Microbial Oil Production Research Articles

Oil-lipids, carotenoids and fatty acids simultaneous production by Rhodotorula mucilaginosa CCT3892 using sugarcane molasses as carbon source

Abstract One of the most important classes of nutritional biomolecules is the oleaginous compounds group, which specially includes the oil-lipids, the carotenoids and the fatty acids. These biocompounds present a wide range of industrial applications because their ability to act as an energy source, antioxidants and metabolic agents for the human body. Therefore, the food industry, mainly focusing on food supplementation, is always searching for new sources of them. In this context, the present study evaluated the total lipids, carotenoids and fatty acids simultaneous production by the Rhodotorula mucilaginosa CCT3892 yeast, using residual sugarcane molasses as carbon source. The results obtained demonstrated that the cultivation of yeast in molasses medium (MC) produced the same content of total lipids and carotenoids (16.50% ± 0.68% and 0.053 ± 0.001 mg g-1, respectively) as the obtained from a synthetic medium (SC) (15.36% ± 1.36% and 0.051 ± 0.001 mg g-1 0.005). Concerning the fatty acids biosynthesis, the MC cultivation generated the most interesting profile once it presented a greater content of oleic acid (74.05%), an unsaturated compound with high nutritional value. The cultivation carried out with the molasses and yeast extract supplementation (MYEC) did not provide an improvement in microbial oil production, what indicated that in this condition there was a predominance of others sorts of substrate metabolization by the yeast cells, as confirmed by the microbial kinetics study.

Oil reservoir simulating bioreactors: tools for understanding petroleum microbiology.

Various aspects of the oil fields in terms of microbial activity (souring, biocorrosion, etc.) and oil production (polymer flooding, etc.) have been evaluated through a variety of experiments. The primary step to study these properties in the laboratory requires the construction and operation of up-flow oil reservoir simulating bioreactors (ORSBs) in real time. Souring by reduction of sulfate to sulfide is a major contributor in enhancing corrosion of metal infrastructure used for oil production and processing. Whether the injection of biocides prevents or remediates reservoir souring can be addressed by flooding up-flow ORSBs. The potential of biopolymers/biosurfactants produced by different microbial strains have also been investigated for the role in maintaining additional oil recovery using ORSB. Additionally, key issues of polymer behavior during flooding of reservoirs could be understood during laboratory studies by monitoring the in situ porous medium rheology. Besides, the change in various ORSB parameters helps in adjudging the effect of different biosurfactants/biopolymers in enhancing oil recovery. Parameters such as permeability reduction, adsorption, interaction with porous matrix, and formation damage can be evaluated using ORSB. The analysis of earlier studies indicated that running bioreactors for longer duration of time can help in drawing conclusion with sharpness and less ambiguity. The current review discusses the construction and application of various types of ORSBs including the experimental studies employing ORSBs.

Techno-economic analysis and life cycle assessment of heterotrophic yeast-derived single cell oil production process

The widespread application and utilization of vegetable oils and fats has led to a significant increase of their annual production. Fats and oils are mainly consumed as food and as animal feed, as raw material in the chemical industry and more recently as raw material for biofuel production. In this work the economics of the biochemical production of microbial oil is investigated and the life cycle assessment is performed for first time in the open literature. The production process is simulated using commercial simulators to perform accurate material and energy balances and all necessary data are collected from the open literature. Following the process simulation step a detailed technoeconomic analysis is performed and the fixed capital investment is estimated for a production that varies from 2 to 40 kt of microbial oil per year. Having completed the technoeconomic analysis the Life Cycle Inventory Analysis is performed, and the environmental impacts are estimated, using the comprehensive Life Cycle Assessment methodology.

Waste fish oil as an alternative carbon source in microbial oil production by Yarrowia lipolytica yeast

Waste fish oil as an alternative carbon source in microbial oil production by Yarrowia lipolytica yeast

Volatile fatty acids as novel building blocks for oil-based chemistry via oleaginous yeastfermentation.

Microbial oils are proposed as a suitable alternative to petroleum-based chemistry in terms of environmental preservation. These oils have traditionally been studied using sugar-based feedstock, which implies high costs, substrate limitation, and high contamination risks. In this sense, low-cost carbon sources such as volatile fatty acids (VFAs) are envisaged as promising building blocks for lipid biosynthesis to produce oil-based bioproducts. VFAs can be generated from a wide variety of organic wastes through anaerobic digestion and further converted into lipids by oleaginous yeasts (OYs) in a fermentation process. These microorganisms can accumulatein the form of lipid bodies, lipids of up to 60% wt/wt of their biomass. In this context, OY isa promising biotechnological tool for biofueland bioproductgeneration using low-cost VFAmedia as substrates. This review covers recent advances in microbial oil production from VFAs. Production of VFAs via anaerobic digestion processes and the involved metabolic pathways are reviewed. The main challenges as well as recent approaches for lipid overproduction are also discussed.

Simultaneous lipid biosynthesis and recovery for oleaginous yeast Yarrowia lipolytica

BackgroundRecent trends in bioprocessing have underlined the significance of lignocellulosic biomass conversions for biofuel production. These conversions demand at least 90% energy upgradation of cellulosic sugars to generate renewable drop-in biofuel precursors (Heff/C ~ 2). Chemical methods fail to achieve this without substantial loss of carbon; whereas, oleaginous biological systems propose a greener upgradation route by producing oil from sugars with 30% theoretical yields. However, these oleaginous systems cannot compete with the commercial volumes of vegetable oils in terms of overall oil yields and productivities. One of the significant challenges in the commercial exploitation of these microbial oils lies in the inefficient recovery of the produced oil. This issue has been addressed using highly selective oil capturing agents (OCA), which allow a concomitant microbial oil production and in situ oil recovery process.ResultsAdsorbent-based oil capturing agents were employed for simultaneous in situ oil recovery in the fermentative production broths. Yarrowia lipolytica, a model oleaginous yeast, was milked incessantly for oil production over 380 h in a media comprising of glucose as a sole carbon and nutrient source. This was achieved by continuous online capture of extracellular oil from the aqueous media and also the cell surface, by fluidizing the fermentation broth over an adsorbent bed of oil capturing agents (OCA). A consistent oil yield of 0.33 g per g of glucose consumed, corresponding to theoretical oil yield over glucose, was achieved using this approach. While the incorporation of the OCA increased the oil content up to 89% with complete substrate consumptions, it also caused an overall process integration.ConclusionThe nondisruptive oil capture mediated by an OCA helped in accomplishing a trade-off between microbial oil production and its recovery. This strategy helped in realizing theoretically efficient sugar-to-oil bioconversions in a continuous production process. The process, therefore, endorses a sustainable production of molecular drop-in equivalents through oleaginous yeasts, representing as an absolute microbial oil factory.

Bioprocess development for the production of novel oleogels from soybean and microbial oils.

This study presents the production of novel oleogels via circular valorisation of food industry side streams. Sugarcane molasses and soybean processing side streams (i.e. soybean cake) were employed as fermentation feedstocks for the production of microbial oil. Fed-batch bioreactor fermentations carried out by the oleaginous yeast Rhodosporidium toruloides led to the production of 36.9 g/L total dry weight with an intracellular oil content of 49.8% (w/w) and 89.4 μg/g carotenoids. The carotenoid-rich microbial oil and soybean oil were evaluated as base oils for the production of wax-based oleogels. The wax esters, used as oleogelators, were produced via enzymatic catalysis, using microbial oil or soybean fatty acid distillate as raw materials. All oleogels presented a gel-like behaviour (G' > G″). However, the highest G' was determined for the oleogel produced from soybean oil and microbial oil-wax esters, which indicated a stronger network. Thermal analysis showed that this oleogel had a melting temperature profile up to 35 °C, which is favorable for applications in the confectionery industry. Also, texture analysis demonstrated that soybean oil-microbial oil wax oleogel was stable (1.9-2.2 N) within 30-days storage period. This study showed the potential of novel oleogels production through the development of bioprocesses based on the valorisation of various renewable resources.

Cultivation modes for microbial oil production using oleaginous yeasts – A review

Microbial oil has been a valuable alternative oil source with applications in the food, cosmetics and biofuels sectors. As concerns for the environment grow, microbially-synthesized oil has emerged as a potential contributor for sustainable production of biodiesel. However, the high costs of its production hinder its large-scale application. Main bottleneck to industrial lipid production is the cost of the fermentation stage. Therefore, it is imperative to design cultivations with little operating requirements and high yields, adjusted to an oleaginous system. This paper provides an overview of the latest advances in oleaginous yeast cultivation in bioreactors. Focus is given to oleaginous yeasts and de novo lipid accumulation due to their high lipid accumulating ability, robustness and versatility across a range of substrates. The advantages and disadvantages of different cultivation modes, feeding patterns and impact on biomass and lipid yield are critically reviewed. The role of biochemical engineering in facilitating understanding of the lipid accumulation kinetics, lipid productivity and its importance as predictive scale up tool is highlighted through a review of existing equations. Perspectives for further bioprocess improvement through kinetic modelling, and valorisation through utilisation of by-products are also discussed.

Development of a Circular Oriented Bioprocess for Microbial Oil Production Using Diversified Mixed Confectionery Side-Streams.

Diversified mixed confectionery waste streams were utilized in a two-stage bioprocess to formulate a nutrient-rich fermentation media for microbial oil production. Solid-state fermentation was conducted for the production of crude enzyme consortia to be subsequently applied in hydrolytic reactions to break down starch, disaccharides, and proteins into monosaccharides, amino acids, and peptides. Crude hydrolysates were evaluated in bioconversion processes using the red yeast Rhodosporidium toruloides DSM 4444 both in batch and fed-batch mode. Under nitrogen-limiting conditions, during fed-batch cultures, the concentration of microbial lipids reached 16.6–17 g·L−1 with the intracellular content being more than 40% (w/w) in both hydrolysates applied. R. toruloides was able to metabolize mixed carbon sources without catabolite repression. The fatty acid profile of the produced lipids was altered based on the substrate employed in the bioconversion process. Microbial lipids were rich in polyunsaturated fatty acids, with oleic acid being the major fatty acid (61.7%, w/w). This study showed that mixed food side-streams could be valorized for the production of microbial oil with high unsaturation degree, pointing towards the potential to produce tailor-made lipids for specific food applications. Likewise, the proposed process conforms unequivocally to the principles of the circular economy, as the entire quantity of confectionery by-products are implemented to generate added-value compounds that will find applications in the same original industry, thus closing the loop.

Biovalorization of vegetable oil refinery wastewater into value‐added compounds by Yarrowia lipolytica

AbstractBACKGROUNDVegetable oil refinery wastewater (VORW) is an environmental pollutant in less developed countries which annually produce 75% of vegetable oils worldwide. Microbial oils from low‐cost substrates are of growing importance for the production of renewable biofuel feedstocks, and food and feed supplements. In the current study, VORW was used as a cheap substrate to produce added‐value compounds including lipase, microbial oil and lipid‐rich biomass using Yarrowia lipolytica. The chemical oxygen demand (COD) reduction of the wastewater was studied after fermentation.RESULTSThe maximum microbial oil concentration and lipase activity were 2.68 g L−1 and 8 U mL−1 after 48 h of inoculation. The microbial oil production increased from 2.86 g L−1 to 7.74 g L−1 after optimization by response surface methodology. Using optimized medium, 11 g L−1 microbial oil and 18.3 g L−1 biomass were obtained in the bioreactor culture. The amount of unsaturated fatty acids (FAs) reached ≤63% in the resulting microbial oil with a suitable FA profile for the production of lipid‐rich biomass. The biomass of Y. lipolytica has 60% lipid content and a significant amount of essential FAs which makes it as a proper feed supplement. The COD level of the wastewater decreased by 80% after 20 h.CONCLUSIONVORW can be used as a promising substrate for the sustainable production of microbial oil and lipid‐rich biomass with useful FAs by Y. lipolytica. This process also can simultaneously reduce environmental pollution of wastewater in a safe and eco‐friendly way. © 2019 Society of Chemical Industry

Screening of Yeast Strains in producing biodiesel

Microbial oil production has many advantages such as rapid proliferation of microbial cells and short production cycle. The raw materials needed for growth are abundant and inexpensive such as starch, sugar, especially the waste which can be utilized in food or paper industry. 170 oil-producing strains were screened by Sudan black staining observation using fat grains as screening index, among which, 6 high lipid yield yeast strains were identified by residual sugar as indirect index. By comparing the oil-producing ability of this strain, it was determined that the first strain was a good strain for oil production by fermentation. The biomass, oil content and oil yield of the strain were 11.58 g/L, 36.68%, 4.25 g/L, respectively. Through analysis on 26S rDNA sequence and observation on morphological characteristics, culture characteristics and physiological and biochemical characteristics, this strain belonged to Cryptococcus spp.

Comparison of two microbial oil extraction technologies

Microbial oil production has many advantages such as rapid proliferation of microbial cells and short production cycle. The raw materials needed for growth are abundant and inexpensive such as starch, sugar, especially the waste which can be utilized in food or paper industry. 170 oil-producing strains were screened by Sudan black staining observation using fat grains as screening index, among which, 6 high lipid yield yeast strains were identified by residual sugar as indirect index. By comparing the oil-producing ability of this strain, it was determined that the first strain was a good strain for oil production by fermentation. The biomass, oil content and oil yield of the strain were 11.58 g/L, 36.68%, 4.25 g/L, respectively. Through analysis on 26S rDNA sequence and observation on morphological characteristics, culture characteristics and physiological and biochemical characteristics, this strain belonged to Cryptococcus spp.

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.

Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars

BackgroundIn the context of sustainable development, yeast are one class of microorganisms foreseen for the production of oil from diverse renewable feedstocks, in particular those that do not compete with the food supply. However, their use in bulk production, such as for the production of biodiesel, is still not cost effective, partly due to the possible poor use of desired substrates or poor robustness in the practical bioconversion process. We investigated the natural capacity of Blastobotrys adeninivorans, a yeast already used in biotechnology, to store lipids under different conditions.ResultsThe genotyping of seven strains showed the species to actually be composed of two different groups, one that (including the well-known strain LS3) could be reassigned to Blastobotrys raffinosifermentans. We showed that, under nitrogen limitation, strains of both species can synthesize lipids to over 20% of their dry-cell weight during shake-flask cultivation in glucose or xylose medium for 96 h. In addition, organic acids were excreted into the medium. LS3, our best lipid-producing strain, could also accumulate lipids from exogenous oleic acid, up to 38.1 ± 1.6% of its dry-cell weight, and synthesize lipids from various sugar substrates, up to 36.6 ± 0.5% when growing in cellobiose. Both species, represented by LS3 and CBS 8244T, could grow with little filamentation in the lipogenic medium from 28 to 45 °C and reached lipid titers ranging from 1.76 ± 0.28 to 3.08 ± 0.49 g/L in flasks. Under these conditions, the maximum bioconversion yield (YFA/S = 0.093 ± 0.017) was obtained with LS3 at 37 °C. The presence of genes for predicted subunits of an ATP citrate lyase in the genome of LS3 reinforces its oleaginous character.ConclusionsBlastobotrys adeninivorans and B. raffinosifermentans, which are known to be xerotolerant and genetically-tractable, are promising biotechnological yeasts of the Saccharomycotina that could be further developed through genetic engineering for the production of microbial oil. To our knowledge, this is the first report of efficient lipid storage in yeast when cultivated at a temperature above 40 °C. This paves the way to help reducing costs through consolidated bioprocessing.

New Method for the Extraction of Single-Cell Oils from Wet Oleaginous Microbial Biomass: Efficiency, Oil Characterisation and Energy Assessment

Culture medium composition and an efficient lipid extraction process have the largest impact on the cost of industrial production of microbial single-cell oil (SCO). In this study, SCO from the yeast Rhodosporidium thoruloides grown in sugarcane medium was successfully produced in a 1 m3 bioreactor. Biomass was concentrated by sedimentation and the acid-hydrolysed cells were extracted using a recurrent extraction (RE) and a newly-proposed wet extraction (WE) process. RE consisted of Soxhlet extraction of dry hydrolysed biomass with different solvents: hexane, chloroform and chloroform/methanol (2:1). The WE process consisted of the extraction of wet hydrolysed biomass with the same solvents. The highest lipid yields (m/m) were achieved by hexane in RE and chloroform in WE. The chloroform WE extract presented similar density, saponification number, iodine value, moisture and acid value as that from RE with hexane, but much lower peroxide content. The fatty acid methyl ester compositions of WE and RE extracts were also very similar. An energy–cost analysis showed that WE used only 13% of the energy used in RE. Moreover, an energy conservation analysis showed that approx. 65% of the energy from molasses was effectively transformed into microbial lipids. The low energy requirement, the high efficiency and the characteristics of the resulting bio-oil achieved by WE evidence the potential of the proposed methodology for use on an industrial scale.

Bioconversion of Poplar Wood Hemicellulose Prehydrolysate to Microbial Oil Using Cryptococcus curvatus.

Poplar wood hemicellulose prehydrolysate was used for microbial oil production using an oleaginous microorganism Cryptococcus curvatus. Initially, the effect of substrate concentration and nitrogen content was investigated on synthetic media. Then poplar wood prehydrolysate without detoxification was used as substrate in the fermentation. The result showed that this strain is capable of consuming both hexose and pentose sugars, a challenge in fermentation of hemicellulosic streams. It was able to accumulate 36.98% of lipid and the fermentation resulted in 13.78g/L of biomass and 5.13g/L of lipid under optimum conditions after 164h of fermentation. The lipid product obtained was characterized in terms of their fatty acid profiles. Overall, this study shows that it is possible to produce microbial oil from a sustainable renewable feedstock like poplar wood hemicellulose prehydrolysate. This robust strain used has the ability to grow on industrially produced hemicellulose which can help in the development of an integrated biorefinery, where all the three components of lignocellulosic biomass are utilized.

Valorisation of sugarcane molasses for the production of microbial lipids via fermentation of two Rhodosporidium strains for enzymatic synthesis of polyol esters

AbstractBACKGROUNDBioprocess development for microbial oil production using cane sugar and sugarcane molasses via fermentation followed by enzymatic conversion into fatty acid esters is presented in this study.RESULTSMicrobial oil was initially evaluated in shake flask fermentations of Rhodosporidium toruloides NRRL Y‐27012 and R. kratochvilovae Y‐43 using either very high polarity (VHP) cane sugar or sugarcane molasses. Supplementation of phosphate salts, trace elements and nitrogen sources in molasses‐based cultures influenced yeast growth and microbial oil production. R. kratochvilovae had different nutrient requirements than R. toruloides. Fed‐batch bioreactor cultures of R. toruloides in molasses‐based cultures without any phosphate salts and trace element supplementation led to a microbial oil concentration of 25 g L−1 and an intracellular lipid content of 61% (w/w). Nutrient supplementation influenced the fatty acid composition of microbial oils indicating that enrichment in oleic acid could be achieved (up to 65.1%). The microbial oil produced by R. toruloides was enzymatically converted into polyol esters with a conversion yield of more than 85% (w/w).CONCLUSIONThis study demonstrates that Rhodosporidium sp. can be cultivated in molasses for the production of oleic acid enriched microbial lipids by adjusting medium composition. The polyol esters derived from microbial lipids could be used as a renewable feedstock for oleochemical production. © 2019 Society of Chemical Industry

Co-utilization of acidified glycerol pretreated-sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain.

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co-utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6g/L glucose from glycerol-pretreated bagasse and 23.4g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5mg/L carotenoid as a by-product. It is expected that microbial oil production can be significantly improved through process optimization.

Intracellular expression of Vitreoscilla haemoglobin improves lipid production in Yarrowia lipolytica.

Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in oleaginous yeast, Yarrowia lipolytica, and its effect on lipid accumulation was analysed. During fermentation, the dissolved oxygen (DO) concentration was controlled at 5, 10, 20 and 30 of full O2 saturation and also uncontrolled by varying the stirring speed and aeration rate. Yarrowia lipolytica harbouring the VHb gene (VHb+ strain) displayed more pseudomycelium than the control strain (VHb- strain), and VHb expression also enhanced the cell density. When DO level was controlled at 30%, the biomass of VHb+ strains reached about 19gl-1 and increased by 27% compared to VHb- strains. Total fatty acid contents, although, were higher in VHb+ strains than in VHb- strains under all DO levels, and maximally increased by c. 40% (from 10·5 to 14·5% of the biomass) when the DO concentration was controlled at 30%. VHb overexpression, however, markedly suppressed citrate secretion. In addition, expression of VHb also induced significant changes in fatty acid composition and increased the oleic acid content. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in Yarrowia lipolytica, which is a most attractive model for microbial oil production because of its excellent lipid accumulation capacity and convenient genetic tools. Expression of VHb resulted in lipid accumulation increased and cell morphology shift, however, markedly suppressed citrate secretion. This study provided the new strategy for fermentation technology to improve lipid production in oleaginous micro-organisms.

Microbial oil production from acidified glycerol pretreated sugarcane bagasse by Mortierella isabellina.

An integrated microbial oil production process consisting of acidified glycerol pretreatment of sugarcane bagasse, enzymatic hydrolysis, microbial oil production by Mortierella isabellina NRRL 1757 and oil recovery by hydrothermal liquefaction (HTL) of fungal biomass in fermentation broth was assessed in this study. Following pretreatment, the effect of residual pretreatment hydrolysate (containing glycerol) on enzymatic hydrolysis was firstly studied. The residual pretreatment hydrolysate (corresponding to 2.0–7.5% glycerol) improved glucan enzymatic digestibilities by 10–11% compared to the enzymatic hydrolysis in water (no buffer). Although residual pretreatment hydrolysate at 2.0–5.0% glycerol slightly inhibited the consumption of glucose in enzymatic hydrolysate by M. isabellina NRRL 1757, it did not affect microbial oil production due to the consumption of similar amounts of total carbon sources including glycerol. When the cultivation was scaled-up to a 1 L bioreactor, glucose was consumed more rapidly but glycerol assimilation was inhibited. Finally, HTL of fungal biomass in fermentation broth without any catalyst at 340 °C for 60 min efficiently recovered microbial oils from fungal biomass and achieved a bio-oil yield of 78.7% with fatty acids being the dominant oil components (∼89%). HTL also led to the hydrogenation of less saturated fatty acids (C18:2 and C18:3) to more saturated forms (C18:0 and C18:1).