Journal of the Energy Institute 90 (2017) 300e315

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Journal of the Energy Institute

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institute

Algal biomass energy carriers as fuels: An alternative green source

Sundaram Arvindnarayan a, Kandasamy K. Sivagnana Prabhu b, *, Sutha Shobana c,
Jeyaprakash Dharmaraja d, A. Pasupathy e
a
Department of Mechanical Engineering, Rohini College of Engineering & Technology, Anjugramam, Kanyakumari 629 851, Tamil Nadu, India
b
Department of Mechanical Engineering, R.M.K. Engineering College, R.S.M. Nagar, Kavaraipettai 601 206, Tiruvallur District, Tamil Nadu, India
c
Department of Chemistry and Research Centre, Aditanar College of Arts and Science, Virapandianpatnam, Tiruchendur 628 216, Thoothukudi District,
Tamil Nadu, India
d
Department of Chemistry, Faculty of Science and Humanities, Sree Sowdambika College of Engineering, Chettikurichi, Aruppukottai 626 134, Tamil Nadu,
India
e
Department of Mechanical Engineering, Varuvan Vadivelan Institute of Technology, Krishnagiri, Gundalapattay, Dharmapuri 636 703, Tamil Nadu, India

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

Article history: In this study, biomass and lipid productivity of an alga namely Botryococcus braunii at dissimilar nitrogen
Received 19 September 2015 cultural environment was observed. In addition, the transesterification of algal feed stock with absolute
Received in revised form ethanol medium in the presence of Ni catalyst with hydrogen environment and Ni(II)-Schiff base chelate
27 November 2015
as promoter was carried out to yield more algal oil as the catalyst along with promoter possesses high
Accepted 1 December 2015
Available online 18 December 2015
specific surface area, strong base strength and high base site concentration. The synthesized Schiff base
and its Ni(II)-Schiff base chelate were characterized by micro elemental, spectral, thermal and XRD/SEM
analyses. The quality of the extracted algal oil was analyzed using ultimate, analytical and spectral
Keywords:
Botryococcus braunii studies. In engine performance test, the algal oilediesel blends showed a slight increase in specific fuel
Ni(II)-Schiff base chelate consumption with lower brake power. The green gas emissions were reduced for all the tested blends
Algal oil engine performance except (NO)x. Moreover, in vitro antimicrobial and antioxidant activities of the algal oil extracted from the
Microbial activity alga of dissimilar nitrogen cultural environment were studied and the results obtained show remarkable
Antioxidant study activity.
© 2015 Energy Institute. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Global air-pollution is a serious problem and the use of vehicles all over the world especially in big cities and towns contribute gaseous
emissions, hence cause the pollution of environment. These gases referred to as green house gases (GHG) that cause global warming. GHG
such as carbon dioxide (CO2), carbon monoxide (CO), nitrogen oxides (NOx) and sulphur oxides (SOx) cause environmental pollution [26].
Bio-diesel is derived from vegetable oils/animal fats in the presence of suitable non polar solvent and a catalyst will be harmful to the diesel
engines [12]. It may be due to their low oxidation stability & volatility, higher viscosity & density, poor fuel atomization and higher green
house gases emission [29]. As our energy demand is increasing rapidly, aquatic biomass is one of the renewable energy sources to produce
bio-oil which is biodegradable and improving lubricity of diesel and there is a considerable reduction of green house gases while applying
algal oil in a conventional diesel engine [1]. Algal oil is an alternative to diesel fuel which has recently attracted huge attention worldwide for
its good exhaust emission, sustainability and biodegradability [24]. The lipids of algal biomass can be converted into superior biofuels
[16,36]. Moreover, an alga is considered as a future feedstock for biorefinery and having the ability to capture CO2, thus creates an eco
friendly environment [33]. Algal oil is formed during two step transesterification of algal feedstock which shows higher fatty acid methyl
esters yield than single/direct stage process transesterification process [38,18,5] and this reaction along with quality of the yield depend
upon the presence of saturated or monounsaturated fatty acids [21]. The majority unsaturated fatty acids produced by algae are linoleic acid,
oleic acid, linolenic acid and palmitoleic acid [11]. The productivity of the algal feedstock surpasses the yield of the best seed crop oil [40].

* Corresponding author. Tel.: þ91 9788833333; fax: þ91 443330 3334.

E-mail address: kksprmk@gmail.com (K.K. Sivagnana Prabhu).

http://dx.doi.org/10.1016/j.joei.2015.12.002
1743-9671/© 2015 Energy Institute. Published by Elsevier Ltd. All rights reserved.
 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 301

Scheme 1. Synthetic route of Schiff base ligand.

Schiff bases derived from an amine and an aldehyde containing hetero hard and soft donor atoms N, O and/or S with transition metal(II)
ions have received a number of prospective technological applications, because of the high p-electron density on donor atoms in the organic
moiety [13]. An attractive application of Schiff base chelates is that they not only act as promoters in the production of bio-oil from biomass
feed stocks much more rapidly than acidic and basic catalysis but also prevent the bio-oil reactor vessel from the corrosion [22,43]. The
Schiff base-metal promoters, with morphological features smaller than a micron at least in one dimension, have very high ratio of surface
area to volume compared to the other promoters with larger grain size [27]. The transition metals included into the essential biochemical
pathways are Ni(II), Cu(II) and Zn(II) ions. They tremendously enhance the activity of Schiff base due to the presence of unfilled d-orbitals
which have an ability to accept electrons. The significance of Ni(II) catalyst is due to its redox chemistry, high natural profusion and low price
in the hydrotreatment of algal feed stock [45]. The Schiff base chelates are more popular and most preferred in the commercial production of
algal oil due to their low cost and availability. Algal oilediesel blends are widely used as power sources in medium and heavy-duty ap-
plications because of their low emission of carbon monoxide (CO) and hydrocarbons (HC) compared to gasoline engines [44].
In this work, the biomass and lipid productivity at dissimilar nitrogen cultural environment the alga namely Botryococcus braunii was
observed. For the first time, a two step transesterification reaction of algal feed stock from an alga namely B. braunii in the presence of Ni
catalyst with hydrogen environment and Ni(II)-Schiff base chelate as promoter in absolute alcohol medium was carried out. The structure of
Ni(II)-Schiff base chelate has been confirmed by micro elemental, spectral, thermal and XRD/SEM analyses. The quality of the algal oil was
analyzed using ultimate, analytical and spectral studies. Moreover, the engine performance and emission tests for the algal oilediesel
blends, in vitro microbial and antioxidant activities of the algal oil were studied.

2. Experimental

2.1. Materials and methods

All the chemical products and solvents were purchased from E. Merck, Sigma Aldrich and S.D. Fine chemicals. B. braunii were collected
and isolated from Kovalam solar salt works in Kanyakumari of India. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (AA) were
purchased from Sigma Aldrich. Schiff base (2-hydroxybenzalidene-p-toluidine) and Ni(II)-Schiff base chelate were synthesized in our
laboratory. Solvents used in the physical measurements were of analytical grade and were purified according to the literature [32]. Melting
point of Schiff base and its metal chelate was determined on Gallenkamp apparatus in open glass capillaries and was uncorrected. Metal
content of the chelate was estimated gravimetrically by standard procedures [42]. Molar conductance of the chelate was measured in DMSO
solvent using Elico CM 180 Conductivity Bridge using 0.01 M KCl solution as calibrant. The micro elemental and ultimate analyses of C, H, N
and S were performed on Elementar Vario EL III CHNS analyzer at STIC, CUSAT, India. Temperature was adjusted to 25 ± 2  C. Infrared spectra
were recorded using KBr pellets on a JASCO FT/IR-410 spectrophotometer in the 4000e400 cm1 range. Fast atomic bombardment mass
spectra (FAB-MS) were recorded using a VGZAB-HS spectrometer in a 3-nitrobenzylalcohol matrix. Magnetic susceptibility measurements
were carried out on a Gouy balance at room temperature using mercuric tetra(thiocyanato)cobaltate(II) as the calibrant. Diamagnetic
corrections were applied in compliance with Pascal's constant [14]. Electronic absorption spectra were recorded with a Hitachi U-2000
double beam spectrophotometer in the 200e1100 nm range. Fatty acid compositions of algal oil were determined using gas chromatography
techniques (GC) and were performed on Shimadzu gas chromatograph equipped with flame ionization detector and capillary column
(30 m  0.25 mm  0.25 mm film). The detector temperature was programmed for 280  C with the flow rate of 0.3 mL/min and the injector
temperature was set at 250  C. Nitrogen gas was used as the carrier. 1H NMR and 13C NMR spectra of the algal oil were carried out in DMSO-
d6 at room temperature using TMS as internal standard on a Perkin Elmer R-32 spectrometer.

Scheme 2. Synthetic route of Ni(II) Schiff base chelate.

302 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

Fig. 1. Proposed mechanism for formation of algal oil from algal feed stock.

Thermal analysis (TGA/DTA) was recorded on a Perkin Elmer (TGS-2 model) thermal analyzer with a heating rate of 10 K/min in dynamic
N2 atmosphere (flow rate 20 mL/min). Powder X-ray diffraction (PXRD) patterns of Schiff base and its metal chelate were recorded on a
Bruker AXS D8 advance powder X-ray diffractometer (Detector: Si(Li)PSD, X-ray source: Cu, Wavelength: 1.5406 Å). The SEM images were
recorded on a JSM-5610 scanning electron microscope. Antimicrobial studies of algal oil were carried out by modified well-diffusion
method. Engine test was carried out using a DI diesel engine with the conventional diesel at standard temperature and pressure. The
emissions at 35 cm from the exhaust valve.

2.2. Effect of nitrogen environment on algal growth for the determination of lipid content

The colonial green alga namely B. braunii accumulates bulk quantities of hydrocarbons among oleaginous micro algae due to its lipid
storage in the extracellular space rather than in the cytoplasm [2]. It is widespread in fresh water reservoirs, ponds and sometime can be
found in brackish lakes. This alga is regarded as a latent source of renewable fuel [41]. Its lipid content is about 25e75 of % dry weight
biomass/oil content [10]. The algal species were collected and weighed (mo). They were cultured as a nineteen series of 250 mL inocula,
corresponding to the concentration which varies from 0.5 M to 3.0 M of nitrogen nutrients viz., (NH4)2SO4: Ammonium sulphate (AS),
NH4NO3: Ammonium nitrate (AN), KNO3: Potassium nitrate (PN), (NH2)2CO: Urea (UR) and NaNO3: Sodium nitrate (SN), agar as medium.
These flasks were incubated at 25 ± 1  C under 5.0 ± 0.2 k lux light intensity with 12:12 hrs of light: a dark cycle at 140 rpm for a week (T),
growth was observed [39,23]. pH of the different nitrogen medium was kept maintained around 7e9.

2.3. General procedure for the synthesis of 2-hydroxybenzalidene-p-toluidine

Schiff base ligand, 2-hydroxybenzalidene-p-toluidine was synthesized by adding salicylaldehyde (0.006 g, 5 mM, in 10 mL of absolute
alcohol) to p-toluidine (0.0054 g, 5 mM, in 10 mL of absolute ethanol) (Scheme 1). To this, ethanolic solution of KOH (0.1%) was added to
 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 303

Fig. 2. (a) Vibrational; (b) Electronic absorption spectra of Schiff base and its Ni(II)-Schiff base chelate.

make pH of the solution to 7e9. The resulting mixture was refluxed for 1e2 h on a water bath and the reaction was monitored through TLC.
After completion of the reaction the volume of the reaction mixture was reduced to half of its original volume and kept aside at room
temperature. The solid product formed was collected by vacuum filtration, washed several times with water, ethanol and finally with diethyl
ether. The Schiff base ligand was recrystallized from alcohol, dried at 100  C and preserved in vacuo over anhydrous CaCl2.

2.4. General procedure for the preparation of Ni(II) Schiff base chelate

Ni(II) Schiff base chelate was synthesized by the dropwise addition of the Schiff base ligand (0.021 g, 10 mM, in 10 mL absolute ethanol)
into the metal salt in the molar ratio of 1:2 (metal:ligand): Ni(CH3COO)2$4H2O (0.012 g, 5 mM, in 10 mL ethanol) (Scheme 2). The pH of the
304 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

Fig. 3. Proposed structure of Ni(II)-Schiff base chelate.

solution was adjusted to 7e9 by introducing ethanolic solution of KOH (0.1%). After the addition the reaction mixture was refluxed for 1e2 h
and the resulting solution was evaporated to half of its original volume and kept aside at room temperature. On standing the solid product
formed was filtered by vacuum filtration, washed several times with water, ethanol, diethyl ether and finally dried in vacuo over anhydrous
CaCl2.

2.5. Extraction of algal feed stock

The cleaned and weighed micro alga namely B. braunii was ground separately in a mortar and mixed with 50% (v/v) hexaneeether
solvent taken in a 500 mL conical flask. The ground alga was kept at 68.97  C for nearly 30 min to remove the moisture content. After 24 h of
incubation at 70  C, the algal feed stock was filtered by using the cloth filter. Thus obtained algal feed stock was kept in a 250 mL beaker for
24 h at room temperature to expel the solvents. The final yield was measured and found to be 98%. The biodiesel yield was expressed in
terms of the relative weight of the biodiesel obtained to that of oil present in the algal biomass. This feed stock was converted into algal oil in
the presence of Schiff base metal chelate as promoter [34].

2.6. Production of algal oil

25 mL of absolute ethanolic solution containing about 0.13 g of metal salt (NiCl2) in 10 mL conductivity water and 0.58 g of Schiff base
metal chelate was added to a solution of 25 mL algal feed stock in 150 mL of ethanol extracted from the alga cultured at KNO3 environment
due to its high lipid productivity. The mixture was stirred well for 30 min and the transesterification reaction was carried out in the presence
of hydrogen environment at the temperature below the boiling point of ethanol with continuous stirring for 1 h. The proposed mechanism
for formation of algal oil from algal feed stock is shown in Fig. 1. Then it was transferred into a separating funnel and kept for some time to
separate algal oil and sediment layers clearly. Then the oil layer was collected, washed thoroughly with conductivity water and finally with
warm water to remove residual promoters. It was dried and stored in an aluminium container for analysis. The yield was found to be 92%.

2.7. Engine performance and emission characteristics

Experiments were carried out in Engine test Laboratory of Mechanical department at R.M.K Engineering College Chennai, Tamilnadu. To
determine the engine performance and the emission characteristics, the biodiesel blends were prepared in laboratory conditions volu-
metrically i.e. 10% with 90% diesel (B10); 50% with 50% diesel (B50). Tests were carried out in a four-stroke, DI (Direct Injection), air cooled
diesel engine with a compression ratio 18:1 for four particular engine speeds. The engine performance was conducted firstly with diesel fuel
(B0) and then with B10 & B50. Exhaust emission tests were performed in the laboratory test bed which consisted of an exhaust gas analyzer.
 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 305

Fig. 4. (a) Thermogram; (b) XRD pattern; (c) SEM pictogram of Schiff base and its Ni(II)-Schiff base chelate.

2.8. In vitro antimicrobial studies

In vitro antimicrobial activity of the five different algal oils, extracted from the algae of five different nitrogen nutrients (AAS, AAN, APN, AUR,
ASN) were tested by well diffusion technique (against three Gram-positive bacteria: Bacillus subtilis, Staphylococcus saphyphiticus and
Staphylococcus aureus, two Gram-negative bacteria: Escherichia coli and Pseudomonas aeruginosa) using Muller Hinton agar nutrient and three
fungi namely Aspergillus niger, Enterobacter species and Candida albicans using potato dextrose agar as the medium. About 2e8 h, older
microorganism species inoculums containing approximately (104e106) colony forming units per mL were used in these analyses [31]. The
standard procedure as described earlier for measuring the zone of inhibition was followed and all the analyses were made in three replicates.

2.9. In vitro antioxidant studies

Further, the five different algal oils (AAS, AAN, APN, AUR, ASN) were tested for their in vitro antioxidant activities under physiological
conditions (303 K) by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay method. DPPH radical scavenging effect was
carried out according to the Blois method [6]. Different volumes of oil (0.1, 0.2, 0.3, 0.4 and 0.5 mL) were shaken vigorously for about
(2e3) min with 1 mL of a 0.1 mM ethanolic DPPH solution in different test tubes. Then each tube was incubated in the dark room for (20e50)
min at room temperature. A blank DPPH solution without oil was used for baseline correction. After incubation, the absorbance value of all
306 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

Fig. 5. The oil productive ability of the green alga Botryococcus braunii at five different nitrogen environments.

the solutions was measured at 517 nm using UVevisible spectrophotometer. The results show a decrease in the absorbance values which
indicate that the oils (B1, C2, D3, P4 and S5) show free radical scavenging activities. Ascorbic acid was used as the reference or positive control.
Free radical scavenging effect in percentage was calculated using the formula as:
2 3
Acontrol  Asample
Scavenging effect ð%Þ ¼ 4 5  100
Acontrol

Fig. 6. Possible pathway of formation of algal oil by hydrotreatment of glycerides.

 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 307

Table 1
Proximate and ultimate analyses of different algal oils at five nitrogen nutrients.

Analyses S. No. Contents Algal oil

AAS AAN APN AUR ASN

Proximate (wt.%) 1. Moisture 5.11 5.02 4.90 5.11 5.09
2. Volatile matter 64.31 64.20 61.93 65.11 66.04
3. Fixed carbon 8.88 8.73 8.99 8.97 8.84
4. Ash 88.65 88.60 88. 01 88.24 66.14

Ultimate (wt.%) 1. Carbon (C) 71.48 71.23 71.94 71.84 71.69

2. Hydrogen (H) 11.11 11.27 11.58 11.47 11.54
3. Nitrogen (N) 0.34 0.37 0.31 0.33 0.33
4. Sulphur (S) 0.08 0.13 0.16 0.16 0.14

C, H, N and S analysis values are within the limit of ±0.3e0.4%.

Table 2
Fuel properties of different algal oils at five nitrogen nutrients.

S. No. Properties Fuel standard AAS AAN APN AUR ASN

Da [9] ASTMb ENc

1. Density (kg/m3) 833 e 860e900 862 862 861 863 862
2. Viscosity (mm2/s) at 40  C 3.21 1.9e6.0 3.5e5.0 4.94 5.13 4.91 5.11 5.12
3. Specific gravity e 0.88 e 0.87 0.83 0.82 0.82 0.88
4. Acid number (mg KOH/g) 0.20 0.5 max 0.5 max 0.41 0.44 0.41 0.45 0.44
5. Calorific value (MJ/kg) 42.23 e e 42.43 42.88 43.88 43.11 43.88
6. Pour point ( C) 17 e e 16 16 16 16 16
7. Flash point ( C) 76 100e170 120 min e e e e e
8. Copper pipe corrosion e 3 max 1 min e e e e e
9. H:C ratio 1.8 e e 1.8 1.8 1.8 1.8 1.8
10. Cold filter plugging point ( C) 3.0 e e 11.11 11.00 10.74 11.00 11.09

() Not detected.

a
D ¼ Diesel.
b
ASTM ¼ ASTM D 6751.
c
EN ¼ EN 14214.

where Acontrol ¼ absorbance of DPPH without oil; Asample ¼ absorbance of the DPPH with oil. All the analyses were made in three replicate
and the results were compared with the control.

3. Results and discussion

3.1. Physico-chemical characterization of Schiff base and its Ni(II)-Schiff base chelate

The synthesised Schiff base derived from salicylaldehyde and p-toluidine and its Ni(II)-Schiff base catalyst are slightly soluble in
methanol and ethanol, highly soluble in DMSO, CHCl3 and acetone and insoluble in water. The purity of the compounds was checked by
micro elemental analysis, FT-IR, electronic and FAB-Mass. Metal chelates of Schiff base are of interest as simple structural models of more
complicated biological process [28]. The observed molecular ion peak with m/z ratio [M þ 1] confirms the proposed formulae and is in good
agreement with the theoretical molecular weight calculated from micro analytical data. The observed molar conductance of these chelate in
DMSO (~103 M) is consistent with the non-electrolytic nature of the chelate [19]. The vibrational, electronic spectra and the proposed
structure are shown in Figs. 2(a), (b) and 3 respectively. The results are given as follows.

3.1.1. Schiff base: 2-[(E)-N-(4-methylphenyl)carboximidoyl]phenol

Yield: 69%; m.p.: 77  C; Colour: Reddish Yellow; Mol. for.: [C14H13NO]; Mol. wt.: 211; C, 78.04 (ca. 79.59): H, 6.12 (ca. 6.20): N, 6.56 (ca.
6.63); Selected FT-IR data in cm1 (KBr disk): v ¼ 3362 (br, n (OeH) vib. of phenolic), 1193 (s, n (CeO) str. of carbonyl), 1597 (s, n (C]N) vib. of
azomethine); 1H NMR (TMS, DMSO-d6) d ppm: 7.67 (d, 1H, CeH aromatic C2), 7.98 (d, 1H, CeH aromatic C3), 2.32 (d, 3H, C4eCH3), 7.95 (d, 1H,

Table 3
Major fatty acid methyl esters (FAME) of algal oil (APN).

S. No. FAME Chemical formula Wt.% of FAME in algal oil (APN)

1. Behinic oil C22H44O2; (22:0) 0.28
2. Caprylic oil C8H16O2; (8:0) 3.92
3. Eicosapentaenoic oil C20H30O2; (20:5) 0.97
4. Lauric oil C12H24O2; (12:0) 1.12
5. Linoleic oil C18H32O2; (18:2) 7.43
6. Linolenic oil C18H30O2; (18:3) 5.67
7. Myristic oil C14H28O2; (14:0) 3.53
8. Oleic oil C18H34O2; (18:1) 17.44
9. Palmitic oil C16H32O2; (16:0) 37.88
10. Palmitoleic oil C16H30O2; (16:1) 9.77
11. Cis-10-Pentadecanoic oil C15H28O2; (15:1) 2.33
12. Stearic oil C18H36O2; (18:0) 3.77
308 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

Fig. 7. (a) Vibrational; (b) 1H NMR spectra of algal oil (APN).

CeH aromatic C5), 7.65 (d, 1H, CeH aromatic C6), 8.13 (s, H, eHC]Ne azomethine C8), 6.88 (d, 1H, CeH aromatic C10 & C11), 7.09 (d, 1H, CeH
aromatic C12), 6.50 (d, 1H, CeH aromatic C13), 9.56 (s, 1H, CeOH phenolic C14); 13C NMR (TMS, DMSO-d6) d ppm: 115.11e129.44 (eCHe,
aromatic C1eC6), 20.38 (eC4HeCH3, aromatic), 181.27 (eHC]Ne azomethine C8), 111.13e152.33 (eCHe, aromatic C9eC13), 152.03 (]CeOH
phenolic C14); FAB-MS: m/z ¼ 212 [M þ 1]; UVevis, (103 M in DMSO): lmax ¼ 39,840 cm1 [INCT (pep*)] and 28,902 cm1 [INCT (nep*)].

3.1.2. Ni(II)-Schiff base chelate [3,30 -bis (4-methylphenyl)-2,20 -spirobi(benzo[e]1-oxa-3l4-aza-2-nickelacycyclohexane)-2,2-diol]

Yield: 67%; m.p.: 147  C; Colour: Reddish Brown; Mol. for.: [NiC28H28N2O4]; Mol. wt.: 514; C, 65.18 (ca. 65.27): H, 5.43 (ca. 5.48): N, 5.38
(ca. 5.44): Ni, 11.32 (ca. 11.39); Selected FT-IR data in cm1 (KBr disk): v ¼ 1237 (s, n (CeO) str. of carbonyl), 1618 (s, n (C]N) vib. of
 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 309

Fig. 8. (a) Maximum engine torque; (b) Change in torque with engine speed.

azomethine), 563 [s, n (NieN)], 454 [w, n (NieO)], 3387, 870 (b, n(H2O) molecule); FAB-MS: m/z ¼ 515 [M þ 1]; UVevis, (103 M in DMSO):
lmax ¼ 33,898 cm1 [LMCT (M ) N)], 26,315 cm1 [3A2g(F) / 3T1g(P)], 16,694 cm1 [3A2g(F) / 3T1g(F)] and 10,256 cm1 [3A2g(F) / 3T2g(F)];
LM (103 M in DMSO) ¼ 12.09 S cm2 mol1; meff BM: 3.02.
The thermogram, Fig. 4(a) of Ni(II)-Schiff base chelate in the first stage shows 7.0% weight loss in the temperature range 37  C to z240  C
due to the removal of two water molecules [7]. In the II stage at z240e487  C, there is a weight loss (23.76%) corresponds to the removal of
salicylaldehyde moiety. In the III stage a loss of 20.85% mass occurs at above the temperature 487  C which corresponds to the removal of p-
toluidine moiety and the air stable metal oxide (14.78%) is the end product.
X-ray analysis of the Schiff base and its Ni(II)-Schiff base chelate was carried out and the diffractograms obtained are given in Fig. 4(b).
The morphology and particle size are illustrated in the Scanning Electron Micrography (SEM) and is shown Fig. 4(c). There is a uniform
matrix of the synthesized chelate in the pictogram. This shows that the compounds are homogeneous phase material [15,20].
310 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

Table 4
Engine performance analysis.

S. No. Characteristics Biodiesel blend (% Vol)

B0 (100% diesel) B10 (90 %diesel þ 10% algal oil) B50 (50% diesel þ 50% algal oil)
1. Speed (N rpm) 1536 1531 1526
Time (s) 134 133 132
2. Fuel consumption rate (FC, mL/100 s) 18.7 19.0 19.8
3. Engine power output (Po, kW) 1.80 2.28 2.37
4. Specific fuel consumption rate (SFC, mL/W) 10.39 8.33 8.31
5. HC emission (ppm) 10.2 7.2 18.3
6. CO emission (%)   0.16
7. CO2 emission (%) 7.1 6.6 6.8

() Not detected.

3.2. Effect of nitrogen environment on algal growth for the determination of lipid content

The incubated cultures from the inocula were washed with distilled water and the purified biomass were oven dried and then weighed
(mt). Now the biomasses were extracted using a mixture of (v/v) non-polar solvents namely chloroform-methanol [23,8]. The lipid fraction
was filtered off through Whatman No.1 filter paper followed by solvent washing and the amount of lipid content was determined by solvent
evaporation, this followed by gravimetric estimation of total lipid productivity. The initial lipid content was taken as Lo and Lt was the total
lipid content after incubation period (T). The oil productive ability of the green alga B. braunii at five different nitrogen environments is
shown in Fig. 5.

Fig. 9. The variations in emissions of (a) NOx; (b) Smoke with different engine speed for the diesel blends.

Table 5
In vitro antimicrobial activities of algal feed stock and five different algal oil by well diffusion method at different times (zone formation in mm).

Complex Diameter of inhibition zone (in mm) for different microorganisms

Escherichia coli Staphylococcus aureus Staphylococcus saphyphiticus Pseudomonas aeruginosa Aspergillus niger Candida albicans Enterobacter species

Time (h)

24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72
Control 27 28 29 31 33 36 28 32 34 28 29 31 24 26 30 28 28 30 25 26 28
Algal feed stock 22 22 23    23 25 26 23 23 24 23 24 25 21 21 23   19
AAS 14 15 17 18 18 19    16 17 18 17 19 20    16 17 
AAN 15 16 16    17 18 19  14  17 17 18 18 19 20 17 18 
APN 20 22 24 25 26 28 26 28 29    23 25 26    21 23 24
AUR 20 21 21 22 24 25    19 20 21 22 23 23 18 19 19   
ASN 17 17 19 21 22 22 20 22 23    21 23 23    20 21 21

(e) denotes less activity.

 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 311

Fig. 10. In vitro antimicrobial activities of algal feed stock and five different algal oil at 72 h by well diffusion method at different times (zone formation in mm).

3.3. Production of algal oil

The algal feed stock consists of C16, C18 & C20 unsaturated and C14, C16 & C18 saturated fatty acids as major constituents and di/triglycerides
as the minor constituents. Ni metal and Ni(II)-chelate on hydrotreatment (30 bar H2) under the above said experimental conditions cat-
alyzes the hydrogenation of unsaturated fatty acids to trans unsaturated & saturated acids which is further hydrogenated to aldehyde and in
turn to alcohol intermediates as a result of liquid alkanes i.e, algal oil production.
For instance, cis-oleic acid on hydrogenation yields stearic (saturated) and trans-vaccenic acid (unsaturated). The di/triglycerides also
involve hydrotreatment to yield higher alkanes and were cracked into lower alkanes [35]. The possible pathway of formation of algal oil by
hydrotreatment of glycerides is given in Fig. 6.

3.4. Physico-chemical properties of algal oil

The physico-chemical properties of algal oil (AAS, AAN, APN, AUR, ASN) such as free fatty acid (FFA) %, acid value, saponification value,
iodine value and unsaponifiable matter were established [41]. Fatty acid composition was determined using gas chromatography (GC).
About 0.1 mL oil in n-hexane was converted to methyl ester before injected into the gas chromatograph. The results of proximate and
ultimate analyses are given in Table 1.
The physico-chemical properties of algal oil along with fatty acid composition are presented in Tables 2 and 3 and compared
with diesel [41,9]. The observed density and corrosion inhibition property of algal oil was low when compared to other bio oils due
to addition of Ni(II)-Schiff base promoter during its production [9,3]. Inhibition efficiency of Schiff base metal chelate is due to the
presence of free electron pairs on nitrogen and oxygen atoms, p-electrons on the aromatic rings of organic moieties, molecular size
and mode of interaction with the metal surface [25]. Vibrational (FT-IR) spectroscopy showed characteristic strong absorption bands
at 1741 cm1 for the ester carbonyl (eC]O) functional group [30] which is shown in Fig. 7(a). The 1H NMR spectra of the five
different algal oils (AAS, AAN, APN, AUR, ASN) show the following principal signals: 0.72 ppm to 0.76 ppm for terminal (eCH3),
1.13e1.19 ppm for methylene protons (eCH2e), 1.87e1.93 ppm for the protons a to the carbonyl groups (eCH2eCOe),
2.59e2.69 ppm for diallylic methylene proton (eC]CeCH2eC]Ce) and 4.19 ppm for protons in a and b position in glyceryl. The
methyl ester content was estimated using the integral value of eOCH3 group peak at 3.66 ppm and the a-CH2 at 2.24 ppm [37]
(Fig. 7(b)).
13
C NMR signal assignments of the algal feedstocks (AAS, AAN, APN, AUR, ASN) also indicated the presence of allylic carbon atoms at
26.7 ppm, methylene carbon atoms at (26.7e29.9) ppm, glyceryl carbon atoms at (61.8e68.8) ppm, olefinic carbon atoms at
(127.0e129.7) ppm and carbonyl carbon atoms at (173.1e178.0) ppm. The iodine value was determined by 1H NMR spectroscopy which is
based on the average molecular weight of the fatty acid methyl esters and the number of double bond [37]. The 13C NMR spectra of diesel
show signals at 20.0e40.0 ppm for aliphatic carbon atoms.
312 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

Fig. 11. (a) Electronic absorption spectra of ascorbic acid, algal feed stock at a concentration of 50 mM; (b) In vitro antioxidant activities of algal feed stock and five different algal oil
at different concentrations (10e50 mg) by DPPH free radical scavenging assay method at room temperature.

3.5. Engine performance

The Engine performance tests were carried out on the DI, air cooled diesel engine with diesel fuel (B0) and then with B10 & B50 of a
compression ratio 18:1 for four particular engine speeds. The Engine Torque, Engine power, average % of Brake Specific Fuel Consumption
(BSFC) and Emissions characteristics were analysed.
 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315 313

Table 6
In vitro antioxidant activities of algal feed stock and five different algal oil by DPPH free radical scavenging assay method at different concentrations (10e50 mg) at room
temperature.

Complex Scavenging activity (%) at different concentrations ± S.D.a

10 mg 20 mg 30 mg 40 mg 50 mg
Control 84 ± 0.17 88 ± 0.18 90 ± 0.20 92 ± 0.20 95 ± 0.21
Algal feed stock 47 ± 0.33 51 ± 0.34 54 ± 0.31 59 ± 0.36 62 ± 0.38
AAS e 39 ± 0.24 41 ± 0.26 44 ± 0.30 48 ± 0.34
AAN 34 ± 0.32 34 ± 0.21 38 ± 0.24 41 ± 0.28 46 ± 0.31
APN 38 ± 0.24 42 ± 0.27 45 ± 0.31 49 ± 0.34 51 ± 0.37
AUR e 45 ± 0.25 49 ± 0.27 52 ± 0.32 56 ± 0.35
ASN 30 ± 0.24 33 ± 0.27 36 ± 0.31 38 ± 0.34 40 ± 0.37
a
S.D.: Standard deviation (average of three replicates) and (e) denotes less activity.

3.5.1. Engine torque

The engine speed was set at 2500, 2000 and 1500 rpm. A torque of 24.5, 25.5 and 22.5 N m were obtained with diesel fuel at 1500, 2000
and 2500 rpm respectively while with B50 fuel, a torque of 21.9, 21.2 and 19.8 N m were observed. The decrement rate of torque was at 16%,
15% and 13% respectively [17]. Maximum engine torque was obtained at 2000 rpm and the change in torque with engine speed is shown in
Fig. 8(a) and (b) respectively. Almost similar results were obtained with B10 fuel.

3.5.2. Brake specific fuel consumption

Brake specific fuel consumption for diesel and B50 fuel was found to be 345 g/kWh and 362 g/kWh at engine speed of 1500 rpm
respectively. The increase in Brake specific fuel consumption for B50 fuel was 4% with respect to diesel fuel. At maximum torque speed at
2000 rpm the brake specific fuel consumption was 390 g/kWh for diesel fuel and 406 g/kWh for B50 fuel. The difference between diesel
fuel and B50 fuel is 3%. A minimum torque speed at 1000 rpm, the brake specific fuel consumption was 402 g/kWh for diesel fuel and
415 g/kWh for B50 fuel. The biodiesel has less heating value in volume than conventional diesel fuel. The average % of Brake Specific Fuel
Consumption (BSFC) increases after endurance test for the diesel (B0) and a blend of fifty percent algal oil (B50). The BSFC of diesel (B10)
was lower than the others which are ascribed to higher heating value and lower viscosity [44,2]. Consequently a lesser amount of diesel
(B0) is desirable to afford an equivalent amount of energy. Engine power output (Po/kW) and specific fuel consumption (SFC/mL W1) are
given in Table 4.

3.6. Emission characteristics

The exhausted emissions from the combustion of blends B10 and B50 at engine speed 1000 rpm showed that the Hydrocarbons (HCs),
Carbon monoxide (CO), Carbon dioxide (CO2), Oxides of Nitrogen (NO)x, Oxygen (O2) and the smoke emissions are less when compared with
B0 except (NO)x emission which is due to the presence of oxygen and lower aromatic HCs in the diesel blend structure [4]. The variations in
emissions of NOx and the smoke with different engine speed for the diesel blends B10 and B50 are shown in Fig. 9(a) and (b) respectively.
The emissions became high as the engine speed increased (Table 4).

3.7. In vitro antimicrobial studies

In vitro antimicrobial activity of the five different algal oil, extracted from the alga of five different nitrogen nutrients (AAS, AAN, APN,
AUR, ASN) were tested at a concentration of 75 mg in DMSO against five pathogenic bacteria and three fungal strains by well diffusion
method using agar as nutrient (Table 5). According to Tweedy's chelation hypothesis, the antimicrobial activities of the algal oil can be
visualized on the basis of the interactions between the hydrocarbons and microorganism.
The commercially available standard drugs ampicillin (antibacterial control) and nystatin (antifungal control) were used as controls. All
the five different oil show remarkable activities towards microorganisms. Variations in the effectiveness of different algal oil against mi-
croorganisms depend on the impermeability to the cell of the microbes or on differences in the ribosome of microbial cells. Higher inhibition
zone shown by the algal oil can be explained on the basis of Overtone's concept and Tweedy's chelation theory [31]. All the oils show more
significant antibacterial and less pronounced antifungal activities and is given in Fig. 10. The observed enhancement in the activity of the oil
may be due to the presence of hydrocarbons in the algal oil composition. The order of activity is as follows; Control > Algal feed
stock > APN > AUR > ASN > AAN > AAS.

3.8. In vitro antioxidant studies

The five different algal oil, extracted from the alga of five different nitrogen nutrients (AAS, AAN, APN, AUR, ASN) were tested for
their in vitro antioxidant studies and 50 mg of algal oil in 50% (v/v) ethanolewater mixture were screened for their in vitro antioxidant
activities. Ascorbic acid (AA) is used as the reference and a blank DPPH solution was used for the baseline correction [6]. The absorption
spectra obtained with DPPH for different oil are shown in Fig. 11(a). Each analysis was made in three replicate and the average is taken
for the analysis. The results were compared with the control and are shown that the algal oils possess potent antioxidant activities
(Table 6). The in vitro antioxidant studies follow the order as Control > Algal feed stock > APN > AUR > AAN > AAS > ASN and is shown
in and Fig. 11(b).
314 S. Arvindnarayan et al. / Journal of the Energy Institute 90 (2017) 300e315

4. Conclusions

In this study, B. braunii a colonial green alga was selected and cultured at five different nitrogen environments (AS, AN, PN, UR and SN) for
the extraction of algal feed stock (AAS, AAN, APN, AUR and ASN). In addition, a Schiff base ligand, 2-hydroxybenzalidene-p-toluidine and its
Ni(II) chelate were synthesized and characterized using micro elemental, spectral, thermal and XRD/SEM analyses. The algal feedstock (APN)
extracted from the alga at PN environment was chosen for algal oil production in the presence of Ni/H2 catalyst and Ni(II)-Schiff base chelate
as promoter. The algal oil extracted was physico-chemically characterized by GC-mass and spectral studies. The observed density and
corrosion inhibition ability of algal oil was low when compared to other bio oils due to addition of Ni(II)-Schiff base promoter during its
production.
Further, the Engine torque, engine power, average % of Brake Specific Fuel Consumption (BSFC) and emissions characteristics were
analysed using algal oilediesel blends (B10 and B50). The results of algal oilediesel blends for B10 and B50 showed that the BSFC of B10
blend was lower than the others which are ascribed to higher heating value and lower viscosity. The Hydrocarbons (HCs), Carbon monoxide
(CO), Carbon dioxide (CO2), Oxides of Nitrogen (NO)x, Oxygen (O2) and the smoke emissions are less when compared with B0 except (NO)x
emission. In vitro antimicrobial activity of the five different algal oils follows the order; Control > Algal feed
stock > APN > AUR > ASN > AAN > AAS. All the oils show more significant antibacterial and less pronounced antifungal activities. The in vitro
antioxidant studies follow the order as Control > Algal feed stock > APN > AUR > AAN > AAS > ASN. The algal oils possess potent antioxidant
activities. Moreover, the B10 and B50 blend fuels are biodegradable and they reduce green gas emission, thus prevent the global warming.

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