Raquel M. Cadete

Spathaspora arborariae sp. nov., a d-xylose-fermenting yeast species isolated from rotting wood in Brazil

Four strains of a new yeast species were isolated from rotting wood from two sites in an Atlantic Rain Forest and a Cerrado ecosystem in Brazil. The analysis of the sequences of the D1/D2 domains of the large-subunit rRNA gene showed that this species belongs to the Spathaspora clade. The new species ferments D-xylose efficiently and is related to Candida jeffriesii and Spathaspora passalidarum, both of which also ferment D-xylose. Similar to S. passalidarum, the new species produces unconjugated asci with a single greatly elongated ascospore with curved ends. The type strain of Spathaspora arborariae sp. nov. is UFMG-HM19.1A(T) (=CBS11463(T)=NRRL Y-48658(T)).

Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian Forest.

Background This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. Methodology/Principal Findings A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L·h to 0.75 g/L·h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L·h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. Conclusions/Significance This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

D-xylose-fermenting and xylanase-producing yeast species from rotting wood of two Atlantic Rainforest habitats in Brazil.

This study investigated the yeast species associated with rotting wood in Brazilian Atlantic Rainforest ecosystems focusing on the identification of D-xylose-fermenting and/or xylanase-producing species. A total of 321 yeast strains were isolated from rotting wood samples collected in two Atlantic Rainforest areas. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Schwanniomyces polymorphus, Scheffersomyces queiroziae, Barnettozyma californica, and Candida (Ogataea) boidinii were the most frequently isolated yeasts. The rarefaction curves for the yeast communities isolated in YNB-D-xylose and YNB-xylan from both areas continued to rise and did not reach an asymptote, indicating that not all yeast diversity had been recovered. Additionally, the yeast composition was variable among the samples and areas, which was confirmed by the values of the Sorensen index. Among the 69 species identified, only 12 were found in both areas sampled. Fifteen possible new species were obtained. Among them, two species (Sugiyamaella sp. 1 and Sugiyamaella xylanicola) showed the ability to ferment D-xylose into ethanol, and three species (Spencermartinsiella sp. 1, Su. xylanicola and Tremella sp.) were able to produce extracellular xylanases. Indeed, most of the xylanase-producing isolates belong to the new species Su. xylanicola, which was also positive for D-xylose fermentation. S.queiroziae and S. stipitis were the main D-xylose-fermenting yeasts identified. The results of this work showed that rotting wood collected from the Atlantic Rainforests is a huge source of yeasts, including new species, with promising biotechnological properties.

Ethanol production by a new pentose‐fermenting yeast strain, Scheffersomyces stipitis UFMG‐IMH 43.2, isolated from the Brazilian forest

The ability of a recently isolated Scheffersomyces stipitis strain (UFMG‐IMH 43.2) to produce ethanol from xylose was evaluated. For the assays, a hemicellulosic hydrolysate produced by dilute acid hydrolysis of sugarcane bagasse was used as the fermentation medium. Initially, the necessity of adding nutrients (MgSO4·7H2O, yeast extract and/or urea) to this medium was verified, and the yeast extract supplementation favoured ethanol production by the yeast. Then, in a second stage, assays under different initial xylose and cell concentrations, supplemented or not with yeast extract, were performed. All these three variables showed significant (p < 0.05) influence on ethanol production. The best results (ethanol yield and productivity of 0.19 g/g and 0.13 g/l/h, respectively) were obtained using the hydrolysate containing an initial xylose concentration of 30 g/l, supplemented with 5.0 g/l yeast extract and inoculated with an initial cell concentration of 2.0 g/l. S. stipitis UFMG‐IMH 43.2 was demonstrated to be a yeast strain with potential for use in xylose conversion to ethanol. The establishment of the best fermentation conditions was also proved to be of great importance to increasing the product formation by this yeast strain. These findings open up new perspectives for the establishment of a feasible technology for ethanol production from hemicellulosic hydrolysates. Copyright

Candida materiae sp. nov., a yeast species isolated from rotting wood in the Atlantic Rain Forest.

Three strains of a novel yeast species, Candida materiae sp. nov., were isolated from rotting wood in an Atlantic rain forest site in Brazil. Analysis of the sequences of the D1/D2 domains of the large-subunit rDNA showed that this species belonged to the Spathaspora clade and was related to Candida jeffriesii and Spathaspora passalidarum. Unlike C. jeffriesii and S. passalidarum, C. materiae sp. nov. did not ferment xylose. The type strain of C. materiae sp. nov. is UFMG-07-C15.1BT (=CBS 10975T=CBMAI 956T).

Selection, growth, and chemo-sensory evaluation of flocculent starter culture strains of Saccharomyces cerevisiae in the large-scale production of traditional Brazilian cachaça

The physiological and kinetic capabilities of 233 Saccharomyces cerevisiae isolates, originating from traditional Brazilian cachaça fermentation, were evaluated under laboratory conditions to select flocculent and non-H2S producing strains to be employed in beverage production. Three flocculent S. cerevisiae strains were selected, two non-H2S producing and one H2S producing, and their kinetic performances were analysed during two large-scale fermentation experiments in a traditional cachaça distillery. One non-flocculent H2S-producing S. cerevisiae strain was also used for comparison with the flocculent strains. The results of mitochondrial DNA restriction analysis showed that the three flocculent starter S. cerevisiae strains, as well as the non-flocculent strain, remained in the process during the whole fermentation period, with cells numbering around 10(7) cfu/ml. All selected strains produced ethanol yields that were typically higher in the distillery than in the laboratory conditions, except for strain UFMGA-1240. The greatest diversity of non-Saccharomyces yeasts was observed prior to day 21 of cachaça fermentation; Pichia membranifaciens and Hanseniaspora guilliermondii were the most frequently isolated species. These yeasts were present in lower densities throughout the whole process. The cachaça produced by the selected strains contained concentrations of chemical compounds in accordance with current Brazilian legislation, and all cachaças scored well in sensory effective tests. In addition to the advantage of being flocculent, the strain UFMGA-1031 is non-H2S producing and also produces cachaça with good sensory acceptance. Therefore, this flocculent and non-H2S producing S. cerevisiae strain is highly suitable as a starter for production of high quality traditional cachaça.

Genomic analysis and D-xylose fermentation of three novel Spathaspora species: Spathaspora girioi sp. nov., Spathaspora hagerdaliae f. a., sp. nov. and Spathaspora gorwiae f. a., sp. nov.

Three novel D-xylose-fermenting yeast species of Spathaspora clade were recovered from rotting wood in regions of the Atlantic Rainforest ecosystem in Brazil. Differentiation of new species was based on analyses of the gene encoding the D1/D2 sequences of large subunit of rRNA and on 642 conserved, single-copy, orthologous genes from genome sequence assemblies from the newly described species and 15 closely-related Debaryomycetaceae/Metschnikowiaceae species. Spathaspora girioi sp. nov. produced unconjugated asci with a single elongated ascospore with curved ends; ascospore formation was not observed for the other two species. The three novel species ferment D-xylose with different efficiencies. Spathaspora hagerdaliae sp. nov. and Sp. girioi sp. nov. showed xylose reductase (XR) activity strictly dependent on NADPH, whereas Sp. gorwiae sp. nov. had XR activity that used both NADH and NADPH as co-factors. The genes that encode enzymes involved in D-xylose metabolism (XR, xylitol dehydrogenase and xylulokinase) were also identified for these novel species. The type strains are Sp. girioi sp. nov. UFMG-CM-Y302(T) (=CBS 13476), Sp. hagerdaliae f.a., sp. nov. UFMG-CM-Y303(T) (=CBS 13475) and Sp. gorwiae f.a., sp. nov. UFMG-CM-Y312(T) (=CBS 13472).

Candida amazonensis sp. nov., an ascomycetous yeast isolated from rotting wood in the Amazonian forest.

Five strains of a novel yeast species were isolated from rotting wood samples collected in an Amazonian forest site in the state of Roraima, northern Brazil. The sequences of the D1/D2 domains of the large subunit of the rRNA gene showed that this species belongs to the Scheffersomyces clade and is related to Candida coipomoensis, Candida lignicola and Candida queiroziae. The novel species Candida amazonensis sp. nov. is proposed to accommodate these isolates. The type strain of C. amazonensis sp. nov. is UFMG-HMD-26.3(T) ( = CBS 12363(T) = NRRL Y-48762(T)).

Cyberlindnera xylosilytica sp. nov., a xylitol-producing yeast species isolated from lignocellulosic materials.

Independent surveys of yeasts associated with lignocellulosic-related materials led to the discovery of a novel yeast species belonging to the Cyberlindnera clade (Saccharomycotina, Ascomycota). Analysis of the sequences of the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit rRNA gene showed that this species is related to C. japonica, C. maesa and C. easanensis. Six isolates were obtained from different sources, including rotting wood, tree bark and sugar cane filter cake in Brazil, frass from white oak in the USA and decayed leaf in Taiwan. A novel species is suggested to accommodate these isolates, for which the name C. xylosilytica sp. nov. is proposed. The type strain of C. xylosilytica sp. nov. is NRRL YB-2097(T) ( = CBS 13984(T) = UFMG-CM-Y347(T)) and the allotype is UFMG-CM-Y409 ( = CBS 14083). The novel species is heterothallic and complementary mating types are represented by the type and allotype strains. The MycoBank number is MB 811428.

The yeasts of the genus Spathaspora: potential candidates for second-generation biofuel production

Yeasts of the Spathaspora clade have the ability to convert d‐xylose to ethanol and/or xylitol. This is an important trait, as these yeasts may be used to produce bioethanol from lignocellulosic biomass or as a source of new d‐xylose metabolism genes for recombinant industrial strains of Saccharomyces cerevisiae. The core group of the genus Spathaspora has 22 species, both formally described and not yet described. Other species, such as Sp. allomyrinae, Candida alai, C. insectamans, C. lyxosophila, C. sake, Sp. boniae and C. subhashii are weakly associated with this clade, based on LSU rRNA gene D1/D2 sequence analyses. Spathaspora passalidarum, Sp. arborariae, Sp. gorwiae and Sp. hagerdaliae produce mostly ethanol from d‐xylose, whereas the remaining species within the Spathaspora clade already tested for this property may be considered xylitol producers. Among the d‐xylose‐fermenting Spathaspora species, Sp. passalidarum is the best ethanol producer, displaying high ethanol yields and productivities when cultured in media supplemented with this pentose under oxygen‐limited or anaerobic conditions. The species also exhibits rapid d‐xylose consumption and the ability to ferment glucose, xylose and cellobiose simultaneously. These characteristics suggest that Sp. passalidarum is a potential candidate for domestication and use in the fermentation of lignocellulosic materials. Copyright