Academic literature on the topic 'Odpadní substráty'

Aktivovaný kal na čistírnách odpadních vod je neustále vystavován nízkým koncentracím antimikrobiálních látek a dalších léčiv. To vyvolává otázku, jak mikroorganismy k těmto látkám na čistírně odpadních vod přistupují. Zda jsou schopny se v tomto prostředí na tyto látky adaptovat, degradovat je, případně je využít jako substrát. Nebo jestli jsou tyto látky aktivovaným kalem opomíjeny. Pro posouzení adaptace aktivovaného kalu byla využita metoda PCR pro sledování genů resistence a testy biologické rozložitelnosti. Pro testy byl využit aktivovaný kal z ČOV a kal adaptovaný v laboratorních SBR modelech při koncentracích antibiotik 500 ng∙l−1 a 500 μg∙l−1. Biologická rozložitelnost byla posuzována dle normy ČSN ISO 14593. Testované látky byly sledovány pomocí skupinového stanovení celkového anorganického uhlíku. Jako testované látky byly vybrány: benzylpenicilin, ampicilin, streptomycin, erythromycin, chloramfenikol, sulfamethoxazol a trimetoprim. Aktivovaný kal z čistírny odpadních vod neměl vyvinutou aktivitu k biodegradaci testovaných antibiotik. Je pravděpodobné, že vysoké zatížení snadno biologicky rozložitelným substrátem a krátké zdržení odpadní vody na ČOV, vede k tomu, že mikroorganismy aktivovaného kalu nejsou nuceny tyto látky aktivně utilizovat a brání se jim pouze tvorbou obranných mechanismů pomocí genů antibiotické resistence. Nízké koncentrace antibiotik v SBR modelech vytvářely selekční tlak na mikroorganismy a podněcovaly šíření genů antibiotické resistence. English Activated sludge in wastewater treatment plants is constantly exposed to low concentrations of antimicrobials and other drugs. This raises the question of how microorganisms approach to these substances in the sewage treatment plant. Whether they can adapt, degrade, or use antibiotics as a substrate in this environment or the activated sludge neglects these substances. To assess the adaptation of activated sludge, the PCR method for monitoring antibiotic resistance genes and biodegradability tests were used. These tests were carried out with activated sludge from WWTP and sludge adapted in laboratory SBR models at 500 ng∙l−1 and 500 μg∙l−1 of chosen antibiotics. Their biodegradability was assessed according to ČSN ISO 14593. The tested substances were monitored by group determination of total inorganic carbon. The chosen substances were: benzylpenicillin, ampicillin, streptomycin, erythromycin, chloramphenicol, sulfamethoxazole and trimethoprim. Activated sludge had no developed activity for biodegradation of tested antibiotics. It is likely that the high load of readily biodegradable substrate and the short retention of the wastewater at the WWTP lead to the activated sludge not being forced to actively utilize these substances and will only prevent from them by forming defence mechanisms using antibiotic resistance genes. Low concentrations of antibiotics in SBR models produced selective pressure on microorganisms and stimulated the spread of antibiotic resistance genes.

Aktivovaný kal na čistírnách odpadních vod je neustále vystavován nízkým koncentracím antimikrobiálních látek a dalších léčiv. To vyvolává otázku, jak mikroorganismy k těmto látkám na čistírně odpadních vod přistupují. Zda jsou schopny se v tomto prostředí na tyto látky adaptovat, degradovat je, případně je využít jako substrát. Nebo jestli jsou tyto látky aktivovaným kalem opomíjeny. Pro posouzení adaptace aktivovaného kalu byla využita metoda PCR pro sledování genů resistence a testy biologické rozložitelnosti. Pro testy byl využit aktivovaný kal z ČOV a kal adaptovaný v laboratorních SBR modelech při koncentracích antibiotik 500 ng∙l−1 a 500 μg∙l−1. Biologická rozložitelnost byla posuzována dle normy ČSN ISO 14593. Testované látky byly sledovány pomocí skupinového stanovení celkového anorganického uhlíku. Jako testované látky byly vybrány: benzylpenicilin, ampicilin, streptomycin, erythromycin, chloramfenikol, sulfamethoxazol a trimetoprim. Aktivovaný kal z čistírny odpadních vod neměl vyvinutou aktivitu k biodegradaci testovaných antibiotik. Je pravděpodobné, že vysoké zatížení snadno biologicky rozložitelným substrátem a krátké zdržení odpadní vody na ČOV, vede k tomu, že mikroorganismy aktivovaného kalu nejsou nuceny tyto látky aktivně utilizovat a brání se jim pouze tvorbou obranných mechanismů pomocí genů antibiotické resistence. Nízké koncentrace antibiotik v SBR modelech vytvářely selekční tlak na mikroorganismy a podněcovaly šíření genů antibiotické resistence. English Activated sludge in wastewater treatment plants is constantly exposed to low concentrations of antimicrobials and other drugs. This raises the question of how microorganisms approach to these substances in the sewage treatment plant. Whether they can adapt, degrade, or use antibiotics as a substrate in this environment or the activated sludge neglects these substances. To assess the adaptation of activated sludge, the PCR method for monitoring antibiotic resistance genes and biodegradability tests were used. These tests were carried out with activated sludge from WWTP and sludge adapted in laboratory SBR models at 500 ng∙l−1 and 500 μg∙l−1 of chosen antibiotics. Their biodegradability was assessed according to ČSN ISO 14593. The tested substances were monitored by group determination of total inorganic carbon. The chosen substances were: benzylpenicillin, ampicillin, streptomycin, erythromycin, chloramphenicol, sulfamethoxazole and trimethoprim. Activated sludge had no developed activity for biodegradation of tested antibiotics. It is likely that the high load of readily biodegradable substrate and the short retention of the wastewater at the WWTP lead to the activated sludge not being forced to actively utilize these substances and will only prevent from them by forming defence mechanisms using antibiotic resistance genes. Low concentrations of antibiotics in SBR models produced selective pressure on microorganisms and stimulated the spread of antibiotic resistance genes.

Proces částečná nitritace-anammox odstraňuje amoniakální dusík z odpadních vod s polovičními náklady na aeraci, až o 80 % nižší produkcí přebytečného kalu a bez spotřeby organického substrátu. Jde o zavedený proces pro odstraňování dusíku z kalových vod z anaerobní fermentace, a podobně koncentrovaných a teplých odpadních vod. Na tyto vody se částečná nitritace-anammox aplikuje již déle než deset let, a to např. pod názvy ANAMMOX®, ANITA™ Mox, DEMON®, nebo TERRAMOX®. Optimalizované provozy těchto technologií dusík běžně odstraňují při zatížení 0,5–2,3 kg∙m–3∙d–1 (30–35 °C). Současnou výzvou pro výzkum je implementace částečné nitritace-anammox do hlavního proudu studené splaškové odpadní vody, přičemž konkrétními problémy jsou (i) potlačení nežádoucích nitratačních mikroorganismů (NOB) a (ii) adaptace mikroorganismů anammox na nízké teploty. Náš výzkum jsme začali s jednostupňovým procesem, a poté nitritaci a anammox rozdělili do dvou reaktorů. Prezentujeme strategii, která v laboratorním měřítku NOB účinně potlačila i při 12 °C a dále i v pilotním měřítku při 13–30 °C. Dále ukazujeme, že anammox je možné na nízké teploty adaptovat studenými šoky. Tyto výsledky umožní rozšířit úsporné odstraňování dusíku i do hlavního proudu splaškové odpadní vody na ČOV. English: Partial nitritation-anammox (PN/A) process removes nitrogen from wastewater with 50% reduction of aeration costs, 80% less excess sludge and no consumption of organic carbon. PN/A is an established process for the removal of nitrogen from reject water from anaerobic digestion and other similarly warm and concentrated streams. On such wastewater, PN/A has been applied in full scale for over 10 years under names such as ANAMMOX®, ANITA™ Mox, DEMON® or TERRAMOX®, whose optimized installations consistently achieve nitrogen removal loading rates of 0,5–2,3 kg∙m–3∙d–1. The current challenge for research is to implement PN/A into the main stream of cold municipal wastewater, the specific challenges being (i) suppression of undesirable nitrite oxidizing bacteria (NOB) and (ii) adaptation of anammox microorganisms to low temperatures. Our initial experiences with one-stage PN/A in the main stream led us to the separation of PN/A in two subsequent reactors. Subsequently, we developed a strategy for NOB suppression in partial nitritation even under 12 °C, which we then successfully tested in the pilot scale. Furthermore, we found that anammox can be adapted to low temperatures using cold shocks. In sum, these results will enable extending the savings for nitrogen removal into the main stream of wastewater at WWTP.

Abstract The subject of the investigation was the model solution containing 50 g/dm³ waste light ends from PVC production, dissolved in the ethanol-water (1:1) mixture. The main components of light ends were as follows: trichloromethane, tetrachloromethane, and chloroderivatives of ethane. Granular ferric-chromic catalyst (TZC 3/1) was used in the investigation. The temperature range applied in experiments was 400-600ºC and the contact time was 0.27 s. Gaseous products of the reaction were analysed in order to determine among others concentration of chlorine, formaldehyde, oxygen, carbon monoxide and dioxins. The content of total organic carbon (TOC), chloride ions and formaldehyde was determined in a condensate. Oxidation of the mixture proceeded in the all temperature range with high efficiency in regard to initial TOC value of the solution. The concentration of dioxins in the combustion gases obtained in the process carried out in temperature 450ºC amounted to 0.021-0.027 ng TEQ/m3, and was significantly lower than the admissible value of 0.1 ng TEQ/m3. Congeners of polychlorinated dibenzofuranes (PCDFs) predominated in the combustion gases. Tested catalyst did not undergo deactivation during 150 h substrate oxidation.

Carotenoids are naturally occurring pigments of plants also produced by microbes. The area of their application concerns mainly food industry; however, they are used in chemical, pharmaceutical, and cosmetics industry as well. Currently, the isolation of carotenoids from plants is markedly regulated by legislation, so the study of their production is greatly emphasised, where the microbiological, instead of the synthetic, production of carotenoids is being prioritized. This work was made as a comparative study of carotenogenic yeasts of the genes Rhodotorula, Sporobolomyces, and Cystofilobasidium. Their ability to use various waste substrates as a carbon and nitrogen source and source of other nutrition factors was tested. In this work, conditions of random mutagenesis were optimized. Particular yeast strains were also subjected to the effect of mutagen ethyl methanesulfonate (EMS) in order to increase the production of biomass and specific metabolites – carotenoids and other lipid-soluble substances. Random mutagenesis and mutant strain selection was performed using waste subtrates as glycerol, pasta and some pasta hydrolyzed by fungal extracellular enzymes. Subsequently, a control of specific DNA sequences in pigments overproducing mutants was analyzed by PCR/DGGE (denaturating gradient gel electrophoresis). Increased production of -carotene was achieved in a mutant of Sporobolomyces roseus strain growing on glycerol, pasta, and hydrolyzed pasta. Overproduction of carotenoids by mutant strain of Rhodotorula glutinis was observed in glucose medium only. Mutants of Cystofilobasidium capitatum exhibited a decrease of biomass production; on the other hand, the production of carotenoids increased especially in pasta medium hydrolyzed by enzyme preparative from Fusarium solani. In this work it was confirmed that using random mutagenesis strains capable to utilize waste substrates can be selected. In mutant strains increased carotenoids biosynthesis was observed, which enables effective use of cheap substrates and reduction of the negative effects of wastes on the environment.

Yeasts are like other organisms constantly exposed to environmental influences. Their survival depends on the skills to adapt to environmental changes, including the ability to use various alternative sources of nutrients. In presented PhD thesis carotenogenic yeast belonging to the genera Rhodotorula, Sporobolomyces and Cystofilobasidium were tested for ability to use of selected waste substrates, and also subjected to several types of exogenous stress effects and mutations in order to increase the production of microbial biomass enriched with specific metabolites. As alternative nutrient sources derived from waste substrates from agricultural and farm production apple peel, pulp, corn germ and more were tested. Yeasts were also exposed to osmotic, oxidative and combined stress (benefits of various concentrations of NaCl and H2O2 to the culture media), followed by metal ions of selenium and chromium in concentrations of 0.01 mM, 0.1 mM and 1 mM. The effect of mutagen methanesulfonic acid ethyl ester was tested too. In all experiments the adaptivity of cells, morphological changes, color pigments produced by the media while some important fungal metabolites production and changes in chromosomal DNA fragmentation were analyzed. In order to evaluate potential changes in the yeast genome after treatment with mutagen and stress factors methods for isolation of intact chromosomal DNA and DNA analysis by pulsed field gel electrophoresis was optimized. The amount of produced metabolites was mainly analyzed by RP-HPLC with UV/VIS and MS detection. The work has been shown that most strains are able to use waste substrates and produced selected target metabolites. Biomass, for example, in R. aurantiaca on apple fiber was about 7 g/l and in C. capitatum cultivated on modified whey reached to 9 g/l. Amount of produced carotenoids by R. aurantiaca cultivated on wheat germ and maize after enzymatic hydrolysis by F. solani was 1.01 mg/g and S. roseus on pasta 4.3 mg/g. The values of ergosterol synthesis in R. aurantiaca are on the apple shells around 4.8 mg/g, in S. roseus on pasta with the enzymatic hydrolysis of P. chrysosporium 8.9 mg/g. The best substrate for biomass production and induction of carotenoids are waste substartes containing a mixture of simple and complex carbohydrates enriched with the addition of nitrogen compounds. Potential cytotoxic effect of stress factors of low concentrations was demonstrated. Red yeast genome was able to distribute by optimized PFGE, the karyotype of tested yeasts contain 11 or more chromosomes with visible differences between yeast species and genera. During exchange internship the ability of recombinant yeast S. cerevisiae to convert xylose to xylitol, which would be achieved by increasing the production of bioethanol as alternative fuel sources was studied. It turned out that both ligninocellulose materials to bioethanol production, as well as various waste substrates for microbial synthesis of carotenoids would reduce costs for industrial production of yeast metabolites, as well as to reduce the negative burden on the environment.

Pro zpracování a nakládání s odpadními substráty lze použít řadu postupů a možností. Stále se rozšiřující spektrum metod a technologií umožňuje další využití materiálů a energie ve formě obnovitelných zdrojů. Jedním z řešení pro zpětné získávání některých odpadních materiálů je využití tzv. bílé (průmyslové) biotechnologie, která zahrnuje praktickou aplikaci metabolických aktivit celé řady různých mikroorganizmů včetně jejich specifických biologických drah k produkci látek s vysokou přidanou hodnotou. V předložené práci screeningového typu bylo pro zhodnocení odpadních surovin využito několik druhů mikroorganizmů kultivovaných za různých specifických podmínek včetně kultivace na odpadních materiálech získaných zejména ze zemědělství a potravinářství. Cílem bylo získání vybraných typů průmyslově cenných metabolitů, případně energie. Předložená studie byla zaměřena na srovnání růstu a produkčních vlastností několika kmenů karotenogenních kvasinek rodu Rhodotorula, Sporobolomyces a Cystofilobasidium, kultivovaných v médiích s obsahem glycerolu (technický a odpadní glycerol), dále v médiích obsahujících pšeničnou slámu, hydrolyzovanou slámu zpracovanou v hydrotermálním procesu při vysoké teplotě a zbytky po filtraci hydrolyzátu. Dalším testovaným odpadním substrátem byla syrovátka. Všechny testované kvasinky byly schopny využít glycerol jako jediný zdroj uhlíku. Produkce biomasy při kultivaci na technickém glycerolu se více či méně přibližovala kontrole (cca 7 - 10 gl-1), zatímco při kultivaci na odpadním glycerolu byla produkce vyšší (10.9 - 14.5 gl-1). Produkce karotenoidů a ergosterolu byla vyšší v glukózovém médiu než v médiu s obsahem glycerolu. Všechny testované kvasinky byly rovněž schopny produkovat neutrální lipidy, a to v rozmezí 11 - 15 %, s výjimkou C. capitatum, kde produkce dosahovala více než 22 % obsahu neutrálních lipidů. Pšeničná sláma a produkty z ní připravené se ukázaly být využitelnými substráty s vysokým potenciálem pro produkci biomasy i metabolitů, a to zejména u kmene S. roseus. Syrovátka, jako odpadní produkt mlékarenství, byla účinně využita jako substrát pro kokultivaci karotenogenních kvasinek a bakterií mléčného kvašení. Kokultivační proces může vyvolat nadprodukci pigmentů a ergosterolu, přičemž získaná biomasa díky obohacení o bakterie L. casei dosahovala vyšší kvality. Za účelem energetického využití mikrobiálního metabolismu formou mikrobiálních palivových článků, tzv. „Microbial Fuel Cell“ byla aplikována směsná kultura bakterií získaných z čistírny odpadních vod. Tyto mikroorganizmy hrají významnou roli při výrobě elektrické energie a současně také při čištění odpadních vod. Elektřina je generována přímo z organických látek přítomných v kultivačním médiu a lze ji použít pro provoz čistírny samotné a případně i pro další aplikace.

Proposed doctoral thesis deals with microbial production of biopolymers (polysaccharides and polyesters) using renewableble inexpensive resources as carbon and nitrogen source. At first, production of extracellular metabolites, mainly pullulan and poly-L-malic acid, by polymorphic microorganism Aureobasisium pullulans was studied. Further part of thesis was focused on production of bacterial biopolyesters polyhydroxyalkanoates (PHA). PHA are produced in the form of intracellular granules by various bacteria species. During the study of PHA production, sythesis of poly(3-hydroxybutyrate) and technologically significant copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was investigated as well with regard to waste substrate utilization in biorafinery concept. Spent coffee ground seemed to be applicable waste in biorefinery proces. One of the coffee wastes used for PHA production was spent coffee ground hydrolysate which was converted to PHA by Burkholderia cepacia. Moreover, copolymer of 3HB and 3HV was accumulated when SCGH was used as a sole carbon source, no precursor had to be added. Another coffee waste, which was utillized by Cupriavidus necator H16, is represented by oil extracted from spent coffee grounds. Hydrolysis proces of waste substrates had to be optimized, to reach the highest possible saccharide concentration. Due to the hydrolysis process several toxic compounds can be formed, hence, influence of hydrolysis procesings on polyphenols content in hydrolysates was monitored. Waste substrates were also tested as potentional complex nitrogen source for PHA production. Hydrolysates of cheese whey and chicken feather were used as inexpensive complex nitrogen source for PHA production by C. necator, when waste frying oil was used as a carbon substrate. The finnal part of the thesis is dealing with possibilities how the PHA can be processed into atractive aplication forms. Liposomes with partial content of PHB had been prepared, moreover, for improving of antioxidant activity and antimicrobial effect the waste coffee oil was added. Micro-fibrous PHB materials were prepared for the purposes of water filtration. Higher filtration effectivity was observed when composite PHB fibrous material containing active charcoal or metal oxides was used. Important part of thesis is represented by study of degradation processes of prepared PHA-based aplication forms. Various microorganisms were tested on PHA depolymerases production. Biodegradability of PHA materials was tested in of composting conditions by standard composting test IS/ISO 20200. Changes in mechanical properties of polyurethan, where polyol was partialy replaced by PHB, were tested depending on the exposure of composting condition.

Carotenoids are naturally occurring pigments produced by bacteria, yeasts, filamentous fungi and plants. They exhibit significant biological effects and are widely used in the food industry, pharmacy and cosmetics. The aim of this diploma thesis proposed as a comparative study was regulation of carotenoid and ergosterol production in red yeasts using several waste substrates as whey, corn germs, wheat, apple fiber and pasta. To selected production media extracellular hydrolytic enzymes degrading polysaccharide were added. These enzymes were obtained from the cultivation media of four fungal strains. In this study three carotenogenic yeast strains were used: Rhodotorula glutinis, Sporobolomyces roseus and Cystofilobasidium capitatum. All strains were cultivated simultaneously and changes in biomass and carotenoid production in different production media were monitored and compared. As the best waste substrate apple fiber was utilized, particularly in Rhodotorula glutinis, which exhibited mainly biomass production increase. In Sporobolomyces roseus increased production of biomass and carotenoids have been reported in media with hydrolyzed fiber and pasta as well. Beta-carotene production in this strain reached 4776,38 mg/g of dry weight. The strain Cystofilobasidium capitatum exhibited in waste media a decerase of biomass production accompanied with increased production of carotenoids, especially in wheat mush and pasta medium hydrolyzed by enzyme preparative from Aureobasidium pullulan. It can be concluded that hydrolyzed waste substrates are very hopeful as cheap nutrient sources for yeast strains producing carotenoids and ergosterol. Nevertheless, further study of substrate processing for individual strains is needed.

The aim of Diploma thesis was the cultivation of microorganisms Lactobacillus plantarum CCM 7039 and Bacillus coagulans CCM 2658 on hydrolysed spent coffee grounds, production of lactid acid and modelling of these processes to find out more detailed characteristics. First, both microorganisms were cultivated in Erlenmeyer flasks on spent coffee grounds hydrolysate containing lipids and processed by extraction of lipids, e.g. lipid-free. The effect of nitrogen source and inoculation medium were monitored in the course of cultivation. The optimal conditions found were used for subsequent fermentation in bioreactors. Further, microorganisms were cultivated in the Erlenmeyer flasks in the media with individual carbohydrates typical for hydrolysed spent coffee grounds to determine its ability to utilize these carbohydrates. By fermentation in the bioreactors, the yield of lactic acid from carbohydrates was 97 % in Lactobacillus plantarum and 99 % in Bacillus coagulans, respectively. Models for growth and productivity of microorganisms were processed in MATLAB using logistic, generalized logistic and Monod functions. In general, the best data fit was achieved using a logistic function.

The aim of this work is to study the possibility of microbial production of polyhydroxyalkanoates (PHA). PHA can be used as biodegradable materials. Bacterial strain Cupriavidus necator was used for laboratory production of PHA. This bacterium was cultivated in medium with various precursors to produce copolymers of 3HB with 3HV or 4HB. Another part of the work was aimed at cultivation of C. necator on different waste substrates, especially oils, with the aim to achieve the highest production of polymer. Another large part of the thesis is dedicated to isolation strategies of PHA using enzymes. Commercially used proteases – alcalase and pancreatin – can be used with advantages for digestion of bacterial cells. A number of optimization experiments showed that application of proteases leads to enhancement of PHA purity to about 13%. Purity increase up to 90 % was achieved by adding a surfactant, which promotes the solubility of non-PHA forming polymer. This surfactant increases the purity of 20 % when compared to control. The last part of presented work deals with the use of enzyme solution isolated from Bacillus subtilis medium. Its application to C. necator culture led to the yield of polymer at a purity exceeding 95 %. These results could represent the basis for new isolation strategies, which can lead to more efficient yield of PHA.

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.

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.

The scope of thesis was the optimization of methods for the study of yeast and their metabolites using flow cytometry and fluorescence microscopy. Red yeasts are characterized by overproduction of carotenoids and lipids, which are used in food, pharmaceutical and feed industries. Currently, intensive research is being carried on to find appropriate microbiological alternatives for synthesis of these substances. Present thesis is focused on selected yeast genera: Rhodotorula, Sporobolomyces, Cystofilobasidium and strain Phaffia rhodozyma. Yeasts were cultivated on different nutrient media, in which glucose was used as a nutritional source, and also on glycerol and whey as waste material. In two strains - Cystofilobasidium macerans and Rhodotorula mucilaginosa growth characteristics were determined on a synthetic glucose production medium. All studied strains were able to use waste substrates as a source of nutrients. Some of the strains displayed increased production of carotenoids, and, additionally, in some cases also relatively high production of lipids. In classical cultivation in lipid and glucose medium supplemented with vitamins the best production characteristics displayed Rhodotorula glutinisstrain. In glycerol medium the highest amount of carotenoids and lipidic substances produced Sporobolomyces shibatanus strain. Strain Sporobolomyces roseus showed the best production characteristics on whey as the main source of carbon. The results show use of whey and glycerol seems like appropriate option for potential carbon source to cultivate carotenogenic yeasts and production of carotenoids and selected lipidic substances as products with higher added value. Further optimization of nutrient medium on the given substrates is needed for higher production of selected metabolites. Fluorescence microscopy and flow cytometry have proved to be suitable options for determination of the observed metabolites in the cells, their amount and viability.