## 4 Immobilized Systems in Microbiological Degradation of EDCs
The TiO\({}_{2}\) immobilization technique shows a significant ability to coordinate with amine and carboxyl groups with a good biocompatibility and low price as well. Titanium dioxide also exhibits possibilities for great modification. One of the techniques including TiO\({}_{2}\) usage is coating by TiO\({}_{2}\) sol-gel of montmorillonite (O-MMT) electrospun nanofibrous membrane to prepare a functional composite nanofibrous material as a lacase immobilization matrix. [PERSON] et al. [71] reported a significant protective effect of titanium dioxide during immobilization of lacase from _Pleutoruts ostreatus_. In that study, the authors used two micro-pollutants, including bisphenol-A known as EDCs compound. Immobilization had a positive influence on the enzyme activity at a pH range of 2.5-7 and stability during 12 h incubation (at pH 3 as a benchmark). Also, the temperature influence on lacase activity was lower in the immobilized system. Both, free and immobilized enzymes work at a temperature range of 25-70 \({}^{\circ}\)C, but free lacase reaches a lower activity compared to the immobilized one. It shows that immobilization on advanced carriers may stabilize enzymes and allows the system to reuse them. However, it should be noted that the degradation rate after each cycle was lower. In the 24 h degradation cycle in hybrid membrane reactors (pH 5.5, 25 \({}^{\circ}\)C, and 5 L/m\({}^{2}\) h of flux) [PERSON] et al. (2017) reported a 50% decrease of bisphenol-A decomposition [71]. In a similar experiment, using lacase from _P. ostreatus_, [PERSON] et al. [72] used monoaminoethyl-N-aminoethyl (MANAE-agarose) as a reusable immobilization carrier. Modified agarose carriers like MANAE-agarose are widely used in the physical adsorption of enzymes characterized by good immobilization efficiency with a significant influence on enzyme activity [73]. In comparison to free lacase pH, thermal and storage stability were improved. Both, free and immobilized lacase have optimal activity at pH 5, however, immobilized lacase at pH 8 loses 40% of its initial activity compared to the free enzyme which loses 80%. Significantly, the temperature stability was higher. In comparison to free lacase, relative activity was 2.3- and 7-fold higher at the temperatures of 40 and 55 \({}^{\circ}\)C, respectively. Also, storage at 4 \({}^{\circ}\)C was much improved by immobilization. After 20 and 40 days, free enzymes lose 50 and 60% of its initial activity, whereas immobilized enzyme loses only 20 and 30% of its initial activity after 40 and 170 days of storage, respectively. It shows the importance of carrier choice to the immobilized system. During another study, [PERSON] et al. [74] showed a high level of EDCs degradation by fungal lacase from _Trametes versicolor_ and _Myciophthora thermophila_ immobilized on acrylic beads. After 24 h free _T. versicolor_ lacase decomposes 27.8% of 17\(\beta\)-estradiol while the immobilized enzyme degraded 100% of the dose used. Free _M. thermophila_ lacase had a better removal rate--63.2%--however, the immobilized system degrades 100% of the 17\(\beta\)-estradiol as well. Experiments with other EDCs, such as bisphenol A (BPA), nonylphenol (NP), benzylbutylphthalate (BBP) or propylparaben (PrPb) show very similar dependence. _T. versicolor_ lacase degraded 20.9, 51.5, 1.16 and 8.54% of BPA, NP, BBP and PrPb, respectively, whereas the immobilized enzyme showed removal of 82.0, 81.7, 95.5 and 100%, respectively. For lacase from _M. thermophila_ similar results were obtained. Nevertheless, absorption of EDCs on acrylic beads was significant, and in most cases comparable, with the degradation results. The authors correlate a high level of absorption with lacase activity as a positive combination of EDCs removal, however, this point of view could be questioned [74]. Nevertheless, immobilization on nanobeads or nanoparticles becamemore popular in EDCs degradation as well. [PERSON] et al.