Role of the mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina.

OBJECTIVES: The transport of citrate from the mitochondria to the cytoplasm is essential during lipid accumulation. This study aimed to explore the role of mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina. RESULTS: Homologous MaYHM (the gene encoding the mitochondrial citrate-oxoglutarate carrier) was overexpressed in M. alpina. The fatty acid content of MaYHM-overexpressing recombinant strains was increased by up to 30% compared with the control. Moreover, the intracellular α-ketoglutarate level in recombinant strains was increased by 2.2 fold, together with a 23-35% decrease in NAD+-isocitrate dehydrogenase activity compared with the control. The overexpression of MaYHM altered the metabolic flux in the glutamate dehydrogenase shunt and 4-aminobutyric acid shunt during metabolic reprogramming, supplying more carbon to synthesize fatty acids. CONCLUSIONS: Overexpression of MaYHM resulted in more efflux of citrate from mitochondria to the cytoplasm and enhanced lipid accumulation. These findings provide new perspectives for the improvement of industrial lipid production in M. alpina.

Production of arachidonic acid by Mortierella alpina using wastes from potato chips industry.

AIMS: This research aimed to determine the potential use of wastes from the potato chips industry as a carbon source to develop an economical culture medium for the production of biomass, lipids and arachidonic acid (ARA) by Mortierella alpina. METHODS AND RESULTS: A synthetic culture medium was optimized using a Plackett-Burman and central composite rotatable design, and used as a base to evaluate and characterize the potential use of wastes from the potato chips industry as carbon sources for the production of biomass, lipids and ARA by M. alpina. The waste was selected among other solid and liquid hydrolysed residues/by-products, and local low-cost alternatives for nitrogen sources were also evaluated. After 6 days of fermentation, the biomass concentration reached 20 g l-1 with 40% of total lipids, and a 35% ARA content in the lipids fraction. Savings in production were calculated using a sensitivity analysis for the alternative culture medium in different scenarios. CONCLUSIONS: This study showed a 7% savings in culture media expenses in the production of ARA-enriched biomass of M. alpina, compared to the conventional synthetic culture medium, when waste from the potato chips industry was used as an alternative source of carbon and macro/microelements, supplemented with a low-cost yeast extract alternative. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstration of the use of potato chips wastes as a low-cost carbon source for the biomass, lipids and ARA production, suggesting an eco-friendly alternative for the use of agri-food wastes for valuable metabolites production.

Adsorption-Degradation of Polycyclic Aromatic Hydrocarbons in Soil by Immobilized Mixed Bacteria and Its Effect on Microbial Communities.

The combined action of biosorption and biodegradation can achieve a remarkable reduction of organic pollutants. In this study, Pseudomonas sp. SDR4 and Mortierella alpina JDR7 were selected as the representative microorganisms to investigate adsorption and degradation of polycyclic aromatic hydrocarbons (PAHs) in soil using immobilization technology and the subsequent change of the microbial community structure. The results showed that the adsorption capacity of immobilized carriers was much higher than that of dead microorganisms and that the addition of dead microorganisms did not affect the adsorption characteristics of immobilized carriers. The chemical reaction was the major factor controlling the adsorption rate of PAHs in sterilized soil (CK), nonsterilized soil (CK-1), and soil amended with dead body immobilized JDR7 and SDR4 mixed bacteria (MB-D). The growth and metabolism of Pseudomonas sp. SDR4 and M. alpina JDR7 are the main reason for enhanced PAH degradation in the soil amended with living body immobilized JDR7, SDR4 mixed bacteria (MB).

Two-stage pH control combined with oxygen-enriched air strategies for the highly efficient production of EPA by Mortierella alpina CCFM698 with fed-batch fermentation.

Dissolved oxygen and pH are critical factors influencing cell growth and metabolism. In our previous work, we constructed the recombinant strain Mortierella alpina CCFM698, which has the ability to produce EPA at room temperature. However, our experiments showed that the dissolved oxygen produced by the aeration and agitation of the fermenter was insufficient for cell growth and EPA synthesis by this recombinant strain. Moreover, the optimum pH for cell growth was incompatible with that of EPA accumulation. This study introduced a combined strategy of two-stage pH control with oxygen-enriched air in fed-batch fermentation to facilitate both cell growth and EPA production in M. alpina CCFM698. After 10 days of fermentation in a 7.5 L tank, the biomass production reached 41.2 g/L, with a lipid content of 31.5%, and EPA accounting for 26.7% of total lipids. The final EPA production reached 3.47 g/L, which is the highest yet achieved by M. alpina. This study reveals the critical role of dissolved oxygen and pH control for EPA production of M. alpina, and provides an easy and efficient strategy for industrial production of EPA.

Application of the cbh1 promoter in Mortierella alpina and optimization of induction conditions.

Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.

Geographic Object-Based Analysis of Airborne Multispectral Images for Health Assessment of Capsicum annuum L. Crops.

Vegetation health assessment by using airborne multispectral images throughout crop production cycles, among other precision agriculture technologies, is an important tool for modern agriculture practices. However, to really take advantage of crop fields imagery, specialized analysis techniques are needed. In this paper we present a geographic object-based image analysis (GEOBIA) approach to examine a set of very high resolution (VHR) multispectral images obtained by the use of small unmanned aerial vehicles (UAVs), to evaluate plant health states and to generate cropland maps for Capsicum annuum L. The scheme described here integrates machine learning methods with semi-automated training and validation, which allowed us to develop an algorithmic sequence for the evaluation of plant health conditions at individual sowing point clusters over an entire parcel. The features selected at the classification stages are based on phenotypic traits of plants with different health levels. Determination of areas without data dependencies for the algorithms employed allowed us to execute some of the calculations as parallel processes. Comparison with the standard normalized difference vegetation index (NDVI) and biological analyses were also performed. The classification obtained showed a precision level of about 95 % in discerning between vegetation and non-vegetation objects, and clustering efficiency ranging from 79 % to 89 % for the evaluation of different vegetation health categories, which makes our approach suitable for being incorporated at C. annuum crop's production systems, as well as to other similar crops. This methodology can be reproduced and adjusted as an on-the-go solution to get a georeferenced plant health estimation.

Role of Adenosine Monophosphate Deaminase during Fatty Acid Accumulation in Oleaginous Fungus Mortierella alpina.

In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.

Algal-fungal symbiosis leads to photosynthetic mycelium.

Mutualistic interactions between free-living algae and fungi are widespread in nature and are hypothesized to have facilitated the evolution of land plants and lichens. In all known algal-fungal mutualisms, including lichens, algal cells remain external to fungal cells. Here, we report on an algal-fungal interaction in which Nannochloropsis oceanica algal cells become internalized within the hyphae of the fungus Mortierella elongata. This apparent symbiosis begins with close physical contact and nutrient exchange, including carbon and nitrogen transfer between fungal and algal cells as demonstrated by isotope tracer experiments. This mutualism appears to be stable, as both partners remain physiologically active over months of co-cultivation, leading to the eventual internalization of photosynthetic algal cells, which persist to function, grow and divide within fungal hyphae. Nannochloropsis and Mortierella are biotechnologically important species for lipids and biofuel production, with available genomes and molecular tool kits. Based on the current observations, they provide unique opportunities for studying fungal-algal mutualisms including mechanisms leading to endosymbiosis.

Macro-morphological characterization and kinetics of Mortierella alpina colonies during batch cultivation.

An effective method for research of macro-morphological characterization and its kinetics was developed by studying the macro-morphological characteristics of Mortierella alpina, an oleaginous zygomycete widely used to produce lipids rich in PUFA, in function of culture medium composition and to link morphological features of fungus with the level of lipid production. A number of distinct morphological forms including hollow pellets, fluffy pellets and freely dispersed mycelia were obtained by changing the fermentation factors. By fitting a Logistic curve, the maximum specific growth rate (µmax)was obtained, which determined the final mycelia morphology. µmax of 0.6584 in three kind of morphological forms is the more appropriate. According to the Luedeking-Piret equation fitting, α≠0 and ß≠0, lipid production was partially associated with the hyphal growth, fluffy pellets which turn glucose into lipidwas more effective than the other two kinds of morphological forms.

Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms.

Recent developments in molecular biology and metabolic engineering have resulted in a large increase in the number of strains that need to be tested, positioning high-throughput screening of microorganisms as an important step in bioprocess development. Scalability is crucial for performing reliable screening of microorganisms. Most of the scalability studies from microplate screening systems to controlled stirred-tank bioreactors have been performed so far with unicellular microorganisms. We have compared cultivation of industrially relevant oleaginous filamentous fungi and microalga in a Duetz-microtiter plate system to benchtop and pre-pilot bioreactors. Maximal glucose consumption rate, biomass concentration, lipid content of the biomass, biomass, and lipid yield values showed good scalability for Mucor circinelloides (less than 20% differences) and Mortierella alpina (less than 30% differences) filamentous fungi. Maximal glucose consumption and biomass production rates were identical for Crypthecodinium cohnii in microtiter plate and benchtop bioreactor. Most likely due to shear stress sensitivity of this microalga in stirred bioreactor, biomass concentration and lipid content of biomass were significantly higher in the microtiter plate system than in the benchtop bioreactor. Still, fermentation results obtained in the Duetz-microtiter plate system for Crypthecodinium cohnii are encouraging compared to what has been reported in literature. Good reproducibility (coefficient of variation less than 15% for biomass growth, glucose consumption, lipid content, and pH) were achieved in the Duetz-microtiter plate system for Mucor circinelloides and Crypthecodinium cohnii. Mortierella alpina cultivation reproducibility might be improved with inoculation optimization. In conclusion, we have presented suitability of the Duetz-microtiter plate system for the reproducible, scalable, and cost-efficient high-throughput screening of oleaginous microorganisms.

Lipid production and characterization by Mortierella (Umbelopsis) isabellina cultivated on lignocellulosic sugars.

AIMS: To study and characterize the lipids produced by Mortierella (Umbelopsis) isabellina, during its growth on mixtures of glucose and xylose. METHODS AND RESULTS: Glucose and xylose were utilized as carbon sources, solely or in blends, under nitrogen-limited conditions, in batch-flask trials (initial sugars at 80 g l-1 ). Significant lipid production (maximum lipid 17·8 g l-1 ; lipid in DCW 61·0% w/w; lipid on glucose consumed 0·23 g g-1 ) occurred on glucose employed solely, while xylose concentration in the growth medium was conversely correlated with lipid accumulation. With increasing xylose concentrations into the blend, lipid storage decreased while xylitol in significant concentrations (up to 24 g l-1 ) was produced. Irrespective of the sugar blend employed, significant quantities of endopolysaccharides were detected in the first growth steps (in the presence of nitrogen into the medium or barely after its disappearance) while lipids were stored thereafter. Neutral lipids, mainly composed of triacylglycerols, were the main microbial lipid fraction. Phospholipids were quantified both through fractionation and subsequent gravimetric determination and also through determination of phosphorus, and it seemed that the second method was more accurate. Phospholipids were mainly composed of phosphatidylcholine and another nonidentified compound presumably being phosphatidyldimethylethanolamine. CONCLUSIONS: Mortierella isabellina is suitable to convert lignocellulosic sugars into lipids. SIGNIFICANCE AND IMPACT OF THE STUDY: Differentiations between metabolism on xylose and glucose were reported. Moreover, this is one of the first reports indicating extensive analysis of microbial lipids produced by M. isabellina.

Expression of Vitreoscilla hemoglobin enhances production of arachidonic acid and lipids in Mortierella alpina.

BACKGROUND: Arachidonic acid (ARA, C20:4, n-6), which belongs to the omega-6 series of polyunsaturated fatty acids and has a variety of biological activities, is commercially produced in Mortierella alpina. Dissolved oxygen or oxygen utilization efficiency is a critical factor for Mortierella alpina growth and arachidonic acid production in large-scale fermentation. Overexpression of the Vitreoscilla hemoglobin gene is thought to significantly increase the oxygen utilization efficiency of the cells. RESULTS: An optimized Vitreoscilla hemoglobin (VHb) gene was introduced into Mortierella alpina via Agrobacterium tumefaciens-mediated transformation. Compared with the parent strain, the VHb-expressing strain, termed VHb-20, grew faster under both limiting and non-limiting oxygen conditions and exhibited dramatic changes in cell morphology. Furthermore, VHb-20 produced 4- and 8-fold higher total lipid and ARA yields than those of the wild-type strain under a microaerobic environment. Furthermore, ARA production of VHb-20 was also 1.6-fold higher than that of the wild type under normal conditions. The results demonstrated that DO utilization was significantly increased by expressing the VHb gene in Mortierella alpina. CONCLUSIONS: The expression of VHb enhances ARA and lipid production under both lower and normal dissolved oxygen conditions. This study provides a novel strategy and an engineered strain for the cost-efficient production of ARA.

The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis.

Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process.

Experimental Climate Change Modifies Degradative Succession in Boreal Peatland Fungal Communities.

Peatlands play an important role in global climate change through sequestration of atmospheric CO2. Climate-driven changes in the structure of fungal communities in boreal peatlands that favor saprotrophic fungi can substantially impact carbon dynamics and nutrient cycling in these crucial ecosystems. In a mesocosm study using a full factorial design, 100 intact peat monoliths, complete with living Sphagnum and above-ground vascular vegetation, were subjected to three climate change variables (increased temperature, reduced water table, and elevated CO2 concentrations). Peat litterbags were placed in mesocosms, and fungal communities in litterbags were monitored over 12 months to assess the impacts of climate change variables on peat-inhabiting fungi. Changes in fungal richness, diversity, and community composition were assessed using Illumina MiSeq sequencing of ribosomal DNA (rDNA). While general fungal richness reduced under warming conditions, Ascomycota exhibited higher diversity under increased temperature treatments over the course of the experiment. Both increased temperature and lowered water table position drove shifts in fungal community composition with a strong positive effect on endophytic and mycorrhizal fungi (including one operational taxonomic unit (OTU) tentatively identified as Barrenia panicia) and different groups of saprotrophs identified as Mortierella, Galerina, and Mycena. These shifts were observed during a predicted degradative succession in the decomposer community as different carbon substrates became available. Since fungi play a central role in peatland communities, increased abundances of saprotrophic fungi under warming conditions, at the expense of reduced fungal richness overall, may increase decomposition rates under future climate scenarios and could potentially aggravate the impacts of climate change.

Proteomic analysis of Mortierella isabellina M6-22 during cold stress.

We aimed to gain a better understanding of cold adaption in Mortierella isabellina M6-22 by using proteomics approaches. The temperature range and optimal temperature for M6-22 growth were investigated, and composition changes in fatty acids were analyzed. Accompanied with the 2-D gel electrophoresis, MALDI-TOF/TOF-MS analysis was conducted to characterize alterations in protein profiling in M6-22 cultured at 30 °C for 24 h and 15 °C for another 24 h when compared with those cultured at 30 °C for 48 h. Gene Ontology (GO) cluster analysis was finally conducted for successfully identified proteins. M6-22 cells could grow well at temperatures ranging from 15 to 30 °C. As temperature decreased from 30 to 15 °C, LA and GLA significantly increased from 11.63 to 17.85 % and from 9.12 to 13.19 %, respectively, while oleic acid significantly decreased from 47.25 to 36.53 %. Proteomics analyses revealed 111 differentially expressed protein spots, among which 5 unique proteins (A38, A40, A47, A49 and A58), 29 up-regulated proteins and 10 down-regulated proteins were identified by MALDI-TOF/TOF-MS. GO enrichment analysis demonstrated that these proteins mainly involved in glycolytic pathway (A34 and A50), electron transport (A28), ATP production (A35 and B39) and protein modification (A38). A total of 44 differentially expressed proteins have been successfully identified in M. isabellina M6-22 cultured at 15 °C. These proteins may play important roles in cold adaption via regulation of ATP synthesis, activation of cold-adaptive proteins, degradation of needless protein, accumulation of PUFAs, etc.

Lipid Fraction and Intracellular Metabolite Analysis Reveal the Mechanism of Arachidonic Acid-Rich Oil Accumulation in the Aging Process of Mortierella alpina.

The mechanism of arachidonic acid (ARA) content increase during aging of Mortierella alpina was elucidated. Lipid fraction analysis showed that ARA content increased from 46.9% to 66.4% in the triacylglycerol (TAG) molecule, and ARA residue occupation increased in the majority of TAG molecules during the aging process. For the first time, intracellular metabolite analysis was conducted to reveal the pathways closely associated with ARA biosynthesis during aging. The main reason for the increased ARA content was not only at the expense of other fatty acids degradation but also at the expense of further ARA biosynthesis during aging. Furthermore, translocation played a vital role in ARA redistribution among the glycerol moiety, and mycelium did not die immediately with key pathways activated to maintain a relatively stable intracellular environment. This study lays a foundation for further improvement of ARA content in the oil product obtained from M. alpina.

Efficient production of arachidonic acid by Mortierella alpina through integrating fed-batch culture with a two-stage pH control strategy.

Arachidonic acid (ARA) yield and productivity of Mortierella alpina mutant D20 were enhanced by integrating a fed-batch culture combined with a two-stage pH control strategy. Following a kinetic analysis of the whole fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 5.5 for the first 48 h and then shifted to 6.5 till the end of fermentation. Using this strategy, a maximum ARA production of 8.12 g/L was achieved. On the basis of pH control, the effects of fed-batch cultures on ARA productivity were further investigated. A maximum ARA productivity of 1.40 g/L/d was obtained with a two-stage constant-speed glucose feeding strategy, starting with a glucose concentration of 50 g/L. This strategy was simple and economical to operate, and it may be possible to apply this approach for large-scale industrial production of ARA.

Effects of inhibitory compounds in lignocellulosic hydrolysates on Mortierella isabellina growth and carbon utilization.

Oleaginous fungus Mortierella isabellina showed excellent lipid conversion on non-detoxified lignocellulosic hydrolysate. This study investigated the effects of inhibitory compounds (furfural, hydroxymethylfurfural, and ferulic and coumaric acids) in lignocellulosic hydrolysate on M. isabellina growth and lipid production. M. isabellina can tolerate furfural (∼1 g/L), hydroxymethylfurfural (∼2.5 g/L), ferulic (∼0.5 g/L) and coumaric acid (∼0.5 g/L) with normal growth rates. Synergistic effect of these inhibitors (2 g/L furfural, 0.4 g/L hydroxymethylfurfural, 0.02 g/L ferulic acid and 0.02 g/L coumaric acid) moderately reduces total fungal growth (by 28%), while the presence of these inhibitors has minor impact on cell lipid contents and lipid profiles. In the presence of inhibitory compounds, (13)C-tracing has revealed that M. isabellina can simultaneously utilize glucose and acetate, and acetate is mainly assimilated for synthesis of lipid and TCA cycle amino acids. The results also demonstrate that glucose has strong catabolite repression for xylose utilization for biomass and lipid production in the presence of inhibitors.

Arachidonic acid production by Mortierella alpina using raw crop materials.

BACKGROUND: Arachidonic acid (ARA) is one of the three essential fatty acids, and it is important for human body to keep healthy and is widely used. At present, expensive materials such as glucose and yeast extract are generally reported to be optimal for ARA production. A new cost-effective fermentation process including cheaper material for ARA production is of great significance. METHODS: Feasibility of using corn meal and powdered soybean for fungal growth and lipid accumulation was evaluated by means of single factor test. N-hexadecane concentration was optimized, and the effect of temperature on biomass and ARA content was examined. RESULTS: Mortierella alpina made better use of the aforementioned material as carbon and nitrogen sources for both hyphae growth and ARA production compared with glucose and yeast extract. Maximal levels of 10.9 g/L ARA and 26.1 g/L total lipids were obtained when 66 g/L corn meal, 54 g/L soybean meal and 6% (v/v) n-hexadecane were supplemented. A temperature-shift strategy involved three steps, namely, 30°C (3 days) - 25°C (4 days) - 20°C (4 days), which further improved ARA production by 24.7%. CONCLUSIONS: Several factors such as carbon and nitrogen sources, temperature and dissolved oxygen had great influence on biomass and microbial oil production. Mortierella alpina preferred corn and soybean meal compared with glucose and yeast extract, which would surely alleviate the high cost of ARA production. Based on this study, the new process is both low cost and practicable.

Microbial lipid production from corn stover via Mortierella isabellina.

Microbial lipid is a promising source of oil to produce biofuel if it can be generated from lignocellulosic materials. Mortierella isabellina is a filamentous fungal species featuring high content of oil in its cell biomass. In this work, M. isabellina was studied for lipid production from corn stover. The experimental results showed that M. isabellina could grow on different kinds of carbon sources including xylose and acetate, and the lipid content reached to 35 % at C/N ratio of 20. With dilution, M. isabellina could endure inhibition effects by dilute acid pretreatment of corn stover (0.3 g/L furfural, 1.2 g/L HMF, and 1 g/L 4-hydroxybenozic acid) and the strain formed pellets in the cell cultivations. An integrated process was developed combining the dilute acid pretreatment, cellulase hydrolysis, and cell cultivation for M. isabellina to convert corn stover to oil containing fungal biomass. With 7.5 % pretreated biomass solid loading ratio, the final lipid yield from sugar in pretreated biomass was 40 % and the final lipid concentration of the culture reached to 6.46 g/L.