Andrea Haronikova

Use of several waste substrates for carotenoid-rich yeast biomass production

Carotenoids are industrially significant pigments produced in many bacteria, fungi, and plants. Carotenoid biosynthesis in yeasts is involved in stress response mechanisms. Thus, controlled physiological and nutrition stress can be used for enhanced pigment production. Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques including use of liquid waste substrates as carbon and/or nitrogen source. In this work several red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were enrolled into a comparative screening study. To increase the yield of these pigments at improved biomass production, several types of exogenous as well as nutrition stress were tested. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Synthetic media with addition of complex substrates (e.g. yeast extract) and vitamin mixtures as well as some waste materials (whey, potato extract) were used as nutrient sources. Peroxide and salt stress were applied too. The production of carotene enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. All strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. The highest yields were obtained in R. glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/L of beta-carotene) and in R. mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement.

Influence of Culture Media on Microbial Fingerprints Using Raman Spectroscopy.

Raman spectroscopy has a broad range of applications across numerous scientific fields, including microbiology. Our work here monitors the influence of culture media on the Raman spectra of clinically important microorganisms (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans). Choosing an adequate medium may enhance the reproducibility of the method as well as simplifying the data processing and the evaluation. We tested four different media per organism depending on the nutritional requirements and clinical usage directly on a Petri dish. Some of the media have a significant influence on the microbial fingerprint (Roosvelt-Park Institute Medium, CHROMagar) and should not be used for the acquisition of Raman spectra. It was found that the most suitable medium for microbiological experiments regarding these organisms was Mueller-Hinton agar.

Morphological and Production Changes in Stressed Red Yeasts Monitored Using SEM and Raman Spectroscopy

We report on our investigations of the influence of different cultivation conditions on lipids, using scanning electron microscopy (SEM) and Raman spectroscopy techniques. Here, SEM uses electron beam to gain information about morphology of cells which reflects cells response on the applied stress. Consequently, Raman spectroscopy [1] was used for the determination of carotenoids and lipids present in the biomass. Thus, our study targets some factors which could lead to efficient industrial production of carotenoids and lipids in selected biotechnological production.

Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth

Abstract Red yeast Cystofilobasidium capitatum autofluorescence was studied by means of confocal laser scanning microscopy (CLSM) to reveal distribution of carotenoids inside the cells. Yeasts were cultivated in 2L fermentor on glucose medium at permanent light exposure and aeration. Samples were collected at different times for CLSM, gravimetric determination of biomass and HPLC determination of pigments. To compare FLIM (Fluorescence Lifetime Imaging Microscopy) images and coupled data (obtained by CLSM) with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of beta-carotene. Liposomes lecithin:ergosterol:beta-carotene were investigated too. Two different intracellular forms of carotenoids were observed during most of cultivations, with third form appeared at the beginning of stationary phase. Observed behavior is probably due to formation of some kind of carotenoid protective system in membranes of different compartments of yeast cell, especially cytoplasmic membrane.

Monitoring of growth and production characteristics of red yeasts cultivated on hydrothermally pretreated lignocellulosic pine material

The aim of this work was to compare the production of carotenes and ergosterol by red yeasts grown on pine lignocellulose substrates. The yeast strains Rhodotorula aurantiaca and Sporobolomyces shibatanus were grown on the liquid fraction of steam pretreated pine (210 °C, catalyst SO2). Biomass production on a pine hydrolysate was lower than on glucose. The highest content of carotenoids and ergosterol in the cells of R. aurantiaca grown on pine hydrolysate was about 1.7 mg g-1 and 0.8 mg g-1 (dwt), respectively, and in S. shibatanus about 0.9 mg g-1 and 0.1 mg g-1, respectively. Hemicellulose hydrolysates may contain many compounds that have inhibitory effects on microorganisms. In this work, the influences of some inhibitors were assessed by cultivating yeasts on media with a representative addition of the selected compounds. From these tests, furfural appears to be the most critical inhibitor, whereas acetic acid and 5-hydroxymethyl furfural (HMF) do not affect the growth so much. (Less)

Raman spectroscopy to monitor the effects of temperature regime and medium composition on micro-organism growth

A biomass of yeast strains has been studied using Raman spectroscopy due to their potential applications in the field of biofuel generation, food industry and biotechnological applications. In order to utilize biomass for efficient industrial/biotechnological production, the optimal cultivation parameters have to be determined which in turn lead to high production of desired substances such as oil, carotenoids, and pigments in the selected cell line of yeast. Therefore, we focused on different cultivation conditions (the effects of temperature regime and medium composition) and their influence on microorganisms growth and metabolic changes.

SEM and Raman Spectroscopy Applied to Biomass Analysis for Application in the Field of Biofuels and Food Industry

A biomass of algal (Trachydiscus minutus, Botryococcus sudeticus, and Chlamydomonas sp.) and red yeast strains (Rhodotorula spp., Cystofilobasidium spp. and Sporobolomyces spp.) has been studied due to their potential applications in the field of biofuel generation and food industry [1-2]. In order to utilize biomass for efficient industrial production, the optimal cultivation parameters have to be determined which in turn lead to high production of desired substances such as oil and carotenoids in the selected cell line [2]. Main aim of our investigations was to study – using scanning electron microscopy (SEM) and Raman spectroscopy techniques – how different cultivation conditions influence production of oil and carotenoids. Raman spectroscopy can be used for the determination of the oil present in the biomass and also for the determination of carotenoids as the intensity ratios of specific, selected Raman bands [1]. SEM uses electron beams to gain information about morphology of cells (biomass structure) which is very important factor to study cells response on the applied stress.

Time-resolved study of microorganisms by Raman spectroscopy

The main goal of our investigations is to focus on the basic physiological mechanisms of microorganisms (yeast and bacteria), exposed to different conditions, by time-resolved Raman spectroscopy. This study provides an insight into the mechanism of targeted stress factors or the influence of different cultivation times on species metabolism in vivo, in realtime and label free. We also focused on time-course study of physico-chemical properties of bacterial cells and cell cytoplasm with respect to the intracellular content of polyhydroxyalkanoates and to the production of yeast lipids or carotenoids.