Journal of Environmental Management 325 (2023) 116595 Contents lists available at ScienceDirect Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman Research article Fungal treatment of agricultural washing wastewater: Comparison between two operational strategies Eduardo Beltrán-Flores a, Martí Pla-Ferriol b, Maira Martínez-Alonso b, Núria Gaju b, Montserrat Sarrà a, *, Paqui Blánquez a a Departament d’Enginyeria Química Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Agricultural washing wastewater (AWW) is an important source of pesticides that, given its intrinsic charac­ Fungal bioremediation teristics, has a high potential to be treated by fungal bioremediation using white rot fungi. In the present study, Pesticides two AWW treatment strategies were compared: a fluidized-bed reactor (FBR) with T. versicolor pellets and a Agricultural wastewater rotating drum bioreactor (RDB) with T. versicolor immobilized on wood. The RDB effluent showed better results Fluidized-bed reactor Rotating-drum bioreactor in all studied parameters compared to those of the FBR, including pesticide removal (87%), toxicity, laccase activity, COD, absorbance and microbial communities. Additionally, the fungal assemblage showed that T. versicolor was successfully immobilized in the RDB, which triggered a major shift in the initial community. Afterwards, solid by-products were treated in a fungal biopile-like system reaching high biodegradation rates. Therefore, this study validates the fungal RDB as a viable alternative for AWW treatment, opening up the pos­ sibility of a further in-situ and full-scale application. 1. Introduction also known as rinsate, which is generated on farms when washing agricultural machinery and equipment. Afterwards, AWW is usually The main effects of climate change, i.e., growth in atmospheric CO2 deposited in collection ponds, avoiding dilution by rainfall and leaks levels, temperature rise and changes in precipitation patterns, have been during handling that could contaminate the surrounding soil (European shown to lead to increased pest activities on crops. Changes in the dy­ Commission, 2009a). Finally, accumulated AWW should be treated by namics of pest influx and crop disease threaten to cause significant an in-situ process specifically designed for this application before agricultural production and economic losses, and compromise global discharge. food supply and security (Singh, 2015). In addition, a growing world Over the last few decades, various physical, chemical and biological population and rising living standards require a greater effort from the technologies have already been implemented for pesticide elimination primary sector to meet global food demand. In this context, the use of from wastewater. However, there are several limitations that affect the pesticides is necessarily unavoidable to ensure pest control and food treatment of pesticides by physical-chemical methods, such as the production worldwide (Popp et al., 2013). transfer of these compounds from liquid to solid phase without real Nevertheless, pesticides are becoming a major global concern owing degradation (e.g. sorption), high operational costs (e.g. membrane to their overuse in agricultural activities, environmental persistence, filtration) and the potential formation of transformation products (e.g. high mobility, bioaccumulation and toxic effects (Sharma et al., 2019). ozonation) (Ahmed et al., 2017). Among them, sorption has been one of For these reasons, good practices in the agricultural sector should be the most widely applied techniques for pesticide removal due to its easy subjected to clear regulations regarding the correct management and manipulation, high removal efficiency and relative low cost (depending application of pesticides, comprising the prohibition of highly toxic on the sorbent), whereas this technology has significant drawbacks such pesticides, the selection of specific pesticides for each type of pest and as critical pH dependence and difficulties in sorbent preparation and crop, and the application of reasonable doses. In this respect, particular regeneration (Mojiri et al., 2020; Titchou et al., 2021). emphasis should be placed on agricultural washing wastewater (AWW), In contrast, bioremediation is gaining increasing attention in recent * Corresponding author. E-mail address: montserrat.sarra@uab.cat (M. Sarrà). https://doi.org/10.1016/j.jenvman.2022.116595 Received 12 June 2022; Received in revised form 13 October 2022; Accepted 19 October 2022 Available online 2 November 2022 0301-4797/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 decades as an environmentally friendly, efficient and low-cost approach 2. Materials and methods (Marican and Durán-Lara, 2018). In particular, bioremediation using white rot fungi (WRF) seems to be a very promising technology for AWW 2.1. Fungal strain and reagents treatment. AWW is usually produced at relatively low volumes, which is desirable in biological processes using WRF as these microorganisms T. versicolor ATCC 42530 was acquired from the American Type generally require relatively long hydraulic residence times. Further­ Culture Collection. The fungus was maintained on malt extract (2% w/v) more, AWW is generated at high concentrations of multiple pesticides, at 25 ◦ C and routinely subcultured every 30 days. Blended mycelial which can be treated by WRF due to their excellent resistance to suspension and T. versicolor pellets were prepared as previously elevated toxicities. Consequently, WRF do not require prior acclimati­ described elsewhere (Blánquez et al., 2004). T. versicolor immobilized on sation as in other biological processes such as conventional activated Q. ilex wood chips was prepared as reported by Beltrán-Flores et al. sludge. The powerful enzyme system of WRF allows the removal of a (2021). Acetonitrile and formic acid were purchased from Merck wide range of organic compounds, including pesticides, considered (Darmstadt, Germany). These chemicals and others used for T. versicolor recalcitrant in other treatment systems (Mir-Tutusaus et al., 2018). subculture were of high purity grade. Among WRF, Trametes versicolor has recently proved to be one of the best candidates in terms of degradation activity for several pesticides (Hu 2.2. AWW characterization et al., 2020). Fungal reactors have been extensively studied for wastewater treat­ AWW was taken in September 2020 from an artificial pond designed ment (Espinosa-Ortiz et al., 2016). During the last years, the scientific to collect wastewater produced after washing spraying equipment and community seems to have reached a certain consensus on the benefits agricultural machinery in the Sustainable Plant Protection program of provided by biomass immobilization in fungal reactors, as it overcomes the Agrifood Research and Technology Center in the Institute of Agri­ some of the most common limitations in fungal treatments, including food Research and Technology (IRTA) in association with the Mas Badía fungal growth on reactor walls and accessories, foam production and Foundation (Catalonia, Spain). Wastewater was stored in the fridge at biomass washout by decoupling the cellular retention time (CRT) from 4 ◦ C until use. the hydraulic retention time (HRT) (Mir-Tutusaus et al., 2018). There are basically two types of fungal immobilization: autoimmobilization as 2.3. Bioreactor setup pellets and immobilization on carriers. Carriers can be either inert (e.g., polyurethane foam cubes) or non-inert (e.g., wood) (Zhuo and Fan, 2.3.1. Rotating drum bioreactor 2021). The graphical abstract shows the RDB, whose characteristics have In this regard, one potentially applicable system for AWW treatment been described in a recent publication (Beltrán-Flores et al., 2022). The is an air-pulsed fluidized-bed reactor (FBR) using T. versicolor pellets, RDB (2.3 L) was fed in sequential batches for two periods of 17 days and which is a well-established fungal reactor that has been successful in the inner tube was rotated one and a half turns every 24 h. An external treating micropollutants at lab-scale even for a long-term period (Mir-­ recirculation loop (4.7 L day− 1) was required for pH adjustment and DO Tutusaus et al., 2019). However, operating an FBR under non-sterile measurement, which were performed in a recirculation tank (≈0.4 L). conditions has several operational limitations, such as bacterial The pH was automatically controlled at 4.5 by adding either 1 M HCl or contamination (since glucose is added as a carbon source) and foaming NaOH. The DO level was monitored using a CyberScan 600 Series (as forced aeration is required) (Mir-Tutusaus et al., 2018). Waterproof Handheld (Eutech Instruments). The DO level remained Alternatively, a rotating-drum reactor (RDB) with T. versicolor above 30% in the recirculation tank throughout the treatment. The inner immobilized on wood has shown good performance in treating pesti­ tube contained 1100 g DW of colonized wood. Liquid samples were cides from agricultural wastewater and its long-term viability has also taken from the reactor effluent for analyses of pesticide concentration, been previously demonstrated (Beltrán-Flores et al., 2020). Immobili­ laccase, absorbance, chemical oxygen demand (COD), heterotrophic zation on lignocellulosic materials such as wood has some advantages: plate count (HPC), toxicity, phytotoxicity and microbial communities. sustainability, low cost, use of a specific substrate for fungi (limiting Solid samples were withdrawn at the end of the treatment to measure bacterial competition) and micropollutant sorption (Hu et al., 2020; the remaining pesticide content and microbial communities. The Torán et al., 2017). Furthermore, the design of the RDB has been spe­ removal efficiency of the studied pesticides was calculated according to cifically conceived for in-situ application in agricultural fields. However, Eq. (1): special emphasis should be placed on the DOC increase resulting from C0 − Ct the release of soluble organic compounds into the water and the sorption Pesticide removal (%) = ⋅100 (1) capacity of wood (Beltrán-Flores et al., 2022). The RDB is a bioreme­ Ci diation system where very complex physicochemical and biological in­ Variables: C0 is the initial concentration (or content inside the wood) teractions coexist, thus pesticide removal can occur by different of the studied pesticide (mg⋅L− 1 for the liquid phase and mg⋅g− 1 for the mechanisms: sorption on wood, sorption by the fungal biomass and solid phase) and Ct is the concentration (or content inside the wood) at a biodegradation by the fungal enzyme system (both extracellular and specific time (mg⋅L− 1 for the liquid phase and mg⋅g− 1 for the solid intracellular). In this regard, previous studies have evaluated the phase). removal kinetics and mechanisms of various pesticides by wood and T. versicolor (Beltrán-Flores et al., 2021; Hu et al., 2022). 2.3.2. Fluidized-bed reactor Therefore, a comparative study between both reactors is required to A total of 2.2 g L− 1 DW pellets (WW/DW = 23 ± 3 for triplicate elucidate whether fungal immobilization strategy, i.e., pellets (auto- measurement) were transferred to an FBR (see graphical abstract) con­ immobilization) or immobilization on lignocellulosic material, is more sisting of a 7.5 cm diameter body and a considerably wider 13.5 cm viable for AWW treatment. The aim of the present work was to set up an diameter head. The FBR useful volume was 1.5 L, but only 1.3 L of AWW FBR and an RDB using T. versicolor as inoculum for treating AWW from was added in anticipation of possible foaming. The AWW was previously pesticide application equipment, and compare their performances and autoclaved at 121 ◦ C for the experiment under sterile conditions. The applicability considering different perspectives. reactor was operated in batch mode for 17 days at 25 ◦ C. The pH was controlled at a constant value of 4.5 by adding 1 M NaOH or 1 M HCl. Fluidized conditions were achieved by introducing a 1 s air pulse every 4 s through a solenoid valve placed at the bottom part of the reactor. The aeration rate was set at 0.8 L min– 1. Glucose and NH4Cl were fed for 2 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 T. versicolor maintenance at a molar C/N ratio of 7.5 (Mir-Tutusaus et al., was used to carry out non-metric multidimensional scaling (NMDS) 2017). The same analyses performed for the liquid samples in the RDB using the Bray-Curtis distance and similarity indices (Oksanen et al., (see Section 2.3.1) were replicated for the FBR. 2020). Colour, Bacterial counts, COD and diversity indexes were further visualized by NMDS using envfit function (pmax = 0.05). 2.4. Solid-phase treatment in a biopile-like system 2.7. Vibrio fischeri bioluminescence inhibition test (Microtox® test) Solid by-products produced in the RDB were piled inside Scott glass bottles (Duran, Inc; 250 mL, 95 × 105 mm) equipped with an open-port Toxicity test was performed using the acute toxicity bioassay kit from screw cap opened with a passive air inlet through a 0.45 μm filter. A total Modern Water (London, UK). In brief, the test is based on the attenuation of 30 g DW of by-products (in triplicate) obtained at the end of the of Vibrio fischeri bioluminescence after 5 and 15 min of exposure to second batch in the RDB were treated under non-sterile conditions and selected dilutions of the samples, previously adjusted to pH 7. Toxicity 25 ◦ C for 27 days. Afterwards, the solid samples were withdrawn to was expressed as toxicity units (TU). measure the remaining pesticide content. 2.8. Seed germination test 2.5. Pesticide identification and quantification The phytotoxicity of the treated/untreated agricultural wastewater A proposal for the identification of the pesticides detected in the was analysed through a seed germination test of tomato (Solanum agricultural wastewater was performed by high-performance liquid lycopersicum) by exposing 10 seeds to 10 mL of each sample (triplicate) chromatography (HPLC, 1200RR, Agilent Technologies). The HPLC was in Petri dishes for 24 h. Subsequently, each group of 10 seeds was placed equipped with a diode-array detector (DAD) and a microTOF-Q Mass on a Whatman Nº1 filter of 70 mm diameter inside Petri dishes for 10 Spectrometer (Bruker Daltonics, Bremen, Germany). Briefly, 1 mL of days of incubation at room temperature and exposure to natural light. sample was withdrawn and filtered through a 0.22 μm Millipore Millex- The filters were pre-humidified with 3 mL distillate water. Relative seed GV PVDF filter. Chromatographic separation was performed using an germination (SG), relative root elongation (RE) and germination index C18 reversed-phase column (Phenomenex®, Kinetex® EVO C18 100 Å, (GI) were determined as described elsewhere (Rodriguez-Rodriguez 5 μm, 4.6 mm × 150 mm) with the following operating conditions: et al., 2011). 30 ◦ C, eluent flow rate of 0.9 mL⋅min-1 and injection volume of 40 μL. The mobile phase consisted of 0.01% formic acid solution and acetoni­ trile, which was pumped isocratically at a ratio of 50:50. 2.9. Other analyses Analytes were first detected by DAD using a wavelength of 252 nm. Afterwards, the eluent was split using a T-type splitter before entering Pesticides were extracted from the wood as recently reported the QTOF (split = 1:2). Thus, the flow rate reaching the ESI-Q-TOF-MS (Beltrán-Flores et al., 2022). Ergosterol analysis was performed by the detector was 0.3 mL min− 1. Samples were analysed in the positive homogenization and extraction of the colonized wood per triplicate, as electrospray mode (ESI). MS data were acquired at a m/z range of described by Rodríguez-Rodríguez et al. (2010a). Conductivity was 50–1000 Da. Spray voltage was set at 2000 V and the drying gas tem­ measured using a CRISON MicroCM 2100 conductometer. Colour was perature was 210 ◦ C. The capillary voltage of the ion source was 5000 V. determined at a wavelength of 650 nm with a UNICAM 8625 UV/VIS. The dry gas flow rate was set at 8.0 L min− 1 and the nebulizer gas COD and ammonia concentrations were measured using the commercial pressure was 4.0 bar. Nitrogen was used as both drying and nebulized kits LCK114 or LCK314m and LCH303, respectively (Hach Lange, Ger­ gas. The pre-pulse storage time was 8 μs and the source transfer time was many). Total suspended solids (TSSs) and volatile suspended solids 65.0 μs Instrument calibration was performed by using a 10 mM sodium (VSSs) were measured according to the standard methods 2540 D and formate solution. 2540 E, respectively, and HPC were determined following the standard The MS data were processed using Bruker Compass DataAnalysis 4.2 method 9215 (Rice et al., 2017). Chloride, nitrite and sulfate concen­ (Bruker Daltonics, Bremen, Germany). Candidate compounds were trations were quantified by ion chromatography using a Dionex identified on the Chemspider website by their exact masses (Royal So­ ICS-2000. ciety of Chemistry, 2022). Finally, confirmatory analyses and quantifi­ T. versicolor superficial growth on wood was evaluated by scanning cation were conducted using the analytical standards of the detected electron microscopy (SEM). Solid samples were fixed on a 2.5% pesticides. In this case, the analysis was performed using the same glutaraldehyde solution with 0.1 M phosphate buffer (PB). Post-fixation analytical method in a Dionex Ultimate 3000 HPLC-UV. was performed using 1% osmium vapour containing 0.8% ferrocyanide in 0.1 M PB overnight. Afterwards, the samples were washed with ul­ 2.6. DNA extraction and PCR-DGGE of the fungal community trapure water and dehydrated in increasing concentrations of ethanol (50, 70, 90, 96 and 100%). Finally, the samples were subjected to the All liquid and solid samples were collected, immediately stored at critical point drying method (Baltec CPD030) and metallized with AuPd. 4 ◦ C, and processed as described earlier by the authors (Beltrán-Flores et al., 2022). Briefly, total DNA extraction was performed on the sam­ 3. Results and discussion ples, and the DNA extracts were subsequently amplified by nested PCR to reduce non-specific products and increase sensitivity, using two sets 3.1. AWW characteristics of specific primers targeting fungal ITS (Internal Transcribed Spacer) region (Gardes and Bruns, 1993; White et al., 1990). Afterwards, all Physicochemical characteristics of the AWW are summarized in amplification products were subsequently studied by denaturing Table S1 (Supplementary material). The main characteristics of this gradient gel electrophoresis (DGGE) to assess fungal genetic variations AWW compared to other agricultural and urban wastewater was the throughout the experiment and provide an estimate of richness and high content of organic matter in the form of COD or TOC detected, and abundance of prominent members of the fungal assemblage. as a consequence of the eventual decomposition of this organic matter, After visualization of DGGE profiles, prominent bands were cut, slightly higher levels of ammonium and bacteria counts (Beltrán-Flores recovered, and sequenced at external facilities by Macrogen, Inc. (South et al., 2021; Metcalf and Eddy, 2003). This study analyses not only the Korea). Sequences determined in this study are available at the National treatment efficiency concerning the removal of pesticides, but also the Center for Biotechnology Information (NCBI) GenBank database under evolution of other characteristic parameters to determine the final accession numbers ON116618 through ON116654. The Vegan package effluent quality. 3 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 3.2. Pesticide identification and quantification in Fig. 1. The FBR operation was conducted under both sterile (FBR-S) and non-sterile (FBR) conditions to evaluate the effect of microbial Liquid chromatography coupled to time-of-flight mass spectrometry contamination on the treatment performance. Although non-sterility (LC-qTOF-MS) is a useful technique for qualitative analysis and identi­ can benefit the efficiency of fungal treatment owing to the positive fication of organic micropollutants (Arsand et al., 2018). High mass synergies created by the microbial consortium, e.g. by metabolite resolution along with the acquisition of mass profile data allowed the degradation, non-sterile conditions can also exert pressure on the WRF screening of non-targeted compounds. survival, which in turn can reduce the fungal activity and process Unknown compounds were identified through exact mass analysis. duration (Mir-Tutusaus et al., 2018). In this case, the FBR-S was more LC-qTOF-MS analysis shows high specificity, allowing the pesticide efficient than the FBR concerning total pesticide removal, reaching 88 identification by the characterization of the monoisotopic mass with a and 51%, respectively. This result confirms that other microorganisms, maximum error of ±2.5 mDa. The detected exact masses were close to such as bacteria, can compete for substrate and reduce fungal metabolic those of the following common pesticides: thiacloprid (THIA), chlorto­ activity over time (Mir-Tutusaus et al., 2019). However, from a practical luron (CHLOR), azoxystrobin (AZO) and tebuconazole (TEBU). More­ point of view, the FBR can be considered a better option than the FBR-S over, the isotope pattern validated the identification of such pesticides. given that sterilisation is a costly process that should be avoided at In this regard, the experimental isotopic patterns match the predicted full-scale applications, and negligible bacterial proliferation was isotope distributions as shown in Table S2 (Supplementary materials). observed in the FBR (see Section 3.3.3.) while maintaining high removal Afterwards, commercial reagents of each pesticide were spiked to the efficiency. AWW to verify that the chromatogram peaks of the standard compounds Alternatively, the AWW was also treated by T. versicolor immobilized matched those detected in the original matrix. After confirmatory on wood in an RDB, which has shown good results in treating agricul­ analysis, initial pesticide concentrations were quantified obtaining a tural wastewater under non-sterile conditions (Beltrán-Flores et al., total of 40.55 mg L− 1 of pesticides, consisting of 19.17 mg L− 1 THIA, 2022). As shown in Fig. 1, higher removal yields were obtained in the 7.42 mg L− 1 CHLOR, 4.47 mg L− 1 AZO and 9.49 mg L− 1 TEBU. Although RDB, reaching up to 85% of total removal, compared to the FBR (51%). these concentrations are relatively high for agricultural water In fact, equilibrium was not reached in the FBR during the 17 days of (Köck-Schulmeyer et al., 2019), they are reasonable considering that the treatment, while the RDB showed maximum removals after the first 5 AWW comes from the washing of agricultural equipment that had been days. This result is particularly interesting for future full-scale reactor in direct contact with pesticides. THIA is a neonicotinoid insecticide that applications, as operating periods, and thus reactor volumes, could be acts by disrupting the nervous system of these organisms. This pesticide considerably reduced. However, a significant amount of pesticides could was included in the watch list of substances in Decision (2015)/495/EU be sorbed on the wood in the RDB (Beltrán-Flores et al., 2021), which (European Commission, 2015), as it was suspected to pose a high risk to motivated the solid phase study presented in Section 3.4. human health as endocrine disrupting, neurotoxic and carcinogenic THIA was the pesticide with the lowest removal performances in compounds. Recently, the EC banned THIA by not renewing its license both systems, being 38% and 80% for the FBR and RDB, respectively. In for use in the EU (European Commission, 2019). CHLOR is an herbicide contrast, both the FBR (86%) and the RDB (100%) achieved high TEBU belonging to the phenylurea class that acts by the inhibition of photo­ removals. Thus, although WRF have been reported to remove both synthetic electron transport. It is moderately toxic to most aquatic spe­ pesticides (Chan-Cheng et al., 2020; Mori et al., 2021), in the present cies, birds and worms, although toxicity to mammals is considered low. study T. versicolor was clearly more efficient removing TEBU than THIA. AZO is a broad-spectrum systemic fungicide that acts on the target or­ Since the RDB showed the best results in terms of pesticide removal, ganism by inhibiting spore germination and has been recognized to have as well as other parameters studied in Section 3.3.3, such as COD, colour physiological impact on some aquatic organisms. TEBU is a triazole and bacterial count studied in Section 3.3.3, a second batch was con­ fungicide that affects the biosynthesis of the phytohormone gibberellin, ducted in this system to evaluate its performance in a sequential treat­ thus inhibiting seed germination and plant growth. It is classified as a ment. In this regard, pesticide removal yields (87% total removal) possible carcinogen (rating C) on the U.S. Environmental Protection comparable to those achieved in the first batch (85% total removal) Agency’s Office of Pesticide Programs list of carcinogens. These last were obtained, demonstrating the viability of the RDB for sequential/ three pesticides are currently approved for use in the EU under EC continuous treatments. The ability of the RDB to operate for extended Directive 1107/2009 (European Commission, 2009b), but AZO and periods has been previously reported for the treatment of spiked agri­ TEBU are currently under study. AZO has been included in the watch list cultural wastewater by (Beltrán-Flores et al., 2022), which was main­ of substances in Decision (2022)/1307/EC and TEBU was first included tained for up to 225 days. in the watch list of substances in Decision (2020)/1161/EC, but due to insufficient high-quality monitoring data TEBU has remained on the 3.3.2. Toxicity watch list in Decision (2022)/1307/EC. (European Commission, The capacities of both reactors to remove the pesticides detected in 2022;2020). the AWW were evaluated in Section 3.3.1. However, other undetected compounds or potential metabolites may also contribute to the overall 3.3. Comparison between bioreactors toxicity of the effluent (Marco-Urrea et al., 2009), thus acute toxicity and phytotoxicity analyses were also required (Table 1). Initial toxicity 3.3.1. Pesticide removal performance of 13.6 TU was considerably reduced in both reactors. As occurred in the Two different fungal immobilization strategies were used: auto- case of the detected pesticides (see Section 3.3.1), sterile conditions immobilization (pellets) in the FBR and immobilization on wood chips favoured the reduction of toxicity in the FBR, denoting a better degra­ in the RDB. In the RDB, the initial ergosterol content was approximately dation activity of the fungal consortium in the absence of competing 0.121 ± 0.018 mg g DW− 1 of wood. Considering that 1100 g of colo­ microorganisms. However, even under non-sterile conditions, the nized wood chips were transferred to the reactor and the ergosterol- removal capacity of the RDB prevailed over that of the FBR-S. In any biomass correlation for the selected strain is 6.61 mg g DW− 1 of case, toxicity values were lower than the wastewater discharge limit (25 fungal biomass (Rodríguez-Rodríguez et al., 2010b), a total of 20 g DW TU) established in Catalonia (DOGC, 2003). of biomass were introduced to the reactor. However, only 30% of the Similar results were reflected in the phytotoxicity test. The RDB biomass was continuously submerged in the reactor, which is 2.2 g effluent caused less impact on tomato seed germination and growth than biomass DW⋅L− 1. Hence, an equivalent pellet inoculum was used in each that of the FBR. Actually, the RDB effluent achieved even better SG and FBR experiment. GI results than distillate water, which was probably ascribed to the Pesticide removal profiles in the FBR and RDB treatments are shown release of some nutrients that considerably stimulate seedling growth, 4 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 Fig. 1. Pesticide removals achieved for THIA (a), CHLOR (b), AZO (c) and TEBU (d) in each experiment. Table 1 Toxicity and phytotoxicity of the treated and original agricultural wastewaters. Wastewater Operating conditions Toxicity (TU) Phytotoxicity Relative seed germination SG (%) Relative root elongation RE (%) Germination index GI (%) Pond – 13.6 ± 1.0 52.9 ± 5.2 69.6 ± 4.7 36.9 ± 3.2 FBR-S Sterile 6.0 ± 0.3 88.2 ± 20.6 82.28 ± 15.5 72.6 ± 17.8 FBR Non-sterile 8.6 ± 2.8 76.5 ± 31.8 82.9 ± 11.9 63.4 ± 11.8 RDB-1 Non-sterile 2.2 ± 0.3 111.8 ± 40.9 93.7 ± 20.2 104.7 ± 29.2 RDB-2 Non-sterile 2.9 ± 0.1 61.8 ± 27.0 78.5 ± 1.7 48.5 ± 4.3 such as NH+4 and K (Loffredo et al., 2016). However, partial inhibition + was obtained in the RDB-2 compared to the RDB-1, indicating a certain deterioration of the effluent quality throughout the treatment. Since pesticide concentrations were even lower in the RDB-2 than in the RDB-1, the increased toxicity was related to the formation of some metabolites. In this respect, a possible metabolite of THIA with 200-fold higher toxicity in vertebrates has been previously reported (Casida, 2011). This phenomenon should be further investigated in future works. 3.3.3. Monitoring of bioreactors As shown in Sections 3.3.1 and 3.3.2, the RDB was clearly more efficient than the FBR concerning toxicity abatement. However, there are other parameters to consider in selecting the best reactor for AWW treatment, such as laccase activity, COD, absorbance and microbial counts. Laccase is a typical indicator used to evaluate enzyme activity in fungal treatments (Mir-Tutusaus et al., 2017). The RDB reached sub­ stantially higher laccase activity levels than the FBR during the treat­ ment, with activity peaks of 16 and 2 AU⋅L− 1, respectively (Fig. 2). Furthermore, growing patterns were observed for the RDB and FBR-S, Fig. 2. Time-course profile of laccase activity in each experiment. 5 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 while the FBR showed a decreasing trend from day 5 onwards, of some organic compounds that give the characteristic dark colour to evidencing the negative effect of contamination on the enzyme meta­ Q. ilex wood. In the case of the FBR, a higher turbidity was also observed bolism of the pelleted fungus. Interestingly, this laccase depletion was probably as a consequence of a significant loss of pellet morphology not observed in the RDB under non-sterile condition, indicating that the (Espinosa-Ortiz et al., 2016). immobilization on wood chips played a vital role in maintaining the The HPC results are presented as the logarithm of CFU in Table 2. The enzymatic activity of the fungal consortium. HPCs remained constant in both reactors, indicating that bacterial Laccase production was mainly attributed to Trametes sp. and Pha­ contamination was successfully controlled. The addition of glucose in naerochaete sp., which are the only two genera of WRF detected in the the FBR did not result in significant bacterial proliferation, contrary to fungal consortium (see Section 3.5) capable of synthesising this enzyme what was reported in the same reactor by Hu et al. (2021), probably (Yang et al., 2017). In this regard, Torán et al. (2017) reported low caused by the inhibitory effect of the relatively high toxicity RW on the laccase activity for reactors based on both pelleted and colonized-wood, bacterial community. but in the latter case, lower extent of fungal colonization (according to Considering the remarkable good results of pesticide removal (Sec­ ergosterol test) had been achieved during the fungal growth stage. Hu tion 3.3.1), toxicity reduction (Section 3.3.2) and the stable values of the et al. (2021) obtained slightly higher laccase levels in an FBR with characteristic AWW parameters obtained in this section, the RDB T. versicolor pellets compared to a trickle-bed reactor (TBR) with colo­ effluent was found to present an overall quality clearly superior to that nized wood, but this fact was mainly attributed to poor aeration inside from the FBR and the original AWW. Furthermore, unlike the continuous the TBR. Furthermore, laccase activity was significantly higher in the glucose supplementation required in the FBR, the RDB only demanded second batch of the RDB, which evidenced that the immobilized fungal the initial low-cost wood chips as substrate. Therefore, it is concluded community was able to increase its enzyme activity as it adapted to that the RDB is the most promising treatment for further full-scale ap­ non-sterile conditions. Laccase has been reported to be involved in plications. This reactor could be used for the in-situ treatment of the pesticide degradation, although other enzymes or the intracellular AWW produced in the IRTA’s agricultural fields (Mas Badía). In these P-450 enzyme system may also play a key role (Hu et al., 2022). fields, several pesticide applications are performed for each type of crop COD, absorbance and HPCs of the treated effluents are presented in throughout the summer campaign. To avoid cross-contamination of Table 2. Contradictory results regarding the COD reduction capacity of pesticides between crops, the machinery is washed after each applica­ T. versicolor have been previously reported (Cruz-Morató et al., 2013; tion, and the produced wastewater is accumulated in the collection Souza et al., 2014). In this study, COD increased significantly in both pond. Accordingly, at the end of the campaign period, the collected reactors, but to a lesser extent in the case of the RDB. In the RDB, the wastewater could be treated in batch mode by a full-scale RDB for main contribution to COD was probably the release of organic matter pesticide and toxicity abatement. from the wood chips (Lacorte et al., 2003). In the case of the FBR, COD increase was attributed to the addition of antifoam Tween 80 (also 3.4. Solid-phase study and treatment known as polysorbate 80, theoretical COD = 2207 mg O2 mL− 1). In this respect, fungal treatments involving forced aeration are known to cause Studies involving sorption processes should not only evaluate foaming (Font et al., 2003). Consequently, antifoam was only required micropollutant transfer from the liquid to the solid phase, but also the in the FBR, as the RDB was an open system with aerobic conditions. fate of the polluted sorbent. Hence, solid by-product obtained from the More antifoam was required in the case of the FBR-S (7 mL) compared to RDB were analysed to determine the contributions of the sorption and the FBR (5 mL) probably owing to the more intensive metabolic activity biodegradation to the overall removal by means of pesticide mass bal­ demonstrated by T. versicolor under sterile conditions, which in turn ance. Sorption was found to be a crucial mechanism of pesticide elimi­ increased COD level in the FBR-S. Regarding the RDB, COD was sub­ nation, being around 81% THIA, 83% CHLOR, 83% AZO and 25% TEBU. stantially lower in the second batch (RDB-2), which indicates that a This remarkable sorption contribution of pesticides by wood had pre­ significative part of the soluble organic compounds of the wood were viously been reported in the literature (Hu et al., 2021). Sorption ca­ extracted during the first cycle. Moreover, some of the soluble organic pacity of wood has been related to pesticide hydrophobicity, and matter of the wood was probably consumed by T. versicolor and the therefore, affinity of these compounds for wood fibres (Beltrán-Flores associated fungal consortium instead of being released into the liquid et al., 2021). In the present study, CHLOR and AZO showed slightly phase (Beltrán-Flores et al., 2022). In that study, the COD of the inoc­ higher sorption than THIA, which may be due to the fact that they are ulated reactor was found to be substantially lower than that of the more hydrophobic compounds. However, TEBU was the most apolar control reactor, with this difference becoming more noticeable over (log Kow = 3.7) and simultaneously the least sorbed pesticide, which was time. Therefore, these results are a positive sign for the RBD application mainly attributed to its high biodegradability in the early stages of in large-scale during long-term operations. treatment, as shown in Fig. 1 (d). In other words, biodegradation was the Wastewater colouration increased throughout the treatment in the fastest removal mechanism for TEBU, preventing its potential sorption RDB, being substantially lower in the second batch (Table 2). As with the on wood. COD, the colour gain in the RDB was mainly attributed to the extraction Solid by-products resulting from the RDB were subsequently treated Table 2 COD, colour and microbial counts of the agricultural wastewater throughout the treatment in the three different fungal reactors. Operation time (day) COD (mg O2 ⋅ L− 1) Colour Microbial counts [log (CFU⋅mL− 1)] FBR-S FBR RDB_1 RDB_2 FBR-S FBR RDB_1 RDB_2 FBR-S FBR RDB_1 RDB_2 0 2088 2088 2088 2088 0.171 0.171 0.171 0.171 – 6.47 6.47 6.47 0* 5190 4802 – 1 5600 5160 3709 3228 0.079 0.183 0.197 0.156 – 3 6380 7392 4407 3517 0.080 0.170 0.296 0.172 – 5 8360 10,024 5340 3727 0.052 0.155 0.390 0.191 – 6.42 6.85 6.46 7 11,448 10,404 6006 3754 0.046 0.129 0.413 0.197 – 10 13,128 9296 6336 4068 0.039 0.212 0.427 0.228 – 6.48 7.02 6.10 12 13,104 8120 6643 4158 0.040 0.310 0.302 0.247 – 17 10,476 8140 7076 4785 0.041 0.372 0.243 0.212 – 6.61 6.54 6.51 * Antifoam was initially added to prevent excessive foaming resulting from aeration. 6 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 in a biopile-like reactor during 27 days to evaluate the ability of the Table 3 remaining fungus to biodegrade sorbed pesticides. Fig. 3 shows the re­ Biodegradation of pesticides contained in polluted waste achieved by sults of the pesticide removal balance in the two-stage treatment con­ T. versicolor after 27 days of treatment. sisting of the RDB and the biopile. The analysis of the biomass after the Pesticide Initial Biodegradation Biodegradation rate biopile treatment allowed the quantification of the pesticide biodegra­ concentration ± SD ± SD (%) ± SD (mg⋅g− 1⋅day− 1) dation in the solid biopile, being 5% THIA, 26% CHLOR, 13% AZO, 2% (mg⋅g− 1) x 102 x 105 TEBU. Although these percentages are apparently low, the total amount Thiacloprid 3.83 ± 0.40 4.56 ± 0.47 7.96 ± 0.82 of pesticides removed is remarkable given their high content in the wood Chlortoluron 1.51 ± 0.12 26.20 ± 1.76 17.67 ± 1.19 Azoxystrobin 0.91 ± 0.14 12.54 ± 2.54 5.10 ± 1.03 (≈28 mg g wood DW− 1). Thus, biodegradation rates of the pesticides Tebuconazole 0.57 ± 0.08 2.06 ± 0.91 1.78 ± 0.79 were also calculated to facilitate comparison with other literature results (Table 3). In this regard, a similar biodegradation rate of total pesticides (3.25⋅10− 4 mg g wood DW− 1⋅day− 1) was achieved in a previous fungal sp. and Phanaerochaete sp. have been reported to degrade pesticides treatment of wood in a biopile system (Beltrán-Flores et al., 2021). (Chen et al., 2021; Zehra et al., 2017). Meyerozyma sp. has also been Longer operational periods, such as those commonly applied in documented to degrade other micropollutants such as textile dyes (Ali compost-like systems, could considerably reduce the remaining pesti­ et al., 2021). It should be noted that these samples present an array of cide content of the solid by-products (Rodriguez-Rodriguez et al., 2011). minor bands that could not be studied due to their small representation In addition, future research should explore other operational strategies (grouped under “Others”). Furthermore, Trametes sp. was not identified to improve removal efficiency, such as the addition of fresh colonized in any of the three replicates, indicating its absence from the initial wood, re-inoculation of solid by-products, adjustment of the moisture AWW. Regarding those three prominent bands, Penicillium sp. is a content, modification of the inoculation method and wood crushing. ubiquitous genus, found as part of the mycobiome of many aquatic and terrestrial environments playing a paramount role as a decomposer 3.4.1. Fungal community assemblage (Park et al., 2018), and it has a major importance in the natural envi­ In order to assess the fungal diversity of both reactors and the ronment. The second one, Phanaerochaete sp., is of special interest since persistence of T. versicolor after being inoculated, a PCR-DGGE analysis it is also a WRF with similar ligninolytic capabilities to Trametes was performed. The DGGE band profiles and UPGMA cluster analysis of (Köck-Schulmeyer et al., 2019). Finally, the yeast Meyerozima sp. has the fungal populations from the effluent of the reactors can be observed (among other capabilities) a Chitinase enzyme activity of great interest in Fig. S1 (Supplementary materials). This figure shows remarkable in plant growth promoting (Kapoor 2021). differences between the final samples from both reactors, FBR and RDB, Other genera detected in this initial sample include: Rhodotorula, one as they are clearly separated in the cluster analysis, while replicates of of the most studied yeast genera, that can utilize a wide range of sub­ the same sample show a high degree of homogeneity. Besides, FBR strates (mono- and polysaccharides, waste materials, etc.) while pro­ samples cluster closer to the AWW, indicating fewer changes concerning ducing high-value substances (Kot 2016); Didymella (a fungal plant the initial fungal community than after the RDB treatment. pathogen) (Corlett, 1981), Cladosporium (a common mold, occasionally To study the presence of T. versicolor in the effluent of both reactors, found as a fruit pathogen) (Carolina Virginia et al. 2021), Cystobasidium and rule out its previous presence in the AWW, prominent bands were (a parasite of other fungi, isolated from soils rich in cellulosic waste) excised from the gels and sequenced. This allowed determination of the (Vyas and Chhabra, 2017), and Paraglomus (a mycorrhizal fungus typi­ phylogenetic affiliations of every recovered band and calculation of the cally found in arable soils) (Hijri et al., 2006). relative abundance of each phylotype present in all samples analysed After treatment with RDB, the most obvious change in the commu­ (Fig. 4 and Table S3, Supplementary materials). nity is the reduction of fungal diversity, with only 4 and 3 prominent Samples corresponding to AWW display high fungal diversity, with 3 bands left in the first and second batch samples respectively (Fig. 4). The prominent genera representing more than half of the fungal community appearance of Trametes is also noteworthy: it is detected in the effluent (Penicillium sp., Phanaerochaete sp. and Meyerozima sp.). Both Penicillium of the first batch, and its presence is increased after the second batch, representing in the end more than a 10% of the fungal diversity. How­ ever, when comparing with the fluidized reactor samples, it can be noted how the presence of Trametes sp. in the effluent of the RDB treatment is actually minor: in the final FBR effluent sample, Trametes sp. alone represents almost 50% of the fungal diversity. This result suggests that the immobilization on wood used in the RDB was more effective in retaining biomass than in the case of auto-immobilization (pellets) of the FBR. This statement is also supported by the clustering (dendrogram, Fig. S1) data that shows how the AWW community has a greater simi­ larity to the FBR effluent community than to the RDB effluent samples. This indicates that fungal immobilization in the RDB allows for a greater change in the fungal community inside the reactor, influenced by the inoculated T. versicolor. However, in the FBR, although there is more presence of Trametes sp. in the liquid phase, the rest of the fungal community does not seem to have significantly changed, which may be due to a poor consolidation of Trametes sp. in pellet form, leading to a lower capacity for proliferation and influence on the rest of the species. Finally, to better relate sample clustering with phylotypes present and environmental variables, a NMDS was plotted using DGGE band profiles and environmental parameters (colour, bacterial count Fig. 3. Balance of pesticide removals in the RDB: biodegradation from liquid phase (green), adsorption on wood (blue) and biodegradation from wood in a (CFU⋅ml− 1) and COD (mg O2⋅L− 1)) (Fig. S1 and Table S3). As can be seen biopile reactor (blue line pattern). Results are shown as mean values and cor­ from NMDS (Fig. 5), liquid samples show equivalent grouping to that responding standard deviations for triplicate measurements. (For interpretation observed in the dendrograms. Both RDB samples (first batch and second of the references to color in this figure legend, the reader is referred to the Web batch), appear close together in the plot, mostly influenced by three version of this article.) genera: Rhodotorula, Meyerozyma and Cystobasidium, and distantly 7 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 Fig. 4. Relative abundance of fungal populations in the original AWW and in the FBR and RDB effluents, corresponding to the liquid samples (left), and the RDB and the biopile lignocellulosic support samples at the end of each experiment (right). Values for Shannon Diversity and Evenness Indices are shown at the bottom of each sample. Fig. 5. Non-metric multidimensional scaling (NMDS) ordination for all liquid samples based on Bray-Curtis similarity index with abiotic parameters and diversity indexes imaging. Symbols: red squares represent AWW, green circles correspond to final FBR effluent samples, and blue and lilac triangles belong to RDB effluent samples from the first and second batch, respectively. Grey dots represent species scores. Light grey vectors represent relevant (p.max = 0.05) pa­ rameters: C = “Colour”, BC = “Bacterial Count”, COD = “Chemical Oxygen Demand”, H’ = “Shannon Di­ versity Index). 2D Stress = 0.01764. (For interpreta­ tion of the references to color in this figure legend, the reader is referred to the Web version of this article.) related from AWW and FBR. In the same way, AWW and FBR samples similar fungal genera are detected in both systems (Fig. 4). Most bands form two separated groups depending on fungal populations composi­ seem to coincide in both treatments, with a significant difference: tion and abundances, the former mostly influenced by differences in the Meyerozima sp. seems not to be present in the biopile sample, although it community (involving Phanaerochaete, Didymella and Cladosporium), was prominent in the RDB. and the latter by Aspergillus and Trametes, respectively. All replicates of Interestingly, Trametes sp. decreases its presence in the community each sample seem to cluster together, indicating consistency in the re­ once the wood is treated in the biopile, while other genera seem to thrive sults. Regarding environmental parameters, it can be observed how the and significatively increase their abundance. Those genera include AWW sample is greatly represented by the high values of their Shannon Phanaerochaete sp. and Cystobasidium sp. (already mentioned in the diversity indices. This fact can also be appreciated in Fig. 4, where it is previous section) but also Vishniacozyma sp., a cosmopolitan yeast, shown how this sample has much greater fungal diversity (Shannon previously isolated from soil, wood, and fruit trees. Finally, values for Index), as it was expected for an environmental sample, when compared Shannon Diversity and Evenness indices present no significant differ­ to the “post-treatment” samples, both RDB and FBR effluents. ence between them, indicating no major shifts in the community, aside Wooden samples from RDB and the biopile were analysed together. from those already mentioned. DGGE band profiles and cluster analysis corresponding to the fungal community from extracted wood chips are shown in Fig. S1 (Supple­ 4. Conclusions mentary materials). Samples from the RBD and the biopile appear completely separated and grouped with their corresponding replicas, as AWW with an inherently high pesticide content was treated in two they are two different systems. When studying these solid samples, different fungal reactors. The RDB proved to be a better candidate than however, it must be noted that fungal diversity is relatively low, and the FBR according to all studied parameters, including 87% versus 51% 8 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 in pesticide removal, respectively. Fungal community study showed that Blánquez, P., Casas, N., Font, X., Gabarrell, X., Sarrá, M., Caminal, G., Vicent, T., 2004. Mechanism of textile metal dye biotransformation by Trametes versicolor. Water T. versicolor was especially dispersed in the FBR, while this fungus was Res. 38, 2166–2172. https://doi.org/10.1016/j.watres.2004.01.019. successfully immobilized in the RDB. In addition, solid by-products were Casida, J.E., 2011. Neonicotinoid metabolism: compounds, substituents, pathways, treated with T. versicolor in a biopile reactor achieving remarkable enzymes, organisms, and relevance. J. Agric. Food Chem. 59, 2923–2931. https:// biodegradation rates of pesticides of up to 17.67 ± 1.19 ⋅ 10− 5 mg g− 1 doi.org/10.1021/jf102438c. Chan-Cheng, M., Cambronero-Heinrichs, J.C., Masís-Mora, M., Rodríguez-Rodríguez, C. day − 1. These results suggest that the RDB is a promising approach for E., 2020. Ecotoxicological test based on inhibition of fungal laccase activity: further AWW full-scale treatments. application to agrochemicals and the monitoring of pesticide degradation processes. Ecotoxicol. Environ. Saf. 195, 1–10. https://doi.org/10.1016/j. ecoenv.2020.110419. Autorship contribution statement Chen, A., Li, W., Zhang, X., Shang, C., Luo, S., Cao, R., Jin, D., 2021. Biodegradation and detoxification of neonicotinoid insecticide thiamethoxam by white-rot fungus Eduardo Beltrán Flores: Investigation, Formal analysis, Writing- Phanerochaete chrysosporium. J. Hazard Mater. 417, 1–10. https://doi.org/ 10.1016/j.jhazmat.2021.126017. original draft, Writing-Review & Editing. Martí Pla-Ferriol: Investiga­ DOGC, 2003. Decret 130/2003. D. Of. la General. Catalunya 5, 11143–11158. tion, Formal analysis, Writing-original draft, Writing-review & editing of Espinosa-Ortiz, E.J., Rene, E.R., Pakshirajan, K., Van Hullebusch, E.D., Lens, P.N.L., fungal community analysis. Maira Martínez-Alonso: Supervision, 2016. Fungal pelleted reactors in wastewater treatment: applications and perspectives. Chem. Eng. J. 283, 553–571. https://doi.org/10.1016/j. Conceptualization, Resources, Writing-review & editing of fungal com­ cej.2015.07.068. munity analysis. Núria Gaju: Supervision, Conceptualization, Re­ European Commission, 2022. Commission Implementing Decision (EU) 2022/1307 of 22 sources, Writing-Review & Editing of fungal community analysis. July 2022 Establishing a Watch List of Substances for Union-wide Monitoring in the Field of Water Policy Pursuant to Directive 2008/105/EC of the European Montserrat Sarrà: Supervision, Conceptualization, Resources, Writing- Parliament and of the Council. Official Journal of the European Union. Review & Editing. Paqui Blánquez: Supervision, Conceptualization, European Commission, 2020. Commision Implementing Decision (EU) 2020/1161-4 Resources, Writing-Review & Editing. August 2020-establishing a Watch List of Substances for Union-wide Monitoring in the Field of Water Policy Pursuant to Directive 2008/105/EC of the European Parliament and of the Council. Official Journal of the European Union. Declaration of competing interest European Commission, 2019. Thiacloprid - Final Renewal Report for the Active Substance Thiacloprid. OJ. https://doi.org/10.2903/j.efsa.2019.5595. European Commission, 2015. Commission implementing decision (EU) 2015/495 of 20 The authors declare that they have no known competing financial March 2015 establishing a watch list of substances for Union-wide monitoring in the interests or personal relationships that could have appeared to influence field of water policy pursuant to directive 2008/105/EC of the European parliament and of the co. Off. J. Eur. Union 40–42. the work reported in this paper. European Commission, 2009a. Directive 2009/127/EC of the European Parliament and of the Council of 21 October 2009 amending Directive 2006/42/EC with regard to Data availability machinery for pesticide application (Text with EEA relevance). Off. J. Eur. Union L 310, 29–33. European Commission, 2009b. The Placing of Plant Protection Products on the Market Data will be made available on request. and Repealing Council Directives 79/117/EEC and 91/414/EEC. Official Journal of the European Union. Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning. Acknowledgements Font, X., Caminal, G., Gabarrell, X., Romero, S., Vicent, M.T., 2003. Black liquor detoxification by laccase of Trametes versicolor pellets. J. Chem. Technol. Project PID2019-103989RB-100 financed by MCIN/AEI/10.13039/ Biotechnol. 78, 548–554. https://doi.org/10.1002/jctb.834. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes - 501100011033. The authors acknowledge Servei d’Anàlisi Química at application to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113–118. the Universitat Autònoma de Barcelona for its contribution to the https://doi.org/10.1111/j.1365-294X.1993.tb00005.x. pesticide identification. Eduardo Beltrán-Flores acknowledges support Hu, K., Barbieri, M.V., López-García, E., Postigo, C., López de Alda, M., Caminal, G., Sarrà, M., 2022. Fungal degradation of selected medium to highly polar pesticides by from a MINECO predoctoral research grant (BES-2017-080500). Martí Trametes versicolor: kinetics, biodegradation pathways, and ecotoxicity of treated Pla Ferriol acknowledges the predoctoral grant from UAB. waters. Anal. Bioanal. Chem. 414, 439–449. https://doi.org/10.1007/s00216-021- 03267-x. Hu, K., Sarrà, M., Caminal, G., 2021. Comparison between two reactors using Trametes Appendix A. Supplementary data versicolor for agricultural wastewater treatment under non-sterile condition in sequencing batch mode. J. Environ. Manag. 293, 1–7. https://doi.org/10.1016/j. Supplementary data to this article can be found online at https://doi. jenvman.2021.112859. Hu, K., Torán, J., López-García, E., Barbieri, M.V., Postigo, C., de Alda, M.L., Caminal, G., org/10.1016/j.jenvman.2022.116595. Sarrà, M., Blánquez, P., 2020. Fungal bioremediation of diuron-contaminated waters: evaluation of its degradation and the effect of amenable factors on its References removal in a trickle-bed reactor under non-sterile conditions. Sci. Total Environ. 743, 1–9. https://doi.org/10.1016/j.scitotenv.2020.140628. Köck-Schulmeyer, M., Postigo, C., Farré, M., Barceló, D., López de Alda, M., 2019. Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., Thomaidis, N.S., Xu, J., 2017. Progress in Medium to highly polar pesticides in seawater: analysis and fate in coastal areas of the biological and chemical treatment technologies for emerging contaminant Catalonia (NE Spain). Chemosphere 215, 515–523. https://doi.org/10.1016/j. removal from wastewater: a critical review. J. Hazard Mater. 323, 274–298. https:// chemosphere.2018.10.049. doi.org/10.1016/j.jhazmat.2016.04.045. Lacorte, S., Latorre, A., Barceló, D., Rigol, A., Malmqvist, A., Welander, T., 2003. Organic Ali, S.S., Sun, J., Koutra, E., El-Zawawy, N., Elsamahy, T., El-Shetehy, M., 2021. compounds in paper-mill process waters and effluents. TrAC, Trends Anal. Chem. 22, Construction of a novel cold-adapted oleaginous yeast consortium valued for textile 725–737. https://doi.org/10.1016/S0165-9936(03)01009-4. azo dye wastewater processing and biorefinery. Fuel 285, 1–13. https://doi.org/ Loffredo, E., Castellana, G., Taskin, E., 2016. A two-step approach to eliminate pesticides 10.1016/j.fuel.2020.119050. and estrogens from a wastewater and reduce its phytotoxicity: adsorption onto plant- Arsand, J.B., Hoff, R.B., Jank, L., Dallegrave, A., Galeazzi, C., Barreto, F., Pizzolato, T.M., derived materials and fungal degradation. Water, Air, Soil Pollut. 227, 1–12. https:// 2018. Wide-scope determination of pharmaceuticals and pesticides in water samples: doi.org/10.1007/s11270-016-2883-2. qualitative and confirmatory screening method using LC-qTOF-MS. Water, Air, Soil Marco-Urrea, E., Pérez-Trujillo, M., Vicent, T., Caminal, G., 2009. Ability of white-rot Pollut. 229, 399–419. https://doi.org/10.1007/s11270-018-4036-2. fungi to remove selected pharmaceuticals and identification of degradation products Beltran-Flores, E., Pla-Ferriol, M., Martínez-Alonso, M., Gaju, N., Blanquez, P., Sarra, M., of ibuprofen by Trametes versicolor. Chemosphere 74, 765–772. https://doi.org/ 2022. Fungal bioremediation of agricultural wastewater in a long-term treatment: 10.1016/j.chemosphere.2008.10.040. biomass stabilization by immobilization strategy. J. Hazard Mater. 439, 1–11. Marican, A., Durán-Lara, E.F., 2018. A review on pesticide removal through different https://doi.org/10.2139/ssrn.3991524. processes. Environ. Sci. Pollut. Res. 25, 2051–2064. https://doi.org/10.1007/ Beltrán-Flores, E., Sarra, M., Blánquez, P., 2021. Pesticide bioremediation by Trametes s11356-017-0796-2. versicolor: application in a fixed-bed reactor, sorption contribution and Metcalf, Eddy, 2003. Wastewater Engineering: Treatment and Reuse, fourth ed. McGraw- bioregeneration. Sci. Total Environ. 794, 1–11. https://doi.org/10.1016/j. Hill, Boston. scitotenv.2021.148386. Mir-Tutusaus, J.A., Baccar, R., Caminal, G., Sarrà, M., 2018. Can white-rot fungi be a real Beltrán-Flores, E., Torán, J., Caminal, G., Blánquez, P., Sarrà, M., 2020. The removal of wastewater treatment alternative for organic micropollutants removal? A review. diuron from agricultural wastewaters by Trametes versicolor immobilized on Water Res. 138, 137–151. https://doi.org/10.1016/j.watres.2018.02.056. pinewood in simple channel reactors. Sci. Total Environ. 728, 1–10. https://doi.org/ 10.1016/j.scitotenv.2020.138414. 9 E. Beltrán-Flores et al. Journal of Environmental Management 325 (2023) 116595 Mir-Tutusaus, J.A., Parladé, E., Llorca, M., Villagrasa, M., Barceló, D., Rodriguez- Rodríguez-Rodríguez, C.E., Marco-Urrea, E., Caminal, G., 2010b. Degradation of Mozaz, S., Martinez-Alonso, M., Gaju, N., Caminal, G., Sarrà, M., 2017. naproxen and carbamazepine in spiked sludge by slurry and solid-phase Trametes Pharmaceuticals removal and microbial community assessment in a continuous versicolor systems. Bioresour. Technol. 101, 2259–2266. https://doi.org/10.1016/j. fungal treatment of non-sterile real hospital wastewater after a coagulation- biortech.2009.11.089. flocculation pretreatment. Water Res. 116, 65–75. https://doi.org/10.1016/j. Sharma, A., Kumar, V., Shahzad, B., Tanveer, M., Sidhu, G.P.S., Handa, N., Kohli, S.K., watres.2017.03.005. Yadav, P., Bali, A.S., Parihar, R.D., Dar, O.I., Singh, K., Jasrotia, S., Bakshi, P., Mir-Tutusaus, J.A., Parladé, E., Villagrasa, M., Barceló, D., Rodríguez-Mozaz, S., Ramakrishnan, M., Kumar, S., Bhardwaj, R., Thukral, A.K., 2019. Worldwide Martínez-Alonso, M., Gaju, N., Sarrà, M., Caminal, G., 2019. Long-term continuous pesticide usage and its impacts on ecosystem. SN Appl. Sci. 1, 1–16. https://doi.org/ treatment of non-sterile real hospital wastewater by Trametes versicolor. J. Biol. 10.1007/s42452-019-1485-1. Eng. 13, 1–13. https://doi.org/10.1186/s13036-019-0179-y. Singh, J., 2015. Emerging crop pest scenario under the impact of climate change – a brief Mojiri, A., Zhou, J.L., Robinson, B., Ohashi, A., Ozaki, N., Kindaichi, T., Farraji, H., emerging crop pest scenario under the impact of climate change – a brief review. Vakili, M., 2020. Pesticides in aquatic environments and their removal by adsorption J. Agric. Phys. 11, 13–20. methods. Chemosphere 253, 126646. https://doi.org/10.1016/j. Titchou, F.E., Zazou, H., Afanga, H., El Gaayda, J., Akbour, R.A., Hamdani, M., 2021. chemosphere.2020.126646. Removal of Persistent Organic Pollutants (POPs) from water and wastewater by Mori, T., Ohno, H., Ichinose, H., Kawagishi, H., Hirai, H., 2021. White-rot fungus adsorption and electrocoagulation process. Groundw. Sustain. Dev. 13, 100575 Phanerochaete chrysosporium metabolizes chloropyridinyl-type neonicotinoid https://doi.org/10.1016/j.gsd.2021.100575. insecticides by an N-dealkylation reaction catalyzed by two cytochrome P450s. Torán, J., Blánquez, P., Caminal, G., 2017. Comparison between several reactors with J. Hazard Mater. 402, 1–10. https://doi.org/10.1016/j.jhazmat.2020.123831. Trametes versicolor immobilized on lignocellulosic support for the continuous Oksanen, J., Blanchet, F., Kindt, R., Legendre, P., Minchin, P., , R., O., Simpson, G., treatments of hospital wastewater. Bioresour. Technol. 243, 966–974. https://doi. Solymos, P., Stevens, M., Wagner, H., 2020. vegan: community ecology package. R org/10.1016/j.biortech.2017.07.055. package version 2.5–7. The R Project for Statistical Computing, Vienna, Austria White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. In: Innis, M., Gelfand, D., Sninsky, J., [WWW Document]. http://cran.r-project.org/web/packages/vegan/index.html. White, T. (Eds.), Amplification and Direct Sequencing of Fungal Ribosomal RNA Popp, J., Pető, K., Nagy, J., 2013. Pesticide productivity and food security. A review. Genes for Phylogenetics” in “PCR Proto- Cols: A Guide to Methods and Applications. Agron. Sustain. Dev. 33, 243–255. https://doi.org/10.1007/s13593-012-0105-x. Acad. Press. Inc., New York, pp. 315–322. Rice, C.E.W., Baird, R.B., Eaton, A., 2017. Standard Methods for the Examination of Yang, J., Li, W., Bun Ng, T., Deng, X., Lin, J., Ye, X., 2017. Laccases: production, Water and Wastewater. American Public Health Association, American Water Works expression regulation, and applications in pharmaceutical biodegradation. Front. Association, Water Environment Federation. https://doi.org/10.1016/0003-2697 Microbiol. 8, 1–24. https://doi.org/10.3389/fmicb.2017.00832. (90)90598-4. Zehra, A., Dubey, M.K., Meena, M., Aamir, M., Patel, C.B., Upadhyay, R.S., 2017. Role of Rodriguez-Rodriguez, C.E., Jelić, A., Llorca, M., Farré, M., Caminal, G., Petrović, M., Penicillium Species in Bioremediation Processes, New and Future Developments in Barceló, D., Vicent, T., 2011. Solid-phase treatment with the fungus Trametes Microbial Biotechnology and Bioengineering: Penicillium System Properties and versicolor substantially reduces pharmaceutical concentrations and toxicity from Applications. Elsevier B. https://doi.org/10.1016/B978-0-444-63501-3.00014-4. sewage sludge. Bioresour. Technol. 102, 5602–5608. https://doi.org/10.1016/j. Zhuo, R., Fan, F., 2021. A comprehensive insight into the application of white rot fungi biortech.2011.02.029. and their lignocellulolytic enzymes in the removal of organic pollutants. Sci. Total Rodríguez-Rodríguez, C.E., Marco-Urrea, E., Caminal, G., 2010a. Naproxen degradation Environ. 778, 1–38. https://doi.org/10.1016/j.scitotenv.2021.146132. test to monitor Trametes versicolor activity in solid-state bioremediation processes. Royal Society of Chemistry. ChemSpider | Search and share chemistry [WWW J. Hazard Mater. 179, 1152–1155. https://doi.org/10.1016/j.jhazmat.2010.02.091. Document]. URL. http://www.chemspider.com/. (Accessed 28 August 2022). accessed. 10