Biomass and lipid production from indigenous Nannochloropsis sp. by employing stress factors for improved biodiesel production

Abstract

The marine microalgae Nannochloropsis sp. was grown in a different stress factor to produce maximum biomass and lipid production. The experimental stress factors were light, salinity and pH. The result showed that the maximum growth rate of Nannochloropsis sp. was observed best in photoautotrophic, the salinity of 30 ppt and pH of 8 at 25 ºC ± 1. Under the optimized conditions, biomass and lipid productivity was at 1.37 ± 0.08 g L−1 d−1 and 9.45 ± 0.96 g L−1 d−1, respectively. The fatty acids in the microalgae lipid were found to be as follows (%, w/w of total lipids) ΣMUFA74.08%, ΣPUFA 8.86% and ΣSFA 16.86%. The present study suggested Nannochloropsis sp. promising indigenous marine algae for twin uses in aquaculture as well as in biodiesel production. The dominance of unsaturated fatty acids makes the lipid from Nannochloropsis sp. one of the novel sources for biodiesel production. Unsaturated fatty acids in a higher ratio will obviously make the green fuel readily meet the critical specifications for biodiesel, especially in conforming to the cold properties of the fuel.

Full text

Vol.:(0123456789)
Environment, Development and Sustainability
https://doi.org/10.1007/s10668-021-01910-2
1 3
Biomass andlipid production fromindigenous
Nannochloropsis sp. byemploying stress factors forimproved
biodiesel production
PrimillaParamasivam1· KarthianiKanagesan1· PrakashBhuyar1,2·
NatanamurugarajGovindan1,3· MohdHasbiAb.Rahim1,3·
GaantyPragasManiam1,2,3,4
Received: 27 August 2021 / Accepted: 15 October 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
The marine microalgae Nannochloropsis sp. was grown in a different stress factor to pro-
duce maximum biomass and lipid production. The experimental stress factors were light,
salinity and pH. The result showed that the maximum growth rate of Nannochloropsis sp.
was observed best in photoautotrophic, the salinity of 30 ppt and pH of 8 at 25 ºC ± 1.
Under the optimized conditions, biomass and lipid productivity was at 1.37 ± 0.08g L−1
d−1 and 9.45 ± 0.96g L−1 d−1, respectively. The fatty acids in the microalgae lipid were
found to be as follows (%, w/w of total lipids) ΣMUFA74.08%, ΣPUFA 8.86% and ΣSFA
16.86%. The present study suggested Nannochloropsis sp. promising indigenous marine
algae for twin uses in aquaculture as well as in biodiesel production. The dominance of
unsaturated fatty acids makes the lipid from Nannochloropsis sp. one of the novel sources
for biodiesel production. Unsaturated fatty acids in a higher ratio will obviously make the
green fuel readily meet the critical specifications for biodiesel, especially in conforming to
the cold properties of the fuel.
Keywords Nannochloropsis sp.· Biodiesel· Unsaturated fatty acid· Marine algae
* Gaanty Pragas Maniam
gaanty@ump.edu.my
1 Faculty ofIndustrial Sciences andTechnology, Universiti Malaysia Pahang, Lebuhraya Tun Razak,
26300Gambang,Kuantan, Pahang, Malaysia
2 School ofRenewable Energy, Maejo University, 50290, ChiangMai, Thailand
3 Earth Resources & Sustainability Centre, Universiti Malaysia Pahang, Lebuhraya Tun Razak,
26300Gambang,Kuantan, Pahang, Malaysia
4 Centre forResearch inAdvanced Tropical Bioscience, Universiti Malaysia Pahang, Lebuhraya Tun
Razak, 26300Gambang,Kuantan, Pahang, Malaysia

P.Paramasivam et al.
1 3
1 Introduction
The typical application of fossil petroleum has expressed a question mark in continuous
supply to human demand. Even after the discoveries of several substitute energies, specifi-
cally renewable energy from biofuels and solar, fossil fuels remain a significant contributor
to the global energy demand (Bhuyar etal., 2020; Saengsawang etal., 2020; Yustinadiar
etal., 2020). Based on data extracted from British Petroleum, in 2018, the total primary
energy consumption indicated in the 68th edition of World Energy Statistics 2019 was
13,864.9 million tons of oil equivalent (power, natural gas, coal, nuclear, hydroelectric,
renewable energy). The overall consumption of oil was 4662.1 million tons, while renew-
able energy was still low at 561.3 million tons (Mendes, 2020). Hence, the use of renew-
able energies must be increased to counter the fossil fuel confines (Lawrence etal., 2011;
Ramaraj etal., 2021; Whangchai etal., 2021).
Biodiesel is a green fuel that can replace fossil petroleum fuel. Biodiesel comprises a
variety of fatty acid esters (Nurfitri etal., 2013; Ma’arof etal., 2021). Vegetable oil (canola,
palm, castor, soybean, corn oil), animal fat and spent cooking oil are widely recognized
sources of commercial biodiesel (Deepanraj etal., 2017; Kapor etal., 2017). Biodiesel raw
materials produced from these plants are contained in low quantities and present limita-
tions in their production, affecting the food crop budget (Yustinadiar etal., 2020). Hence,
there is the need for new biodiesel raw materials to combine higher production with more
sustainable land use. Microalgae have been considered promising renewable feedstock to
supplant fossil fuels since the 1970s (Jayakumar etal., 2021).
An earlier study using microalgae as an alternative source as biodiesel feedstock showed
high oil yields, less agricultural land needed and reduced algae cultivation costs (Contre-
ras-Pool etal., 2016). In addition to the raw materials of bio-based products (biodiesel,
animal feed, dietary supplements), marine microalgae are a valuable source of biomass
and address environmental issues like global warming since they consume CO2 (Yustina-
diar etal., 2020). Microalgae can be grown in various conditions (fresh, brackish or salty
water), which are incompatible with traditional farming (Bhuyar etal., 2021c; Khammee
etal., 2021). In addition, these can be cultivated on farms or in bioreactors. Because of this
non-selective processing, microalgae produce a superior yield per hectare with improved
environmental performance (Bhuyar etal., 2021a; Trejo etal., 2021). In addition, microal-
gae biomass production influences significant carbon dioxide fixation, which inhibits waste
greenhouse gas emissions (1kg of dry algal biomass requires approximately 1.8kg CO2)
(Chandrakant etal., 2021; Ma etal., 2016).
Among marine microalgae, Nannochloropsis sp. is widely recognized for its capacity
to accumulate a considerable amount of triacylglycerol (TAG) for biodiesel production
(Ma etal., 2018). Nannochloropsis belongs to Phylum Heterokontophyta, Class Eustig-
matophyceae and Eustigmataceae family of unicellular and nonmobile marine microalgae.
Nannochloropsis sp. has become a prime for lipid and biofuel research as these marine
microalgae proliferate in open ponds or photobioreactors and can be grown in seawater
with high lipid yields up to 60% of dry weight (DW) (Embong etal., 2021; Rodolfi etal.,
2009). Nannochloropsis sp. is also enriched with high-value, polyunsaturated fatty acids
(PUFAs) such as omega-3 eicosapentaenoic acid (EPA), which have a small, compact hap-
loid genome (~ 30 Mbp) (Ashour etal., 2019; Khazaai etal., 2021).
Besides, microalgae can be grown under photoautotrophic and heterotrophic growth
modes and can be easily modified under the influence of several abiotic stressors such
as high salinity, pH and cultivation conditions (light and dark) for the desired result end

Biomass andlipid production fromindigenous Nannochloropsis…
1 3
products. Therefore, a slight variance of these factors may cause massive fluctuations in
microalgae’s growth and lipid composition (Abd Malek etal., 2021; Nithin etal., 2020;
Zbakh etal. 2012). Fluctuations in salinity can also affect the marine microalgae’s growth
and lipid accumulation as they are resistant to salinity variations. According to Ashour
et al. (2019), salinity adaptability varies from one microalga to another. The research-
ers had classified them as halophilic, where the microalgae cells require salt for optimum
growth, whereas halotolerant is a saline medium survival response. Despite the evolving
salinity function in starch metabolism representing its species-specific and condition-
dependent existence, limiting salinity is a novel way to alter marine microalgae’s biochemi-
cal composition (Bartley etal., 2013a). Recent studies have reported that high salinity in
Dunaliella sp. inhibits growth and accumulates lipid (Ishika etal., 2019; Sundararaju etal.,
2020).
Another critical consideration is the pH (hydrogen ion concentration) of the culture
medium, which also highly affects the prime growth of microalgae (Bartley etal., 2013b).
According to (Chen & Durbin, 1994), pH significantly affects algal metabolism. The domi-
nance of unsaturated fatty acids makes the lipid from Nannochloropsis sp. one of the novel
sources for biodiesel production. Unsaturated fatty acids in a higher ratio will obviously
make the green fuel readily meet the key specifications for biodiesel, especially in con-
forming to the cold properties of the fuel. This present study explores the optimization
of light conditions (phototrophic and heterotrophic), pH range and salinity on microalgae
(Nannochloropsis sp.) for biodiesel production and the effect of the growth rate, chloro-
phyll a, microalgal biomass and lipid content, respectively.
2 Material andmethods
2.1 Collection ofmicroalgae sample
For this research analysis, the microalgae samples were collected from seawater at Balok
Coast (3° 56′ 59″ N, 103° 22′ 3″ E), Peninsular Malaysia East Coast area, bordering the
South China Sea. Microalgae samples were obtained using a 5-μm underground mesh scale
(~ 0.1m) to collect 5000-mL water samples.
2.2 Isolation andidentification ofspecies
The collected water samples were filtered through Whatman qualitative filter papers with
a specific pore size of 0.45µm that hold specific filter membranes to retain the organisms
of interest while permeating others. After that, the filtered membranes were washed with
500mL sterile freshwater several times. Govindan etal. (2019) defined that the microalgae
samples were cleaned and isolated, respectively. Concisely, a sterile inoculation loop was
dipped from the filter medium in the resuspended cell sample and spread over Conway
agar plate and further incubated under 20μmol photons m−2 s−1 for 12h at 25ºC ± 2. The
grown cells were taken from each colony and examined for morphological characteristics
and other features under a fluorescence microscope Olympus BX 53 (UK). The samples
were then imaged by field emission scanning electron microscope (FESEM) using a Joel
(Japan) JSM-7008 FESEM. Following the taxonomy clarifications of Hibberd (1981), the
isolated microalgae were further described. An entire 22 different species of microalgae

P.Paramasivam et al.
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were isolated and recognized. Among these, Nannochloropsis sp. was the potential green-
algae, was selected for further analysis as their capacity to grow fast to a higher concentra-
tion of cells and comparatively capable of accumulating high lipid levels in the biomass.
2.3 Conway medium preparation
Conway medium preparation includes the preparation of macronutrients, trace metal and
vitamin solutions for stock solutions. The chemical compositions were dissolved in sterile
distilled water to prepare stock solutions. Table 1 shows the composition of the Conway
medium. 1mL of solution A (macronutrient), 0.5mL of solution B (trace element), 0.1mL
of solution C (vitamins) were transferred to 1000mL of filtered and sterilized seawater
(30ppt).
2.4 Nannochloropsis sp. Inoculum
The pure culture of Nannochloropsis was well preserved on the plates of Conway agar Petri
dish. 250-mL Erlenmeyer flask was used for the preparation of the inocula. An aseptic sus-
pending a loop-full of cells from the agar was immersed in a 200-mL Conway medium.
The culture flask was continuously aerated with 5% (v/v) CO2-mixed sterilized air. At
Table 1 Composition of modified Conway medium (Oo etal., 2017)
Stock No Substances Volume
I Solution A (per liter distilled water)
MnCl2.4H2O 0.36g
H3BO333.6g
EDTA 45g
NaH2PO4.2H2O 20g
NaNO310g
FeCl3.6H2O 1.3g
Solution B 1mL
II Solution B (per 100mL distilled water)
ZnCl22.1g
CoCl2.6H2O 2g
(NH4)6Mo7O24.4H2O 0.9g
CuSO4.5H2O 2g
Concentrated HCL 10ml
III Solution C (per 200mL distilled water)
Vitamin B1 0.2g
Vitamin B12 10mg
Medium
Solution A 1mL/L
Solution B 0.1mL/L
Solution C 100µL
Sterile Seawater (30ppt) 1L

Biomass andlipid production fromindigenous Nannochloropsis…
1 3
standard temperature and pressure, the flow rate of the aeration gas was 75mL min−1. The
cultivation temperature was maintained at 25 ± 1°C. The illumination was maintained at
35μmol m−2 s−1 and in the flask. The cultures conditions were monitored for 14days.
2.5 Optimization ofNannochloropsis sp. understress factor
Microalgae growth is highly dependent on the conditions of the environment, and vari-
ables in the condition of culture are different from one species to another. Light, salinity
and pH are being the most studied variables. Under the right stress factors, it is expected
that the species can secrete the highest lipid, which maximizes the benefits of choosing this
particular species of Nannochloropsis sp. for the production of biodiesel. In this study, the
most vital factors were chosen, such as light, salinity and pH, to observe the influence of
these factors in extracting the amount of lipid from the species (Bhuyar etal., 2021b).
2.6 Effect oflight
In this present study, the influence of light has been studied in two different conditions:
photoautotrophic (light) and heterotrophic (dark), respectively. Glucose is used as a car-
bon source in heterotrophic conditions. Three different concentrations of glucose (2, 4 and
6g/L) were added into Conway medium, respectively. Meanwhile, cultures in phototrophic
maintained under fluorescent light at 35µmol photons m−2 s−1. The cultures were main-
tained at 25 ± 1ºC and aerated continuously.
2.7 Effect ofsalinity
In this study, the effect of salinity has been studied in different salinities (20, 30 and 40ppt)
to maximize biomass and lipid production. Each culture condition was studied under stand-
ard laboratory temperature of 25ºC ± 1 ºC with continuous illumination of 35µmol pho-
tons m−2 s−1.
2.8 Effect ofpH
The pH is a critical factor since it determines CO2’s solubility and availability along with
other nutrients. Therefore, the impact of pH for growth and lipid production was studied
using pH ranging from 5 to 8, respectively. The temperature was constant at 25ºC ± 1ºC
with the continuous illumination of 35µmol photons m−2 s−1.
2.9 Determination ofdry biomass concentration andgrowth rate
In the experiment, the final concentration of biomass was determined gravimetrically. The
dry cell weight of the microalgae biomass was determined using the method of (Chiu etal.,
2009), where the algal cells were collected during the late log phase and centrifuged for
5 min at 6500 g. Pellets were extracted by centrifugation and washed twice again with
distilled water. The samples were dried to freeze for 24h. The weighed pellet mass and
the initial culture sample volume were used to determine the dry biomass concentration in
the original sample (Govindan etal., 2019). The biomass concentration was assessed by

P.Paramasivam et al.
1 3
calculating the optical density at 680nm to plot the growth curves (Marudhupandi etal.,
2016) Genesys 10S UV–Vis spectrophotometer. Specific growth rate (µ d−1) was calcu-
lated as follows:
where µ is the specific growth rate, t2 and t1 (day), X2 and X1 represent cell number param-
eters at a time, respectively.
2.10 Lipid extraction fromNannochloropsis sp.
The procedure used by Bligh and Dyer (1959) was to obtain the lipids from freeze-dried
biomass. Concisely, chloroform (1mL), methanol (2mL) and deionized water (0.8mL)
were added to 3g dried freeze biomass. After 2min of the vortex step, the mixture was
placed at room temperature for 4h. Then, 1 mL of chloroform was added and 30s vor-
tex mixture again. Finally, 1 mL of deionized water was added, and 30s of the vortex
was applied to the mix. The resulting suspension was centrifuged for 10min at 4150g to
form three layers. The third layer (chloroform) was extracted from the surface. The solvent
obtained was evaporated in a fume hood at 60°C. The lipid extract was measured gravi-
metrically, expressing total lipids by dry cell weight (DCW) in grams per liter (g L−1).
2.11 Lipid composition analysis
The freeze-dried biomass containing triglycerides was converted using insitu transesteri-
fication to fatty acid methyl esters (FAME), where methanol was used as the solvent. Fur-
ther, the FAME was extracted to evaluate the lipid fatty acid profile. The dry biomass was
mixed with 5mL of methanol (1:2 v/v; methanol was by combining 1.8mL of concen-
trated potassium hydroxide(catalyst) with 100mL of methanol) and held at 50°C for over-
night. GCMS, gas chromatography-mass spectroscopy (Agilent 7890A), dissolved the oil
residue in 2ml of hexane. Helium gas was used as carrier gas while injection of the sample
was 1μL (Bouyam etal., 2017). The column was Mega-Wax MS (length 30m× internal
diameter 0.32mm × film thickness 0.50µm), using methyl heptadecanoate as an internal
standard.
3 Results anddiscussion
3.1 Isolation andidentification ofmicroalgae
The morphology of single-cell green algae similar to plant cells forms the basis for their
identification (Ma et al., 2016). The Nannochloropsis sp. fluorescence micrograph and
FESEM image are shown in Fig.1. Concerning identified morphological topographies
(Hibberd, 1981; Santos & Leedale, 1995) and high-resolution images (Fig.1), the isolate
𝜇
=
InX
2−
InX1
t
2
−t
1

Biomass andlipid production fromindigenous Nannochloropsis…
1 3
was spherical to oval cells with a smooth cell wall with 2–5µm diameter. The green algae
were able to grow in Conway medium formulated with seawater.
3.2 Optimization ofNannochloropsis sp. understress factors
3.2.1 Effect oflight condition (photoautotrophic andheterotrophic)
The photoautotrophic and heterotrophic growth curve of Nannochloropsis sp. is shown
in Fig.2. The exponential growth was observed from day 2 up to day 6 of culture time.
The maximum biomass of cultivated Nannochloropsis sp. was obtained under photoauto-
trophic condition (1.39 ± 0.23g/L) and lipid accumulation (44.6 ± 0.75g/L) at the same
time 4g/L of glucose. In heterotrophic conditions, Nannochloropsis sp. obtained biomass
(1.12 ± 0.14g/L) and lipid accumulation (21.4 ± 0.39g/L) significantly lower than the het-
erotrophic condition. The growth period of heterotrophic cultivation of Nannochloropsis
sp. was slow while comparing to the photoautotrophic condition. Ma etal. (2016) said
photoautotrophic is one of the most critical factors determining the microalgae’s growth
rate. Nannochloropsis sp. cultivated under photoautotrophic conditions had significantly
higher biomass and lipid accumulation than microalgae grown under heterotrophic condi-
tions. Throughout the experiment, those results were strongly correlated with the reading
Fig. 1 Fluorescence micrograph of Nannochloropsis sp. under the magnification of 10× a and FESEM
under 3000× b magnification
Fig. 2 Growth rate of Nanno-
chloropsis sp. under photoau-
totrophic and heterotrophic at dif-
ferent growth phases (lag phase,
log phase, stationary phase and
decline phase)
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Photoautotrophic Heterotrophic

P.Paramasivam et al.
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of optical density. In 1998, Vazhapilly and Chen tried to grow N. oculata with glucose
or acetate, and the result was that this particular strain could not use these two organic
carbon sources for heterotrophic conditions. There are only a few records of heterotrophic
development in Nannochloropsis cultivation (Chini-Zittelli etal., 1999). Nannochloropsis
sp. biomass was attained at 326mg/L in heterotrophic cultures where glucose was used
as a source of carbon, while in phototrophic conditions, the total biomass (392mg/L) was
significantly higher than the heterotrophic level. Heterotrophic condition’s lipid yield was
significantly lower compared to photoautotrophic condition (Cheirsilp & Torpee, 2012;
Vazhappilly & Chen, 1998).
3.2.2 Effect ofsalinity (ppt)
The growth rate of Nannochloropsis sp. cultured under three different salinities showed
significant cell growth (Fig.3). The exponential phase was observed from day 2 to day
6 of culture time. The biomass and lipid were measured in two different phases: the log
Fig. 3 Growth rate of Nanno-
chloropsis sp. in three different
salinities (20, 30 and 40ppt)
0
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20ppt 30ppt 40ppt
Table 2 Effect of stress factors on lipid production from Nannochloropsis sp
Values are shown as mean ± standard deviation (n = 4)
Log phase Stationary phase
Factors Dry wt. (g/L) Lipid (g/L) Lipid (%) Dry wt. (g/L) Lipid (g/L) Lipid (%)
Light
Photoautotrophic 1.35 ± 0.36 0.58 ± 0.06 41.8 ± 0.82 1.39 ± 0.23 0.62 ± 0.05 44.6 ± 0.75
Heterotrophic 0.99 ± 0.28 0.21 ± 0.08 21.1 ± 0.47 1.12 ± 0.14 0.24 ± 0.02 21.4 ± 0.39
Salinity (ppt)
20 0.84 ± 0.04 0.23 ± 0.02 27.3 ± 0.67 1.17 ± 0.03 0.36 ± 0.01 30.7 ± 0.27
30 1.19 ± 0.03 0.45 ± 0.01 37.8 ± 1.48 1.24 ± 0.04 0.52 ± 0.07 41.9 ± 0.78
40 0.53 ± 0.03 0.09 ± 0.03 12.9 ± 0.31 0.45 ± 0.05 0.06 ± 0.01 13.3 ± 0.22
pH
5 0.92 ± 0.28 0.21 ± 0.04 22.8 ± 1.69 0.98 ± 0.17 0.26 ± 0.06 26.5 ± 1.54
6 1.14 ± 0.08 0.28 ± 0.07 24.6 ± 1.08 1.25 ± 0.28 0.33 ± 0.09 26.4 ± 1.08
7 1.17 ± 0.05 0.36 ± 0.03 30.2 ± 2.46 1.16 ± 0.03 0.28 ± 0.07 30.7 ± 1.48
8 1.19 ± 0.07 0.42 ± 0.05 35.2 ± 2.57 1.26 ± 0.00 0.53 ± 0.02 42.0 ± 1.87

Biomass andlipid production fromindigenous Nannochloropsis…
1 3
phase and the stationary phase. Based on Table2, the maximum biomass production at
the dry weight (1.24 ± 0.04g/L) and lipid production (41.9 ± 0.78g/L) were significantly
higher at 30ppt. At 20ppt, the stationary phase ended on day 10 with biomass production
of 1.17 ± 0.03g/L and 30.7 ± 0.27g/L of lipid production. However, at 40ppt, the growth
rate started dropping after day 8, and the maximum biomass and lipid production was
0.45 ± 0.05g/L and 13.3 ± 0.22g/L.
This present study observed that optimal salinity ranges for Nannochloropsis sp. growth
(20-30ppt) were constant with preceding research (22–34 PSU) and (20–40 PSU) (Bartley
etal., 2013a, 2013b; Renaud etal., 1994). Further research has shown that higher salinity
(40 PSU) has led to lower cell abundance, in line with our findings of decreased Nan-
nochloropsis sp. at 40 PSU (Pal etal., 2011). Bartley etal. (2013a, 2013b) indicated that
microalgae cell growth tends to drop at higher salinity due to the accumulation of com-
patible solutes, which function as an osmoprotective to stabilize metabolism enzymes.
Meanwhile, Hu (2004) had confirmed that a slight increase in salinity resulted in total lipid
accumulation of algae, as salinity in response to osmotic pressure rises from 10 to 35%.
According to Abu-Rezq etal. (1999), N. salina was found algae cells to develop rapidly
with salinity between 20 and 40ppt after treatment. The obtained data of this present study
indicated that at 30ppt, the lipid percentage of dry cell weight was reported as 41.9%. This
result follows Ashour etal. (2019), where they studied N. oculata for biodiesel production,
and the outcome of lipid percentage of dry cell weight was reported as 37.7% at 35ppt of
salinity. Optimal salinity leads to increased lipid content as altering the fatty acid metabo-
lism has been its prime role (Abu-Rezq etal., 2010). Bartley etal. (2013a, 2013b) had
specified that stressing the algae cell by providing salinity one time may cause significant
accumulation in lipid than stress by nutrient limitation by itself. Hence, it can be suggested
that under 30ppt, Nannochloropsis sp. grow better and produce maximum biomass and
lipid.
3.2.3 Effect ofpH
Based on Fig.4, the maximum growth was observed at pH 8. The exponential phase started
after day 2 up to day 6 consecutively. Accumulation of lipids tended to be uninfluenced by
pH. However, the most significant mean accumulation occurred in the pH 8 treatment aver-
age of (42.0 ± 1.87g/L), whereas at pHs 5 and 6 growth rate was significantly lower than
pHs 7 and 8. Earlier study was justified by Bartley etal. (2013a, 2013b) where the pH
range for N. salina growth was 8–9. They proved that N. Salina could not grow at pH 10
Fig. 4 Growth curve of Nan-
nochloropsis sp. cultivated under
four pH 5, 6, 7 and 8
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pH 5 pH 6 pH 7 pH 8

P.Paramasivam et al.
1 3
while well growing at pH 7. Other studies suggesting pH manipulation by CO2 supply typi-
cally finds lower pH optimal (7–7.8) in the case of Nannochloropsis sp.
Meanwhile, in the present study, Nannochloropsis sp grew well in pH 8 compared to
another pH. In the most recent studies, the researcher demonstrated that the highest growth
rate of N. gaditana was 218 × l05 cells/ml measured at pH 8 on the 20th day, with a maxi-
mum oil yield of 34.6% at the same pH (log phase) (MarKose etal., 2020). Nannochlo-
ropsis sp. showed the highest growth in the present study, and oil yield was recorded in
alkaline pH. The justification for this may be that the alkaline pH inhibits cell release and
thus causes lipid accumulation. In earlier research of Guckert and Cooksey (1990), a study
on chlorella CHLOR-1 claimed that autospore forms the alkaline pH, thus decreasing cell
release resulting in lipid accumulation. Meanwhile, in earlier studies, Rodolfi etal. (2009)
found that the overall lipid content of N. gaditana ranged from 24.4 to 35.7% at pH 8.
Hence, pH 8 appeared to be the most optimum for Nannochloropsis sp. to maximize lipid
production.
3.3 Growth rate, biomass production andbiochemical composition ofoptimized
condition
Like other microorganisms, microalgae develop in four growth phases: lag, exponen-
tial(development),stationary,anddeathorlysis,asshown in Fig.5. They transform pho-
tonic energy, water and CO2 into sugars and convert sugars to macromolecules like lipids
or/and triacylglycerols (TAG) (Moazami etal., 2012). Figure2 shows the growth curve of
Nannochloropsis sp., which was cultivated in Conway medium for 14days. The exponen-
tial phase began after 1day of incubation and ended on the 14th day with a maximum dry
weight of 0.710 gL−1 showing evident impact in the stationary (9th day) phase. Thus, the
9th day is considered as the end of the exponential phase. At this point, microalgae cells
showed 64.3 dw% of lipid. After the 9th day, the declining phase was started.
Fig. 5 Nannochloropsis sp.
growth curve displaying dry
weight at various stages of
growth: lag phase, exponential
phase, stationary phase and death
phase
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1234567891011121314
Dry weight g L -1
)086DO(etarhtworG
Cultivation days
Dry weight (g L-1) Growth rate (OD 680)
Table 3 Productivity values
and lipid content of the biomass
(n = 4)
Variable Results
Biomass productivity (g L−1 d−1)1.37 ± 0.08
Lipid productivity (g L−1 d−1)9.45 ± 0.96
Lipid content of biomass (g L−1 d−1)0.643 ± 2.08

Biomass andlipid production fromindigenous Nannochloropsis…
1 3
Moreover, under controlled conditions, Nannochloropsis sp. showed biomass and lipid
productivities at 0.15 gL−1d−1 and 9.45 gL−1, d−1, respectively (Table3). At the end of
the experiment, Nannochloropsis sp. showed the specific growth rate and lipid production
were significantly higher at phototrophic cultivation with 30ppt and pH of 8. A similar
result has been observed by MarKose etal. (2020), where they studied N. gaditana under
optimized physical parameters, which are photoautotrophic (2000lx), salinity (30ppt) and
pH 8 to maximum the lipid accumulation. As an outcome of the study, the lipid percentage
of dry cell weight was 40%, significantly lower than our present study.
3.4 Lipid composition ofNannochloropsis sp.
For sustainable biodiesel production, the ideal range of microalgal spe-
ciesrequireshighlipid productivity andappropriatecharacteristicsofthegeneratedFAMEs
(Bajwa etal., 2020). The results showed that Nannochloropsis sp. fatty acid profiles were
altered under these cultured conditions (Table4). Many researchers reported that fatty acid
profile. The predominant fatty acids of Nannochloropsis sp., as shown in Table4, were
oleic acid (C18:1), palmitic acid (C16:0) and linoleic acid (C18:2). The high percentage
of oleic acid in the fatty acid compositions makes it entirely suitable for developing bio-
diesel of good quality (Moazami etal., 2012). In this experiment, nitrate was added to
the Walnes medium to maximize the lipid accumulation in Nannochloropsis sp. Pal etal.
(2011) concluded that the percentage of oleic acid depends on the nitrate concentration
used in the experiment. The produced methyl esters for Nannocholoropsis sp. were tested
on properties of biodiesel using an Atago RX 5000 refractometer whereby the outcome
was 1.44718th, in agreement with the literature of Jung etal. (2018) where the Nannochlo-
ropsis sp. reading of refractive index showed 1.46. Hence, Nannochloropsis sp. is the most
suitable candidate for biodiesel production.
Table 4 Fatty acid profile of
Nannochloropsis sp. (Chini-
Zittelli etal., 1999; Moazami
etal., 2012; Sukenik etal., 1993)
FFA content = 0.5 wt.% (as oleic acid)
Fatty acid Structure Composition (%)
Present work Previous work
(FAME range)
Oleic acid C18:1 72.60 14.10–45.40
Palmitic acid C16:0 13.35 4.63–18.20
Linoleic acid C18:2 8.860 1.19–12.20
Stearic acid C18:0 3.070 1.10–7.10
Palmitoleic acid C16:1 1.200 0.11–17.80
Eicosanoic acid C20:0 0.440 0.63–1.52
Gadoleic acid C20:1 0.280 0.87–1.50
ΣMUFAs 74.08
ΣSFAs 16.86
ΣPUFAs 8.86

P.Paramasivam et al.
1 3
4 Conclusion
Nannochloropsis sp. developed under different light conditions, salinities and pH exhibited
significant changes under their growth and biochemical composition. These stress factors
had the most substantial impact on biomass concentration and lipid content. The oleic acid
comprises a maximum of 72.6% and palmitic acid 13.35%, demonstrating the best candi-
date for biodiesel production. Therefore, Nannochloropsis sp. has the extreme lipid accu-
mulation capacity under relevant factors, and the fatty acid profile produced may be suit-
able for producing biodiesels. The dominance of unsaturated fatty acids makes the source
one of the most suitable to meet the requirement for fuel, particularly in meeting the cold
properties specifications. Nannochloropsis sp. has become a suitable lipid source for bio-
diesel production with a sufficient fraction of unsaturated fatty acids and lipid content.
Acknowledgements The authors gratefully acknowledge Universiti Malaysia Pahang (UMP) for financial
support through Flagship Research Grant (RDU182205).
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