Journal of Hazardous Materials 439 (2022) 129614 Contents lists available at ScienceDirect Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat Research Paper Fungal bioremediation of agricultural wastewater in a long-term treatment: biomass stabilization by immobilization strategy Eduardo Beltrán-Flores a, Martí Pla-Ferriol b, Maira Martínez-Alonso b, Núria Gaju b, Paqui Blánquez a, *, Montserrat Sarrà 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 H I G H L I G H T S G R A P H I C A L A B S T R A C T • A fungal bioremediation treatment was successfully operated for more than 7 months. • Using the suitable substrate and oper­ ating conditions improved biomass retention. • Inoculation of T. versicolor led to the establishment of a stable fungal consortium. • Fungal bioremediation technology is ready for full-scale application. A R T I C L E I N F O A B S T R A C T Editor: Jianhua Guo Fungal bioremediation emerges as an effective technology for pesticide treatment, but its successful imple­ mentation depends on overcoming the problem of microbial contamination. In this regard, fungal immobilization Keywords: on wood seems to be a promising strategy, but there are two main drawbacks: the predominant removal of Pesticides pesticides by sorption and fungal detachment. In this study, agricultural wastewater with pesticides was treated Wood chips by Trametes versicolor immobilized on wood chips in a rotary drum bioreactor (RDB) for 225 days, achieving Continuous treatment fungal consolidation and high pesticide biodegradation through two main improvements: the use of a more Trametes versicolor Rotating drum bioreactor favorable substrate and the modification of operating conditions. Fungal community dynamic was assessed by denaturing gradient gel electrophoresis (DGGE) analysis and subsequent prominent band sequencing, showing a quite stable community in the RDB, mainly attributed to the presence of T. versicolor. Pesticide removals were up to 54 % diuron and 48 % bentazon throughout the treatment. Afterwards, pesticide-contaminated wood chips were treated by T. versicolor in a solid biopile-like system. Hence, these results demonstrate that the microbial contamination constraint has definitely been overcome, and fungal bioremediation technology is ready to be implemented on a larger scale. * Corresponding author. E-mail address: paqui.blanquez@uab.cat (P. Blánquez). https://doi.org/10.1016/j.jhazmat.2022.129614 Received 21 February 2022; Received in revised form 13 May 2022; Accepted 13 July 2022 Available online 21 July 2022 0304-3894/© 2022 The Authors. Published by Elsevier B.V. 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 Hazardous Materials 439 (2022) 129614 1. Introduction 2. Materials and methods Pesticides are a broad group of chemical compounds used to improve 2.1. Fungal strain and culture conditions agricultural production. However, pesticides have a high potential to drift into aquatic environments after application through storm-water T. versicolor ATCC 42530 was purchased from the American Type discharges, surface runoff and leaching from agricultural areas (Vry­ Culture Collection. T. versicolor was maintained on malt extract (2 % w/ zas, 2018). Thus, water bodies located near intensive agricultural fields v) at 25 ◦ C and routinely subcultured every 30 days. A mycelial sus­ are more vulnerable to pesticide contamination (Deknock et al., 2019). pension of T. versicolor was prepared as previously described elsewhere Furthermore, most pesticides are considered persistent compounds that, (Blánquez et al., 2004). T. versicolor immobilized on Q. ilex wood chips depending on their solubility, can remain dissolved in water or bio­ was prepared as described by Beltrán-Flores et al. (2021). accumulate in biota (Andrade et al., 2021). Long-term exposure to pesticides can lead to undesirable and harmful effects on the environ­ 2.2. Agricultural wastewater ment and human health. Even at low concentrations, chronic exposure to pesticides can pose a serious health threat not only to agricultural Wastewater was collected from an agricultural drainage channel in workers but also to the general population (Leong et al., 2020). the Llobregat River Basin in Gavà (Catalonia, Spain) in July (AW I), In this scenario, the development of effective treatments for the October (AW II) and December (AW III) 2020, and subsequently stored removal of pesticides in agricultural wastewater (AW) is mandatory. at 4 ºC. In the case of the first water collection, a coagulation- Physical-chemical methods have the advantage of being well- flocculation pre-treatment was required to erase algae. Pretreated AW established, fast and effective processes against pesticides. However, I was treated in Period I (P-I), while AW II & III in Period II (P-II) and III these processes generate by-products (such as in adsorption) or toxic (P-III), respectively. Coagulant ferric chloride (40 mg L– 1) and floccu­ transformation products (such as in single ozonation) and they are lant PROSEDIM CS 209 (2 mg L– 1) were added according to the considered relatively expensive (Ahmed et al., 2017). In contrast, following process: 2 min coagulation at 200 rpm, 15 min flocculation at bioremediation has emerged as a cost-effective, sustainable, and effi­ 20 rpm and 30 s sedimentation. The physicochemical characteristics of cient alternative, although it typically requires longer treatment times the AW are summarized in Table S1 (Supplementary material). The and involves more complications related to biomass maintenance wastewater was adjusted to pH 4.5 and spiked with 10 ppm diuron and (Azubuike et al., 2020). bentazon to allow detection by HPLC-UV. Stock solutions of diuron and Among other microorganisms, white-rot fungi (WRF) have attracted bentazon were previously prepared in acetonitrile of high purity grade attention for their unique and versatile metabolism, which is capable of at 10 mg⋅mL− 1. degrading a wide range of xenobiotics through the activity of their intracellular and extracellular enzyme systems (Zhuo and Fan, 2021). In addition, WRF have shown excellent resistance to highly toxic waters 2.3. Chemicals and reagents without any prior acclimatisation process, unlike other biological treatments such as conventional activated sludge (Mir-Tutusaus et al., Diuron was purchased from Sigma Aldrich (Barcelona, Spain) and 2017). Nevertheless, fungal treatment still presents major challenges: KAOS-B (48 % bentazon) was acquired from SAPEC AGRO (Barcelona, nutrient addition, foaming, biomass ageing and microbial contamina­ Spain). Acetonitrile and formic acid were supplied by Merck (Darmstadt, tion (Mir-Tutusaus et al., 2018). In this regard, immobilization on Germany). These chemicals and others used for T. versicolor subculture lignocellulosic materials has been proposed as a promising strategy to were of high purity grade. address some of these operational limitations. Immobilization on sup­ ports prevents mycelial dispersion, which frequently causes foaming and 2.4. AW treatment blocking, and allows separation between hydraulic retention time (HRT) and cellular retention time. In addition, lignocellulosic materials, such Two rotating drum bioreactors (RDB) were operated in parallel, one as wood, provide a specific source of nutrients for fungi that facilitates of the reactors was assembled with wood initially colonized by operation under non-sterile conditions (Torán et al., 2017). T. versicolor, which is the experimental reactor (E-RDB), while the other A previous scientific paper used T. versicolor immobilized on wood to reactor was set up only with wood as a control (C-RDB). Each RDB was treat agricultural wastewater in a rotating drum bioreactor (RDB) constructed with a methacrylate tube (length: 45.0 cm x diameter: 8.4 (Beltrán-Flores et al., 2020). This reactor was operated for a short period cm) supported on a polyvinylchloride gutter, which contained 2.3 L of of time to evaluate the feasibility of the treatment, but some critical agricultural wastewater. The remaining RDB characteristics have been drawbacks, such as the loss of immobilized biomass, were detected. described in a recent study (Beltrán-Flores et al., 2020). In this case, the However, these problems may be solved by implementing favorable inner tube rotated one and a half turns every either 4 or 24 h (depending operational strategies to consolidate biomass immobilization, thus on the experiment) to ensure aerobic conditions. An external recircu­ enhancing pesticide degradation, in a long-term continuous treatment. lation loop (4.7 L⋅day− 1) was required for pH adjustment and dissolved Therefore, the aim of the present study was to set up a RDB using Tra­ oxygen measurement, which were performed in a recirculation tank (≈ metes versicolor immobilized on Quercus ilex wood chips to remove 0.4 L). The pH was automatically controlled at 4.5 by adding either 1 M diuron and bentazon from AW over a long-term period. Throughout the HCl or NaOH. The dissolved oxygen (DO) level was monitored using a treatment, rotation frequency and HRT were modified to analyse their CyberScan 600 Series Waterproof Handheld (Eutech Instruments). The effect on the bioreactor performance, including biomass maintenance DO level remained above 30 % in the recirculation tank throughout the and removal efficiency. A comprehensive study of the microbial pop­ treatment. ulations was also conducted to evaluate the relative abundance of Liquid samples were withdrawn from the reactor effluent for analysis T. versicolor as well as other prominent members of the fungal commu­ of pesticide concentration, laccase, color, chemical oxygen demand nity throughout the treatment, until a stable community with degrading (COD), heterotrophic plate count (HPC), and microbial community capability was achieved. analysis. Solid samples were taken at the beginning and end of each period for ergosterol, fiber and microbial population characterizations. The reactors were successfully operated in continuous mode during 225 days. However, the treatment can be divided into three stages according to the different operational conditions implemented in each period, as shown in Table 1. 2 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 Table 1 richness and abundance of prominent members of the fungal Values of the main operating variables in each treatment period. assemblage. Period Time Rotation Wood amount (g) HRT All DGGEs were performed in a “Dcode Universal Mutation Detection (days) frequency (d) System” (Bio-Rad). Desired PCR products were loaded into 6 % (wt/vol) P-I 0–93 1.5 turns every 4 315 3 polyacrylamide gels with a 15–55 % chemical denaturing gradient (100 h % denaturant contained 7 M urea and 40 % (v/v) deionized formamide) P-II 93–186 1.5 turns every 210 (old) + 335 (fresh) = 3 and submerged in 1x TAE (40 mM Tris acetate at pH 7.4, 20 mM sodium 24 h 545 acetate, 1 mM EDTA). P-III 186–225 1.5 turns every 545 (old) 5 24 h Electrophoresis was performed in all cases at a constant 75 V and a temperature of 60 ◦ C for 16 h using a Bio-Rad Power Pac 1000 power P-I, P-II and P-III are periods I, II and III, respectively. supply. The gels were inspected under UV illumination using a BioRad Universal Hood II Gel Doc™ XR System (BioRad) and photographed 2.5. Pesticide analysis in liquid phase after staining them with ethidium bromide. After visualization of DGGE profiles, prominent bands were cut, Residual concentrations of diuron and bentazon were quantified by recovered, and stored at − 20ºC in a buffer solution. Later, all excised high-performance liquid chromatography (HPLC), with a lower limit of bands were reamplified and sequenced at external facilities by Macro­ detection of 0.5 ppm. Liquid samples were initially filtered through gen, Inc. (South Korea). The resulting sequences were compared with Millipore Millex-GV PVDF filters (0.22 µm). Analyses were performed those in the Gen-Bank nucleotide database by using the Basic Local using a Dionex Ultimate 3000 HPLC system equipped with a UV detector Alignment Search Tool (BLAST) to identify the closest known relatives operating at 254 nm. The separation was conducted in a C18 reversed- (Altschul et al., 1990). Sequences determined in this study are available phase column (Phenomenex®, Kinetex® EVO C18 100 Å, 5 µm, 4.6 at the National Center for Biotechnology Information (NCBI) GenBank mm × 150 mm) at 30 ºC with a mobile phase consisting of 0.01 % formic database under accession numbers OL684714 through OL684779. acid solution and acetonitrile. The organic gradient for chromatographic InfoQuest™ FP (v4.5) software (Bio-rad, USA) was used to analyse the separation was described elsewhere (García-Vara et al., 2021). The flow DGGE profiles and perform hierarchical cluster analysis by an un­ rate and sample injection volume were 0.9 mL⋅min− 1 and 40 µL, weighted pair group with mathematical averages (UPGMA) based on respectively. Euclidean distances. Subsequent analyses and statistics were performed using R version 4.1.2. 2.6. DNA extraction and PCR-DGGE of the fungal community 2.7. Fiber content analysis Effluent and lignocellulosic support samples from the bioreactors were externally collected at regular intervals, immediately stored at 4ºC Cellulose, hemicellulose and lignin contents were analysed by the and delivered within a few hours. Liquid samples were then filtered method of Van Soest et al. (1991). The fiber content study was per­ through 0.22 µm filters (Durapore®: PDVF membrane, 47 mm diam­ formed using an Ankom200 Fiber Analyzer incubator (Ankom Tech­ eter), using a vacuum filtration pump (Millipore). After that, dry filters nology, USA) by continuously introducing sulfuric acid (96 %), an acid were stored at − 80ºC until DNA extraction. Solid samples, instead, were detergent solution (consisting of sulfuric acid and cetyl­ processed to separate the fungal mycelium from the wood chips by trimethylammonium bromide) and a neutral detergent solution (con­ mechanical force (sterile forceps), followed by agitation (30 min, 200 taining SDS, disodium EDTA dihydrate, 10-hydrated sodium RPM), and sonication (7 min) in phosphate buffer (0.1 M, pH 7.0). tetraborate, anhydrous dibasic sodium phosphate and triethylene glycol, Total DNA extraction was then performed on filters using DNeasy® pH 6.9–7.1). Ash was obtained after at least 3.5 h incineration in a PowerWater DNA extraction kit (Qiagen), and on solid samples using muffle at 550 ºC. Fiber components were calculated as follows: DNeasy® PowerSoil DNA extraction kit (Qiagen) always following Hemicellulose (%) = NDF – ADF (1) manufacturer instructions. After extraction, all DNA samples were stored at − 20ºC until further processing. These samples were subse­ Cellulose (%) = ADF – ADL (2) quently amplified by nested PCR to reduce non-specific products and increase sensitivity, using two sets of specific primers targeting fungal Lignin (%) = ADL – Ash (3) ITS (Internal Transcribed Spacer) region. The reaction mix was prepared where NDF represents the natural detergent fiber, ADF corresponds to on a final volume of 50 µL, containing 10–50 ng of DNA template, 20 acid detergent fiber and ADL means acid detergent lignin. mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP premixed, 12.5 pmoles of each primer and 0.5 U 2.8. Fungal treatment in a biopile system of Taq DNA polymerase enzyme (Invitrogen, Waltham, Massachusetts, USA). A total of 105 g of polluted wood chips and colonized wood chips The primer pair used for the first round of the nested PCR was EF4 were withdrawn from the control and experimental RDBs, respectively, and ITS4 (Gardes and Bruns, 1993), while primers ITS1F and ITS2R after 93 days of treatment. Afterwards, 30 g of contaminated wood was (White et al., 1990) were utilized for the second round. A GC-clamp (5′ - treated by T. versicolor in a biopile system per duplicate using three CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC C − 3′ ) different strategies: inoculation on wood, and re-inoculation and non-re- was attached to the 5′ end of the primer ITS1F to prevent complete inoculation on wood containing pre-grown fungus. For this purpose, 7 denaturation for DGGE profiling. All PCR reactions were carried out in a mL of the mycelial suspension was inoculated onto the wood in Scott Bio-Rad Thermocycler (model S1000) programmed for 35 cycles, each glass bottles (Duran, Inc; 250 mL, 95 ×105 mm) equipped with one port of 30 s at 94 ◦ C, 30 s at 55 ◦ C and 30 s at 72 ◦ C, an initial denaturation screw cap opened with a passive air intake through a filter (0.45 µm). step of 94 ◦ C for 5 min, and a final extension step at 72 ◦ C for 7 min. The The culture was maintained at a constant temperature of 25 ºC under specific amplification products were analysed using agarose gel (1.5 %) static and non-sterile conditions for up to 48 days. Solid samples were in 1x TBE buffer (Trisborate–EDTA) stained with 0.5 µg/mL ethidium withdrawn after 27 and 48 days. bromide. Afterwards, all amplification products were further studied by denaturing gradient gel electrophoresis (DGGE) to assess fungal genetic variations throughout the experiment and provide an estimate of 3 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 2.9. Other analyses observed after 78 days of treatment, when only 20 % of the initial fungal content remained immobilized on the wood (see Table 2). P-I was Both pesticides, diuron and bentazon, were extracted from wood maintained until 93 days to give to the fungus a chance to recolonize the following a modified method of Köck-Schulmeyer et al. (2013) as pre­ wood, as occurred in a previous study after significant decreases in viously done in another study (Beltrán-Flores et al., 2021). Ergosterol biomass (Rodríguez-Rodríguez et al., 2010b), but the ergosterol level content was monitored to quantify fungal biomass. The ergosterol was not recovered. analysis was conducted by homogenization and extraction of colonized The substantial loss of biomass in P-I was compensated by replacing wood in triplicate as described elsewhere (Rodríguez-Rodríguez et al., part of the polluted wood with fresh inoculated wood chips to the E- 2010a). Laccase activity was determined through the absorbance RDB. The same procedure was performed in the C-RDB, but in this case changes induced by the oxidation of 2,6-dymetoxyphenol (DMP) and replacing the contaminated wood by fresh wood chips. Subsequently, expressed in activity units per litre (UA⋅L− 1), as described elsewhere the rotation frequency was reduced to 1.5 turns every 24 h (6 times less (Wariishi et al., 1992). than in P-I) during the next 93 days of P-II (186 days in total). As shown Conductivity was determined using a CRISON MicroCM 2100 con­ in Table 2, the ergosterol level was successfully maintained during the ductometer. Color was measured as absorbance at a wavelength of 650 first 78 days of P-II. This result showed evidence that static conditions nm by an UNICAM 8625 UV/VIS. COD and ammonia concentrations favored the preservation and growth of the fungal biomass. By reducing were measured with the commercial kits LCK114 or LCK314m and the turning frequency to 24 h, the non-submerged biomass fraction LCH303, respectively (Hach Lange, Germany). Total suspended solids probably had a longer recovery period from micropollutant exposure (TSSs) and volatile suspended solids (VSSs) were measured according to and submergence, which probably benefited fungal immobilization and the standard methods 2540 D and 2540 E, respectively, and HPC were growth. determined following the standard method 9215 (Baird et al., 2017). In the 186-day sample, a significant drop in ergosterol level was Chloride, nitrite and sulfate concentrations were quantified by ion detected. However, this decrease was attributed to an incident in the chromatography using a Dionex ICS-2000. control system on day 181, which caused the pH of the water to drop Surface growth of T. versicolor on wood was assessed by scanning from 4.5 to 3.3 for 24 h. After this period, this technical failure was electron microscopy (SEM). Solid samples were fixed on a 2.5 % solved and the pH reset to 4.5. It is well known that a too acidic pH glutaraldehyde solution with 0.1 M phosphate buffer (PB). Afterwards, inhibits the enzymatic production and fungal growth (Mir-Tutusaus post-fixation was performed using 1 % osmium vapor containing 0.8 % et al., 2018). In P-III, the HRT was increased from 3 to 5 days, thus ferrocyanide in 0.1 M PB overnight. Afterwards, the samples were keeping the water longer in the reactor. In this case, a remarkable fungal washed with ultrapure water and dehydrated in increasing concentra­ colonization of the wood was achieved after only 44 days of P-III (225 tions of ethanol (50 %, 70 %, 90 %, 96 % and 100 %). Finally, the days in total). This rapid colonization is probably a result of the samples were subjected to the critical point drying method (Baltec consolidation of the established fungal community (see section 3.2.2), CPD030) and metallized with AuPd. and an enhanced reduction in pesticide concentration (see section 3.2.4). 3. Results and discussion Variations in the fungal biomass content throughout the treatment detected via ergosterol analysis were also visualized by SEM. As shown 3.1. Colonization of wood chips in Fig. 1(a) and (b), wood chips were densely colonized by T. versicolor mycelial biomass under sterile conditions. Afterwards, the colonized Q. ilex wood chips presented the following characteristics: apparent wood was used to set up a RDB under non-sterile environment in the and real densities of 0.27 g⋅mL− 1 ± 0.01 and 0.89 g⋅mL− 1 ± 0.02, operational conditions of P-I. As demonstrated by the ergosterol results, respectively, and a porosity of 69.3 % ± 0.5 % (Beltrán-Flores et al., the fungal mycelial density was considerably reduced after 93 days of 2021). Afterwards, T. versicolor was inoculated on Q. ilex wood in a treatment (see Fig. 1(c)). However, the same extent of colonization was sterile box, reaching 0.26 mg⋅g− 1 DW of ergosterol content after 17 days recovered after implementing the modifications of the operational of culture. This value was considerably higher than that obtained in a conditions in P-II and P-III (see Fig. 1(d)). screening assay by Torán et al. (2017), in which up to 0.031 mg⋅g− 1 DW were reported using pine bark as immobilization support after 9 days of 3.3. Fungal community assemblage culture. These results can be explained by the fact that, in nature, T. versicolor is more frequently found on hardwood species, such as In order to assess the fungal diversity of both reactors and the Q. ilex, than in conifers (Cotter, 2014). behavior and persistence of T. versicolor after being inoculated, a DGGE analysis was performed. In Fig. S1 (Supplementary materials), the DGGE 3.2. Reactor performance Table 2 3.2.1. Biomass content Fungal biomass presented on the wood chips, in absolute and relative terms, The initial treatment conditions are those indicated as P-I in Table 1, throughout the treatment. corresponding to the first 93 days of treatment. The main objective of P-I Period Time Ergosterol amount ± SD (mg⋅g Remaining biomass ± was to evaluate the adaptability of the fungus to the real environmental (days) DW wood− 1) SD (%) conditions found in an RDB. In this regard, an RDB had been previously I 0 0.26 ± 0.02 100 used for the treatment of AW by T. versicolor pre-grown on lignocellu­ I 48 0.12 ± 0.02 44 ± 6 losic substrate (Beltrán-Flores et al., 2020). However, the fungus I 78 0.05 ± 0.01 20 ± 2 apparently detached from the wood surface throughout the treatment. I 93 0.05 ± 0.01 21 ± 1 In that case, the loss of biomass was mainly attributed to the application II 93 0.15 ± 0.01 56 ± 4 II 123 0.12 ± 0.01 45 ± 2 of an excessive continuous rotation speed (6 rpm). High rotation speeds II 141 0.13 ± 0.02 50 ± 8 can cause undesirable damage to the mycelial structure of fungi (Zhong, II 171 0.15 ± 0.01 59 ± 0 2010). Consequently, in P-I of the present study, the rotation frequency II 186 0.09 ± 0.01 36 ± 1 was considerably reduced from 6 rpm to 1.5 turns every 4 h. The III 186 0.09 ± 0.01 36 ± 1 time-course of ergosterol content was monitored to quantify the pres­ III 225 0.25 ± 0.03 97 ± 13 ence of fungal biomass over the studied period. *Results are shown as mean values and corresponding standard deviations for Despite this adjustment, a significant decrease in fungal biomass was triplicate measurements. 4 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 Fig. 1. SEM images of initial colonized wood for magnifications of 200 X (a) and 1500 X (b), after 93 days for 200 X (c) and 230 days for 200 X (d). band profiles and UPGMA cluster analysis of the fungal populations RDB, further supporting the hypothesis that inoculated T. versicolor has a from the lignocellulosic support of the reactors can be observed. These stabilizing effect on the community, reducing diversity as it samples were taken at four different times: at the start of the experiment consolidates. (“t0”), at the end of period I (“t93”), at the end of period II (“t186”) and In order to assess the presence of T. versicolor throughout the finally, near the end of the experiment (“t216”). This figure shows experiment prominent bands of the gels were sequenced, allowing the remarkable differences between samples from both reactors, control (C) calculation of the relative abundance of each phylotypes present in solid and experimental (E), as they are clearly separated in the cluster anal­ and liquid samples from both reactors (Figs. 2 and 3 respectively). In the ysis, while replicates of the same sample show a high degree of wooden substrate from the E-RDB (Fig. 2), it could be observed how homogeneity. relative abundance of T. versicolor decreased over time from 90 % of the It can also be noted that the fungal profiles corresponding to the C- total fungal diversity, down to a 30 % at the end of stage II, and from this RDB exhibit a higher degree of complexity than those belonging to the E- point to the end of the experiment it remained constant. This suggests RDB, thus revealing a higher fungal diversity in the former in compar­ that the reactor reached a stable state where Trametes coexisted with two ison with the latter. This suggests that inoculation of T. versicolor in the more evenly distributed fungal phylotypes: Dipodascus sp. and Rhodo­ E-RDB contributes to a more stable, less diverse community of fungal torula sp. (note that Evenness index is close to 1). Dipodascus sp. is found species, as it dominates the competition for space and nutrients. in the wastewater used as the influent (Fig. 3) and Rhodotorula sp. was To fully comprehend fungal dynamics in the system, effluent of the already present in the wood inoculated with T. versicolor (Fig. 2). Both reactors was also analysed along the experiment. The analysis of these genera have been described in biodegradation studies (De Hoog and samples provides data about those fungi that were washed away from Smith, 2011; Dusengemungu et al., 2020), and Rhodotorula sp. was also the lignocellulosic support. This, coupled with the previous analysis of detected in a reactor inoculated with T. versicolor (del Álamo et al., the lignocellulosic support, allows for a better understanding of how 2022). Regarding the C-RDB, a clear dominance of Penicillium is detected fungal populations evolve inside the system throughout the treatment. in the early stages, (Fig. 2). An increase of fungal diversity was observed Fig. S2 (Supplementary materials) shows the DGGE band profiles and over time, so that at the end of the stage III, 8 different phylotypes were cluster analysis corresponding to the fungal community from the detected, with no evidence of having reached a fungal community sta­ effluent. Unlike in the lignocellulosic matrix study, in this case both bility, as opposed to the E-RDB. RDBs presented a common dominant phylotype throughout the entire Relative abundance of fungal populations in influent and effluent experiment. Interestingly, samples taken from the same time tend to samples was also calculated at different stages of the experiment (Fig. 3). cluster together regardless of which reactor they come from. This can be Expectedly, T. versicolor was detected at all stages of the E-RDB, but not explained by shifts in the composition of the influent fungal commu­ in the C-RDB. However, it can be observed that T. versicolor relative nities that could affect survival and adherence of different species inside abundance decreased in the effluent as the experiment progressed. This, both reactors. However, in the same way as in the lignocellulosic sup­ coupled with the results obtained from the wooden matrix analysis, port analysis, diversity in the C-RDB tends to be higher than in the E- suggests that the inoculated fungus consolidated over time in the E-RDB, 5 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 Fig. 2. Relative abundance of fungal populations in the control reactor (left), and in the experimental reactor (right), corresponding to the lignocellulosic support samples from the stages studied (0, initial; I, t93; II, t186; III, t216). Values for Shannon Diversity and Evenness Indices are shown at the bottom of each sample. Fig. 3. Relative abundance of fungal populations in the control reactor (left), and in the experimental reactor (right), corresponding to the effluent samples from the stages studied (0, initial; I, t60; II, t120 and t186; III, t216). Values for Shannon Diversity and Evenness Indices are shown at the bottom of each sample. 6 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 keeping attached to the lignocellulosic matrix and therefore reducing its washout from the system. As the experiment progresses, a clearly dominant phylotype appears in the effluent of both reactors: Dipodascus (De Hoog and Smith, 2011; Dusengemungu et al., 2020; Kawai and Hu, 2009). This fungal genus, ends up representing a 40 % of the fungal diversity in the C-RDB, and more than 90 % in the E-RDB. Dipodascus was, in fact, the dominant genus detected in the original AW used as influent for this experiment before pre-treatment was performed, when its presence decreased in favor of the ubiquitous Penicillium. This com­ mon fungal genus colonizes the lignocellulosic substrate in the C-RDB, and it seems that Dipodascus is not able to remain attached to it, and so it is washed from the system. Interestingly, in the E-RDB Penicillium is not part of the dominant population, probably displaced by the inoculated Trametes. Finally, it can be observed how Shannon indexes corresponding to the E-RDB are usually lower than in the C-RDB for both lignocellulosic support and liquid samples (Shannon Diversity Index data is shown in Figs. 2 and 3). This is consistent with the previous hypothesis that inoculation of T. versicolor in the E-RDB contributes to a more stable and less diverse fungal community, as it consolidates in the wooden matrix. Therefore, the present study has demonstrated the ability of T. versicolor immobilized on wood to survive under non-sterile condi­ Fig. 4. Time-course profiles of diuron and bentazon removals grouped in 15- tions during long-term treatments. Although the relative abundance of days periods obtained in the E-RDB and the C-RDB. The error bars represent this fungus is reduced, a stable consortium is established that succeeds in the standard deviations for the specified periods. maintaining its degrading capability, as shown in Section 3.2.4. maintained its treatment capacity for a longer period of time (225 days). 3.3.1. Evolution of bacterial counts In contrast, the C-RDB even showed bentazon from 60 to 75 days on­ The HPC results are presented as the logarithm of colony-forming wards in P-I, and from 165 to 180 days onwards in P-II. Therefore, units (CFU) per mL in Fig. S3 (Supplementary materials). Bacterial inoculation of T. versicolor improved treatment efficiency while counts remained at a lower level in the E-RDB compared to the C-RDB extending the useful operational period of the system. throughout the entire treatment, especially during P-II and P-III. Of particular interest is the maintenance of diuron removal capacity Therefore, bacterial contamination was successfully controlled in the E- by the E-RDB in P-II and P-III, being considerably more stable over time RDB, indicating that the immobilization on Q. ilex wood can be an than in P-I. In contrast to P-I, where E-RDB and C-RDB removals seemed appropriate strategy to limit bacterial growth. In other systems where to follow similar trends, a divergence was observed in the last two pe­ glucose is used instead of wood for fungal maintenance, high bacterial riods. This phenomenon was mainly attributed to a higher amount of growths have been frequently reported (Hu et al., 2021; Mir-Tutusaus fungal biomass immobilized on the wood in the E-RDB as a consequence et al., 2017). of the reduction of the reactor rotation frequency in P-II and the increase of the HRT from 3 to 5 days in P-III. In P-I, the E-RDB achieved up to 12.6 3.3.2. Pesticide removal % more removal than the C-RDB (days 45–60). In P-II, biomass main­ Time-course profiles of diuron and bentazon concentrations are tenance was consolidated and increased by up to 13.9 % (days presented in Fig. S4 (Supplementary material). However, to facilitate 165–180). Finally, increasing HRT prolonged treatment time, resulting interpretation of the results, pesticide concentrations were converted in an improvement of up to 25.1 % in the E-RDB compared to the C-RDB. into relative removals and grouped into 15-days periods in Fig. 4. As can In the case of bentazon, its removal was probably influenced by the be seen, the elimination efficiencies achieved in the RDB-E were clearly presence of laccase, which was only detected at the beginning of P-I and higher than those in the RDB-C for both pesticides throughout the P-II, when fresh biomass was introduced into the reactor (see Fig. S5 of treatment, demonstrating the favorable contribution of T. versicolor to Supplementary material). Recently, laccase has been reported to be the removal capacity of the system, reaching removal peaks of 54 % involved in bentazon biodegradation (García-Vara et al., 2021), while diuron and 48 % bentazon. there is no clear consensus for diuron (Coelho-Moreira et al., 2018; Hu Comparing pesticides, the diuron removal was clearly higher to that et al., 2020b). Nevertheless, the lack of laccase does not necessarily of bentazon, even in the case of the C-RDB. This phenomenon was partly entail fungal inactivation, as T. versicolor possesses an intracellular related to the different sorption capacity of Q. ilex for each pesticide. enzyme system capable of degrading this type of compounds (Mir-Tu­ Beltrán-Flores et al. (2020) showed that diuron had higher affinity for tusaus et al., 2017). Q. ilex than bentazon, which was related to the different hydrophobic properties of the compounds. Thus, wood not only serves as an immo­ 3.3.3. COD and color bilization support and a substrate for fungus, but can also play a sig­ The initial COD of the AW was considerably increased from 144 and nificant role in pesticide removal by sorption process. However, the 44 mg O2⋅L− 1 in P-I and P-II&III, respectively, to approximately removal in the C-RDB cannot be attributed exclusively to sorption, as 3000 mg O2⋅L− 1 upon addition of the stock solutions of diuron and other fungi detected in the microbial community (such as Dipodascus sp. bentazon (10 mg⋅mL− 1). This increase was mainly attributed to the use and Rhodotorula sp.) have been described in bioremediation studies (De of acetonitrile as solvent for the stock solutions of each pesticide Hoog and Smith, 2011; Dusengemungu et al., 2020). (1 mg⋅mL− 1), since its theoretical contribution to the COD of the AW A partial renovation of the colonized wood was required to recover (2451 mg O2⋅L− 1) is much higher than that of diuron (13 mg O2⋅L− 1) the biomass washed out of the reactor and the treatment capacity of the and bentazon (15 mg O2⋅L− 1). However, as shown in Fig. 5, COD was system from 93 days onwards (see Section 2.2.1). In this regard, the rapidly reduced by about half in the first days of treatment in both re­ highest removal efficiencies were achieved at the beginning of P-I and P- actors, indicating that acetonitrile was easily removed. However, after II, when fresh biomass was added to the system. Changes in operating this drastic decrease, the COD values of both reactors were still conditions enhanced biomass retention, and as a result, the E-RDB considerably higher than the COD of the original wastewater. This 7 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 Table 3 Comparison of fiber composition between the wood chips from the E-RDB after 225 days of treatment and the original wood. Components Composition (%⋅DW− 1) Reduction (%) E-RDB (t = 0 days) E-RDB (t = 225 days) NDF (%) 84.34 ± 0.25 83.60 ± 0.01 – ADF (%) 60.51 ± 0.03 60.62 ± 0.11 – ADL (%) 15.53 ± 0.31 13.13 ± 0.08 – Hemicellulose 23.83 ± 0.28 22.98 ± 0.12 8.55 ± 0.10 Cellulose 44.98 ± 0.34 47.49 ± 0.19 -0.12 ± 0.01 Lignin 11.68 ± 0.32 9.07 ± 0.09 26.36 ± 0.72 Ash 3.85 ± 0.01 4.06 ± 0.01 0 Wood mass – – 5.17 ± 0.01 *Results are shown as mean values and corresponding standard deviations for triplicate measurements. which are relatively easily biodegradable polysaccharides used as sub­ Fig. 5. COD and color profiles of the AW during the continuous treatment. Blue strates in the primary metabolism of multiple fungi and other microor­ and green triangles are the COD concentrations of the effluent streams in the E- ganisms, lignin is an extremely recalcitrant compound and only seems to RDB and C-RDB, respectively, while the black and red squares are the color be biodegradable through secondary metabolism by the ligninolytic levels of the effluent streams in the E-RDB and C-RDB, respectively. Vertical system of WRF. Since lignin oxidation results in no energy gain, the WRF dashed lines divide each operating period (I: Period I, II: Period II and III: Period III). main objective in secreting lignocellulosic enzymes (primally lignin peroxidase, manganese peroxidase and laccase) is to access the internal polysaccharides of the wood (Pointing, 2001). The fact that the highest difference was associated with the extraction of some soluble com­ wood consumption seems to be lignin indicates that T. versicolor, as the pounds from the wood: initially, a large amount of these extracts was only WRF detected in the E-RDB, was probably the main microorganism released, but as the treatment progressed, their contribution to COD consuming wood components. became less important. In P-III, COD level rose slightly as a consequence This analysis also showed a slight loss of wood mass during the of the increase in HRT from 3 to 5 days. treatment. This result supports the hypothesis that the increase in COD Contradictory results have been previously reported regarding the was caused by the release of soluble organic matter from wood. How­ COD reduction capacity of T. versicolor (Cruz-Morató et al., 2013; Souza ever, since wood decomposition was relatively low, substrate replace­ et al., 2014). In the present study, a better performance was observed in ment was found to be unnecessary to ensure survival of T. versicolor. the E-RDB compared to the C-RDB, suggesting that T. versicolor along Therefore, fresh wood would only be required to restore the adsorption with the microbial consortium contributed to reduce COD generated by capacity of the system. the extraction of organic compounds from wood. In this regard, although COD remained above the original COD throughout the treat­ ment, the E-RDB showed considerably lower levels than the C-RDB, with 3.4. Solid-phase treatment in a biopile system average relative removal of 14 %, 23 % and 51 % in P-I, P-II and P-III, respectively. In treatments involving sorption, not only the removal performance Similar results were obtained with respect to color analysis. A in the liquid phase, but also the fate of the polluted sorbent should be considerable increase in color was detected when the wastewater came studied. Thus, additional remediation approaches should be explored to in contact with the wood owing to the extraction of some soluble com­ reduce the pesticide content in the solid by-products. Accordingly, a pounds that give the characteristic dark color to Q. ilex wood. In P-II and solid-phase treatment was performed in a biopile-like system by inoc­ P-III, considerable colouration increases were shown in the C-RDB as a ulating T. versicolor onto the by-products. Samples were withdrawn from result of the introduction of fresh wood and the increased HRT, the E-RDB (inoculated wood) and C-RDB (non-inoculated wood) after 93 respectively. However, the E-RDB showed higher decolorization over days of treatment (at the end of P-I), to be subsequently treated in the time, reaching average eliminations of 62 % in P-I, 82 % in P-II and 84 % biopile for up to 48 days. In this regard, three different strategies were in P-III compared to the C-RDB. As shown in Fig. 5, the addition of fresh assessed: wood inoculation with T. versicolor (C-I), no re-inoculation (E- wood in P-II and the increase of HRT in P-III leaded to more intense N) and re-inoculation of colonized wood (E-R). colors in the C-RDB, while remaining approximately constant in the E- Although a similar experiment was performed in a recent study RDB. Similar results were obtained by Torán et al. (2017) using (Beltrán-Flores et al., 2021), the by-products had been subjected to a T. versicolor immobilized on pine wood in a trickle-bed reactor, in which treatment period of only 20 days and the biopile treatment period had approximately constant colouration levels were reported during the been limited to 27 days. The use of more realistic operational periods treatment. Therefore, the E-RDB proved to be efficient in terms of COD both in the RDB (93 days), which in turn change the characteristics of and color removal. the wood and therefore the post-treatment performance, and in the biopile (48 days), can provide more reliable results and useful infor­ 3.3.4. Variation of fiber content in wood mation for further full-scale application. A fiber analysis was conducted to assess the wood consumption by Fig. 6 shows the results of the biopile experiment after 27 and 48 the fungal biomass. The chemical composition of each fiber type is given days of treatment. As expected, higher residual concentrations were in Table 3. For a better interpretation of the results, the inert ash fraction detected for diuron, which as a hydrophobic compound tends to be was considered inert and used as a basis to calculate the relative content sorbed by wood. Interestingly, higher initial concentrations were found of each fiber referred to the initial wood content (t = 0), and thus obtain in the colonized wood than in the wood without fungus. This fact evi­ the percentage reduction of each component. denced a slight sorption contribution of the fungal biofilm to the overall As can be seen, the degradation capacity of the fungal consortium removal, which has been previously reported (Hu et al., 2020a). changes depending on the fiber type. Interestingly, the most highly A mass balance of the total amount of both pesticides introduced into degraded compound was lignin. Unlike cellulose and hemicellulose, the reactor showed that the experimental sorbed amount was found to be considerably lower, 0.207 mg⋅g− 1 for diuron and 0.010 mg⋅g− 1 for 8 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 Fig. 6. Evolution of residual concentration of diuron (a) and bentazon (b), and ergosterol content in the biowastes during the biopile treatment. Residual con­ centrations are represented with bars after 0 (blue), 27 (green) and 48 (yellow) days of treatment (n = 2). Black line is the average ergosterol level for three rep­ licates. Biopile conditions: C-I corresponds with wood inoculated; E-N with biowastes without re-inoculation; E-R with biowastes with re-inoculation. Results are shown as mean values and corresponding standard deviations for duplicate measurements. bentazon, compared to those reported in the above-mentioned study, significant decrease after 48 days (Fig. 6). The partial decomposition of being of up to 0.240 mg⋅g− 1 for diuron and 0.020 mg⋅g− 1 for bentazon the wood along with the presence of pre-grown fungus probably (Beltrán-Flores et al., 2021). These results suggest that the wood reduced benefited the adaptation and growth of the fresh inoculum. part of its adsorption capacity when subjected to a long-term treatment, These results suggest that T. versicolor-based biopile can be a useful probably as a result of the consumption and degradation of this substrate tool for the bioremediation of polluted substrate, thus allowing its reuse by the fungal consortium. as immobilization support. This technology would reduce substrate re­ Fig. 6 shows that the relative elimination of bentazon is higher than quirements in full-scale applications, while maintaining remarkable that of diuron throughout the treatment. For example, for E-I and 48 removal efficiencies and biomass growths. Wood could also be acquired days, relative removals were 95 % for bentazon and 45 % for diuron. as a by-product or waste from timber industries in the case of full-scale However, biodegradation kinetics of both pesticides was also estimated application, making the process even more sustainable, especially to compare the treatment efficiency with the literature. Table 4 shows compared to other materials commonly used for fungal immobilization the biodegradation kinetic constants and rates for both pesticides, which such as polyurethane foam (Mir-Tutusaus et al., 2018). were found to be clearly higher for bentazon, as shown in Fig. 6(b). It is feasible to assume that bentazon, being a more polar molecule, has a 4. Conclusions lower diffusion capacity inside the wood, and thereby higher bioavail­ ability to interact with the fungus. Agricultural wastewater was successfully treated by T. versicolor Table 4 also shows that E-R was the most efficient strategy in terms of immobilized on Q. ilex wood chips in a rotating drum bioreactor during a degradation rate, followed by E-N and C-I. However, the degradation long-term process (> 7 months). Inoculation of T. versicolor on Q. ilex performance of the E-R was only slightly higher than that achieved by wood chips and adjustment of the operational parameters led to the the E-N, which questions the viability of reinoculation. In this regard, consolidation of a fungal consortium in the reactor, composed of similar results of diuron degradation rates were reported in another T. versicolor, Dipodascus sp. and Rhodotorula sp., with enhanced degra­ solid-phase biopile treatment with T. versicolor (Beltrán-Flores et al., dative capacity. Decreasing rotation and increasing HRT improved total 2021). With regard to bentazon, the removal efficiency was notably biomass retention and thus, pesticide removal over a longer operational higher, but in that case the wood had been previously subjected to a period. Significant COD and color removals were also obtained much shorter period of treatment, 20 days instead of 93 days used in the compared to the control reactor. Afterwards, solid by-products exposed present study. This result may be due to the fact that older wood may to the long-term process were treated by T. versicolor in a biopile-like constitute a worse substrate for the fungus, thus depressing its metabolic system achieving high biodegradation yields. Therefore, unlike previ­ activity in the biopile treatment. ous studies, biomass was maintained, high biodegradation (not only In any case, the application of the E-R was justified considering sorption) was achieved, contaminated by-products were successfully ergosterol content, being more stable than in the E-N, which showed a treated after a long period of exposure, and in addition, a detailed Table 4 Biodegradation rates and first-order kinetic constants of diuron and bentazon removals by T. versicolor using different inoculation strategies in a solid-phase biopile treatment. Biopile conditions Pesticide: diuron Pesticide: bentazon 1 Degradation rate (mg⋅g− day− 1) x R2 k x 102 (days− 1) R2 Degradation rate (mg⋅g− 1 day− 1) x R2 k x 102 (days− 1) R2 103 103 Inoculated (C-I) 0.70 ± 0.03 1.00 0.37 ± 0.03 0.19 ± 0.05 0.94 4.19 ± 1.92 0.86 0.99 Non-re-inoculated (E- 1.73 ± 0.42 0.94 0.93 ± 0.18 0.25 ± 0.08 0.90 5.30 ± 0.37 1.00 N) 0.96 Re-inoculated (E-R) 2.22 ± 0.41 0.97 1.28 ± 0.15 0.26 ± 0.07 0.94 6.29 ± 0.74 0.99 0.99 *Results are shown as mean values and corresponding standard deviations for duplicate measurements. 9 E. Beltrán-Flores et al. Journal of Hazardous Materials 439 (2022) 129614 microbial study was included confirming fungal consolidation in the runoff from soybean dominated agricultural areas in Pampean streams, Argentina. Sci. Total Environ. 788, 147676 https://doi.org/10.1016/j.scitotenv.2021.147676. reactor. These results demonstrated that the use of the appropriate Azubuike, C.C., Chikere, C.B., Okpokwasili, G.C., 2020. 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