Systematic screening strategy for fungal laccase activity of endophytes from Otoba gracilipes with bioremediation potential.

Fungal laccases are promising for biotechnological applications, including bioremediation and dye biotransformation, due to their high redox potential and broad substrate specificity. However, current bioprospecting methods for identifying laccase-producing fungi can be challenging and time-consuming. For early detection, it was developed a three-step, multi-criteria weighting system that evaluates fungal strains based on: First, the biotransformation capacity of three dyes (i.e., Congo red, brilliant blue G-250, and malachite green), at three different pH values, and with a relative weighting supported for the redox potential of each colorant. The relative decolorization coefficient (RDC), used as th2e first classification criterion, expressed their potential performance. Second, under the same conditions, laccase activity was estimated by observing the different degrees of oxidation of a given substrate. The selection criterion was the relative oxidation coefficient (ROC). Finally, laccase activity was quantified in submerged fermentations using three inducers (i.e., loofah sponge, Tween 80, and veratyl alcohol). This multicriteria screening strategy evaluated sixteen isolated endophytic fungal strains from Otoba gracilipes. The system identified Beltraniopsis sp. ET-17 (at pH values of 5.00 and 5.50) as a promising strain for dye biotransformation, and Phlebia floridensis as the best laccase producer, achieving a high activity of 116 µmol min-1 L-1 with loofah sponge as an inducer. In-vitro testing confirmed the efficacy of P. floridensis, with 53.61 % decolorization of a dye mixture (brilliant blue-Congo red. ratio 1:1) after 15 days of incubation. Thus, with the proposed screening strategy it was possible to highlight two species of interest at an early bioprospecting stage on a Colombian native tree poorly explored.

Water Quality, Heavy Metals, and Antifungal Susceptibility to Fluconazole of Yeasts from Water Systems.

Aquatic environments could be reservoirs of pathogenic yeasts with acquired antifungal resistance. The susceptibility to antifungal agents of yeasts present in the wastewater and natural waters of the city of Cali was evaluated. Samples were taken from two types of water: drinking water (Meléndez River, drinking water treatment plant "Puerto Mallarino" in the Cauca River) and wastewater (South Channel of the Cauca River, "Cañaveralejo-PTAR" wastewater treatment plant). Physico-chemical parameters, heavy metal concentration, and yeast levels were determined using standard procedures. Yeasts were identified using API 20 C AUX (BioMérieux) and sequence analysis of the ITS1-5.8S-ITS2 and D1/D2 regions of the large subunit of the ribosome. Susceptibility assays against fluconazole and amphotericin B using the minimum inhibitory concentration (MIC) test were determined using the microdilution method. The influence of physico-chemical parameters and heavy metals was established using principal component analysis (PCA). Yeast counts were higher at WWTP "PTAR" and lower at Melendez River, as expected. A total of 14 genera and 21 yeast species was identified, and the genus Candida was present at all locations. Susceptibility tests showed a 32.7% resistance profile to fluconazole in the order DWTP "Puerto Mallarino = WWTP "PTAR" > South Channel "Navarro". There were significant differences (p < 0.05) in the physico-chemical parameters/concentration of heavy metals and yeast levels between the aquatic systems under study. A positive association was observed between yeast levels and total dissolved solids, nitrate levels, and Cr at the "PTAR" WWTP; conductivity, Zn, and Cu in the South Channel; and the presence of Pb in the "Puerto Mallarino" DWTP. Rhodotorula mucilaginosa, Candida albicans, and Candida sp. 1 were influenced by Cr and Cd, and Diutina catelunata was influenced by Fe (p < 0.05). The water systems explored in this study showed different yeast levels and susceptibility profiles, and, therefore, possible genetic differences among populations of the same species, and different physico-chemical and heavy metals concentrations, which were probably modulating the antifungal-resistant yeasts. All these aquatic systems discharge their content into the Cauca River. We highlight the importance to further investigate if these resistant communities continue to other locations in the second largest river of Colombia and to determine the risk posed to humans and animals.

Pigment Production Improvement in Rhodotorula mucilaginosa AJB01 Using Design of Experiments.

The discovery of biopigments has received considerable attention from the industrial sector, mainly for potential applications as novel molecules with biological activity, in cosmetics or if aquaculture food supplements. The main objective of this study was to increase the production of carotenoid pigments in a naturally pigmented yeast by subjecting the yeast to various cellular stresses using design of experiments. The fungal strain Rhodotorula mucilaginosa AJB01 was isolated from a food sample collected in Barranquilla, Colombia, and one of the pigments produced was ß-carotene. This strain was subjected to various stress conditions, including osmotic stress using different salts, physical stress by ultraviolet (UV) light, and light stress using different photoperiods. The optimal growth conditions for carotenoid production were determined to be 1 min of UV light, 0.5 mg/L of magnesium sulfate, and an 18:6 h light/dark period, which resulted in a carotenoid yield of 118.3 µg of carotenoid per gram of yeast.

Rapid classification of chromoblastomycosis agents genera by infrared spectroscopy and chemometrics supervised by sequencing of rDNA regions.

Chromoblastomycosis (CBM) is a skin and subcutaneous infection caused by species of seven fungal genera. Identification of CBM species is performed by DNA sequencing of one or more genes, which becomes a time-consuming work. Fourier Transform Infrared Spectroscopy (FTIR) has been used for the identification of other microorganisms, however, only one CBM genus was evaluated by FTIR analysis to date. Therefore, the study is aimed to differentiate the CBM agents for identification at genera level using FTIR supervised by Internal Transcribed Spacer (ITS) rDNA region. Seventy-seven isolates of the main five CBM genera were prepared for Attenuated Total Reflection FTIR (ATR-FTIR) with a new methodology using slices of dry fungus in glass fixing-modeling proposed in this study. The algorithm Hierarchical Cluster Analysis (HCA) was used to analyze the differences and similarities between species through the spectra. Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) allowed to correctly classify all samples of five CBM genera. The ATR-FTIR/OPLS-DA models highlighted important contributions of regions attributed to NH and OH stretching, amide I of proteins, polysaccharides bands and fingerprint region for the complete differentiation of the genera investigated. Thus, FTIR can be a fast and inexpensive alternative for identification of CBM agents.

Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification.

High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

Yeast carotenoids: production and activity as antimicrobial biomolecule.

Carotenoids are a large group of organic, pigmented, isoprenoid-type compounds that play biological activities in plants and microorganisms (yeasts, bacteria, and microalgae). Literature reported it as vitamin A precursors and antioxidant activity. Carotenoids also can act as antimicrobial agents and few reports showed quantitative measurements of Minimal Inhibitory Concentrations against different pathogens. In this sense, some carotenoids were added to medical-surgical materials. The demand for scale-up of different naturally obtained carotenoids has increased due to the concern about the detrimental health effects caused by synthetic molecules and antimicrobial resistance. In this review, we reported the variability in pigment production and culture conditions, extraction methods used in laboratory, and we discussed the antimicrobial activity carried out by these molecules and the promising acting as new molecules to be scaled-up to industry.

Producción de carotenoides en levaduras nativas aisladas de sistemas acuáticos en Cali, Colombia / Carotenoids production in wild yeasts isolated from aquatic systems in Cali, Colombia

Introducción: Los carotenoides son fuente importante de actividades biológicas funcionales, tales como antioxidantes o antimicrobianas, además de tener gran impacto a nivel industrial, ya sea en cosmética o suplementación animal en acuacultura. Se han reportado varias moléculas novedosas a partir de aislamientos en Latinoamérica, principalmente en la Patagonia, Argentina. Sin embargo, no hay reportes en Colombia que evalúen la producción de carotenoides en levaduras nativas pigmentadas. Objetivo: Se evaluó la capacidad de producción de carotenoides en levaduras nativas aisladas de lagos, ríos y aguas residuales de la ciudad de Cali, Colombia. Materiales y métodos: Se caracterizaron 30 levaduras provenientes de dos colecciones. De estas se obtuvo su biomasa, rendimiento de carotenoides totales y producción de ß-caroteno. Las cepas promisorias fueron identificadas secuenciando la región ITS1-5.8S-ITS2. Resultados: El mayor rendimiento en la extracción de pigmentos se obtuvo para las cepas P11A (84,36 ± 5,24 µg/g) y Rhodotorula paludigena CS13 (56,26 ± 7,08 µg/g), mientras que las concentraciones más altas de ß-caroteno fueron 10,2 µg/mL (R. paludigena CS13) y 9,7 µg/mL (R. mucilaginosa/alborubescens P10A). La cinética de crecimiento y producción de pigmentos durante cinco días fue óptima para la cepa P11A, ya que hubo un aumento en el rendimiento de carotenoides totales 10 veces mayor (48 h: 109,62 µg/g, 120 h: 1403,10 µg/g). Conclusiones: En este estudio se encontró que levaduras aisladas de sistemas acuáticos son promisorias para la producción de pigmentos carotenoides (incluyendo ß-caroteno), siendo su extracción y caracterización viable para futuros estudios biotecnológicos.

Introduction: Carotenoids are an important source of biological activities, such as antioxidant or antimicrobial. Also, carotenoids impact the cosmetic or food supplement industry, mainly in aquaculture. Several reports in Latin America showed novel molecules, mainly in isolated strains in Patagonia, Argentina. However, in Colombia, there are not reports about carotenoid production from pigmented wild yeasts. Objective: We assessed the carotenoid production ability in wild yeasts isolated from lakes, wastewater and rivers located in Cali, Colombia. Materials and methods: 30 yeasts were selected from two collections, each of them was characterized by the biomass, yield of total carotenoids and ß-carotene production. Promisor strains were identified with sequence analysis of ITS1-5.8S-ITS2 region. The highest yield in pigment extraction was obtained by strains P11A (84,36 ± 5,24 µg/g) and Rhodotorula paludigena CS13 (56,26 ± 7,08 µg/g), while higher concentrations of ß-carotene were 10,2 µg/mL (R. paludigena CS13) and 9,7 µg/mL (R. mucilaginosa/alborubescens P10A). The kinetics of growth and pigment production for five days was optimal for the P11A strain, where we found an increasing 10-fold higher (48 h: 109,62 µg/g, 120 h: 1403,10 µg/g). Conclusions: We suggest that yeasts isolated from aquatic systems are promising for the production of carotenoid pigments (including ß-carotene), making their extraction and characterization viable for future biotechnological studies.

Dominant yeasts associated to mango (Mangifera indica) and rose apple (Syzygium malaccense) fruit pulps investigated by culture-based methods.

The biotechnological potential of yeasts associated to different habitats in Colombia has been poorly studied, especially the yeasts associated with different plant structures. Fruit pulps are interesting substrates mainly for the growth of yeast species, that can positively affect the productivity and quality of some bioeconomic species. Therefore, the objective of this study was to identify the dominant yeast species associated with mango and rose apple fruit pulps in Cali, Colombia. A total of 90 isolates were obtained, which were grouped considering their colony morphology. The D1/D2 domain of the large ribosomal RNA gene (LSU rRNA gene) or internal transcribed spacer (ITS) 1, ribosomal gene 5.8S and ITS 2 (ITS) regions of one to several representative isolates from each group was sequenced and compared with type strains for identification. The species Hanseniaspora thailandica, H. opuntiae and Clavispora lusitaniae were reported as shared by both fruits, specific for rose apple (H. uvarum, Pichia terricola, Rhodosporidiobolus ruineniae and Candida albicans), or for Mango (Meyerozyma caribbica, M. guilliermondii, C. natalensis, Aureobasidium pullulans, Pichia sp., Saturnispora diversa and C. jaroonii). Two morphotypes were not identified at the taxonomic level of species and were reported as candidates for new species, belonging to the genera Wickerhamomyces and Pichia.

Epidemiological aspects and characterization of the resistance profile of Fusarium spp. in patients with invasive fusariosis.

Introduction. The remarkable intrinsic resistance of Fusarium species to most antifungal agents results in high mortality rates in the immunocompromised population.Aims. This study aimed to investigate the epidemiology, clinical features and antifungal susceptibility of Fusarium isolates in patients with invasive fusariosis.Methodology. A total of 27 patients admitted to a referral hospital from January 2008 to June 2017 were evaluated. Antifungal susceptibility testing of isolates was performed by broth microdilution according to the Clinical and Laboratory Standards Institute guidelines.Results. Haematological malignancy was the predominant underlying condition, with an incidence of invasive fusariosis of 14.8 cases per 1000 patients with acute lymphoid leukaemia and 13.1 cases per 1000 patients with acute myeloid leukaemia. The Fusarium solani species complex (FSSC) was the most frequent agent group, followed by the Fusarium oxysporum species complex (FOSC). Voriconazole showed the best activity against Fusarium, followed by amphotericin B. Itraconazole showed high minimum inhibitory concentration values, indicating in vitro resistance. Clinical FSSC isolates were significantly (P<0.05) more resistant to amphotericin B and voriconazole than FOSC isolates.Conclusion. The present antifungal susceptibility profiles indicate a high incidence of fusariosis in patients with haematological malignancy. Species- and strain-specific differences in antifungal susceptibility exist within Fusarium in this setting.

In Vitro Interactions of Amphotericin B Combined with Non-antifungal Agents Against Rhodotorula mucilaginosa Strains.

Rhodotorula species are emerging as opportunistic pathogens, causing catheter-associated fungemia in patients with compromised immunity. R. mucilaginosa is considered the most common species involved in human infections. Correct identification and susceptibility testing of Rhodotorula isolates recovered from the blood stream or central nervous system are essential to determine the best management of this unusual infection. The antifungal susceptibility tests showed that Rhodotorula was susceptible to low concentrations of amphotericin B (AMB) but was less susceptible to voriconazole. Combinations of AMB plus several non-antifungal medications were evaluated against 35 susceptible (Rm AMB-S) and resistant (Rm AMB-R) clinical Rhodotorula isolates using the broth microdilution checkerboard technique. We showed that in vitro exposure to increasing concentrations of AMB changed the susceptibility profile to these strains, which were named the Rm AMB-R group. The most synergistic interactions were AMB + simvastatin, followed by AMB + amlodipine and AMB + warfarin. Synergism and antagonism were observed in both groups for the combination AMB + cyclosporine A. AMB combined with a fluoroquinolone (AMB + levofloxacin) also demonstrated antagonism for the Rm AMB-S strains, but a high percentage of synergistic interactions was observed for the Rm AMB-R group. A combination drug approach can provide a different strategy to treat infections caused by AMB-resistant R. mucilaginosa.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

The Oleaginous Yeast Meyerozyma guilliermondii BI281A as a New Potential Biodiesel Feedstock: Selection and Lipid Production Optimization.

A high throughput screening (HTS) methodology for evaluation of cellular lipid content based on Nile red fluorescence reads using black background 96-wells test plates and a plate reader equipment allowed the rapid intracellular lipid estimation of strains from a Brazilian phylloplane yeast collection. A new oleaginous yeast, Meyerozyma guilliermondii BI281A, was selected, for which the gravimetric determination of total lipids relative to dry weight was 52.38% for glucose or 34.97% for pure glycerol. The lipid production was optimized obtaining 108 mg/L of neutral lipids using pure glycerol as carbon source, and the strain proved capable of accumulating oil using raw glycerol from a biodiesel refinery. The lipid profile showed monounsaturated fatty acids (MUFA) varying between 56 or 74% in pure or raw glycerol, respectively. M. guilliermondii BI281A bears potential as a new biodiesel feedstock.

Chromoblastomycosis caused by Rhinocladiella similis: Case report.

We report a case of chromoblastomycosis in lesions on the chest and foot. Itraconazole was chosen as the initial treatment for this patient, who was followed up for 8 months before becoming noncompliant. The pathogenic fungal species was identified as Rhinocladiella similis by ITS region sequencing. In vitro analyses indicate that the fungus was sensitive to posaconazole and itraconazole. This report presents R. similis as a new agent of chromoblastomycosis and raises the hypothesis that this species could be more resistant to some antifungals than R. aquaspersa.

South Brazilian wines: culturable yeasts associated to bottled wines produced in Rio Grande do Sul and Santa Catarina.

A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of "Rio Grande do Sul" (RS) accounts for 90% of Brazilian wines. The state of "Santa Catarina" (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.

Actualización en caracterización molecular de levaduras de interés industrial / Update on molecular characterization for yeast of industrial interest

Las levaduras, además de ser un modelo de la investigación biomédica, tienen diversas aplicaciones en la industria alimentaria, en agricultura y la producción de etanol combustible. Dado que la calidad y la cantidad del producto dependen de la dinámica y la frecuencia de los microorganismos presentes en la fermentación, el uso de herramientas de caracterización molecular se ha incrementado y popularizado en las industrias que emplean levaduras. Estas técnicas se basan en la amplificación o análisis por enzimas de restricción de una porción del ADN genómico de levadura y se clasifican de acuerdo a su capacidad de resolución taxonómica para discriminar a nivel inter o intra-específica. La primera parte de la revisión incluye pruebas interespecíficas tales como, análisis de restricción o RFLP para las regiones ITS2, ITS1-5.8, D1 / D2 de los genes 26S ribosomal DNA. La segunda parte incluye, pruebas de uso común para caracterización nivel de cepa, tales como: la amplificación aleatoria del ADN polimórfico (RAPD), análisis cromosómico por electroforesis en gel de campo pulsado (PFGE), análisis de restricción del ADN mitocondrial (ADNmt- RFLP) análisis por mini / micro satélites y la huella genética de ADN por amplificación de regiones interdelta de los transposones Ty. Esta revisión describe y discute los detalles técnicos de los métodos más utilizados para la caracterización molecular de las levaduras y algunos ejemplos de sus aplicaciones en el contexto industrial.

Yeasts, besides being a model of biomedical research, have various applications in the food industry, in agriculture and the production of ethanol fuel. Since the quality and quantity of the product depend on the dynamics and frequency of microorganisms present in the fermentation, the use of molecular characterization tools has been increased and popularized in the industries that use yeast. These techniques are based on amplification or analysis by restriction enzyme of a portion of yeast genomic DNA and are classified according their ability of taxonomic resolution to discriminate at inter- or intra-specific level. The first part of this review includes interspecific tests such as, restriction analysis or RFLP for the ITS2 regions, ITS1-5.8, D1 / D2 of ribosomal DNA 26S genes. The second part includes, tests commonly used for characterization at strain level, such as random amplified DNA polymorphism (RAPD), Chromosome analysis by pulsed gel field electrophoresis (PFGE), restriction analysis of mitochondrial DNA (ADNmt- RFLP), analysis of the mini / micro satellites and DNA fingerprinting by amplifying interdelta regions of Ty transposons. This review describes and discuses technical details of the most commonly used methods for molecular characterization of yeast and some examples of their applications in the industrial context.

Molecular Epidemiology of Agents of Human Chromoblastomycosis in Brazil with the Description of Two Novel Species.

The human mutilating disease chromoblastomycosis is caused by melanized members of the order Chaetothyriales. To assess population diversity among 123 clinical strains of agents of the disease in Brazil we applied sequencing of the rDNA internal transcribed spacer region, and partial cell division cycle and ß-tubulin genes. Strains studied were limited to three clusters divided over the single family Herpotrichiellaceae known to comprise agents of the disease. A Fonsecaea cluster contained the most important agents, among which F. pedrosoi was prevalent with 80% of the total set of strains, followed by 13% for F. monophora, 3% for F. nubica, and a single isolate of F. pugnacius. Additional agents, among which two novel species, were located among members of the genus Rhinocladiella and Cyphellophora, with frequencies of 3% and 1%, respectively.

Enzymatic Activity and Susceptibility to Antifungal Agents of Brazilian Environmental Isolates of Hortaea werneckii.

Four strains of Hortaea werneckii were isolated from different substrates in Brazil (a salt marsh macrophyte, a bromeliad and a marine zoanthid) and had their identification confirmed by sequencing of the 26S rDNA D1/D2 domain or ITS region. Most of the strains were able to express amylase, lipase, esterase, pectinase and/or cellulase, enzymes that recognize components of plant cells as substrates, but did not express albuminase, keratinase, phospholipase and DNAse, whose substrates are animal-related. Urease production was positive for all isolates, while caseinase, gelatinase and laccase production were variable among the strains. All the strains grew in media containing up to 30% NaCl. We propose that the primary substrate associated with H. werneckii is plant-related, in special in saline environments, where the fungus may live as a saprophyte and decomposer. Infection of animal-associated substrates would be secondary, with the fungus acting as an opportunistic animal pathogen. All strains were resistant to fluconazole and presented high MIC for amphotericin B, while they were susceptible to all the other antifungal agents tested.

(GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.

Diversidad de levaduras asociadas a chichas tradicionales de Colombia / Yeast diversity associated to Colombian traditional chichas

En Colombia el conocimiento de la comunidad levaduriforme ha sido limitado, ya que los estudios se han enfocado principalmente en especies de interés clínico. Las fermentaciones espontáneas a partir de diversos sustratos representan hábitats de gran importancia para el estudio de la dinámica de las poblaciones de levaduras nativas, por esta razón, en el presente estudio se aislaron e identificaron las levaduras asociadas a las chichas de maíz, piña y arracacha, que son bebidas fermentadas de manera artesanal en Colombia. Se realizó el aislamiento de las levaduras más representativas de la chicha durante sus tres fases de fermentación: inicial, tumultuosa y final. Inicialmente, se hizo una caracterización parcial de los aislados, que incluyó pruebas fisiológicas, y medición de su capacidad para producir filamentos y esporas. Sin embargo, debido a que estas técnicas no fueron suficientes para identificar los aislados hasta el nivel taxonómico de género o de especie, se complementó el estudio de cada aislado empleando técnicas moleculares basadas en el análisis de restricción del gen rRNA 5.8S y los espaciadores transcritos internos (ITS1 e ITS2). Cuando el empleo de esta técnica no permitió obtener resultados definitivos y para confirmar las asignaciones realizadas usando PCR-RFLPs, se secuenció el dominio D1/D2 del gen 26S rRNA de los aislados más representativos. Mediante estas técnicas se lograron identificar las especies más representativas de los tres tipos de chicha: Candida tropicalis, Pichia kluyveri, Pichia guilliermondii, Hanseniapora guilliermondii, Pichia fermentans, Saccharomyces cerevisiae, Candida maltosa, Rhodotorula glutinis, Torulaspora delbrueckii, Hanseniaspora uvarum, Kazachstania exigua, Kluyveromyces marxianus, Yarrowia lypolitica, Candida parapsilosis, Debaromyces hansenii, Cryptococcus arboriformis, Saccharomyces martiniae, Dekkera anomala, Aureobasidium pullulans y Candida pseudointermedia. La caracterización preliminar de los aislados...

In Colombia, knowledge about yeast communities has been limited because most reports have focused on yeast species with clinical relevance. The spontaneous fermentation of different substrates creates important habitats for analyzing wild yeast populations; for this reason, in this study we isolated and identified yeasts associated with the “chichas” of corn, pineapple, and “arracacha,” which are traditional fermented Colombian beverages. The most representative yeasts were isolated from “chicha” during its three phases of fermentation: initial, tumultuous and final. Initially, we made a partial characterization of isolated yeasts, including macroscopic and microscopic descriptions, physiological tests, and measurement of capacity for producing spores and filaments. However, because these techniques were not sufficient for identification of isolated yeasts to the level of genus and species, the study was complemented by using molecular techniques based on restriction analysis of the ITS1-5.8S rRNA gene-ITS2. When this technique did not permit us to obtain positive results and confirm the PCR-RFLP results, we used the sequence of the D1/D2 domain of the 26S rRNA gene instead for most representative isolates. With these techniques, we identified the most representative yeast species of the three classes of “chicha”: Candida tropicalis, Pichia kluyveri, Pichia guilliermondii, Hanseniapora guilliermondii, Pichia fermentans, Saccharomyces cerevisiae, Candida maltosa, Rhodotorula glutinis, Torulaspora delbrueckii, Hanseniaspora uvarum, Kazachstania exigua, Kluyveromyces marxianus, Yarrowia lypolitica, Candida parapsilosis, Debaromyces hansenii, Cryptococcus arboriformis, Saccharomyces martiniae, Dekkera anomala, Aureobasidium pullulans and Candida pseudointermedia. The preliminary characterization of isolated yeasts, based on ethanol-tolerance and salt-tolerance tests, permitted recognition of wild yeasts for possible biotechnological uses in industry.