Yeasts in the attine ant-fungus mutualism: Diversity, functional roles, and putative biotechnological applications.

Insects interact with a wide variety of yeasts, often providing a suitable substrate for their growth. Some yeast-insect interactions are tractable models for understanding the relationships between the symbionts. Attine ants are prominent insects in the Neotropics and have performed an ancient fungiculture of mutualistic basidiomycete fungi for more than 55-65 million years. Yeasts gain access to this sophisticated mutualism, prompting diversity, ecological, and biotechnological studies in this environment. We review half a century research in this field, surveying for recurrent yeast taxa and their putative ecological roles in this environment. We found that previous studies mainly covered the yeast diversity from a small fraction of attine ants, being Saccharomycetales, Tremellales, and Trichosporonales as the most frequent yeast or yeast-like orders found. Apiotrichum, Aureobasidium, Candida, Cutaneotrichosporon, Debaryomyces, Meyerozyma, Papiliotrema, Rhodotorula, Trichomonascus, and Trichosporon are the most frequent recovered genera. On the other hand, studies of yeasts' ecological roles on attine ant-fungus mutualism only tapped the tip of the iceberg. Previous established hypotheses in the literature cover the production of lignocellulosic enzymes, chemical detoxification, and fungus garden protection. Some of these roles have parallels in biotechnological processes. In conclusion, the attine ant environment has a hidden potential for studying yeast biodiversity, ecology, and biotechnology, which has been particularly unexplored considering the vast diversity of fungus-growing ants.

Yeasts in the nests of the leaf-cutter ant Acromyrmex balzani in a Savanna biome: exploitation of community and metabolic diversity.

The leaf-cutter ant Acromyrmex balzani is responsible for causing important losses in reforestation areas, crops, and pastures, and is frequently found in the Brazilian savanna (Cerrado). So far, there is no information regarding the yeast communities that occur in their nests. Here, we evaluated the diversity, composition, and structure of yeast communities in both fungus gardens (FG) and external refuse dump (RD) of this ant species (Palmas, Tocantins, northern Brazil). A total of 720 yeasts were isolated, comprising 52 species distributed in 29 genera. The RDs have significantly richer and more diverse yeast communities than the fungus gardens, regardless of the season and the level of preservation in the area. The isolates produced a wide range of carbon polymer-degrading enzymes and were able to assimilate carbon-sources present in plant materials. We observed a different proportion of enzyme-producers and carbon-assimilation found in external refuse dump and fungus gardens from preserved and disturbed areas, suggesting that this interaction may vary depending on the environmental conditions. A. balzani nests in the savanna biome are a hotspot of yeast species with ecological, clinical, and biotechnological implications.

Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments.

Antarctica is the coldest, windiest, and driest continent on Earth. In this sense, microorganisms that inhabit Antarctica environments have to be adapted to harsh conditions. Fungal strains affiliated with Ascomycota and Basidiomycota phyla have been recovered from terrestrial and marine Antarctic samples. They have been used for the bioprospecting of molecules, such as enzymes. Many reports have shown that these microorganisms produce cold-adapted enzymes at low or mild temperatures, including hydrolases (e.g. α-amylase, cellulase, chitinase, glucosidase, invertase, lipase, pectinase, phytase, protease, subtilase, tannase, and xylanase) and oxidoreductases (laccase and superoxide dismutase). Most of these enzymes are extracellular and their production in the laboratory has been carried out mainly under submerged culture conditions. Several studies showed that the cold-adapted enzymes exhibit a wide range in optimal pH (1.0-9.0) and temperature (10.0-70.0 °C). A myriad of methods have been applied for cold-adapted enzyme purification, resulting in purification factors and yields ranging from 1.70 to 1568.00-fold and 0.60 to 86.20%, respectively. Additionally, some fungal cold-adapted enzymes have been cloned and expressed in host organisms. Considering the enzyme-producing ability of microorganisms and the properties of cold-adapted enzymes, fungi recovered from Antarctic environments could be a prolific genetic resource for biotechnological processes (industrial and environmental) carried out at low or mild temperatures.

Wickerhamiella kiyanii f.a., sp. nov. and Wickerhamiella fructicola f.a., sp. nov., two yeasts isolated from native plants of Atlantic rainforest in Brazil.

Two novel species, Wickerhamiella kiyanii f.a., sp. nov. (type strain FB1-1DASP(T) = CBS 12905(T) = CBMAI 1613(T)) and Wickerhamiella fructicola f.a., sp. nov. (type strain H10Y(T) = CBS 12902(T) = CBMAI 1614(T)) are proposed in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales) to accommodate three strains isolated from flowers and fruits typical of the Brazilian Atlantic rainforest. The novel status of these yeast species was established by sequence divergence observed in the D1/D2 domains of the LSU rRNA gene from the most closely related, described species as well as by phylogenetic analysis. Wickerhamiella kiyanii sp. nov. differs from its nearest phylogenetic neighbours W. pagnoccae CBS 12178(T), Candida jalapaonensis CBS 10935(T) and Candida drosophilae CBS 8459(T) by 2.2-4.2% in the D1/D2 sequences. By contrast, a sequence divergence of 13.2-13.8% was observed between W. fructicola sp. nov. and its closest, described phylogenetic relative Candida kazoui JCM 12558(T) and Candida hasegawae JCM 12559(T). Taxonomic descriptions of the two novel species are given.

Yeasts found on an ephemeral reproductive caste of the leaf-cutting ant Atta sexdens rubropilosa.

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.

Microbial culture collections as pillars for promoting fungal diversity, conservation and exploitation.

Fungi are a diverse group of organisms with an overall global number of 1.5M up to 3.3M species on Earth. Besides their ecological roles as decomposers, fungi are important in several aspects of applied research. Here, we review how culture collections may promote the knowledge on diversity, conservation and biotechnological exploitation of fungi. The impact of fungi diversity on biotechnological studies is discussed. We point out the major roles of microbial repositories, including fungal preservation, prospecting, identification, authentication and supply. A survey on the World Data Center for Microorganisms (WDCM) powered by the World Federation for Culture Collections and on the Genetic Heritage Management Council (CGEN) database revealed that 46 Brazilian culture collections registered in these databases are dedicate to preserving fungi. Most of these culture collections are located in the Southeast of Brazil. This scenario also demonstrates that Brazil has many collections focused on fungal strains, but the lack of up-to-date information in WDCM as well as of a solid national platform for culture collections registration do not allow accurate assessment of fungal preservation.

In vitro susceptibility of environmental isolates of Exophiala dermatitidis to five antifungal drugs.

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.

Black Fungi and Hydrocarbons: An Environmental Survey for Alkylbenzene Assimilation.

Environmental pollution with alkylbenzene hydrocarbons such as toluene is a recurring phenomenon. Their toxicity and harmful effect on people and the environment drive the search for sustainable removal techniques such as bioremediation, which is based on the microbial metabolism of xenobiotic compounds. Melanized fungi present extremophilic characteristics, which allow their survival in inhospitable habitats such as those contaminated with hydrocarbons. Screening methodologies for testing the microbial assimilation of volatile organic compounds (VOC) are scarce despite their importance for the bioremediation of hydrocarbon associated areas. In this study, 200 strains of melanized fungi were isolated from four different hydrocarbon-related environments by using selective methods, and their biodiversity was assessed by molecular and ecological analyses. Seventeen genera and 27 species from three main orders, namely Chaetothyriales, Cladosporiales, and Pleosporales, were identified. The ecological analysis showed a particular species distribution according to their original substrate. The isolated strains were also screened for their toluene assimilation potential using a simple and inexpensive methodology based on miniaturized incubations under controlled atmospheres. The biomass produced by the 200 strains with toluene as the sole carbon source was compared against positive and negative controls, with glucose and with only mineral medium, respectively. Nineteen strains were selected as the most promising for further investigation on the biodegradation of alkylbenzenes.

The isolation of pentose-assimilating yeasts and their xylose fermentation potential.

For the implementation of cellulosic ethanol technology, the maximum use of lignocellulosic materials is important to increase efficiency and to reduce costs. In this context, appropriate use of the pentose released by hemicellulose hydrolysis could improve de economic viability of this process. Since the Saccharomyces cerevisiae is unable to ferment the pentose, the search for pentose-fermenting microorganisms could be an alternative. In this work, the isolation of yeast strains from decaying vegetal materials, flowers, fruits and insects and their application for assimilation and alcoholic fermentation of xylose were carried out. From a total of 30 isolated strains, 12 were able to assimilate 30gL-1 of xylose in 120h. The strain Candida tropicalis S4 produced 6gL-1 of ethanol from 56gL-1 of xylose, while the strain C. tropicalis E2 produced 22gL-1 of xylitol. The strains Candida oleophila G10.1 and Metschnikowia koreensis G18 consumed significant amount of xylose in aerobic cultivation releasing non-identified metabolites. The different materials in environment were source for pentose-assimilating yeast with variable metabolic profile.

Heavy metals and TPH effects on microbial abundance and diversity in two estuarine areas of the southern-central coast of São Paulo State, Brazil.

Coastal areas may be impacted by human and industrial activities, including contamination by wastewater, heavy metals and hydrocarbons. This study aimed to evaluate the impact of hydrocarbons (TPH) and metals on the microbiota composition and abundance in two estuarine systems in the coast of São Paulo: the Santos (SE) and Itanhaém (IE) estuaries. The SE was found to be chronically contaminated by heavy metals and highly contaminated by hydrocarbons. This finding was correlated with the increased density of cyanobacteria in sediments and suggests the possible use of cyanobacteria for bioremediation. These contaminants influence the density and composition of estuarine microbiota that respond to stress caused by human activity. The results are troubling because quantitative and qualitative changes in the microbiota of estuarine sediments may alter microbiological processes such as decomposition of organic matter. Moreover, this pollution can result in damage to the environment, biota and human health.

Survival of Atta sexdens workers on different food sources.

Leaf-cutting ants belonging to the tribe Attini are major herbivores and important agriculture pests in the neotropics, these ants being thought to feed on the sap which exudes from the plant material which they cut and also on the mycelium of a symbiotic fungus that grows on plant material inside their nests in what is called "the fungus garden". However, we have found that the survival of Atta sexdens worker ants on leaves, on mycelium of the ants' symbiotic fungus, Leucoagaricus gongylophorus, or on plant polysaccharides was the same as that of starved A. sexdens, while, conversely, significantly longer survival was achieved by ants fed on the fungus garden material or on some of the products (especially glucose) of the hydrolysis of plant polysaccharides. We found that the fungus garden contained glucose at a higher concentration than that found in leaves or fungal mycelium, and that this glucose was consumed by the ant to the extent that it was probably responsible for up to 50% of the nutritional needs of the workers. The fungus garden contained polysaccharide degrading enzymes (pectinase, amylase, xylanase and cellulase) in proportions similar to that observed in laboratory cultures of L. gongylophorus. It thus appears that A. sexdens workers obtain a significant part of their nutrients from plant polysaccharide hydrolysis products produced by the action of extracellular enzymes released by L. gongylophorus. In this paper we discuss the symbiotic nutrition strategy of A. sexdens workers and brood and the role played by plant polysaccharides in the nutrition of attine ants.

A metabolic pathway assembled by enzyme selection may support herbivory of leaf-cutter ants on plant starch.

Mutualistic associations shape the evolution in different organism groups. The association between the leaf-cutter ant Atta sexdens and the basidiomycete fungus Leucoagaricus gongylophorus has enabled them to degrade starch from plant material generating glucose, which is a major food source for both mutualists. Starch degradation is promoted by enzymes contained in the fecal fluid that ants deposit on the fungus culture in cut leaves inside the nests. To understand the dynamics of starch degradation in ant nests, we purified and characterized starch degrading enzymes from the ant fecal fluid and from laboratory cultures of L. gongylophorus and found that the ants intestine positively selects fungal α-amylase and a maltase likely produced by the ants, as a negative selection is imposed to fungal maltase and ant α-amylases. Selected enzymes are more resistant to catabolic repression by glucose and proposed to structure a metabolic pathway in which the fungal α-amylase initiates starch catalysis to generate byproducts which are sequentially degraded by the maltase to produce glucose. The pathway is responsible for effective degradation of starch and proposed to represent a major evolutionary innovation enabling efficient starch assimilation from plant material by leaf-cutters.

The isolation of pentose-assimilating yeasts and their xylose fermentation potential

ABSTRACT For the implementation of cellulosic ethanol technology, the maximum use of lignocellulosic materials is important to increase efficiency and to reduce costs. In this context, appropriate use of the pentose released by hemicellulose hydrolysis could improve de economic viability of this process. Since the Saccharomyces cerevisiae is unable to ferment the pentose, the search for pentose-fermenting microorganisms could be an alternative. In this work, the isolation of yeast strains from decaying vegetal materials, flowers, fruits and insects and their application for assimilation and alcoholic fermentation of xylose were carried out. From a total of 30 isolated strains, 12 were able to assimilate 30 g L-1 of xylose in 120 h. The strain Candida tropicalis S4 produced 6 g L-1 of ethanol from 56 g L-1 of xylose, while the strain C. tropicalis E2 produced 22 g L-1 of xylitol. The strains Candida oleophila G10.1 and Metschnikowia koreensis G18 consumed significant amount of xylose in aerobic cultivation releasing non-identified metabolites. The different materials in environment were source for pentose-assimilating yeast with variable metabolic profile.

Bandoniozyma gen. nov., a genus of fermentative and non-fermentative tremellaceous yeast species.

BACKGROUND: Independent surveys across the globe led to the proposal of a new basidiomycetous yeast genus within the Bulleromyces clade of the Tremellales, Bandoniozyma gen. nov., with seven new species. METHODOLOGY/PRINCIPAL FINDINGS: The species were characterized by multiple methods, including the analysis of D1/D2 and ITS nucleotide sequences, and morphological and physiological/biochemical traits. Most species can ferment glucose, which is an unusual trait among basidiomycetous yeasts. CONCLUSIONS/SIGNIFICANCE: In this study we propose the new yeast genus Bandoniozyma, with seven species Bandoniozyma noutii sp. nov. (type species of genus; CBS 8364(T)  =  DBVPG 4489(T)), Bandoniozyma aquatica sp. nov. (UFMG-DH4.20(T)  =  CBS 12527(T)  =  ATCC MYA-4876(T)), Bandoniozyma complexa sp. nov. (CBS 11570(T)  =  ATCC MYA-4603(T)  =  MA28a(T)), Bandoniozyma fermentans sp. nov. (CBS 12399(T)  =  NU7M71(T)  =  BCRC 23267(T)), Bandoniozyma glucofermentans sp. nov. (CBS 10381(T)  =  NRRL Y-48076(T)  =  ATCC MYA-4760(T)  =  BG 02-7-15-015A-1-1(T)), Bandoniozyma tunnelae sp. nov. (CBS 8024(T)  =  DBVPG 7000(T)), and Bandoniozyma visegradensis sp. nov. (CBS 12505(T)  =  NRRL Y-48783(T)  =  NCAIM Y.01952(T)).

Selection of Xilose-Fermenting Yeast Strains

ABSTRACT In Brazil, ethanol is obtained by fermentat of sugar cane juice using Saccharomyces cerevisiae. The cane juice extraction generates the bagasse that has been used for obtaining generation biofuel. However, the sugarcane bagasse has 30% pentose that cannot be fermented to ethanol by S. cerevisiae. Thus the aim of this study was to isolate a yeast able to ferment xylose to ethanol. Samples of cane juice and flowers were used for the isolation of 165 strains that were then screened for ethanol production using plate testing. Among them, the ethanol positive strains Wickerhamomyces anomalus, Schizosaccharomyces pombe and Starmerella meliponinorum were selected for a xylose fermentation assay, using a semi-synthetic and bagasse hydrolysate as must. S. meliponinorum and S. pombe produced 0.63 and 2.7 gL-1 of ethanol, respectively, from xylose in a semisynthetic medium. In the medium consisting of bagasse hydrolysate must, 0.67 and 1.1 gL-1 of ethanol were obtained from S. meliponinorum and S. pombe, respectively. All the yeasts produced xylitol from xylose in the semisynthetic medium and S. meliponinorum was that which produced the highest quantity (14.5 g L-1).

Purification of Candida guilliermondii and Pichia ohmeri killer toxin as an active agent against Penicillium expansum.

An antifungal assay with cell-free culture supernatant of Pichia ohmeri 158 and Candida guilliermondii P3 was tested against Penicillium expansum strain #2 at 25 degrees C by measuring hyphal length and percentage conidia germination. C. guilliermondii was more effective against P. expansum conidia germination (58.15% inhibition), while P. ohmeri showed higher inhibition of mycelial growth (66.17%), indicating a probable mechanism associated with killer activity. This killer toxin (molecular mass <3 kDa) was partially purified by normal phase HPLC, using TSKgel Amide-80 analytical and preparative columns. Compared with crude extract, the killer toxin eluted from the post analytical column significantly inhibited P. expansum:% inhibition rose from 42.16 to 90.93% (C. guilliermondii) and 39.32 to 91.12% (P. ohmeri) (p < 0.05). The one-step purification process was adequate in isolating killer toxin from culture supernatant and also increased anti-Penicillium activity.

Endophytic yeasts and filamentous fungi associated with southern Brazilian apple (Malus domestica) orchards subjected to conventional, integrated or organic cultivation.

Endophytic fungi were isolated from leaves, flowers and fruit of healthy apple trees (Malus domestica , Borkh.) growing in southern Brazilian orchards under three different cultivation systems (conventional, integrated and organic), during two vegetative cycles. The greatest total number of endophytic isolates was obtained from the orchards under organic cultivation when compared to integrated and conventional cultivation systems. Filamentous fungi from the genera Colletotrichum , Xylaria and Botryosphaeria were the most frequent ones and the most representative yeast genera were Sporobolomyces , Rhodotorula , Debaryomyces and Cryptococcus . It is suggested that some isolates may be used as indicators of the different management systems.

Cryptococcus haglerorum, sp. nov., an anamorphic basidiomycetous yeast isolated from nests of the leaf-cutting ant Atta sexdens.

A yeast strain (CBS 8902) was isolated from the nest of a leaf-cutting ant and was shown to be related to Cryptococcus humicola. Sequencing of the D1/D2 region of the 26S ribosomal DNA and physiological characterization revealed a separate taxonomic position. A novel species named Cryptococcus haglerorum is proposed to accommodate strain CBS 8902 that assimilates n-hexadecane and several benzene compounds. Physiological characteristics distinguishing the novel species from some other members of the C. humicola complex are presented. The phylogenetic relationship of these strains to species of the genus Trichosporon Behrend is discussed.

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Taking into account the preliminary antagonistic/biodegradation property showed by Pichia membranifaciens and Sporobolomyces roseus, which decreased the initial patulin concentration of 588.4 to 290.0 mug/mL, ability of P. ohmeri 158 in biocontrol against Penicillium expansum and patulin decrease in vitro was performed. The culture supernatant of P. ohmeri 158 was effective against 66.17 percent micelial growth, indicating antibiosis related with the killer phenomenon. The initial patulin concentration of 223 mug in the presence of P. ohmeri 158 cells was decreased over 83 percent of the original concentration, when incubated at 25°C/2 days and > 99 percent after 5 days incubation time, with undetectable patulin level after 15 days. The initial pH 4.0 decreased to pH 3.3 along 15 days experiment, suggesting that patulin decrease was an active process and a consequence of yeast metabolism. The results suggested that P. ohmeri 158 could be a promising alternative for the inhibition of P. expansum growth and patulin degradation.

Considerando o antagonismo e degradação de patulina detectados em Pichia membranifaciens e Sporobolomyces roseus no estudo preliminar, este trabalho avaliou o efeito antagônico de Pichia ohmeri 158 no desenvolvimento de Penicillium expansum e a degradação de patulina "in vitro". O sobrenadante do cultivo de P. ohmeri 158 inibiu 66,17 por cento do desenvolvimento micelial, indicando antibiose relacionada ao fator killer. A concentração inicial de patulina (223 mug) na presença de células íntegras de P. ohmeri foi reduzida em mais de 83 por cento após dois dias de incubação a 25°C e superior a 99 por cento após 5 dias, com níveis indetectáveis no 15° dia. O decréscimo do pH 4,0 inicial para pH 3,3 sugeriu que a eliminação de patulina é um processo ativo e uma conseqüência do metabolismo da levedura. Os resultados obtidos concluem que P. ohmeri 158 é uma alternativa promissora na inibição do desenvolvimento de P. expansum e na degradação de patulina.