Ultrasound-assisted extraction (UAE) is also widely considered as an advanced extraction technique. This technique uses high-frequency sounds, usually higher than 16 kHz and a limited amount of solvent in order to produce an effective extrac­tion of the compounds of interest in the solvent employed, increasing their mass transfer and solubility, by disrupting the food matrix being extracted. As in PLE, the selection of the suitable solvent for extraction by UAE will be made depending on the compounds of interest. For instance, a mixture of dichloromethane/methanol (2:1) was employed to extract lipids from microalgae using UAE [140]. For more polar compounds, such as chlorophylls, methanol was demonstrated as a more effective solvent [175]. This technique has the advantage of providing faster extrac­tion processes compared to conventional techniques. UAE was compared to other solvent-based extraction of pigments and fatty acids from several algae samples. It was demonstrated that UAE was simple, allowed extraction of interesting com­pounds and did not produce alteration or breakdown products [197]. However, when this technique was directly compared to SFE for the extraction of carotenoids from

D. salina, it was shown that SFE was more effective for the extraction of these low polarity compounds, above all in terms of selectivity [98]. At certain conditions, in which a complex sample is being extracted containing the interesting compounds as well as other polar compounds, SFE was demonstrated to be more selective than UAE [98]. UAE has been also employed to extract polysaccharides derived from

Chlorella pyrenoidosa [171].

When sonicating the samples for a given period of time, an increase in the tem­perature of the sample can be observed as a result of the vibration of the molecules. For this reason, considering that most of bioactives are thermally labile compounds, it is common to proceed in a temperature controlled environment. For instance, pig­ments and fatty acids were obtained from algae at -4°C using 35 kHz and 80 W for 90 min [197]. The use of temperatures below 4-5°C allows a better preservation of the extracted compounds, that otherwise, could be degraded.

The last advanced extraction technique also used for bioactives extraction from algae is MAE. In MAE, the sample is heated by using microwaves, at typical pow­ers of 700 W for a short time. Compared to traditional extraction techniques, the use of microwaves allows the decreasing of extraction times significantly limiting also the amount of solvent needed. Again, the temperature will be an important param­eter to be controlled. Once selected the extraction solvent for the extraction of bio­actives from algae, the microwaves power as well as the extraction time has to be defined. Experimental designs can be useful in determining the best extraction con­ditions. For instance, response surface methodology was employed to optimize the MAE of astaxanthin from H. pluvialis [203]. By using this statistical approach, the microwave power (141 W), extraction time (83 s), solvent volume (9.8 mL), and number of extracting cycles (4 cycles) were optimized. At present, MAE has not been extensively applied to extraction of bioactives from algae, although given its success in the extraction of plant materials, it can be easily inferred the great pos­sibilities for its application to algae samples.