See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/259473806

Cultivation of Scenedesmus obliquus in Photobioreactors: Effects of Light

Intensities and Light–Dark Cycles on Growth, Productivity, and Biochemical
Composition

Article  in  Applied Biochemistry and Biotechnology · December 2013

DOI: 10.1007/s12010-013-0679-z · Source: PubMed

CITATIONS READS

61 1,237

5 authors, including:

Barbara Gris Tomas Morosinotto

University of Padova University of Padova
9 PUBLICATIONS   140 CITATIONS    196 PUBLICATIONS   5,347 CITATIONS   

SEE PROFILE SEE PROFILE

Eleonora Sforza
University of Padova
63 PUBLICATIONS   1,042 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Risinnova and PRIN2015 View project

Regulation of photosynthetic electron transport View project

All content following this page was uploaded by Barbara Gris on 14 June 2016.

The user has requested enhancement of the downloaded file.

Appl Biochem Biotechnol (2014) 172:2377–2389
DOI 10.1007/s12010-013-0679-z

Cultivation of Scenedesmus obliquus in Photobioreactors:

Effects of Light Intensities and Light–Dark Cycles
on Growth, Productivity, and Biochemical Composition

Barbara Gris & Tomas Morosinotto &

Giorgio M. Giacometti & Alberto Bertucco &
Eleonora Sforza

Received: 3 October 2013 / Accepted: 5 December 2013 /

Published online: 28 December 2013
# Springer Science+Business Media New York 2013

Abstract One of the main parameters influencing microalgae production is light, which
provides energy to support metabolism but, if present in excess, can lead to oxidative stress
and growth inhibition. In this work, the influence of illumination on Scenedesmus obliquus
growth was assessed by cultivating cells at different light intensities in a flat plate
photobioreactor. S. obliquus showed a maximum growth rate at 150 μmol photons m−2 s−1.
Below this value, light was limiting for growth, while with more intense illumination
photosaturation effects were observed, although cells still showed the ability to duplicate.
Looking at the biochemical composition, light affected the pigment contents only while
carbohydrate, lipid, and protein contents remained stable. By considering that in industrial
photobioreactors microalgae cells are subjected to light–dark cycles due to mixing, algae were
also grown under pulsed illumination (5, 10, and 15 Hz). Interestingly, the ability to exploit
pulsed light with good efficiency required a pre-acclimation to the same conditions, suggesting
the presence of a biological response to these conditions.

Keywords Light intensity . Pulsed light . Biodiesel . Microalgae . Photosynthesis .

Light use efficiency

Highlights
• S.obliquus showed significant productivity at different light intensities
• Saturation of photosynthesis was observed with irradiations over 150 μmol photons m−2 s−1
• Pulsed light effect was tested to simulate mixing in a photobioreactor
• Cells grow efficiently with pulsed light only if acclimated to these conditions
• Lipid accumulation is not affected by light regimes
Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0679-z)
contains supplementary material, which is available to authorized users.
B. Gris : A. Bertucco : E. Sforza (*)
Department of Industrial Engineering, University of Padua, Via Marzolo 9, 35131 Padua, Italy
e-mail: eleonora.sforza@unipd.it

T. Morosinotto : G. M. Giacometti
Department of Biology, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy
2378 Appl Biochem Biotechnol (2014) 172:2377–2389

Introduction

Fossil fuel reserves are exhausting and their massive exploitation is also causing the accumu-
lation of carbon dioxide in the atmosphere with possible consequences on the climate. There
is, thus, the necessity to develop new clean sources of energy, alternative to fossil fuels.
Biofuels, in particular biodiesel produced from algal biomass, have an interesting potential as a
nontoxic, renewable, and biodegradable fuel [1]. Microalgae are among the most efficient
photosynthetic organisms [2], and some species have much higher lipid yields than agricultural
oleaginous crops, since their oil content may exceed 70 % (w/w), compared with the maximum
5 % of crops [3, 4]. Furthermore, microalgae do not require high-quality agricultural land, thus
avoiding possible competition with food or feed production [5]. In addition, microalgae require
only water, nutrients, CO2, coupled with sunlight, which are all available at low cost [2, 4].
Despite these advantages, several challenges remain to be addressed to make this technol-
ogy competitive for commercial production of biodiesel [6–8]. Improved strains, in terms of
volumetric productivity; appropriate lipid profile and metabolic convenience; improved
photobioreactors, in terms of operation under high cell densities; gaseous mass transfer;
transmittance of light; and less expensive downstream processing are all needed to make this
technology feasible for large-scale production of biodiesel [4].
Among the several factors affecting microalgal productivity [9–11], light is one of the major
parameters to be considered since it provides all the energy required to support metabolism,
but, if present in excess, it can damage cells, leading to oxidative stress and photoinhibition
and thus lower photosynthetic efficiency. To respond to excess light, photosynthetic organisms
evolved two types of mechanisms. The first one consists in the repair of photosynthetic
apparatus components damaged by excess illumination. This is known to involve in particular
Photosystem II, through the continuous degradation and re-synthesis of its D1 subunit, which
is preferentially damaged under illumination [12, 13]. The second mechanism involves the
reduction of oxidative damage through thermal dissipation of energy in excess. Both mech-
anisms cause a reduction in light use efficiency and they must be minimized to reach an
optimal productivity [14].
In photobioreactors, algal cultures reach high optical densities, which cause inhomogeneity
in light distribution. As a consequence, cells on the surface, directly exposed to light, absorb
most of the available radiation, but must also activate mechanisms of energy dissipation, to
avoid oxidative damage. Instead, the cells in the dark zone of the photobioreactor (PBR) receive
only a small part of the radiation, which is limiting for their growth [15]. The consequence of the
light distribution is thus that algae in PBR use available energy with reduced efficiency. To
avoid this issue, a reduction of light path could be beneficial, but thin reactors are unlikely to be
economically sustainable on a large scale. In addition, in thin reactors, problems of
photosaturation and inhibition are enhanced. Thus, finding other design solutions is crucial to
enhance photosynthetic efficiency.
A further source for complexity to be considered is that in photobioreactors, cells are actively
mixed and move between the dark and light regions. As a consequence, they experience fast
alternation of light and dark. Such dark–light cycles have been suggested to increase the
photosynthetic efficiency in several cases [16–22]. Pulsating involves condensation of the
whole energy into shorter periods, and in certain conditions, it causes no overall energy
compromise. In particular, the kinetic of the photon absorption is in the range of 1–15 ms, a
time needed to reset the system, prior to being ready to receive another photon [23]. However,
the effect is strongly dependent on the frequency, and in some cases, these cycles can even be
detrimental for productivity [14]. The culture system is also crucial to verify the positive effect
on growth. For instance, Park and Lee [24] reported that cell concentration is a key parameter in
Appl Biochem Biotechnol (2014) 172:2377–2389 2379

the exploitation of pulsed light, based on observation that at lower cell densities the instanta-
neous high light intensity caused photoinhibition anyway [24]. The technical feasibility of
forcing the light regime in actual photobioreactors is currently under investigation [25, 26], with
the aim of assuring mixing cycle up to 10 Hz which could yield increased microalgal
productivity [27].
Scenedesmus obliquus is one of the most promising species as feedstock for biodiesel
production [28, 29], since it presents several favorable features such as a fast growth, efficient
CO2 fixation, and the ability to grow in wastewaters and accumulate lipids. In this paper, the
influence of different light intensities, provided both continuously and at high-frequency
pulsation, on S. obliquus growth and biochemical composition was assessed.

Materials and Methods

Algae Strain and Cultivation

S. obliquus 276.7, from SAG, was grown in sterile BG11 medium [30]. The medium and all
materials were sterilized in an autoclave for 20 min at 121 °C in order to prevent any
contamination. Cultures were maintained and propagated in the same medium with the
addition of 10 g/L of plant agar, under continuous light. Pre-cultures for the inoculum were
grown in flasks at 120 μmol photons m−2 s−1 and maintained in exponential phase. Cells with
an optical density (OD) of 0.50 at 750 nm were inoculated in a 1.2-cm wide flat bed
photobioreactor. The flat plate photobioreactors were built with transparent materials
(polycarbonate) for maximum utilization of light energy. The working volume was 150 mL
and the culture was mixed by an air-CO2 flow of 0.055 L/min of gas per liter of culture at 5 %
of CO2, from a sparger placed in the bottom of the panel. The amount of CO2 in air was
regulated by two flow meters. The gas flow supplied a non-limiting CO2 content to the culture,
which was also responsible of cells mixing (see Sforza et al. [14] for the schematic of the
reactor). Reactors were exposed to different constant light intensities ranging from 10 to
1,000 μmol m−2 s−1.
For low light intensities (up to 150 μmol m−2 s−1), illumination was provided with a
fluorescence lamp, while for higher intensities (from 200 to 1,000 μE m−2 s−1) and pulsed
light, a LED lamp (Light Source SL 3500, Photon System Instruments) was employed. The
latter was also employed for low light intensities to verify that the difference in light source did
not affect growth kinetics. Light intensity was measured using a photo-radiometer (HD 2102.1,
Delta Ohm). In experiments with pulsed light, the LED light source was programmed to
generate square wave dark–light cycles with the desired intensity and frequency.
Flashes were described from parameters as flash time (tf), dark time (td), flash light intensity
(I0), and integrated light intensity (Ia), as reported in Table 2. In the experiments with pulsed
light, three different frequencies were used (5, 10, and 15 Hz) and consistent with mixing cycle
in the photobioreactor [31].
Temperature in growth chambers was kept at 23±1 °C. The medium was buffered with
HEPES 10 mM, pH 8, to avoid acidification due to CO2 excess and to maintain the pH in the
range of algal viability (between 7 and 8). Algal growth was measured by daily changes in OD
at 750 nm (UV 500 UV-Visible Spectro, Spectronic Unicam), and cell number was monitored
using Burker Counting Chamber (HBG, Germany). The specific growth rate was calculated
from experimental measures in exponential phase, where nutrients were still not limiting, as
the slope of the logarithmic phase for the number of cells or of the dry weight. The dry weight
was measured by filtering a known volume of culture by cellulose acetate filters of 0.2 μm
2380 Appl Biochem Biotechnol (2014) 172:2377–2389

(Whatman). Filters were pre-dried for 10 min at 80 °C in order to remove any moisture.
Biomass filtered was dried for 2 h at 80 °C and then weighed to calculate the dry weight in
terms of grams per liter. All experiments were performed in at least two independent biological
replicates and each measurement has at least three replicates.

Analytical Procedures

Microalgae composition, in terms of proteins, total sugar, and lipids, was measured for each
light intensity at the end of batch culture, once the stationary phase was reached. Total sugar
content was estimated using the Anthrone method [32, 33]: the reaction with sulfuric acid
transforms all the sugars present in the sample in 5-hydroxymethylfurfural, which reacts with
Anthrone to generate a blue-green complex absorbing light at 625 nm.
The concentration of solubilized proteins extracted from about 35 mg of dry biomass was
determined using bicinchoninic acid assay (BCA) [34]. Cells were ground with quartz powder
in lysis buffer (1.5 mL, 50 mM Tris–HCl pH 8.0, 20 % guanidine hydrochloride, 10 mM
EDTA, 10 mM DTT, 0.4 μM PMSF) and, after the addition of 0.05 % Triton X-100,
centrifuged at 10,000 rpm for 10 min [35]. The supernatant was used for protein determination
by the BCA assay; dilutions of bovine serum albumin were used to prepare a series of protein
standards. Total protein was measured by Smith analysis with a commercially available kit
(Sigma-Aldrich).
Total lipids were extracted overnight from dried cells using chloroform/methanol (1:2 vol/vol)
in accordance with Bligh&Dyer method, in a Soxhlet apparatus [36]. The lipid mass was
measured gravimetrically after solvent removal using a rotary evaporator.
Pigments were extracted from the cell sampled in the exponential phase, using 10 × 106
cells and DMSO as extraction solvent, after grinding with quartz powder (this protocol was
specifically set up for the species) and incubation at 65 °C for 15 min to facilitate pigment
extraction. Total pigment content was evaluated spectrophotometrically by absorbance in the
spectrum from 350 to 750 nm. Absorbance at 480, 649, and 665 nm were used to calculate
concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids [37].
The efficiency of Photosystem II was measured as Fv/Fm parameter, on the same day of
pigment analysis, using Dual-Pam-100 (Measuring System), with previous acclimation of the
sample in the dark for 20 min.

Statistical Analysis

Student’s t tests were applied to ascertain significant differences in specific growth rate, final
cell concentration, final biomass concentration in terms of grams per liter, and cellular weight.
The level of statistical significance was P<0.05.

Results

Different Light Intensities

Growth and Productivity of S. obliquus at Various Light Intensities

All the experiments reported here were performed in flat photobioreactors and with low optical
density (starting at OD750 of 0.2), to reduce as much as possible the effect of self-shading on
growth kinetics. CO2 and nitrogen, in the form of nitrate, were provided in excess to avoid
Appl Biochem Biotechnol (2014) 172:2377–2389 2381

growth limitation due to lack of nutrients and highlight the effect of different illumination
intensities (Fig. 1 and Table 1).
Figure 1a, b shows the effect of light intensity on growth and final cell concentration. At
low light intensities (Fig. 1a), ranging from 10 to 150 μmol m−2 s−1, the specific growth rate
and cell concentration increased linearly with light intensity, with a peak at 150 μmol m−2 s−1
showing that, in this range, light is limiting for cell growth. At 150 μmol m−2 s−1, growth rate
and final cell concentration both reached their maximal values. Over this limit (Fig. 1b), the
increase of light intensity did not result in any enhancement of the growth rate nor increased
final cell concentration, suggesting that the saturation point of photosynthesis was reached.
These results are different from those reported by Liu et al. [38], who observed a maximal
growth rate at higher intensities (250–400 μmol photons m−2 s−1) for Scenedesmus sp.
However, this could be explained by the different reactor depths (about 4 cm [38]) that likely
caused some self-shading effect. If this is the case, the average irradiation in the culture is far
lower than the incident light. It is also worth mentioning that other species grown in thin flat
panels similarly showed photosaturation at irradiances over 150 μmol photons m−2 s−1 [14]. It
is worth underlining that in all cases S. obliquus cell growth is exponential from the beginning

a b

c d

Fig. 1 S. obliquus growth under different light intensities in a flat plate photobioreactor. a Growth kinetics of
algae exposed to low light intensities ranging from 10 to 150 μmol m−2 s−1, and data are shown in triangles,
circles, and rhombus. b Growth kinetics at high light intensities, 200, 350 and 1,000 μmol m−2 s−1, represented in
circles, rhombus, and triangles, respectively. c Growth parameters determined from curves reported in a and b,
specific growth rate (squares) and biomass concentration (rhombus). d Final concentration, expressed as dry
biomass, normalized to the total amount of light impinging the panel during the total duration of the experiment,
used as approximate estimate of biomass production
2382 Appl Biochem Biotechnol (2014) 172:2377–2389

Table 1 Summary of results obtained (cellular concentration, dry weight, and cellular weight) at various light
intensities at the end of growth curves

Light condition Cell concentration [106 cells/mL] Specific growth rate [day−1] Dry weight [g/L]

Mean SD Mean SD Mean SD

10 μmol m−2 s−1 7.5 3.2 0.102 0.040 0.17 0.127

50 μmol m−2 s−1 61.0 3.4 0.482 0.065 1.22 0.361
150 μmol m−2 s−1 113.9 17.2 0.863 0.104 3.30 0.673
200 μmol m−2 s−1 56.0 4.2 0.548 0.073 1.71 0.198
350 μmol m−2 s−1 70.0 6.4 0.493 0.004 4.18 0.742
1,000 μmol m−2 s−1 79.0 0.007 0.571 0.133 4.92 0.401
5 Hz 44.58 5.816 0.273 0.058 1.17 0
10 Hz 73 0.422 0.333 0.046 1.86 0.11
15 Hz 30.30 0.331 0.366 0.054 0.83 0.01
10 Hz adapted 87.31 0.356 0.552 0.062 1.54 0.08

of the growth curve, thus without any detectable initial lag phase, suggesting that this species is
able to grow under a wide range of light conditions without suffering an extensive stress that
makes necessary a cell adaptation. At the limiting range of light intensities
(from 10 to 150 μmol m−2 s−1), the final biomass concentration, in terms of grams per liter,
follows the same trend of one of the specific growth rates (Fig. 1c). However, above
150 μmol m−2 s−1, the final biomass dry weight increases with the light intensity even if the
number of cells does not, suggesting that in these conditions cells increased their weight, while
stopping duplication. In fact, despite of a decrease in the cellular concentration of 30 to 40 %,
at high light intensities (350 and 1,000 μmol m−2 s−1), there is an increase of the dry weight,
from about 25 to 50 % compared to 150 μmol m−2 s−1. Cellular weights for cultures exposed
to 350 and 1,000 μmol m−2 s−1 (about 59 and 63 pg/cell, respectively) were found significantly
higher than those at 150 μmol m−2 s−1 (29 pg/cell). This increased cell size induced by strong
illumination was also confirmed by qualitative observations using a microscope. The final dry
weight (Fig. 1c) also suggests that at the stationary phase the cells are still capable of
increasing their biomass. A likely explanation for this observation is that cells experience less
stress towards the end of the curve because with the increase of cell concentration there is the
appearance of self-shading effects which allows the growth to continue. However, stress is still
perceived by the cells which in fact stopped duplication, and all biomass production goes into
the increase of cellular weight.
Photosynthetic efficiency in different conditions can be estimated by the biomass produced,
expressed as dry weight biomass, normalized to the total amount of light supplied (moles of
photons that reached the panel surface during the whole time of the experiment) (Fig. 1d). In
this case, two regions are identifiable: from 10 to 150 μmol m−2 s−1, this value was roughly
constant, suggesting that in this interval all light reaching cultures is exploited with a similar
efficiency. On the other hand, over 150 μmol m−2 s−1, this ratio strongly decreased suggesting
that, although cells are still capable of producing biomass, light is used with lower efficiency. It
is worth underlining the case of cells exposed to the strongest light which, as mentioned above,
reached the higher final biomass concentration. Even if they produce about 45 % more
biomass than cells under 150 μmol m−2 s−1, they are also exposed to far more light, and
therefore, their light use efficiency is much lower than in the other conditions.
Appl Biochem Biotechnol (2014) 172:2377–2389 2383

Effects of Various Light Intensities on Pigment Content and Maximum Photosynthetic

Quantum Efficiency

By considering the analysis of pigment concentration, shown in Fig. 2a, cells exposed to
different light intensities showed a decrease in Chl a content per cell and a relative increase in
carotenoids content. Pigment absorption spectra, normalized to 10 × 106 cells/mL, are reported
in Fig. S1 that allows appreciating the relative increase in carotenoids evidenced by the
stronger absorption in the 350–500-nm range. These spectra also show no relevant effect on
Chl a/b ratio, suggesting that antenna size is not affected by incident light, as observed in other
algae [39]. Thus, S. obliquus, similar to other photosynthetic organisms, showed an acclima-
tion response by decreasing the Chl a content to reduce light-harvesting ability and accumu-
lating carotenoids which have an antioxidant activity.
The parameter Fv/Fm gives an estimation of the maximum quantum efficiency of PSII and
is useful to show the presence of photoinhibition at high irradiancies [40, 41]. Fv/Fm was
monitored for all light conditions during the exponential phase (5th–6th day of growth curves).
Different from the expected and also observed for other species like Nannochloropsis salina
[14], S. obliquus did not show significant changes in this parameter at different light intensities,
as reported in Fig. 2b, suggesting that cells are capable of keeping the Photosystem II efficient
even under intense illumination.

Influence of Light Intensity on Biochemical Composition

Biochemical composition (Fig. 3), in terms of the content of total proteins, lipids, and
carbohydrates, was analyzed for all light conditions, except for 10 μmol m−2 s−1 where the
low biomass yields limited the measurements. The protein, carbohydrate, and lipid contents of
S. obliquus cells showed no major variations at the different light intensities. In particular, it is
interesting to observe that lipid content is similar under all conditions, at about 40 % of DW.
Different from other algal species such as Nannochloropsis [42, 43], light excess does not
induce overproduction of lipids, suggesting that these responses are not common for all algae
but depend on the species [44, 45]. These data are consistent with those reported by Breuer
et al. [46], who reported that the maximum TAG content in Scenedesmus was independent of
light intensity, while strongly affected by pH and temperature.

a b

Fig. 2 Effects of different light intensities on photosynthetic apparatus. a Pigment contents of S. obliquus under
different light intensities, Chl a content at various light intensities is shown in circles and carotenoids/Chl ratio is
shown in squares. b Fv/Fm parameter, used as a proxy for the photosynthetic efficiency, at different light intensities
2384 Appl Biochem Biotechnol (2014) 172:2377–2389

Fig. 3 Biochemical composition of S. obliquus under different light intensities. Protein, carbohydrate, and lipid
contents are reported in micrograms per milligram for each light intensity

Pulsed Light

Effects of Pulsed Light on the Growth of S. obliquus

As mentioned previously, algae in a photobioreactor can move from fully exposed regions to
darkness, because of mixing. In order to investigate the illumination influence on
photobioreactors at an industrial scale, it is seminal to understand how these conditions affect
algal growth. For this reason, S. obliquus cells were grown under dark–light cycles at several
frequencies. The effect of high-frequency light–dark cycle on S. obliquus was previously studied
by Grobbelaarl et al. [47], but it is not clear how that cycles could affect biomass productivity of
this species. For this reason, the specific growth rate and final biomass concentrations are reported
as a function of different frequencies of dark–light cycles. As shown in Table 2 and schematized
in Fig. S2, flashes at different intensities, 1,000 and 1,500 μmol m−2 s−1 were used, as well as
different durations of light and dark phases, always providing a total amount of energy corre-
sponding to the optimal intensity of 150 μmol m−2 s−1. In contrast to what was observed for
N. salina [14], S. obliquus showed in all cases retarded growth under pulsed light with respect to
the cells exposed to the same amount of photons provided continuously (Fig. 4). As shown in
Fig. 4, S. obliquus can grow better at 10 Hz than in other conditions. At 5 and 15 Hz, growth was
even more inhibited. Several works reported [26, 48] that under the same time-averaged light

Table 2 Description of pulsed light conditions employed to Scenedesmus obliquus growth

Condition Light intensity (I0), Frequency of light Flash time Dark time Integrated light intensity
μmol m−2 s−1 change, Hz (tf), ms (td), ms (Ia), μmol m−2 s−1

150 150 – ∞ – 150

1,500—5 1,500 5 20 180 150
1,500—10 1,500 10 10 90 150
1,000—15 1,000 15 10 56.6 150
Appl Biochem Biotechnol (2014) 172:2377–2389 2385

Fig. 4 S. obliquus growth under different frequencies of pulsed light. S. obliquus growth curves at different
intensities and frequencies, 1,500 μmol m−2 s−1 (5, 10 Hz) and 1,000 μmol m−2 s−1 (15 Hz). Kinetics with 150
and 1,000 μmol m−2 s−1 continuous light reported for comparison

intensity the growth rate of microalgae was determined by the L/D frequency, and the
higher the frequency, the greater the growth rate would be. Concerning Scenedesmus,
Xue et al. [27] reported an increased growth under 10 Hz with respect to 0.1 Hz, but no
comparison with continuous illumination was carried out. In the present work, we
observed that even if using 10 Hz, S. obliquus growth was higher than in the other cases
and this was still far from the kinetics at 150 μmol m−2 s−1 of continuous light (reported
in Fig. 4 for comparison). It is also interesting to observe that pulsed light was even more
detrimental than the same amount of strong light (1,000 μmol m−2 s−1) provided
continuously (see Fig. 4). In addition, in all the cases analyzed, cultures showed a long
initial lag phase, suggesting that cells required some time to adapt to these non-optimal
conditions before being able to start duplication.

Acclimation to Pulsed Light

Since cells exposed to pulsed light showed a substantial lag phase, cultures previously grown
at 10 and 15 Hz were re-diluted to the starting concentration in fresh medium and re-inoculated
in the same conditions of light regime, in order to evaluate the possible effect of a pre-
acclimation to pulsed light. As shown in Fig. 5a, b under both frequencies (10 and 15 Hz),
when cells were already acclimated to pulsed light, no lag phase was observed after the re-
inoculation, and algae growth was much higher with respect to the corresponding non-
acclimated cells in Fig. 4. These results clearly show that cells acclimated to the pulsed light
conditions, and only when this response is activated, they are able to effectively use the light
provided.
This is also confirmed by analyzing light use efficiency, again estimated by
normalizing final dry weight on the available light. In fact, non-acclimated cultures
showed an energy conversion of about 0.103 g/mol photons, while after an acclima-
tion period, the conversion increased to 0.142 g/mol photons. Even if the efficiency is
2386 Appl Biochem Biotechnol (2014) 172:2377–2389

a b

Fig. 5 Effects of acclimation to pulsed light on growth of S. obliquus. a Comparison between growth in pulsed
light before (dotted line) and after acclimation, at the frequency of 10 Hz. b Comparison between growth at
15 Hz before (dotted line) and after acclimation period. Measured data of growth curve shown as dotted line are
reported in Fig. 4

still lower than the one determined for cells exposed to optimal conditions of continuous light of
150 μmol m−2 s−1 (corresponding to 0.354 g/mol photons), acclimation to pulsed light causes a
clear increase.

Photosynthetic Apparatus, Pigment Contents, and Biochemical Composition of S. obliquus

in Pulsed Light

Pigment content and biochemical composition were analyzed only for 10 Hz, before and after
acclimation, because of the low biomass concentration obtained at the end of the growth curve
at 5 and 15 Hz. As shown in Fig. 6a, Chl a content and carotenoids/Chl ratio were slightly
lower than in cells grown under 150 μmol m−2 s−1 of continuous light, but not significantly
different. Also for biochemical composition (Fig. 6b), no significant difference was observed
in the acclimated experiment compared to the non-acclimated, and in pulsed light compared to

a b

Fig. 6 Chl a and carotenoids/Chl ratio and biochemical composition of S. obliquus under different frequencies
of pulsed light. a Pigment contents of S. obliquus under different frequencies, Chl a is shown in black and
carotenoids/Chl ratio is shown in gray. b Protein, carbohydrate, and lipid contents are reported in micrograms per
milligram for each frequency
Appl Biochem Biotechnol (2014) 172:2377–2389 2387

continuous light. These results suggest that acclimation to pulsed light do not require a major
reorganization of the photosynthetic apparatus which would be evidenced by an alteration of
pigment composition. A deeper investigation of the adaptation responses allowing better
growth under pulsed light is clearly needed.

Discussion

The experimental results presented show that the green microalga S. obliquus shows a
substantial growth rate even at intensities much higher than the optimal one. These results
confirm the observation of Masojidek et al. [49] who reported a low sensitivity of
Scenedesmus to photoinhibition and a higher capacity to adapt to higher irradiances due to
an effective quenching mechanism and high photosynthetic capacity. This ability of using non-
optimal light makes the exploitation of S. obliquus particularly suitable for an outdoor large-
scale production, in which light conditions cannot be controlled at a laboratory scale. In
particular, in an industrial cultivation of microalgae for biodiesel production carried out
continuously during the year, cells are exposed to different light intensities along with days
and seasons, and the ability of the selected species to maintain substantial growth in all
conditions is a valuable feature. However, it must be underlined that, although cells are indeed
capable of growing even with the highest light intensities, in these conditions, they also have a
much lower productivity in terms of biomass produced per light available. In a perspective of a
large-scale cultivation of algae, it should always be considered that in a photobioreactor, most
of the light is absorbed by the first layers of cells which shade the ones underneath. If the first
layer absorbs most of the light and uses it with poor efficiency, it causes a decrease in overall
productivity and therefore should be avoided as much as possible. Photobioreactor design can
help in this direction by optimizing the mixing system, and cells could be exposed to intense
light for a limited amount of time, increasing light use efficiency and therefore overall
productivity. In this work, it was demonstrated that S. obliquus can grow under high-
frequency pulsation and use this radiation with good efficiency. This ability, however, is
achieved only after an acclimation period, in contrast to other species such as Nannochloropsis
where this time was not necessary [14]. Nevertheless, it should be underlined that an industrial
scale photobioreactor will work in a long-term continuous operation, thus providing the
required time for acclimation to growing conditions.
An additional major conclusion is that S. obliquus showed a steady high lipid content
(≈40 %) in all light conditions both continuous and pulsed. Such a scarce influence of light
intensity on the biochemical composition of S. obliquus is promising in the perspective of a
large-scale application. In fact, while other species show higher lipid contents, these are
normally achieved by applying nutritional stress to the culture, which often also affects overall
biomass productivity. A good stable lipid accumulation under a variety of conditions without
the need of applying any stress is a valuable property because it would result in a steady
production without the need of precise control of operating conditions of the photobioreactor
which is difficult and costly to achieve.

Conclusions

The effect of different illumination on S. obliquus was investigated. The species shows a
maximum growth rate at 150 μmol m−2 s−1. Above this value, the growth is inhibited, and
although algae show substantial biomass accumulation, they exploit light with a lower
2388 Appl Biochem Biotechnol (2014) 172:2377–2389

efficiency. Cultures exposed to pulsed light show reduced growth compared to continuous
light, but if cells are allowed the time to acclimate to alteration of dark–light cycles, they
strongly enhance their productivity. Finally, it was found that the lipid content of S. obliquus is
not affected by the variation of light intensity.

Acknowledgments TM acknowledges ERC starting grant no. 309485 (BioLEAP).

References

1. Demirbas, A., & Fatih Demirbas, M. (2011). Energ. Convers. Manage., 52(1), 163–170.
2. Heilmann, S. M., Jader, L. R., Harned, L. A., Sadowsky, M. J., Schendel, F. J., Lefebvre, P. A., von Keitz, M.
G., & Valentas, K. J. (2011). Appl. Energ., 88(10), 3286–3290.
3. Chisti, Y. (2007). Biotechnology Advances, 25(3), 294–306.
4. Malcata, F. X. (2011). Trends in Biotechnology, 29(11), 542–549.
5. Greenwell, H. C., Laurens, L. M. L., Shields, R. J., Lovitt, R. W., & Flynn, K. J. (2010). Journal of the Royal
Society Interface, 7(46), 703–726.
6. Scott, S. A., Davey, M. P., Dennis, J. S., Horst, I., Howe, C. J., Lea-Smith, D. J., & Smith, A. G. (2010).
Curr. Opin. Biotech., 21(3), 277–286.
7. Demirbas, A. (2011). Appl. Energ., 88(10), 3541–3547.
8. Koller, M., Salerno, A., Tuffner, P., Koinigg, M., Böchzelt, H., Schober, S., Pieber, S., Schnitzer, H.,
Mittelbach, M., & Braunegg, G. (2012). Journal of Cleaner Production, 37, 377–388.
9. Munn, M., Frey, J., & Tesoriero, A. (2010). Environmental Management, 45(3), 603–615.
10. Kitaya, Y., Azuma, H., & Kiyota, M. (2005). Advances in Space Research, 35(9), 1584–1588.
11. Stephens, E., Ross, I. L., Mussgnug, J. H., Wagner, L. D., Borowitzka, M. A., Posten, C., Kruse, O., &
Hankamer, B. (2010). Trends in Plant Science, 15(10), 554–564.
12. Murata, N., Takahashi, S., Nishiyama, Y., & Allakhverdiev, S. I. (2007). Biochim. Biophy. Acta, 1767(6),
414–421.
13. Nixon, P. J., Michoux, F., Yu, J., Boehm, M., & Komenda, J. (2010). Annals of Botany, 106(1), 1–16.
14. Sforza, E., Simionato, D., Giacometti, G. M., Bertucco, A., & Morosinotto, T. (2012). PloS One, 7(6),
e38975.
15. Richmond, A., Cheng-Wu, Z., & Zarmi, Y. (2003). Biomolecular Engineering, 20(4–6), 229–236.
16. Kok, B. (1956). Biochimica et Biophysica Acta, 21(2), 245–258.
17. Matthijs, H. C., Balke, H., van Hes, U. M., Kroon, B. M., Mur, L. R., & Binot, R. A. (1996). Biotechnology
and Bioengineering, 50(1), 98–107.
18. Nedbal, L., Tichy, V., Xiong, F., & Grobbelaar, J. U. (1996). Journal of Applied Phycology, 8, 325–333.
19. Kim, Z.-H., Kim, S.-H., Lee, H.-S., & Lee, C.-G. (2006). Enzyme Micro. Tech., 39(3), 414–419.
20. Gordon, J. M., & Polle, J. E. W. (2007). Appl. Microbiol. Biot., 76(5), 969–975.
21. Grobbelaar, J. U. (2010). Photosynthesis Research, 106(1–2), 135–144.
22. Xue, S., Su, Z., & Cong, W. (2011). Journal of Biotechnology, 151(3), 271–277.
23. Carvalho, A. P., Silva, S. O., Baptista, J. M., & Malcata, F. X. (2011). Appl. Microbiol. Biot., 89(5), 1275–1288.
24. Park, K.-H., & Lee, C.-G. (2001). Biotechnol. Bioproc. E., 6(3), 189–193.
25. Brindley, C., & Fernández, F. G. A. (2011). Bioresource Technol., 102(3), 3138–3148.
26. Janssen, M., Bresser, L. D., Baijens, T., Tramper, J., Mur, L. R., Snel, F. H., & Wijffels, H. (2000). Journal of
Applied Phycology, 12(3–5), 225–237.
27. Xue, S., Zhang, Q., Wu, X., Yan, C., & Cong, W. (2013). Bioresource Technol, 138, 141–147.
28. Tang, D., Han, W., Li, P., Miao, X., & Zhong, J. (2011). Bioresource Technol., 102(3), 3071–3076.
29. Kaewkannetra, P., Enmak, P., & Chiu, T. (2012). Biotechnol. Bioproc. E, 17(3), 591–597.
30. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., & Stanier, R. Y. (1979). J. Gen. Microb., 111(1), 1–61.
31. Perner-Nochta, I., & Posten, C. (2007). Journal of Biotechnology, 131(3), 276–285.
32. Morris, D. L. (1948). Science, 107(2775), 254–255.
33. Trevelyan, W. E., & Harrison, J. S. (1952). Biochemical Journal, 50(3), 298–303.
34. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E.
K., Olson, B. J., & Klenk, D. C. (1985). Analytical Biochemistry, 150(1), 76–78.
35. Msanne, J., Xu, D., Konda, A. R., Casas-Mollano, J. A., Awada, T., Cahoon, E. B., & Cerutti, H. (2012).
Phytochemistry, 75, 50–59.
36. Bligh, E. G., & Dyer, W. J. (1959). Can. J. Biochem. Phys., 37(8), 911–917.
Appl Biochem Biotechnol (2014) 172:2377–2389 2389

37. Wellburn, A. R. (1994). Journal of Plant Physiology, 144(3), 307–313.

38. Liu, J., Yuan, C., Hu, G., & Li, F. (2012). Appl. Biochem. Biotech., 166(8), 2127–2137.
39. Bonente, G., Pippa, S., Castellano, S., Bassi, R., & Ballottari, M. (2011). Journal of Biological Chemistry,
287, 5833–5847.
40. Baker, N. R. (2008). Annual Review of Plant Biology, 59, 89–113.
41. Maxwell, K., & Johnson, G. N. (2000). J. Experim. Bot., 51(345), 659–668.
42. Simionato, D., Sforza, E., Corteggiani Carpinelli, E., Bertucco, A., Giacometti, G. M., & Morosinotto, T.
(2011). Bioresource Technol., 102(10), 6026–6032.
43. Cheirsilp, B., & Torpee, S. (2012). Bioresource Technol., 110, 510–516.
44. Solovchenko, A. E. (2008). Journal of Applied Phycology, 20, 245–251.
45. Ruangsomboon, S. (2012). Bioresource Technol., 109, 261–265.
46. Breuer, G., Lamers, P. P., Martens, D. E., Draaisma, R. B., & Wijffels, R. H. (2013). Bioresource Technol.,
143, 1–9.
47. Grobbelaarl, J. U., Nedbal, L., & Tichy, V. (1996). Journal of Applied Phycology, 8(4–5), 335–343.
48. Vejrazka, C., Janssen, M., Streefland, M., & Wijffels, R. H. (2011). Biotechnology and Bioengineering,
108(12), 2905–2913.
49. Masojidek, J., Torzillo, G., Koblizek, M., Kopecky, J., Bernardini, P., Sacchi, A., & Komenda, J. (1999).
Planta, 209(1), 126–135.

View publication stats