Background Crude glycerol (CG) and hemicellulose hydrolysate (HH) are low—value side-products of biodiesel transesterification and pulp—and paper industry or lignocellulosic ethanol production, respectively, which can be converted to microbial lipids by oleaginous yeasts. This study aimed to test the ability of oleaginous yeasts to utilise CG and HH and mixtures of them as carbon source. Results Eleven out of 27 tested strains of oleaginous yeast species were able to grow in plate tests on CG as sole carbon source. Among them, only one ascomycetous strain, belonging to Lipomyces starkeyi, was identified, the other 10 strains were Rhodotorula spec. When yeasts were cultivated in mixed CG/ HH medium, we observed an activation of glycerol conversion in the Rhodotorula strains, but not in L. starkeyi. Two strains—Rhodotorula toruloides CBS 14 and Rhodotorula glutinis CBS 3044 were further tested in controlled fermentations in bioreactors in different mixtures of CG and HH. The highest measured average biomass and lipid concentration were achieved with R. toruloides in 10% HH medium mixed with 55 g/L CG—19.4 g/L and 10.6 g/L, respectively, with a lipid yield of 0.25 g lipids per consumed g of carbon source. Fatty acid composition was similar to other R. toruloides strains and comparable to that of vegetable oils. Conclusions There were big strain differences in the ability to convert CG to lipids, as only few of the tested strains were able to grow. Lipid production rates and yields showed that mixing GC and HH have a stimulating effect on lipid accumulation in R. toruloides and R. glutinis resulting in shortened fermentation time to reach maximum lipid concentration, which provides a new perspective on converting these low-value compounds to microbial lipids.

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Background Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast. Methods The FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts Rhodotorula toruloides CBS 14, Lipomyces starkeyi CBS 1807 and Yarrowia lipolytica CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The R. toruloides model was additionally tested on Rhodotorula babjevae DBVPG 8058 and Rhodotorula glutinis CBS 2387. Results The R 2 of the FT-NIR model for R. toruloides was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of R. toruloides samples with a root mean square error of prediction (RMSEP) of 3.21. The R 2 of the Lipomyces model was 96%, with RMSECV 2.4 and RMSEP 3.8. The R 2 of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods. Conclusions Using FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.

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Microbial lipids produced from lignocellulose and crude glycerol (CG) can serve as sustainable alternatives to vegetable oils, whose production is, in many cases, accompanied by monocultures, land use changes or rain forest clearings. Our projects aim to understand the physiology of microbial lipid production by oleaginous yeasts, optimise the production and establish novel applications of microbial lipid compounds. We have established methods for fermentation and intracellular lipid quantification. Following the kinetics of lipid accumulation in different strains, we found high variability in lipid formation even between very closely related oleaginous yeast strains on both, wheat straw hydrolysate and CG. For example, on complete wheat straw hydrolysate, we saw that one Rhodotorula glutinis strain, when starting assimilating D‐xylosealso assimilated the accumulated lipids, while a Rhodotorula babjevae strain could accumulate lipids on D‐xylose. Two strains (Rhodotorula toruloides CBS 14 and R. glutinis CBS 3044) were found to be the best out of 27 tested to accumulate lipids on CG. Interestingly, the presence of hemicellulose hydrolysate stimulated glycerol assimilation in both strains. Apart from microbial oil, R. toruloides also produces carotenoids. The first attempts of extraction using the classical acetone‐based method showed that β‐carotene is the major carotenoid. However, there are indications that there are also substantial amounts of torulene and torularhodin, which have a very high potential as antioxidants.