Cultivation of carotenogenic yeasts on waste substrates usinf solid state fermentation

„Semi-solid fermentation“ je čoraz viac využívanou technikou k produkcii významných metabolitov, či obohatenej biomasy za nízkych finančných nákladov, nízkej spotreby kultivačnej vody a zároveň s malou záťažou na životné prostredie. Problémom tejto techniky ale môže byť voľba vhodného mikroorganizmu, ktorý je schopný rásť a produkovať pri nízkej aktivite vody. Predložená práca je poňatá ako pilotná štúdia troch kvasinkových kmeňov Rhodotorula glutinis, Cystofilobasidium capitatum a Sporobomomyces roseus kultivovaných technikou „semi-solid state fermentation“ (semi-SSF). Kvasinky boli kultivované v sériách produkčných médií s postupným znižovaním obsahu kultivačnej vody. Ako uhlíkatý zdroj sacharidov boli použité cestoviny a hydrolyzované cestoviny, v kontrolných médiách glukóza. Všetky študované kmene boli schopné rásť a produkovať sledované lipidické metabolity i pri nízkych obsahoch vody. Produkcie karotenoidov a sterolových látok boli vyššie práve v médiách s nízkou aktivitou vody. Bunky sa tak pravdepodobne chránili pred väčším osmotickým tlakom. Ako najlepší producent karotenoidov i biomasy sa ukázal kmeň Cystofilobasidium capitatum, kultivovaný na médiu s hydrolyzovanými cestovinami a obsahom vody 40%. Semi-SSF sa javí ako dobrá technika pre selekciu kmeňov s nadprodukčnými vlastnosťami. Výberom vhodného produkčného média a koncentrácie vody sa dá optimalizovať stimulácia produkcie sledovaných metabolitov v kvasinkových bunkách.Semi-solid fermentation is an eco - friendly technique more and more used for production of significant metabolites or enriched biomass at low entrance cost and low comsumption of water. The problem of this technique might be the right choice of microorganism able to grow and produce at low water activity. This work is a pilot study of three red yeast strains – Rhodotorula glutinis, Cystofilobasidium capitatum and Sporobomomyces roseus cultivated by semi-solid state fermentation (semi-SSF). Yeasts were cultivated in series of production media with gradual reduction of cultivation water content. Pasta and hydrolyzed pasta were used as source of sacharides, glucose served as the carbon source in control media. All studied strains we able to grow and produce observed lipidic metabolites also at low water contents. Production of carotenoids and sterols was higher in semi-solid media. Cystofilobasidium capitatum strain was identified as the best producer of carotenoids and biomass. This strain was cultivated on hydrolyzed pasta media with 40% water content. Semi-SSF seems to be an adequate technique for selection of strains having over-productive properties. Observed metabolites production in yeast cells can be optimized by choosing the appropriate production media and water activity.

Study of Metabolic Adaptation of Red Yeasts to Waste Animal Fat Substrate

Carotenogenic yeasts are non-conventional oleaginous microorganisms capable to utilize various waste substrates. In this work 4 red yeast strains (Rhodotorula, Cystofilobasidium and Sporobolomyces sp.) were cultivated in media containing crude, emulsified and enzymatically hydrolysed animal waste fat, compared with glucose and glycerol as single C-sources. Cell morphology (cryo-SEM, TEM), production of biomass, lipase, biosurfactants, lipids (GC/FID) carotenoids, ubiquinone, ergosterol (HPLC/PDA) in yeast cells was studied depending on medium composition, C-source and C/N ratio. All studied strains are able to utilize solid and processed fat. Biomass production at C/N=13 was higher on emulsified/hydrolysed fat than on glucose/glycerol. Production of lipids and lipidic metabolites was enhanced for several times on fat; the highest yields of carotenoids (24.8 mg/l) and lipids (54.5%/CDW) were found in S.pararoseus. Simultaneous induction of lipase and biosurfactants was observed on crude fat substrate. Increased C/N ratio (13-100) led to higher biomass production in fat media. Production of total lipids increased in all strains to C/N 50. Oppositely, production of carotenoids, ubiquinone and ergosterol dramatically decreased with increased C/N in all strains. Compounds accumulated in stressed red yeasts are having great application potential and can result from valorization of animal waste fat in the biorefinery concept.Karotenogenní kvasinky jsou schopny utilizovat řadu různých substrátů včetně odpadů. V této práci je popsána kultivace vybraných kmenů karotenogenních kvasinek rodů Rhodotorula, Cystofilobasidium, and Sporobolomyces sp. v médiu obsahujícícm surový odpadní živočišný tuk a upravený substrát emulsifikací a enzymovou hydrolýzou. Výsledky byly srovnány s kultivací na glukóze a glycerolu jako jednoduchých zdrojích uhlíku

Revealing the Potential of Lipid and beta-Glucans Coproduction in Basidiomycetes Yeast

Beta (beta)-glucans are polysaccharides composed of D-glucose monomers. Nowadays, beta-glucans are gaining attention due to their attractive immunomodulatory biological activities, which can be utilized in pharmaceutical or food supplementation industries. Some carotenogenicBasidiomycetesyeasts, previously explored for lipid and carotenoid coproduction, could potentially coproduce a significant amount of beta-glucans. In the present study, we screened elevenBasidiomycetesfor the coproduction of lipids and beta-glucans. We examined the effect of four different C/N ratios and eight different osmolarity conditions on the coproduction of lipids and beta-glucans. A high-throughput screening approach employing microcultivation in microtiter plates, Fourier Transform Infrared (FTIR) spectroscopy and reference analysis was utilized in the study. Yeast strainsC. infirmominiatumCCY 17-18-4 andR. kratochvilovaeCCY 20-2-26 were identified as the best coproducers of lipids and beta-glucans. In addition,C. infirmominiatumCCY 17-18-4,R. kratochvilovaeCCY 20-2-26 andP. rhodozymaCCY 77-1-1 were identified as the best alternative producers of beta-glucans. Increased C/N ratio led to increased biomass, lipid and beta-glucans production for several yeast strains. Increased osmolarity had a negative effect on biomass and lipid production while the beta-glucan production was positively affected

Regulation of production of lipids and lipid compounds in yeasts

U oleogénnych kvasiniek dochádza za vhodných podmienok k nadprodukcii a následnej akumulácii lipidov a lipidických látok. Touto vlastnosťou sa vyznačujú aj červené kvasinky, ktoré okrem lipidov produkujú aj karotenoidy – prírodné pigmenty využívané v potravinár-stve a doplnkoch stravy. Práca bola zameraná na skríning produkčných vlastností kmeňov Cystofilobasidium capitatum, Rhodotorula glutinis, Sporobolomyces roseus a Sporobolomy-ces shibatanus. Ako zdroje uhlíka boli zvolené glukóza a glycerol ako odpadný produkt pri produkcii biopalív. Najlepšie produkčné vlastnosti sa prejavili pri kmeňoch Cystofilobasidium capitatum a Rhodotorula glutinis. Pri nich došlo k zvýšeniu akumulovaných lipidov za súčasnej produkcie vyšších koncentrácii karotenoidov. Kmene boli testované metódou FTIR spektroskopie, ktorá umožňuje vysokovýkonnú, nenáročnú a presnú analýzu.Oleogenic yeasts under appropriate conditions produce and accumulate lipids and lipid-soluble metabolites in high amounts. This attribute is characteristic also for red yeasts that except lipids accumulate also carotenoids – natural pigments used in food industry and dietary supplements. The aim of this diploma thesis was designed as a comparative screening study of production properties of strains Cystofilobasidium capitatum, Rhodotorula glutinis, Sporobolomyces roseus and Sporobolomyces shibatanus. Choosen carbon sources were glucose and glycerol as waste by-product in biofuel industry. The best production properties were found in Cystofilobasidium capitatum and Rhodotorula glutinis. These two strains produced increased amounts of lipids as well as higher amounts of carotenoids. Strains were tested by FTIR spectroscopy that enables high-throughput, uncomplicated and accurate analysis.

On the Electronic Structure of mer,trans−[RuCl3(1H−indazole)2(NO)]mer,trans-[RuCl_{3}(1 H -indazole)_{2}(NO)], a Hypothetical Metabolite of the Antitumor Drug Candidate KP1019: An Experimental and DFT Study

The study reported herein focused on the electronic structure of the {Ru(NO)}6 fragment and characterization of the oxidation state of ruthenium in mer,trans-[RuCl3(Hind)2(NO)] (1; Hind = 1H-indazole) resulting from the reaction of mer,trans-[RuCl3(H2O)(Hind)2] (2) with NO in acetone or solid-state Anderson rearrangement of (H2ind)2[RuCl5(NO)] at 180 °C. The results of X-ray diffraction, 1H, 13C, and 15N NMR, EPR, IR, and UV/Vis spectroscopy, cyclic voltammetry, magnetic susceptibility, and XANES/EXAFS as well as theoretical data have been critically analyzed. The localized orbitals, domain-averaged Fermi holes, frontier orbitals, Mulliken population, and quantum theory of atoms-in-molecules (QTAIM) analyses are presented. In addition, mer,trans-[RuIIICl3(H2O)(Hind)2] (2) and trans-[RuIICl2(Hind)4] (3) were experimentally and theoretically investigated as reference compounds. A complete active space SCF calculation was performed to estimate the extent of antiferromagnetic spin–spin coupling in 1. We found that the closed-shell structure {RuIII(NO)0}6 fits better to the physical/spectroscopic properties of 1, although {RuII(NO)+}6 is formally still suitable for describing the oxidation state of Ru in this entit