**2.2 Laccase**

*Advances in Microfluidics and Nanofluids*

analyzers are commercially available for wastewater monitoring, and they are based on different detection techniques, such as colorimetric, chemical, electrochemical or optical [11]. Here, a growing trend is emerging where microfluidic technologies are considered for environmental detection mainly due to their lower investment and operation costs, as well as reduced infrastructure requirements. Moreover, it has been shown that microreactors, help to maximize biodegradation processes due to the absence of dead volume, allow to perform continuous reactions, and enable to control the contact between the reagents by changes in the microchannel geometries [9, 12]. By handling fluids in microchannels, it is possible to achieve high production yields, and minimize waste generation. Moreover, with this approach it is feasible to operate under short reaction and analysis times, is relatively cheap and enable high-throughput schemes [13]. However, the manufacture of microfluidic devices generally relies on sophisticated cleanroom techniques [14], which is disadvantageous due to their high costs. This issue has been overcome with low-cost manufacturing methods such as polymer laminates, 3D printing, and laser cutting [15, 16]. In this approach, devices are often manufactured by cutting a piece of polymethylmethacrylate (PMMA) followed by engraving a predesigned microchannel pattern on a separate PMMA. The device is then assembled by gluing the two pieces together. PMMA is one of the preferred thermoplastics for the manufacture of microfluidic devices, due to its optical transparency, superior mechanical properties, low cost and good workability in conjunction with its ease for prototyping and mass manufacturing [17]. In this study, we will how PMMA can be used to manufacture micromixers and we will analyze and compare the potential environmental impact of implementing them for wastewater treatment. Life cycle assessment (LCA) has been widely applied in the wastewater treatment industry due to its important role as a tool for the sustainability assessment of new technologies, processes and the improvement of waste management practices. On this, inputs, such as raw materials and energy, and outputs, such as waste and emissions, are collected in the form of elementary flows for the whole life cycle (Life Cycle Inventory – LCI step) and then converted into environmental impact indexes by means of characterization factors (Life Cycle Impact Assessment – LCIA step) [18]. According to Corominas et al. [19], LCA can be a useful decision-support tool for examining alternative future operational scenarios during strategic planning within the water sector. Also, LCA evaluates beyond the limit imposed by the trade-off between process efficiency and final effluent quality because it considers resource and energy consumption, air emissions and waste generation [20].

In this study, we explore the design and manufacture of micromixers for wastewater treatment to enable the enzyme-based degradation of dyes. In this regard, we propose a LCA assessment to establish the potential environmental impact of implementing these devices. Also, this analysis integrates the required chemical supplies, energy, and water needed for wastewater treatment. Through life cycle assessment (LCA), we compared six different designs of micromixers to identify the one providing the least environmental impact during operation. LCA analysis might therefore contribute significantly to improving wastewater treatment process by coupling micromixers capable of remediating wastewaters with high efficiencies.

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)

**94**

**2. Materials and methods**

**2.1 Materials**

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.
