Bioresource Technology 102 (2011) 5770

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production
T. 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 Africa
b

a r t i c l e

i n f o

a b s t r a c t
Global 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: faizalb@dut.ac.za (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;

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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 choosing the species. Low-cost harvesting requires large cell size, high specic gravity compared to the medium and reliable autoocculation. The schematic outline of the procedures involved in upstream to downstream processing of microalgal lipids is presented in Fig. 1. 3.1. Sampling environments The mega biodiversity of microalgae entails them as suitable candidates for biofuel production. It is estimated that there are between one and ten million algal species, most of them being microalgae. Microalgae require light, CO2, appropriate pH, suitable salinity, macronutrients (nitrates and phosphates), vitamins and trace elements for their growth (Chisti, 2007; Brennan and Owende, 2010). These nutritional and environmental requirements are variable under natural conditions. Microalgae are found in diverse environmental conditions and habitats such as lacustrine, brackish, freshwater, hyper saline, wastewater maturation ponds, dams, rivers, marine and coastal areas. These natural ecosystems have immeasurable value as sources of hyper-lipid producing microalgae and it has been reported that many microalgal species tolerate brackish and saline waters. Table 3 depicts lipid content of some microalgal strains collected from different aquatic environments. Due to selection pressure and changing environmental conditions, there are a wide range of microalgal species worldwide found in extreme environ-

Table 2 The four most important groups of algae in terms of abundance (Khan et al., 2009). Algae Diatoms (Bacillariophycea) Green algae (Chlorophyceae) Bluegreen algae (Cyanophyceae Golden algae (Chrysophyceae) Known species (approx.) 100,000 8000 2000 1000 Storage material Chyrsolaminarin (polymer of carbohydrates) and TAGs Starch and TAGs Starch and TAGs TAGs and carbohydrates Habitat Oceans, fresh and brackish water Fresh water Different habitats Fresh water

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

59

Collection of microalgal samples from different aquatic environments

Enrichment of culture sample

Isolation of microalgae using traditional and advanced techniques

Purification

Strain Identification by microscopic and molecular tools

Strain maintenance and Storage in repository

Assessment of Growth/ Physiology

Qualitative and Quantitative analysis of lipids

Evaluation in open and closed systems

Recommendation for mass production

Biofuel production
Fig. 1. Steps involved and outcome of bioprospecting of microalgae for biofuels production.

ments (Rodol et al., 2009). In any habitat, microalgae have been shown to have successional tendencies due to variable nutrient availability, inclement weather and seasonal variations (Bernal et al., 2008). In bioprospecting it is important to collect microalgal samples temporally and spatially so as to determine if there are any successional tendencies in the habitat. According to Anandraj et al. (2008), microalgal biomass has shown clear temporal and spatial patterns during the heterogeneous conditions of the open and closed phases in estuaries. The microalgae are found as a mixed consortium and the population dynamics of the microalgae in any habitat is complex (Bernal et al., 2008). Different types of microalgal strains require different habitats, hence the diversity of environmental conditions where microalgae can be collected. In tropical and subtropical countries, inland dams, lakes and rivers have seasonal variations in ambient water temperature ranging

from 10 to 30 C. Lower winter temperatures promote the growth of benthic microalgal strains such as Senedesmus sp. whereas summer temperatures enhance the growth of the Chlorophytes such as Chlorella strains. The average pH in these habitats also varies from 3 to 8.5 depending on the prevailing edaphic conditions and agricultural practices in the area. In addition, the nutritional composition of the aquatic system is a major factor for microalgae to thrive in these habitats. The major macronutrients required for microalgal growth are phosphates, nitrates and ammonium and these are required in suitable concentrations to promote lipid synthesis by the microalgae (Celekli and Yavuzatmaca, 2009; Chen et al., 2009; Hsieh and Wu, 2009). Brackish aquatic systems constitute a mixture of seawater and fresh water and this usually occurs at the river mouth on the coastline. The ecological and physico-chemical parameters in this habi-

60 Table 3 Habitats and oil content of some microalgae. Microalga Botryococcus braunii Monodus subterraneus UTEX 151 Chlorella vulgaris CCAP 211/11b Chlorococcum sp. UMACC112 Scenedesmus sp. F&M-M19 Scenedesmus sp. DM Chlorella sp. Neochloris oleoabundans Crypthecodinium cohnii Tetraselmis sueica Monallanthus salina Dunaliella primolecta Phaeodactylum tricornutum Isochrysis sp. Nannochloris sp. Cylindrotheca sp. Pavlova salina CS49 Skeletonema sp. CS252 Chaetoceros muelleri F&M-M43 Pavlova lutheri CS182 Chaetoceros calcitrans CS178 Nitzschia sp. Nannochloropsis sp. Schizochytrium sp. Oil content (% dry weight) 2575 16.1 19.2 19.3 19.6 21.1 2832 3554 20 1523 >20 23 2030 2533 2035 1637 30.9 31.8 33.6 35.5 39.8 4547 3168 5077

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

Habitat Fresh water/estuary Freshwater Freshwater Freshwater Freshwater Freshwater Freshwater Fresh water Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine

positioning system (GPS) coordinates of the sampling location must be recorded for future reference and resampling (Woelfel et al., 2007).

3.3. Sampling equipment and procedure The microalgal sampling and selection process is well established although it requires specialised equipment and may be time consuming (Anandraj et al., 2007). The equipment required for microalgal sampling includes a knife, mesh net (0.7 lm mesh), scooping jar, vials for collecting samples, scalpels, dissolved CO2 and O2 analyser with a data logger, light meter, GPS, salinity meter, multi probe system (measuring pH, temperature, turbidity, conductivity and light intensity simultaneously). Heavy duty equipment includes a suitable vehicle for rough terrain with enough space for the collected samples. There is no denite sampling procedure documented in literature though researchers can follow simple and cheap methods of collecting microalgal samples. Ideally samples can be collected from the natural substrata by chipping, scrapping, and by brushing from rock surfaces and bottom sediments. The brushing method was reported to be effective and reproducible method of collecting microalgal cells and also that it does not damage them (MacLulich, 1986). Sampling in deep freshwater lakes and dams requires systematic sampling whereby water samples are scooped from at least three depth levels to the bottom of the lake or dam. This will allow for collection of microalgae which prefer different light intensities. Bottom sediments are also major habitats of benthic microalgae and therefore should be collected together with pieces of detritus and mud. Stringent regimes and protocols need to be exercised when sampling. Therefore an all encompassing sampling regime is essential to isolate microalgae from aquatic environments.

Modied from Chisti (2007), Rodol et al., (2009).

tat are mainly a result of the dissolved CO2, O2 and dissolved salts due to eddies and turbulence created as the two water bodies mix. A number of microalgal strains prefer brackish conditions because of the nutritional composition of the aquatic system and the warmer temperatures (Woelfel et al., 2007). Most of the microalgae found in brackish conditions are found in suspension as a consequence of the rapid water movements (Anandraj et al., 2008). Hypersaline environments (halophilic), thermophilic springs and maturation ponds are ideal extreme environments for the isolation and selection of lipid producing microalgae with novel properties. Isolates collected from these aquatic habitats are robust and have rare and unique characteristics and therefore possibly better adapted to specic conditions (Sheehan et al., 1998). The water collected from hypersaline aquatic bodies can be used for media formulation for microalgal growth in photobioreators and raceway ponds under controlled laboratory conditions. For the purpose of bioprospecting, it is imperative to screen as many aquatic environments as possible to increase the potential to isolate and select unique hyper-lipid producing microalgae. 3.2. Parameters to measure on site It is critical to measure some parameters on the collection site in order to simulate these conditions when culturing specimens under laboratory conditions. Elementary factors inuencing microalgal growth are measured on the sampling site. These include abiotic factors such as light (quality and quantity), water temperature, nutrient concentration (nitrates and phosphates), dissolved O2, dissolved CO2, pH, and salinity. Technically it is impractical to measure biotic factors on site such as pathogens (bacteria, fungi and viruses) and any competitors in the habitat. These can be analysed microscopically and using conventional microbiological methods in the laboratory after collecting the water samples. However if resources and time are available it is desirable to measure the operational factors such as shear produced by mixing, dilution rates, depth and water velocity i.e. if the water is owing. The global

4. Isolation and purication techniques 4.1. Culture media Various culture media have been developed for isolation and cultivation of microalgae (Table 4). Some of them are modications formulated after detailed study on the nutrient requirement of the organism. In case of marine algae, though articial sea water media is common, good growth of algae can be achieved by adding small quantities of nonpolluted natural seawater (less than 14%) to the articial seawater (Andersen, 2005). Schreiber solution consisting of a mixture of nitrate and phosphate was devised, based on the minimum requirement for the two elements shown by a diatom culture. Soil extracts were added to Schreibers medium for growing the green dasycladalean Acetabularia and unicellular and benthic marine algae. Earlier algal media were devised to include antibiotics, vitamins, trace metals and later with EDTA, a metabolically inert chelator, to replace organic chelators such as citrate (Andersen, 2005). Likewise modied media supporting growth of a wide range of microalgae can be formulated by carefully manipulating major nutrients (Scott et al., 2010). Antibiotics can be added to the growth medium to discourage growth of contaminating cyanobacteria and other bacteria. Addition of germanium dioxide inhibits the growth of diatoms. Axenic cultures can be obtained by treating isolated algae to an extensive washing procedure, and/ or with one or more antibiotics. In addition, bacterial contamination in microalgal purication can be prevented by employing a procedure involving treatment with a detergent and phenol (Abu et al., 2007). Therefore media composition is vital and most importantly, media has to be inclusive of essential components that are

T. Mutanda et al. / Bioresource Technology 102 (2011) 5770 Table 4 Common media used for microalgal strains from different aquatic environments. Media AF6 medium, modied Freshwater + Marine + Brackish Suitable for Euglenophyceae, volvocalean algae, xanthophytes, many cryptophytes, dinoagellate and green ciliate; specic for algae requiring slightly acidic medium Broad spectrum marine algae Freshwater Chlorophyceae Freshwater soil Cyanophyceae Broad spectrum medium for freshwater Chlorophyceae, Xantophyceae, Chrysophyceae, and Cyanophyceae; unsuitable for algae with vitamin requirements Cyanobacteria, cryptophytes, green algae, and diatoms Freshwater diatom Freshwater Chrysophyceae Broad spectrum medium for coastal and open ocean algae Broad spectrum medium for oligotrophic marine algae For oligotrophic (oceanic) marine phytoplankters Broad spectrum medium for marine algae Broadspectrum medium General medium for marine algae especially coccolithophores Reference(s) Watanabe et al. (2000)

61

AK medium Beijerinck medium BG-11 Bolds basal medium

+ + +

+ +

+ +

Barsanti Barsanti Barsanti Barsanti

and and and and

Gualtieri Gualtieri Gualtieri Gualtieri

(2006) (2006) (2006) (2006)

COMBO Medium Diatom Medium, modied DY-III medium ESAW medium K medium L1 medium Medium F Medium G MNK medium

+ + +

Kilham et al. (1998) Cohn et al. (2003) Barsanti and Gualtieri (2006) Berges et al. (2001) Barsanti and Gualtieri (2006) Guillard and Hargraves (1993) Jeffrey and LeRoi (1997) Blackburn et al. (2001) Nol et al. (2004)

+ + + + + +

+ + + + ND ND

ND, not determined; +, can be used; , cannot be used.

found in the natural environment to support and not exclude the growth of isolates of interest. 4.2. Conventional methods 4.2.1. Single-cell isolation Perhaps the most common method for single-cell isolation is by micropipette, although automation is more advantageous. Micropipette isolation is usually performed with a Pasteur pipette or a glass capillary having a straight or bent or curved tip. The goal of micropipette isolation is to pick up a cell from the sample, deposit it without damage into a series of sterile droplets, until a single algal cell, free of all other protists, can be condently placed into the culture medium. Subsequently, the sample can be examined microscopically in a glass or plastic dish, in a multiwell plate, or on a microscope slide. However, the microalgal droplets can be placed on agar to reduce evaporation but this step depends on the size of the cells. Furthermore, the single cell can be pipetted and discharged into the sterile rinsing droplet and before the cell can settle, it should be picked up and transferred. Skill of the technique is important not to shear or damage the cell. For agellates, cessation of swimming sometimes indicates damage. For diatoms, broken frustules can refract light differently than for intact cells. Leakage of protoplasm is an obvious sign of severe damage. Rogerson and co-workers, (1986) employed repeated introduction and ejection of cells, suspended in a 1% crude papain solution, into and from a micropipette to generate ca. 10% naked cells of Coscinodiscus asteromphalus. These naked cells were reisolated via micropipette into fresh medium. The traditional method of micropipette isolation can be successfully employed with certain precautions. Ultraclean droplets for rinsing are necessary, because the tiny cells cannot be easily distinguished from particles, especially when working with seawater. 4.2.2. Isolation using agar plates Isolation of cells on agar plates is also an old and common method. It is the preferred isolation method for many coccoid algae and most soil algae, not only for ease of use but also because axenic cultures can often be directly established without further treatment. For successful isolation onto agar, the alga must be able to

grow on agar as streak or pour plates (Brahamsha, 1996). In most cases, the concentration of agar is not an important factor, assuming the agar is between 0.8% and 1.52.0%. Some algae do not grow on the surface of agar plates, but they do grow embedded in agar. 4.2.3. Atomized cell spray technique A ne or atomized spray of cells can be used to inoculate agar plates. The technique can vary, but in general a liquid cell suspension is atomized with forced sterile air so that cells are scattered onto the plate (Andersen, 2005). For best results, the spray can be administered in a sterile hood or clean environment so that airborne bacteria and fungi are not dispersed onto the plate. The plates are incubated, and after colony formation, selected cells are removed and inoculated. 4.2.4. Dilution techniques The goal of the dilution method is to deposit only one cell into a test tube, ask, or well of a multiwell plate, thereby establishing a single-cell isolate (Andersen, 2005). If the approximate cell concentration is known, then it is easy to calculate the necessary dilution so that, on the basis of probability, a small volume contains a single cell. The technique can be altered in several ways like dilution with culture medium, distilled water, seawater, ltered water from the sample site, or some combination of these. Also, ammonium, selenium, or another element can be added to some isolation tubes or cell wells to specically select for species that require these nutrients (Andersen, 2005). 4.2.5. Gravimetric separation Gravity separation can be effective for separating larger and smaller organisms, and the two primary methods are centrifugation and settling. Gravity is perhaps more frequently used to concentrate the target organisms rather than to establish unialgal cultures. Usually, the goal is to separate the larger and heavier cells from smaller algae and bacteria. Gentle centrifugation for a short duration brings large dinoagellates and diatoms to a loose pellet, and the smaller cells can be decanted. Centrifugation with use of density gradients (e.g., Silica sol, Percoll) has been used to separate mixed laboratory cultures where each species was separated into a sharp band (Andersen, 2005). Settling is effective for non-swim-

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ming large or heavy cells. Both centrifugation and settling techniques are effective for concentrating larger cells, but single-cell isolation is nearly impossible and hence combined with other techniques. 4.2.6. Media enrichment Enrichment cultures have long been used as a preliminary step towards single-cell isolations. Common enriching substances include culture medium, soil water extract, or nutrients like nitrate, ammonium, and phosphate or a trace metal. One strategy of bacteria-free algal cultures is to make the medium as acidic as possible without killing the alga. Peat moss can be substituted for soil water extract when enriching for desmids and some other algae from acid habitats. Organic substances, such as yeast extract, casein, or urea can be added in low concentrations when isolating osmotrophic algae. Tiny amounts of various fruits and vegetables were added to the culture in trying to isolate Oxyrrhis in a mixed culture of phytoplankton and it was discovered that unltered lemon juice, and subsequently extracts from lemon rind, led to success, because of the availability of ubiquinone or plastiquinone (Andersen, 2005). Natural samples are often decient in one or more nutrients, but in nature algae survive, because bacterial action, grazing, and death of organisms recycle those nutrients. Sampling reduces recycling, and nutrient stress can cause death to the target species. Sometimes enrichments can also be detrimental, if the target species is rare and unable to compete with weedy species. The enriching substance can be varied and usually added in minimum quantity and in stages. Selective culturing is an exceptional tool for enrichment culturing of hyper-lipid producing microalgae. Since the goal is to isolate microalgae growing at a high CO2 concentration, then an enrichment culture can be aerated with 15% CO2 and thus select for species with high CO2 tolerance (de Morais and Costa, 2007; Ramanan et al., 2010). Although conventional methods for isolation has sufced, there are limitations and therefore continued need for developing novel methods for isolation of microalgae. 4.3. Advanced methods Algal cultures may be unialgal which means they contain only one kind of alga, usually a clonal population (but which may contain bacteria, fungi, or protozoa), or cultures may be axenic meaning that they contain only one alga. Streaking and spraying are useful conventional techniques for single-celled, colonial, or lamentous algae growing on agar surface. Many agellates, however, as well as other types of algae must be isolated by singleorganism isolations using advanced techniques. Among algal cell manipulation techniques, the techniques for cell separation and isolation with high specicity are limiting because cell populations are frequently heterogeneous and the cells are suspended in a solution of different chemicals, biomolecules, and cells. Compared with traditional separation methods, microfabricated devices have small working volume and subsequently reduced throughput. 4.3.1. Micromanipulation A micromanipulator allows the selection of single cells from liquid culture. This would mean that a single cell can be selected from an enriched environmental sample and grown in liquid monoculture as well as plates. This will afford a time saving of $60% over the current serial plating technique. This equipment is stipulated in literature as the ideal tool for algal screening and isolation (Kacka and Donmez, 2008; Moreno-Garrido, 2008). The successful application of micromanipulation techniques requires the expertise and experience. It requires the handling of an Inverted

microscope or stereo microscope with magnication up to 200. Phase contrast or dark eld optics is an advantage. Capillary tubes or haematocrit tubes of approximately 1 mm diameter 100 mm long are used for picking individual cells (Godhe et al., 2002; Knuckey et al., 2002).

4.3.2. Flow cytometry Flow cytometric analysis permits the investigator to perform a rapid and quantitative version of the experiments that could otherwise be performed by uorescent microscopy. Fluorescence-Activated Cell Sorting (FACS) allows the process to be taken one very important step further. Cells with a specic characteristic or indeed a combination of characteristics can be separated from the sample for further analysis or growth. Electronic cell sorting for isolation and culture of dinoagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette (Sinigalliano et al., 2009). Trauma to electronically sorted cells was not a limiting factor, as fragile dinoagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (>4 l) cultures was higher for manual picking than for electronic cell sorting (2% vs. 0.5%, respectively). However, manual picking of cells is more laborintensive and time consuming. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium, which was optimized for axenic dinoagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual post-sort culture maintenance. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains (Sinigalliano et al., 2009).

4.4. Other methods Immunological and non-immunological methods to separate cells of interest are available. Conventional cell separation can be carried out by immunoreactions of membrane protein with the capturing antibodies because the type of integrated proteins is specic for their function. Immunological technique is a mainstay of commercialized cell separation methods such as the uorescence-activated cell sorting (Takahashi et al., 2004); magnetic-activated cell sorting (Han and Frazier, 2005); afnity based cell sorting (Chang et al., 2005). One of the advantages of this method is high specicity and selectivity. However, the disadvantage is that the immunologically isolated cells may suffer from damages and overall separation system involves high cost and complicated processes such as immunoreactions and elution of cells from the capturing antibodies. On the other hand, non-immunological methods are relatively fast and simple techniques. They include techniques such as dielectrophoresis (Doh and Cho, 2005); hydrodynamic separation (Shevkoplyas et al., 2005); aqueous two-phase system (Yamada et al., 2004) and ultrasound separation (Petersson et al., 2004). These exploit an interactive physical property of cell with the surrounding media. However, a disadvantage is its low specicity for cell separation, as cells do not show remarkable differences between each cell type with the exception of immunological properties. In this method, the type of cells is determined and separated according to their cell size, shape and other physical properties. The advantages and disadvantages of the microalgal purication techniques described in this section are summarised in Table 5.

T. Mutanda et al. / Bioresource Technology 102 (2011) 5770 Table 5 Advantages and disadvantages of microalgal purication techniques. Techniques Pringsheims micropipette method Advantages Single cells can be successively transferred and puried Disadvantages Laborious and time-consuming method requiring considerable manual skills. The method often fails with small non-agellate cells, which are more difcult to recognise during serial transfers Some delicate agellates are easily damaged during successive micropipette transfers. Cannot be used with most agellate taxa which fail to grow on solid substrates Unsuccessful when the numerical ratio between algae and bacteria is unfavourable. Costly method It is problematical with small algal cells and with cells secreting mucilage because of bacteria embedded in the mucilage which may also clog lters. Damage the alga as well as leads to increased resistance levels in contaminating bacteria Require considerable costs for equipment and its operation Require multi-user or central facilities. Reference(s) Melkonian (1990)

63

Agar plating (or spraying) Serial dilution Differential centrifugation Filtration

Relatively easy Relatively easy Less damaging to sensitive cells Less damaging to sensitive cells and usually gives better separation of algae from bacteria than differential centrifugation. Relatively easy Low cost Precise and rapid method Simultaneous measurements of individual particle volume, uorescence and light scatter properties Highly suitable for separating bacteria from algae to establish axenic algal cultures Can be used directly in natural samples Useful for small and delicate taxa Useful for separating attached bacteria from algal cell walls or mucilage

Brahamsha (1996)

Melkonian (1990)

Use of antibiotics Flow cytometry

Sensen et al. (1993)

Axenic cultures are difcult to obtain from algae to which bacteria are physically attached.

Ultrasonication

Not a standalone method Should be coupled subsequent to cell sorting

Steup and Melknonian (1981)

5. Screening of microalgae The screening stage of bioprospecting focuses on isolation and identication of algal species capable of substantial lipid production, targeting organisms with rapid growth rate and tolerance to environmental parameters. The conventional method used for lipid determination involves solvent extraction and gravimetric determination (Bligh and Dyer, 1959). A major disadvantage of the conventional method is that it is time consuming, labor-intensive and it has a low throughput screening rate. Moreover, approximately 1015 mg wet weight of cells, (Akoto et al., 2005) must be cultured for the extraction and derivatization. Consequently, there is greater interest on a rapid in situ measurement of the lipid content (Cooksey et al., 1987). Nile Red (9-diethylamino-5H-benzo[a]phenoxazine-5-one), a lipid-soluble uorescent dye, has been commonly used to evaluate the lipid content of animal cells and microorganisms (Genicot et al., 2005) and especially microalgae (Cooksey et al., 1987; Elsey et al., 2007). Nile Red possesses several characteristics advantageous to in situ screening. It is relatively photostable, intensely uorescent in organic solvents and hydrophobic environments. The emission maximum of Nile Red is blue-shifted as the polarity of the medium decreases, (Cooksey et al., 1987; Greenspan and Fowler, 1985; Laughton, 1986; Lee et al., 1998) which allows one to differentiate between neutral and polar lipids at the excitation and emission wavelengths. Elsey et al. (2007), showed the technical emission spectra for Nile Red in various solvents. The peak emission intensity of Nile Red in hexane occurs near 576 nm when excited at 486 nm. The chloroform and ethanol peak excitation was recorded at 600 and 632 nm, respectively (Elsey et al., 2007). In acetone, Nile Red is excited at 488525 nm and the uorescent emission measured at 570600 nm using various instruments (Cooksey et al., 1987). Measurement of neutral lipids using the Nile Red application requires the instrument to be calibrated using the stain dissolved in an organic solvent and account for the nonlinear intensity emission with respect to time. Measurements of lipid per

unit cell, requires a calibration curve that correlates uorescence to lipid content, whether determined gravimetrically or by use of lipid standards (Elsey et al., 2007). Thick cell walls of microalgae inhibit the permeation of Nile Red and may indicate the absence of oil, even though gravimetric analysis shows high yields of neutral lipids. It has been noted that the permeation of Nile Red dye is also variable among algal species, requiring the use of high levels of DMSO (2030% vol./vol.) and elevated temperatures (40 C) (Chen et al., 2009). Alternatively, the lipophilic uorescent dye BODIPY 505/515 (4,4-diuoro-1,3,5,7-tetramethyl-4-bora-3a,4adiaza-s-indacene) has recently been used as a vital stain to monitor algal oil storage within viable cells. Lipid bodies are stained green and chloroplasts red and visualized in live oleaginous (oil-containing) algal cells (Cooper et al., 2010) (Fig. 2). The advantage of BODIPY is that high lipid yielding cells may be identied and isolated microscopically using a micromanipulator system, ow cytometry or a uorescence-activated cell sorter (Cooper et al., 2010). Subsequently, pure cultures may be propagated from the isolated viable cells. BODIPY 505/515 has been shown to have a narrower emission spectrum than Nile Red, making it potentially more useful for confocal imaging, where uorescence contrast enhancement of lipid bodies is important for image resolution (Cooper et al., 2010). Unlike Nile Red, BODIPY 505/515 has the advantage that it does not bind to cytoplasmic compartments other than lipid bodies and chloroplasts. A recent study (Dean et al. 2010) demonstrated the use of Fourier transform infrared micro-spectroscopy (FTIR) to determine lipid and carbohydrate content of freshwater microalgae. FTIR was shown to be an efcient and rapid tool for monitoring lipid accumulation of microalgae. This study had reported highly signicant correlations between the FTIR- and Nile Red-based lipid measurements. For the purposes of bioprospecting for high lipid yielding microalgae, a rapid throughput of sample processing is required. The semi-quantication of neutral lipids using Nile Red or BODIPY and uorescence microscopy allows for an initial rapid screening and visualization of lipid globules. FTIR spectroscopy may be used

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

Fig. 2. (A) Nile Red stained Chlorella sp. viewed at 1000 using a uorescence microscope at 490 nm excitation and 585 nm emission lters. Neutral lipid globules in the cytosol are stained yellow (unpublished data). (B) Oil-containing lipid bodies can be vitally stained and visualized in live oleaginous (oil-containing) algal cells using the green uorescent dye, BODIPY 505/515. In this picture, vitally stained lipid bodies (green) are easily distinguished from chloroplasts (red) in an O. maius Naegeli freshwater lamentous algal cell using a Zeiss Axioskop epiuorescence microscope (Cooper et al., 2010). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

thereafter to quantify the yield of lipids. Once the high lipid producing microalgae have been identied, isolated and puried, a further step in the screening would be to determine the photosynthetic efciency of the culture. Subsequent to screening, understanding the physiology of the algal isolate is imperative. Pulse Amplitude Modulated (PAM) chlorophyll-a uorescence measurements are widely used as a simple, rapid, and non-invasive method to assess the physiological state of microalgae (Schreiber et al., 1994). It is also a valuable tool to assess the optimum growth conditions required to maximize the biomass yield and to quantify the effect of nutrient or other extreme environmental stresses (salinity, temperature, PAR and pH) on the algal culture. Neutral lipid synthesis is stimulated under nutrient depleted or limited conditions. Many microalgae have the ability to produce up to almost 80% dry cell weight of triacylglycerols (TAG) as a storage lipid (Chisti, 2007; Spolaore et al., 2006) under nutrient or other environmental stress. The PAM uorometer parameters (Electron transport rate [ETR], maximum quantum efciency of Photosystem II [FV/Fm], and non-photochemical quenching [NPQ]) may be used as indicators of nutrient stress and consequently the possibility of neutral lipid synthesis and can be a valuable instrument in the screening process. Neutral lipid synthesis is likely to occur during the stationary phase of growth due to nutrient limitation (Li et al., 2008). The screening process of microalgae bioprospecting has to be comprehensive in assessing the lipid producing potential as well as the kinetics of growth and tolerance. The success of downstream processing is dependent on reliable biochemical and physiological screening tools such as the BODIPY lipid stain, FTIR spectroscopy and PAM Fluorometry.

acceleration. They recorded the highest cell recovery at 13,000g ($95%) followed by 6000 g (60%) and 1300 g (40%). Highest cell recovery obtained at 13,000g ($95%) followed by 6000 g (60%) and 1300 g (40%). Cell viability was found to depend on the microalgal species and the method of centrifugation. Before extracting the algae oil, high moisture present in algae biomass must be removed by means of drying. The temperature used in microalgae drying is crucial factor for separating the oil from dried algae biomass. Higher drying temperature decreases both concentration of triacylglycerides and lipid yield (Widjaja et al., 2009). The algae biomass dried at 60 oC under vacuum (Widjaja et al., 2009) or freeze drying gave the best results for extraction of algae lipid. The lipids present in the dried/freeze dried biomass must be extracted and analysed for quantication of the fatty acids. The lipids produced by algae are often accumulated intracellularly, which could require extraction of the lipids from crude cell pastes (Grima et al., 2003). These techniques are used for rupturing the algae cells to release the lipid compounds of algae biomass. Different cell disruption techniques such as autoclaving, microwaves, sonication, osmotic shock and bead beating have been evaluated in order to increase lipid extraction efciency (Lee et al., 1998, 2010). They concluded that microwave oven method was most simple, easy, and efcient method for lipid extraction from microalgae. Although many methods for algal lipid extraction have been recommended, the most popular is a slightly modied method of Bligh and Dyer (1959). The solvent extraction was still the main extraction method used by many researchers due to its simplicity and relatively inexpensive requiring almost no investment for equipment (Letellier and Budzinski, 1999). 6.2. Sample preparation for lipid analysis

6. Lipid analyses 6.1. Pre-treatment of algae biomass for lipid extraction The harvesting of algae biomass can be achieved by physical, chemical or biological methods or combination of any two of these methods. The techniques currently employed in microalgae harvesting cum cell recovery include centrifugation, occulation, ltration, gravity sedimentation, otation and electrophoresis. Among these methods, centrifugation is found to be the most efcient. Heasman et al. (2000) studied algae cell recovery at three different centrifugation conditions for ten microalgal species and reported that efciency of recovery decreased with decreasing

Qualitative and quantitative composition of lipids can be investigated by established techniques. Thin-layer chromatography (TLC), high pressure liquid chromatography (HPLC) or gas chromatography (GC) or any chromatography with mass spectrometry are some of the different techniques employed for quantication of lipid from microalgae. Lipid proling of biodiesel feedstock is commonly done by GC with a Flame Ionization Detector (FID) according to ASTM D6584 and EN 14105 methods. Before the fatty acid components of algae lipids can be analysed by GC, it is necessary to convert them to low molecular weight non-polar derivatives, such as fatty acid methyl esters (FAME). Direct transesterication (simultaneous extraction and transesterication) and transesterication (transesterication after lipid extraction) were

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

65

adopted in sample preparation for algae lipid conversion to biodiesel (Johnson and Wen, 2009). Triglycerides in oils are reacted with methanol in presence of base/acidic catalyst in transesterication reaction to produce FAME. Acid catalysts used in transesterication of microalgal oil includes sulphuric acid and hydrochloric acid, while alkali catalysts like sodium hydroxide and potassium hydroxide are suitable for vegetable oil and animal fat respectively (Huang et al., 2010). After transesterication, the microalgal fatty esters develop slight green colour due to presence of chlorophylls. Before chromatographic analysis, any traces of chlorophylls, catalyst or water must be removed to avoid GC column contaminations (Sheehan et al., 1998). 6.3. Characterization of algal oil for biodiesel production The fatty acid compositions of microalgae oil may vary to individual species/strains and their environmental conditions. The fatty acid esters compositions of microalgae sources are different from plant oils. Algal oils contain a high degree of polyunsaturated fatty acids with four or more double bonds when compared to vegetable oils (Zittelli et al., 2006; Rodol et al., 2009). The identication of lipid composition in selected algae strain is essential for determining the suitability to biodiesel and fuel quality. The most important fuel properties considered to assess the potential of biodiesel as substitute of diesel fuel are viscosity, cetane number (CN), density, cold lter plugging point, oxidative stability, lubricity, ignition quality and combustion heat (Xiaoling and Qingyu, 2006). The physical characteristics of both fatty acids and triglycerides can be determined by chain length, number of double bonds and amount of each fatty ester components in both fatty acids and triglycerides (Mittelbach and Remschmidt, 2004; Ramos et al., 2009). The saponication values (SV) and iodine value (IV) represent the ignition quality of fuel and presence of unsaturated fatty acid component in FAMEs. Higher CN values indicate better ignition properties of the fuel (Meher et al., 2006). Higher unsaturated fatty acids (UFA) present in the oil gave higher iodine values and heating of higher UFA caused the polymerization of glycerides, which leads to formation of deposits or deterioration of the lubricating (Mittelbach, 1996; Ramos et al., 2009). FAMEs with higher degree of unsaturation are not suitable for biodiesel. The CN, SV and IV can be predicted from fatty acid methyl esters of oils by using empirical equations (Krisnangkura 1986; Kalayasiri et al., 1996). According to biodiesel standard EN 14214 methods, the concentration of linolenic acid and acid containing four double bonds in FAMEs should not exceed the limit of 12% and 1%, respectively. Higher oleic acid content increases the oxidative stability for longer storage (Knothe, 2005) and decreases the cold lter plugging point (CFPP) for use in cold regions (Stournas et al., 1995). The pour and cloud points of feedstock decrease with increasing
Table 6 Some of the target genes and the primers used for phylogenetic studies of microalgae. Target region 18S rDNA Primers used

chain length and branching of the alcohol moiety (Foglia et al., 1997). Biodiesel feedstock with a high degree of saturation is more resistant to oxidation and more stable in presence of light, oxygen, high temperatures, and metals (Canakci and Sanli, 2008; Knothe, 2005). The determination of fatty acid composition of algae oil is essential for assessing the fuel quality of biodiesel. Different culture conditions play an important role in the lipid composition, like C:N ratio, which is the major factor (Papanikolaou et al., 2004). In order to improve the fuel properties of algae biodiesel, suitable culture conditions may be used and the properties of biodiesel produced from algae oils must meet the International Biodiesel Fuel Standards. 7. Microalgal identication strategies using molecular approach Identication and enumeration of the algae of interest is a major challenge faced by researchers worldwide. Microscope-based microalgal cell identication methods are usually the standard procedures used in laboratories for the rapid screening of algal samples. Conventional light microscopy has been extended to the use of phase contrast microscopy, uorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for species level identication. However, conventional microscopy techniques used to analyse microalgae can give misleading results since they lack morphological markers for precise identication; further microalgae can often change cell size and shape during their life cycle (Godhe et al., 2002; Bertozzini et al., 2005). Mostly algae do not survive during xation or they shrink, lose pigmentation and agella so that a proper identication is impossible. In addition, some species constitute very minor fraction of the total planktonic community, which leads to tedious analysis of distinct samples. The identication of microalgae in eld samples using microscopy is also time consuming and requires both experience and signicant taxonomic and technical skills (Godhe et al., 2002). The molecular-based techniques developed in the last two decades have led to rapid and accurate monitoring, identication and quantication of microalgae species in mixed phytoplankton samples. Without complete DNA sequence, the analysis of small conserved genes has proved to be very helpful in the clarication of the relationships between algae. The most common DNA regions analysed today for phylogenetic purposes are ribosomal RNA genes (rRNA), mitochondria genes, plastid genes (rbcL), ITS (Internal Transcribed Sequences) and microsatellite DNA sequences (Table 6). Ribosomal RNAs are one of the widely used and are exceptionally useful for the comparative analysis of organisms (Rasoul-Amini et al., 2009; Moro et al., 2009). They are very slowly altering molecules and major elements in the protein synthetic machinery of all cells. Small rRNA (SSU rRNA) genes are more highly con-

References Tinti et al. (2007) Moro et al. (2009) Auinger et al. (2008) Rasoul-Amini et al. (2009) Scholin and Anderson (1996) and Kamikawa et al. (2007) Novis et al. (2009) Richlen et al. (2007) and Auinger et al. (2008) Kamikawa et al. (2007) Coleman (2003, 2007) and Jrgen et al. (2008) Kamikawa et al. (2007) Henrichs et al. (2007)

Large subunit (LSU rDNA) Plastid rbcL Small-subunit (SSU rDNA) Mitochondrial cytochrome c oxidase subunit (cox2cox1/cox2cox1 Internal Transcribed Spacer (ITS) ITS1+5.8S+ITS2 Microsatellite locus

16S1 N/16S2 N ChloroF/ChloroR EK82F/Proto5R 519F/1406R FD8/RB, D1/D2 18ScomF1/Dino18SR1, EK82f/Proto 5r coxF/coxR ITS1/ITS2 ITS3/ITS4 Kbr1Kbr10

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served than large rRNA (LSU rRNA) genes, and are therefore more useful for analysis of more distantly related species. Analysis of the LSU rRNA gene has been very useful for sorting out closely related species concepts, like species groups and geographical origin of different clonal isolates (Scholin and Anderson, 1996). When using the microalgae for Polymerase Chain reaction (PCR) studies, the genomic DNA has to be extracted and puried from samples, removing potential inhibitors that often cause PCR inhibition and low yields of PCR products (Godhe et al., 2002; Bertozzini et al., 2005). The efciency of the genomic DNA extraction step is very important for the subsequent PCR assay, especially when it is to be used in quantitative investigation on cultured or environmental samples. To overcome this, direct PCR amplication from few cells without the need for DNA extractions has been reported (Godhe et al., 2002; Auinger et al., 2008). Furthermore, in the sampling of long-term monitoring programmes the PCR is also applied to preserved natural samples. Fixatives, such as Lugols solution, formalin and glutaraldehyde, are used as preserving agents for the long term storage of phytoplankton material without destroying the DNA needed as template in morphological and molecular identication (Godhe et al., 2002; Auinger et al., 2008). Quantitative analysis of planktonic protists and microalgae from preserved eld samples combining morphological and small-subunit (SSU) rRNA gene sequence using single cell PCR has been reported recently (Auinger et al., 2008). A further promising molecular approach is the application of DNA microarrays, which are applied generally for gene expression and have been used with oligonucleotide probes of conserved genes for species identication at all taxonomic levels (Gescher et al., 2008).The technique is based on a minimized but, high throughput form of a dot blot through application of sequences or probes in an ordered array on the chip. The chip is made of glass and has special properties. The microarray offers the potential to facilitate the analysis of multiple targets from one sample in one experiment (Gescher et al., 2008). A combination of molecular probes/primers and DNA microarrays could serve as a rapid and reliable tool for rapid screening of microalgae. The application of novel molecular techniques has the potential to revolutionise microalgal classication and especially identifying hyper-lipid producing microalgae. 8. Microalgal maintenance One of the major limitations with regards to culture maintenance is effective technology for long term storage of cultures (Moreno-Garrido, 2008). As living microorganisms, microalgal samples must be stored under suitable controlled laboratory conditions so that they do not degenerate and lose viability. Bioprospecting for microalgal strains is costly and time consuming; therefore it is crucial to store the cultures properly so that the bioprospecting exercise is not put to waste. The best way to store puried microalgal cells is to have the cells in an aqueous system under suboptimal temperature and light regimens. According to Lorenz et al. (2005) standard light intensities between 10 30 mmol photons m2 s1 have proved appropriate in combination with subdued temperatures for long-term culturing of most microalgal taxa. Over-illumination of the stored microalgal cultures should be avoided at all costs to prevent from photo-oxidative stress in microalgae and also the problem of localised heating. Light and dark photoperiods are required for the maintenance of most cultures with 12:12 and 16:8 h light: dark regimens recommended for a wide range of microalgal strains (Lorenz et al., 2005). For long term storage, microalgal specimens should be kept at low temperatures of 2 C but however most microalgal strains can be kept at 1520 C. The stored microalgal cultures should be routinely sub cultured to get metabolically active inocula and this should be done at least 13 weeks employing conventional aseptic

microbiological techniques to avoid contaminating the puried cultures. Generally it is difcult to establish storage conditions for newly isolated microalgal strains and as a rule of thumb, it is recommended to do trial and error of different light intensities and temperatures (Lorenz et al., 2005). The viability of these cultures is then tested periodically and a suitable maintenance regime is established for future application. It is prudent to appropriately label the stored cultures using waterproof labels and permanent ink and also to routinely check for contamination using light microscopy. Media for each strain must be carefully chosen so that the microalgal cells are not stressed to an extent of altering their morphology and development of deleterious effects. A wide range of media are available such as BBM, BG-11, ASW and so on for long term maintenance of microalgae. The different media that can be used for long term maintenance of microalgae are described is Section 4 and Table 4. These media types can support the growth of microalgae from specic aquatic environments such as fresh water, marine and brackish. The media chosen can either be in liquid or solid agar medium. However slants can prolong the viability of microalgal cells in storage. Microalgae can be lyophilised and stored as a powder at 2 C. In addition, cryopreservation of microalgal cells has been found to be effective as a culture maintenance strategy (Day and Brand, 2005). The ability to routinely cryopreserve microalgal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift (Rhodes et al., 2006).Currently microalgae from diverse aquatic environments are maintained by serially subculturing which is labor intensive and therefore repeatedly exposes the culture to contamination and handling error (Cox et al., 2009). To date, standard cryogenic techniques are documented as best methods for long term microalgal storage (Cox et al., 2009). 9. Biofuel production 9.1. Exploring possibility of algae based biofuels The algae oil and spent biomass are considered as potential sources for biofuels production. Microalgal biomass can be produced using either open raceway ponds or photobioreactors. The pros and cons of these two systems are described in Table 7. The biodiesel produced from algae oil by using transesterication process can be used for replacing the conventional diesel fuel. The algae oil cake produced in algae biofuel industry is ca. of 12 times of lipid yield. For example, 1000 kg of algae biomass having 35% oil content can yield 650 kg of algae deoiled cake. Biomass Energy Conversion Technologies (BECT) can be used to convert the algae biomass into different kinds of energy fuels. BECT is divided into thermo-chemical and biochemical methods. The BECT includes combustion (for heat energy), pyrolysis (pyrolytic gas, bio-oil, biochar), gasication (syngas), thermo-chemical liquefaction (biocrude oil), biomethanation (biogas), photobiological hydrogen production (hydrogen) and alcoholic fermentation (ethanol). Algae oil can be converted into biodiesel by the transesterication process as depicted in Fig. 3a and b. The recent developments in research on algae biofuels production has been extensively investigated (Harun et al., 2010). 9.2. Biodiesel production The viscosity of the raw microalgal oil is usually higher than that of diesel fuel. Raw oil can be converted into biodiesel by means of transesterication process. The transesterication reaction can be catalyzed by alkalis, acids, enzymes or supercritical methanol. Free fatty acid (FFA) content of algae is in the range of

T. Mutanda et al. / Bioresource Technology 102 (2011) 5770 Table 7 Generalized comparisons of two different cultivation methods of algae production. Factors Cultivation Cultivation Contamination Cleaning Controlling of growth conditions Temperature Automatic cooling system Automatic heating system Microbiology safety Biomass production Biomass quality Biomass productivity Lipid productivity Light utilization efciency Operational mode Air pump Shear CO2 transfer rate Mixing efciency Water loss Evaporation O2 concentration Open ponds Multi strain cultivation High None Very difcult Highly variable None None None Variable Low Low Low Photobioreactors Well suitable for single strain cultivation Less to none Required due to wall growth and dirt Easy Required cooling Built in Built in

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(a)
Algae cultivation

Harvesting of algae

Drying of algae

Dried algae

Oil extraction
UV Reproducible High High High

Deoiled cake

Algae oil

Transesterification reactor

CO2 loss Economics Space required Periodical maintenance Capital investment Operating cost Harvesting cost Scale up technology for commercial level

Built in Low Poor Poor Very high High Low due to continuous spontaneous out gassing High, depending on pond depth High Less Low Lower High Easy to scale up

Built in High Excellent Excellent Low No evaporation Build-up occurred requires gas exchange device Low

Algae biodiesel

Glycerol

(b)

Low More High Higher Lower Most of photobioreactor models are difcult to scale up due to limitations

Fig. 3. Schematic representation of biodiesel production from microalgae oil (a) and Biodiesel production by transesterication reaction (b).

Modied from Pulz (2001) and Harun et al. (2010).

2050% (Kosaric and Velikonja, 1995; Mansour et al., 2005), which is the main factor for formation of soap in alkaline catalyst based transestercation process and separation of biodiesel and glycerol is difcult. Due to these problems, the alkaline catalyst based biodiesel production is not suitable for algae oil with high FFA content. For transesterication of high free fatty acid feedstocks, acid catalysts were found to be useful for pre-treating, but its reaction conversion rates are very slow (Um and Kim, 2009). Enzymes are also used for feedstock with higher free fatty acid, but these catalysts are expensive and unable to provide the degree of reaction completion required to meet the American Society for Testing and Materials (ASTM) fuel specication. The use of supercritical transesterication process method for algae biodiesel production is also restricted due to process economics and safety concerns related to the reaction conditions (Ehimen et al., 2010). Ehimen and co-workers (2010) studied the effect of operational parameters such as alcohol volume, moisture content, temperature, reaction time, and mixing on an acid-catalyzed in situ transesterication process for production of biodiesel from microalgae lipids. They found that water content in the algae biomass was greater than 115% w/w (based on oil weight) and this inhibited the in situ transesterication reaction. The Mcgyan process is continuous transesterication process for biodiesel production from various feedstocks. In this process, the combination of alcohol and lipid

is sent into a continuous xed-bed reactor lled with a sulfated metal oxide catalyst at elevated temperature and pressure to perform the transesterication and esterication reactions simultaneously. Added advantages of this technology are: no catalyst is used, less reaction time and uses no water or dangerous chemicals. (Um and Kim, 2009). Adapting a biorenery approach i.e. utilising the oil for biodiesel, spent biomass, glycerol, etc. as value added products has certainly made the technology attractive and therefore attracted much attention in the eld. 9.3. Thermo-chemical and biochemical conversion Thermo-chemical conversion process involves thermal decomposition of organic components present in algae biomass into different kind of energy fuels. The end products may vary from gas or liquid or solid fuel depending on temperature used in the thermo-chemical conversion process. The thermal decomposition of organic components can be achieved by different processes such as direct combustion, gasication, thermo-chemical liquefaction, and pyrolysis. Gasication is the conversion of biomass into a combustible gas mixture by the partial oxidation of biomass at high temperatures, typically in the range 800900 C (McKendry, 2002) and the produced syngas can be used for thermal applications or fuel for diesel/gas turbine engines. Hirano et al. (1998) studied gasication of Spirulina at temperatures ranging from 850 to 1000oC for methanol production. They estimated that algae biomass gasication at 1000oC produced the highest theoretical yield of 0.64 g methanol from 1 g of algae biomass.

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T. Mutanda et al. / Bioresource Technology 102 (2011) 5770 Akoto, L., Pel, R., Irth, H., Brinkman, U.A.T., Vreuls, R.J.J., 2005. Automated GCMS analysis of raw biological samples. Application to fatty acid proling of aquatic micro-organisms. J. Anal. Appl. Pyrol. 73, 6975. Anandraj, A., Perissinotto, R., Nozais, C., 2007. A comparative study of microalgal production in a marine versus a river-dominated temporarily open/closed estuary, South Africa. Estuar. Coast. Shelf. Sci. 73, 768780. Anandraj, A., Perissinotto, R., Nozais, C., Stretch, D., 2008. The recovery of microalgal production and biomass in a South African temporarily open/closed estuary, following mouth reaching. Estuar. Coast. Shelf. Sci. 79, 599606. Andersen, R., 2005. Algal Culturing Techniques. Elsevier Academic Press, Burlington. Auinger, B.M., Pfandl, K., Boenigk, J., 2008. Improved methodology for identication of protists and microalgae from plankton samples Preserved in Lugols iodine solution: combining microscopic analysis with single-cell PCR. Appl. Environ. Microb. 74, 25052510. Balat, M., Balat, M., Kirtay, E., Balat, H., 2009. Main routes for the thermo-conversion of biomass into fuels and chemicals. Part 1: pyrolysis systems. Energ. Convers. Manag. 50, 31473157. Barsanti, L., Gualtieri, P., 2006. Algae Anatomy, Biochemistry, and Biotechnology. CRC Press, Taylor and Francis Group, Boca Raton, Florida. pp. 215235. Berges, J.A., Franklin, D.J., Harrison, P.J., 2001. Evolution of an articial seawater medium: Improvements in enriched seawater, articial water over the past two decades. J. Phycol. 37, 11381145. Bernal, C.B., Vzquez, G., Quintal, I.B., Bussy, A.L., 2008. Microalgal dynamics in batch reactors for municipal wastewater treatment containing dairy sewage water. Water Air Soil Poll. 190, 259270. Bertozzini, E., Penna, A., Pierboni, E., Bruce, I., Magnani, M., 2005. Development of new procedures for the isolation of phytoplankton DNA from xed samples. J. Appl. Phycol. 17, 223229. Blackburn, S.I., Bolch, C.J.S., Haskard, K.A., Hallegraeff, G.M., 2001. Reproductive compatibility among four global populations of the toxic dinoagellate Gymnodinium catenatum (Dinophyceae). Phycologia 40 (1), 7887. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purication. Can. J. Biochem. Phys. 37, 911917. Borowitzka, M.A., 1997. Microalgae for aquaculture: opportunities and constraints. J. Appl. Phycol. 9, 393401. Brahamsha, B., 1996. A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus. Appl. Environ. Microb. 62, 17471751. Brennan, L., Owende, P., 2010. Biofuels from microalgae a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14, 557577. Bridgwater, A.V., 2003. Renewable fuels and chemicals by thermal processing of biomass. Chem. Eng. J. 91, 87102. Canakci, M., Sanli, H., 2008. Biodiesel production from various feedstocks and their effects on the fuel properties. J. Ind. Microbiol. Biotechnol. 35, 431441. Celekli, A., Yavuzatmaca, M., 2009. Predictive modelling of biomass production by Spirulina platensis as function of nitrate and NaCl concentrations. Bioresour. Technol. 100, 18471851. Chang, W.C., Lee, L.P., Liepmann, D., 2005. Biomimetic technique for adhesion-based collection and separation of cells in a microuidic channel. Lab Chip 5, 6473. Chen, W., Zhang, C., Song, L., Sommereld, M., Hu, Q., 2009a. A high throughput Nile Red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Meth. 77, 4147. Chen, W., Zhang, Q., Dai, S., 2009b. Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa. J. Appl. Phycol. 21, 701706. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294306. Clark, J., Deswarte, F., 2008. Introduction to chemicals from biomass, Wiley Series in Renewable Resources, ISBN978-0-470-05805. Cohn, S.A., Farrell, J.F., Munro, J.D., Ragland, R.L., Weitzell Jr., R.E., Wibisono, B.L., 2003. The effect of temperature and mixed species composition on diatom motility and adhesion. Diatom Res. 18, 225243. Coleman, A.W., 2003. ITS2 is a double edged tool for eukaryote evolutionary comparisons. Trends Genet. 19, 370375. Coleman, A.W., 2007. Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucl. Acids Res. 35, 33223329. Cooksey, K.E., Guckert, J.B., Williams, S.A., Callis, P.R., 1987. Fluorometricdetermination of the neutral lipid-content of microalgal cells using Nile red. J. Microbiol. Meth. 6, 333345. Cooper, M.S., Hardin, W.R., Petersen, T.W., Cattolico, R.N., 2010. Visualizing green oil in live algal cells. J. Biosci. Bioeng. 109, 198201. doi:10.1016/ j.jbiosc.2009.08.004. Cox, S.L., Hulston, D., Mass, E.W., 2009. Cryopreservation of marine thraustochytrids (Labyrinthulomycetes). Cryobiology 59, 363365. Day, J.G., Brand, J.J., 2005. Cryopreservation methods for maintaining microalgal cultures. In: Andersen, R.A. (Ed.), Algal Culturing Techniques. Elsevier Academic Press, UK. De Morais, M.G., Costa, J.A.V., 2007. Carbon dioxide xation by Chlorella kessleri, C. Vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in asks and vertical tubular photobioreactors. Biotechnol. Lett. 29, 13491352. Dean, A.P., Sigee, D.C., Estrada, B., Pittmann, J.K., 2010. Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae. Bioresour. Technol. 101, 44994507. Doh, I., Cho, Y.H., 2005. A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process. Sensor Actuat. A-Phys. 12, 5965. Dote, Y., Sawayama, S., Inoue, S., Minowa, T., Yokoyama, S.Y., 1994. Recovery of liquid fuel from hydrocarbon rich microalgae by thermochemical liquefaction. Fuel 73, 18551857.

Thermo-chemical liquefaction is a process that can be employed to convert wet algal biomass material into liquid fuel. Added advantages of this technology are conversion of wet biomass into useful energy and no feedstock drying process involved (Clark and Deswarte, 2008). Dote et al.(1994) found that thermochemical liquefaction of microalgae species like Botryococcus braunii, Dunaliella tertiolecta and Spirulina platensis yielded 64, 42 and 3048% dry wt. basis of oil and fuel properties of biocrude oil (3045.9 MJ/kg) which was close to that of petroleum based heavy oil (42 MJ/kg) (Jena and Das, 2009). Pyrolysis is the thermal decomposition of materials in the absence of oxygen or when signicantly less oxygen is present than required for complete combustion (Balat et al., 2009). Slow pyrolysis of biomass is associated with high charcoal content (350 700 C), but the fast pyrolysis is associated with liquid fuels, at low temperature (675775 K) (Bridgwater, 2003), and/or gas, at high temperature (Encinar et al., 1998). Fast pyrolysis process for conversion of algae biomass is used to produce 6075 wt.% of liquid bio-oil, 1525 wt.% of solid char, and 1020 wt.% of non condensable gases, depending on the feedstock used (Mohan et al., 2006). Miao et al. (2004) studied fast pyrolysis of Chlorella protothecoides and Microcystis aeruginosa grown phototrophically. They reported that bio-oil yields of 18% (higher heating value [HHV] of 30 MJ kg1) and 24% (HHV of 29 MJ kg1) for microalgae C. prothothecoides and Microcystis aeruginosa, respectively. Researchers have also reported using anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable (Sialve et al., 2009). The chemical composition of microalgae shows variation according to species, seasons and habitats (Ruprez, 2002). Due to high protein content of microalgae, increased ammonia is produced upon microbial protein degradation, which inhibit anaerobic microorganisms in anaerobic digestion. The overall performance of anaerobic digestion system is affected due to low C:N ratio of the microalgae, but it can be improved by co-digestion with a high C:N ratio material (Brennan and Owende, 2010). Physico-chemical pre-treatment and co-digestion are strategies that can signicantly and efciently increase the conversion yield of the algal organic matter into methane. 10. Conclusions Due to the increasing global appetite for renewable liquid fuels, the need for developing suitable technology to meet this demand is imperative. Comparative analysis of producing oil from algae and crop based biomass show the former to be more advantageous on various aspects. However, conducting an extensive search of environments for unique hyper-lipid producers is crucial to the success of the technology. The application of suitable and efcient technology for sampling, isolation and screening and culture maintenance is important. Identication of selected strains using novel molecular techniques is more accurate than current conventional methods. Producing the right quality and quantity of oil and FAME product will largely determine the techno-economics of the technology. In addition thermo-chemical and biochemical conversion process can improve the economics. Most of the literature to date has largely focused on downstream processing of oil to biodiesel. As shown in the current communication, the upstream processing of biomass to maximize the downstream yields is crucial for the overall success of the technology. References
Abu, G.O., Ogbonda, K.H., Aminigo, R.E., 2007. Optimization studies of biomass production and protein biosynthesis in a Spirulina sp. Isolated from an oil polluted ame pit in the Niger delta. Afr. J. Biotechnol. 6, 25502554.

T. Mutanda et al. / Bioresource Technology 102 (2011) 5770 Ehimen, E.A., Sun, Z.F., Carrington, C.G., 2010. Variables affecting the in situ transesterication of microalgae lipids. Fuel 89, 677684. Elsey, D., Jameson, D., Raleigh, B., Cooney, M.J., 2007. Fluorescent measurement of microalgal neutral lipids. J. Microbiol. Meth. 68, 639642. Encinar, J.M., Beltran, F.J., Ramiro, A., Gonzalez, J.F., 1998. Pyrolysis/gasication of agricultural residues by carbon dioxide in the presence of different additives: inuence of variables. Fuel Process. Technol. 55, 219233. Foglia, T.A., Nelson, L.A., Dunn, R.O., Marmer, W.N., 1997. Low temperature properties of alkyl esters of tallow and grease. J. Am. Oil Chem. Soc. 74, 951 955. Genicot, G., Leroy, J.L.M.R., Van Soom, A., 2005. The use of a uorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes. Theriogenology 63, 11811194. Gescher, C., Metes, K., Medlin, L.K., 2008. The ALEX CHIP development of a DNA chip for identication and monitoring of Alexandrium. Harmful Algae 7, 485 494. Godhe, A., Anderson, D.M., Rehnstam-Holm, A.S., 2002. PCR amplication of microalgal DNA for sequencing and species identication: studies on xatives and algal growth stages. Harmful Algae 27, 18. Greenspan, P., Fowler, S.D., 1985. Spectrouorometric studies of the lipid probe, Nile Red. J. Lipid Res. 26, 781788. Greenwell, H.C., Laurens, M.L., Shields, R.J., Lovitt, R.W., Flynn, K.J., 2009. Placing microalgae on the biofuels priority list: a review of the technological challenges. J. Roy. Soc. Interface. doi: 10.1098/rsif.2009.0322. Grifths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 276, 2325. Grima, E.M., Belarbi, E.H., Acin Fernndez, F.G., Medina, A.R., Chisti, Y., 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 20, 491515. Guillard, R.R.L., Hargraves, P.E., 1993. Stichochrysisimmobilis is a diatom, not a chrysophyte. Phycologia 32, 234236. Han, K.H., Frazier, A.B., 2005. A microuidic system for continuous magnetophoretic separation of suspended cells using their native magnetic properties. Proc. Nanotech. 1, 187190. Harun, R., Singh, M., Forde, G.M., Danquah, M.K., 2010. Bioprocess engineering of microalgae to produce a variety of consumer products. Renew. Sust. Energ. Rev. 14, 10371047. Heasman, M., Diemar, J., OConnor, W., Sushames, T., Foulkes, L., 2000. Development of extended shelf-life microalgae concentrate diets harvested by centrifugation for bivalve molluscs a summary. Aquac. Res. 31, 637659. Henrichs, D.W., Renshaw, M.A., Santamaria, C.A., Richardson, B., Gold, J.R., Campbell, L., 2007. PCR Amplication of Microsatellites from Single Cells of Karenia brevis Preserved in Lugols Iodine Solution. Mar. Biotechnol. 10, 122127. Hirano, A.K., Hon-Nami, K., Kunito, S., Hada, M., Ogushi, Y., 1998. Temperature effect on continuous gasication of microalgal biomass: theoretical yield of methanol production and its energy balance. Catal. Today 45, 399404. Hsieh, C., Wu, W., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100, 39213926. Huang, G., Chen, F., Wei, D., Zhang, X., Chen, G., 2010. Biodiesel production by microalgal biotechnology. Appl. Energ. 87, 3846. Jeffrey, S.W., LeRoi, J.M., 1997. Simple procedures for growing SCOR reference microalgal cultures. In: Jeffrey, S.W., Mantoura, R.F.C., Wright, S.W. (Eds.), Phytoplankton Pigments in Oceanography; Monographs on Oceanographic Methodology 10. UNESCO, France, pp. 181205. Jena, U., Das, K.C. 2009. Production of biocrude oil from microalgae via thermochemical liquefaction process. Bioenergy Engineering, Bellevue, Washington, DC, BIO-098024. American Society of Agricultural and Biological Engineers, St. Joseph, Michigan, 1114 October 2009. Johnson, M.B., Wen, Z., 2009. Production of biodiesel fuel from microalga Schizochytrium limacinum by direct transesterication of algal biomass. Energy Fuels 23, 51795183. Jrgen, P.E., Lena Struwe, W.I., Jin, E., 2008. Identication and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea. J. Microbiol. Biotechnol. 18, 821827. Kacka, A., Donmez, G., 2008. Isolation of Dunaliella spp. from a hypersaline lake and their ability to accumulate glycerol. Bioresour. Technol. 99, 83488352. Kalayasiri, P., Jayashke, N., Krisnangkura, K., 1996. Survey of seed oils for use as diesel fuels. J. Am. Oil Chem. Soc. 73, 471474. Kamikawa, R., Masuda, I., Oyama, K., Yoshimatsu, S., Sako, Y., 2007. Genetic variation in mitochondrial genes and intergenic spacer region in harmful algae Chattonella species. Fisheries Sci. 73, 871880. Khan, S.A., Rashmi, Hussain, M.Z., Prasad, S., Banerjee, U.C., 2009. Prospects of biodiesel production from microalgae in India. Renew. Sust. Energ. Rev. 13, 23612372. Kilham, S.S., Kreeger, D.A., Lynn, S.G., Goulden, C.E., Herrera, L., 1998. COMBO: a dened freshwater culture medium for algae and zooplankton. Hydrobiologia 377, 147159. Knothe, G., 2005. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process. Technol. 86, 10591070. Knuckey, R.M., Brown, M.R., Barrett, S.M., Hallegraeff, G.M., 2002. Isolation of new nanoplanktonic diatom strains and their evaluation as diets for juvenile Pacic oysters (Crassostrea gigas). Aquaculture 211, 253274. Kosaric, N., Velikonja, J., 1995. Liquid and gaseous fuels from biotechnology: challenge and opportunities. FEMS Microbiol. Rev. 16, 111142. Krisnangkura, K., 1986. A simple method for estimation of cetane index of vegetable oil methyl esters. J. Am. Oil Chem. Soc. 63, 552553.

69

Laughton, C., 1986. Measurement of the specic lipid content of attached cells in microtitre cultures. Anal. Biochem. 156, 307314. Lee, S.J., Yoon, B.D., Oh, H.M., 1998. Rapid method for the determination of lipid from the green alga Botryococcus braunii. Biotechnol. Tech. 12, 553556. Lee, J. Yoo, C., Jun, S., Ahn, C., Hee-Mock O., 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol 101(1) Supplement 1:S75-S77. doi:10.1016/j.biortech.2009.03.058. Letellier, M., Budzinski, H., 1999. Microwave assisted extraction of organic compounds. Analysis 27, 259. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81, 629636. Lorenz, M., Friedl, T., Day, J.G., 2005. Perpetual maintenance of actively metabolizing microalgal cultures. In: Andersen, R.A. (Ed.), Algal Culturing Techniques. Elsevier Academic Press, UK. MacLulich, J.H., 1986. Experimental evaluation of methods for sampling and assaying intertidal epilithic microalgae. Marine Ecol. Prog. Ser. 34, 275 280. Mansour, M.P., Frampton, D.M.F., Nichols, P.D., Volkman, J.K., Blackburn, S.I., 2005. Lipid and fatty acid yield of nine stationary-phase microalgae: applications and unusual C24-C28 polyunsaturated fatty acids. J. Appl. Phycol. 17, 287300. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energ. Rev. 14, 217232. McKendry, P., 2002. Energy production from biomass (part 2): conversion technologies. Bioresour. Technol. 83, 4754. Meher, L.C., Vidya, S.D., Naik, S.N., 2006. Technical aspects of biodiesel production by transesterication a review. Renew. Sust. Energ. Rev. 10, 248268. Melkonian, M. 1990. Phylum chlorophyta: class chlorophyceae. In: Margulis, L., Corliss, J.O., Melkonian, M. Chapman, D.J., (Eds), Handbook of Protoctista, 608016. Jones and Bartlett Publishers, Boston. Miao, X.L., Wu, Q.Y., Yang, C.Y., 2004. Fast pyrolysis of microalgae to produce renewable fuels. J. Anal. Appl. Pyrolysis 71, 855863. Mittelbach, M., 1996. Diesel fuel derived from vegetable oils, VI: specications and quality control of biodiesel. Bioresour. Technol. 56, 711. Mittelbach, M., Remschmidt, C., 2004. Biodiesel: The Comprehensive Handbook. Boersedruck Ges. M.B.H, Vienna. Mohan, D., Pittman, C.U., Steele, P.H., 2006. Pyrolysis of wood/biomass for bio-oil: a critical review. Energy Fuel 20, 848889. Moreno-Garrido, I., 2008. Microalgae immobilization: current techniques and uses. Bioresour. Technol. 99, 39493964. Moro, C.V., Crouzet, O., Rasconi, S., Thouvenot, A., Coffe, G., Batisson, I., Bohatier, J., 2009. New design strategy for development of specic primer sets for PCRbased detection of Chlorophyceae and Bacillariophyceae in environmental samples. Appl. Environ. Microbiol. 17, 57295733. Nol, M.H., Kawachi, M., Inouye, I., 2004. Induced dimorphic life cycle of a coccolithophorid, Calyptrosphaera sphaeroidea (Prymnesiophyceae, Haptophyta). J. Phycol. 40, 112129. Novis, P.M., Halle, C., Wilson, B., Tremblay, L.A., 2009. Identication and characterization of freshwater algae from a pollution gradient using rbcL sequencing and toxicity testing. Arch. Environ. Con. Toxicol. 57, 504514. Olaizola, M., 2003. Commercial development of microalgal biotechnology: from the test tube to the market place. Biomol. Eng. 20, 459466. Papanikolaou, S., Komaitis, M., Aggelis, G., 2004. Single Cell Oil (SCO) production by Mortierella isabellina grown on high-sugar content media. Bioresour. Technol. 95, 287291. Petersson, F., Nilsson, A., Holm, C., Jnsson, H., Laurell, T., 2004. Separation of lipids from blood utilizing ultrasonic standing waves in microuidic channels. Analyst 129, 938943. Pulz, O., 2001. Photobioreactors: production systems for phototrophic microorganisms. Appl. Microbiol. Biotehnol. 57, 287293. Ramanan, R., Kanan, K., Deshkar, A., Yadav, R., Chakrabarti, T., 2010. Enhanced algal CO2 sequestration through calcite deposition by Chlorella sp. and Spirulina platensis in a mini-raceway pond. Bioresour. Technol. 101, 26162622. Ramos, M.J., Fernndez, C.M., Casas, A., Rodrguez, L., Prez, A., 2009. Inuence of fatty acid composition of raw materials on biodiesel properties. Bioresour. Technol. 100, 261268. Rasoul-Amini, S., Ghasemi, Y., Morowvat, M.H., Mohagheghzadeh, A., 2009. PCR amplication of 18S rRNA, single cell protein production and fatty acid evaluation of some naturally isolated microalgae. Food Chem. 116, 129 136. Rhodes, L., Smith, J., Tervit, R., Roberts, R., Adamson, J., Adams, S., Decker, M., 2006. Cryopreservation of economically valuable marine micro-alga in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology 52, 152156. Richlen, M.L., Morton, S.L., Barber, P.H., Lobel, P.S., 2007. Phylogeography, morphological variation and taxonomy of the toxic dinoagellate Gambierdiscus toxicus (Dinophyceae). Harmful Algae 7, 614629. Rodol, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100112. Rogerson, A., DeFreitas, A.S.W., McInnes, A.C., 1986. Observations on wall morphogenesis in Coscinodiscus asteromphalus (Bacillariophyceae). Trans. Am. Microsci. Soc. 105, 5967. Ruprez, P., 2002. Mineral content of edible marine seaweeds. Food Chem. 79, 23 26.

70

T. Mutanda et al. / Bioresource Technology 102 (2011) 5770 Steup, M., Melknonian, M., 1981. C-l,4-Glucan phosphorylase forms in the green alga Eremosphaera viridis. Physiol. Plant 51, 343348. Stournas, S., Lois, E., Serdari, A., 1995. Effects of fatty acid derivatives on the ignition quality and cold ow of diesel fuel. J. Am. Oil Chem. Soc. 72, 433437. Takahashi, K., Hattori, A., Suzuki, I., Ichiki, T., Yasuda, K., 2004. Non-destructive onchip cell sorting system with real-time microscopic image processing. J. Nanobiotechnol. 2, 5. Tinti, F., Boni, L., Pistocchi, R., Riccardi, M., Guerrini, F., 2007. Species-specic probe, based on 18S rDNA sequence, could be used for identication of the mucilage producer microalga Gonyaulax fragilis (Dinophyta). Hydrobiologia 580, 259263. Um, B.H., Kim, Y.S., 2009. Review: a chance for Korea to advance algal-biodiesel technology. J. Ind. Eng. Chem. 15, 17. Watanabe, M.M., Kawachi, M., Hiroki, M., Kasai, F., 2000. NIES collection list of strains. sixth ed. In: Microalgae and Protozoa. Microbial Culture Collections, National Institute for Environmental Studies, Tsukuba, Japan, p. 159. Widjaja, A., Chien, C., Ju, Y., 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan (Chin) Inst. Chem. Eng. 40, 1320. Woelfel, J., Schumann, R., Adler, S., Hubener, T., Karsten, U., 2007. Diatoms inhabiting a wind at of the Baltic Sea: species diversity and seasonal variations. Estuar. Coast. Shelf. Sci. 75, 296307. Xiaoling, M., Qingyu, W., 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97, 841846. Yamada, M., Kasim, V., Nakashima, M., Edahiro, J., Seki, M., 2004. Continuous cell partitioning using an aqueous two-phase ow system in microuidic devices. Biotechnol. Bioeng. 88, 489494. Zittelli, G.C., Rodolfo, L., Biondi, N., Tredici, M.R., 2006. Productivity and photosynthetic efciency of outdoor cultures of Tetraselmis suecica in annular columns. Aquaculture 261, 932943.

Scholin, C.A., Anderson, D.M., 1996. LSU rDNA-based RFLP assays for discriminating species and strains of Alexandrium (Dinophyceae). J. Phycol. 32, 1022 1035. Schreiber, U., Bilger, W., Neubauer, C., 1994. Chlorophyll uorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Schulze, E.D., Caldwell, M.M. (Eds.), Ecophysiology of Photosynthesis. Springer, Berlin, pp. 4970. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., Smith, A., 2010. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 21, 110. doi:10.1016/j.copbio.2010.03.005. Sensen, C.W., Kirsten, H., Melkonian, M., 1993. The production of clonal and axenic cultures of microalgae using uorescence-activated cell sorting. Eur. J. Phycol. 28, 9397. Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998. A look back at the US Department of Energys aquatic species program Biodiesel from Algae. Report NREL/TP-58024190. National Renewable Energy Laboratory, Golden, CO. Shevkoplyas, S.S., Yoshida, T., Munn, L.L., Bitensky, M.W., 2005. Biomimetic autoseparation of leukocytes from whole blood in a microuidic device. Anal. Chem. 77, 933937. Sialve, B., Bernet, N., Bernard, O., 2009. Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol. Adv. 27, 409416. Sinigalliano, C.D., Winshell, J., Guerrero, M.A., Scorzetti, G., Fell, J.W., Eaton, R.W., Brand, L., Rein, K.S., 2009. Viable cell sorting of dinoagellates by multiparametric ow cytometry. Phycologia 48, 249257. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 8796.