The principle of extracting lipids from micro-algae is based on the basic chemistry concept of “like dissolving like”. According to Halim et al. (Halim, et al., 2012) the extraction process can be divided into five steps:
1. Penetration of organic solvent through the organic solvent mixture 2. Interaction of organic solvent with the lipids
3. Formation of organic solvent-lipid-complex
4. Diffusion of organic solvent-lipids complex across the cell membrane
5. Diffusion of organic solvent-lipids complex across the static organic solvent film into the bulk organic solvent
The use of a specific solvent depends on various criteria:
The targeted molecule needs to be soluble in the used solvent/solvent mixture
Low boiling point
Non-toxic
Easy recovery of the solvent
Hexane is cited as a highly efficient solvent. It is characterised by high extraction capability and low cost (Harun et al. 2010). In addition, Fajardo et al. (Fajardo et al. 2007) investigated a two-stage process by which the lipid extraction could be improved. In the first stage ethanol was used as the solvent, and in the second stage hexane, in order to clean the extracted lipids and to attain oil yields of over 80 %.
Chloroform/methanol (1:2 v/v) is the most frequently used solvent system for lipid extraction from any living tissues. Folch et al. developed this method for isolating brain tissue. The residual water in algal cells acts as ternary component. That enables the complete extraction of both neutral and polar lipids.
The use of a chloroform/methanol mixture leads to a fast and quantitative extraction of lipids but due to its toxicity chloroform implies a great environmental and health risk. Alternatively hexane/isopropanol (3:2 v/v) (HIP) has been suggested as a low-toxic substitute (Guckert, et al., 1988; Lee, et al., 1998;
Nagle, et al., 1990). For micro-algae lipid extraction HIP showed more affinity towards neutral lipids compared to chloroform/methanol. Guckert and collegues attributed this towards HIP’s inability to extract membrane-bound polar lipids (Guckert, et al., 1988).
Alcohols have a strong affinity towards membrane-bound lipid complexes as they are able to form hydrogen bonds. Their polar nature, however, limits the extractability of neutral lipids. For this reason, alcohols are usually combined with non-polar solvents such as hexane or chloroform (Halim, et al., 2011).
Studies on TAG extraction efficiency of single solvents and solvent mixtures from Selenastrum rinoi were carried out at the University of Applied Science at Senftenberg. The standard extraction method with chloroform/methanol was used as reference.
Figure 4.6 Solvent screening (alga: Selenastrum rinoi) (HS Lausitz 2013)
Compared to the chloroform/methanol extraction method especially short-chained alcohols show great potential for extracting TAGs from the used alga. Increasing of chain length resulted in a drop of the TAG content in the entire extract.
For solvent mixtures an increasing ratio of non-polar solvent leads to a higher TAG content in the extract when compared to the single solvents.
Studies, carried out by Nagle and Lemke, came to similar results. They evaluated lipid extracting efficiency of three solvents (1-butanol, hexane/isopropanol, ethanol) from Chaetoceras muelleri (Nagle, et al., 1990). The ternary water/methanol/chloroform mixture served as control. The control was found to be the most effective extraction mixture. Compared to this all used solvents were fairly effective at extracting a pure lipid product with 1-butanol being the best of all three.
Most of the laboratory-scale solvent extractions reported in literature were performed as batch-processes, although they are limited by lipid mass transfer equilibrium. Continuous extraction processes however require a large amount of solvent (Halim, et al., 2012).
0 10 20 30 40 50 60 70 80 90 100
Extracted fatty acids as percentage of total fatty acids in %
Figure 4.7 Solvent mixture screening (alga: Selenastrum rinoi) (HS Lausitz 2013)
A special form of solvent extraction is soxhlet extraction, which is used in particular on a laboratory scale. In a special apparatus (Figure 4.8) the FA and TAG are extracted by repeated washing (percolation) with the recycled organic solvent, such as hexane or petroleum ether (Oilgae 2011). As a result of the repeated evaporation and condensation of the solvent, uncharged solvent is always brought into contact with the material being extracted and the mass transfer equilibrium is by-passed.
Figure 4.8 Soxhlet apparatus
0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00
Extracted fatty acids as percentage of total fatty acids in %
Guckert et al. conducted a comparative study (using Chlorella) of various procedures featuring different solvents and techniques:
- Soxhlet extraction with methyl chloride/methanol,
- Extraction according to Bligh and Dryer (Bligh and Dyer 1959) with methyl chloride/methanol and
- Extraction with hexane/isopropyl/water (Guckert et al. 1988)
It was demonstrated that the procedures, as expected, led to differing lipid yields. They differ also, however, in their selectivity with regard to certain lipid classes (neutral lipids, glycolipids, polar lipids).
Consequently, only the Bligh and Dryer method delivers maximum extraction for all lipid classes (Guckert et al. 1988). These results are less interesting for technical applications to produce FAs and TAGs, but certainly are of significance for biochemical analysis relating to lipid formation in the micro-alga Chlorella.
Frenz et al. subjected 18 organic solvents with differing polarity to screening for the production of hydrocarbons from a 'living' culture of the micro-alga Botryococcus braunii (Frenz et al. 1989). A key assessment criterion – alongside the highest possible hydrocarbon yield – was the so-called biocompatibility. This refers to the sustaining of photosynthesis activity in the extraction process. The studies showed that high hydrocarbon yields are attainable with strongly polarised solvents (alcohols, ketones). At the same time, however, the ability of the cells to regrow and again produce hydrocarbons following extraction is lost. By contrast, photosynthesis activity is largely retained when using weakly polarised or non-polar solvents (n-alkanes). Any water present inhibits the extraction process, so that only low oil yields are attained. If the algal biomass is dewatered (centrifuging, filtration) high extraction yields can be attained with weakly polarised solvents while at the same time maintaining biocompatibility (Frenz et al. 1989). Table 4.4 sets out the relevant results for seven solvents.
Table 4.4 Hydrocarbon yields and photosynthesis activity following extraction (Frenz et al. 1989)
Extraction agent Yield1) Activity2)
n-hexane 70.6 % 80.7 %
n-heptane 63.7 % 83.2 %
n-octane 64.8 % 86.7 %
n-dodecane 63.1 % 90.7 %
Dodecyl acetate 45.5 % 69.7 %
Dihexyl ether 61.5 % 68.3 %
Dodecanedioic acid-diethyl ester 47.5 % 29.2 %
1) Hydrocarbon yield referred to total hydrocarbon content (dry)
2) Photosynthesis activity (24 h after solvent treatment) referred to original activity
A procedure of the kind is interesting for continuous or periodic removal of products from living cell cultures and offers the following advantages:
- Improved cost-effectiveness and
- Fewer inhibiting influences on the product (Frenz et al. 1989).