Using of waste substrates for the lipid production by Metschnikowia yeasts

Diplomová práca bola zameraná na štúdium biotechnologického využitia odpadných substrátov k produkcii lipidov kvasinkami rodu Meschnikowia. Odpadné suroviny a ich následná transformácia na produkty s pridanou hodnotou akými sú napríklad mikrobiálne lipidy sú v súčasnosti považované za alternatívny zdroj výroby biopalív. Experimentálna časť bola preto zameraná na skúmanie vplyvu uhlíkatého zdroja k riadenej nadprodukcii lipidov prostredníctvom kvasiniek rodu Metschnikowia. Celkovo bolo vybraných dvanásť zástupcov tohto rodu. Pre časť kmeňov, konkrétne kvasinky M. pulcherrima 145, M. pulcherrima 147, M. pulcherrima 149, M. andauensis 129 a M. fructicola 15, zo zbierky kultúr kvasiniek (CCY - Culture Collection of Yeast) boli prevedené aj rastové charakteristiky, ktoré, môžu slúžiť pre pochopenie fyziológie samotných kvasinkových kmeňov a v budúcnosti napomôcť pri ďalšej analýze produkovaných metabolitov. Zvyšné kmene M.chrysoperlae 1158, M. pulcherrima 1232, M. fructicola 1235, M. andauensis 1241, M. sinensis 1244, M. zizyphicola 1247 a M. shanxiensis 1250 pochádzali zo zbierky CBS (Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands). Kultivácia bola prevádzaná na odpadnom živočíšnom tuku, glycerole a srvátke s rôznymi pomermi C/N. Pre vyššie výťažky lipidov boli jednotlivé kmene kultivované za podmienok zníženej teploty (15°C) po dobu 14 dní. Obsah vyprodukovaných lipidov bol stanovovaný technikou plynovej chromatografie a Ramanovou spektroskopiou. Ako najvhodnejší substrát pre tvorbu mikrobiálnych lipidov bolo vyhodnotené médium s obsahom glycerolu, kde množstvo naakumulovaných lipidov predstavovalo v bunkách kvasinky M. pulcherrima 1232 36,31%. U kvasiniek rodu Metschnikowia bol zároveň prevedený aj kvantitatívny screening enzýmovej aktivity lipáz za použitia spektrofotometrickej metódy s p-NPP. Riadená produkcia lipolytických enzýmov bola sledovaná s využitím dvoch typov médií: odpadný tuk a odpadný tuk s prídavkom emulzifikátoru (Tween 80). Záver práce bol doplnený o analýzu karyotypu kvasiniek rodu Metschnikowia pomocou techniky pulznej gélovej elektroforézy.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.

Determination of basic analytical parameters of paprika of different geographic origin

Cieľom tejto bakalárskej práce bolo stanoviť základné analytické parametre mletej papriky rôzneho geografického pôvodu ako ASTA farbivosť, pH, obsah vlhkosti a popola. Teoretická časť sa predovšetkým zameriava na charakteristiku papriky, jej rozdelenie, chemické zloženie a výrobu mletej papriky. Taktiež sa zaoberá aj problematikou korenia konkrétne jeho rozdelením, významom a uskladnením. V experimentálnej časti sú načrtnuté jednotlivé postupy stanovenia parametrov (ASTA, pH, vlhkosť, popol) desiatich druhov mletých paprík. Hodnoty ASTA farbivosti vzoriek sa pohybovali v rozmedzí 80 – 175 ASTA jednotiek. Analýzou pH bolo preukázané, že všetky vzorky paprikových extraktov vykazovali kyslý charakter s pH okolo 5. Obsah celkovej vlhkosti a popola prítomného vo vzorkách predstavoval 8 – 15 % a 5 – 7 % hm.The aim of this bachelor thesis was to determine the basic analytical parameters of paprika of different geographic origin such as ASTA colour, pH, moisture content and ash. The theoretical part primarily focuses on the characteristic of paprika, it´s division, the chemical composition and the production of ground red pepper. It also deals with spice issues, it´s division, meaning in and storage. In the experimental part there are outlined the individual procedures of determination of parameters (ASTA, pH, moisture, ash) of ten samples of ground red pepper. ASTA values ranged from 80 – 175 ASTA units. Analysis of pH showed that all samples of pepper extracts showed an acidic pH with value around 5. The total moisture and ash content in the samples was 8 – 15 % and 5 – 7 %.

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