3.1.1 Organic Solvent Extraction

Intracellular chlorophyll has traditionally been extracted from microalgal biomass using organic solvents [49]. The extraction process involves the organic solvent molecules penetrating through the cell membrane and dissolving the lipids as well as the lipoproteins of chloroplast membranes. Parameters which affect the yield of chlorophyll extraction by organic solvents include polarity of the organic solvents, storage conditions of the microalgal biomass, extraction duration, and number of extraction steps [44, 47]. Since chlorophyll is easily oxidized, extraction yield is also affected by the formation of degradation products. Chlorophyll starts degrading as soon as their molecules are exposed to excess light, atmospheric oxygen, high temperature, and acidic or basic pH condition [11 ] .

Acetone, methanol, and ethanol are three of the most common solvents used to recover chlorophyll from microalgae. Table 3 summarizes key findings from previ­ous studies on organic solvent extraction of microalgal chlorophyll. Jeffrey et al.

[24] , Simon and Helliwell [49], Sartory and Grobbelaar [44] found methanol and ethanol to be more efficient at extracting chlorophyll from microalgal biomass than acetone. Simon and Helliwell [49] conducted their sonication-assisted chlorophyll extractions in an ice bath and in the dark to prevent the formation of degradation products. They found that, with sonication, methanol recovered three times more chlorophyll than 90% acetone. Despite these findings, acetone remains the select primary solvent for chlorophyll extractions due to its known propensity to inhibit any chlorophyll degradation.

Macias-Sanchez et al. [28] recently used dimethyl formamide (DMF) to extract chlorophyll from microalgae and revealed the superiority of this solvent compared to the more traditional organic solvents, such as methanol, ethanol, and acetone. Extraction using DMF did not require prior cellular disruption as pigments were completely extracted after a few steps of soaking. Additionally, the chlorophyll remained stable for up to 20 days when stored in the dark at 5°C [32, 47]. The high toxicity associated with DMF, however, substantially decreased its appeal as an extraction solvent.

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Storage of the dried microalgal biomass at low temperatures (-18 or -20°C) was found to assist cell disruption and to promote chlorophyll extraction. In a study by Schumann et al. [47], freezing the biomass in liquid nitrogen followed by lyophili — sation and then storage at -18°C was found to be the optimal storage procedure.

Sartory and Grobbelaar [44] found the efficiency of chlorophyll extraction from fresh water microalgae to be optimal when the extraction was carried out at the solvent’s boiling point. It was shown that boiling the biomass for 3-5 min in metha­nol or acetone prior to 24-h extraction led to the complete recovery of chlorophyll a without the formation of any degradation products.

Such findings are contradictory to the general assumption that chlorophyll degraded upon slight temperature elevation.

The amount of chlorophyll extracted from a particular microalgal species was found to be highly dependent on its growth stage. Microalgae extracted in the sta­tionary growth phase were shown to have significantly higher amount of chloro­phyll a compared to the same species in the logarithmic phase [47] .