Physiological characterization of polyextremotolerant yeasts from cold environments of Patagonia.

Yeasts from cold environments have a wide range of strategies to prevent the negative effects of extreme conditions, including the production of metabolites of biotechnological interest. We investigated the growth profile and production of metabolites in yeast species isolated from cold environments. Thirty-eight strains were tested for their ability to grow at different temperatures (5-30 °C) and solute concentrations (3-12.5% NaCl and 50% glucose). All strains tested were able to grow at 5 °C, and 77% were able to grow with 5% NaCl at 18 °C. We were able to group strains based on different physicochemical/lifestyle profiles such as polyextremotolerant, osmotolerant, psychrotolerant, or psychrophilic. Five strains were selected to study biomass and metabolite production (glycerol, trehalose, ergosterol, and mycosporines). These analyses revealed that the accumulation pattern of trehalose and ergosterol was related to each lifestyle profile. Also, our findings would suggest that mycosporines does not have a role as an osmolyte. Non-conventional fermentative yeasts such as Phaffia tasmanica and Saccharomyces eubayanus may be of interest for trehalose production. This work contributes to the knowledge of non-conventional yeasts with biotechnological application from cold environments, including their growth profile, metabolites, and biomass production under different conditions.

Contaminated Perry in Patagonia Argentina: A Case Study.

Perry is a beverage obtained by fermentation of pear juice, popular in the North Hemisphere. In Argentina it is an emerging market, particularly in the Patagonian region. The aim of this work is to describe and to evaluate the spoilage yeasts associated to six perry samples showing signs of microbiological contamination from a local craft perry company in North Patagonian region. Eighteen yeasts were isolated from four of the six perry samples where Brettanomyces custersianus, Brettanomyces bruxellensis and Zygosaccharomyces parabailii were identified. The growth capacity of these isolates in the presence of antimicrobial agents (sulfite and potassium sorbate) was analyzed in solid media. Growth parameters in sterile perry must was evaluated and the production of undesirable compounds were evaluated, products were characterized in terms of their aromatic and physicochemical features. The yeasts Z. parabailii NPCC1791 was able to grow on plates containing sulfite concentrations of up to 4 mM and produced high methanol concentrations in perry. Additionally, B. bruxellensis NPPC1792 was able to produce methanol as well as high concentrations of volatile phenols including 4-ethylphenol and 4-ethylguaiacol. These results demonstrate the potential of these species as perry contaminants. Given the lack of studies describing the contaminating yeasts in perry production, this work represents the first report about perry spoilage yeasts in Argentina, with this knowledge, control strategies can be developed to prevent microbiological contamination and minimize product loss.

[Microbial contaminants in bottled craft beer of Andean Patagonia, Argentina]. / Contaminantes microbianos en cervezas artesanales embotelladas de la Patagonia andina argentina.

The brewing activity in Andean Patagonia plays a very important role in the region's economy, being microbial contamination one of the main problems in terms of quality. The presence of contaminant bacteria and wild yeasts in beer generate microbiological, physical and chemical changes that impact on its sensory attributes. However, few breweries establish criteria and policies to guarantee the quality of their products in a microbiological sense. The purpose of this work was to study for the first time the incidence of microbial contaminants in bottled craft beers from Andean Patagonia, identify the main microorganisms involved and establish relationships between contamination and the physicochemical variables of beer. We analyzed 75 beers from 37 breweries from 12 different Patagonian cities. Our results showed that 69.3% of the analyzed beer exhibited contaminant microorganism growth. Bacteria Levilactobacillus brevis and wild yeasts of Saccharomyces were the main microorganisms responsible for these contaminations. In addition, we found that microbial contamination had an impact on beer sensory profile and also that pH was correlated with the presence of lactic acid bacteria in beer, being an indicator of contamination for these bacteria. In conclusion, we observed that 8 out of 10 breweries studied showed contamination problems, highlighting the need to design prevention and control strategies in microbreweries.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Novel yeast taxa from the cold: description of Cryolevonia giraudoae sp. nov. and Camptobasidium gelus sp. nov.

Twenty-one psychrophilic yeast isolates related to the Camptobasidiaceae family in the Microbotryomycetes class were obtained from ice collected from cold environments worldwide. A new psychrophilic species from the recently described genus Cryolevonia, Cryolevania giraudoae is proposed to accommodate 18 isolates from Patagonia (Argentina) and Antarctica (holotype CRUB 2086T). In addition, a new psychrophilic species in the genus Camptobasidium is described as Camptobasidium gelus sp. nov. (holotype CBS 8941T), based on three isolates from glacial ice in the Russel glacier (Greenland ice sheet) and Antarctica. The strict psychrophilic profile is the salient feature of both novel species.

Sex in the cold: taxonomic reorganization of psychrotolerant yeasts in the order Leucosporidiales.

Species of Leucosporidiales are a group of psychrotolerant yeasts with biotechnological potential. In the present work, we studied the phenotypic, genetic and sexual characteristics of three species of this genus (Leucosporidium scottii, Leucosporidiella creatinivora and Le. yakutica) to clarify the evolutionary relationship among these closely related taxa. From the results obtained, it becomes clear that these yeasts can interbreed. Although genetic delimitation is possible for the three species, the extent of nucleotide substitutions and phenotypic differences observed between them are lower than that expected for species that have ended the speciation process. Our taxonomic conclusion is to maintain the three taxa until further genomic data are gathered. However, the concept of L. scottii species complex is proposed for this group of species. Finally, we transfer all Leucosporidiella and Mastigobasidium species to Leucosporidium (Leucosporidiales), and, in order to end the polyphyly condition of these taxa, we propose the new genus Pseudoleucosporidium gen. nov. and the new combination Peudoleucosporidium fasciculatum comb. nov.

Yeasts from glacial ice of Patagonian Andes, Argentina.

Glacial ice and snow are known habitats for cold-adapted microorganisms. Research on cold-adapted yeast biodiversity from Perito Moreno and Mount Tronador glaciers (Patagonia, Argentina), and production of extracellular enzymatic activity at low temperatures (5 and 18 °C), was performed and described in this study. Ninety percent (90%) of the isolates were basidiomycetous; 16 genera and 29 species were identified. Twenty-five percent (25%) of total isolates corresponded to psychrophilic yeasts, whereas 75% were psychrotolerant yeasts. Eighty-five percent (85%) of all isolates had at least one enzymatic activity. Multiple correspondence analysis and cluster classification revealed a relationship between certain genera and some enzymatic activities. Cold-adapted yeast isolates were able to hydrolyze natural compounds (casein, lipids, starch, pectin, and carboxymethylcellulose) at low temperatures, suggesting a significant ecological role of these organisms as organic matter decomposers and nutrient cyclers. These yeasts are especially relevant for metabolic and ecological studies, as well as for yeast-based biotechnological process at low temperatures.

Cryptococcus species (Tremellales) from glacial biomes in the southern (Patagonia) and northern (Svalbard) hemispheres.

Cryptococcus species (Basidiomycota) were isolated as the predominant yeast from glacial biomes of both Patagonia (Argentina) and the Svalbard archipelago (Norway). For a selected group of Cryptococcus belonging to Tremellales, assimilative profile, production of extracellular hydrolytic enzymes and ribosomal DNA internal transcribed spacer and large subunit (D1/D2) sequences were analysed. Cryptococcus victoriae, which was originally described from Antarctica, was the most frequently found species at both locations. High variability within the species was observed and described at the genotypic and phenotypic levels, two newly described species were found in both Patagonia and Svalbard: Cryptococcus fonsecae and Cryptococcus psychrotolerans. Two other new species were found only in Patagonia: Cryptococcus frias and Cryptococcus tronadorensis. Three additional new taxa were found, but they are not named as they were only represented by single isolates.

Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity, distribution and synthesis of photoprotective compounds and extracellular enzymes.

Nahuel Huapi (NH) Lake is an oligotrophic temperate lake of glacial origin with high transparency, surrounded by well-developed forests and located at San Carlos de Bariloche, Nahuel Huapi National Park, in Patagonia, Argentina. In this lake, we characterized yeast distribution and diversity along a south-to-north transect and established a relationship between the ability to produce photoprotective compounds (PPCs) (carotenoid pigments and mycosporines) and the occurrence of yeast at different collection points. Subsurface water samples were filtered for yeast isolation. Total yeast counts ranged between 22 and 141 CFU L(-1) , and the highest values corresponded to the most impacted sites. Littoral sites had a low proportion of yeast-producing PPCs and this group prevailed in pelagic sites. This is probably a result of the high transparency of the water and the increased UV exposure. The yeast community from NH Lake showed a high species richness and a uniform distribution of taxa between pelagic and border collection points. Yeasts were identified as belonging to 14 genera and 34 species. Rhodotorula mucilaginosa and Cryptococcus victoriae were the most frequently found species, representing 14.4% and 13.6% of the total yeast isolates, respectively. Most of the yeast isolates demonstrated at least one extracellular enzymatic activity (mainly cellulase and lipase activities), which suggested that these microorganisms are metabolically active in the lake.

The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica.

The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates.

Yeasts Occurring in Surface and Mouth Cavity of Two Chelonian Species, Podocnemis expansa Schweigger and P. unifilis Troschel (Reptilia: Chelonia: Pelomedusidae), in the Javaés River Border of Araguaia National Park in Brazil.

Thirty-eight specimens of free-ranging Podocnemis expansa (Amazon turtle) and 22 of P. unifilis (Tracajá) were screened for yeast isolation from surface (plastron, skin, and nails), eye, and mouth cavity. A hundred and eighteen yeast isolates belonging to 39 species were obtained. Debaryomyces hansenii, Candida galli, C. sake, and Rhodotorula mucilaginosa were the most frequent species isolated from these chelonians. Species diversity measured by Shannon's index was shown to be low and a degree of dominance could be detected as species known as potential pathogens were commonly isolated. The effective number of species in plastron of P. expansa was higher than in mouth samples, but not in P. unifilis probably due to dietary factors. P. expansa animals were captured on the beaches, and the superficial yeast populations may include terrestrial species. P. unifilis animals were captured in the water and the yeasts from superficial sites may represent species from river water.

Wickerhamomyces patagonicus sp. nov., an ascomycetous yeast species from Patagonia, Argentina.

Eight strains of a novel yeast species were isolated from tree saps of 'Coihue' (Nothofagus dombeyi, Nothofagaceae) and glacial meltwater (Castaño Overo River) in the Nahuel Huapi National Park, Patagonia, Argentina. The sequences of the D1/D2 domains of the large subunit of the rRNA gene showed that this novel yeast species belongs to the Wickerhamomyces genus (Order Saccharomycetales, Family Wickerhamomycetaceae). The closest related species were Candida ponderosae and Wickerhamomyces chambardii. Wickerhamomyces patagonicus sp. nov. is proposed to accommodate these novel strains, with the type strain CRUB 1724(T) (=CBS 11398(T) =JCM 16381(T)).

Cryptococcus spencermartinsiae sp. nov., a basidiomycetous yeast isolated from glacial waters and apple fruits.

Seven strains representing a novel yeast species belonging to the genus Cryptococcus were isolated from different substrates from Patagonia, Argentina, and The Netherlands. Three strains were isolated from a meltwater river draining from the Frias glacier at Mount Tronador situated in Nahuel Huapi National Park (Patagonia) and four were isolated from apple surfaces in Randwijk, The Netherlands. Analysis of the D1/D2 large-subunit rRNA gene and ITS region sequences indicated that these strains represent a single species that is distinct from other species of the Tremellales clade. The name Cryptococcus spencermartinsiae sp. nov. is proposed to accommodate these strains. The type strain is CRUB 1230(T) (=CBS 10760(T) =DBVPG 8010(T)).

Cryptococcus agrionensis sp. nov., a basidiomycetous yeast of the acidic rock drainage ecoclade, isolated from an acidic aquatic environment of volcanic origin.

Seventy-one strains were isolated from the River Agrio-Lake Caviahue acidic aquatic environment in Argentina. Strains were isolated mainly from the most acidic section of the river (pH 1.8-2.7). According to the mini/microsatellite-primed PCR technique and physiological data, these strains are representatives of a single novel species of the genus Cryptococcus. Analysis of the D1/D2 region of the large-subunit rRNA gene showed that the strains belong to the order Filobasidiales of the subphylum Agaricomycotina. The novel isolates formed a phylogenetic group with Cryptococcus ibericus, Cryptococcus aciditolerans and Cryptococcus metallitolerans, the most closely related species. This group, which is phylogenetically related to the Gastricus clade, is considered to be an ecoclade due to its peculiar ecology and physiology. The name Cryptococcus agrionensis sp. nov. is proposed to accommodate these isolates, with strain CRUB 1317(T) (=CBS 10799(T)=JCM 15321(T)) as the type strain.

Characterization of a novel South American population of the astaxanthin producing yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

A novel population of the biotechnologically important yeast Xanthophyllomyces dendrorhous, the sexual stage of Phaffia rhodozyma, has been recently isolated for the first time in the southern Hemisphere (Patagonia, Argentina). The aim of the present work was to phenotypically and genotypically characterize two representative strains of this new population, and assess such strains as a potential biotechnological source of astaxanthin, fatty acids and extracellular enzymes. Minor variations were found in physiological tests. PCR fingerprinting studies (MSP-PCR) showed the main differences between X. dendrorhous Patagonian and Type strains. Patagonian strains accumulated a xanthophyll-like pigment, which was identified as astaxanthin. These strains showed low fatty acids content (mainly polyunsaturated fatty acids) and, of a total of six extracellular enzymes tested, only produced amylase. Genetic differences between Patagonian and collection X. dendrorhous strains could be explained by geographic isolation and habitat specificity.

Extracellular enzymatic activities of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina).

As part of a project aimed at the selection of cold-adapted yeasts expressing biotechnologically interesting features, the extracellular enzymatic activity (EEA) of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina) was investigated. Ninety-one basidiomycetous yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Dioszegia, Mrakia, Rhodotorula, Rhodosporidium, Sporobolomyces, Sporidiobolus, Cystofilobasidium, and Udeniomyces) were screened for extracellular amylolytic, proteolytic, lipolytic, esterasic, pectinolytic, chitinolytic, and cellulolytic activities. Over 15% of the strains exhibited three or more different EEAs at 4 degrees C and more than 63% had at least two EEAs at the same temperature. No chitinolytic or cellulolytic activities were detected at 4 and 20 degrees C. Cell-free supernatants exhibited significantly higher (P < 0.01) protease and lipase activities at < or = 10 degrees C, or even at 4 degrees C. In light of these findings, cold environments of Patagonia (Argentina) may be considered a potential source of cold-adapted yeasts producing industrially relevant cold-active enzymes.

Biodiversity of cold-adapted yeasts from glacial meltwater rivers in Patagonia, Argentina.

The occurrence of culturable yeasts in glacial meltwater from the Frías, Castaño Overo and Río Manso glaciers, located on Mount Tronador in the Nahuel Huapi National Park (Northwestern Patagonia, Argentina) is presented. Subsurface water samples were filtered for colony counting and yeast isolation. The total yeast count ranged between 6 and 360 CFU L(-1). Physiologic and molecular methods were employed to identify 86 yeast isolates. In agreement with yeast diversity data from studies for Antarctic and Alpine glaciers, the genera Cryptococcus, Leucosporidiella, Dioszegia, Rhodotorula, Rhodosporidium, Mrakia, Sporobolomyces, Udeniomyces and Candida were found. Cryptococcus and Leucosporidiella accounted for 50% and 20% of the total number of strains, respectively. Among 21 identified yeast species, Cryptococcus sp. 1 and Leucosporidiella fragaria were the most frequent. The typically psychrophilic Mrakia yeast strain and three new yeast species, yet to be described, were also isolated. All yeast strains were able to grow at 5, 10, and 15 degrees C. Among yeast strains expressing extracellular enzymatic activity, higher proteolytic and lipolytic activities were obtained at 4 degrees C than at 20 degrees C.