**2. Materials and methods**

#### **2.1 Materials**

Iron (II) chloride tetrahydrate (98%) (FeCl2\*4H2O), Iron (III) chloride hexahydrate (97%) (FeCl3\*6H2O), and dye Eriochrome Black T (EBt) (C.I. 14645) were obtained from PanReac AppliChem (Spain). 2,2-azino-bis(3-ethylbenzothiazoline-6)

**95**

*Micromixers for Wastewater Treatment and Their Life Cycle Assessment (LCA)*

cylindrical magnets (ϕ: 6 mm x h: 7 mm) were purchased at a local shop.

**2.3 Synthesis of magnetite and laccase immobilization**

magnetism and washed thoroughly with MilliQ water.

**2.5 Experimental test for wastewater treatment**

**2.4 Geometry design and fabrication**

vertical loops (See **Figure 1**).

sulphonic acid (ABTS), glutaraldehyde (25%), sodium hydroxide (NaOH) (98%), tetramethylammonium hydroxide (TMAH) (25%), 3-Aminopropyl-triethoxysilane (APTES) (98%) were purchased from Sigma-Aldrich (USA). Polymethyl methacrylate (PMMA), Methyl methacrylate, Ethanol (96%) and 345 mT Neodymium

Laccases (*P. sanguineus* CS43) (EC 1.10.3.2) were obtained from tomato medium as described elsewhere [21]. Briefly, mycelia were removed from the culture supernatant by filtration using two tangential flow filters in series, one of them with pore size of 0.5 mm while the other of 0.2 mm. The obtained laccase cocktail was ultra-filtered using a membrane with a molecular weight cut-off of 10 kDa.

Magnetite nanoparticles were synthesized by coprecipitation of 20 mL of 1 M FeCl2 and 20 mL of 2 M FeCl3 under agitation at 1,500 rpm and 90 °C. 40 mL of 8 M NaOH and 40 mL of 2% (v/v) TMAH were then added to the mixture during 3.5 h at a flow rate of 12 mL/h. Nanoparticles (Magnetite) were magnetically separated aided by a strong permanent magnet, then washed thoroughly with 2% (v/v) TMAH, and

Magnetite nanoparticles were buffered by adding a NaOH solution until pH approached 11, then sonicated for 10 min. 50 μL of 2% (v/v) TMAH was added and dispersed and then the mixture was sonicated for 10 min. Silanization of the nanoparticles was carried out by adding 50 μL of 2% (v/v) APTES followed by sonication for 20 more min. 50 μL of 2% (v/v) glutaraldehyde was added to the mixture as the crosslinker, and left to react for 30 min. Finally, 50 μL of 960 U/L laccase was added and left overnight to immobilize the enzyme on the nanoparticles. The resulting bionanocompounds (i.e., Lac-Magnetite) were separated by

Six different prototypes of micromixers were designed with different microchannel geometries for the reaction chambers. This was achieved by varying the number of layers of PMMA sheets required to create the channel. In the case of one layer, the channel geometry was circular and triangular. In contrast the assembly of multiple layers enabled rectangular-3D, one loop, two horizontal loops, and two

For the micromixers manufacture, each design was engraved and cut on sheets of polymethylmethacrylate (PMMA), with a thickness of 3 mm and an area of 75x25 mm, using a Speedy 100, 60 W laser cutting system (TROTEC, Germany). Sheets were glued together to assemble the devices by applying a few drops of 96% ethanol on the contacting surfaces and maintaining a constant pressure for 8 minutes at 105 °C.

To estimate the dye biodegradation, we selected the EBt dye as a model. The EBt solutions were prepared at pH 5.48 and three different concentrations, namely, 5 mg/L, 10 mg/L and 20 mg/L. Biodegradation tests were conducted by introducing 5 mg of the bionanocompound and 5 mL of dye solution into each micromixer for 25 minutes at a constant rate of 12 mL/h. A neodymium permanent magnet, of

finally sonicated for 100 min in a VibraCell ultrasonic bath (Sonics, USA).

*DOI: http://dx.doi.org/10.5772/intechopen.96822*

**2.2 Laccase**

sulphonic acid (ABTS), glutaraldehyde (25%), sodium hydroxide (NaOH) (98%), tetramethylammonium hydroxide (TMAH) (25%), 3-Aminopropyl-triethoxysilane (APTES) (98%) were purchased from Sigma-Aldrich (USA). Polymethyl methacrylate (PMMA), Methyl methacrylate, Ethanol (96%) and 345 mT Neodymium cylindrical magnets (ϕ: 6 mm x h: 7 mm) were purchased at a local shop.
