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The impact of global warming on biological communities colonizing European alpine ecosystems was recently studied. Hexagonal open top chambers (OTCs) were used for simulating a short-term in situ warming (estimated around 1°C) in some alpine soils to predict the impact of ongoing climate change on resident microbial communities. Total microbial DNA was extracted from soils collected either inside or outside the OTCs over 3 years of study. Bacterial and fungal rRNA copies were quantified by qPCR. Metabarcoding sequencing of taxonomy target genes was performed (Illumina MiSeq) and processed by bioinformatic tools. Alpha- and beta-diversity were used to evaluate the structure of bacterial and fungal communities. qPCR suggests that, although fluctuations have been observed between soils collected either inside and outside the OTCs, the simulated warming induced a significant (p < 0.05) shift only for bacterial abundance. Likewise, significant (p < 0.05) changes in bacterial community structure were detected in soils collected inside the OTCs, with a clear increase of oligotrophic taxa. On the contrary, fungal diversity of soils collected either inside and outside the OTCs did not exhibit significant (p < 0.05) differences, suggesting that the temperature increase in OTCs compared to ambient conditions was not sufficient to change fungal communities.

Microplastics are emerging pollutant found in many ecosystems including soil. Within them, polyvinyl chloride (PVC) is one of the most toxic polymers and is known for its remarkable resistance to degradation. The recalcitrant nature of PVC and the improper waste disposal could cause serious environmental concerns. In addition, the possible fragmentation and accumulation of small plastic particles in agricultural soils might have impacts on soil chemical and microbiological properties. Based on these considerations, a microcosm experiment was set up to investigate the effects of PVC microplastics (0.021% w/w) on soil chemical properties and soil bacterial and fungal communities at different incubation times (from 3 to 360 days).Among chemical parameters, soil CO2 emissions, fluorescein diacetate hydrolysis (FDA), total organic C (TOC), total N, water extractable organic C (WEOC), water extractable N (WEN) and SUVA254 were investigated, while the structure of soil microbial communities was studied at different taxonomic levels (phylum and genus) by sequencing bacterial 16S and fungal ITS2 rDNA (Illumina MiSeq). The number and the dimensions of PVC particles were also evaluated after one year of experiment.The results showed that the presence of PVC particles in soil caused significant (p < 0.05) variations in chemical parameters in short- and medium-term, thus suggesting that the presence of this polymer in soil can affect the turnover of organic matter. In the long period, instead, an increase of the soil enzymatic activity (FDA) was observed.The analysis of microbiological parameters showed that PVC microplastics significantly affected (p < 0.05) the structure of soil microbial communities changing the abundances of specific bacterial and fungal taxa: Acidobacteria, Actinobacteria, Bacteroides, Candidatus_Saccharibacteria, and Proteobacteria, , among bacteria, and Ascomycota, Basidiomycota, and Mortierellomycota among fungi, suggesting that the impact of this polymer could be taxa-dependent.A significant (p < 0.05) decrease of the number and dimensions of PVC particles was also detected after one year of incubation, supposing a possible role of microbial community on polymer degradation.

In the ice-free areas of Victoria Land in continental Antarctica, where the conditions reach the limits for life sustainability, highly adapted and extreme-tolerant microbial communities exploit the last habitable niches inside porous rocks (i.e. cryptoendolithic communities). These guilds host the main standing biomass and principal, if not sole, contributors to environmental/biogeochemical cycles, driving ecosystem processes and functionality in these otherwise dead lands. Although knowledge advances on their composition, ecology, genomic and metabolic features, a large-scale perspective of occurring interactions and interconnections within and between endolithic fungal assemblages is still lacking to date. Unravelling the tight relational network among functional guilds in the Antarctic cryptoendolithic communities may represent a main task. Aiming to fill this knowledge gap, we performed a correlation-network analysis based on amplicon-sequencing data of 74 endolithic microbiomes collected throughout Victoria Land. Endolithic communities' compositional pattern was largely dominated by Lichenized fungi group (83.5%), mainly represented by Lecanorales and Lecideales, followed by Saprotrophs (14.2%) and RIF+BY (2.4%) guilds led by Tremellales and Capnodiales respectively. Our findings highlighted that fungal functional guilds' relational spectrum was dominated by cooperative interactions led by lichenised and black fungi, deeply engaged in community trophic sustain and protection, respectively. On the other hand, a few negative correlations found may help in preserving niche boundaries between microbes living in such strict spatial association.

Five yeast strains were isolated from soil and sediments collected from Alps and Apennines glaciers during sampling campaigns carried out in summer 2007 and 2017, respectively. Based on morphological and physiological tests and on phylogenetic analyses reconstructed with ITS and D1/D2 sequences, the five strains were considered to belong to two related but hitherto unknown species within the genus Mrakia, in an intermediate position between Mrakia cryoconiti and Mrakia arctica. The names Mrakia stelviica (holotype DBVPG 10734T) and Mrakia montana (holotype DBVPG 10736T) are proposed for the two novel species and a detailed description of their morphological, physiological and phylogenetic features are presented. Both species fermented glucose, sucrose and trehalose, which is an uncommon feature in basidiomycetous yeasts, and showed septate hyphae with teliospore formation.

A perennially frozen lake at Boulder Clay site (Victoria Land, Antarctica), characterized by the presence of frost mounds, have been selected as an in situ model for ecological studies. Different samples of permafrost, glacier ice and brines have been studied as a unique habitat system. An additional sample of brines (collected in another frozen lake close to the previous one) was also considered. Alpha- and beta-diversity of fungal communities showed both intra- and inter-cores significant (p < 0.05) differences, which suggest the presence of interconnection among the habitats. Therefore, the layers of frost mound and the deep glacier could be interconnected while the brines could probably be considered as an open habitat system not interconnected with each other. Moreover, the absence of similarity between the lake ice and the underlying permafrost suggested that the lake is perennially frozen based. The predominance of positive significant (p < 0.05) co-occurrences among some fungal taxa allowed to postulate the existence of an ecological equilibrium in the habitats systems. The positive significant (p < 0.05) correlation between salt concentration, total organic carbon and pH, and some fungal taxa suggests that a few abiotic parameters could drive fungal diversity inside these ecological niches.

With great interest, we read the article by Moshirfar et al. [...]

The diversity and composition of endolithic bacterial diversity of several locations in McMurdo Dry Valleys (Continental Antarctica) were explored using amplicon sequencing, targeting the V3 and V4 of the 16S region. Despite the increasing interest in edaphic factors that drive bacterial community composition in Antarctic rocky communities, few researchers focused attention on the direct effects of sun exposure on bacterial diversity; we herein reported significant differences in the northern and southern communities. The analysis of β-diversity showed significant differences among sampled localities. For instance, the most abundant genera found in the north-exposed rocks were Rhodococcus and Blastococcus in Knobhead Mt.; Ktedonobacter and Cyanobacteria Family I Group I in Finger Mt.; Rhodococcus and Endobacter in University Valley; and Segetibacter and Tetrasphaera in Siegfried Peak samples. In south-exposed rocks, instead, the most abundant genera were Escherichia/Shigella and Streptococcus in Knobhead Mt.; Ktedonobacter and Rhodococcus in Finger Mt.; Ktedonobacter and Roseomonas in University Valley; and Blastocatella, Cyanobacteria Family I Group I and Segetibacter in Siegfried Peak. Significant biomarkers, detected by the Linear discriminant analysis Effect Size, were also found among north- and south-exposed communities. Besides, the large number of positive significant co-occurrences may suggest a crucial role of positive associations over competitions under the harsher conditions where these rock-inhabiting microorganisms spread. Although the effect of geographic distances in these extreme environments play a significant role in shaping biodiversity, the study of an edaphic factor, such as solar exposure, adds an important contribution to the mosaic of microbial biodiversity of Antarctic bacterial cryptoendolithic communities.

The impact of climate change in the European Alps has been roughly twice the global average, dramatically reducing permafrost extent and thickening of its active layer. Therefore, the study of the abiotic factors (i.e. chemical/physical parameters) affecting the microbial diversity inhabiting Alpine permafrost appears to be of dramatic relevance. Within the European Alps, the Stelvio area exhibits these effects in a particularly evident way, with important consequences on microbial ecosystems. Therefore, microbial communities inhabiting a permafrost core collected in the Scorluzzo active rock glacier (Stelvio Pass, Italian Central Alps) were investigated along a depth gradient (410 to 524 cm from the surface). The taxonomic structures of bacterial and fungal communities were investigated via a next-generation sequencing (NGS) approach (Illumina MiSeq), targeting the bacterial V3-V4 regions of 16S rDNA and the fungal ITS2 region. Abiotic soil factors (grain size, electrical conductivity, ice/water content, pH, Loss-on-Ignition - LOI, total and organic carbon, nitrogen and phosphorous) were analysed. Richness and Shannon-H diversity indices were correlated to abiotic factors. Bacterial diversity was significantly (p

This is the accepted manuscript of the paper: Borruso L., Sannino C., Selbmann L., Battistel D., Zucconi L., Azzaro M., Turchetti B., Buzzini P., Guglielmin M., (2018). A thin ice layer segregates two distinct fungal communities in Antarctic brines from Tarn Flat (Northern Victoria Land). Scientific Reports 8: 6582 | DOI:10.1038/s41598-018-25079-3 Abstract Brines are hypersaline solutions which have been found within the Antarctic permafrost from the Tarn Flat area (Northern Victoria Land). Here, an investigation on the possible presence and diversity of fungal life within those peculiar ecosystems has been carried out for the first time. Brines samples were collected at 4- and 5-meter depths (TF1 and TF2, respectively), from two brines separated by a thin ice layer. The samples were analyzed via Illumina MiSeq targeting the ITS region specific for both yeasts and filamentous fungi. An unexpected high alpha diversity was found. Beta diversity analysis revealed that the two brines were inhabited by two phylogenetically diverse fungal communities (Unifrac value: 0.56, p value < 0.01; Martin’s P-test p-value < 0.001) characterized by several specialist taxa. The most abundant fungal genera were Candida sp., Leucosporidium sp., Naganishia sp. and Sporobolomyces sp. in TF1, and Leucosporidium sp., Malassezia sp., Naganishia sp. and Sporobolomyces sp. in TF2. A few hypotheses on such differentiation have been done: i) the different chemical and physical composition of the brines; ii) the presence in situ of a thin layer of ice, acting as a physical barrier; and iii) the diverse geological origin of the brines.

The involvement of DNA methylation in the response to cold stress of two different yeast species (Naganishia antarctica, psychrophilic, and Naganishia albida, psychrotolerant), exhibiting different temperature aptitudes, has been studied. Consecutive incubations at respective optimum temperatures, at 4 &deg, C (cold stress) and at optimum temperatures again, were performed. After Methylation Sensitive Amplified Polymorphism (MSAP) fingerprints a total of 550 and 423 clear and reproducible fragments were amplified from N. antarctica and N. albida strains, respectively. The two Naganishia strains showed a different response in terms of level of DNA methylation during cold stress and recovery from cold stress. The percentage of total methylated fragments in psychrophilic N. antarctica did not show any significant change. On the contrary, the methylation of psychrotolerant N. albida exhibited a nonsignificant increase during the incubation at 4 &deg, C and continued during the recovery step, showing a significant difference if compared with control condition, resembling an uncontrolled response to cold stress. A total of 12 polymorphic fragments were selected, cloned, and sequenced. Four fragments were associated to genes encoding for elongation factor G and for chitin synthase export chaperon. To the best of our knowledge, this is the first study on DNA methylation in the response to cold stress carried out by comparing a psychrophilic and a psychrotolerant yeast species.

The effect of altitude and season on abundance and diversity of the culturable heterotrophic bacterial and yeast community was examined at four forest sites in the Italian Alps along an altitude gradient (545–2000 m). Independently of altitude, bacteria isolated at 0 °C (psychrophiles) were less numerous than those recovered at 20 °C. In autumn, psychrophilic bacterial population increased with altitude. The 1194 bacterial strains were primarily affiliated with the classes Alpha-, Beta-, Gammaproteobacteria, Spingobacteriia and Flavobacteriia. Fifty-seven of 112 operational taxonomic units represented potential novel species. Strains isolated at 20 °C had a higher diversity and showed similarities in taxa composition and abundance, regardless of altitude or season, while strains isolated at 0 °C showed differences in community composition at lower and higher altitudes. In contrast to bacteria, yeast diversity was season-dependent: site- and altitude-specific effects on yeast diversity were only detected in spring. Isolation temperature affected the relative proportions of yeast genera. Isolations recovered 719 strains, belonging to the classes Dothideomycetes, Saccharomycetes, Tremellomycetes and Mycrobotryomycetes. The presence of few dominant bacterial OTUs and yeast species indicated a resilient microbial population that is not affected by season or altitude. Soil nutrient contents influenced significantly abundance and diversity of culturable bacteria, but not of culturable yeasts. Electronic supplementary material The online version of this article (doi:10.1007/s00792-016-0874-2) contains supplementary material, which is available to authorized users.

Since immemorial time beer is the product of wort fermentation catalyzed by the cells of two main yeast species, namely, Saccharomyces cerevisiae and Saccharomyces pastorianus, with hop addition. However, in recent years the beer market changed completely and novel types of beers, such as low-alcohol and low-calorie beers and spontaneously fermented or flavored beers, have become very popular. These different beers vary for the nature and amounts of water, cereals, hops, and other additives, techniques used for production, storage, and consumption, and also yeasts used as starter. Indeed, some non-conventional yeasts have been recently proposed as starter cultures for brewing.

Tree fluxes are sugar-rich, sometimes ephemeral, substrates occurring on sites where tree sap (xylem or phloem) is leaking through damages of tree bark. Tree sap infested with microorganisms has been the source of isolation of many species, including the biotechnologically relevant carotenoid yeast Phaffia rhodozyma. Tree fluxes recently sampled in Germany yielded 19 species, including several psychrophilic yeasts of the genus Mrakia. Four strains from tree fluxes represented a potential novel Mrakia species previously known from two isolates from superficial glacial melting water of Calderone Glacier (Italy). The Italian isolates, originally identified as Mrakia aquatica, and two strains from Germany did not show any sexual structures. But another culture collected in Germany produced clamped hyphae with teliospores. A detailed examination of the five isolates (three from Germany and two from Italy) proved them to be a novel yeast species, which is described in this manuscript as Mrakia fibulata sp. nov. (MB 830398), holotype DSM 103931 and isotype DBVPG 8059. In contrast to other sexually reproducing Mrakia species, M. fibulata produces true hyphae with clamp connections. Also, this is the first psychrotolerant Mrakia species which grows above 20 °C. Spring tree fluxes are widespread and can be recognized and sampled by amateurs in a Citizen Science project. This substrate is a prominent source of yeasts, and may harbor unknown species, as demonstrated in the present work. The description of Mrakia fibulata is dedicated to our volunteer helpers and amateurs, like Anna Yurkova (9-years-old daughter of Andrey Yurkov), who collected the sample which yielded the type strain of this species.

Supraglacial debris of Miage Glacier (Mont Blanc, Italy) was used as an in situ model for monitoring growth and modification of the taxonomic structure of fungal populations using an in-growth mesh bag approach over three consecutive years. Sterilized debris was placed in mesh bags (MB) and buried in the debris layer. Pristine debris (D) covering the surface of the glacier was also studied for comparative purposes. Quantitative PCR revealed that after a time of between one and two years the fungal abundance in the sterilized supraglacial debris contained in MB increased to reach a level comparable with those found in D samples. Likewise, the analysis of alpha-diversity exhibited similar dynamics during the three years of study. On the contrary, beta-diversity and Linear discriminant analysis Effect Size (LEfSe), showed apparently fluctuating dynamics from the first to the third year of study and a reduction of the number of fungal taxa discriminating MB and D samples. These observations confirm the hypothesis that fungal cells deposited on MB either by the surrounding debris or continuously brought via wind or atmospheric precipitations, can be subjected to an in situ growth which leads the abundance of the fungal communities occurring in MB to levels found in pristine debris (D) but showing fluctuating taxonomic structures.

Due to the increasing consumer demand, the production of low alcoholic and non alcoholic beer is the new goal of the present brewing producers. Although the beer with reduced alcohol content is currently obtained by physical methods, the use of non-Saccharomyces yeast, with low fermentations capacities, may represent an interesting biological approach. In this study the ethanol content and the volatile profile of a beer obtained using the basidiomycetous psychrophilic yeast strain Mrakia gelida DBVPG 5952 was compared with that produced by a commercial starter for low alcohol beers, Saccharomycodes ludwigii WSL17. The two beers were characterized by a low alcohol content (1.40% and 1.32% v/v) and by a low diacetyl production (5.04 and 5.20 μg/L). However, the organoleptic characteristics of the beer obtained using M. gelida are more appreciated by the panelists, in comparison to the analogous produced with the commercial strain of S. ludwigii.

Background: Some lignocellulosic biomass feedstocks occur in Mediterranean Countries. They are still largely unexploited and cause considerable problems due to the lack of cost-effective harvesting, storage and disposal technologies. Recent studies found that some basidiomycetous yeasts are able to accumulate high amount of intracellular lipids for biorefinery processes (i.e., biofuels and biochemicals). Accordingly, the above biomass feedstocks could be used as carbon sources (after their pre-treatment and hydrolysis) for lipid accumulation by oleaginous yeasts. Results: Cardoon stalks, stranded driftwood and olive tree pruning residues were pre-treated with steam-explosion and enzymatic hydrolysis for releasing free mono- and oligosaccharides. Lipid accumulation tests were performed at two temperatures (20 and 25 °C) using Leucosporidium creatinivorum DBVPG 4794, Naganishia adeliensis DBVPG 5195 and Solicoccozyma terricola DBVPG 5870. S. terricola grown on cardoon stalks at 20 °C exhibited the highest lipid production (13.20 g/l), a lipid yield (28.95%) close to the maximum theoretical value and a lipid composition similar to that found in palm oil. On the contrary, N. adeliensis grown on stranded driftwood and olive tree pruning residues exhibited a lipid composition similar to those of olive and almonds oils. A predictive evaluation of the physical properties of the potential biodiesel obtainable by lipids produced by tested yeast strains has been reported and discussed. Conclusions: Lipids produced by some basidiomycetous yeasts grown on Mediterranean lignocellulosic biomass feedstocks could be used as supplementary sources of oils for producing biofuels and biochemicals. © 2018 The Author(s).

Extremely halophilic archaea (EHA) might play an important role in salted fish production. So far, limited information has been available on the effect of EHA and salt concentration on the safety and quality characteristics of salted anchovies. Eight Halobacterium salinarum strains were isolated from different sea salt samples and subjected to phenotypic and genotypic characterization. The strains were then inoculated into fresh salt before addition to anchovies. A total of 18 experimental productions were performed. The inoculated trials showed the lowest counts of undesired microorganisms. In particular, salted anchovies produced with Hbt. salinarum H11 showed the lowest histamine concentration as well as the highest sensory scores. Differences in terms of volatile organic compounds (VOCs) were estimated among trials. Furthermore, multivariate analysis showed that experimental production performed with a reduced amount of salt (175 g of sea salt per kg of anchovies) did not affect the final quality of salted anchovies. The strain Hbt. salinarum H11 produced salted anchovies with well-appreciated organoleptic features. Thus, the addition of EHA and the use of a lower amount of sea salt might represent a valuable alternative to the traditional method for production of salted anchovies.

Yeasts play a relevant role as starter cultures in traditional foods and beverages, as well as in innumerable biotechnological applications for obtaining high-value bulk and fine biochemicals. Despite a considerable number of studies on yeasts have been performed by using almost exclusively the species Saccharomyces cerevisiae (otherwise labeled as baker’s yeast), the number of yeast species described so far accounts for more than 1600, belonging to over 130 ascomycetous and basidiomycetous genera. This huge yeast diversity includes many non-Saccharomyces species possessing useful, and sometimes uncommon, metabolic features potentially interesting for both food and nonfood industries. Like other organisms, cold-adapted yeasts include species able to survive and grow in cold environments. They are usually labeled as psychrophiles or psychrotolerants on the basis of their cardinal growth temperatures. Among them, yeasts belonging to the phylum Basidiomycota apparently exhibit a superior adaptation to cold. This apparent superiority, which could be the result of some metabolic strategies implemented for adapting life to different thermal conditions in order to overcome the adverse effect of cold, can be considered worthwhile for implementing their biotechnological application at low temperatures. Accordingly, cold-adapted basidiomycetous yeasts have attracted considerable attention for their biotechnological potential, because they have developed the ability to synthesize cold-active enzymes, as well as other important biochemicals, namely, cryoprotectant compounds, polymers, lipids, and other miscellaneous compounds.

The main terrestrial cold areas (outside Antarctica and the Arctic and subarctic regions, viz., the Earth strip running approximately between 60°N and 60°S) are those covered by glaciers and permafrost soil, primarily confined in the Himalayas, Andes, and European high mountains and only little portions in other parts of the globe.

The contribution of two starter ( Lactobacillus delbrueckii and Streptococcus thermophilus ) and nine non-starter ( Enterococcus casselliflavus , Enterococcus faecalis , Enterococcus durans , Enterococcus gallinarum , Lactobacillus casei , Lactobacillus paracasei , Lactobacillus rhamnosus , Pediococcus acidilactici and Pediococcus pentosaceus ) species of lactic acid bacteria (LAB) to the volatile organic compounds (VOCs) of Caciocavallo Palermitano cheese was investigated. The strains used in this study were isolated during the production/ripening of the stretched cheese and tested in a cheese-based medium (CBM). The fermented substrates were analyzed for the growth of the single strains and subjected to the head space solid phase micro-extraction (HS-SPME) and gas chromatography – mass spectrometry (GC–MS). The 11 strains tested were all able to increase their numbers in CBM, even though the development of the starter LAB was quite limited. GC–MS analysis registered 43 compounds including seven chemical classes. A lower diversity of VOCs was registered for the unfermented curd based medium (CuBM) analyzed for comparison. The class of ketones represented a consistent percentage of the VOCs for almost all LAB, followed by alcohols and esters. The volatile profile of Pediococcus acidilactici and Lactobacillus delbrueckii was mainly characterized by 2-butanol, butanoic acid and hexanoic acid and their esters, while that of Lactobacillus casei and Lactobacillus rhamnosus was characterized by 2,3-butanedione and 2-butanone, 3-hydroxy. In order to correlate the VOCs produced by Caciocavallo Palermitano cheeses with those generated by individual LAB, the 4-month ripened cheeses resulting from the dairy process monitored during the isolation of LAB were also analyzed for the volatile chemical fraction and the compounds in common were subjected to a multivariate statistical analysis. The canonical analysis indicated that the VOCs of the ripened cheeses were mainly influenced by E. gallinarum , L. paracasei , L. delbrueckii , L. rhamnosus and L. casei and that 1-hexanol, o -xylene and m -xylene were the cheese VOCs highly correlated with LAB.

The aim of the present work was to investigate the microbiological, chemical, and sensory characteristics of red wine subjected to post-fermentation maceration that was extended to 90 days. For this purpose, the ‘Aglianico di Taurasi’ grape was used as a case study. The total yeast concentration increased until day 40 of maceration and decreased thereafter, whereas the concentration of lactic acid bacteria slightly increased. Dekkera/Brettanomyces spp. and acetic acid bacteria were not detected. The yeast community was composed of Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Metschnikowia pulcherrima, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Pichia guilliermondii, Aureobasidium pullulans and Debaryomyces carsonii. Nine S. cerevisiae strains were detected at high levels at different times of maceration. The results of all the conventional chemical analyses of the wines were in agreement with the regulations of commercial production and, interestingly, the changes in terms of concentration demonstrated the presence of yeast and LAB populations that were not only alive but also in a metabolically active state until day 90 of maceration. The alcohol and glycerol contents slightly increased until day 90. The concentrations of malic acid decreased, whereas those of lactic acid slightly increased throughout the maceration process. Furthermore, different durations of maceration resulted in significant differences in the total polyphenol content, which was higher at 40–50 days. The main phenolic compounds were benzoic and cinnamic acids and catechins. Interestingly, the highest ratio between (+)-catechin and (−)-epicatechin was found on day 40. In addition, the highest antioxidant activity was observed between days 40 and 50. The concentration of volatile organic compounds, which were mainly represented by alcohols, increased until the end of the maceration process. Sensory analysis revealed that samples that were subjected to maceration for a long period of time showed the highest odour and taste complexity and no off-odours and/or off-flavours were detected. These data confirmed that extending post-fermentation maceration to 90 days has no negative impact on the microbiological, chemical and sensory composition of wines, but affects the polyphenol content and potential health benefits of the resulting wine.

The present work was undertaken to evaluate the effect of natural wine making on the microbial and chemical composition of Marsala base wine. To this purpose, a large-scale vinification process of Grillo grape cultivar was monitored from the grape harvest to the final product. Total yeasts (TY) showed a rapid increase after must pressings and reached values that were almost identical to those registered during conventional wine makings. Lactic acid bacteria (LAB) were registered at the highest levels simultaneous to those of yeast growth at the beginning of the process. Saccharomyces cerevisiae was the species found at the highest concentrations in all samples analysed. Several strains (n = 16) was registered at high levels during the alcoholic fermentation (AF) and/or aging of the wine; only two of them were detected on the grape surface. Lactobacillus plantarum was the LAB species most frequently isolated during the entire vinification process. The ethanol content was approximately 14 % (v/v) at the end of vinification. The pH value did not greatly vary during the process, and the volatile acidity (VA) was detected at low concentrations during the entire transformation. The concentration of malic acid rapidly decreased during the AF; on the other hand, lactic acid showed an irregular trend during the entire process. Trans-caffeoyl tartaric acid was the most abundant hydroxycinnamoyl tartaric acid, and volatile organic compounds (VOC) were mainly represented by isoamylic alcohol and isobutanol.

The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB), Streptococcus thermophilus and some lactobacilli, mainly Lactobacillus crispatus and Lactobacillus reuteri , were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as Enterococcus casseliflavus , Enterococcus faecium , Enterococcus faecalis , Enterococcus lactis , Lactobacillus delbrueckii , and Streptococcus thermophilus , while the other strains, all belonging to the genus Enterococcus , could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.

In this study, the composition of the cultivable microbial populations of 38 nectar honey and honeydew honey samples of different botanical and geographical origin were assessed. After growth in specific media, various colonies with different appearance were isolated and purified before phenotypic (morphological, physiological and biochemical traits) and genotypic [randomly amplified polymorphic DNA (RAPD), repetitive DNA elements-PCR (rep-PCR) and restriction fragment length polymorphism (RFLP)] differentiation. The identification was carried out by 16S rRNA gene sequencing for bacteria and, in addition to RFLP, by sequencing the D1/D2 region of the 26S rRNA gene for yeasts and the 5.8S-ITS rRNA region for filamentous fungi. The results showed the presence of 13 species of bacteria, 5 of yeasts and 17 of filamentous fungi; the species most frequently isolated were Bacillus amyloliquefaciens, Zygosaccharomyces mellis and Aspergillus niger for the three microbial groups, respectively. The highest microbial diversity was found in multifloral honeys. No correlation among the microbial species and the botanical/geographical origin was found, but some strains were highly adapted to these matrices since they were found in several samples of different origin.

Nine yeast species belonging to genera Candida , Cryptococcus , Phaffomyces , Rhodotorula and Wickerhamomyces , and one species of Aureobasidium genus were isolated from the cloaca of migratory birds. Candida glabrata and C. inconspicua were the species most frequently isolated and Wickerhamomyces sylviae , which has recently been described as a new species isolated from bird cloaca, was again found. The majority of isolates showed the ability to grow up to 40 °C and/or at pH 3.0, two environmental conditions typical of the digestive tract of birds. The phylogenetic analysis of the D1/D2 domain of 26S rRNA gene placed the cultures of Phaffomyces in a new lineage that differed from the closest species, P. opuntiae , by 13 nucleotide substitutions. The new species was able to grow at 40 °C and at pH 2.5, which suggests a possible adaptation to the bird cloaca. Moreover, the ability to grow in the presence of digitonin at pH 3.7 and the assimilation of ethyl acetate indicates a potential cactophilic origin. For the first time, the presence of yeasts belonging to the Phaffomyces clade in Europe and also in non-cactus environments is reported. The new species is formally described as P. usticensis sp. nov. (PYCC 6346T = CBS 12958T).

In the present work, we investigated the phylogenetic position and phenotypic characteristics of eight yeast isolates collected from migratory birds on the island of Ustica, Italy. A phylogenetic analysis based on the D1/D2 region of the large-subunit rRNA gene showed that all isolates clustered as a single separate lineage within the Wickerhamomyces clade. They exhibited distinct morphological and physiological characteristics and were clearly separated from their closest relatives, Wickerhamomyces lynferdii, Wickerhamomyces anomalus and Wickerhamomyces subpelliculosus, in blastn searches. On the basis of the isolation source, physiological features and molecular strain typing carried out with randomly amplified polymorphic DNA (RAPD)-PCR and minisatellite-primed (MSP)-PCR analysis, the isolates were identified as strains of the same species. The name Wickerhamomyces sylviae f.a., sp. nov. is proposed to accommodate these novel strains; the type strain is U88A2T ( = PYCC 6345T = CBS 12888T). The MycoBank number is MB 804762.

Two traditional sausage products (“salsiccia” and “salame”) processed from the raw meat of the Black Sicilian swine “Suino Nero dei Nebrodi” were microbiologically investigated during the manufacturing and ripening stages. Both products were dominated by lactic acid bacteria (LAB), especially rod-shaped types. The concentration of enterococci was consistent in salame. Coagulase-negative cocci increased slower than LAB. Yeasts showed an increasing trend during the ripening of both products. Enterobacteriaceae were counted at a constant level of about 105 CFU/g in both products, while pseudomonads diminished during ripening. Coagulase-positive staphylococci, Listeria monocytogenes and Salmonella spp. were not detected at the end of the ripening process. Characterisation of LAB at the strain and species level revealed that Lactococcus lactis was found only in the meat mixture, while Lactobacillus sakei and various enterococci persisted during the monitoring period. Some LAB strains isolated from sausages were also identified on the surface of the factory equipment. Two strains (Lactobacillus sakei SS106A and Enterococcus faecalis SS91) were characterised by their anti-Listeria properties due to bacteriocin-like inhibitory substance production. A multiple strain starter composed of Lactobacillus sakei and enterococci has been proposed to maintain the typical characteristics of the two fermented meat products microbiologically investigated in this study.

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes’ milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the nonstarter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5- month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes.

The composition of yeast and lactic acid bacteria (LAB) communities and the chemical evolution of the large-scale commercial vinification of Catarratto IGT Sicilia, carried out under the biological regime, was followed from grape harvest until bottling. Simultaneously to the maximum growth of yeasts, LAB counts reached high level of concentration (6-7 log CFU mL(-1)) during the first steps of the alcoholic fermentation. Yeast identification was determined applying different molecular methods. The highest species biodiversity was observed on grape and must samples taken soon after pressing. Saccharomyces cerevisiae was detected at dominant concentrations during the entire winemaking process. LAB cultures were grouped and identified by a combined phenotypic and genotypic approach. Leuconostoc mesenteroides, Lactobacillus hilgardii and Lactobacillus plantarum species were identified; the last was the main LAB recognized during vinification. The winemaking process was also chemically monitored. The alcoholic content was approximately 12.67% (v v(-1)) at bottling; pH, volatile acidity and total acidity showed a moderate increase during vinification. Tartaric, citric and malic acids decreased until bottling, while lactic acid showed a rapid increase at the end of maceration and bottling. Trans-caffeil tartaric acid was the most abundant phenolic compound and volatile organic compounds (VOC) were mainly represented by isoamylic alcohol, isobutanol, ethyl acetate and octanoic acid.

The potential for the transport and diffusion of some pathogenic microorganisms by migratory birds is of concern. Migratory birds may be involved in the dispersal of microorganisms and may play a role of mechanical and biological vectors. The efficiency of dispersal of pathogenic microorganisms depends on a wide range of biotic and abiotic factors that influence the survival or disappearance of a given agent in a geographical area. In the present study, 349 migratory birds were captured in four sites (Mazara del Vallo, Lampedusa, Ustica and Linosa), representing the main stop-over points during spring and autumnal migration, and analyzed for the presence of filamentous fungi. A total of 2,337 filamentous fungi were isolated from 216 birds and identified by a combined phenotypic-genotypic approach to species level. Twelve species were identified in the study, with Cladosporium cladosporioides, Alternaria alternata, and Aspergillus niger as the most abundant. The transport of these fungal species isolated in this study is of considerable importance because some of these species can create dangers to human health.

In this work, the yeast ecology associated with the spontaneous fermentation of Grillo cultivar grapes from 10 vineyards was analyzed from grape harvest till complete consumption of must sugars. The microbiological investigation started with the plate count onto two culture media to distinguish total yeasts (TY) and presumptive Saccharomyces (PS). Yeasts were randomly isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 14 species belonging to 10 genera. The distribution of the yeasts within the vineyards showed some differences in species composition and concentration levels among 2008 and 2009 vintages. Due to the enological relevance, all Saccharomyces cerevisiae isolates were differentiated applying two genotypic tools (interdelta analysis and microsatellite multiplex PCR of polymorphic microsatellite loci) that recognized 51 strains. Based on the low production of H(2)S, acetic acid and foam, ethanol resistance, growth in presence of high concentrations of potassium metabisulphite (KMBS) and CuSO(4) and at low temperatures, 14 strains were selected and used as starter to ferment grape must at 13 °C and 17 °C in presence of 100 mg/L of KMBS. Three strains (CS160, CS165 and CS182) showed optimal technological aptitudes.

https://www.nature.com/articles/s41598-018-25079-3