2004 Kimura K. Kamisaka Y. J. Microbiol. Methods

8/14/2019 2004 Kimura K. Kamisaka Y. J. Microbiol. Methods

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Rapid estimation of lipids in oleaginous fungi and yeasts using

Nile red fluorescence

K. Kimura*, M. Yamaoka, Y. Kamisaka

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology,

AIST Tsukuba Central 6, 1-1 Higashi 1-Chome, Tsukuba, Ibaraki, 305-8655 Japan

Received 1 September 2003; received in revised form 31 October 2003; accepted 31 October 2003

Abstract

A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was

established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide

range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24–

0.47 Ag/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488

nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid

amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of

various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid

amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple andeasy pretreatment of shaking with glass beads for 5–10 min was added to the protocol. The established method was applicable

to estimate the lipid content of a wide range of microorganism cultures containing 2–5000 Ag-lipid/ml-broth.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Lipids; Oleaginous fungi and yeasts; Nile red fluorescence

1. Introduction

Some oleaginous microorganisms accumulate lip-

ids in the cell at more than 50% of the dry cell weight.They have the possibility to be commercial oil pro-

ducers for food and energy resources (Ratledge, 1989;

Ratledge and Wynn, 2002). Since accumulated lipids

are localized in the intracellular organelle, the so-

called ‘‘lipid body’’ (Murphy and Vance, 1999), the

lipid body formation and maturation are important

processes for the lipid production in oleaginous

microorganisms. How lipid bodies are formed is

crucial to the design of microbial oils. We have

studied lipid transport pathways into lipid bodies of the oleaginous Mortierella fungus using fluorescent

lipid analogues (Kamisaka et al., 1999; Kamisaka and

Noda, 2001). Further studies are intended to screen

compounds or mutants affecting the lipid body for-

mation. For these screening procedures, a rapid and

simple method to determine lipids is indispensable.

Conventional methods of lipid determination have

many complicated steps, i.e., extraction, purification,

concentration, and determination, which are time-con-

suming. A spectrophotometric method using Sudan

0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2003.10.018

* Corresponding author. Tel.: +81-298-861-6666; fax: +81-298-

861-6171.

E-mail address: [email protected] (K. Kimura).

www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 56 (2004) 331 – 338



black B (Thakur et al., 1989) and fluorescence spec-

trometric methods using Luminor 490PT (Pomoshch-

nikova et al., 1981) and Nile red (Cooksey et al., 1987;

Lee et al., 1998; Cole et al., 1990) were reported todetermine the lipid content of yeasts, algae, and ciliates,

all of which were dispersed cells without forming large

aggregations. For oleaginous f ungi, Luminor 490PT

(Pomoshchnikova et al., 1983) and Nile blue (Vijaya-

lakshmi et al., 2003) were used as the only examples.

Among several dyes, Nile red seems preferable for the

intracellular lipid determination but has not been wide-

ly used. Although Nile red can stain most lipids, its

fluorescence character varies depending on the situa-

tion of the lipids. The maximum wavelength of Nile red

emission with neutral lipids is shorter than that with

polar lipids and the former intensity is higher than the

latter (Greenspan and Fowler, 1985). The fluorescence

intensity of lipids composed of unsaturated fat ty acids

is stronger than that of the saturated fatty acids (Fowler

et al., 1987). The emission maximum shifts to a shorter

wavelength depending on the hydrophobicity of the

lipid molecules and their surroundings (Greenspan and

Fowler, 1985). Previous studies (Cooksey et al., 1987;

Lee et al., 1998; Vijayalakshmi et al., 2003) used

special conditions for their microbes so that the fluo-

rescence was measured at the fixed emission wave-

length. They were difficult to apply to variousmicroorganisms and their culture conditions without

optimization. We have overcome these problems in

order to obtain a rapid lipid determination method with

a wide range of applicability. In this study, the mea-

surement condition for the lipid determination using

fluorescence by Nile red was examined in detail and

optimized. The established method can be applicable to

various oleaginous microorganisms including mycelia

which produce various types of pellets.

2. Materials and methods

2.1. Organism and culture

Oleaginous yeasts ( Lipomyces starkeyi IFO-10381,

Rhodosporidium toruloides IFO-0559, Cryptococcus

curvatus IFO-1159) and fungi ( Mortierella isabellina

IFO-7884, Mortierella nana IFO-8794, Mortierella

ramanniana var. angulispora IFO-8187) were

obtained from the Institute for Fermentation (Osaka,

Japan) and maintained on YM agar (glucose 10 g/l,

peptone 5 g/l, yeast extract 3 g/l, malt extract 3 g/l, agar

20 g/l). All the microorganisms were precultured in

YM broth (glucose 10 g/l, peptone 5 g/l, yeast extract 3g/l, malt extract 3 g/l) and cultured for observation of

the lipid accumulation in medium A (glucose 30 g/l,

yeast extract 1.5 g/l, NH4Cl 0.5 g/l, KH2PO4 7.0 g/l,

Na2HPO412H2O 5.0 g/l, MgSO47H2O 1.5 g/l,

FeCl3 6H2O 0 . 0 8 g / l , Z n S O47H2O 0 . 0 1 g / l ,

CaCl22H2O 0 . 1 g / l , M n S O45H2O 0 . 1 m g / l ,

CuSO45H2O 0.1 mg/l, Co(NO3)26H2O 0.1 mg/l; pH

5.5 (Suutari et al., 1993)). For cultivation of the yeasts,

1 ml of the 2-day preculture in YM broth was inocu-

lated to 100 ml of medium A in a 500 ml Erlenmeyer

flask with three baffles. For cultivation of the Mortier-

ella fungi, a 2-day preculture in YM broth was pre-

treated by shaking with the same volume of about 3-

mm diameter glass beads for 5 min to provide a

homogeneous suspension for inoculation. Ten milli-

liters of medium A broth with a 2% inoculation was

pipetted into a 50-ml plastic conical tube with a

ventilation plug. The entire broth in a tube was har-

vested at once for the sampling. Flasks and tubes were

shaken in a rotary shaker at 120 rpm at 27 jC. Lipid

accumulation profiles of oleaginous yeasts and fungi

were demonstrated as average data of two batches.

2.2. Nile red staining and fluorescence spectrometry

Nile red (9-diethylamino-5 H -benzo[a]phenoxa-

]phenoxazine-5-one) obtained from the Aldrich Chem-

ical (Milwaukee, USA) was dissolved in 0.1 mg/ml

with acetone. Fluorescence spectra were measured with

the spectrofluorometer FP-750 with PC control equip-

ment (JASCO, Tokyo, Japan). The acquiring and

processing of the data were done using the PC software

(JASCO). In order to eliminate the effect of auto-

fluorescence of the cells and scattering of the cellsuspension, the fluorescence spectra were obtained as

follows. A 100-Al aliquot of the culture broth of was

mixed with 2 ml of 10 mM potassium phosphate buffer

with 0.15M KCl (pH 7.0; PBS) in a 10-mm acryl

cuvette. The spectrum in a wavelength region of 400

to 700 nm for the cell suspension without Nile red was

recorded. The 10-Al Nile red solution was then added

and mixed well. Five minutes later, the spectrum in the

same wavelength region was recorded again. The cell

suspension in a cuvette was mixed well by an upside-

K. Kimura et al. / Journal of Microbiological Methods 56 (2004) 331–338332



down inversion just before measurement in order to

avoid cell sedimentation. Spectra were corrected by

subtracting the spectra before and after the Nile red

addition using the PC software. The fluorescenceintensity corresponding to the lipid amount was deter -

mined at the peak of the corrected spectrum (see Fig. 1).

2.3. Time course of fluorescence development and

fading

Spectra of the cell suspension were recorded at 1, 5,

10, 15, and 20 min after the Nile red addition in various

amounts (2–100 Al). To avoid cell sedimentation, the

cell suspension was well mixed just before every data

acquisition. To minimize emission fading, irradiation

of the cell suspension was done only during the data

acquisition.

2.4. Microscopy

Microscopic photographs were taken with a Nikon

E600 microscope (Nikon, Tokyo, Japan) equippedwith a color CCD digital camera (DP12, Olympus,

Tokyo, Japan) using a 450–490-nm excitation filter, a

505-nm diachronic mirror and a 520-nm barrier filter

with 40 or 60 objective lens. The obtained digital

color pictures were corrected to grey scale figures by

PC software.

2.5. Lipid analysis

The total lipid concentration was determined by gas

chromatographic analysis of the total fatt y acids direct-

ly transmethylesterified from dried cells (Kumon et al.,

2002). One milliliter of 10% methanolic HCl and 0.5

ml methylene chloride were added to the dried cells and

kept at 60 jC for 3 h for the direct methylesterification.

The reaction was stopped by the addition of 2 ml

saturated NaCl solution and 1 ml hexane. The resultant

methyl esters recovered in the hexane layer were then

applied to a gas chromatograph (GC-17-A; Shimadzu,

Kyoto, Japan) equipped with a TC-70 capillary column

(30 m 0.25 mm i.d., GL Science, Tokyo, Japan)

under temperature programming (180– 220 jC a t 4

jC/min increments). Peanut oil (Nacalai Tesque,Kyoto, Japan) was transmethylesterified and used as

the reference material.

2.6. Other Analytical Methods

The glucose concentration was determined using

Glucose CII Test Wako (Wako, Osaka, Japan). Cell

growth was based on the dry cell weight. Cells in the

1– 3-ml culture broth were collected after washing with

the same volume of water by centrifugation, and

weighed after being dried at 105 j

C overnight.

3. Results and discussion

3.1. Optimization of Nile red staining and fluores-

cence data collection

To develop a widely applicable method, optimiza-

tion of the Nile red staining for fluorescence measure-

ment of intracellular lipids was studied. Lipid

Fig. 1. Emission spectra of Nile red-stained cell suspension. Culture

broth (100 Al)of L. starkeyi inlogphasewasmixedwith 2 mlof PBS.

Before (: : :) and after (- - -) the addition of 10 Al Nile red solution,

emission spectra of the cell suspension were recorded with excitation

wavelengthat 488 nm (A) and 552 nm (B). The corrected spectra (—)

were derived by subtracting the spectra before and after the Nile red

addition. The fluorescence intensity ( X ) was derived at the peak of

the corrected spectrum. The emission spectra were recorded at 5 min

after the Nile red addition.

K. Kimura et al. / Journal of Microbiological Methods 56 (2004) 331–338 333



detection by Nile red has been commonly measured

with excitation at 480–490 and at 510–560 nm. The

former target is neutral lipids to fluoresce and the

latter target is polar lipids (Greenspan et al., 1985).Emission spectra of typical excitation wavelengths at

488 and at 552 nm are compared in Fig. 1. Both

emission spectra, corrected as described in the Mate-

rials and methods, showed similar peaks at 565–575

nm, but had different peak fluorescence intensities.

Fluorescence with 488-nm excitation showed a higher

intensity than with 522-nm excitation. In addition, the

spectrum with the 488-nm excitation was much easier

and more reproducible to separate the effect of scat-

tering by excitation than the spectrum with 552-nm

excitation. In all the tested microorganisms, the peak

wavelengths of emission varied between 565 and 585

nm. Therefore, we chose the excitation wavelength of

488 nm and observed the emission of 565–585 nm as

lipids fluorescence. The fluorescence of Nile red by

itself without lipids existed at around 600– 605nm

with a slight peak, but was negligible. Nile red is

nearly insoluble in water and its fluorescence imme-

diately quenches in aqueous solution (Sackett and

Wolff, 1987). Based on these results, removal of

excess Nile red from the suspension after the staining

is not necessary.

Fig. 2a shows the time course of the fluorescenceof the Lipomyces cell suspension with different Nile

red concentrations. Upon the microscopic observa-

tion, fluorescence of the Nile red stained cells rapidly

faded in the first few seconds of irradiation. For the

spectrometric measurement, fluorescence of the cell

suspension did not fade so rapidly. At every Nile red

concentration, full-staining was achieved between 1

and 5 min. After 5 min, fluorescence fading was

observed especially at high Nile red concentrations

above 2.3 Ag/ml. Therefore, we chose 5 min as the

most efficient staining period.The fluorescence intensity increased with an in-

crease in the Nile red concentration up to 2.3 Ag/ml,

but not linearly with the Nile red concentration. This

means that an increase in the Nile red concentration can

amplify the fluorescence intensity, but its amplification

will not linearly increase with the Nile red concentra-

tion. Fig. 2b shows the fluorescence with various

amount of culture broth at 5 min after the Nile red

addition. The fluorescence intensity increased with an

increase in culture broth amount; the fluorescence

Fig. 2. Fluorescence of Lipomyces cell broth stained with Nile red

with excitation at 488 nm. Into the suspension of 2 ml PBS and

culture broth of L. starkeyi in stationary phase, 0.1 mg/ml Nile redsolution in acetone was added. (a) Time course of staining of 100 Al

culture broth with Nile red in various concentrations. Spectra were

recorded at time intervals and the fluorescence with time was shown

as intensity of the corrected spectrum peak. (b) Fluorescence with

various amount of culture broth of Lipomyces at 5 min. Final Nile

red concentration in the suspension was as follows: o, Nile red

0.095 Ag/ml; 4, 0.24 Ag/ml; 5, 0.47 Ag/ml; . , 0.94 Ag/ml;E, 2.3

Ag/ml; n, 4.5 Ag/ml. Values are average of two determinations. (c)

Variations of emissions spectra of the same culture broth of

Lipomyces in early stationary phase at 5 min after 10 Al Nile red

addition. Average fluorescence intensity at the peak wavelength

(569 nm) was 120.1 unit with S.D. of 2.6 unit for n =12.




intensity of suspension with more than 100 Al culture

broth did not linearly increase with lipid amount in cell

suspension. Usually we used 100 Al culture broth

throughout the one-batch culture for the relative com- parison. The minimum lipid amount by this protocol

was 20Ag in a 2.1-ml final suspension at 0.095– 2.3 Ag/

ml Nile red for Lipomyces cells (data not shown).

Sensitivity for the low lipid concentration was not

enhanced by an increase in the Nile red concentration.

The fluorescence fading became remarkable for high

Nile red concentrations above 2.3 Ag/ml with time. For

the various lipid concentrations tested, the Nile red

concentration of 0.24–0.47 Ag/ml showed reproduc-

ible fluorescence since higher Nile red concentrations

could produce higher experimental errors.

As a result, the rapid estimation method of intra-

cellular lipids was optimized as follows. Nile red (0.1

mg/ml, 10 Al) was added to the cell suspension, the

mixture of 50– 100 Al culture broth and 2 ml PBS, andheld for 5 min after mixing well. Spectra in the

emission wavelength 400–700 nm with excitation at

488 nm were recorded before the Nile red addition

and 5 min after the addition. The corrected fluores-

cence spectrum was derived by subtracting the spectra

before and after the Nile red addition. The fluores-

cence intensity at the peak of the corrected spectrum

corresponded to the intracellular lipid amount. With

the established method described above, the emission

spectra of the culture broth was analyzed repeatedly in

Fig. 2c; it shows the good reproducibility.

Fig. 3. Time course of lipid accumulation of oleaginous fungi. (a) L. starkeyi IFO-10381; (b) R. toruloides IFO-0559; (c) C. curvatus IFO-1159;

(d) M. rammanniana var. angulispora IFO-8187; (e) M. isabellina IFO-7884; (f) M. nana IFO-8794. – n – , Glucose concentration in the culture

broth; – E – , dry cell weight (DCW); – . – , lipid concentration; – x – , fluorescence intensity. Lipid concentration and fluorescence intensity

were the calculated values originally contained in 1 ml of culture broth. Values are average of two determinations.




3.2. Rapid estimation of lipid concentration in the

culture of various oleaginous fungi

We then investigated whether the established meth-od was applicable to various oleaginous yeasts and

fungi having different cell and lipid body morpholo-

gies. The time courses of the cultivation wit h lipid

accumulation of yeasts and fungi are shown in Fig. 3.

The lipid concentration of the culture broth was

determined as the total fatty acids in 1 ml culture

broth, as described in the Materials and methods (Fig.

3a–c). In oleaginous yeasts, the total lipid concentra-

tion and fluorescence intensity increased with an

increase in the dry cell weight, following the glucose

consumption from the culture broth. Lipid bodies, in

which lipids accumulate in oleaginous microorgan-

isms, have different shapes and development depend-ing on the species and culture conditions. Fig. 4 is

microscopic photographs of the tested microorgan-

isms at t he beginning of the lipid accumulation. L.

starkeyi (Fig. 4a) produced one or two large lipid

bodies in the spherical cells, as previously shown

(Naganuma et al., 1986). Small lipid bodies with less

than a 0.5-Am diameter in the cell with a 2–3-Am

diameter at the beginning of the culture increased in

size to more than 5 Am after a 100-h cultivation. The

Fig. 4. Microscopic photographs of differential interference contast ( 1) and Nile red fluorescence ( 2) of oleaginous fungi at the beginning

of lipid accumulation. (a) L. starkeyi IFO-10381; (b) R. toruloides IFO-0559; (c) C. curvatus IFO-1159; (d) M. rammanniana var. angulispora

IFO-8187; (e) M. isabellina IFO-7884; (f) M. nana IFO-8794. In the fluorescence photograph, the lipid bodies which are fluoresced by Nile red

are reflected as white circlets in the field identical to the differential interference contrast photograph. Bar indicates 10 Am.




cell expanded in diameter followed by growth of the

lipid body. The largest lipid body reaching to 10 Am in

diameter was produced in cells with more than a 12-

Am diameter. R. toruloides (Fig. 4b) produced two tofour lipid bodies with 1–3-Am diameter in the ellip-

soidal cells. Small cells with small lipid bodies of less

than 0.5-Am diameter became bigger cells with two to

three lipid bodies up to 2–3 Am and some smaller

ones around 1 Am. C. curvatus (Fig. 4c) had initially

many small lipid bodies with less than 0.5 Am in long

rod-shaped cells. After a 140-h cultivation, about half

of the cells transformed into the enlarged spherical or

ellipsoidal cells contained one or two 2–3-Am lipid

bodies and many smaller ones of less than 1 Am.

Fig. 3d–f shows the time course of the cultivation

of mycelia which made various types of pellets

depending on the species and its cultivation condition.

For the application of our protocol to mycelial grown

fungi, a pretreatment was required. Into the conical

tube culture, the same volume of glass beads (3-mm

diameter) was added to break down the pellets by

shaking for 10 min. A nearly homogeneous suspen-

sion was used for the fluorescence measurement. The

fluorescence intensity increased with an increase in

the cell biomass and lipid concentration. Mortierella

rammanniana var. angulispora (Fig. 4d) formed an

irregular soft pellet mixture, from small particles of less than 0.5 mm to flattened pellets like torn-off clay

with long side of more than 5 mm. In the hyphae lipid

bodies with less than a 0.5-Am diameter, initially

enlarged up to 3-Am diameter, but did not grow

thereafter and increased in number after a 50-h culti-

vation. M. isabellina (Fig. 4e) formed small soft

particulate pellets like a wet powder of less than 1-

mm diameter having lipid bodies with up to 1-Am

diameter. M. nana (Fig. 4f) formed irregular pellets of

soft particles with a 0.5 –3-mm diameter. In the

hyphae, small lipid bodies with less than 0.5-Amdiameters at the beginning of the culture enlarged to

2–3-Am diameters and increased in number. In all the

Mortierella fungi tested, the inside of the hyphae

became filled with lipid bodies as the lipid accumu-

lation proceeded. In all the fungi tested, the change in

fluorescence intensity well corresponded to the

change in the lipid concentration during the cultures.

In Fig. 5, the fluorescence intensity is plotted

versus the lipid concentration based on the data in

Fig. 3. Linear relationships between the lipid concen-

tration and fluorescence intensity were obtained up to

a 2.5 mg/ml lipid concentration among the various

microorganisms which had different types of cells and

lipid bodies in size, shape, and number. The relationcoefficients (r ), mentioned in the legend of the figure,

were high irrespective of the variation of cells and

lipid bodies throughout the culture. The slopes of the

linear lines were not identical among species but

similar among them except for R. toruloides. Similar

slopes with high relation coefficients were also

obtained in other Mortierella fungi such as Mortier-

ella alpine (data not shown). On the other hand, R.

toruloides was the exception which showed a lower

slope than the other species.

Fig. 5. Relationship between lipid concentration and fluorescence in

oleaginous yeast (a) and fungi (b) throughout the cultivation. – . – ,

L. starkeyi IFO-10381 (r = 0.959); – n – , R. toruloides IFO-0559

(r = 0.861); – E – , C. curvatus IFO-1159 (r = 0.913); – o – , M.

rammanniana var. angulispora IFO-8187 (r = 0.983); – 5 – , M.

isabellina IFO-7884 (r = 0.980); – D – , M. nana IFO-8794

(r = 0.873). Plots were taken from the time course data of Fig. 3.

Lipid concentration and fluorescence intensity were the calculated

values originally contained in 1 ml of culture broth. Values are

means of two runs.




With our protocol, 20 Ag lipids in a 2.1-ml final

suspension were detected. In the case of cells with a

lower lipid content, the condensation of cells by

centrifugation of the culture up to 10 times enabledthe lipid measurement. On the other hand, cell sus-

pension which was diluted up to 41 times did not lead

to a noticeable error in the case of a higher lipid

content. The established method is considered to be

applicable to a wide range of microorganisms in a

lipid concentrations range of 2–5000 Ag/ml. In the

case of cells accumulating lipids more than 20% of

dry cell weight, our protocol could detect lipids in the

culture broth during the early stage of lipid accumu-

lation. The Nile red addition in high concentration

could increase the fluorescence intensity but could not

enhance the lipid sensitivity although it would require

the attention to the staining period and fading.

Previous studies on lipid estimations using the

fluorescence of Nile red and Nile blue were applica-

tions specialized for some target microbes. On the

other hand, our method has been designed for a wide

range of microorganisms and represented the applica-

bility of more practical use. A wide range of applica-

tions including the mycelial pellet culture has not

documented. The method is very rapid and easy

compared with the conventional methods, which re-

quire complicated procedures such as extraction, pu-rification, and determination of lipids.

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2004 Kimura K. Kamisaka Y. J. Microbiol. Methods