The Use of Raman Spectroscopy to Monitor Metabolic Changes in Stressed Metschnikowia sp. Yeasts

Raman spectroscopy is a universal method designed for the analysis of a wide range of physical, chemical and biological systems or various surfaces. This technique is suitable to monitor various components of cells, tissues or microorganisms. The advantages include very fast non-contact and non-destructive analysis and no or minimal need for sample treatment. The yeasts Metschnikowia can be considered as industrially usable producers of pulcherrimin or single-cell lipids, depending on cultivation conditions and external stress. In the present study, Raman spectroscopy was used as an effective tool to identify both pulcherrimin and lipids in single yeast cells

Use of Waste Substrates for the Lipid Production by Yeasts of the Genus Metschnikowia—Screening Study

Oleogenic yeasts are characterized by the ability to accumulate increased amounts of lipids under certain conditions. These microbial lipids differ in their fatty acid composition, which allows them to be widely used in the biotechnology industry. The work focuses on the influence of various stress factors in the cultivation process, such as reduced temperature or nutritional stress through the use of various waste substrates, together with manipulating the ratio of carbon and nitrogen sources in the medium. The ability of yeast to produce significant amounts of unsaturated fatty acids was also demonstrated in the work. The most suitable substrate for lipid production was a medium containing glycerol, where the amount of accumulated lipids in the yeast M. pulcherrima 1232 was up to 36%. In our work, the crude animal fat was used for the production of high-value lipids, which to the best of our knowledge is a new result

Generacija novih genotipskih i fenotipskih svojstava prirodnih i umjetnih hibrida kvasaca

Evolution and genome stabilization have mostly been studied on the Saccharomyces hybrids isolated from natural and alcoholic fermentation environments. Genetic and phenotypic properties have usually been compared to the laboratory and reference strains, as the true ancestors of the natural hybrid yeasts are unknown. In this way the exact impact of different parental fractions on the genome organization or metabolic activity of the hybrid yeasts is difficult to resolve completely. In the present work the evolution of geno- and phenotypic properties is studied in the interspecies hybrids created by the cross-breeding of S. cerevisiae with S. uvarum or S. kudriavzevii auxotrophic mutants. We hypothesized that the extent of genomic alterations in S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii should affect the physiology of their F1 offspring in different ways. Our results, obtained by amplified fragment length polymorphism (AFLP) genotyping and karyotyping analyses, showed that both subgenomes of the S. cerevisiae x S. uvarum and of S. cerevisiae × S. kudriavzevii hybrids experienced various modifications. However, the S. cerevisiae × S. kudriavzevii F1 hybrids underwent more severe genomic alterations than the S. cerevisiae × S. uvarum ones. Generation of the new genotypes also influenced the physiological performances of the hybrids and the occurrence of novel phenotypes. Significant differences in carbohydrate utilization and distinct growth dynamics at increasing concentrations of sodium chloride, urea and miconazole were observed within and between the S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii hybrids. Parental strains also demonstrated different contributions to the final metabolic outcomes of the hybrid yeasts. A comparison of the genotypic properties of the artificial hybrids with several hybrid isolates from the wine-related environments and wastewater demonstrated a greater genetic variability of the S. cerevisiae × S. kudriavzevii hybrids. Saccharomyces cerevisiae × S. uvarum artificial and natural hybrids showed considerable differences in osmolyte tolerance and sensitivity to miconazole, whereas the S. cerevisiae × S. kudriavzevii hybrids exhibited differences also in maltotriose utilization. The results of this study suggest that chromosomal rearrangements and genomic reorganizations as post-hybridization processes may affect the phenotypic properties of the hybrid progeny substantially. Relative to their ancestors, the F1 segregants may generate different phenotypes, indicating novel routes of evolution in response to environmental growth conditions.Evolucija i stabilizacija genoma kvasca uglavnom se proučavaju s pomoću interspecijskih hibrida roda Saccharomyces, izoliranih iz prirodnih staništa ili tijekom alkoholnih fermentacija. Njihova genetska i fenotipska svojstva obično se uspoređuju sa svojstvima laboratorijskih i referentnih sojeva, budući da su izvorni roditeljski sojevi prirodnih hibrida kvasaca nepoznati. Na ovaj je način teško u potpunosti razumjeti utjecaj različitih roditeljskih frakcija na organizaciju genoma ili metaboličku aktivnost hibrida kvasaca. U ovom je radu proučena evolucija genotipskih i fenotipskih svojstava interspecijskih hibrida, nastalih križanjem kvasca S. cerevisiae s auksotrofnim mutantima kvasaca S. uvarum i S. kudriavzevii. Naša je hipoteza bila da bi genomske promjene nastale u hibridima S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii trebale na različite načine utjecati na fiziologiju njihovih F1 segreganata. Rezultati dobiveni genotipizacijom, tj. analizom polimorfizma duljine umnoženih fragmenata (engl. amplified fragment length polymorphism - AFLP) i kariotipizacijom pokazuju da su oba subgenoma hibrida S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii izmjenjena. Međutim, promjene genoma segreganata F1 hibrida S. cerevisiae × S. kudriavzevii bile su znatnije od onih segreganata hibrida S. cerevisiae × S. uvarum. Novi su genotipovi utjecali na fiziološke značajke hibrida te nastanak novih fenotipova. Bitna je razlika među hibridima S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii opažena u potrošnji šećera i različitoj dinamici rasta kod povećanih koncentracija natrijevog klorida, uree i mikonazola. Roditeljski su sojevi različito utjecali na konačnu metaboličku sliku hibrida kvasaca. Usporedbom genotipskih svojstava umjetnih hibrida s nekoliko hibrida izoliranih iz prirodnih staništa (vinograda i otpadnih voda) utvrđena je veća genetska raznolikost hibrida S. cerevisiae × S. kudriavzevii. Umjetni i prirodni hibridi Saccharomyces cerevisiae × S. uvarum bili su različito osjetljivi prema osmolitima i mikonazolu, dok su se hibridi S. cerevisiae × S. kudriavzevii razlikovali i u potrošnji maltotrioze. Iz dobivenih se rezultata može zaključiti da kromosomalna rekombinacija i genomska reorganizacija kao post-hibridizacijski procesi mogu značajno utjecati na fenotipska svojstva hibridnih potomaka. U usporedbi s roditeljskim sojevima, segreganti F1 mogu generirati različite fenotipove, što upućuje na zaključak da su specifični uvjeti rasta kvasaca uzrokovali nastanak novih evolucijskih tokova

Next-Generation Sequencing Reveals Significant Bacterial Diversity of Botrytized Wine

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies

Taxonomic Revision of the pulcherrima Clade of Metschnikowia (Fungi): Merger of Species

The type strains of 10 small-spored species of the ascomycetous yeast genus Metschnikowia usually form a compact group on the phylogenetic trees inferred from barcode sequences. Based on the name of the species, which was described first (Metschnikowia pulcherrima), the group is frequently referred to as the pulcherrima clade. All strains produce the iron-chelate pigment pulcherrimin and have antagonistic effects on many microorganisms. Recent results of molecular phylogenetic, genetic, and genomic research raised doubts about the taxonomic division of the clade. Those data—combined with results obtained in this study by comprehensive analysis of primary and secondary barcode sequences, physiological tests, and hybridisation experiments—demonstrate that the species cannot be distinguished from each other by the criteria of any of the phenotypic, phylogenetic, and biological species concepts. Therefore, I propose that the species of the pulcherrima clade be merged into one species under the oldest species name, M. pulcherrima

Wine Yeasts 1.0

The conversion of grape juice into wine is a complex biochemical process involving alcoholic fermentation, production of wide range of metabolites and interactions of yeast strains, bacteria and fungi [...