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Bioresource Technology 102 (2011) 5770

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Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel productionT. Mutanda a, D. Ramesh a, S. Karthikeyan b, S. Kumari a, A. Anandraj c, F. Bux a,*a

Institute for Water and Wastewater Technology, Durban University of Technology, Durban 4001, South Africa Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India c Department of Nature Conservation, Mangosuthu University of Technology, Durban 4026, South Africab

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a b s t r a c tGlobal petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 2050% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purication and identication of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identication of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identication, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 30 March 2010 Received in revised form 9 June 2010 Accepted 17 June 2010 Available online 10 July 2010 Keywords: Biofuel Bioprospecting Microalgae Sampling Strain identication

1. Introduction The depletion of fossil fuel reserves has caused an increase in demand and price of diesel. The uncertainty in their availability is considered to be the important trigger for researchers to search for alternative sources of energy, which can supplement or replace fossil fuels (Harun et al., 2010; Mata et al., 2010). In recent years, research has been directed to explore alternate fuels from various biological renewable sources. Biodiesel is an alternative to diesel fuel, which is produced from oils via transesterication. It is nontoxic, biodegradable and has the potential to replace the conventional diesel fuel. The use of biodiesel will ultimately leads to reduction of harmful emissions of carbon monoxide, hydrocarbons and particulate matter and to the elimination of SOx emissions, which can also help in reducing the greenhouse effects and global warming. Presently, biodiesel is produced from different crops, such as, soybean, rapeseed, sunower, palm, coconut, jatropha, karanja, used fried oil and animal fats (Spolaore et al., 2006; Khan et al., 2009). There will be certain limitations in the use of these oils* Corresponding author. Tel.: +27 31 3732597; fax: +27 31 3732778. E-mail address: (F. Bux). 0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.077

as alternate fuels because of its food demand, life span, lower yield/ ha, higher land usage and higher price inter alia (Mata et al., 2010). It is necessary to search for non food based alternate feedstocks for biodiesel production. Selection of biodiesel feedstock is based on higher yields, short duration, lower production cost and less land usage. Among the various biodiesel feedstocks, the microalgae oil has the potential to replace the conventional diesel fuel. In order to avert fuel shortages in the future, a substantial amount of nancial resources (more than 300 million dollars) has been set aside to facilitate basic research in phycology to enable researchers in the tropics and subtropical regions to search and collect microalgae for evaluation of their feasibility for biofuel production (Sheehan et al., 1998). Microalgae are desirable for biofuel production as compared to plants because of the following reasons: (1) microalgae have fast growth rates, high biomass yield potential using non-fresh water streams as substrate, (2) microalgal based biofuels do not interfere with food security concerns, (3) biofuels generated from microalgal lipids have less emissions and contaminants as compared to petroleum based fuels therefore reduced greenhouse gas emissions and (4) microalgae require non-arable land for their cultivation and can utilise industrial ue gas as carbon source and moreover it can be harvested daily (Chisti, 2007;


T. Mutanda et al. / Bioresource Technology 102 (2011) 5770

Table 1 Potential of microalgae as primary PUFA resources (Spolaore et al., 2006). PUFA Docosahexaenoic acid (DHA) Eicosapentaenoic acid (EPA) c-Linolenic acid (GLA) Arachidonic acid (AA) Potential application Infant formulas; nutritional supplements; aquaculture Nutritional supplements, aquaculture Infant formulas; nutritional supplements Infant formulas; nutritional supplements Microalgal producer Crypthecodinium, chizochytrium Nannochloropsis, Phaeodactylum Nitzschia, Pavlova Spirulina Porphyridium

Greenwell et al., 2009; Grifths and Harrison, 2009; Rodol et al., 2009; Mata et al., 2010). To date, the main focus of research in the eld of biofuels from microalgae has been centred on downstream aspects such as bioreactor designs, biomass and lipid production from microalgae, biomass harvesting techniques and the chemistry of biofuel production. Microalgal bioprospecting encompasses searching and collection of unique microalgal strains from different aquatic environments for exploiting the potential applications of value added products such as polyunsaturated fatty acids (Olaizola, 2003; Spolaore et al., 2006) (Table 1). A lot of literature is available on the mass production and sustainable use of microalgae for biodiesel production and little emphasis placed on an in-depth study of microalgal bioprospecting. Therefore, the main objective of this review paper is to report current strategies focusing on bioprospecting for microalgae with the main aim of producing biofuels. This manuscript investigates current developments in microalgae bioprospecting. Protocols and procedures employed for successful microalgal bioprospecting are presented and described in-depth. Microalgal sampling, storage conditions and isolation and strain selection procedures are also discussed in detail. 2. Microalgae Microalgae are unicellular microscopic (2200 lm), polyphyletic, noncohesive, articial assemblage of CO2 evolving, autotrophic organisms which grow by photosynthesis and are the eukaryotic representatives although the prokaryotic cyanobacteria are frequently included with the algae (Greenwell et al., 2009). Algae are also dened as thallophytes (plants lacking roots, embryos, vascular system, stems and leaves) that have chlorophyll-a as their primary photosynthetic pigment and lack a sterile covering of cells around the reproductive organs (Brennan and Owende, 2010). Algae can either be autotrophic or heterotrophic; the former require only inorganic compounds such as CO2, salts and a light energy source for growth; while the latter are nonphotosynthetic therefore require an external source of organic compounds as well as nutrients as an energy source (Brennan and Owende, 2010). The evolutionary history and taxonomy of microalgae is complex due to constant revisions as a result of new genetic and ultrastructural evidence. The main criteria for categorising microalgae are pigmentation, life cycle and basic cellular structure (Brennan and Owende, 2010). Microalgae are classied into two prokaryotic divisions (Cyanophyta and Prochlorophyta) and nine eukaryotic divisions (Glaucophyta, Rhodophyta, Heterokontophyta, Haptophyta, Cryptophyta, Dinophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta). However according to Khan et al. (2009), the most

important groups of algae in terms of abundance are: diatoms, green algae, bluegreen algae and golden algae (Table 2). There is potential for further exploitation of these organisms for production of value added products and biofuels. 3. Sampling Microalgal collection is mainly inuenced by environmental factors (both biotic and abiotic), parameters measured onsite, type of aquatic system and sampling equipment. The collection method adopted is crucial for success, because damaged or dead cells may lead to failure. Therefore the temporal and spatial collection strategy should be adopted to cater for any succession that can occur at the sampling site (Anandraj et al., 2008; Bernal et al., 2008). For successful biofuel production using microalgae as feedstock for biomass and lipid accumulation, the crucial step is to search, collect and identify hyper-lipid producing strains. Selection of fast-growing, productive strains, optimized for the local climatic conditions are of fundamental importance to the success of any algal mass culture and particularly for low-value products such as biodiesel. According to Borowitzka (1997), it is also important to evaluate harvesting costs at the time of choos