Effect of Salinity and Photoperiod on Growth of Microalgae Nannochloropsis sp. and Tetraselmis sp. Nature Environment and Pollution Technology
In this present study, the effect of salinity and photoperiod on the growth of Nannochloropsis sp. and Tetraselmis sp. was examined to optimize microalgal growth. Different salinities (5, 10, 15 and 20‰) for Nannochloropsis sp. and (10, 15, 20 and 25‰) for Tetraselmis sp. were studied. Both microalgae were cultivated at two different photoperiod cycles (24:0 and 12:12 h light:dark). Maximum growth rates and cell concentrations for Nannochloropsis sp. and for Tetraselmis sp. were achieved at salinities of 10 and 15‰, respectively. In terms of photoperiod, the maximum growth rates and cell concentrations for both microalgae were obtained at photoperiod of 24:0 h light:dark. This study shows that the cell concentrations and growth rates of both microalgae are highly dependent on salinity and photoperiod.
Effect of Salinity and Photoperiod on Growth of Microalgae Nannochloropsis
sp. and Tetraselmis sp.
M. Fakhri*(**)†, N. B. Arifin*, B. Budianto*, A. Yuniarti* and A. M. Hariati**
*Department of Aquaculture, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang 65145, Indonesia
**Laboratory of Fish Nutrition, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang 65145, Indonesia
†Corresponding author: M. Fakhri
In this present study, the effect of salinity and photoperiod on the growth of Nannochloropsis sp. and
Tetraselmis sp. was examined to optimize microalgal growth. Different s alinities ( 5, 10, 15 and 20‰)
for Nannochloropsis sp. and (10, 15, 20 and 25‰) for Tetraselmis sp. were studied. Both microalgae
were cultivated at two diff erent photoperiod cycles (24:0 and 12:12 h light:dark). Maximum growth
rates and cell concentrations for Nannochloropsis sp. and for Tetraselmis sp. were achieved at
salinities of 10 and 15‰, respectively. In terms of photoperiod, the maximum growth rates and cell
concentrations for both microalgae were obtained at photoperiod of 24:0 h light:dark. This study
shows that the cell concentrations and growth rates of both microalgae are highly dependent on
salinity and photoperiod.
Nat. Env. & Poll. Tech.
2015pp. 563 -5 66Vol. 14
ISSN: 0972-6268 No. 3
Nature Environment and Pollution Technology
An Internation al Quarterly Scientific Journal
Original Research Paper
Microalgae are photosynthetic organisms that are one of
the most promising primary producers (Pruvost et al. 2009).
Marine microalgae are used for many purposes, such as for
animal feed (De Pauw & Persoone 1988), industrial applica-
tions (Singh & Gu 2010) and biofuel production (Hossain
et al. 2008). Nannochloropsis sp. and Tetraselmis sp. are
known to be an important food source for aquaculture (De
Pauw & Persoone 1988, Borowitzka 1997) because of their
rapid growth rates and useful chemical properties (Singh &
Gu 2010). Fast growth rate of microalgae generally depends
on their abilities to double their cells during exponential
phase within 24 h or in a short time (210 min) (Chisti 2007).
Adjusting the environmental conditions could become
an effective way to optimize the growth rates of microalgae
(Mata et al. 2010). A combination of environmental factors
such as salinity, light intensity, temperature (Ak et al. 2008),
photoperiod and nutrient composition in the culture sys-
tem are known to affect the microalgal growth rates (Kitaya
et al. 2008). Photoperiod cycle is one of the main factors
that influence the growth rate of photoautotrophic
microalgae culture (Parmar et al. 2011). Besides that, varia-
tions in salinity also affect the growth rate of marine
microalgae (Adenan et al. 2013).
A study on the effect of photoperiod on growth of
Nannochloropsis sp. and Tetraselmis sp. have been reported
(Wahidin et al. 2013, Alsull & Omar 2012). Moreover, the
effect of salinity on Nannochloropsis sp. and Tetraselmis
suecica growth have also been studied (Pal et al. 2011, Alsull
& Omar 2012). However, algal growth varies from one spe-
cies to another and depends on the environmental condi-
tions and the species origin (Banerjee et al. 2011).
The purpose of this study was to determine cell concen-
tration and the cell growth rate of Nannochloropsis sp. and
Tetraselmis sp. under different salinities and different pho-
toperiod regimes (light:dark cycles).
MATERIALS AND METHODS
Algal cultures: Nannochloropsis sp. and Tetraselmis sp.
were obtained from the Laboratory of Fish Nutrition, Fac-
ulty of Fisheries and Marine Sciences, University of
Brawijaya. Stock cultures were cultivated under the labora-
tory conditions (29±2°C, 4,500 lux) in a Walne medium.
Experimental culture conditions: Four-day old culture of
Nannochloropsis sp. and Tetraselmis sp. was applied as in-
oculum. Initial cell concentration in all treatments was ad-
justed at 5 × 105 cells/mL. The inoculum was cultivated
into 0.5 L flasks containing 0.35 L of Walne medium at four
different salinities (5, 10, 15 and 20‰) for Nannochloropsis
sp. and (10, 15, 20 and 25‰) for Tetraselmis sp. and at two
different photoperiod regimes (24:0 and 12:12 h light:dark
cycles). All treatments were conducted in triplicates. Cul-
Vol. 14, No. 3, 2015 Nature Env ironment and Pollution Technology
564 M. Fakhri et al.
tures were mixed by an air pump and illuminated by fluo-
rescent lamp at a light intensity of 4,500 lux with tempera-
ture of 29±2°C. The experiment was conducted for 6 days.
Growth analysis: Cell count using a 0.1 mm deep Neubauer
Haemocytometer (BOECO, Hamburg, Germany) was used
to monitor algal growth. At a logarithmic growth phase, the
increase in cell concentration (dx) is proportional to the
amount (x) present and to the time interval (dt). The specific
growth rate (µ) was calculated from the following equation:
ln (x2) – ln (x1)
µ = ––––––––––––– ...(1)
Where, µ represents the rate of growth per unit amount
of cell concentration, x1 and x2 = cell concentration at time
l (tl) and time 2 (t2), respectively.
Doubling time (td) of cell represents the mean of the
generation time of biomass. The doubling time (day) of
growth rate was calculated according to the equation:
ln 2 0.693
td = ––––– = –––––– ...(2)
Statistical analysis: Statistical analysis was performed us-
ing SPSS 20.0. Data were analysed using one way analysis
of variance (ANOVA) and t-test to evaluate the existence of
significant differences between treatments. Levels of sig-
nificance were tested at 95% level.
Cell growth of Nannochloropsis sp. and Tetraselmis sp. at
different salinities and photoperiods is given in Figs. 1 and
2. Both the species showed similar growth pattern and the
highest cell concentration was achieved in day 6. In
Nannochloropsis sp., the highest specific growth rate of
1.16 per day (p<0.05) and the maximum cell concentration
of 11.80 × 106 cell mL-1 (p<0.05) on the 6th day of culture
period was obtained at salinity of 10‰ under continuous
illumination. Meanwhile, Tetraselmis sp. achieved the high-
est growth rate of 0.63 per day (p<0.05) and the maximum
cell concentration of 5.25 × 106 cell/mL (p<0.05) on day 6
at salinity of 15‰ under a photoperiod of 24:0 light/dark
cyc l e . The fa s test doubl i n g time re sulted from
Nannochloropsis sp. and Tetraselmis sp. was 0.59 day and
1.09 day, respectively (Tables 1 and 2).
Salinity and photoperiod clearly influenced the cell
concentration in Nannochloropsis sp. and Tetraselmis sp.
Increasing the photoperiod from light:dark of 12:12 to 24:0h
resulted in increasing the specific growth rate and maximum
cell concentration in both the microalgal species (Tables 1
and 2). Tables 1 and 2 also show that Nannochloropsis sp.
and Tetraselmis sp. grew much faster at low salinities as
compared with high salinities.
Table 1: Specific growth rates, doubling times, and max imum cell concentrations at different salinities and photoperiods of Nan nochloropsis
Photope riod Salin ity (‰) Specific growth Dou bling time Maximum cell conce ntratio n
(L:D cycle) h rate (day-1 ) (d ay) 106 cell mL-1)
24 :0 5 0. 8 2 0.8 5 7. 7 5
1 0 1.1 6 0.5 9 11 .8 0
1 5 0.8 5 0.8 1 7. 33
2 0 0.7 6 0.9 1 6. 92
12 :1 2 5 0 .75 0 . 9 2 6.67
1 0 0.9 0 0.7 7 9. 58
1 5 0.7 8 0.8 8 7. 08
2 0 0.7 4 0.9 3 6. 75
Table 2: Specific growth rates, doubling times, and maximum cell concentrations at diffe rent salin itie s and photoperiods of Tetras elmis sp.
Photope riod Salin ity (‰) Specific growth Dou bling time Maximum cell conce ntratio n
(L:D cycle) h rate (day-1 ) (d ay) (106 cell mL-1)
24:0 10 0. 53 1 .31 4. 08
1 5 0 .6 3 1 .0 9 5. 25
2 0 0 .5 3 1 .3 0 4. 25
2 5 0 .4 8 1 .4 4 3. 25
12 :1 2 1 0 0 .4 4 1 .5 6 3. 33
1 5 0 .5 5 1 .2 4 4. 67
2 0 0 .4 7 1 .4 6 3. 75
2 5 0 .4 1 1 .5 1 2. 75
Nature Environment and Pollution Technology Vol. 14, No. 3, 2015
EFFECT OF SALINITY AND PHOTOPERIOD ON NANNOCHLOROPSIS AND TETRASELMIS
Environmental parameters such as salinity, light, photope-
riod and temperature have been reported to affect the growth
of microalgae (Wahidin et al. 2013, Adenan et al. 2013,
Banerjee et al. 2011, Ak et al. 2008). In the present study,
the effec t of salinity and photoperiod on growth of
Nannochloropsis sp. and Tetraselmis sp. was determined.
This study showed that a specific growth rate increased with
the increasing salinity in both the species with optimum
values of 10‰ for Nannochloropsis sp. and 15‰ for
Tetraselmis sp. (Tables 1 and 2). However, the growth rates
turned to drop significantly (p<0.05) when salinity was
above 10‰ and 15‰ for Nannochloropsis sp. and
Tetraselmis sp., respectively. This phenomenon agreed with
Adenan et al. (2013), who reported that increasing salinity
from 20 to 25‰, increased the growth rate of Chlorella sp.,
however, increasing salinity to 30‰ decreased the specific
growth rate. On the contrary to our results, Alsull and Omar
(2012) reported that the optimum salinity for growth of both
species was 33‰. On the other hand, Pal et al. (2011) found
that Nannochloropsis sp. grew best at salinity of 13‰. We
suggest that the results are probably different because each
species has a different growth response to salinity (Sudhir &
Murthy 2004, Ak et al. 2008).
The maximum specific growth rates of Nannochloropsis
sp. and Tetraselmis sp. obtained in this study were 1.16
day-1 and 0.63 day-1, respectively at the optimum salinity.
The specific growth rate of Nannochloropsis sp. in this study
was significantly higher than the values of 0.80 and 0.86
day-1 as reported by Pal et al. (2011) and Alsull & Omar
(2012), respectively. In Tetraselmis sp., the growth rate in
this study is in agreement with that of (Michels et al. 2014),
who found that the highest growth rate of Tetraselmis
suecica was 0.68 day-1.
Fig. 1: Incre ase in cell concentration (n = 3) o f Nannochloropsis sp. unde r different pho toperiod lengths (a) 24:0 and (b) 12:12 light and
dark cycles and different salinitie s (5, 10, 15 and 20‰) during the culture period .
Fig. 2: Increase in cell concentration (n = 3) of Tetraselmis sp. und er different photoperiod lengths (a) 24:0 and (b) 12:12 light and dark
cycles and dif ferent salinities (10, 15, 20 and 25‰) during the culture peri od.
Vol. 14, No. 3, 2015 Nature Env ironment and Pollution Technology
566 M. Fakhri et al.
In the present study, the growth rates of both microalgal
species increased with increasing photoperiod (Tables 1 and
2). The optimum photoperiod for the growth of both species
was 24:0 h light:dark cycle. This result agrees with the find-
ings of Wahidin et al. (2013), who found that photoperiod
of 24:0 light:dark cycle resulted the highest growth rate of
Nannochloropsis sp. In addition, Alsull & Omar (2012)
showed that Tetraselmis sp. grew best under continuous il-
lumination. This is probably due to the fact that insufficient
light causes photolimitation of microalgal growth (Wahidin
et al. 2013).
Salinity and photoperiod are two major factors that signifi-
cantly affect the growt h of Nannochl oro psis sp. and
Tetraselmis sp. Both microalgae show a similar growth pat-
tern under different salinity and different photoperiod dur-
ing the culture period. Both species grow best under con-
tinuous illumination, however, each species has a specific
optimum salinity. These findings are very important in or-
der to prepare a mass culture of these two species of
This research was financially supported in part by Faculty
of Fisheries and Marine Sciences, University of Brawijaya,
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