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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,*
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
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 20–50% 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, puriﬁcation and identiﬁcation 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. Identiﬁcation 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 identiﬁcation, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail. Ó 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 identiﬁcation
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 transesteriﬁcation. 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, sunﬂower, 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: email@example.com (F. Bux). 0960-8524/$ - see front matter Ó 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 ﬁnancial 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 ﬂue gas as carbon source and moreover it can be harvested daily (Chisti, 2007;
3. the main focus of research in the ﬁeld of biofuels from microalgae has been centred on downstream aspects such as bioreactor designs.. noncohesive. 2006) (Table 1). collect and identify hyper-lipid producing strains. The collection method adopted is crucial for success. There is potential for further exploitation of these organisms for production of value added products and biofuels.. According to Borowitzka (1997). most of them being microalgae. 2010). fresh and brackish water Fresh water Different habitats Fresh water .) 100. 2009. Euglenophyta. nutritional supplements Infant formulas. These nutritional and environmental requirements are variable under natural conditions. nutritional supplements. type of aquatic system and sampling equipment. Phaeodactylum Nitzschia. Low-cost harvesting requires large cell size. dams. Dinophyta. Rodolﬁ et al. Rhodophyta. 2010). 2009. because damaged or dead cells may lead to failure. For successful biofuel production using microalgae as feedstock for biomass and lipid accumulation. 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. Microalgae Microalgae are unicellular microscopic (2–200 lm). However according to Khan et al. Sampling Microalgal collection is mainly inﬂuenced by environmental factors (both biotic and abiotic).1. CO2. 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. artiﬁcial assemblage of CO2 evolving. Therefore. Cryptophyta. 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. brackish. the former require only inorganic compounds such as CO2. suitable salinity. it is also important to evaluate harvesting costs at the time of choosing the species. Haptophyta. vascular system.. This manuscript investigates current developments in microalgae bioprospecting. polyphyletic. Sampling environments The mega biodiversity of microalgae entails them as suitable candidates for biofuel production. 2009). (2009). Algae are also deﬁned as thallophytes (plants lacking roots. 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. 3. chizochytrium Nannochloropsis. biomass and lipid production from microalgae. 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.. nutritional supplements Microalgal producer Crypthecodinium.58 T. Selection of fast-growing. Brennan and Owende. blue–green algae and golden algae (Table 2). hyper saline. Due to selection pressure and changing environmental conditions. while the latter are nonphotosynthetic therefore require an external source of organic compounds as well as nutrients as an energy source (Brennan and Owende. rivers.. 2006). 2008). Spolaore et al.. aquaculture Nutritional supplements. Chlorarachniophyta and Chlorophyta). Microalgae are found in diverse environmental conditions and habitats such as lacustrine. vitamins and trace elements for their growth (Chisti. green algae. high speciﬁc gravity compared to the medium and reliable autoﬂocculation. 2. biomass harvesting techniques and the chemistry of biofuel production. Protocols and procedures employed for successful microalgal bioprospecting are presented and described in-depth. autotrophic organisms which grow by photosynthesis and are the eukaryotic representatives although the prokaryotic cyanobacteria are frequently included with the algae (Greenwell et al. Mutanda et al.. The main criteria for categorising microalgae are pigmentation. 2003. the crucial step is to search. parameters measured onsite. embryos. 2010). 2010). 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. Bernal et al. Pavlova Spirulina Porphyridium Greenwell et al. the most important groups of algae in terms of abundance are: diatoms. 2008. 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. Mata et al. 2009. To date. macronutrients (nitrates and phosphates). the main objective of this review paper is to report current strategies focusing on bioprospecting for microalgae with the main aim of producing biofuels. Table 3 depicts lipid content of some microalgal strains collected from different aquatic environments. Grifﬁths and Harrison. appropriate pH.000 8000 2000 1000 Storage material Chyrsolaminarin (polymer of carbohydrates) and TAGs Starch and TAGs Starch and TAGs TAGs and carbohydrates Habitat Oceans. life cycle and basic cellular structure (Brennan and Owende. Microalgae require light. freshwater. Algae can either be autotrophic or heterotrophic. Heterokontophyta. Microalgae are classiﬁed into two prokaryotic divisions (Cyanophyta and Prochlorophyta) and nine eukaryotic divisions (Glaucophyta. / Bioresource Technology 102 (2011) 57–70 Table 1 Potential of microalgae as primary PUFA resources (Spolaore et al. Microalgal sampling. Algae Diatoms (Bacillariophycea) Green algae (Chlorophyceae) Blue–green algae (Cyanophyceae Golden algae (Chrysophyceae) Known species (approx. PUFA Docosahexaenoic acid (DHA) Eicosapentaenoic acid (EPA) c-Linolenic acid (GLA) Arachidonic acid (AA) Potential application Infant formulas. salts and a light energy source for growth. wastewater maturation ponds. The schematic outline of the procedures involved in upstream to downstream processing of microalgal lipids is presented in Fig. 2010).. The evolutionary history and taxonomy of microalgae is complex due to constant revisions as a result of new genetic and ultrastructural evidence. 2007. 2009). It is estimated that there are between one and ten million algal species. storage conditions and isolation and strain selection procedures are also discussed in detail. 1. aquaculture Infant formulas.. productive strains. marine and coastal areas.
In addition.5 depending on the prevailing edaphic conditions and agricultural practices in the area. Steps involved and outcome of bioprospecting of microalgae for biofuels production. Different types of microalgal strains require different habitats.. In tropical and subtropical countries. 2009). microalgae have been shown to have successional tendencies due to variable nutrient availability. The major macronutrients required for microalgal growth are phosphates. 1. 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. Lower winter temperatures promote the growth of benthic microalgal strains such as Senedesmus sp. The microalgae are found as a mixed consortium and the population dynamics of the microalgae in any habitat is complex (Bernal et al. (2008). 2009). Hsieh and Wu. Mutanda et al. 2008).. ments (Rodolﬁ et al. / Bioresource Technology 102 (2011) 57–70 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. lakes and rivers have seasonal variations in ambient water temperature ranging from 10 to 30 °C. 2009.. The ecological and physico-chemical parameters in this habi- . Chen et al. inclement weather and seasonal variations (Bernal et al. whereas summer temperatures enhance the growth of the Chlorophytes such as Chlorella strains. In any habitat. microalgal biomass has shown clear temporal and spatial patterns during the heterogeneous conditions of the open and closed phases in estuaries. 2008). The average pH in these habitats also varies from 3 to 8.T. Brackish aquatic systems constitute a mixture of seawater and fresh water and this usually occurs at the river mouth on the coastline.. the nutritional composition of the aquatic system is a major factor for microalgae to thrive in these habitats. According to Anandraj et al. nitrates and ammonium and these are required in suitable concentrations to promote lipid synthesis by the microalgae (Celekli and Yavuzatmaca. inland dams. hence the diversity of environmental conditions where microalgae can be collected. 2009.
2005). Oil content (% dry weight) 25–75 16. multi probe system (measuring pH.7 lm mesh). scooping jar. There is no deﬁnite sampling procedure documented in literature though researchers can follow simple and cheap methods of collecting microalgal samples. 2010). DM Chlorella sp.. Technically it is impractical to measure biotic factors on site such as pathogens (bacteria.3.3 19. vials for collecting samples. 1986). salinity meter. 2007). turbidity. dissolved CO2. Neochloris oleoabundans Crypthecodinium cohnii Tetraselmis sueica Monallanthus salina Dunaliella primolecta Phaeodactylum tricornutum Isochrysis sp. Modiﬁed from Chisti (2007). light meter.8 45–47 31–68 50–77 T. dissolved CO2 and O2 analyser with a data logger. Isolation and puriﬁcation techniques 4.60 Table 3 Habitats and oil content of some microalgae. 1998).1 19. bacterial contamination in microalgal puriﬁcation can be prevented by employing a procedure involving treatment with a detergent and phenol (Abu et al. dilution rates. This will allow for collection of microalgae which prefer different light intensities.2. 2008). Isolates collected from these aquatic habitats are robust and have rare and unique characteristics and therefore possibly better adapted to speciﬁc conditions (Sheehan et al. a metabolically inert chelator. Cylindrotheca sp.. 2007). 2005). / Bioresource Technology 102 (2011) 57–70 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. Schreiber solution consisting of a mixture of nitrate and phosphate was devised. Some of them are modiﬁcations formulated after detailed study on the nutrient requirement of the organism. For the purpose of bioprospecting. Heavy duty equipment includes a suitable vehicle for rough terrain with enough space for the collected samples. 3. good growth of algae can be achieved by adding small quantities of nonpolluted natural seawater (less than 1–4%) to the artiﬁcial seawater (Andersen. and/ or with one or more antibiotics. Schizochytrium sp. 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. Pavlova salina CS49 Skeletonema sp.6 35. Earlier algal media were devised to include antibiotics. media has to be inclusive of essential components that are . Ideally samples can be collected from the natural substrata by chipping. fungi and viruses) and any competitors in the habitat.1 28–32 35–54 20 15–23 >20 23 20–30 25–33 20–35 16–37 30. 3.. In addition. Stringent regimes and protocols need to be exercised when sampling. CS252 Chaetoceros muelleri F&M-M43 Pavlova lutheri CS182 Chaetoceros calcitrans CS178 Nitzschia sp. temperature. water temperature. tat are mainly a result of the dissolved CO2.2 19.. scalpels. Nannochloropsis sp. pH.e. Culture media Various culture media have been developed for isolation and cultivation of microalgae (Table 4). Therefore an all encompassing sampling regime is essential to isolate microalgae from aquatic environments.9 31. O2 and dissolved salts due to eddies and turbulence created as the two water bodies mix.8 33. Likewise modiﬁed media supporting growth of a wide range of microalgae can be formulated by carefully manipulating major nutrients (Scott et al. vitamins. Rodolﬁ et al. it is imperative to screen as many aquatic environments as possible to increase the potential to isolate and select unique hyper-lipid producing microalgae. The brushing method was reported to be effective and reproducible method of collecting microalgal cells and also that it does not damage them (MacLulich. trace metals and later with EDTA. Soil extracts were added to Schreiber’s medium for growing the green dasycladalean Acetabularia and unicellular and benthic marine algae. scrapping. Therefore media composition is vital and most importantly. 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. 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. Microalga Botryococcus braunii Monodus subterraneus UTEX 151 Chlorella vulgaris CCAP 211/11b Chlorococcum sp. A number of microalgal strains prefer brackish conditions because of the nutritional composition of the aquatic system and the warmer temperatures (Woelfel et al. Antibiotics can be added to the growth medium to discourage growth of contaminating cyanobacteria and other bacteria. and salinity. 2007). UMACC112 Scenedesmus sp. depth and water velocity i. Hypersaline environments (halophilic).5 39... and by brushing from rock surfaces and bottom sediments.6 21. thermophilic springs and maturation ponds are ideal extreme environments for the isolation and selection of lipid producing microalgae with novel properties.. 2007). Elementary factors inﬂuencing microalgal growth are measured on the sampling site. F&M-M19 Scenedesmus sp. 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. Mutanda et al.. In case of marine algae. Addition of germanium dioxide inhibits the growth of diatoms.1. However if resources and time are available it is desirable to measure the operational factors such as shear produced by mixing. if the water is ﬂowing. conductivity and light intensity simultaneously). (2009). Most of the microalgae found in brackish conditions are found in suspension as a consequence of the rapid water movements (Anandraj et al. based on the minimum requirement for the two elements shown by a diatom culture. mesh net (0. The global 4. though artiﬁcial sea water media is common. Nannochloris sp. Bottom sediments are also major habitats of benthic microalgae and therefore should be collected together with pieces of detritus and mud. nutrient concentration (nitrates and phosphates). The equipment required for microalgal sampling includes a knife. GPS. These can be analysed microscopically and using conventional microbiological methods in the laboratory after collecting the water samples. dissolved O2. to replace organic chelators such as citrate (Andersen. Axenic cultures can be obtained by treating isolated algae to an extensive washing procedure. These include abiotic factors such as light (quality and quantity).
Gravity is perhaps more frequently used to concentrate the target organisms rather than to establish unialgal cultures. dinoﬂagellate and green ciliate. (2000) 61 AK medium Beijerinck medium BG-11 Bolds basal medium À + + + + À + À + À + À Barsanti Barsanti Barsanti Barsanti and and and and Gualtieri Gualtieri Gualtieri Gualtieri (2006) (2006) (2006) (2006) COMBO Medium Diatom Medium. volvocalean algae. because the tiny cells cannot be easily distinguished from particles. the spray can be administered in a sterile hood or clean environment so that airborne bacteria and fungi are not dispersed onto the plate. seawater. Ultraclean droplets for rinsing are necessary. 1996). Rogerson and co-workers. on the basis of probability. modiﬁed Freshwater + Marine + Brackish À Suitable for Euglenophyceae. 4. free of all other protists.4.2. speciﬁc for algae requiring slightly acidic medium Broad spectrum marine algae Freshwater Chlorophyceae Freshwater soil Cyanophyceae Broad spectrum medium for freshwater Chlorophyceae. Atomized cell spray technique A ﬁne or atomized spray of cells can be used to inoculate agar plates. or on a microscope slide. Also.2. modiﬁed DY-III medium ESAW medium K medium L1 medium Medium F Medium G MNK medium À + + À À À À + À + + Kilham et al. found in the natural environment to support and not exclude the growth of isolates of interest. suspended in a 1% crude papain solution. Isolation using agar plates Isolation of cells on agar plates is also an old and common method. a small volume contains a single cell. 4. (2004) À + + + + + + À + + + + ND ND ND. Gentle centrifugation for a short duration brings large dinoﬂagellates and diatoms to a loose pellet. Xantophyceae. the single cell can be pipetted and discharged into the sterile rinsing droplet and before the cell can settle.2. If the approximate cell concentration is known. (2001) Noël et al. the goal is to separate the larger and heavier cells from smaller algae and bacteria. 2005). 2005). then it is easy to calculate the necessary dilution so that. the alga must be able to grow on agar as streak or pour plates (Brahamsha. Centrifugation with use of density gradients (e. It is the preferred isolation method for many coccoid algae and most soil algae. the microalgal droplets can be placed on agar to reduce evaporation but this step depends on the size of the cells. broken frustules can refract light differently than for intact cells. ﬂask. In most cases. Conventional methods 4. Dilution techniques The goal of the dilution method is to deposit only one cell into a test tube. (2003) Barsanti and Gualtieri (2006) Berges et al.1. (2001) Barsanti and Gualtieri (2006) Guillard and Hargraves (1993) Jeffrey and LeRoi (1997) Blackburn et al.0%. selenium. but they do grow embedded in agar. Chrysophyceae. Micropipette isolation is usually performed with a Pasteur pipette or a glass capillary having a straight or bent or curved tip. These naked cells were reisolated via micropipette into fresh medium. and Cyanophyceae. 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. Gravimetric separation Gravity separation can be effective for separating larger and smaller organisms. For ﬂagellates. although automation is more advantageous. cessation of swimming sometimes indicates damage. The technique can be altered in several ways like dilution with culture medium. The technique can vary. especially when working with seawater. deposit it without damage into a series of sterile droplets. À. or well of a multiwell plate.2. but in general a liquid cell suspension is atomized with forced sterile air so that cells are scattered onto the plate (Andersen. distilled water.5. or another element can be added to some isolation tubes or cell wells to speciﬁcally select for species that require these nutrients (Andersen.2. until a single algal cell. and the smaller cells can be decanted. into and from a micropipette to generate ca. For diatoms. 4.2. green algae. Subsequently. 4. ﬁltered water from the sample site.T.5–2. xanthophytes.8% and 1. and after colony formation. Mutanda et al. 4. Skill of the technique is important not to shear or damage the cell. / Bioresource Technology 102 (2011) 57–70 Table 4 Common media used for microalgal strains from different aquatic environments. Media AF6 medium. (1986) employed repeated introduction and ejection of cells. selected cells are removed and inoculated. 2005). 10% naked cells of Coscinodiscus asteromphalus. The goal of micropipette isolation is to pick up a cell from the sample. 2005). cannot be used. unsuitable for algae with vitamin requirements Cyanobacteria. can be conﬁdently placed into the culture medium.. it should be picked up and transferred. thereby establishing a single-cell isolate (Andersen. the sample can be examined microscopically in a glass or plastic dish. The traditional method of micropipette isolation can be successfully employed with certain precautions.2. and the two primary methods are centrifugation and settling. +. Usually. Some algae do not grow on the surface of agar plates. Silica sol.3. not only for ease of use but also because axenic cultures can often be directly established without further treatment. the concentration of agar is not an important factor. Settling is effective for non-swim- . cryptophytes. However. The plates are incubated. assuming the agar is between 0. ammonium. For best results.g. Leakage of protoplasm is an obvious sign of severe damage. many cryptophytes. in a multiwell plate. Furthermore. (1998) Cohn et al. not determined. can be used. or some combination of these. Percoll) has been used to separate mixed laboratory cultures where each species was separated into a sharp band (Andersen. For successful isolation onto agar. Single-cell isolation Perhaps the most common method for single-cell isolation is by micropipette.
In this method. They include techniques such as dielectrophoresis (Doh and Cho. 4. biomolecules. as fragile dinoﬂagellates. the techniques for cell separation and isolation with high speciﬁcity are limiting because cell populations are frequently heterogeneous and the cells are suspended in a solution of different chemicals. 2002.. fungi. as well as other types of algae must be isolated by singleorganism isolations using advanced techniques. which was optimized for axenic dinoﬂagellate cultures. / Bioresource Technology 102 (2011) 57–70 ming large or heavy cells. 2008). the type of cells is determined and separated according to their cell size. This equipment is stipulated in literature as the ideal tool for algal screening and isolation (Kacka and Donmez.1. 2009). 2007.2. non-immunological methods are relatively fast and simple techniques. However. Cells with a speciﬁc characteristic or indeed a combination of characteristics can be separated from the sample for further analysis or growth. such as Karenia brevis (Dinophyceae). 2005). 4. On the other hand. 2008. The enriching substance can be varied and usually added in minimum quantity and in stages. Sampling reduces recycling. The advantages and disadvantages of the microalgal puriﬁcation techniques described in this section are summarised in Table 5. 2004).3. 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. and cells.2. soil water extract. then an enrichment culture can be aerated with 1–5% CO2 and thus select for species with high CO2 tolerance (de Morais and Costa. Knuckey et al. Capillary tubes or haematocrit tubes of approximately 1 mm diameter Â 100 mm long are used for picking individual cells (Godhe et al. 2009). Compared with traditional separation methods. there are limitations and therefore continued need for developing novel methods for isolation of microalgae. and subsequently extracts from lemon rind. Among algal cell manipulation techniques. afﬁnity based cell sorting (Chang et al. Peat moss can be substituted for soil– water extract when enriching for desmids and some other algae from acid habitats. Phase contrast or dark ﬁeld optics is an advantage.62 T. Seventy percent of isolated cells were recovered in a new medium. such as yeast extract. but single-cell isolation is nearly impossible and hence combined with other techniques. Media enrichment Enrichment cultures have long been used as a preliminary step towards single-cell isolations. Common enriching substances include culture medium. however. usually a clonal population (but which may contain bacteria.. magnetic-activated cell sorting (Han and Frazier.. 2004) and ultrasound separation (Petersson et al. One of the advantages of this method is high speciﬁcity and selectivity. The appropriate recovery medium may enhance the rate of successful isolations. Mutanda et al. Trauma to electronically sorted cells was not a limiting factor. 2010). . aqueous two-phase system (Yamada et al. Streaking and spraying are useful conventional techniques for single-celled.. because of the availability of ubiquinone or plastiquinone (Andersen. The rate of successful isolation of large-scale (>4 l) cultures was higher for manual picking than for electronic cell sorting (2% vs. and phosphate or a trace metal. 0.. It requires the handling of an Inverted microscope or stereo microscope with magniﬁcation up to 200Â. However. One strategy of bacteria-free algal cultures is to make the medium as acidic as possible without killing the alga.4. led to success. 2005). grazing. a disadvantage is its low speciﬁcity for cell separation.. because bacterial action.. but in nature algae survive. Ramanan et al. Selective culturing is an exceptional tool for enrichment culturing of hyper-lipid producing microalgae. Conventional cell separation can be carried out by immunoreactions of membrane protein with the capturing antibodies because the type of integrated proteins is speciﬁc for their function. This will afford a time saving of $60% over the current serial plating technique.. Since the goal is to isolate microalgae growing at a high CO2 concentration. Although conventional methods for isolation has sufﬁced. Natural samples are often deﬁcient in one or more nutrients. Sometimes enrichments can also be detrimental. 2004). However. 2005). casein. hydrodynamic separation (Shevkoplyas et al. Micromanipulation A micromanipulator allows the selection of single cells from liquid culture. manual picking of cells is more laborintensive and time consuming. 4. Organic substances. These exploit an interactive physical property of cell with the surrounding media. This would mean that a single cell can be selected from an enriched environmental sample and grown in liquid monoculture as well as plates. The successful application of micromanipulation techniques requires the expertise and experience. or ﬁlamentous algae growing on agar surface. 4. Flow cytometry Flow cytometric analysis permits the investigator to perform a rapid and quantitative version of the experiments that could otherwise be performed by ﬂuorescent microscopy. 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 unﬁltered lemon juice. respectively). Immunological technique is a mainstay of commercialized cell separation methods such as the ﬂuorescence-activated cell sorting (Takahashi et al. ammonium. Other methods Immunological and non-immunological methods to separate cells of interest are available. and nutrient stress can cause death to the target species.6. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual post-sort culture maintenance. 2002).3. Fluorescence-Activated Cell Sorting (FACS) allows the process to be taken one very important step further. Many ﬂagellates. or protozoa).5%. Moreno-Garrido.. Electronic cell sorting for isolation and culture of dinoﬂagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette (Sinigalliano et al. electronic single-cell sorting has the potential to accelerate the discovery of new algal strains (Sinigalliano et al.3. Advanced methods Algal cultures may be unialgal which means they contain only one kind of alga. and death of organisms recycle those nutrients. colonial. shape and other physical properties. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. or urea can be added in low concentrations when isolating osmotrophic algae. as cells do not show remarkable differences between each cell type with the exception of immunological properties. 4. survived electronic cell sorting to yield viable cells. if the target species is rare and unable to compete with weedy species. or nutrients like nitrate. 2005). when combined with newly developed automated methods for growth screening. microfabricated devices have small working volume and subsequently reduced throughput. 2005). or cultures may be axenic meaning that they contain only one alga. Both centrifugation and settling techniques are effective for concentrating larger cells. However..
.. 1987). Techniques Pringsheim’s micropipette method Advantages Single cells can be successively transferred and puriﬁed Disadvantages Laborious and time-consuming method requiring considerable manual skills. Ultrasonication Not a standalone method Should be coupled subsequent to cell sorting Steup and Melknonian (1981) 5.. The chloroform and ethanol peak excitation was recorded at 600 and 632 nm. The emission maximum of Nile Red is blue-shifted as the polarity of the medium decreases. approximately 10–15 mg wet weight of cells. FTIR spectroscopy may be used . The method often fails with small non-ﬂagellate cells. 1987. 2009)... Relatively easy Low cost Precise and rapid method Simultaneous measurements of individual particle volume. 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. labor-intensive and it has a low throughput screening rate.. a rapid throughput of sample processing is required. BODIPY 505/515 has been shown to have a narrower emission spectrum than Nile Red. respectively (Elsey et al. Nile Red (9-diethylamino-5H-benzo[a]phenoxazine-5-one). Elsey et al. 2). A major disadvantage of the conventional method is that it is time consuming. Greenspan and Fowler. where ﬂuorescence contrast enhancement of lipid bodies is important for image resolution (Cooper et al. pure cultures may be propagated from the isolated viable cells.4adiaza-s-indacene) has recently been used as a vital stain to monitor algal oil storage within viable cells. 1985. making it potentially more useful for confocal imaging. (Cooksey et al./vol. intensely ﬂuorescent in organic solvents and hydrophobic environments. Nile Red possesses several characteristics advantageous to in situ screening.... Consequently. even though gravimetric analysis shows high yields of neutral lipids. 1959). Moreover. 2010). / Bioresource Technology 102 (2011) 57–70 Table 5 Advantages and disadvantages of microalgal puriﬁcation techniques. 2007). (Akoto et al. 2007). targeting organisms with rapid growth rate and tolerance to environmental parameters. 1987. Thick cell walls of microalgae inhibit the permeation of Nile Red and may indicate the absence of oil. The semi-quantiﬁcation of neutral lipids using Nile Red or BODIPY and ﬂuorescence microscopy allows for an initial rapid screening and visualization of lipid globules. which are more difﬁcult to recognise during serial transfers Some delicate ﬂagellates are easily damaged during successive micropipette transfers. A recent study (Dean et al. 2005) must be cultured for the extraction and derivatization. requires a calibration curve that correlates ﬂuorescence to lipid content.and Nile Red-based lipid measurements. In acetone. has been commonly used to evaluate the lipid content of animal cells and microorganisms (Genicot et al. It is relatively photostable. Lee et al.. Cannot be used with most ﬂagellate taxa which fail to grow on solid substrates Unsuccessful when the numerical ratio between algae and bacteria is unfavourable. Nile Red is excited at 488–525 nm and the ﬂuorescent emission measured at 570–600 nm using various instruments (Cooksey et al. there is greater interest on a rapid in situ measurement of the lipid content (Cooksey et al. 2010). the lipophilic ﬂuorescent dye BODIPY 505/515 (4.. whether determined gravimetrically or by use of lipid standards (Elsey et al. FTIR was shown to be an efﬁcient and rapid tool for monitoring lipid accumulation of microalgae. 1986. Screening of microalgae The screening stage of bioprospecting focuses on isolation and identiﬁcation of algal species capable of substantial lipid production. Unlike Nile Red. (2007). Measurements of lipid per unit cell. ﬂow cytometry or a ﬂuorescence-activated cell sorter (Cooper et al.3. 1987). Subsequently. ﬂuorescence 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. 2010) (Fig. Alternatively. 2005) and especially microalgae (Cooksey et al. Mutanda et al. For the purposes of bioprospecting for high lipid yielding microalgae. The advantage of BODIPY is that high lipid yielding cells may be identiﬁed and isolated microscopically using a micromanipulator system.. Laughton. 2007). 1998) which allows one to differentiate between neutral and polar lipids at the excitation and emission wavelengths. Lipid bodies are stained green and chloroplasts red and visualized in live oleaginous (oil-containing) algal cells (Cooper et al. The peak emission intensity of Nile Red in hexane occurs near 576 nm when excited at 486 nm. (1993) Axenic cultures are difﬁcult to obtain from algae to which bacteria are physically attached. a lipid-soluble ﬂuorescent dye. 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 ﬁlters.5.. The conventional method used for lipid determination involves solvent extraction and gravimetric determination (Bligh and Dyer. BODIPY 505/515 has the advantage that it does not bind to cytoplasmic compartments other than lipid bodies and chloroplasts. showed the technical emission spectra for Nile Red in various solvents.7-tetramethyl-4-bora-3a. This study had reported highly signiﬁcant correlations between the FTIR. 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.. 2010) demonstrated the use of Fourier transform infrared micro-spectroscopy (FTIR) to determine lipid and carbohydrate content of freshwater microalgae. 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 (20–30% vol.) and elevated temperatures (40 °C) (Chen et al.4-diﬂuoro-1. Elsey et al. 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.T.
Sample preparation for lipid analysis 6. The lipids produced by algae are often accumulated intracellularly. Before the fatty acid components of algae lipids can be analysed by GC. 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. 2010).2. viewed at 1000Â using a ﬂuorescence microscope at 490 nm excitation and 585 nm emission ﬁlters.. Neutral lipid globules in the cytosol are stained yellow (unpublished data). rapid. Once the high lipid producing microalgae have been identiﬁed. These techniques are used for rupturing the algae cells to release the lipid compounds of algae biomass. / Bioresource Technology 102 (2011) 57–70 Fig. 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. gravity sedimentation.. 2006) under nutrient or other environmental stress. understanding the physiology of the algal isolate is imperative. Higher drying temperature decreases both concentration of triacylglycerides and lipid yield (Widjaja et al. They recorded the highest cell recovery at 13... which could require extraction of the lipids from crude cell pastes (Grima et al. 2. BODIPY 505/515.. Cell viability was found to depend on the microalgal species and the method of centrifugation. The success of downstream processing is dependent on reliable biochemical and physiological screening tools such as the BODIPY lipid stain. The PAM ﬂuorometer parameters (Electron transport rate [ETR]. Spolaore et al. and efﬁcient method for lipid extraction from microalgae. acceleration. 1994). Among these methods. 2010). sonication.64 T. 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. microwaves. 2007.. centrifugation is found to be the most efﬁcient.000g ($95%) followed by 6000 g (60%) and 1300 g (40%). Thin-layer chromatography (TLC). In this picture. (B) Oil-containing lipid bodies can be vitally stained and visualized in live oleaginous (oil-containing) algal cells using the green ﬂuorescent dye. maximum quantum efﬁciency of Photosystem II [FV/Fm]. a further step in the screening would be to determine the photosynthetic efﬁciency of the culture. 1999). 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.000g ($95%) followed by 6000 g (60%) and 1300 g (40%). Heasman et al. (For interpretation of the references to colour in this ﬁgure legend.1. it is necessary to convert them to low molecular weight non-polar derivatives. 1998. temperature. (A) Nile Red stained Chlorella sp. the most popular is a slightly modiﬁed method of Bligh and Dyer (1959). Pulse Amplitude Modulated (PAM) chlorophyll-a ﬂuorescence measurements are widely used as a simple. They concluded that microwave oven method was most simple. Direct transesteriﬁcation (simultaneous extraction and transesteriﬁcation) and transesteriﬁcation (transesteriﬁcation after lipid extraction) were . and non-invasive method to assess the physiological state of microalgae (Schreiber et al. FTIR spectroscopy and PAM Fluorometry. ﬂocculation. osmotic shock and bead beating have been evaluated in order to increase lipid extraction efﬁciency (Lee et al. chemical or biological methods or combination of any two of these methods. Pre-treatment of algae biomass for lipid extraction The harvesting of algae biomass can be achieved by physical. Many microalgae have the ability to produce up to almost 80% dry cell weight of triacylglycerols (TAG) as a storage lipid (Chisti.. Neutral lipid synthesis is stimulated under nutrient depleted or limited conditions. Subsequent to screening. The lipids present in the dried/freeze dried biomass must be extracted and analysed for quantiﬁcation of the fatty acids. 6. Lipid analyses 6. isolated and puriﬁed. 2009). vitally stained lipid bodies (green) are easily distinguished from chloroplasts (red) in an O.) thereafter to quantify the yield of lipids. Highest cell recovery obtained at 13.. Lipid proﬁling of biodiesel feedstock is commonly done by GC with a Flame Ionization Detector (FID) according to ASTM D6584 and EN 14105 methods. Before extracting the algae oil. (2000) studied algae cell recovery at three different centrifugation conditions for ten microalgal species and reported that efﬁciency of recovery decreased with decreasing Qualitative and quantitative composition of lipids can be investigated by established techniques. Different cell disruption techniques such as autoclaving. 2008). maius Naegeli freshwater ﬁlamentous algal cell using a Zeiss Axioskop epiﬂuorescence microscope (Cooper et al. easy. 2003). high moisture present in algae biomass must be removed by means of drying. The algae biomass dried at 60 oC under vacuum (Widjaja et al. Mutanda et al. ﬁltration. ﬂotation and electrophoresis. high pressure liquid chromatography (HPLC) or gas chromatography (GC) or any chromatography with mass spectrometry are some of the different techniques employed for quantiﬁcation of lipid from microalgae. the reader is referred to the web version of this article. PAR and pH) on the algal culture. The temperature used in microalgae drying is crucial factor for separating the oil from dried algae biomass. Although many methods for algal lipid extraction have been recommended. The techniques currently employed in microalgae harvesting cum cell recovery include centrifugation. Neutral lipid synthesis is likely to occur during the stationary phase of growth due to nutrient limitation (Li et al. such as fatty acid methyl esters (FAME). 2009) or freeze drying gave the best results for extraction of algae lipid.
.8S+ITS2 Microsatellite locus 16S1 N/16S2 N ChloroF/ChloroR EK82F/Proto5R 519F/1406R FD8/RB. further microalgae can often change cell size and shape during their life cycle (Godhe et al. Mutanda et al. The identiﬁcation of microalgae in ﬁeld samples using microscopy is also time consuming and requires both experience and signiﬁcant taxonomic and technical skills (Godhe et al. The most important fuel properties considered to assess the potential of biodiesel as substitute of diesel fuel are viscosity. lose pigmentation and ﬂagella so that a proper identiﬁcation is impossible.. oxidative stability.T. FAMEs with higher degree of unsaturation are not suitable for biodiesel. Characterization of algal oil for biodiesel production The fatty acid compositions of microalgae oil may vary to individual species/strains and their environmental conditions. 2009).. some species constitute very minor fraction of the total planktonic community. Biodiesel feedstock with a high degree of saturation is more resistant to oxidation and more stable in presence of light. Small rRNA (SSU rRNA) genes are more highly con- References Tinti et al. 2009. 2009). Triglycerides in oils are reacted with methanol in presence of base/acidic catalyst in transesteriﬁcation reaction to produce FAME. The CN. EK82f/Proto 5r coxF/coxR ITS1/ITS2 ITS3/ITS4 Kbr1–Kbr10 . 2002). Ramos et al. (2007) Novis et al. lubricity. which leads to tedious analysis of distinct samples. 1996).. The fatty acid esters compositions of microalgae sources are different from plant oils.. the analysis of small conserved genes has proved to be very helpful in the clariﬁcation of the relationships between algae. Bertozzini et al. Higher CN values indicate better ignition properties of the fuel (Meher et al. 2002. The identiﬁcation of lipid composition in selected algae strain is essential for determining the suitability to biodiesel and fuel quality.. The molecular-based techniques developed in the last two decades have led to rapid and accurate monitoring. According to biodiesel standard EN 14214 methods. suitable culture conditions may be used and the properties of biodiesel produced from algae oils must meet the International Biodiesel Fuel Standards. Moro et al.. oxygen. density.. Before chromatographic analysis. Microscope-based microalgal cell identiﬁcation methods are usually the standard procedures used in laboratories for the rapid screening of algal samples. Higher oleic acid content increases the oxidative stability for longer storage (Knothe. Rodolﬁ et al. identiﬁcation and quantiﬁcation of microalgae species in mixed phytoplankton samples. number of double bonds and amount of each fatty ester components in both fatty acids and triglycerides (Mittelbach and Remschmidt. 2004). 2009). Higher unsaturated fatty acids (UFA) present in the oil gave higher iodine values and heating of higher UFA caused the polymerization of glycerides. Algal oils contain a high degree of polyunsaturated fatty acids with four or more double bonds when compared to vegetable oils (Zittelli et al. Acid catalysts used in transesteriﬁcation of microalgal oil includes sulphuric acid and hydrochloric acid. 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. 2009). 1995). ﬂuorescence microscopy.. 2010). Different culture conditions play an important role in the lipid composition. 2005).. After transesteriﬁcation. 2005). Ribosomal RNA’s are one of the widely used and are exceptionally useful for the comparative analysis of organisms (Rasoul-Amini et al. scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for species level identiﬁcation. (2007) Moro et al. conventional microscopy techniques used to analyse microalgae can give misleading results since they lack morphological markers for precise identiﬁcation. mitochondria genes. Knothe. (2008) Rasoul-Amini et al. (2009) Scholin and Anderson (1996) and Kamikawa et al. However. (2008) Kamikawa et al. Kalayasiri et al. D1/D2 – 18ScomF1/Dino18SR1.. 1996. In addition. Mostly algae do not survive during ﬁxation or they shrink. / Bioresource Technology 102 (2011) 57–70 65 adopted in sample preparation for algae lipid conversion to biodiesel (Johnson and Wen. 2005) and decreases the cold ﬁlter plugging point (CFPP) for use in cold regions (Stournas et al. They are very slowly altering molecules and major elements in the protein synthetic machinery of all cells. any traces of chlorophylls. (2007) Coleman (2003. 2004. 1997). Ramos et al. Conventional light microscopy has been extended to the use of phase contrast microscopy. (2008) Kamikawa et al. (2007) Large subunit (LSU rDNA) Plastid rbcL Small-subunit (SSU rDNA) Mitochondrial cytochrome c oxidase subunit (cox2–cox1/cox2–cox1 Internal Transcribed Spacer (ITS) ITS1+5. The saponiﬁcation values (SV) and iodine value (IV) represent the ignition quality of fuel and presence of unsaturated fatty acid component in FAMEs. 7. The physical characteristics of both fatty acids and triglycerides can be determined by chain length. 2007) and Jürgen et al... which is the major factor (Papanikolaou et al. 2009). 2008. 1998). (2009) Richlen et al. which leads to formation of deposits or deterioration of the lubricating (Mittelbach. 2006. high temperatures. (2007) Henrichs et al. In order to improve the fuel properties of algae biodiesel. SV and IV can be predicted from fatty acid methyl esters of oils by using empirical equations (Krisnangkura 1986. Microalgal identiﬁcation strategies using molecular approach Identiﬁcation and enumeration of the algae of interest is a major challenge faced by researchers worldwide. ignition quality and combustion heat (Xiaoling and Qingyu.. plastid genes (rbcL). ITS (Internal Transcribed Sequences) and microsatellite DNA sequences (Table 6). while alkali catalysts like sodium hydroxide and potassium hydroxide are suitable for vegetable oil and animal fat respectively (Huang et al. respectively. the microalgal fatty esters develop slight green colour due to presence of chlorophylls.3. (2007) and Auinger et al. and metals (Canakci and Sanli. 6.. 2006). Without complete DNA sequence. The determination of fatty acid composition of algae oil is essential for assessing the fuel quality of biodiesel. catalyst or water must be removed to avoid GC column contaminations (Sheehan et al. cold ﬁlter plugging point. 2006). (2009) Auinger et al. like C:N ratio.. Target region 18S rDNA Primers used chain length and branching of the alcohol moiety (Foglia et al. the concentration of linolenic acid and acid containing four double bonds in FAMEs should not exceed the limit of 12% and 1%. cetane number (CN). The most common DNA regions analysed today for phylogenetic purposes are ribosomal RNA genes (rRNA).
The stored microalgal cultures should be routinely sub cultured to get metabolically active inocula and this should be done at least 1–3 weeks employing conventional aseptic microbiological techniques to avoid contaminating the puriﬁed cultures. A combination of molecular probes/primers and DNA microarrays could serve as a rapid and reliable tool for rapid screening of microalgae.. According to Lorenz et al. of 1–2 times of lipid yield. 9. 2008). The efﬁciency of the genomic DNA extraction step is very important for the subsequent PCR assay. The transesteriﬁcation reaction can be catalyzed by alkalis.. are used as preserving agents for the long term storage of phytoplankton material without destroying the DNA needed as template in morphological and molecular identiﬁcation (Godhe et al. The recent developments in research on algae biofuels production has been extensively investigated (Harun et al. 2008). These media types can support the growth of microalgae from speciﬁc aquatic environments such as fresh water. However slants can prolong the viability of microalgal cells in storage. Free fatty acid (FFA) content of algae is in the range of . The viability of these cultures is then tested periodically and a suitable maintenance regime is established for future application. Bertozzini et al. The different media that can be used for long term maintenance of microalgae are described is Section 4 and Table 4. The chip is made of glass and has special properties. Microalgae can be lyophilised and stored as a powder at 2 °C. Bioprospecting for microalgal strains is costly and time consuming. Raw oil can be converted into biodiesel by means of transesteriﬁcation process. it is recommended to do trial and error of different light intensities and temperatures (Lorenz et al. ASW and so on for long term maintenance of microalgae. 8. To overcome this. The best way to store puriﬁed microalgal cells is to have the cells in an aqueous system under suboptimal temperature and light regimens. in the sampling of long-term monitoring programmes the PCR is also applied to preserved natural samples.. Biomass Energy Conversion Technologies (BECT) can be used to convert the algae biomass into different kinds of energy fuels. standard cryogenic techniques are documented as best methods for long term microalgal storage (Cox et al.. 2005).. 2002. A further promising molecular approach is the application of DNA microarrays.The technique is based on a minimized but. Auinger et al. When using the microalgae for Polymerase Chain reaction (PCR) studies. 3a and b. 2010). removing potential inhibitors that often cause PCR inhibition and low yields of PCR products (Godhe et al..1. 1996). 2008). The application of novel molecular techniques has the potential to revolutionise microalgal classiﬁcation and especially identifying hyper-lipid producing microalgae. Generally it is difﬁcult to establish storage conditions for newly isolated microalgal strains and as a rule of thumb. Furthermore. BG-11... (2005) standard light intensities between 10– 30 mmol photons mÀ2 sÀ1 have proved appropriate in combination with subdued temperatures for long-term culturing of most microalgal taxa. microalgal samples must be stored under suitable controlled laboratory conditions so that they do not degenerate and lose viability. gasiﬁcation (syngas). formalin and glutaraldehyde. For example. The microarray offers the potential to facilitate the analysis of multiple targets from one sample in one experiment (Gescher et al. 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. direct PCR ampliﬁcation from few cells without the need for DNA extractions has been reported (Godhe et al. thermo-chemical liquefaction (biocrude oil). 2008). biomethanation (biogas). especially when it is to be used in quantitative investigation on cultured or environmental samples. marine and brackish. 2008).. 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. A wide range of media are available such as BBM. Fixatives. such as Lugol’s solution. 2009).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. The BECT includes combustion (for heat energy).. high throughput form of a dot blot through application of sequences or probes in an ordered array on the chip. 2002. the genomic DNA has to be extracted and puriﬁed from samples. For long term storage.66 T. 2009). Analysis of the LSU rRNA gene has been very useful for sorting out closely related species concepts. enzymes or supercritical methanol. Biofuel production 9.. BECT is divided into thermo-chemical and biochemical methods. Microalgal biomass can be produced using either open raceway ponds or photobioreactors. 2005). acids. 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. microalgal specimens should be kept at low temperatures of ±2 °C but however most microalgal strains can be kept at 15–20 °C. Biodiesel production The viscosity of the raw microalgal oil is usually higher than that of diesel fuel.. cryopreservation of microalgal cells has been found to be effective as a culture maintenance strategy (Day and Brand. Auinger et al. To date. 2006). Mutanda et al. The pros and cons of these two systems are described in Table 7.2. 2002. 2005). The biodiesel produced from algae oil by using transesteriﬁcation process can be used for replacing the conventional diesel fuel. pyrolysis (pyrolytic gas. Algae oil can be converted into biodiesel by the transesteriﬁcation process as depicted in Fig. Quantitative analysis of planktonic protists and microalgae from preserved ﬁeld samples combining morphological and small-subunit (SSU) rRNA gene sequence using single cell PCR has been reported recently (Auinger et al. 2005). In addition. therefore it is crucial to store the cultures properly so that the bioprospecting exercise is not put to waste. The media chosen can either be in liquid or solid agar medium. Exploring possibility of algae based biofuels The algae oil and spent biomass are considered as potential sources for biofuels production. 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. biochar). like species groups and geographical origin of different clonal isolates (Scholin and Anderson. The algae oil cake produced in algae biofuel industry is ca. As living microorganisms. Microalgal maintenance One of the major limitations with regards to culture maintenance is effective technology for long term storage of cultures (Moreno-Garrido. 1000 kg of algae biomass having 35% oil content can yield 650 kg of algae deoiled cake.. / Bioresource Technology 102 (2011) 57–70 served than large rRNA (LSU rRNA) genes. and are therefore more useful for analysis of more distantly related species. 2008). 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.. photobiological hydrogen production (hydrogen) and alcoholic fermentation (ethanol). bio-oil. 9.. which are applied generally for gene expression and have been used with oligonucleotide probes of conserved genes for species identiﬁcation at all taxonomic levels (Gescher et al.
less reaction time and uses no water or dangerous chemicals. as value added products has certainly made the technology attractive and therefore attracted much attention in the ﬁeld. acid catalysts were found to be useful for pre-treating. 2009). which is the main factor for formation of soap in alkaline catalyst based transesterﬁcation process and separation of biodiesel and glycerol is difﬁcult.3. etc. Modiﬁed from Pulz (2001) and Harun et al. 2005). 9. Mutanda et al. (2010). Schematic representation of biodiesel production from microalgae oil (a) and Biodiesel production by transesteriﬁcation reaction (b). The thermal decomposition of organic components can be achieved by different processes such as direct combustion. and pyrolysis. utilising the oil for biodiesel. reaction time. / Bioresource Technology 102 (2011) 57–70 Table 7 Generalized comparisons of two different cultivation methods of algae production. moisture content. In this process. 2009). and mixing on an acid-catalyzed in situ transesteriﬁcation process for production of biodiesel from microalgae lipids. The Mcgyan process is continuous transesteriﬁcation process for biodiesel production from various feedstocks. Ehimen and co-workers (2010) studied the effect of operational parameters such as alcohol volume. 20–50% (Kosaric and Velikonja. 3. 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 difﬁcult to scale up due to limitations Fig. 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. Enzymes are also used for feedstock with higher free fatty acid. 1995. 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 efﬁciency Operational mode Air pump Shear CO2 transfer rate Mixing efﬁciency Water loss Evaporation O2 concentration Open ponds Multi strain cultivation High None Very difﬁcult 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 67 (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. (1998) studied gasiﬁcation of Spirulina at temperatures ranging from 850 to 1000oC for methanol production. They estimated that algae biomass gasiﬁcation at 1000oC produced the highest theoretical yield of 0. The end products may vary from gas or liquid or solid fuel depending on temperature used in the thermo-chemical conversion process. typically in the range 800–900 °C (McKendry. temperature. Added advantages of this technology are: no catalyst is used.T. but its reaction conversion rates are very slow (Um and Kim.. Gasiﬁcation is the conversion of biomass into a combustible gas mixture by the partial oxidation of biomass at high temperatures.e. The use of supercritical transesteriﬁcation process method for algae biodiesel production is also restricted due to process economics and safety concerns related to the reaction conditions (Ehimen et al. gasiﬁcation. Mansour et al.. 2002) and the produced syngas can be used for thermal applications or fuel for diesel/gas turbine engines. Due to these problems. thermo-chemical liquefaction. glycerol. For transesteriﬁcation of high free fatty acid feedstocks. Adapting a bioreﬁnery approach i. 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 transesteriﬁcation reaction. the alkaline catalyst based biodiesel production is not suitable for algae oil with high FFA content. Hirano et al. . 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 speciﬁcation. spent biomass. (Um and Kim. 2010). the combination of alcohol and lipid is sent into a continuous ﬁxed-bed reactor ﬁlled with a sulfated metal oxide catalyst at elevated temperature and pressure to perform the transesteriﬁcation and esteriﬁcation reactions simultaneously.64 g methanol from 1 g of algae biomass.
215–235.A.. pp.. M. Deswarte.W. Dean. Cooksey. 29..S. Balat... 19. R. D. K. M. 2003).. J. 431–441.A. Borowitzka. ISBN978-0-470-05805. Burlington. 911–917. B. and Biotechnology. I. Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Recovery of liquid fuel from hydrocarbon rich microalgae by thermochemical liquefaction. A. C. Biotechnol. the need for developing suitable technology to meet this demand is imperative..L. Ind. Reproductive compatibility among four global populations of the toxic dinoﬂagellate Gymnodinium catenatum (Dinophyceae). D. 2005. J. Franklin. Harrison.W.08. Thermo-chemical liquefaction is a process that can be employed to convert wet algal biomass material into liquid fuel. 37...2009. 2010)... Bruce.L. Environ. E. Pfandl. C. J. E. Sanli. L. 259–270. Phycol. Added advantages of this technology are conversion of wet biomass into useful energy and no feedstock drying process involved (Clark and Deswarte.. Biochemistry. Munro.F. Magnani. doi:10. Sigee. Anandraj. Renewable fuels and chemicals by thermal processing of biomass. 4499–4507. The effect of temperature and mixed species composition on diatom motility and adhesion. 2006). Renew. Cohn. (Ed. Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae. M. Petersen. Dai. Callis. Estuar.W. J. 50. increased ammonia is produced upon microbial protein degradation. Clark. Doh. 2009). 2009).L.. Wiley Series in Renewable Resources. 1987. Day. Fluorometricdetermination of the neutral lipid-content of microalgal cells using Nile red. P.. 18. Zhang. Estrada.... 15–25 wt. Appl. Pierboni. K.R.L.. 6.. Introduction to chemicals from biomass.. D. Dunaliella tertiolecta and Spirulina platensis yielded 64. L. G.. Convers. Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa. Quintal. 64–73. S.. 2009). 2008. Kirtay. 2003. Due to high protein content of microalgae. Q. 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