Barrel Usage on the Microbial Community Composition of Red Wines.

Wine aged in barrels or bottles is susceptible to alteration by microorganisms that affect the final product quality. However, our knowledge of the microbiota during aging and the factors modulating the microbial communities is still quite limited. The present work uses high-throughput sequencing (HTS) techniques to deal with the meta-taxonomic characterization of microbial consortia present in red wines along 12 months aging. The wines obtained from two different grape varieties were aged at two different cellars and compared based on time of wine aging in the barrels, previous usage of the barrels, and differences between wine aging in oak barrels or glass bottles. The aging in barrels did not significantly affect the microbial diversity but changed the structure and composition of fungal and bacterial populations. The main microorganisms driving these changes were the bacterial genera Acetobacter, Oenococcus, Lactobacillus, Gluconobacter, Lactococcus, and Komagataeibacter and the fungal genera Malassezia, Hanseniaspora, and Torulaspora. Our results showed that the oak barrels increased effect on the microbial diversity in comparison with the glass bottles, in which the microbial community was very similar to that of the wine introduced in the barrels at the beginning of the aging. Furthermore, wine in the bottles harbored higher proportion of Lactobacillus but lower proportion of Acetobacter. Finally, it seems that 1 year of previous usage of the barrels was not enough to induce significant changes in the diversity or composition of microbiota through aging compared with new barrels. This is the first meta-taxonomic study on microbial communities during wine aging and shows that the microorganism composition of barrel-aged wines was similar at both cellars. These results hint at the possibility of a common and stable microbiota after aging in the absence of exogenous alterations. Further corroborations on the current outcome would be valuable for the comparison and detection of microbial alterations during aging that could potentially prevent economic losses in the wine industry.

Qualitative Factor-Based Comparison of NMR, Targeted and Untargeted GC-MS and LC-MS on the Metabolomic Profiles of Rioja and Priorat Red Wines.

Wine origin and ageing are two factors related to wine quality which in turn is associated to wine metabolome. Currently, new metabolomic techniques and proper statistics procedures allow accurate profiling of wine metabolome. Thus, the main goal was to evaluate different metabolomic methodologies on their ability to provide patterns on the wine metabolome based on selected factors, such as ageing of barrel-aged wine (factor time), prior usage of the barrels (factor barrel-type), and differences between wine ageing in barrels or glass bottles (factor bottled-wine). In the current study, we implement NMR, targeted and untargeted GC-MS and LC-MS metabolomic analytical techniques so as to gain insights into the volatile and nonvolatile wine metabolome composition of red wines from two cellars located in the only two Spanish Qualified Appellations of Origin; DOQ Priorat and DOCa Rioja regions. Overall, 95 differentially significant metabolites were identified facilitating the evaluation of the analytical methodologies performance and finding common trends of those metabolites depending on the considered factor. The results did not favor NMR as an effective technique on the current dataset whereas suggested LC-MS as an adequate technique for revealing differences based on the factor time, targeted GC-MS on the factor barrel-type, and untargeted GC-MS on the factor bottled-wine. Thus, a combination of different metabolomic techniques is necessary for a complete overview of the metabolome changes. These results ease the selection of the correct methodology depending on the specific factor investigated.

Geographical Origin Has a Greater Impact on Grape Berry Fungal Community than Grape Variety and Maturation State.

We used barcoded sequencing to analyze the eukaryotic population in the grape berries at different ripening states in four Australian vineyards. Furthermore, we used an innovative compositional data analysis for assessing the diversity of microbiome communities. The novelty was the introduction of log-ratio balances between the detected genera. Altogether, our results suggest that fungal communities were more impacted by the geographical origin of the Australian vineyards than grape variety and harvest time. Even if the most abundant genera were Aureobasidium and Mycosphaerella, they were ubiquitous to all samples and were not discriminative. In fact, the balances and the fungal community structure seemed to be greatly affected by changes of the genera Penicillium, Colletotrichum, Aspergillus, Rhodotorula, and Botrytis. These results were not evident from the comparison of relative abundance based on OTU counts alone, remarking the importance of the balance analysis for microbiome studies.

Brettanomyces bruxellensis wine isolates show high geographical dispersal and long persistence in cellars.

Brettanomyces bruxellensis is the main wine spoiler yeast all over the world, yet the structure of the populations associated with winemaking remains elusive. In this work, we considered 1411 wine isolates from 21 countries that were genotyped using twelve microsatellite markers. We confirmed that B. bruxellensis isolates from wine environments show high genetic diversity, with 58 and 42% of putative triploid and diploid individuals respectively distributed in 5 main genetic groups. The distribution in the genetic groups varied greatly depending on the country and/or the wine-producing region. However, the two possible triploid wine groups showing sulfite resistance/tolerance were identified in almost all regions/countries. Genetically identical isolates were also identified. The analysis of these clone groups revealed that a given genotype could be isolated repeatedly in the same winery over decades, demonstrating unsuspected persistence ability. Besides cellar residency, a great geographic dispersal was also evidenced, with some genotypes isolated in wines from different continents. Finally, the study of old isolates and/or isolates from old vintages revealed that only the diploid groups were identified prior 1990 vintages. The putative triploid groups were identified in subsequent vintages, and their proportion has increased steadily these last decades, suggesting adaptation to winemaking practices such as sulfite use. A possible evolutionary scenario explaining these results is discussed.

Analysis of the NCR Mechanisms in Hanseniaspora vineae and Saccharomyces cerevisiae During Winemaking.

There is increasing interest in the use of non-Saccharomyces yeasts in winemaking due to their positive attributes. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good fermentation capacity and an increase in aromatic properties. However, this yeast represents a concern in mixed culture fermentation because of its nutrient consumption, especially nitrogen, as its mechanisms of regulation and consumption are still unknown. In this study, we analyzed the nitrogen consumption, as well as the nitrogen catabolism repression (NCR) mechanism, in two genome-sequenced H. vineae strains, using synthetic must fermentations. The use of synthetic must with an established nitrogen content allowed us to study the NCR mechanism in H. vineae, following the amino acid and ammonia consumption, and the expression of genes known to be regulated by the NCR mechanism in S. cerevisiae, AGP1, GAP1, MEP2, and PUT2. H. vineae exhibited a similar amino acid consumption and gene expression profile to S. cerevisiae. However, the wine strain of S. cerevisiae QA23 consumed ammonia and valine more quickly and, in contrast, tyrosine and tryptophan more slowly, than the H. vineae strains. Our results showed a similar behavior of nitrogen regulation in H. vineae and S. cerevisiae, indicating the presence of the NCR mechanism in this Hanseniaspora yeast differentiated before the whole genome duplication event of the Saccharomyces complex. Future study will elucidate if the NCR mechanism is the only strategy used by H. vineae to optimize nitrogen consumption.

Cell size distributions of soil bacterial and archaeal taxa.

Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes in situ, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 µm to 0.2 µm) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 µm in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including Acidobacteria, Gemmatimonadetes, Crenarchaeota, Verrucomicrobia, and Elusimicrobia). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized.

Temporal variability in the diversity and composition of stream bacterioplankton communities.

Bacterioplankton in freshwater streams play a critical role in stream nutrient cycling. Despite their ecological importance, the temporal variability in the structure of stream bacterioplankton communities remains understudied. We investigated the composition and temporal variability of stream bacterial communities and the influence of physicochemical parameters on these communities. We used barcoded pyrosequencing to survey bacterial communities in 107 streamwater samples collected from four locations in the Colorado Rocky Mountains from September 2008 to November 2009. The four sampled locations harboured distinct communities yet, at each sampling location, there was pronounced temporal variability in both community composition and alpha diversity levels. These temporal shifts in bacterioplankton community structure were not seasonal; rather, their diversity and composition appeared to be driven by intermittent changes in various streamwater biogeochemical conditions. Bacterial communities varied independently of time, as indicated by the observation that communities in samples collected close together in time were no more similar than those collected months apart. The temporal turnover in community composition was higher than observed in most previously studied microbial, plant or animal communities, highlighting the importance of stochastic processes and disturbance events in structuring these communities over time. Detailed temporal sampling is important if the objective is to monitor microbial community dynamics in pulsed ecosystems like streams.

Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities.

The microbial world has been shown to hold an unimaginable diversity. The use of rRNA genes and PCR amplification to assess microbial community structure and diversity present biases that need to be analyzed in order to understand the risks involved in those estimates. Herein, we show that PCR amplification of specific sequence targets within a community depends on the fractions that those sequences represent to the total DNA template. Using quantitative, real-time, multiplex PCR and specific Taqman probes, the amplification of 16S rRNA genes from four bacterial species within a laboratory community were monitored. Results indicate that the relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template. Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the 'rare biosphere'. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa which are required to decipher the complete extent of microbial diversity in nature.

Moonmilk deposits originate from specific bacterial communities in Altamira Cave (Spain).

The influence of bacterial communities on the formation of carbonate deposits such as moonmilk was investigated in Altamira Cave (Spain). The study focuses on the relationship between the bacterial communities at moonmilk deposits and those forming white colonizations, which develop sporadically throughout the cave. Using molecular fingerprinting of the metabolically active bacterial communities detected through RNA analyses, the development of white colonizations and moonmilk deposits showed similar bacterial profiles. White colonizations were able to raise the pH as a result of their metabolism (reaching in situ pH values above 8.5), which was proportional to the nutrient supply. Bacterial activity was analyzed by nanorespirometry showing higher metabolic activity from bacterial colonizations than uncolonized areas. Once carbonate deposits were formed, bacterial activity decreased drastically (down to 5.7% of the white colonization activity). This study reports on a specific type of bacterial community leading to moonmilk deposit formation in a cave environment as a result of bacterial metabolism. The consequence of this process is a macroscopic phenomenon of visible carbonate depositions and accumulation in cave environments.

Differential effects of distinct bacterial biofilms in a cave environment.

Current microbial surveys using molecular methods provide us with critical information on the major components of natural bacterial communities. However, limited investigation has been performed on the influence of bacterial metabolism on the environment. In this study, we analyzed the pH generated by distinct bacterial communities in a cave environment. Different bacterial biofilms developing on the walls of the cave were visually distinguished by their colorations (e.g., white, yellow, and gray) and mineral depositions, and previous studies have reported on their bacterial diversity and distribution. Using pH microelectrodes, we carried out in situ measurements and were able to detect differences among these bacterial biofilms. White biofilms and carbonate depositions resulted in alkaline pH values. Gray biofilms also increased the pH although these values remained lower than in white biofilms. A combination of gray-white biofilms resulted in alkaline pH values with highest values at the white edge of the colonies. Yellow biofilms generated a slightly acid pH. These results suggest that different bacterial communities can lead to distinct effects on their environment, for instance, precipitation or dissolution of carbonates in caves. These results add information about metabolic response to current knowledge from bacterial diversity surveys, providing information on the interaction between complex bacterial communities and the geological substrate.

Assessment of bacterial and fungal growth on natural substrates: consequences for preserving caves with prehistoric paintings.

The most representative bacterium (Pseudonocardia sp.) and fungus (Fusarium sp.) from the microbial communities of a cave containing paleolithic paintings were isolated and their growth on natural substrates assessed. Growth was tested at the in situ and optimal, laboratory growth temperature. Development was analyzed with and without supplemented nutrients (glucose, ammonium, phosphate, peptone). Results showed that the assayed bacterium on natural substrate was able to develop best at in situ temperature and the addition of organic nutrients and/or phosphate enhanced its growth. The growth of the assayed fungus, however, was limited by low temperature and the availability of ammonium. These results confirm a differential behavior of microorganisms between the laboratory and the natural environments and could explain previous invasion of fungi reported for some caves with prehistoric paintings.

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow-pigmented, oxalotrophic bacteria.

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1(T), YOx(T) and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1(T), YOx(T) and NS13 belong to the Betaproteobacteria, most closely related to Oxalicibacterium flavum TA17(T) (97.2-99.7% sequence similarity). The major whole-cell fatty acids were C(16:0), C(16:1)omega7c and C(17:0) cyclo. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1(T) and YOx(T) from O. flavum TA17(T) and from each other. Therefore, it is concluded that the strains OD1(T) and YOx(T) represent novel species within the genus Oxalicibacterium, for which the names Oxalicibacterium horti sp. nov. (type strain OD1(T)=DSM 21640(T)=NBRC 13594(T)) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx(T)=DSM 21641(T)=CCM 2767(T)) are proposed.