Microalgae appear to be the only source of biodiesel that have the potential to com­pletely replace fossil diesel (Table 11.2). Unlike other oil crops, microalgae grow rapidly and many of them are exceedingly rich in oil (Griffith and Harrison, 2009). Microalgae commonly double their biomass within 18 to 24 h (Sheehan et al., 1998).

OS 4^

TABLE 11.1 С02 Biofixation and Biomass Productivity of Various Microalgae in Different Reactor Configurations C02 Fixation Rate (g m_3h_’) or Specific Growth Rate11 (h_1) or Microalgal Species C02 Feed Gas (%) Removal Efficiency (%) Biomass Productivity0 (g m_3h_’) Reactor Type Chlamydomonas reinhardtii 30* NA 0.08-0.01d Batch hotobioreactors Chlorella pyrenoidosa too* NA 0.09-0.09d Chlorogleopsis sp. 5 0.8-1.9a 0.0007-0.00604 Scenedesmus obliquus 60* NA 0.06-0.04d Spirulina platensis 0.03 NA 0.0082-0.002d C. kessleri 18 NA 0.84d Open photobioreactor Chlorella sp. 6-8 10-50% NA Chlorella sp. 10 46% 0.09f N. salina 5 NA 1.25е N. salina 15 NA 4.1е Spirulina LEB18 6 0.218 0.2475f Spirulina platensis 10 39% 0.1164f Closed photobioreactor Botryococcus braunii 2-3 3-18a NA Chlorella sp. 0.03 1.3784h 0.75 llf Chlorella sp. 0.03 NA 0.099f 3 NA 0.212f 10 NA 0.045f 15 NA 0.030f

Source: Table is updated version of published work of Kumar, A. et al. (2010); Fulke, A. et al. (2010); Ramanan, R. et al. (2009); Zhao, B. et al. (2011); Ho, S. H. et al.

(2010); Cheng, L. et al. (2006).

Note: Abbreviation: NA, not available, з, ь, c prom Kumar A. et al., 2010. d Specific growth rate (h_1). e Biomass productivity (g mr3/d_1). f Biomass productivity, calculated (g L-1d-1). g Calculated C02 fixation rate (g g_1d_1). h C02 fixation rate (g L-1d-1).

1 C02 fixation rate (mg L-1d-1). j Biomass productivity (mg L-1d-1).

*mM in medium.

TABLE 11.2

Comparison of Some Biodiesel Sources

Oil Yield (L ha-1y-1)

172

446

1,190

1,892

5,950

Microalgaea

Microalgaeb a 70% oil (by wt.) in biomass. b 30% oil (by wt.) in biomass.

Source: Adapted from Chisti (2007) and Mata et al. (2010).

The oil content in microalgae can exceed 80% by weight of dry biomass (Spolaore et al., 2006). The biofuel production potentials of various algal strains reported are summarized in Table 11.3. Depending on the species, microalgae produce many different kinds of lipids, hydrocarbons, and other complex oils. Hexadecanoic acid methyl ester (16:0), palmitoleic acid methyl ester (16:1), octadecanoic acid methyl ester (18:1), and stearic acid methyl ester (18:0) are some of the major FAMEs found to be suitable for biodiesel production derived from microalgal lipids (Dayananda et al., 2007; Francisco et al., 2010; Fulke et al., 2010). Using microalgae to produce biodiesel will not compromise the production of food, fodder, and other products derived from crops (Griffith and Harrison, 2009). The strain Botryococcus braunii, however, grows slowly and produces about 30% to 73% hydrocarbons under labora­tory conditions (Dayananda et al., 2006; 2007; 2010).

For cost-effective commercial biodiesel production, appropriate strain selection according to the suitability for site of cultivation and local environmental conditions is imperative (Sheehan et al., 1998; Griffith and Harrison, 2009; Chanakya et al., 2012). The key challenge for microalgal biodiesel production is the screening and selection of microalgal species that can maintain a high growth rate with high lipid content in addition to a high metabolic rate (Griffith and Harrison, 2009). The spe­cies that are metabolically rigorous can tolerate high concentrations of salt, CO2, high alkalinity, and high temperature; and have the ability to grow and replicate under nutritional stress by altering their metabolic pathways—these are the species that are found to be most promising in this regard (Verma et al., 2010). Nitrogen limi­tations have been found to enhance lipid accumulation in the microalgae (Griffith and Harrison, 2008). Yeesang and Cheirsilp (2011) studied the effect of nitrogen deprivation and iron (Fe3+) enhancement with higher light intensity on lipid con­tent. They observed an increase in lipid content from 25.8% to 35.9% (Yeesang and Cheirsilp, 2011). The findings of Liu et al. (2008) also confirmed that lipid content in Chlorella vulgaris increased by three — to sevenfold when the growth medium was supplemented with 0.012 mM Fe3+.

Enhancement of CO2 sequestration and lipid accumulation is one of the major challenges that can be duly addressed by an extensive search for the new genes involved in the process (bio-prospecting) or targeted genetic engineering, both of which are promising approaches (Kumar et al., 2010).

Genetic and metabolic engineering transformations in microalgae are lim­ited to very few microalgal species. The use of molecular biology techniques as a toolkit to engineer microalgae for biodiesel production is a demanding strategy. Understanding, incorporation, and expression of the gene encoding rate-limiting enzyme of inorganic carbon uptake and lipid biosynthetic pathways are of more importance (Badger and Price, 2003; Verma et al., 2010). With the advancements in genome sequencing with sequence availability of Anabaena, Ostreococcus tauri, Thalassiosira pseudonana, and other algal species (Beer et al., 2009; Verma et al., 2010), the genetic transformation of microalgal species for various purposes is now promising. Cyclotella cryptica and Navicula saprophila were genetically trans­formed with the acetyl-CoA carboxylase (acc) gene isolated from Cyclotella cryptica for enhanced lipid synthesis. Such efforts could successfully enhance the activity of the acc gene; however, no significant lipid content was found to increase in trans­genic species, indicating that acc activity by itself cannot increase lipid biosynthesis and accumulation (Dunahay et al., 1996). More holistic approaches were forwarded for lipid enhancement through genetic engineering. Studies on the insights of various regulatory steps of the lipid biosynthetic pathway (Courchesne et al., 2009), expres­sion, and regulatory analysis of genes and enzymes (such as fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA, diacylglycerol acyltransferase) involved in triac — ylglycerol (TG) formation have been carried out (Bouvier-Nave et al., 2000; Dehesh et al., 2001; Jako et al., 2001).

Genetic transformations, which influence TG biosynthesis, may enhance bio­diesel production in transgenic microalgae (Verma et al., 2010). There have been considerable enhancements in the genetic engineering aspects of algae to improve the performance of transgenic microalgae, including (1) the efficient expression of transgenes, (2) riboswitches for gene regulation in algae, (3) inducible nuclear promoters and reporter genes (luciferase), as well as (4) inducible chloroplast gene expression (Beer et al., 2010).

Transcription-level regulations by transcription factors can also be used as a strat­egy to control the overall metabolite flux. The effect of transcription regulatory pro­teins has also been studied with respect to their expression levels to increase the production of secondary metabolites of interest in plants (Verma et al., 2010). In addi­tion to the approaches discussed above, further genome sequencing efforts need time. Advancements in existing tools and the development of new genetic transformation tools and screening methods will add further rigor to the efforts to optimize the accumulation of lipid and/or other metabolites alongside improving the economics of its production (Beer et al., 2010; Verma et al., 2010). Looking at the current interest in microalgae-based biofuels and microalgae/prototrophs, fundamental research will indisputably provide further advances in the near future (Beer et al., 2010). However, with respect to the utilization of genetically modified crops in India, that country has already accepted the release of Bacillus thuringiensis (Bt) cotton, which is suc­cessfully growing without causing any environmental problems. We (the authors) are of the opinion that in the near future, the scientific community will be exploring genetically modified microalgae in both open ponds as well as closed photobioreac­tors. But prior to doing that, several scientific issues should be addressed, and risk assessment (to ecosystem) studies must be performed to determine the legitimacy of using genetically modified microalgal strains to produce biodiesel.