Bhaskar Bhadra

Yeasts and yeast-like fungi associated with tree bark: diversity and identification of yeasts producing extracellular endoxylanases.

A total of 239 yeast strains was isolated from 52 tree bark samples of the Medaram and Srisailam forest areas of Andhra Pradesh, India. Based on analysis of D1/D2 domain sequence of 26S rRNA gene, 114 strains were identified as ascomycetous; 107 strains were identified as basidiomycetous yeasts; and 18 strains were identified as yeast-like fungi. Among the ascomycetous yeasts, 51% were identified as members of the genus Pichia, and the remaining 49% included species belonging to the genera Clavispora, Debaryomyces, Kluyveromyces, Hanseniaspora, Issatchenkia, Lodderomyces, Kodamaea, Metschnikowia, and Torulaspora. The predominant genera in the basidiomycetous yeasts were Cryptococcus (48.6%), Rhodotorula (29%), and Rhodosporidium (12.1%). The yeast-like fungi were represented by Aureobasidium pullulans (6.7%) and Lecythophora hoffmanii (0.8%). Of the 239 yeast strains tested for Xylanase, only five strains of Aureobasidium sp. produced xylanase on xylan-agar medium. Matrix-assisted laser desorption ionization-time of flight analysis and N-terminal amino-acid sequence of the xylanase of isolate YS67 showed high similarity with endo-1-4-β-xylanase (EC 3.2.1.8) of Aureobasidium pullulans var. melanigenum.

Isolation and characterization of ethanol-producing yeasts from fruits and tree barks.

Aims:  Isolation and identification of yeasts converting xylose to ethanol.

Diversity of Yeasts from Puddles in the Vicinity of Midre Lovénbreen Glacier, Arctic and Bioprospecting for Enzymes and Fatty Acids

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C18:1 (w9C) and C18:2 (w9,12C) were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8°C compared to 22°C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.

Isolation and Characterization of Xylitol-Producing Yeasts from the Gut of Colleopteran Insects

A total of 35 yeasts were isolated from the gut of beetles collected from Hyderabad city, India. Twenty of these yeasts utilized xylose as a sole carbon source but only 12 of these converted xylose to xylitol. The ability to convert xylose to xylitol varied among the isolates and ranged from 0.12 to 0.58 g/g xylose. Based on the phenotypic characteristics and phylogenetic analysis of the D1/D2 domain sequence of 26S rRNA gene, these isolates were identified as members of Pichia, Candida, Issatchenkia, and Clavispora. Strain YS 54 (CBS 10446), which was phylogenetically similar to Pichia caribbica and which formed hat-shaped ascospore characteristics of the genus Pichia, was the best xylitol producer (0.58 g xylitol/g xylose). YS 54 was also capable of producing xylitol from sugarcane bagasse hydrolysate and the efficiency of conversion was 0.32 g xylitol/g xylose after 20 cycles of adaptation in medium containing sugarcane bagasse hydrolysate.

Exiguobacterium soli sp. nov., a psychrophilic bacterium from the McMurdo Dry Valleys, Antarctica

A novel psychrophilic bacterium, designated strain DVS 3Y(T), was isolated from a moraine sample from the McMurdo Dry Valleys, Antarctica. Phenotypic and chemotaxonomic characteristics and data from a phylogenetic analysis based on 16S rRNA gene sequences indicated that strain DVS 3Y(T) was related to the genus Exiguobacterium. Strain DVS 3Y(T) exhibited >97 % gene sequence similarity with respect to Exiguobacterium acetylicum DSM 20416(T) (97.4 %), Exiguobacterium oxidotolerans DSM 17272(T) (97.4 %), Exiguobacterium indicum IAM 15368(T) (97.6 %), Exiguobacterium undae DSM 14481(T) (98.2 %), Exiguobacterium sibiricum DSM 17290(T) (98.6 %) and Exiguobacterium antarcticum DSM 14480(T) (99.6 %). However, the levels of DNA-DNA relatedness between strain DVS 3Y(T) and E. acetylicum DSM 20416(T), E. oxidotolerans JCM 12280(T), E. indicum IAM 15368(T), E. undae DSM 14481(T), E. sibiricum DSM 17290(T) and E. antarcticum DSM 14480(T) were less than 70 % (52, 25, 16, 33, 16 and 38 %, respectively). Strain DVS 3Y(T) also differed from these six closely related species in a number of phenotypic traits. Phenotypic, chemotaxonomic and phylogenetic data suggest that strain DVS 3Y(T) represents a novel species of the genus Exiguobacterium, for which the name Exiguobacterium soli sp. nov. is proposed. The type strain is DVS 3Y(T) (=MTCC 4816(T)=JCM 14376(T)).

Rhodotorula himalayensis sp. nov., a novel psychrophilic yeast isolated from Roopkund Lake of the Himalayan mountain ranges, India

Twenty-five psychrophilic yeasts were isolated from the soil of Roopkund Lake, Himalayas, India. Two colony morphotypes were identified and representatives of ‘morphotype 1’ were identified as Cryptococcus gastricus. Representatives of ‘morphotype 2’, namely 3AT, 4A, 4B and Rup4B, showed similar phenotypic properties and are identical with respect to the nucleotide sequence of the ITS1-5.8S rRNA gene-ITS2 region and D1/D2 domain of the 26S rRNA gene. The sequence of D1/D2 domain of 3AT shows 97.6–98.8% similarity with Rhodotorulapsychrophila CBS10440T, Rhodotorulaglacialis CBS10437T and Rhodotorulapsychrophenolica CBS10438T and in the neighbour-joining phylogenetic tree strains; 3AT, 4A, 4B and Rup4B form a cluster with Rhodotorula glacialis and Rhodotorula psychrophila. Strains 3AT, 4A, 4B and Rup4B also differ from their nearest phylogenetic relatives in several biochemical characteristics such as in assimilation of d-galactose, l-sorbose, maltose, citrate, d-glucuronate and creatinine. Thus, based on the phylogenetic analysis and the phenotypic differences 3AT, 4A, 4B and Rup 4B are assigned the status of a new species of Rhodotorula for which the name Rhodotorula himalayensis sp. nov. is proposed with 3AT as the type strain (=CBS10539T =MTCC8336T).

Thiomonas bhubaneswarensis sp. nov., an obligately mixotrophic, moderately thermophilic, thiosulfate-oxidizing bacterium.

A novel aerobic, obligately mixotrophic, moderately thermophilic, thiosulfate-oxidizing bacterium, S10T, was isolated from hot-spring sediment samples collected from Atri, Bhubaneswar, India. The cells of this isolate stained Gram-negative and were strictly aerobic, non-sporulating, rod-shaped and motile with a single polar flagellum. Strain S10T was positive for oxidase and catalase activities. It was capable of utilizing thiosulfate under mixotrophic growth conditions. Mixotrophic growth was observed at pH 6.0-8.5 and 25-45 degrees C; optimum growth occurred at pH 7.5-8.0 and 30-37 degrees C. The major cellular fatty acids were C12:0 3-OH, C16:1omega7c, C16:0, C17:0 cyclo, C18:1omega7c and C19:0 cyclo omega8c. The DNA G+C content of strain S10T was 64.8 mol%. 16S rRNA gene sequence analysis indicated that the bacterium clustered within the radiation of the genus Thiomonas and showed 98.0% similarity with Thiomonas perometabolis ATCC 23370T and Thiomonas intermedia ATCC 15466T. However, DNA-DNA reassociation values of strain S10T with Thiomonas perometabolis JCM 20426T and Thiomonas intermedia JCM 20425T, its nearest phylogenetic relatives, were 46 and 39%, respectively. On the basis of phenotypic, physiological and chemotaxonomic properties, 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, it is proposed that strain S10T represents a novel species of the genus Thiomonas, Thiomonas bhubaneswarensis sp. nov.; the type strain is S10T (=DSM 18181T=JCM 14806T).

Pichia garciniae sp. nov., isolated from a rotten mangosteen fruit (Garcinia mangostana L., Clusiaceae).

Ascogenous yeasts were isolated from a decaying mangosteen fruit (Garcinia mangostana L., Clusiaceae). Based on colony morphology and RAPD analysis, the strains were grouped into four groups (G-I, G-II, G-III and G-IV). Phenotypic properties and analysis of the D1/D2 domain sequence of the 26S rRNA gene identified representative members of groups G-II, G-III and G-IV as Pichia manshurica (=Pichia galeiformis), Pichia fermentans and Pichia anomala, respectively. Representatives of group G-I, YS110(T) and YS111, showed similar phenotypic traits, 99.9 % similarity in the sequence of the D1/D2 domain of the 26S rRNA gene and ITS1-5.8S rRNA gene-ITS2 sequence and 92 % DNA-DNA relatedness. Hence, YS110(T) and YS111 are regarded as members of the same species. Based on D1/D2 domain and ITS region sequences, the nearest phylogenetic neighbours of YS110(T) and YS111 were identified as Pichia membranifaciens and P. manshurica. However, YS110(T) differs from these two nearest phylogenetic neighbours by >3 % in the D1/D2 domain sequence and by >18 % with respect to the ITS region sequence. In addition, YS110(T) and YS111 differ from P. membranifaciens NRRL Y-2026(T) and P. manshurica NRRL Y-27978(T) with respect to a number of phenotypic traits. The strains show 52-57 % DNA-DNA relatedness with P. membranifaciens NRRL Y-2026(T) and P. manshurica NRRL Y-27978(T). Strains YS110(T) and YS111 are proposed as two strains of a novel species, for which the name Pichia garciniae sp. nov. is proposed. The type strain is YS110(T) (=NRRL Y-48422(T) =CBS 10758(T)).

RESEARCH ARTICLE: Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae)

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae)

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.