The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

Abstract High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production.

High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51g/L and 33.84g/L at 37°C and 40°C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42°C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

Two Gut-Associated Yeasts in a Tephritid Fruit Fly have Contrasting Effects on Adult Attraction and Larval Survival.

Yeast-insect interactions have been well characterized in drosophilid flies, but not in tephritid fruit flies, which include many highly polyphagous pest species that attack ripening fruits. Using the Queensland fruit fly (Bactrocera tryoni) as our model tephritid species, we identified yeast species present in the gut of wild-collected larvae and found two genera, Hanseniaspora and Pichia, were the dominant isolates. In behavioural trials using adult female B. tryoni, a fruit-agar substrate inoculated with Pichia kluyveri resulted in odour emissions that increased the attraction of flies, whereas inoculation with Hanseniaspora uvarum, produced odours that strongly deterred flies, and both yeasts led to decreased oviposition. Larval development trials showed that the fruit-agar substrate inoculated with the 'deterrent odour' yeast species, H. uvarum, resulted in significantly faster larval development and a greater number of adult flies, compared to a substrate inoculated with the 'attractive odour' yeast species, P. kluyveri, and a yeast free control substrate. GC-MS analysis of volatiles emitted by H. uvarum and P. kluyveri inoculated substrates revealed significant quantitative differences in ethyl-, isoamyl-, isobutyl-, and phenethyl- acetates, which may be responsible for the yeast-specific olfactory responses of adult flies. We discuss how our seemingly counterintuitive finding that female B. tryoni flies avoid a beneficial yeast fits well with our understanding of female choice of oviposition sites, and how the contrasting behavioural effects of H. uvarum and P. kluyveri raises interesting questions regarding the role of yeast-specific volatiles as cues to insect vectors. A better understanding of yeast-tephritid interactions could assist in the future management of tephritid fruit fly pests through the formulation of new "attract and kill" lures, and the development of probiotics for mass rearing of insects in sterile insect control programs.

Potential probiotic Pichia kudriavzevii strains and their ability to enhance folate content of traditional cereal-based African fermented food.

With the aim of selecting starter cultures with interesting probiotic potential and with the ability to produce folate in a food matrix, yeast strains isolated from fermented cereal-based African foods were investigated. A total of 93 yeast strains were screened for their tolerance to pH 2 and 0.3% of bile salts. Pichia kudriavzevii isolates gave the best results. Selected P. kudriavzevii strains were tested for survival to the simulated human digestion and for adhesion to Caco-2 cells. Moreover, presence of folate biosynthesis genes was verified and production of extra and intra-cellular folate determined during growth in culture medium. 31% of yeast strains could tolerate pH 2, while 99% bile salts. Survival rate after simulated digestion ranged between 11 and 45%, while adhesion rate between 12 and 40%. Folate production was mainly intracellular, maximum after 24 h of growth. To be closer to traditional cereal-based fermentations, a P. kudriavzevii strain with good probiotic potential was co-inoculated with Lactobacillus fermentum strains in a pearl millet gruel. This resulted in in situ folate production that peaked after 4 h. The use of strains with both probiotic and nutritional enrichment properties may have a greater impact for the consumers.

Isolation, selection and evaluation of antagonistic yeasts and lactic acid bacteria against ochratoxigenic fungus Aspergillus westerdijkiae on coffee beans.

UNLABELLED: In this study, yeasts and lactic acid bacteria (LAB) were isolated from coffee fruits and identified via biochemical and molecular approaches. The isolates represented the Pichia, Debaryomyces, Candida, Clavispora, Yarrowia, Sporobolomyces, Klyveromyces, Torulaspora and Lactobacillus genera. Four isolates, namely Pichia fermentans LPBYB13, Sporobolomyces roseus LPBY7E, Candida sp. LPBY11B and Lactobacillus brevis LPBB03, were found to have the greatest antagonist activity against an ochratoxigenic strain of Aspergillus westerdijkiae on agar tests and were selected for further characterization. Applications of P. fermentans LPBYB13 in coffee cherries artificially contaminated with A. westerdijkiae showed efficacy in reducing ochratoxin A (OTA) content up to 88%. These results highlight that P. fermentans LPBYB13 fulfils the principle requirements of an efficient biological control of aflatoxigenic fungi in coffee beans and may be seen as a reliable candidate for further validation in field conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies based on microbial ecology and antagonistic interactions are important for the development of new strategies in controlling aflatoxin contamination of crops and are relevant to further biotechnological applications. This study shows that coffee fruit is a potential source for the isolation of microbial strains with antifungal ability. A new yeast strain, Pichia fermentans LPBYB13, showed efficacy in reducing growth and ochratoxin A production of Aspergillus westerdijkiae in coffee beans. Our results should encourage the use of this yeast strain on a large scale for biocontrol of aflatoxigenic fungi in coffee beans.

Preparative separation of helvolic acid from the endophytic fungus Pichia guilliermondii Ppf9 by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.

Actividad in vitro de 5 antifúngicos contra aislados clínicos y ambientales de pichia anomala / In vitro activity of 5 antifungal against environmental and clinical isolates of pichia anomala

Las infecciones fúngicas han aumentado en el último tiempo, entre ellas, las infecciones por levaduras emergentes, cuyos patrones de sensibilidad no han sido estudiados debido a la inexistencia de patrones de temperatura y medios de cultivo apropiados para realizar test de sensibilidad. El presente trabajo, fue propuesto como un modelo experimental de Pichia anomala, aislada de pacientes con MICs superiores que los aislados ambientales, siendo la anfotericina B el antifúngico con la mejor actividad antifúngica.

Styrene formation by the decomposition by Pichia carsonii of trans-cinnamic acid added to a ground fish product.

It is not well known how the formation of styrene by microorganisms can occur in foods. In this study, we described and characterized the production of styrene by a yeast isolated from chikuwa fish paste. The styrene was not detected in fresh and normal food products nor in the food package's plastic film. The food containing styrene contained cinnamic acid as an antimicrobial agent and spice, and it was contaminated by 5.4 x 10(6) CFU of a yeast per gram. On the basis of morphological and biochemical features, the yeast isolated was determined to be a strain of Pichia carsonii, now designated strain CHI. Strain CHI, which was able to grow on cinnamic acid, had the ability to form styrene from trans-cinnamic acid via trans-p-coumaric and caffeic acids. The MIC of trans-cinnamic acid against strain CHI was 230 micrograms/ml. Strain CHI thrived well at pH 5.0 and 26.0 degrees C and was tolerant to 20% NaCl. Styrene was subsequently produced in ground fish meat containing cinnamic acid into which strain CHI had been inoculated. The yeast was found to be an environmental contaminant in food processing plants of the chikuwa manufacturer.

[Species composition and biochemical properties of yeasts from the water of the Bratsk reservoir]. / Vidovoi sostav i biokhimicheskie svoistva drozhzhei iz vody Bratskogo vodokhranilishcha.

The specific composition of 370 yeast strains isolated from the water of the Bratsk Reservoir was studied. The strains were assigned to 7 genera and 16 species according to their morphological, cultural, and physiologo-biochemical properties. Asporogenous forms prevailed; 52 strains possessed the amylolytic activity. The proteolytic activity was found in the cultures very seldom. The capability to assimilate organic phosphorus compounds was detected in 8% of the strains, and the ability to use inorganic phosphorus compounds was registered in 38% of the strains. Up to 95% of the strains utilized oil; 88%--engine oil; 43%--phenol. The specific yeast composition varied depending on the biotype.