The efficiency of the extraction of microalgal valuable components like lipids, proteins, pigments, polysaccharides is closely linked to the level of cells disintegration. When the intact cells are disintegrated, the intracellular molecules are released from the cell structures and then diffuse into the surrounding medium. The methods of disintegrating cells are classified according to their mechanical or non-mechanical mode of action (Figure 17).
Mechanical and physical methods include bead mill, ultrasound, autoclave, freeze-drying and microwaves, while non-mechanical methods involve cell lysis by enzymes, acidic or basic treatments, or osmotic shock. These different techniques can be used to assist the solvent extraction or can constitute a pretreatment before the actual extraction step.
41
Figure 17- The processes of microalgal cell disintegration.
1.8.2.1. Non-mechanical cell wall disintegration techniques
These methods are labelled as environmental friendly. The first method involves the use of enzymes which allow to break the cell wall with a release of the cellular content without the alteration of the present molecules (Figure 18). However, the difficulty is to choose the appropriate enzyme to hydrolyze the microalgae cells. For this reason, it is desirable to know the composition of the cell wall to be treated. Enzymes like chitinase, lysozyme, pectinase, sulfatase, β- glucuronidase, and laminarinase had the widest effect across Nannochloropsis strains. Chlorella strains are typically more sensitive to chitinases and lysozymes which degrade the outer surface of the cell wall and removes hair-like fibers protruding from the surface [11].
Figure 18- Disruption of cell walls using degrading enzymes for enhanced lipid recovery [128].
Cell disintegration methods
Mechanical/Physical Microwaves Pulsed electric field Bead beating Autoclave Ultrasound Chemical/Biological Acid or base Enzymes Osmotic shock
42 A second non-mechanical method is “Osmotic shock”. The increase in salinity in an aqueous medium is at the origin of a hypertonic medium which constitutes an osmotic shock for marine microalgae. As a result, the cells will contract, thus promoting the diffusion of cellular content to the outside. This technique proved to be effective for the extraction of lipids from wet
Chlamydomonas reinhardtii with NaCl or sorbitol as osmotic agents at different concentrations as
reported by Yoo et al. [129].
1.8.2.2. Mechanical cell wall disintegration techniques 1.8.2.2.1 The “bead beating"
Bead beating, also known as bead mill, is a very simple technique of cell degradation. It consists of a very strong agitation of the medium comprising the solution of microalgae and quartz or metal beads [130]. The collision and the friction of these beads with the cells cause a disintegration of the latter. It is a very advantageous technique in terms of efficiency and energy consumption and can be applied on an industrial scale (Figure 19).
For the extraction of lipids from Botryococcus braunii, among all the techniques tested, bead beating was very effective in pretreatment followed by chloroform / methanol (2/1: v / v) extraction [131].
Figure 19- Cell disruption by Bead Milling [132].
Over-disruption may impact and cause denaturation of the desired component. The time to disrupt the cells and the reproducibility of the method become more important factors for production scale processes. It is usually necessary to establish the minimum force of the disruption method that will
43 yield the best product [133]. Additionally, once the cells are disrupted, it is often essential to protect the desired product from normal biological processes and from oxidation or other chemical events.
1.8.2.2.2 Microwaves
Microwaves have the particularity of instantaneously heating polar molecules and especially water molecules. Since microalgae cells are made up of 65-85% of water, following exposure to microwaves, the cells are subject to significant constraints that result in cells disintegration and thus favoring the release of molecules of interest (Figure 20). The phenomenon can therefore lead to increase the efficiency of microalgae lipid extraction procedures. However, microwaves require working from wet biomass. The study of Lee et al [27] also highlighted the effectiveness of microwaves compared to other techniques (such as bead beating, osmotic shock and autoclave) on different microalgae such as Botryococcus sp., Chlorella vulgaris and Scenedesmus. This process can reduce the production costs of microalgae biodiesel as reported by Yong-Ming Dai et al, [15].
Figure 20- Microalgae extraction mechanism using microwave irradiation [134].
1.8.2.2.3. Pulsed electric fields (PEF)
PEF is another potential technique in which cells are exposed to strong electric fields for very short periods of time. Electrical pulses make the cells permeable, thus implying an increase in the transfer of material through the membranes. This very promising technique can be applied in the
44 pretreatment of microalgal biomass before solvent extraction. Zbinden et al., [127] showed the effectiveness of PEF on lipid extraction of the microalgae Ankistrodesmus falcatus
1.8.2.2.4. Ultrasound Assisted Extraction (UAE)
Ultrasound Assisted Extraction has the advantage of being a technique of disintegration at low temperature, thus leading to a decrease of the thermal denaturation of the sensitive compounds. Ultrasound is easily transposable at industrial scale and can operate continuously [135].
When low frequency ultrasound is applied in a liquid, a cavitation phenomenon occurs, the formation of tiny bubbles within the liquid medium, which is responsible for cell damage [136]. When these bubbles reach resonance size, they collapse releasing mechanical energy in the form of shock waves equivalent to several thousand atmospheres of pressure. The shock waves disrupt cells present in suspension. As ultrasound breaks the cell wall mechanically by cavitation shear forces, and facilitates the transfer of inner components like lipids, proteins, pigments and carbohydrates from the cell into the solvent (Figure 21). In ultrasonic applied field, a frequency range between 20-25 to 600 kHz (mega Sonics) is commonly used for cell disruption and separation of particles from biomass as reported by Pablo Juliano et al., Chemat et al., [31], [137]. The duration of ultrasound needed depends on the cell type, the sample size and the cell concentration. In microalgal field, Jaeschke et al., [138] find ultrasound as an alternative technology to extract carotenoids and lipids from Heterochlorella luteoviridisultra. Prabakaram and Ravindram [139] carried out a study on three species of microalgae on which different methods of disintegration were tested (autoclave, bead beating, microwaves, osmotic shock and ultrasound) where ultrasound appears to be the technique of choice to obtain the best lipid yield.
In 2016, Ferreira et al [141], studied the effect of low frequency acoustic ultrasound on microalgae
Chlorella vulgaris, Nannochloropsis oculata and Scenedesmus obliquus for carbohydrates,
proteins, lipids and pigments valorization. But the study did not exclude the use of toxic and harmful organic and high volatile solvent like n-hexane, chloroform, 2-butanol, isopropanol, ethanol and methanol.
As reported by Oohashi et al. [142], it is generally accepted that humans cannot perceive sounds in the frequency range above 20 kHz, and the use of such "inaudible" high-frequency components may significantly affect the brain activity of listeners. High noise levels require hearing protection
45 and sonic enclosures. Thus using ultrasound may cause harmful effect on human health. Therefore, during this thesis, low ultrasonic frequency (having a low acoustic non-hurt able frequency 12 kHz) will be carried out on Spirulina sp. and Chlorella vulgaris in the absence of any toxic solvent
(Figure 22).
Figure 21- Graphical representation of cavitation-bubble collapsing and releasing inner components in three steps (bubble and cell representation, breakdown of the cell wall and bubble collapse, finally diffusion of the solvent through the cellular disruption and release of the compounds) and the principle of acoustic cavitation [140].
46 Figure 22- Schematic diagram of experimental apparatus used for microalgal extraction under
ultrasound irradiation [143].