Yeasts from Lesotho: their classification and possible applications
In view of the decline of natural habitats due to urban and industrial development, the need to search for new yeasts is pressing since few natural habitats have been thoroughly investigated for yeast species. Yeasts have not been isolated from Lesotho before despite being an ideal environment for yeasts. In addition, isolation of yeasts able to utilize complex substrates, similar to intermediates in the degradation of chlorophenols would be important in their detoxification. Yeasts with these abilities are usually isolated from polluted environments and their presence from the pristine environment in Lesotho would be unexpected. The species Lipomyces starkeyi and L. tetrasporus were found distributed throughout the various habitats while Debaryomyces hansenii, D. hansenii var. fabryi and D. occidentalis were found in 60% - 70% of the different regions. Debaryomyces polymorphus, Dipodascus spicifer, Galactomyces geotrichum, G. reessii, Kluyveromyces lactis , L. kononenkoae, L. mesembrius, L. spencermartinsiae, Pichia anomala, P. fabianii, P. guilliermondii and Yarrowia lipolytica were site specific. The fatty acid profiles of the isolated lipomycetous yeasts are similar to those reported in literature. This corroborates the value of this phenotypic characteristic in the taxonomy of these yeasts. The PCR products of the ITS region of some of the type strains of the family Lipomycetaceae showed high length variation enabling rapid identification. The type strains, the sub-species and the varieties of the family Lipomycetaceae could be differentiated from each other using RFLP profiles obtained with the restriction enzymes used in combination i.e. Cfo I, HaeIII and MboI. The RFLP profiles for the five Lesotho isolates with atypical carbohydrate patterns could be separated into three groups. The first group, comprising of three isolates (52b, 73, 93), gave similar restriction patterns suggesting that they are the same species. Isolate 58b yielded a distinct profile while isolate 97 had similar sized ITS-PCR (1000bp) to that of L. lipofer and L. tetrasporus . It is important to assess the variation in RFLP profiles between various strains from different habitats of a particular species in order to determine the conserved status of this genotypic character. More strains of a species representing the Lipomycetaceae should be subjected to similar RFLP analysis to further determine its conserved status. The D1/D2 sequence data enabled separation of the five isolates into three groups. The first group, comprising of three isolates, showed 1% nucleotide substitutions to L. starkeyi suggesting that these isolates are probably known Lipomyces spp. The other two isolates yielded sequences that were 99% identical to L. kononenkoae subsp. kononenkoae (isolate 58b) and 100% identical to that of L. tetrasporus (isolate 97) further suggesting that these isolates are probably known Lipomyces species. Isolation of basidiomycetous yeasts from unusual carbon sources has been reported, however, this is the first report of isolation of ascomycetous yeasts able to grow on unusual substrates. The substrate that yielded the highest number of isolates was 1,4 cyclohexanedimethanol. Pichia anomala and Y. lipolytica strains (identity confirmed with D1/D2 sequencing) isolated from 2-chlorobutyric acid could not degrade the 2-chlorobutyric acid probably due to toxicity of low molecular weight organic acids at low pH. The ability of P. anomala isolated from 1,4 cyclohexanedimethanol to grow on dodecane and pristane is unusual and this strain should also be subjected to sequencing to confirm identification. Debaryomyces hansenii, P. anomala, P. fabianii, P. guilliermondii and Y. lipolytica could grow on the cyclohexane derivatives although they preferred a shorter straight chain hydrocarbon as confirmed by higher growth rates in the presence of dodecane. The isolates could not grow in the presence of monoterpenes, although they had been isolated from substrates that were supposed to mimic the structures of monoterpenes and which previously had yielded limonene utilizing Rhodotorula species. The inability of these strains to grow on monoterpenes might be due to toxicity of the monoterpenes, because of their hydrophobicity and lipophilicity resulting in partitioning into the lipid bilayer of cell membranes.