Effect of organic acids on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans
© Huang et al; licensee BioMed Central Ltd. 2012
Received: 24 October 2011
Accepted: 19 January 2012
Published: 19 January 2012
Microbial lipids have drawn increasing attention in recent years as promising raw materials for biodiesel production, and the use of lignocellulosic hydrolysates as carbon sources seems to be a feasible strategy for cost-effective lipid fermentation with oleaginous microorganisms on a large scale. During the hydrolysis of lignocellulosic materials with dilute acid, however, various kinds of inhibitors, especially large amounts of organic acids, will be produced, which substantially decrease the fermentability of lignocellulosic hydrolysates. To overcome the inhibitory effects of organic acids, it is critical to understand their impact on the growth and lipid accumulation of oleaginous microorganisms.
In our present work, we investigated for the first time the effect of ten representative organic acids in lignocellulosic hydrolysates on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans cells. In contrast to previous reports, we found that the toxicity of the organic acids to the cells was not directly related to their hydrophobicity. It is worth noting that most organic acids tested were less toxic than aldehydes to the cells, and some could even stimulate the growth and lipid accumulation at a low concentration. Unlike aldehydes, most binary combinations of organic acids exerted no synergistic inhibitory effects on lipid production. The presence of organic acids decelerated the consumption of glucose, whereas it influenced the utilization of xylose in a different and complicated way. In addition, all the organic acids tested, except furoic acid, inhibited the malic activity of T. fermentans. Furthermore, the inhibition of organic acids on cell growth was dependent more on inoculum size, temperature and initial pH than on lipid content.
This work provides some meaningful information about the effect of organic acid in lignocellulosic hydrolysates on the lipid production of oleaginous yeast, which is helpful for optimization of biomass hydrolysis processes, detoxified pretreatment of hydrolysates and lipid production using lignocellulosic materials.
Keywordslignocellulosic hydrolysate organic acid inhibition lipid production Trichosporon fermentans
Biodiesel, a mixture of long-chain monoalkyl fatty acid esters, has been considered a good alternative to conventional petrodiesel oil because of its green and renewable characteristics . Although it has been used in many countries around the world, the high production cost, of which oil feedstock accounts for about 75%, has become a hurdle, and the sustainable and stable supply of cheap lipids is crucial for their further development and wide application . Nowadays, the most commonly used feedstocks in biodiesel production are vegetable oils and waste oils from restaurants or industry. However, vegetable oils such as rapeseed oil and corn oil contribute to the world's food supply, and thus their use as feedstock for biodiesel production has brought about the food versus biofuel debate . The amount of waste oils is limited and cannot meet the increasing demand for biofuel. Microbial oils, namely, single-cell oils (SCOs), which have long been used as substitutes for high-added-value lipids [4, 5] such as cocoa butter [6, 7], are now believed to be a promising candidate as biodiesel feedstock because of their fatty acid composition, which is similar to those of vegetable oils . At present, however, the high fermentation cost of SCOs limits their use for biodiesel production [7, 9]. The adoption of inexpensive media, such as molasses , industrial glycerol , monosodium glutamate wastewater  and lignocellulosic hydrolysates  for lipid fermentation is one of the possible resolutions of this problem. Recently, the use of lignocellulosic materials for SCO production has attracted increasing attention because these materials are the most abundant and renewable biomass resources in nature [8, 12].
Lignocellulosic biomass consists of cellulose, hemicellulose and lignin, whose relative proportion depends on their material sources . The hydrolysis of lignocellulosic materials into soluble, fermentable sugars is necessary for their efficient utilization by microorganisms. However, a variety of by-products, mainly organic acids, aldehydes and alcohols such as acetic acid, furfural from decomposition of pentoses, 5-hydroxymethylfurfural from degradation of hexoses and aromatics (aromatic alcohols, acids and aldehydes) from lignin, are inevitably generated during hydrolysis with dilute acid . In most cases, these by-products, known as "inhibitors," exert negative effects on the growth, metabolism and product formation of microorganism cells in the fermentation process .
Recently, we reported that despite the oleaginous yeast Trichosporon fermentans's production of a poor lipid yield on nondetoxified, sulfuric acid-treated rice straw hydrolysate (SARSH), it grew well with efficient lipid accumulation on detoxified SARSH , suggesting that the inhibitors in the lignocellulosic hydrolysate do have great effects on lipid fermentation. Among the inhibitors, organic acids are generally the most abundant, and ten kinds of organic acids, including aliphatic acids (acetic acid, formic acid, levulinic acid and caproic acid), aromatic or furan acids (4-hydroxybenzoic acid, syringic acid, vanillic acid, furoic acid, ferulic acid and gallic acid) have been found in lignocellulosic hydrolysate. Little is known about their inhibition on lipid fermentation, however [16, 17]. To provide some interesting information necessary for lipid fermentation on lignocellulosic hydrolysates, we systematically investigated, for the first time, the inhibitory effects of the above-mentioned organic acids on the growth and lipid accumulation of T. fermentans with a mixture of glucose and xylose at a ratio of 2:1 (wt/wt) as the carbon source, owing to its similarity to lignocellulosic hydrolysates.
Results and discussion
Effect of fermentation conditions on the cell growth and lipid accumulation of Trichosporon fermentans on the medium without inhibitor
Lipid yield (g/L)
24.0 ± 0.7
61.7 ± 1.6
14.8 ± 0.5
22.4 ± 1.2
58.6 ± 1.7
13.1 ± 1.0
21.6 ± 1.1
54.3 ± 1.2
11.7 ± 0.9
18.4 ± 0.9
57.2 ± 1.7
10.5 ± 0.8
21.5 ± 0.7
56.3 ± 1.4
12.1 ± 0.7
19.9 ± 0.9
55.8 ± 1.1
11.1 ± 0.7
23.6 ± 1.2
58.9 ± 1.2
13.9 ± 1.0
Effects of organic acids on the growth and lipid accumulation of T. fermentans
Concentration of organic acids required to inhibit the lipid yield of Trichosporon fermentans
Relative lipid content
Relative lipid content (%)
The sugar consumption in the medium containing the selected organic acid was also recorded after fermentation for seven days at which the control without inhibitor gave the maximum lipid yield and the residual sugar was xylose with a concentration of about 15.7 g/L (Figure 3). Interestingly, except for furoic and caproic acids, the relative sugar consumption was above 100% in the presence of a small amount of organic acids. However, improved sugar utilization did not necessarily lead to an enhanced lipid yield. For example, T. fermentans could utilize more sugars than the control in the presence of 5 mM syringic acid, but the corresponding relative lipid yield was only 81.1%. At higher concentrations, however, all the tested organic acids suppressed the sugar utilization, and the higher the concentration, the more pronounced the suppression. Among the organic acids examined, levulinic acid showed the least influence on sugar utilization, which is in accordance with the observation that levulinic acid displayed the lowest toxicity to lipid production of T. fermentans.
In our previous studies [10, 12], we found that when glucose is almost exhausted, cellular lipids can be used as a carbon source to maintain the growth of T. fermentans. In general, microorganisms consume their accumulated lipids mainly through the glyoxylate bypass pathway, and, more specifically, different microbes might preferentially consume different kinds of fatty acids to maintain their growth . In this work, there is also apparent cellular lipid degradation between the seventh and tenth days for control without inhibitors. A similar tendency was observed in the culture of T. fermentans on the media containing various organic acids (data not shown). The lipid degradation rate is slower than the control, however, because of the presence of acids, especially the aromatic acids, suggesting that organic acids repress lipid turnover as well. This is an interesting phenomenon because repression of accumulated lipid degradation has been observed only in multiple limited media .
Effect of organic acids on the fatty acid composition of lipids
Fatty acid composition (%)
Palmitic acid (C16:0)
Linoleic acid (C18:2)
Oleic acid (C18:1)
Stearic acid (C18:0)
Acetic acid (2 g/L)
Syringic acid (0.5 g/L)
Vanillic acid (0.5 g/L)
Caproic acid (0.25 g/L)
Furoic acid (0.5 g/L)
4-hydroxybenzoic acid (0.5 g/L)
Formic acid (1 g/L)
Levulinic acid (4 g/L)
Ferulic acid (2 g/L)
Gallic acid (4 g/L)
Effects of inoculum size, temperature, and initial pH on the inhibition by organic acids
Effects of binary combinations of organic acids on cell growth and lipid accumulation of T. fermentans
It has been reported that the synergistic effect of different inhibitors present in the lignocellulosic hydrolysates is complex [32–34]. Therefore, the effects of binary combinations of organic acids on the cell growth and lipid accumulation of T. fermentans were tested at their respective IC25 concentrations listed in Table 2. Acetic and 4-hydroxybenzoic acids, the typical aliphatic and aromatic acids in lignocellulosic hydrolysate, respectively, were chosen for binary combinations with other organic acids. In the experiments, whenever two acids were combined, the predicted relative biomass, lipid content and lipid yield represented the values after deduction of the summed inhibition on biomass, lipid content and lipid yield by each of the two tested inhibitors at their IC25 concentrations. If the actual experimental value exceeded the predicted value, the inhibition was referred to as "synergistic."
The inhibitory effect of organic acids on the cell growth and lipid accumulation of T. fermentans can be relieved or even eliminated at low concentrations. Thus, more efforts should be made to improve the stress assistance of T. fermentans to inhibitors by genetic engineering and/or domestic methods. The development of cost-effective hydrolytic or detoxification processes to obtain lignocellulosic hydrolysates with lower inhibitor concentrations would also be useful. The inhibition of organic acids can also be reduced by optimizing culture conditions, such as inoculum size, temperature and initial pH of the medium.
Microorganism and chemicals
Oleaginous yeast T. fermentans CICC 1368 was obtained from the China Center of Industrial Culture Collection and kept on wort agar at 4°C. Levulinic acid, 4-hydroxybenzoic acid, syringic acid, vanillic acid and furoic acid were purchased from Alfa Aesar (Heysham, UK). Acetic acid, formic acid, caproic acid, ferulic acid, gallic acid and other chemicals were obtained from commercial sources and were of the highest purity available.
Medium, precultivation and cultivation
The composition of the precultivation medium (pH 6.0) was as follows: glucose and xylose 20 g/L (ratio 2:1 wt/wt), 10 g/L peptone and 10 g/L yeast extract. The composition of the fermentation medium (pH 6.5) was as follows: 100 g/L glucose and xylose (ratio 2:1 wt/wt), 1.8 g/L peptone, 0.5 g/L yeast extract, 0.4 g/L MgSO4·7H2O, 2.0 g/L KH2PO4, 0.003 g/L MnSO4·H2O and 0.0001 g/L CuSO4·5H2O.
The preculture was performed in a 250-ml conical flask containing 50 ml of precultivation medium in a rotary shaker at 28°C and 160 rpm for 24 hours. Next, 5% seed culture (2.5 ml) was inoculated in a 250-ml conical flask containing 47.5 ml of fermentation medium, and cultivation was carried out in a rotary shaker at 25°C and 160 rpm for seven days.
Effect of sugar concentration on the growth and lipid accumulation of T. fermentans
A mixture of glucose and xylose at a ratio of 2:1 (wt/wt) was used as the carbon source. The medium, at an initial sugar concentration of 25, 50, 75, 100, 125, 150, 200, 300 or 400 g/L, respectively, was used for the substrate inhibition study. After five days' fermentation, the biomass, lipid content, lipid yield and sugar concentration of T. fermentans on the medium with different initial sugar concentrations were compared.
Effects of organic acids on the growth and lipid accumulation
After precultivation, 2.5 ml of seed culture were inoculated in 47.5 ml of fermentation medium containing the selected organic acid. To facilitate averaging, results throughout the text are expressed as percentages of the control values without addition of the tested inhibitor (with the biomass, lipid content, lipid yield and residual sugar concentration after seven days' fermentation being 24.0 g/L, 61.7%, 14.8 g/L and 15.7 g/L, respectively). IC25 and IC50, defined as the molar concentrations of the tested organic acids that cause 25% and 50% inhibition of the lipid yield of T. fermentans, respectively, were measured according to the data shown in Figure 3. All reported data were averages of experiments performed at least in triplicate.
Effects of inoculum size, temperature and initial pH on the inhibition of organic acids
The effects of inoculum size, temperature or initial pH on the potency of organic acids were examined using acid concentrations of IC50. For inoculum size, 5%, 10% and 15% seed culture were inoculated in the fermentation media containing the selected acids (IC50). For temperature, the cultures with 5% inoculum size were maintained at 22°C, 25°C and 28°C, respectively. Fermentation media containing the assayed acids were adjusted to pH 5.5, 6.5 or 7.5 prior to inoculation to test the effect of initial pH. Biomass, lipid content and lipid yield were all measured after seven days' fermentation.
Binary combinations of organic acids
Two selected organic acids with each concentration at IC25 were added to the fermentation medium. Cultures were inoculated as described above and incubated for seven days (5% inoculum size, pH 6.5, and 25°C). Cultures grown without adding organic acids were used as the control.
Effects of organic acids on sugar utilization and malic enzyme activity
The effects of organic acids on sugar utilization were examined with the acid concentration being IC25. The relative sugar consumption was defined as the ratio of the amount of glucose and xylose consumed by the yeast cells grown on the culture medium containing the selected organic acid for seven days to that without the acid. The malic enzyme activity of T. fermentans was measured according to our previous work  with a SHIMADZU UV-2550 spectrophotometer (Kyoto, Japan).
Biomass was harvested by centrifugation and weighed in its lyophilized form . Extraction of lipid from lyophilized biomass was performed according to a procedure modified from the one described by Folch et al. , with a mixture of chloroform and methanol (2:1 vol/vol). The extracted lipid was centrifuged to obtain a clear supernatant, and the solvent was removed by evaporation under a vacuum at 100 hPa, 55°C and 100 rpm (EYELA NE series rotary evaporator; Tokyo Rikakikai Co, Ltd, Tokyo, Japan). Lipid yield is expressed as the amount of lipid extracted from the cells in per liter of fermentation broth (g/L), and lipid content is defined as the percentage of lipid to dry biomass (% wt/wt). The fatty acid profile of the lipid from T. fermentans was determined by gas chromatography (GC-2010 Plus; Tokyo Rikakikai Co, Ltd) with an ionization detector and a DB-1 capillary column (0.25 cm × 30 m; Agilent Technologies Inc, Santa Clara, CA, USA) according to previously published procedures . D-xylose and D-glucose were measured by high-performance liquid chromatography (Waters Corp, Milford, MA, USA) with a differential refractive index detector (Waters 2410; Waters Corp) and an Aminex HPX-87P column (300 mm × 7.8 mm; Bio-Rad Laboratories, Hercules, CA, USA) at 85°C. Deionized water was used as the mobile phase at 0.5 mL/minute.
- IC25 and IC50:
the molar concentrations of the tested organic acids that causes 25% and 50% inhibition on the lipid yield of T. fermentans
Control trial on the medium without inhibitor
We acknowledge the Major State Basic Research Development Program "973" (grant 2010CB732201), the National Natural Science Foundation of China (grants 31071559, 21072065 and 20876059), the Science and Technology Project of Guangdong Province (grants 2009B080701085 and 2009B030801004), the Doctoral Program of Higher Education (grant 20090172110019), the Open Project Program of the State Key Laboratory of Pulp and Paper Engineering, SCUT (grant 201138), and the Open Project Program for Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology for financial support.
- Demirbas A: Progress and recent trends in biofuels. Prog Energy Combust Sci 2007, 33: 1-18. 10.1016/j.pecs.2006.06.001View ArticleGoogle Scholar
- Xue FY, Zhang X, Luo H, Tan TW: A new method for preparing raw material for biodiesel production. Process Biochem 2006, 41: 1699-1702. 10.1016/j.procbio.2006.03.002View ArticleGoogle Scholar
- Adamczak M, Bornscheuer U, Bednarski W: The application of biotechnological methods for the synthesis of biodiesel. Eur J Lipid Sci Technol 2009, 111: 800-813. 10.1002/ejlt.200900078View ArticleGoogle Scholar
- Moreton RS (Ed): Single Cell Oil. Harlow, UK: Longman Scientific & Technical; 1988.Google Scholar
- Moreton R: Modification of fatty acid composition of lipid accumulating yeasts with cyclopropene fatty acid desaturase inhibitors. Appl Microbiol Biotechnol 1985, 22: 42-45.Google Scholar
- Papanikolaou S, Aggelis G: Yarrowia lipolytica : A model microorganism used for the production of tailor-made lipids. Eur J Lipid Sci Technol 2010, 112: 639-654. 10.1002/ejlt.200900197View ArticleGoogle Scholar
- Wu S, Hu C, Zhao X, Zhao ZB: Production of lipid from N-acetylglucosamine by Cryptococcus curvatus . Eur J Lipid Sci Technol 2010, 112: 727-733. 10.1002/ejlt.201000005View ArticleGoogle Scholar
- Zhao X, Kong XL, Hua YY, Feng B, Zhao ZB: Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi . Eur J Lipid Sci Technol 2008, 110: 405-412. 10.1002/ejlt.200700224View ArticleGoogle Scholar
- Li Q, Du W, Liu DH: Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol 2008, 80: 749-756. 10.1007/s00253-008-1625-9View ArticleGoogle Scholar
- Zhu LY, Zong MH, Wu H: Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation. Bioresour Technol 2008, 99: 7881-7885. 10.1016/j.biortech.2008.02.033View ArticleGoogle Scholar
- Papanikolaou S, Aggelis G: Biotechnological valorization of biodiesel derived glycerol waste through production of single cell oil and citric acid by Yarrowia lipolytica . Lipid Technol 2009, 21: 83-87. 10.1002/lite.200900017View ArticleGoogle Scholar
- Huang C, Zong MH, Wu H, Liu QP: Microbial oil production from rice straw hydrolysate by Trichosporon fermentans . Bioresour Technol 2009, 100: 4535-4538. 10.1016/j.biortech.2009.04.022View ArticleGoogle Scholar
- Reddy N, Yang Y: Biofibers from agricultural byproducts for industrial applications. Trends Biotechnol 2005, 23: 22-27. 10.1016/j.tibtech.2004.11.002View ArticleGoogle Scholar
- Palmqvist E, Hahn-Hagerdal B: Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol 2000, 74: 25-33. 10.1016/S0960-8524(99)00161-3View ArticleGoogle Scholar
- Almeida J, Modig T, Petersson A, Hahn-Hägerdal B, Lidén G, Gorwa-Grauslund M: Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae . J Chem Technol Biotechnol 2007, 82: 340-349. 10.1002/jctb.1676View ArticleGoogle Scholar
- Chen X, Li Z, Zhang X, Hu F, Ryu D, Bao J: Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds. Appl Biochem Biotechnol 2009, 159: 1-14. 10.1007/s12010-009-8550-yView ArticleGoogle Scholar
- Hu C, Zhao X, Zhao J, Wu S, Zhao ZB: Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides . Bioresour Technol 2009, 100: 4843-4847. 10.1016/j.biortech.2009.04.041View ArticleGoogle Scholar
- Economou CN, Aggelis G, Pavlou S, Vayenas D: Modeling of single-cell oil production under nitrogen-limited and substrate inhibition conditions. Biotechnol Bioeng 2011, 108: 1049-55. 10.1002/bit.23026View ArticleGoogle Scholar
- Mussatto S, Dragone G, Roberto I: Influence of the toxic compounds present in brewer's spent grain hemicellulosic hydrolysate on xylose-to-xylitol bioconversion by Candida guilliermondii . Process Biochem 2005, 40: 3801-3806. 10.1016/j.procbio.2005.06.024View ArticleGoogle Scholar
- Tran A, Chambers R: Red oak wood derived inhibitors in the ethanol fermentation of xylose by Pichia stipitis CBS 5776. Biotechnol Lett 1985, 7: 841-845. 10.1007/BF01025567View ArticleGoogle Scholar
- Papanikolaou S, Galiotou-Panayotou M, Fakas S, Komaitis M, Aggelis G: Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media. Bioresour Technol 2008, 99: 2419-2428. 10.1016/j.biortech.2007.05.005View ArticleGoogle Scholar
- Aggelis G, Komaitis M: Enhancement of single cell oil production by Yarrowia lipolytica growing in the presence of Teucrium polium L. aqueous extract. Biotechnol Lett 1999, 21: 747-749. 10.1023/A:1005591127592View ArticleGoogle Scholar
- Sarris D, Galiotou-Panayotou M, Koutinas AA, Komaitis M, Papanikolaou S: Citric acid, biomass and cellular lipid production by Yarrowia lipolytica strains cultivated on olive mill wastewater-based media. J Chem Technol Biotechnol 2011, 86: 1439-1448. 10.1002/jctb.2658View ArticleGoogle Scholar
- Zaldivar J, Ingram LO: Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol Bioeng 1999, 66: 203-210. 10.1002/(SICI)1097-0290(1999)66:4<203::AID-BIT1>3.0.CO;2-#View ArticleGoogle Scholar
- Huang C, Wu H, Liu QP, Li YY, Zong MH: Effects of aldehydes on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans . J Agric Food Chem 2011, 59: 4606-4613. 10.1021/jf104320bView ArticleGoogle Scholar
- Narendranath N, Thomas K, Ingledew W: Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium. J Ind Microbiol Biotechnol 2001, 26: 171-177. 10.1038/sj.jim.7000090View ArticleGoogle Scholar
- Papanikolaou S, Sarantou S, Komaitis M, Aggelis G: Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media. J Appl Microbiol 2004, 97: 867-875. 10.1111/j.1365-2672.2004.02376.xView ArticleGoogle Scholar
- Ratledge C: Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie 2004, 86: 807-815. 10.1016/j.biochi.2004.09.017View ArticleGoogle Scholar
- Wynn JP, Ratledge C: Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans . Microbiology 1997, 143: 253-257. 10.1099/00221287-143-1-253View ArticleGoogle Scholar
- Papanikolaou S, Gortzi O, Margeli E, Chinou I, Galiotou-Panayotou M, Lalas S: Effect of Citrus essential oil addition upon growth and cellular lipids of Yarrowia lipolytica yeast. Eur J Lipid Sci Technol 2008, 110: 997-1006. 10.1002/ejlt.200800085View ArticleGoogle Scholar
- Aggelis G, Athanassopoulos N, Paliogianni A, Komaitis M: Effect of a Teucrium polium L. extract on the growth and fatty acid composition of Saccharomyces cerevisiae and Yarrowia lipolytica . Antonie van Leeuwenhoek 1998, 73: 195-198. 10.1023/A:1000673426077View ArticleGoogle Scholar
- Duarte LC, Carvalheiro F, Neves I, Gírio FM: Effects of aliphatic acids, furfural, and phenolic compounds on Debaryomyces hansenii CCMI 941. Appl Biochem Biotechnol 2005, 121: 413-425. 10.1385/ABAB:121:1-3:0413View ArticleGoogle Scholar
- Oliva J, Negro M, Sáez F, Ballesteros I, Manzanares P, González A, Ballesteros M: Effects of acetic acid, furfural and catechol combinations on ethanol fermentation of Kluyveromyces marxianus . Process Biochem 2006, 41: 1223-1228. 10.1016/j.procbio.2005.12.003View ArticleGoogle Scholar
- Oliva JM, Ballesteros I, Negro MJ, Manzanares P, Cabañas A, Ballesteros M: Effect of binary combinations of selected toxic compounds on growth and fermentation of Kluyveromyces marxianus . Biotechnol Progr 2004, 20: 715-720. 10.1021/bp034317pView ArticleGoogle Scholar
- Kavadia A, Komaitis M, Chevalot I, Blanchard F, Marc I, Aggelis G: Lipid and γ-linolenic acid accumulation in strains of Zygomycetes growing on glucose. J Am Oil Chem Soc 2001, 78: 341-346. 10.1007/s11746-001-0266-3View ArticleGoogle Scholar
- Folch J, Lees M, Sloane-Stanley G: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957, 226: 497-509.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.