Bioactive lipids containing polyunsatured fatty acids (PUFA), especially n-3 PUFA such as a–linolenic acid (ALA, C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3), and docosahexaenoic acid (DHA, C22:6n-3), have been shown to be effective in preventing or treating several diseases including cardiovascular disorders, cancer, type 2 diabetes, inflammatory bowel disorders, asthma, arthritis, kidney and skin disorders, depression and schizophrenia. Marine fish lipid is the main conventional source of n-3 PUFA used in functional food, nutraceuticals and pharmaceuticals, but microalgae are recognised as an additional important source. Although the oil extraction from lipid-bearing biomasses is normally carried out by using organic solvents, supercritical CO2 (ScCO2) is regarded with interest being safer than hexane and offering a negligible environmental impact, a shorter extraction time and a high quality final product, above all under a toxicological point of view. Whilst some experimental papers are available on the supercritical fluid extraction (SFE) of oil from microalgal strains, only limited information exists on the kinetics of the process. In such a contest, a mathematical model able to describe the kinetics of a SFE process was applied to the extraction with ScCO2 of oil from the Nannochloropsis sp, a marine microalga commonly used in aquaculture and characterised by an oil with a high proportion of n-3 PUFA.

To optimise the extraction parameters, a kinetic approach developed during a previous research activity was adopted. This approach, based on the Fick’s law, uses the following exponential equation to describe the evolution of extracted oil over time (t):

where: Oe = amount (g) of oil extracted at a random time t per gram of microalgal biomass submitted to SFE (adimensional); H* = adimensional constant, ranging from 0 to 1, related to the equilibrium constant H (H* = H/(H+1)); [Os] = oil concentration in starting material (adimensional); k = kinetic constant (s-1).

The extraction rate (R) calculated as first derivative of the exponential equation (1):

reaches its maximum value (Rmax) at the beginning of extraction, when t is close to 0:

the value of Rmax (s-1) was assumed as an index to evaluate the efficiency of the SFE system vs the oil fraction of microalga. In particular, while the constant k gives information on the kinetics of the SFE, the product H*×[Os], representing the asymptotic value of the extraction curve when t ® ¥, measures the maximum amount of oil extractable in the working conditions adopted. In presence of a highly efficient SFE process, H* tends to 1 and therefore the maximum amount of oil extractable per unit of biomass is equal to the concentration of oil in the starting material. The identification of the best values to be assigned to the equation parameters k and H*×[Os] was carried out by a commercially available statistical program.

Table 26: Amount (mg) of oil extracted per gram of Nannochloropsis submitted to SFE, as a

function of run time and working conditions (temperature and pressure) adopted

On the basis of the data tabled, the following remarks can be done:

a) ScCO2 is proven to be a good solvent for Nannochloropsis oil, as testified by the values assumed by the equation parameter H*×[Os]. This parameter, which represents the mgs of oil extracted from 1 g of microalga when the equilibrium is reached (extraction time = ¥), assumes in fact for all SFE runs values close to the concentration of oil in starting material and to that obtained when percolation with n-hexane is adopted;

b) P highly affects the kinetics of SFE, as confirmed by the values assumed by the constant k when working at the same T.

c) also the increase of T affects the kinetics of SFE, as testified by the value that k assumes when working at the same P, but such influence appears relatively low;

d) the binomial pressure-temperature is more crucial than T and P alone in determining the kinetics of the SFE process.

e) in all conditions adopted, the extraction with ScCO2 resulted faster than that carried out by percolation with n-hexane, as testified by the values assumed by k and/or Rmax. For example, when working at the hardest SFE conditions, the rate at the beginning of extraction (Rmax) is about seven times that measured using the Soxhlet apparatus.

To relate Rmax to density of ScCO2, the value of which is influenced by both the pressure and temperature adopted, the following equation introduced was adopted:

where: R*max is Rmax expressed in grams of extracted oil per litre of ScCO2 flowed through the bed of microalga; r = density of ScCO2 (g×l-1); a, b, c = equation parameters; T = temperature (°K). The identification of the best values to be assigned to the equation parameters a, b and c was carried out by the statistical program introduced above. Table 27 reports the value of the parameters a, b and c calculated using the experimental values of Rmax reported in Table 2. Each value was previously expressed in g×l-1 by using the following equation:

where: m = amount of lyophilised microalga submitted to each SFE run (g); r = density of ScCO2 at T and P adopted (g×l-1); F = flow rate of ScCO2 (g×s-1);

Table 27: Values of parameters involved in the Chrastil equation 4 adopted to relate the solvent power of ScCO2 (evaluated by R*max) to its density (r). c.i. = confidence interval (p = 0.05); r = correlation coefficient