Microbial diversity in sherry wine biofilms and surrounding mites.

Sherry wines are film wines produced in the Jerez-Xérès-Sherry and Montilla-Moriles regions in southern Spain which require an aging process under flor biofilms, known as "biological aging". The presence of mites in Sherry wine wineries has been reported and associated with improved wine volatile properties. This work analyzes the microbial diversity in flor biofilms and mites in Sherry wine wineries using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) and ITS/gene amplification. Two mite species, Carpoglyphus lactis and Tyrophagus putrescentiae, were spotted in the sampled winery and 32 microorganism species were identified in their exoskeleton or surrounding biofilms. To our knowledge, 26 of these species were never described before in sherry wine environments. We hypothesized that mites feed on the flor biofilms as well as another type of biofilm located in barrel cracks, known by winemakers as "natas" (cream in English). These non-studied biofilms showed the highest microbiome diversity among all samples (followed by C. lactis spotted nearby) thus, representing a niche of microorganisms with potential biotechnological interest. Besides mites, Drosophila flies were spotted in the sampling areas. The role of flies and mites as vectors that transport microorganisms among different niches (i.e., flor biofilms and natas) is discussed.

Flor yeast immobilization in microbial biocapsules for Sherry wine production: microvinification approach.

Sherry wine is a pale-yellowish dry wine produced in Southern-Spain which features are mainly due to biological aging when the metabolism of biofilm-forming yeasts (flor yeasts) consumes ethanol (and other non-fermentable carbon sources) from a previous alcoholic fermentation, and produces volatile compounds such as acetaldehyde. To start aging and maintain the wine stability, a high alcohol content is required, which is achieved by the previous fermentation or by adding ethanol (fortification). Here, an alternative method is proposed which aims to produce a more economic, distinctive Sherry wine without fortification. For this, a flor yeast has been pre-acclimatized to glycerol consumption against ethanol, and later confined in a fungal-based immobilization system known as "microbial biocapsules", to facilitate its inoculum. Once aged, the wines produced using biocapsules and free yeasts (the conventional method) exhibited chemical differences in terms of acidity and volatile concentrations. These differences were evaluated positively by a sensory panel. Pre-acclimatization of flor yeasts to glycerol consumption was not successful but when cells were immobilized in fungal pellets, ethanol consumption was lower. We believe that immobilization of flor yeasts in microbial biocapsules is an economic technique that can be used to produce high quality differentiated Sherry wines.

Diversity of Cellulolytic Microorganisms Associated with the Subterranean Termite Reticulitermes grassei.

Reticulitermes grassei is a subterranean termite species that forages on woody structures of the Iberian Peninsula, and is often a building and crops pest. A total of 23 microorganisms associated with the activity of R. grassei were isolated from colonized ecosystems in southern Spain. They were morphologically and molecularly characterized, with fungi being the most prevalent ones. The fungi showed high values of optimum growth temperature, suggesting that they could be able to survive and develop in warm regions. Their cellulolytic activity was tested in carboxymethylcellulose (CMC) agar, concluding that all fungal isolates produce cellulases, and the enzymatic index (EI) was revealed in CMC agar with Gram's iodine solution, with Penicillium citrinum showing the highest EI and Trichoderma longibrachiatum the highest mycelial growth rate on CMC. A preliminary microorganism dispersion assay was carried out with the termites, concluding that these insects may have a positive influence on fungal dispersion and the subsequent colonization of new substrates. Our study suggests that fungi associated with R. grassei may potentially be of interest in biotechnological fields such as biofuel production and the food industry.

Revealing the Yeast Diversity of the Flor Biofilm Microbiota in Sherry Wines Through Internal Transcribed Spacer-Metabarcoding and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Flor yeast velum is a biofilm formed by certain yeast strains that distinguishes biologically aged wines such as Sherry wine from southern Spain from others. Although Saccharomyces cerevisiae is the most common species, 5.8 S-internal transcribed spacer (ITS) restriction fragment length polymorphism analyses have revealed the existence of non-Saccharomyces species. In order to uncover the flor microbiota diversity at a species level, we used ITS (internal transcribed spacer 1)-metabarcoding and matrix-assisted laser desorption/Ionization time of flight mass spectrometry techniques. Further, to enhance identification effectiveness, we performed an additional incubation stage in 1:1 wine:yeast extract peptone dextrose (YPD) before identification. Six species were identified: S. cerevisiae, Pichia manshurica, Pichia membranifaciens, Wickerhamomyces anomalus, Candida guillermondii, and Trichosporon asahii, two of which were discovered for the first time (C. guillermondii and Trichosporon ashaii) in Sherry wines. We analyzed wines where non-Saccharomyces yeasts were present or absent to see any potential link between the microbiota and the chemical profile. Only 2 significant volatile chemicals (out of 13 quantified), ethanol and ethyl lactate, and 2 enological parameters (out of 6 quantified), such as pH and titratable acidity, were found to differ in long-aged wines. Although results show a low impact where the non-Saccharomyces yeasts are present, these yeasts isolated from harsh environments (high ethanol and low nutrient availability) could have a potential industrial interest in fields such as food microbiology and biofuel production.