António F. Palavra

Supercritical carbon dioxide extraction of compounds with pharmaceutical importance from microalgae

Abstract Four microalgae ( Botryococcus braunii , Chlorella vulgaris , Dunaliella salina , Arthrospira maxima ) were object of supercritical CO 2 extraction studies, which were carried out in a flow apparatus at temperatures between 313.1 and 333.1 K and pressures up to 35.0 MPa. The microalga Botryococcus braunii produces extracellular alkadienes. Supercritical extracts obtained at 313.1 K, and pressures of 12.5, 20.0 and 30.0 MPa, were golden and limpid, unlike those obtained with organic solvents. The hydrocarbons of this microalga were selectively extracted and this selectivity increased with pressure. The microalga Chlorella vulgaris is a carotenoid producer of canthaxanthin and astaxanthin. The extraction yields of lipids and carotenoids were compared for whole, crushed and slightly crushed algae at a pressure of 35.0 MPa and a temperature of 328.1 K. The yield of supercritical extraction of carotenoids was also compared at several conditions of pressure and temperature. It increased with pressure at constant temperature, remained practically constant with temperature, at pressures of 27.5 and 35.0 MPa, and decreased with temperature at 20.0 MPa. The third alga studied was the Dunaliella salina , which produces β-carotene in high yield. Natural β-carotene ( cis and trans mixture) from this alga was submitted to supercritical carbon dioxide and it was assessed, at pressures up to 30.0 MPa and temperature of 313.1 K, that both isomers presented higher solubility than the synthetic trans -β-carotene and that the cis isomer was much more soluble in supercritical CO 2 than the trans isomer. Moreover, it was shown that the cis / trans ratio, in the supercritical extracts, increased significantly, relatively to the original one in the microalga, when the Dunaliella was submitted to supercritical CO 2 . The cyanobacteria Arthrospira ( Spirulina ) maxima can produce in large amounts the γ-linolenic acid (GLA), C18:3 ω6. The yield and selectivity of the supercritical extraction, using CO 2 and CO 2 doped with ethanol, of the lipids and GLA were determined and compared with those obtained with organic solvents.

Supercritical CO2 extraction of carotenoids and other lipids from Chlorella vulgaris

Freeze-dried samples of the microalga Chlorella vulgaris were submitted to supercritical CO2 at temperatures of 40 and 55 °C and pressures up to 35.0 MPa. This study was carried out on whole and crushed algae. The extraction yields of carotenoids and other lipids were low in the former case and improved significantly in the latter one. Extraction yields of carotenoids and other lipids increased with pressure. The fraction of carotenoids in the oil was also greater at higher pressures. On the other hand, supercritical CO2 extraction of carotenoids compared favourably with hexane and acetone extractions.

A biorefinery from Nannochloropsis sp. microalga - extraction of oils and pigments. Production of biohydrogen from the leftover biomass.

The microalga Nannochloropsis sp. was used in this study, in a biorefinery context, as biomass feedstock for the production of fatty acids for biodiesel, biohydrogen and high added-value compounds. The microalgal biomass, which has a high lipid and pigment content (mainly carotenoids), was submitted to supercritical CO2 extraction. The temperature, pressure and solvent flow-rate were evaluated to check their effect on the extraction yield. The best operational conditions to extract 33 g lipids/100 g dry biomass were found to be at 40 °C, 300 bar and a CO2 flow-rate of 0.62 g/min. The effect of adding a co-solvent (ethanol) was also studied. When supercritical CO2 doped with 20% (w/w) ethanol was used, it was possible to extract 45 g lipids/100 g dry biomass of lipids and recover 70% of the pigments. Furthermore, the remaining biomass after extraction was effectively used as feedstock to produce biohydrogen through dark fermentation by Enterobacter aerogenes resulting in a hydrogen production yield of 60.6 mL/g dry biomass.

Supercritical CO2 extraction of Thymus zygis L. subsp. sylvestris aroma

The supercritical fluid extraction (SFE) of aroma compounds from Thyme (Thymus zygis L. subsp. sylvestris) was studied. Extraction was conducted at different temperatures (300–323 K) and pressures (8–20 MPa). The extraction time was varied between 40 and 100 min. The study was carried out according to the response surface methodology (RSM). The extracts were analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS). The yield and the composition of the extracts obtained by supercritical CO2 were compared with those obtained by steam distillation (SD). SFE produced higher extraction yields than SD. This may be explained by simultaneous extraction of both aroma and non aroma compounds of higher molecular weight. Higher yields were produced for the higher values of pressure (20 MPa) and extraction time (120 min). With respect to extract composition, the content of hydrocarbon monoterpenes was shown to be mainly related to the extraction temperature, higher values being produced between 308 and 310 K. Phenols, including thymol, the major component of T. zygis aroma, were also extracted at the higher tested levels of the parameters. The production of the terpenic alcohols (TA) was found to be mainly dependent on the extraction time. This study allows the conclusion that pressure is the most important extraction parameter.

Comparison of pennyroyal oils obtained by supercritical CO2 extraction and hydrodistillation

Flowers and leaves of pennyroyal (Mentha pulegium L.) were extracted using supercritical CO2 followed by a two-stage fractional separation technique. Using a flow apparatus with an extraction vessel (1 l) and two separators (0.27 l), the best conditions of extraction (P=100 bar and T=50°C) and of separation (P=80 bar, T=−16°C for the first separator and P=18 bar, T=0°C for the second) were found and used to investigate the effect of different mean particle diameters. The pennyroyal oil obtained under these conditions was compared with that obtained by hydrodistillation. Twenty-one components were identified by GC–MS and quantified by GC. Both the hydrodistillation and supercritical oils of Mentha pulegium L. contain the oxygenated monoterpenes pulegone (∽80%) and menthone (∽9%) as the main components. The aroma of the pennyroyal oil obtained with supercritical CO2 was closer to that of the starting vegetable material than the aroma of the essential oil produced by hydrodistillation. Copyright

Composition and antioxidant activity of Thymus vulgaris volatiles: Comparison between supercritical fluid extraction and hydrodistillation

Supercritical fluid extraction (SFE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO(2) flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC-MS and 52 components were identified. The main volatile components obtained were p-cymene (10.0-42.6% for SFE and 28.9-34.8% for HD), gamma-terpinene (0.8-6.9% for SFE and 5.1-7.0% for HD), linalool (2.3-5.3% for SFE and 2.8-3.1% for HD), thymol (19.5-40.8% for SFE and 35.4-41.6% for HD), and carvacrol (1.4-3.1% for SFE and 2.6-3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0-1.2% for HD versus t-0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.

An apparatus for vapor—liquid equilibrium at elevated temperatures and pressures and selected results for the water—ethanol and methanol—ethanol systems

Abstract Niesen, V., Palavra, A., Kidnay, A.J. and Yesavage, V.F., 1986. An apparatus for vapor—liquid equilibrium at elevated temperatures and pressures and selected results for the water—ethanol and methanol—ethanol systems. Fluid Phase Equilibria , 31: 283–298. This paper presents a description of a system that is used to study vapor—liquid equilibrium at elevated temperatures (to 625 K) and pressures (to 10 MPa) for mixtures of compounds that are liquid at room temperature. In the evaluation of the equipment, results were obtained for the water—ethanol system at 150, 200 and 250°C and the methanol—ethanol system at 100 and 140°C. Liquid phase activity coefficients for these two systems were calculated using corrections determined from the modified Soave equation of state. The data obtained for these systems appear to be thermodynamically consistent.

A biorefinery from Nannochloropsis sp. microalga – Energy and CO2 emission and economic analyses

Are microalgae a potential energy source for biofuel production? This paper presents the laboratory results from a Nannochloropsis sp. microalga biorefinery for the production of oil, high-value pigments, and biohydrogen (bioH2). The energy consumption and CO2 emissions involved in the whole process (microalgae cultivation, harvest, dewater, mill, extraction and leftover biomass fermentation) were evaluated. An economic evaluation was also performed. Oil was obtained by soxhlet (SE) and supercritical fluid extraction (SFE). The bioH2 was produced by fermentation of the leftover biomass. The oil production pathway by SE shows the lowest value of energy consumption, 177-245 MJ/MJ(prod), and CO2 emissions, 13-15 kgCO(2)/MJ(prod). Despite consuming and emitting c.a. 20% more than the SE pathway, the oil obtained by SFE, proved to be more economically viable, with a cost of 365€/kg(oil) produced and simultaneously extracting high-value pigments. The bioH2 as co-product may be advantageous in terms of product yield or profit.

Herbicidal activity of volatiles from coriander, winter savory, cotton lavender, and thyme isolated by hydrodistillation and supercritical fluid extraction.

The volatiles from Coriandrum sativum L., Satureja montana L., Santolina chamaecyparissus L., and Thymus vulgaris L. were isolated by hydrodistillation (essential oil) and supercritical fluid extraction (volatile oil). Their effect on seed germination and root and shoot growth of the surviving seedlings of four crops ( Zea mays L., Triticum durum L., Pisum sativum L., and Lactuca sativa L.) and two weeds ( Portulaca oleracea L. and Vicia sativa L.) was investigated and compared with those of two synthetic herbicides, Agrocide and Prowl. The volatile oils of thyme and cotton lavender seemed to be promising alternatives to the synthetic herbicides because they were the least injurious to the crop species. The essential oil of winter savory, on the other hand, affected both crop and weeds and can be appropriate for uncultivated fields.

Enrichment of the thymoquinone content in volatile oil from Satureja montana using supercritical fluid extraction

Supercritical fluid extraction (SFE) of the volatile oil from Satureja montana L. was performed under different conditions of pressure (90 and 100 bar), temperature (40 and 50 degrees C), mean particle sizes (0.4, 0.6 and 0.8 mm) and CO(2) flow rate (0.8, 1.1 and 1.3 kg/h) to understand the influence of these parameters on the composition and yield of this oil. The results were compared with those obtained for the essential oil isolated by hydrodistillation (HD). The volatile and the essential oil were analysed by GC and GC-MS. The main compounds are carvacrol (52.2-62.0% for HD vs. 41.7-64.5% for SFE), thymol (8.6-11.0% for HD vs. 6.0-11.3% for SFE), p-cymene (6.9-12.8% for HD vs. 6.0-17.8% for SFE), gamma-terpinene (6.4-9.4% for HD vs. 2.3-6.0% for SFE) and beta-bisabolene (2.0-2.7% for HD vs. 2.2-3.5% for SFE). The major difference between SFE and HD was the relative amount of thymoquinone, an oxygenated monoterpene with important biological activities, which can be ten-fold higher in volatile oil (1.6-3.0 for SFE vs. 0.2% for HD). The morphology of the glandular trichomes of S. montana and the effect of the grinding process on them was also evaluated by SEM.