Insights into the Enhanced Lipid Production Characteristics of a Fresh

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Insights into the Enhanced Lipid Production Characteristics of a Fresh Water Microalga under High Salinity Conditions Neha Arora,† Alok Patel,† Meenakshi Sharma,† Juhi Mehtani,† Parul A. Pruthi,† Vikas Pruthi,*,† and Krishna Mohan Poluri*,†,‡ †

Department of Biotechnology, and ‡Center for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India S Supporting Information *

ABSTRACT: Bioprospecting of microalgae capable of growing and accumulating high amounts of lipids in high salinity conditions such as seawater can substantially improve the economic vaibaility of algal biodiesel production. In view of this, a fresh water microalga, Scenedesmus sp. IITRIND2, was cultivated under saline conditions to assess its halotolerant behavior and potential as biodiesel feedstock. The microalga efficiently adapted to 100% seawater salinity, enhanced its lipid content by 52%, thus yielded ∼3.2 fold higher lipid productivity as compared to the Bold’s basal media (BBM). The increase in the lipid content was balanced by a sharp decrease in its protein and carbohydrate content. Biochemical analysis evidenced that salinity induced oxidative stress resulted in reduced levels of photosynthetic pigments, elevated levels of reactive oxygen species (H2O2, thiobarbituric acid reactive substances), osmolytes (proline, glycine betaine), and activity of antioxidant enzymes (catalase, ascorbate peroxidase). These studies suggested that microalga efficiently modulated its metabolic flexibility in order to acclatamize the salanity induced stress. Further, the FAME analysis revealed the dominance of C14:0, C16:0, C18:0, C18:1, and C18:2 fatty acids under halotolerant conditions, and the properties of the resulting biodiesel were in compliance with ASTM (American Society for Testing Materials) D6751 and EN 14214 (European) fuel standards. These results consolidate that the lipid augmented halotolerant algal strains capable of growing in saline/seawater can be formulated as environmental sustainable and economic viable sources for biodiesel production.

1. INTRODUCTION Microalgae are one of the most diverse group of species that have gained immense interest due to their potential use in biofuel industry.1 Oleaginous microalgae are green gold mines for producing bio-oil as they can accumulate significant amounts of triglycerides (TAGs) in their lipid bodies.2,3 The biodiesel derived from microalgae lipid is nontoxic, biodegradable and contributes to no net CO2 or sulfur emissions.4 Their emergence as propitious feedstocks for biodiesel production can be owed to their ease of cultivation and large scale deployment. Nevertheless, the high cost associated with large scale production infers a long road ahead for their commercialization as sustainable biodiesel.5 To bridge this gap, various efforts are being made by using low cost feed stocks such as waste/sea waters, metobolically altered algal strains, enhancing lipid concentrations by using environmental stress factors etc.6−8 Among those, bioprospecting of high TAG accumulating strains capable of growing in sea/waste waters is of central attention. The unique ability of microalgae to grow © XXXX American Chemical Society

on such waters can reduce their dependence on fresh water reserves and nutrients, thus making the biodiesel production environmentally sustainable and economically viable.6 Moreover, utilization of seawater for cultivation of microalgae reduces the contamination risk and competition by other microorganisms compared to fresh and waste waters. Nonhalophilic microorganisms such as algae that can grow in the absence as well in the presence of salts are designated as halotolerant. These halotolerants have a great metabolic flexibility to cope and adapt to high saline environments.9 They achieve this by accumulating high amounts of organic solutes to maintain the osmotic balance.9 Once exposed to salinity, microalgal cells restore turgor pressure by eliminating Na+, accumulating K+ while maintaining the intracellular level Received: February 27, 2017 Revised: May 18, 2017 Accepted: June 7, 2017

A

DOI: 10.1021/acs.iecr.7b00841 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research of Ca2+ constant. Accumulation of metabolites such as glycerol, fructose, sucrose, and trehalose maintains osmolality, while charged molecules including proline and glycine betaine readjust the osmotic equilibrium by preventing water loss.10−13 Such a readjustment of metabolites within microalgae generates stress, resulting in the accumulation of large amounts of either carbohydrate or lipid content for their survival. The carbohydrate accumulated in the microalgal cells is an important energy source and can be potentially used for bioethanol, biohydrogen, and bioplastic generation.14,15 However, the strains with incremented lipid contents can act as an energy reserve material and considered to be the novel candidates for producing algal oils/biodiesel.3,7,8,11 Hence, understanding the mechanism of enhanced lipid accumulation in response to salinity stress would provide useful insights on key regulators of lipid metabolism and halotolerance in microalgae, leading to directed pathway engineering for reducing biodiesel production costs. To this end, a fresh water oleaginous microalga Scenedesmus sp. IITRIND2 (GenBank accession no. KT932960) was cultivated in artificial seawater (ASW) to test its halotolerance capabilities. The potential of the novel microalga was analyzed by estimating its biomass, lipid productivity, and subsequent biodiesel production. The results obtained were further correlated with the alterations observed in the cell size, photosynthetic pigments, lipid, carbohydrate, protein content, stress related enzymes, and osmolytes under saline conditions to shed light on the adaptive strategies of microalga. The results unveil that the metabolic alterations occurred in the microalga when exposed to seawater salinity commensurate with enhanced TAG accumulation with an acceptable fatty acid profile for biodiesel production. The study suggests that adaptation mechanism of microalgae under high saline conditions can be exploited to deploy seawater as an economically viable feed stock for biodiesel production.

kept constant, and pH was set at 7.2−7.4. Modified BBM was used as control. The detailed recipe of the different ASW (%) is listed in Table S1 in Supporting Information. The microalga were pregrown for a period of 4 days in 250 mL shake flasks in different concentrations of ASW in order to acclimitize the cells to the different salinity. The seed cultures were taken from the respective flasks and then inoculated in 1 L shake flasks in triplicates with initial inoculum of 0.02 g/L (fresh weight) for 7 days. All flasks were shaken periodically after every 6 h on a magnetic stirrer for 20 min at 130 rpm. Biomass and Lipid Productivity Estimation. Microalga growth phases were monitored after every 24 h by measuring the absorbance at 750 nm. The dry cell weight (DCW; g/L) was calculated by harvesting the cells (centrifuged at 5000 rpm for 10 min at 25 °C) and washing the pellet thrice with distilled water to remove any medium components. The pellet was then dried at 60 °C for 24 h and gravimetrically measured to obtain the DCW. Biomass productivity ((mg/L)/d) was calculated by the following equation: Biomass productivity =

Final DCW − Initial DCW Cultivation time

Lipid accumulation was visualized by Nile red staining. Briefly, microalga cells suspension (200 μL) was incubated with 10 μL of Nile red (0.1 mg/mL in dimethyl sulfoxide) for 15 min in the dark. The cell suspension was then centrifuged; the pellet obtained was washed thrice with 0.9% saline solution and visualized under a fluorescent microscope (EVOS-FL, Advance Microscopy Group, AMG, USA) equipped with the RFP light cube. Lipid was extracted from the cells cultivated in ASW and BBM after 7 days of inoculation using earlier established protocol.18 Lipid productivity ((mg/L)/d) was calculated using the following equation: Lipid productivity

2. MATERIALS AND METHODS 2.1. Materials. Chemicals used for the preparation of artificial seawater (ASW) and modified Bold’s basal media (BBM) were purchased from Himedia, India. All solvents and reagents were HPLC grade. Nile red stain (9-diethylamino-5Hbenzo[α]phenoxazine-5-one) was procured from Invitrogen (Life Technology, USA) 2.2. Experimental Design. Algal Strain Cultivation. Scenedesmus sp. IITRIND2 (GenBank accession no. KT932960) isolated previously by our group from a fresh water lake was used to investigate effects of ASW on its growth and lipid accumulation.16 Initially, the microalga was adapted to ASW and was maintained at 25 °C, at 130 rpm with photoperiod of 18:6 h light/dark cycle of 7 days under white light illumination (200 (μmol/m2)/s). The modified ASW had the following composition (g/L): 6.29 MgSO4·7H2O, 1.0 NaNO3, 0.07 KH2PO4, 0.18 NaHCO3, 0.098 KBr, 0.026 H3BO3, 0.003 NaF with 4.66 MgCl2·6H2O, 1.02 CaCl2·2H2O, 28.65 NaCl, 0.67 KCl as sea salts, respectively.17 The medium was vacuum filtered using 0.45 μm syringe filters to avoid precipitation of sea salts. Microalgal Cultivation. To understand the resistance to salinity stress and evaluate the maximum lipid productivity of microalga in brackish water and seawater, cells were grown in four different salinity percentages of ASW (0, 30, 50, 80, and 100%) corresponding to 0, 10.5, 17.5, 28, and 35 g/L of sea salts, respectively. The rest of the nutrients concentrations were

=

(Final lipid concentration − Initial lipid concentration) Cultivation time

2.3. Estimation of Fatty Acid Profile and Biodiesel Properties. Fatty acid methyl esters (FAMEs) were obtained by transesterification of the total lipid extracted using 6% H 2 SO 4 . 18 The FAMEs were then analyzed using gas chromatography−mass spectroscopy (GC−MS; Agilent Technologies, USA) with electron ionization (70 eV), DB-5 capillary column (30 mm × 0.25 mm × 1 μm) using helium (1 mL/ min) as carrier gas.16 The biodiesel physical properties including iodine value (IV), saponification value (SV), cetane number (CN), degree of unsaturation (DU), long chain saturation factor (LCSF), high heating value (HHV), cold filter plugging property (CFPP), kinematic viscosity (kV), density (D), and oxidative stability (OS), which determine the vehicular quality of biodiesel, were evaluated using derived empirical formulas (Table S2).18 2.4. Cell Size and Biochemical Composition Estimation. Cell diameters of approximately 100 microalga cells were measured using “ImageJ 1.49a” software. Nitrogen content was estimated using an elemental CHNS elemental analyzer (Thermo Fischer, USA). Crude protein was estimated using the following equation: Crude protein (%) = 6.25 × Total nitrogen (%)

Total carbohydrate in the lipid extracted microalga sample was estimated using phenol sulfuric acid method.19 Briefly, 100 B


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Figure 1. (A) Growth curve. (B) Biomass productivity ((mg/L)/d) and lipid productivity ((mg/L)/d) of Scenedesmus sp. IITRIND2 grown in different percentages of artificial seawater (ASW) and BBM for a period of 7 days.

1 M). Absorbance at 390 nm was recorded, and H2O2 content was expressed as l mol of H2O2 per gram of fresh weight (FW).21 The calibration curve was obtained using H2O2 (0−14 nmol) standard solutions, prepared in 0.1% TCA. Lipid peroxidation was determined in terms of thiobarbituric acid reacting substance (TBARS) as per the protocol described by Tian and Yu, 2009.22 The standard curve for TBARS estimation was generated by using 1,1,3,3-tetraethoxypropane (TMP) in 0.2−20 μM concentration range. TBARS content was measured by using the following formula:

mg of lipid extracted biomass was treated with 2% H2SO4 and autoclaved at 120 °C for 30 min. The cell suspension was then centrifuged, and the supernatant obtained was used for estimation of sugar using glucose as standard. Pigments (chlorophyll a, chlorophyll b, and carotenoids) in microalgae cultivated in ASW after 7 days (early stationary phase) were estimated using the protocol of Lichtenthaler, 1987.20 Briefly, 2 mL of cell suspension was harvested and then the pellet was suspended in 2 mL of methanol (99%) and incubated for 24 h at 45 °C. The supernatant was used for estimation of pigments (μmol) using the following equations:

TBARS content = (A532 − A 600)EC

μg ⎞ ⎛ ⎟ = 16.72A Chlorophyll a ⎜Chl a; 665.2 − 9.16A 652.4 ⎝ mL ⎠

where A is the absorbance in nm and EC is the extinction coefficient (1.56 × 105 M/cm). Osmolytes. Total proline in microalgal cells grown in ASW was extracted using 3% sulfosalicyclic acid and estimated using L-proline as standard. The optical density was measured at 520 nm for determining total proline content in cells.23 Glycine betaine content in microalga was estimated by taking 0.5 g cells and homogenizing it in equal volumes (200 μL) of deionized water, 2 N H2SO4 and incubated in ice bath for 2 h. After incubation, 200 μL of cold KI−I2 reagent (1.75 g of I2 and 2 g of KI in 10 mL of deionized water) was added and kept at 4 °C overnight. The cell suspension was then centrifuged at 10 000 rpm for 10 min and the supernatant was removed. Betaine periodic complexes formed were extracted by 1,2-dichloroethane and incubated in dark for 2 h. Absorbance at 365 nm was measured using glycine betaine as standard.24 All the photosynthetic pigments, ROS, and osmolytes were calculated using per gram of fresh weight (FW). Antioxidant Enzymes. To estimate the antioxidant potential of microalga grown in ASW, enzymatic extract was prepared and activities of assay catalase (CAT) and ascorbate peroxidase (APX) were recorded.25 CAT activity was determined by mixing 0.1 mL of enzymatic extract with 3% H2O2 and phosphate buffer (pH 7.0) and measuring the change in the

μg ⎞ ⎛ ⎟ = 34.09A Chlorophyll b ⎜Chl b; 652.4 − 15.28A 665.2 ⎝ mL ⎠

⎛ μg ⎞ ⎟ Caratenoids ⎜ ⎝ mL ⎠ = (1000A470 − 1.63 Chl a − 104.9 Chl b)/221 Pigments in extract (μmol) =

Pigments in the extract (μg) Molecular weight of the pigment

where standard molecular weight of chlorophyll a, chlorophyll b, and carotenoids were 894, 908, and 570 g/mol, respectively. 2.5. Stress Metabolites Estimation. Reactive Oxygen Species (ROS). To estimate the ROS content in microalgae after 7 days grown in ASW, changes in H2O2 and lipid peroxidation were recorded. For H2O2 estimation, the cell suspension was centrifuged at 5000 rpm for 10 min. Cell pellet (0.5 g) was homogenized with 0.1% trichloroacetic acid (TCA; w/v) and centrifuged at 10 000 rpm for 10 min. Supernatant (0.5 mL) obtained was mixed with 0.5 mL of 10 mM phosphate buffer saline (PBS), pH 7.0, and 1 mL of potassium iodide (KI; C


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Industrial & Engineering Chemistry Research initial rate of disappearance of H2O2 at 240 nm for 150 s using an extinction coefficient of 0.0436 mM/cm. APX activity was estimated by monitoring changes in absorbance at 290 nm and using an extinction coefficient of 2.8 mM/cm.25 One unit of the enzyme activity was defined as the enzyme amount that transforms 1 μmol of the substrate per minute. H2O2 and ascorbate were used as substrate for measuring CAT and APX activities. The enzyme specific activity (U/mg protein) was calculated using the following equation: Specific activity =

{(Change in OD per min) × 1000 × Reaction volume} EC × Volume of enzyme in sample × Protein concn

The reaction volume used was 3 mL, while the volume of enzyme was 0.1 mL. 2.6. Statistical Analysis. The experiments were carried out in triplicates (n = 3), and all the data values are expressed as the mean ± standard deviation. Figure 2. Scenedesmus sp. IITRIND2 viewed under a light microscope (panel I); and visualized by epifluorescent microscope using Nile red staining (panel II) on the 7th day.

3. RESULTS AND DISCUSSION 3.1. Dry Cell Weight (DCW), Biomass Productivity, and Lipid Productivity. Microalgae can alter their metabolic infrastructure in order to acclimatize to various adverse environments ranging from physiological (nutrient, heavy metal, temperature, pH) to operational (batch, contionous, fed-batch or two stage).2,3,26 Bioprospecting of novel microalgal strains capable of growing in waste/seawater along with enhanced lipid accumulation can pave a path for commercialization of algal oils. Thus, to gain mechanistic insights on the lipid accumulation of microalgae under high salinity stress condition, it is imperative to use sea waters as a sustainable source, as this would advance our knowledge in both the applied and basic research areas of algal biodiesel production. Given this interest, Scenedesmus sp. IITRIND2 was cultured in ASW with different sea salt concentrations (0, 30, 50, 80, and 100%) and evaluated DCW, biomass productivity, and lipid productivity as compared to BBM. An increase in optical density (OD) was recorded in microalgal cells grown in ASW as compared to BBM with two distinct growth phases, namely, log phase (1−5 days) and early stationary phase (5−7 days) as shown in Figure 1A. Further an increase in the sea salts concentration from 0 to 35 g/L, the cells showed a negative growth rate (decrease in OD), which could be easily visualized by the change in the color of cultures from dark green to light green (Figure 1A and Figure S1). Maximum DCW (1.7 ± 0.3 g/L) and biomass productivity (236 ± 4 (mg/L)/d) were obtained in cells cultivated in 0 g/L sea salts as compared to BBM (0.8 ± 0.1 g/L; 111 ± 4 (mg/L)/d) after 7 days as shown in Figure 1B. This increase in DCW in ASW could be due to the presence of sodium bicarbonate (0.18g/L) and high sodium nitrate (1 g/L) as compared to BBM.27,28 In parallel to decrease in the DCW (due to salt stress), an increase in the sea salts concentration led to an apparent lipid augmentation. Data showed about 3.2-fold higher lipid productivity (82.9 ± 1.8 (mg/L)/d) in 100% sea salt medium compared to BBM (25.7 ± 1.2 (mg/L)/d) as depicted in Figure 1B and Figure 2. Comparison of this microalga with other reported freshwater microalgae grown in different salinities (either exclusively in NaCl or in sea salts) showed an increase in lipid content (%) and lipid productivity ((mg/L)/d) as to Nannochloris

oleobundans, Scenedesmus sp. CCNM 1077, C. sorokiniana HS1, and Chlorococcum sp. RAP 13 (Table 1). 3.2. Changes in Biochemical Composition. The holistic effects of salinity were analyzed by estimating changes in cell size, carbohydrate, protein, lipid content, and photosynthetic pigments. Positive correlation between cell size and different percentages of salinity was observed (Figure 2 and 3A). The maximum cell size (12.6 ± 1.6 μm) was obtained in 100% ASW, which was 2.7-fold higher than BBM (4.6 ± 1.2 μm) and indicated the adaptation of the microalga to saline conditions, thus contributing to osmotic stability of cells (Figure 3A). Such an increase in cell size could be attributed to the enhanced degree of vacuolization with increasing salt concentration. Further, Na+ gets sequestered in the vacuole to maintain the cell’s tugour pressure.29 Our results were in line with those reported recently by Kaewkannetra et al., 2012, Pelah et al., 2014, and Kim et al., 2016.29−31 Nile red staining of algal cells cultivated in different percentages of ASW evidenced the increase of bright-yellow lipid droplets inside the cells (Figure 2). A gradual increase in lipid content (%) was observed with increase in salinity, attaining maximum lipid content (51.8 ± 3.2%) in 100% seawater followed by 80% > 50% > 30% > 0% > BBM, respectively (Figure 3A). This could be due to salinity stress and presence of sodium bicarbonate (dissolved inorganic carbon),27 which triggered the accumulation of neutral lipids preferably as triacylglycerols (TAGs) inside the cells. Indeed, escalation in lipid content (%) was accompanied by a decrease in protein content with maximum protein content in BBM (40.6 ± 4.2%) as compared to 22.4 ± 1.2% in 100% seawater while the carbohydrate content in all the microalga cells ranged between ∼26% and 33%, respectively. These results suggest that under salinity stress, microalga down-regulates protein synthesis as also reported in brown alga Ectocarpus siliculosus.32 The changes in photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) in Scenedesmus IITRIND2 cultivated in different concentrations of sea salts are shown in D


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Table 1. Comparison of DCW (g/L), Lipid Content (%), Biomass Productivity ((mg/L)/d), and Lipid Productivity ((mg/L)/d) Obtained from Different Fresh Water Microalgae Species in the Related Studies Grown in Different Saline Waters salt (g/L)

DCW (g/L)

lipid content (%)

biomass productivity ((mg/L)/d)

lipid productivity ((mg/L)/d)

0.2 23.4

1.5 0.4

14.8 33.1

123.3 19

56.4 6.3

Popovich et al., 201210 Pancha et al., 201539

Chlorella sorokiniana CYI C. sorokiniana HS1 Desmodesmus abundans Chlorococcum sp. RAP 13

Enriched natural seawatera BG-11 (blue green media) (two stage culture)b Deep seawater (20%)a BG-11b BG-11b Natural seawater (50%)a

30 20

2.40 1.0 1.8 2.3

51.7 35.6 40.4 20.8

176.6 101 200.2 152.5

140.8 36 67.1 31

Scenedesmus sp. IITRIND2

Artificial seawatera

0 10.5 17.5 28 35

1.35 1.44 1.32 1.21 1.12

27.4 38.9 40.9 45.5 51.8

235.7 205.7 188.6 172.9 160

52.9 80 77.1 78.6 82.8

Chen et al., 201340 Kim et al., 201631 Xia et al., 201412 Sabeela Beevi and Sukumaran, 201541 This study

microalgae Nannochloris oleobundans Scenedesmus sp. CCNM 1077

a

growth media

ref

Studies performed using sea salts. bStudies performed exclusively using NaCl.

Figure 3. Changes in (A) cell size (μm), total protein (%), total carbohydrates (%), and total lipid content (%) and (B) chlorophyll a, chlorophyll b, and carotenoids of Scenedesmus sp. IITRIND2 cultivated in different percentages of artificial seawater (ASW) and BBM as on 7th day.

μM/g FW) was also enhanced upon increasing the concentration of sea salts. The algal cells also showed an elevation in antioxidant enzymes (CAT and APX) activities (Figure 4). CAT activity ranged from 8.19 × 103 ± 0.12 to 47.7 × 103 ± 1.78 U/mg protein while the activity of APX ranged from 0.43 ± 0.02 to 2.54 ± 0.04 U/mg protein when the concentration of sea salts was increased from 0 to 35 g/L, respectively. In general, microalgae when cultivated under environmental stress conditions produce various reactive oxygen species (ROS) such as H2O2, superoxide (O2−), and hydroxyl (OH−) that cause oxidative damage to the cells. ROS target lipids, carbohydrates, proteins, and DNA, which lead to the cell’s demise.25 TBARS is the product of lipid peroxidation and indicates presence of free radicals.33 In order to mitigate the oxidative stress, microalgae had developed efficient intrinsic antioxidant systems involving enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase

Figure 3B. Data revealed an apparent decline in chlorophyll a, chlorophyll b, and carotenoids with an increase in sea salts concentration, thus demonstrating the inhibition of its photosynthetic apparatus. Chlorophyll a and chlorophyll b were majorly affected as reduction of ∼4-fold and ∼6-fold was observed, while ∼2.5 fold decrease in carotenoids was recorded in cells cultivated in 100% ASW. 3.3. Changes in Stress Metabolites. In order to throw light on the mechanistic aspects of the halotolerence behavior of the Scenedesmus sp. IITRIND2, we also investigated the modulation of the stress metabolites (ROS), osmolytes (proline and glycine betaine), and antioxidant enzymes (CAT and APX) cultivated under ASW with different sea salt concentrations and BBM (Figure 4). The levels of H2O2 and TBARS progressively increased with the concentration of sea salts, subsequently reaching 38.8 ± 4.5 μM/g FW and 0.18 ± 0.03 μM/g FW, which were 10.3- and 4.7-fold higher than BBM. The content of proline (178 ± 7 μM/g FW) and glycine betaine (4.54 ± 0.05 E


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Figure 4. Changes in H2O2, lipid peroxidation (TBARS), osmolytes (proline and glycine betaine) content, and antioxidant enzymes (CAT and APX) activity in Scenedesmus sp. IITRIND2 cultivated in BBM and different percentages of ASW as on 7th day.

(APX).22 SOD is responsible for scavenging ROS, then CAT decomposes H2O2 to H2O and O2, while APX degrades H2O2 with ascorbate oxidation to dehydroascorbate and water.22 Along with the generation of antioxidant enzymes, microalgae start accumulating osmolytes mainly proline and glycine betaine, which maintains the osmoregulation by scavenging excess ROS, and re-establish cellular redox balance, cytosolic pH buffer, and stabilize subcellular structures as depicted in the schematic (Figure 5).25

cultivated in different percentages of salinity revealed the presence of myristic acid (C14:0), palmitic acid (C16:0), 7,10hexadecadienoic acid methyl ester (C16:2), 7,10,13-hexadecatrienoic acid methyl ester (C16:3), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) as the major fatty acids (Table 2). Presence of heptadecanoic acid (C17:0) in 0%, 30%, and 80% salinity cultivated cells was detected, while no traces of arachidic acid (C20:0) was obtained in 100% salinity. Data showed that linolenic acid (C18:3) was only present in 0% and 30% seawater grown cells. Increasing sea salts from 0 to 35 g/L resulted in an increase in the proportion of oleic acid from ∼29% to 54% (Table 2). Synthesis of oleic acid requires large amounts of NAD(P)H and oxygen which eases the effect of reactive oxygen species (accumulated due to salinity stress), aiding in cell survival with enhanced lipid production.18 Increase in MUFA’s content was recorded under saline conditions (Table 2). Such an increase in MUFA’s content in microalgae is responsible for maintaining the fluidity of cell membrane and also prevents its disruption toward unsaturated, short, and branched fatty acids having a lower temperature than saturated, straight, and long chain fatty acids.34 Also, an oil containing high amounts of oleic acid can improve the oxidative stability and the cold flow properties of biodiesel.35 Similar results have also been reported in Desmodesmus abundans when it was cultivated in different concentrations of NaCl ranging from 0 to 30 g/L which showed increase in its MUFAs with substantial decrease in its PUFAs content.12 Physical properties of biodiesel derived from of Scenedesmus sp. IITRIND2 cultivated in ASW and BBM were shown to be in compliance with ASTM D6751-02 (American Society for Testing Materials) and EN 14214 (European) biodiesel standards (Table 3). The microalga irrespective of the cultivation media used showed better physical properties as compared to Jatropha or Palm oil methyl esters (Table 3). Iodine value (67 g I2/100 g) with cetane number of 62 was recorded in biodiesel derived from cells cultivated in 100% salinity. The iodine value obtained was lower than 120 g I2/100 g, making the biodiesel less susceptible to gum formation.

Figure 5. Systematic representation of physiological and metabolic changes occurring in Scenedesmus sp. IITRIND2 when grown in ASW.

3.4. Fatty Acid Profile and Biodiesel Properties. The enhanced lipid content thus obtained from Scenedesmus sp. IITRIND2 under saline conditions was analyzed for its fatty acid composition and biodiesel physical properties to enumerate the vehicular quality of biodiesel. Microalgae contain both saturated fatty acids (SFAs) and unsaturated fatty acids (MUFAs, PUFAs), which get altered by both biotic and abiotic factors. The GC−MS profile of Scenedesmus sp. IITRIND2 F


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Table 2. FAME Composition (%) of Scenedesmus sp. IITRIND2 Grown in Different Percentages of ASW and BBM on the 7th Daya relative percentage of FAMEs cultivation medium BBM 0% ASW 30% ASW 50% ASW 80% ASW 100% ASW a

C14:0 2.53 ± 0.01 2.13 ± 0.02 2.01 ± 0.01 2.19 ± 0.05 1.09 ± 0.01 0.84 ± 0.01

C16:0 9.53 ± 0.02 11.75 ± 0.1 13.97 ± 0.16 5.2 ± 0.1 8.25 ± 0.5 15.05 ± 1.21

C16:2

C16:3

C17:0

2.79 ± 0.08 1.46 ± 0.3 1.86 ± 0.04 1.61 ± 0.2 ND

ND

ND

6.1 ± 0.5

0.13 ± 0.02 0.14 ± 0.03 ND

0.55 ± 0.01

2.51 ± 0.1

5.88 ± 0.03 3.65 ± 0.07 ND

0.12 ± 0.01 ND

C18:0 2.18 ± 0.01 2.27 ± 0.01 4.24 ± 0.06 5.18 ± 0.01 3.32 ± 0.01 4.2 ± 0.3

C18:1

C18:2

33.33 ± 2.41 29.31 ± 0.23 30.3 ± 2.5 40.2 ± 1.4 52.26 ± 7.1 53.67 ± 0.76

6.01 ± 0.4 8.62 ± 0.2 10.59 ± 1.5 7.03 ± 0.01 5.69 ± 0.8 7.42 ± 0.6

C18:3

C20:0

ND

ND

0.33 ± 0.01 0.33 ± 0.02 ND

0.1 ± 0.01 0.2 ± 0.01 0.21 ± 0.02 0.55 ± 0.05 ND

ND ND

SFA 14.24 ± 1.21 16.38 ± 2.1 20.56 ± 2.3 12.78 ± 0.8 13.33 ± 0.5 20.09 ± 1.1

MUFA 33.33 ± 2.3 29.31 ± 0.12 30.3 ± 0.9 40.2 ± 1.4 52.26 ± 2.5 53.67 ± 4.3

PUFA 8.8 ± 0.3 16.51 ± 0.1 18.66 ± 0.3 12.29 ± 0.02 5.69 ± 0.03 10.48 ± 0.6

ND: not detected.

Table 3. Comparison of Biodiesel Physical Properties of Scenedesmus sp. IITRIND2 Cultivated in Different Percentages of ASW and BBM with ASTM D6751, EN 14214 Fuel Standards, and Plant Oil Methyl Esters (Jatropha and Palm)a standard fuel parameters

plant oil methyl esters

artificial seawater

physical properties

ASTM D6751-02

EN 14214

control, BBM

0%

30%

50%

80%

100%

JME

PME

Saponification value (mg KOH) Iodine value (g I2/100g) Cetane number Degree of unsaturation (% wt) Long chain saturation factor (% wt) High heating value (MJ/kg) Cold flow plugging property (° C) Kinematic viscosity (mm2/s) Density (g/cm3 ) Oxidative stability (h)

47 min 1.9−6.0 -

120 (max) 51 (min) ≤5/≤ −20 3.5−5.0 0.86−0.90 ≥6

110 44 85 51 2 44 −9.5 3.99 0.87 22

123 61 75 62 2.4 44 −8.2 3.99 0.88 16

137 65.68 70 68 3.7 42.84 −3.8 3.99 0.88 13

127 60.25 74 65 3.3 42 −5.1 4.02 0.87 20

137 54.67 72 64 3.1 43 −6.1 4.65 0.87 23

163 67 62 75 3.6 41.73 −4.2 3.72 0.88 19

96.55 54 −2 4.33 0.88 3.86

49.56 61 13 4.43 0.87 16.5

a

The dash (-) indicates no standard limit designated by ASTM D6751-02 and EN 14214 biodiesel standards. JME= Jatropha methyl ester, PME= Palm oil methyl ester.

content, in order to counter salinity stress. The fatty acid profiles and biodiesel physical properties obtained here are abiding with ASTM D6751 and EN 14214 biodiesel standards, signifying their applicability in diesel engines. The utilization of such saline/seawater feedstocks along with halotolerant strains can reduce the fresh water footprint and nutrient consumption leading to suitable biodiesel production. However, before deployment of seawater for commercial cultivation of salt tolerant microalgae, high quality geological assessment of saline waters needs to be investigated to understand the strengths and limitations of this source. Moreover, in order to grow microalgae on commercial scale, large amounts of seawater is essential which requires establishment of proper recycling and water pumping systems, which can pump the onshore seawater continuously to the cultivation facility.38 One major issue with pumping systems is biofouling of the seawater that can increase the contamination risk of microalgal cultures. Thus, the collected seawater has to be processed to remove any sediments/microorganisms before using it for algal cultivation. The mechanistic insights obtained on Scenedesmus sp. IITRIND2 halotolerance by systematic monitoring of various biochemical components can open new avenues to identify hyper salt responsive gene(s) and enhanced lipid accumulating algae for high quality biodiesel production. Further, these concepts have to be strengthened using transcriptiomics, metabolic engineering approaches, and pilot commercial level

Further, high cetane number obtained here could ensure less ignition delay, smooth engine run, better cold start properties with reduced gaseous and particulate emissions from the obtained biodiesel.36 The biodiesel derived from these cells showed the high heating value of 42 MJ/kg, cold filter plugging property of −4 °C, kinematic viscosity of 3.7 mm2/s, and oxidative stability of 19 h. The density of the derived biodiesel ranged between 0.87 and 0.88 g/cm3. Cold filter plugging point (CFPP) of the biodiesel derived from microalgae grown in ASW was lower as compared to plant oil methyl esters (Jatropha and Palm oil), suggesting its usage at low temperatures. CFPP is directly dependent on the content of SFA’s mainly C16:0 and C18:0 as these two fatty acids precipitate faster under low temperatures.37 The high salanity condition did not affect the kinematic viscosity and density of obtained biodiesel as both were within the set range of acceptable standards for biodiesel (Table 3). The enhanced oxidative stability of microalgae cultivated under the saline conditions was higher than plant oil methyl esters, indicating a long shelf life of the obtained biodiesel.

5. CONCLUDING REMARKS In the present investigation, we reported the role of salt induced stress and the mechanism of salt tolerance using a fresh water microalga (Scenedesmus sp. IITRIND2). The microalga acclimatized to 100% saline conditions by enhancing its lipid G


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biodiesel production plants as a next step action plan, in order to achieve biodiesel production in an economically viable and envriornmentally sustainable mode.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.7b00841. Recipe of ASW (Table S1), empirical formulas for biodiesel properties (Table S2), and flask cultures (Figure S1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*V.P.: e-mail, [email protected], [email protected]; phone, 091-1332-285530; fax, 091-1332-273560. *K.M.P.: e-mail, [email protected], [email protected]; phone, 091-1332-284779; fax, 091-1332-273560. ORCID

Krishna Mohan Poluri: 0000-0003-3801-7134 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors are thankful for financial support to P.A.P. under DBTBioCare Programme (Grant 102/IFD/SAN/3539/2011-2012), DBT-SRF to N.A. (Grant 7001-35-44), and UGC-SRF to M.S. K.M.P. acknowledges the receipt of DBT-IYBA fellowship and SERB-LS Young Scientist Award.

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REFERENCES

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Insights into the Enhanced Lipid Production Characteristics of a Fresh

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