Fatty Acid Biosynthesis from a New Isolate Meyerella sp. N4

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Fatty acid biosynthesis from a new isolate Meyerella sp., N4: molecular characterization, nutrient starvation and fatty acid profiling for lipid enhancement Rathinasamy Karpagam, Ranjan Preeti, Kalimuthu Jawahar Raj, Saseeswaran Saranya, Balasubramaniem Ashokkumar, and Perumal Varalakshmi Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/ef501969a • Publication Date (Web): 05 Dec 2014 Downloaded from http://pubs.acs.org on December 11, 2014

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Fatty acid biosynthesis from a new isolate Meyerella sp., N4: Molecular characterization, nutrient starvation and fatty acid profiling for lipid enhancement Rathinasamy Karpagam1, Ranjan Preeti1, Kalimuthu Jawahar Raj 1, Saseeswaran Saranya 1, Balasubramaniem Ashokkumar2 and Perumal Varalakshmi1* 1

Department of Molecular Microbiology, School of Biotechnology,

Madurai Kamaraj University, Madurai, Tamil Nadu, India. 2

Department of Genetic Engineering, School of Biotechnology,

Madurai Kamaraj University, Madurai, Tamil Nadu, India. Keywords: Biodiesel, Meyerella sp., Nutrient starvation, Lipid content, Microalgae, Fatty acid methyl esters.

ACS Paragon Plus Environment

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ABSTRACT Microalgae are considered as potential feedstock for biodiesel production. In this study, a new green microalga was isolated from a freshwater pond in India and identified as Meyerella sp. N4. Nutrients starvation strategy was employed in this strain to understand the enhancement of the lipid accumulation. The increase in accumulated lipid was further confirmed by Nile red staining followed by confocal laser scanning microscopic observation. Nitrogen (NaNO3) starvation increased the lipid accumulation up to 60.4 ± 3.7 % of dry cell weight at 314 ± 13mg total lipid/L in 23 days, when compared with the other conditions tested. Quantification of total fatty acid content by Gas Chromatography-Mass Spectrometry showed the presence of two major fatty acids, C16 at 34 ± 4.8 wt%, and C18:1 at 26 ± 3.6 wt%. Biodiesel quality parameters such as Cetane number at 57.7 ± 0.3 Iodine value at 77.7 ± 1.1 g I2 100 g-1 oil, Saponification value at 190.5 ± 1.8 mg KOH g-1 oil and the Cold filter plugging point at -1.5 ± 1.0°C are in good agreement with the international standards, American Standard ASTMD6751, and European Standards (EN 14214) of biodiesel, thus making Meyerella sp. N4, a potential candidate for biodiesel production.

INTRODUCTION Microalgae are considered one of the most promising renewable feedstock for biodiesel production as they can grow in a wide range of environmental conditions, utilizing atmospheric carbon dioxide (CO2) and sunlight as carbon and energy sources, respectively, for biomass production and triacylglycerols (TAGs) accumulation.1,2 TAGs can be converted to biodiesel by a transesterification process using either acid or base catalyst.3 Thus, fuel from microalgae would be environment-friendly and sustainable. However, inadequate accumulation of TAG is one of


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the major concerns in biodiesel production. Under optimal conditions, most microalgae synthesize fatty acids, which constitute about 5–20% of their dry cell weight (DCW). Nitrogen, phosphorus, and iron sufficiency in microalgae increases the rate of photosynthetic CO2 ﬁxation by seven to ten times, while their deﬁciency in most microalgae results in decreased photosynthetic ability, altered lipid metabolism and a significant increase in their TAG accumulation.4–7 This study aimed to enhance the lipid biosynthesis and characterization of fatty acid methyl ester (FAME) for biodiesel application in a fresh water indigenous green algal isolate Meyerella sp. N4 in nutrient-complete as well as nutrients-starved conditions.

MATERIALS AND METHODS Sample collection, isolation and cultivation of microalgae Microalgal samples were collected using sterile poly bags from freshwater pond at Nagercoil, Kanyakumari District, Tamil Nadu, India. Geographical co-ordinates of the location are 8° 10' 0" North, 77° 26' 0" East. The medium used for cultivation of microalgae was BG11 medium consisting of Sodium nitrate (NaNO3)- 1.5 g/L, Sodium carbonate (Na2CO3) - 0.02 g/L, Disodium magnesium EDTA (Na2MgEDTA) – 0.001 g/L, ammonium ferric citrate (C6H8FeNO7) – 0.006 g/L, Calcium chloride (CaCl2.2H2O) –0.036 g/L, Citric acid (C6H8O7)0.006 g/L, Magnesium sulphate (MgSO4.7H2O) – 0.075 g/L, Dipotassium hydrogen phosphate (K2HPO4) – 0.0305 g/L, Boric acid (H3BO3) – 2.8 mg/L, Manganese chloride (MnCl2.4H2O) – 1.81 mg/L, Zinc sulphate (ZnSO4.7H20) – 0.222 mg/L, Copper sulphate (CuSO4.5H2O) – 0.079 mg/L, Cobaltous chloride (CoCl2.6H2O)– 0.05 mg/L, Sodium Molybdate (NaMoO4.2H2O) – 0.391 mg/L, MoO4 – 0.018 mg/L (pH 6.8±0.2). Samples were serially diluted in distilled water and inoculated on BG11 medium by spread plate method and incubated at 25°C for 14 days. The plates were maintained under constant illumination 1500 lux of photoperiod (light: dark -12:12


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hours cycle) and the purity of the isolate N4 was monitored under light microscope (Labomed, Germany). Scanning Electron Microscopy (SEM) Air dried microalgal sample was spread on double sided conductive carbon tap fixed on the stub. After attaining high vacuum (3.99e-4 Pa), the filament was activated and the sample was examined under SEM (Quanta 250- FEI, Czech Republic, 20 kW, magnification 50,000X). SEM images were taken at different locations of the carbon tape that were used for the analysis of the cell surface structures. DNA Extraction Genomic DNA from the isolate Meyerella sp. N4 was extracted at exponential growth phase according to a modified protocol.9 Cell pellets were collected after centrifugation, washed with TEN buffers (10 mmol/L Tris-HCl, 10 mmol/L Na2EDTA, 150 mmol/L NaCl), resuspended in 150 µL icecold water and vortexed for one minute. Further, 350 µL SDS-EB buffer was added to the pellet (2% SDS, 400 mmol/L NaCl, 40 mmol/L Na2EDTA, 100 mmol/L Tris-HCl) and vortexed for 30 seconds to lyse the cells and incubated for 5 min. After cell lysis, 350 µL of phenol: chloroform: isoamylalcohol (25:24:1) was added and the sample was incubated for 5 min. DNA was extracted from the aqueous phase with 250 µL chloroform: isoamylalcohol (24:1), precipitated with 0.6 volume of isopropanol at -20°C for 1 h, and then spun down at 12,000 rpm for 15 min. Finally, the extracted DNA was washed with 1 mL of 70% ethanol, airdried and then resuspended in 20 µL of MilliQ water for polymerase chain reaction (PCR). DNA Amplification


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PCR was performed from 100 ng of genomic DNA as template using ITS primer.10 Four µL of genomic DNA was taken as template to 50 µL of PCR reaction mixture consisting of 25 µL of PCR Ready Mix (Sigma Aldrich, USA), 2 µL of forward and reverse primer each (0.4 µM), 17 µL of molecular biology grade water (Himedia, Mumbai). The ITS1-5.8S-ITS2 region was amplified with the following primer sequences 5’-ACCTAGAGGAAGGAGAAGTCGTAA3’, 5’-TTCCTCCGCTTATTGATATGC-3’ as forward and reverse primer, respectively. Reactions

were cycled using Nexus Gradient Master cycler (Epperndorf, Germany) with the following conditions: initial denaturation at 94°C for 4 min followed by 32 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec, final extension at 72°C for 10 min. Amplified PCR products were electrophoresed on 1.2% agarose gel for 30 min at 100 V and the positive amplicons were purified using the Genelute kit (Sigma Aldrich, USA), as per the manufacture’s protocol. A negative control was run along with each PCR reaction to evaluate contamination. Further PCR products, were sequenced by Sanger’s dideoxy method of sequencing. Sequences obtained were subjected to BLAST analysis to identify the boundaries of ITS1-5.8S-ITS2 from published sequences. A phylogenetic tree was constructed (neighbor joining tree method of phylogeny test with 1000 bootstrap replications using MEGA 6.06 software) using the multiple sequence alignment (Clustal W) of the ribosomal sequences of Meyerella sp. N4 and the existing Chlorella sp. to resolve the taxonomic pattern of the related microalgae. The ITS1-5.8S-ITS2 sequences were submitted to GenBank (KJ414314.1) (www.ncbi.nlm.nih.gov). Nutrient Starvation and Growth Kinetics All the nutrient starvation experiments were performed using BG11 media inoculated with 6 mg (dry wt.) of microalga in 100 mL of media (initial cell density was 0.4OD = 6.25 X


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106 cells / mL). For N-, P- and Fe-starvations, the three components (NaNO3; K2HPO4; ferric citrate and 0.061 g/L KOH was given as potassium source) were not added in the BG-11 medium. Complete BG-11 medium was maintained as control. Each starved conditions and control were maintained separately in duplicates and were incubated under constant illumination 1500 lux of alternate photoperiod (light: dark -12:12 hours cycle) at 25°C with continuous agitation at 90 rpm without providing aeration exogenously. The specific growth rate of microalgae under normal and starved condition was monitored at four day intervals by measuring the optical density of the cell suspension at 670 nm using colorimeter (Elico, CL 63, India) and the cell density through cell counting using hemocytometer (Neubauer, Germany). Optical and cell density measurements were done in duplicates for the three independent cultures. The specific growth rates were obtained from the difference in cell density to the corresponding time interval at exponential phase, calculated using the following equation (Eq. 1)11. Specific growth rate, µ (day-1)

= 2.303 (log N2 − log N1)/ (t2 – t1)

(1)

In the equation, N1 and N2 were the cell density at exponential phase. Confocal imaging of lipid bodies in live cells Cells in stationary phase were stained with Nile Red to investigate lipid body accumulation.12 For staining, 100 µL of each microalgal culture from nutrient-starved condition and control was added to 25 µL of Dimethyl sulphoxide (DMSO) and 4 µL of Nile Red (Nile blue A oxazone) solution at a concentration of 5 mg/mL of acetone (Hi-Media, Mumbai, India), mixed well and incubated in dark for 10min. Images were acquired using a confocal laser scanning microscope (LSM 510-META-ZEISS - Germany). Nile red signal as bright


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fluorescence was captured using a laser excitation and emission line at 530 nm and 575 nm, respectively. Lipid extraction and quantification Total lipids were extracted from microalgal biomass during the stationary phase at 23rd day of incubation.13 The dried microalgal biomass was pre-weighed (Shimadzu, Germany) and ground in mortar and pestle with muffled glass beads in 1 mL of chloroform-methanol (2:1, v/v). The lipids were extracted using chloroform-methanol (2:1, v/v) by repeated centrifugation at 8000 rpm for 10 min until the pellet becomes colorless. In order to facilitate phase separation, 1% NaCl was added to the supernatant in a separating flask with vigorous shaking. Finally, the organic lower phase was allowed for solvent evaporation in a glass plate (pre-weighed) at room temperature and dry weight of the lipid was then measured gravimetrically. The lipid quantifications were done in duplicates in two independent analyses. Qualitative analysis of lipids by thin layer chromatography (TLC) Total lipids were separated by TLC using the developing solvent (hexane; diethyl ether; acetic acid in the ratio of 70:30:1).10 Triolein (Hi-Media) was used as TAG standard. To visualize different fractions of total lipid, the TLC plate was sprayed with 10% copper sulphate (in 8% phosphoric acid) and charred at 180 ˚C for 10 min. Fatty acid methyl esters (FAME) Preparation and GC-MS analysis FAME was prepared through transesterification by acid catalysis.14 The extracted lipid sample was diluted to 1.5 - 2.5 mg and added in to the GC vial. To this, equal volume of methanol (300 µL) and 15 µL of 35% conc. HCl, which corresponds to final concentration of 0.39 mol/L, were added and the transesterification reaction was performed by incubating the


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contents at 100°C for 90 min. After incubation, the transmethylated sample (FAME) was extracted using the same volume of hexane (300 µL) transferred to GC vial (Cyberlab). To this pentadecanoic acid methyl ester (C 15:0) at a concentration of 10 µg/µl as internal standard was added for the quantification and separation of FAME detected by GC (Agilent Technologies 6890, N Series , USA) and m/z value detected by EI-MS (Electron impact ionization mass spectrometry, JEOL GC MATE-II, JEOL Ltd, Tokyo, Japan) equipped with column HP-5 MS, photon multiplier tube detector and quadruple double focusing mass analyzer. Highly pure helium was used as carrier gas at a flow rate 1 mL/min. The ion chamber and GC interface temperature were maintained at 250°C. The initial temperature of the oven was set to 50°C and then increased to 250°C at a rate of 10°C per minute, functioning at 70 eV ionisation voltage with a scan range from 50 to 600 amu. The m/z value of FAME compounds of the standard Supelco 37 component FAME mix (Sigma-Aldrich, USA), and the Meyerella sp. N4 were compared with National Institute for Standards and Technology (NIST) database at Sophisticated Analytical Instrument Facility, Indian Institute of Technology- Madras, Chennai, India (www.saif.iitm.ac.in). Relative percentage of each fatty acid was calculated by area normalization method and FAME yields were calculated using the following equation (Eq. 2)7,15. FAME concentration (mg g-1 Lipid) = (Ax × Wis)/ (Ais × Ws ) × 1000

(2)

Where Ax is the area of fatty acid methyl ester peak in the chromatogram of the sample, Ais is the area of methyl ester peak of internal standard in the chromatogram of the sample, Wis is the weight of internal standard (in mg) added to the sample and Ws is the lipid weight (in mg). Evaluation of fuel properties of biodiesel The FAME profile of Meyerella sp. N4 were used to calculate iodine value (IV), Saponification value (SV), cetane number (CN), Degree of Unsaturation (DU), Cold filter


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plugging point (CFPP) and Long chain saturation factor (LCSF) using the following empirical equations (Eq. 3 to Eq. 8).14-18 IV = ∑(254×F×D)/MW

(3)

SV = ∑(560×F)/ MW

(4)

CN = (46.3+[5458/SV]) - (0.225×IV)

(5)

DU= MUFA wt%+ (2×PUFA wt %)

(6)

Where F is the percentage of each type of fatty acid, MW is the molecular weignt of corresponding fatty acid, and D is the number of double bonds. CFPP= (3.1417× LCSF) - 16.477

(7)

Whereas LCSF is Long chain saturation factor, LCSF= (0.1×C16) + (0.5×C18) + (1×C20) + (1.5×C22) + (2×C24)

(8)

Statistical analysis The data was analyzed using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test, and statistical significance was set at 0.05 (P < 0.05). All the experiments were carried out in duplicates with two independent analyses (n = 4).


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RESULTS AND DISCUSSION Isolation and Identification of Microalgae A freshwater microalga predominantly growing in a freshwater pond located in Nagercoil, Tamil Nadu, India was isolated and screened for lipid production based on its luxurious growth in that environment. The axenic culture was raised in liquid BG-11 medium after repeated plating in BG-11 agar plates and the purity was monitored (Supplementary Fig. A). Microscopic observation of this microalgal isolate indicated their colonial existence and purity. Preliminary morphological identification of microalgal cultures by microscopic analysis showed that the isolate N4 belonged to Chlorella-related coccoid green algae in the family Chlorellaceae. Smooth cell wall without flagella or spines is an important feature of the genera Chlorella and Meyerella. Of the two microalgae, a parietal and cup-shaped chloroplast without any pyrenoids is an essential characteristic for distinguishing Chlorella and Meyerella species. 19 The N4 cells were solitary, 3 µm in diameter, spherical in shape, and devoid of flagella or spines as observed under SEM (Fig. 1). Based on the presence of aforementioned features, it was confirmed that our isolate N4 belonged to genera Meyerella. To further confirm the morphological observations, phylogenetic analysis of ITS1-5.8S-ITS2 rRNA gene sequences of N4 isolate was performed. ITS1-5.8S-ITS2 rRNA regions of N4 isolate was PCR-amplified, sequenced and submitted to Genbank (Acc. No. KJ414314.1). BLAST hits of ITS1-5.8S-ITS2 rRNA gene sequences of algal isolates N4 showed 87% sequence identity with Meyerella sp. (Acc. Nos. AY543045.1 and AY543046.1). This sequence information of Meyerella sp. was recently recorded in the samples collected during winter and spring seasons from Lake Itasca, Itasca State Park, MN, USA.20 Thus, our microalgal isolate N4 identified by ITS region showed close similarity with the only available sequence information of Meyerella sp. in the NCBI


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database (Supplementary Fig. B). It is noteworthy that so far, fatty acid profiling of this strain has not been reported. Thus, the potential of Meyerella sp. N4 for lipid production was investigated further in the present study. Growth kinetics of Meyerella sp. N4 The nutrient availability in the culture medium is an important factor that influences growth kinetics, which directly affect lipid accumulation. Hence, the growth rate of Meyerella sp. N4 was determined in nutrient starvation and control conditions through continuous monitoring of its growth by measuring the OD and cell count till the alga reached the stationary phase. Meyerella sp. N4 reached the stationary phase on Day 23 in nutrients-starved conditions (lack of nitrogen, phosphorous and iron) without further increase in the growth rate whereas the alga grown under nutrient-complete condition (control) attained early stationary phase on Day 23 but the biomass continued to increase with extended incubation (Fig. 3). The specific growth rate of Meyerella sp. N4 correlated with nutrient availability; the growth rate increased when the nutrients were sufficient in the medium whereas the growth rate decreased in nutrient-starved medium. The specific growth rate (µ) of Meyerella sp. N4 was different in different culture conditions. The growth rate in control was 0.224 day-1, while that in nitrogen-, phosphorous-, and iron-starved conditions was 0.06 day-1, 0.123 day-1, and 0.19 day-1, respectively. These results are consistent with the earlier reports, where Neochloris oleoabundans and Chlorella sp. had shown relatively lower growth rate in nutrient-limited conditon.21-23 Apparently, Botrycoccus braunii had shown lower growth rate (0.14 day-1) even with 10% external carbon supply in nutrient sufficient medium. 24


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Cytochemical analysis of lipid bodies As a preliminary screening, Nile red staining followed by confocal laser scanning microscopic observation was performed to assess the potential of Meyerella sp. N4 for lipid bodies accumulation in nutrients-starvation conditions. Meyerella sp. N4 stained with Nile Red showed lipid bodies indicated by strong fluorescence during nitrogen starvation. Previously, similar observations were made in Chlorella sp. and Chlamydomonas sp.25,26 The cells of Meyerella sp. N4 grown in nutrient-starved condition and control showed qualitative differences in size and numbers of the lipid bodies. Specifically, cells in nitrogen starvation showed higher accumulation of lipids (3–5 lipid bodies with ~2 µm diameter) when compared to phosphorous-, iron-starvation and control (1–2 lipid bodies with ~1 µm diameter) (Fig. 2). Several green algae are known to accumulate large amounts of TAG in lipid bodies, particularly in abiotic stresses, like nitrogen starvation and light exposure.27 Effect of nutrient starvation in lipid yield and biomass production Lipid production is the most important characteristic for the use of microalgae in biodiesel production. In the present study, the total lipids extracted from Meyerella sp. N4 in different starvation conditions were qualitatively analyzed by TLC for the presence of triacylglycerol (TAG) and diacylglycerol (DAG); and triolein was used as a standard for this experiment (Supplementary Figure. C). The results revealed that among the nutrient starvation studies, the nitrogen starvation induced higher accumulation of TAG in Meyerella sp. N4 indicated by the presence of thick bands with high intensity whereas it was negligible with the other starvation conditions and control. In addition, the lipid production and lipid content of Meyerella sp. N4 was quantified in stationary phase (23rd day) in different nutrients-starved conditions and control. Earlier studies


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have very well documented that microalgae can accumulate neutral lipids in stationary phase than in the exponential phase. 28, 29 Moreover, Chlorella pyrenoidosa accumulates lipid during stationary phase, which increases from 11.23% in nitrogen-rich to 34.29% in nitrogen-starved media.7 However, longer incubation (20 days) of Chlorella vulgaris in nitrogen-starved media produced higher lipid content (29.53%) than that in shorter (15 days) incubation (26.71%). Meyerella sp. N4 showed significant (P < 0.05) increase in lipid content (60.4%) and lipid production (13.64 mg/L/day or 314 mg/L) in nitrogen starvation than control (31.2%, 8.80 mg/L/day or 202 mg/L) (Fig. 4 and 5). Nitrogen is an important nutrient for the microalgal cell proliferation and growth. Thus, it is not surprising that nitrogen starvation arrests biomass growth and the metabolic flux would be redirected to the accumulation of TAG as most of the reducing equivalents such as ATP and NADPH formed by photosynthesis would be utilized for biosynthesis of neutral lipids. 2,6,30 The biosynthesis of neutral lipids (TAG) could be a protective mechanism for the cells against stressful environments.31 However, there were no significant changes observed in the lipid content and lipid production of Meyerella sp. N4 during phosphorous starvation (34.5%, 7.87 mg/L/day or 181 mg/L) and iron starvation (33.4%, 8.72 mg/L/day or 200 mg/L), respectively than the control (Fig. 4 and 5). Phosphorus plays a key role in the transport of metabolic energy and is an essential component of nucleotides and phospholipids for all living cells.32 In addition, phosphorus requirements for optimal algal growth may differ considerably from species to species even in absence of other additional factors.33 During nutrient starvation, biomass production was also examined and found to be decreased in N (22.6 mg/L/day), P (22.80 mg/L/day), Fe (25.36 mg/L/day) starvation than the control (29.05 mg/L/day). Thus the accumulation of high lipid content is usually accompanied by


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lower biomass production in nutrient starvation approaches and the findings of the current study corroborate the same. Interestingly, the lipid production of Meyerella sp. N4 was achieved only at 23rd day (stationary phase) of cultivation without supply of any external CO2 and aeration during the study. The observed findings pertaining to biomass, lipid production, and lipid content of Meyerella sp. N4 was also consistent with the earlier reports, in which similar experimental conditions were employed. For example, the lipid production in Botryococcus sp., Chlorella vulgaris, and Scenedesmus sp. were only 160.3 mg/L, 77.9 mg/L, and 66.5 mg/L, respectively after 14 days of incubation in BG-11 medium34, whereas lipid production in Chlorella vulgaris YSL04 (440 mg/L) and Chlamydomonas pitschmannii YSL04 (540 mg/L) increased only after 21 days of cultivation.35 It is generally known that the supply of CO2 under nitrogen starvation could tremendously influence the biomass and lipid production of the microalgae.36 However, Meyerella sp. N4 has clearly demonstrated its potential as a photosynthetic organism that can accumulate high lipid content even without CO2 supply, which may augment higher biomass and lipid production for biodiesel production. FAME analysis It is known that the carbon chain length of saturated and unsaturated fatty acids affects the quality of biodiesel properties like cetane number and oxidative stability.37 Among the fatty acids, medium chain saturated and mono unsaturated fatty acids (C 14 to C 18) are suitable for the biodiesel with better oxidative stability.7 The algal lipids are mainly composed of glycerol, sugars, or saturated or unsaturated fatty acids, which usually have 12–22 carbon atoms in their fatty acids.38 Consequently, the FAME of Meyerella sp. N4 grown in control and starved conditions were quantified by GC-MS analysis, and the relative percentage of fatty acid was


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plotted (Fig. 6). The aforementioned analysis revealed that the total saturated fatty acid of Meyerella sp. N4 was highest (27%) in P-starvation conditions and lowest in N-starved condition (23%). Similarly, the maximum MUFA synthesis by Meyerella sp. N4 was noticed in P-starved media (46%) than the other starvations (42% for P and 41% for Fe) and control (44%). It was also found that the PUFA contents from the cells grown in control, N-, P-, and Fe-starved conditions were in the range of 20–28% and noticeable increase in PUFA were recorded in Nstarved (27%) and Fe-starved (28%) conditions than the control (21%) and P- starved condition (20%) (Supplementary Fig. D). Usually high percentage of SFA and MUFA are preferential for increasing energy yield and oxidative stability; however, oils containing MUFAs are more prone to solidification at low temperatures. Oils containing high PUFAs showed very good cold-flow properties, and the biodiesel with such properties are more vulnerable to oxidation.39 Moreover, FAME quantification of Meyerella sp. N4 revealed that the concentration of medium chain FAMEs C 16:0, C 16:1, C 18:1 and C 18:2 were increased during nutrient starvations (Table.2). Especially the concentration of FAME of C 18:2 were noticeably increased in N starved condition (97 ± 18.0 mg g-1 Lipid ) than the control (43.3 ± 2.3 mg g-1 Lipid ).Whereas, C 18:1 FAME content were increased noticeably in P starved conditions (76.3 ± 5.6 mg g-1 Lipid ) than the control (62.8 ± 0.9 mg g-1 Lipid) which corroborates the previous reports.40 Similarly total FAME yield was also increased to 1.2-2.0 folds in N and P starved conditions when compared with Fe starvation and control (Table.2). Fuel properties of biodiesel The quality of biodiesel is greatly influenced by the fatty acid profile of the algae. The total amount of saturated, monounsaturated, and polyunsaturated FAME would determine the fuel properties. Moreover, the degree of unsaturation (DU) is the summation of the mass of


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monounsaturated and polyunsaturated fatty acid, and is one of the key properties that would highly influence the oxidative stability of biodiesel.16 Therefore, in this study, various parameters for assessing the biodiesel fuel quality like saponification value (SV), iodine value (IV), cetane number (CN), degree of unsaturation (DU), long chain saturation factor (LCSF), and cold filter plugging point (CFPP) of FAME of Meyerella sp. N4 were calculated (Table.1). The increase in CN corresponding to the increase of carbon number is correlated to the delay in ignition of fuel, and the requirement of CN (minimum 47 according to ASTM) is usually linked with higher degree of unsaturation.41 Hence, CN value was calculated for biodiesel obtained from Meyerella sp. N4 grown in control and starvation conditions. These values were found to be in accordance with international standards (ASTM). The IV of biodiesel is used to determine the number of double bonds present in the fatty acids (no. of grams of iodine consumed by 100 g of oil), which determines the oxidative stability of the fuel. Similarly, in our study the IV of Meyerella sp. N4 was found to meet the international standards and would be used as biodiesel with higher combustion quality. As per the standards, the maximum limit of IV -120 g Iodine/100 g is accepted for determining the fuel quality.7,8 SV is used to calculate the CN of the fuel, (the no. of milligrams of KOH required to saponify one gram of oil) according to ASTM and EN 14214. The SV of Meyerella sp.N4 was found to be nearly equal in nutrients-starvation (187.9 ± 2.3, 187.5 ± 0.5 & 193.2 ± 2.7 mg KOH g-1 oil in N-, P- and Fe-starvation, respectively) than the control (190.5 ± 1.8 mg KOH g-1 oil). Likewise, SV values obtained for Scenedesmus (202.02 mg KOH g-1 oil) and Chlorella luteoviridis (207.91 mg KOH g-1 oil) were almost equal to that for Meyerella sp. N4.8 Cold filter plugging point (CFPP) and degree of unsaturation (DU) were evaluated according to ASTMD6751 and EN 14214 standards.42,7 Considering the international standards and definitions, the CFPP (-4.1 °C to -1.5 °C in nutrient starvation and control) and


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Energy & Fuels

DU (86.1 ± 0.7) wt % in P starvation) of Meyerella sp. N4 is highly suited for better oxidative stability (Table.2). Hence, the fuel properties of fatty acids obtained from Meyerella sp. N4 had ensured this alga can be a promising alternative for biodiesel feedstock. CONCLUSION An indigenous microalga N4 was isolated from freshwater pond and was identified as Meyerella sp. N4 that showed significant increase in the lipid content, lipid production, and FAME profile in nutrients starvation than the control. Nitrogen starvation led to an accumulation of total lipids significantly as high as 60.4 % (DCW) than the control 31.2% (DCW). Consequently, the fuel properties of biodiesel obtained from Meyerella sp. N4 met the International Standards (ASTMD6751 and EN 14214). To the best of our knowledge the oleaginous microalgal isolate Meyerella sp. N4 screened in the current study has been explored for its potential in the accumulation of high lipid for the first time and the results indicate that this alga can be a suitable feedstock for biodiesel production.

AUTHOR INFORMATION Corresponding Author Author to whom correspondence should be addressed: E-mail: [email protected] Author Contributions All the authors have contributed to the writing of this manuscript and have given approval to the final version of the manuscript preparation. Funding Sources Authors thank Department of Science and Technology (DST), India for the financial assistance (Ref: SR/ FT/LS-20/2012 dt.18.9.2012).


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Acknowledgement We also acknowledge SAIF, IITM Chennai, India for GC-MS analysis. Conflict of interest Authors have no conflict of interest to declare.

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26. Huang, GH., Chen, G.; Chen, F. Biomass and Bioenergy, 2009, 33 (10), 1386-1392. 27. Rochaix, J.D. FEBS Letters. 2002, 529 (1) 34-38. 28. Bell, M.V., Pond, D., Phytochemistry. 1996, 41, 465–471. 29. Dunstan, G.A., Volkman, J.K., Barrett, S.M., Garland, C.D., J. Appl. Phycol. 1993, 5, 71– 83. 30. Msanne, D.; Xu, A. R.; Konda, J. A.; Casas-Mollano, T.; Awada, E. B.; Cahoon.; Cerutt, H. Phytochemistry. 2012, 75, 50-59. 31. White, S.; Anandraj, A.; Bux, F. Bioresour. Technol. 2011, 102, 1675-1682. 32. Tillberg, J.-E. & Rowle, J. R.1989, Physiol. Plant.75, 315-324. 33. Kuhl, A. Algal physiology and biochemistry, Univ. California Press.1962, 636-654. 34. Lee J.-Y.; Yoo,C.; Jun, -Y.S.; Ahn ,-Y. C.; Oh, –M. H. Bioresour. Technol. 2010. 101, S75-S77. 35. Abou-Shanab,R.A.I.; Ibrahim, A. M.; Kim,S,-N.; Oh, Y.-K.; Choi, J., Jeon, B.-H. Biomass and Bioenergy. 2011, 35, 3079-3085. 36. Morita M, Watanabe Y, Saiki H. Appl Biochem Biotechnol. 2000. 87 (3), 203-18. 37. Islam, M.A.; Magnusson, M.; Brown R.J,; Ayoko,; A.G.; Nabi. N.M.; Heimann, K. Energies, 2013, 6, 5676-5702. 38. Spolaore, P.; Joannis-Cassan, C.; Duran,E and Isambert, A. Journal of bioscience and bioengineering . 2006, 101 (2) 87–96. 39. Musharraf, S. G.; Ahmed, M. A.; Zehra, N,; Kabir.; N, Choudhary.; M. I.; Rahman, A. Chemistry Central Journal. 2012, 6, 149. 40. Singh, D. K.; Mallick, N. J. Microbiol. Biotech. Res., 2014, 4 (1), 37-44.


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41. Sokoto, M.A.; Hassan, L.G., Dangoggo, S.M.; Ahmad, H.G.; Nigerian, U. J. Basic and Applied Science. 2011, 19 (1), 81- 86. 42. Knothe, G. Fuel Process. Technol. 2005, 86 (10), 1059–1070.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Energy & Fuels

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Energy & Fuels

Control N starvation P starvation Fe starvation

3000

2500

2000

6

Cell density ( x 10 per ml)

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1500

1000

500

0 0

5

10

15

20

25

Time (Days)


30

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Biomass productivity (mg/l/day)

Biomass/ Lipid productivity (mg/l/day)

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Energy & Fuels

Lipid productivity (mg/l/day)

30

25 20

*

15 10

5 0 Control

N starvation

P starvation Fe starvation


Energy & Fuels

70

**

60

Lipid content ( %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

50 40 30

20 10 0 Control

N starvation P starvation Fe starvation


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35 Complete Nutrient N starvation

30

P starvation

Fatty acid Composition (in %)

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Energy & Fuels

Fe starvation

25 20 15 10

5 0 C14:0 C16:0 C16:1 C16:2 C17:0 C18:0 C 18:1 C18:2 C 20:1 C20:2 C 22:1 Type of Fatty acid


Energy & Fuels

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FAME Concentration (mg g-1 Lipid) C14:0 C16:0 C16:1 C16:2 C17:0 C18:0 C18:1 C18:2 Total FAME

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Complete Nutrient

N starvation

P starvation

Fe starvation

13.8 ± 3.8 51.4 ± 0.8 34.2 ± 8.9 12.4 ± 3.2 3.2 ± 1.7 22.7 ± 1.3 62.8 ± 0.9 43.3 ± 2.3 244 ± 23

7.9 ± 3.0 54.8 ± 10.5 34 ± 9.0 4.5 ± 1.2 3.9 ± 0.08 22.4 ± 2.9 58.1 ± 9.0 97 ± 18.0 308 ± 61

9.0 ± 1.5 54.6 ± 9.5 33.6 ± 0.5 13.3± 0.9 13 .0± 4.0 32.6 ± 6.7 76.3 ± 5.6 58.0 ± 10 291 ± 47

6.1 ± 1.3 40.2 ± 3.0 29.5 ± 1.9 17 ± 2.4 7.1± 1.2 14.3 ± 1.2 61.3 ± 2.8 50.3 ± 0.7 226 ± 14


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Media conditions

Energy & Fuels

Iodine value (g I2 100 g-1 oil)

Cetane number

Degree of Unsaturation (wt. %)

Long chain saturation factor (°C)

Cold filter plugging point (°C)

Complete Nutrient

Saponification value (mg KOH g-1 oil) 190.5 ± 1.8

77.7 ± 1.1

57.7 ± 0.3

86.4 ± 1.9

4.8 ± 0.5

-1.5 ± 1.0

N starvation

187.9 ± 2.3

88 ± 4.1

55.5 ± 1.3

97 ± 4

4 ± 0.4

-3.9 ± 1.3

P starvation

187.5 ± 0.5

77 ± 1.1

58.1 ± 0.3

86.1 ± 0.7

5.0 ± 0.09

-0.6 ± 0.3

Fe starvation

193.2 ± 2.7

89 ± 0.2

54.5 ± 0.3

97.4 ± 0.2

3.9 ± 0.09

-4.1 ± 0.3


Energy & Fuels

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Figure captions Figure 1: Meyerella sp.N4 under scanning electron microscope (50,000X Magnification) showing 3 µm cell diameter and smooth cell surface topology. Figure 2: Visualization of lipid bodies of Meyerella sp. N4 (stained with Nile red) under confocal microscopy (63X) in various conditions. (I) Control (V) N starved BG-11 medium (3 to 5 numbers of lipid bodies with a size of ~2 µm in diameter) (III) P starved BG-11 medium (VII) Fe starved BG-11 medium ( I, III and VII showed 1 to 2 numbers lipid bodies with a size of ~1µm in diameter). (II) Bright field image of control, (VI) Bright field image of N starved cells, (IV) Bright field image of P starved cells, and (VIII) Bright field image of Fe starved cells. Scale bar- 5 µm and 10 µm. Figure 3: Growth curve of Meyerella sp.N4. Cell density of Meyerella sp.N4 in control (Complete BG-11 medium), nitrogen starved (devoid of NaNO3), phosphorous (devoid of K2HPO4, instead KOH was given as potassium source), and iron-starved (devoid of C6H8FeNO7) BG-11 media at standard growth conditions plotted against time as described in Materials and Methods. Data represent the mean values of two independent duplicate analyses (n=4), and error bars show standard deviations. Figure 4: Biomass productivity and lipid productivity (mg/l/day) of Meyerella sp.N4 under control, nitrogen, phosphorous and iron starved conditions after 23 days of cultivation. Data represent the mean values of two independent duplicate analyses (n=4), and error bars show standard deviations. *P value < 0.05 indicates the statistical significant difference. (Tukey’s honestly significant difference (HSD) test)


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Energy & Fuels

Figure 5: Lipid content of Meyerella sp.N4 in control, nitrogen, phosphorous and iron starved conditions: Lipid content was expressed as percentage dry weight of lipid per 100 mg of biomass. Data represent the mean values of two independent duplicate analyses (n=4), and error bars show standard deviations. **P value < 0.01 denotes the statistical significant difference. Figure 6: Fatty acid composition (expressed in %) in control and starvation conditions. (calculated by area normalization method (i.e) the area of fatty acid peak divided by total peak area in the chromatogram of GC-MS analysis). Data represent the mean values of independent duplicate analyses and error bars show standard deviations. Supplementary Figure. A. Meyerella sp. N4 under light microscopic view (100X objective). Scale Bar-7 µm. Supplementary Figure B: Phylogenetic tree analysis of Meyerella sp. N4 with existing species of Chlorella based on ITS sequences: Bootstrap values are indicated as percentage at the nodes of the tree (with 1000 bootstrap replications). The distance between the related species is being measured by the scale bar. Supplementary Figure C: Separation of neutral lipids by thin layer chromatography. Triolein was used as the standard, separated into different neutral lipid moieties, TAG (triacylglycerol), DAG- (diacyl glycerol) and MAG- (monoacyl glycerol). Lipids of N starved cultures of Meyerella sp.N4 showed distinct TAG band. Supplementary Figure D: Relative percentage of saturated fatty acid (SFA), mono unsaturated fatty acid (MUFA) and poly unsaturated fatty acid (PUFA) of Meyerella sp.N4 in (a) Control (b) N starvation (c) P starvation (d) Fe starvation.


Energy & Fuels

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Table 1: Total yield of FAME obtained from Meyerella sp. N4 grown in nutrient complete starved condition. Data represent the mean values of independent duplicate analyses and error bars show standard deviations. Table 2: Empirical parameters of biodiesel fuel properties of Meyerella sp.N4 with control and nutrient starvations.


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Fatty Acid Biosynthesis from a New Isolate Meyerella sp. N4

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