Ecotoxicology and Environmental Safety ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production R. Karpagam a, R. Preeti a, B. Ashokkumar b, P. Varalakshmi a,n a b
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
art ic l e i nf o
a b s t r a c t
Article history: Received 11 October 2014 Received in revised form 11 March 2015 Accepted 16 March 2015
Lipid from microalgae is one of the putative oil resources to facilitate the biodiesel production during this era of energy dissipation and environmental pollution. In this study, the key parameters such as biomass productivity, lipid productivity and lipid content were evaluated at the early stationary phase of Chlamydomonas reinhardtii, CC1010 cultivated in nutrient starved (nitrogen, phosphorous), glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 supplementation (0.001%, 0.002% and 0.003%) in Tris-AcetatePhosphate (TAP) medium. The lipid content in nitrogen starved media was 61% which is 2.34 folds higher than nutrient sufficient TAP medium. Glucose supplementation has lead to proportional increase in biomass productivity with the increasing concentration of glucose whereas vitamin B12 supplementations had not shown any influence in lipid and biomass production. Further, fatty acid methyl ester (FAME) profiling of C. reinhardtii, CC 1010 has revealed more than 80% of total SFA (saturated fatty acid) and MUFA (mono unsaturated fatty acid) content. Quality checking parameters of biodiesel like cetane number, saponification value, iodine number and degree of unsaturation were analyzed and the biodiesel fuel properties were found to be appropriate as per the international standards, EN 14214 and ASTM D6751. Conclusively, among all the treatments, nitrogen starvation with 0.1% glucose supplementation had yielded high lipid content in C. reinhardtii, CC 1010. & 2015 Elsevier Inc. All rights reserved.
Keywords: Biodiesel Microalgae Profile Lipid productivity Fuel quality parameters
1. Introduction Fuels contribute to 70% of the total global energy demand. Due to exhaustion of fossil fuels and increased CO2 emission, the biofuel production has gained significance. Biodiesel is a biofuel obtained from transesterification of oil or fat from any biological resources (Gouveia and Oliveira, 2009; Yilancioglu et al., 2014). Oleagineous microalgae have greater potential advantages for biodiesel production which includes higher efficiency in photosynthesis, high lipid productivity per unit of land and also it can grow in wide range of environment (Wang et al., 2008).The growth of microalgae by photosynthesis can also lead to the reduction of CO2 in the atmosphere. Waste water primarily contains nitrates and phosphates that can be easily removed by algae during its growth that would augment in waste water treatment (Wang et al., 2008; Sheehan et al., 1998). Thus the microalgaebased fuel production has feasible benefits with respect to environmental safety (Sheehan et al., 1998; Doan et al., 2011). In microalgae, photosynthesis through CO2 fixation produces n
Corresponding author. Fax: þ 91 452 2459105. E-mail address: [email protected] (P. Varalakshmi).
glyceraldehyde-3-phosphate (G3P) which is the precursor of storage molecules such as polysaccharides and lipids. G3P is further converted to pyruvate and then to acetyl – CoA by oxidative decarboxylation. In spite of the significance of acetyl-CoA biosynthesis, the carboxylation of acetyl-CoA to malonyl-CoA is the committing step in fatty acid biosynthesis, catalyzed by the acetyl-CoA carboxylase in the plastid or in the cytosol (Bellou et al., 2014). In these consequences, the appropriate media and cultivation conditions for microalgae to direct the metabolic pathway towards lipid biosynthesis is the quest of research for biodiesel production. Several studies have been reported for lipid synthesis could be attained in nutrient stress by various microalgae like Chlorella vulgaris, Scenedesmus obliquus (Breuer et al., 2012), Organic carbon supplementation in Neochloris oleoabundans (Daniela et al., 2013), nutrient starvation and acetate supplementation in Chlamydomonas reinhardtii (Siaut et al., 2011; Ramanan et al., 2013). The heterotrophic conditions can significantly increase the growth rates and biomass than the photoautotrophic growth of microalgae (Morales-Sánchez et al., 2013). However triacylglycerol (TAG) accumulation in most of the microalgae usually occurs when major nutrients are depleted in the media (Duong et al., 2012; Hu et al., 2013). The level of lipid accumulation is highly influenced by growth cycle, media composition, culturing time and most
http://dx.doi.org/10.1016/j.ecoenv.2015.03.015 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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importantly, species as well as strain specific (Yeh and Chang, 2012; Sharma et al., 2012; Tao et al., 2013). In this study the key parameters such as biomass concentration, lipid productivity, lipid content and the biodiesel quality were evaluated for C. reinhartdii, CC 1010 under control, nitrogen starved (TAPN ), phosphorous starved media (TAPP ) conditions. The fatty acid methyl ester (FAME) of C. reinhartdii, CC 1010 was examined by GC–MS analysis. Further, biodiesel fuel qualities were determined by evaluating the parameters like, saponification value (SV), cetane number (CN), iodine value (IV) and degree of unsaturation (DU) from the empirical equations relating to molecular structures of FAME (Islam et al., 2013). Consequently, in this study the role of lipid accumulation was investigated in C. reinhartdii, CC1010 under glucose, vitamin B12 supplementation and photoheterotrophic nitrogen starvation with glucose which revealed that C. reinhardtii, CC 1010 can be a potential feedstock for biodiesel production.
2. Materials and methods 2.1. Cultivation of microalgae in nutrient starved and supplemented media C. reinhardtii, CC1010 obtained from University of Minnesota was cultivated in 100 ml of TAP medium with 11.8 mg70.08 (SD) with the cell density of 3.5 105 cells ml 1 as initial inoculum. Normal TAP medium includes 7.5 mM NH4Cl was used in this study (Harris., 2009). C. reinhardtii, CC1010 inoculated in normal TAP media was maintained as control. For nitrogen and phosphorous starvation studies, TAP media devoid of NH4Cl [TAPN ] and TAP media devoid of phosphate solution (K2HPO4 and KH2PO4) [TAP P ] were prepared, adjusted to pH 7.0 with HCl. Nutrient supplementation studies were carried out with the addition of glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 (0.001%, 0.002% and 0.003%) to the TAP media. The aforementioned nutrient supplemented TAP media were made from filter sterilized 10% glucose and 1% vitamin B12 stocks. Photoheterotrophic nitrogen starvation studies with 0.1 and 0.2% glucose supplementations [TAPN þ 0.1% glucose] and [TAPN þ0.2% glucose] respectively were also performed. All the above mentioned nutrient conditions were performed twice in independent duplicates under constant illumination (2000 lux) at a distance of 50 cm for alternate photoperiod (light: dark – 12:12 h cycle) at 25 °C under shaking (90 rpm). 2.2. Analysis of lipid bodies through confocal microscopy Cells grown in nutrient starvation and control conditions were stained with 4 ml of Nile Red (Nile blue A oxazone, a lipophilic stain) solution at a concentration of 5 mg/ml of acetone (Hi-media, Mumbai, India), 25 ml of dimethyl sulphoxide (DMSO) was added to aid dye penetration, mixed well by vortexing and incubated in dark for 10 min. Images of the cells with bright fluorescence signal of nile red stained lipid bodies were captured using a laser excitation and emission line at 530 nm and 575 nm respectively using the confocal laser scanning microscope (LSM 510-METAZEISS-Germany). 2.3. Lipid extraction and quantification Total lipids were extracted from microalgal biomass grown in different nutrient conditions at early stationary phase as per the modified method of Folch et al., 1957. The dried microalgal biomass was preweighed (Shimadzu, Germany) and pulverized in mortar and pestle with glass beads in 1 ml of chloroform–methanol (2:1, v/v). The lipids were extracted using chloroform–
Table 1 Biomass concentration (g/L), lipid content (%) and lipid productivity (mg/L/day) of nutrient starved cultures. Total lipids were extracted from C. reinhardtii, CC 1010 at 12th day of growth for control and TAPP where as 5th day in TAPN . Data represent mean 7 S.D. (n¼ 4) of two independent duplicate analyzes. Media conditions
TAP
TAP N
TAP P
Biomass concentration (g/L) Lipid content (%) Lipid productivity (mg/L/day)
0.7770.04 23.8 71.6 15.2 70.42
0.2647 0.04 617 3.6 32.1** 74.2
0.39 7 0.05 27.9 7 6.3 14.6 7 2.2
nn
Asterisk denotes the significant difference with a P value o0.01.
methanol (2:1, v/v) by centrifugation at 8000 rpm for 10 min. The centrifugation step was repeated 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 evaporated in a preweighed glass plate and dry weight of the lipid was measured gravimetrically. 2.4. Separation of neutral lipids by TLC Thin layer chromatographic technique was adopted to separate the lipids extracted from different nutrient conditions, using the developing solvent (hexane; diethyl ether; acetic acid in the ratio of 70:30:1) (Herrera-Valencia et al., 2012). Triolein (HIMEDIA, Mumbai, India) was used as TAG standard. To visualize the various type of lipids, the TLC plate was sprayed with 10% copper sulfate (in 8% phosphoric acid) and dried at 180 °C for 10 min. 2.5. FAME preparation and GC–MS analysis The lipid sample was dissolved in 0.5 ml of toluene in a screwcapped glass test tube. To this 1.5 ml methanol and 50 ml of 35% conc. HCl (0.39 M) were added and the transesterification reaction was performed by incubating the contents at 100 °C for 1 h and 30 min. One ml of hexane was added and vortexed further. The hexane layer (FAME) was collected and transferred to GC vial (Cyberlab, USA). Thus the transesterfication was done through acid catalysis (HCl) (Ichihara and Fukubayashi, 2010). Further the FAME was separated using gas chromatograph (JEOL GC MATE-II, JEOL Ltd, Tokyo, Japan) equipped with column HP-5 MS; photo multiplier tube detector. High 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 min. Electron impact ionization mass spectrometry (EI-MS) was used to detect the m/z value of the separated fatty acid compound. The mass Spectrum data of each peak of the chromatogram was compared with the NIST database for the identification of the fatty acid compound. The relative percentage of fatty acids were calculated by area normalization method. 2.6. Determination of fuel properties Fuel properties such as saponification value (SV), iodine value (IV), cetane number (CN) and degree of unsaturation (DU) were calculated from the fatty acid profile of C. reinhardtii, CC 1010 using the following empirical formula (Nascimento et al., 2013; Karpagam et al., 2015a; Hu et al., 2008).
IV =
∑ (254 × F × D)/MW
SV =
∑ (560 × F)/MW
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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Fig. 1. Observation of oil bodies in Nile red stained cells of C. reinhardtii, CC 1010 under confocal laser scanning microscope (at 63 objective). (I) Control showing 0.5– 1.0 mm in size of oil bodies. (II) Oil bodies with the size of 2–4 mm accumulated in TAPN medium (III) TAPP medium showing 0.5–1.0 mm size of oil bodies. Scale bar – 5 and 10 mm. Table 2 Biomass concentration (g/L), Lipid productivity (mg/L/day) and lipid content (%) of C. reinhardtii, CC-1010 in TAP without nitrogen and 0.1% and 0.2% glucose. Data represent mean of two independent duplicate analyzes7 S.D (n ¼4). Media conditions
TAP N
TAP N þ 0.1% glucose
Biomass concentration (g/L) Lipid content (%) Lipid productivity (mg/ L/day)
0.26 7 0.04 0.377 0.035
0.377 0.04
61.2 7 3.6 32.17 4.2
477 9.25 33.87 3.1
58.7 7 4.6 42.9 7 4.7
TAP N þ 0.2% glucose
CN = (46.3 + [5458/SV]) − (0.225 × IV)
DU = MUFA wt% + (2×PUFA wt%) Where F is the % of each type of fatty acid, MW is the molecular weight of corresponding fatty acid and D is the no. of double bonds. 2.7. Statistical analysis One-way ANOVA was employed to resolve the statistical significance followed by Tukey’s honestly significant difference (HSD) test (Lowry, 1998-2015).
3. Results and discussion 3.1. Growth parameters and lipid productivity under nutrient starvation In order to assess the phase of growth in control and starved media, the growth curve was plotted against cell density and time. C. reinhardtii, CC1010 attained stationary phase after five days of cultivation in TAPN , seven days in TAPP media, in turn the cells grown in control attained only early stationary phase even after 12 days of cultivation (Supplementary Fig. S1(b)). However, under P
Fig. 2. Relative percentage of fatty acid of C. reinhardtii, CC1010 in control and nutrient starved cultures (calculated from the area of fatty acid peak divided by total peak area in the chromatogram of GC–MS analysis by area normalization method). Table 4 Quality parameters of biodiesel for control and nutrient starved cultures of C. reinhardtii, CC 1010. Biodiesel fuel Properties
TAP
TAPN
TAPP
SN (mg KOH g-1 oil) IV (g I2 100 g-1 oil) CN DU (wt%)
165.9 64.8 64.6 73
190.7 61.4 61.1 65.3
172.5 50.2 66.6 57.1
limitation, phosphates are replenished by certain physiological mechanisms such as P mobilization via starch synthesis from existing cultured inoculum, secretion of phosphatases and ribonucleases, activation of transporters, increasing the affinity of enzymes for nutrient assimilation (Yehudai-Resheff et al., 2007; Wykoff et al., 1999). Optimal concentration of macronutrients that incorporates in to the microalgae for the biosynthesis of cellular components is represented by a constant known as Redfield ratio
Table 3 Biomass concentration (g/L), lipid productivity (mg/L/day) and lipid content (%) of C. reinhardtii, CC 1010 in TAP media supplemented with glucose and vitamin. Data represent mean7 S.D (n¼ 4) of two independent duplicate analyzes. Supplementation of media
Control
0.05% glucose
0.1% glucose
0.15% glucose
0.2% glucose
0.001% vitamin
0.002% vitamin
0.003% vitamin
Biomass concentration (g/L) Lipid productivity (mg/L/day) Lipid content (%)
0.777 0.04 15.2 7 0.42 23.8 7 1.6
0.8 70.02 15.4 70.3 23.2 70.6
0.81 7 0.01 15.83 7 0.25 23.62 7 0.54
0.84 7 0.02 14.13 7 0.84 20.18 7 1.7
0.85 7 0.03 15.5 7 0.8 227 1.5
0.75 70.3 15.4 70.7 24.571.6
0.79 7 0.04 14.4 7 1.1 227 1.4
0.767 0.04 14.5 7 0.6 23.17 2.01
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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(C:N:P – 106:16:1) (Redfield, 1934) that explains the least growth in TAPN followed by TAPP media. Lipid productivity of C. reinhardtii, CC1010 cultivated in TAPN and TAPP media were 32.1 mg/L/day and 14.6 mg/L/day respectively, when compared to control (15.2 mg/L/day) (Table 1). Biomass concentration and lipid content of C. reinhardtii, CC1010 in nutrient starved media were given in Table 1. Lipid content of CC1010 in TAPN was about 61% as revealed by prominent oil bodies in Nile red staining (Fig. 1) and high intense TAG band was observed in TLC (Supplementary Fig. S2), than in TAPP and control. Hence under nitrogen depleted conditions, the unavailability of nitrogen reduces the photosynthesis and respiration rate due to damaged PS II with concomitant increase in cyclic phosphorylation for TAG synthesis (Zhang et al., 2013). 3.2. Photoheterotrophic nitrogen starvation with glucose for enhanced lipid production In contrast to photoautotrophic microalgal cells, heterotrophic cells are directing the excessive carbon for the biosynthesis of neutral lipids (Liu et al., 2011a; Kong et al., 2013). In our study, the supply of 0.1% glucose in the TAPN medium has led to increased TAG accumulation than TAPN (Supplementary Fig. S2). There is a noticeable increase in lipid productivity of 42.9 mg/L/day in TAPN supplemented with 0.1% glucose than TAPN (32.1 mg/L/ day) and 33.8 mg/L/day in TAPN supplemented with 0.2% glucose (Supplementary Fig. S2 and Table S2). Similar increase in lipid content of around 10.64% at 10 g/L of glycerol and 2 g/L of glucose was obtained in C. vulgaris, than in autotrophic mode with the lowest lipid content of 7.48% (Chandra et al., 2014). In Tetraselmis suecica cultures, the rate of lipid synthesis appeared to be significantly enhanced by adding bicarbonate in a dose dependent manner with rates of 0.5, 0.8 and 1.3 pg cell 1day 1 for 0, 1 and 2 g L 1 respectively (White et al., 2012). Consequently, in our study glucose supplementation in nutrient starvation resulted in higher lipid storage with noticeable increase in biomass concentration (Table 2). 3.3. Effect of glucose and vitamin supplementation Glucose promoted the donation of electrons to the plastoquinone pool from the respiratory substance, and the transformation of energy was promoted by photosynthetic system, which provided the energy needed for the anabolism of cells caused by addition of glucose to the medium (Wang et al., 2000). Partial organic carbon supplementation has increased the biomass and lipid productivity in Chlorella sorokiniana and Coelastrella sp. M60 (Wan et al., 2012; Karpagam et al., 2015b). Based on the earlier reports mentioned above, the effect of different concentrations of glucose supplementation in TAP media (0.05%, 0.1%, 0.15%, and 0.2%) on biomass concentration, lipid productivity and lipid content of C. reinhardtii, CC1010 were evaluated after 12 days of growth. Lipid content of glucose supplemented C. reinhardtii, CC1010 did not reflect any significant change (Supplementary Fig. S3 (a) and Table S3). The increased biomass in glucose supplementation had shown the moderate increase in total chlorophyll concentration (mg/g DCW) in glucose supplementation when compared to control and the constant chlorophyll a/b ratio also implies that the biosynthesis and degradation of Chl a and b were balanced (Yang et al., 1995). Thus glucose supplementation would support the biomass increase (Supplementary Fig. S4 and Supplementary Table S1) ( Table 3). Cyanocobalamin (Vitamin B12) is the key growth promoter for living organism and about 50% of microalgal population requires vitamin B12 for its growth (Droop, 2007; Helliwell et al., 2014). In order to evaluate the effects of vitamin B12, TAP media was
supplemented with different concentrations of vitamin B12 (0.001%, 0.002% and 0.003%). The significant influence of vitamin B12 supplementation on biomass and lipid production was not found in C. reinhardtii, CC 1010 and lipids were analyzed by the TLC (Supplementary Fig. S3 (b) and Table S3). Suggestions from the earlier reports revealed that the Chlamydomonas cells have the capacity to synthesis cobalamin and the exogenous cobalamin in the media would be generally accumulated in the cells (Watanabe et al., 1991). 3.4. Determination of fuel properties through FAME profiling Increased saturated fatty acid (SFA) and mono unsaturated fatty acid (MUFA) especially C 16:0 (11% in control to 18% in TAPN ) and C18:1 (37% in control to 40.5% in TAPN ) respectively under nitrogen starvation which are similar to the previous report (Zhang et al., 2013) (Fig. 2). Fuel properties such as saponification value (SV), cetane number (CN), Iodine value (IV) and degree of unsaturation (DU) were calculated from the fatty acid compositions using the empirical equations as given in materials and methods. DU is strongly associated to the fuel properties such as kinematic viscosity, specific gravity, cloud point including cetane number and Iodine value. CN is the measure of ignition quality and it increases with the increase in saturated fatty acid content in the oil and the iodine value gives the measure of double bond numbers (Nascimento et al., 2013). Kinematic viscosity, cetane number, oxidation stability, cold flow properties of the biodiesel increases with decreasing DU whereas iodine value and the density of the biodiesel increases with increased unsaturation. Thus the fuel parameters of C. reinhardtii, CC1010 were found in accordance with the biodiesel fuel specifications given by the regulatory international standards, ASTM D6751 (U.S.) and EN 14214 (Europe) (Hoekman et al., 2012). (Table 4).
4. Conclusion In this study, the biomass, lipid productivity and FAME profile of C. reinhardtii CC1010 grown in normal, nitrogen and phosphorous starvation were analyzed. TAP media without nitrogen with the supply of glucose increases the lipid productivity in C. reinhardtii CC1010. Biodiesel fuel properties of C. reinhardtii CC1010 based on FAME profile were found to have met with the international biofuel standards ASTM D6751 (U.S.) and EN 14214 (Europe). Conclusively, strategies for enhancement of lipid production in C. reinhardtii CC1010 through photoheterotrophic media conditions could enhance the lipid accumulation for the production of biodiesel.
Acknowledgment Authors thank Department of Science and Technology (SERB) New Delhi, India (Ref: SR/ FT/LS-20/2012 dt.18.9.2012) for the financial assistance to PV and DST-PURSE program for instrumentation facility. We also acknowledge SAIF, IITM Chennai, India for GC–MS analysis.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.03. 015.
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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References Bellou, S., Baeshen, N.M., Elazzazy, M.A., Aggeli, D., Sayegh, F., Aggelis, G., 2014. Microalgal lipids biochemistry and biotechnological perspectives. Biotechnol. Adv. 32, 1476–1493. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 79, 217–226. Chandra, R., Rohit, M.V., Swamy, Y.V., Venkata Mohan, S., 2014. Regulatory function of organic carbon supplementation on biodiesel production during growth and nutrient stress phases of mixotrophic microalgae cultivation. Bioresour. Technol. 165, 279–287. Daniela, M.S., Tinoco-Valencia, R., Kyndt, J., Martinez, A., 2013. Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source. Biotechnol. Biofuels 6, 100. Doan, T.T.Y., Sivaloganathan, B., Obbard, J.P., 2011. Screening of marine microalgae for biodiesel feedstock. Biomass Bioenergy 35, 2534–2544. Droop, M.R., 2007. Vitamins, phytoplankton and bacteria: symbiosis or scavenging. J. Plankton Res. 29, 107–113. Duong, V.T., Li, Y., Nowak, E., Schenk, P.M., 2012. Microalgae isolation and selection for prospective biodiesel production. Energies 5, 1835–1849. Folch, J., Lees, M., Stanley, G.H.S., 1957. Simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509. Gouveia, L., Oliveira, A.G., 2009. Microalgae as a raw material for biofuels production. J. Ind. Microbiol. Biotechnol. 36, 269–274. Harris, E.H., 2009. The genius Chlamydomonas The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and its Laboratory Use, 1. Elsevier, pp. 1–18, 2nd edition, Kindle edition, Academic Press, USA. Helliwell, K.E., Scaife, M.A., Sasso, S., Araujo, A.P., Purton, S., Smith, A.G., 2014. Unraveling vitamin B12-responsive gene regulation in algae. Plant Physiol. 165 (1), 388–397. Herrera-Valencia, V.A., Us-Vázquez, R.A., Larqué-Saavedra, F.A., Barahona-Pérez, L.F., 2012. Naturally occurring fatty acid methyl esters and ethyl esters in the green microalga Chlamydomonas reinhardtii. Ann. Microbiol. 62, 865–870. Hoekman, S.K., Broch, A., Robbins, C., Ceniceros, E., Natarajan, M., 2012. Review of biodiesel composition, properties, and specifications. Renew. Sustain. Energy Rev. 16, 143–169. Hu, C.W., Chuang, L.T., Yu, P.C., Chen, C.N.N., 2013. Pigment production by a new thermotolerant microalga Coelastrella sp. F50. Food Chem. 138, 2071–2078. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Ichihara, K., Fukubayashi, Y., 2010. Preparation of fatty acid methyl esters for gas– liquid chromatography. J. Lipid Res. 51 (3), 635–640. Islam, M.A., Magnusson, M., Brown, R.J., Ayoko, A.G., Nabi, N.M., Heimann, K., 2013. Energies 6, 5676–5702. Karpagam, R., Raj, K.J., Ashokkumar, B., Varalakshmi, P., 2015b. Characterization and fatty acid profiling in two fresh water microalgae for biodiesel production: lipid enhancement methods and media optimization using response surface methodology. Bioresour. Technol., http://dx.doi.org/10.1016/j.biortech.2015.01.053 Karpagam, R., Raj, K.J., Saranya, S., Ashokkumar, B., Varalakshmi, P., 2015a. Fatty acid biosynthesis from a new isolate Meyerella sp. N4: molecular characterization, nutrient starvation, and fatty acid profiling for lipid enhancement. Energy Fuels 29, 143–149. Kong, W.B., Yang, H., Cao, T.Y., Song, H., Hua, F.S., Xia, G.C., 2013. Effect of glycerol and glucose on the enhancement of biomass, lipid and soluble carbohydrate production by Chlorella vulgaris in mixotrophic culture. Food Technol. Biotechnol. 51 (1), 62–69. Liu, J., Huang, J., Sun, Z., Zhong, Y., Jiang, Y., Chen, F., 2011a. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production. Bioresour. Technol. 102, 106–110. Morales-Sánchez, D., Tinoco-Valencia, R., Kyndt, J., Martinez, A., 2013. Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source. Biotechnol. Biofuels 6, 100.
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Nascimento, I.A., Marques, S.S.I., Cabanelas, I.T.D., Pereira, S.A., Druzian, J.I., De souza, C.O., Vich, D.V., De Carvalho, G.C., Nacimento, M.A., 2013. Screening microalgae strains for biodiesel production: lipid productivity and estimation of fuel quality based on fatty acid profiles as selective criteria. Bioenergy Res. 6, 1–13. Richard Lowry, 1998-2015. One-Way Analysis of Variance for Independent or Correlated Samples. 〈http://vassarstats.net/anova1u.html〉. Ramanan, R., Kim, B.H., Cho, D.H., Ko, S.R., Oh, H.M., Kim, H.S., 2013. Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii. FEBS Lett. 587 (4). http://dx.doi.org/10.1016/j.febslet.2012.12.020, Epub 2013 Jan 10. PubMed PMID: 23313852. Redfield, A.C., 1934. On the proportions of organic derivations in sea water and their relation to the composition of plankton In: Daniel, R.J. (Ed.), James Johnstone Memorial Volume. University Press of Liverpool, UK, pp. 177–192. Sharma, K.K., Schuhmann, H., Schenk, P.M., 2012. High lipid induction in microalgae for biodiesel production. Energies 5, 1532–1553. Sheehan, J., Camobreco, V., Duffield, J., Graboski, M., Shapouri, H., 1998. Life cycle inventory of biodiesel and petroleum diesel for use in an urban bus. U.S. Department of Energy and U.S. Department of Agriculture, USA, Technical report NREL/SR-580-24089. Siaut, M., Cuiné, S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Beyly, A., Beisson, F., Triantaphylidès, C., Li-Beisson, Y., Peltier, G., 2011. Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol. 11, 7. Tao, L., Linglin, W., Aifen, L., Chengwu, Z., 2013. Responses in growth, lipid accumulation, and fatty acid composition of four oleaginous microalgae to different nitrogen sources and concentrations. Chin. J. Oceanol. Limnol. 31 (6), 1306–1314. Wan, M.X., Wang, R.M., Xia, J.L., Rosenberg, J.N., Nie, Z.Y., Kobayashi, N., Oyler, G.A., Betenbaugh, M.J., 2012. Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures. Biotechnol. Bioeng. 109, 8. Wang, B., Li, Y., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79, 707–718. Wang, Y.H., Ye, J.Y., Mi, H.L., Li, Y.G., Zhang, C.L., 2000. Relationship between the growth of Synechococcus sp. PCC6803 on medium with glucose and the photosynthetic energy transformation. Acta Bot. Sin. 42, 1122–1125. Watanabe, F., Nakona, Y., Tamura, Y., Yamanaka, H., 1991. Vitamin B12 metabolism in a photosynthesizing green alga, Chlamydomonas reinhardtii. Biochim. Biophys. Acta 1075 (1), 36–41. White, D.A., Pagarette, A., Rooks, P., Ali, S.T., 2012. The effect of sodium bicarbonate supplementation on growth and biochemical composition of marine microalgae cultures. J. Appl. Phycol., 10811–012-9849-6. Wykoff, D.D., Grossman, R.A., Weeks, D.P., Usuda, H., Shimogawara, K., 1999. Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas. Proc. Natl. Acad. Sci. USA 96 (26), 15336–15341. Yang, C.–M., Hsu, J.-C., Chen, Y.-R., 1995. Light-sensitivity of chlorophyll formation in the leaves of Ficus microcarpa cv Golden-leaves. Bot. Bull. Acad. Sin. 36, 215–221. Yeh, K.-L., Chang, J.-S., 2012. Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. Bioresour. Technol. 105, 120–127. Yehudai-Resheff, S.L., Zimmer, Y., Komine, Stern, B.D., 2007. Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii. Plant Cell 19, 1023–1038. Yilancioglu, K., Cokol, M., Pastirmaci, I., Erman, B., Cetiner, S., 2014. Oxidative stress is a mediator for increased lipid accumulation in a newly isolated Dunaliella salina strain. PLoS ONE 9 (3), e91957. Zhang, Y.M., Chen, H., He, C.L., Wang, Q., 2013. Nitrogen starvation induced oxidative stress in an oil-producing green alga Chlorella sorokiniana C3. PLoS ONE 8 (7), e69225.
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production R. Karpagam a, R. Preeti a, B. Ashokkumar b, P. Varalakshmi a,n a b
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
art ic l e i nf o
a b s t r a c t
Article history: Received 11 October 2014 Received in revised form 11 March 2015 Accepted 16 March 2015
Lipid from microalgae is one of the putative oil resources to facilitate the biodiesel production during this era of energy dissipation and environmental pollution. In this study, the key parameters such as biomass productivity, lipid productivity and lipid content were evaluated at the early stationary phase of Chlamydomonas reinhardtii, CC1010 cultivated in nutrient starved (nitrogen, phosphorous), glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 supplementation (0.001%, 0.002% and 0.003%) in Tris-AcetatePhosphate (TAP) medium. The lipid content in nitrogen starved media was 61% which is 2.34 folds higher than nutrient sufficient TAP medium. Glucose supplementation has lead to proportional increase in biomass productivity with the increasing concentration of glucose whereas vitamin B12 supplementations had not shown any influence in lipid and biomass production. Further, fatty acid methyl ester (FAME) profiling of C. reinhardtii, CC 1010 has revealed more than 80% of total SFA (saturated fatty acid) and MUFA (mono unsaturated fatty acid) content. Quality checking parameters of biodiesel like cetane number, saponification value, iodine number and degree of unsaturation were analyzed and the biodiesel fuel properties were found to be appropriate as per the international standards, EN 14214 and ASTM D6751. Conclusively, among all the treatments, nitrogen starvation with 0.1% glucose supplementation had yielded high lipid content in C. reinhardtii, CC 1010. & 2015 Elsevier Inc. All rights reserved.
Keywords: Biodiesel Microalgae Profile Lipid productivity Fuel quality parameters
1. Introduction Fuels contribute to 70% of the total global energy demand. Due to exhaustion of fossil fuels and increased CO2 emission, the biofuel production has gained significance. Biodiesel is a biofuel obtained from transesterification of oil or fat from any biological resources (Gouveia and Oliveira, 2009; Yilancioglu et al., 2014). Oleagineous microalgae have greater potential advantages for biodiesel production which includes higher efficiency in photosynthesis, high lipid productivity per unit of land and also it can grow in wide range of environment (Wang et al., 2008).The growth of microalgae by photosynthesis can also lead to the reduction of CO2 in the atmosphere. Waste water primarily contains nitrates and phosphates that can be easily removed by algae during its growth that would augment in waste water treatment (Wang et al., 2008; Sheehan et al., 1998). Thus the microalgaebased fuel production has feasible benefits with respect to environmental safety (Sheehan et al., 1998; Doan et al., 2011). In microalgae, photosynthesis through CO2 fixation produces n
Corresponding author. Fax: þ 91 452 2459105. E-mail address: [email protected] (P. Varalakshmi).
glyceraldehyde-3-phosphate (G3P) which is the precursor of storage molecules such as polysaccharides and lipids. G3P is further converted to pyruvate and then to acetyl – CoA by oxidative decarboxylation. In spite of the significance of acetyl-CoA biosynthesis, the carboxylation of acetyl-CoA to malonyl-CoA is the committing step in fatty acid biosynthesis, catalyzed by the acetyl-CoA carboxylase in the plastid or in the cytosol (Bellou et al., 2014). In these consequences, the appropriate media and cultivation conditions for microalgae to direct the metabolic pathway towards lipid biosynthesis is the quest of research for biodiesel production. Several studies have been reported for lipid synthesis could be attained in nutrient stress by various microalgae like Chlorella vulgaris, Scenedesmus obliquus (Breuer et al., 2012), Organic carbon supplementation in Neochloris oleoabundans (Daniela et al., 2013), nutrient starvation and acetate supplementation in Chlamydomonas reinhardtii (Siaut et al., 2011; Ramanan et al., 2013). The heterotrophic conditions can significantly increase the growth rates and biomass than the photoautotrophic growth of microalgae (Morales-Sánchez et al., 2013). However triacylglycerol (TAG) accumulation in most of the microalgae usually occurs when major nutrients are depleted in the media (Duong et al., 2012; Hu et al., 2013). The level of lipid accumulation is highly influenced by growth cycle, media composition, culturing time and most
http://dx.doi.org/10.1016/j.ecoenv.2015.03.015 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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importantly, species as well as strain specific (Yeh and Chang, 2012; Sharma et al., 2012; Tao et al., 2013). In this study the key parameters such as biomass concentration, lipid productivity, lipid content and the biodiesel quality were evaluated for C. reinhartdii, CC 1010 under control, nitrogen starved (TAPN ), phosphorous starved media (TAPP ) conditions. The fatty acid methyl ester (FAME) of C. reinhartdii, CC 1010 was examined by GC–MS analysis. Further, biodiesel fuel qualities were determined by evaluating the parameters like, saponification value (SV), cetane number (CN), iodine value (IV) and degree of unsaturation (DU) from the empirical equations relating to molecular structures of FAME (Islam et al., 2013). Consequently, in this study the role of lipid accumulation was investigated in C. reinhartdii, CC1010 under glucose, vitamin B12 supplementation and photoheterotrophic nitrogen starvation with glucose which revealed that C. reinhardtii, CC 1010 can be a potential feedstock for biodiesel production.
2. Materials and methods 2.1. Cultivation of microalgae in nutrient starved and supplemented media C. reinhardtii, CC1010 obtained from University of Minnesota was cultivated in 100 ml of TAP medium with 11.8 mg70.08 (SD) with the cell density of 3.5 105 cells ml 1 as initial inoculum. Normal TAP medium includes 7.5 mM NH4Cl was used in this study (Harris., 2009). C. reinhardtii, CC1010 inoculated in normal TAP media was maintained as control. For nitrogen and phosphorous starvation studies, TAP media devoid of NH4Cl [TAPN ] and TAP media devoid of phosphate solution (K2HPO4 and KH2PO4) [TAP P ] were prepared, adjusted to pH 7.0 with HCl. Nutrient supplementation studies were carried out with the addition of glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 (0.001%, 0.002% and 0.003%) to the TAP media. The aforementioned nutrient supplemented TAP media were made from filter sterilized 10% glucose and 1% vitamin B12 stocks. Photoheterotrophic nitrogen starvation studies with 0.1 and 0.2% glucose supplementations [TAPN þ 0.1% glucose] and [TAPN þ0.2% glucose] respectively were also performed. All the above mentioned nutrient conditions were performed twice in independent duplicates under constant illumination (2000 lux) at a distance of 50 cm for alternate photoperiod (light: dark – 12:12 h cycle) at 25 °C under shaking (90 rpm). 2.2. Analysis of lipid bodies through confocal microscopy Cells grown in nutrient starvation and control conditions were stained with 4 ml of Nile Red (Nile blue A oxazone, a lipophilic stain) solution at a concentration of 5 mg/ml of acetone (Hi-media, Mumbai, India), 25 ml of dimethyl sulphoxide (DMSO) was added to aid dye penetration, mixed well by vortexing and incubated in dark for 10 min. Images of the cells with bright fluorescence signal of nile red stained lipid bodies were captured using a laser excitation and emission line at 530 nm and 575 nm respectively using the confocal laser scanning microscope (LSM 510-METAZEISS-Germany). 2.3. Lipid extraction and quantification Total lipids were extracted from microalgal biomass grown in different nutrient conditions at early stationary phase as per the modified method of Folch et al., 1957. The dried microalgal biomass was preweighed (Shimadzu, Germany) and pulverized in mortar and pestle with glass beads in 1 ml of chloroform–methanol (2:1, v/v). The lipids were extracted using chloroform–
Table 1 Biomass concentration (g/L), lipid content (%) and lipid productivity (mg/L/day) of nutrient starved cultures. Total lipids were extracted from C. reinhardtii, CC 1010 at 12th day of growth for control and TAPP where as 5th day in TAPN . Data represent mean 7 S.D. (n¼ 4) of two independent duplicate analyzes. Media conditions
TAP
TAP N
TAP P
Biomass concentration (g/L) Lipid content (%) Lipid productivity (mg/L/day)
0.7770.04 23.8 71.6 15.2 70.42
0.2647 0.04 617 3.6 32.1** 74.2
0.39 7 0.05 27.9 7 6.3 14.6 7 2.2
nn
Asterisk denotes the significant difference with a P value o0.01.
methanol (2:1, v/v) by centrifugation at 8000 rpm for 10 min. The centrifugation step was repeated 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 evaporated in a preweighed glass plate and dry weight of the lipid was measured gravimetrically. 2.4. Separation of neutral lipids by TLC Thin layer chromatographic technique was adopted to separate the lipids extracted from different nutrient conditions, using the developing solvent (hexane; diethyl ether; acetic acid in the ratio of 70:30:1) (Herrera-Valencia et al., 2012). Triolein (HIMEDIA, Mumbai, India) was used as TAG standard. To visualize the various type of lipids, the TLC plate was sprayed with 10% copper sulfate (in 8% phosphoric acid) and dried at 180 °C for 10 min. 2.5. FAME preparation and GC–MS analysis The lipid sample was dissolved in 0.5 ml of toluene in a screwcapped glass test tube. To this 1.5 ml methanol and 50 ml of 35% conc. HCl (0.39 M) were added and the transesterification reaction was performed by incubating the contents at 100 °C for 1 h and 30 min. One ml of hexane was added and vortexed further. The hexane layer (FAME) was collected and transferred to GC vial (Cyberlab, USA). Thus the transesterfication was done through acid catalysis (HCl) (Ichihara and Fukubayashi, 2010). Further the FAME was separated using gas chromatograph (JEOL GC MATE-II, JEOL Ltd, Tokyo, Japan) equipped with column HP-5 MS; photo multiplier tube detector. High 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 min. Electron impact ionization mass spectrometry (EI-MS) was used to detect the m/z value of the separated fatty acid compound. The mass Spectrum data of each peak of the chromatogram was compared with the NIST database for the identification of the fatty acid compound. The relative percentage of fatty acids were calculated by area normalization method. 2.6. Determination of fuel properties Fuel properties such as saponification value (SV), iodine value (IV), cetane number (CN) and degree of unsaturation (DU) were calculated from the fatty acid profile of C. reinhardtii, CC 1010 using the following empirical formula (Nascimento et al., 2013; Karpagam et al., 2015a; Hu et al., 2008).
IV =
∑ (254 × F × D)/MW
SV =
∑ (560 × F)/MW
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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Fig. 1. Observation of oil bodies in Nile red stained cells of C. reinhardtii, CC 1010 under confocal laser scanning microscope (at 63 objective). (I) Control showing 0.5– 1.0 mm in size of oil bodies. (II) Oil bodies with the size of 2–4 mm accumulated in TAPN medium (III) TAPP medium showing 0.5–1.0 mm size of oil bodies. Scale bar – 5 and 10 mm. Table 2 Biomass concentration (g/L), Lipid productivity (mg/L/day) and lipid content (%) of C. reinhardtii, CC-1010 in TAP without nitrogen and 0.1% and 0.2% glucose. Data represent mean of two independent duplicate analyzes7 S.D (n ¼4). Media conditions
TAP N
TAP N þ 0.1% glucose
Biomass concentration (g/L) Lipid content (%) Lipid productivity (mg/ L/day)
0.26 7 0.04 0.377 0.035
0.377 0.04
61.2 7 3.6 32.17 4.2
477 9.25 33.87 3.1
58.7 7 4.6 42.9 7 4.7
TAP N þ 0.2% glucose
CN = (46.3 + [5458/SV]) − (0.225 × IV)
DU = MUFA wt% + (2×PUFA wt%) Where F is the % of each type of fatty acid, MW is the molecular weight of corresponding fatty acid and D is the no. of double bonds. 2.7. Statistical analysis One-way ANOVA was employed to resolve the statistical significance followed by Tukey’s honestly significant difference (HSD) test (Lowry, 1998-2015).
3. Results and discussion 3.1. Growth parameters and lipid productivity under nutrient starvation In order to assess the phase of growth in control and starved media, the growth curve was plotted against cell density and time. C. reinhardtii, CC1010 attained stationary phase after five days of cultivation in TAPN , seven days in TAPP media, in turn the cells grown in control attained only early stationary phase even after 12 days of cultivation (Supplementary Fig. S1(b)). However, under P
Fig. 2. Relative percentage of fatty acid of C. reinhardtii, CC1010 in control and nutrient starved cultures (calculated from the area of fatty acid peak divided by total peak area in the chromatogram of GC–MS analysis by area normalization method). Table 4 Quality parameters of biodiesel for control and nutrient starved cultures of C. reinhardtii, CC 1010. Biodiesel fuel Properties
TAP
TAPN
TAPP
SN (mg KOH g-1 oil) IV (g I2 100 g-1 oil) CN DU (wt%)
165.9 64.8 64.6 73
190.7 61.4 61.1 65.3
172.5 50.2 66.6 57.1
limitation, phosphates are replenished by certain physiological mechanisms such as P mobilization via starch synthesis from existing cultured inoculum, secretion of phosphatases and ribonucleases, activation of transporters, increasing the affinity of enzymes for nutrient assimilation (Yehudai-Resheff et al., 2007; Wykoff et al., 1999). Optimal concentration of macronutrients that incorporates in to the microalgae for the biosynthesis of cellular components is represented by a constant known as Redfield ratio
Table 3 Biomass concentration (g/L), lipid productivity (mg/L/day) and lipid content (%) of C. reinhardtii, CC 1010 in TAP media supplemented with glucose and vitamin. Data represent mean7 S.D (n¼ 4) of two independent duplicate analyzes. Supplementation of media
Control
0.05% glucose
0.1% glucose
0.15% glucose
0.2% glucose
0.001% vitamin
0.002% vitamin
0.003% vitamin
Biomass concentration (g/L) Lipid productivity (mg/L/day) Lipid content (%)
0.777 0.04 15.2 7 0.42 23.8 7 1.6
0.8 70.02 15.4 70.3 23.2 70.6
0.81 7 0.01 15.83 7 0.25 23.62 7 0.54
0.84 7 0.02 14.13 7 0.84 20.18 7 1.7
0.85 7 0.03 15.5 7 0.8 227 1.5
0.75 70.3 15.4 70.7 24.571.6
0.79 7 0.04 14.4 7 1.1 227 1.4
0.767 0.04 14.5 7 0.6 23.17 2.01
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(C:N:P – 106:16:1) (Redfield, 1934) that explains the least growth in TAPN followed by TAPP media. Lipid productivity of C. reinhardtii, CC1010 cultivated in TAPN and TAPP media were 32.1 mg/L/day and 14.6 mg/L/day respectively, when compared to control (15.2 mg/L/day) (Table 1). Biomass concentration and lipid content of C. reinhardtii, CC1010 in nutrient starved media were given in Table 1. Lipid content of CC1010 in TAPN was about 61% as revealed by prominent oil bodies in Nile red staining (Fig. 1) and high intense TAG band was observed in TLC (Supplementary Fig. S2), than in TAPP and control. Hence under nitrogen depleted conditions, the unavailability of nitrogen reduces the photosynthesis and respiration rate due to damaged PS II with concomitant increase in cyclic phosphorylation for TAG synthesis (Zhang et al., 2013). 3.2. Photoheterotrophic nitrogen starvation with glucose for enhanced lipid production In contrast to photoautotrophic microalgal cells, heterotrophic cells are directing the excessive carbon for the biosynthesis of neutral lipids (Liu et al., 2011a; Kong et al., 2013). In our study, the supply of 0.1% glucose in the TAPN medium has led to increased TAG accumulation than TAPN (Supplementary Fig. S2). There is a noticeable increase in lipid productivity of 42.9 mg/L/day in TAPN supplemented with 0.1% glucose than TAPN (32.1 mg/L/ day) and 33.8 mg/L/day in TAPN supplemented with 0.2% glucose (Supplementary Fig. S2 and Table S2). Similar increase in lipid content of around 10.64% at 10 g/L of glycerol and 2 g/L of glucose was obtained in C. vulgaris, than in autotrophic mode with the lowest lipid content of 7.48% (Chandra et al., 2014). In Tetraselmis suecica cultures, the rate of lipid synthesis appeared to be significantly enhanced by adding bicarbonate in a dose dependent manner with rates of 0.5, 0.8 and 1.3 pg cell 1day 1 for 0, 1 and 2 g L 1 respectively (White et al., 2012). Consequently, in our study glucose supplementation in nutrient starvation resulted in higher lipid storage with noticeable increase in biomass concentration (Table 2). 3.3. Effect of glucose and vitamin supplementation Glucose promoted the donation of electrons to the plastoquinone pool from the respiratory substance, and the transformation of energy was promoted by photosynthetic system, which provided the energy needed for the anabolism of cells caused by addition of glucose to the medium (Wang et al., 2000). Partial organic carbon supplementation has increased the biomass and lipid productivity in Chlorella sorokiniana and Coelastrella sp. M60 (Wan et al., 2012; Karpagam et al., 2015b). Based on the earlier reports mentioned above, the effect of different concentrations of glucose supplementation in TAP media (0.05%, 0.1%, 0.15%, and 0.2%) on biomass concentration, lipid productivity and lipid content of C. reinhardtii, CC1010 were evaluated after 12 days of growth. Lipid content of glucose supplemented C. reinhardtii, CC1010 did not reflect any significant change (Supplementary Fig. S3 (a) and Table S3). The increased biomass in glucose supplementation had shown the moderate increase in total chlorophyll concentration (mg/g DCW) in glucose supplementation when compared to control and the constant chlorophyll a/b ratio also implies that the biosynthesis and degradation of Chl a and b were balanced (Yang et al., 1995). Thus glucose supplementation would support the biomass increase (Supplementary Fig. S4 and Supplementary Table S1) ( Table 3). Cyanocobalamin (Vitamin B12) is the key growth promoter for living organism and about 50% of microalgal population requires vitamin B12 for its growth (Droop, 2007; Helliwell et al., 2014). In order to evaluate the effects of vitamin B12, TAP media was
supplemented with different concentrations of vitamin B12 (0.001%, 0.002% and 0.003%). The significant influence of vitamin B12 supplementation on biomass and lipid production was not found in C. reinhardtii, CC 1010 and lipids were analyzed by the TLC (Supplementary Fig. S3 (b) and Table S3). Suggestions from the earlier reports revealed that the Chlamydomonas cells have the capacity to synthesis cobalamin and the exogenous cobalamin in the media would be generally accumulated in the cells (Watanabe et al., 1991). 3.4. Determination of fuel properties through FAME profiling Increased saturated fatty acid (SFA) and mono unsaturated fatty acid (MUFA) especially C 16:0 (11% in control to 18% in TAPN ) and C18:1 (37% in control to 40.5% in TAPN ) respectively under nitrogen starvation which are similar to the previous report (Zhang et al., 2013) (Fig. 2). Fuel properties such as saponification value (SV), cetane number (CN), Iodine value (IV) and degree of unsaturation (DU) were calculated from the fatty acid compositions using the empirical equations as given in materials and methods. DU is strongly associated to the fuel properties such as kinematic viscosity, specific gravity, cloud point including cetane number and Iodine value. CN is the measure of ignition quality and it increases with the increase in saturated fatty acid content in the oil and the iodine value gives the measure of double bond numbers (Nascimento et al., 2013). Kinematic viscosity, cetane number, oxidation stability, cold flow properties of the biodiesel increases with decreasing DU whereas iodine value and the density of the biodiesel increases with increased unsaturation. Thus the fuel parameters of C. reinhardtii, CC1010 were found in accordance with the biodiesel fuel specifications given by the regulatory international standards, ASTM D6751 (U.S.) and EN 14214 (Europe) (Hoekman et al., 2012). (Table 4).
4. Conclusion In this study, the biomass, lipid productivity and FAME profile of C. reinhardtii CC1010 grown in normal, nitrogen and phosphorous starvation were analyzed. TAP media without nitrogen with the supply of glucose increases the lipid productivity in C. reinhardtii CC1010. Biodiesel fuel properties of C. reinhardtii CC1010 based on FAME profile were found to have met with the international biofuel standards ASTM D6751 (U.S.) and EN 14214 (Europe). Conclusively, strategies for enhancement of lipid production in C. reinhardtii CC1010 through photoheterotrophic media conditions could enhance the lipid accumulation for the production of biodiesel.
Acknowledgment Authors thank Department of Science and Technology (SERB) New Delhi, India (Ref: SR/ FT/LS-20/2012 dt.18.9.2012) for the financial assistance to PV and DST-PURSE program for instrumentation facility. We also acknowledge SAIF, IITM Chennai, India for GC–MS analysis.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.03. 015.
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
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References Bellou, S., Baeshen, N.M., Elazzazy, M.A., Aggeli, D., Sayegh, F., Aggelis, G., 2014. Microalgal lipids biochemistry and biotechnological perspectives. Biotechnol. Adv. 32, 1476–1493. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 79, 217–226. Chandra, R., Rohit, M.V., Swamy, Y.V., Venkata Mohan, S., 2014. Regulatory function of organic carbon supplementation on biodiesel production during growth and nutrient stress phases of mixotrophic microalgae cultivation. Bioresour. Technol. 165, 279–287. Daniela, M.S., Tinoco-Valencia, R., Kyndt, J., Martinez, A., 2013. Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source. Biotechnol. Biofuels 6, 100. Doan, T.T.Y., Sivaloganathan, B., Obbard, J.P., 2011. Screening of marine microalgae for biodiesel feedstock. Biomass Bioenergy 35, 2534–2544. Droop, M.R., 2007. Vitamins, phytoplankton and bacteria: symbiosis or scavenging. J. Plankton Res. 29, 107–113. Duong, V.T., Li, Y., Nowak, E., Schenk, P.M., 2012. Microalgae isolation and selection for prospective biodiesel production. Energies 5, 1835–1849. Folch, J., Lees, M., Stanley, G.H.S., 1957. Simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509. Gouveia, L., Oliveira, A.G., 2009. Microalgae as a raw material for biofuels production. J. Ind. Microbiol. Biotechnol. 36, 269–274. Harris, E.H., 2009. The genius Chlamydomonas The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and its Laboratory Use, 1. Elsevier, pp. 1–18, 2nd edition, Kindle edition, Academic Press, USA. Helliwell, K.E., Scaife, M.A., Sasso, S., Araujo, A.P., Purton, S., Smith, A.G., 2014. Unraveling vitamin B12-responsive gene regulation in algae. Plant Physiol. 165 (1), 388–397. Herrera-Valencia, V.A., Us-Vázquez, R.A., Larqué-Saavedra, F.A., Barahona-Pérez, L.F., 2012. Naturally occurring fatty acid methyl esters and ethyl esters in the green microalga Chlamydomonas reinhardtii. Ann. Microbiol. 62, 865–870. Hoekman, S.K., Broch, A., Robbins, C., Ceniceros, E., Natarajan, M., 2012. Review of biodiesel composition, properties, and specifications. Renew. Sustain. Energy Rev. 16, 143–169. Hu, C.W., Chuang, L.T., Yu, P.C., Chen, C.N.N., 2013. Pigment production by a new thermotolerant microalga Coelastrella sp. F50. Food Chem. 138, 2071–2078. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621–639. Ichihara, K., Fukubayashi, Y., 2010. Preparation of fatty acid methyl esters for gas– liquid chromatography. J. Lipid Res. 51 (3), 635–640. Islam, M.A., Magnusson, M., Brown, R.J., Ayoko, A.G., Nabi, N.M., Heimann, K., 2013. Energies 6, 5676–5702. Karpagam, R., Raj, K.J., Ashokkumar, B., Varalakshmi, P., 2015b. Characterization and fatty acid profiling in two fresh water microalgae for biodiesel production: lipid enhancement methods and media optimization using response surface methodology. Bioresour. Technol., http://dx.doi.org/10.1016/j.biortech.2015.01.053 Karpagam, R., Raj, K.J., Saranya, S., Ashokkumar, B., Varalakshmi, P., 2015a. Fatty acid biosynthesis from a new isolate Meyerella sp. N4: molecular characterization, nutrient starvation, and fatty acid profiling for lipid enhancement. Energy Fuels 29, 143–149. Kong, W.B., Yang, H., Cao, T.Y., Song, H., Hua, F.S., Xia, G.C., 2013. Effect of glycerol and glucose on the enhancement of biomass, lipid and soluble carbohydrate production by Chlorella vulgaris in mixotrophic culture. Food Technol. Biotechnol. 51 (1), 62–69. Liu, J., Huang, J., Sun, Z., Zhong, Y., Jiang, Y., Chen, F., 2011a. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production. Bioresour. Technol. 102, 106–110. Morales-Sánchez, D., Tinoco-Valencia, R., Kyndt, J., Martinez, A., 2013. Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source. Biotechnol. Biofuels 6, 100.
5
Nascimento, I.A., Marques, S.S.I., Cabanelas, I.T.D., Pereira, S.A., Druzian, J.I., De souza, C.O., Vich, D.V., De Carvalho, G.C., Nacimento, M.A., 2013. Screening microalgae strains for biodiesel production: lipid productivity and estimation of fuel quality based on fatty acid profiles as selective criteria. Bioenergy Res. 6, 1–13. Richard Lowry, 1998-2015. One-Way Analysis of Variance for Independent or Correlated Samples. 〈http://vassarstats.net/anova1u.html〉. Ramanan, R., Kim, B.H., Cho, D.H., Ko, S.R., Oh, H.M., Kim, H.S., 2013. Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii. FEBS Lett. 587 (4). http://dx.doi.org/10.1016/j.febslet.2012.12.020, Epub 2013 Jan 10. PubMed PMID: 23313852. Redfield, A.C., 1934. On the proportions of organic derivations in sea water and their relation to the composition of plankton In: Daniel, R.J. (Ed.), James Johnstone Memorial Volume. University Press of Liverpool, UK, pp. 177–192. Sharma, K.K., Schuhmann, H., Schenk, P.M., 2012. High lipid induction in microalgae for biodiesel production. Energies 5, 1532–1553. Sheehan, J., Camobreco, V., Duffield, J., Graboski, M., Shapouri, H., 1998. Life cycle inventory of biodiesel and petroleum diesel for use in an urban bus. U.S. Department of Energy and U.S. Department of Agriculture, USA, Technical report NREL/SR-580-24089. Siaut, M., Cuiné, S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Beyly, A., Beisson, F., Triantaphylidès, C., Li-Beisson, Y., Peltier, G., 2011. Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol. 11, 7. Tao, L., Linglin, W., Aifen, L., Chengwu, Z., 2013. Responses in growth, lipid accumulation, and fatty acid composition of four oleaginous microalgae to different nitrogen sources and concentrations. Chin. J. Oceanol. Limnol. 31 (6), 1306–1314. Wan, M.X., Wang, R.M., Xia, J.L., Rosenberg, J.N., Nie, Z.Y., Kobayashi, N., Oyler, G.A., Betenbaugh, M.J., 2012. Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures. Biotechnol. Bioeng. 109, 8. Wang, B., Li, Y., Wu, N., Lan, C.Q., 2008. CO2 bio-mitigation using microalgae. Appl. Microbiol. Biotechnol. 79, 707–718. Wang, Y.H., Ye, J.Y., Mi, H.L., Li, Y.G., Zhang, C.L., 2000. Relationship between the growth of Synechococcus sp. PCC6803 on medium with glucose and the photosynthetic energy transformation. Acta Bot. Sin. 42, 1122–1125. Watanabe, F., Nakona, Y., Tamura, Y., Yamanaka, H., 1991. Vitamin B12 metabolism in a photosynthesizing green alga, Chlamydomonas reinhardtii. Biochim. Biophys. Acta 1075 (1), 36–41. White, D.A., Pagarette, A., Rooks, P., Ali, S.T., 2012. The effect of sodium bicarbonate supplementation on growth and biochemical composition of marine microalgae cultures. J. Appl. Phycol., 10811–012-9849-6. Wykoff, D.D., Grossman, R.A., Weeks, D.P., Usuda, H., Shimogawara, K., 1999. Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas. Proc. Natl. Acad. Sci. USA 96 (26), 15336–15341. Yang, C.–M., Hsu, J.-C., Chen, Y.-R., 1995. Light-sensitivity of chlorophyll formation in the leaves of Ficus microcarpa cv Golden-leaves. Bot. Bull. Acad. Sin. 36, 215–221. Yeh, K.-L., Chang, J.-S., 2012. Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. Bioresour. Technol. 105, 120–127. Yehudai-Resheff, S.L., Zimmer, Y., Komine, Stern, B.D., 2007. Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii. Plant Cell 19, 1023–1038. Yilancioglu, K., Cokol, M., Pastirmaci, I., Erman, B., Cetiner, S., 2014. Oxidative stress is a mediator for increased lipid accumulation in a newly isolated Dunaliella salina strain. PLoS ONE 9 (3), e91957. Zhang, Y.M., Chen, H., He, C.L., Wang, Q., 2013. Nitrogen starvation induced oxidative stress in an oil-producing green alga Chlorella sorokiniana C3. PLoS ONE 8 (7), e69225.
Please cite this article as: Karpagam, R., et al., Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production. Ecotoxicol. Environ. Saf. (2015), http://dx.doi.org/10.1016/j.ecoenv.2015.03.015i
