Yeast communities of primary and secondary peat swamp forests in southern Thailand.

Khanthuli peat swamp forest (PSF) is one of a few fertile peat swamp forests that remain in Thailand. It is composed of primary PSF and some areas which have been degraded to secondary PSF due to drought, wildfires and land conversion, which have resulted in a decrease in peat layers and change in the species of the plant community. In this study, diversity of yeasts in peat from both primary and secondary PSF areas of the Khanthuli PSF was determined based on culture-dependent approaches, using dilution plate and enrichment techniques. A total of 66 yeast isolates were identified by the analysis of sequence similarity of the D1/D2 region of the large subunit rRNA gene or the combined analysis of sequence of the D1/D2 region and internal transcribed spacer region and confirmed by phylogenetic analysis of the D1/D2 region to belong to 22 known yeast species and six potential new species in the genera Candida (Kurtzmaniella, Lodderomyces, Ogataea, Pichia and Yamadazyma clades), Clavispora, Cyberlindnera, Galactomyces, Hanseniaspora, Metschnikowia, Saturnispora, Schwanniomyces, Cryptotrichosporon, Pichia, Curvibasidium, Papiliotrema, Rhodotorula, and Saitozyma. The most prevalent yeasts in the primary PSF were Cyberlindnera subsufficiens and Galactomyces candidus, while Saitozyma podzolica was the most frequently found in peat from the secondary PSF. Common yeast species in both, primary and secondary PSF, were Cy. subsufficiens, G. candidus and Rhodotorula mucilaginosa.

Torulaspora nypae sp. nov., a novel yeast species isolated from nipa (Nypa fruticans Wurmb.) inflorescence sap in southern Thailand.

Two strains (YSP-384 and YSP-399), representing a novel Torulaspora species, were isolated from two nipa inflorescence sap samples collected in Trang province in the southern part of Thailand. The two strains had identical sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions. The two strains were closest to Torulaspora maleeae CBS 10694T, but with 1.1 % nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 5.2 % nucleotide substitutions in the ITS regions. Phylogenetic analysis based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene supported that the two strains represented a distinct species in the genus Torulaspora. Some phenotypic characteristics of the two strains differed from T. maleeae including the two strains have ability to assimilate d-xylose, d-glucono-δ-lactone and melizitose, and inability to ferment maltose and raffinose, whereas T. maleeae has opposite results. Therefore, the two strains are described as representing a novel species, for which the name Torulaspora nypae sp. nov. was proposed.

Chemical consequences of three commercial strains of Oenococcus oeni co-inoculated with Torulaspora delbrueckii in durian wine fermentation.

This work evaluated for the first time the chemical consequences of three commercial strains of Oenococcus oeni co-inoculated with Torulaspora delbrueckii in durian wine fermentation. Compared with the control (yeast only, 5.70% v/v ethanol produced), samples co-inoculated with T. delbrueckii and O. oeni PN4 improved ethanol production (6.06% v/v), which was significantly higher than samples co-inoculated with Viniflora (4.78% v/v) or Enoferm Beta (5.01% v/v). Wines co-fermented with the respective latter two oenococci contained excessive levels of ethyl acetate (>80mg/L) that were likely to affect negatively wine aroma. In addition, they led to significantly higher acetic and lactic acid production relative to PN4. O. oeni PN4 seemed to be the most suitable strain to co-inoculate with T. delbrueckii for simultaneous alcoholic and malolactic fermentation in durian wine by contributing moderately increased concentrations of higher alcohols, acetate esters and ethyl esters that would have positive sensory impacts.

TdPIR minisatellite fingerprinting as a useful new tool for Torulaspora delbrueckii molecular typing.

Torulaspora delbrueckii yeast strains are being increasingly applied at the industrial level, such as in the winemaking process, and so their identification and characterisation require effective, fast, accurate, reproducible and reliable approaches. Therefore, the development of typing techniques that allow discrimination at the strain level will provide an essential tool for those working with T. delbrueckii strains. Here, 28 T. delbrueckii strains from various substrates were characterised using different PCR-fingerprinting molecular methods: random amplified polymorphic DNA with polymerase chain reaction (RAPD-PCR), minisatellites SED1, AGA1, DAN4 and the newly designed T. delbrueckii (Td)PIR, and microsatellites (GAC)5 and (GTG)5. The aim was to determine and compare the efficacies, reproducibilities and discriminating powers of these molecular methods. RAPD-PCR using the M13 primers and the newly designed TdPIR3 minisatellite primer pair provided discrimination of the greatest number of T. delbrueckii strains. TdPIR3 clustered the 28 strains into 16 different groups with an efficiency of 100%, while M13 clustered the strains into 17 different groups, although with a lower efficiency of 89%. Moreover, the TdPIR3 primers showed reproducible profiles when the stringency of the PCR protocol was varied, which highlighted the great robustness of this technique. In contrast, variation of the stringency of the M13 PCR protocol resulted in modification of the amplified profiles, which suggested low reproducibility of this technique.

Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast Torulaspora delbrueckii.

The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼ 1900 years ago), and to the Neolithic era for bioprocesses (∼ 4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.

Characterisation of commercial and natural Torulaspora delbrueckii wine yeast strains.

Forty-three South African Torulaspora delbrueckii yeast isolates from the ARC Infruitec-Nietvoorbij yeast culture collection, the T. delbrueckii type strain (CBS 1146), one reference T. delbrueckii strain (CBS 4663), two T. delbrueckii strains isolated from commercial yeast blends (Viniflora® Harmony.nsac and Viniflora® Melody.nsac), and a commercial Saccharomyces cerevisiae yeast (VIN 13) had their identities confirmed and were characterised using conventional and molecular microbiological techniques. These included a selection of growth media as well as CHEF electrophoretic karyotyping and PCR-RFLP analyses. Based on the biochemical and physiological results the strains were divided into 13 groups. The performances of the yeasts were also monitored by means of laboratory-scale fermentations in grape must at 15 °C and 22 °C. The fermentation kinetic data showed that at 22 °C, the yeasts were divided into two distinct groups, a faster and a slower fermenting group. The fermentation curves of the laboratory-scale study at 15 °C showed that, at this lower temperature, the yeasts also fermented at different speeds, but the fermentation curves showed greater separation. The biochemical and physiological grouping did not coincide with the fermentation abilities and good fermenters could be found in more than one group. Chemical analyses of the resultant wines (alcohol, volatile acidity, glycerol, total SO2, residual sugar) were used in Principle Component Analyses. The yeasts that grouped close to the S. cerevisiae reference strain (VIN 13) showed more acceptable wine chemical profiles, while those further away displayed less acceptable profiles. Three locally isolated strains and one commercial T. delbrueckii yeast strain, Viniflora® Harmony.nsac. produced wines with acceptable chemical profiles at both temperatures. These strains also had comparable fermentation kinetics to the S. cerevisiae reference. Therefore, depending on the fermentation temperature, different T. delbrueckii strains will be suitable for specific wine styles and some may even be considered for single inoculations without S. cerevisiae in industrial fermentations.

Torulaspora indica a novel yeast species isolated from coal mine soils.

Four yeast strains (APSS 805, APSS 806, APSS 815 and AP-18) belonging to a novel Torulaspora species were isolated from coal mine soils of Singareni in Andhra Pradesh state, India. Another strain (PBA-22) was isolated from agricultural field soil from Gujarat state, India. The vegetative cells of all these strains were round, haploid and produced asci by conjugation between independent cells or mother cell and bud, with rough ascospores, suggesting their possible relation to ascomycetous yeast genus Torulaspora. Phylogenetic analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene and Internal Transcribed Spacer (ITS) regions revealed that, among the five strains, three viz. APSS 805, APSS 806 and APSS 815 have identical sequences. The other two strains (AP-18 and PBA-22) differed from the other three strains in less than 1% nucleotide substitutions in the combined D1/D2 domain and ITS sequences, indicating that all of them (five strains) may belong to the same species. These five strains were closely related to Torulaspora globosa, but showed more than 3-7% sequence divergence from T. globosa and all other species in the genus Torulaspora in the combined sequence analysis of D1/D2 domain and ITS region of rRNA gene. In addition, these strains also showed distinct microsatellite finger-printing pattern from related species and differed in several physiological responses suggesting that these strains belong to a novel species of Torulaspora. We propose to name these strains as Torulaspora indica sp. nov., and designate APSS 805(T) = MTCC 9772 (T) = CBS 12408 (T) as the type strain of this novel species. The Mycobank number of the novel species is MB 563738.

Torulaspora quercuum sp. nov. and Candida pseudohumilis sp. nov., novel yeasts from human and forest habitats.

Strains XZ-46A, XZ-105, XZ-129 and XZ-281(T) isolated from the oral cavities of healthy Tibetan volunteers were revealed to represent two novel ascomycetous yeast species by molecular taxonomic characterizations. Strain XZ-281(T) was most closely related to Candida humilis, but differed from the type strain of the species by eight (1.2%) substitutions in the 26S rRNA gene D1/D2 domain and by >100 (>20%) mismatches in the internal transcribed spacer (ITS) region. Strains XZ-46A, XZ-105 and XZ-129 had identical or similar D1/D2 and ITS sequences with each other and with strain 17YF(T) isolated from a leaf of an oak tree (Quercus sp.). The closest relative of this group was Torulaspora microellipsoides. They differed from the type strain of the species by five (0.9%) substitutions in the D1/D2 domain and >70 (>15%) mismatches in the ITS region. A sexual state was observed in strain 17YF(T), but not in the other four oral strains. An anamorphic name Candida pseudohumilis sp. nov. is proposed for strain XZ-281(T) (=AS 2.3956(T)=CBS 11404(T)) and a teleomorphic name Torulaspora quercuum sp. nov. is proposed for strain 17YF(T) (=AS 2.3768(T)=CBS 11403(T)) and the other three oral strains.

Genetic characterization and phenotypic variability in Torulaspora delbrueckii species: Potential applications in the wine industry.

In this study, several strains of Torulaspora delbrueckii yeast species were evaluated in the laboratory for their enological properties. In a preliminary step, the ability of different molecular methods to discriminate among T.delbrueckii strains was compared. A combination of 7 PCR methods was able to separate 21 strains into 18 groups, while an REA-PFGE method allowed, in one experiment, the separation into 19 groups. The T.delbrueckii strains used presented a wide phenotypic variability in fermentation behaviour, e.g. Lag Phase (LP) duration, T50 parameter (time necessary to ferment half the sugar), and ethanol production. These 3 parameters have to be considered for industrial selection, particularly the LP duration. The majority of T.delbrueckii strains produced 8 to 11% and 7 to 10% ethanol vol. at 17 degrees C and 24 degrees C, respectively, with a maximum ethanol concentration of 12.35 at 17 degrees C and 10.90% vol. at 24 degrees C. The phenotypic variability of this species was also reflected in volatile acidity, glycerol, and aroma production. These experiments confirmed the low volatile acidity and glycerol production of this species and revealed a difference in osmotic stress response, compared to Saccharomyces cerevisiae. T.delbrueckii presented high fermentation purity and produced low levels of undesirable volatile compounds, such as hydrogen sulphide and volatile phenols.