Methods for Oxygenation of Continuous Cultures of Brewer’s Yeast, Saccharomyces cerevisiae

Maintaining steady-state, aerobic cultures of yeast in a bioreactor depends on the configuration of the bioreactor system as well as the growth medium used. In this paper, we compare several conventional aeration methods with newer filter methods using a novel optical sensor array to monitor dissolved oxygen, pH, and biomass. With conventional methods, only a continuously stirred tank reactor configuration gave high aeration rates for cultures in yeast extract peptone dextrose (YPD) medium. For filters technologies, only a polydimethylsiloxan filter provided sufficient aeration of yeast cultures. Further, using the polydimethylsiloxan filter, the YPD medium gave inferior oxygenation rates of yeast compared to superior results with Synthetic Complete medium. It was found that the YPD medium itself, not the yeast cells, interfered with the filter giving the low oxygen transfer rates based on the volumetric transfer coefficient (KLa). The results are discussed for implications of miniaturized bioreactors in low-gravity environments.

A comparative study for production of germ tube in Candida albicans of various pulmonary samples, by different methods in a tertiary care hospital of south west Rajasthan

Background: Systemic candidiasis is associated with a high crude mortality rate, even with first line antifungal therapy. C. albicans is the predominant cause of invasive fungal diseases which is a serious public health issue. The main objective was to assess the reliability of different media for germ tube production in Candida albicans isolated from various clinically diagnosed pulmonary samples.Methods: All Candida isolates were identified and speciated by conventional methods such as Gram’s staining, germ tube test, chlamydospore formation on corn meal agar, sugar fermentation test, sugar assimilation test, and growth on Hi-chrome candida agar.Results: Out of 108 clinical isolates of Candida albicans, 5 different methods were used for germ tube production. Pooled human sera showed 93/108 (86.1%) was the most sensitive method wherein YEPD (yeast extract peptone dextrose) broth 91/108 (84.7%) was the reliable and easy method for detection of germ tube, followed by trypticase soy broth 81/108 (81.4%); peptone water 80/108 (74.7%) and 2% sucrose 71/108 (65.7%).Conclusions: YPED broth is found to be a better serum free substrate and subsequently for the presumptive differentiation of C. albicans from non-albicans candida (NAC), without the extensive time required for the preparation and testing of pooled human serum. Furthermore, this medium is commercially available, more stable, effective, and is not bio hazardous.

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Effect of Choline Acetate on the Yeast Cells During Fermentation: Kinetics Approach

Background: Ionic liquids (ILs) are claimed as green solvents and have demonstrated ability in the dissolution of several biomaterials and polymers, thus promoting their applications in wide scope. The aim of this study was to evaluate the yeast cells in the ionic liquid (IL), choline acetate [Cho]OAc as an eco-friendlier IL for their growth and ability of fermentation of the empty fruit bunch (EFB) biomass for ethanol production via monitoring the cell growth curve and kinetics. Methods: The Saccharomyces cerevisiae was cultured for five days on yeast extract-peptone-dextrose (YPD) agar and the sub-culturing was carried out every two weeks to evaluate the growth of yeast in [Cho]OAc at different concentrations; 5%, 10%, and 20%, where it was pre-cultivated for 24 h at 30 °C and 150 rpm in a shaking incubator as a control culture. Yeast cell number was determined by counting using a hemocytometer. Also, the kinetic growth of yeast during the fermentation of EFB was evaluated. Result: Cell concentration increased, ethanol production increased along with the optical density with the peak reached at 72 h, then declined. Enzymatic hydrolysis process followed by fermentation of EFB using [Cho][OAc] improved the yield of sugar, saccharification and ethanol around 1.938, 2.879, and 2.165 times in comparison with untreated EFB with IL. Furthermore, values of specific growth rates obtained in IL-medium and non-IL-medium are applicable for ethanol production to take place. The results showed the ability of yeast cells for fermentation even in the presence of IL. Conclusion: The results showed that the presence of the IL did not suppress the growth of the yeast cells and did not impair the fermentation productivity.

Effect of phenotypic switching on the susceptibility of Candida krusei towards amphotericin B, nystatin and Piper betle aqueous extract

Candida krusei (C. krusei) is associated with oral candidiasis, particularly in immunecompromised patients. The objective of the study was to determine the effect of phenotypic switching to the susceptibility of C. krusei towards amphotericin B, nystatin and aqueous extract of Piper betle (P. betle). To induce phenotypic switching, C. krusei was inoculated in yeast extract peptone dextrose (YEPD) broth supplemented with 5 mg mL-1 phloxine B andincubated for five hours at 25 °C. Later, 100 µL of the suspension was inoculated on YEPD agar supplemented with 5 mg mL-1 phloxine B and incubated for five days at 25 °C. Disc diffusion and minimum inhibitory concentration (MIC) assays were conducted to determine the susceptibility of C. krusei. The results showed that all C. krusei switched generations were susceptible towards amphotericin B and nystatin with the 3rd and 4th generations significantly more susceptible than the un-switched, respectively (P<0.05). All C. krusei switched generations were also observed to be susceptible towards the aqueous extract of P. betle. The MIC of amphotericin B, nystatin and P. betle were determined at 10 µg mL-1, 10 µg mL-1 and 12.5 mg mL-1, respectively for all generations of C. krusei. In conclusion, the susceptibility of C. krusei was phenotypically switched generation dependent towards amphotericin B, nystatin, and Piper betle aqueous extract.

The Role of Oxidoreductase-Like Protein Olp1 in Sexual Reproduction and Virulence of Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete human fungal pathogen causing lethal meningoencephalitis, mainly in immunocompromised patients. Oxidoreductases are a class of enzymes that catalyze redox, playing a crucial role in biochemical reactions. In this study, we identified one Cryptococcus oxidoreductase-like protein-encoding gene OLP1 and investigated its role in the sexual reproduction and virulence of C. neoformans. Gene expression patterns analysis showed that the OLP1 gene was expressed in each developmental stage of Cryptococcus, and the Olp1 protein was located in the cytoplasm of Cryptococcus cells. Although it produced normal major virulence factors such as melanin and capsule, the olp1Δ mutants showed growth defects on the yeast extract peptone dextrose (YPD) medium supplemented with lithium chloride (LiCl) and 5-fluorocytosine (5-FC). The fungal mating analysis showed that Olp1 is also essential for fungal sexual reproduction, as olp1Δ mutants show significant defects in hyphae growth and basidiospores production during bisexual reproduction. The fungal nuclei imaging showed that during the bilateral mating of olp1Δ mutants, the nuclei failed to undergo meiosis after fusion in the basidia, indicating that Olp1 is crucial for regulating meiosis during mating. Moreover, Olp1 was also found to be required for fungal virulence in C. neoformans, as the olp1Δ mutants showed significant virulence attenuation in a murine inhalation model. In conclusion, our results showed that the oxidoreductase-like protein Olp1 is required for both fungal sexual reproduction and virulence in C. neoformans.

Exploring potentials of indigenous yeasts in fermentation of Chambourcin Grapes

Background: Indigenous yeasts present on grape berries have been shown to impact the winemaking process and final wine quality. In this study, we used Chambourcin, a hybrid grape cultivar, to isolate indigenous yeasts for potential use in winemaking. Hybrid grapes are of particularly interest due to higher resistance to cold temperature and fungal diseases. Methods: Yeasts were isolated from spontaneous fermentation of crushed grapes by plating on Dichloran Rose Bengal Chloramphenicol agar and identified by Sanger sequencing. Yeast candidates were selected for their ability to grow in Yeast Extract-Peptone-Dextrose broth supplemented with varying ethanol concentration. Candidate yeasts were chosen for mock fermentation using sterile Chambourcin juice. Volatile and non-volatile compounds that predict wine quality (flavor and aroma) were measured by UPLC and GC-MS. Results: Hanseniaspora uvarum, Starmerella bacillaris, Candida californica, and Zygoascus meyerae were predominantly isolated from Chambourcin. S. bacillaris isolate 180002 and C. californica isolate 180004 were able to tolerate up to 10% ethanol compared to S. cerevisiae (ethanol tolerance up to 12%). Our results demonstrate that isolate 180002 grown in ethanol supplemented medium is comparable to the commercial S. cerevisiae. Various aroma profiles of three main chemical families – esters, higher alcohol and volatile acids in products fermented by S. bacillaris and other isolates were observed. Conclusion: Starmerella bacillaris isolate 180002 demonstrates potential for use in winemaking with hybrid grapes based on ethanol tolerance and production of wine related chemical compounds. This study further supports the application of indigenous grape-associated yeasts in creating flavor and aroma diversity of wine.

Identification of Yeasts with Mass Spectrometry during Wine Production

The aim of the present study was to identify yeasts in grape, new wine “federweisser” and unfiltered wine samples. A total amount of 30 grapes, 30 new wine samples and 30 wine samples (15 white and 15 red) were collected from August until September, 2018, from a local Slovak winemaker, including Green Veltliner (3), Mūller Thurgau (3), Palava (3), Rhein Riesling (3), Sauvignon Blanc (3), Alibernet (3), André (3), Blue Frankish (3), Cabernet Sauvignon (3), and Dornfelder (3) grapes; federweisser and unfiltered wine samples were also used in our study. Wort agar (WA), yeast extract peptone dextrose agar (YPDA), malt extract agar (MEA) and Sabouraud dextrose agar (SDA) were used for microbiological testing of yeasts. MALDI-TOF Mass Spectrometry (Microflex LT/SH) (Bruker Daltonics, Germany) was used for the identification of yeasts. A total of 1668 isolates were identified with mass spectrometry. The most isolated species from the grapes was Hanseniaspora uvarum, and from federweisser and the wine—Saccharomyces cerevisiae.

Isolation, Identification, and Characterization of Pectinolytic Yeasts for Starter Culture in Coffee Fermentation

This experiment was carried out to identify and select pectinolytic yeasts that have potential use as a starter culture for coffee fermentation during wet processing. The coffee fruit was fermented for 48 h at 28 °C and a sample was taken from the fermented solution and spread onto yeast extract-peptone-dextrose agar (YPDA) media and incubated at 28 °C. A total of 28 yeasts were isolated, eight of which had the ability to produce pectinase enzymes. The species of those eight yeasts were molecularly identified and confirmed. These yeasts are Wickerhamomyces anomalus (strain KNU18Y3), Saccharomycopsis fibuligera (strain KNU18Y4), Papiliotrema flavescens (strain KNU18Y5 and KNU18Y6), Pichia kudriavzevii (strain KNU18Y7 and KNU18Y8), and Saccharomyces cerevisiae (strain KNU18Y12 and KNU18Y13). The pectin degradation index of S. fibuligera (strain KNU18Y4), W. anomalus (strain KNU18Y3), and P. flavescens (strain KNU18Y6) were higher compared to the others, at 178%, 160%, and 152%, respectively. The pectinase enzyme assays were made on two growth media: coffee pulp media (CPM) and synthetic pectin media (SPM). S. fibuligera (strain KNU18Y4) and W. anomalus (strain KNU18Y3) had great potential in producing polygalacturonase (PG) and pectin lyase (PL) compared to others in both media. However, S. cerevisiae strains (KNU18Y12 and KNU18Y13) produced higher pectin methylesterase (PME). Using MEGA 6 software, the phylogenetic trees were constructed to determine the evolutionary relationship of newly identified yeasts from our experiment and previously published yeast species. The sequences of the yeasts were deposited in the National Center for Biotechnology Information (NCBI) database.