In this diploma thesis we investigate how wine characteristics is influenced by the apiculate wine yeast Metschnikowia pulcherrima. For this purpose, two wines of a grape variety Welschriesling were manufactured using an identical technological approach with the only distinction: two separated musts were supplied with broth containing different yeasts. The literary part of the thesis discusses yeasts used in winery in general. We describe both apiculate yeasts and Saccharomyces. In this part, we also further discuss the polymer chain reaction and similar methods. The experimental part deals with possibilities of Metschnikowia pulcherrima DNA isolation from fermenting must and the subsequent quantification of yeasts with help of the real-time PCR method. After evaluation and comparison of the wines, where both general and expert public participated, it was concluded that the yeasts substantially influence the wine cha-racteristics.

The oleaginous yeasts have an ability to accumulate an increased number of lipids, under certain circumstances. These microbial lipids differentiate in the number of fat acids present, which enables their wide application in biotechnological industry. This master’s thesis is aimed on lipid production, number of the fat acid groups present, and squalene production by Metschnikowia yeasts, based on the cultivating conditions. Biomass and lipid production was observed in separate cultivation media, with the addition of the different waste substrates. Production properties were observed by method of the gas chromatography. For the squalene production observation, a HLPC method was chosen. All production groups were able to accumulate lipids on the waste substrate, although in different values. These values were very individual, especially in the areas of the specific groups and growth on the given substrate. The lipid composition was different, which was caused by differences in the waste substrates. With regards to the squalene production – the yeasts from the Metschnikowia family were not able to produce squalene in the presence of the terbinafine and its increasing concentration. Therefore, the same procedure was chosen, as it was for the Yarrowia lipolytica yeast, with the difference in the sterol synthesis, however squalene was still not produced this way.

This thesis was focused on study of biotechnological utilization of waste substrates to produce lipids by yeast of the genus Metschnikowia. Waste materials and their subsequent transformation into high value-added products such as microbial lipids are currently considered as an alternative source for biofuel production. Therefore, the experimental part was aimed at investigating the influence of a carbon source to the controlled overproduction of lipids by yeast Metschnikowia. Total of 12 yeast strains of the genus Metschnikowia were selected. Yeast strains M. pulcherrima , M. pulcherrima 147, M. pulcherrima 149, M. andauensis 129 a M. fructicola 15 were purchased from Culture Collection of Yeasts (CCY, Bratislava, Slovakia). The growth characteristics of this yeast strains were also studied. It may serve to better understanding of the physiology of the yeast strains and also to help in further analysis of the produced metabolites. The other strains M. chrysoperlae 1158, M. pulcherrima 1232, M. fructicola 1235, M. andauensis 1241, M. sinensis 1244, M. zizyphicola 1247 a M. shanxiensis 1250 were purchased from CBS (Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands).Yeast strains were cultivated on crude animal fat, glycerol and cheese whey under conditions of different C/N ratios. Because of higher lipid yields, cultivation was carried out at 14°C for 14 days. The accumulated lipid content was determined by gas chromatography and Raman spectroscopy. The glycerol-containing medium was evaluated as the most suitable for microbial lipids production. The total amount of lipids present in cells of M. pulcherrima 1232 was 36,31%. At the same time, quantitative screening of lipase enzymatic activity in Metschnikowia yeast was performed using spectrophotometric method with p-NPP. Controlled production of lipolytic enzymes has been monitored by using two types of media: crude animal fat and crude animal fat with addition of emulsifier (Tween 80). The conclusion of the work was supplemented by analysis of the karyotype of yeasts of the genus Metschnikowia using the technique of pulsed gel electrophoresis.

2-phenylethanol (2PE) is a valuable fragrance compound which gives a rose-like aroma. As such 2PE is one the highest used fragrances globally. While 2PE is predominantly produced from petrochemical resources, there is a growing market for a naturally derived alternative for food products. 2PE from natural sources is priced so highly due to limited supply from rose petals. Recently, a few reports have demonstrated the production in yeasts through both the de novo production from glucose and ex novo biosynthesis with L-phenylalanine as a precursor. While these are promising most of the yeasts used can only produce low titres under optimal conditions, and the fermentation still appears to be too expensive. In this investigation the wine yeast M. pulcherrima was selected to be assessed for 2PE production. M. pulcherrima is known to produce 2PE in small titres in wine production though has yet to be explored as a platform for this product. M. pulcherrima has several advantages as a yeast platform, in that it produces a range of antimicrobials which can ward off invasive species, allowing for less sterile control in any large scale fermentation. M. pulcherrima was demonstrated to be able to produce 2PE in high titres in the batch mode through de novo synthesis of glucose, producing up to 1 g/L in shake flasks on the lab scale. Arabitol was also observed in the fermentation broth and was produced up to 20 g/L. The fermentation was then scaled up to 2L in batch mode. From these experiments, up to 700 mg/L of 2PE was produced. This is substantially more than any other yeast in the literature to date. Though when xylose or glycerol was present then 2PE production was severely limited. M. pulcherrima was also demonstrated to be able to produce 2PE by bioconversion from phenylalanine up to 1.5 g/L. This 2PE concentration is suggested to be threshold of toxicity to M. pulcherrima by the toxicity study. The production of 2PE could be increased substantially by introducing an absorbent into the process. Liquid solvents and solid adsorbents were assessed to increase 2PE production, used as in-situ 2PE adsorbents. Oleyl alcohol was found to be a good solvent for in-situ extractive solvent in M. pulcherrima culture and increase the production to 3.3 g/L which is higher than 2PE tolerance threshold of the yeast. Activated carbon was also found to be an excellent 2PE adsorbents, with maximum Langmuir adsorption capacity up to 0.807 g/g. 2PE synthesis with activated carbon as an in-situ adsorbent can increase 2PE production to 14 g 2PE/L. Finally, the process was scaled to 2L and run in batch, continuous and semi-continuous modes. This study demonstrates that not only is M. pulcherrima a viable organism to produce 2PE but it has the potential to be scaled up and run in a more cost effective semi-continuous mode when coupled to a continuous extraction technique.

Palm Oil is the highest produced edible oil globally, with over 66 million tonnes produced annually. It has been estimated that up to 50% of all products sold in the supermarket contain palm oil in some form. Palm oil has attractive properties such as a high melting point and texture due to a balanced ratio of unsaturated and saturated fatty acids. It contains approximately 40% oleic acid (monounsaturated fatty acid), 10% linoleic acid (polyunsaturated fatty acid), 45% palmitic acid and 5% stearic acid (saturated fatty acid), that results in an edible oil that is suitable for use in a variety of food, detergent and cosmetics products. In addition, palm oil is the least expensive oil produced due to its high productivity and extensive production. Due to the high demand for the product, vast amounts of rainforest have been cleared to make way for more plantations, reducing biodiversity and releasing huge levels of carbon dioxide into the atmosphere. There is a clear need for an alternative lipid that can match palm oils properties but can be produced sustainably. Recent work suggests that some yeasts are capable of producing a similar oil to palm oil and can be grown on waste resources. In this thesis a novel bioprospecting protocol was developed to isolate yeasts that can survive the harsh conditions necessary for industrial biotechnology. In this way a vineyard and the local area was sampled for yeasts which were then cultured under extremes of pH, multiple sugars and inhibitors caused from the breakdown of lignocellulose. The wild yeast were cultured in four stages: minimal medium with Lysine; minimal medium with inhibitors; minimal medium with xylose as sole carbon-source; and lastly minimal medium with only arabinose and cellobiose as carbon-sources. Only strains that survived each stage were taken forward to the next, to isolate species that were truly suited to these conditions. Out of the estimated 1000s of strains screened this resulted in 12 strains of yeast, mostly in the Metschnikowia pulcherrima, group being able to cope with the conditions. The 12 strains were further analyzed by culturing them in an array of 4 different model lignocellulosic feedstocks namely wheat straw, corn Stover, sugarcane bagasse, and palm kernel cake hydrolysates. Other conditions incorporated in these analysis were a range of pH from pH 1.5 to pH 7.0; four levels of a mixture of 5 inhibitors; and two different temperatures. All of the 12 strains showed similar behaviour where inhibitor tolerance was only marked at higher pH, and at low pH the strains could not grow at all. Though all strains were able to grow on the hydrolysate models, even those with little glucose and/or xylose content. The lipid profile of the strains was also assessed and proved to be similar to most terrestrial crops, with suitable lipid profiles for a rapeseed oil, and in some cases palm oil substitute. Lastly, to further evaluate the accurate identification of the strains as there are some ambiguity in the Metschnikowia pulcherrima group, we applied an approach only widely used for Pathogenic Bacteria/Yeast identification, Multilocus Sequence Typing (MLST). Using 25 strains (7 of this collection), 6 type species and some isolates from the original culture collection in Bath. Sequences of 6 genes was analysed using the Bayesian statistical method. The result showed grouping of M. pulcherrima into 3-4 groups 9 different for each gene. M. Corniflorae being the outgroup. In all 3 genes successfully sequenced: M. Fruticola; R6; Mp DAH 3; and ICS48 were consistently shown to be clonal. The work presented here demonstrates a new method for bioprospecting strains capable of isolating strains for industrial biotechnology, and for characterisation of the yeast in the Metschnikowia genus. Some of the yeasts identified were oleaginous, and could potentially be used as a novel source of palm oil substitute.

Oleaginous yeasts posses the ability to accumulate increased amount of lipids under appropriate conditions. These microbial lipids vary in the composition of fatty acids which results in their wide application in the biotechnological industry. This master thesis focuses on the lipid production and fatty acids composition from waste substrates by the yeasts Metschnikowia depending on various cultivation conditions. The influence of temperature, the ratio of carbon and nitrogen in medium, and the concentration of different carbon sources was studied. The cheap and easy available waste substrates as glycerol and animal fat were used for the cultivation. The production characteristics of the yeasts were monitored by various technique including gas chromatography, Raman spectroscopy and fluorescence microscopy FLIM. Moreover, the partial optimalization of the pulse field gel electrophoresis was applied in order to characterize the karyotype of the yeasts Metschnikowia. All the studied strains were able to use the waste substrates and at the same time to produce lipids. The amount of lipids and mainly their compositions vary depending on the yeast strain and on the culture conditions. Nevertheless, the ability of the yeasts to produce significant amount of unsaturated fatty acids by manipulation of culture conditions was proved. The maximum lipid yield was achieved by M. pulcherrima 149 on glycerol medium and by M. andauensis 129 on medium containing waste animal fat.

L'élimination des protéines est une étape clé lors de la production du vin blanc afin d'éviter l'apparition éventuelle d'un voile inoffensif mais inesthétique. Des solutions de rechange à l'utilisation de la bentonite sont activement recherchées en raison des problèmes technologiques, organoleptiques et de durabilité associés à son utilisation. Dans cette étude, MpAPr1, une protéase aspartique extracellulaire préalablement isolée et partiellement caractérisée à partir de la levure Metschnikowia pulcherrima, a été clonée et exprimée de manière hétérologue dans la levure Komagataella pastoris. Les propriétés enzymatiques de MpAPr1 ont été initialement caractérisées dans un extrait brut. Après plusieurs essais faisant appel à différentes techniques, MpAPr1 a été purifié avec succès par chromatographie échangeuse de cations. Son activité contre les protéines de raisin a été initialement testée dans une solution modèle dans des conditions environnementales optimales pour l'activité de MpAPr1 puis dans celles qui règnent lors de la vinification. Ensuite, l'activité de MpAPr1 a été évaluée dans du moût de raisin et au cours de la fermentation alcoolique. La présence de MpAPr1, supplémenté au moût de raisin, a entraîné une dégradation partielle des protéines de raisin tout au long de la fermentation et une légère différence dans la composition en composés volatils du vin. L'étude a confirmé que les protéases aspartiques pourraient représenter une alternative à la bentonite pour l'industrie du vin et que les levures non-Saccharomyces telles que M. pulcherrima pourraient avoir un impact bénéfique sur les propriétés du vin. / Protein removal is a key step during the production of white wine in order to avoid the possible appearance of a harmless but unsightly haze. Alternatives to the use of bentonite are actively sought because of technological, organoleptic and sustainability issues associated with its use. In this study, MpAPr1, an extracellular aspartic protease previously isolated and partially characterised from the yeast Metschnikowia pulcherrima, was cloned and expressed heterologously in Komagataella pastoris. Enzymatic properties of MpAPr1 were initially characterised in a crude extract. After several attempts using different techniques, MpAPr1 was successfully purified via cation exchange chromatography. Its activity against haze-forming grape proteins was initially tested in a model solution under optimal environmental conditions for MpAPr1 activity and under those occurring during winemaking. Thereafter, MpAPr1 activity was evaluated in grape must and throughout alcoholic fermentation. The presence of MpAPr1, supplemented to grape must, resulted in the partial degradation of grape proteins throughout fermentation and ultimately in a slight difference in the wine’s composition in volatile compounds. The study provides further evidence that aspartic proteases could represent a potential alternative to bentonite for the wine industry and that non-Saccharomyces yeasts such as M. pulcherrima could have a beneficial impact on wine properties.

<p dir="ltr">In this dissertation, I explore how human actions (climate change, road salt, land use change, species invasions) interact with and influence morphology, disease, and population dynamics in freshwater organisms (amphibians and aquatic crustaceans). First, I examined how the incidence and timing of disease epidemics in native species (<i>Daphnia dentifera</i>) caused by a generalist parasite (<i>Metschnikowia bicuspidata</i>) influenced the success and impact of an invasive species (<i>Daphnia lumholtzi</i>) in freshwater zooplankton (Chapter 1). In the following chapter, I explored how host-parasite interactions are affected by the interactive effects of multiple environmental stressors, focusing on American bullfrog tadpoles (<i>Lithobates catesbeianus</i>), two of their common parasites (<i>Batrachochytrium dendrobatidis</i> (<i>Bd</i>) and trematode parasites in the family Echinostomatidae), and two common stressors (fluctuating temperatures and sublethal road salt pollution; Chapter 2). Finally, I investigated how the combination of climate (temperature and precipitation) and land use (developed and/or forested area) change have influenced the body size of a common toad (Fowler’s toad, <i>Anaxyrus fowleri</i>) from 1930 – 2020 utilizing museum specimens (Chapter 3). Together, this research establishes how emerging and persistent anthropogenic environmental stressors will interact to affect morphology, disease, and population dynamics in vulnerable freshwater organisms.</p>