Trichosporon Oleaginosus Research Articles

Monitoring Yeast Cultures Grown on Corn Stover Hydrolysate for Lipid Production

Microbial oils can be used as an alternative sustainable and renewable feedstock to fossil reserves for producing lubricants and polyurethane materials. Two oleaginous yeasts were grown on non-detoxified corn stover hydrolysate supplemented with corn steep liquor and mineral medium in shake flasks. Trichosporon oleaginosus DSM 11815 displayed the highest lipid production. This strain was further cultivated in a bench bioreactor, using the same culture medium, under a batch regime. Flow cytometry was used to monitor the T. oleaginosus culture using the dual staining technique (SYBR Green and PI) for cell membrane integrity detection. Values of 42.28% (w/w) and 0.06 g/Lh lipid content and lipid productivity, respectively, were recorded for T. oleaginosus cultivated in the bench bioreactor operated under a batch regime. During the cultivation, most of the yeast cells maintained their integrity. T. oleaginosus has the potential to be used as an oil microbial source for a wide range of industrial applications. In addition, it is robust in adverse conditions such as lignocellulosic hydrolysate exposure and oxygen-limiting conditions. Flow cytometry is a powerful and useful tool for monitoring yeast cultivations on lignocellulosic hydrolysates for cell count, size, granularity, and membrane integrity detection.

Comprehensive Exploration of the Growth and Lipid Synthesis Phases of T. oleaginosus Cultures Implementing Design of Experiments and Response Surface Methodology.

Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability.

Optimization of Pretreatment Conditions and Enzymatic Hydrolysis of Corn Cobs for Production of Microbial Lipids by Trichosporon oleaginosus

Microbial lipids produced from lignocellulosic biomass are sustainable alternative feedstock for biodiesel production. In this study, corn cobs were used as a carbon source for lipid production and growth of oleaginous yeast Trichosporon oleaginosus. Lignocellulosic biomass was subjected to alkali and acid pretreatment using sulfuric acid and sodium hydroxide under different temperatures, catalyst concentrations and treatment times. Pretreatment of corn cobs was followed by cellulase hydrolysis. Hydrolysis of alkali pretreated (2% NaOH at 50 °C for 6 h, 1% NaOH at 50 °C for 16 h, 2% NaOH at 121 °C for 1 h, 1% NaOH at 121 °C for 2 h) and acid pretreated (1% H2SO4 120 °C for 20 min, and 2% H2SO4 120 °C for 10 min) corn cobs resulted in more than 80% of the theoretical yield of glucose. The effect of substrate (5, 10, 15 and 20%, g g−1) and cellulase loading (15 and 30 Filter Paper Units per gram of glucan, FPU g−1) on fermentable sugar yield was also studied. The maximal glucose concentration of 81.64 g L−1 was obtained from alkali-pretreated corn cobs (2% NaOH at 50 °C for 6 h) at 20% substrate loading and 30 FPU of Cellic CTec2 g−1 of glucan. Enzymatic hydrolysates of pretreated biomasses and filtrates of lignocellulosic slurries obtained after pretreatment were used for growth and lipid synthesis by T. oleaginosus. The highest lipid concentration of 18.97 g L−1 was obtained on hydrolysate of alkali-pretreated corn cobs (with 1% NaOH at 50 °C for 16 h) using a 15% (g g−1) substrate loading and 15 FPU g−1 of cellulase loading. Significant lipid accumulation was also achieved using undetoxified filtrates of pretreated slurries as substrates. Results showed that pretreated corn cobs and undetoxified filtrates are suitable carbon sources for the growth and efficient accumulation of lipids in T. oleaginosus.

Strategies for Improvement of Lipid Production by Yeast Trichosporon oleaginosus from Lignocellulosic Biomass.

Microbial lipids have similar fatty acid composition to plant oils, and therefore, are considered as an alternative feedstock for biodiesel production. Oleaginous yeasts accumulate considerable amounts of lipids intracellularly during growth on low-cost renewable feedstocks such as lignocellulosic biomass. In this study, we cultivated yeast Trichosporon oleaginosus on hydrolysate of alkaline pretreated corn cobs. Different process configurations were evaluated and compared, including separate hydrolysis and fermentation (SHF) with cellulase recycle and simultaneous saccharification and fermentation (SSF) in batch and fed-batch mode. At low enzyme loading, the highest lipid concentration of 26.74 g L−1 was reached in fed-batch SSF fed with 2.5% (g g−1) substrate. Batch SHF was conducted for four rounds with recycling the cellulase adsorbed on unhydrolyzed lignocellulosic biomass. Thirty percent of cellulase saving was achieved for rounds 2–4 without compromising productivity and lipid yield. The addition of Tween 80 to lignocellulosic slurry improved the hydrolysis rate of structural carbohydrates in pretreated lignocellulosic biomass. Furthermore, supplementing the growth medium with Tween 80 improved lipid yield and productivity without affecting yeast growth. Oleaginous yeast T. oleaginosus is a promising strain for the sustainable and efficient production of lipids from renewable lignocellulosic feedstock.

Pretreatments for enhancing sewage sludge reduction and reuse in lipid production

BackgroundConverting wastewater sludge to lipid is considered as one of the best strategies of sludge management. The current problem of lipid production from wastewater sludge is the low yield (0.10–0.16 g lipid/g dry sludge) due to the low availability of easily uptaken materials (such as soluble monosaccharide and oligosaccharide) in sludge to oleaginous microorganism (Rhodotorula glutinis, Trichosporon oleaginosus, Lipomyces starkeyi). Pretreatments are efficient methods to improve sludge bioavailability. This study is aimed to achieve high lipid production from sludge and high sludge reduction.ResultsIn this study, it was observed that the soluble chemical oxygen demand (SCOD) had significantly increased after different pretreatment. The SCOD in the supernatant was increased from 32.64 to 180.25 mg/L, 924.16 mg/L, 1029.89 mg/L and 3708.31 mg/L after acidic (pH 2 for 2 h), alkaline (pH 12 for 2 h), microwave irradiation (15 min with 5 min interval), and ultrasonication (30 min at 450 W and 20 kHz frequency with 5 s on and 2 s off mode) pretreatment, respectively. Pretreatments have also increased the release of total nitrogen (TN) and total phosphorus (TP) from solids. The sludge after different pretreatments were used as a medium for lipid production, and the highest lipid content (36.67% g/g) was obtained in the fermentation with ultrasonication pretreatment sludge, and the sludge reduction was 63.10%. For other pretreatments, the lipid content and sludge reduction were 18.42% and 32.63% in acid pretreatment case, 21.08% and 36.44% in alkaline pretreatment case, and 26.31% and 43.03% in microwave pretreatment case, respectively.ConclusionIt was found that ultrasonication pretreatment was the most efficient way to increase the sludge biodegradability (SCOD) and to release TN and TP from solid phase to liquid phase. Pretreated sludge for lipid production achieved significant improvement in lipid yield and sludge reduction. Lipids produced from pretreated sludge were transesterified to biodiesel and the analysis showed that biodiesel had a similar composition as commercial biodiesel. The study reveals that pretreatment on sludge is a promising method for enhancing biological sludge management efficiency.

Impact of nitrogen on the industrial feasibility of biodiesel production from lipid accumulated in oleaginous yeast with wastewater sludge and crude glycerol

In this study, oleaginous yeast Trichosporon oleaginosus was cultivated in wastewater sludge and crude glycerol medium for lipid production. The sufficiency of nitrogen in sludge was verified by conducting the experiments with and without the addition of NH4Cl in the medium. Biomass production, lipid production and lipid content of the fermentation with NH4Cl addition were 55.33 g/L, 26.98 g/L and 48.77% (w/w), respectively; however, they were 42.66 g/L, 17.88 g/L and 41.91% (w/w) in the one without addition of NH4Cl, respectively. The process of biodiesel produced from the lipid cultivated with sludge and crude glycerol could provide net energy gain and greenhouse gas emission reduction. The estimated biodiesel production cost in this study was 630 US $/tonne with NH4Cl addition and 860 $/tonne without NH4Cl addition, respectively. Compared to the current commercial biodiesel price (900 US $/tonne), biodiesel production from wastewater sludge and crude glycerol is very competitive. The study provides an insight of sustainable bioenergy production system from wastewater sudge.

Contact-free infrared OD measurement for online monitoring of parallel stirred-tank bioreactors up to high cell densities

Miniaturized stirred-tank bioreactor systems provide a scalable platform for high-throughput bioprocess development. Online measurement of process variables is a major demand to enable efficient process monitoring and control in parallel operated bioreactors. One miniaturized laser light source and two photodiodes were placed around a cylindrical disposable bioreactor made of polystyrene for individual and contact-free measurement of optical density (OD). One photodiode was positioned at an angle of 28° to the laser for measuring the scattered light, a second photodiode was positioned face to face with the laser for measuring the transmitted light. Miniaturized lasers with wavelengths of 650 nm (orange), 780 nm (NIR) and 850 nm (IR) were evaluated. The best results were achieved with a laser emitting at 850 nm. Both signals (scattered and transmitted light) were influenced by the stirrer speed (usually constant) and the microorganisms under study. After individual calibration, online OD monitoring of pH-controlled fed-batch processes was successfully shown with Escherichia coli, Corynebacterium glutamicum, and Trichosporon oleaginosus. Online OD measurements based on the transmitted light signals showed low standard deviation at low cell densities, whereas scattered light signals were more accurate at higher cell densities. Correlations were reliable up to cell dry weight concentrations of 46 g L−1 in stirred-tank bioreactors on a milliliter scale.

Cell Factories for Industrial Production Processes: Current Issues and Emerging Solutions

Despite all the progresses made by metabolic engineering, still only a few biotechnological processes are running at an industrial level. In order to boost the biotechnological sector, integration strategies as well as long-term views are needed. The aim of the present review is to identify the main drawbacks in biotechnological processes, and to propose possible solutions to overcome the issues in question. Novel cell factories and bioreactor design are discussed as possible solutions. In particular, the following microorganisms: Yarrowia lipolytica, Trichosporon oleaginosus, Ustilago cynodontis, Debaryomyces hansenii along with sequential bioreactor configurations are presented as possible cell factories and bioreactor design solutions, respectively.

Non-conventional yeasts as superior production platforms for sustainable fermentation based bio-manufacturing processes

Non-conventional yeasts are an excellent option for a number of different industrial bioprocesses. They possess beneficial natural phenotypes, which translates to several fermentation advantages when compared to traditional hosts, like <i>Saccharomyces cerevisiae</i>. The non-conventional yeasts <i>Yarrowia lipolytica</i>, <i>Trichosporon oleaginosus</i>, <i>Kluyveromyces marxianus</i>, <i>Dekkera bruxellensis</i>, <i>Pichia kudriavzevii</i>, <i>Debaryomyces hansenii</i> and <i>Hansenula polymorpha</i>, are considered desirable industrial hosts due to their natural characteristics, including tolerance to several by-products and inhibitors, thermotolerance, salt resistance or osmo- and xerotolerance. Therefore, they are a great alternative for the industrial production of bioethanol, fine chemicals, lipids and recombinant proteins, among others. In this review, we summarize the best natural characteristics of those seven non-conventional yeasts and their use in industrial biotechnology, as well as the molecular/synthetic biology tools available for their genetic modification. Moreover, possible limitations regarding their performance in industrial fermentations and a list of challenges to overcome in the future are also discussed.

Co-production of ethanol and biodiesel from sweet sorghum juice in two consecutive fermentation steps

Sugars from sweet sorghum stalks can be used to produce ethanol and also to grow oleaginous yeasts. Instead of two separate processes, in this paper we propose a different route producing ethanol and microbial oil in two consecutive fermentation steps. Three yeasts were compared in the first ethanol producing step. In the second step four different oleaginous yeasts were tested. Sweet sorghum juice was first clarified and concentrated. High gravity ethanol fermentation was carried out with concentrated juice with 23.7 g/100 mL of total sugars and without added nutrients. Total sugars were 2.5 times more than the original clarified juice. One yeast gave the best overall response over the two other tested; relative high ethanol productivity, 1.44 g ethanol/L·h−1, and 90% of sugar consumption. Aeration by flask agitation produced superior results than static flasks for all yeasts. Microbial oil production was done employing the residual liquid left after ethanol separation. The pooled residual liquid from the ethanol distillation contained 7.08 g/mL of total carbohydrates, rich in reducing sugars. Trichosporon oleaginosus and Lipomyces starkeyi produced higher dry biomass, total sugar consumption and oil productivity than the other two oleaginous yeasts tested; with values around 25 g/L, 80%, and 0.55 g oil/L·h−1 respectively. However, the biomass oil content in all yeasts was relatively low in the range of 14 to 16%. The two step process is viable and could be considered an integral part of a consolidated biorefinery from sweet sorghum. How to cite: Rolz C, de León R, Mendizábal de Montenegro AL. Co-production of ethanol and biodiesel from sweet sorghum juice in two consecutive fermentation steps. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.05.002.

Engineering Trichosporon oleaginosus for enhanced production of lipid from volatile fatty acids as carbon source

Trichosporon oleaginosus is one of the most promising hosts for microbial lipid production owing to its high-productivity; In an effort to develop an economical production process, we engineered T. oleaginosus towards high-lipid production from volatile fatty acids (VFA) derived from anaerobic fermentation of food waste. First, we established a method for labeling intracellular lipid with lipophilic BODIPY fluorescent dye. Next, a random library was constructed by treatment with a chemical mutagen, and high-lipid producers were screened using fluorescence-activated cell sorting. Subsequently, one clone, N14, was successfully isolated, which exhibited 3-fold higher lipid production (19.4%) in VFA (6 g/L) media than the wild-type strain, and also showed increased lipid production in higher concentrations of VFA (18 or 24 g/L). Based on fatty acid methyl ester (FAME) analysis, N14 contained higher stearic acid (C18:0) and oleic acid (C18:1) content compared with those of the wild-type strain.

Correction to: Engineering Trichosporon oleaginosus for enhanced production of lipid from volatile fatty acids as carbon source

The article Engineering Trichosporon oleaginosus for enhanced production of lipid from volatile fatty acids as carbon source, written by Kyungsoo Lee*,‡, Yong Jae Lee**,‡, Ho Nam Chang***, and Ki Jun Jeong*,****,†, was originally published on the publisher’s internet portal (currently SPringerLink) on 08 February 2019 with misprinted DOI number, 10.1007/s11814-018-0229-7, due to the technical error from converting manuscript file from Microsoft Word to PDF. The correct DOI number for the article is 10.1007/s11814-019-0312-0.

Economical lipid production from Trichosporon oleaginosus via dissolved oxygen adjustment and crude glycerol addition

The effect of dissolved oxygen concentration on lipid accumulation in Trichosporon oleaginosus has been investigated. The experiment was performed in 15 L fermenters. The dissolved oxygen concentration varied by adjusting the agitation and aeration. High dissolved oxygen level at 50%–60% enhanced cell growth. Maintaining low dissolved oxygen concentration at 20%–30% during lipogenesis phase led to high final lipid content (51%) in Trichosporon oleaginosus. The consumptions of energy and cost of the process were evaluated. The energy consumption in the dissolved oxygen level optimized process was 41% less than that with dissolved oxygen level at 50%–60%. In addition, the cost was also reduced around one time in the dissolved oxygen level optimized process compared to the one with dissolved oxygen level at 50%–60%. The study provided a feasible way of enhancing lipid accumulation in Trichosporon oleaginosus and reducing the consumption of energy and cost of lipid production from Trichosporon oleaginosus.

Lipid Production by Yeast Trichosporon oleaginosus on the Enzymatic Hydrolysate of Alkaline Pretreated Corn Cobs for Biodiesel Production

Biodiesel is still mainly produced from different vegetable (e.g., rapeseed, palm, soybean, sunflower, and used cooking oils) oils and animal fats, and therefore it has a negative impact on food and feed prices. For biodiesel production as an alternative feedstock, lipids from oleaginous microorganisms could be used. One of the oleaginous microorganisms is yeast Trichosporon oleaginosus that has the capacity to grow and accumulate lipids on different lignocellulosic hydrolysates. In this study, corn cobs were pretreated by alkali at loading from 0.08 to 1.6 g/g dry weight of untreated (raw) corn cobs (g NaOH/gDW_UCC) at 121 °C for 30 min. In a further step, alkaline pretreated corn cobs were subjected to the enzymatic hydrolysis by using commercial multienzymes mixtures. The optimal alkali loading (0.16 g/gDW_UCC) was related to the highest glucose and xylose yields which were observed during enzymatic hydrolysis of substrate. In this study, T. oleaginosus was applied for lipid production on the enzymatic...

Enhanced Triacylglycerol Production With Genetically Modified Trichosporon oleaginosus.

Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21–0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

Lipid accumulation from Trichosporon oleaginosus with co-fermentation of washed wastewater sludge and crude glycerol

In this study, two types of organic waste including sludge and crude glycerol were employed for lipid production from oleaginous yeast Trichosporon oleaginosus. As wastewater sludge contains metals and other substances, which could inhibit growth of the microorganisms, sludge washing was performed to reduce/remove the substance in order to enhance the lipid production in this study. The results revealed that the lipid production by Trichosporon oleaginosus was higher in the medium prepared using washed sludge fortified with glycerol compared to that of the unwashed sludge fortified with glycerol. The maximum biomass and lipid concentrations were 44.48 ± 2.34 g/L and 17.37 ± 1.22 g/L, respectively in the fermentation with washed sludge and glycerol and 39.17 ± 2.06 g/L and 14.26 ± 1.17 g/L in the fermentation with unwashed sludge and glycerol. The study showed that sludge washing had improved lipid production from sludge.

The pH-based fed-batch for lipid production from Trichosporon oleaginosus with crude glycerol

In this study, it was found that the optimal pH for the growth of Trichosporon oleaginosus was related to the fermentation medium. A neutral or weak acid pH condition was optimal for the growth of Trichosporon oleaginosus in the extract-peptone-dextrose and wastewater sludge medium. Significant inhibition was observed at neutral pH in the wastewater sludge + crude glycerol medium due to the high soap content of the crude glycerol. By converting the soap to free fatty acid (FFA) at pH 5, the soap inhibition could be prevented. Fed-batch fermentation was employed to produce lipid from Trichosporon oleaginosus at pH 5 controlled by feeding crude glycerol. A remarkably high biomass (65.63 g/L) and lipid (35.79 g/L) concentration were achieved from the pH-based fed-batch fermentation in this study.

Utilization of methanol in crude glycerol to assist lipid production in non-sterilized fermentation from Trichosporon oleaginosus

In this work, methanol in crude glycerol solution was used to assist the lipid production with oleaginous yeast Trichosporon oleaginosus cultivated under non-sterilized conditions. The investigated methanol concentration was 0%, 1.4%, 2.2%, 3.3% and 4.4% (w/v). The results showed that methanol played a significant role in the non-sterilized fermentation for lipid production. The optimal methanol concentration was around 1.4% (w/v) in which the growth of T. oleaginosus was promoted and overcame that of the contaminants. The non-sterilized fed-batch fermentation with initial methanol concentration of 1.4% (w/v) was then performed and high biomass production (43.39 g/L) and lipid production (20.42 g/L) were achieved.

Metabolism of aromatics by Trichosporon oleaginosus while remaining oleaginous

BackgroundThe oleaginous yeast, Trichosporon oleaginosus, has been extensively studied for its ability to metabolize non-conventional feedstocks. These include phenol-containing waste streams, such as distillery wastewater, or streams consisting of non-conventional sugars, such as hydrolyzed biomass and various bagasse. An initial BLAST search suggests this yeast has putative aromatic metabolizing genes. Given the desirability to valorize underutilized feedstocks such as lignin, we investigated the ability of T. oleaginosus to tolerate and metabolize lignin-derived aromatic compounds.ResultsTrichosporon oleaginosus can tolerate and metabolize model lignin monoaromatics and associated intermediates within funneling pathways. Growth rates and biomass yield were similar to glucose when grown in 4-hydroxybenzoic acid (pHBA) and resorcinol, but had an increased lag phase when grown in phenol. Oleaginous behavior was observed using resorcinol as a sole carbon source. Fed-batch feeding resulted in lipid accumulation of 69.5% on a dry weight basis.ConclusionsThough the exact pathway of aromatic metabolism remains to be determined for T. oleaginosus, the results presented in this work motivate use of this organism for lignin valorization and phenolic wastewater bioremediation. Trichosporon oleaginosus is the first yeast shown to be oleaginous while growing on aromatic substrates, and shows great promise as a model industrial microbe for biochemical and biofuel production from depolymerized lignin.

Chemical and biological conversion of crude glycerol derived from waste cooking oil to biodiesel

In this study, crude, purified, and pure glycerol were used to cultivate Trichosporon oleaginosus for lipid production which was then used as feedstock of biodiesel production. The purified glycerol was obtained from crude glycerol by removing soap with addition of H3PO4 which converted soap to free fatty acids and then separated from the solution. The results showed that purified glycerol provided similar performance as pure glycerol in lipid accumulation; however, crude glycerol as carbon source had negatively impacted the lipid production of T. oleaginosus. Purified glycerol was later used to determine the optimal glycerol concentration for lipid production. The highest lipid yield 0.19g/g glycerol was obtained at 50g/L purified glycerol in which the biomass concentration and lipid content were 10.75g/L and 47% w/w, respectively. An energy gain of 4150.51MJ could be obtained with 1tonne of the crude glycerol employed for biodiesel production through the process proposed in this study. The biodiesel production cost estimated was 6.32US$/gal. Fatty acid profiles revealed that C16:0 and C18:1 were the major compounds of the biodiesel from the lipid produced by T. oleaginosus cultivated with crude and purified glycerol. The study found that purified glycerol was promising carbon source for biodiesel production.