Characterization of fatty acids and hydrocarbons of chlorophycean microalgae towards their use as biofuel source

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

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Characterization of fatty acids and hydrocarbons of chlorophycean microalgae towards their use as biofuel source Srivatsan Vidyashankar a, Kodur Shankaramurthy VenuGopal a, Gadde Venkata Swarnalatha a, Mysore Doddaiah Kavitha a, Vikas Singh Chauhan a, Ramasamy Ravi b, Ashwini Kumar Bansal c, Ranjit Singh c, Anil Pande c, Gokare Aswathanarayana Ravishankar a, Ravi Sarada a,* a

Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute (CFTRI), Mysore 570 020, India b Sensory Sciences Department, CSIR-Central Food Technological Research Institute (CFTRI), Mysore 570 020, India c Oil and Natural Gas Corporation Limited (ONGC), Keshava Deva Malaviya Institute of Petroleum Exploration (KDMIPE), 9 Kaulagarh Road, Dehradun 248 195, India

article info

abstract

Article history:

Microalgae accumulate important biofuel precursors such as fatty acids and hydrocarbons.

Received 4 June 2014

Identification of microalgal strains with ideal fuel quality precursor profile is important

Received in revised form

during bioprospection studies. In this direction, thirty two freshwater green microalgae

27 February 2015

were characterized for their biomass productivity, fatty acid and hydrocarbon composition

Accepted 1 March 2015

under autotrophic growth conditions. Scenedesmus dimorphus CFR 1-05/FW, Oocystis pusilla

Available online

CFR 6-01/FW and Quadrigula lacustris CFR 7-01/FW were high biomass producing strains with shorter doubling time. Lipid accumulation was monitored by nile red staining with

Keywords:

mild acetic acid pretreatment (at 7 m mol L
1) to microalgal cells. Six strains viz., S.

Microalgae

dimorphus CFR 1-05/FW, Scenedesmus obtusus CFR 1-09/FW, Chlorococum sp. CFR 2-01/FW, C.

Fatty acid methyl esters

humicola CFR 2-03/FW, Chlorella sorokiniana CFR 3-01/FW, Dictyosphaerium CFR 5-01/FW

Hydrocarbons

showed lipid accumulation of >20% mass fraction at stationary phase. Palmitic, oleic and

Principal component analysis

alpha linolenic acids were major fatty acids in all the chlorophycean species. Fuel grade

Biofuels

hydrocarbons which can be directly blended with petroleum fuels were identified. Fourteen strains showed hydrocarbon content of >10% mass fraction of dry biomass. n-Paraffins of chain length between C15 to C20 were predominant hydrocarbons in all the strains. Branched isoprenoid hydrocarbons were detected in Scenedesmus sp. CFR 1- 13/FW constituting 49% mass fraction of total hydrocarbons. High quantities of n-tetradecane (40%) was detected in Kirchneriella cornuta CFR 8-01/FW. The similarity of microalgal hydrocarbon profiles with paraffinic and isoparaffinic fraction of petroleum diesel and compliance of FAME based biodiesel to international standards indicate the suitability of algae derived biofuels for blending with conventional petroleum fuels. © 2015 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ91 821 2516501; fax: þ91 821 2517233. E-mail address: [email protected] (R. Sarada). http://dx.doi.org/10.1016/j.biombioe.2015.03.001 0961-9534/© 2015 Elsevier Ltd. All rights reserved.

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1.

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

Introduction

Microalgae could be a potential carbon neutral energy source owing to their higher photosynthetic efficiency (ten folds) and CO2 utilization compared to terrestrial crop plants, simple growth requirements and adaptability to variety of environmental conditions [1,2]. Microalgae produce biofuel precursors such as lipids (long chain fatty acids), long chain hydrocarbons and fatty alcohols that can be directly utilized as fuels or as additives in petroleum fuels. The most commonly followed method of biofuel production from microalgal oil involves conversion of lipids or triacylglycerols (TAG) into fatty acid alkyl esters, also known as biodiesel, by the process of transesterification. The main precursors involved in this process are fatty acids. Fatty acids of microalgae show a wide variation in their distribution pattern among different groups, thus making them as genus or species specific chemical marker [3]. Characterization of fatty acids in microalgae is important since certain key fuel parameters such as cetane number (ignition quality) and cold flow properties are directly dependent upon the fatty acid profile [4]. Many bioprospection studies on microalgae involve lipid/fatty acid quantification overlooking the quality criteria [5] which is very essential in determining the utility of lipid as biofuel substrate. Therefore, profiling microalgal species for their fatty acid composition becomes necessary. Hydrocarbons produced by microalgae offer a biofuel that could be converted to gasoline by hydrocracking or can be blended directly with diesel and aviation fuel [6]. Microalgal hydrocarbon appears to be attractive as biofuel since transesterification process is energy intensive and accounts for 70%e80% of cost during biodiesel production [7]. In addition to high costs, the final product recovery from transesterification, devoid of toxic catalysts, is low, affecting the economics of biofuel production [8]. Hence, screening microalgal species for fuel grade hydrocarbons is important. Certain microalgae such as Botryococcus braunii have been identified with presence of long chain hydrocarbons and ether lipids. These hydrocarbons have odd carbon number (C23eC33) alkadiene/trienes, triterpenes (botryococcenes, C30eC37) and methylated squalenes (C31eC34) with potential fuel applications [9]. However, slow growth rate of B. braunii with an average doubling time of 36.5 h and low biomass productivity (34 mg L
1 day
1) have been the major limiting factors for its industrial exploitation [10,11]. Selection of microalgal strains with high biomass productivity is important for successful algal biomass production for low-value products such as biofuels. In this process, the first critical step is the establishment of a germplasm of microalgal strains from the natural environment. Though several reports are available on the screening of microalgae for biofuel production [12e14] only few species such as Scenedesmus obliquus, Dunaliella sp., Chlorella vulgaris and Nannochloropsis sp. have been well studied [15]. The vast biodiversity of India provides a valuable resource for development of indigenous microalgal strains as biofuel feedstock. In addition, the ambient climatic conditions present are largely favorable (temperature approximately between 25  C and 30  C and light illumination) for mass outdoor production of algae.

Hence, in the present study an effort was made towards establishment of a germplasm of freshwater microalgae with biofuel potential. The culture collection mainly focused on green microalgae (Chlorophyceae) since they are ubiquitous, fast growing and oleaginous compared to other groups [2]. Green algae respond to abiotic stress conditions better than other taxa with their average lipid content increasing by two to three folds making them a suitable feedstock for biofuel production [16]. In the present work, the term biofuel refers to microalgae derived biodiesel (fatty acid methyl esters) and long chain hydrocarbon extracts.

2.

Materials and methods

2.1.

Isolation and cultivation of microalgae

Microalgae were collected from different fresh water bodies of India using commercial plankton net, called as nylo-bolt cloth, with pore size of 7e10 mm. The survey was undertaken before the onset of monsoon during JanuaryeMarch and the ambient temperature and pH of the water bodies were recorded. Microalgae growing as biofilms on rock surfaces, walls on banks of water bodies, temple walls were collected as surface scrapes. The collected microalgae were stored in modified Knop's enrichment solution and purified by the method described by Vidyashankar et al., [17].

2.1.1.

Maintenance of axenic cultures

The bacterial and fungal contaminants in monoalgal cultures were overcome by antibiotic treatment. The microalgal cultures were plated in nutrient agar (SRL, Mumbai) and incubated at 37  C along with the antibiotic containing Hexa disc™ (HiMedia Laboratories, Mumbai). The antibiotics giving maximum inhibitory zone were selected for treatment viz., ampicillin (at 100 mg L
1), cefotoxime (at 100 mg L
1), gentamycin (at 100 mg L
1) and erythromycin (20 mg L
1) for overcoming bacterial contamination. Fungal contamination was overcome with use of Amphotericin-B (at 25 mg L
1). The purity of the cultures was ensured by repeated sub-culturing and regular microscopic observation. The purified algal isolates were identified and characterized based on morphological characteristics and cell dimensions [18] (Table 1) using light microscope (Olympus BX 51, Japan) and imaging software (ProgRes C5, Germany) (Fig. 1). The purified microalgal strains are perpetually maintained in both liquid and solidified Bold basal medium (BBM) at CSIR-CFTRI, Mysore, India. The morphological characterization of microalgal isolates and the geographical co-ordinates of their natural habitat are presented in Table 1.

2.1.2.

Microalgal growth and productivity measurements

The microalgae strains were incubated at a temperature of 25 ± 1  C under 30 mE m
2 s
1 light intensity with a photoperiod of 16:8 h light and dark cycles. The microalgal growth was monitored constantly by measuring the optical density at 560 nm using spectrophotometry [17]. The specific growth rate (m) was measured by Equation (1) and the doubling time (D) was measured by the Equation (2) as described by Lee and Shen [19] and Converti et al. [20].

Table 1 e Geographical co-ordinates of place of isolation and morphological characterization of freshwater chlorophycean microalgae. Genus

Codea

Scenedesmus sp.

Cell dimensionsb

Morphological description

A

Scenedesmus sp. CFR 1-01/FW

Unicellular cells without spines, older cells occur in diads

8.39 mm (Length) x 4.37 mm (Width)

B

Scenedesmus sp. CFR 1-02/FW

6.76 ± 1.01 mm (L) x 11.44 ± 0.72 mm (W) Apical spines- 4.85 mm, central spines - 2.63 mm

C

Scenedesmus sp. CFR 1-03/FW

D

Scenedesmus sp. CFR 1-04/FW

E

S. dimorphus CFR 1-05/FW

F

Scenedesmus sp. CFR 1-06/FW

G

Scenedesmus sp. CFR 1-07/FW

H

Scenedesmus sp. CFR 1-08/FW

I

S. obtusus CFR 1-09/FW

J

Scenedesmus sp. CFR 1-10/FW

K

Scenedesmus sp. CFR 1-11/FW

L

S. perforatus CFR 1-12/FW

Cells predominantly in tetrad coenobia, spines present on the apical ends and centre of coenobia, thick pyrenoids present, no mucilagenous sheath Cells predominantly in diad coenobia, spines present only in apical ends, thick pyrenoids present, no mucilagenous sheath Cells predominantly in tetrads, thick pyrenoids present, mucilagenous sheath absent Cells predominantly in tetrads, thick pyrenoids present, no mucilagenous sheath Cells predominantly tetrad, thick pyrenoid present, mucilagenous sheath absent Cells predominantly tetrad, thick pyrenoid present, mucilagenous sheath absent Cells predominantly tetrad, thick pyrenoids present, mucilagenous sheath absent Unicellular, circular shape, thick pyrenoids, mucilagenous sheath absent Cells predominantly tetrad, spines present on apical ends and centre of the tetrad, thick pyrenoid present, mucilagenous sheath absent Cells predominantly tetrad, thick pyrenoid present, mucilagenous sheath absent Cells predominantly in tetrads, thick pyrenoids present, mucilagenous sheath absent

Place of isolation Unkal lake, Hubli 15 220 53.4500 N, 75 060 39.6500 E, elevation 629 m Sri Sathya Sai University garden pond, Puttaparthi, 14 090 36.7800 N, 77 480 44.4800 E, elevation 463 m

8.73 mm (L) x 3.59 mm (W) Apical spines- 0.98 mm

Kabini water reservoir, Mysore, 11 580 24.6100 N, 76 210 09.5600 E, elevation 684 m

6.39 ± 0.05 mm (L) x 5.64 ± 0.25 mm (W)

Karanji lake, Mysore 12 180 17.0200 N, 76 400 34.4000 E, elevation 749 m University pond, University of Madras, Chennai, India 13 50 200 N, 80 160 1200 E Karanji lake, Mysore 12 180 17.0200 N, 76 400 34.4000 E, elevation 749 m Karanji lake, Mysore 12 180 17.0200 N, 76 400 34.4000 E, elevation 749 m Karanji lake, Mysore 12 180 17.0200 N, 76 400 34.4000 E, elevation 749 m IGM Hospital pond, Agartala, 23 490 52.5600 N, 91 160 36.11E, elevation 40 m Kandaswamy temple pond, Chennai, 13 050 08.6700 N, 80 160 43.0200 E, elevation 11 m

7.764 mm (L) x 5.231 mm (W)

7.33 ± 0.99 mm (L) x 12.20 ± 1.81 mm (W)

7.47 ± 0.44 mm (L) x 12.56 ± 1.07 mm (W)

6.49 ± 0.41 mm (L) x 10.14 0.83 mm (W)

6.16 ± 0.47 mm

4.84 ± 0.84 mm (L) x 3.73 ± 0.47 mm (W) Apical spines: 3.54 mm Central spines- 2.25 mm

10.70 ± 2.50 mm (L) x 17.12 ± 1.92 mm (W)

13.93 ± 1.09 mm (L) x 20.19 ± 1.66 mm (W)

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Microalgal isolates

Naini lake, Nainital 29  220 50.8100 N, 79 270 48.6100 E, elevation 1943m National Institute of Technology, garden pond, Surathkal 13  000 38.6700 N, 74 470 38.4500 E, elevation 25m (continued on next page)

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78

Table 1 e (continued ) Genus

Chlorella sp.

Microalgal isolates

M

Scenedesmus sp. CFR 1-13/FW

N

S. bijugatus CFR 1-14/FW

O

Scenedesmus sp. CFR 1-15/FW

P

Chlorococum sp. CFR 2-01/FW

Q

Chlorococcum sp. CFR 2-02/FW

R

C. humicola CFR 2-03/FW

S

Chlorococcum sp. CFR 2-04/FW

T

Chlorococcum sp. CFR 2-05/FW

U

C. sorokiniana CFR 3-01/FW

V

C. vulgaris CFR 3-02/FW

W

C. vulgaris CFR 3-03/FW

Morphological description Cells predominantly tetrad, spines present on apical ends and centre of the tetrad, thick pyrenoid present, mucilagenous sheath absent Cells predominantly tetrad, thick pyrenoid present, mucilagenous sheath absent Cells predominantly in tetrad coenobia, spines present on the apical ends of coenobia, thick pyrenoids present, no mucilagenous sheath Cells colonial, circular, cells dissociate when agitated, pyrenoid present, mucilagenous sheath present Cells colonial, circular, cells dissociate when agitated, pyrenoid present, mucilagenous sheath present Cells circular, colonial arrangement, 4 celled colony, cells do not dissociate when agitated, thick mucilagenous sheath present Cells colonial, 4 to 8 to16 cell colony, irregular arrangement of cells, cells dissociate when agitated, thick pyrenoid present, mucilagenous sheath present. Cells circular, colonial arrangement, 4 celled colony, cells do not dissociate when agitated, thick mucilagenous sheath present Cells unicellular, circular, pyrenoids present, cup shaped chloroplast present Cells unicellular, circular, pyrenoids present, cup shaped chloroplast present Cells unicellular, circular, pyrenoids present, cup shaped chloroplast present, mucilagenous sheath absent

Cell dimensionsb

Place of isolation

9.55 ± 1.42 mm (L) x 3.24 ± 0.58 mm (W) Apical spines- 2.82 mm, Central spines: 1.87 mm

Pallikaranai marshy waters, Chennai 12  560 54.1800 N, 80 120 06.7700 E, elevation 1m

7.16 ± 0.82 mm (L) x 11.03 ± 2.29 mm (W)

Hairige lake, Hunsur, Mysore 12.31 N, 76.29 E, Elevation 792 m Karanji lake, Mysore 12 180 17.0200 N, 76 400 34.4000 E, elevation 749 m

8.02 ± 1.31 mm (L) x 13.90 ± 1.18 mm (W)

Individual cells - 3.15 ± 0.45 mm

Vishnutank, Mahablaipuram, 12  360 57.3300 N, 80 110 41.1700 E, elevation 11m

Individual cells
8.46 ± 0.98 mm

Water storage tank, LGB nagar, Coimbatore 11 030 14.2500 N, 76 590 28.0400 E elevation 432 m Kapil Teertham, Tirupati 13 390 2500 N, 79 250 1600 E

Individual cells - 14.42 ± 1.26 mm

Individual cells
8.69 ± 0.32 mm

Shore temple pond, Mahabalipuram, 12  370 05.8100 N, 80 110 45.8800 E, elevation 9m

Individual cells - 9.15 ± 0.64 mm

Naini lake, Nainital, 29 230 21.3200 N, 79 270 33.36E, elevation 1951 m

3.52 ± 0.11 mm

INDIA GATE stagnant water pond, New Delhi, 28 360 4600 N, 77 130 4600 E Singanallur tank, Coimbatore, 10 590 26.4300 N, 77 010 17.8800 E, elevation 390 m Raghavendra swami temple pond, Mantralayam, 15 560 32.8800 N, 77 250 25.1300 E, elevation 323 m

4.02 ± 0.33 mm

3.23 ± 0.46 mm

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

Chlorococcum sp.

Codea

Ankistrodesmus sp.

Other Chlorophycean microalgae

b

C. vulgaris CFR 3-04/FW

Y

A. convolutus CFR 4-01/FW

Z

A. convolutus CFR 4-02/FW

AA

Dictyosphaerium CFR 5-01/FW

BB

Oocystis pusilla CFR 6-01/FW

Cells unicellular, ellipsoid, pyrenoid present

8.39 ± 1.46 mm (L) x 4.37 ± 0.69 mm (W)

CC

Quadrigula lacustris CFR 7-01/FW

Cells unicellular, lengthy cells with acicular tips, pyrenoids absent, mucilagenous sheath absent

17.54 ± 1.66 mm (L) x 4.16 ± 0.46 mm (w)

DD

Kircheneriella cornuta CFR 8-01/FW

10.50 ± 1.37 mm (L) x 4.77 ± 0.94 mm (W) width at cell centre

EE

Selenastrum gracile CFR 9-01/FW

Cells unicellular, spindle/lunate shaped, pyrenoids absent, no mucilagenous sheath Cells spindle/sickle shaped, unicellular, pyrenoids absent, mucilagenous sheath absent

FF

Coelastrum asteroidum CFR 10-01/FW

Cells colonial, 8 celled colony, star shaped colony, cells do not dissociate when agitated mucilagenous sheath present

Individual cells - 5.31 ± 0.202 mm

26.81 ± 4.58 mm (L) x 6.37 ± 0.7 mm (W) (width at cell centre) 28.61 ± 5.58 mm (L) x 6.37 ± 0.79 mm (W) (width at cell centre) 4.76 ± 0.508 mm

29.95 ± 4.48 mm (L) x 6.80 ± 1.37 mm (W) at centre

Barathidhasan university garden pond, Trichy, 10 460 36.6800 N, 78 410 43.5600 E, elevation 92 m Hadinaru lake, Mysore, 12 100 17.4400 N, 76 440 33.2700 E, elevation: 674 m Raghavendra swami temple pond, Mantralayam, 15 560 32.8800 N, 77 250 25.1300 E, elevation 323 m Barathidhasan university garden pond, Trichy, 10 460 36.6800 N, 78 410 43.5600 E, elevation 92 m Water body near NIOT campus, Chennai 12  560 53.9500 N, 80 130 20.1800 E elevation 1m Barathidhasan university garden pond, Trichy, 10 460 36.6800 N, 78 410 43.5600 E, elevation 92 m Raghavendra swami temple pond, Mantralayam, 15 560 32.8800 N, 77 250 25.1300 E, elevation 323 m Sri Guru Ram Rai Gurudwara temple pond, Dehradun, 30 190 06.9900 N, 78 010 52.2700 E, elevation 654 m Sengeniamman temple pond, Chennai, 12  560 47.4700 N, 80 150 12.1200 E, elevation 6m

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a

Unicellular, circular shaped, pyrenoids present, cup shaped chloroplast, mucilagenous sheath absent Cells spindle/sickle shaped, unicellular, pyrenoids absent, mucilagenous sheath absent Cells spindle/sickle shaped, unicellular, pyrenoids absent, mucilagenous sheath absent Unicellular, circular shape, mucilagenous sheath present, pyrenoid present

5.63 þ 0.75 mm

X

- The alphabetical codes and order of strain listing are maintained uniformly as in main text. - The cell measurements are an average of 50 individual cells in the mid log phase of the cultivation.

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b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

Fig. 1 e Photomicrographs of some representative freshwater green microalgae 1- Kirchneriella cornuta (DD), 2- Chlorellasp. (W), 3- Chlorococcum sp. (T), 4- Scenedesmus perforatus (L), 5- Quadrigula lacustris (CC), 6- Scenedesmus sp. (D), 7- Oocystis pusilla (BB), 8- Coelastrum asteroidum (FF), 9- Selenastrum gracile (EE), 10- Scenedesmus obtusus (I), 11- Ankistrodesmus convolutus (Y), 12- Scenedesmus dimorphus (E).


 1 Xm m ¼ ln t Xo

(1)

where Xm and Xo are the concentrations of biomass (g L
1) at the end and beginning of a batch run, respectively, and t is the duration of the run. For estimation of biomass concentration, aliquots of cultures at the beginning and end of a batch run were harvested by centrifugation at 2912 RCF for 5 min and washed twice with de-ionized water and freeze dried. D¼

0:693 m

(2)

The microalgal cultures were allowed to reach stationary phase (four weeks incubation). The biomass yield (B) (g L
1) and productivity PB (mg L-1 day
1) was calculated by Equations (3) and (4). B ¼ Xm
Xo

(3)

Xm
Xo T2
T1

(4)

PB ¼

T2
T1 represent the incubation period of an experiment where T1 is the initial time i.e. day 0 and T2 is the final day (in number) of incubation.

2.2. Nile red fluorescence for qualitative monitoring of lipid accumulation Nile red fluorescence was used for qualitative monitoring of lipid accumulation in algal cells. Aliquots of microalgae culture were mixed with nile red dye solution (100 mg L
1 in acetone) at a ratio of 100:1 to obtain a final concentration of 1 mg L
1 nile red dye [17]. To facilitate the dye penetration in cultures with thick mucilaginous sheath, an additional pretreatment step was included. The pre-treatment was either vigorous cell agitation by vortexing or heating of cell and dye mixture at 40  C or incubation with mild acid (acetic acid). Among the evaluated pretreatments, mild acid treatment at a final concentration of 7m mol L
1 was selected based on the extent of emission (relative fluorescence units, RFU) (Supplementary data, Figs. S1, S2). The incubation time with dye solution was optimized at 20 min with intermittent mixing of the cell suspension. The fluorescence emission was

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

recorded with spectrofluorometer (RF5301, Shimadzu, Japan) with an excitation wavelength of 480 nm and slit width of 10 nm. Lipid fluorescence was observed at 570 nm (Supplementary data, Fig. S3). The chlorophyll auto fluorescence of each microalgal aliquot was recorded and subtracted with emission fluorescence. The microalgal cells incubated with nile red dye were imaged under fluorescence microscopy (Olympus BX 51, Japan) with ProgRes C5 (Germany) imaging software (Supplementary data, Fig. S4).

2.3.

Lipid extraction and fatty acid composition analysis

Lipid extraction and estimation was carried out with chloroform: methanol (2:1) solvent mixture as described by Christie [21]. The lipid content (CL, % mass fraction), lipid productivity (PL, mgL-1 day
1) was calculated using Equations (5) and (6). CL ¼

WL WB

(5)

where WL and WB are the weights of the extracted lipids and of the dry algae biomass taken respectively. PL ¼

CL t

(6)

where CL is the concentration of lipids at the end of the batch run and t is the duration of the run. The fatty acid composition of the microalgal biomass was determined by converting crude lipid extracts to fatty acid methyl esters as described by Christie [21]. In brief, 5 cm3 of cold methanol containing 5% volume fraction of acetyl chloride was added to the dry lipid sample and refluxed for 2 h. After refluxing, 15 cm3 of 0.85 mol L
1 NaCl solution was added to the mixture and FAMEs were extracted in excess of hexane. The hexane layer was neutralized with equal volumes of 0.20 mol L
1 KHCO3 solution and dried over anhydrous Na2SO4. FAMEs were dissolved in HPLC grade n-hexane and 0.5 mm3 of FAME extract was injected in GC (Shimadzu 2010 plus, Japan) equipped with flame ionization detector (FID). The peaks were confirmed by GCeMS (Perkin Elmer, Turbomass Gold, Mass spectrometer, USA). A poly(dimethyl)siloxane capillary column (30 m  0.32 mm ID  0.25 mm film thickness) (Rtx-1, Restek Inc. USA) was used for FAME separation with a temperature program of 120  C (5 min hold) to 280  C (10 min hold) at a ramp rate of 5 K min
1. Nitrogen was used as carrier gas and injection port temperature was set at 220  C [17]. The FAMEs were identified by comparing their retention times with standard FAME mixture and fragmentation pattern with authentic standards (C-8 to C-24 FAME mix, SUPELCO). The fatty acids composition was expressed as relative percentage composition of total FAMEs.

2.4.

Hydrocarbon extraction, purification and profiling

Hydrocarbons were extracted from dry algal biomass (100 mg) using n-hexane as described by Dayananda et al. [22]. The crude extracts were purified using a glass column of dimensions 10 mm (diameter) x 150 mm (height) packed with silica gel (60e120 mesh, SRL, Mumbai) in n-hexane. Pure hydrocarbons were eluted with n-hexane and all other polar components such as lipids, pigments were retained in the

81

column. The extracts were dried under vacuum and estimated gravimetrically. The hydrocarbon content (CH, % mass fraction) of the biomass was measured by the Equation (6a). CH ¼

WH WB

(6a)

where WH and WB are the weights of the extracted hydrocarbons and of the dry algae biomass taken respectively. The purified extracts were analyzed for the hydrocarbon profiles by GC-FID and GCeMS equipped with Rtx-5, low polarity phase; crossbond diphenyl dimethyl polysiloxane capillary column (30 m  0.32 mm ID x 0.25 mm film thickness) (Restek Inc. USA). A gradient temperature program was followed with an initial column temperature of 130  C with 5 min hold followed by an increase in temperature to 200  C with 2 min hold at the ramp rate of 8 K min
1 and finally raised to 280  C at the rate of 5 K min
1 with a 15 min hold. Helium was used as carrier gas at a flow rate of 1 cm3 min
1. The injector port temperature was maintained at 240  C [22,23]. The hydrocarbons were identified based on their relative retention time compared to that of a standard mixture of hydrocarbons pentadecane, eicosane and triacontane (Fluka, Switzerland). Mass spectra were recorded under electron impact ionization at 70ev electron energy with a mass range from 40 to 600 Da at a rate of one scan per second. Mass spectra were identified by matching their fragmentation pattern with literature data.

2.5.

Evaluation of biodiesel quality

Biodiesel quality (fuel characteristics) such as saponification value (SV), cetane number (CN), iodine value (IV), long chain saturated factor (LCSF) and cold filter plugging point (CFPP) were determined based on the fatty acid composition of microalgal strains using empirical Equations (7)e(11) described by Shekh et al. [5]; Ramos et al. [24]; Fransisco et al. [25] and Talebi et al. [26]. SV ¼

IV ¼

X 560N M X 254DN M

CN ¼ 46:3 þ

5458
0:225IV SV

(7)

(8)

(9)

LCSF ¼ ½0:1:C16 ðwt%Þ þ 0:5:C18 ðwt%Þ þ 1:C20ðwt%Þ þ 1:5:C22ðwt%Þ þ 2:C24ðwt%Þ CFPP ¼ ð3:1417  LCSFÞ
16:477

(10) (11)

Where D, M and N denotes number of double bonds, molecular mass and % mass fraction of each fatty acid component, respectively.

2.6.

Statistical analysis

Principal Component Analysis (PCA) was performed using samples (microalgal strains) over their attributes (fatty acid methyl esters) to explore the underlying interrelationships between them. PCA biplots were generated using Statistica v

82

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5.5 [27] statistical software package. Results were expressed as the mean ± standard deviation (SD) of three replicates. Difference between the groups were statistically analyzed by using one way ANOVA followed by Tukey Kramer multiple comparison test at significance level of P < 0.05.

3.

Results and discussion

3.1.

Isolation and identification of microalgae

A germplasm of 32 microalgal strains was established following a survey covering one hundred and seventy five natural fresh water bodies located in 10 states of India during the period 2009e2011 (Table 1). Most of the fresh water habitats surveyed in the study were located in moderate to high human activity zone. Samples were collected before the onset of monsoon and in early summer coinciding with the seasonal growth triggers. To capture the biodiversity of a given water body, sub-samples were collected from different regions of a lake such as near shore region, sediment material and submerged region and were thoroughly mixed. The pH of the water bodies was either neutral or slightly alkaline between 7.5 and 8.5. Photomicrographs of representative microalgal strains are presented in Fig. 1. The strains are represented with alphabetical codes for easier identification throughout the text. Among the isolated microalgae, Scenedesmus sp. was dominant (15 strains) followed by Chlorococcum sp. (5 strains), Chlorella sp. (4 strains) and, Ankistrodesmus sp. (2 strains), Selenastrum gracile, Dictyosphaerium sp., Oocystis pusilla, Quadrigula lacustris, Kirchneriella cornuta, and Coelastrum asteroidum. Scenedesmus sp. was found to be distributed in variety of habitats such as temple ponds (high human activity), marshy waters, large water reservoirs (low human activity) indicating their ubiquitous presence and adaptation to wide temperature variations ranging from 30  C ± 3  C (Chennai, Tamil Nadu), to 25  C ± 2  C (Mysore, Karnataka) to 17  C ± 2  C (Nainital, Uttharkhand).

3.2. Growth and biomass concentration of microalgal strains The growth of the microalgal strains was monitored by measuring the specific growth rate (m day
1), which is the increase in cell number per unit time. The average specific growth rate and doubling time of microalgal strains is represented in Table 2. Higher specific growth rate is an important criteria in selection of microalgal strains as it represents a shorter doubling time. In the present study, highest specific growth rate was observed for Scenedesmus dimorphus (E), 0.105 ± 0.001; O. pusilla (BB), 0.091 ± 0.001; Q. lacustris (CC), 0.091 ± 0.0009; and lowest for Scenedesmus sp. (F, G), 0.021 ± 0.0005. The doubling time ranged from 6.6 h in S. dimorphus (E) to 33.01 h in Scenedesmus sp (F, G). Species with higher growth rate (shorter doubling time) and higher biomass productivity are essential for successful biomass production. These properties impart selective advantage such as reduced risk of contamination from competing species and high biomass yield per volume of harvest, reducing the production costs appreciably during mass cultivation [16]. However these

parameters (specific growth rate and biomass productivity), vary with growth conditions such as light intensity, culture pH, nutrient e.g., nitrogen and phosphorus concentration, external CO2 supplementation. Comparison of algal species across various cultivation conditions is beyond the scope of this study. The BBM was used as the growth medium and all the strains were incubated under uniform growth conditions as described in Section 2.1.2 above. The biomass yields of the microalgal strains in a batch run are summarized in Table 2 and biomass productivity of the strains are represented in Fig. 2. Scenedesmus sp. (A), S. dimorphus (E), Scenedesmus sp. (B), Scenedesmus sp. (K), Chlorococcum sp. (T), O. pusilla (BB), Q. lacustris (CC) were high biomass producing strains (>0.75 g L
1) under the autotrophic conditions with basal medium components containing initial nitrogen and phosphorous concentration of 2.94 m mol L
1 and 1.71 m mol L
1 respectively [28].

3.3.

Lipid content and fatty acid composition

In the present study, all the strains were cultivated under uniform growth conditions in nutrient replete BBM. The cells were harvested after four weeks when the stationary phase was reached and nile red staining showed maximum lipid accumulation. Most of the strains accumulated lipids during early stationary phase in the mid third week of incubation. The intracellular lipids were observed as golden yellow droplets on staining (Supplementary data, Fig. S4). Lipid content is generally reported as % mass fraction of dry biomass. Among the 32 microalgal strains, the lipid content varied between 8.9% in Scenedesmus sp. (A) to 40.9% mass fraction of dry biomass in Chlorella sp. (V) (Table 2) with an average lipid content of 16.79% and a median value of 16.05%. The average lipid content observed for the microalgal strains were in agreement with the range of 11%e31% mass fraction of dry biomass as observed by Griffiths and Harrison [16] and Talebi et al. [26]. In the present study, six strains viz., S. dimorphus CFR 1-05/FW, Scenedesmus obtusus CFR 1-09/FW, Chlorococum sp. CFR 2-01/FW, C. humicola CFR 2-03/FW, Chlorella sorokiniana CFR 3-01/FW, Dictyosphaerium CFR 5-01/FW showed lipid accumulation >20% mass fraction at stationary phase and were considered as high lipid producing strains. The presence of high lipid content could improve the efficiency of biomass processing resulting in higher product (biodiesel) yield and reduce the production costs significantly [12]. The feasibility of microalgal biodiesel production will be significantly affected by the average biomass productivity of strains and lipid content alone may not be a determining factor. Several authors have reported an inverse relationship between biomass productivity and lipid accumulation [12,16,17,20]. For example, Botryococcus sp. has been reported to accumulate high lipid/hydrocarbon content (>25% mass fraction) but with low growth rate and biomass productivity (34 mg L
1 day
1) [11,29] limiting its industrial feasibility. Similar observations were made in the present study with high lipid containing strains viz., Scenedesmus sp. (I), Chlorococcum sp. (P), C. vulgaris (V), Dictyosphaerium sp. (AA) and S. gracile (EE). Therefore, strains with optimal biomass productivity and lipid content would be more favorable for biodiesel

Table 2 e Growth, biomass, lipid and hydrocarbon content of microalgal isolates. Code

Scenedesmus sp.

A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA BB CC DD EE FF

Chlorococcum sp.

Chlorella sp.

Ankistrodesmus sp. Other Chlorophycean genera

Microalgal isolates

Scenedesmus sp. CFR 1-01/FW Scenedesmus sp. CFR 1-02/FW Scenedesmus sp. CFR 1-03/FW Scenedesmus sp. CFR 1-04/FW S. dimorphus CFR 1-05/FW Scenedesmus sp. CFR 1-06/FW Scenedesmus sp. CFR 1-07/FW Scenedesmus sp. CFR 1-08/FW S. obtusus CFR 1-09/FW Scenedesmus sp. CFR 1-10/FW Scenedesmus sp. CFR 1-11/FW S. perforatus CFR 1-12/FW Scenedesmus sp. CFR 1-13/FW Scenedesmus sp. CFR 1-14/FW Scenedesmus sp. CFR 1-15/FW Chlorococum sp. CFR 2-01/FW Chlorococcum sp. CFR 2-02/FW C. humicola CFR 2-03/FW Chlorococcum sp. CFR 2-04/FW Chlorococcum sp. CFR 2-05/FW C. sorokiniana CFR 3-01/FW C. vulgaris CFR 3-02/FW C. vulgaris CFR 3-03/FW C. vulgaris CFR 3-04/FW A. convolutus CFR 4-01/FW A. convolutus CFR 4-02/FW Dictyosphaerium CFR 5-01/FW Oocystis pusilla CFR 6-01/FW Quadrigula lacustris CFR 7-01/FW Kircheneriella cornuta CFR 8-01/FW Selenastrum gracile CFR 9-01/FW Coelastrum asteroidum CFR 10-01/FW

Specific growth rate (m day
1) 0.073 ± 0.068 ± 0.087 ± 0.048 ± 0.105 ± 0.021 ± 0.021 ± 0.048 ± 0.076 ± 0.072 ± 0.076 ± 0.053 ± 0.048 ± 0.053 ± 0.047 ± 0.074 ± 0.081 ± 0.038 ± 0.04 ± 0.087 ± 0.032 ± 0.046 ± 0.046 ± 0.048 ± 0.047 ± 0.043 ± 0.053 ± 0.091 ± 0.091 ± 0.054 ± 0.029 ± 0.054 ±

0.003 0.003 0.004 0.002 0.005 0.001 0.001 0.002 0.003 0.003 0.004 0.003 0.003 0.002 0.001 0.003 0.003 0.001 0.001 0.003 0.002 0.002 0.002 0.002 0.003 0.003 0.002 0.004 0.004 0.003 0.001 0.003

Doubling time (hours) 9.50 10.19 7.97 14.44 6.60 33.01 33.01 14.44 9.12 9.63 9.12 13.08 14.44 13.08 14.75 9.37 8.56 18.24 17.33 7.97 21.66 15.07 15.07 14.44 14.75 16.12 13.08 7.62 7.62 12.84 23.90 12.84

Biomass yield (g L
1) 0.76 ± 0.74 ± 0.49 ± 0.56 ± 0.74 ± 0.38 ± 0.39 ± 0.57 ± 0.51 ± 0.73 ± 0.78 ± 0.59 ± 0.39 ± 0.46 ± 0.66 ± 0.40 ± 0.58 ± 0.44 ± 0.47 ± 0.8 ± 0.25 ± 0.23 ± 0.6 ± 0.60 ± 0.61 ± 0.52 ± 0.53 ± 0.81 ± 0.95 ± 0.59 ± 0.33 ± 0.61 ±

0.034b 0.04b 0.013e 0.07d 0.02b 0.08f 0.08f 0.06d 0.03d 0.02b 0.07b 0.04c 0.02f 0.04e 0.04c 0.07ef 0.02c 0.04e 0.07e 0.02b 0.02g 0.02g 0.02c 0.06c 0.02c 0.02d 0.018d 0.02b 0.02a 0.012c 0.03f 0.032c

Total lipid content (% mass fraction of dry biomass) 8.9 11.8 13.09 20 21.6 12.6 13 12.8 32.8 10.4 11.75 10.75 10.57 10.5 15.94 24.55 16.7 22 11.86 16.33 21.00 40.29 16.17 10.25 10.3 16.74 23.95 18.1 19.8 14.51 19.76 12.65

± 1.5a ± 1.7a ± 0.29a ± 5.77c ± 1.99c ± 0.23a ± 0.8a ± 0.6a ± 3.69d ± 1.8a ± 1.6a ± 0.57a ± 0.75a ± 0.10a ± 0.9b ± 3.77c ± 0.57b ± 0.2c ± 1.86a ± 0.33b ± 2.0c ± 4.2e ± 1.7b ± 1.25a ± 0.3a ± 1.74b ± 1.85c ± 5.89b ± 3.30c ± 1.51b ± 1.76c ± 0.62a

Hydrocarbon content (% mass fraction of dry biomass) 1.81 2.1 7.98 5.5 1.69 1.23 0.56 10.5 4.16 14.66 5.44 4.8 19.89 13.08 5.47 10.86 6.6 1 0.0 17.61 10.64 14.06 3.63 11.48 9.34 7.8 6.53 18.38 4.52 2.16 11.36 10.36 8.8

± 0.01a ± 0.32a ± 0.78b ± 1.44c ± 0.09a ± 0.03a ± 0.05a ± 1.14d ± 1.45c ± 2.64e ± 0.78c ± 1.63c ± 2.19f ± 1.08e ± 0.57c ± 1.82d ± 0.87c ± 0.05a ± 2.26f ± 1.20d ± 1.06e ± 0.72c ± 1.76d ± 1.27d ± 1.66c ± 0.53c ± 2.60f ± 0.87c ± 0.96a ± 0.3d ± 0.6d ± 1.47d

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

Genus

Data represents mean ± SD of three replicates. Mean values in a column sharing common superscripts are statistically not significant at P < 0.05 by one way ANOVA. Data recorded for 28 day old culture.

83

84

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Fig. 2 e Biomass productivity (mgL-1 day¡1) of microalgal strains under autotrophic growth conditions. Strains are represented in alphabetical codes, refer Table 1 for details.

applications. A balanced parameter accounting for both biomass productivity and lipid content such as volumetric lipid productivity (mg L-1 day
1) could be applied for selection of such strains [16]. In the present study, the strains such as S. dimorphus (E), O. pusilla (BB), Q. lacustris (CC), meet the criteria of both high biomass yield and stationary phase lipid content

as indicated by their higher lipid productivity (>15 mg L
1 day
1) (Fig. 3). The lipid contents reported in the present study are obtained under nutrient replete conditions which can be further enhanced by altering the culture conditions mainly nutritional regime such as nutrient deprivation (nitrate,

Fig. 3 e Lipid productivity (mgL-1 day¡1) of microalgal strains under autotrophic growth conditions. Strains are represented in alphabetical codes, refer Table 1 for details.

85

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

phosphate) [30]. Therefore a two phase cultivation system could be suggested for improved lipid accumulation. The first stage comprising of biomass enhancing nutrient replete conditions is followed by the incubation of obtained biomass under nutrient deplete conditions for lipid enhancement [12,16,17]. Though higher lipid content and volumetric lipid productivity are important criteria for selection of microalgae for production of biofuels, the lipid quality i.e., the fatty acid composition determines the suitability of the lipid as substrate for biofuel production. Many investigations over look this phenomenon, hence a detailed evaluation of fatty acid composition was carried out in the present study. The fatty acid (FA) composition of the microalgal strains is presented in Table 3. Palmitic (C16:0), oleic (C18:1, D9) and alpha linolenic acid (ALA, C18:3, D9, D12, D15) were the predominant FA in most of the strains contributing more than 60e92.08 % mass fraction of total fatty acids except in Chlorococcum sp. (Q and R), and Scenedesmus sp. (H). The position of

double bonds in the fatty acid chain length is represented with D. Traces (less than 0.5% mass fraction of total fatty acids) of 7, 10, 13-hexadecatrienoic acid; 11, 14, 17-eicosatrienoic acid; 7hexadecenoic acid; 11-hexadecenoic acid were observed in Chlorococcum sp. (T). The concentration of lauric and myristic acid showed wide variations in FA composition among the strains. Concentration of lauric acid varied from <2% mass fraction of total FA in most of the strains to >5% in Scenedesmus strains (F, G and H), S. gracile (EE) and >15% in Chlorococcum sp (Q and R). Similar variations were observed in myristic acid distribution, with it contributing less than 3% mass fraction of total FA in most strains except in Chlorococcum sp. (Q, 22%), Chlorella sp. (W, 12%) and Ankistrodesmus sp. (Z, 13%) (Table 2). In the present study, no genus specific distribution of FA was observed. However FAME analysis could provide an insight to the strain's ecological adaptation. FA profiles could have been acquired over a prolonged exposure to a particular environmental condition [13]. For instance in the present

Table 3 e Fatty acid composition of microalgal strains. Fatty acid methyl esters (FAME)a (% mass fraction of total FAMEs)

Microalgal strains

Scenedesmus sp.

Chlorococcum sp.

Chlorella sp.

Ankistrodesmus sp. Dictyosphaerium sp. O. pusilla Q. lacustris K. cornuta S. gracile C. asteroidum Standard deviation

A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA BB CC DD EE FF

C-12:0

C-14:0

C-16:1

C-16:0

C-18:1

C-18:2

C-18:3

C-18:0

C-20:0

C:22:0

e e 1.09 2.04 e 10.91 7.77 9.36 0.46 e e e 1.39 0.98 2.13 0.73 17.22 15.21 e 0.94 e 2.95 1.81 0.72 e 1.87 e 1.37 e e 5.58 e 0.02e2.7

1.04 2.29 1.52 2.67 0.99 4.11 4.11 3.77 0.85 0.73 1.63 7.93 1.8 0.82 1.52 1.95 22.12 2.67 3.21 1.05 2.88 2.02 12 3.76 2.68 13.22 1.37 1.79 3 2.95 3.16 0.97 0.2e2.24

2.81 6.09 3.96 2.08 1.00 12.43 1.57 11.28 3.14 2.04 2.65 2.19 1.26 1.79 Traces 1.52 3.39 Traces 4.07 7.39 5.61 Traces 3.2 Traces 3.55 3.7 1.84 2.57 3.21 2.24 Traces 2.6 0.14- 1.82

28.81 41.48 39.13 35.78 49.17 18.93 29.15 17.73 30.59 40.39 25.71 43.85 32.75 27.92 29.02 27.83 20.78 35.52 33.29 20.73 51.99 45.86 36.22 28.24 46.3 27.01 27.82 39.93 49.08 56.26 26.58 31.81 1.37e5.1

23.31 16.89 22.28 11.88 23.91 27.77 24.41 9.68 36.38 33.11 17.71 26.72 36.07 36.62 43.31 26.8 8.12 8.99 7.38 17.64 15.06 46.22 13.25 32.5 6.76 24.67 64.08 25.0 36.87 19.11 39.59 47.92 0.67e4.86

14.14 8.62 14.66 Traces 8.5 0.52 11.26 11.85 14.81 3.77 10.95 5.74 8.7 10.16 2.89 10.64 5.2 7.86 9.77 18.95 8.49 Traces 19.19 5.66 12.38 4.99 Traces Traces 2.02 5.15 4.27 2.06 0.2e1.86

27.02 21.0 11.43 28.15 18.00 13.53 11.87 22.04 7.8 13.78 39.04 8.38 13.85 13.99 18.08 21.45 5.55 14.69 27.69 29.89 11.39 Traces 14.3 26.91 24.39 16.86 Traces 23.21 Traces 9.94 17.11 11.61 0.5e2.99

2.43 3.01 2.64 5.42 e e e e 3.08 5.26 1.87 5.18 4.17 5.17 e 4.68 3.91 e 5.5 2.46 3.59 e e e 3.37 e 4.47 3.81 3.33 4.31 e 5.5 0.13e0.85

e e e e e

e e e e e e

5.33 3.81 4.74 e e e e e 0.4 0.15

4.67 4.07 2.24 e e e e 0.42 0.15

e e 5.45 e e e 1.29 e e e e e e e e 0.56 e 0.18e0.45

e 13.71 2.72 e e e 0.37 e e e 2.22 e e e e 0.37 e 0.02e1.71

Values for trace fatty acids such as C14:1, C20:1 and C24:0 are not represented in the table. Fatty acids detected less than 0.5% mass fraction of total FAMEs are considered as traces. Data expressed as mean ± SD (n ¼ 3). Data recorded for 28 day old culture. a Only major fatty acids are expressed herein.

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study two strains viz., Scenedesmus sp. (K) and Chlorococcum sp (T) isolated from high altitude Nainital lake, Nainital, Uttarakhand (Refer Table 1) showed high ALA content (Table 2). This could be attributed to the prolonged exposure of the strains to colder conditions where the temperatures range between 5  C and 18  C in winter. Guschina and Harwood [31] reported increased unsaturation of FA in algae grown under colder conditions (10  Ce25  C). Thus FA profiling of algal strains could provide important information on a strain's ecological adaptation and application potential.

3.4.

PCA of FAMEs

PC analysis was performed using FAME composition data to understand the fatty acid distribution among various strains. The FA distribution is represented as PCA biplots for Scenedesmus strains (Fig. 4a) and other strains Fig. 4b. From a total of 13 fatty acids detected among algal strains, only 6 fatty acids contributed to the major variations in the fatty acid composition. Based on these variations, 4 distinctly different groups were identified viz., ALA, oleic acid, palmitic acid and lauric/myristic/arachidic acid groups. Remaining fatty acids such as C16:1, C24:0, C14:1, C18:0 were considered insignificant variables. The principal axis 1 (PC 1) explained about 28.6% of the variance followed by PC-2 which explained about 16.44% of the variance. ALA group included Chlorococcum sp. (S, T), Scenedesmus sp. (A, B, D and K) and Ankistrodesmus convolutus (Y). The Oleic acid group included Dictyosphaerium sp. (AA), S. gracile (EE), C. asteroidum (FF), Chlorella sp. (V) and Scenedesmus sp. (O). The Palmitic acid group was comprised of K. cornuta (DD), Q. lacustris (CC), Chlorella sp. (U), and Scenedesmus sp. (J, L). The lauric/myristic/arachidic acid group was constituted by Chlorococcum sp. (Q, R) and Scenedesmus sp. (F, G, and H).

3.5.

Hydrocarbon analysis of microalgal strains

Direct use of hydrocarbon extracts reduces the cost associated with energy consumption during transesterification of lipids to fatty acid alkyl esters. Further, alkyl esters of fatty acids have relatively poor oxidative stability and poor cold flow properties [32,33]. Hence, identifying fuel quality hydrocarbons in microalgae becomes important. The hydrocarbon extracts of microalgae can be directly blended with petroleum diesel or can be used as an additive to diesel or aviation fuel at optimal levels without affecting the fuel properties. In addition, the hydrocarbons could be converted to gasoline by hydrocracking [6]. The hydrocarbon content was highest in Scenedesmus sp. (M) and Chlorococcum sp. (S) with 19.8% and 18% mass fraction of total biomass, respectively. Hydrocarbon content showed wide variation among the Scenedesmus isolates, ranging from 19% (M) to 2% (A) mass fraction of biomass (Table 2). Based on the carbon chain length, the hydrocarbons were grouped into four categories viz., C30 with reference to the retention time of the standard hydrocarbons pentadecane, eicosane, triacontane [22]. The hydrocarbon composition of microalgal isolates as analyzed by GCeMS is presented in Table 4. The analysis revealed straight chain alkanes (n-paraffins) of chain length between C15 to C20 such

as hexadecane (C16), heptadecane (C17), octadecane (C18), nonadecane (C19), eicosane (C20) as predominant in all the strains. Among the higher chain hydrocarbons, heneicosane (C21), docosane (C22), tricosane (C23), tetracosane (C24), pentacosane (C25) and heptacosane (C27) were detected. Interestingly branched isoprenoid hydrocarbons such as 2, 6, 10, 14 - tetramethyl pentadecane (pristane), 2, 6, 10, 14 - tetramethyl hexadecane (phytane) and 2, 6, 10, 14- tetramethyl heptadecane were detected only in Scenedesmus sp. (M) contributing up to 42% mass fraction of total hydrocarbons. On the other hand, strains such as Scenedesmus sp. (F, G), Kirchneriella sp. (DD) contained higher proportions of short chain hydrocarbons mainly C14 to C16 contributing to 60% mass fraction of the total hydrocarbons. Kirchneriella sp. (DD) showed highest C14 content (40.1%) compared to other strains. Further to the branched hydrocarbons, traces (less than 1% mass fraction of total hydrocarbons) of long chain alcohols such as 1-hexacosanol, 1-heptacosanol, 1-eicosanol and 2-butyl-1-octanol and monounsaturated alkenes such as 11-tricosene, 1-hexacosene and 10-heneicosene were detected in hydrocarbon fractions of microalgae. Similar type of saturated linear hydrocarbons ranging between C12 and C32 and branched hydrocarbons ranging between C14 to C28 were reported in B. braunii by Dayananda et al. [34]; Banerjee et al. [10] and Volova et al. [35]. An important observation during GC-FID profiling of hydrocarbon extracts was the detection of plasticizer phthalate as an artefact. Use of plastic-ware was minimized during extraction and sample preparation steps to avoid interference of plasticizer (Supplementary data, Fig. S5). The wide variation in the hydrocarbon content among the isolates from same genera can be attributed to the physiological adaptations of the alga to different geographical locations. In case of widely studied alga B. braunii, the hydrocarbon content has been reported in the range of 2.0%e86.0 % mass fraction of dry biomass due to the differences in the strains, the race it belongs to, cultural and physiological conditions [36,37].

3.6. Comparison of microalgae derived biofuels (FAME/ hydrocarbons) with international standards Fatty acid structure and composition influence the quality characteristics of biodiesel [38]. Some of the essential quality parameters of biodiesel such as CN, IV and CFPP indicate the ignition quality, oxidative stability and cold flow properties respectively. The fuel properties of microalgal FAMEs are presented in Table 5. The CN value of most of the microalgal FAMEs meet the international standards of minimum 51 [39]. This could be attributed to the presence of higher proportions of SFA, mainly palmitic acid. However higher SFA ratios affect the flow properties causing crystallization/solidification of fuel, blocking the fuel filters of engine under colder climatic conditions [32]. Therefore the lowest acceptable temperature limit for CFPP is recommended at
5  C to
13  C [24,40]. With reference to above mentioned CFPP limits most of the microalgal FAMEs in the present study failed to meet this criteria. However, presence of PUFAs may improve the cold flow properties. Although beneficial in terms of flow properties, higher proportions of PUFAs affect the oxidative stability of

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

87

Fig. 4 e PCA biplots for the distribution of microalgal strains in relation to their FAME composition. Scenedesmus sp. (4A), other microalgal genera (4B). Strains are represented in alphabetical codes, refer Table 1 for details.

88

b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

Table 4 e Hydrocarbon composition of microalgae strains. Hydrocarbon compositiona (% mass fraction of total hydrocarbons)

Microalgal strains

Scenedesmus sp.

Chlorococcum sp.

Chlorella sp.

Ankistrodesmus sp. Dictyosphaerium sp. O. pusilla Q. lacustris K. cornuta S. gracile C. asteroidum

A B C D E F G H I J K L Mb N O P Q R S T U V W X Z AA BB CC DD EE FF

C14

C15

C16

C17

C18

C19

C20

C21

C22

C23

C24

C27

2.40 5.82 10.15 6.12 7.36 20.46 20.10 4.08 2.11 4.18 5.62 4.39 2.17 5.04 e 6.42 7.77 6.42 5.10 6.41 5.94 7.54 9.97 6.13 1.73 2.17 1.51 1.81 40.12 13.18 3.44

7.03 25.57 20.49 17.70 21.25 20.67 22.31 10.09 6.24 16.83 9.02 8.26 8.75 7.86 28.83 9.26 10.10 9.21 7.57 9.91 9.38 8.83 20.06 26.65 6.33 19.68 5.68 10.56 e 14.19 7.49

10.58 0.00 8.42 6.33 4.92 e 20.55 16.62 11.34 32.11 11.53 11.49 16.69 11.02 e 13.25 12.64 12.36 11.03 12.51 11.84 12.73 e 6.26 10.66 17.52 10.70 12.96 23.48 10.03 11.26

16.60 17.36 13.70 15.89 15.65 e 37.03 10.51 13.22 2.31 11.95 14.57 1.21 15.77 18.07 2.54 13.51 11.01 14.95 13.04 12.52 12.00 12.88 20.19 12.35 e 14.69 9.01 23.92 6.72 11.05

12.11 7.50 5.94 8.52 7.32 e e 9.46 12.35 2.09 11.71 11.68 1.09 11.95 3.89 14.66 12.28 12.88 11.96 12.63 11.60 11.57 6.12 20.47 12.65 17.52 12.22 14.13 9.02 4.43 13.69

11.52 18.05 15.38 14.98 16.14 e e 9.00 11.63 2.09 10.91 11.34 e 9.84 16.77 11.07 11.36 8.63 9.05 3.04 10.58 10.44 11.82 20.31 12.30 17.32 11.81 13.81 e 8.87 10.95

11.31 12.74 13.49 12.52 12.92 21.49 e 9.15 11.39 2.44 11.70 11.06 1.26 11.25 13.88 13.52 11.18 11.86 10.89 11.83 11.24 10.78 22.19 e 12.43 11.28 11.39 11.70 3.39 22.41 13.00

9.95 12.96 12.44 11.78 12.39 e e 7.13 9.73 e 9.29 9.51

8.16 e e 2.72 0.91 e e 5.92 8.00 e 7.97 7.71

6.21 e e 2.07 0.69 e e 4.41 5.96 e 6.00 5.70

8.67 6.09 9.63 9.44 9.26 7.92 10.05 9.19 8.86 6.00 e 10.74 14.51 9.81 11.69 e 5.74 9.67

7.77 12.47 9.02 7.72 8.09 7.36 8.28 7.72 7.49 10.95 e 9.04 e 7.94 5.66 e 9.95 8.85

5.36 e 5.99 e 4.27 4.95 6.13 5.79 5.43 e e 6.91 e 5.93 4.28 e e 6.09

3.67 e e 1.22 0.41 e e 7.23 4.15 16.55 4.29 3.93 8.62 4.03 e 4.63 4.01 4.21 3.80 4.41 4.21 3.90 e e 4.86 e 4.08 2.98 e 4.49 4.51

0.46 e e 0.15 0.05 e e 1.06 3.88 e e 0.35 11.12 1.43 e e e 1.80 5.43 1.76 e 0.43 e e e e 4.23 1.41 e e e

Strains are represented in alphabetical codes (refer Table 1 for codes). Data recorded for 28 day old culture. a - Prominent hydrocarbons as confirmed by GCeMS and values are expressed as % mass fraction of total hydrocarbons. b Branched hydrocarbons were detected only in Scenedesmus sp. (M) - 2, 6, 10, 14, Tetramethyl pentadecane (17.38%); 2, 6, 10, 14, Tetramethyl hexadecane (18.15%); 2, 6, 10, 14, Tetramethyl heptadecane (6.28%); Hexatricontane (7.29%).

fuel as indicated by IV [41]. Strains belonging to ALA group (from PCA) such as Scenedesmus sp. (A, K), Chlorococcum sp. (T) have high IV than recommended international standards of 120 gI2100 g
1 lipid owing to higher proportions of ALA (>12% mass fraction of total FAME) [UNE 14214 39]. With due considerations to fuel quality in terms of ignition, cold flow properties and oxidative stability, an ideal composition of SFA and MUFA is essential. For example, methyl esters of fatty acids C14:0, C16:1 and C18:1 in a ratio 5:4:1 would be ideal in meeting international biodiesel standards [42]. However such ratios may not occur naturally in microalgal lipids, thus requiring blending of FAMEs obtained from different strains. Based on the fatty acid composition of microalgae and PCA in the present study, strains belonging to oleic acid group such as S. dimorphus (E), O. pusilla (BB), C. asteroidum (FF), C. vulgaris (V) may be more suitable for biofuel production. This may be because, higher quantities of methyl oleate or its addition has been suggested to improve the properties of biodiesel fuel, mainly oxidative stability and low melting temperature [29,43]. Further, oleic acid has been

identified as the precursor of the non-isoprenoid hydrocarbons in certain microalgae such as Botryococcus [44]. In addition to FAME, the present study indicated that hydrocarbon fractions of microalgae were similar to n-paraffinic fraction of diesel where the hydrocarbons, mainly paraffins (both branched and linear) of chain length between C9 and C25 were predominant [45]. However diesel contains many other cyclic and polar compounds such as napthalenes, alkyl benzenes, alkyl napthalenes and aromatics which were not detected in microalgae. The hydrocarbons from microalgae could be targeted primarily as fuel additives for improving the fuel properties mainly CN and oxidative stability. Blending of medium chain saturated alkanes such as tetradecane, pentadecane or hexadecane to PUFA rich biodiesel would be ideal for improving the oxidative stability. Secondly, these hydrocarbons can be converted to gasoline by hydrocracking or blended directly with diesel or jet fuels [46,47]. The quality of microalgae derived biofuel (FAME derived biodiesel and hydrocarbon extracts) in the present study are acceptable as per international standards for vegetable oil based biodiesel

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b i o m a s s a n d b i o e n e r g y 7 7 ( 2 0 1 5 ) 7 5 e9 1

Table 5 e Biodiesel fuel properties of microalgal strains. Strain

Scenedesmus sp.

Chlorococcum sp.

Chlorella sp.

Ankistrodesmus sp. Other Chlorophycean Genera

A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA BB CC DD EE FF

SFA % Mass fraction of FAMEs

MUFA % Mass fraction of FAMEs

PUFA % Mass fraction of FAMEs

SV

32.28 46.78 44.37 45.91 50.17 39.28 49.51 39.67 37.23 46.38 29.21 56.96 40.11 35.71 34.49 35.18 77.74 61.55 42.00 25.64 58.46 52.49 50.03 32.72 52.35 46.14 33.66 46.90 55.42 63.52 38.11 38.28

26.59 23.60 26.24 14.54 24.91 40.20 25.98 23.15 40.16 35.94 20.78 28.91 37.33 40.13 44.52 28.32 11.51 11.48 11.45 25.47 21.65 46.59 16.45 32.50 10.89 28.92 66.34 27.91 40.08 21.35 40.52 50.87

41.14 29.62 26.10 28.15 26.50 14.05 23.13 33.89 22.61 17.55 49.99 14.12 22.55 24.15 20.97 32.09 10.75 22.55 37.46 48.84 19.89 0.00 33.49 32.57 36.76 21.79 0.00 23.20 2.02 15.09 21.37 13.67

196.16 199.47 192.42 177.76 201.99 195.33 197.57 194.32 195.69 197.37 196.03 200.83 197.24 196.12 195.64 187.86 211.02 196.91 180.68 196.11 201.44 197.99 203.02 192.70 200.12 194.33 195.00 195.33 194.99 200.98 198.23 201.31

International Standardsa

IV gI2100 g
1 lipid 117.83 90.46 77.91 86.09 82.98 71.69 72.74 98.34 80.68 73.53 138.71 56.69 83.18 88.76 90.02 98.62 33.58 61.25 99.16 133.12 63.57 39.85 84.73 107.71 94.64 77.39 57.08 84.65 38.11 53.27 86.47 77.68 Max. 120

CN

LCSF % Mass fraction of FAMEs

CFPP  C

47.61 53.31 57.13 57.63 54.65 58.11 57.56 52.26 56.04 57.41 42.93 60.72 55.26 54.16 53.94 53.17 64.61 60.24 54.20 44.18 59.09 64.90 54.12 50.39 52.28 56.97 61.45 55.20 65.72 61.47 54.38 55.93 Min. 51

4.09 5.65 5.23 6.29 4.92 10.76 13.73 12.62 7.96 6.67 3.51 6.98 5.36 6.41 6.32 5.12 24.60 13.05 6.08 4.22 6.99 6.43 3.62 2.82 6.31 9.67 5.02 5.90 6.57 7.78 7.49 5.93


3.61 1.29
0.04 3.28
1.03 17.34 26.66 23.16 8.53 4.47
5.46 5.44 0.36 3.65 3.37
0.39 60.80 24.53 2.62
3.21 5.50 3.73
5.10
7.60 3.36 13.91
0.72 2.05 4.18 7.97 7.06 2.16
5 to
13b

SFA - Saturated Fatty Acids, MUFA - Monounsaturated fatty acids, PUFA - Polyunsaturated fatty acids, SV - Saponification value, IV - Iodine value, CN - Cetane Number, LCSF - Long Chain Saturation Factor, CFPP - Cold Filter Plugging Point. Values are average of three replicates. Strains are represented in alphabetical codes (for details refer Table 1). Data recorded based on FAME profile obtained from 28 days old culture. a Based on UNE eEN 14214, b Based on Ramos et al. [24] and Islam et al. [40].

except for poor cold flow properties. From the present study it can be concluded that microalgal biofuels could be an alternative to vegetable oil derived biodiesel.

4.

Conclusion

The study led to the establishment of a germplasm of freshwater chlorophycean microalgae for biofuel application. The strains were screened for growth, biomass, lipid content, productivity and fuel quality under autotrophic growth conditions. Three best strains viz., S. dimorphus (E), Q. lacustris (CC) and O. pusilla (BB) were identified in terms of shorter doubling time (6.6 he7.6 h), higher biomass productivity (>73 mg L
1 day
1), lipid content (>20% mass fraction) and volumetric lipid productivity (>15 mg L
1 day
1). The fatty acid profiles of these strains were rich in saturated (palmitic) and

monounsaturated fatty acid (oleic acid) indicating the potential of their lipids as biodiesel substrate. The strains were also characterized for fuel grade hydrocarbons. The medium chain (C15 to C20) saturated alkanes and isoparaffins constituted the major hydrocarbon fraction in all the strains. The similarity of microalgal hydrocarbon profile to commercial diesel indicates their suitability as additives to transportation fuel and for blending.

Acknowledgments Financial support (KDMIPE/CR/GC/BIOFUEL/2(389)2009) from KDMIPE-ONGC is gratefully acknowledged. Authors acknowledge Prof. Hosmani, Dept. of Biotechnology, Mahajana's College, Mysore and Dr. Krishnamurthy, Head,

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Krishnamurthy Institute of Algology, Chennai for the help extended in identification of algal species. The authors thank Director, CSIR-CFTRI for encouragement and support. Dr. S.R.Bhowmik, HOI, Dr. R.R.Singh, GM Geochemistry and Dr.A.K.Jain DGM Chemistry, ONGC- KDMIPE, Dehradun are gratefully acknowledged for their constant support and encouragement. VS, GVS and MDK acknowledge the financial support by UGC, CSIR and ICMR, Govt. of India respectively, in the form of senior research fellowship.

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biombioe.2015.03.001.

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