International Biodeterioration & Biodegradation 95 (2014) 11e18

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International Biodeterioration & Biodegradation

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Effects of different salinities and pH on the growth and proximate

composition of Nannochloropsis sp. and Tetraselmis sp. isolated from
South China Sea cultured under control and natural condition
Helena Khatoon a, *, Norazira Abdu Rahman a, Sanjoy Banerjee b, Nazurah Harun a,
Siti Suhada Suleiman a, Nur Hazwani Zakaria a, Fathurrahman Lananan c,
Siti Hajar Abdul Hamid c, Azizah Endut d
a
School of Fisheries and Aquaculture Sciences, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia
b
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c
School of Ocean Engineering, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia
d
East Coast of Environmental Research Institute, Gong Badak Campus, Sultan Zainal Abidin University, 21300 Kuala Terengganu, Terengganu, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Nannochloropsis sp. and Tetraselmis sp. are widely used in aquaculture as a source of protein, lipid and
Received 14 January 2014 carbohydrate. The growth and proximate composition of microalgae could be affected by different cul-
Received in revised form ture conditions especially salinity, temperature and light. Thus, this study was aimed to compare the
24 June 2014
growth and proximate composition of Nannochloropsis sp. and Tetraselmis sp. cultured in different sa-
Accepted 29 June 2014
Available online 19 July 2014
linities and pH under different culture conditions. In this study Nannochloropsis sp. and Tetraselmis sp.
were isolated from South China Sea and cultured at different salinities of 20, 30 and 40 ppt and different
pH of 5.5, 7.5, 8.5 and 9.5 under natural and control condition until stationary phase. Results showed that
Keywords:
Salinity
Nannochloropsis sp. and Tetraselmis sp. had significantly higher (p < 0.05) cell density, lipid and carbo-
Growth rate hydrate content under control condition at 30 ppt. However, protein content was significantly higher
Proximate composition (p < 0.05) in Nannochloropsis sp. when cultured under natural condition at 30 ppt. High cell density,
pH protein, lipid and carbohydrate content was obtained when cultured at pH 7.5 and 8.5 for both species.
Nannochloropsis sp. The output of this study could be considered for Nannochloropsis sp. and Tetraselmis sp. cultivation to
Tetraselmis sp. provide appropriate levels of protein, lipid and carbohydrate as feed supplement for aquaculture
organisms.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction industry application (Hemaiswarya et al., 2011). In addition, its high

Many microalgae species are potential producers of value-added
bioactive compounds such as pigments, vitamins, and long-chain
polyunsaturated fatty acids (Pal et al., 2011). Thus, microalgae are
highly potential to synthesize marine drugs such as antibiotics,
vitamin and antioxidants for cosmetics, pharmacological and food
industry (Hu et al., 2008). In aquaculture, only microalgae with
valuable properties were used and the composition of the algal
biomass with regards to lipid, carbohydrate and protein determines
its overall economic potential (Williams and Laurens, 2010).
Nannochloropsis sp. and Tetraselmis sp. are common microalgae
species that have promising potential especially in aquaculture
* Corresponding author. Tel.: þ60 96685027; fax: þ60 9668 5002.
E-mail addresses: hlnkhatoon@gmail.com (H. Khatoon), enazizah@unisza.edu.
my (A. Endut).
nutritional value, ease of culturing, lack of toxicity, correct cell size
and digestible cell wall fulfil the selection criteria to be use in
aquaculture purpose (Hemaiswarya et al., 2011). For instance,
Nannochloropsis sp. itself is able to yields high amounts of triglyc-
eride fatty acid (TFA) and polyunsaturated fatty acid (PUFA) lipids
which can reach up to 65e70% of total dry weight (Rodolfi et al.,
2009) which makes it a species with a high potential for the pro-
duction of biofuels and as well as for feeding of the hatcheries
grown larval and juveniles of bivalves and fish.
These beneficial fatty acid and nutrients are currently extracted
almost exclusively from fish and is considered as unsustainable
because more than 1 kg fish is needed to produce 1 kg carnivorous
farmed fish (Taelman et al., 2013). In addition, small pelagic species
are co-captured and used to produces fish oil and fish meal, which
are mainly used as feed in aquaculture system and this threatens
the sustainability of the wild stock because of the rising

http://dx.doi.org/10.1016/j.ibiod.2014.06.022
0964-8305/© 2014 Elsevier Ltd. All rights reserved.
12 H. Khatoon et al. / International Biodeterioration & Biodegradation 95 (2014) 11e18
aquaculture production (Taelman et al., 2013). The increasing in the
prices for these fish-based products leads to the search of alter-
natives to these sources (Shepherd, 2013). A good alternative is a
commercial-scale production of microalgae biomass because this
could reduce the cost and ecological impact of intensive fish
farming (Muller-Feuga, 2000).
However, the growth and nutrient content of microalgae is
affected and influenced by the culture conditions such as light in-
tensity, nutrient limitation, temperature, pH, and salinity (Cho
et al., 2007). Thus, even a slight fluctuation in those factors can
caused changes in the growth and proximate composition of the
microalgae either by increasing the nutritional value or decreasing
it and affect the overall value of the microalgae.
Variations in salinity also influence the growth and proximate
composition of the marine microalgae even though it is tolerant to
changes in salinity. According to Rao et al. (2007), adaptability to
salinity differs between algae and they are grouped as halophilic
(salt requiring for optimum growth) and halotolerant (having
response mechanism to survive in saline medium). Decreasing
salinity is a unique way to change the biochemical composition of
marine microalgae although the changeable role of salinity on
starch metabolism indicates its species-specific and cultivation
condition-dependent nature (Yao et al., 2013). High salinity has
been reported to inhibit the growth, lipid and triacylglyceride
accumulation of Dunaliella sp. (Takagi et al., 2006).
Another important factors that also seriously affect the optimal
growth of algal cultures is the hydrogen ion concentration (pH) of
the culture medium (Khalil et al., 2010). Algal species can only grow
well when the pH is within a certain range, without taking into
account other environmental factors (Wang et al., 2011). Never-
theless, any changes in the pH can cause changes either by inhib-
iting, change the biochemical composition or causing cell death to
the microalgae. For example, the dry weight gain and the
biochemical components of Dunaliella bardawil were greatly
enhanced at pH 7.5 while Chlorella ellipsoidea attained their
maximum dry weight and carbohydrate content at alkaline pH
(Khalil et al., 2010).
This study was aimed to determine the optimum salinity, pH
and culture condition that can results in higher growth and prox-
imate composition (protein, lipid and carbohydrate) of Nanno-
chloropsis sp. and Tetraselmis sp. These results can be applied by
farmers and industry in culturing microalgae with a targeted
growth and proximate composition that can only be achieved un-
der certain culture conditions.
2. Material and methods
2.1. Sample collection, culture and maintenance
Nannochloropsis sp. and Tetraselmis sp. were isolated from the
South China Sea and maintained at the laboratory of Faculty of
Fisheries and Aqua-Industry, Universiti Malaysia Terengganu. The
pure Nannochloropsis sp. and Tetraselmis sp. was cultured in Con-
way culture medium (Tompkins et al., 1995). Sub-culturing was
done every two weeks to maintain pure and healthy stock culture.
Pure seed culture of Nannochloropsis sp. and Tetraselmis sp. were
maintained at 30 ppt salinity in Conway medium.
2.2. Media preparation
Preparation of Conway medium involves the preparation of
stock solutions which consisted of the macronutrients, trace metal
solutions and vitamins. For the preparation of stock solutions, the
chemical compositions were diluted in distilled water. Chemical
composition of Conway medium was shown in Table 1. One
Table 1
Chemical composition of Conway medium.
Conway medium (Tompkins et al., 1995)
Nitrate KNO3 (100 g m3)
Phosphate Na3PO4 (20 g m3)
Trace metal Na2H2EDTA$2H2O (45 g m3)
FeCl3$6H2O (1.3 g m3)
ZnCl2 (4.2 g m3)
MnCl2$4H2O (0.36 g m3)
CoCl2$6H2O (4.0 g m3)
CuSO4$5H2O (4.0 g m3)
(NH4)6Mo7O24$4H2O (1.8 g m3)
H3BO3 (33.4 g m3)
Vitamin Thiamin HCl (200 mg m3)
Cyanocobalamin (10 mg m3)
milliliter of macronutrient, 0.5 mL of trace metal, and 0.1 mL of
vitamins were added to 1000 mL of filtered and sterilized seawater
at 30 ppt salinity prior to the inoculation and maintenance of pure
microalgae seed culture.
2.3. Experimental design
Experiments were carried out in 1 L working volume under
natural and control condition in three different salinities which
were 20, 30 and 40 ppt. For the maximum density of Nanno-
chloropsis oculata, the optimal salinity condition was 30 ppt ac-
cording to Cho et al. (2007). Hence, 20 ppt and 40 ppt salinity were
included in this study to see the performance on both species when
cultured under variable range of salinity other than the normal
30 ppt seawater salinity.
Four different pH of 5.5, 7.5, 8.5 and 9.5 were varied. The pH was
adjusted using hydrochloric acid and sodium bicarbonate. Indoor
culture condition was maintained inside the room at 25  C with a
light intensity of 1588 lux using cool fluorescent light and 24 h
photoperiod. The microalgae culture under natural condition was
kept outdoor under shade and relied on natural illumination. The
initial cell density was 1  105 cells mL1 for each treatment.
Growth parameters in terms of cell density and optical density
were measured daily. Cells were harvested at their stationary phase
by centrifugation at 8000 rpm for 10 min followed by washing
twice with sterilize distilled water. Samples were then freeze-dried
and kept at 20  C until further analysis. Batch experiments for
each microalgae species at various salinities, pH and culture con-
ditions were carried out in three replicates. Filtered aeration was
provided to all culture flasks throughout the experimental period to
provide agitation and facilitate gas exchange.
2.4. Growth parameters analysis
Microalgae growth was determined based on the cell density
and optical density techniques. Microalgae cells were sampled and
counted daily using a rhodium-coated haemacytometer (Hawksley
AC1000, UK). The specific growth rate (SGR) of microalgae was
calculated with Eq. (1).
SGR/day ¼ ln (X2/X1)/t2  t1 (1)
where, X1, biomass concentration at the beginning of the selected
time interval; X2, biomass concentration at the end of the selected
time interval; t2  t1, selected treatment period (in days) for the
determination of biomass of microalgae species.
The optical density was determined using a spectrophotometer
(Shimadzu UV-1601, Japan) at the wavelength of 540 nm and
530 nm, respectively, for Nannochloropsis sp. and Tetraselmis sp.
 H. Khatoon et al. / International Biodeterioration & Biodegradation 95 (2014) 11e18 13

2.5. Protein analysis

Protein was analyzed according to Lowry et al. (1951). Samples

were freeze-dried and made into 25 mL solution by mixing with
distilled water. From the 25 mL of sample prepared, 0.5 mL was
taken from each sample and analyzed by adding 1 N NaOH, fol-
lowed by an alkaline copper solution and 0.5 mL of FolineCiocalteu
reagent. The mixed solution added with FolineCiocalteu reagent
was kept in dark places for 30 min. Standard protein concentrations
were prepared using bovine serum albumin. The absorbance was
measured using a spectrophotometer (Shimadzu UV-1601, Japan)
at a wavelength of 750 nm.

2.6. Lipid analysis

Centrifuged microalgae biomass with a volume of 1 mL was

initially washed with 0.9% saline solution and freeze-dried. Then
lipid analysis was conducted by the sulfuric acid-charring method
based on Marsh and Weinstein (1966), following the carbonization
method using tripalmitin as the standard after extracting lipids
according to the method of Bligh and Dyer (1959). The samples
were extracted from 4.5 mL of chloroform: methanol (concentra-
tion ratio of 1:2) and it was then centrifuged at 10 000 rpm for
10 min. After separating the supernatant from the biomass, 1.5 mL
of chloroform and 1.5 mL of distilled water was added and the
sample was centrifuged again. Polar phase was removed with a
pipette and evaporated under vacuum and incubated a water-bath
at 35  C. After completely dried, 2 mL of concentrated sulfuric acid
was added. The carbonization process was carried out at 200  C for
15 min, then the tubes were cooled and 3 mL of water was added
into each tubes. The absorbance was measured at 375 nm.
2.7. Carbohydrate analysis
Carbohydrate analysis was conducted based on the method of
Dubois et al. (1956). For each sample, 5e6 mg was taken and made
into 25 mL solution by mixing with distilled water. Prior to analysis,
5% phenol solution and concentrated sulfuric acid was prepared.
Samples were analyzed by adding 1 of 5% phenolic solution and
5 mL of concentrated sulfuric acid. The standard was prepared
using glucose. The optical density was measured at 488 nm in a
spectrophotometer (Shimadzu UV-1601, Japan).
Fig. 2. Cell density (cells mL1  107) versus culture period (day) for Nannochloropsis
sp. and Tetraselmis sp. cultured in four different pH (5.5, 7.5, 8.5, 9.5) under natural and
control condition (values are mean ± standard error).
2.8. Statistical analysis
Collected data were analyzed using two-way analysis of vari-
ance (ANOVA). Significant differences amongst treatments were
determined using Tukey test at 95% confidence interval level. Pro-
tein, lipid and carbohydrate percentages were arcsine transformed
before the statistical analysis. All statistical analysis was done using
the Minitab 16™ software.
3. Results
3.1. Growth of Nannochloropsis sp. and Tetraselmis sp.
Cell density and optical density of Nannochloropsis sp. and Tet-
raselmis sp. cultured under control and natural condition in
response to various salinities and pH are shown in Figs. 1, 2, 3 and 4,
respectively. The maximum density between Nannochloropsis sp.
and Tetraselmis sp. cultured in 30 ppt, with the respective cell
density of 4.877  107 cells mL1 and 3.543  107 cells mL1, was
not significantly different to each other. Tetraselmis sp. showed
significantly lower cell density when cultivated at the salinity of
20 ppt. At 20 and 40 ppt, the cell density of Nannochloropsis sp.
were significantly lower than those cultivated in the salinity of
30 ppt with the value of 4.380  107 cells mL1 and

Fig. 1. Cell density (cells mL1  107) versus culture period (day) for Nannochloropsis Fig. 3. Optical density (abs) versus culture period (day) for Nannochloropsis sp. and
sp. and Tetraselmis sp. cultured in three different salinities (20, 30 and 40 ppt) under Tetraselmis sp. cultured in three different salinities (20, 30 and 40 ppt) under natural
natural and control condition (values are mean ± standard error). and control condition (values are mean ± standard error).
14 H. Khatoon et al. / International Biodeterioration & Biodegradation 95 (2014) 11e18

Fig. 4. Optical density (abs) versus culture period (day) for Nannochloropsis sp. and
Tetraselmis sp. cultured in four different pH (5.5, 7.5, 8.5, 9.5) under natural and control
condition (values are mean ± standard error).

Fig. 5. Total protein content (% dry weight) of Nannochloropsis sp. and Tetraselmis sp.
2.443  107 cells mL1, respectively. The differences of cell density cultured in three different salinities (20, 30 and 40 ppt) under natural and control
of Tetraselmis sp. in other salinity were insignificant. condition (values are mean ± standard error).
At the end of the experiment, the specific growth rate of both
Nannochloropsis sp. and Tetraselmis sp. was found to be significantly
higher when cultured at 30 ppt under control condition (Table 2). In
addition to salinity stress, the differences in pH were also showed to
lead to the change in the specific growth rate of both microalgae. In
this study, both Nannochloropsis sp. and Tetraselmis sp. cultured at
pH 5.5 and 9.5 showed a significantly lower cell density as
compared to pH 7.5 and 8.5.

3.2. Protein content of Nannochloropsis sp. and Tetraselmis sp.

cultivated in natural and control condition

The protein contents of Nannochloropsis sp. and Tetraselmis sp. in

response to 20, 30, 40 ppt salinities under natural and control
condition were shown in Fig. 5. Under control condition at 30 ppt,
both Nannochloropsis sp. and Tetraselmis sp. showed a significantly
higher protein content as compared to those of 20 and 40 ppt sa-
linities. Nannochloropsis sp. cultured under control condition at 20,
30 and 40 ppt yield 35.6%, 44.3%, 37% of total protein content,
meanwhile Tetraselmis sp. yield 47.5%, 55.2%, 30.1% of total protein
content, respectively. At the salinity of 40 ppt in control condition,
Fig. 6. Total protein content (% dry weight) of Nannochloropsis sp. and Tetraselmis sp.
Tetraselmis sp. showed significantly lower total protein content as
cultured in four different pH (5.5, 7.5, 8.5, 9.5) under natural and control condition
compared to 20 ppt and 30 ppt. In case of Nannochloropsis sp., there (values are mean ± standard error).
were no differences of the protein content between 20 and 40 ppt

in control condition. However, at 30 ppt protein content was

Table 2
significantly higher (p < 0.05) compared to 40 ppt. The protein
Specific growth rate and harvested culture period of marine microalgae, Nanno-
chloropsis sp. and Tetraselmis sp. using different salinities and pH under natural and contents of Nannochloropsis sp. and Tetraselmis sp. in response to
control condition. pH 5.5, 7.5, 8.5 and 9.5 under natural and control condition were
shown in Fig. 6. The output of this study showed that the optimum
Parameter Nannochloropsis sp. Tetraselmis sp.
pH for the growth of Nannochloropsis sp. and Tetraselmis sp. were at
Control Natural Control Natural pH 7.5 and 8.5, respectively.
Culture period 12 7 8 9
(days)
3.3. Lipid content of Nannochloropsis sp. and Tetraselmis sp.
SGR (/day) Salinity (ppt) cultivated in natural and control condition
20 0.699 ± 0.025 0.876 ± 0.019 0.681 ± 0.004 0.584 ± 0.017
30 0.708 ± 0.028 0.946 ± 0.006 0.734 ± 0.010 0.598 ± 0.019
Lipid content of Nannochloropsis sp. and Tetraselmis sp. were
40 0.644 ± 0.004 0.846 ± 0.028 0.665 ± 0.016 0.557 ± 0.011
pH described in Fig. 7. In the control condition, both species showed
5.5 0.220 ± 0.003 0.301 ± 0.005 0.330 ± 0.005 0.316 ± 0.007 extremely high sensitivity in response towards the changes in
7.5 0.290 ± 0.005 0.508 ± 0.005 0.435 ± 0.019 0.346 ± 0.049 salinity. Nannochloropsis sp. showed significantly higher (p < 0.05)
8.5 0.234 ± 0.001 0.623 ± 0.003 0.352 ± 0.042 0.359 ± 0.025 lipid content when cultured in 30 ppt under control condition
9.5 0.213 ± 0.005 0.462 ± 0.000 0.320 ± 0.009 0.264 ± 0.023
compared to 20 and 40 ppt. Tetraselmis sp. also showed higher lipid
 H. Khatoon et al. / International Biodeterioration & Biodegradation 95 (2014) 11e18
Fig. 7. Total lipid content (% dry weight) of Nannochloropsis sp. and Tetraselmis sp.
cultured in three different salinities (20, 30 and 40 ppt) under natural and control
condition (values are mean ± standard error).
content in 30 ppt under control condition followed by 40 ppt and
20 ppt. The difference in the pH value caused changes in the total
lipid content of both Nannochloropsis sp. and Tetraselmis sp. (Fig. 8).
Both culture under natural and control condition showed a signif-
icantly high (p<0.05) lipid content when cultured at pH 7.5 which is
also the optimal pH for higher growth.
3.4. Carbohydrate content of Nannochloropsis sp. and Tetraselmis
sp. cultivated in natural and control condition
The carbohydrate content of Nannochloropsis sp. and Tetraselmis
sp. in response to 20, 30 and 40 ppt salinities under natural and
control condition was shown in Fig. 9. Results showed that there
was no significant difference (p > 0.05) between the different sa-
linities for Nannochloropsis sp. when cultured in control condition.
On the other hand, Tetraselmis sp. cultured in both natural and
control condition at 30 ppt had significantly higher (p < 0.05)
carbohydrate when cultured in 30 ppt compared to 40 ppt. But
there was no significant difference between 30 and 20 ppt. At 20, 30
and 40 ppt, Nannochloropsis sp. accumulated significantly higher
carbohydrate content as compared to Tetraselmis sp.
At another perspective, accumulation of carbohydrate in natural
and control condition was apparent. Tetraselmis sp. and
Fig. 8. Total lipid content (% dry weight) of Nannochloropsis sp. and Tetraselmis sp.
cultured in four different pH (5.5, 7.5, 8.5, 9.5) under natural and control condition
(values are mean ± standard error).
15
Fig. 9. Total carbohydrate content (% dry weight) for Nannochloropsis sp. and Tetra-
selmis sp. cultured in three different salinities (20, 30 and 40 ppt) under natural and
control condition (values are mean ± standard error).
Nannochloropsis sp. cultured at 20, 30 and 40 ppt under control
condition yields significantly higher total carbohydrate content
compared to natural condition. Nannochloropsis sp. culture at 20, 30
and 40 ppt under control condition (31%, 34.5% and 33.5%,
respectively) also yielded significantly higher total carbohydrate
content as compared to the natural condition (16.5%, 15%, 6.9%,
respectively).
Nannochloropsis sp. and Tetraselmis sp. cultured at different pH
also showed significant changes on the total carbohydrate content
of both microalgae (Fig. 10). Increase or decrease in the normal
seawater pH of 7.5e8.5 towards acidic and alkaline pH leads to the
decrease in the carbohydrate content. Nannochloropsis sp. showed a
significantly higher carbohydrate content when cultured at pH 8.5
whereas Tetraselmis sp. showed the highest value at pH 7.5 under
control condition. For significantly higher yield of carbohydrate,
natural condition at the salinity of 30 ppt and pH 7.5 to 8.5 favors all
other treatment conditions for both Nannochloropsis sp. and Tet-
raselmis sp.
Fig. 10. Total carbohydrate content (% dry weight) of Nannochloropsis sp. and Tetra-
selmis sp. cultured in four different pH (5.5, 7.5, 8.5, 9.5) under natural and control
condition (values are mean ± standard error).
16 H. Khatoon et al. / International Biodeterioration & Biodegradation 95 (2014) 11e18
4. Discussion
4.1. Growth of Nannochloropsis sp. and Tetraselmis sp.
Parmar et al. (2011) stated that light intensity and photoperiod
(light and dark) cycles were one of the important factors that
determined the growth rate of the microalgae. At 20, 30 and 40 ppt,
Nannochloropsis sp. and Tetraselmis sp. cultured under control
condition had significantly higher (p < 0.05) cell density as
compared to the microalgae cultured in the natural condition.
These data were highly correlated with the optical density reading
throughout the experimental period. At high salinity, the growth of
microalgae was suspended due to the accumulation of compatible
solutes that act as osmoprotectant to stabilize metabolism enzymes
(Fatma et al., 2007). Research by Bartley et al. (2013) demonstrated
that higher salinity resulted in lower cell abundance which was
consistent with the present findings of decreased Nannochloropsis
sp. and Tetraselmis sp. cultivated at 40 ppt. Culture under natural
condition was exposed to high light intensities (1260e39 878 lux)
and 12:12 h lightedark period compared to control condition
(1588 lux). According to Cheirsilp and Torpee (2012), the growth of
Nannochloropsis sp. continuously increased to the maximum level
with the increase in light intensity up to 10 000 lux. Finding from
this study was in accordance with Chen et al. (2011) which reported
that the growth of microalgae had been inhibited as the light in-
tensity increased beyond the saturation light intensity.
For the pH stress, the output of study was supported by Khalil
et al. (2010) where it was reported that the highest yield of dry
biomass of D. bardawil was obtained at pH 7.5 (control culture) and
a sharp decline in the biomass was observed at pH 4 and 10.
Another study by Liu et al. (2007) also showed that the growth of
Chattonella marina remained unchanged in the normal range of pH
in seawater (pH 7.5e8.5), while a significant reduction in growth
was observed as the pH increased beyond pH 9. According to
Hodaifa et al. (2009), the concentration of free carbon dioxide in the
medium was highly available at pH 5e6. Yet, as the pH increase
above pH 6, this concentration begins to transform into bicarbon-
ate, reducing the free CO2 available and therefore, contributes to the
decreasing in microalgae growth.
From this study, Nannochloropsis sp. culture under control
condition at the salinity of 30 ppt and pH 8.5 showed the maximum
growth performance in terms of cell density and optical density as
compared to other salinities, pH and exposure to natural condition.
Thus, it was advisable that cultivation of Nannochloropsis sp. and
Tetraselmis sp. for mass culture purposes such as for aquaculture
industries should be maintained under control condition.
4.2. Protein content of Nannochloropsis sp. and Tetraselmis sp.
cultivated in natural and control condition
Any fluctuation in the salinity would result in production of
microalgae biomass with low total protein content. This result was
in accordance with Gu et al. (2012) who reported that the final
protein content of Nannochloropsis sp. decreased with the increase
in salinity. According to Allakhverdiev et al. (2005), high NaCl
concentration may have caused the inactivation of ATP-synthase
which results in inhibited protein synthesis. Thus, it was advis-
able for the Nannochloropsis sp. and Tetraselmis sp. cultivation to be
carried out under control condition at the salinity of 30 ppt to yield
high protein content.
Surprisingly, the effects of different levels of salinities in the
natural condition on the total protein content of Tetraselmis sp.
were not significant. On the other hand Nannochloropsis sp. had
significantly higher protein content (p < 0.05) at 30 ppt compared
to 20 and 40 ppt. This crucial finding could lead to a low cost
Nannochloropsis sp. and Tetraselmis sp. protein targeted culture
because no artificial continuous lighting and salinity adjustment
were required. Slight fluctuation in salinities (20e40 ppt) that
usually occur under natural condition as a result of evaporation was
not significant. In fact, Nannochloropsis sp. showed a significantly
higher protein content when cultured under natural condition as
compared to control condition. The protein contents of Nanno-
chloropsis sp. (51e60.7%) in natural condition were much higher
than the reported value which was approximately 40% in the same
species (Chiu et al., 2009). This significantly high protein content of
natural condition could be caused by the higher outdoor temper-
ature, which range from 27 to 39  C and high light intensity
(1260e39 878 lux) as compared to control condition. The high
protein content of microalgae culture in natural condition could
also due to the lightedark period of the natural condition enables
the production of the protein synthesis during dark period
(Allakhverdiev et al., 2005).
Effects of different pH on the microalgae protein and carbohy-
drate contents in this study were found to be in accordance with
Khalil et al. (2010). It was reported that the protein and carbohy-
drate of D. bardawil attained their maximum values at pH 7.5.
Shifting the pH value towards the acidic or alkaline side signifi-
cantly decreased the content of protein when cultured under con-
trol condition. This study showed that the highest protein content
was evident in Nannochloropsis sp. cultured at 30 ppt under natural
condition as compared to Tetraselmis sp. and all other treatments.
The optimal pH value for high protein content production was pH
7.5e8.5 under control condition.
4.3. Lipid content of Nannochloropsis sp. and Tetraselmis sp.
cultivated in natural and control condition
Based on the results, factors affecting lipid content of these
species could be elaborated through several perspectives. Accord-
ing to Mata et al. (2010), Nannochloropsis sp. and Tetraselmis sp.
were among the most common microalgae known that have high
concentration of lipid (20e50% of dry mass). Each algal species have
an optimal salinity level for growth and lipid production and
depending on the physiological state this level might be different
for different species (Chaffin et al., 2012; Fuentes-Grünewald et al.,
2012). Salinity stress affects various physiological and bio-chemical
mechanisms related with the growth and development of micro-
algae. It can lead to the increase in lipid content of microalgae due
to its important role in causing changes in the fatty acid meta-
bolism (Kalita et al., 2011).
For the lipid content, this result was supported by Hu (2004)
who reported that an increase in salinity leads to a slight increase
in total lipid content of algae when salinity increased from 10% to
35% in response to osmotic pressure. When cells were exposed to
salinity, specific processes such as, regulation of the uptake and
export of ions through the cell membrane, restoration of turgor
pressure, and accumulation of osmo-protecting solutes and stress
proteins was activated leading to a stable growth state
(Allakhverdiev et al., 2000; Talebi et al., 2013). These mechanisms
lead to an increase in the total lipid content which act as a reserve
energy material until favorable conditions arise (Asulabh et al.,
2012; Talebi et al., 2013). Thus, it can be suggested that under
control condition, Tetraselmis sp. and Nannochloropsis sp. should
maintained at 30 ppt due to high lipid content produced in the
treatment.
Under natural condition, there were no significant differences
(p > 0.05) among the different salinities for both species. Thus, it
could be suggested that changes of salinity in the range of
20e40 ppt in natural condition on the accumulation of lipid were
negligible. However, many studies reported that the lipid
 H. Khatoon et al. / International Biodeterioration & Biodegradation 95 (2014) 11e18
accumulation in algae occurs during environmental stress (Asulabh
et al., 2012). The output of this study were in contrary since total
lipid under the natural condition of microalgae exposed to envi-
ronmental stress was lower than those of the control condition.
According to the results, the difference in temperature and light
intensity of both conditions had great effects on the cell density and
lipid content of microalgae. In control condition, the microalga was
exposed to continuous low light intensities and temperature as
compared to the natural condition. According to Khotimchenko and
Yakovleva (2005), light normally stimulates fatty acid synthesis,
growth and formation of chloroplast membrane. In the natural
condition, the cells accumulated lipid and grew well during the day
but there was a sharp decrease in the biomass concentration and
lipid content during the night which was up to 30% (Han et al.,
2012). The physiological changes due to the variation of natural
illumination may be partly responsible for the lower cell density
and lipid productivity of the microalgae as compared to those
cultured under control condition with continuous illumination.
In the present study, the difference in the pH value also caused
changes in the total lipid content of both Nannochloropsis sp. and
Tetraselmis sp. The similar result also has been observed by Khalil
et al. (2010) which indicated that culturing microalgae at an
optimal pH can increase the biomass concentration and lipid con-
tent. In the combination of all treatments, it was found that Nan-
nochloropsis sp. was the best species to have the highest lipid
content when cultured under control condition at 30 ppt and pH
7.5.
4.4. Carbohydrate content of Nannochloropsis sp. and Tetraselmis
sp. cultivated in natural and control condition
For the carbohydrate content, increased salinity has been re-
ported to enhance intracellular starch content in freshwater
microalgae such as Chlamydomonas reinhardtii (Siaut et al., 2011).
However, starch content was shown to decrease with the increase
of salinity in the marine microalga Dunaliella sp. (Chen and Jiang,
2009). According to Yao et al. (2013), the inconsistent role of
salinity on starch metabolism indicates its species-specific and
cultivation condition-dependent nature. In this study, both marine
Nannochloropsis sp. and Tetraselmis sp. cultivated at salinity of
30 ppt showed significantly higher carbohydrate content than
culture at 20 and 40 ppt under natural condition. The present
findings were in contrast with Khalil et al. (2010) which showed
that the carbohydrate of Chlorella ellipsoidea attained its highest
value at pH 9 and then significantly decreased at pH 10 and the
lowest value of total carbohydrate was obtained at pH 4.
Control condition yield higher total carbohydrate content as
compared to natural condition and study by Radakovits et al. (2010)
showed that the carbohydrate productivity of the microalgae was
higher than lipids in the condition without intensive stress. This
could explain the lower lipid content of control condition as
compared to carbohydrate content found in this study.
5. Conclusions
Nannochloropsis sp. and Tetraselmis sp. cultivated at various sa-
linities and pH and culture condition exhibited remarkable changes
in their growth and biochemical composition. High growth rate,
lipid, carbohydrate and protein content were shown by both spe-
cies when cultured in the salinity of 30 ppt. However, Tetraselmis sp.
exhibited the highest protein content under control condition as
compared to Nannochloropsis sp. In the production of Nanno-
chloropsis sp. and Tetraselmis sp. for specific target for instance, high
lipid content for biofuel production, the optimum salinity, pH and
culture condition resulted from this study can be used.
17
Acknowledgements
This study was supported by the Ministry of Higher Education,
Malaysia, through Fundamental Research Grant Scheme (FRGS)
project no. FRGS/1/2013/STWNO3/UMT/03/7 and Geran Galakan
Penyelidikan (GGP), Universiti Malaysia Terengganu, project no.
68007/2013/43.
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