**4. Microalgal lipids**

178 Biodiesel – Feedstocks and Processing Technologies

The term 'algae' is used to describe a huge variety of prokaryotic (strictly termed Cyanobacteria) and eukaryotic organisms with a range of morphologies and phylogenies. They represent a wide array of species, inhabiting environments from deserts to the Arctic Ocean, including both salt and fresh water. They vary in colour, shape and size, from picoplankton (0.2 to 2 m) to giant kelp fronds up to 60 m in length (Barsanti & Gualtieri, 2006). Macroalgae (e.g. seaweeds) are generally large (can be seen without the aid of a microscope), multicellular and often show some form of cellular specialisation. Microalgae are usually less than 2 mm in diameter and unicellular or colonial. Microalgae have been investigated for a variety of commercial applications. Annual global microalgal production is currently estimated at about 10 000 metric tons, with the main algae cultivated being *Spirulina* (accounting for roughly half of the worldwide algal production), *Chlorella*,

Algae have been investigated as a source of energy in many different contexts, from direct combustion to the production of hydrogen gas. Anaerobic digestion can be applied for the generation of methane or biogas (Golueke et al., 1957). Algal species with high oil content are particularly attractive as a feedstock for biodiesel production. Research into algae for the mass-production of oil has focused on the microalgae due to their high lipid content compared to macroalgae. Most algal species considered for biodiesel production are either green algae (Chlorophyta) or diatoms (Bacillariophyta) (Sheehan et al., 1998). They are generally photosynthetic, but several species are able to grow heterotrophically or

Microalgae have higher growth rates than land-based plants. Due to their simple cellular structure and existence in an aqueous environment, the entire cell surface is available for light capture and mass transfer, leading to high rates of substrate uptake and photosynthetic efficiency (Miao & Wu, 2006; Sheehan et al., 1998). In contrast to land-based oil crops, where only the seeds are harvested, each algal cell contains lipid and hence the yield of product from biomass is much higher (Becker, 1994). Due to these differences, the oil yield per area

of microalgal cultures potentially exceeds that of the best oilseed crops (Table 1).

Algae 4.7 to 14 Sheehan et al., 1998 Palm 0.54 Mata et al., 2010 Jatropha 0.19 Sazdanoff, 2006 Rapeseed 0.12 Sazdanoff, 2006 Sunflower 0.09 Sazdanoff, 2006 Soya 0.04 Sazdanoff, 2006 Table 1. Average productivities of some common oil seed crops compared to algae

Microalgal lipids can be extracted to yield oil similar to that from land-based oilseed crops. The amount and composition of the oil varies between algal species. Algal oil can be converted to biodiesel through the same methods applied to vegetable oil. The idea of using microalgae as a source of transportation fuel is not new. Research in this field has been

**Oil source Yield (L.m-2.yr-1) Reference**

**2. Microalgae** 

*Dunaliella* and *Haematococcus*.

mixotrophically (Barsanti & Gualtieri, 2006).

**3. Biodiesel from microalgae** 

The main components of algae cells are proteins, carbohydrates and lipids (Becker, 1994). Microalgae naturally produce lipids as part of the structure of the cell (e.g. in cell membranes and as signalling molecules), and as a storage compound, similar to fat stores in animals and humans (Tsukahara & Sawayama, 2005). The term lipid encompasses a variety of compounds with different chemical structures (e.g. esters, waxes, cholesterol). The most common lipids are composed of a glycerol molecule bound to three fatty acids, known as triacylglycerol or TAG, or to two fatty acids with the third position taken up by a phosphate (phospholipids) or carbohydrate (glycolipids) group. Fatty acids consist of a long unbranched carbon chain. They are classified according to the number of carbon atoms in the chain and the number of double bonds, for example saturated (no double bonds), monounsaturated (one double bond) or polyunsaturated (more than one double bond). Microalgae commonly contain fatty acids ranging from C12 to C24, often with C16 and C18 unsaturates. Certain species contain significant amounts of polyunsaturated fatty acids.

Storage lipids, generally in the form of TAG, accumulate in lipid vesicles called oil bodies in the cytoplasm. Most fast-growing species have relatively low lipid content during normal growth, with these lipids mainly consisting of phospho- or glycolipids associated with cell membranes. Under certain conditions, generally triggered by stress or the cessation of growth, lipid content can increase to over 60% of cell dry weight (DW), mostly composed of

Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel 181

Open systems consist of natural waters such as lakes, ponds and lagoons, or artificial ponds and containers that are open to the atmosphere. Most commercial production to date has taken place in open ponds as these systems are easy and cheap to construct (Pulz, 2001). The most common technical design is the raceway pond: an oblong, looped pond mixed by a paddlewheel, with water depths of 15 to 20 cm (Becker, 1994). Biomass concentrations of between 0.1 and 1 g.L-1 and biomass productivities of between 50 and 100 mg.L-1.day-1 are possible (Chisti, 2007; Pulz, 2001). The main advantages of open systems are their low cost and ease of construction and operation. They also offer the potential for integration with

Disadvantages of open systems include contamination with unwanted species such as foreign algae, yeast, bacteria and predators, evaporation of water, diffusion of CO2 to the atmosphere and low control over environmental conditions, particularly temperature and solar irradiation (Becker, 1994; Pulz, 2001). In addition, the relatively low cell densities achieved can lead to higher cost of cell recovery (Chen, 1996). Only a few microalgal species have been successfully mass cultivated in open ponds. These tend to be either fast-growers that naturally outcompete contaminating algae (e.g. *Chlorella* and *Scenedesmus*), or species that grow in a specialised environment such as high salt (e.g. *Dunaliella salina*) or high pH (*Spirulina platensis*), which limits growth of competitors and predators (Chen, 1996). Due to the lack of control over cultivation conditions resulting in low productivity, and the fact that many desirable species cannot be effectively maintained in open systems, attempts have been made to overcome some of these limitations through the use of enclosed reactor

Closed systems, or photobioreactors, consist of containers, tubes or clear plastic bags of various sizes, lengths and orientations (Pulz, 2001). Commonly used designs include vertical flat-plate reactors and tubular reactors, either pumped mechanically or by airlift (Scott et al., 2010). Closed reactors offer a much higher degree of control over process parameters, leading to improved heat and mass transfer, and thus higher biomass yields. They can also offer a much higher surface area to volume ratio for light provision, better control of gas transfer, reduction of evaporation and easier installation in any open space (Chen, 1996). Additionally, the risk of contamination is reduced, CO2 can be contained, production

Productivity in closed systems can be much higher than open systems, with biomass concentrations of up to 8 g.L-1 and productivities of between 800 and 1300 mg.L-1.day-1 (Pulz, 2001). However, they are generally much more costly to build and more energy demanding to operate than open systems (Table 2). Closed systems can also have problems with fouling and oxygen build-up. Large systems can be difficult to clean and sterilize and long sections of enclosed tubing may require oxygen purging. High oxygen concentrations cause the key enzyme Rubisco to bind oxygen instead of carbon dioxide, leading to photorespiration instead of photosynthesis (Dennis et al., 1998). Although closed bioreactors offer a much higher degree of control over process parameters and can have higher yields, it is uncertain whether the increased productivity can offset the higher cost and energy requirements. For a commodity product such as vegetable oil for biodiesel, low cost, high volume production is demanded, while quality is less critical (Pulz, 2001). In this case, the more favourable economics and energy requirements of open ponds may well outweigh the

A hybrid system combining the cost effectiveness of open ponds with the controlled environment of closed systems is appealing and has been tested in a few cases. Generally

wastewater treatment processes or aquaculture systems (Chen, 1996).

conditions can be reproduced and temperature can be controlled.

systems.

advantages of closed reactors.

TAG (Shifrin & Chisholm, 1981; Piorreck et al., 1984; Spoehr & Milner, 1949; De la Pena, 2007; Becker, 1994).

TAGs are the most suitable class of lipids for biodiesel production. Phospholipids are particularly undesirable as they increase consumption of catalyst and act as emulsifiers, impeding phase separation during transesterification (Mittelbach & Remschmidt, 2004; Van Gerpen, 2005). Phospholipids, and some sulphur-containing glycolipids, also increase the phosphorous and sulphur content of the fuel respectively, which must both be below 10 mg.L-1 to meet the European biodiesel standard EN 14214. The type of fatty acids found in the oil can have a profound effect on the biodiesel quality. The fatty acid chain length and degree of saturation (determined by the number of double bonds) affects properties such as the viscosity, cold flow plug point, iodine number and cetane number of the fuel (Ramos et al., 2009). For biodiesel production, it is therefore important to maximize not only total lipid production, but also TAG content and appropriate fatty acid profile.

Lipid synthesis relies on carbon compounds generated from CO2 by photosynthesis, as well as energy and reducing power (in the form of ATP and NAD(P)H respectively). The latter are produced during the light reactions of photosynthesis, while CO2 uptake is mediated by the Calvin cycle during the dark reactions of photosynthesis. The output of the Calvin cycle is a three-carbon compound (glyceraldehyde 3-phosphate), which is converted through glycolysis into acetyl CoA. The conversion of acetyl CoA to malonyl CoA is the first committed step in lipid biosynthesis (Livne & Sukenik, 1992). Throughout metabolism there are a number of branch points at which metabolic intermediates are partitioned between the synthesis of lipids and other products such as carbohydrates and proteins (Lv et al., 2010). For example, acetyl CoA is a substrate for lipid synthesis as well as entry into the TCA cycle, which generates energy and biosynthetic precursors for proteins and nucleic acids. Both external and internal constraints, such as the availability of nutrients and the enzymatic reaction rates, limit the supply of metabolic intermediates. The production of storage lipids is particularly energy and resource intensive (Dennis et al., 1998; Roessler, 1990) and therefore usually occurs at conditions of reduced growth.
