Algal Research 10 (2015) 152163

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Towards the biorenery concept: Interaction of light, temperature and

nitrogen for optimizing the co-production of high-value compounds in
Porphyridium purpureum
Freddy Guihneuf , Dagmar B. Stengel
Botany and Plant Science, School of Natural Sciences, Ryan Institute for Environmental, Marine and Energy Research, National University of Ireland Galway, Galway, Ireland

a r t i c l e

i n f o

Article history:
Received 6 March 2015
Received in revised form 17 April 2015
Accepted 25 April 2015
Available online xxxx
Keywords:
Porphyridium
Batch-cultivation
Phycobiliproteins
Fatty acids
Pigments
Carbohydrates

a b s t r a c t
The interactive effects of light, temperature and nitrogen regime on phycobiliprotein (PB) production and other
compounds such as fatty acids, pigments and carbohydrates, were studied during batch-cultivation of
Porphyridium purpureum, a red microalga, containing multiple compounds of commercial interest. Results indicate that nitrogen-replete modes, such as semi-continuous or continuous regime represent the most suitable culture strategy for PB, carbohydrate, total fatty acid (TFA) and eicosapentaenoic acid (EPA) production in
P. purpureum. Nitrate-deciency causes a strong decrease in growth performance, as well as in its PE, TFA and
EPA contents which may be related to membrane degradation; but induces carbohydrate accumulation.
Nitrate-starved cells of P. purpureum had the ability to restore PB and TFA contents, and specically phycoerythrin
(PE) and EPA levels, after medium refreshment, suggesting an almost complete regeneration of the plastidic
membranes and phycobilisomes. Using response surface methodology (RSM), our results highlight for the rst
time the optimally combined light and temperature conditions necessary to promote growth and compound
production, in particular PB, in P. purpureum batch-cultivated in nitrogen-replete medium. A simultaneous increase in light and temperature causes a strong decrease in cellular PB, TFA, EPA and pigment contents, suggesting a severe damage and possible disruption of thylakoid membranes. The highest PB content (~2.9% d.w.) was
reached under combined low light (30 mol m2 s1) and low temperature (10 C). Despite of this, maximal PB
productivity was obtained at 20 C and under low light intensity, reaching up to 33.3 mg L1 (~2% d.w.). Under
such specic growth conditions, P. purpureum biomass also contained substantial amounts of other valuable
products (i.e., carbohydrates, EPA, Chl. a, zeaxanthin, -carotene) which could therefore be co-extracted, with
PB, by applying a biorenery approach.
2015 Elsevier B.V. All rights reserved.

1. Introduction
The microalgal genus Porphyridium within the Rhodophyta is of
increasing interest as a source of valuable chemical constituents such
as phycobiliproteins [1,2], sulphated exopolysaccharides [3] and longchain polyunsaturated fatty acids [4,5].

Abbreviations: ALA, -linolenic; ANOVA, analysis of variance; APC, allophycocyanin;

ARA, arachidonic acid; BHT, butylated hydroxytoluene; DHA, docosahexaenoic acid; d.w.,
dry weight; EPA, eicosapentaenoic acid; FAMEs, fatty acid methyl esters; GC-FID, gas
chromatography-ame ionization detector; HPLC-DAD/FLD, high performance liquid
chromatography-diode array detector/uorescence detector; LA, linoleic acid; LC-PUFAs,
long-chain polyunsaturated fatty acids; N, nitrogen; OLE, oleic acid; PBs, phycobiliproteins;
PC, phycocyanin; PE, phycoerythrin; RSM, response surface methodology; SD, standard deviation; TAGs, triacylglycerols; TFA, total fatty acid.
Corresponding author.
E-mail addresses: freddy.guiheneuf@nuigalway.ie (F. Guihneuf),
dagmar.stengel@nuigalway.ie (D.B. Stengel).

http://dx.doi.org/10.1016/j.algal.2015.04.025
2211-9264/ 2015 Elsevier B.V. All rights reserved.

Phycobiliproteins (PBs) constitute the major accessory lightharvesting pigments in Cyanophyta, Cryptophyta, and Rhodophyta
[6], and are classied according to their spectroscopic properties
i.e., phycoerythrin (PE, 540570 nm) with a pink/red color, phycocyanin
(PC, 610620 nm) with a blue color and allophycocyanin (APC,
650655 nm) with a bluish-green color. Today, PBs are commercially produced from cyanobacteria including Spirulina and the red microalgae
Porphyridium and Rhodella [710]; PBs are extensively used as nutritive
ingredients and natural dyes for food and cosmetics, potential therapeutic
agents in oxidative stress-induced diseases, and as uorescent markers in
biomedical research [11,12]. The PB content of Porphyridium purpureum
has been reported to reach 4.8% of dry weight (d.w.), mainly consisting
of 70% PE, and smaller proportions of PC (20%), and APC (10%) [1]. Although Porphyridium spp. are suitable candidates for PB production,
their extracellular polysaccharides are more commonly studied [3,
1317]. Indeed, Porphyridium spp. can synthesize and secrete sulfated
polysaccharides [18,19] with antiviral, anti-radiation and antioxidant activities, antitumor and immunomodulatory activities, as well as reducing
blood cholesterol level [2024]. Their cellular carbohydrates (57% d.w.)

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

could also, alternatively, provide the carbon necessary for bioethanol

production by fermentation. Porphyridium spp. additionally contain a
substantial amount of long-chain polyunsaturated fatty acids (LCPUFAs), in particular eicosapentaenoic acid (EPA, 20:5 n 3) [4,25],
which plays a major role in preventing medical disorders including:
heart and circulation [26], inammation [27], and cancer [28]. Their biomass contains as well substantial levels of protein (2839% d.w.), tocopherol, vitamin K, and pigments such as carotenoids [7].
Despite the chemical richness of Porphyridium spp. and the
potential for the co-production of multiple products, a biorenery
approach has never been fully implemented and remains a challenge. In microalgae bioactive levels and composition strongly vary
with growth conditions (e.g. temperature, light, nutrients) [1,29,
30], providing the potential for manipulation and induction for specic applications [31]. However, albeit this important physiological
feature, most previous studies on Porphyridium spp. are limited to independent growth parameters or environmental factors investigating one specic bioactive compound or chemical group at a time [1,
5,3235]. More intensive research is thus needed to explore the interactive effects of multiple abiotic factors on this algal genus in
order to develop multi-product cultivation strategies that retain
and enhance the production and functionality of several different
cell components.
The aim of our study was therefore to investigate the combined
effects of light, temperature and nitrogen regime on PB production,
as major high-value component in P. purpureum, while also considering other bioactives or chemicals of commercial interest such as
fatty acids, pigments and carbohydrates. Using batch-cultures
different nitrogen regimes were applied in the rst instance. Then,
a full factorial design experiment was conducted which allowed a
response surface methodology (RSM) treatment of the data, to assess the interactive effects of light and temperature under high
nitrogen-supply, aiming to optimize the cultivation strategy. To the
best of our knowledge this is the rst study investigating the simultaneous response of several high-value components of a red
microalga to combined changes of two factors or growth conditions.
Applying the biorenery concept, biomass is exploited for coproduction of multiple compounds and possible co-extraction with
potential applications for several industries (e.g., human and animal
nutrition, pharmaceuticals and biofuels), thus reducing energy input
and biomass wastage.

2. Materials and methods

2.1. Strain, medium and pre-cultivation conditions
The red microalga P. purpureum PLY#539 (synonym: Porphyridium
cruentum) was obtained from the Plymouth Culture Collection of
Marine Microalgae at the Marine Biological Association of the United
Kingdom (UK). Prior to experiments, P. purpureum were maintained in
controlled growth chambers (Binder GmbH, Germany) under continuous illumination (100 mol m2 s1), provided by lumilux cool daylight uorescent lamps (OSRAM L18W/865, Germany), a temperature
of 20 C, and batch-cultivated on F/2-RSE medium as described by
Guihneuf and Stengel [36]. This medium contains 0.1 g L1 sodium nitrate and 0.174 g L1 sodium bicarbonate (2.07 mM) which is the normal bicarbonate concentration in articial seawater [37], and was
adjusted to pH 8.0 with HCl before to be sterilized by autoclaving at
121 C for 20 min. Aeration and mixing were provided by air-bubbling
at a received net inow of ~ 1.52 L min1 using a KOI AIR KA25 air
pump (maximum air pump output of 25 mL min1, Blagdon, England).
Each experiment was performed using an initial biomass dry weight
concentration comprised between 0.1 and 0.25 g L1 and obtained
from daily-diluted cultures with fresh medium in order to maintain
the cells in the exponentially growing stage.

153

2.2. Experimental design

2.2.1. Nitrogen regime experiment
The effect of nitrogen availability was initially investigated in
batch-mode using three different sodium nitrate (NaNO3) regimes.
Pre-cultivated daily-diluted cultures were centrifuged (1200 g for
5 min) using a Hettich Rotina 38R centrifuge (Andreas Hettich GmbH,
Germany), washed twice, and re-suspended in nitrogen-replete (N+)
or nitrogen-limited (N+/) media using, respectively, 1 and 0.1 g L1
NaNO3 as initial concentrations, or in nitrogen-starved (N) medium
by omitting its addition. After 10 days of cultivation under specic regimes, nitrogen-replete and nitrogen-starved cultures were diluted
ve times with full medium (1 g L1 NaNO3) and allowed to grow for
7 additional days. These experiments were performed in 2000 mL
glass Erlenmeyer asks, using a working volume of 1600 mL, and
under conditions similar to pre-cultivation (continuous illumination
of 100 mol m2 s1 and a temperature of 20 C). Three independent
replications (individual asks) were performed for each nitrogen regime over 17 days of cultivation. Growth performance was determined
and biomass harvested every 23 days for further cellular biochemical
analyses (see Section 2.3).
2.2.2. Combined light and temperature experiment
A full factorial design with two factors (irradiance and temperature)
was used. Each response variable was tested at 3 different levels of irradiance (30, 100 and 200 mol m2 s1) and 5 temperatures (10, 15, 20,
25 and 30 C). A total of 45 asks were used, treatments representing 15
points of the factorial design, and triplicates for each point to determine
the experimental errors (n = 3). Using a second degree polynomial
(i.e., quadratic model), the response surfaces of the main variables of
interest, biomass dry weight, total PB, carbohydrate and EPA productivities, inside the experimental domain were evaluated by analysis of
variance (ANOVA) using SigmaPlot software version 11.0 (Systat
Software Inc., USA). The quadratic model proposed for each response
variable (Yi) was
Yi 0 1 XL 2 XT 1;1 XL 2 2;2 XT 2 1;2 XL XT

where 0 is the intercept, 1 and 2 the linear coefcients, 1,1 and 2,2
the quadratic coefcients, 1,2 the interaction coefcient, and XT and XL
are the factors temperature and light, respectively. The coefcient of regression of the equations was used to determine the goodness of t of
the model with each experimental dataset. Statistical signicance of
light intensity and temperature effects on variables was assessed by
means of an F-test. This approach allowed the determination of the
optimal conditions as well as the effects on each variable of putative
interactions between factors.
The experiment was performed using nitrogen-replete medium
containing a high initial NaNO3 concentration of 1 g L1, and conducted
in 250 mL glass Erlenmeyer asks using a working volume of 150 mL.
Samples were collected after 10 days of cultivation to determine
the nal biomass and to conduct further biochemical analyses (see
Section 2.3).
2.3. Dry weight, biomass harvesting and storage
P. purpureum cells were harvested gently by centrifuging (1200 g for
5 min). The pellets obtained were then frozen, and stored at 20 C
prior to analysis.
Growth performance and biomass productivity were assessed by dry
weight (d.w.) measurements according to Zhu and Lee [38], and
expressed in g L1. Cells harvested by centrifugation were washed
twice with 20 mL 0.5 M ammonium formate to eliminate salt residues.
Then, algal cell suspensions were ltered through pre-weighed
glassber lters (Whatman GF/F, 47 mm, nominal pore size 0.7 m)

154

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

and the lters were dried at 95 C until constant weight (~ 56 h),

cooled down in a vacuum desiccator, and weighed again.
2.4. Nitrate determination
Nitrate concentrations in the media were measured according to
the method reported by Collos et al. [39] and modied as follows;
after 10-fold or 100-fold dilution with distilled water, residual nitrate
concentration in the medium was directly determined according to
optical density measured at 220 nm using a Cary 50 Scan UV-Visible
spectrophotometer (Varian Inc., Palo Alto, CA, USA).
2.5. PB extraction and determination
PBs were extracted using a modied version of the method developed by Chopin et al. [40]. The frozen pellet of algal cells was ground
in liquid nitrogen before resuspension in 0.1 M phosphate buffer
(pH 6.8) for overnight extraction at 4 C in the dark. The samples
were then centrifuged at 5000g for 10 min and the supernatant was
used for PB measurements. Absorbance of the extracts was read at
455, 564, 592, 618 and 645 nm using a Cary 50 Scan UV-Visible
spectrophotometer.
PE and PC concentrations were determined using the equations of
Beer and Eshel [41].


PE mg mL1 OD564

nm OD592 nm OD455 nm OD592 nm

 0:2  0:12

B


PC mg mL1 OD618

nm OD645 nm OD592 nm OD645 nm

 0:51  0:15

pigments from a frozen algal pellet were extracted twice by grinding in

cold 90% aqueous acetone (HPLC grade, Fischer Scientic, UK). After
overnight extraction at 4 C, the samples were then centrifuged
(5000g for 10 min at 4 C) and ltered before analysis. All extraction
processes were conducted on ice and low light conditions to avoid degradation of algal pigments. Pigments were then analyzed and quantied
using a High Performance Liquid Chromatography (HPLC, Agilent 1200
series) equipped with a Diode Array Detector (DAD) and a Fluorescence
Detector (FLD), and separated using a C18 column (150 mm 4.6 mm
inner diameter, Eclipse XDB-C18, Agilent Technologies). The gradient
mobile phase consisted of 80:20 (v/v) methanol/0.5 M ammonium acetate (pH = 7.2, with 0.1% (w/v) added butylated hydroxytoluene
(BHT)), 87.5:12.5 (v/v) acetonitrile/MillQ water (0.1% (w/v) added
BHT) and ethyl-acetate. Identication of algal pigments was obtained
by comparison of retention times and spectra with commercial pigment
standards (DHI, Hrsholm, Denmark; Sigma-Aldrich Co., St. Louis, USA).
Pigment concentrations were calculated using standard curves obtained
by injection of precisely quantied amounts of commercial standards.
2.9. Statistical data analysis
All experiments were carried out in triplicate i.e., measurements
were conducted on three biological replicates (n = 3). Results are
expressed as means standard deviation (SD). The effects of the
xed factors light intensity and temperature on the different
variables or parameters investigated were analyzed using a two-way
analysis of variance (ANOVA). Post-hoc multiple comparisons were
conducted using a Tukey test to identify homogeneous subgroups that
differed signicantly (level of signicance: P b 0.05). All statistics were
performed using SigmaPlot software version 11.0 (Systat Software
Inc., USA).

C
3. Results and discussion
where OD is the optical density of the pigment at the particular
wavelength.
The total PB content was calculated by adding values from
Eqs. (B) and (C).
2.6. Total carbohydrate content determination
Total cell-bound carbohydrate content was analyzed according to
the phenol-sulfuric acid method [42] using D-galactose (Merck) as a
standard. In brief, the harvested cells were hydrolyzed in sulfuric acid
(1 N) in a boiling water bath for 1 h [18]. The cell debris was then removed by centrifugation, and total carbohydrates were determined in
the supernatant.
2.7. Fatty acid analysis
After direct transmethylation of the freeze-dried cells, fatty acid
methyl esters (FAME) were analyzed by gas chromatography (GC)
using an Agilent 7890A GC/5975C MSD Series (Agilent Technologies, Santa Clara, CA, USA) equipped with a ame ionization
detector (FID) and a fused silica capillary column (DB-WAXETR,
0.25 mm 30 m 0.25 m, Agilent Technologies). Protocols for
direct-transmethylation and GC-FID conditions were identical to
those used by Guihneuf and Stengel [36]. Authentic commercially
available FAME standards (Supelco, Bellefonte, PA, USA) were used to
identify the fatty acids by comparing the peak retention times of the
samples and standards. Pentadecanoic acid 15:0 (Pentadecanoic acid,
99%, Alfa Aesar, UK) was added as internal standard.
2.8. Pigment analysis
Pigments were extracted and analyzed according to the method described by Wright [43] and modied by Bidigare and Trees [44]. In brief,

3.1. Nitrogen regime controls PB, carbohydrate, and fatty acid production
Effect of different nitrogen (N) regimes on P. purpureum growth
performance and major cellular components such as cellular PB,
carbohydrates and fatty acids was examined. Three different regimes
(N-replete, N-limited and N-starved conditions) were tested using nitrate (NaNO3) as N source. After 10 days of cultivation, N-replete and
N-starved cultures were resupplied with nutrients by dilution with
full medium (1 g L1 NaNO3) in order to assess the recovery capacity
of P. purpureum cells after N-starvation.
3.1.1. Nitrogen regime and biomass production
The highest biomass (d.w.) was obtained in the N-replete cultures,
increasing linearly to reach 3.4 g L 1 by day 10 (Fig. 1B). The Nreplete culture displayed a constant nitrate uptake rate and a constant
biomass productivity, equal to 27 mg NaNO3 L 1 day1 and
0.33 g L1 day 1, respectively. Previously, comparing over thirty
microalgal strains cultivated in nutrient-replete medium, Rodol et al.
[45] reported P. cruentum as the best biomass producer, with a productivity slightly higher but close to our data (0.37 g L1 day1). Under the
N-limited regime, nitrate level in the medium approached zero on day
9, and the biomass concentration attained a maximum of 1.9 g L 1
after 17 days (Fig. 1A). As a result, nitrate uptake and biomass productivity were strongly reduced (11 mg NaNO3 L 1 day 1 and
0.11 g L1 day1 respectively). Surprisingly, the biomass concentration
of P. purpureum N-starved cultures reached 1.8 g L1 after only 10 days
(Fig. 1C), which corresponds to a biomass productivity of
0.15 g L1 day1, demonstrating the ability of this species to sustain
growth during nitrate-starvation. This may be explained by the use of
P. purpureum intracellular N or by the reassignment of its N pool usually
allocated to the synthetize of PB (see following Section 3.1.2, decrease in
PB content under N-starvation) to other cellular functions involved in

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

N+/-

N+

N+

N+

N-

10

50

Total PB [g.L-1] (

40

N+/-

N+

N+

N+

N-

7.5

2.5

1.5

30
1
0.5

10

0
)

1.0
Carbohydrates [g.L-1] (

25

20

N+/-

N+

N+

N+

N-

60
50

0.8

40

0.6

30

0.4

20

0.2

10
0

0.0
60

8.5

Total PB [% DW] ( )

50
1

Nitrate [%] (

75
2

60

9.5

Carbohydrates [% DW] ( )

DW [g.L-1] (

100
3

pH (

155

N+/-

N+

N+

N+

N-

3.0
2.5

40

2.0

30

1.5

20

1.0

10

TFA [% DW] ( )

TFA [mg.L-1] (

50

3.5

0.5
0.0

11

14

17

Culture time (days)

10

10

17

10

10

17

Culture time (days)

Culture time (days)

Fig. 1. Time courses of biomass d.w. concentration (g L1 of culture, white bars), nitrate uptake (% of initial NaNO3, ), medium pH (), PB volumetric concentration (g L1 of culture, light
grey bars) and content (% d.w., ), cellular carbohydrate volumetric concentration (g L1 of culture, dark grey bars) and content (% d.w., ), and cellular total TFA concentration (g L1 of
culture, black bars) and content (% d.w., ) of P. purpureum cultivated under N-limited (N+/, 0.1 g L1 NaNO3; A, E, C and K), N-replete (N+, 1 g L1 NaNO3; B, F, E and L), and Ndepleted (N, 0 g L1 NaNO3; C, G, J and M) conditions, and at an irradiance of 100 mol m2 s1 and temperature of 20 C. The arrow represents the time of Nitrate-depletion
when cultivated in N-limited medium. The vertical dash-line indicates the time when cultures grown under N-replete and N-depleted conditions were diluted by ve with fresh
N-replete medium i.e., semi-continuous and N-starvation recovery experiments. All measurements represent the mean SD of three individual replicates.

Table 1
Changes of PB volumetric concentration (mg L1), content (% d.w.) and composition (% total PB) in cultures of P. purpureum cultivated under different N regimes at an irradiance of 100
mol m2 s1 and a temperature of 20 C. All data represent the mean SD of three individual replicates.
Nitrogen regimea

Daysb

mg L1
Total PB

% d.w

% total PB
PE

PC

N+/

0
7
9
17

0.8 0.1
7.7 0.9
10.5 0.3
10.4 0.9

0.7 0.1
6.8 1.0
9.0 0.3
9.4 0.8

0.12 0.01
0.84 0.18
1.48 0.07
1.03 0.21

1.27 0.08
1.16 0.04
1.41 0.16
0.56 0.06

1.09 0.08
1.03 0.07
1.21 0.14
0.51 0.07

0.18 0.01
0.13 0.03
0.18 0.02
0.06 0.02

85.8 0.7
88.9 3.4
86.1 0.4
90.2 1.5

14.2 0.7
11.2 3.4
13.9 0.4
9.8 1.5

N+

0
7
10
17

4.7 0.3
34.6 5.8
47.2 6.6
21.8 1.1

4.0 0.3
29.8 4.4
38.8 4.5
19.1 0.9

0.75 0.11
4.85 1.40
8.45 2.39
2.68 0.15

1.73 0.19
1.80 0.36
1.44 0.06
1.55 0.05

1.45 0.15
1.54 0.28
1.18 0.01
1.36 0.05

0.27 0.06
0.25 0.08
0.26 0.05
0.21 0.01

84.2 2.0
86.2 2.1
82.3 2.9
87.7 1.1

15.8 2.0
13.8 2.1
17.7 2.9
12.3 1.1

0
7
10
17

4.7 0.3
1.4 0.1
0.6 0.2
26.4 0.1

4.0 0.3
1.4 0.1
0.6 0.3
22.6 0.1

0.75 0.11
0.03 0.02
0.03 0.01
3.84 0.02

1.73 0.19
0.14 0.03
0.04 0.01
1.50 0.15

1.45 0.15
0.14 0.03
0.03 0.01
1.31 0.12

0.27 0.06
0.00 0.00
0.00 0.00
0.26 0.02

84.2 2.0
98.3 1.7
94.8 2.4
85.5 1.2

15.8 2.0
1.7 1.7
5.2 3.3
14.5 1.2

Before dilution
N+ semi-continuous
N
Before dilution
N+ recovery

PE

PC

Total PB

PE

PC

Cultivated under N-limited (N+/, 0.1 g L1 NaNO3), N-replete (N+, 1 g L1 NaNO3), and N-depleted (N, 0 g L1 NaNO3) conditions.
Nitrate-depletion was reached after 9 days when cultivated in a N-limited medium. Cultures grown under N-replete and N-depleted conditions were diluted by ve in day 10 with
fresh N-replete medium.
a

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F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

the maintenance of cell growth. The cellular N content is around 710%

of d.w. for most microalgae during balanced growth conditions [46].
Therefore, biomass of some species may still increase 8-fold once extracellular N is fully depleted [47]. In previous culture optimization studies,
a concentration close to 1 g L1 NaNO3 has similarly been reported to
maximize P. purpureum growth [1,48].
In summary, unlimited N-supply such as N-replete regime or continuous cultivation seems to be the best culture strategy for P. purpureum
growth and biomass production.

3.1.2. Nitrogen regime and PB production

PB volumetric productivity, content and composition of P. purpureum
were analyzed for cells grown under the three regimes tested (Fig. 1 and
Table 1). Results clearly demonstrate that N availability represents a
limiting factor for PB accumulation and productivity. The highest PB volumetric concentration (47 mg L1) and content (1.8% d.w.) were obtained in N-replete cultures (Fig. 1F); N-starvation always induced a strong
decrease in PB content, from 1.4% (day 9) to 0.6% (day 17) under Nlimitation (Fig. 1E) and more drastically from 1.7% (day 0) to almost 0%
(day 10) under N-starvation (Fig. 1G). Similarly, PE content was reduced
by 99% over a period of 14 days under N-limitation [49]. Indeed, detachment of phycobilisomes from the thylakoid membranes and their reduction in number may explain the strong decrease in PB content caused by
N-starvation [50].
Our results also clearly highlight the ability of P. purpureum cells to
recover from N-starvation and rebuild almost completely their PB content, reaching 1.5% d.w. in 7 days after medium refreshment (Fig. 1G).
Indeed, recovery upon addition of N resulted in increased PB content
and appearance of phycobilisomes attached to the thylakoids [49].
Under the N-replete regime, medium refreshment allowed the maintenance of a constant PB content (~ 1.6% d.w.), suggesting that semicontinuous or continuous cultivation may be the most suitable modes

for PB production in P. purpureum (Fig. 1F). The concentration of PE

has been previously shown to be directly controlled by N availability
in the culture medium, increasing with renewal rate during semicontinuous cultivation [19]. On the other hand, the substantial decrease
in PB content observed in day 4 (Fig. 1E and F) may be explained by light
stress occurring after cell dilution of the inoculum culture. Typically
transfer of cells from low to high light results in an adaptation of
the PB content to the new light conditions [32]. Hence, PB content
decreased, as did the phycobilisome number in cells exposed to
high light [51,52]. Interactive effects of light and temperature on
P. purpureum growth and biochemical composition will be discussed
in detail in Section 3.2.
PE is known to be the major component and main economically
valuable component of PBs in P. purpureum [1,53]. It always accounted
for more than 80% of the total PBs in our study (Table 1). Fig. 2 illustrates
the red uorescence emitted by the PE-rich cells and observed by uorescence microscopy, as well as the decrease in total PB content induced
by N-depletion and N-starvation.

3.1.3. Nitrogen regime and total cellular carbohydrate production

Generally, in Porphyridium spp., cellular carbohydrate content increases under reduced N availability [35], and depletion of nitrate
from the medium has been described to promote the production of
cell-wall polysaccharide and starch [29,50]. Here, when P. purpureum
was grown in N-limited medium (Fig. 1H), the cellular carbohydrate
content (on d.w. basis) increased during the logarithmic phase of
growth reaching a maximum and stable level of 40% after nitratedepletion (between days 11 and 17). The carbohydrate content of Nstarved cultures (Fig. 1J) similarly peaked at 48% d.w., before stabilizing
at ~40%. The highest cellular carbohydrate production and volumetric
concentration were however obtained in P. purpureum grown in Nreplete medium (0.92 g L1 after 10 days; Fig. 1I). This can be explained

Fig. 2. Light and uorescence micrographs of P. purpureum cells cultivated for 10 days under N-replete (A and B), N-limited (C) or N-depleted (D) conditions, showing the decrease in
cellular PB content induced by N-depletion. Red uorescence emitted by the R-phycoerythrin-rich cells (maximum emission at 580 nm) was observed using a 450500 nm band-pass
exciter lter. (For interpretation of the references to color in this gure legend, the reader is referred to the electronic version of this article.)

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

by the relatively high biomass obtained under N-repletion (3.3 g L1) in

association with a low, lowest but non-negligible, carbohydrate content
(28% d.w.). Comparing three regimes of supply (batch-mode, continual
mode and decient mode), Arad et al. [18] similarly obtained a maximum cell-bound polysaccharide production in batch cultures, demonstrating the highest nal cell densities and growth performance. Here,
refreshment of the medium was also shown to induce a decrease in cellular carbohydrate content after both N-replete and N-starved cultures
(Fig. 1IJ). For an optimal carbohydrate production, cellular accumulation and growth performance therefore need to be considered
simultaneously.

157

excessive production of smooth endoplasmic reticulum under normal

growth conditions, or by a collapse of phycobilisome-depleted thylakoids under nutrient-starvation [55]. In our study, medium refreshment
of N-starved cells induced an increase in TFA content (Fig. 1M), suggesting de novo fatty acid synthesis to rebuild the degraded cellular membranes. Supplementation of the N-starved cells has been shown to
lead to an almost complete regeneration of the plastid and
phycobilisome number; lipid bodies were degraded and their fatty
acids were used for membrane formation [50]. Our results suggest a
similar recovery process from N-starvation with an almost complete
restoration of the PB and TFA content. Wanner and Kst [55] proposed
lipid bodies and TAG function as a membrane component store
which is used for rapid membrane synthesis during regeneration.
Later on, Khozin-Goldberg et al. [56] demonstrated that TAG can contribute to the biosynthesis of eukaryotic galactolipids constituting the
thylakoid membranes.
Table 2 displays the fatty acid composition of P. purpureum cultivated under the different N-regimes. Levels of both major n3 PUFAs presented, -linolenic (ALA, 18:3 n3) and eicosapentaenoic (EPA, 20:5
n3) acids, decreased after N-depletion and N-starvation. No change
in the fatty acid composition was observed in N-replete cultures.
Using a semi-continuous culture, Fbregas et al. [19] reported that the
percentage of EPA increased with increasing medium renewal rates at
the expense of linoleic (LA, 18:2 n 6) and arachidonic (ARA, 20:4
n 6) acids, highlighting the role played by N availability on EPA
level. By contrast, the decrease in n 3 PUFAs was mainly correlated
to an increase in palmitic (16:0) and oleic (OLE, 18:1 n 9) acids in
this study. The maximal EPA volumetric concentration, and therefore
productivity, was obtained under N-repletion, while the highest EPA
content (0.44% d.w.) was obtained after medium refreshment of the
same regime (i.e., refreshment like semi-continuous culture). Semicontinuous or continuous cultivation mode represents therefore the
best production system for high EPA content and productivity in
P. purpureum.

3.1.4. Nitrogen regime and fatty acid content and composition

Many microalgal strains have the ability to accumulate large quantities of lipids in the form of triacylglycerols (TAGs) under environmental
stress conditions such as N-starvation, reaching up to ~80% in some species [47,54]. However, lipid or total fatty acid (TFA) contents reported in
red microalgae such as Porphyridium spp. are usually low (57% [34], 9
14% [7], 47% [4], 5.9% [5], 9.5% [45] d.w.), suggesting an inferior ability
of this genus to accumulate lipids. After cultivation in our chosen conditions (Fig. 1 and Table 2), the TFA content (1.12.8% d.w.) was lower
than other previously reported data. The highest TFA content (2.8%
d.w. after medium refreshment, day 17), volumetric concentration
(50.3 mg L1, day 10) and productivity (5 mg L1 day1) were obtained under N-replete condition (Fig. 1L). Indeed, Rodol et al. [45] showed
that despite a high biomass productivity in N-replete cultures, lipid content in P. cruentum was rather low and thus lipid productivity was not
among the highest of the microalgal species investigated. Our results
also demonstrate the inability of P. purpureum to accumulate fatty
acids after N-depletion (Fig. 1K) and during N-starvation (Fig. 1M), possibly caused by its low ability to accumulate TAGs, especially in cultures
mixed by air-bubbling without CO2 enrichment. As observed in Fig. 1
(A-C), the slight increase in medium pH associated to cell growth may
suggest a decrease in dissolved CO2 and therefore a limitation in inorganic carbon present in the cultures. In both cases, TFA content decreased under N-deciency to a level as low as 1.1% d.w. Similarly,
Cohen [4] did not observe any increase in TFA content in the stationary
phase of seven P. cruentum strains when batch-cultivated. The decrease
in TFA content may be explained by chloroplastic membrane degradation, as supported by the parallel decrease in PB content observed during N-deciency. Indeed, TAGs have been shown to increase 2.5-fold
under nutrient-starvation but 80% of the TAGs formed were recovered
from isolated lipid bodies which exhibited an unusual high phospholipid content suggesting their origin from membrane degradation [50].
Lipid bodies in Porphyridium spp. are formed by aggregation and fusion
of cellular membranes; these membranes are derived either from

3.2. Interactive effects of light and temperature on P. purpureum high-value

components
Large-scale production of microalgae such as Porphyridium spp. requires optimization of the major parameters affecting growth as well
as bioactive production, with particular reference to temperature and
light [57]. To date, most studies on this algal genus investigated the effect of one of these factors, or the interactive effect of both, but generally
only on growth performance or one compound group of interest [5,15,
25,5761]. To the best of our knowledge, no studies have so far investigated the combined effects of light and temperature on both

Table 2
Fatty acid composition of P. purpureum cultivated under different N regimes at an irradiance of 100 mol m2 s1 and a temperature of 20 C. All data represent the mean SD of three
individual replicates.
Nitrogen regimea

Daysb

TFA

EPA

Fatty acid composition (% of TFA)

mg L1

% d.w.

mg L1

% d.w.

16:0

2.4 0.1
1.3 0.1
1.9 0.2
1.1 0.2

0.2 0.0
1.4 0.1
1.9 0.1
3.0 0.2

16:1 n7

18:0

18:1 n9

18:2 n6

18:3 n3

20:4 n6

20:5 n3

Others

N+/

0
7
9
17

1.4 0.1
8.7 0.8
13.5 1.8
22.4 1.0

0.37 0.04
0.21 0.03
0.25 0.02
0.15 0.02

29.1
32.3
32.4
36.3

4.1
4.9
3.2
1.9

3.4
2.9
1.7
1.2

7.6
7.2
10.7
17.7

5.4
5.3
5.6
8.4

22.9
24.2
28.4
16.5

2.8
2.0
1.2
0.5

15.8
15.7
13.9
13.4

7.9
5.6
2.9
4.1

N+

0
7
10
17

6.7 0.3 2.7 0.2 1.0 0.0 0.41 0.05

33.2 1.2 2.1 0.2 5.2 0.3 0.33 0.03
50.3 6.2 2.2 0.2 7.9 0.8 0.34 0.05
33.9 1.6 2.8 0.1 5.4 0.1 0.44 0.01

31.4
31.4
31.2
32.7

1.2
1.4
1.0
2.0

2.3
1.4
1.7
2.0

12.1
12.3
12.9
11.9

6.0
6.4
6.6
4.9

22.4
22.1
22.5
22.6

3.6
1.6
1.6
1.1

15.1
15.7
15.8
15.8

5.9
7.7
6.8
7.0

0
7
10
17

6.7 0.3 2.7 0.2 1.0 0.0 0.41 0.05

12.9 3.1 1.6 0.3 1.5 0.2 0.19 0.04
15.3 2.5 1.1 0.1 1.4 0.5 0.10 0.03
32.0 2.7 2.3 0.2 4.3 0.6 0.30 0.02

31.4
36.6
41.0
36.6

1.2
1.4
1.9
3.4

2.3
3.3
1.2
3.3

12.1
14.1
16.4
11.2

6.0
6.1
6.2
4.9

22.4
17.5
15.3
18.8

3.6
1.8
1.4
0.8

15.1
12.0
8.8
13.5

5.9
7.2
7.7
7.4

Before dilution
N+ semi-continuous
N
Before dilution
N+ recovery
a,b

See footnotes in Table 1.

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F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

simultaneously, growth and production of Porphyridium major highvalue chemicals. In this context, we assessed the interactive effects of
light and temperature on growth, PB production and others of
P. purpureum batch-cultivated under N-replete condition, accordingly
to our initial results (Section 3.1). This was done by response surface
methodology (RSM) applying a second degree polynomial
(i.e., quadratic model) in order to determine the optimal culture conditions for biomass d.w., total PB, carbohydrate and EPA production.
Meanwhile, detailed analyses of the fatty acid and pigment content
and composition were performed to gain biochemical and physiological
insights into the algal response to these combined abiotic factors.
3.2.1. Optimization of P. purpureum growth for maximal high-value compound production
The conventional method of culture optimization, one factor at a
time, is time-consuming, expensive and often leads to misinterpretations of results when interactions between different factors occur.
Therefore, a full factorial design with two factors (light intensity and
temperature) was used to evaluate their interactive effects on variables
and to determine optimal culture conditions for each compound of
interest.
By applying regression analysis on the experimentally determined
data, the coefcients of the quadratic model to predict the response variables (Y) were estimated for the following second degree polynomial
equations:
Y Biomass 4:7830 0:0235XL
0:5133XT 0:000051973XL 2 0:0092XT 2 0:0006XL XT 1
Y Total

PB

28:35230:0758XL 6:0861XT
0:0005XL 2 0:1424XT 2 0:0066XL XT

Y Carbo 1:2680 0:0034XL

0:1609XT 0:00001013XL 2 0:0034XT 2 0:00005987XL XT
3
Y EPA 0:8693 0:0022XL
0:4067XT 0:000005766XL 2 0:0114XT 2 0:0001XL XT

When the values of XL to XT were substituted in the above equations,

the model predicted values (Y) were obtained (Table 3). The predicted
values were in reasonably close agreement when compared with the
experimentally obtained data. Indeed, analysis of variance revealed
that the quadratic models derived from RSM could adequately be used

to describe the culture conditions for each variable. The four R2-values
0.8462, 0.9320, 0.7926 and 0.7037, respectively, determined for biomass productivity response (YBiomass), total PB productivity response
(YTotal PB), carbohydrate productivity response (YCarbo) and EPA productivity response (YEPA) indicate a relatively good t between the model
and experimental data. As an example, the R2-value implied that the
sample variation of 93.2% for total PB was attributed to the factors,
and also that only 6.8% of the total variation was not explained by the
model. Moreover, the respective F-values of 15.4110, 38.3970, 9.9366
and 6.6483 revealed that regression was statistically signicant
(P b 0.05) at the 95% condence level.
Fig. 3 shows the interactive effects between light intensity and
temperature represented by isoresponse contour plots (i.e., RSM)
of biomass d.w., total PB, carbohydrate and EPA productivities of
P. purpureum batch-cultivated under N-replete condition.
3.2.1.1. Interaction of light and temperature on biomass production. The
results of the regression analysis and the two-way ANOVA indicate
that temperature (P b 0.0001) and light (P b 0.0001) had signicant independent effects, as well as an interactive effect (P b 0.0001), on biomass production. As suggested by Dermoun et al. [57], these two
factors, which are generally considered independent, are in fact closely
related and interact on Porphyridium growth, especially under nonlimited nutrient conditions. Under optimal light intensity estimated
around 90 mol m2 s1, the biomass productivity increased with increasing temperature in the range of 1025 C reaching a maximum of
3.1 g L1 (experimental data) and 2.7 g L1 (predicted data) at 25 C
(Fig. 3 and Table 3). Similar ranges in light and temperature levels
have been previously described to be optimal for Porphyridium growth,
such as ~2530 C and ~50100 mol m2 s1 [57,58,6264].
3.2.1.2. Interaction of light and temperature on PB production. Statistical
analyses also indicated signicant independent effects of light and temperature on PB production (P b 0.0001 and P b 0.0001, respectively), as
well as a signicant interactive effect (P b 0.0001). Optimal PB production was obtained at ~ 20 C and low light intensity
(30 mol m 2 s1) reaching up to 33.3 mg L1 (experimental data)
and 30.6 mg L 1 (predicted data). On the other hand, increases in
light and temperature were negatively correlated (P b 0.001 and
P b 0.001, respectively) with PB content, with a maximum of 2.89%
d.w. observed under low light and low temperature (Fig. 3 and
Table 3). Our results are similar to those for P. purpureum, P. cruentum
and Porphyridium aerugineum [32,51], indicating that cells cultured at
low light possess up to three times more PBs than cells grown under
high light. Indeed, high-light grown cells had the smallest

Table 3
Combined effects of light and temperature on biomass, total PB and carbohydrates, and EPA content and productivity of P. purpureum cultivated in a N-replete medium (day 10) with the
real experimental and the model-based prediction values of the response variable. All data represent the mean SD of three individual replicates.
Light
intensity

T C

mol m2 s1
30

100

200

10
15
20
25
30
10
15
20
25
30
10
15
20
25
30

Biomass

Total PB

Experimental
data

Model
predictions

Carbohydrates

Experimental
data

Model
predictions

EPA

Experimental
data

Model
predictions

g L1

% d.w.

mg L1

mg L1

% d.w.

mg L1

mg L1

% d.w.

mg L1

mg L1

0.4 0.1
1.1 0.1
1.7 0.1
2.6 0.4
2.6 0.3
0.6 0.1
1.1 0.1
2.6 0.1
3.1 0.3
2.6 0.3
0.9 0.1
1.3 0.1
2.5 0.1
2.2 0.1
0.9 0.1

2.89 0.34
2.80 0.30
1.96 0.09
0.96 0.15
0.73 0.04
1.53 0.09
1.32 0.10
0.81 0.06
0.49 0.14
0.21 0.03
1.04 0.20
1.09 0.04
0.39 0.03
0.32 0.02
0.07 0.00

11.6 0.7
29.3 1.8
33.3 0.9
24.5 0.6
18.7 1.4
9.8 0.4
15.1 0.9
20.7 1.3
15.2 2.0
5.6 0.7
9.3 0.3
13.9 0.3
9.8 0.9
7.0 0.2
1.8 0.1

14.4
26.1
30.6
28.0
18.3
8.6
18.0
20.2
15.3
3.3
8.0
14.1
13.1
4.9
1.5

45.0 1.3
33.0 5.0
32.5 2.6
27.7 1.0
23.3 4.3
31.0 6.2
26.3 2.5
30.1 2.9
22.6 3.7
17.0 1.5
25.4 3.8
24.0 0.7
29.9 2.1
26.6 1.1
n.d.

183 23
344 28
552 30
715 96
587 57
196 25
300 06
768 69
701 50
441 23
228 03
306 09
747 44
592 45
n.d.

76
446
646
676
536
181
531
710
719
558
159
479
628
607
n.d.

0.40 0.05
0.27 0.05
0.21 0.05
0.09 0.02
0.08 0.01
0.33 0.03
0.29 0.02
0.14 0.05
0.08 0.02
0.08 0.02
0.19 0.03
0.27 0.06
0.13 0.02
0.07 0.01
n.d.

1.5 0.2
3.6 0.6
2.8 0.6
2.3 0.4
2.1 0.8
2.1 0.1
3.3 0.2
3.6 0.6
2.5 0.4
2.2 0.5
1.7 0.2
3.0 0.6
3.2 0.6
1.6 0.2
n.d.

2.1
2.7
2.7
2.2
1.0
2.1
2.7
2.8
2.1
0.9
2.0
2.5
2.5
1.8
n.d.

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

159

Fig. 3. Isoresponse contour plots of biomass d.w., total PB, carbohydrate and EPA productivities as a function of irradiance and temperature when P. purpureum cultivated under N-replete
medium (day 10).

photosynthetic unit size (PB plus Chl.), the highest photosynthetic capacity, and the highest growth rates [51]. Gurin-Dumartralt et al. [65]
showed that under white light the Chl. and PE contents decreased
with increasing light intensity while the ratio PE/Chl. was little affected.
PB content also decreased in response to an increase in light intensity in
another red microalga Rhodella reticulata and the Cryptophyte
Rhodomonas sp. [66,67]. Additional details on pigment content and
composition are provided in Section 3.2.3.
3.2.1.3. Interaction of light and temperature on carbohydrate production.
Temperature was the only factor exhibiting a signicant effect on carbohydrate production (P b 0.001). Carbohydrate productivity was not signicantly affected by light (P = 0.291) nor the interaction between light
and temperature (P = 0.474). Maximum carbohydrate productivity
was obtained under a light intensity of 100 mol m2 s1 and temperatures between 20 and 25 C, reaching up to 768 mg L1 (experimental
data) and 719 mg L1 (predicted data) (Fig. 3 and Table 3). In addition,
carbohydrate content decreased signicantly with both light (P b 0.001)
and temperature (P b 0.001), and accounted for a maximum of 45% d.w.
under combined low light (30 mol m 2 s1) and low temperature
(10 C) (Table 3). Previously Friedman et al. [68] showed speciesspecic responses of cellular polysaccharides to light intensity; in
P. aerugineum they increased with increasing light but cell number
remained almost constant; Porphyridium sp. responded to varying
light by changing cell density, but changes in cell polysaccharides
were much smaller. This was conrmed by the highest carbohydrate

content in P. purpureum cells N-starved or N-depleted which exhibited

reduced cell growth (Fig. 1). Low irradiance and temperature inducing
a longer cellular cycle also proved to be greater for carbohydrate accumulation even under N-replete condition. Indeed, Cohen et al. [69] indicate that when growth is reduced by any limiting factor, such as light
restriction, lipid and carbohydrate synthesis may be enhanced at the expense of proteins.
3.2.1.4. Interaction of light and temperature on EPA production. Temperature alone, and interaction between both factors, also signicantly affected EPA production (P b 0.001, P = 0.001, respectively) but no
signicant variations were observed with changes in light alone (P =
0.283). According to the quadratic model (Eq. (4)) used to create the
isoresponse contour plot, the optimal growth conditions for EPA
production were estimated to be around 17.5 C and
50 mol m 2 s1, suggesting a maximum EPA productivity of
2.8 mg L1 (predicted data), while the measured highest value reached
3.6 mg L1 (experimental data) (Fig. 3 and Table 3). Our results also
demonstrated that EPA content decreased signicantly with both light
(P b 0.001) and temperature (P b 0.001). Therefore, the highest EPA
content, 0.40% d.w., was obtained under low light (30 mol m2 s1)
and low temperature (10 C) (Table 3). Nuutila et al. [48] previously
demonstrated that the EPA content per cell decreased with increasing
temperature while the total EPA production is mainly affected by the
size of inoculum, especially under low temperature. Their highest EPA
production, 298 g per 50 mL (i.e., ~5.96 mg L1), was achieved after

160

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

Table 4
Combined effects of light and temperature on the fatty acid content and composition of P. purpureum cultivated in a N-replete medium (day 10). All data represent the mean SD of three
individual replicates.
Light intensity

T C

mol m2 s1
30

100

200

10
15
20
25
30
10
15
20
25
30
10
15
20
25
30

TFA

Fatty acid composition (% of TFA)

mg L1

% d.w.

16:0

16:1 n7

18:0

18:1 n9

18:2 n6

18:3 n3

20:4 n6

20:5 n3

Others

13.9 2.3
33.4 3.8
29.9 2.2
35.1 8.4
30.9 4.7
14.2 1.5
26.9 3.8
38.8 6.5
30.9 4.3
28.4 2.5
11.7 1.7
26.0 2.8
33.9 3.8
22.8 1.6
n.d.

3.7 0.6
2.5 0.3
2.2 0.2
1.4 0.3
1.2 0.2
2.2 0.2
2.3 0.3
1.5 0.3
1.0 0.2
1.1 0.1
1.3 0.2
2.3 0.2
1.3 0.1
1.0 0.1
n.d.

38.0
36.3
36.2
35.8
34.3
35.7
35.9
35.8
34.8
34.2
34.0
33.9
32.8
32.6
n.d.

0.6
0.7
0.7
0.6
0.9
0.6
0.8
0.7
1.0
2.1
0.7
0.4
0.6
0.6
n.d.

1.8
2.8
2.5
2.7
2.7
2.5
2.3
2.5
2.8
2.8
2.2
3.5
3.3
3.4
n.d.

13.0
13.8
15.8
16.5
16.9
14.4
15.9
17.2
17.8
17.4
16.6
17.4
20.6
20.8
n.d.

4.9
5.6
6.5
7.5
10.1
4.1
5.9
6.8
11.1
13.2
5.4
7.2
8.9
12.5
n.d.

22.6
22.0
21.1
21.3
19.2
18.8
17.4
17.8
15.3
13.3
19.3
16.3
15.3
14.8
n.d.

3.9
3.4
3.2
3.7
3.4
3.7
3.9
3.9
3.8
4.2
2.5
4.4
4.0
3.7
n.d.

10.7
10.7
9.4
6.6
6.9
14.8
12.4
9.4
8.4
7.6
14.8
11.6
9.5
6.8
n.d.

4.6
4.7
4.6
5.3
5.6
5.4
5.5
5.9
5.0
5.2
4.5
5.3
5.0
4.8
n.d.

21 days of cultivation (~0.28 mg L1 day1) using high inoculum concentration of 5 105 cell mL1 at 8 C. With the lowest inoculum concentration of 2 105 cell mL1 at a temperature range 825 C, it
ranged from 51 to 85 g per 50 mL (i.e., 1.031.70 mg L1, 0.05
0.08 mg mL1 day1). Equally, in our study, the EPA productivity of
P. purpureum, when batch-cultivated, ranged from 1.5 to 3.6 mg L 1
after 10 days of cultivation (i.e., 0.150.36 mg L1 day1). Increasing
the cultivation temperature was also shown to lower the yield of ARA
per cell but increased the rate of its production per unit volume and
time [62]. This is also true for EPA to a certain extent, up to a temperature of 20 C. Additional details on the combined effect of light and temperature on fatty acid content and composition follow in Section 3.2.2.
3.2.2. Light and temperature effects on total fatty acid content and
composition
Details on the combined effects of light and temperature on the TFA
content and composition are displayed in Table 4. As observed for EPA
content, the TFA content (3.7% d.w.) was maximal under low light
(30 mol m2 s1) and low temperature (10 C), and decreased significantly with increasing light and temperature (P b 0.001, P b 0.001,
respectively). This could be explained by a drastic reduction in thylakoid
membrane area under high light [52], and the low ability of
P. purpureum to accumulate lipids even under N-starvation as previously described (Section 3.1.4). Our result is also in agreement with Cohen
and Heimer [69], suggesting that slow growth induced by limiting factors, such as low light and low temperature, could promote lipid synthesis. Nonetheless, due to the common low TFA content in P. purpureum
(1.03.7% d.w.), culture conditions reaching the highest TFA productivities (3538 mg L1) were slightly different and closer to those required
to reach the highest biomass productivities (2025 C and between 30
and 100 mol m2 s1), demonstrating again the importance of taking
into consideration growth and content to assess optimal productivity.
Table 4 also displays the interactive effects of light and temperature
on the distribution of the major fatty acids present in P. purpureum
batch-cultivated under N-replete medium. EPA has been described as
the main LC-PUFA in P. cruentum cultivated under optimal growth conditions, such as optimal temperature under non-limiting light conditions [25,30]. When the growth rate was reduced by decreased light
intensity, increased cell density, suboptimal temperature, suboptimal
pH, or increased salinity; the content of EPA decreased and that of
ARA increased, the latter becoming the major LC-PUFA [25].
In our study, the proportions of ARA stayed surprisingly low (3.2
4.4% TFA) and the main PUFAs were LA (4.112.5% TFA), ALA (13.3
22.6% TFA) and EPA (6.614.8% TFA). This may be explained by the difference in strains used in different studies. Both n3 PUFAs, ALA and
EPA, increased signicantly under low temperature (10 C), while OLE

and LA decreased. Thus, our results also demonstrate an increase in

n6 PUFAs related to a decrease in n3 PUFAs. The highest percentage of EPA (14.8% TFA) was obtained under 100200 mol m2 s1 at
10 C; and the highest percentage of ALA (22.6% TFA) under both low
light (30 mol m2 s1) and low temperature (10 C). Our results differ
therefore from those obtained by others [25,30], with a maximum EPA
level in cells grown under low light and/or low temperature which
demonstrated the lowest growth. A decrease in n3 PUFAs, in particular EPA, with increasing light or temperature is a common trend observed in Porphyridium spp. [63,70] and numerous other microalgal
species [37,7173].
Acclimation to low temperatures generally results in PUFA enrichment, mostly in complex polar lipids constituting the membranes, leading to an increase in membrane uidity [7274]. In their patent,
Thepenier et al. [75] relate an invention for the selective production of
PUFAs, especially EPA, by P. cruentum by subjecting a biomass grown
under optimal conditions to a temperature decrease. Light intensity
also regulates LC-PUFA synthesis in microalgae [76,77], inducing accumulation of n 3 LC-PUFAs such as EPA under low light [37] which
may facilitate thylakoid membrane uidity and, therefore, the velocity
of electron ow involved in photosynthesis [78]. As a consequence,
the slightly lower percentage of EPA (10.7% TFA) observed under low
light and low temperature is probably induced by light-stress and the
relative high light availability per cell occurring under reduced cell
density.
Combined modications of more than one abiotic factor, such as
light and temperature, appear therefore to be an effective additional
strategy to regulate and maintain high n3 PUFA level and content in
P. purpureum.
3.2.3. Light and temperature effects on pigment content and composition
The combined effects of light and temperature on the major pigment
content and productivity as well as composition have also been investigated and are displayed in Table 5 and Fig. 4. In addition to changes in
PB, a large number of studies already investigated independently the effect of these factors on pigments (i.e. Chl. a and carotenoids) and photosynthetic activity of Porphyridium spp. [51,52,64,65,68,7981]. Again,
high light-grown cells had the smallest photosynthetic unit size (PB
plus Chl.), the highest photosynthetic capacity, and the highest growth
rates [51]. Cunningham et al. [52] showed that P. cruentum cells exposed
to increasing growth irradiance exhibited up to a three-fold reduction in
photosystems I and II and phycobilisomes, associated with a decrease in
Chl. a and -carotene per cell, and a drastic reduction in thylakoid membrane area [52]. This is in agreement with our results showing a general
decrease in all contents, PB (Table 3, from 2.89 to almost 0% d.w.) and
other major pigments of P. purpureum (Table 5), with combined

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

161

Table 5
Combined effects of light and temperature on the pigment content and productivity of P. purpureum cultivated in a N-replete medium (day 10). All data represent the mean SD of three
individual replicates.
Light intensity

T C

Chlorophyll a

mol m2 s1
30

10
15
20
25
30
10
15
20
25
30
10
15
20
25
30

100

200

g g1 d.w.

g L1

g g1 d.w.

g L1

g g1 d.w.

364 37
1192 40
1084 49
1176 78
1048 43
209 38
387 34
764 93
837 16
315 19
201 16
389 8
357 18
291 15
99 7

900 42
1134 46
646 30
460 59
413 50
325 31
339 24
334 26
270 37
121 9
223 27
306 10
143 11
131 2
108 10

78 3
283 12
337 27
411 13
327 2
60 9
149 12
267 8
341 24
163 6
119 12
204 2
182 25
196 28
68 6

194 20
269 11
198 18
162 27
129 14
94 6
130 10
105 7
110 23
63 6
132 10
160 2
73 11
88 9
74 9

136 5
256 8
237 11
252 19
258 14
84 17
137 11
187 39
189 8
96 5
120 5
163 4
130 13
111 5
38 2

338 37
244 24
139 10
98 12
102 16
131 14
120 11
73 14
61 9
37 5
134 20
128 1
52 5
50 4
41 6

increasing light and temperature (i.e., Chl. a, from 1134 to 108 g g1

d.w.; zeaxanthin, from 269 to 63 g g 1 d.w.; and -carotene, from
338 to 37 g g1 d.w.). Despite the reduced cell growth under low
light (30 mol m2 s1), highest productivities of Chl. a, zeaxanthin
and -carotene occurred at this light intensity but specically within
30 mol m-2 s-1

100 mol m-2 s-1

31

32

10 C
17

200 mol m-2 s-1

34

34

47

51
21

21

32

27

28
46

15 C
22

20
26

33

21

25

28

33

19

24

50

52
29

42

51

54

19

25 C

40

58
28

20 C

37

32
39

3222

22

30 C

4351

53
26

17
35

Chlorophyll

-carotene

Zeaxanthin

g L1

Zeaxanthin

24

37

40

-carotene

Fig. 4. Combined effects of light and temperature on the pigment composition (% molar) of
P. purpureum cultivated in a N-replete medium (day 10). All data represent the mean SD
of three individual replicates.

the optimal growth temperature range of 2025 C (Table 5). Again,

the low cell density at 30 mol m2 s1 and 10 C, and therefore the relative high light availability per cell, may have caused the unexpected
decreases in pigment contents and productivities obtained under such
conditions. Our results show a decrease in Chl. a/Car ratio, based on
the % molar of each pigment (Fig. 4) with increasing light intensity, suggesting a role other than light-harvesting for the two carotenoids. Regardless of light intensity, this was also observed under suboptimal
temperatures resulting, most of the time, in reduced cell-growth and,
therefore, highest light stress per cell, particularly at the low temperature of 10 C.
Carotenoids such as zeaxanthin and -carotene have long been recognized as key constituents of photoprotective defenses, in particular to
high light stress, in microalgal eukaryotes [82,83]. For example, carotene and astaxanthin are, respectively, overproduced in the
microalgae Dunaliella salina and Haematococcus pluvialis in response
to high light intensities [84,85]. The combined stressors light and temperature therefore cause signicant changes in the pigment composition of light harvesting apparatus of P. purpureum which may be
attributed to the disruption of thylakoid membranes. This suggestion
is supported by the decrease in PB, Chl. a and carotenoid content
under high light intensity which is promoted by elevated temperature.
4. Conclusions
For optimal bioactive production, cell content and biomass yield
have to be considered simultaneously. Nitrate-deciency causes a
strong decrease in P. purpureum growth, as well as in its PE, TFA and
EPA contents which may be caused by membrane degradation but induces cellular carbohydrate accumulation. Due to high biomass productivity, a N-replete regime represents however the best culture strategy
for PB, carbohydrate, TFA and EPA production in batch-cultivated
P. purpureum. Our results also demonstrate the ability of nitratestarved cells of P. purpureum to restore PB and TFA content (especially
their PE and EPA levels) after medium refreshment, suggesting an
almost complete regeneration of the plastidic membranes and
phycobilisomes. Nutrient-replete modes, such as semi-continuous or
continuous regime may therefore represent the most suitable production strategies for PE and other valuable compounds of the red
microalga P. purpureum.
The application of such culture strategies applied to large-scale production, especially outdoors, requires optimization of other major abiotic parameters that affect growth as well as bioactive production. Using
RSM, our results highlight for the rst time the optimal combined
light and temperature conditions necessary to promote growth and
compound production in P. purpureum, in particular PB as the most

162

F. Guihneuf, D.B. Stengel / Algal Research 10 (2015) 152163

economically valuable product. A simultaneous increase in light and

temperature causes a strong decrease in cellular PB, TFA, EPA and pigment contents, suggesting a severe damage and possible disruption of
thylakoid membranes of chloroplast. The highest PB content (~ 2.9%
d.w.) was reached under combined low light (30 mol m2 s1) and
low temperature (10 C). Despite of this, maximal PB productivity was
obtained at 20 C under low light intensity, reaching up to
33.3 mg L 1 (~ 2% d.w.) after 10 days of batch-cultivation using Nrepleted medium. Under such specic growth conditions,
P. purpureum biomass also showed to be able to accumulate substantial
amounts of other valuable products (i.e., ~ 32.5% d.w. carbohydrates,
~0.21% d.w. EPA, ~646 g g1 d.w. Chl. a, ~198 g g1 d.w. zeaxanthin,
~ 139 g g1 d.w. -carotene) which could therefore be co-extracted,
with PBs, by implementation of a biorenery approach.

Acknowledgements
This work was supported by NutraMara, the Irish Marine Functional
Foods Research Initiative. The authors gratefully acknowledge nancial
support by the Irish Marine Institute and the Department of Agriculture,
Food and the Marine (DAFM); and thank Matthias Schmid and Udo
Nitschke (Botany and Plant Science, School of Natural Sciences, Ryan Institute, NUI Galway) for their constructive comments during the preparation of the manuscript.

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