Research Article Efficacy of EDTA and Phosphorous on Biomass Yield and Total Lipid Accumulation in Two Green Microalgae with Special Emphasis on Neutral Lipid Detection by Flow Cytometry Gour Gopal Satpati, Prakash Chandra Gorain, and Ruma Pal Phycology Laboratory, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, West Bengal 700019, India Correspondence should be addressed to Ruma Pal; rpalcu@redifmail.com Received 10 August 2015; Revised 15 December 2015; Accepted 24 December 2015 Academic Editor: Antonio Randazzo Copyright © 2016 Gour Gopal Satpati et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chlorella ellipsoidea and Chlorococcum infusionum, promising microalgae for biodiesel feedstock production, were treated with ethylenediaminetetraacetate (EDTA) and phosphorous to induce stress which was then followed by fow cytometry to study the enhanced intracellular neutral lipid content. Treatment resulted in up to a threefold increase in total lipid content of Chlorella (41.8 ± 1.9% at 16 days of incubation period) and more than twofold increases in Chlorococcum (31.3 ± 1.0% at 18 days of incubation period) under phosphorous starvation in the culture. It was observed that maximum biomass yields in Chlorella and Chlorococcum were 1.56 ± 0.06 and 2.17 ± 0.12 g/L at 1.5 g/L of phosphorous afer 20 and 18 days of incubation periods, respectively. Te qualitative analyses of neutral lipid bodies under stress conditions were performed by confocal microscopy and revealed bright golden-yellow lipid droplets in stress exposed cells. Signifcant increase of monounsaturated fatty acids under the nutrient limited conditions was suitable to produce biodiesel. Te maximum biomass (g/L) and lipid content (% dry cell weight) at diferent stresses showed signifcant results ( < 0.05) by single-factor Analysis of Variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). 1. Introduction Continued exploitation of the world’s fossil fuel reserves is unsustainable due to their depleted status and simultaneous steady accumulation of greenhouse gases in the atmosphere. Development of sustainable energy sources is therefore nec- essary to overcome this problem. Te production of biodiesel from photosynthetic microorganisms is considered as an efective strategy to produce renewable energy. One of the most promising candidates for production of alternative energy is microalgae because of their higher growth rate and oil content than those of the conventional energy crops such as jatropha, soybean, palm, canola, and corn [1–3]. Addition- ally, some conditions of cultivation should be strategically changed to enhance microalgal lipid. Most recent studies have found that the lipid accumulation in microalgae could be increased through altering parameters such as temperature, light, CO 2 , salinity, and nutrient concentration [4–10]. Nutrient is the most important regulator of microalgal growth and lipid accumulation. Many microalgal species can synthesize large quantities of lipid and carbohydrates along with other bioactive molecules under varied nutrient supplement conditions. Tese newly synthesized lipids and carbohydrates can be directly converted to biodiesel and bioethanol, respectively [1, 11, 12]. Under diferent nutrient starvation, microalgae can accumulate large quantities of neutral lipid in the form of triacylglycerol (TAG) within the cell cytoplasm [11]. A few reports were available on the efect of ethylenediaminetetraacetate (EDTA) on growth and lipid productivity in microalgae. In Nannochloropsis oculata the dry weight of the biomass and lipid gradually increased with increased concentration of EDTA [10]. Te marine diatom Amphiprora paludosa synthesized 65.64% lipid under 0.026 mM of Fe-EDTA [13]. Simultaneous efect of phosphorous (P) infuences the growth and lipid synthesis in microalgae [9, 14–17]. Efect of P defciency in Chlorella vulgaris was studied by Mutlu et al. [9]. Te lipid accumulation and growth characteristics of Chlorella kessleri under diferent concentration of P were Hindawi Publishing Corporation Advances in Biology Volume 2016, Article ID 8712470, 12 pages http://dx.doi.org/10.1155/2016/8712470