Xylitol production by Candida species grown on a grass hydrolysate

Xylitol was produced by selected species of the yeast Candida after growth on a medium containing a hydrolysate of the North American perennial prairie grass big bluestem. The grass was hydrolysed by a combination of dilute acid and enzymatic treatments. After growth on the medium for 120 h at 30 °C, Candida tropicalis ATCC 750 produced a 1.4-fold higher level of xylitol than did C. tropicalis ATCC 20215 while biomass production by C. tropicalis ATCC 750 was 1.7-fold higher than Candida guilliermondii ATCC 20216. The xylitol yields observed for C. tropicalis ATCC 750, Candida mogii ATCC 18364 and C. guilliermondii ATCC 20216 were at least 1.4-fold higher than the yield observed for C. tropicalis ATCC 20215 after growth for 120 h at 30 °C.     

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture, from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at 28 °C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.     

The shelf life and effectiveness of granular formulations of Metschnikowia pulcherrima and Pichia guilliermondii yeast isolates that control postharvest decay of citrus fruit

Our overall objectives were to prepare commercially acceptable formulations of the postharvest biological control yeasts, Metschnikowia pulcherrima and Pichia guilliermondii, which have a long storage life and to determine the effectiveness of these formulations to control postharvest green and blue moulds on citrus fruit. Yeasts, grown on a cane molasses-based medium, were combined with talc or kaolin carriers and various adjuvants and the viability of yeast in 12 formulations was determined over a 6 month period. Formulation no. 11, containing talc, sodium alginate, sucrose, and yeast extract, for both yeasts had a significantly higher viable yeast cell content over a 6 month storage period. Among the formulations, three formulations (formulations no. 5, 6, and 11) were selected for additional in vivo testing because they had higher levels of viability amongst yeast cell populations during storage and were easier to resuspend remained in suspension more easily. These formulations were tested on Satsuma mandarin and grapefruit to control green and blue moulds. Formulations no. 5, 6, and 11 for both yeasts effectively controlled green mould, while only formulation no. 11 with either yeast isolate M. pulcherrima (isolate M1/1) or P. guilliermondii (isolate P1/3) effectively controlled both blue and green moulds.     

L-Arabinose metabolism in Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation

l-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603^T and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to d-glucose and d-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from d-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated—arabitol from l-arabinose and xylitol from d-xylose. Different l-arabinose concentrations and oxygen conditions were tested to better understand l-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from l-arabinose (and also xylitol from d-xylose) than C. arabinofermentans PYCC 5603^T. In contrast to the latter, P. guilliermondii PYCC 3012 did not produce any traces of ethanol in complex l-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in l-arabinose-assimilating yeasts.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

Production of bioethanol by direct bioconversion of oil-palm industrial effluent in a stirred-tank bioreactor

The purpose of this study was to evaluate the feasibility of producing bioethanol from palm-oil mill effluent generated by the oil-palm industries through direct bioconversion process. The bioethanol production was carried out through the treatment of compatible mixed cultures such as Thrichoderma harzianum, Phanerochaete chrysosporium, Mucor hiemalis, and yeast, Saccharomyces cerevisiae. Simultaneous inoculation of T. harzianum and S. cerevisiae was found to be the mixed culture that yielded the highest ethanol production (4% v/v or 31.6 g/l). Statistical optimization was carried out to determine the operating conditions of the stirred-tank bioreactor for maximum bioethanol production by a two-level fractional factorial design with a single central point. The factors involved were oxygen saturation level (pO₂%), temperature, and pH. A polynomial regression model was developed using the experimental data including the linear, quadratic, and interaction effects. Statistical analysis showed that the maximum ethanol production of 4.6% (v/v) or 36.3 g/l was achieved at a temperature of 32°C, pH of 6, and pO₂ of 30%. The results of the model validation test under the developed optimum process conditions indicated that the maximum production was increased from 4.6% (v/v) to 6.5% (v/v) or 51.3 g/l with 89.1% chemical-oxygen-demand removal.     

Evaluation of the Pro-Lab ID ring system for the identification of medically important yeasts

We evaluated 151 coded isolates of medically important yeast species belonging to the genera Candida, Cryptococcus, Geotrichum, Rhodoturula, Saccharomyces and Torulopsis using the newly developed rapid Pro-Lab Identification Ring, PL 960 system (PLID-Ring). All isolates were concurrently identified by the API 20C and conventional procedures comprising macro- and micromorphology, assimilation and fermentation of various carbon and nitrogen compounds. The PLID-Ring system identified isolates of Candida albicans, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, Rhodotorula rubra, and Torulopsis glabrata with 100% accuracy in 24 h. This system identified C guilliermondii and S. cerevisiae isolates with an accuracy of 90% and 86%, respectively, while those belonging to Cr. neoformans, T. candida (= C. famata), C. rugosa and C. tropicalis were identified with 38.4%, 50%, 12.5% and 50% accuracy, respectively. Three isolates of Cr. laurentii were not identified by the PLID-Ring system. The overall accuracy of the PLID-Ring system was 81.45% (123 of 151 isolates). However, the system does not include species such as Cr. laurentii in its data base. When these three Cr. laurentii isolates were excluded from the evaluation, the accuracy of the PLID-Ring system increased from 81.45% to 83.1%.     

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (<Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt.) and white (<Emphasis Type="Italic">Tuber magnatum</Emphasis> Pico) truffles

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated l-methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.     

Screening and identification of yeast strains from fruits and vegetables: Potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici)

Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4%, respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis, and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature. The application of P. guilliermondii is more effective for preserving chilli fruits than conventional preservation with chlorinated water.     

Characteristics of Egyptian boza and a fermentable yeast strain isolated from the wheat bread

A sample of indigenous beer, boza was collected at Cairo, Egypt and analysed. Boza was an off-white porridge-like slurry containing 3.8% (v/v) ethanol. Volatile esters and higher alcohols such as ethyl acetate and isoamyl alcohol were detected in the boza by gas chromatography. The pH of the boza was 3.7. Organoleptically, this alcoholic beverage had an estery flavour and a sour taste. A fermentable yeast strain EG1 was isolated from the material wheat bread and identified, and was considered to resemble Candida krusei. The rice sake made with the yeast strain C. krusei EG1 at 30 °C contained 11.7% ethanol, 74.1 mg/l ethyl acetate and its pH value was 4.2.     

Restoration of the Wild-Type Phenotype in Pichia guilliermondii Transformants

Pichia guilliermondii strain with blocked GTP cyclohydrolase II was transformed using replicative plasmids and their fragments containing the structural gene RIB1 of this enzyme. Experiments showed that the presence of the ARS element and the promoter region of this gene in the genome of transformants reduces the probability of their reversion to the wild-type phenotype. Different types of recombination in the yeast P. guilliermondiiare discussed.     

Several new substrates for Desulfovibrio vulgaris strain Marburg and a spontaneous mutant from it

The ability of Desulfovibrio vulgaris strain Marburg (DSM 2119) to oxidize alcohols was surveyed in the presence and absence of hydrogen-scavenging anaerobes, Acetobacterium woodii and Methanospirillum hungatei. In the presence of sulfate, D. vulgaris grew not only on ethanol, 1-propanol, and 1-butanol, but also on isobutanol, 1-pentanol, ethyleneglycol, and 1,3-propanediol. Metabolism of these alcohols was simple oxidation to the corresponding acids, except with the last two substrates: ethyleneglycol was oxidized to glycolate plus acetate, 1,3-propanediol to 3-hydroxypropionate plus acetate. Experimental evidence was obtained, suggesting that 2-methoxyethanol was not utilized by all the cells of strain marburg, but by a spontaneous mutant. 2-Methoxyethanol was oxidized to methoxyacetate by the mutant. Co-culture of strain Marburg plus A. woodii grew on ethanol, 1-propanol, 1-butanol, and 1,3-propanediol in the absence of sulfate. Co-culture of strain Marburg plus M. hungatei grew on ethanol, 1-propanol, and 1-butanol, but not on ethyleneglycol and 1,3-propanediol, Co-culture of the mutant plus A. woodii or M. hungatei did not grow on 2-methoxyethanol.     

The Photodynamic Inactivation of the Yeast Candida guilliermondii in the Presence of Photodithazine

Photodithazine, a glucosamine salt of chlorin e ₆, is highly effective in sensitization of Candida guilliermondii cells to visible light. The sensitizing effect of photodithazine was found to be related to free or cell surface–bound molecules of this dye. Sodium azide (a singlet oxygen quencher) and propyl gallate (an inhibitor of lipid peroxidation) protected yeast cells from the photodithazine-enhanced photoinactivation.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> age,cultivation field location and yeast strain on tequila fermentation process

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.     

URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量

CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae) 1211半乳糖代谢负调控基因GAL80,获得菌株S. cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S. cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S. cerevisiae 1211-3。S. cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青...     

The types of bioreactors used for shoots and embryos

Plant regenerated organs such as shoots, bulbs, microtubers, corms, embryos, etc. have been successfully proliferated in the bioreactor. The use of a bioreactor leads to the development of technology suitable for large scale plant propagation. The basic construction and characteristics of various types of bioreactor systems are reviewed in relation to shoot and embryo cultures. A pilot scale 500 liter bioreactor system was applied to the production of large scale Stevia rebaudiana shoots.Abbreviations DW dry weight - EC electrical conductivity - FW fresh weight - ORP oxidation-reduction potential     

Evaluation of Saccharomyces cerevisiae grown in a multistage chemostat environment under increasing levels of glucose

The concept of net consumption and/or production rates of fermentation metabolites has been proposed and applied to evaluate the performance of Saccharomyces cerevisiae grown in a multistage chemostat environment under increasing glucose concentrations (increased gravity). Results show that both rates are strongly affected by high glucose doses (osmotic pressure) and that ethanol inhibition has a profound influence on the performance of yeast cells.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.