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World J Microbiol Biotechnol (2009) 25:921925 DOI 10.

1007/s11274-009-9960-2

SHORT COMMUNICATION

A quick isolation method for mutants with high lipid yield in oleaginous yeast
Jufang Wang Renmin Li Dong Lu Shuang Ma Yaping Yan Wenjian Li

Received: 12 September 2008 / Accepted: 7 January 2009 / Published online: 24 January 2009 Springer Science+Business Media B.V. 2009

Abstract A novel method has been developed to easily isolate the mutants with high lipid yield after irradiating oleaginous yeast cells with carbon ions of energy of 80 MeV/u. Pre-selection of the mutants after ion irradiation was performed with culture medium in which the concentration of cerulenin, a potent inhibitor of fatty acid synthetase, was at 8.96 lmol/l. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by the sulfo-phospho-vanillin reaction instead of the conventional methanolchloroform extraction. Two mutants with high lipid yield have been successfully selected out by the combined method. This easy and simple method is much less time-consuming but very efcient in the mutant isolation, and it has demonstrated great potential on mutation breeding in oleaginous microorganism. Keywords Oleaginous yeast Mutant Cerulenin Sulfo-phospho-vanillin reaction

Introduction The commercial application of microbial oils, i.e. single cell oils (SCO), has become commonplace worldwide and the demand is continuously increasing (Spolaore et al. 2006). It has long been a subject of both research and
This work was supported by the Western Light Program of Talent Cultivation of Chinese Academy of Sciences (O606180XBO). J. Wang (&) R. Li D. Lu S. Ma Y. Yan W. Li Radiobiology Laboratory, Institute of Modern Physics, Chinese Academy of Sciences, 730000 Lanzhou, Peoples Republic of China e-mail: jufangwang@impcas.ac.cn

industrial interest for many years to produce microbial oils through oleaginous microorganisms that involve bacteria, yeasts, moulds and algae. Microbial polyunsaturated fatty acids, such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), are very important in nutrition (Azeem et al. 1999; Ratledge 2004). Because of their similar composition of fatty acids to that of vegetable oils, microbial oils are now the potential feedstock for biodiesel production (Li et al. 2007; Zhu et al. 2008). Therefore any signicant enhancement of the lipid yields in oleaginous strain will offer great opportunity for industrial production. Strain improvement has been achieved mainly through mutagen or genetic recombination in which the improved strains are randomly screened that result in low efciency. The conventional methods to determine lipid contents in microorganisms typically require solvent extraction and weighing (Bligh and Dyer 1959; Gerhardt et al. 1994; Somashekar et al. 2001) in which the extraction often manipulates a signicant amount of biological material and the process is very tedious and time-consuming. Attempts have been made to develop newer and better methods as evidenced by the reported measurement of absorbance in oleaginous yeast cells stained with Sudan Black B (Thakur et al. 1989; Patnayak and Sree 2005) and a colorimetric method based on the sulfo-phospho-vanillin reaction to quantify the lipids from bacteria samples (Izard and Limberger 2003). Although these methods allow a quick and direct estimation of intracellular lipid, developed methods are still in need. New and efcient isolation procedures that can signicantly enhance the selection of the improved oleaginous strains would greatly speed up the industrial application of SCO. Fatty acid biosynthesis is catalyzed by type II fatty acid synthase (FAS) in most bacterium and by type I FAS in eukaryotes. One of the key features for mutant isolation is

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to identify the good inhibitors that form tight complexes in the enzyme active site so that only improved mutants with high fatty acid synthase activity can grow in the inhibitor incorporated medium. Cerulenin [(2S)(3R)2,3-epoxy-4oxo-7,10-dodecadienoylamide], isolated from the culture broth of the fungus Cephalosporium caerulens, is a potent inhibitor because it targets specically the b-ketoacyl-ACP synthases by becoming covalently attached to the active site cysteine so that type I and type II fatty acid synthase systems can be irreversibly inhibited (Heath et al. 2001). It had been reported that the content of intracellular polyunsaturated fatty acid produced in bacteria could be enhanced by cerulenin treatment (Morital et al. 2005). Based on the reports and our analyses, we have carried out a study of using cerulenin on the isolation and detection of mutants with high lipid yield in oleaginous microorganisms after mutagen treatment. In this article, we will present the development of an easy method that combines cerulenin incorporated medium for pre-selection and sulfo-phospho-vanillin reaction for estimation of the lipid yield to quickly select mutant strains after irradiating the oleaginous yeast cells with carbon ions, a very powerful mutation inducer.

Pre-selection with cerulenin In order to determine the proper concentration of cerulenin for pre-selection, yeast culture of the control sample was planted on the YEPD agar supplemented with cerulenin (Biomol, 2240 lmol/l) at a concentration gradient from 2.24 to 11.20 lmol/l. According to the survival fraction and cerulenin concentration, the irradiated cultures were properly diluted and seeded on the YEPD agar incorporated with 8.96 lmol/l cerulenin for 5 days at 28C. Large colonies (diameter [ 2 mm) were picked out from the YEPD agar and transferred to fresh medium for preservation. Estimation of lipid concentration by sulfo-phospho-vanillin reaction Ten milliliters of fermentation broth was centrifuged. The yeast cell pellet was washed free of nutrients and resuspended in 2 ml of distilled water. To a test tube, 100 ll cell suspension or 100 ll distilled water was added. After mixing with 2 ml of 18 mol sulfuric acid, the tubes were incubated in a boiling water bath for 10 min, and cooled for 5 min in a water bath at room temperature. Five milliliters of phosphoric acidvanillin (Sangon) reagent were added to each tube and incubated for 15 min at 37C. Details for the preparation of the phosphoric acidvanillin reagent are the same as described in the publication (Izard and Limberger 2003). The absorption at 530 nm was measured using the distilled water as the control sample. A reference curve was obtained by plotting absorbance against the corresponding lipid concentration ranging from 0.09 to 0.34 g/l determined by conventional gravimetric method. The lipid concentration in the fermentation broth of pre-selected colonies was estimated from the reference curve. All curves were plotted by Origin 7.0 (Origin Lab., America). Conventional methanol-chloroform extraction One gram of dried cells was hydrolyzed with 10 ml of 4 mol HCl in a boiling water bath for 1 h and then the sample was washed free of acid. Afterwards, methanol:chloroform:water at a ratio of 2:1:0.8 (v/v) were kept in the sample. After mixing and 10 min incubation at 4C, the lipids were separated from the water-soluble material by diluting the sample with one volume of chloroform followed by one volume of water. The layer of chloroform and lipid mixture was completely removed with a pipette. Finally, the chloroform in the extracted mixture was evaporated away and the total lipid was quantied by a gravimetric method.

Materials and methods Microorganism and cultivation The red yeast Rhodotorula glutinis AY 91015 purchased from China Center for Type Culture Collection (CCTCC) was cultivated for 23 days at 28C in YEPD medium (g/ l): glucose 20, peptone 10, yeast extract 10 and agar 20 for solid medium. Fermentation was performed in the modied YEPD medium with the following composition (g/l): glucose 30, peptone 10, yeast extract 10, MgSO4 7H2O 1.5 and KH2PO4 1.0, pH is 5.5. The cultures were grown in 100 ml media in 250 ml asks for 6 days at 28 1C on a rotary shaker at 150 rev/min. Irradiation Exponential growing yeast cultures were irradiated with carbon ions of energy of 80 MeV/u at the heavy ion research facility of Lanzhou (HIRFL, Institute of Modern Physics, Lanzhou, China). The irradiation doses were 5, 15, 40 and 55 Gy, calculated from particle uencies and linear energy transfer (LET). For more irradiation technical details please refer to the publication (Wang et al. 2008). The survival fraction of the yeast cells after irradiation was determined by a standard colony formation assay. In all experiments controls were sham-irradiated.

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Results After irradiation of the exponential growing culture of Rhodotorula glutinis AY 91015 to 80 MeV/u carbon ions, the survival fraction of the yeast cells was determined by the colony formation assay. The results are listed in Table 1 and these measured data clearly show the cell survival fraction decreases with increasing irradiation dose. During the determination of the proper concentration of cerulenin for pre-selection, it was found that cerulenin suppressed efciently the colony formation of Rhodotorula glutinis AY 91015. The size of colony became smaller and smaller as the concentration of cerulenin in the culture medium increased. The relationship between large colony (diameter [ 2 mm) formation efciency and cerulenin concentration incorporated in medium is shown in Fig. 1. At a concentration of 8.96 lmol/l nearly all colonies became very small (diameter B 2 mm), while the colony size of the control sample without cerulenin treatment was pretty large (diameter [ 5 mm). Therefore YEPD agar supplemented with 8.96 lmol/l cerulenin is sufcient for the pre-selection of mutants with high fatty acid synthase activity. In general, high mutation frequency is usually found at low survival. Thus the pre-selection was performed mainly in yeast cultures irradiated with 40 and 55 Gy carbon ions. After spreading the irradiated samples on the agar containing cerulenin and then incubating the culture at 28C for 5 days, most of parent cells were prevented from growing into normal large colonies. Only a few of possible mutants with enhanced lipid synthesis capacity grew into large ones that can be visibly isolated. As the result of visible large size, 33 large colonies (diameter [ 2 mm) were selected out from thousands of small colonies (diameter B 2 mm). The colorimetric method based on the sulfo-phosphovanillin reaction has been used for the determination of total serum lipids in humans (Frings and Dunn 1970; Tietz 1982), even though the detailed chemical reactions remain unknown. One assumption is that the unsaturated
Large colony formation efficiency

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Fig. 1 Large colony formation efciency of Rhodotorula glutinis AY 91015 grown on YEPD agar supplemented with cerulenin at a concentration gradient

components of a lipid specimen rst become oxidized to ketones, and then the ketones condense with vanillin or a derivative of the vanillin under the inuence of acid catalysis. Following the assumed condensation reaction, dehydration of an aldol-type intermediate is further assumed to yield a more highly unsaturated product that absorbs visible light (Tietz 1982). The maximum absorption of sulfo-phospho-vanillin stained yeast cells as well as lipid extracted from the yeasts was 530 nm. The absorbance was therefore measured at this wavelength. The relationship between the absorbance of stained cells and the lipid concentration of the yeast fermentation broth was tted by least square. As shown in Fig. 2, a linear equation y = 38.2257x ? 0.67314 well ts the data with a correlation coefcient of 0.995. Thus a

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0.30 0.25 0.20 0.15 0.10 0.05 0.1

Table 1 The survival fraction of Rhodotorula glutinis AY 91015 cells irradiated with 80 MeV/u carbon ions Dose (Gy) 0 (control) 5 15 40 55 Colonies 92 9.8 67 6.9 54 4.5 19 6.6 8 2.2 Survival fraction 1.00 0.11 0.73 0.08 0.59 0.05 0.21 0.07 0.09 0.02

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The number of colonies represents the mean value of six independent samples

Fig. 2 The relationship between the absorbance of stained cells and the lipids concentration measured by the sulfo-phospho-vanillin method

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World J Microbiol Biotechnol (2009) 25:921925 Table 2 Biomass and lipid production in the control and mutant strains Strain Control M5 M 16 Biomass (g l-1) 2.03 0.022 2.17 0.017 2.21 0.020 Lipid content (%) 18.19 1.281 28.78 3.036 30.67 2.938 Lipid concentration (g l-1) 0.34 0.028 0.60 0.054 0.65 0.061

reference curve for estimation of the lipid concentration in the fermentation broth of the pre-selected colonies by sulfo-phospho-vanillin reaction was obtained. To nd out the correlation between the sulfo-phosphovanillin reaction and conventional methanol-chloroform extraction for lipid quantication, lipid concentrations in the fermentation broth from several experiments were measured by both methods. As shown in Fig. 3, lipid concentration measured by the sulfo-phospho-vanillin reaction method are in very good agreement with those determined by the conventional method, giving an average error of 2.3% and a maximum error of 5.2%. The estimation of lipid concentration with the reference curve has shown that 22 colonies, of the total isolation of 33, with increased lipid concentration compared with the control sample. The positive selection rate is 66%. Among the improved 22 colonies, mutant M 5 with lipid concentration of 0.60 g/l and M 16 with lipid concentration of 0.65 g/l was outstanding in comparison with the control with lipid concentration of 0.34 g/l. It has to be noted that the reference curve in Fig. 2 was drown with control yeast cells giving a relatively low lipid concentration in the fermentation medium (0.34 g/l) while the lipid concentration of mutants with high lipid yield was surely higher than 0.34 g/l. The higher lipid requires the fermentation sample be diluted to get a reasonable estimate. Based on the biomass of the dry weight, calculations of the lipid yield of individual cell have shown that the lipid content of the control, M 5 and M 16 were 18.19, 28.78 and 30.67%, respectively, as listed in Table 2. Lipid content of the two mutants is substantially enhanced too.

All data are the means of three parallel samples

Discussion The SCO production through oleaginous microorganisms usually starts from the selection of the improved strains with high lipid yield. As described here, the novel method developed by us has a number of advantages over others reported in literatures. The pre-selection with cerulenin incorporated medium provides a rapid, visible and labor saving isolation of improved mutant strains with high fatty acid synthase activity. Although the positive selection rate is of only 66%, it can be increased by selecting colonies with diameter [ 2 mm. As it is conrmed in later experiments, the positive selection rate could be 71% if colonies with diameter [ 3 mm were selected out. This may be due to the subjective criterion (diameter [ 2 mm) is not an optimum for the colony selection. Considering our results and the report that cerulenin treatment is effective among certain kinds of eukaryotic microorganisms, such as thraustochytrids that are the potential polyunsaturated fatty acid producers for industrial use (Singh and Ward 1997), it can be expected that the use of cerulenin for isolation could be expanded into other oleaginous fungi after mutagen treatment. Since the estimation of lipid concentration in the fermentation broth of the pre-selected colonies is based on the sulfo-phospho-vanillin reaction in the whole yeast cells, it does not require the breakage of cells involved in other chemical and enzymatic methods. Furthermore, no extraction and separation step is necessary and only a small amount of biological material is required while the typical extraction of lipids uses a two-phase separation that requires a signicant amount of biological material as well as a signicant amount of manipulation of the biological material preceding the quantitation (Gerhardt et al. 1994). With the advantage of easy handling, the sulfo-phosphovanillin method is ideal to select mutants with high lipid yield in oleaginous yeast after the pre-selection with cerulenin. As the sulfo-phospho-vanillin method can be used in conjunction with the quantitation of other cell components using the same sample, the analysis of the different ratios of lipid/DNA and lipid/protein can be used as an indicator of signicant physiological conditions altering lipid, protein, or DNA synthesis in the mutant strain when

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Fig. 3 Comparison of lipid concentration measured by the sulfophospho-vanillin reaction and those determined by the conventional methanol-chloroform extraction method

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925 Beltran G, Novo M, Guillamon JM, Mas A, Rozes N (2008) Effect of fermentation temperature and culture media on the yeast lipid composition and wine volatile compounds. Int J Food Microbiol 121:169177. doi:10.1016/j.ijfoodmicro.2007.11.030 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purication. Can J Biochem Physiol 37:911917 Frings CS, Dunn RT (1970) A colorimetric method for determination of total serum lipids based on the sulfo-phospho-vanillin reaction. Am J Clin Pathol 53:8991 Gerhardt P, Murray RGE, Wood WA, Krieg NR (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC Heath RJ, White SW, Rock CO (2001) Lipid biosynthesis as a target for antibacterial agents. Prog Lipid Res 40:467497. doi: 10.1016/S0163-7827(01)00012-1 Izard J, Limberger RJ (2003) Rapid screening method for quantitation of bacterial cell lipids from whole cells. J Microbiol Methods 55:411418. doi:10.1016/S0167-7012(03)00193-3 Li YH, Zhao ZB, Bai FW (2007) High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microb Technol 41:312317. doi:10.1016/j.enzmictec.2007. 02.008 Morital N, Nishida T, Tanaka M, Yano Y, Okuyama H (2005) Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria. Biotechnol Lett 27:389393 Papanikolaou S, Galiotou-Panayotou M, Fakas S, Komaitis M, Aggelis G (2007) Lipid production by oleaginous Mucorales cultivated on renewable carbon sources. Eur J Lipid Sci Technol 109:10601070. doi:10.1002/ejlt.200700169 Parekh S, Vinci VA, Strobel RJ (2000) Improvement of microbial strains and fermentation processes. Appl Microbiol Biotechnol 54:287301. doi:10.1007/s002530000403 Patnayak S, Sree A (2005) Screening of bacterial associates of marine sponges for single cell oil and PUFA. Lett Appl Microbiol 40:358363. doi:10.1111/j.1472-765X.2005.01671.x Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86:807815. doi: 10.1016/j.biochi.2004.09.017 Singh A, Ward OP (1997) Microbial production of docosahexaenoic acid (DHA, C22:6). Adv Appl Microbiol 45:271312. doi: 10.1016/S0065-2164(08)70266-1 Somashekar D, Venkateshwaran G, Srividya C, Krishnanand, Sambaiah K, Lokesh BR (2001) Efcacy of extraction methods for lipid and fatty acid composition from fungal cultures. World J Microbiol Biotechnol 17:317320. doi:10.1023/A:101679 2311744 Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:8796. doi:10.1263/jbb.101.87 Thakur MS, Prapulla SG, Karanth NG (1989) Estimation of intracellular lipid by the measurement of absorbance of yeast cells stained with Sudan Black B. Enzyme Microb Technol 11:252254. doi:10.1016/0141-0229(89)90101-4 Tietz NW (1982) Fundamentals of clinical chemistry. Saunders, Philadelphia Wang JF, Li RM, Guo CL, Fournier C, K-Weyrather W (2008) The inuence of fractionation on cell survival and premature differentiation after carbon ion irradiation. J Radiat Res (Tokyo) 49:391398. doi:10.1269/jrr.08012 Zhu LY, Zong MH, Wu H (2008) Efcient lipid production with Trichosporon fermentans and its use for biodiesel preparation. Bioresour Technol 99:78817885. doi:10.1016/j.biortech.2008. 02.033

compared to the wild-type strain (Izard and Limberger 2003). This technique may lead to further investigation of the nature and distribution of lipids in the cell. It is reported that the content of lipid in some microorganisms can exceed 70% of their biomass (Ratledge 2004), while the lipid content of the mutants and control presented in this report is relatively low. Many factors including medium components, such as carbon source, nitrogen source and C/N molar ratio etc. as well as culture temperature and pH have signicant inuences on cell growth and lipid accumulation of oleaginous microorganism (Parekh et al. 2000; Papanikolaou et al. 2007; Beltran et al. 2008). Thus the low lipid content in our measurement could well be due to the different origin of the oleaginous strain and the fermentation conditions. To improve biomass and lipid content in the control and M 16 strain, fermentation conditions as well as medium components have been optimized that has resulted in the lipid content of the control and M 16 strain to 24.57 and 42.38%, respectively. This has demonstrated that there is still room to reach higher lipid contents in these strains. Further experiment for improvement of the lipid yield has been planned in the future.

Conclusion The newly developed method provides a quick isolation of mutants with high lipid yield in oleaginous yeast cells after irradiation treatment. Pre-selection performed with cerulenin incorporated medium leads to a visible colony detection that is easy, efcient and time-saving in comparison with random selection. Afterwards, lipid concentration in the fermentation broth of the pre-selected colonies was estimated by colorimetric measurement of the intact cells based on sulfo-phospho-vanillin reaction which is much less tedious than the conventional methanol-chloroform extraction. Moreover, only a small amount of biological material is required. This method is very promising on mutation breeding in oleaginous microorganisms.
Acknowledgments The authors are grateful to the staff of the HIRFL facility for providing the beams, and to Professor Dang Bingrong of Institute of Modern Physics, Chinese Academy of Sciences, for his assistance in planning the radiation procedure. Especially, the authors are very grateful to Prof. Gao Qingxing of Life Science School, Lanzhou University, Gansu Province, P.R.China, for his great help in manuscript preparation.

References
Azeem A, Neelagund YF, Rathod V (1999) Biotechnological production of oil: fatty acid composition of microbial oil. Plant Foods Hum Nutr 53:381386. doi:10.1023/A:1008011603096

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