Biodegration of endocrine-disrupting chemicals by a fungal consortium in synthetic wastewater.
Kasonga, Kabeya
Kasonga, Kabeya
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Abstract
Over the years, research on the impact of chemicals on the environment has mainly focused on nutrient pollution, heavy metals, active ingredients of pesticides products and other persistent organic pollutants (POPs). However, more recently, there has been increasing concern due to the environmental and health risks associated with endocrine-disrupting chemicals (EDCs) such as pharmaceutical compounds (PhCs), active pharmaceutical ingredients (APIs), pharmaceutical and personal care products (PPCPs). The incomplete removal of EDC (considered as emerging pollutants) in conventional wastewater treatment processes has been reported, due to the recalcitrant nature of these compounds. As a result, the aim of the current study was to assess the capability of South African indigenous fungal strains in removing EDCs from wastewater. To achieve this aim, five objectives were pursued.
In the first objective, five South African indigenous fungi were isolated and identified; their growth conditions on solid and liquid media were optimised for further utilisation in the degradation of EDCs such as PhCs. Conventional PCR was carried out using the ITS1 and ITS4 universal primers to amplify the internal transcribed spacer 2 (ITS1/ITS4) region as well as ß-tubulin-F/R for identification of the isolates. This step was followed by phylogenetic analysis of partial ITS gene sequences from the selected isolates. A field emission gun scanning electron microscope (FEG-SEM) was used for characterisation of the fungal isolates. Five fungal strains were found to be closely related to Aspergillus niger, Mucor circinelloides, Trichoderma longibrachiatum, Trametes polyzona, and Rhizopus microsporus. The growth of these fungal strains was optimised on solid and liquid media in terms of C/N ratio and incubation times at various temperatures (28±1°C, 30±1.5°C and 37±1°C). A C/N ratio of 5.5:1 and 2.14:1 gave better proliferation of the mycelial mat at 30±1.5 °C, while the liquid low nitrogen medium (LN-m) with C/N ratio of 50:1 at 30±1.5 °C yielded better fungal dry biomass. Preliminary results of growth optimisation in batch flasks [aerated batch flask (ABFs), shaken batch flasks (SBFs) and stationary batch flasks (StBFs)] inoculated with 10% of fungal mycelium solution revealed the importance of the carbon source (glucose) and nitrogen source (ammonium tartrate dibasic: AT) for suitable growth in StBFs under daylight at room temperature.
In the second objective, the five isolated fungal strains were used to investigate their diclofenac sodium (DCF) degradation potential using synthetic wastewater, taking into consideration the operating conditions (the supply of air, glucose and AT, the daylight and the non-sterile conditions). Results revealed that, in the presence of glucose, R. microsporus removed the highest percentage of DCF in ABFs (95%), followed by A. niger (80%). Mucor circinelloides, T. polyzona, and T. longibrachiatum were found to remove 53.85% of DCF in SBFs, 77.15% in StBFs and 25.3% in ABFs. Low DCF pourcentage removal were recorded in StBFs without glucose for R. microsporus (65.44%), A. niger (50.97%), M. circinelloides (25.47%), in SBFs for T. longibrachiatum (8.79%) and in non-sterile StBFs for T. polyzona (36.09%). A DCF removal of only 41.88% was recorded for the consortium of these five fungal strains in StBFs. The supply of glucose in batch flasks and daylight enhanced the DCF removal efficiency of the fungal strains.
The third objective focused on the fungal tolerance to PhCs, namely carbamazepine (CBZ), DCF and ibuprofen (IBP), the fungal ligninolytic enzyme activities and the simultaneous PhC biodegradation capability by the five isolated fungal strains. The tolerance was performed by monitoring the fungal mycelium mat diameters in solid media and its dry biomass in liquid media, at the drug concentration range of 0.1 to 15 mg/L. The fungal enzymatic activities were assessed in terms of production of the lignin-degrading enzymes, lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac), respectively. The removal efficiency of the fungi for the PhCs and their metabolites was assessed in ABFs. The drug concentrations and the formation of intermediate compounds were determined by using SPE-UPLC/MS. A tolerance of over 70% was observed for all the fungi at a drug concentration of 0.1 mg/L. All the fungi were capable of producing MnP and LiP at very low concentrations, while all the lignin-degrading enzymes were produced by T. polyzona. The pH 4.3, the temperature of 37±1.5 °C and the incubation time of six (6) days were the optimum conditions for the fungal enzymatic activities. The DCF removal rates ranging between 78% and 100% were observed for all the fungi after 24 h, while 98% of IBP was removed after two (2) days by M. circinelloides. The best removal of CBZ (87%) was achieved by R. microsporus after ten (10) days. The intermediate compounds (CBZ-10,11-epoxide, 2-hydroxy-CBZ, 3-hydroxy-CBZ, 10,11-dihydro-10,11-dihydroxy-CBZ, acridine and 9-hydroxy-acridine and acridone were suggested to originate from CBZ, while 2,6-dicholorobenzoic, 2,4-dichlorobenzoic and 3,5-dichlorobenzoic acid were proposed to originate from DCF, and 1-hydroxy-IBP and 2-hydroxy-IBP from IBP) were identified. Nevertheless, they disappeared after 24 h (from DCF) and after ten (10) days of incubation for others. The removal of PhCs through biodegradation and mineralisation was proposed to be due not only to the intracellular ligninolytic enzymes Lac, LiP and MnP, but also to the synergistic role of extracellular fungal metabolites such as cytochrome P450 and others like xylanase enzymes, and fungal biosorption and adsorption processes have also to be taken into account. This study demonstrated that apart from fungi of the phylum Basidiomycota, fungi belonging to the phyla Ascomycota and Zygomycota are also producers of ligninolytic enzymes and have the ability to biodegrade emerging pollutants such as PhCs.
In the fourth objective, a non-sterile stirred fluidized bioreactor (NSFB) inoculated by a fungal consortium of the five isolated fungi was used to assess the removal of selected PhCs and their metabolites. The outcomes of the study revealed that the fungal consortium was capable of producing all the ligninolytic enzymes (Lac, MnP and LiP) at room temperature, pH 4.5±0.5 and a continuous air supply with 8.5±0.5 mg/L dissolved oxygen. A pH 4.5 was unfavourable for high biomass generation, while the pH < 3 favoured high biomass growth. A supply of sufficient nutrient (glucose and ammonium tartrate) at a C/N ratio of 5:1 enhanced fungal biomass growth and the production of ligninolytic enzymes in the following order: MnP (502.45 ± 20.18 U/L) > Lac (252.12 ± 45.07 U/L) > and LiP (145.21 ± 25.71 U/L). The PhC removal efficiency of the fungal consortium within 24 h was observed in the following order: DCF (95.7% and 93.9%) > IBP (90% and 78.6%) > CBZ (89.9% and 65.7%) with pH 4.5 and pH < 3, respectively, and more than 98% was removed after five (5) days. This was found to be higher than the individual PhC removal capability of isolated fungi in the ABFs. With the exception of 2,4- dichlorobenzoic, 2;5- dichlorobenzoic and 2,6-dichlorobenzoic acid, all the metabolites found in ABFs were also identified in NSFB. The presence of all these metabolites was detected particularly in the ABF, however, their removal was effectively accomplished in the NSFB. This study highlights the rapid and high degree of degradation of the selected PhCs by a fungal consortium in the NSFB compared to their level of elimination in conventional biological wastewater treatment systems.
In the fifth objective, the biodegradation of CBZ, DCF and IBP was evaluated through the development of granules using a consortium of the five fungal strains. A sequencing batch reactor (SBR) run with a consortium of five fungal isolated at room temperature, air supply with dissolved oxygen (DO) concentration of 8.35 ± 0.35 mg/L, the pH range of 3.5 to 6, the shear force at a stirring speed of 120 rpm and the retention time (RT) of two (2) days and one (1) day was used. Granules were developed in terms of different C/N ratios (0.5:1, 1:1, 2:1, 2.5:1, 5:1, 10:1, 25:1, 50:1, 100:1). Ligninolytic enzymatic activity was determined spectrophotometrically for Lac, LiP and MnP. Removal of PhCs was assessed and metabolites were identified using the SPE-UPLC/MS methods. The C/N ratio of 5:1 was found to be suitable for the formation of compact granules over the pH range of 3 - 4.6 from, and ligninolytic enzyme production from day 6 onwards. Compared to the RT of two (2) days, the RT of one (1) day was found to enhance the wash-out of suspended biomass and the produced fungal metabolites. With an initial concentration of 1 mg/L PhC, the removal was found to occur in the following order: 92.6% of DCF > 78.4% of IBP > 70.7% of CBZ in the SBR at an RT of two (2) days and 93.2% of DCF > 80.5% of IBP > 71.2% of CBZ at an RT of one (1) day. With the development of the fungal consortium granules from day 6, onwards, the fungal removal efficiency of PhCs increased at steady state up to 97.41±0.25%, 99.83±0.14% and 99.91±0.08 for CBZ, DCF and IBP in the SBR at an RT of one (1) day, respectively. For an RT of two (2) days, the removal efficiency for CBZ, DCF and IBP was found to be 91.94±0.05%, 99.31±0.12% and 97.72±0.23%, respectively. Regardless of granule formation, a variety of chemical reactions (oxidation, hydroxylation, hydrolysis, methoxylation and demethoxylation) have been proposed for the degradation pathways catalysed by enzyme-producing fungi. These chemical reactions generated fragment ions of intermediate compounds mentioned above. Both parent compounds and intermediates could be quantified at very low concentrations until 17 days in the SBR.
In the last objective, a consortium of the five fungal strains was used in the SBR to evaluate their capability to degrade the selected PhCs with emphasis on the reduction of the estrogenic activities of the PhCs and intermediate compounds. The in vitro estrogenic activities were assessed using the T47D-KBluc reporter gene assay. The estradiol equivalent (EEq) values of 1.707 ± 0.298 ng/L and 2.687 ± 0.174 ng/L were recorded at the start-up time and after 4 h, respectively. The presence of intermediates was found to induce estrogenic activity after 4 h. After 24 h of incubation, the EEq values were found to be below the limit of quantification (LoQ) and below the limit of detection (LoD) of the assay. None of the samples exhibited any anti-estrogenic activity. The fungal consortium inoculum was found to induce toxicity at a 0.4x concentration, as observed under a microscope. This study revealed that the fungal consortium is capable of reducing the estrogenic activity of PhC metabolites to below the LoD, which appeared to make the most significant contribution to the endocrine-disrupting activity of the wastewater effluents.
Based on the findings of the current study, it can be concluded that a consortium of South African indigenous fungal strains Aspergillus niger, Mucor circinelloides, Trichoderma longibrachiatum, Trametes polyzona and Rhizopus microsporus can be used for the bioremediation of recalcitrant PhCs in pharmaceutical industry wastewater. The benefit of the fungal granules acting like a biofilm is the application of the ability of the fungal consortium to not only biodegrade/remove PhCs, but also to reduce the estrogenic activity and toxicity of a variety of unknown metabolites from wastewater treatment plants (WWTP) effluents. Therefore, this study suggests the implementation of a consortium of South African indigenous fungal strains in the management of polluted wastewater containing emerging pollutants such as EDCs and PhCs.
Description
Submitted in partial fulfilment of the requirements for the degree, Philosophiae Doctor in the Department of Environmental, Water and Earth Sciences, Faculty of Science at the Tshwane University of Technology.
Date
2019-04-05
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Tshwane University of Technology
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Keywords
Wastewater, Biodegration, Fungal strains, Methoxylation, PhC metabolites