- Email: [email protected]
Influence of citrate on Chlorella vulgaris for biodiesel production
Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Contents lists available at ScienceDirect
Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Original Research Paper
Influence of citrate on Chlorella vulgaris for biodiesel production Thangapandi Marudhupandi a,n, Velusamy Gunasundari a, Thipramalai Thankappan Ajith Kumar a,c, Kapila R.A. Tissera b a
Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences Annamalai University, Parangipettai 608502, Tamil Nadu, India E-tropical Fish International (Pvt.) Ltd., Sri Lanka c National Bureau of Fish Genetic Resources, Indian Council of Agriculture Research, Lucknow, Uttar Pradesh 226002, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 6 February 2014 Received in revised form 22 March 2014 Accepted 23 March 2014
The present study was carried out on microalgae Chlorella vulgaris to investigate the effect of citrate metabolite as a nutrient for biodiesel production. The oil obtained from C. vulgaris is a promising fuel substitute and can be used as biodiesel. C. vulgaris was cultured in conway medium at different concentrations of citrate, namely 0.5, 1.0 and 2 g/L were added to the culture medium in different experimental groups. Subsequently, the growth, proximate composition and biomass content of the cultured cells were analyzed. The maximum biomass (1.68 70.044 g/L) in the cultured cells was obtained at a citrate concentration of 2.0 g/L in the culture medium. However, the highest oil yield (28.62%) was obtained at the 1owest citrate concentration. Physico-chemical parameters such as pH, viscosity, density, acid and iodine values of the extracted oil were analyzed. In addition, a freeze-dried sample of the algae was analyzed to ascertain its biochemical composition. The maximum lipid content (37.037 7 0.88%) was obtained at the minimum experimental concentration of citrate (0.5 g/L) in the culture media. An increase in citrate concentration in the media resulted in a decrease in total lipid content of the cells, but increased the carbohydrate content up to 29.43 7 0.72%. Fatty acid profiles of the control and experimental groups were analyzed. The present study was suggested that the presence of lowest concentration of citrate in the culture media can improve the accumulation of lipids in C. vulgaris. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Chlorella vulgaris Citrate Lipid Biodiesel Biomass
1. Introduction The diminishing reserves of crude oil, liquid fuels derived from the plant material -biofuels are an attractive source of energy (Scott et al., 2010). Biodiesel, which consisting of fatty acid methyl ester, is renewable, biodegradable, non-toxic as it produces less sulfur dioxide and unburned gases than fossil fuel (Fu et al., 2013). According to a World Bank Report (2008), about 6.5 billion liters of biodiesel were produced globally in 2006. The European Union had contributed 75% and the United State of America (USA), 13%. However, the present contribution of biodiesel to the global transportation fuel consumption was only 0.14% and its favorable policies of the major countries in the world are expected to increase this contribution by 5 times in 2020 (Courchesne et al., 2009). In comparison with conventional terrestrial plants, oil-rich microalgae have shown to be a promising alternative source for lipids for used as biodiesel. They are also widespread and have the capacity to yield comparatively higher oil contents (Rodolfi
n
Corresponding author: Mobile: þ91 8870366413. E-mail address: [email protected] (T. Marudhupandi).
et al., 2010). In comparison with terrestrial biomass, the advantage of microalgae is much higher due to its photosynthetic competence, higher growth rates and improved CO2 alleviation (Brennan and Owende, 2009). Fatty acids are the building blocks for TAG. All other cellular lipids synthesized in the chloroplast use a separate set of enzymes, of which acetyl CoA carboxylase (ACCase) is the key in regulating the synthesis of fatty acids (Hu et al., 2008). The lower lipid content and biomass of microalgae is considered as major obstacles in biodiesel production from microalgae, thus increasing the production cost (Sheehan et al., 1998). Many studies have been conducted with a view of increasing the lipid content in algal cells such as nitrogen deprivation and phosphate limitation (Rodolfi et al., 2010), silicon deficiency (Griffiths and Harrison, 2009), iron supplementation (Liu et al., 2008) and application of varying carbon dioxide (CO2) concentrations (Tsuzuki, 1990) have been tested to achieve a higher yield of fatty acids in algal cells. In addition, Feng et al. (2005) reported that the glucose was added initially to the culture medium to achieve an enhanced fatty acid production in Chlorella vulgaris. Citrate plays a vital role in the intermediary metabolism activity by catalyzing the efflux of citrate from the mitochondrial matrix
http://dx.doi.org/10.1016/j.bcab.2014.03.008 1878-8181/& 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
T. Marudhupandi et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
to the cytosol in exchange for tricarboxylates, dicarboxylates or phosphoenolpyruvate (Kramer and Palmieri, 1992). In the cytosol, citrate yields acetyl-CoA, which represses adenosin triphosphate dependent (ATP) citrate lyase activity. This in turn modulates the glycolytic flux by inhibiting phosphofructokinase. This is a positive allosteric affecter of acetyl-CoA carboxylase, and it activates fatty acid synthesis. With this background, the present study was conducted to evaluate the effect of citrate on biomass and lipid content in C. vulgaris. 2. Materials and methods 2.1. Microalgae and culture conditions An inoculant of marine microalgae C. vulgaris was obtained from the marine ornamental fish hatchery of the Centre for Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, India. The algae was cultured in Conway medium (Walne, 1974) with 1 ml citrate solutions at different concentrations, (0.5, 1 and 2 g/L) in different groups. The control group did not contain citrate. All cultures were maintained at 287 1 1C in 250 ml flasks containing 100 ml culture under illuminated at 2500 l with 12 h light, 12 h dark and shaken at 120 rpm on an orbital shaker. Cultures were harvested on day 4 (96 h). 2.2. Cell growth and dry cell weight The growth status of the culture was measured by using a UV–visible spectrophotometer (Thermoscientific, Evolution 201, USA) at an absorbance of 660 nm. The dry cell weight of microalgal biomass was analyzed by the method proposed by Chiu et al. (2009) with minor modification. Microalgal cells were harvested and centrifuged at 2000 rpm for 10 min. Centrifuged samples were washed twice with distilled water and freeze-dried. The specific growth rate was calculated by using the following formula proposed by Tang et al. (2011). Specific growth rate m (d 1) was calculated from the following equation: Specific growth rate ðmÞ ¼ ðln X 1 ln X 0 Þ=t 1 –t 0 where, X1 and X0 were the biomass concentration (g L 1) on day's t1 and t0, respectively. 2.3. Determination of proximate composition Triplicate samples of freeze-dried cells (10 mg) from each of the experimental cultures were analyzed for total lipid, protein and carbohydrate contents. Total lipid contents were estimated by the method of Folch et al. (1957), protein was estimated by the method of Lowry et al. (1951) and carbohydrates were analyzed by Anthrone method (Seifter et al., 1950). 2.4. Oil extraction Oil extraction from microalgal biomass (2 g) was carried out by the method of Bligh and Dyer (1959). The percentage of oil yield was calculated as Yield of oil ð%Þ ¼ Weight of the oil extracted ðgÞ= Weight of the dried biomass ðgÞ 2.5. Physico-chemical parameters of algal oil The physico-chemical parameters of algae oil such as pH, viscosity, density, acid and iodine values were analyzed by standard methods of analysis (AOCS, 1998).
2.6. Analysis of fatty acid profile by GC–MS The fatty acid components were analyzed with the help of GC–MS. Fatty acid methyl esters (FAME) were obtained by esterification of the lipids (Ichihara and Fukubayashi, 2010). One micro litter of the sample was injected into capillary columns of 25 mm 2 mm 0.33 m film thickness. The equipment used was the Gas Chromatograph model 6890 N of Agilent Technologies, USA. Injection temperature was 220 1C. The initial column temperature was maintained at 60 1C for 4 min and the temperature was subsequently increased to 250 1C with a gradient of 20 1C min 1. Hydrogen was used as the carrier gas at a flow rate of 30 ml/min. Fatty acid profiles of the samples were identified by comparing the commercial Eucary data base with MIS software package (MIS Ver. No. 3.8, Microbial ID. Inc., Newark, Delaware).
3. Results and discussion The proximate composition of the control and experimental groups of C. vulgaris given in Table 1. The protein content of the control and experimental groups were ranged from 44.147 0.92 to 46.117 1.24%. No considerable deviations were present in the protein content among the different groups. However, the carbohydrate and lipid contents showed perceptible deviations depending upon the concentration of citrate in the culture media. In comparison with the control, the maximum cell-carbohydrate content (29.43 70.72%) was observed with the highest tested concentration of citrate (2 g/L) in the media. In contrast, the maximum lipid content of cells (37.03 70.88%) was seen to be associated with the minimum tested concentration of citrate (0.5 g/L). The similar tendancy that the reduced lipid content ranged from 22.5% to 15.9% was observed in C. vulgaris while increasing the concentration of KNO3 ranged from 0.2 to 5.0 mM (Ming et al., 2010). The present study suggested that the variation in lipid and carbohydrate contents in the various tested groups could be the influence of different levels of citrate in the culture media and the high carbohydrate content seen at 2 g/L might be due to high ATP and nicotinamide adinine dinucleotide (NADH) in C. vulgaris that could have repressed glycolysis. The effect of citrate concentrations on the growth of C. vulgaris was estimated by measuring turbidity of the culture solution at 660 nm with a spectrophotometer (Fig. 1). Control showed the minimum growth and the experimental groups growth rate was increased with increase in concentration. The maximum biomass concentrations and high specific growth rates of control and experimental groups were shown in Table 2. Maximum biomass concentration (1.68 70.004 g/L) and highest specific growth rate (1.10770.016 d 1) was obtained in the group containing 2 g/L of citrate in the culture media. It was higher than the maximum reported biomass concentration in C. vulgaris which, is 1.2 g/L (Ming et al., 2010). In the present study, the biomass concentration and specific growth rate had increased at the higher concentration (2 g/L) of citrate but the lipid content in that group was lower. At the lowest concentration (0.5 g/L) of citrate that enhanced Table 1 Biochemical composition (% dry weight biomass) of C. vulgaris at different concentration of citrate and control. Value indicates mean 7 SD. Samples (g/L)
Carbohydrates (%)
Protein (%)
Lipid (%)
Control 0.5 1.0 2.0
9.767 0.3 12.067 0.66 21.717 0.52 29.43 7 0.72
44.147 0.92 43.78 7 0.82 45.25 7 1.06 46.117 1.24
23.137 0.24 37.03 7 0.88 24.37 0.70 16.047 0.38
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
T. Marudhupandi et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 1.0
OD at 660 nm
Table 4 Peak area percentage (%) of fatty acid content of control and various concentration of citrate (0.5, 1 and 2 g/L) in C. vulgaris.
CON 0.5 g/L 1.0 g/L 2.0 g/L
0.8
0.6
0.4
0.2
0.0 0
12
24
36
48
60
72
84
96
108
Culture time (h) Fig. 1. Growth curve of Chlorella vulgaris by using various concentrations of citrate (0.5, 1 and 2 g/L) and control (Con).
Table 2 The maximum biomass concentration (Xmax) and maximum specific growth rate (mmax) of C. vulgaris under different concentrations of citrate and control. Values indicate mean7 SD. Samples (g/L)
Xmax (g L 1)
lmax (d 1)
Control 0.5 1.0 2.0
0.377 0.02 0.747 0.043 1.177 0.020 1.687 0.044
0.7297 0.007 0.903 7 0.004 1.0167 0.012 1.1077 0.016
Table 3 Physico-chemical properties of algae oil from C. vulgaris at different concentration of citrate and control. Samples Oil yield (g/L) percentage (%)
pH Density (g/cm3)
Viscosity at Iodine value (g of I2/100 g 40 1C (mm2/s) of oil)
Acid value (mg KOH/g of acid)
Control 0.5 1.0 2.0
7 7 7 7
4.1 4.3 4.4 4.4
0.39 0.45 0.41 0.38
19.23 28.62 21.84 16.47
3
0.8991 0.9088 0.8993 0.8978
31.2 34.6 32.4 29.8
the lipid level in C. vulgaris but the biomass was low (1.34 7 70.043 g/L). Physicochemical characteristics of the oil samples are important from a shelf life point of view. They determine the ease or difficulties in storing. Physicochemical parameters and the yield percentages of the algal oil obtained in the present study are shown in Table 3. The oil yield varied in different experimental groups from 16.47 to 28.62%. In the present study, the maximum oil yield was obtained at a citrate concentration of 0.5 g/L in the culture media. Oil density, pH, viscosity, iodine and acid values were within the range between, 0.8991 and 0.9088 g/cm3, 7, 4.1 and 4.4 mm2/s, 29.8 and 31.2 g of I2/100 g of oil and 0.38 and 0.45 mg KOH/g of acid. In both control and experimental groups, densities of algal oil obtained are within the accepted values from 0.86 to 0.90 g/cm. The values for viscosity were between the standard range between 3.5 and 5.0 mm2/s. This compares with the ISO 15607 standard. An iodine value less than 120 gI/100 g of oil and an acid value less than 5 mg KOH/g of acid are considered most suitable values defining the quality of biodiesel. Thus, acid and iodine values of the algal oil obtained in the present study readily meet the standard values required by the European Standard (2003) for biodiesel.
Fatty acid
Control (%)
0.5 (%)
1.0 (%)
2.0 (%)
C10:0 C12:0 C14:0 C15:0 C16:0 C16:1 C16:1ω5 C16:1ω6 C16:1ω9 C16:2 C16:3 C17:0 C18:0 C18:1 C18:1ω7 C18:1ω9 C18:2 C18:3 C20:0 C20:1 C20:4ω6 C20:5ω3 C22:4ω6 C22:5ω3 C22:6ω3 Unknown
2.13 1.65 0.43 0.69 29.14 1.37 1.74 0.32 1.02 2.32 0.41 0.71 1.63 3.23 3.46 1.34 24.59 8.60 1.66 0.53 1.39 4.11 1.18 1.2 1.83 3.32
1.26 1.51 0.49 0.07 41.12 0.71 0.53 0.27 0.56 1.78 0.6 0.32 0.72 1.85 1.24 0.98 25.64 4.63 1.51 0.36 1.16 3.84 0.74 1.39 1.31 2.41
1.33 1.26 0.12 0.6 28.43 1.62 0.57 0.34 0.87 1.02 0.55 0.6 1.28 1.16 0.82 0.52 31.14 8.53 1.63 0.41 1.37 8.37 0.71 1.17 1.8 3.78
0.39 0.98 0.18 0.78 22.12 1.42 0.64 0.19 0.88 0.98 0.32 0.61 1.78 1.38 2.55 0.83 35.17 10.22 1.02 0.43 1.69 9.22 0.16 1.56 1.83 2.67
Fatty acid profile of the microalgae samples were presented in Table 4. Unsaturated fatty acids such as eicosapentaenoic acid (EPA, 20:5ω-3) and docosahexaenoic acid (DHA, 22:6ω-3) were obtained in the range between 3.84–9.22% and 1.31–1.83% in both control and experimental groups. In the case of 0.5 g/L citrate level, a notable variation was observed in the quantity of palmatic acid (C16:0), relative quantity of which had gone up to 41.12%. Total fatty acid level in that group also had increased to 37.03 7 70.88% (Table 1). It is interesting to note that Venkata Mohan et al. (2011) had reported that the palmatic or hexadecanoic acid (C16:0) were endowed with the best bio-fuel properties among the saturated fatty acids. It was suggested that low citrate concentration (0.5 g/L) in the culture media encouraged the accumulation of saturated fatty acid C16:0. Similarly, Ming et al. (2010) have observed a higher lipid content at a lower concentration of 0.2 mM KNO3 and 1.0% of CO2. In this instance, the lipid content was 22.5% and 20.0% respectively. In the present study, significant increment in poly unsaturated fatty acids (C18:2) was observed in experimental groups (0.5, 1 and 2 g/L) 25.64, 31.14 and 35.17% respectively.
4. Conclusion Citrate play an important intermediatory metabolic role in both lipogenesis and sterol biosynthetic pathway, as well as a supply of NAD þ and NADPH which support the glycolysis and lipid biosynthesis, respectively. Because of its metabolic importance some studies has been carried out on other organisms for understating its role on fatty acid metabolism. However, to the best of our knowledge this is the first kind of study for investigating the influence of citrate on microalgae C. vulgaris for biofuel aspects. Based on the present findings it concluded that we can use the lowest concentration of citrate for enhancing the fatty acid level. In addition, increasing concentration of citrate favours the carbohydrate accumulation on C. vulgaris, it could be also used as an alternate bio-fuels production. It was suggested that the lower concentration stimulate the lipid biosynthesis on C. vulgaris, while
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
T. Marudhupandi et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
increasing the concentration of citrate, which in turn stimulate the glycolysis. However, further study is needed in the direction of molecular mechanism of citrate on microalgae. Authors contributions TM and TTA is the main investigator for designing, conducted the experiment and drafted the manuscript. VG assisting in experiment and sample analysis. KRAT is a advisor of this study and assisting in language improvement of this MS. Conflict of interest Authors have declared that there are no conflicts of interest. Acknowledgments The authors are grateful to the authorities of Annamalai University for providing facilities to carry out the experiment. The first author extends his sincere thanks to the University Grants Commission, India for financial support under the scheme of CPEPA. Further, we are deeply thankful to Dr. Arthur M.A. Pistorius, Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Netherlands for his valuable suggetion in completing this study. References AOCS, 1998. Official methods and recommended practices of the American oil chemist's Society, 5th ed. AOCS, Champaign, Illinois. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2009. Biofuels from microalgae – a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 14, 557–577. Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 100, 833–838. Courchesne, N.M.D., Parisien., A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J Biotechnol. 141, 31–41.
European Standard EN 14111, 2003. Fat and Oil Derivatives – Fatty Acid Methyl Esters (FAME) Determination of Iodine Value. Feng, F.Y., Yang, W., Jiang, G.Z., Xu, Y.N., Kuang, T.Y., 2005. Enhancement of fatty acid production of Chlorella vulgaris. (Chlorophyceae) by addition of glucose and sodium thiosulphate to culture medium. Proc. Biochem. 40, 1315–1318. Folch, J., Ees, M.I., Stanely, S.G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Fu, X., Li, D., Chen, J., Zhang, Y., Huang, W., Zhu, Y., Yang, J., Zhang, C., 2013. A microalgae residue based carbon solid acid catalyst for biodiesel production. Bioresour. Technol. 146, 767–770. Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507. 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. Kramer, R., Palmieri, F., 1992. Metabolite carriers in mitochondria. In: Ernster, L. (Ed.), Molecular Mechanism in Bioenergetics. Elsevier Science, Amsterdam, pp. 359–384. Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99, 4717–4722. Lowry, O.H., Rosenbrough, W.J., Fair, H.C., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 195, 265–275. Ming, L.J., Cheng, H.L., Hua, H.X., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of C. vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., 2010. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Smith, D.J.L., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277–286. Seifter, S., Dayton, S., Novic, B., Muntwylar, E., 1950. The estimation of glycon with the anthrone reagent. Arch. Biochem. Biophys. 25, 190–200. Sheehan, J., Dunahay, T., Benemann, J., Roessler, P.A., 1998. Look Back at the US Department of Energy's Aquatic Species Program-Biodiesel from Algae. National Renewable Energy Laboratory, USA. Tang, D., Han, W., Li, P., Miao, X., Zhong, J., 2011. CO2 bio fixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels. Bioresour. Technol. 102, 3071–3076. Tsuzuki, M., 1990. Effects of CO2 concentration during growth on fatty acid composition in microalgae. Plant Physiol. 93, 851–856. Venkata Mohan, S., Prathima Devi, M., Mohanakrishna, G., Amarnath, N., Lenin Babu, M., Sarma, P.N., 2011. Potential of mixed microalgae to harness biodiesel from ecological water-bodies with simultaneous treatment. Bioresour. Technol. 102, 1109–1117. Walne, P.R., 1974. Culture of bivalve molluscs. 50 years experience at Conway. Fishing News Books Ltd, United Kingdom. World Bank Report, 2008. Focus B, Biofuels: Promise and Risks.
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
Contents lists available at ScienceDirect
Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Original Research Paper
Influence of citrate on Chlorella vulgaris for biodiesel production Thangapandi Marudhupandi a,n, Velusamy Gunasundari a, Thipramalai Thankappan Ajith Kumar a,c, Kapila R.A. Tissera b a
Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences Annamalai University, Parangipettai 608502, Tamil Nadu, India E-tropical Fish International (Pvt.) Ltd., Sri Lanka c National Bureau of Fish Genetic Resources, Indian Council of Agriculture Research, Lucknow, Uttar Pradesh 226002, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 6 February 2014 Received in revised form 22 March 2014 Accepted 23 March 2014
The present study was carried out on microalgae Chlorella vulgaris to investigate the effect of citrate metabolite as a nutrient for biodiesel production. The oil obtained from C. vulgaris is a promising fuel substitute and can be used as biodiesel. C. vulgaris was cultured in conway medium at different concentrations of citrate, namely 0.5, 1.0 and 2 g/L were added to the culture medium in different experimental groups. Subsequently, the growth, proximate composition and biomass content of the cultured cells were analyzed. The maximum biomass (1.68 70.044 g/L) in the cultured cells was obtained at a citrate concentration of 2.0 g/L in the culture medium. However, the highest oil yield (28.62%) was obtained at the 1owest citrate concentration. Physico-chemical parameters such as pH, viscosity, density, acid and iodine values of the extracted oil were analyzed. In addition, a freeze-dried sample of the algae was analyzed to ascertain its biochemical composition. The maximum lipid content (37.037 7 0.88%) was obtained at the minimum experimental concentration of citrate (0.5 g/L) in the culture media. An increase in citrate concentration in the media resulted in a decrease in total lipid content of the cells, but increased the carbohydrate content up to 29.43 7 0.72%. Fatty acid profiles of the control and experimental groups were analyzed. The present study was suggested that the presence of lowest concentration of citrate in the culture media can improve the accumulation of lipids in C. vulgaris. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Chlorella vulgaris Citrate Lipid Biodiesel Biomass
1. Introduction The diminishing reserves of crude oil, liquid fuels derived from the plant material -biofuels are an attractive source of energy (Scott et al., 2010). Biodiesel, which consisting of fatty acid methyl ester, is renewable, biodegradable, non-toxic as it produces less sulfur dioxide and unburned gases than fossil fuel (Fu et al., 2013). According to a World Bank Report (2008), about 6.5 billion liters of biodiesel were produced globally in 2006. The European Union had contributed 75% and the United State of America (USA), 13%. However, the present contribution of biodiesel to the global transportation fuel consumption was only 0.14% and its favorable policies of the major countries in the world are expected to increase this contribution by 5 times in 2020 (Courchesne et al., 2009). In comparison with conventional terrestrial plants, oil-rich microalgae have shown to be a promising alternative source for lipids for used as biodiesel. They are also widespread and have the capacity to yield comparatively higher oil contents (Rodolfi
n
Corresponding author: Mobile: þ91 8870366413. E-mail address: [email protected] (T. Marudhupandi).
et al., 2010). In comparison with terrestrial biomass, the advantage of microalgae is much higher due to its photosynthetic competence, higher growth rates and improved CO2 alleviation (Brennan and Owende, 2009). Fatty acids are the building blocks for TAG. All other cellular lipids synthesized in the chloroplast use a separate set of enzymes, of which acetyl CoA carboxylase (ACCase) is the key in regulating the synthesis of fatty acids (Hu et al., 2008). The lower lipid content and biomass of microalgae is considered as major obstacles in biodiesel production from microalgae, thus increasing the production cost (Sheehan et al., 1998). Many studies have been conducted with a view of increasing the lipid content in algal cells such as nitrogen deprivation and phosphate limitation (Rodolfi et al., 2010), silicon deficiency (Griffiths and Harrison, 2009), iron supplementation (Liu et al., 2008) and application of varying carbon dioxide (CO2) concentrations (Tsuzuki, 1990) have been tested to achieve a higher yield of fatty acids in algal cells. In addition, Feng et al. (2005) reported that the glucose was added initially to the culture medium to achieve an enhanced fatty acid production in Chlorella vulgaris. Citrate plays a vital role in the intermediary metabolism activity by catalyzing the efflux of citrate from the mitochondrial matrix
http://dx.doi.org/10.1016/j.bcab.2014.03.008 1878-8181/& 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
T. Marudhupandi et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
to the cytosol in exchange for tricarboxylates, dicarboxylates or phosphoenolpyruvate (Kramer and Palmieri, 1992). In the cytosol, citrate yields acetyl-CoA, which represses adenosin triphosphate dependent (ATP) citrate lyase activity. This in turn modulates the glycolytic flux by inhibiting phosphofructokinase. This is a positive allosteric affecter of acetyl-CoA carboxylase, and it activates fatty acid synthesis. With this background, the present study was conducted to evaluate the effect of citrate on biomass and lipid content in C. vulgaris. 2. Materials and methods 2.1. Microalgae and culture conditions An inoculant of marine microalgae C. vulgaris was obtained from the marine ornamental fish hatchery of the Centre for Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, India. The algae was cultured in Conway medium (Walne, 1974) with 1 ml citrate solutions at different concentrations, (0.5, 1 and 2 g/L) in different groups. The control group did not contain citrate. All cultures were maintained at 287 1 1C in 250 ml flasks containing 100 ml culture under illuminated at 2500 l with 12 h light, 12 h dark and shaken at 120 rpm on an orbital shaker. Cultures were harvested on day 4 (96 h). 2.2. Cell growth and dry cell weight The growth status of the culture was measured by using a UV–visible spectrophotometer (Thermoscientific, Evolution 201, USA) at an absorbance of 660 nm. The dry cell weight of microalgal biomass was analyzed by the method proposed by Chiu et al. (2009) with minor modification. Microalgal cells were harvested and centrifuged at 2000 rpm for 10 min. Centrifuged samples were washed twice with distilled water and freeze-dried. The specific growth rate was calculated by using the following formula proposed by Tang et al. (2011). Specific growth rate m (d 1) was calculated from the following equation: Specific growth rate ðmÞ ¼ ðln X 1 ln X 0 Þ=t 1 –t 0 where, X1 and X0 were the biomass concentration (g L 1) on day's t1 and t0, respectively. 2.3. Determination of proximate composition Triplicate samples of freeze-dried cells (10 mg) from each of the experimental cultures were analyzed for total lipid, protein and carbohydrate contents. Total lipid contents were estimated by the method of Folch et al. (1957), protein was estimated by the method of Lowry et al. (1951) and carbohydrates were analyzed by Anthrone method (Seifter et al., 1950). 2.4. Oil extraction Oil extraction from microalgal biomass (2 g) was carried out by the method of Bligh and Dyer (1959). The percentage of oil yield was calculated as Yield of oil ð%Þ ¼ Weight of the oil extracted ðgÞ= Weight of the dried biomass ðgÞ 2.5. Physico-chemical parameters of algal oil The physico-chemical parameters of algae oil such as pH, viscosity, density, acid and iodine values were analyzed by standard methods of analysis (AOCS, 1998).
2.6. Analysis of fatty acid profile by GC–MS The fatty acid components were analyzed with the help of GC–MS. Fatty acid methyl esters (FAME) were obtained by esterification of the lipids (Ichihara and Fukubayashi, 2010). One micro litter of the sample was injected into capillary columns of 25 mm 2 mm 0.33 m film thickness. The equipment used was the Gas Chromatograph model 6890 N of Agilent Technologies, USA. Injection temperature was 220 1C. The initial column temperature was maintained at 60 1C for 4 min and the temperature was subsequently increased to 250 1C with a gradient of 20 1C min 1. Hydrogen was used as the carrier gas at a flow rate of 30 ml/min. Fatty acid profiles of the samples were identified by comparing the commercial Eucary data base with MIS software package (MIS Ver. No. 3.8, Microbial ID. Inc., Newark, Delaware).
3. Results and discussion The proximate composition of the control and experimental groups of C. vulgaris given in Table 1. The protein content of the control and experimental groups were ranged from 44.147 0.92 to 46.117 1.24%. No considerable deviations were present in the protein content among the different groups. However, the carbohydrate and lipid contents showed perceptible deviations depending upon the concentration of citrate in the culture media. In comparison with the control, the maximum cell-carbohydrate content (29.43 70.72%) was observed with the highest tested concentration of citrate (2 g/L) in the media. In contrast, the maximum lipid content of cells (37.03 70.88%) was seen to be associated with the minimum tested concentration of citrate (0.5 g/L). The similar tendancy that the reduced lipid content ranged from 22.5% to 15.9% was observed in C. vulgaris while increasing the concentration of KNO3 ranged from 0.2 to 5.0 mM (Ming et al., 2010). The present study suggested that the variation in lipid and carbohydrate contents in the various tested groups could be the influence of different levels of citrate in the culture media and the high carbohydrate content seen at 2 g/L might be due to high ATP and nicotinamide adinine dinucleotide (NADH) in C. vulgaris that could have repressed glycolysis. The effect of citrate concentrations on the growth of C. vulgaris was estimated by measuring turbidity of the culture solution at 660 nm with a spectrophotometer (Fig. 1). Control showed the minimum growth and the experimental groups growth rate was increased with increase in concentration. The maximum biomass concentrations and high specific growth rates of control and experimental groups were shown in Table 2. Maximum biomass concentration (1.68 70.004 g/L) and highest specific growth rate (1.10770.016 d 1) was obtained in the group containing 2 g/L of citrate in the culture media. It was higher than the maximum reported biomass concentration in C. vulgaris which, is 1.2 g/L (Ming et al., 2010). In the present study, the biomass concentration and specific growth rate had increased at the higher concentration (2 g/L) of citrate but the lipid content in that group was lower. At the lowest concentration (0.5 g/L) of citrate that enhanced Table 1 Biochemical composition (% dry weight biomass) of C. vulgaris at different concentration of citrate and control. Value indicates mean 7 SD. Samples (g/L)
Carbohydrates (%)
Protein (%)
Lipid (%)
Control 0.5 1.0 2.0
9.767 0.3 12.067 0.66 21.717 0.52 29.43 7 0.72
44.147 0.92 43.78 7 0.82 45.25 7 1.06 46.117 1.24
23.137 0.24 37.03 7 0.88 24.37 0.70 16.047 0.38
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
T. Marudhupandi et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 1.0
OD at 660 nm
Table 4 Peak area percentage (%) of fatty acid content of control and various concentration of citrate (0.5, 1 and 2 g/L) in C. vulgaris.
CON 0.5 g/L 1.0 g/L 2.0 g/L
0.8
0.6
0.4
0.2
0.0 0
12
24
36
48
60
72
84
96
108
Culture time (h) Fig. 1. Growth curve of Chlorella vulgaris by using various concentrations of citrate (0.5, 1 and 2 g/L) and control (Con).
Table 2 The maximum biomass concentration (Xmax) and maximum specific growth rate (mmax) of C. vulgaris under different concentrations of citrate and control. Values indicate mean7 SD. Samples (g/L)
Xmax (g L 1)
lmax (d 1)
Control 0.5 1.0 2.0
0.377 0.02 0.747 0.043 1.177 0.020 1.687 0.044
0.7297 0.007 0.903 7 0.004 1.0167 0.012 1.1077 0.016
Table 3 Physico-chemical properties of algae oil from C. vulgaris at different concentration of citrate and control. Samples Oil yield (g/L) percentage (%)
pH Density (g/cm3)
Viscosity at Iodine value (g of I2/100 g 40 1C (mm2/s) of oil)
Acid value (mg KOH/g of acid)
Control 0.5 1.0 2.0
7 7 7 7
4.1 4.3 4.4 4.4
0.39 0.45 0.41 0.38
19.23 28.62 21.84 16.47
3
0.8991 0.9088 0.8993 0.8978
31.2 34.6 32.4 29.8
the lipid level in C. vulgaris but the biomass was low (1.34 7 70.043 g/L). Physicochemical characteristics of the oil samples are important from a shelf life point of view. They determine the ease or difficulties in storing. Physicochemical parameters and the yield percentages of the algal oil obtained in the present study are shown in Table 3. The oil yield varied in different experimental groups from 16.47 to 28.62%. In the present study, the maximum oil yield was obtained at a citrate concentration of 0.5 g/L in the culture media. Oil density, pH, viscosity, iodine and acid values were within the range between, 0.8991 and 0.9088 g/cm3, 7, 4.1 and 4.4 mm2/s, 29.8 and 31.2 g of I2/100 g of oil and 0.38 and 0.45 mg KOH/g of acid. In both control and experimental groups, densities of algal oil obtained are within the accepted values from 0.86 to 0.90 g/cm. The values for viscosity were between the standard range between 3.5 and 5.0 mm2/s. This compares with the ISO 15607 standard. An iodine value less than 120 gI/100 g of oil and an acid value less than 5 mg KOH/g of acid are considered most suitable values defining the quality of biodiesel. Thus, acid and iodine values of the algal oil obtained in the present study readily meet the standard values required by the European Standard (2003) for biodiesel.
Fatty acid
Control (%)
0.5 (%)
1.0 (%)
2.0 (%)
C10:0 C12:0 C14:0 C15:0 C16:0 C16:1 C16:1ω5 C16:1ω6 C16:1ω9 C16:2 C16:3 C17:0 C18:0 C18:1 C18:1ω7 C18:1ω9 C18:2 C18:3 C20:0 C20:1 C20:4ω6 C20:5ω3 C22:4ω6 C22:5ω3 C22:6ω3 Unknown
2.13 1.65 0.43 0.69 29.14 1.37 1.74 0.32 1.02 2.32 0.41 0.71 1.63 3.23 3.46 1.34 24.59 8.60 1.66 0.53 1.39 4.11 1.18 1.2 1.83 3.32
1.26 1.51 0.49 0.07 41.12 0.71 0.53 0.27 0.56 1.78 0.6 0.32 0.72 1.85 1.24 0.98 25.64 4.63 1.51 0.36 1.16 3.84 0.74 1.39 1.31 2.41
1.33 1.26 0.12 0.6 28.43 1.62 0.57 0.34 0.87 1.02 0.55 0.6 1.28 1.16 0.82 0.52 31.14 8.53 1.63 0.41 1.37 8.37 0.71 1.17 1.8 3.78
0.39 0.98 0.18 0.78 22.12 1.42 0.64 0.19 0.88 0.98 0.32 0.61 1.78 1.38 2.55 0.83 35.17 10.22 1.02 0.43 1.69 9.22 0.16 1.56 1.83 2.67
Fatty acid profile of the microalgae samples were presented in Table 4. Unsaturated fatty acids such as eicosapentaenoic acid (EPA, 20:5ω-3) and docosahexaenoic acid (DHA, 22:6ω-3) were obtained in the range between 3.84–9.22% and 1.31–1.83% in both control and experimental groups. In the case of 0.5 g/L citrate level, a notable variation was observed in the quantity of palmatic acid (C16:0), relative quantity of which had gone up to 41.12%. Total fatty acid level in that group also had increased to 37.03 7 70.88% (Table 1). It is interesting to note that Venkata Mohan et al. (2011) had reported that the palmatic or hexadecanoic acid (C16:0) were endowed with the best bio-fuel properties among the saturated fatty acids. It was suggested that low citrate concentration (0.5 g/L) in the culture media encouraged the accumulation of saturated fatty acid C16:0. Similarly, Ming et al. (2010) have observed a higher lipid content at a lower concentration of 0.2 mM KNO3 and 1.0% of CO2. In this instance, the lipid content was 22.5% and 20.0% respectively. In the present study, significant increment in poly unsaturated fatty acids (C18:2) was observed in experimental groups (0.5, 1 and 2 g/L) 25.64, 31.14 and 35.17% respectively.
4. Conclusion Citrate play an important intermediatory metabolic role in both lipogenesis and sterol biosynthetic pathway, as well as a supply of NAD þ and NADPH which support the glycolysis and lipid biosynthesis, respectively. Because of its metabolic importance some studies has been carried out on other organisms for understating its role on fatty acid metabolism. However, to the best of our knowledge this is the first kind of study for investigating the influence of citrate on microalgae C. vulgaris for biofuel aspects. Based on the present findings it concluded that we can use the lowest concentration of citrate for enhancing the fatty acid level. In addition, increasing concentration of citrate favours the carbohydrate accumulation on C. vulgaris, it could be also used as an alternate bio-fuels production. It was suggested that the lower concentration stimulate the lipid biosynthesis on C. vulgaris, while
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i
T. Marudhupandi et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
increasing the concentration of citrate, which in turn stimulate the glycolysis. However, further study is needed in the direction of molecular mechanism of citrate on microalgae. Authors contributions TM and TTA is the main investigator for designing, conducted the experiment and drafted the manuscript. VG assisting in experiment and sample analysis. KRAT is a advisor of this study and assisting in language improvement of this MS. Conflict of interest Authors have declared that there are no conflicts of interest. Acknowledgments The authors are grateful to the authorities of Annamalai University for providing facilities to carry out the experiment. The first author extends his sincere thanks to the University Grants Commission, India for financial support under the scheme of CPEPA. Further, we are deeply thankful to Dr. Arthur M.A. Pistorius, Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Netherlands for his valuable suggetion in completing this study. References AOCS, 1998. Official methods and recommended practices of the American oil chemist's Society, 5th ed. AOCS, Champaign, Illinois. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Brennan, L., Owende, P., 2009. Biofuels from microalgae – a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 14, 557–577. Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 100, 833–838. Courchesne, N.M.D., Parisien., A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J Biotechnol. 141, 31–41.
European Standard EN 14111, 2003. Fat and Oil Derivatives – Fatty Acid Methyl Esters (FAME) Determination of Iodine Value. Feng, F.Y., Yang, W., Jiang, G.Z., Xu, Y.N., Kuang, T.Y., 2005. Enhancement of fatty acid production of Chlorella vulgaris. (Chlorophyceae) by addition of glucose and sodium thiosulphate to culture medium. Proc. Biochem. 40, 1315–1318. Folch, J., Ees, M.I., Stanely, S.G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Fu, X., Li, D., Chen, J., Zhang, Y., Huang, W., Zhu, Y., Yang, J., Zhang, C., 2013. A microalgae residue based carbon solid acid catalyst for biodiesel production. Bioresour. Technol. 146, 767–770. Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507. 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. Kramer, R., Palmieri, F., 1992. Metabolite carriers in mitochondria. In: Ernster, L. (Ed.), Molecular Mechanism in Bioenergetics. Elsevier Science, Amsterdam, pp. 359–384. Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99, 4717–4722. Lowry, O.H., Rosenbrough, W.J., Fair, H.C., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 195, 265–275. Ming, L.J., Cheng, H.L., Hua, H.X., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of C. vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., 2010. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Smith, D.J.L., Smith, A.G., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 277–286. Seifter, S., Dayton, S., Novic, B., Muntwylar, E., 1950. The estimation of glycon with the anthrone reagent. Arch. Biochem. Biophys. 25, 190–200. Sheehan, J., Dunahay, T., Benemann, J., Roessler, P.A., 1998. Look Back at the US Department of Energy's Aquatic Species Program-Biodiesel from Algae. National Renewable Energy Laboratory, USA. Tang, D., Han, W., Li, P., Miao, X., Zhong, J., 2011. CO2 bio fixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels. Bioresour. Technol. 102, 3071–3076. Tsuzuki, M., 1990. Effects of CO2 concentration during growth on fatty acid composition in microalgae. Plant Physiol. 93, 851–856. Venkata Mohan, S., Prathima Devi, M., Mohanakrishna, G., Amarnath, N., Lenin Babu, M., Sarma, P.N., 2011. Potential of mixed microalgae to harness biodiesel from ecological water-bodies with simultaneous treatment. Bioresour. Technol. 102, 1109–1117. Walne, P.R., 1974. Culture of bivalve molluscs. 50 years experience at Conway. Fishing News Books Ltd, United Kingdom. World Bank Report, 2008. Focus B, Biofuels: Promise and Risks.
Please cite this article as: Marudhupandi, T., et al., Influence of citrate on Chlorella vulgaris for biodiesel production. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.03.008i