Abstract
Recalcitrant pollutants in water and wastewater systems threaten the current water scarcity posed by drought in the sub-Saharan region. One of the leading persistent organic pollutants is antiviral drugs (ARVs), introduced into aquatic systems as pharmaceutical waste from the healthcare and poorly disposed of medicines that find their way into wastewater treatment plants (WWTPs). The increased environmental exposure is caused by increased demand for ARVs, primarily to control HIV/AIDS. However, not limited to HIV/AIDS, viral outbreaks like Ebola and the current COVID-19 pandemic skyrocketed the use of ARVs.
The alarming concern is the increased environmental pollution, especially water pollution and the inefficiency of existing wastewater treatment systems. In South Africa, there have been reports of ARVs being inefficiently removed by current wastewater systems and, thus, being released to aquatic bodies. Therefore, the desired need to develop inexpensive yet efficient strategies to remove persistent pollutants like ARVs from wastewater. The introduction of an additional treatment step in the conventional techniques has been investigated. For example, a combination of a biological system with a photocatalytic treatment (use of semiconductor materials) post the biological treatment, and promising results have been obtained.
In the light of a combined system, the study aimed to design an environmentally friendly biocatalyst which will be used in a simulated integrated biological and biocatalytic system, a green chemistry approach in removing recalcitrant pollutants like ARVs from wastewater. Biocatalytic degradation involves using living organisms (i.e. plants, microorganisms etc.) or their metabolites (i.e. enzymes) to convert pollutants into smaller compounds. The study targeted enzymes (laccases) as potential biocatalysts, but their applications are hindered by chemical stress, temperature instability and poor recyclability. Hence, as a solution, covalent immobilisation of the enzymes on two-dimensional (2D) nanoparticle supports (MXenes) was investigated.
The study used Ti2N MXene to immobilise the laccase enzyme to remove efavirenz as a model ARV in wastewater. The selective etching of the Ti2AlN MAX phase using in situ produced HF (LiF and HCl mixture) produced Ti2N MXene. The crystal structure of the prepared materials was investigated by Raman, selected area diffraction (SAED), and powder X-Ray diffraction (XRD), which assigned the materials to Ti2AlN MAX phase and Ti2N MXene. Possible surface terminations, chemical states and elemental compositions of the MXene were obtained from Fourier-transform infrared spectroscopy (FTIR), energy-dispersive X-Ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) analysis. The EDS spectra of the MXene showed the presence of new elements F and O. The elements were ascribed to Ti-F, Ti-O and O-F functional groups by FTIR and affirmed by XPS. Moreover, XPS confirmed the partial oxidation of the MXene and disregarded the formation of TiO2. Hence, the prepared Ti2NTx MXene have the following surface termination; Tx= O, F, and OH groups.
Glutaraldehyde, a bifunctional linker used in conjunction with hexamethylene diamine (HMDA), was investigated to enhance enzyme activity and laccase loadings after immobilisation on the MXene. Our findings revealed that HMDA improved laccase activity and had an insignificant role in immobilisation. The study utilised laccase isoenzymes with plant and fungal origin from the Rhus vernificera and Trametes versicolor species, respectively. Studies from the Rhus vernificera laccase obtained from 2 mg/mL initial concentration gave 7.81x10−3 U/mg enzyme activity at 11.6% laccase loading. At the same initial concentration, 256 U/mg enzyme activity and 27.2% laccase loadings were obtained from the Trametes versicolor laccase. Therefore, Trametes versicolor showed superiority over Rhus vernificera laccase, thus, being used in application studies.
Laccase-assisted (mediator: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) biocatalytic degradation of efavirenz was explored after the poor degradation of efavirenz by laccase in 4 hours. The laccase biocatalytic degradation of efavirenz without ABTS (not assisted degradation) achieved <30% degradation by the immobilised enzyme, while <50% was achieved for the free laccase at optimal conditions (pH, temperature, enzyme loadings, and efavirenz concentration). Significant improvement was observed in the assisted degradation as ~63% and ~60% degradation was obtained by immobilised and free laccase, respectively. Six degradation fragments were observed from the LCMS chromatograms from the
assisted and non-assisted biocatalytic degradation by the free and immobilised laccase.
The optimal pH for Trametes versicolor is between pH 4.5 and 5.5, but the immobilisation improved the pH tolerance of the enzyme. An enzyme activity above 80% was attained at pH 7, which allowed the application of the enzyme in pH conditions of wastewater. Therefore, a 3L bioreactor was integrated into a biological wastewater treatment system operated at pH 7 using the immobilised laccase to remove efavirenz from wastewater. The combined degradation of efavirenz was ~40% and ~73% at 5 and 25 ppm efavirenz concentration, respectively. The results showed effective removal of efavirenz in wastewater compared to the literature on biological treatment of efavirenz by WWTPs.