Bioremoval of estrogens by laccase immobilized onto polyacrylonitrile/polyethersulfone material: Effect of inhibitors and mediators, process characterization and catalytic pathways determination

Highlights

• Laccase immobilization using electrospun polyacrylonitrile/polyethersulfone fibers.

• Efficient removal of estrogens over wide concentration, pH and temperature range.

• Significant effect of wastewater constituents on removal efficiency of estrogens.

• Meaningful reduction of estrogenic activity of the solution after treatment.

• Mineralization as the final stage of enzymatic conversion of estrogens.

Abstract

The presence of micropollutants in water, wastewater and soil are a global problem due to their persistent effect on ecosystems and human health. Although there are many methods of removal of environmental pollutants, they are often ineffective for degradation of pharmaceuticals, including estrogens. In presented work we proposed fabrication of electrospun material from polyacrylonitrile/polyethersulfone (PAN/PES) as a support for laccase immobilization by covalent binding. Oxidoreductase was attached to the electrospun fibers using polydopamine as a linker and produced system was used for degradation of two estrogens: 17β-estradiol (E2) and 17α-ethynylestradiol (EE2). It was shown that 92% of E2 and 100% of EE2 were degraded after 24 h of the process. Moreover, the effect of surfactants, metal ions and mediators on conversion efficiencies of estrogens was investigated and it was confirmed that immobilized enzyme possessed higher resistance to inhibitory agents as well as thermal and storage stability, compared to its native form. Finally, estrogenic activities of E2 and EE2 solutions decreased around 99% and 87%, respectively, after enzymatic conversion, that corresponds to significant reduction of the total organic carbon and formation of low-toxic final products of estrogens degradation.

Introduction

Due the fact that most drugs are not fully metabolised in the human body, human’s secretions are the main source of wastewater contamination with active pharmaceutical compounds. Among other pharmaceuticals, estrogens should be of particular interest. Four estrogens commonly occurred in wastewaters among which three of them are natural steroids produced by living organisms: estrone (E1), 17β-estradiol (E2) and estriol (E3), whereas 17α-ethynylestradiol (EE2) is classified as a synthetic compound (Adeel et al., 2017, Gallouze et al., 2021, Fuentes and Silveyra, 2019). Estrogens affect the body’s endocrine system by influencing the synthesis, release, transport, metabolism and excretion of hormones. However, after entering the body from the outside, at unknow concentration, they can cause negative responses from human hormonal systems causing diseases related with immune, reproductive or neurological systems (Fredj et al., 2015). Estrogens are also dangerous for animals leading to collapse of local fish populations, sex reversal of males or inhibition of fish eggs production (Kallel et al., 2020).

Taking into account the fact that estrogens could have a negative impact on living organisms, new methods for removal of these compounds are still being sought because traditional ways of their degradation are usually inefficient, cost-non-effective and possess negative environmental impact (Akpotu et al., 2019). This is related mainly to the presence of numerous of various compounds in wastewaters, such as salts, surfactants or organic matter, which interfere with removal of estrogens, by for example ozonation or photocatalysis (Xia et al., 2013). It should be also noted that among estrogens, the most stable and toxic is 17β-estradiol (Bilal and Iqbal, 2019, Budeli et al., 2020), therefore its monitoring and efficient removal is of the highest interest.

A sustainable, cost-effective, and environmentally friendly solution of this problem might be an enzyme-mediated degradation. Biocatalysts could operate under mild conditions without the need for high energy input, and exhibit greater biodegradation potential than microorganisms. Biotransformation of estrogens and toxic micropollutants can be performed using naturally occurring enzymes such as laccases, tyrosinases, or various peroxidases. These oxidoreductase enzymes are capable of converting the aniline and phenolic functional groups present in estrogen compounds into free radicals, usually leading to formation of insoluble oligomers susceptible to precipitation or final mineralization leading to formation of low-toxic effluents (Beck et al., 2018). Moreover, available studies confirm the potential of oxidoreductases to effectively detoxify wastewater simultaneously reducing estrogenic activity (Torres-Duarte et al., 2012, Wang et al., 2012, Zdarta et al., 2021). For example, Shreve et al. (2016) showed that after conversion of E2 and EE2 by free laccase, the estrogenic activities of estrogens’ solutions were decreased around 98%, whereas in work presented by Lloret et al. (2011), the estrogenic activity of mixture of estrogens decreased up to 37% after treatment by laccase immobilized in a sol-gel matrix.

To improve the practical properties of the oxidoreductases, such as reusability and long-term stability, an immobilization method could be applied. Immobilization extends catalytic properties of the enzymes and provides greater enzyme activity under a wide range of process conditions (Mohammadi et al., 2018). A widely used method of enzyme immobilization is a covalent binding, characterized by the formation of permanent and stable chemical bonds between the amino acid residues contained in the enzyme structure and the functional groups on the support surface. The formation of chemical bonds between the biocatalyst and the matrix is crucial and usually requires appropriate reaction conditions and/or the presence of functional groups located on the surface of the material (Bilal et al., 2018, Zhang et al., 2018a). To improve support affinity to the biomolecules, surface modification is frequently used. One of the widely used surface modifiers in enzyme immobilization is polydopamine (PDA). The quinone groups present in PDA allow relatively easy surface functionalization due to creation of stable PDA layer onto support surface (Zhang et al., 2018b). In recently published studies, laccase immobilized on various supports such as poly(vinylidene fluoride) membrane, bacterial cellulose or Fe₃O₄ nanoparticles, modified by polydopamine, was used for removal of water pollutants such as dyes or chlorophenols with efficiencies exceeding 80% (Chen et al., 2018, Zhai et al., 2019, Zhu et al., 2020, Zdarta et al., 2019). However, there is lack of information regarding application of electrospun fibers modified by PDA for laccase immobilization and degradation of estrogens in available literature.

Thus, in the presented work, electrospun fibers produced from polyacrylonitrile and polyethersulfone (PAN/PES) were applied as support for laccase immobilization by covalent binding using polydopamine as surface modifier to create stable and reusable biocatalytic system. Next, a proof-of-concept for the application of as produced biocatalytic system in biodegradation of two estrogens: naturally produced 17β-estradiol (E2) and synthetic 17α-ethinylestradiol (EE2) at various process conditions, such as pH, temperature, estrogens concentrations and reaction time was presented to examine robustness of the produced systems. The important parts were a study on effect of the presence of wastewater matrix ingredients such as surfactants, metal ions as well as compound accelerating reaction on the biodegradation efficiency of estrogens. Additionally, in order to evaluate possible catalytic pathways and to determine toxicity of the final effluents, biodegradation products were characterized and total organic carbon and estrogenic activity of solutions after enzymatic treatment were examined. It was found that proposed heterogeneous biocatalyst should be considered as promising alternative for efficient E2 and EE2 degradation at various process conditions.