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DOI 10.1007/s00425-013-1946-5

Original Article

Conversion of membrane lipid acyl groups to triacylglycerol

and formation of lipid bodies upon nitrogen starvation
in biofuel green algae Chlorella UTEX29
EltonC.Goncalves JodieV.Johnson
BalaRathinasabapathi

Received: 11 May 2013 / Accepted: 1 August 2013

Springer-Verlag Berlin Heidelberg 2013

AbstractAlgal lipids are ideal biofuel sources. Our

objective was to determine the contributors to triacylglycerol (TAG) accumulation and lipid body formation in Chlorella UTEX29 under nitrogen (N) deprivation. A fivefold
increase in intracellular lipids following N starvation for
24h confirmed the oleaginous characteristics of UTEX29.
Ultrastructural studies revealed increased number of lipid
bodies and decreased starch granules in N-starved cells
compared to N-replete cells. Lipid bodies were observed
as early as 3h after N removal and plastids collapsed after
48h of stress. Moreover, the identification of intracellular pyrenoids and differences in the expected nutritional
requirements for Chlorella protothecoides (as UTEX29
is currently classified) led us to conduct a phylogenetic
study using 18S and actin cDNA sequences. This indicated
UTEX29 to be more phylogenetically related to Chlorella
vulgaris. To investigate the fate of different lipids after N
starvation, radiolabeling using 14C-acetate was used. A significant decrease in 14C-galactolipids and phospholipids
matched the increase in 14C-TAG starting at 3h of N starvation, consistent with acyl groups from structural lipids
as sources for TAG under N starvation. These results have
important implications for the identification of key steps
Electronic supplementary materialThe online version of this
article (doi:10.1007/s00425-013-1946-5) contains supplementary
material, which is available to authorized users.
E.C.Goncalves B.Rathinasabapathi(*)
Plant Molecular and Cellular Biology Program, Horticultural
Sciences Department, University of Florida, Gainesville,
FL 326110690, USA
e-mail: brath@ufl.edu
J.V.Johnson
Chemistry Department, University of Florida, Gainesville,
FL 326117200, USA

controlling oil accumulation in N-starved biofuel algae

and demonstrate membrane recycling during lipid body
formation.
Keywords Biofuel Chlorella Galactolipid
Radiolabeling Triacylglycerol Ultrastructure

Introduction
The development of alternative sources of renewable
energy is of utmost importance as world energy consumption is projected to grow by 53% from 2008 to 2035 (Hu
etal. 2008). Although renewable fuels are the fastest growing form of energy, its contribution to total energy production is projected to increase a mere 4% by 2035 (Hu
etal. 2008). In addition, the fact that US transportation
fuels must contain 36billion gallons of renewable fuels by
2022 (Chisti 2007) highlights the importance of all efforts
put into developing renewable fuels to an affordable, sustainable and scalable level. Algal lipid from microalgae is
one of the best sources for biofuels due to the following
reasons: impressively high algal growth rates (commonly
doubling its biomass within 24h) (Chisti 2007), tolerance
to extreme conditions (desert and arid lands) (Hu etal.
2008), their abilities to recycle wastewater (agricultural
run-off, industrial and municipal wastewater) and CO2
from flue gases emitted from power plants (Hu etal. 2008;
Wilkie etal. 2011), reduced competition with food crops
(Griffiths and Harrison 2009) and, finally, the production
of multiple value-added co-products (e.g., polymers, surfactants, proteins and pigments) (Foley etal. 2011; Wilkie
etal. 2011) in parallel to the accumulation of large quantities of neutral lipids (up to 80% by weight of dry biomass)
(Chisti 2007).

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Green microalgae, especially within the genus Chlorella,

are known for their great potential for high-quality biofuel
production as they can grow in a variety of environmental
conditions, use different carbon sources, achieve high biomass and lipid content (up to 55% of cellular dwt) with
adequate fatty acid composition (Chen and Walker 2011;
Guarnieri etal. 2011; Heredia-Arroyo etal. 2010; Miao
and Wu 2004; OGrady and Morgan 2011; Xu etal. 2006).
Several studies have investigated the effect of nitrogen starvation as an inducer for triacylglycerol (TAG) accumulation in green algae, with most of the studies conducted in
the model green algae Chlamydomonas reinhardtii (Goodson etal. 2011; James etal. 2011; Miller etal. 2010; Mujtaba etal. 2012; Nguyen etal. 2011; Yeh and Chang 2011).
However, many aspects of TAG accumulation in response
to nitrogen starvation remain unresolved. For instance,
most studies on TAG accumulation examined cells after
24h or later time points of N starvation, not clarifying
the early events of the process. Moreover, it is not known
whether what is observed in Chlamydomonas is conserved
in other green algae with greater potential for outdoor,
large-scale production of biofuels.
A study on Chlamydomonas suggested that the de
novo synthesis of fatty acids in the chloroplast is a limiting factor for TAG production, although the recycling of
membrane lipids could account for up to 30% of the TAG
produced under N starvation (Fan etal. 2011). Moreover,
a tracer experiment with 13C-labeled glucose in Chlorella
protothecoides indicated overall stability in global carbon
flux during N starvation but did not assess the contribution
of recycling of membrane lipids into TAG (Xiong etal.
2010). In this study, a series of radiolabeling experiments
with 14C-acetate was performed to track membrane lipids
remodeling into TAG during N starvation in a green algal
strain with potential for outdoor cultivation, with further
identification of lipids through HPLC/UV/(+)ESIMS2.
Molecular phylogeny of cDNAs was used to confirm the
taxonomic status of the strain. A time-course transmission
electron microscopy (TEM) study revealed major ultrastructural changes occurring during the early and late events
of lipid droplet formation.

medium (BAM) (Sorokin and Krauss 1958) was prepared

with the following composition (in g/L): KNO3, 1.25;
KH2PO4, 1.25; MgSO47H20, 1.0; CaCl2, 0.0835; H3BO3,
0.1142; FeSO47H20, 0.0498; ZnSO47H20, 0.0882;
MnCl24H2O, 0.0144; MoO3, 0.0071; CuSO45H2O,
0.0157; Co(NO3)26H20, 0.0049; EDTA, 0.5; and filtersterilized glucose, 9. The pH was adjusted to 6.8 before
autoclaving. For the nitrogen-free (N) medium, KNO3
was replaced by KCl for the same final potassium concentration. To induce nitrogen deficiency, algal cells previously grown in N-replete medium were washed once in
N medium, suspended in fresh N medium and cultured
for 348h. Cell counting was done using a hemocytometer counting chamber (Fisher Scientific, USA) and turbidity measurements were done using a spectrophotometer
(DU730, Beckman Coulter, Pasadena, CA). The relationship between OD600 and cell density was determined as 1
OD600=8.3108cells/mL within a maximum deviation
of 14% of that relationship in both nitrogen-replete and
nitrogen-deficient cultures. For calculations, we therefore
employed OD600 for normalizing for cell numbers.
Lipid staining using Nile red
Detection of intracellular lipid bodies was conducted as
previously described (Chen etal. 2009). Corresponding
amounts of cells were treated with a solution of the vital fluorescent stain Nile Red (Greenspan etal. 1985) (50g/L)
and DMSO (20% v/v) to increase membrane permeability.
After 10min of incubation, the relative fluorescence (excitation at 530nm and emission at 570nm) was measured in a
Synergy HT microplate reader (Biotek, USA).
Gravimetric method to quantify lipid productivity

Materials and methods

Cells from nitrogen-replete or nitrogen-depleted cultures

were collected by centrifugation at 3,500rpm at 10C for
15min in a Heraeus Labofuge 400R centrifuge in 50-mL
tubes. The cell pellets were extracted for total lipids as
described elsewhere. The solvent in the lipid extract was
evaporated in a nitrogen evaporator (Organomation, MA)
and the total lipid fraction was dried in an oven set at 50C
until two consecutive weighings were constant. The lipid
yield was expressed as g/100g dry weight of cells.

Algal cultures and nitrogen starvation

Transmission electron microscopy

UTEX29 (as of Jan 2012, named as C. protothecoides by

the stock center) was purchased from the algae culture
collection at the University of Texas, Austin, and grown
in liquid medium under a light:dark regime of 18:6h
(80 mol photons/m2/s), under continuous agitation at
130rpm. A nitrogen-replete (+N) mineral basal algal

Algal cells were collected by centrifugation (5min,

3,500rpm in swing-out rotors of a Heraeus Labofuge
400R centrifuge) fixed in 3% (v/v) glutaraldehyde in BAM
medium overnight and washed three times with 0.1M
sodium cacodylate, 2mM MgCl2, 1mM CaCl2, 0.25%
(wt/v) NaCl pH 7.23. Fixed cells were processed with the

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aid of a Pelco BioWave laboratory microwave (Ted Pella,

Redding, CA, USA). The samples were buffer washed,
post-fixed with 2% (wt/v) OsO4, water washed, dehydrated
in a graded ethanol series 25, 30, 40, 50, 55, 60, 65, 80,
90, 100% (v/v), infiltrated with LRWhite acrylic resin
(Electron Microscopy Sciences, Hatfield, PA) and cured at
60C for 2days. Cured resin blocks were trimmed, thin
sectioned and collected on formvar copper 200 mesh grids,
post-stained with 2% aqueous uranyl acetate and Reynolds lead citrate. Sections were examined with a Hitachi
H-7000 TEM (Hitachi High Technologies America, Inc.,
Schaumburg, IL) and digital images were acquired with
a Veleta 2k2k camera and iTEM software (Olympus
Soft-Imaging Solutions Corp., Lakewood, CO). The TEM
experiments were done in triplicates and repeated twice.
Three slides were prepared from each replicate and 30cells
were analyzed from each slide.

Analysis of lipids using thin layer chromatography

Total lipid extracts were separated in baked ammonium
sulfate-impregnated silica plates (J.T. Baker, USA) by thin
layer chromatography (TLC) using a double-development
solvent system (2/3 in acetonetoluenewater (91:30:3 by
volume) followed by full development in hexanediethyl
etheracetic acid (70:30:1 by volume) (Fan etal. 2011).
TLC plates were briefly stained with iodine vapor for quick
visualization of lipid bands. Bands were identified based on
their relative mobilities (Rf) compared to standard lipids.
For the quantification of radioactivity, the bands were
scrapped off the plate, mixed with 2mL distilled water and
2mL Ready Gel liquid scintillation cocktail, homogenized
and radioactivity counted in a LS6500 liquid scintillation
counter (Beckman, USA).
HPLC/UV/(+)ESImass spectrometrymass spectrometry

Radiotracer labeling using 14Cacetate

[2-14C]Acetic acid (Na salt in water) was purchased from
American Radiolabeled Chemicals Inc (St. Louis, MO). For
labeling studies, the cells were initially grown in 250mL
flasks containing 100mL of +N medium until the mid-logarithmic phase, transferred to 4.5mL glass vials for a 24-h
incubation with 3mL of +N medium containing [14C]-acetate (50nci/L), followed by centrifugation for 10min at
3,500rpm in a swing-out rotor of a Heraeus Labofuge 400R
centrifuge, removal of supernatant, and finally addition of
+N or N, label-free medium, for a 48-h growth period.
In the pulse-chase experiment, cells were incubated for
1h in +N medium containing [14C]-acetate (50nci/L),
centrifuged for 10min at 3,500rpm in a swing-out rotor
of a Heraeus Labofuge 400R centrifuge, supernatant was
removed, cell pellets were washed with N medium and
suspended in N medium containing 0.5mM unlabeled
acetate. The experiments were conducted in triplicate.
Extraction of total lipids
Equal number of cells were harvested from different treatments by centrifugation (10min, 8,000rpm, in an Eppendorf 5418 centrifuge) and frozen in liquid nitrogen. The
lipid extraction method reported by Fan etal. (2011) was
used with minor modifications. Total lipids were extracted
with methanolchloroformformic acid (2:1:0.1, v:v:v) in
the presence of glass beads under maximum agitation for
5min using a Vortex Genie 2 vortex (Fisher Scientific).
Maximum extraction efficiency was achieved by repeating
this step 7, followed by phase separation using 1M KCl,
0.2M H3PO4 solution. This procedure was sufficient to
extract all lipids detectable by iodine staining in thin layer
chromatography (TLC).

Lipid bands isolated from the TLC plates were dissolved

in isopropanol and separated using an HPLC (1100 series
model G1312A binary pump, Agilent, Palo Alto, CA)
equipped with a C8 column (Hypurity 5m; 2.1100mm
with a C8 guard column; Thermo Scientific) and gradients
utilizing a binary mobile phase system of water and isopropanol (both were HPLC-grade; Honeywell Burdick &
Jackson, Muskegon, MI) with a flow rate of 0.15mL/min.
For the data presented here, the gradient was set to 30%
isopropanol at time 0 and then increased linearly to 60%
isopropanol in 15min and then to 95% isopropanol in
50min and then held at 95% isopropanol for 20min. All
mass spectrometric data were obtained with a ThermoFinnigan (San Jose, CA) LCQ fitted with the conventional electrospray ionization source. The sheath and auxiliary gases
were nitrogen (65 and 5, respectively, instrumental unitless
parameter) and the heated capillary temperature was set
at 250C. For (+)ESIMSn, the spray voltage was set at
3.3kV, the heated capillary voltage at +12.5V and the tube
lens offset at 0V. Collision-induced dissociation (CID) tandem mass spectrometry (MSn) of [M+Na]+ ions was performed with 37u precursor ion isolation, 30ms activation
time and either 37.5% normalized CID energy at 0.25qCID
or 40% normalized CID energy at 0.30qCID. Ultraviolet/
visible (UV) detection at 210 or 220nm was obtained with
an Agilent 1100 series G1314A UV/Vis detector.
Cloning 18s rRNA and actin mRNA partial sequences
from UTEX29
RNA extraction was done using NucleoSpin RNA Plant
Kit (MachereyNagel, Germany). Synthesis of cDNA
and RT-PCR were performed using Superscript One-Step
RT-PCR with Platinum Taq (Invitrogen, USA) according

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to the manufacturers instructions. The primers for 18s

rRNA were 5-WACCTGGTTGATCCTGCCAGT-3 and
5-GATCCTTCYGCAGGTTCACCTAC-3. The primers
for actin were 5-GTGACCAACTGGGACGAC-3 and
5-CGGGCAGCTCGTAIGTCTT-3. The gel-extracted
PCR products were cloned into pCR 2.1-TOPO vector and
sequenced (Sambrook 2001).
Molecular phylogeny of UTEX29 partial sequences
Database searches were performed using UTEX29 18s
rRNA and actin cDNA sequences as queries. Homologous
sequences within the taxa green algae were obtained by
a Basic Local Alignment Search Tool (BLAST) search at
the National Center for Biotechnology Information. The
sequences with the best e value, coverage and supporting literature were chosen for further analysis (Table S1). Multiple
sequence alignment was conducted using ClustalW software
in MEGA5 (Tamura etal. 2011). Phylogenetic trees were
inferred from the aligned sequence data using the neighborjoining method (Saitou and Nei 1987) in MEGA5, with the
tree being tested by bootstrapping with 1,000 replicates.
Statistical analysis
Quantitative data were analyzed using JMP software, Version 7 (SAS Institute Inc., Cary, NC). Means were compared using Students t test, =0.05. All experiments were
conducted in triplicate.

Results
Lipid accumulation
Upon arrival of strain 29 from the UTEX algae collection, cells were repeatedly streaked in sterile agar plates
Fig.1Ultrastructure of
nitrogen-starved cells indicated
lipid accumulation and plastid
degradation. a Cells grown in
N-replete medium for 48h. b
Cells grown in N-free medium
for 48h. p Pyrenoid, s starch,
LB lipid body, Ch Chloroplast.
See Materials and methods
for details on processing the
cells

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containing BAM to guarantee that all experiments were

done using axenic cultures. An evaluation of lipid accumulation using Nile Red staining of cells (Greenspan
etal. 1985) confirmed that Chlorella sp. UTEX29 is an
oil-accumulating species and demonstrated the effect of
its cultivation under photoheterotrophic conditions (under
light and addition of glucose into the medium). There was a
~fivefold increase in relative intracellular neutral lipids (as
measured by Nile Red fluorescence) upon 24h of growth
under N-free medium compared to N-replete cultures (Supplemental Fig.1a). Total lipid content of N-deplete cultures, evaluated by gravimetric method, was 68% by dry
weight (Fig.1b).
Ultrastructure of nitrogenstarved cells
In order to investigate the event of membrane recycling
and the formation of lipid bodies under N-starvation stress,
sample cells were fixed for TEM after growing them in
the presence or absence of N. The microscopic observations supported and confirmed the increased formation of
lipid bodies after 48h of stress, first detected by the Nile
Red assay (Supplemental Fig.1). The ultrastructure of
cells from N-replete medium indicated abundant presence
of starch granules and pyrenoid structures in a single large
chloroplast, with rare presence of small LBs (Fig.1a), as
previously described in ultrastructural studies in other
Chlorella species (Bertagnolli and Nadakavukaren 1970;
Pyliotis and Goodchild 1975). Cells in the N-free medium
displayed very abundant and large lipid bodies and morphological symptoms of programmed cell death (Zuo
etal. 2012), with most organelles undergoing fragmentation (Fig.1b). Of 30 cells randomly chosen for observation from 3 independent microscopic preparations, only 2
of the control cells (under +N condition) had small LBs,
whereas 29 of the 30 observed cells under 48h of N starvation contained large LBs. This result led us to investigate

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Fig.2Lipid bodies could be
observed as early as 3h of N
starvation. a, b Cells grown in
N-replete medium for 3h. c, d
Cells grown in N-free medium
for 3h. ch Chloroplast, LB
lipid body, m mitochondria, n
nucleus, p pyrenoid, s starch

the early time points in N starvation and determine the

initial moment of lipid body formation. Cells were fixed
after 3, 6, 12, and 24h following the transfer of the cells
to N medium. The cells under N-replete conditions (control) for 3h presented large single chloroplasts filled with
starch granules and pyrenoid structures, and organelles
such as nucleus and mitochondria but were absent in LBs
(Fig. 2a, b). In contrast, a great increase in LB formation
was detected after 3h under N starvation (Fig.2c, d). Of
the 30 cells randomly chosen for observation in 3 independent microscopic preparations, 18 cells under N contained LBs and none under +N contained LBs. The lipid
bodies were mainly found outside of the chloroplast, with
a single observation at 6h of N starvation in which LB-like
structures previously described as plastoglobuli (Goodson
etal. 2011; Murphy 2012) were observed inside the chloroplast (Fig.3).

suggested that a more in-depth taxonomic characterization of UTEX29 strain was required. We first conducted a
growth experiment to test this strains nutritional requirements. In contrast with the nutritional requirements
reported for C. protothecoides (Huss etal. 1999), UTEX29
was able to grow in the absence of thiamine and with
nitrate as the only N source in the medium (Supplemental Fig.2). Furthermore, UTEX29 partial 18s rRNA and
actin cDNA sequences were obtained and compared to
other green microalgae to infer their evolutionary relationship. The phylogenetic trees for both 18s rRNA and actin
(Fig. 4a, b) indicate that UTEX29 strain is, in fact, more
phylogenetically related to Chlorella vulgaris than to Auxenochlorella protothecoides, highlighting the difficulties
and the importance of correctly classifying and identifying
microalgae using a combination of ultrastructure, physiology and molecular phylogeny.

Taxonomic characterization of Chlorella sp. UTEX29

14

The presence of pyrenoid structures has never been

reported in C. protothecoides (Guiry 2013) and, thus,

The ultrastructural observation of chloroplast membrane

degradation upon long periods of N starvation (Fig.1a)

Cacetate labeling of fatty acids

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not shown). The band intensities in Fig.5b were due to the

radioactivity in the different lipid fractions, revealed by the
autoradiograph of the TLC plate. Qualitative assessment of
autoradiograms showed that N-starved cells had increased
14
C in TAG and decreased 14C in other fractions containing different species of polar lipids (P1P6), compared to
parallel cells that were grown under N-replete conditions
(Fig. 5b). This result suggested a substrateproduct relationship between polar lipids and TAG. Quantitative analysis of the different lipid fractions confirmed this observation (Fig.5c). The significant reduction of radioactivity
in P3 and P5 upon N starvation and the fact that we were
not able to definitely identify them using lipid standards
for TLC led us to prioritize the identification of those lipid
fractions using mass spectrometry.
Identification of TLC lipid fractions
Fig.3Ultrastructure of lipid bodies (LB) and plastoglobuli (arrow)
in a cell exposed to N starvation for 6h

suggested that recycling of the acyl groups from membrane

lipids could be a contributor to the newly synthesized TAG
upon such stress. In order to verify that, an experiment
was designed to label all fatty acids by incorporation of
14
C-acetate under N-replete conditions (Fig.5a). Cells were
incubated with 14C-acetate for 24h to permit unbiased
labeling of all classes of fatty acids regardless of its synthesisdegradation kinetics. Following this step, the cells were
transferred into fresh N-replete or N-free media for 48h,
the total lipids were extracted and separated by TLC (plate

Fig.4Phylogenetic trees of
Chlorella sp. UTEX29 partial
nucleotide sequences compared to other green algae.
Identification of sequences
used for this analysis is listed
in supplemental Table2.
The percentage of replicate
trees in which the associated
taxa clustered together in the
bootstrap test (1,000 replicates)
is shown next to the branches.
The tree is drawn to scale, with
branch lengths in the same
units as those of the evolutionary distances used to infer the
phylogenetic tree. a 18s rRNA,
b actin cDNA

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P3 and P5 bands from N-replete cell samples were independently scrapped off the TLC plate and isopropanol
solutions of these bands were further characterized via
C8 HPLC/(+)ESIMSn. With (+)ESIMS, the lipids
produced mainly [M+Na]+ ions with the molecular
weights (M) being consistent with that of galactolipids,
digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG), respectively (Fig.6). For the
P3 fraction, compounds eluted between 30 and 60min
with m/z 887.6, 957.3, 953.6, 955.7, 931.6, 937.5, 939.6,
941.6, 764.2 and 806.3 [M+Na]+ ions consistent with
the expected species of DGDG with differing lengths of
fatty acids (Fig.6a). Product ions from dissociation of

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(a)
*Centrifuge
*Wash (-N) +N for 48h
*Resuspend

Total Lipids
extracted
and separated
by TLC

+N for 24h
+N
TAG

P:6
P:5
P:4
P:3
P:2

-N

-N for 48h

(c)
Radioactivity (%)

(b)

80

Plus N

Minus N

70
60
50
40
30
20
10

0
P1

P:1

P2

*
P3

*
P4

*
P5

P6

TAG

Lipid fraction

Fig.5Tracking membrane remodeling by 14C-acetate labeling of

fatty acids. a Experimental scheme: cells in +N medium were incubated with 14C-acetate for 24h, the culture was then divided into two,
centrifuged (5min, 5,000g), pellets were washed with N medium
and resuspended in +N or N medium. After 48h of growth, total
lipids were extracted and different lipid classes were separated by
thin layer chromatography. b Autoradiography of TLC plate in which
lipid extracts from cells under +N and N were loaded side by side
(only 1 of the 3 replicates is shown). The arrows indicate the TAG
band, in which the intensity increases under N, and two lipid frac-

tions, referred as P3 and P5, in which the band intensity decreases

under N compared to control. c TLC zones were scrapped off and
radioactivity measured by liquid scintillation counter. Radioactivity in
each band was expressed as percentage of total radioactivity for each
specific treatment. (Experiment was done in triplicates. Bars represent mean and standard error for six observations from two experiments.) Total radioactivity in lipid fractions (100%) was 3.9nCi for
+N and 7.5nCi for N treatment, respectively. The asterisk indicates significant differences between means of control and corresponding N treatments by a t test (=0.05)

the [M+Na]+ ions confirmed the parent molecules to

be DGDGs (Table S2). For the P5 fraction, compounds
eluted between 30 and 60min with m/z 785.4, 787.4,
787.5, 769.6, 771.6, 773.6, 779.5, 619.6 and 806.2
[M +Na]+ ions consistent with the expected species of
MGDG with differing lengths of fatty acids (Fig.6b). The
patterns of the product ions from parent [M+Na]+ ions
observed in the P5 fraction were consistent with the presence of MGDG in the fraction P5 (Table S2). The regiochemical distribution of the acyl chains in MGDG and
DGDG (Tables S2, S3, respectively), considering no modifications occurred to the molecules while processing the
samples, were tentatively deduced as previously described
(Guella etal. 2003). Using the same strategy, phosphatidylcholine (PC) was identified as the major contributor to
TLC fraction P1. Phosphatidylethanolamine (PE) is also
expected to be present in P1, by comparing to the previously described Rf values in this solvent system (Fan
etal. 2011), but its low abundance may have hindered

detection. We tentatively identified P2 as sulfoquinovosyldiacylglycerol as its mobility in the TLC plate (Rf
values) matched the previously reported values in Chlamydomonas (Fan etal. 2011). Fraction P4 co-migrated
with P3 and was analyzed as a single sample, reflecting
the wide variety of DGDG species detected (Table S3).
Fraction P6 co-migrated with pigments such as chlorophylls and was not identified in this study.
Pulsechase of 14Cacetatelabeled fatty acids
A pulse-chase experiment was done to investigate the
moment in which the remodeling of polar lipids into
TAG began after N starvation. After incubating the cell
cultures for 1h with 14C-acetate, fatty acid labeling was
stopped by transferring the cell cultures to fresh N-replete
or N-free medium containing excess unlabeled acetate.
The cells were harvested after 1, 3 and 12h following this
transfer. Total lipids were extracted and fractions were

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BP6

(a)

BP7

100
BP1

95
90
85

BP8
BP2

80

Relative Abundance

Fig.6Identification of P3 and
P5 TLC bands by C8 HPLC/
(+)ESIMS. HPLC/(+)ESI
MS (m/z 700900) base peak
chromatograms for P5 (a) and
base peaks (m/z 8501,100) for
P3 (b). Sample-related peaks
whose MSn product spectra
are consistent with the MGDG
class of lipids in P5 (a) and with
the DGDG class of lipids in P3
(b) are shown as BP1BP11
for P5 and BP18 for P3. The
m/z values with the retention times, shown minutes in
brackets are as follows: For P5,
BP1BP11 were 785.6 (25.1),
787.5 (25.9), 789.4 (27.3),
789.6 (28.4), 793.6 (29.3),
769.5 (31.4), 771.6 (32.9),
773.5 (34.1), 775.5 (35.6),
777.5 (36.5), and 778.6 (38.0),
respectively. For P3, BP1BP8
were 953.6 (25.9), 955.7 (27.3),
931.6 (29.5), 1,085.3 (30.47),
937.6 (32.82), 939.6 (33.9),
941.6 (35.6) and 941.7 (36.33).
respectively. See Tables S2 and
S3 for the details of MS/MS
products

75
70
65
60

BP10

BP3

55
50

BP9

45
40
BP11

35
30

BP4

25

BP5

20
15
10
5
0

10

12

14

16

18

20

22

24

26

30

28

32

34

36

38

40

42

Time (min)

(b)

BP6

BP7

100
95
90
85

Relative Abundance

80

BP2

75
70
65
BP5

60
55
BP1

50
45
40
35

BP3

30

BP4

25

BP8

20
15
10
5
0

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

Time (min)

separated in a TLC plate, which was exposed to an X-ray

sheet. There was a significant increase in the percentage
of radioactivity in TAG and decreases in the percentage
of radioactivity in MGDG and in the fraction containing
phospholipids as early as 3h of N starvation compared to
cells in the N-replete condition (Fig.7). There was also a
further decrease in radioactivity in MGDG from 3 to 12h
of N. No significant changes were observed in DGDG
(Fig. 7) or in the fraction containing the remaining unidentified 14C lipids, which co-migrated with pigments
(data not shown).

13

Discussion
The accumulation of LBs by microalgae is a metabolic
response that may enable the cells to efficiently store
energy, especially under unfavorable environmental conditions, and to rapidly utilize the acyl groups from TAG
to synthesize new membranes or other metabolites once
favorable growth conditions resume (Murphy 2012; Waltermann and Steinbuchel 2005). In this study, ultrastructural observations clearly showed the effect of N removal
in the collapse of membrane systems with concomitant

Planta
60

TAG

40
30

bc

ab

20

% radioactivity

% radioactivity

50

DGDG

50

60

40
30

ab

ab

ab

ab

20
10

10

0
N+ 1h N-1h

N+3h N- 3h

N+ 1h N-1h

N+12h N-12h

MGDG

N+3h N- 3h

N+12h N-12h

Phospholipids

60
60

40
30
20
10

ab

ab
d

% radioactivity

50

% radioactivity

Fig.7Pulse-chase of 14C-acetate-labeled fatty acids suggests

galactolipids being recycled into
TAG as early as 3h upon N.
After a short-term labeling (1h)
of fatty acids with 14C-acetate,
unlabeled acetate was added,
cells were centrifuged down,
washed and transferred into
N-free medium. Sampling was
done at 1, 3 and 12h after transfer into N-free medium. Bars
represent the mean and standard
errors for three replicates. Columns not marked by the same
letters are significantly different
(=0.05). Total radioactivity
in lipid fractions (100%) for
+N treatments was 1.2, 1.31,
and 0.9nCi for 0, 3, and 12h,
respectively. For N treatments,
the values were 1.62, 1.63,
and 1.2nCi for 0, 3, and 12h,
respectively

50
40

ab

a
bc

30

20
10
0

N+ 1h N-1h

N+3h N-3h

accumulation of LBs after 48h (Fig.1). While the pulsechase experiment suggested TAG accumulation as early as
3h of N (Fig.7), no significant difference (=0.05) in
TAG was detected using the Nile Red fluorescence assay
(data not shown). However, the early event of LB formation (after 3h of N) was confirmed by the transmission
electron microscopic observations (Fig.2), demonstrating
the importance of this approach to overcome the limitations in detection levels of indirect methods for measuring
intracellular TAG, such as the Nile Red fluorescence assay.
Although some studies have shown ultrastructural changes
in response to N deprivation in different algal species
(Garca-Ferris and de los Ros 1996; Goodson etal. 2011;
Pyliotis and Goodchild 1975), this is the first study in commercially important biofuel green algae to investigate the
ultrastructural changes occurring in the early events (3h) of
N deprivation. Another interesting ultrastructural observation was that the starch granules observed in abundance and
filling large areas of the single chloroplast in the control
cells (Figs.1a, 2a, b) were not observed in the cells under
N deficiency (Figs.1b, 2c, d). This result is consistent with
previous observations (Wang etal. 2009), in which it was
demonstrated that starchless mutants of Chlamydomonas
accumulate up to 30-fold the amount of TAG (approximately 400mg/109cells) compared to the wild-type strain,
after 48h of N starvation. This indicates that starch and
TAG are competing carbon sinks and that degradation of
starch during N may increase the carbon flux into acetylCoA and TAG de novo synthesis. Since the presence of
plastoglobuli was observed in one of the examined cells
(Fig. 3) as also observed by Orus and Martinez (1991), it

N+12h N-12h

N+ 1h N-1h

N+ 3h N-3h

N+12h N-12h

is possible that a chloroplastic pathway for TAG synthesis may also be present in Chlorella as it was described in
Chlamydomonas (Fan etal. 2011; Goodson etal. 2011).
However, the fact that it was observed only once indicates
it may not be the preferential route for TAG synthesis under
the experimental conditions employed. Interestingly, N
deficiency in the higher plant Arabidopsis thaliana induces
the partial mobilization of acyl groups from galactolipids
to plastoglobules in the chloroplast, where fatty acid phytyl
esters accumulate (Gaude etal. 2007), but the enzyme systems involved have not been identified.
The correct identification of green microalgae, especially within the Chlorella genus, which comprises
more than 100 species described (Krienitz etal. 2004),
is a complex task that may be overlooked in some studies and that is key for drawing correct conclusions when
studying axenic cultures. The identification of pyrenoids,
intracellular structures found in C. vulgaris, in our TEM
study (Figs.1, 2) prompted us to examine if Chlorella sp.
UTEX29 is correctly assigned as C. protothecoides. The
nutritional requirements experiment (Supplemental Fig.2)
showed that UTEX29 does not require thiamine and can
utilize ammonium as N source for normal growth. The
phylogenetic studies with UTEX29 18S rRNA and actin
cDNA partial sequences (Fig.4) indicate that UTEX29
strain is more structurally, nutritionally and phylogenetically related to C. vulgaris than to C. protothecoides. This
result raises the question of how reliable is the species-level
identification and the importance of using complementary
approaches when investigating the identity of algae used in
biofuel industry.

13

 Planta

This study demonstrates that the oil-accumulating

characteristic of the green algae Chlorella sp. UTEX29 is
reproducibly triggered by N removal from the medium,
with a ~fivefold increase in detectable lipid content within
24h of N (Supplemental Fig.1). Several studies have
investigated the effect of nitrogen starvation as an inducer
for TAG accumulation in both the genus Chlorella (Hortensteiner etal. 2000; Pyliotis and Goodchild 1975; Xiong
etal. 2010; Yeh and Chang 2011) and a related species, the
model green algae C. reinhardtii (Fan etal. 2011; Goodson
etal. 2011; James etal. 2011; Miller etal. 2010; Nguyen
etal. 2011; Yeh and Chang 2011). However, these studies
focus at 24h or later time points of N starvation, without
clarifying the early events on lipid body formation. Moreover, information regarding the contribution of membrane
lipids toward TAG synthesis is also very limited to date. A
study on Chlamydomonas indicated that the de novo synthesis of fatty acids in the chloroplast is a limiting factor for
TAG production, and assuming that the decreases in polar
lipid amounts during N are entirely mobilized into TAG,
Fan etal. (2011) suggested that recycling of membrane
lipids could account for up to 30% of the TAG synthesis
after 48h of N deprivation. By labeling the fatty acids with
14
C-acetate and tracking the radioactivity shifts after 24h
of N removal (Fig.5), Fan etal. (2011)s assumption was
confirmed, as a 34% decrease in polar lipids radioactivity was detected, which accounted for the same percentage
increase in TAG radioactivity (Fig.5c). The 14C-acetatelabeling experiment in this study provides evidence in vivo
to demonstrate a substrateproduct relationship between
TAG and structural polar lipids upon N deficiency, providing a key support for the hypothesis of membrane recycling
into TAG. While our study was in progress, Li etal. (2012)
reported that the activity of a galactoglycerolipid lipase
(PGD1) was critical in TAG accumulation under N starvation in C. reinhardtii (Li etal. 2012), consistent with our
findings in Chlorella.
The TLC lipid zones P3 and P5, which showed a significant decrease in radioactivity under N conditions, were
identified by ESIMS/MS as the galactolipids MGDG and
DGDG, which account for up to 80% of membrane lipids
in green plant tissues (Guella etal. 2003). This result suggests that galactolipids may be a major contributing source
of acyl groups for the synthesis of TAG under N. Although
GCMS is routinely used to identify fatty acid composition
of lipid compounds, the LC(+)ESIMS/MS approach performed in this study has the advantage of generating highresolution lipid profiles without the fatty acid methyl esterification (FAME) step, and still obtain information about the
acyl chains. The fatty acid composition of the lipids was possible by analyzing the CID products of the [M+Na]+ ions
(Tables S1, S2), and the putative regiochemical distribution
was deduced as previously described (Guella etal. 2003). The

13

possible identification of odd-chain fatty acids C19:1, and

C19:2 at the sn-1 position of galactolipids needs further study.
The significant increase in radioactivity ( =0.05) in
TAG observed as early as 3h of N starvation with concomitant decreased radioactivity in both MGDG and phospholipids observed with the pulse-chase experiment (Fig.7) suggests that lipases and acyltransferases may be involved in the
response to this nutrient stress at an early time point following N starvation. In fact, two recent transcriptomic studies
in Chlamydomonas under N detected upregulated genes
encoding putative lipases that could play a role in releasing fatty acids from membrane lipids (Boyle etal. 2012;
Miller etal. 2010). Moreover, a recent proteomics study in
Chlamydomonas identified lipase-like oil body associated
proteins, which may be localized at contact sites between
LBs and chloroplasts, and thus, be involved in the degradation of plastidial membranes (Nguyen etal. 2011). In Chlamydomonas, one of the enzymes involved in this process is
phospholipid:diacylglycerol acyltransferase (PDAT), which
was recently shown to have both acyltransferase and lipase
activities in a variety of substrates, such as phospholipids,
galactolipids, and TAG (Yoon etal. 2012). While PDAT and
PGD1 homologs would be expected to play a similar role in
Chlorella, other enzymes which have not been fully characterized may also contribute to this phenomenon of membrane
recycling of fatty acids into TAG. In fact, the replacement of
the membrane lipid phosphatidylcholine present in Chlorella
by diacylglycerol-N, N, N-trimethylhomoserine (Fan etal.
2011) in Chlamydomonas, confirmed in this study, illustrates
the evolutionary divergences between the two genera.
The present study shows that the recycling of structural
membrane lipids significantly contributes to the increased
flux of acyl moieties into TAG starting as early as 3h of
N starvation in Chlorella. It also provides key ultrastructural information that will serve as a basis for subsequent
high-throughput proteomic and transcriptomic studies to
investigate the initial and late metabolic changes, leading
to the trigger of LB formation and TAG accumulation in
oleaginous green algae.
Acknowledgments We thank Dr. Byung-Ho Kang, Karen Kelley, and Kim Backer-Kelley (University of Florida, Interdisciplinary
Center for Biotechnology Research, Electron Microscopy and BioImaging lab) for help with transmission electron microscopy. The
HPLCMS-MS analyses of lipid fractions were done at Chemistry
Department, University of Florida. EG thanks the Plant Molecular
and Cellular Biology program, the College of Agriculture and Life
Sciences and the Horticultural Sciences Department, University of
Florida for a graduate research assistantship.

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