Martín Moliné

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 from high-altitude lakes: influence of UV radiation

Mountain lakes located at a high elevation are typically exposed to high UV radiation (UVR). Little is known about the ecology and diversity of yeasts inhabiting these extreme environments. We studied yeast occurrence (with special emphasis on those producing carotenoid pigments) at five high-altitude (>1400 m a.s.l.) water bodies located in the Nahuel Huapi National Park (Bariloche, Argentina). Isolates were identified using a polyphasic approach. Production of photoprotective compounds (carotenoids and mycosporines) by yeast isolates, and UVB resistance of selected species were studied. All water samples contained viable yeast cells in variable numbers, generally ranging from 49 to 209 cells L(-1). A total of 24 yeast species was found; at least four represented novel species. Carotenogenic yeasts prevailed in lakes with low water conductivity and higher transparency and chlorophyll a levels. Apparently, the ability to produce photoprotective compounds in yeasts was related to the transparency of mountain lake waters, and strains from more transparent waters developed increased UVB resistance. Our results indicate that UVR is an important environmental factor affecting the yeast community structure in aquatic habitats.

Photoprotective role of carotenoids in yeasts: Response to UV-B of pigmented and naturally-occurring albino strains.

In this work, the photoprotective role of carotenoids in yeasts was analysed by contrasting the responses to UV-B of pigmented and naturally occurring albino strains of Sporobolomyces ruberrimus and Cystofilobasidium capitatum in different conditions. Albino and pigmented strains were confirmed to be conspecific by PCR fingerprinting and rDNA sequencing. Experimental exposure to UV-B conducted with both yeast species showed that the pigmented strains were more tolerant to UV-B than the albino strains and that the increment in carotenoid contents during the stationary growth phase enhance survivorship. These results indicated that carotenoid pigments afford UV-B protection in yeasts.

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.

Production of torularhodin, torulene, and β-carotene by Rhodotorula yeasts.

Yeasts of the genera Rhodotorula are able to synthesize different pigments of high economic value like β-carotene, torulene, and torularhodin, and therefore represent a biotechnologically interesting group of yeasts. However, the low production rate of pigment in these microorganisms limits its industrial application. Here we describe some strategies to obtain hyperpigmented mutants of Rhodotorula mucilaginosa by means of ultraviolet-B radiation, the procedures for total carotenoids extraction and quantification, and a method for identification of each pigment.

UVB Photoprotective Role of Mycosporines in Yeast: Photostability and Antioxidant Activity of Mycosporine-Glutaminol-Glucoside

Abstract Several yeast species are able to synthesize and accumulate UV-radiation-absorbing mycosporine metabolites that are of unclear physiological function. In this work we analyzed the relationship between mycosporine-glutaminol glucoside (MGG) production, cell survival after UVB irradiation, and formation of cyclobutane pyrimidine dimers (CPDs). We also assessed the photostability and singlet oxygen quenching activity of MGG. A set of nine isolates of the basidiomycetous yeast Cryptococcus steppossus cultured in both dark and light conditions was used for the studies. Survival of the UVB-irradiated isolates and MGG concentration had a linear relationship when the concentration was over 2.5 mg g−1. CPD accumulation and MGG accumulation were inversely related. MGG in aqueous solution was photostable with a photodecomposition quantum yield of 1.16 × 10−5. MGG quenching of singlet oxygen was also observed, and the rate constant for the process in D2O was 5.9 × 107 M−1 s−1. Our results support the idea that MGG plays an important role as a UVB photoprotective metabolite in yeasts by protecting against direct damage on DNA and probably against indirect damage by singlet oxygen quenching.

Production of the UVB-absorbing compound mycosporine-glutaminol-glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.

Occurrence of photoprotective compounds in yeasts from freshwater ecosystems of northwestern Patagonia (Argentina).

Abstract In this paper we present the results of research on the occurrence, induction and role of photoprotective compounds (PPCs) present in native aquatic yeasts from freshwater Patagonian ecosystems. We focus on the effect of UV radiation (UVR) as a factor that controls the level of photoprotection of yeasts, and explore its potential significance in shaping yeast distributional patterns. The research presented here combines field surveys and laboratory work, including the isolation and culture of native yeasts strains, and laboratory assays under different radiation conditions. The results obtained suggest that yeasts are common dwellers of oligotrophic Patagonian water bodies, and provide the first evidence of the distribution of PPC (carotenoid and mycosporine)–producing yeasts in temperate freshwaters. A greater proportion of carotenogenic yeasts were observed in high-elevation lakes. The yeast strains isolated from these environments were found to produce higher amounts of mycosporines (MYCs), and to present higher tolerance to UVB exposure than those from piedmont lakes. Patagonian yeasts have only one type of MYC, mycosporine-glutaminol-glucoside (myc-glu-glu), which seems common to all other yeasts. By analyzing the production of myc-glu-glu in a large number of yeasts belonging to different taxonomic groups, we propose that this compound may have potential use as a chemotaxonomic marker in yeast systematics. Collectively, our work reveals that in Patagonian freshwater yeasts there is an apparent relationship between the ability to produce PPCs, their tolerance to UV exposure and their success in colonizing habitats highly exposed to UVR.

Favored isolation and rapid identification of the astaxanthin-producing yeast Xanthophyllomyces dendrorhous(Phaffia rhodozyma) from environmental samples.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) yeasts are biotechnologically exploited as a natural source of astaxanthin for aquaculture. Based on results of recent studies, it has become clear that this species possesses a greater genetic variability generating the necessity to uncover it and assess its potential for the astaxanthin industry. However, difficulties for the isolation of the X. dendrorhous hinder extensive environmental surveys which need to be carried out to better understand the habitat, distribution and genetic diversity of this species. We extensively searched for distinctive physiological traits of X. dendrorhours by testing phenotypic properties simultaneously with a panel of common sympatric fungi. As a result we obtained a new and innovative strategy for improving X. dendrorhous recovery rate and identification from environmental samples. This strategy involved the use of trehalose‐based media, and a rapid X. dendrorhous identification method based on the simultaneous spectrophotometric detection of astaxanthin and UV‐absorbing compounds (mycosporines). The proposed procedures proved effective in field trials conducted in natural environments of Patagonia (Argentina) and thus represent an important tool for the discovery of new astaxanthin‐producing strains of X. dendrorhous useful for the aquaculture industry.