| 1. |
- Bakeeva, Albina, et al.
(författare)
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The Potential of Kluyveromyces marxianus to Produce Low-FODMAP Straight-Dough and Sourdough Bread: a Pilot-Scale Study
- 2021
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Ingår i: Food and Bioprocess Technology. - : Springer Science and Business Media LLC. - 1935-5130 .- 1935-5149. ; 14, s. 1920-1935
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Tidskriftsartikel (refereegranskat)abstract
- Diets low in fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) can help reduce symptoms in 50 to 80% of patients suffering from irritable bowel syndrome. Patients are, therefore, often advised to avoid products contributing to FODMAP intake, such as cereal grain products. However, these products are nutritious staple foods and avoiding their consumption may result in nutritional deficiencies. The development of low-FODMAP, high-fiber cereal grain products is therefore desirable. This pilot-scale study shows that Kluyveromyces marxianus CBS6014 (K. marxianus) results in more fructan hydrolysis and a significantly lower final fructan level in white and whole-grain toast bread as well as in rye sourdough bread compared to a commercial Saccharomyces cerevisiae baking strain. Moreover, combined fructan and fructose levels in white and whole-grain bread prepared with K. marxianus remained well below the threshold concentration for low-FODMAP products. In addition to reducing fructan levels, K. marxianus in rye sourdough bread also positively impacted bread height. Whereas further follow-up studies are needed to assess the potential of K. marxianus for bread production fully, our study suggests that this yeast species may open exciting novel routes for the production of low-FODMAP, high-fiber products.
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| 2. |
- Chmielarz, Mikolaj
(författare)
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Conversion of lignocellulose and crude glycerol to lipids by oleaginous yeasts - physiology and diversity
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oleaginous yeasts are organisms capable of accumulating lipids. Some of them can grow on various substrates considered to be too toxic for many other microorganisms. Crude glycerol (CG) and hemicellulose hydrolysate (HH) are two examples of such substrates. Lipid quantification by extraction is a time-consuming process which requires usage of organic solvents and strong acids when applied on yeasts. To address this, we developed a new method using Fourier-transform near infra-red (FT-NIR) spectroscopy to quantify lipids within yeast cells. The resulting model for Rhodotorula toruloides had a R2 of 98% and a 5% error in prediction when compared with the traditional lipid extraction method. The method was used to follow lipid formation kinetics in subsequent experiments.Out of 27 tested oleaginous yeast strains, less than half could grow on crude glycerol. Two strains, R. toruloides CBS14 and Rhodotorula glutinis CBS3044 grew well on this substrate, and were even activated when it was supplemented with hemicellulose hydrolysate (producing up to 12.5 g/L lipids). RNA sequencing in R. toruloides CBS14 revealed increased transcription of genes related to energy metabolism, mitochondrial enzymes and genes involved in protein synthesis. There were only little differences in genes related to glycerol metabolic pathways. Probably, cells grown in CGHH have a more efficient energy metabolism and thus, more ATP to build up biomass and lipids, and to take up substrate from the medium.The new method for lipid quantification and the identified regulated genes can be the basis for further manipulations of yeast metabolism to reach sustainable microbial oil production from residual substrates.
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| 3. |
- Chmielarz, Mikolaj, et al.
(författare)
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FT-NIR: a tool for rapid intracellular lipid quantification in oleaginous yeasts
- 2019
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLipid 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.MethodsThe 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.ResultsThe 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.ConclusionsUsing 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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| 4. |
- Chmielarz, Mikolaj, et al.
(författare)
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Microbial lipid production from crude glycerol and hemicellulosic hydrolysate with oleaginous yeasts
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 5. |
- Martín Hernández, Giselle De La Caridad, et al.
(författare)
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Chromosome-level genome assembly and transcriptome-based annotation of the oleaginous yeast Rhodotorula toruloides CBS 14
- 2021
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Ingår i: Genomics. - : Elsevier BV. - 0888-7543 .- 1089-8646. ; 113, s. 4022-4027
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Tidskriftsartikel (refereegranskat)abstract
- Rhodotorula toruloides is an oleaginous yeast with high biotechnological potential. In order to understand the molecular physiology of lipid synthesis in R. toruloides and to advance metabolic engineering, a high-resolution genome is required. We constructed a genome draft of R. toruloides CBS 14, using a hybrid assembly approach, consisting of short and long reads generated by Illumina and Nanopore sequencing, respectively. The genome draft consists of 23 contigs and 3 scaffolds, with a N50 length of 1,529,952 bp, thus largely representing chromosomal organization. The total size of the genome is 20,534,857 bp and the overall GC content is 61.83%. Transcriptomic data from different growth conditions was used to aid species-specific gene annotation. We annotated 9464 genes and identified 11,691 transcripts. Furthermore, we demonstrated the presence of a potential plasmid, an extrachromosomal circular structure of about 11 kb with a copy number about three times as high as the other chromosomes.
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| 6. |
- Martín Hernández, Giselle De La Caridad, et al.
(författare)
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Enhanced glycerol assimilation and lipid production in Rhodotorula toruloides CBS14 upon addition of hemicellulose primarily correlates with early transcription of energy-metabolism-related genes
- 2023
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Ingår i: Biotechnology for Biofuels and Bioproducts. - : Springer Science and Business Media LLC. - 2731-3654. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLipid formation from glycerol was previously found to be activated in Rhodotorula toruloides when the yeast was cultivated in a mixture of crude glycerol (CG) and hemicellulose hydrolysate (CGHH) compared to CG as the only carbon source. RNA samples from R. toruloides CBS14 cell cultures grown on either CG or CGHH were collected at different timepoints of cultivation, and a differential gene expression analysis was performed between cells grown at a similar physiological situation.ResultsWe observed enhanced transcription of genes involved in oxidative phosphorylation and enzymes localized in mitochondria in CGHH compared to CG. Genes involved in protein turnover, including those encoding ribosomal proteins, translation elongation factors, and genes involved in building the proteasome also showed an enhanced transcription in CGHH compared to CG. At 10 h cultivation, another group of activated genes in CGHH was involved in beta-oxidation, handling oxidative stress and degradation of xylose and aromatic compounds. Potential bypasses of the standard GUT1 and GUT2-glycerol assimilation pathway were also expressed and upregulated in CGHH 10 h. When the additional carbon sources from HH were completely consumed, at CGHH 36 h, their transcription decreased and NAD(+)-dependent glycerol-3-phosphate dehydrogenase was upregulated compared to CG 60 h, generating NADH instead of NADPH with glycerol catabolism. TPI1 was upregulated in CGHH compared to cells grown on CG in all physiological situations, potentially channeling the DHAP formed through glycerol catabolism into glycolysis. The highest number of upregulated genes encoding glycolytic enzymes was found after 36 h in CGHH, when all additional carbon sources were already consumed.ConclusionsWe suspect that the physiological reason for the accelerated glycerol assimilation and faster lipid production, was primarily the activation of enzymes that provide energy.
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| 7. |
- Passoth, Volkmar, et al.
(författare)
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Oleaginous yeasts for biochemicals, biofuels and food from lignocellulose-hydrolysate and crude glycerol
- 2023
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 40
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Forskningsöversikt (refereegranskat)abstract
- 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 beta-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.
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