The manuscript prepared for Journal of Applied Phycology:

Exergy based screening of biocompatible solvents for in situ lipid extraction from Chlorella vulgaris

Hassan Nezammahalleha, MohsenNosratia, FaezehGhanatib, SeyedAbbasShojaosadatia

aBiotechnology Group, Faculty of Chemical Engineering, TarbiatModares University, Tehran, Iran

bDepartment of Plant Science, Faculty of Biological Sciences, TarbiatModares University, Tehran, Iran

Permeation of solvents through cell membrane

Permeation of a solvent through cytoplasmic membrane can be described by diffusive flux as. Where,,, and are permeability, solvent concentration in aqueous medium, and the one in the membrane, respectively. The higher solvent solubility led to higher diffusive flux of the solvent. The permeability of the solvent is defined as follow:

Where, shows the partition coefficient of membrane/water system ( in Table 2 of the main text), represents the diffusion coefficient of a solvent through membrane, and is the thickness of the membrane. The diffusion coefficient of a solvent can be obtained by the Stokes-Einstein expression as:

Where, , , , , are Boltzmann constant, temperature, boundary condition parameter depending on the relative size of solute and solvent, the radius of diffusing solvent, and viscosity of the membrane, respectively.As an example, the diffusion coefficient of n-octane and 1-hexanol from hydrated cell wallwith a thickness of about 20 nm (Yamamoto 2004)are about 0.7 ×10-9 and 0.8×10-9 m2s-1 and their permeabilities are 796.2 and 3.2, respectively.Thus, the diffusive flux of these hydrophobic n-octane and hydrophilic 1-hexanol solvents are 0.0006 and 0.037 mol m-2s-1. The hydrophilic solvents are about two orders of magnitude more diffusive than the hydrophobic solvents mainly because of higher solubility.

Frenz et al.(1988) have shown that the kinetic limitation to solvent permeation from algal cell wall resulted in least lipid recovery by apolar alkane solvents. This limitation is imposed by the presence of water molecules surrounding the algal cells.However, the alkanes were of higher lipid recovery from filtered algae than polar solvents due to higher permeability.

Solvent screening guidelines for in situ lipid extraction from microalgae

High distribution coefficient of lipid in solvent / Low boiling point
High selectivity for the target compound / Inexpensive
Low emulsion forming tendency / Bulk availability and handling ease
Immiscibility with water / Nonhazardous nature
High thermal and chemical stability / High extracting efficiency
Non biodegradable / Density difference

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Model of microalgal total lipid

Table S1a Physicochemical properties and chemical exergies of the neutral lipids modeled for C. vulgaris

Lipid type / Composition, % / Molecular weight g mol-1 / Molar entropy Jmol-1K-1 / Higher heating value, kJ mol-1 / Molar chemical exergy, kJ mol-1
TAG-14:00 / 8.99 / 722 / 4968.66 / 28937.6 / 28019.93
TAG-16:00 / 25.11 / 806 / 5088.14 / 32855.6 / 31948.55
TAG-16:01 / 2.00 / 800 / 5095.03 / 32357.6 / 31480.91
TAG-16:02 / 10.01 / 794 / 5101.92 / 31859.6 / 31013.27
TAG-16:03 / 8.99 / 788 / 5108.81 / 31361.6 / 30545.63
TAG-18:00 / 0.91 / 890 / 5207.62 / 36773.6 / 35877.17
TAG-18:01 / 4.99 / 884 / 5194.54 / 36275.6 / 35409.53
TAG-18:02 / 19.99 / 878 / 5221.4 / 35777.6 / 34941.89
TAG-18:03 / 19.00 / 872 / 5228.29 / 35279.6 / 34474.25

aData obtained from (Peralta-Ruiz 2013)

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Microalgae staining

FigS1Microscopy image of stained Chlorella vulgaris by Evans blue dye. Red arrows show cell debris formed by lysed cells. Blue arrows show themicroalgae species stained blue due to loss of membrane selective permeability. Green arrows show the unstained ones with cell membranes impermeable to such macromolecules as Evans blue.Scale bar corresponds to 10 µm.

Fig S2 Microscopy image of clumped microalgae cells during Evans blue staining. Scale bar corresponds to 10 µm.

Measurement of microalgal lipid content

Microalgal total lipid content was measured gravimetrically by the modified Bligh and Dyer method (Zhu 2002). In brief, the microalgae were collected by centrifugation at 5000 rpm for 4 min. The concentrated algae were washed with deionized water three times prior to being freeze dried. One gram dried microalgae was pulverized in a mortar and 0.1 g of the sample was mixed with 2 mL chloroform/methanol (2:1). The mixture was ultrasonicated for 10 min at 60 W and agitated by vortex for 2 min at room temperature. To separate the phases, the sample was centrifuged at 5000 rpm for 5 min. The lipid containing lower phase was separated and poured in a test tube in order to evaporate all the solvent. These extraction and separation processes were repeated three times in order to maximize lipid extraction. Total lipid content was calculated as follow:

Where,, , and are the dry microalgal biomass, the weight of empty test tube, and the weight of test tube containing the extracted lipids, respectively.

Percentage of extracted lipid from viable cells

In situ lipid extraction process from algal suspension can be done by three possible mechanisms, namely, products excretion, cell permeabilization, and cell death. To find out the amount of extracted lipids by product excretion mechanism without any detrimental effect on cell, the percentage of extracted lipids from live cells was estimated by the following equation:

Where,is the percentage of in situ extracted lipids, and are the percentage of the extracted lipids from viable and dead cells, respectively. , , and are respectively the number of live cells, the number of dead ones, and the total number of cells. The number of live cells is related to cell viability percentage () which was determined by microscopy examination. Accordingly, the equation can be simplified to the following relation:

The extraction yield from dead cells can be taken xd = 0.32, the maximum yield that can be reached by the solvents. Thus, the lipid extraction from live cells by apolar solvents was calculated and presented in Table S2. This calculation was conducted for in situ lipid extraction by only apolar solvents because no viable cells would be left in the column during in situ extraction by polar solvents, as find from biological activity investigations.

Table S2 Percentage of extracted lipids from viable cells by different solvents after distinct extraction times

Percentage of
Extracted Lipids / Extraction time
Solvent type / 5 / 10 / 15 / 20
n-hexane / 3.814 / 5.281 / 6.792 / NVa
n-octane / 1.292 / 2.899 / 4.329 / 5.829
n-decane / 0 / 0 / 1.627 / 3.026

aNV: No viable cells

References

Frenz J, Largeau, C., Casadevall, E., Kollerup, F., Daugulis, A. J. (1988) Hydrocarbon recovery and biocompatibility of solvents for extraction from cultures of Botryococcus braunii Biotechnology and Bioengineering 34:755-762

Peralta-Ruiz Y, Gonzalez-Delgado, A.-D., Kafarov, V. (2013) Evaluation of alternatives for microalgae oil extraction based on exergy analysis Applied Energy 101:226-236

Yamamoto M, Fujishita, M, Hirata, A., Kawano, S. (2004) Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlorella vulgaris (Chlorophyta, Trebouxiophyceae) Journal of Plant Research 117:257-264

Zhu M, Zhou, P. P., Yu, L. J. (2002) Extraction of lipids from Mortierella alpina and enrichment of arachidonic acid from the fungal lipids Bioresource Technology 84:93-95

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