Realizing the oil-yielding potentialities with much faster growth rate and efficient CO2 fixation, microalgae appear to be the best option as a renewable source of biodiesel that has the potentiality to completely replace the petroleum diesel fuel. However, the lipid content in the selected microalga/strain is required to be high; otherwise the economic performance would be hard to achieve.

Each species of microalga produces different ratios of lipids, carbohydrates and proteins. Nevertheless, these tiny organisms have the ability to manipulate their metabolism by simple manipulations of the chemical composition of the culture medium (Behrens and Kyle, 1996); thus, high lipid productivity can be achieved. Physiological stresses such as nutrient limitation/defi — ciency, salt stress and high light intensity have been employed for directing metabolic fluxes to lipid biosyn­thesis of microalgae. Many reports are available, where attempts have been made to raise the lipid pool of various microalgal species. Table 11.1 summarizes those studies.

Exceptionally, an oil content of 86% of dry cell weight (dcw) was reported in the brown resting state colonies of Botryococcus braunii, while the green active state colonies were found to account for 17% only (Brown et al., 1969). However, the major obstacle in focusing B. braunii as an industrial organism for biodiesel production is its poor growth rate (Dayananda et al., 2007). Nitrogen limita — tion/deficiency has been found to raise the lipid content of a number of microalgal species profoundly. For instance, Piorreck and Pohl (1984) reported an increased lipid pool from 12% to 53% (dcw) in Chlorella vulgaris under nitrogen-limited condition. Unlike the green algae, the blue-green algae viz. Anacystis nidulans and Oscillato — ria rubescens contained the same quantities of lipid at different nitrogen concentrations. It was observed by Illman et al. (2000) that four species of Chlorella (Chlorella emersonii, Chlorella minutissima, C. vulgaris and Chlorella pyrenoidosa) could accumulate lipid up to 63, 57, 40 and 23% (dcw), respectively, in low N-medium. These values in control vessels were, respectively, 29%, 31%, 18% and 11% in the above order. In the same year, Takagi et al. (2000) observed an increase in intracellular lipid pool up to 51% (dcw) against 31% control in 3% CO2-purged cultures of Nannochloris sp. UTEX LB1999 grown in continuous low nitrate (0.9 mM)-fed medium. Chlorella protothecoides also showed a rise in lipid pool from 15% to 55% (dcw), when grown heterotrophically with glucose (1%) under reduced nitrogen concentration (Miao and Wu, 2004). Similarly, C. protothecoides depicted a lipid pool of 55% (dcw) when grown heterotrophically with corn powder hydrolysate under nitrogen limitation (Xu et al., 2006).

Gao et al. (2010) used sweet sorghum hydrolysate instead of corn powder for C. protothecoides culture, and lipid yield of 50% (dcw) was recorded. Nitrogen limitation/starvation also enhanced the lipid content in Neochloris oleoabundans, Nannochloropsis oculata,

C. vulgaris, Chlorella zofingiensis, Dunaliella tertiolecta and Thalassiosira pseudonana (Converti et al., 2009; Feng et al., 2011a; Gouveia and Oliveira, 2009; Jiang et al., 2012). However, the marine microalga Isochrysis zhang — jiangensis was found to accumulate lipid under high nitrate concentration, rather than limitation or depletion (Feng et al., 2011b).

Limitation of phosphate was also found to enhance lipid accumulation in Ankistrodesmus falcutus and Mono — dus subterraneus (Kilham et al., 1997; Khozin-Goldberg and Cohen, 2006). Rodolfi et al. (2009) screened 30 microalgal strains for lipid production, among which the marine genus Nannochloropsis sp. F&M-M24 emerged as the best candidate for oil production (50% under phosphorus deficiency against 31% control). Sobczuk and Chisti (2010) observed an increase in intra­cellular lipid content up to 60% (dcw) against 27% control in Choricystis minor under simultaneous nitrate and phosphate deficiencies. In Scenedemus obliquus, lipid accumulation up to 58% (dcw) was recorded when subjected to simultaneous nitrate and phosphate limita­tions in presence of sodium thiosulphate (against 13% under control condition, Mandal and Mallick, 2009). Simultaneous nitrate, phosphate and iron limitations have also been reported to stimulate lipid accumulation in a microalga C. vulgaris (57% against 8% control, Mallick et al., 2012).

In addition to nutrient limitations/deficiencies, other stress conditions may also enhance lipid accumulation in microalgae. Takagi et al. (2006) studied the effect of NaCl on accumulation of lipids and triacylglycerides in the marine microalga Dunaliella sp. Increase in initial NaCl concentration from 0.5 M (seawater) to 1.0 M resulted in a higher intracellular lipid accumulation (71% dcw). Damiani et al. (2010) studied the effects of continuous high light intensity (300 mmol photons/ m2 s) on lipid accumulation in Haematococcus pluvialis grown under nitrogen-sufficient and nitrogen-deprived conditions. A lipid yield of 33—35% was recorded under the high light intensity as compared to 15% yield in con­trol cultures. Nitrogen deprivation was, however, not found to raise the lipid content of H. pluvialis cultures.

Nutrient limitations/deficiencies or physiological stresses required for accumulation of lipids in microal­gal cells is associated with reduced cell division (Ratledge, 2002). The overall lipid productivity is there­fore compromised due to the low biomass productivity. For instance, Scragg et al. (2002) studied the energy recovery from C. vulgaris and C. emersonii grown in com­plete Watanabe medium and also in low nitrogen medium. The results showed that the low nitrogen medium, although induced higher lipid accumulation in both the test algae with high calorific values, the overall energy recovery was lower in comparison to Watanabe’s medium. A commonly suggested counter measure is to use a two-stage cultivation strategy, dedi­cating the first stage for cell growth/division in nutrient sufficient medium, and the second stage for lipid accu­mulation under nutrient starvation or other physiolog­ical stresses. To get maximal biomass and lipid yield, CO2 can also be utilized. Chiu et al. (2009) reported an increased accumulation of lipid (from 31% to 50% dcw) in the stationary phase cultures of N. oculata NCTU-3 grown under 2% CO2 aeration.