Physiological and molecular analysis of 
carbon source supplementation and pH 
stress-induced lipid accumulation in the 
marine diatom Phaeodactylum tricornutum
Authors: Florence Mus, Jean-Paul Toussaint, Keith E. 
Cooksey, Matthew W. Fields, Robin Gerlach, Brent 
M. Peyton, & Ross P. Carlson.
NOTICE: The final publication is available at Springer via http://dx.doi.org/10.1007/
s00253-013-4747-7. 
Mus F, Toussaint J-P, Cooksey KE, Fields MW, Gerlach R, Peyton BM, Carlson RP, 
"Physiological and molecular analysis of carbon source supplementation and pH stress-induced 
lipid accumulation in the marine diatom Phaeodactylum tricornutum," Applied Microbiology and 
Biotechnology, March 2013 97(8):3625–3642.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Physiological and molecular analysis of carbon source 
supplementation and pH stress-induced lipid accumulation in 
the marine diatom Phaeodactylum tricornutum
Florence Mus, Jean-Paul Toussaint, Keith E. Cooksey, Matthew W. Fields, Robin 
Gerlach, Brent M. Peyton, & Ross P. Carlson
Abstract 
A detailed physiological and molecular analysis of lipid accumulation under a suite of conditions including 
nitrogen limitation, alkaline pH stress, bicarbonate supple-mentation, and organic acid supplementation was 
performed on the marine diatom Phaeodactylum tricornutum. For all tested conditions, nitrogen limitation was a 
prerequisite for lipid accumulation and the other culturing strategies only enhanced accumulation highlighting 
the importance of com-pounded stresses on lipid metabolism. Volumetric lipid lev-els varied depending on 
condition; the observed rankings from highest to lowest were for inorganic carbon addition (15 mM 
bicarbonate), organic acid addition (15 carbon mM acetate), and alkaline pH stress (pH9.0). For all lipid-
accumulating cultures except acetate supplementation, a common series of physiological steps were observed. 
Upon extracellular nitrogen exhaustion, culture growth con-tinued for approximately 1.5 cell doublings with 
decreases in specific protein and photosynthetic pigment content. As nitrogen limitation arrested cell growth, 
carbohydrate con-tent decreased with a corresponding increase in lipid con-tent. Addition of the organic carbon 
source acetate appeared to activate alternative metabolic pathways for lipid accumu-lation. Molecular level data 
on more than 50 central metab-olism transcripts were measured using real-time PCR. Analysis of transcripts 
suggested the central metabolism pathways associated with bicarbonate transport, carbonic anhydrases, and C4 
carbon fixations were important for lipid accumulation. Transcriptomic data also suggested that repurposing of 
phospholipids may play a role in lipid accu-mulation. This study provides a detailed physiological and 
molecular-level foundation for improved understanding of diatom nutrient cycling and contributes to a metabolic 
blue-print for controlling lipid accumulation in diatoms.
Keywords: Diatom . Phaeodactylum tricornutum . Alkaline pH stress . Nitrogen limitation . Bicarbonate addition . 
Lipids . Biodiesel
Introduction
Diatoms, unicellular microalgae possessing a silicon-based cell wall and belonging to the class 
Bacillariophyceae, are  an ecologically successful taxonomic group of phytoplankton. These microalgae represent 
a large fraction of global primary productivity (Falkowski et al. 1998; Brzezinski et al. 2002; Granum et al. 
2005) and play fundamental roles in global
F. Mus : K. E. Cooksey : M. W. Fields
Department of Microbiology, Montana State University, Bozeman, MT 59717, USA
F. Mus : J.-P. Toussaint : R. Gerlach : B. M. Peyton : R. P. Carlson Department of Chemical and Biological Engineering, Montana State University, Bozeman, MT 59717, 
USA
F. Mus : J.-P. Toussaint : M. W. Fields : R. Gerlach :
B. M. Peyton : R. P. Carlson (*)
Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA
F. Mus (*)
Department of Biochemistry, Montana State University, Bozeman, MT 59717, USA
nutrient cycling of carbon, nitrogen, phosphorus, and silicon
(Werner 1977; Tréguer et al. 1995; Søndergaard and Jeppesen
2005; Jeppesen et al. 2005; Wilhelm et al. 2006). In addition
to their fundamental role in global nutrient cycles, diatoms
represent a potential bioprocess platform for synthesizing
biofuels and other value-added products (Hildebrand et al.
2012). Interest in renewable biofuels has increased in recent
years due to instability in petroleum costs and climate change
data correlated to greenhouse gases. Microalgae are of con-
siderable interest because many accumulate significant
amounts of energy-rich compounds, such as triacylglycerol
(TAG) or other lipids that can be harvested as biofuel precur-
sors (Sheehan et al. 1998; Dismukes et al. 2008).
Increased levels of TAG ssynthesis occur when oleaginous
algae are subjected to nutrient imbalances or culturing stress
imposed by chemical or physical stimuli. Nitrogen is a com-
monly reported nutrient-limiting factor triggering lipid accu-
mulation in microalgae (e.g., Shifrin and Chisholm 1981;
Tornabene et al. 1983; Roessler 1990a, b). Deficiencies of
other nutrients including phosphate (Khozin-Goldberg and
Cohen 2006), sulfur (Sugimoto et al. 2008), silicon (Coombs
et al. 1967; Roessler 1990a, and b) iron (Liu et al. 2008) or
combinations thereof (Valenzuela et al. 2012) have been
reported to stimulate lipid/TAG biosynthesis. The major
chemical stimuli linked to TAG accumulation are salinity
and growth medium pH (Cohen et al. 1988; Guckert and
Cooksey 1990; Tatsuzawa et al. 1996; Azachi et al. 2002). A
physiological role for TAG is to serve as a carbon and energy
storage material, particularly in stressed or aged algae.
Additionally, TAG synthesis can consume excess electrons,
alleviating an over-reduced electron transport chain during
culturing periods with high photon fluxes. A comprehensive
understanding of lipid and TAG metabolism and its role in
microalgal ecology is essential for informed culturing and
engineering strategies to optimize biofuel potential.
Lipid metabolism, particularly the biosynthetic pathways
of fatty acids (FAs) and TAGs, is poorly understood in micro-
algae as compared to higher plants. However, based upon
gene sequence homology and some shared enzymatic charac-
teristics, it is believed generally that microalgae and higher
plants share many basic pathways for FA and TAG biosyn-
thesis (Ratledge et al. 1988; Roessler 1988; Andre et al. 2007;
Hu et al. 2008). Biosynthesis of FAs, the building blocks for
TAGs and membrane lipids, occurs in the chloroplast and is
catalyzed by two conserved enzymes: acetyl-CoA carboxyl-
ase and type 2 fatty acid synthase (Riekhof et al. 2005;
Riekhof and Benning 2009; Moellering et al. 2009). The
resulting FAs can be used directly in the chloroplast to se-
quentially produce lysophosphatidic acid and phosphatidic
acid (PA). PA and its dephosphorylated product diacylglycerol
(DAG), when generated in the chloroplast, serve primarily as
precursors for structural lipids of the photosynthetic mem-
brane system (Browse and Somerville 1991; Ohlrogge and
Browse 1995). Alternatively, FAs can be exported into the
cytosol and used to produce membrane lipids or energy stor-
ing TAGs (Browse and Somerville 1991; Ohlrogge and
Browse 1995). TAGs are believed to be assembled in the
endoplasmic reticulum by the acylation of DAG by acyl-
CoA dependent diacylglycerol acyltransferase (DGAT;
Cases et al. 1998; Li-Beisson et al. 2010), and then seques-
tered in cytosolic lipid bodies. Alternative pathways to con-
vert membrane lipids and/or carbohydrates to TAG have
recently been demonstrated in bacteria, plants, and yeast using
acyl-CoA-independent routes (Dahlqvist et al. 2000; Stahl et
al. 2004; Arabolaza et al. 2008). Such pathways have not yet
been studied in microalgae.
To better understand microalgal lipid metabolism and
associated carbon fluxes, a physiological and molecular
analysis of lipid accumulation was performed in a model
oleaginous marine diatom, Phaeodactylum tricornutum.
Lipid accumulation was investigated as a function of indi-
vidual and compounded culturing perturbations including
nitrogen limitation, alkaline pH stress, and the addition of
inorganic or organic carbon sources. The use of pH stress to
induce microalgae lipid accumulation has been reported
previously with chlorophytes (Guckert and Cooksey 1990;
Gardner et al. 2011; Santos et al. 2012), but the effect of pH
on lipid accumulation in the genealogically and physiolog-
ically distinct diatoms has not been previously reported.
This study contrasts pH-induced lipid accumulation in a
diatom with nitrogen limitation (Pal et al. 2011; Gong et
al. 2012), bicarbonate salt addition (Gardner et al. 2012a;
White et al. 2012) and addition of three different organic
acids (Cooksey 1974; Cerón García et al. 2005, 2006; Wang
et al. 2012). For each of the tested culturing conditions,
fundamental physiological data including cell number, pho-
tosynthetic pigment, protein content, carbohydrate content,
and dissolved inorganic carbon levels was collected and
correlated to lipid synthesis. In addition, transcript levels
for approximately 50 genes involved with carbon fixation,
carbon concentrating mechanisms, fatty acid and TAG syn-
thesis, polysaccharide synthesis/degradation, as well as a
number of other physiological indicators were measured.
The physiological data coupled with transcript analysis pro-
vided insights into the complex P. tricornutum metabolic
pathways involved in lipid accumulation.
Materials and methods
Cell culture P. tricornutum strain Culture of Marine
Phytoplankton (CCMP) 2561 (referred to here as Pt1) was
acquired from the Provasoli–Guillard CCMP. Pt1 was cul-
tured on modified ASPII media (24×10−3M Tris, 8.61×
10−5M K2HPO4) pH7.8 (Provasoli et al. 1957). Modified
ASPII media allowed better pH control during growth and
prevented phosphate limitation. Pt1 cultures were checked
for bacterial contamination by inoculation into respective
medium supplemented with 0.1 % proteose peptone and
1 mM acetate, and incubated in the dark (Cooksey 1974).
Axenic status of the cultures was confirmed by PCR ampli-
fication of 16S rDNA of both eubacteria (primers 27F and
192R) and archaea (primers 21F and 915R; DeLong 1992;
Su et al. 2007).
Growth conditions Experiments were conducted in at least
triplicate in batch using 70×500 mm glass tubes reactors
containing 1.25 L modified ASPII media submersed in a
temperature controlled water bath. Temperature was main-
tained at 21±1 °C and light (400 μmolm−2s−1) was main-
tained on a 14:10 light/dark cycle by a T5 light bank.
Rubber stoppers, containing ports for aeration and sampling,
were used to seal the tubes. Aeration (400 mLmin−1) was
supplied by humidified compressed air and controlled using
individual rotameters on each bioreactor (Cole-Parmer,
Vernon Hills, IL, USA). Glass tubes reactors were inoculat-
ed during the dark period at 5×105cellmL−1 using expo-
nentially growing cultures (2×106cellmL−1) that were never
nitrate or phosphate depleted. Samples were collected every
24 h for 7 days at the transition from light to dark cycle.
Cell counting Cell concentrations were determined using an
optical hemacytometer (Hausser Scientificm, Horsham, PA,
USA) with a minimum of 400 cells counted for statistical
reliability.
Chlorophyll and carotenoid measurements Chlorophylls a
and c content and carotenoid content were determined spec-
trophotometrically (spectrophotometer Genesys 6, Thermo
Electron Corporation) in 100 % methanol (Lichtenthaler and
Wellburn 1983; Wellburn 1994; Ritchie 2006, 2008).
Nitrate assay Nitrate content in ASPII mediumwas measured
using a colorimetric assay based on the reaction of Szechrome
NAS reagent (Polysciences Inc., Warrington, PA, USA) with
nitrate ions. One milliliter of culture was centrifuged at
16,000×g for 15 min. The supernatant was collected for nitrate
quantification. Of the samples, 0.1 mL was gently mixed with
1 mL of reagent solution prepared as described by the manu-
facturer (Polysciences Inc., Warrington, PA, USA) and incu-
bated for 20 min at room temperature. The absorbance was
measured at 570 nm using a spectrophotometer (Genesys 6,
Thermo Electron Corporation), and nitrate concentration of the
sample was calculated using a nitrate standard curve.
Phosphate assay Inorganic phosphate content in ASPII me-
dium was measured using a colorimetric assay based on the
change in absorbance of the dye malachite green in the pres-
ence of phosphomolybdate complexes (Innova Biosciences
Ltd., UK). One milliliter of culture was centrifuged at
16,000×g for 15 min. The supernatant was collected for
phosphate quantification. Of the sample, 0.1 mL was gently
mixed with reagents prepared as described by the manufac-
turer (Innova Biosciences Ltd., UK) and incubated 30 min at
room temperature. The absorbance was measured at
630 nm using a microplate reader (FL600, BioTek
Instruments Inc., USA) and inorganic phosphate concen-
tration of the sample was calculated using an inorganic
phosphate standard curve.
Protein and carbohydrate assays P. tricornutum cultures
(10 mL) grown in ASPII media were harvested by centrifu-
gation and resuspended in 0.7 mL 50 mM Tris, 500 mM
NaCl, pH7.2 buffer supplemented with protease inhibitor
cocktail from Roche (Complete Mini, Roche Diagnostics,
In, USA). Cells were disrupted by three cycles of sonication
(7 W, 30 s; Q-Sonica LLC, XL 2000 series, Misomix
Sonicators, Newtown, CT, USA). The homogenate was
centrifuged at 10,000×g for 1 min. The pellet, containing
insoluble debris, was discarded. Protein assay and carbohy-
drate assays were performed on the same cell lysate for each
time point and tested condition. Protein was quantified using
the Quick Start Bradford Protein Assay from BioRad
(Hercules, CA, USA). Twenty microliter of sample were
mixed with 1 mL of BioRad reagent and incubated at room
temperature for 20 min. The absorbance was measured at
595 nm using a spectrophotometer (Genesys 6, Thermo
Electron Corporation). Protein content of the sample was
calculated using a standard curve (gamma-globulin stand-
ards used as described by the manufacturer).
For carbohydrate quantification, 200 μL of supernatant
were mixed with 1 mL of reagent (86 % v/v sulfuric acid;
700 mgL−1L-cysteine) and incubated at 100 °C for 3 min.
The mixture was rapidly cooled to room temperature and the
absorbance was measured at 415 nm using a spectropho-
tometer (Genesys 6, Thermo Electron Corporation).
Carbohydrate content of the sample was calculated using a
standard curve (glucose standards).
DIC measurements Seven milliliter of culture was filtered
(0.2 μm) and used to measure dissolved inorganic carbon
(DIC) using a FormacsHT TOC/TN analyzer (Skalar,
Netherlands). A standard curve was performed as described
by the manufacturer.
Lipids analysis Cellular TAG accumulation was estimated
using Nile Red (9-diethylamino-5H-benzo(α)phenoxazine-
5-one; Sigma-Aldrich, St. Louis, MO USA) fluorescence
method (Cooksey et al. 1987). The protocol was consistent
with previously described methods (Gardner et al. 2011).
Nile Red fluorescence intensity values are reported on a per
10,000 cells basis.
HPLC analysis Organic acid analysis was performed by
liquid chromatography (Agilent Technologies 1200 series
high performance liquid chromatography (HPLC), Santa
Clara, CA, USA) using an Aminex HPX-87H (BioRad,
Hercules, CA, USA) ion exchange column. Pt1 cells were
collected at the indicated times and centrifuged, and the
supernatant was transferred to a new vial and frozen at
−20 °C for subsequent analysis. Samples were thawed and
filtered prior to HPLC injection. Twenty microliters of su-
pernatant was injected onto the column and eluted with
8 mM filtered sulfuric acid at a flow rate of 0.5 mlmin−1
at 45 °C. UV detector peaks were recorded using Agilent
ChemStation software, and quantification was performed
using standard curves for each organic acid.
Extraction of RNA for real-time PCR Total RNA was iso-
lated from Pt1 cells using the Plant RNA kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s
instructions. Genomic DNA was removed from RNA sam-
ples by two successive DNase treatments. Approximately
40 μg of isolated RNAwas treated with five units of RNase-
free turbo DNase (Ambion, Grand Island, NY, USA) for
30 min at room temperature. The Qiagen RNeasy MinElute
kit (Qiagen) was used to purify DNase-treated total RNA
from degraded DNA, DNase, contaminating proteins, and
potential inhibitors of the reverse transcriptase reaction. The
concentration of the eluted RNA was determined with a
Nanodrop analyzer. Absence of genomic DNA contamina-
tion was checked by PCR.
Reverse transcription reactions First-strand cDNA synthe-
sis was primed from purified total RNA template using
(dT)12–18 primers. The reverse transcription reaction was
performed using the reverse transcriptase Superscript III
kit (Invitrogen, Grand Island, NY, USA) as described by
the manufacturer. (dT)12–18 primers were annealed to 150 ng
of total RNA and extended for 1 h at 50 °C using 200 units
of reverse transcriptase Superscript III.
Quantitative real-time PCR Levels of specific mRNA tran-
scripts from each sample were quantified by real time PCR
using the Engine RotorGeneQ system (Qiagen). One micro-
liter of single-stranded cDNA from the reverse transcriptase
reaction (see above) was used as template for the real-time
PCR experiments. The real-time PCR amplifications were
performed using reagents from the DyNAmo TM SYBR
green real-time PCR kit (Finnzymes, Lafayette, CO,
USA). Specific primers were designed to amplify gene
regions consisting of 150–200 nucleotides. The primers
used for real time PCR are described in supplemental
Table 1 and were designed using Primer3 software.
Amplification by RT-PCR of single products of the expected
sizes was verified on 2 % (w/v) agarose gels and specificity
of PCR products was verified by sequencing. Melting curve
analyses were performed on all PCR products to ensure that
single DNA species were amplified. Real-time PCR ampli-
fications were performed using the following cycling
parameters: an initial single step at 95 °C for 10 min
(denaturation) was followed by 40 cycles of the following:
(a) 94 °C for 10 s (denaturation), (b) 60 °C for 15 s (primer
annealing), and (c) 72 °C for 15 s (elongation). A final
single step at 72 °C for 10 min followed these 40 cycles.
The relative expression ratio of a target gene was calculated
based on the 2−ΔΔCT method (Livak and Schmittgen 2001),
using the average cycle threshold (CT) calculated from trip-
licate measurements. Relative expression ratios from three
independent experiments are reported. RPS (ribosomal pro-
tein small subunit 30S; 10847), TBP (TATA box binding
protein; 10199), CdkA (protein kinase; 20262) genes, previ-
ously described (Siaut et al. 2007), were used as constitutive
control gene for normalization. Relative abundances for
each tested culture conditions were then standardized to
the ASPII media pH=7.8 control condition.
Chemicals and enzymes 9-Diethylamino-5H-benzo(α)phe-
noxazine-5-one (Nile Red) stock solution was made in ace-
tone. Inhibitors ethoxyzolamide (EZA), acetazolamide
(AZA) stock solutions were made in DMSO. Nile Red and
EZA were purchased from Sigma-Aldrich; AZA was pur-
chased from Alfa Aesar (Ward Hill, MA, USA).
Software Clustering heat maps were generated with R sta-
tistical package 2.15.1 software. Transit peptide predictions
were determined using ChloroP (Emanuelsson et al. 1999),
iPSOT (Bannai et al. 2002), and TargetP (Emanuelsson et al.
2000). Nucleotide and protein sequences were obtained
from JGI P. tricornutum data bank (http://genome.jgi-
psf.org/Phatr2/Phatr2.home.html). Primers were designed
using Primer3 software (http://frodo.wi.mit.edu/primer3/).
Results
Physiological analysis of lipid accumulation
in P. tricornutum
To expand the understanding of lipid accumulation in the
marine diatom P. tricornutum Pt1, lipid accumulation under
conditions of nitrogen limitation, alkaline pH stress, dis-
solved inorganic carbon supplementation, and organic acid
supplementation was analyzed. For all reported compari-
sons, Pt1 cultures grown on ASPII only medium were
considered the control culture and all references to lipid
and TAG are based on the Nile Red assay. The cultures
were followed for 7 days with a 14:10 light–dark cycle.
Samples were collected for physiological analysis every
24 h at the light-to-dark transition. Due to the large number
of tested conditions, physiological analyses focused on a
single culture time point which correspond with the diel
cycle TAG content maxima. Transcript levels for chosen
genes were assessed during peak lipid accumulation. To
distinguish different types of data, figures reporting volu-
metric culture properties (e.g., cell number per volume) are
presented as line graphs and specific culture properties (e.g.,
carbohydrate per cell) are presented as bar graphs. Specific
culture properties are not reported during the first 2 days due
to low cell numbers introducing large parameter variability.
N-limitation-based lipid accumulation
Inducing lipid accumulation via nutrient limitation is a
widely utilized strategy. Nitrate limitation was used as the
base case lipid induction method. P. tricornutum cultures
were grown with different initial NaNO3 concentrations:
5.90×10−4M NaNO3 considered here to be the control
condition and 1.18×10−4M NaNO3 considered here to be
the reduced nitrate condition, both medium compositions
are nitrate-limited. Initial nitrate levels did not affect cell
growth rates but did impact final cell densities (Fig. 1a).
Medium pH increased slightly for the control culture due to
higher cell density (Fig. 1b). P. tricornutum Pt1 cells grown
in low nitrate medium showed a lipid accumulation peaking
at day 4 (Fig. 1d), approximately 24 h after medium nitrate
was depleted (Fig. 1c). The specific cellular lipid content
under nitrate limitation increased about fivefold relative to
the control culture (Table 1). Specific carbohydrate content
in the reduced nitrate culture decreased as lipid content
increased (Fig. 1h). Cellular photosynthetic pigment (chlor-
ophylls a+c and carotenoids) and cellular protein decrease
after the exhaustion of nitrate (Fig. 1e–g). A major differ-
ence between the experimental reduced nitrate culture and
the control culture is the fivefold difference in stationary
phase cell concentration which changed the relative avail-
ability of DIC at the time of nitrate depletion (Fig. 2a).
Extracellular phosphate concentrations were also measured
for this culture and every other tested condition. The cul-
tures were never phosphate limited; data can be found in
supplementary material (Electronic supplementary material
(ESM) Table 3).
Additional nutrient limitation conditions were explored
including sulfur and silicon limitation. None of these con-
ditions induced significant lipid accumulation with Pt1 (data
not shown).
pH stress-based lipid accumulation
pH stress has been used to induce lipid accumulation in
chlorophytes and the presented data is the first report on
pH stress and lipid accumulation in a marine diatom.
Control P. tricornutum cultures were grown at pH=7.8;
cultures were grown at pH=7.0 to establish a “low” pH
Table 1 Comparison of peak specific lipid content in P. tricornutum Pt1 grown under different conditions (n=3)
Conditions Nile Red Fluorescence per 104 cells, day4 Nile Red Fluorescence per 104 cells, day 5
Control (ASPII pH=7.8) 17.67±4.12 11.21±5.57
Reduced nitrate 99.11±6.08 26.92±4.89
Nile Red Fluorescence per 104 cells, day5 Nile Red Fluorescence per 104 cells, day6
Control (ASPII pH=7.8) 11.21±5.57 11.78±6.25
pH=7.0 3.46±0.38 2.32±0.56
pH=8.5 38.54±5.96 18.79±3.85
pH=9.0 55.49±7.23 74.39 ±1.89
NaHCO3 added at day0 NaHCO3 added at day4 NaHCO3 added at day0 NaHCO3 added at day4
5 mM NaHCO3 7.86±2.76 50.15±9.56 3.18±1.75 26.41±3.97
15 mM NaHCO3 40.71±3.45 49.62±8.19 63.71±15.0 114.30±6.60
25 mM NaHCO3 27.47±5.67
a 50.55±25.23a 22.21±13.09a 36.02±14.38a
Conditions Nile Red Fluorescence per 104 cells, day6 Nile Red Fluorescence per 104 cells, day7
Control (ASPII pH=7.8) 11.78±6.25 11.56±3.12
Formate Formate Formate Formate
Acetate Acetate Acetate Acetate
Lactate added at day0 Lactate added at day4 Lactate added at day0 Lactate added at day4
Formate (15 mM) 5.07±1.53 10.33±3.40 3.17±0.45 6.55±0.64
Acetate (7.5 mM) 47.06±8.75 43.85±3.99 75.81±19.27 51.20±7.79
Lactate (5 mM) 9.32±0.45 7.80±3.76 8.04±2.87 6.20±2.58
a Average and standard deviation calculated on six independent experiments
stress; and cultures were grown at pH=8.5 and 9.0 to
establish “high” pH stress conditions. The low and high
pH stresses reduced culture specific growth rates (Fig. 3a).
The Tris buffer was sufficient to maintain relatively stable
pH values over the course of the 7-day experiments.
Volumetric and specific Nile Red fluorescence intensity
increased significantly at elevated pH values, indicating an
increase in lipid accumulation (Fig. 3d and Table 1, respec-
tively). As seen in Fig. 3d, the maximum lipid accumulation
was observed at day5 for pH=8.5 and at day6 for pH=9.0.
Both of these maxima correspond closely with nitrate de-
pletion (Fig. 3c). Analogous to the nitrate-limited cultures,
the pH=9 cultures’ lipid maxima demonstrated a transitory
profile, the pH=8.5 cultures’ lipid profiles demonstrate a
Time (days)
Ni
le
 
Re
d 
flu
o
re
sc
e
nc
e
 
in
re
ns
ity
0
500
1000
1500
2000
Time (days)
N
aN
O 3
 
(m
g/L
)
0
10
20
30
40
50
60
70
Time (days)
Ce
ll n
um
be
r (
ce
ll/m
L)
1e+4
1e+5
1e+6
1e+7
1e+8
a
c
d
Time (days)
Ca
rb
o
hy
dr
a
te
s 
(pg
/ce
ll)
0
2
4
6
8
10
Time (days)
Pr
o
te
in
s 
(pg
/ce
ll)
0
5
10
15
20
25
Time (days)
To
ta
l c
ar
ot
en
oi
ds
 (p
g/c
ell
)
0.00
0.05
0.10
0.15
0.20
0.25
Time (days)
To
ta
l c
hl
or
op
hy
lls
 (p
g/c
ell
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6e
f
g
h
Time (days)
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
2 3 4 5 6 7
2 3 4 5 6 7
2 3 4 5 6 7
2 3 4 5 6 7
0 1 2 3 4 5 6 7
pH
7.0
7.5
8.0
8.5
9.0b
Control
Reduced nitrate
Control
Reduced nitrate
Fig. 1 P. tricornutum cultures
grown under nitrate limitation.
Control condition (ASPII pH7.8;
5.90×10−4M NaNO3; filled
circles, black bars) and reduced
nitrate condition (ASPII pH7.8,
1.18×10−4M NaNO3; circles,
gray bars). a Cell concentration,
b medium pH, c medium nitrate
concentration, d volumetric Nile
Red fluorescence, e specific
cellular chlorophyll content,
f specific cellular carotenoids
content, g specific cellular
protein content, and h specific
cellular carbohydrate content.
Average and standard deviation
calculated from three biological
replicates
more complicated trend with a peak transitioning to sus-
tained elevated lipid levels. No significant lipid accumula-
tion was observed at pH=7 (Fig. 3d). Specific carbohydrate
content in the high pH cultures decreased as volumetric lipid
levels increased. Elevated pH did not affect specific photo-
synthetic pigment (chlorophylls a+c and carotenoids) con-
tent although the cultures grown at pH=7 had a stable, high
specific pigment content presumably because the reduced
growth rate postponed nitrate depletion. The specific protein
content decreased with pH stress in comparison to the con-
trol condition. The elevated pH increased the DIC levels
over the control cultures due to the strong effect of pH on
bicarbonate equilibrium chemistry (Fig. 2b).
In addition to pH stress, the chemical stimulus os-
motic stress (i.e., sodium), based on elevated medium
NaCl concentrations, was also investigated as an inducer
of lipid accumulation. No significant increase in lipid
accumulation was observed relative to the control cul-
ture (data not shown).
Bicarbonate supplementation-based lipid accumulation
Addition of bicarbonate salts have been shown to increase
lipid accumulation in a number of microalgae including
diatoms (Gardner et al. 2012a, b). Here, the bicarbonate
effect was contrasted to the pH influenced lipid accumula-
tion and was studied in more detail than previous reports.
Three different concentrations of sodium bicarbonate (5, 15,
and 25 mM) were added at one of two different culturing
phases either day0 (during bioreactor inoculation) or at day
4 (∼24 h before nitrate depletion).
Adding bicarbonate at day0 slowed the Pt1-specific growth
rate possibly due to pH stress while, the addition of bicarbon-
ate on day4 did not appear to affect growth rate (Fig. 4a).
Bicarbonate addition raised the culture pH up to 1.5 pH units
(Fig. 4b). Volumetric and specific Nile Red fluorescence
intensity showed a significant increase when bicarbonate
was added (except the 5 mM treatment added on day0)
indicating stimulated lipid accumulation (Fig. 4d, Table 1).
Time (days)
D
IC
 (m
g/
L)
0
100
200
300
400
500
*
Time (days)
D
IC
 (m
g/
L)
0
2
4
6
8
10
* ** *******
Time (days)
D
IC
 (m
g/
L)
0
10
20
30
40
50
b
* * **
dc
Control 
5 mM NaHCO3 day 0 
5 mM NaHCO3 day 4
15 mM NaHCO3 day 0
15 mM NaHCO3 day 4 
25 mM NaHCO3 day 0
25 mM NaHCO3 day 4
Control 
15 mM Formate day 0 
15 mM Formate day 4
7.5 mM Acetate day 0
7.5 mM Acetate day 4 
5 mM Lactate day 0
5 mM Lactate day 4
Control
pH=7.0 
pH=8.5 
pH=9.0
Time (days)
0 2 4 6 0 2 4 6
0 2 4 60 2 4 6
 
D
IC
 
(m
g/
L)
0
2
4
6
8
10
a
* *
Control
Reduced nitrate
* *
Fig. 2 Medium DIC concentrations. a Dissolved inorganic carbon
concentration/control condition (pH7.8; black bars), reduced nitrate
(medium gray bars). b DIC concentration for cultures grown at differ-
ent medium pH values: control condition (pH7.8; black bars), pH=7.0
condition (medium gray bars), pH=8.5 condition (dark gray bars), pH
=9.0 condition (light gray bars). c DIC concentration for cultures
augmented with NaHCO3; control condition (black bars), 5 mM
NaHCO3 added at day0 condition (medium gray bars), 5 mM
NaHCO3 added at day4 condition (dark gray bars), 15 mM NaHCO3
added at day0 condition (lines on white background bars), 15 mM
NaHCO3 added at day4 condition (lines on dark gray background
bars), 25 mM NaHCO3 added at day0 condition (dots on white
background bars), 25 mM NaHCO3 added at day4 condition (dots
on medium gray background bars). d DIC concentration for cultures
augmented with organic acids: control condition (black bars), 15 mM
formate added at day0 condition (medium gray bars), 15 mM formate
added at day4 condition (dark gray bars), 7.5 mM acetate added at day
0 condition (lines on white background bars), 7.5 mM acetate added at
day4 condition (lines on dark gray background bars). Note the differ-
ent y-axis scales. *Values below the limit of detection
Lipid accumulation associated with bicarbonate was concen-
tration and time of addition dependent. As shown in Table 1,
the maximum specific lipid content increased 10-fold relative
to the control when 15 mM NaHCO3 was added at day4 and
increased fivefold when 5 mM NaHCO3 was added on day4.
Larger increases in lipid accumulation were observed with
day4 additions versus the day0 bicarbonate additions due
partially to CO2 off gassing (Fig. 4d, Table 1). Results
obtained with 25 mM NaHCO3 additions were highly vari-
able, likely a result of precipitation of ASPII medium constit-
uents. Lipid accumulation always occurred close to nitrate
depletion (Fig. 4c) while medium DIC was still measurable
(Fig. 2c). The cultures supplemented with 15 mM NaHCO3
accumulated more carbohydrate than the control culture and
peak specific carbohydrate content occurred approximately
1 day prior to maximum lipid accumulation (Fig. 4h). A
Time (days)
Ni
le
 R
e
d 
flu
o
re
sc
e
nc
e
 in
te
ns
ity
0
1000
2000
3000
4000
5000
6000
7000
Time (days)
N
aN
O 3
 
(m
g/
L)
0
10
20
30
40
50
60
70
Time (days)
Ca
rb
o
hy
dr
a
te
s 
(pg
/ce
ll)
0
2
4
6
8
10
Time (days)
pH
6
7
8
9
10
Time (days)
Ce
ll n
um
be
r (
ce
ll/m
L)
1e+4
1e+5
1e+6
1e+7
1e+8
h
a
b
c
d
Time (days)
Pr
ot
ei
ns
 
(pg
/ce
ll)
0
2
4
6
8
10
12
g
Time (days)
To
ta
l c
ar
ot
en
oi
ds
 (p
g/c
e
ll)
0.00
0.05
0.10
0.15
0.20f
Time (days)
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
4 5 6 7
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
4 5 6 7
4 5 6 7
4 5 6 7
To
ta
l c
hlo
ro
ph
yll
s 
(pg
/ce
ll)
0.0
0.1
0.2
0.3
0.4e
Control
pH=7.0 
pH=8.5
pH=9.0
Control
pH=7.0 
pH=8.5 
pH=9.0
Fig. 3 P. tricornutum cultures
grown at different medium pH
values. Control condition (pH
7.8; filled circles and black
bars), pH=7.0 condition
(circles, medium gray bars), pH
=8.5 condition (filled triangles,
dark gray bars), pH=9.0
condition (triangles, light gray
bars). a Cell concentration, b
medium pH, c medium nitrate
concentration, d volumetric
Nile Red fluorescence, e
specific cellular chlorophyll
content, f specific cellular
carotenoids content, g specific
cellular protein content,
h specific cellular carbohydrate
content. Average and standard
deviation calculated from three
biological replicates
decrease in photosynthetic pigment (chlorophylls a+c and
carotenoids) content was observed following culture nitrate
exhaustion (Fig. 4e and f). Addition of bicarbonate did not
affect specific protein content except in presence of 25 mM
bicarbonate where it was higher for unknown reasons
(Fig. 4g).
Carbonic anhydrase (CA) inhibitors EZA, and AZA (Satoh
et al. 2001; Chen et al. 2006a, b) were used to separate the
function of culture pH stress and the elevated bicarbonate
levels. Lipid accumulation observed in the presence of
15 mM NaHCO3 added at day4 was not affected by AZA
(400 μM) an extracellular CA inhibitor suggesting DIC was
Time (days)
Ni
le
 R
e
d 
flu
o
re
sc
e
nc
e
 in
te
ns
ity
0
5000
10000
15000
20000
Time (days)
N
aN
O 3
 
(m
g/
L)
0
10
20
30
40
50
60
70
Time (days)
pH
7.5
8.0
8.5
9.0
9.5
10.0
b
c
d
Time (days)
Ca
rb
oh
yd
ra
te
s 
(pg
/ce
ll)
0
2
4
6
8
Time (days)
Pr
o
te
in
s 
(pg
/ce
ll)
0
5
10
15
20
25
Time (days)
To
ta
l c
ar
ot
en
oi
ds
 (p
g/c
ell)
0.00
0.05
0.10
0.15
0.20
h
g
f
Time (days)
Ce
ll n
um
be
r (
ce
ll/m
L)
1e+4
1e+5
1e+6
1e+7
1e+8
a
Time (days)
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
4 5 6 7
4 5 6 7
4 5 6 7
0 1 2 3 4 5 6 7 4 5 6 7
To
ta
l c
hlo
ro
ph
yll
s 
(pg
/ce
ll)
0.0
0.1
0.2
0.3
0.4
e
Control 
5 mM NaHCO3 day 0 
5 mM NaHCO3 day 4
15 mM NaHCO3 day 0
15 mM NaHCO3 day 4 
25 mM NaHCO3 day 0
25 mM NaHCO3 day 4
Control
5 mM NaHCO3 day 0
5 mM NaHCO3 day 4
15 mM NaHCO3 day 0
15 mM NaHCO3 day 4
25 mM NaHCO3 day 0
25 mM NaHCO3 day 4
Fig. 4 P. tricornutum cultures
supplemented with sodium
bicarbonate. Control condition
(filled circles and black bars),
5 mM NaHCO3 added at day0
condition (circles, medium gray
bars), 5 mM NaHCO3 added at
day4 condition (filled triangles,
dark gray bars), 15 mM
NaHCO3 added at day0
condition (triangles, lines on
white background bars),
15 mM NaHCO3 added at day4
condition (filled squares, lines
on dark gray background bars),
25 mM NaHCO3 added at day0
condition (squares, dots on
white background bars),
25 mM NaHCO3 added at day4
condition (filled diamonds, dots
on medium gray background
bars). a Cell concentration, b
medium pH, c nitrate
concentration in ASPII media,
d volumetric Nile Red
fluorescence, e specific cellular
chlorophyll content, f specific
cellular carotenoids content, g
specific cellular protein content,
h specific cellular carbohydrate
content. Average and standard
deviation calculated from three
biological replicates
entering the cell as bicarbonate, not CO2 (Fig. 5a). Bicarbonate
is the predominant DIC species at the tested pH values. Lipid
accumulation observed in the presence of 15 mM NaHCO3
added at day4 was inhibited by EZA (400 μM) an extra and
intracellular CA inhibitor (Fig. 5a). EZA reduced lipid accu-
mulation by approximately 80 %. These cultures still experi-
enced pH stress with culture media reaching a pH of
approximately 9. EZA was also shown directly to decrease
DIC consumption while AZA had little to no effect on DIC
utilization (Fig. 5b).
Nitrate availability strongly influenced accumulation
of lipid via bicarbonate supplementation. Pt1 cultures
grown in medium containing twice the standard nitrate
concentration did not accumulate significant amounts
of lipids when subjected to a 15 mM NaHCO3 addition
on day4 (Fig. 5c). The results highlight the importance
of compounded physiological stresses (nitrate limita-
tion, pH stress, and bicarbonate availability) on lipid
accumulation.
Organic carbon-based lipid accumulation
Elevated medium pH and the addition of bicarbonate
both increased either directly or indirectly the medium
DIC (Fig. 2). To test whether the lipid accumulation
effect was specific for inorganic carbon, P. tricornutum
Pt1 were grown at pH=7.8 in the presence of 15 carbon
millimole (CmM) of several organic carbon sources.
The examined organic acids were formate (15 mM),
acetate (7.5 mM), and lactate (5 mM). The magnitude
of the supplements was carbon equivalent to the 15 mM
bicarbonate experiments. The organic carbon sources
were also added at either days0 or 4. Addition of
formate, acetate, or lactate did not affect the maximum
specific growth rate of Pt1 and had only a minor effect
on culture pH (Fig. 6b). No significant lipid accumula-
tion was observed in Pt1 following addition of formate
or lactate, but addition of acetate at both time points
stimulated specific lipid accumulation with a four- to
sixfold increase versus the control culture (Fig. 6d,
Table 1). Acetate addition had no effect on carbohydrate
accumulation unlike the previous pH and bicarbonate
experiments. The addition of lactate, which did not
increase lipid accumulation, significantly increased car-
bohydrate accumulation indicating different metabolic
utilization of acetate and lactate. Organic acid feeding
did not affect significantly photosynthetic pigments or
protein content although the lactate fed culture did seem
to have an increase in cellular protein during mid-
exponential phase (Fig. 6g). HPLC analysis confirmed
consumption of acetate and lactate which corresponded
with culture DIC exhaustion (Fig. 2d) while the formate
concentration did not change significantly (Fig 7).
Considering the apparently different mechanisms for
lipid accumulation under bicarbonate addition and
Time (days)
0
20
40
60
80
100
120
140
c
Control 
15 mM NaHCO3 day 4
15 mM NaHCO3 day 4 under nitrate excess
Time (days)
D
IC
 (m
g/
L)
0
20
40
60
80
100
120
140
160
Time (days)
4 5 6 7
4 5 6 7
4 5 6 7
0
20
40
60
80
100
120
140
a
Control
15 mM NaHCO3 day 4
15 mM NaHCO3 + 400 µM EZA day 4
15 mM NaHCO3 + 400 µM AZA day 4
Control
15 mM NaHCO3 day 4
15 mM NaHCO3 + 400 µM EZA day 4
15 mM NaHCO3 + 400 µM AZA day 4
b
* *
N
ile
 R
ed
 fl
u
or
es
ce
nc
e 
in
te
ns
ity
/1
04
ce
lls
Ni
le
 R
ed
 fl
uo
re
sc
en
ce
 in
te
ns
ity
/1
04
ce
lls
Fig. 5 Effects of carbonic anhydrase inhibitors and nitrate excess on
TAG accumulation. a Effect of carbonic anhydrase inhibitors on lipid
accumulation: fluorescence per 104 cells using the Nile Red assay with
control condition (black bars), 15 mM NaHCO3 added at day4 condi-
tion (medium gray bars), 15 mM NaHCO3+400 μM EZA added at
day4 condition (light gray bars); 15 mM NaHCO3+400 μM AZA
added at day4 condition (dark gray bars). b Effect of carbonic anhy-
drase inhibitors on DIC concentrations: control condition (black bars),
15 mM NaHCO3 added at day4 condition (medium gray bars), 15 mM
NaHCO3+400 μM EZA added at day4 condition (light gray bars);
15 mM NaHCO3+400 μM AZA added at day4 condition (dark gray
bars); c effect of nitrate excess on lipid accumulation: fluorescence per
104 cells using the Nile Red assay with control condition (black bars),
15 mM NaHCO3 added at day4 condition (medium gray bars), 15 mM
NaHCO3 added at day4 under nitrate excess (dots on white back-
ground bar). *Values below the limit of detection
acetate addition, an experiment was performed which
tested the effects of simultaneously adding both carbon
sources. Cultures were supplemented with both organic
and inorganic carbon sources concurrently but, the treat-
ment did not result in an additive effect. Lipid accumu-
lation was similar to the culture supplemented with only
15 mM bicarbonate (data not shown).
Transcript analysis of TAG accumulation-associated
metabolism
P. tricornutum has a robust metabolism capable of a vast
array of metabolic fluxes. To characterize fundamental met-
abolic processes and possible regulatory patterns involved
in lipid accumulation, the abundance of key transcripts were
Time (days)
Ni
le
 R
e
d 
flu
o
re
sc
e
nc
e
 in
te
ns
ity
0
2000
4000
6000
8000
10000
Time (days)
Na
NO
3 
(m
g/
L)
0
10
20
30
40
50
60
70
Time (days)
pH
7.0
7.2
7.4
7.6
7.8
8.0
8.2
8.4
Time (days)
Ce
ll n
um
be
r 
(ce
ll/m
L)
1e+4
1e+5
1e+6
1e+7
1e+8
d
c
b
a
Time (days)
Ca
rb
o
hy
dr
a
te
s 
(pg
/ce
ll)
0
2
4
6
8h
Time (days)
Pr
o
te
in
s 
(pg
/ce
ll)
0
2
4
6
8
10
12
14
Time (days)
To
ta
l c
a
ro
te
no
id
s 
(pg
/ce
ll)
0.00
0.05
0.10
0.15
0.20
Time (days)
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
4 5 6 7
4 5 6 7
4 5 6 7
4 5 6 7
To
ta
l c
hlo
ro
ph
yll
 (p
g/
ce
ll)
0.0
0.1
0.2
0.3
0.4
0.5
e
f
g
Control 
15 mM Formate day 0 
15 mM Formate day 4 
7.5 mM Acetate day 0
7.5 mM Acetate day 4
5 mM Lactate day 0
5 mM Lactate day 4
Control 
15 mM Formate day 0 
15 mM Formate day 4
7.5 mM Acetate day 0
7.5 mM Acetate day 4 
5 mM Lactate day 0
5 mM Lactate day 4
Fig. 6 P. tricornutum cultures
fed either formate, acetate, or
lactate. Control condition (filled
circles and black bars), 15 mM
formate added at day0
condition (circles, medium gray
bars), 15 mM formate added at
day4 condition (filled triangles,
dark gray bars), 7.5 mM
acetate added at day0 condition
(triangles, lines on white
background bars), 7.5 mM
acetate added at day4 condition
(filled squares, lines on dark
gray background bars), 5 mM
lactate added at day0 condition
(squares, dots on medium white
background bars), 5 mM lactate
added at day4 condition (filled
diamonds, dots on medium gray
background bars). a Cell
concentration, b medium pH, c
medium nitrate concentration, d
volumetric Nile Red
fluorescence, e specific cellular
chlorophyll content, f specific
cellular carotenoids content, g
specific cellular protein content,
h specific cellular carbohydrate
content. Average and standard
deviation calculated from three
biological replicates
measured by real-time PCR. Total RNAwas collected at the
peak of lipid accumulation for each culture condition and
used for transcriptomic analysis. Due to cycle threshold (Ct)
value variations in the housekeeping genes (see “Materials
and Methods” sectoin) from the pH9.0 stress condition,
real-time PCR data from that condition was not included;
instead data from pH8.5 was reported. The treatment data
are all compared to the control condition: ASPII media pH=
7.8. A diagram illustrating the different pathway enzymes
included in the analysis can be found in the supplementary
data (ESM Fig. 1). The transcript abundances were investi-
gated at a single time point and therefore do not reflect the
dynamic nature of gene expression.
Carbonic anhydrases and NaHCO3 transporters
qRT-PCR of key transcripts was used to assess the potential
routes of DIC acquisition and transformation. CA play a
fundamental role in DIC metabolism by interconverting
CO2 and bicarbonate. Transcript abundances of the γ-CAs
(20030, 36906) increased significantly under nitrogen limi-
tation and alkaline pH stress (Fig. 8; ESM Table 2). This is
in contrast to the cultures supplemented with bicarbonate
that demonstrated increases in the α-CAs (35370, 44526,
55029, 54251, 42574) transcripts (Fig. 8; ESM Table 2).
The movement of bicarbonate within the cell can be influ-
enced by transporter proteins providing insight into subcel-
lular locations where the bicarbonate may be utilized. The
transcript abundance of the putative external bicarbonate
transporter (1334) and the putative mitochondrial bicarbon-
ate transporter (54405) increased significantly (Fig. 8; ESM
Table 2) although the putative chloroplast bicarbonate trans-
porters (45656, 43194) did not change or decreased under
the tested culture conditions.
Carboxylating/decarboxylating enzymes
Increases in transcript levels for genes essential to C4 me-
tabolism and carbon concentrating mechanisms (CCM)
were observed. At peak lipid accumulation, transcript abun-
dances of carboxylating/decarboxylating enzymes including
both Pt1 phosphoenol pyruvate carboxylases (PEPC: 27976,
51136), both pyruvate carboxylases (PYC: 49339, 30519),
pyruvate dehydrogenase (PDH: 55035), and malic enzymes
(ME: 51970) increased in varying magnitudes based on
culturing conditions including acetate supplementation.
The data suggested an active movement of bicarbonate
between C3 and C4 forms; however, the Rubisco transcripts
levels decreased for all tested conditions suggesting a low
overall fixation of new C3 intermediates. Downregulation of
Rubisco is consistent with the observed changes of tran-
script levels for the plastid bicarbonate transporters (45656,
43194) mentioned previously and published RNA-seq data
(Valenzuela et al. 2012).
Central metabolism
Analysis of central metabolism transcript levels suggested
potentially different adaptations to culturing conditions and
highlighted enzymes that may have important flux roles.
Transcript level increases were observed in the lower por-
tion of the gluconeogenesis pathway. Pyruvate phosphate
Time (days)
La
ct
at
e 
(m
M
)
1
2
3
4
5c
Time (days)
Ac
e
ta
te
 (m
M)
4
5
6
7
Time (days)
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
0 1 2 3 4 5 6 7
Fo
rm
at
e 
(m
M
)
8
10
12
14
16
b
a
15 mM Formate day 0
15 mM Formate day 4
7.5 mM Acetate day 0
7.5 mM Acetate day 4
5 mM Lactate day 0
5 mM Lactate day 4
Fig. 7 Formate, acetate, and lactate concentrations. a Formate con-
centration (formate added at day0 (filled triangles), formate added at
day4 (triangles)). b Acetate concentration (acetate added at day0
(filled circles), acetate added at day4 (circles)). c Lactate concentration
(lactate added at day 0 (filled squares), lactate added at day4
(squares)). Average and standard deviation calculated on three biolog-
ical replicates and two technical replicates
dikinase (PPDK, 21988), phosphoenolpyruvate carboxyki-
nase (PEPCK, 55018), and glyceraldehyde 3-phosphate
dehydrogenase (G3PDH, 22122) transcripts increased in
different combinations for pH stress, bicarbonate addition
pH=8.5
redu
ced-N
acetate 
5 m
M
HCO
3
15 m
M
HCO
3
CA-I (35370)
CA-II (44526)
CA-III (55029)
PTCA1 (51305)
PTCA2 (45443)
CA-VI (54251)
CA-VII (42574)
CA-VIII (20030)
CA-IX (36906)
SLC4A_1 (45656)
SLC4A_2 (43194)
anion exchanger (54405)
HCO3 transporter (1534)
ACC1 (54926)
ACC2 (55209)
FABD (37652)
DGAT (49426)
DGAT (49544)
DGAT (31662)
DGAT (43469)
phospholipase (42683)
phospholipase (39425)
phospholipase (49771)
phospholipase (48445)
phospholipase (45665)
PDAT (8860)
PDAT (49706)
PEPC1 (51136)
PEPC2 (27976)
PYC1 (30519)
PYC2 (49339)
PPDK (21988)
PEPCK (5518)
PDH (55035)
CS (30145)
ME (51970)
G3PDH (33122)
OGDH (29016)
MS (54478)
rubisco rbcL (YP_874418.1)
rubisco rbcL (YP_874417.1)
UDP-GDP (23639)
UDP-GDP (50444)
glucanase (56510)
glucanase (56506)
glucanase (49610)
GS (51092)
GS (22357)
CPS (24195)
fold change
< 0.5
0.5 - 1
1 - 1.5
1.5 - 2.5
>2.5
Fig. 8 Clustering heat map. Hierarchical clustering of transcriptional
fold changes, from triplicate cultures, in response to reduced nitrate
(reduced-N), alkaline pH stress (pH=8.5), acetate supplementation
(7.5 mM) and bicarbonate supplementation (5 and 15 mM HCO3
−) at
day4. The transcripts associated with a fold change less than 1 are
downregulated genes; these data appear in green and blue in the figure.
The transcripts associated with a fold change greater than to 1 are
upregulated genes; these data appear in yellow, orange, and red. Fold
changes are all relative to the control culture (ASPII, pH 7.8). Carbonic
anhydrases: CA-I (35370), CA-II (44526), CA-III (55029), PTCA1
(51305), PTCA2 (45443), CA-VI (54251), CA-VII (42574), CA-VIII
(20030), CA-IX (36906); bicarbonate transporters: SLC4A_1 (45656),
SLC4A_2 (43194), anion exchanger (5440), HCO3 transporter (1534);
acetyl-CoA carboxylases: ACC1 (54926), ACC2 (55209); malonyl CoA-
acyl carrier protein transacylase: FABD (37652); diacylglycerol acyl
transferases: DGAT (49426, 49544, 31662, 43469); phospholipases:
(42683, 39425, 49771, 48445, 45665); phospholipid/diacylglycerol acyl-
transferases: PDAT (8860, 49706); phosphoenol pyruvates: PEPC1
(51136), PEPC2 (27976); pyruvate carboxylases: PYC1 (30519), PYC2
(49339); pyruvate-phosphate dikinase: PPDK (21988); phosphoenolpyr-
uvate carboxylase: PEPCK (55018); pyruvate dehydrogenase PDH
(55035); citrate synthase: CS (30146); malic enzyme: ME (54082);
glyceraldehyde 3-phosphate dehydrogenase: G3PDH (22122); oxogluta-
rate dehydrogenase: OGDC (29016); malate synthase: MS (54478);
ribulose-1,5-bisphosphate carboxylase: RuBp (YP_874418.1, YP_
874417.1); UDP-Glucose-Pyrophosphorylase: UGP (23639, 50444);
exo-1,3-ß-glucanases: glucanase (54926; 55209; 37652); glutamine syn-
thases: GS (51092, 22357); carbomoyl phosphate synthase: CPS (24195)
and nitrogen limitation (Fig. 8). The G3PDH transcripts
decreased during acetate addition suggesting a different
dependence on glycolytic fluxes and likely a direct incor-
poration of acetate into lipid without first being converted
into polysaccharide. Transcripts associated with the TCA
cycle and glyoxylate utilization increased in abundance.
Increases were observed in citrate synthase (CS, 30145),
oxoglutarate dehydrogenase (OGDC, 29016), and malate
synthetase (MS, 54478) for all tested conditions. The tran-
script changes suggest the oxidative portion of the TCA
cycle was being used for energy generation and the incor-
poration of acetyl-CoA into C4 metabolite malate which
could serve as a basic anabolic backbone metabolite or as
a substrate for the CCM process.
Nitrogen cycling
All tested conditions experienced nitrogen limitation.
Increases in expression of carbamoyl phosphate synthase
(24195) and glutamine synthetase (51092) were observed
as logical adaptations for recycling intracellular nitrogen
sources and as a means of adapting to the nitrogen
exhausted conditions.
FA synthesis and TAG synthesis
Transcript levels for genes encoding key FA synthesis
enzymes such as acetyl-CoA carboxylases (ACC: 54926,
55209) and malonyl CoA-acyl carrier protein transacylase
(FABD: 37652) did not change or decreased under all ana-
lyzed culturing conditions (Fig. 8; ESM Table 2; Valenzuela
et al. 2012). Diacylglycerol acyl transferase transcripts
(DGAT: 49462, 49544) involved in the final step of the
TAG synthesis pathway increased under all conditions ex-
cept acetate addition (Fig. 8; ESM Table 2). Alternative
pathways to convert membrane lipids to TAG have been
demonstrated in bacteria, plants, and yeast and would in-
volve phospholipases and/or phospholipid/diacylglycerol
acyltransferase (PDAT) enzymes. Such pathways have not
yet been studied in microalgae. However, transcripts for
some putative phospholipases and PDAT from the P. tricor-
nutum genome, reported here, increased under the tested
conditions (Fig. 8; ESM Table 2), suggesting the repurpos-
ing of fatty acids from membranes may play a role in TAG
synthesis and accumulation.
Carbohydrates metabolism/catabolism
Chrysolaminaran is the principal diatom energy storage
polysaccharide (Kroth et al. 2008). UDP-glucosyl pyrophos-
phorylases (23639, 50444) and 1,3-ß-glucanases (54926;
55209; 37652) are key enzymes in synthesis and degrada-
tion of chrysolaminaran, respectively. Transcripts from
several enzymes involved in chrysolaminaran metabolism
increased under all tested culturing conditions except acetate
feeding; the results corroborate other findings suggesting
lipid accumulation during acetate feeding is distinct from
the other tested conditions (Fig. 8; ESM Table 2). For
instance, in all cases except acetate addition, carbohydrate
levels decrease as lipid levels increase.
Discussion
Physiological and molecular techniques were employed to
study lipid accumulation induced by a suite of conditions
including nitrogen limitation, alkaline pH stress, bicarbonate
addition, and organic acid addition in the model diatom
P. tricornutum Pt1. The results suggest both overlapping and
contrasting physiological responses influence lipid accumu-
lation. First, nitrogen limitation was a necessary prerequisite
for lipid accumulation under all tested conditions indicating
compounded stresses are important for enhanced lipid accu-
mulation. A common series of physiological events were
observed for cultures that accumulated lipid based on pH
stress and bicarbonate addition. After exhausting extracel-
lular nitrogen, the cultures continued growing for approxi-
mately 1.5 doublings using intracellular nitrogen sources.
Growth following extracellular nitrogen exhaustion corre-
sponded with decreases in specific protein and photosyn-
thetic pigment. As nitrogen limitation arrested cell growth,
specific carbohydrate content decreased and specific lipid
content increased suggesting some of the carbon stored as
carbohydrate was repurposed to lipids. This trend was not
observed with cultures fed acetate although nitrogen limita-
tion was a necessary prerequisite for induction of the en-
hanced lipid accumulation. Figure 9 plots the peak specific
lipid content as a function of culture pH. Analogous to
previous work with Chlorophytes, there is a strong correla-
tion between pH and lipid (Guckert and Cooksey 1990;
Gardner et al. 2011). Both elevated medium pH and bicar-
bonate additions increased lipid accumulation (Fig 9a). The
highest lipid accumulation during alkaline stress was ob-
served at pH9.0 with a sixfold increase in specific content
compared to the control (Table 1). Maximal lipid content for
the bicarbonate additions was observed in presence of
15 mM NaHCO3 and specific lipid content increased about
tenfold compared to the control (Table 1). Culture pH
strongly affects the availability of DIC. The mechanisms
of the two lipid-enhancing strategies are both likely based
on a combination of alkaline pH stress and elevated DIC;
the NaHCO3 supplemented cultures had ∼10-fold higher
DIC levels during lipid accumulation as compared to the
pH9 cultures possibly explaining the higher specific lipid
content (Fig. 2). Supplementation with organic acids did not
show a pH dependent relationship (Fig. 9b). The acetate
utilizing pathways are likely independent of external DIC
with the organic acid first being converted to acetyl-CoA. It
is then partitioned between oxidizing TCA cycle fluxes
which produce CO2 (converted to bicarbonate via internal
CA activity) and acetyl-CoA carboxylase which starts bi-
carbonate and acetyl-CoA flux toward lipid synthesis.
Photosynthetic pigment and protein content appear tight-
ly coupled with the accessibility of external nitrogen and
quickly decrease upon nitrogen exhaustion. Downregulation
of photosynthesis is a commonly observed response to
nitrogen starvation (Shelly et al. 2007). Several explanations
are possible. Chlorophyll contains approximately 6 % nitro-
gen on a mass basis which could be reallocated for biosyn-
thetic roles. When extracellular nitrogen is depleted, cells
could utilize chlorophyll as intracellular nitrogen pools to
support synthesis of new cell material. However chlorophyll
represents a relatively small intracellular nitrogen pool com-
pared to protein. A typical diatom is ∼3 % chlorophyll on a
dry mass basis, while protein accounts for ∼40 % of a
typical diatom’s dry mass (Darley 1977; Fernández-Reiriz
et al. 1989). Protein contains approximately 15 % nitrogen
on a mass basis and this macromolecular pool represents 30
times more nitrogen than the chlorophyll pool. Another
potential explanation for chlorophyll decreases during nitro-
gen limitation relates to overproduction of photosynthetic
electrons. Without external nitrogen, the cells have limited
growth potential and reduced availability of anabolic sinks
for photosynthetically derived electrons. Under many cul-
turing conditions, phototrophs actually have an excess of
energy and dissipating the excess energy is a major cellular
challenge (Wilhelm and Jakob 2011). Downregulating pho-
tosynthetic pigments is a potential defense strategy to limit
excess electron flow thus limiting generation of oxygen rad-
icals and reducing equivalents. This strategy could work in
conjunction with the repurposing of the chlorophyll nitrogen.
Interestingly, lipid accumulation only occurred in the pre-
sented studies after specific chlorophyll content dropped be-
low ∼0.1 pg/cell. This is approximately one third the content
of a cell growing under nutrient sufficient conditions.
The formation of a C18 fatty acid consumes approximately
54 NADPH derived from oxygenic photosynthesis. This rep-
resents ∼50 % more electrons than required for the synthesis
of carbohydrate or protein on a carbon mole basis (the degree
of reduction relative to CO2 for a long chain fatty acid is 6, for
a carbohydrate is 4, and for protein is ∼4.2), thus lipids
represent a more carbon efficient electron sink. Palmucci et
al. (2011) have shown that the diatoms P. tricornutum and
Thalassiosira weissflogii reduce carbohydrate stores while
increasing levels of fatty acids under nitrogen starvation.
Further, additional links between starch and lipid metabolism
have been established inmicroalgae (Li et al. 2010; Dean et al.
2010; Gardner et al. 2012b; Markou et al. 2012; Hildebrand,
personal communication). While lipids represent an effective
electron sink, reutilizing lipids is more restrictive than carbo-
hydrates; carbohydrates can be oxidized in the presence of
oxygen or can be fermented anaerobically while lipids can
only be oxidized in the presence of oxygen limiting the con-
ditions when the carbon and energy can be reutilized.
Transport of HCO3
− into the cell can be performed directly
or indirectly. Indirect HCO3
− transport occurs based on CO2
diffusion followed by periplasmic CA activity that catalyzes
conversion of CO2 to HCO3
− (Badger and Price 1994;
Elzenega et al. 2000; Hobson et al. 2001; Chen and Gao
2003), while direct HCO3
− transport can be performed by
HCO3
− uptake proteins (Kaplan and Reinhold 1999;
Giordano et al. 2005). Seven genes encoding carbonic anhy-
drases have been identified in the P. tricornutum Pt1 genome
(Kroth et al. 2008; Tachibana et al. 2011). Two inhibitors of
CA activity, EZA and AZA, were used to determine the
function of P. tricornutum Pt1 CAs in bicarbonate acquisition
and utilization. EZA is highly permeable to biological mem-
branes and will inhibit both internal and external CAs,
Control
Nitrate limitation
pH stress
NaHCO3
A
pH
0
20
40
60
80
100
120
pH
7.0 7.5 8.0 8.5 9.0
7.0 7.5 8.0 8.5 9.0
0
20
40
60
80
100
120
Control
Nitrate limitation
Formate
Acetate
Lactate
Reduced nitrate
 t
3
ont l
Reduced nitrate
r t  
t t  
ct t   
Ni
le
 R
ed
 fl
uo
re
sc
en
ce
 in
te
ns
ity
/1
04
ce
lls
Ni
le
 R
ed
 fl
uo
re
sc
en
ce
 in
te
ns
ity
/1
04
ce
lls
a
b
Fig. 9 Nile Red fluorescence per 104 cells versus culture pH. a The
maximal Nile Red fluorescence per 104 cells for each tested condition
is reported as a function of culture pH. Control condition, ASPII, pH=
7.8 (filled square); pH=7.0, pH=8.5, and pH=9.0 conditions (filled
circles); 5, 15, and 25 mM NaHCO3 supplementation condition
(squares). b Reduced nitrate condition (filled triangle), organic acid
supplementation condition, 15 mM formate (open circle); 7.5 mM
acetate (open diamond); 5 mM lactate (open hexagon). Average and
standard deviation calculated on three biological replicates
whereas AZA is only weakly permeable and inhibits predom-
inately external CAs. Addition of EZA almost completely
abolished lipid accumulation in Pt1 and inhibited DIC con-
sumption, whereas AZA did not affect lipid accumulation or
DIC consumption. These results suggest that internal CA
activities involved in DIC acquisition and utilization are es-
sential for the observed lipid accumulation.
Stress conditions and access to carbon sources are expected
to impact central carbon metabolism. In the presence of bicar-
bonate, α-CA and putative mitochondrial and external bicar-
bonate transporter transcripts increased. In P. tricornutum, α-
CAs are mainly found in the periplasmic space. Increases in
transcripts encoding PPDK, PEPC, PEPCK, ME, and MDH
are in agreement with an active biochemical CO2 concentrat-
ing mechanism in Pt1 (Kroth et al. 2008; Valenzuela et al.
2012). In addition to balancing C3 and C4 intermediates, the
CCM can be used as a futile cycle where metabolites are
transported between different organelles at the expense of
ATP (Kroth et al. 2008; Wilhelm and Jakob 2011). Pt1 grown
in the presence of acetate did not show significant upregula-
tion in CA transcripts except for CA-VII (42574).
TAG biosynthesis in microalgae has been proposed to
occur via the Kennedy pathway described in plants
(Kennedy 1961; Ohlrogge and Browse 1995; Athenstaedt
and Daum 2006; Lung and Weselake 2006). In the present
study, transcripts for DGAT enzymes involved in TAG syn-
thesis increased while transcripts levels of ACC and FABD,
key enzymes of FA synthesis, remained unchanged. The
final step of the TAG synthesis is catalyzed by DGAT, an
enzymatic reaction that is unique to TAG biosynthesis.
Interestingly, Guihéneuf et al. (2011) identified and isolated
a cDNA encoding a novel acyl-CoA/diacylglycerol acyl-
transferase like protein, from P. tricornutum (PtDGAT1).
Alternative splicing consisting of intron retention appears
to regulate the amount of active PtDGAT1 produced, pro-
viding a possible molecular mechanism for increased TAG
biosynthesis in P. tricornutum under certain conditions like
nitrogen starvation. Alternative pathways that convert mem-
brane lipids to TAG have been demonstrated using an acyl
CoA-independent pathway (Arabolaza et al. 2008;
Dahlqvist et al. 2000; Stahl et al. 2004). This pathway uses
phospholipids as acyl donors and DAG as the acceptor with
the reaction catalyzed by PDAT. Microalgae may possess
multiple pathways for TAG synthesis in addition to the de
novo Kennedy pathway such as pathways that convert
membrane phospholipids and glycolipids into TAG. Under
many stress conditions, microalgae undergo rapid degrada-
tion of the photosynthetic membrane with concomitant ac-
cumulation of TAG-enriched lipid bodies (Wang et al. 2009;
Moellering and Benning 2010). The current study showed
increases in Pt1 transcripts for putative phospholipases
and/or phospholipid/diacylglycerol acyltransferases sug-
gesting phosphatidylcholine, phosphatidylethanolamine, or
galactolipids derived from the photosynthetic membrane
could serve as acyl donors for TAG synthesis.
In conclusion, the metabolic capacity of P. tricornutum
Pt1, particularly during environmental changes, is extremely
versatile and complicated. This diatom responded to cultur-
ing conditions in a manner that is just beginning to be
understood. This study, using a combination of physiologi-
cal and molecular approaches, provides a foundation for
further elucidation of the metabolic flexibility and TAG
accumulation potential of this promising diatom.
Acknowledgments The authors of this work acknowledge the Air
Force Office of Scientific Research (AFOSR grant FA9550-09-1-0243)
and US Department of Energy grant DE EE0003136. Dr. Olusegun
Oshota and Dr. Robert Gardner are gratefully acknowledged for tech-
nical assistance. The authors would like also to thank Luis Ramos, Ann
Willis, MSU Center for Biofilm Engineering and MSU Functional
Genomics Core Facility for technical and instrumental support.
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