This article was downloaded by: [RMIT University] On: 27 July 2013, At: 15:11 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae a
a
Prakash Chandra Gorain , Sourav Kumar Bagchi & Nirupama Mallick
a
a
Agricultural and Food Engineering Department , Indian Institute of Technology Kharagpur , Kharagpur , 721302 , India Accepted author version posted online: 12 Jun 2013.Published online: 05 Jul 2013.
To cite this article: Environmental Technology (2013): Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae, Environmental Technology, DOI: 10.1080/09593330.2013.812668 To link to this article: http://dx.doi.org/10.1080/09593330.2013.812668
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2013 http://dx.doi.org/10.1080/09593330.2013.812668
Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae Prakash Chandra Gorain, Sourav Kumar Bagchi and Nirupama Mallick∗ Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
Downloaded by [RMIT University] at 15:11 27 July 2013
(Received 8 March 2013; final version received 1 June 2013 ) Biodiesel from microalgae has the potential as a sustainable fuel, since some species show exceptionally high lipid accumulation potential under various stresses. Effects of different concentrations of Ca, Mg and NaCl in the growth medium on biomass yield and lipid accumulation of Chlorella vulgaris and Scenedesmus obliquus grown under batch culture mode were investigated. Starvation of Mg showed a marginal rise in lipid content for a short period of time. Ca-starved cultures, however, demonstrated a profound rise in lipid content, i.e. 40% of dry cell wt. (dcw) was recorded against 11.9% control for C. vulgaris and 37% (dcw) against 11.3% for S. obliquus. Under increased concentration of Mg, significant rise in biomass and lipid yield was recorded. Effect of NaCl-induced osmotic stress showed lipid accumulation of ∼40% (dcw) in both the test algae, whereas the biomass yield was severely affected. The fatty acid profiles under the above stresses were analysed and discussed. Keywords: biodiesel; microalgae; Ca starvation; lipids; Mg starvation; NaCl; fatty acid methyl esters
1. Introduction Biofuels are arguably more sustainable, technically feasible and environment friendly than the fossil fuels. They are relatively low net emitters of greenhouse gases.[1] Among them, biodiesel and bioethanol have gained mainstream attention. Preference of diesel engines over engines run by other forms of fuel in India carries development of biodiesel technology a higher potential for this nation. Bioethanol, which is used in blends with gasoline, has low cetane numbers and, therefore, is unsuitable for diesel engines. On the other hand, biodiesel can be used to run the conventional diesel engines with only minor adjustments.[2] Biodiesel is formed chemically by reacting lipids (derived from animal or plant sources) with an alcohol, preferably methanol owing to cost effectiveness, to provide fatty acid esters (biodiesel). This chemical reaction, otherwise known as transesterification, requires a strong base such as sodium or potassium hydroxide or an acid such as hydrochloric or sulphuric acid as the catalyst.[3] The first generation of biodiesel was sourced from edible oils such as sunflower and oil palm, whereas the second generation was processed from non-edible oil crops such as jatropha, mahua, pongamia, etc. Currently, the third generation focuses on microalgal biodiesel technology because of several advantages. First of all, microalgae can grow at a very high rate and in some cases biomass doubling time of only 3.5 h was recorded during the exponential growth phase.[4] While the mechanism of photosynthesis ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
in microalgae is similar to that of higher plants, they have the ability to capture solar energy with an efficiency of 10–50 times higher than that of terrestrial plants.[5] They are also capable of producing more amount of oil per unit area of land in comparison with that of all other known oil-producing crops.[6,7] The per hectare yield of microalgal oil has been projected to be 58,700–136,900 L year−1 depending upon the oil content of algae, which is about 10–20 times higher than the best oil-producing crop, i.e. palm (5950 L ha−1 year−1 ).[6] The most acclaimed energy crop, i.e. jatropha, has been estimated to produce only 1892 L ha−1 year−1 . Recent report of Moazami et al. [8] showed that Nannochloropsis sp. PTCC 6016 has the capability of producing 60,000 L of biodiesel ha−1 year−1 . More importantly, due to aquatic in nature, algae do not compete for arable land for their cultivation and can be grown in freshwater or saline or even in wastewaters.[7,9–13] This implies that algae need not compete with other users for freshwater. On the top of these, microalgae grow even faster when fed with extra carbon dioxide, the main greenhouse gas. Production of biodiesel from microalgae primarily depends on the cellular lipid content and biomass productivity of selected species. It has been observed that limitations of major nutrients such as nitrogen and phosphorus triggered lipid accumulation in various microalgal species.[14–19] However, reports on other major nutrients such as Ca and Mg on the lipid accumulation potential
Downloaded by [RMIT University] at 15:11 27 July 2013
2
P.C. Gorain et al.
of microalgae are scarce, except Deng et al. [20] where neutral lipid content was detectably higher in Chlorella vulgaris grown in Ca- and Mg-free medium. It was once suggested that algae that thrive under deficit of these minerals should be classified as lower or more primitive in the phylogenetic tree than those species that cannot survive under such deficit. Studies conducted by Manuel [21] with a Chlorella strain showed remarkable growth up to 10 mM concentration of CaCl2 , but higher concentrations were found inhibitory. Similarly, the process of multiplication was found to require a larger concentration of magnesium in the medium, and this divalent ion significantly influences the chlorophyll biosynthesis.[22] Therefore, here, the aim was set to study growth pattern, corresponding lipid accumulation and variations in fatty acid profile in two green microalgae, C. vulgaris and Scenedesmus obliquus, under starvation as well as high concentrations of calcium and magnesium in the growth medium. For the past few years, biologists are concentrating on the effects of osmotic stress on various aspects of plants and microorganisms. Barghbani et al. [23] reported that with increasing NaCl concentration, there was a decrease in biomass yield of C. vulgaris, and complete suppression of growth was evident at 30 gL−1 NaCl. Reports showing increased lipid productivity, and changes in fatty acids profile in some microalgal species under salinity stress are also available.[24,25] Considering this, the effect of osmotic stress on the two test microalgae was also investigated.
2. Materials and methods 2.1. Organisms and growth conditions Axenic cultures of two green microalgae, S. obliquus (Trup.) Kutz. (SAG 276-3a) and C. vulgaris (an isolate of river Ganges, courtesy: Prof. L. C. Rai, Banaras Hindu University, Varanasi, India), were used in this study, and maintained in a culture room at 25 ± 2◦ C under a photoperiod of 14:10 h at a light intensity of 75 μmol photon m−2 s−1 photosynthetically active radiation (PAR). Both the microalgae were cultured in N11 medium [26] at pH 6.8. For maintenance of batch cultures, the microalgae were grown in 250 mL Erlenmeyer flasks containing 100 mL medium without sparging with air or CO2 . The cultures were hand shaken 2–3 times daily to avoid sticking, and was referred as control culture.
2.2.
Dry weight measurement
Dry cell weight (dcw) was measured in three days intervals, where three flasks, each containing 100 mL of cultures, were withdrawn from the culture rack. The algal cultures were centrifuged at 5000 rpm for 10 min and transferred into preweighed vials for oven-drying at 60◦ C to get a constant weight. Oven-drying was preferred as it was found to be efficient, convenient, cost-effective and the most widely use
method for microalgal drying.[27–30] The biomass yield was estimated by the gravimetric method and expressed as g L−1 .[31] 2.3. Extraction of lipids from algal biomass After estimation of dcw, lipid extraction was done from the same vials following the protocol of Bligh and Dyer [32] with chloroform and methanol as solvents. Lipid content/yields were expressed as % dcw/mg L−1 . 2.4.
Effects of Ca, Mg and NaCl on biomass yield and lipid accumulation in the test microalgae To study the effects of calcium and magnesium starvation on biomass yield and lipid accumulation, C. vulgaris and S. obliquus cells were grown in Ca- and Mg-starved conditions, which were achieved by depriving the medium of the respective salts. To study the effects of increased concentrations of Mg and Ca, microalgal cultures were grown in various increased concentrations of Mg and Ca, and these conditions were achieved by the addition of higher amounts of Mg and Ca salts with respect to the control condition. The various increased concentrations were 0.05 gL−1 (1X: control), 0.1 gL−1 (2X), 0.15 gL−1 (3X), 0.25 gL−1 (5X), 0.5 gL−1 (10X) for MgSO4 · 7H2 O, and 0.01 gL−1 (1X: control), 0.02 gL−1 (2X), 0.03 gL−1 (3X), 0.05 gL−1 (5X), 0.1 gL−1 (10X) for CaCl2 · H2 O. To study the effect of osmotic stress, the stationary phase cultures were subjected to different concentrations of NaCl, i.e. 1.0–9.0 gL−1 of NaCl. Samples were withdrawn on every third day; biomass and lipids were quantified as described above. 2.5.
Transesterification and analysis of fatty acid methyl esters (FAMEs) As acid values of the test microalgal lipids were found to be very high (ranging from 45.2 to 48.3 mg KOH g−1 ), the acid-catalysed transesterification was performed using a molar ratio of oil:methanol:HCl::1:80:4 with a reaction temperature of 60◦ C and reaction time of 6.4 h, as optimized in our laboratory.[33] The top organic phase, which contained FAMEs was pipetted out and the aqueous phase containing residual methanol, glycerol and the catalyst was discarded. The top organic layer was taken for GC-MS analysis using Clarus 680 GC (Perkin-Elmer, Shelton, CT, USA) equipped with an Elite-1 methylpolysiloxane capillary column (30 m × 0.25 mm × 0.25 μm) and MS (Clarus 600 T MS) equipped with a photo-multiplied tube detector which has a phosphor screen. 2.6. Statistical analysis All the experiments were performed in triplicate to check the reproducibility. The results were analysed statistically by Duncan’s new multiple range test (DMRT). DMRT
3
was done by MSTAT-C software (Plant and Soil Sciences Division, Michigan State University, USA).
concentration of Mg lipid yield ranged between 124 and 143 mg L−1 .
3. Results 3.1. Time course of growth and lipid accumulation in C. vulgaris and S. obliquus under control condition The time course of growth and lipid accumulation in C. vulgaris and S. obliquus under the batch culture mode was studied in N11 medium of standard composition. With a lag of 3 days, growth of the test algae increased steadily. The stationary phase was seen at about 21 days (Figure 1(a)). The maximum biomass yield was 1.2 and 1.1 g L−1 , respectively, for C. vulgaris and S. obliquus. Maximum accumulation of lipids (11.3% for S. obliquus and 11.9% dcw for C. vulgaris) was observed at the stationary phase of cultures (Figure 1(b)).
3.3. Effect of Ca on biomass and lipid yield Increased Ca concentration in the growth medium was found to have a little effect on bimass yield of the test microalgae; lipid content, however, showed an increasing trend (Figure 3). Profound rise (∼40% dcw) in cellular lipid pool was observed under Ca-starved condition (Figure 3(c) and 3(d)). The maximum lipid yield reached up to 331 mg L−1 in the case of C. vulgaris. For S. obliquus, the yield was 224 mg L−1 (Table 1).
(a) 1.4
C. vulgaris
1.2
S. obliquus
1 0.8 0.6 0.4 0.2
3.4.
Effect of NaCl on biomass yield and lipid accumulation in C. vulgaris and S. obliquus
Time-course study on the effects of various concentrations of NaCl on biomass yield and lipid accumulation potential of S. obliquus and C. vulgaris were also conducted. Clearcut effects were observed just after 6 days of incubation (Figure 4(a) and 4(b)). Under 5 g L−1 NaCl supplementation, lipid accumulation was raised to ∼40% (dcw) in both the test microalgae. However, biomass yield was affected at increasing NaCl concentration in the medium.
3.5.
Assessment of biomass and lipid yield of the test microalgae under various conditions
Table 1 summarizes the maximum lipid yield vis-à-vis biomass yield of the test microalgae under various conditions (derived from Figures 1 to 4). Biomass yield registered an increase under higher doses of magnesium, whereas a little effect for increased concentrations of calcium in the medium; reduction was recorded under Ca- and Mgstarvation, and NaCl supplementation. Interestingly, lipid yield was found to raise by more than twofold under Mg and NaCl supplementation, and also under Ca-starvation, even threefold under 5 gL−1 NaCl supplementation in S. obliquus against control. Thus, in this study, the maximum lipid yield (b) 14
C. vulgaris
Lipid content (% dcw)
3.2. Effect of Mg on biomass yield and lipid content Figure 2(a–d) shows the time-course study on the effects of Mg on biomass yield and lipid content of C. vulgaris and S. obliquus. The original concentration of Mg in N11 medium was 50 mg L−1 . Increasing the concentration showed positive effects on biomass yield, and at 3X concentration (150 mg L−1 ) biomass yield was raised up to 1.6 g L−1 (33% rise) for C. vulgaris, and 1.5 g L−1 (36% rise) for S. obliquus on the 18th day of incubation (Figure 2(a) and (b)). Cellular lipid content was increased maximum up to 27% and 26% (dcw) for C. vulgaris and S. obliquus, respectively, at 2X concentration (100 mg L−1 ) of Mg on day 15th of incubation (Figure 2(c) and 2(d)). Both the test algae failed to survive longer under Mgstarved condition, though a rise in cellular lipid content was observed at the early phase of growth. Table 1 summarizes the biomass yield and maximum lipid accumulation under different concentrations of Mg. On mg L−1 basis, lipid yield ranged between 328 and 340 mg L−1 in the test microalgae by doubling Mg supplementation, whereas under standard
Biomass yield (gL–1)
Downloaded by [RMIT University] at 15:11 27 July 2013
Environmental Technology
S. obliquus
12 10 8 6 4 2 0
0 0
3
6 9 12 15 18 21 24 27 30 Incubation period (days)
0
3
6 9 12 15 18 21 24 27 30 Incubation period (days)
Figure 1. (a) Biomass yield of C. vulgaris and S. obliquus under control condition, (b) lipid content of C. vulgaris and S. obliquus under control condition.
4
P.C. Gorain et al. (b) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
Biomass yield ( gL–1)
Biomass yield (gL–1)
(a)
Control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
0
3 6
9 12 15 18 21 24 27 30
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
0
3
6
Incubation period (days)
Incubation period (days)
control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
30 25 20 15 10 5
(d) Lipid content (%dcw)
Lipid content (% dcw)
Downloaded by [RMIT University] at 15:11 27 July 2013
(c)
9 12 15 18 21 24 27 30
control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
30 25 20 15 10 5 0
0 0
3
6
9 12 15 18 21 24 27 30
0
3
Incubation period (days)
6
9 12 15 18 21 24 27 30
Incubation period (days)
Figure 2. (a) Biomass yield of C. vulgaris at different concentrations of Mg in the culture medium, (b) biomass yield of S. obliquus at different concentrations of Mg in the culture medium, (c) lipid content of C. vulgaris at different concentrations of Mg in the culture medium and (d) lipid content of S. obliquus at different concentrations of Mg in the culture medium. Table 1.
Summary of biomass yield and maximum lipid accumulation in C. vulgaris and S. obliquus under various culture conditions. Biomass yield
Name of the organism C. vulgaris
S. obliquus
Culture condition Control Mg-starved Ca-starved 2X Mg (0.10 g L−1 ) 2X Ca (0.02 g L−1 ) NaCl (05 g L−1 ) Control Mg-starved Ca-starved 2X Mg (0.10 g L−1 ) 2X Ca (0.02 g L−1 ) NaCl (05 g L−1 )
Days
gL−1
21 09 18 18 18 06 21 12 18 18 21 06
1.2 ± 0.01b 0.6 ± 0.02e 0.9 ± 0.01d 1.4 ± 0.01a 1.4 ± 0.03a 1.02 ± 0.01c 1.1 ± 0.09c 0.5 ± 0.01f 0.8 ± 0.07e 1.4 ± 0.03a 1.2 ± 0.02b 0.98 ± 0.02d
Maximum lipid yield Days
mg L−1
% dcw
21 09 15 15 15 06 21 12 18 15 15 06
143.3 ± 2.1e 91.7 ± 1.7f 331.2 ± 2.9c 339.5 ± 2.2b 232.1 ± 1.9d 398.8 ± 3.7a
11.9 ± 0.7f 15.1 ± 0.2e 40.3 ± 0.9a 27.1 ± 0.3c 20.8 ± 0.4d 39.1 ± 0.7b 11.3 ± 0.6f 14.9 ± 0.4e 37.0 ± 1.1b 26.4 ± 0.7c 20.0 ± 0.8d 38.7 ± 0.6a
124.4 ± 1.8e 75.6 ± 1.4f 303.3 ± 3.7c 327.7 ± 2.8b 224.2 ± 3.7d 377.3 ± 2.8a
Note: Values are mean ± SD, n = 3. Values in each column superscripted by different alphabets (a–f) are significantly different from each other (P < 0.05, DMRT). Separate analysis was done for each column and each microalga.
ranged between 377 and 399 mg L−1 in the test microalgae under 5 gL−1 of NaCl supplementation.
3.6.
Analysis of fatty acid profiles of C. vulgaris and S. obliquus under various conditions
The FAME profile of C. vulgaris biodiesel depicted the presence of four major fatty acids, i.e. palmitic, stearic,
linoleic and linolenic acid methyl esters. For S. obliquus, the biodiesel was rich in palmitic, oleic, linoleic and linolenic acid methyl esters. Both the microalgal oils were found to be rich in saturated/monounsaturated FAMEs with minor proportion of polyunsaturated FAMEs under the control conditions (Tables 2 and 3; peak area magnesium supplementation > calcium starvation. Both the microalgae showed comparable lipid yield with adequate FAME composition under laboratory batch culture condition, which indicates that both S. obliquus and C. vulgaris could be prospective feedstocks for biodiesel production. Furthermore, properties of biodiesel such as viscosity, lubricity, calorific and iodine values, cetane number/index and the storage quality are specifically influenced by the composition of FAMEs. This necessitates extending the study to large scale under the above selected conditions, and assessing the lipid yield as well as various fuel properties for further recommendation.
Acknowledgements Financial support from Indian Institute of Technology Kharagpur, West Bengal, and Indian Council of Agricultural Research, New Delhi, India are thankfully acknowledged.
References [1] Slade R, Bauen A. Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenergy. 2013;53:29–38. [2] Rao PP, Gopalkrishnan KV. Vegetable oils and their methyl esters as fuels for diesel engines. Ind J Technol. 1991;29:292–297. [3] Meher LC, Sagar DV, Naik SN. Technical aspects of biodiesel production by transesterification – a review. Renewable Sustainable Energy Rev. 2006;10:248–268. [4] Moazami N, Ranjbar R, Ashori A, Tangestani M, Nejad AS. Biomass and lipid productivities of marine microalgae isolated from the Persian Gulf and the Qeshm Island. Biomass Bioenergy. 2011;35:1935–1939. [5] Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N. Biofuels from microalgae. Biotechnol Progress. 2008;24:815–820. [6] Chisti Y. Biodiesel from microalgae. Biotechnol Adv. 2007;25:294–306. [7] Haag AL. Algae bloom again. Nature. 2007;447:520–521. [8] Moazami N, Ashori A, Ranjbar R, Tangestani M, Eghtesadi R, Nejad AS. Large-scale biodiesel production using microalgae biomass of Nannochloropsis. Biomass Bioenergy. 2012;39:449–453. [9] Aresta M, Dibenedetto A, Carone M, Colonna T, Fagale C. Production of biodiesel from macroalgae by supercritical CO2 extraction and thermochemical liquefaction. Environ Chem Lett. 2005;3:136–139. [10] Brown L, Zeiler KG. Aquatic biomass and carbon dioxide trapping. Energy Conserv Manage. 1993;34:1005–1013.
Downloaded by [RMIT University] at 15:11 27 July 2013
8
P.C. Gorain et al.
[11] Mallick N. Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. BioMetals. 2002;15:377–390. [12] Mandal S, Mallick N. Waste utilization and biodiesel production by the green microalga Scenedesmus obliquus. Appl Environ Microbiol. 2011;77:374–377. [13] Mandal S, Mallick N. Biodiesel production by the green microalga Scenedesmus obliquus in a recirculatory aquaculture system. Appl Environ Microbiol. 2012;78:5929–5933. [14] Mandal S, Mallick N. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl Microbiol Biotechnol. 2009;84:281–291. [15] Mallick N, Mandal S, Singh AK, Bishai M, Dash A. Green microalga Chlorella vulgaris as a potential feedstock for biodiesel. J Chem Technol Biotechnol. 2012;87:137–145. [16] Nigam S, Rai MP, Sharma R. Effect of nitrogen on growth and lipid content of Chlorella pyrenoidosa. Am J Biochem Biotechnol. 2011;7:124–129. [17] Breuer G, Lamers PP, Martens DE, Draaisma RB, Wijffels RH. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Biores Technol. 2012;124:217–226. [18] Li X, Ying HH, Gan K, Y-xue X. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Biores Technol. 2010;101:5494–5500. [19] Adams C, Godfrey V, Wahlen B, Seefeldt L, Bugbee B. Understanding precision nitrogen stress to optimize the growth and lipid content tradeoff in oleaginous green microalgae. Biores Technol. 2013;131:188–194. [20] Deng X, Fei X, Li Y. The effects of nutritional restriction on neutral lipid accumulation in Chlamydomonas and Chlorella. African J Microbiol Res. 2011;5:260–270. [21] Manuel ME. The cultivation of Chlorella sp. Plant Physiol. 1944;19:359–369. [22] Finkle BJ, Appleman D. The effect of magnesium concentration on growth of Chlorella. Plant Physiol. 1953;28:664–673. [23] Barghbani R, Rezaei K, Javanshir A. Investigating the effects of several parameters on the growth of Chlorella vulgaris using Taguchi’s experimental approach. Int J Biotechnol Wellness Ind. 2012;1:128–133. [24] Sharma KK, Schuhmann H, Schenk PM. High lipid induction in microalgae for biodiesel production. Energies. 2012;5:1532–1553. [25] Rao AR, Dayananda C, Sarada R, Shamala TR, Ravishankar GA. Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Biores Technol. 2007;98:560–564. [26] Soeder CJ, Bolze A. Sulphate deficiency stimulates the release of dissolved organic matter in synchronus culture of Scenedesmus obliquus. Plant Physiol. 1981;52:233–238. [27] Grima EM, Belarbi EH, Ferna’ndez FG, Medina AR, Chisti Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol Adv. 2003;20: 491–515. [28] Griffiths MJ, van Hille RP, Harrison STL. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids. 2010;45:1053–1060. [29] Chinnaswamy S, Bhatnagar A, Hunt RW, Das KC. Microalgae cultivation in a wastewater dominated by carpet
[30]
[31]
[32] [33]
[34] [35] [36]
[37] [38] [39]
[40]
[41] [42] [43] [44] [45] [46] [47] [48]
mill effluents for biofuel applications. Biores Technol. 2010;101:3097–3105. Borges L, Moron-Villarreyes JA, D’Oca M, Abreu PC. Effects of flocculants on lipid extraction and fatty acid composition of the microalgae Nannochloropsis oculata and Thalassiosira weissflogii. Biomass Bioenergy. 2011;35:4449–4454. Rai LC, Mallick N, Singh JB, Kumar HD. Physiological and biochemical characteristics of a copper-tolerant and a wildtype strain of Anabaena doliolum under copper stress. J Plant Physiol. 1991;138:68–74. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917. Mandal S, Patnaik R, Singh AK, Mallick N. Comparative assessment of various lipid extraction protocols and optimization of transesterification process for microalgal biodiesel production. Environ Technol. (accepted). 2013. Nelson DL, Cox MM. Lipid biosynthesis. In: Principles of biochemistry. 4th ed. New York: W. H. Freeman and Company; 2008. p. 805–845. Camp PJ, Randall DD. Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex. Plant Physiol. 1985;77:571–577. Nguemezi JA, Tatchago V. Effects of fertilizers containing calcium and/or magnesium on the growth, development of plants and the quality of tomato fruits in the western highlands of Cameroon. Int J Agric Res. 2010;5:821–831. Hepler PK. Calcium: a central regulator of plant growth and development. Plant Cell. 2005;17:2142–2155. Thompson GA. Lipids and membrane function in green algae. Biochim Biophys Acta. 1996;1302:17–45. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 2008;54:621–639. Dujjanutat P, Kaewkannetra P. Effects of wastewater strength and salt stress on microalgal biomass production and lipid accumulation, World Academy of Science. Eng Technol. 2011;60:1163–1168. Duan X, Ren GY, Liu LL, Zhu WX. Salt-induced osmotic stress for lipid over production in batch culture of Chlorella vulgaris. African J. Biotechnol. 2012;11:7072–7078. Taiz L, Zeiger E. Respiration and lipid metabolism. In: Plant Physiology. 5th ed. New Delhi: Panima Publishing Corporation; 2010. p. 252–253. Knothe G. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process Technol. 2005;86:1059–1070. Behrens PW, Kyle DJ. Microalgae as a source of fatty acids. J Food Lipids. 1996;3:259–272. Frankel EN. Lipid oxidation. Dundee: The Oily Press; 1998. Vol. 10. Knothe G, Dunn RO. Oxidative stability of biodiesel/jet fuel blends by oil stability index (OSI) analysis. J Amer Oil Chem Soc. 2003;80:1047–1048. Bajpai D, Tyagi VK. Biodiesel: source, production, composition properties and its benefits. J Oleo Sci. 2006;55:487–502. Ladommatos N, Parsi M, Knowles A. The effect of fuel cetane improver on diesel pollutant emissions. Fuel. 1996;75:8–14.
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae a
a
Prakash Chandra Gorain , Sourav Kumar Bagchi & Nirupama Mallick
a
a
Agricultural and Food Engineering Department , Indian Institute of Technology Kharagpur , Kharagpur , 721302 , India Accepted author version posted online: 12 Jun 2013.Published online: 05 Jul 2013.
To cite this article: Environmental Technology (2013): Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae, Environmental Technology, DOI: 10.1080/09593330.2013.812668 To link to this article: http://dx.doi.org/10.1080/09593330.2013.812668
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2013 http://dx.doi.org/10.1080/09593330.2013.812668
Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae Prakash Chandra Gorain, Sourav Kumar Bagchi and Nirupama Mallick∗ Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
Downloaded by [RMIT University] at 15:11 27 July 2013
(Received 8 March 2013; final version received 1 June 2013 ) Biodiesel from microalgae has the potential as a sustainable fuel, since some species show exceptionally high lipid accumulation potential under various stresses. Effects of different concentrations of Ca, Mg and NaCl in the growth medium on biomass yield and lipid accumulation of Chlorella vulgaris and Scenedesmus obliquus grown under batch culture mode were investigated. Starvation of Mg showed a marginal rise in lipid content for a short period of time. Ca-starved cultures, however, demonstrated a profound rise in lipid content, i.e. 40% of dry cell wt. (dcw) was recorded against 11.9% control for C. vulgaris and 37% (dcw) against 11.3% for S. obliquus. Under increased concentration of Mg, significant rise in biomass and lipid yield was recorded. Effect of NaCl-induced osmotic stress showed lipid accumulation of ∼40% (dcw) in both the test algae, whereas the biomass yield was severely affected. The fatty acid profiles under the above stresses were analysed and discussed. Keywords: biodiesel; microalgae; Ca starvation; lipids; Mg starvation; NaCl; fatty acid methyl esters
1. Introduction Biofuels are arguably more sustainable, technically feasible and environment friendly than the fossil fuels. They are relatively low net emitters of greenhouse gases.[1] Among them, biodiesel and bioethanol have gained mainstream attention. Preference of diesel engines over engines run by other forms of fuel in India carries development of biodiesel technology a higher potential for this nation. Bioethanol, which is used in blends with gasoline, has low cetane numbers and, therefore, is unsuitable for diesel engines. On the other hand, biodiesel can be used to run the conventional diesel engines with only minor adjustments.[2] Biodiesel is formed chemically by reacting lipids (derived from animal or plant sources) with an alcohol, preferably methanol owing to cost effectiveness, to provide fatty acid esters (biodiesel). This chemical reaction, otherwise known as transesterification, requires a strong base such as sodium or potassium hydroxide or an acid such as hydrochloric or sulphuric acid as the catalyst.[3] The first generation of biodiesel was sourced from edible oils such as sunflower and oil palm, whereas the second generation was processed from non-edible oil crops such as jatropha, mahua, pongamia, etc. Currently, the third generation focuses on microalgal biodiesel technology because of several advantages. First of all, microalgae can grow at a very high rate and in some cases biomass doubling time of only 3.5 h was recorded during the exponential growth phase.[4] While the mechanism of photosynthesis ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
in microalgae is similar to that of higher plants, they have the ability to capture solar energy with an efficiency of 10–50 times higher than that of terrestrial plants.[5] They are also capable of producing more amount of oil per unit area of land in comparison with that of all other known oil-producing crops.[6,7] The per hectare yield of microalgal oil has been projected to be 58,700–136,900 L year−1 depending upon the oil content of algae, which is about 10–20 times higher than the best oil-producing crop, i.e. palm (5950 L ha−1 year−1 ).[6] The most acclaimed energy crop, i.e. jatropha, has been estimated to produce only 1892 L ha−1 year−1 . Recent report of Moazami et al. [8] showed that Nannochloropsis sp. PTCC 6016 has the capability of producing 60,000 L of biodiesel ha−1 year−1 . More importantly, due to aquatic in nature, algae do not compete for arable land for their cultivation and can be grown in freshwater or saline or even in wastewaters.[7,9–13] This implies that algae need not compete with other users for freshwater. On the top of these, microalgae grow even faster when fed with extra carbon dioxide, the main greenhouse gas. Production of biodiesel from microalgae primarily depends on the cellular lipid content and biomass productivity of selected species. It has been observed that limitations of major nutrients such as nitrogen and phosphorus triggered lipid accumulation in various microalgal species.[14–19] However, reports on other major nutrients such as Ca and Mg on the lipid accumulation potential
Downloaded by [RMIT University] at 15:11 27 July 2013
2
P.C. Gorain et al.
of microalgae are scarce, except Deng et al. [20] where neutral lipid content was detectably higher in Chlorella vulgaris grown in Ca- and Mg-free medium. It was once suggested that algae that thrive under deficit of these minerals should be classified as lower or more primitive in the phylogenetic tree than those species that cannot survive under such deficit. Studies conducted by Manuel [21] with a Chlorella strain showed remarkable growth up to 10 mM concentration of CaCl2 , but higher concentrations were found inhibitory. Similarly, the process of multiplication was found to require a larger concentration of magnesium in the medium, and this divalent ion significantly influences the chlorophyll biosynthesis.[22] Therefore, here, the aim was set to study growth pattern, corresponding lipid accumulation and variations in fatty acid profile in two green microalgae, C. vulgaris and Scenedesmus obliquus, under starvation as well as high concentrations of calcium and magnesium in the growth medium. For the past few years, biologists are concentrating on the effects of osmotic stress on various aspects of plants and microorganisms. Barghbani et al. [23] reported that with increasing NaCl concentration, there was a decrease in biomass yield of C. vulgaris, and complete suppression of growth was evident at 30 gL−1 NaCl. Reports showing increased lipid productivity, and changes in fatty acids profile in some microalgal species under salinity stress are also available.[24,25] Considering this, the effect of osmotic stress on the two test microalgae was also investigated.
2. Materials and methods 2.1. Organisms and growth conditions Axenic cultures of two green microalgae, S. obliquus (Trup.) Kutz. (SAG 276-3a) and C. vulgaris (an isolate of river Ganges, courtesy: Prof. L. C. Rai, Banaras Hindu University, Varanasi, India), were used in this study, and maintained in a culture room at 25 ± 2◦ C under a photoperiod of 14:10 h at a light intensity of 75 μmol photon m−2 s−1 photosynthetically active radiation (PAR). Both the microalgae were cultured in N11 medium [26] at pH 6.8. For maintenance of batch cultures, the microalgae were grown in 250 mL Erlenmeyer flasks containing 100 mL medium without sparging with air or CO2 . The cultures were hand shaken 2–3 times daily to avoid sticking, and was referred as control culture.
2.2.
Dry weight measurement
Dry cell weight (dcw) was measured in three days intervals, where three flasks, each containing 100 mL of cultures, were withdrawn from the culture rack. The algal cultures were centrifuged at 5000 rpm for 10 min and transferred into preweighed vials for oven-drying at 60◦ C to get a constant weight. Oven-drying was preferred as it was found to be efficient, convenient, cost-effective and the most widely use
method for microalgal drying.[27–30] The biomass yield was estimated by the gravimetric method and expressed as g L−1 .[31] 2.3. Extraction of lipids from algal biomass After estimation of dcw, lipid extraction was done from the same vials following the protocol of Bligh and Dyer [32] with chloroform and methanol as solvents. Lipid content/yields were expressed as % dcw/mg L−1 . 2.4.
Effects of Ca, Mg and NaCl on biomass yield and lipid accumulation in the test microalgae To study the effects of calcium and magnesium starvation on biomass yield and lipid accumulation, C. vulgaris and S. obliquus cells were grown in Ca- and Mg-starved conditions, which were achieved by depriving the medium of the respective salts. To study the effects of increased concentrations of Mg and Ca, microalgal cultures were grown in various increased concentrations of Mg and Ca, and these conditions were achieved by the addition of higher amounts of Mg and Ca salts with respect to the control condition. The various increased concentrations were 0.05 gL−1 (1X: control), 0.1 gL−1 (2X), 0.15 gL−1 (3X), 0.25 gL−1 (5X), 0.5 gL−1 (10X) for MgSO4 · 7H2 O, and 0.01 gL−1 (1X: control), 0.02 gL−1 (2X), 0.03 gL−1 (3X), 0.05 gL−1 (5X), 0.1 gL−1 (10X) for CaCl2 · H2 O. To study the effect of osmotic stress, the stationary phase cultures were subjected to different concentrations of NaCl, i.e. 1.0–9.0 gL−1 of NaCl. Samples were withdrawn on every third day; biomass and lipids were quantified as described above. 2.5.
Transesterification and analysis of fatty acid methyl esters (FAMEs) As acid values of the test microalgal lipids were found to be very high (ranging from 45.2 to 48.3 mg KOH g−1 ), the acid-catalysed transesterification was performed using a molar ratio of oil:methanol:HCl::1:80:4 with a reaction temperature of 60◦ C and reaction time of 6.4 h, as optimized in our laboratory.[33] The top organic phase, which contained FAMEs was pipetted out and the aqueous phase containing residual methanol, glycerol and the catalyst was discarded. The top organic layer was taken for GC-MS analysis using Clarus 680 GC (Perkin-Elmer, Shelton, CT, USA) equipped with an Elite-1 methylpolysiloxane capillary column (30 m × 0.25 mm × 0.25 μm) and MS (Clarus 600 T MS) equipped with a photo-multiplied tube detector which has a phosphor screen. 2.6. Statistical analysis All the experiments were performed in triplicate to check the reproducibility. The results were analysed statistically by Duncan’s new multiple range test (DMRT). DMRT
3
was done by MSTAT-C software (Plant and Soil Sciences Division, Michigan State University, USA).
concentration of Mg lipid yield ranged between 124 and 143 mg L−1 .
3. Results 3.1. Time course of growth and lipid accumulation in C. vulgaris and S. obliquus under control condition The time course of growth and lipid accumulation in C. vulgaris and S. obliquus under the batch culture mode was studied in N11 medium of standard composition. With a lag of 3 days, growth of the test algae increased steadily. The stationary phase was seen at about 21 days (Figure 1(a)). The maximum biomass yield was 1.2 and 1.1 g L−1 , respectively, for C. vulgaris and S. obliquus. Maximum accumulation of lipids (11.3% for S. obliquus and 11.9% dcw for C. vulgaris) was observed at the stationary phase of cultures (Figure 1(b)).
3.3. Effect of Ca on biomass and lipid yield Increased Ca concentration in the growth medium was found to have a little effect on bimass yield of the test microalgae; lipid content, however, showed an increasing trend (Figure 3). Profound rise (∼40% dcw) in cellular lipid pool was observed under Ca-starved condition (Figure 3(c) and 3(d)). The maximum lipid yield reached up to 331 mg L−1 in the case of C. vulgaris. For S. obliquus, the yield was 224 mg L−1 (Table 1).
(a) 1.4
C. vulgaris
1.2
S. obliquus
1 0.8 0.6 0.4 0.2
3.4.
Effect of NaCl on biomass yield and lipid accumulation in C. vulgaris and S. obliquus
Time-course study on the effects of various concentrations of NaCl on biomass yield and lipid accumulation potential of S. obliquus and C. vulgaris were also conducted. Clearcut effects were observed just after 6 days of incubation (Figure 4(a) and 4(b)). Under 5 g L−1 NaCl supplementation, lipid accumulation was raised to ∼40% (dcw) in both the test microalgae. However, biomass yield was affected at increasing NaCl concentration in the medium.
3.5.
Assessment of biomass and lipid yield of the test microalgae under various conditions
Table 1 summarizes the maximum lipid yield vis-à-vis biomass yield of the test microalgae under various conditions (derived from Figures 1 to 4). Biomass yield registered an increase under higher doses of magnesium, whereas a little effect for increased concentrations of calcium in the medium; reduction was recorded under Ca- and Mgstarvation, and NaCl supplementation. Interestingly, lipid yield was found to raise by more than twofold under Mg and NaCl supplementation, and also under Ca-starvation, even threefold under 5 gL−1 NaCl supplementation in S. obliquus against control. Thus, in this study, the maximum lipid yield (b) 14
C. vulgaris
Lipid content (% dcw)
3.2. Effect of Mg on biomass yield and lipid content Figure 2(a–d) shows the time-course study on the effects of Mg on biomass yield and lipid content of C. vulgaris and S. obliquus. The original concentration of Mg in N11 medium was 50 mg L−1 . Increasing the concentration showed positive effects on biomass yield, and at 3X concentration (150 mg L−1 ) biomass yield was raised up to 1.6 g L−1 (33% rise) for C. vulgaris, and 1.5 g L−1 (36% rise) for S. obliquus on the 18th day of incubation (Figure 2(a) and (b)). Cellular lipid content was increased maximum up to 27% and 26% (dcw) for C. vulgaris and S. obliquus, respectively, at 2X concentration (100 mg L−1 ) of Mg on day 15th of incubation (Figure 2(c) and 2(d)). Both the test algae failed to survive longer under Mgstarved condition, though a rise in cellular lipid content was observed at the early phase of growth. Table 1 summarizes the biomass yield and maximum lipid accumulation under different concentrations of Mg. On mg L−1 basis, lipid yield ranged between 328 and 340 mg L−1 in the test microalgae by doubling Mg supplementation, whereas under standard
Biomass yield (gL–1)
Downloaded by [RMIT University] at 15:11 27 July 2013
Environmental Technology
S. obliquus
12 10 8 6 4 2 0
0 0
3
6 9 12 15 18 21 24 27 30 Incubation period (days)
0
3
6 9 12 15 18 21 24 27 30 Incubation period (days)
Figure 1. (a) Biomass yield of C. vulgaris and S. obliquus under control condition, (b) lipid content of C. vulgaris and S. obliquus under control condition.
4
P.C. Gorain et al. (b) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
Biomass yield ( gL–1)
Biomass yield (gL–1)
(a)
Control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
0
3 6
9 12 15 18 21 24 27 30
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
0
3
6
Incubation period (days)
Incubation period (days)
control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
30 25 20 15 10 5
(d) Lipid content (%dcw)
Lipid content (% dcw)
Downloaded by [RMIT University] at 15:11 27 July 2013
(c)
9 12 15 18 21 24 27 30
control Mg-starved 2X Mg 3X Mg 5X Mg 10X Mg
30 25 20 15 10 5 0
0 0
3
6
9 12 15 18 21 24 27 30
0
3
Incubation period (days)
6
9 12 15 18 21 24 27 30
Incubation period (days)
Figure 2. (a) Biomass yield of C. vulgaris at different concentrations of Mg in the culture medium, (b) biomass yield of S. obliquus at different concentrations of Mg in the culture medium, (c) lipid content of C. vulgaris at different concentrations of Mg in the culture medium and (d) lipid content of S. obliquus at different concentrations of Mg in the culture medium. Table 1.
Summary of biomass yield and maximum lipid accumulation in C. vulgaris and S. obliquus under various culture conditions. Biomass yield
Name of the organism C. vulgaris
S. obliquus
Culture condition Control Mg-starved Ca-starved 2X Mg (0.10 g L−1 ) 2X Ca (0.02 g L−1 ) NaCl (05 g L−1 ) Control Mg-starved Ca-starved 2X Mg (0.10 g L−1 ) 2X Ca (0.02 g L−1 ) NaCl (05 g L−1 )
Days
gL−1
21 09 18 18 18 06 21 12 18 18 21 06
1.2 ± 0.01b 0.6 ± 0.02e 0.9 ± 0.01d 1.4 ± 0.01a 1.4 ± 0.03a 1.02 ± 0.01c 1.1 ± 0.09c 0.5 ± 0.01f 0.8 ± 0.07e 1.4 ± 0.03a 1.2 ± 0.02b 0.98 ± 0.02d
Maximum lipid yield Days
mg L−1
% dcw
21 09 15 15 15 06 21 12 18 15 15 06
143.3 ± 2.1e 91.7 ± 1.7f 331.2 ± 2.9c 339.5 ± 2.2b 232.1 ± 1.9d 398.8 ± 3.7a
11.9 ± 0.7f 15.1 ± 0.2e 40.3 ± 0.9a 27.1 ± 0.3c 20.8 ± 0.4d 39.1 ± 0.7b 11.3 ± 0.6f 14.9 ± 0.4e 37.0 ± 1.1b 26.4 ± 0.7c 20.0 ± 0.8d 38.7 ± 0.6a
124.4 ± 1.8e 75.6 ± 1.4f 303.3 ± 3.7c 327.7 ± 2.8b 224.2 ± 3.7d 377.3 ± 2.8a
Note: Values are mean ± SD, n = 3. Values in each column superscripted by different alphabets (a–f) are significantly different from each other (P < 0.05, DMRT). Separate analysis was done for each column and each microalga.
ranged between 377 and 399 mg L−1 in the test microalgae under 5 gL−1 of NaCl supplementation.
3.6.
Analysis of fatty acid profiles of C. vulgaris and S. obliquus under various conditions
The FAME profile of C. vulgaris biodiesel depicted the presence of four major fatty acids, i.e. palmitic, stearic,
linoleic and linolenic acid methyl esters. For S. obliquus, the biodiesel was rich in palmitic, oleic, linoleic and linolenic acid methyl esters. Both the microalgal oils were found to be rich in saturated/monounsaturated FAMEs with minor proportion of polyunsaturated FAMEs under the control conditions (Tables 2 and 3; peak area magnesium supplementation > calcium starvation. Both the microalgae showed comparable lipid yield with adequate FAME composition under laboratory batch culture condition, which indicates that both S. obliquus and C. vulgaris could be prospective feedstocks for biodiesel production. Furthermore, properties of biodiesel such as viscosity, lubricity, calorific and iodine values, cetane number/index and the storage quality are specifically influenced by the composition of FAMEs. This necessitates extending the study to large scale under the above selected conditions, and assessing the lipid yield as well as various fuel properties for further recommendation.
Acknowledgements Financial support from Indian Institute of Technology Kharagpur, West Bengal, and Indian Council of Agricultural Research, New Delhi, India are thankfully acknowledged.
References [1] Slade R, Bauen A. Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenergy. 2013;53:29–38. [2] Rao PP, Gopalkrishnan KV. Vegetable oils and their methyl esters as fuels for diesel engines. Ind J Technol. 1991;29:292–297. [3] Meher LC, Sagar DV, Naik SN. Technical aspects of biodiesel production by transesterification – a review. Renewable Sustainable Energy Rev. 2006;10:248–268. [4] Moazami N, Ranjbar R, Ashori A, Tangestani M, Nejad AS. Biomass and lipid productivities of marine microalgae isolated from the Persian Gulf and the Qeshm Island. Biomass Bioenergy. 2011;35:1935–1939. [5] Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N. Biofuels from microalgae. Biotechnol Progress. 2008;24:815–820. [6] Chisti Y. Biodiesel from microalgae. Biotechnol Adv. 2007;25:294–306. [7] Haag AL. Algae bloom again. Nature. 2007;447:520–521. [8] Moazami N, Ashori A, Ranjbar R, Tangestani M, Eghtesadi R, Nejad AS. Large-scale biodiesel production using microalgae biomass of Nannochloropsis. Biomass Bioenergy. 2012;39:449–453. [9] Aresta M, Dibenedetto A, Carone M, Colonna T, Fagale C. Production of biodiesel from macroalgae by supercritical CO2 extraction and thermochemical liquefaction. Environ Chem Lett. 2005;3:136–139. [10] Brown L, Zeiler KG. Aquatic biomass and carbon dioxide trapping. Energy Conserv Manage. 1993;34:1005–1013.
Downloaded by [RMIT University] at 15:11 27 July 2013
8
P.C. Gorain et al.
[11] Mallick N. Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. BioMetals. 2002;15:377–390. [12] Mandal S, Mallick N. Waste utilization and biodiesel production by the green microalga Scenedesmus obliquus. Appl Environ Microbiol. 2011;77:374–377. [13] Mandal S, Mallick N. Biodiesel production by the green microalga Scenedesmus obliquus in a recirculatory aquaculture system. Appl Environ Microbiol. 2012;78:5929–5933. [14] Mandal S, Mallick N. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl Microbiol Biotechnol. 2009;84:281–291. [15] Mallick N, Mandal S, Singh AK, Bishai M, Dash A. Green microalga Chlorella vulgaris as a potential feedstock for biodiesel. J Chem Technol Biotechnol. 2012;87:137–145. [16] Nigam S, Rai MP, Sharma R. Effect of nitrogen on growth and lipid content of Chlorella pyrenoidosa. Am J Biochem Biotechnol. 2011;7:124–129. [17] Breuer G, Lamers PP, Martens DE, Draaisma RB, Wijffels RH. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Biores Technol. 2012;124:217–226. [18] Li X, Ying HH, Gan K, Y-xue X. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Biores Technol. 2010;101:5494–5500. [19] Adams C, Godfrey V, Wahlen B, Seefeldt L, Bugbee B. Understanding precision nitrogen stress to optimize the growth and lipid content tradeoff in oleaginous green microalgae. Biores Technol. 2013;131:188–194. [20] Deng X, Fei X, Li Y. The effects of nutritional restriction on neutral lipid accumulation in Chlamydomonas and Chlorella. African J Microbiol Res. 2011;5:260–270. [21] Manuel ME. The cultivation of Chlorella sp. Plant Physiol. 1944;19:359–369. [22] Finkle BJ, Appleman D. The effect of magnesium concentration on growth of Chlorella. Plant Physiol. 1953;28:664–673. [23] Barghbani R, Rezaei K, Javanshir A. Investigating the effects of several parameters on the growth of Chlorella vulgaris using Taguchi’s experimental approach. Int J Biotechnol Wellness Ind. 2012;1:128–133. [24] Sharma KK, Schuhmann H, Schenk PM. High lipid induction in microalgae for biodiesel production. Energies. 2012;5:1532–1553. [25] Rao AR, Dayananda C, Sarada R, Shamala TR, Ravishankar GA. Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Biores Technol. 2007;98:560–564. [26] Soeder CJ, Bolze A. Sulphate deficiency stimulates the release of dissolved organic matter in synchronus culture of Scenedesmus obliquus. Plant Physiol. 1981;52:233–238. [27] Grima EM, Belarbi EH, Ferna’ndez FG, Medina AR, Chisti Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol Adv. 2003;20: 491–515. [28] Griffiths MJ, van Hille RP, Harrison STL. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids. 2010;45:1053–1060. [29] Chinnaswamy S, Bhatnagar A, Hunt RW, Das KC. Microalgae cultivation in a wastewater dominated by carpet
[30]
[31]
[32] [33]
[34] [35] [36]
[37] [38] [39]
[40]
[41] [42] [43] [44] [45] [46] [47] [48]
mill effluents for biofuel applications. Biores Technol. 2010;101:3097–3105. Borges L, Moron-Villarreyes JA, D’Oca M, Abreu PC. Effects of flocculants on lipid extraction and fatty acid composition of the microalgae Nannochloropsis oculata and Thalassiosira weissflogii. Biomass Bioenergy. 2011;35:4449–4454. Rai LC, Mallick N, Singh JB, Kumar HD. Physiological and biochemical characteristics of a copper-tolerant and a wildtype strain of Anabaena doliolum under copper stress. J Plant Physiol. 1991;138:68–74. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917. Mandal S, Patnaik R, Singh AK, Mallick N. Comparative assessment of various lipid extraction protocols and optimization of transesterification process for microalgal biodiesel production. Environ Technol. (accepted). 2013. Nelson DL, Cox MM. Lipid biosynthesis. In: Principles of biochemistry. 4th ed. New York: W. H. Freeman and Company; 2008. p. 805–845. Camp PJ, Randall DD. Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex. Plant Physiol. 1985;77:571–577. Nguemezi JA, Tatchago V. Effects of fertilizers containing calcium and/or magnesium on the growth, development of plants and the quality of tomato fruits in the western highlands of Cameroon. Int J Agric Res. 2010;5:821–831. Hepler PK. Calcium: a central regulator of plant growth and development. Plant Cell. 2005;17:2142–2155. Thompson GA. Lipids and membrane function in green algae. Biochim Biophys Acta. 1996;1302:17–45. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 2008;54:621–639. Dujjanutat P, Kaewkannetra P. Effects of wastewater strength and salt stress on microalgal biomass production and lipid accumulation, World Academy of Science. Eng Technol. 2011;60:1163–1168. Duan X, Ren GY, Liu LL, Zhu WX. Salt-induced osmotic stress for lipid over production in batch culture of Chlorella vulgaris. African J. Biotechnol. 2012;11:7072–7078. Taiz L, Zeiger E. Respiration and lipid metabolism. In: Plant Physiology. 5th ed. New Delhi: Panima Publishing Corporation; 2010. p. 252–253. Knothe G. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process Technol. 2005;86:1059–1070. Behrens PW, Kyle DJ. Microalgae as a source of fatty acids. J Food Lipids. 1996;3:259–272. Frankel EN. Lipid oxidation. Dundee: The Oily Press; 1998. Vol. 10. Knothe G, Dunn RO. Oxidative stability of biodiesel/jet fuel blends by oil stability index (OSI) analysis. J Amer Oil Chem Soc. 2003;80:1047–1048. Bajpai D, Tyagi VK. Biodiesel: source, production, composition properties and its benefits. J Oleo Sci. 2006;55:487–502. Ladommatos N, Parsi M, Knowles A. The effect of fuel cetane improver on diesel pollutant emissions. Fuel. 1996;75:8–14.
