SHORT COMMUNICATION

A quick isolation method for mutants with high lipid yieldin oleaginous yeast

Jufang Wang Æ Renmin Li Æ Dong Lu ÆShuang Ma Æ Yaping Yan Æ 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 con-

centration of cerulenin, a potent inhibitor of fatty acid

synthetase, was at 8.96 lmol/l. Afterwards, lipid concen-

tration in the fermentation broth of the pre-selected

colonies was estimated by the sulfo-phospho-vanillin

reaction instead of the conventional methanol–chloroform

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 efficient in the mutant isolation, and it has demon-

strated 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

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 arachi-

donic 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

significant 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 efficiency.

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 significant 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 Lim-

berger 2003). Although these methods allow a quick and

direct estimation of intracellular lipid, developed methods

are still in need. New and efficient isolation procedures that

can significantly 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

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,

People’s Republic of China

e-mail: jufangwang@impcas.ac.cn

123

World J Microbiol Biotechnol (2009) 25:921–925

DOI 10.1007/s11274-009-9960-2

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-4-

oxo-7,10-dodecadienoylamide], isolated from the culture

broth of the fungus Cephalosporium caerulens, is a potent

inhibitor because it targets specifically 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 poly-

unsaturated 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.

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 2–3 days at 28�C in YEPD medium (g/

l): glucose 20, peptone 10, yeast extract 10 and agar 20 for

solid medium. Fermentation was performed in the modified

YEPD medium with the following composition (g/l): glu-

cose 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 flasks for 6 days at 28 ± 1�C 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 fluencies 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.

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 con-

centration, the irradiated cultures were properly diluted and

seeded on the YEPD agar incorporated with 8.96 lmol/l

cerulenin for 5 days at 28�C. Large colonies (diame-

ter [ 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 resus-

pended 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 mil-

liliters of phosphoric acid–vanillin (Sangon) reagent were

added to each tube and incubated for 15 min at 37�C.

Details for the preparation of the phosphoric acid–vanillin

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, metha-

nol:chloroform:water at a ratio of 2:1:0.8 (v/v) were kept in

the sample. After mixing and 10 min incubation at 4�C, the

lipids were separated from the water-soluble material by

diluting the sample with one volume of chloroform fol-

lowed 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 quantified by a

gravimetric method.

922 World J Microbiol Biotechnol (2009) 25:921–925

123

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 efficiently 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 efficiency 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 sufficient 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 con-

taining cerulenin and then incubating the culture at 28�C

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-phospho-

vanillin 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

components of a lipid specimen first become oxidized to

ketones, and then the ketones condense with vanillin or a

derivative of the vanillin under the influence 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 fitted by least square. As shown in

Fig. 2, a linear equation y = 38.2257x ? 0.67314 well fits

the data with a correlation coefficient of 0.995. Thus a

Table 1 The survival fraction of Rhodotorula glutinis AY 91015

cells irradiated with 80 MeV/u carbon ions

Dose (Gy) Colonies Survival fraction

0 (control) 92 ± 9.8 1.00 ± 0.11

5 67 ± 6.9 0.73 ± 0.08

15 54 ± 4.5 0.59 ± 0.05

40 19 ± 6.6 0.21 ± 0.07

55 8 ± 2.2 0.09 ± 0.02

The number of colonies represents the mean value of six independent

samples

0 2 4 6 8 10 12

0.01

0.1

1

Larg

e co

lon

y fo

rmat

ion

eff

icie

ncy

Cerulenin (µmol/l)

Fig. 1 Large colony formation efficiency of Rhodotorula glutinisAY 91015 grown on YEPD agar supplemented with cerulenin at

a concentration gradient

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Lip

id c

on

cen

trat

ion

(g

/l)

Absorbance

Fig. 2 The relationship between the absorbance of stained cells and

the lipids concentration measured by the sulfo-phospho-vanillin

method

World J Microbiol Biotechnol (2009) 25:921–925 923

123

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 find out the correlation between the sulfo-phospho-

vanillin reaction and conventional methanol-chloroform

extraction for lipid quantification, 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 concen-

tration 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.

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 confirmed in later experi-

ments, 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 thra-

ustochytrids 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 fer-

mentation 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 significant amount of biological material as well

as a significant amount of manipulation of the biological

material preceding the quantitation (Gerhardt et al. 1994).

With the advantage of easy handling, the sulfo-phospho-

vanillin method is ideal to select mutants with high lipid

yield in oleaginous yeast after the pre-selection with ce-

rulenin. As the sulfo-phospho-vanillin method can be used

in conjunction with the quantitation of other cell compo-

nents using the same sample, the analysis of the different

ratios of lipid/DNA and lipid/protein can be used as an

indicator of significant physiological conditions altering

lipid, protein, or DNA synthesis in the mutant strain when

0.1 0.2 0.3 0.4 0.5 0.60.1

0.2

0.3

0.4

0.5

0.6

Lip

id c

on

cen

trat

ion

(g

/l)su

lfo

-ph

osp

ho

-van

illin

Lipid concentration (g/l)methanol-chloroform

Fig. 3 Comparison of lipid concentration measured by the sulfo-

phospho-vanillin reaction and those determined by the conventional

methanol-chloroform extraction method

Table 2 Biomass and lipid production in the control and mutant

strains

Strain Biomass

(g l-1)

Lipid

content (%)

Lipid concentration

(g l-1)

Control 2.03 ± 0.022 18.19 ± 1.281 0.34 ± 0.028

M 5 2.17 ± 0.017 28.78 ± 3.036 0.60 ± 0.054

M 16 2.21 ± 0.020 30.67 ± 2.938 0.65 ± 0.061

All data are the means of three parallel samples

924 World J Microbiol Biotechnol (2009) 25:921–925

123

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 microor-

ganisms 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 significant influences 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 cerule-

nin incorporated medium leads to a visible colony detection

that is easy, efficient and time-saving in comparison with

random selection. Afterwards, lipid concentration in the

fermentation broth of the pre-selected colonies was esti-

mated by colorimetric measurement of the intact cells based

on sulfo-phospho-vanillin reaction which is much less

tedious than the conventional methanol-chloroform extrac-

tion. 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 Sci-

ences, 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 53:381–386. doi:10.1023/A:1008011603096

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

121:169–177. doi:10.1016/j.ijfoodmicro.2007.11.030

Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction

and purification. Can J Biochem Physiol 37:911–917

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 53:89–91

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 40:467–497. 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

55:411–418. 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 41:312–317. 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 27:389–393

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

109:1060–1070. doi:10.1002/ejlt.200700169

Parekh S, Vinci VA, Strobel RJ (2000) Improvement of microbial

strains and fermentation processes. Appl Microbiol Biotechnol

54:287–301. 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

40:358–363. 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 86:807–815. doi:

10.1016/j.biochi.2004.09.017

Singh A, Ward OP (1997) Microbial production of docosahexaenoic

acid (DHA, C22:6). Adv Appl Microbiol 45:271–312. doi:

10.1016/S0065-2164(08)70266-1

Somashekar D, Venkateshwaran G, Srividya C, Krishnanand,

Sambaiah K, Lokesh BR (2001) Efficacy of extraction methods

for lipid and fatty acid composition from fungal cultures. World

J Microbiol Biotechnol 17:317–320. doi:10.1023/A:101679

2311744

Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006)

Commercial applications of microalgae. J Biosci Bioeng

101:87–96. 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

11:252–254. 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

influence of fractionation on cell survival and premature

differentiation after carbon ion irradiation. J Radiat Res (Tokyo)

49:391–398. doi:10.1269/jrr.08012

Zhu LY, Zong MH, Wu H (2008) Efficient lipid production with

Trichosporon fermentans and its use for biodiesel preparation.

Bioresour Technol 99:7881–7885. doi:10.1016/j.biortech.2008.

02.033

World J Microbiol Biotechnol (2009) 25:921–925 925

123