Pretreatments to extraction

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Intracellular oils can be difficult to extract from biomass due to the presence of intact cells. Cellular disruption greatly enhances the bioavailability of compounds and has been found to be necessary for recovering intracellular products from microalgae (Yongmanitchai and Ward, 1989;

Wen and Chen, 2003; Ward and Singh, 2005). A number of physical techniques have been studied and reviewed, including the use of ultrasonifica-tion, high-pressure homogenizers and agitation of biomass in the presence of glass beads (Goto et al., 1993; Mendes-Pinto et al., 2001; Martinez et al., 2003). Most of these techniques are effective in improving product recovery.

The concept of broken and intact cells is introduced to possibly explain the falling extraction rate (FER) period (Barclay, 1997; Belarbi et al., 2000; Kyle, 2001; Molina Grima et al., 2003). This concept takes into account the structure of biological materials where the solute is found in various parts of the cell, especially within the cell walls where the resistance to mass transfer is the largest. In looking at the bulk material being subjected to SFE, a greater number of ruptured cells are seen at the surface since these are vulnerable to the mechanical preparations necessary for successful extractions of the material. The constant extraction rate (CER) period is influenced by these open cells and a fast extraction occurs. This initial highrate extraction decreases (FER) and is followed by the much slower extraction of the oil diffusing from the intact cells diffusion controlled (DC) regime.

Cellular disruption greatly enhances the bioavailability of compounds and has been found to be necessary for recovering intracellular products from microalgae (Kyle, 1996; Robles Medina et al., 1998). High-pressure homogenizers, agitation of biomass with glass beads and even ultrasonifica-tion have been studied (O'Brien et al., 1993; Cheng et al., 1999). Different cellular disruption techniques were assessed for acetone recovery of the carotenoid astaxanthin from cells of Haematococcus pluvialis, a microalga used in the aquaculture industry (Robles Medina et al., 1998) (Fig. 10.4). Biomass that underwent more physical pretreatments, autoclaving and homogenizing, produced three times as much astaxanthin as the other chemical treatments. These physical alterations increased the total amount of carotenoids produced by up to four times compared with the intact cells. The chemical treatments with acid, alkali and enzymes only slightly increased carotenoid extraction from 4 mg/g dry weight of biomass to 6 and 8 mg/g dry biomass. Therefore, mechanical disruptions are effective treatments, resulting in a high yield with no detrimental effects observed during processing.

Steam explosion

Steam explosion is an extensively studied technique for the pretreatment of lignocellulosic materials, such as hard woods and agricultural residues, for ethanol production (Zheng et al., 1998; Sun and Cheng, 2002). In this method, chipped biomass is exposed to high-pressure steam and the pressure is then swiftly reduced, which allows the water molecules that penetrated the substrate structure to escape in an explosive fashion. In this process the lignocellulosic structures are disrupted to increase the


Fig. 10.4 Effects of cellular disruption techniques on the extraction of total caro-tenoids into acetone from Haematococcus biomass. Key: 1, control (intact cells); 2, autoclave; 3, hydrochloric acid, 15 min; 4, hydrochloric acid, 30 min; 5, sodium hydroxide, 15 min; 6, sodium hydroxide, 30 min; 7, enzyme; 8, high-pressure homog-enizer; 9, spray drying (from Robles Medina et al., 1998).

accessibility of cellulose to enzymes and cause hemicellulose degradation. Steam explosion has enhanced the cellulose hydrolysis rate and increased glucose yield from 40 to 80% (Gregg and Saddler, 1996). The effectiveness of steam explosion is influenced by the saturation time, temperature, substrate size and substrate moisture content, with a lower temperature and longer residence time being more favorable (Duff and Murray, 1996; Wright, 1998). The advantages of steam explosion include the low energy requirement and the fact that there are no recycling or environmental costs (Sun and Cheng, 2002). The disadvantages associated with steam explosion when applied to microorganisms would be the high temperatures necessary for implementation, between 160 and 260 °C. This temperature range would alter PUFAs and other thermally labile substances.

CO2 explosion

One novel approach for enhancing CO2 SFE of fungal lipids is through the application of a CO2-explosion pretreatment. This process is analogous to the steam explosion and ammonia fiber explosion (AFEX) processes originally designed to improve the digestibility of lignocellulosic products such as wood and corn stover (Chisti and Mooyoung, 1986; Bermejo Roman et al., 2002). CO2 explosion involves saturation of a substrate with high-pressure CO2 for a given time. This extended contact time allows the CO2

molecules to penetrate the cellular structure. Then, the pressure is instantaneously released, causing the CO2 to flash violently and break the cells apart, theoretically increasing the amount of surface oil for CO2 SFE. Steam explosion requires high temperatures associated with the high pressure required for steam (Dale and Moreira, 1982; Dale et al., 1984; Holtzapple et al., 1991, 1992). AFEX has a lower operation temperature; however, the ammonia can have chemical effects on the substrate and ammonia recycling is necessary (Sun and Cheng, 2002). CO2 explosion can operate at the same low-temperature, high-pressure conditions as SFE and use the same non-toxic, non-reactive solvent, making it more attractive than steam and ammonia solvents.

A CO2-explosion process is similar to steam and ammonia explosion in that there is a saturation phase and a violent release of pressure resulting in a ruptured substrate structure. CO2 explosion uses supercritical CO2 as the saturating solvent. Even though a supercritical fluid possesses a liquid-like density over much of the range of interest, it exhibits gas-like transport properties of diffusivity and viscosity (McHugh and Krukonis, 1994; Brunner, 2005). Figure 10.2 shows the self-diffusivity behavior of CO2 over a range of pressures and temperatures. A characteristic diffusivity value for an SCF at its critical temperature and pressure is 0.7 x 10-3 cm2/s, and a four-fold increase in pressure would result in a decrease of diffusivity to approximately 0.2 x 10-3 cm2/s (Brunner, 1994, 2005). Thereby, an increase in density results in a decrease in diffusivity.

So far, CO2-explosion experiments have been limited to lignocellulosic substrates for potential ethanol production. The effectiveness of this pre-treatment was quantified via enzymatic hydrolysis of the cellulose to yield sugars. CO2-explosion pretreatment on pure cellulose and industrially processed materials improved glucose yield by as much as 50% (Zheng et al., 1995, 1998). With these documented improvements in an extremely structured substrate, there is the potential for microbial cell disruption with the CO2-explosion process.

CO2-explosion effects on pine and aspen and found glucose yield 12 to 14% beyond untreated materials (Kim and Hong, 2001). A major advantage of CO2 explosion is the fact that this inexpensive solvent is already employed in the SFE process in food, pharmaceutical and nutraceutical industries. CO2 explosion can operate at the same low-temperature, high-pressure conditions as SFE.

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