Carotenogenic yeasts are non-conventional oleaginous microorganisms capable of utilizing various waste substrates. In this work, four red yeast strains (Rhodotorula, Cystofilobasidium, and Sporobolomyces sp.) were cultivated in media containing crude, emulsified, and enzymatically hydrolyzed animal waste fat, compared with glucose and glycerol, as single C-sources. Cell morphology (cryo-SEM (cryo-scanning electron microscopy), TEM (transmission electron microscopy)), production of biomass, lipase, biosurfactants, lipids (gas chromatography/flame ionization detection, GC/FID) carotenoids, ubiquinone, and ergosterol (high performance liquid chromatography, HPLC/PDA) in yeast cells was studied depending on the medium composition, the C source, and the carbon/nitrogen (C/N) ratio. All studied strains are able to utilize solid and processed fat. Biomass production at C/N = 13 was higher on emulsified/hydrolyzed fat than on glucose/glycerol. The 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 (cell dry weight)) were found in S. pararoseus. Simultaneous induction of lipase and biosurfactants was observed on crude fat substrate. An increased C/N ratio (13–100) led to higher biomass production in fat media. The production of total lipids increased in all strains to C/N = 50. Oppositely, the production of carotenoids, ubiquinone, and ergosterol dramatically decreased with increased C/N in all strains. Compounds accumulated in stressed red yeasts have a great application potential and can be produced efficiently during the valorization of animal waste fat under the biorefinery concept.

Red yeast strains are ubiquitous microorganisms which accumulate substantial amounts of lipids and lipid-soluble metabolites. Red yeasts utilize many waste substrates of different origin. In this work red yeasts strains (Rhodotorula, Sporobolomyces, Cystofilobasidium) were used for screening of growth and metabolic activity. As a carbon source waste animal fat and its hydrolysis products were used. Hydrolysis of animal fat was tested in alkaline as well as acidic conditions. As the substrate glucose (control), glycerol, crude animal fat, acid fat hydrolyzate and hydrolysate: glucose 1:2 were used. Screening of growth and metabolic activity of red yeasts was performed by flow cytometry. Extracellular lipase production was monitored as adaptation mechanism. Carotenoids, ergosterol and ubiquinone were quantified by HPLC/PDA/MS/ESI and the biomass was evaluated gravimetrically. All tested strains utilized fat hydrolysate and produced red coloured biomass. Cultivation in media containing non-hydrolysed fat led to strain specific induction of extracellular lipase. Amount of lipid metabolites produced by individual strains was depended on glycerol content in medium. The highest increase of lipase production was observed in Cystofilobasidium macerans and Sporobolomyces shibatanus. Valorisation of animal fat can lead to production of unsaturated fatty acids, single cell oils, carotenoid pigments, sterols and enriched red yeast biomass.

Beta (β)–glucans are polysaccharides composed of D-glucose monomers. Nowadays, β-glucans are gaining attention due to their attractive immunomodulatory biological activities, which can be utilized in pharmaceutical or food supplementation industries. Some carotenogenic Basidiomycetes yeasts, previously explored for lipid and carotenoid coproduction, could potentially coproduce a significant amount of β–glucans. In the present study, we screened eleven Basidiomycetes for the coproduction of lipids and β–glucans. We examined the effect of four different C/N ratios and eight different osmolarity conditions on the coproduction of lipids and β–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 strains C. infirmominiatum CCY 17-18-4 and R. kratochvilovae CCY 20-2-26 were identified as the best coproducers of lipids and β-glucans. In addition, C. infirmominiatum CCY 17-18-4, R. kratochvilovae CCY 20-2-26 and P. rhodozyma CCY 77-1-1 were identified as the best alternative producers of β-glucans. Increased C/N ratio led to increased biomass, lipid and β-glucans production for several yeast strains. Increased osmolarity had a negative effect on biomass and lipid production while the β-glucan production was positively affected.

Oleaginous filamentous fungi can accumulate large amount of cellular lipids and biopolymers and pigments and potentially serve as a major source of biochemicals for food, feed, chemical, pharmaceutical, and transport industries. We assessed suitability of Fourier transform (FT) Raman spectroscopy for screening and process monitoring of filamentous fungi in biotechnology. Six Mucoromycota strains were cultivated in microbioreactors under six growth conditions (three phosphate concentrations in the presence and absence of calcium). FT-Raman and FT-infrared (FTIR) spectroscopic data was assessed in respect to reference analyses of lipids, phosphorus, and carotenoids by using principal component analysis (PCA), multiblock or consensus PCA, partial least square regression (PLSR), and analysis of spectral variation due to different design factors by an ANOVA model. All main chemical biomass constituents were detected by FT-Raman spectroscopy, including lipids, proteins, cell wall carbohydrates, and polyphosphates, and carotenoids. FT-Raman spectra clearly show the effect of growth conditions on fungal biomass. PLSR models with high coefficients of determination (0.83–0.94) and low error (approximately 8%) for quantitative determination of total lipids, phosphates, and carotenoids were established. FT-Raman spectroscopy showed great potential for chemical analysis of biomass of oleaginous filamentous fungi. The study demonstrates that FT-Raman and FTIR spectroscopies provide complementary information on main fungal biomass constituents.

Multifunctional biomass is able to provide more than one valuable product, and thus, it is attractive in the field of microbial biotechnology due to its economic feasibility. Carotenogenic yeasts are effective microbial factories for the biosynthesis of a broad spectrum of biomolecules that can be used in the food and feed industry and the pharmaceutical industry, as well as a source of biofuels. In the study, we examined the effect of different nitrogen sources, carbon sources and CN ratios on the co-production of intracellular lipids, carotenoids, β–glucans and extracellular glycolipids. Yeast strain R. kratochvilovae CCY 20-2-26 was identified as the best co-producer of lipids (66.7 ± 1.5% of DCW), exoglycolipids (2.42 ± 0.08 g/L), β-glucan (11.33 ± 1.34% of DCW) and carotenoids (1.35 ± 0.11 mg/g), with a biomass content of 15.2 ± 0.8 g/L, by using the synthetic medium with potassium nitrate and mannose as a carbon source. It was shown that an increased C/N ratio positively affected the biomass yield and production of lipids and β-glucans.

Infrared spectroscopy delivers abundant information about the chemical composition, as well as the structural and optical properties of intact samples in a non-destructive manner. We present a deep convolutional neural network which exploits all of this information and solves full-wave inverse scattering problems and thereby obtains the 3D optical, structural and chemical properties from infrared spectroscopic measurements of intact micro-samples. The proposed model encodes scatter-distorted infrared spectra and infers the distribution of the complex refractive index function of concentrically spherical samples, such as many biological cells. The approach delivers simultaneously the molecular absorption, sample morphology and effective refractive index in both the cell wall and interior from a single measured spectrum. The model is trained on simulated scatter-distorted spectra, where absorption in the distinct layers is simulated and the scatter-distorted spectra are estimated by analytic solutions of Maxwell’s equations for samples of different sizes. This allows for essentially real-time deep learning-enabled infrared diffraction micro-tomography, for a large subset of biological cells.

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.

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