Subburamu Karthikeyan

Department of Agricultural Microbiology Tamil Nadu Agricultural College Coimbatore, India

CONTENTS

3.1 Bioprospecting……………………………………………………………………………………………. 17

3.2 Isolation and Characterization of Naturally Occurring Algae……………………. 21

3.3 Isolation Techniques…………………………………………………………………………………… 22

3.3.1 Media Configuration……………………………………………………………………… 23

3.3.2 Traditional Methods………………………………………………………………………. 23

3.3.3 Advanced Methods………………………………………………………………………… 27

3.4 Screening Criteria and Methods………………………………………………………………….. 28

3.5 Screening and Selection for Lipid Production…………………………………………….. 31

3.6 Preservation………………………………………………………………………………………………… 33

3.6.1 Transfer Techniques………………………………………………………………………. 33

3.6.2 Maintenance Conditions………………………………………………………………… 33

3.6.3 Cryopreservation……………………………………………………………………………. 35

3.7 Role of Repositories……………………………………………………………………………………. 37

3.8 Concluding Remarks………………………………………………………………………………….. 37

References…………………………………………………………………………………………………………… 38

3.1 BIOPROSPECTING

Bioprospecting is the collection of biological material and the exploitation of its molecular, biochemical, and/or genetic content for the development of a commer­cial product. Precisely, bioprospecting relies on the endowment of a bioresource, a stock of novel biodiversity. Bioprospecting is a time-consuming process, where new products and markets must be identified, and a compound that covers com­mercial demands and social needs must be discovered. Algae are ubiquitous and have been evolving as primary biomass producers on the Earth for billions of years. Exploring this existing, self-maintaining, and diverse life form offers a rich base for global biotechnological innovations. Indigenous species are well adapted to pre­vailing regional abiotic and biotic factors, and further local strains provide an ideal platform for additional strain improvement and process optimization. Many algal species remain unknown or unexplored in science, giving logical attention to explore

Lipid Accumulating Algal Groups in Terms of Abundance

TABLE 3.1 Representative Estimated Number of Storage Algae Lipid Producer Described Species Material Habitat Diatoms Chaetoceros ~100,000d Chyrsolaminarin, Oceans, fresh (Bacillariophyceae) calcitrans, Skeletonoma sp., Thalassioria pseudonana, Phaeodactylum tricornutum lipids, polymer of carbohydrates and brackish water, terrestrial Green algaea (Chlorophyceae) Botryococcus braunii, Chlorella spp., Chlorella vulgaris, Dunaliella salina, Scenedemus sp., Ulva sp. 4,053e Starch and TAGs Freshwater, terrestrial, marine Blue-green algae (Cyanophyceae) Spirulina sp. ~2,000d Starch and TAGs Different habitats Golden algae (Chrysophyceae) Isochrysis sp. ~1,000d TAGs, leucosin, chrysolaminarin, carbohydrates Freshwater, marine Red algaeb (Rhodophyta) Lemanea fucina, Gracilaria, Porphyridium cruentum 6,081e Floridean starch Mostly marine, freshwater Brown algaec (Phaeophyceae) Fucus vesiculosus, Ascophyllum nodosum 3,067e Laminarin and mannitol Marine

a Seaweeds are included in the green algae (Chlorophyta); b Red algae (Rhodophyta); and c Brown algae (Ochrophyta or Heterokontophyta). d Adapted from Khan et al. (2009). e The World Conservation Union (2010).

this realm for potential application. To further illustrate this point, only fifteen of the currently known microalgal species are mass cultivated in some applied form for use in nutraceuticals, aquaculture feeds, or for wastewater treatment (Raja et al., 2008). Furthermore, the estimated unknown species for all clades of algae are projected to be two orders of magnitude greater than the currently known species (Norton et al., 1996) (Table 3.1). Of the commercialized algae, only a few species are cultivated

TABLE 3.2

Annual Biomass Potential of Microalgae in Comparison to Major Cultivated Crops

at substantial levels, which is trivial when compared to the annual global produc­tion of cultivated crops (Table 3.2). To propel algal biotechnological applications to commercially significant sustainable levels, regional species should be investigated for potential application to mass-scale cultivation. The idea of bioprospecting indig­enous microalgae for high-value or bioactive products is not innovative. The Aquatic Species Program of National Renewable Energy Laboratory (NREL) stocks more than 3,000 microalgal strains from the United States and Hawaii (Sheehan et al., 1998). Microalgae capable of producing large quantities of docosahexaenoic acid were isolated from marine environments of Western Taiwan (Yang et al., 2010).

Up to now, the key emphasis of microalgal biofuel research has focused on upstream aspects such as bioreactor designs, biomass and lipid production from microalgae, and downstream aspects such as biomass harvesting and the chemistry of oil production.

Microalgal bioprospecting includes isolation of exceptional microalgal strains from aquatic environments for potential value-added products and fine chemicals (Olaizola,
2003; Spolaore et al., 2006). A great deal of literature is accessible on the mass cultivation and sustainable use of microalgae for biofuels; however, relatively few stud­ies have focused on microalgal bioprospecting. Nevertheless, bioprospecting and the establishment of a microalgal collection exclusively for biofuel production have not been reported thus far. Algal bioprospecting or phycoprospecting of indigenous species has an advantage over other methods of sourcing algae from type culture collections and from genetically engineered organisms (Wilkie et al., 2011) (Table 3.3). Screening native algae for species with desirable traits provides a robust biological platform for bioresource production. This biological platform comes equipped with millions of years

TABLE 3.3

Comparison of Different Methods of Sourcing Algae

Source: Adapted from Wilkie et al. 2011.

FIGURE 3.1 Schematic outline of procedures in bioprospecting algae for biodiesel production.

of adaptation to the local climate and biota, meaning less energy expended on methods of environmental control and sterile techniques. Specific criteria for the production of biofuels from indigenous algae should include biomass and lipid productivity, harvesting the cells, and oil extractability. Further, the algal oil derived should contain 20%-25% C16 and C18 saturated fatty acid methyl esters and high amounts of unsaturated fatty acid chains, thus offering more cleavage sites to produce hydrocarbons (Gunstone and Harwood, 2007). Phycoprospecting may improve the efficiency of lipid extraction by yielding organisms with traits amenable to oil recovery. For specific objectives such as algal biodiesel, feedstocks for wastewater utilization or mitigation of greenhouse gases (GHGs), the chosen algal strains should satisfy requirements such as the ability to survive in wastewater, capability to grow robustly with higher cell densities, hyper­lipid content as triacylglycerol, and be capable of heterotrophic or mixotrophic growth as wastewater provides both organic and inorganic carbon sources. Until now, research on screening and acclimation of microalgae to adapt to wastewater environments is very sporadic (Zhou et al., 2011). A schematic outline and procedures in bioprospecting algae for biofuel production are outlined in Figure 3.1.