Appl Biochem Biotechnol

DOI 10.1007/s12010-015-1717-9

Effect of Light Intensity and Photoperiod on Growth

and Biochemical Composition of a Local Isolate
of Nostoc calcicola

Fateme Khajepour 1 & Seyed Abbas Hosseini 1 &

Rasoul Ghorbani Nasrabadi 1 & Giorgos Markou 2

Received: 13 May 2015 / Accepted: 11 June 2015

# Springer Science+Business Media New York 2015

Abstract A study was conducted to investigate the effect of light intensity (21, 42, and
63 μmol photons m−2 s−1) and photoperiod (8:16, 12:12, and 16:8 h light/dark) on the
biomass production and its biochemical composition (total carotenoids, chlorophyll a,
phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC), total protein, and
carbohydrates) of a local isolate of Nostoc calcicola. The results revealed that
N. calcicola prefers dim light; however, the most of the levels of light intensity and
photoperiod investigated did not have a significant impact on biomass production.
Increasing light intensity biomass content of chlorophyll a, PE, PC, APC, and total
protein decreased, while total carotenoids and carbohydrate increased. The same be-
havior was observed also when light duration (photoperiod) increased. The interaction
effect of increasing light intensity and photoperiod resulted in an increase of carbohy-
drate and total carotenoids, and to the decrease of chlorophyll a, PE, PC, APC, and
total protein content. The results indicate that varying the light regime, it is capable to
manipulate the biochemical composition of the local isolate of N. calcicola, producing
either valuable phycobiliproteins or proteins under low light intensity and shorter
photoperiods, or producing carbohydrates and carotenoids under higher light intensities
and longer photoperiods.

Keywords Nostoccalcicola . Light regime . Pigments . Phycobiliprotein . Protein . Carbohydrate

* Fateme Khajepour
fatemekhajepour@yahoo.com

1
Fisheries Faculty, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
2
Department of Natural Resources Management and Agricultural Engineering, Agricultural University
of Athens, Iera Odos 75, 11855 Athens, Greece
 Appl Biochem Biotechnol

Introduction

Cyanobacteria (CB) (previously known as blue-green algae) are the largest group of photo-
synthetic prokaryotes that exist, with large morphological and habitual diversity, and a wide
distribution in the world [1]. They are a rich source for novel chemicals, with potential
application in medicine and industry [2]. Interesting biomass compounds that could be
produced by CB include phycobiliproteins, carotenoids, fatty acids, proteins, polysaccharides,
vitamins, and phenolic compounds, which exhibit antioxidant, antimicrobial,
antiinflammatory, hepatoprotective, immunomodulation, and anticancer activities [3]. Gener-
ally, CB express unique features for the development of value products for the pharmaceutical,
nutraceutical, cosmetic, and biofuel industry [4, 5].
Various environmental and cultivation factors, such as temperature, light intensity, photo-
period, nutrient limitation etc., affect not only cell growth rates but cell composition as well,
affecting the biomass content of particular compounds. Several studies have shown that the
photoacclimation ability of algae depends on changes in both, thylakoid number and content of
pigments [6–10]. Because variation in irradiance is a typical event in cultivation systems under
natural conditions (open ponds, indoor or outdoor photobioreactors irradiated with solar
energy etc.), its influence on the biochemical composition of CB has considerable ecological,
physiological, and biotechnological importance [11]. Given that presently, microalgae and CB
are produced commercially under natural conditions (mainly open ponds), which are subjected
to seasonal and daily fluctuation of light intensity and duration, it is important to determine the
effect of irradiance and photoperiod on biomass production and its composition. Moreover,
these information could be used for the optimization of biomass production under controlled
cultivation conditions. Day length affects the circadian rhythm of photosynthesis, respiration,
cell division, and subsequently growth rates [12]. Besides the pigment contents, other algal cell
components such as carbohydrates and proteins can also be influenced by the light regime [6].
The cyanobacterium N. calcicola is a rich source of pigments with medicinal, industrial,
and biotechnological significance. Important cyanobacterial pigments include phycocyanin
(PC), phycoerythrin (PE), allophycocyanin (APC), and carotenoids [3, 13]. Nostocacean
cyanobacteria are of particular significance because they exhibit the additional capability of
nitrogen fixation [14], reducing the need of nitrogen fertilizers. Biotechnological studies for
N. calcicola investigating biomass production and biomass composition are rarely reported.
Therefore, in this study, N. calcicola was studied to determine the effect of light regime on
biomass production and biomass content in total protein, carbohydrate, chlorophyll a, total
carotenoids, phycocyanin, phycoerythrin, and allophycocyanin.

Materials and Methods

Organism and Culture Condition

The cyanobacterium N. calicola (ISC89) used in the study was obtained from the Department of
Bioscience, Shahid Beheshti University, Iran. The experiment was carried out in 500-ml Erlen-
meyer flasks and 250-ml growth medium. Cultures were carried out in triplicates at constant
aeration and temperature (25±2 °C) in a room culture of phycolaboratory of the Agriculture
Science and Natural Resource, Gorgan University, Gorgan, Iran. For the cultivation of N. calcicola
the growth medium Zehnder-8 was used with the following composition [15]: 59 mg l−1Ca(NO3)2.
Appl Biochem Biotechnol

4H2O, 250 mg l−1NaNO3, 25 mg l−1MgSO4 ·7H2O, 31 mg l−1K2HPO4, 21 mg l−1Na2CO3,

90 mg l−1FeCl3.6H2O, 146 mg l−1EDTA-Na, 146 ml HCl, and 80 ml per liter of the following
stock solution: 50 mg l−1Na2SiO3.9H2O, 100 mg l−1CuSO4 ·5H2O, 80 mg l−1(NH4)6Mo7O24 ·
4H2O, 280 mg l−1ZnSO4 ·7H2O, 230 mg l−1MnCl2 ·4H2O, 3100 mg l−1H3BO3, 480 mg l−1Al2
(SO4)3 ·18H2O, 120 mg l−1KBr, 80 mg l−1KI, 140 mg l−1Co(NO3)2.6H2O, and 40 mg l−1LiCl.H2O
(Sigma-Aldrich). The pH of the growth media was adjusted to 7.0±0.2 before autoclaving. Then,
growth media were sterilized through autoclaving (121 °C, 20 min) and inoculated after 24 h [7]
with N. calcicola at an initial cell density of 0.15±0.05 gDM l−1. The cultivation was performed
using light intensities of 21, 42, and 63 μmol photons m−2 s−1 and 8/16, 12/12, and 16/8 h light/
dark photoperiods.

Analytical Methods

Determination of Biomass Production, Chlorophyll a, and Total Carotenoid Content

Biomass (g l−1 dry weight) was measured every second day following the method of Premila
and Umamaheswara Rao [16]. For chlorophyll a determination, a sample of culture broth was
filtered by membrane filter (GF/C, 0.45 μm, Germany) and grinded for 1 min (using mortar
and pestle). After grinding, 10 ml acetone was added and stored at 4 °C for 24 h. After this
time, suspension was centrifuged for 10 min at 4000 rpm, and chlorophyll a was determined
spectrophotometrically on the supernatant, according to equation 1, using the spectrophotom-
eter Libra S12 (England). Acetone 90 % was used as blank [17]:
Ve
Chlorophyll að11:85*ðA664 −A750 Þ−1:54*ðA647 −A750 Þ−0:08*ðA630 −A750 ÞÞ ð1Þ
VfZ

where Ve is the extraction volume in ml, Vf the filtered volume in l, and Z the cuvette length-
path in cm.
For total carotenoids the same procedure as for chlorophyll a was used. The supernatant
was transferred to a glass cuvette and total carotenoids were measured spectrophotometrically
according to equation 2 [17]:

Ve
Total carotenoids ¼ ð10*ðA480 −3*A750 ÞÞ ð2Þ
VfZ

Determination of Phycobiliprotein Content

Phycobiliproteins were extracted after osmotic shock using glycerol as the osmoticum [18].
Aliquots of 10 ml were centrifuged (for 10 min and 4000 rpm) and the pellets homogenized
with glycerol and incubated in the dark at 4 °C for 2 h. Water (9:1 water to homogenized
pellets) was then added for the lysis of the cells. The samples were centrifuged, and the
supernatant was used for the estimation of phycobiliproteins according to Bogorad [19].

Determination of Carbohydrate and Total Protein Content

Content of carbohydrates were estimated using the anthrone method [20] after hot alkaline
extraction [21]. Pellets after centrifugation were resuspended in dH2O and heated in 40 %
 Appl Biochem Biotechnol

(w/v) KOH at 90 °C for 1 h. After cooling, cold absolute ethanol (1.2 ml) was added and stored
in a fridge at −20 °C overnight. The sample was centrifuged and the supernatant was
discarded. The pellet was then resuspended in 1.5 ml dH2O. Then, anthrone reagent was
added to the sample followed by the addition of sulphuric acid and boiled at 100 °C for 15 min
[20]. After cooling, the absorbance was read at 578 nm. The amount of carbohydrate was
estimated using a standard curve created using D-glucose.
Total protein was extracted according to Meijer and Wijffels [22]. Pellets (50 ml) of
microalgae were frozen at −20 °C. Cells were centrifuged for 10 min at 4 °C at 4000 rpm.
Cell extraction was at 4 °C. Pellets were washed in 25 mM phosphate buffer, pH 7.0, and then
resuspended in 10 ml phosphate buffer with 1 % (w/v) SDS. Cells were disrupted by ultra-
sonic device. Total protein was determined according to Bradford [23] using bovine serum
albumen standards.

Statistical Analysis

Multi-factor analysis of variance (MANOVA) was used to investigate the effects of photope-
riod and irradiance on the respond variables. For the determination of the significant effect by
independent variables (p<0.05), a multiple range test, using least significant differences, was
performed. One-way ANOVA was used to determine if there was a significant difference
between the dependent variables. Least significant difference’s test at reliability level of 5 %
was used to identify differences between each level of treatment. All data were checked for
normality and homogeneity.

Results and Discussion

Biomass Production

As shown in Fig. 1, the growth patterns of N. calcicola under the three light intensities and
three photoperiods investigated are illustrated. As shown, for the majority of the cultures, there
was no difference in the biomass production, except cultures with 21 and 42 μmol photons
m−2- s−1 in photoperiod of 12/12, which displayed higher biomass production. Between the
two light intensities, the cultures with 21 μmol photons m−2- s−1 had the highest final biomass
density, suggesting that the optimum light regime for N. calcicola is 21 μmol photons m−2- s−1

(a) (b) (c)

1.4 1.4 1.4
Biomass producon (g l-1)

1.2 1.2 1.2

1.0 1.0 1.0
0.8 0.8 0.8
0.6 0.6 0.6
0.4 0.4 0.4
0.2 0.2 0.2
0.0 0.0 0.0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Time (d)

Fig. 1 Biomass production of N. calcicola under three different light intensities (filled square indicates 21, filled
circle indicates 42, and filled diamond indicates 63 μmol photons m−2- s−1) and three different photoperiods (a
8/16, b 12/12, and c 16/8 day/night)
Appl Biochem Biotechnol

and 12/12 photoperiod. Different species of microalgae or cyanobacteria respond in a different

way to light intensity and photoperiod [7,19]. Some species are characterized as Bsun^ and
other as Bshade^ species, depending on the required light intensities required for maximum
growth. Some species, increasing light intensity, increase their growth rates until a saturation
point is reached. At higher light intensities than this saturation point, due to excess light that
cannot be absorbed by the photosynthetic apparatus, photooxidation and photoinhibition can
occur [8,12, 24]. The results of the present study perhaps indicate that N. calcicola prefers dim
light, being a Bshade^ species [25]. However, it is well known that the limiting growth rate of
nitrogen fixing cyanobacteria is the process of the nitrogen fixing itself [26], a fact that may
explain that in overall, there was no significant influence of the light regime on the final
biomass density of N. calcicola. It is generally known that changes in light intensity, light
duration, and in light quality affect the growth and the biochemical composition of microalgae
and cyanobacteria [6, 27].

Biomass Composition

Effect of Light Intensity

In Figs. 2–4, the variations of the determined biomass compounds during the cultivation of
N. calcicola are illustrated. In all cases studied, the biomass content of the compounds
increased with time. At day 4, the only significant effect of the light intensity was on total
protein and APC. The minimum total protein was associated with 63 μmol photons m−2 s−1,
while minimum APC was observed at light intensities of 21. At day 8, light intensity affected
significantly (p<0.05) all the measured parameters. The response of chlorophyll a, total
carotenoid, total protein, and APC to light intensity were similar and decreased when light

(a) (b) (c)

5 5 5
Chlorophyll a (mg gDM )
-1

4 4 4

3 3 3

2 2 2

1 1 1

0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14

(a) (b) (c)

Total carotenoids (mg gDM-1)

8 8 8

6 6 6

4 4 4

2 2 2

0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14
Time (d)

Fig. 2 Variation of chlorophyll a and total carotenoids during growth under three different light intensities (filled
square indicates 21, filled circle indicates 42, and filled diamond indicates 63 μmol photons m−2- s−1) and three
different photoperiods (a 8/16, b 12/12, and c 16/8 day/night)
 Appl Biochem Biotechnol

intensity increased to 63 μmol photons m−2 s−1. In contrast, carbohydrate content increased
when light intensity increased to 63 μmol photons m−2 s−1. PE and PC contents were
maximum (4.27 and 7 mg gDM−1, respectively) when light intensity was 21 μmol photons
m−2 s−1. Also at day 12, light intensity affected significantly (p<0.05) all of the measured
parameters. The results of chlorophyll a and total protein were similar to the results of day 8. In
contrast, total carotenoid and carbohydrate content increased when light intensity increased to
63 μmol photons m−2 s−1. For the phycobiliprotein fraction (PE, PC, and APC), the maximum
of its content was observed in cultures with 21 and 42 μmol photons m−2 s−1, while increasing
light intensity to 63 μmol photons m−2 s−1, the content decreased (Table 1).

Effect of Photoperiod

At day 4, the photoperiod did not have any significant affect (p>0.05) on any of the measured
parameters, while at day 8, with exception of carbohydrate content, the photoperiod had a
significant effect (p<0.05) on all the other measured parameters (Figs. 2 and 3). The maximum
content of total carotenoid of 7.18 and 7.12 mg gDM−1 were observed in cultures with 12/12
and 8/16 h photoperiod, respectively. Also, the minimum content of protein was associated

(a) (b) (c)

6 5 5
Phycoerithrin (mg gDM-1)

5 4 4
4
3 3
3
2 2
2

1 1 1

0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14

(a) (b) (c)

10 8 7
Phycocyanin (mg gDM-1)

6
8
6 5
6 4
4
4 3

2 2
2
1
0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14

(a) (b) (c)

Allophycocyanin (mg gDM-1)

5 5 5

4 4 4

3 3 3

2 2 2

1 1 1

0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14
Time (d)

Fig. 3 Variation of phycoerithrin, phycocyanin, and allophycocyanin during growth under three different light
intensities (filled square indicates 21, filled circle indicates 42, and filled diamond indicates 63 μmol photons
m−2- s−1) and three different photoperiods (a 8/16, b 12/12, and c 16/8 day/night)
Appl Biochem Biotechnol

(a) (b) (c)

500 500 500
Proteins (mg gDM )
-1

400 400 400

300 300 300

200 200 200

100 100 100

0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14

(a) (b) (c)

600 600 600
Carbohydrates (mg gDM-1)

500 500 500

400 400 400

300 300 300

200 200 200

100 100 100

0 0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14
Time (d)

Fig. 4 Variation of proteins and carbohydrates during growth under three different light intensities (filled square
indicates 21, filled circle indicates 42, and filled diamond indicates 63 μmol photons m−2- s−1) and three different
photoperiods (a 8/16, b 12/12, and c 16/8 day/night)

with 16/8 h. Response of PE, PC, and APC was similar; decreasing light duration, resulted to
the significantly increase of their content. At day 12, higher chlorophyll a (3.51 mg gDM−1)
and protein (342 mg gDM−1) content were related to 12/12 h photoperiod (Figs. 2 and 4).
Increasing photoperiod, the total carotenoid and carbohydrate content increased. The maxi-
mum content of total carotenoid (6.75 mg gDM−1) and carbohydrate (519.55 mg gDM−1)
observed in cultures grown on 16/8 h photoperiod. Regarding the phycobiliprotein fraction,
photoperiod had no effect on APC (p>0.05), but PC and PE content increased when
photoperiod was 8/16 h (Fig. 3).

Interaction of Irradiance and Photoperiod

At day 4, the interacted effect of light intensity and photoperiod was significant (P<
0.05) on chlorophyll a and total carotenoid. At day of 8, these effects were significant
on chlorophyll a, total carotenoid, PE, PC and APC. At the end of the cultivation
period (day 12), the interacted effect of light intensity and photoperiod was significant
in some cultures regarding the content of total carotenoid, carbohydrate, PE, and PC.
Content of total carotenoid increased as light intensity and duration increased to the
cultures with 21 and 42 μmol photons m−2 s−1, while the duration did not affect the
content in cultures with 63 μmol photons m−2 s−1, in which the maximum content of
total carotenoids was obtained. High light intensities and light durations affected
negatively the protein, PE, PC, and APC content. Maximum protein concentration
was 440.8 mg gDM−1, while maximum PE and PC was 5.20 and 7.54 mg gDM−1, at
42 μmol photons m−2 s−1 and 8/16 h photoperiod, respectively. In contrast, highlight
intensity and light duration positively affected the carbohydrate content in the cultures
with 21 and 42 μmol photons m−2 s−1, while with 63 μmol photons m−2 s−1, the
duration did not affect it.
Table 1 Biochemical composition (mg gDM−1) of N. calcicola cultured under experimental treatments for 12 of culture

Light intensity μmol 21 42 63

photons m2− s−1

Photoperiod 8/16 12/12 16/8 8/16 12/12 16/8 8/16 12/12 16/8
Light/dark
Chlorophyll a 3.41±0.07bc 3.47±0.13b 3.47±0.09b 3.47±0.14b 3.77±0.22a 3.45±0.08b 3.21±0.09c 3.28±0.05bc 3.21±0.03c
Total carotenoids 6.34±0.06c 6.4±0.11c 6.73±0.08ab 6.4±0.13c 6.66±0.2b 6.71±0.07ab 6.83±0.08ab 6.88±0.05a 6.83±0.03ab
Phycoerithrin 5.1±0.1a 4.11±0.14c 4.16±0.06c 5±0.1a 4.5±0.04b 4.2±0.03c 3.22±0.25d 3.4±0.07d 3.4±0.1d
a bc d b cd cd cd cd
Phycocyanin 7.41±0.42 6.31±0.35 5.55±0.15 6.8±0.61 6.41±0.31 6.05±0.15 5.83±0.37 6.2±0.2 5.94±0.09cd
a ab ab ab bc a c c
Allophycocyanin 4.27±0.15 4.07±0.11 4.08±0.2 4.13±0.09 3.84±0.18 4.2±0.17 3.54±0.22 3.72±0.25 3.73±0.2c
ab a cd bc bc cd cd d
Proteins 354.66±22.26 382.53±29.51 312.86±2.19 339.46±21.6 345.8±10.05 317.93±13.34 319.2±7.6 297.66±25.3 243.2±15.2e
cd cd bc cd d a ab a
Carbohydrates 470.66±17.24 469.33±25.48 495.33±9.86 476±19.07 457.33±23.18 529.33±14.46 511.33±16.16 531.33±14.74 534±10a

Each value is the mean of three samples

Values in the same row different superscripts are significantly different (p<0.05)
Data are mean±standard deviation
Appl Biochem Biotechnol
Appl Biochem Biotechnol

In overall, total protein, chlorophyll a, APC, PE, and PC content was lower at high
irradiance (63 μmol photons m−2 s−1), while total carotenoids and carbohydrate content
showed an inverse trend. The changes in pigments are considered to be an adaptation
mechanism to high light [24]. Low light induce synthesis of more photosynthetic units to
aid light harvesting, while at high light, algae synthesize less photosynthetic units to
prevent photo damage [27–29]. Poza-Carrión et al. [30] report that high light intensity
significantly decreased the content of phycobiliproteins (PBP) in Nostoc sp., while Post
et al. [31] have shown that the light harvesting pigments chlorophyll a and PC in
Osillatoria agardhii increased when light became limited. Vernotte et al. [32] have
shown that when light intensity during growth is low or when cells are cultured
heterotrophically in darkness with glucose for a long time, PBP of Microcystism sp.
appeared in excess over chlorophyll. Kim et al. [33] in their work stated that growth at
higher light intensity was faster, so pigment accumulation could not be promoted. Also,
adaptation to increase in irradiance results in a decrease in the number of thylakoids per
cell accompanied by alteration in the number of photosynthetic units and PBPs of cell
rather than to a change in the size of the photosynthetic unit or PBPs which might result
from chromatic adaptation [33]. Moreover, due to their antioxidant properties, caroten-
oids are believed to protect organisms against oxidative stress caused by excess light
[34]. Carotenoids due to their antioxidant properties display interesting capabilities for
treating several diseases, and considering that N. calcicola is a rich source for this
pigment [13], it is worth studying its adaptation to excess light by altering its carotenoid
content. In the present study, the total carotenoid content was higher at 42 and 63 (μmol
photons m−2 s−1) irradiance and 12 and 16 h photoperiod, confirming that carotenoids are
accumulated under higher light intensities. Additionally, it has been reported that in
N. calcicola, higher photoperiods cause a metabolic shift resulting in the accumulation of
carbohydrate and carotenoids [6]. It has been reported that carbohydrate content also to
be accumulated during the stationary phase [35, 36] as was also observed in the present
study. High light intensity could also be used to induce carbohydrate accumulation,
which could be used as feedstock for bioethanol production [37].
A significant difference observed on the present study was in the amount of protein
produced at the various light conditions. This is in agreement with other studies, in
which that biochemical composition is being affected by light conditions was reported
[35, 38, 39]. In the present study, protein content increased with increase in light
intensity, and the maximum protein content was observed at 21 μmol photons
m−2 s−1 and 12/12 h photoperiod. This is in agreement to the related published works
[40, 41]. Moreover, the observation that protein content was higher during the expo-
nential growth phase was also reported for the microalga Isochrysis sp. [6]. In general,
the regulation of carbohydrate and protein storage results from the regulation of
photosynthetic activities. With shorter light periods, the rates of synthesis of proteins
in the dark are substantially lowered, allowing for an increased pigment synthesis,
enhanced photosynthetic carbon fixation, and thereby the flow of carbon to carbohy-
drate storage [42]. During the following dark period, pigment synthesis is depressed
due to increased rates of protein synthesis at the expense of carbohydrates [43]. Since
2-oxoglutarate and glutamate are intermediates of both pigment and protein synthesis, it
has been suggested that pigment synthesis results from competition for these interme-
diates controlled by photophosphorylation. Long dark periods will have similar effects
on the energy charge as low irradiances [43].
 Appl Biochem Biotechnol

Conclusions

In conclusion, the light regime under which N. calcicola will be cultivated should be taken into
consideration because it affects the biomass biochemical composition. It is possible to
manipulate in an extent the biochemical composition by changing the light regime. The
investigated local isolate of N. calcicola under low light intensity and photoperiod increased
the phycobiliprotein fraction, which is a source of high-value products. In contrast, increasing
light intensity total carotenoids are accumulated, which display also some potential interest as
antioxidant agent. Total carotenoids were affected by photoperiod only in cultures with 21 and
42 μmol photons m−2 s−1. High light intensity could also be used to induce carbohydrate
accumulation, which could be used as feedstock for bioethanol production.

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