Gandham S. Prasad

The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic

Ten different versions of the D1/D2 divergent domain of the large-subunit ribosomal DNA were identified among interbreeding members of the yeast species Clavispora lusitaniae. One major polymorphism, located in a 90-bp structural motif of the D2 domain, exists in two versions that differ by 32 base substitutions. Three other polymorphisms consist of a two-base substitution, a two-base deletion, and a single-base deletion, respectively. The polymorphisms are independent of one another and of the two mating types, indicating that the strains studied belong to a single, sexually active Mendelian population. Several strains were heterogeneous for one or more of the polymorphisms, and one strain was found to be automictic and capable of producing asci on its own by isogamous conjugation or by bud-parent autogamy. These observations suggest circumspection in the use of sequence divergence as the principal criterion for delimiting yeast species.

Apophysomyces elegans: an Emerging Zygomycete in India

ABSTRACT Apophysomyces elegans was considered a rare but medically important zygomycete. We analyzed the clinical records of eight patients from a single center in whom zygomycosis due to A. elegans was diagnosed over a span of 25 months. We also attempted a DNA-based method for rapid identification of the fungi and looked for interstrain polymorphism using microsattelite primers. Three patients had cutaneous and subcutaneous infections, three had isolated renal involvement, one had rhino-orbital tissue infection, and the final patient had a disseminated infection involving the spleen and kidney. Underlying illnesses were found in two patients, one with diabetes mellitus and the other with chronic alcoholism. A history of traumatic implantation was available for three patients. All except two of the patients responded to surgical and/or medical therapy; the diagnosis for the two exceptions was made at the terminal stage of infection. Restriction enzyme (MboI, MspI, HinfI) digestion of the PCR-amplified internal transcribed spacer region helped with the rapid and specific identification of A. elegans. The strains could be divided into two groups according to their patterns, with clustering into one pattern obtained by using microsatellite [(GTG)5 and (GAC)5] PCR fingerprinting. The study highlights the epidemiology, clinical spectrum, and diagnosis of emerging A. elegans infections.

Utilization of corn steep liquor for biosynthesis of pullulan, an important exopolysaccharide.

Five different agricultural wastes viz. rice bran oil cake, soya bean oil cake, cotton seed oil cake, mustard seed oil cake and corn steep liquor (CSL) were evaluated for their use as nutrient along with 15% (w/v) glucose as carbon source for biosynthesis of pullulan using Aureobasidium pullulans RBF 4A3. Among the selected agricultural wastes, CSL was found to be the best and supported production of 77.92gL(-1) pullulan under un-optimized conditions. Single point optimization technique resulted in increase in 18% pullulan (88.59gL(-1)) production. The process was successfully validated in a 7-L fermenter and a process economic analysis has suggested that use of CSL as nutrient may result in 3-fold reduction of cost of raw materials for pullulan production as compared to a process where conventional nitrogen sources were used. These observations may be helpful in development of a cost effective process for pullulan production.

Malassezia arunalokei sp. nov., a Novel Yeast Species Isolated from Seborrheic Dermatitis Patients and Healthy Individuals from India.

ABSTRACT The majority of species within the genus Malassezia are lipophilic yeasts that colonize the skin of warm-blooded animals. Two species, Malassezia globosa and Malassezia restricta, are implicated in the causation of seborrheic dermatitis/dandruff (SD/D). During our survey of SD/D cases, we isolated several species of Malassezia and noticed vast variations within a few lipid-dependent species. Variations observed in the phenotypic characteristics (colony morphology, absence of catalase activity, growth at 37°C, and precipitation surrounding wells containing Tween 20 or Cremophor EL) suggested the possible presence of a novel species. Sequence divergence observed in the internal transcribed spacer (ITS) region, the D1/D2 domain, and the intergenic spacer 1 (IGS1) region of rDNA and the TEF1 gene, PCR-restriction fragment length polymorphism (RFLP) analysis of the ITS2 region, and fluorescent amplified fragment length polymorphism analysis support the existence of a novel species. Based on phenotypic and molecular characterization of these strains, we propose a new species, namely, M. arunalokei sp. nov., and we designate NCCPF 127130 (= MTCC 12054 = CBS 13387) as the type strain.

Cost effective production of pullulan from agri-industrial residues using response surface methodology.

Response surface methodology was used to develop an economically feasible process for the fermentative production of pullulan using agri-industrial residues, jaggery, de-oiled jatropha seed cake (DOJSC) and corn steep liquor (CSL), as sole media components. A second order polynomial model was obtained using central composite design to understand the effects of interactions among these substrates on pullulan biosynthesis. Results indicated that, lower concentrations of CSL and DOJSC and higher concentrations of jaggery favoured pullulan production. The optimal nutrient composition (18% jaggery, 3% DOJSC and 0.97% CSL) as suggested by the model resulted in production of 66.25 g/L pullulan with a productivity of 0.92 g/Lh. Analysis of raw material cost component for pullulan production suggested that sole utilization of agri-residues may lead to development of cost effective process for pullulan production.

Gleaning evolutionary insights from the genome sequence of a probiotic yeast Saccharomyces boulardii

BackgroundThe yeast Saccharomyces boulardii is used worldwide as a probiotic to alleviate the effects of several gastrointestinal diseases and control antibiotics-associated diarrhea. While many studies report the probiotic effects of S. boulardii, no genome information for this yeast is currently available in the public domain.ResultsWe report the 11.4 Mbp draft genome of this probiotic yeast. The draft genome was obtained by assembling Roche 454 FLX + shotgun data into 194 contigs with an N50 of 251 Kbp. We compare our draft genome with all other Saccharomyces cerevisiae genomes.ConclusionsOur analysis confirms the close similarity of S. boulardii to S. cerevisiae strains and provides a framework to understand the probiotic effects of this yeast, which exhibits unique physiological and metabolic properties.

Deoiledjatropha seed cake is a useful nutrient for pullulan production

BackgroundEver increasing demand for fossil fuels is a major factor for rapid depletion of these non-renewable energy resources, which has enhanced the interest of finding out alternative sources of energy. In recent years jatropha seed oil has been used extensively for production of bio-diesel and has shown significant potential to replace petroleum fuels at least partially. De-oiled jatropha seed cake (DOJSC) which comprises of approximately 55 to 65% of the biomass is a byproduct of bio-diesel industry. DOJSC contains toxic components like phorbol esters which restricts its utilization as animal feed. Thus along with the enhancement of biodiesel production from jatropha, there is an associated problem of handling this toxic byproduct. Utilization of DOJSC as a feed stock for production of biochemicals may be an attractive solution to the problem.Pullulan is an industrially important polysaccharide with several potential applications in food, pharmaceuticals and cosmetic industries. However, the major bottleneck for commercial utilization of pullulan is its high cost. A cost effective process for pullulan production may be developed using DOJSC as sole nutrient source which will in turn also help in utilization of the byproduct of bio-diesel industry.ResultsIn the present study, DOJSC has been used as a nutrient for production of pullulan, in place of conventional nutrients like yeast extract and peptone. Process optimization was done in shake flasks, and under optimized conditions (8% DOJSC, 15% dextrose, 28°C temperature, 200 rpm, 5% inoculum, 6.0 pH) 83.98 g/L pullulan was obtained. The process was further validated in a 5 L laboratory scale fermenter.ConclusionThis is the first report of using DOJSC as nutrient for production of an exopolysaccharide. Successful use of DOJSC as nutrient will help in finding significant application of this toxic byproduct of biodiesel industry. This in turn also have a significant impact on cost reduction and may lead to development of a cost effective green technology for pullulan production.

The role of culture collections as an interface between providers and users: the example of yeasts

The importance and species diversity of yeasts in food production are described, including a listing of agricultural applications. Two yeast species were selected for case studies on distribution patterns from microbial culture collections: the high representation of Saccharomyces cerevisiae in culture collections enabled global comparison, while Dekkera bruxellensis deposits and distributions were analyzed from the perspective of a single culture collection. In conclusion, culture collections need to cover temporal gaps between deposit and application of strains. The further development of culture collections in countries of high but underexplored species diversity should facilitate the conservation and management of microbial resources.

Complete genome sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii

The probiotic yeast, Saccharomyces boulardii (Sb) is known to be effective against many gastrointestinal disorders and antibiotic-associated diarrhea. To understand molecular basis of probiotic-properties ascribed to Sb we determined the complete genomes of two strains of Sb i.e. Biocodex and unique28 and the draft genomes for three other Sb strains that are marketed as probiotics in India. We compared these genomes with 145 strains of S. cerevisiae (Sc) to understand genome-level similarities and differences between these yeasts. A distinctive feature of Sb from other Sc is absence of Ty elements Ty1, Ty3, Ty4 and associated LTR. However, we could identify complete Ty2 and Ty5 elements in Sb. The genes for hexose transporters HXT11 and HXT9, and asparagine-utilization are absent in all Sb strains. We find differences in repeat periods and copy numbers of repeats in flocculin genes that are likely related to the differential adhesion of Sb as compared to Sc. Core-proteome based taxonomy places Sb strains along with wine strains of Sc. We find the introgression of five genes from Z. bailii into the chromosome IV of Sb and wine strains of Sc. Intriguingly, genes involved in conferring known probiotic properties to Sb are conserved in most Sc strains.

Understanding the effect of interaction among aeration, agitation and impeller positions on mass transfer during pullulan fermentation by Aureobasidium pullulans

Pullulan is a non-ionic, water-soluble homopolysaccharide produced via fermentation using Aureobasidium pullulans, a black yeast. The unique physicochemical properties of pullulan have made it a potential candidate for a diverse range of applications in various industrial sectors such as food, pharmaceutical, cosmetics, and biomedicine. Low yield and productivity are major challenges for the extensive commercialization of this biopolymer, and they are often found to be associated with poor mass transfer during the fermentative production of pullulan. The present study is an attempt to address this challenge by a unique multivariate approach. The interactive influence of air-flow rates, agitation speed and impeller spacing on volumetric mass transfer coefficient (kLa) was investigated using a statistical design. The design has demonstrated a unique correlation between mass transfer capabilities and process variables, whereas pullulan fermentation kinetics has revealed a dependence of microbial activity on kLa values in a bioprocess. Furthermore, the resultant mass transfer conditions were applied for pullulan production, which enhanced the productivity significantly (1.61 g L−1 h−1). These observations might help in obtaining improved mass transfer conditions for the economic production of pullulan.