2.2. Methods

Bromide-based ILs (1-butyl-3-methylimidazolium bromide [Bmim][Br]) was synthesized using preexisting protocol [17]. 1-methylimidazole was reacted with 1-bromobutane in a 3-neck round bottom flask. For acetate-based ILs, similarly to [Bmim][Br], pre-existing protocol was referred to synthesize [Bmim][OAc].

[Omim][Br] was reacted with glacial acetic acid in a 3-neck round bottom flask. The 3-neck round bottom flask was then attached to a reflux condenser and refluxed for 48 h at 40 °C in neat conditions. The resulting mixture was then washed with diethyl ether and ethyl acetate. The residual solvent was removed with a rotary evaporator (Buchi Rotavapor R-300, Switzerland). NMR analysis was conducted to determine the purity of the IL. Similar synthesis route was used for the synthesis of 1-octyl-3-methylimidazolium bromide [Omim][Br], 1-dodecyl-3-methylimidazlium bromide [Domim][Br], 1- octyl-3-methylimidazolium acetate [Omim][OAc] and 1-dodecyl-3-methylimidazlium acetate [Domim][OAc]. Given the long alkyl chain of [Domim], the synthesis time was doubled from 48 h to 96 h to ensure complete reaction of starting chemicals.

The synthesized IL were mixed with water at differing ratio (from 0.1% to 20% w/v). Surface tension of the mixtures was measured via the pendant drop method using OCA Contact Angle System (DataPhysics Instrument, Germany). 1 ml of sample was taken with a syringe to be analyzed. All experiments were conducted in triplicates.

Water and IL mixtures were subjected to viscosity and density measurement using SVM 3000 Viscometer (Anton Paar, Austria). Approximately 3 or 4 ml of the sample were used for each measure. The instrument was set to M5-Single temperature mode and the measurement was carried out at 25 °C. All experiments were conducted in triplicates.

In order to determine the impact of the viscosity and surface tension towards the acoustic cavitation, chemical dosimetry using potassium iodide was performed as a simple and robust method [18]. The chemical dosimetry to monitor triiodide formation may be adequate to quantitatively prove if the small addition of ILs into water either disturbs or enhances the water sonolysis producing HO° radicals. The full chemical equation is as shown below[19]:

0.1 mol of potassium iodide was dissolved in 1L of deionized water. 50 ml of solution was subjected to ultrasonication for 30 min with using a 20 kHz ultrasonic probe at 50% amplitude (Sonics VCX 750, USA). The calorimetric power released by the probe was measured to be at 30 W. The spectrophotometer absorbance at 355 nm was measured every 5 min using Biochrome Libra S60 UV-Spectrophotometer [18].

IL mediated ultrasonication of the microalgae were conducted based on an optimized protocol. Each IL was sampled at a volume / volume (v/v) concentration of 0.5%, 1%, 1.5%, 2%, 2.5%, 5%, 10% and 20%, and completed with 50 ml distilled water containing 0.2 g of microalgae. The mixtures were then subjected to a 20 kHz irradiation with a 13 mm ultrasonic probe at 50% amplitude for 20 min at 2 s:1s pulse mode. A circulating water bath (Julabo SW22, Germany) was attached to the double-jacket ultrasonic vessel to ensure a constant temperature of 15 °C, to avoid any temperature changes due to the sonication effect. A first control experiment without the presence of IL was also conducted.

The well-established Bligh and Dyer [20] method was used as extraction method. However, due to the addition of IL into the system, the original method has been slightly modified to accommodate the addition of the IL as follow: 50 ml of a methanol/chloroform (2:1) mixture was added into the algae mixture. The resulting solution was then centrifuged (Hettich Rotofix 32A, Germany) at 4000 rpm for 15 min. The separated bottom layer was transferred into a new flask and dried at 55 °C. The dried sample was then mixed with a hexane/water (1:1) solution to remove any residual IL eventually dissolved in the chloroform mixture. The hexane layer containing lipid was then transferred into a new glass vial and dried. Then, the lipids contents were weighed and expressed in terms of algae dry weight percentage (% DW).