Methods of extraction

Fatty acid composition is vital to determining the quality and physical properties of biodiesel.

It is therefore beneficial to have a rapid FAME extraction and screening for algae from environmental samples (Guzman et al., 2010). Accurate lipid profiling of algae is usually carried out using GC-MS, which not only requires a large sample of biomass but is also coupled with lengthy sample preparation often involving lyophilisation, extraction, purification and transesterification (Mutanda et al., 2011). Comparisons of different lipid extraction methods on different algal species have been reported, in attempts to shorten and simplify the extraction of FAMEs to more rapidly obtain experimental results.

The extraction of FAMEs is usually a 2-step process. Firstly the extraction of the TAGs from biomass, followed by a transesterification reaction yielding FAMEs (and glycerol as a by-product) commonly analysed by GC-MS (MacDougall et al., 2011) yet can also be analysed with FT-IR spectroscopy (Pistorius et al., 2009). After transesterification water is added to remove methanol and other impurities (Cooney et al., 2009).

There are many ways to extract lipids from microalgae (Figure 7.1), pressing, homogenization, milling, solvent extraction, supercritical fluid, enzymes, ultrasonic-assisted extraction, osmotic shock and novel methods such as ‘milking’ (Packer 2009). Depending on moisture content, different processing techniques may be required. For example dry biomass is suitable for gasification, pyrolysis or combustion, whereas wet material is more suited to enzymatic fermenting (McKendry, 2002).

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Figure 7.1: A generalised flow diagram showing the variety of extraction methods which can be applied to wet and dry algal biomass.

Adapted from Mercer and Armenta (2011).

All extraction methods have their benefits and drawbacks and may be suitable for certain applications only or used in conjunction with one another (Table 7.1). Physical and chemical methods usually involve the addition of large volumes of solvent (Mercer and Armenta, 2011). Water associated with biomass can block solvent from getting in and out of cells (Cooney et al., 2009). Drying of material can reduce the quantity of solvents needed (Hass and Wagner, 2011), yet requires a higher energy input (dewatering algal biomass) and is undesirable. Solvent must be able to permeate biomass fully and match polarity of the product to be extracted. Benzene, cyclohexane, hexane, acetone and chloroform have proven effective on microalgal pastes (Mercer and Armenta, 2011). A mixture of two or more solvents can drastically alter the extraction properties of a solvent. Co-polar solvents can make membranes more porous to better extract lipid from inside cells (Cooney et al., 2009).

Chloroform:methanol (2:1) as a solvent routinely yields the highest FAME content after transesterification (Johnson and Wen, 2009). Solvent extraction alone (i.e. in a soxhlet) is rarely sufficient to extract all oil (Mercer and Armenta, 2011). Increased temperature can increase solvation power of a solvent (by overcoming solute-solute and solute-matrix interactions). Additionally, increased pressure helps transport solvent to products trapped in pores and matrices. Mechanical methods can offset the need for higher temperature and pressure (Cooney et al., 2009).

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Table 7.1: Advantages and disadvantages of a variety of lipid extraction methods.

Adapted from Mercer and Armenta (2011).

Extraction Method Advantages Disadvantages

Solvent extraction Solvents are relatively inexpensive results are reproducible

Most organic solvents are flammable and toxic, solvent recovery is expensive, large volumes required

Pressing Easy to use, no solvent needed Large amount of sample required, slow process

Supercritical fluid Non-toxic, so solvent residue in product, non-flammable, simple operation

High power consumption, difficult to scale up.

Microwave Rapid extraction, high yields Needs optimisation, difficult to scale up, may affect FAMEs

Enzymes Could be efficient, safe Expensive

Enzymes have the potential to partially or fully degrade the cell wall, yet are reliant on the nature of the species in use and often too expensive to use on an industrial scale. Enzymes for transesterification whilst expensive show good tolerance to free fatty acid chain levels. As such, immobilization of enzymes may prove useful in process integration (Um and Kim, 2009). Ultra-sonication at high frequencies causes ‘cavitation’ (creating bubbles in solvent which violently collapse) producing shockwaves which can disrupt the cell wall. However this is an energy intensive process, which also heats the solvent very quickly and is difficult to scale up (Mercer and Armenta, 2011).

Supercritical fluid extraction is a rapid method of extraction, eliminating the use of solvents and as such, gives a clean product without the need for purification (no heavy metals present, free of inorganic salts). Alterations to its specificity can be made with the addition of solvents, pressure, temperature, CO₂ flow rate and extraction time. Yields are high, yet the process is expensive and algae must be dry as moisture acts as barrier, reducing contact algae and CO₂ (Mercer and Armenta, 2011).

Microwaving is a rapid extraction method that works on principle of selective heating.

Microwaves directly affect polar materials and when used on biomass, trace quantities of moisture evaporate generating a significant amount of pressure that stresses the cell to rupture and release contents (Mercer and Armenta, 2011). However the process may be difficult to scale up and still requires optimisation. Microwaves may also produce oxidative agents which could damage algal products. Preliminary results seem to suggest a huge reduction in extraction time and sufficiently more oil recovered (Balasubramanian et al., 2011).

182 Knowledge of intact lipid profiles could be useful such as selection of a strain for further research (e.g. by UHPLC-MS, currently under development at Bath). In situ transesterification has been shown to improve oil extraction (Hass and Wagner, 2011).Transesterification requires the addition of alcohol (acts as a catalyst) and heat, performed at a high pH. Unsaturated fatty acids can be problematic as they can create cross links (Singh et al., 2011).

Extraction followed by transesterification often gives incomplete recovery of lipids (Cooney et al., 2009). Direct transesterification of algal biomass has been proven to be very effective (at least 15-20% over extraction-transesterification reactions) (Mercer and Armenta, 2011); in particular for use with microwave extraction (Balasubramanian et al., 2011 and Patil et al., 2012). With this process it is important to include a cooling system (to keep temperatures

<100°C) for the solvent and uniform mixing (Patil et al., 2010). Small changes in temperature (5°C) and extraction time (min) can make a large difference in the quantity of recovered algaenan-producing species (Chapter 3.1.3) and the production of algaenan in Botryococcus spp. is believed to be linked to synthesis and exportation of oleic acids to the cell wall (Templier et al., 1993). Theoretically, other algaenan-producing species (such as C. emersonii) may also be suitable for culture in a biphasic ‘milking’ PBR.

Efficacy of the extraction method varies enormously depending on species and exact conditions deployed. Cell shape and structure of different species, no matter how minor can have an effect (Mercer and Armenta, 2011). For example a study by Lee et al. (2010), compared C. vulgaris, B.brauni, and Scenedesmus sp. over a variety of extraction methods;

soxhlet, autoclaving, bead beater, microwave, sonication and osmotic shock. Microwaving was consistently most successful in this study for maximum oil extraction, however other methods varied in their efficacy in extracting oils depending on species (Figure 7.2). Similar results were found in a study by Prabakaran and Ravindran (2011).

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Figure 7.2: A comparison of different lipid extraction methods using Botryococcus sp., C. vulgaris and Scenedesmus sp.

From Lee et al. (2010).

The extraction methods described are also commonly used for extraction of other products (such as β-carotene) (Hejazi et al., 2004). It is important to note that extraction methods used in the laboratory for recovery of microalgal oils, may not apply to industrial scale systems and certain methods of extraction will be more or less efficient depending on the details of operation (Mercer and Armenta, 2011). Upon scaling up an extraction process, the vast quantities of solvent required become impractical. Residual solvent contamination not only affects the final product but also machinery parts. Inorganic salts within solvents can reduce selectivity (i.e. additional polar extracts can result in poor flow characteristics). Recovery and recycling of solvents also adds to the expense (Cooney et al., 2009).

184 7.2 Results