This is the accepted version of the article: Hu, Kaidi; Torán, Josefina; López-García, Ester; [et al.]. «Fungal bioremediation of diuron-contaminated waters : evaluation of its degradation and the effect of amendable factors on its removal in a trickle-bed reactor under non-sterile conditions». Science of the Total Environment, Vol. 743 (November 2020), art. 140628. DOI 10.1016/j.scitotenv.2020.140628 This version is available at https://ddd.uab.cat/record/238003 under the terms of the license Fungal bioremediation of diuron-contaminated waters: evaluation of its degradation and the effect of amendable factors on its removal in a trickle-bed reactor under non-sterile conditions Kaidi Hu1, Josefina Torán1, Ester López-García2, Maria Vittoria Barbieri2, Cristina Postigo2, Miren López de Alda2, Gloria Caminal3, Montserrat Sarrà1*, Paqui Blánquez1 1Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain 2Water, Environmental and Food Chemistry Unit (ENFOCHEM), Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain 3Institut de Química Avançada de Catalunya (IQAC), CSIC. Jordi Girona 18-26, 08034 Barcelona, Spain *Corresponding author: Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel: + 34- 34935812789; E-mail: montserrat.sarra@uab.cat 1 Graphic abstract Cytochrome P450 Homogenized Immobilized Fungal pellets Cl NH 2 Fungal trickle-bed reactor Cl 3,4-DCA Non-sterile Removal > 80% 2 Highlights  T. versicolor could effectively degrade both diuron and 3,4-dichloroaniline.  Cytochrome P450 is involved in diuron degradation by T. versicolor.  Degradation pathway proposed based on metabolites identification.  Diuron was largely removed in a trickle-bed bioreactor under non-sterile conditions.  Contact time is a crucial factor according to reactor evaluation. 3 Abstract The occurrence of the extensively used herbicide diuron in the environment poses a severe threat to the ecosystem and human health. Four different ligninolytic fungi were studied as biodegradation candidates for the removal of diuron. Among them, T. versicolor was the most effective species, degrading rapidly not only diuron (83%) but also the major metabolite 3,4-dichloroaniline (100%), after 7-day incubation. During diuron degradation, five transformation products (TPs) were found to be formed and the structures for three of them are tentatively proposed. According to the identified TPs, a hydroxylated intermediate 3-(3,4-dichlorophenyl)-1-hydroxymethyl-1-methylurea (DCPHMU) was further metabolized into the N-dealkylated compounds 3-(3,4-dichlorophenyl)-1- methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The discovery of DCPHMU suggests a relevant role of hydroxylation for subsequent N-demethylation, helping to better understand the main reaction mechanisms of diuron detoxification. Experiments also evidenced that degradation reactions may occur intracellularly and be catalyzed by the cytochrome P450 system. A response surface method, established by central composite design, assisted in evaluating the effect of operational variables in a trickle-bed bioreactor immobilized with T. versicolor on diuron removal. The best performance was obtained at low recycling ratios and influent flow rates. Furthermore, results indicate that the contact time between the contaminant and immobilized fungi plays a crucial role in diuron removal. This study represents a pioneering step forward amid techniques for bioremediation of pesticides-contaminated waters using fungal reactors at a real scale. 4 1 Keywords: White-rot fungi; herbicide removal; transformation products; bioreactor; response 2 surface methodology, high-resolution mass spectrometry 3 4 1. Introduction 5 Over the last few decades, pesticide usage has shown a sustained growing tendency as a result of 6 the exponential increase of the human population that brings along the need for intensifying food 7 production. In this context, herbicides act as a key component of modern global agricultural systems 8 (Bilal et al., 2019). Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a phenylurea herbicide, is 9 used extensively to control weeds throughout the world, not only in agriculture but also in urban 10 and industrial scenarios (Liu, 2014). This herbicide is stable to hydrolysis at neutral pH (pH 5–9) 11 and is generally persistent in soil with a half-live of 320–330 days, and thus, can reach water bodies 12 through leaching or surface runoff (Giacomazzi and Cochet, 2004; Langeron et al., 2014; Liu, 2014). 13 Moreover, its main degradation product, 3,4-dichloroaniline (3,4-DCA) exhibits high toxicity and 14 persistence in soils (Giacomazzi and Cochet, 2004; Tasca and Fletcher, 2018). The presence of 15 diuron in the environment may pose a severe threat to human health and aquatic organisms in several 16 ways (Giacomazzi and Cochet, 2004; Huovinen et al., 2015; Liu, 2014; Mansano et al., 2018). To 17 reduce diuron risks and protect water ecosystems, the European Commission has included this 18 herbicide in the priority hazardous substance list (Directive 2013/39/EU) and established 19 environmental quality standards (EQS) in surface waters. Due to its potential effects, diuron is also 20 one of the 92 substances that are currently being assessed by the European Chemicals Agency 21 (ECHA) as a potential endocrine disruptor. Despite worldwide efforts to control its environmental 22 presence, diuron is still frequently detected in surface and ground waters (Kaonga et al., 2015; Rippy 5 23 et al., 2017; Santos et al., 2015). Thus, scientific efforts are needed to overcome its persistence and 24 recalcitrance (Liu et al., 2018; López-Ramón et al., 2019; Santos et al., 2019). In this setting, the 25 development of techniques to remove diuron and 3,4-DCA from the environment is strongly 26 motivated and urgent. 27 Bioremediation techniques present two major advantages over both chemical and physical 28 remediation approaches. sustainability and low cost (Azubuike et al., 2016). To date, various 29 microorganisms have been reported to harbor the capacity to simultaneously degrade diuron and 30 3,4-DCA (Ellegaard-Jensen et al., 2013; Sørensen et al., 2008; Sharma et al., 2010; Villaverde et al., 31 2017). Most of the studies conducted in this line have focused on bacterial species, while white-rot 32 fungi (WRF), that are metabolically versatile and capable of degrading a wide spectrum of 33 xenobiotics due to their nonspecific lignin-degrading enzymes, have only been marginally 34 investigated (Singh and Singh, 2014). Although different reactors based on WRF have been 35 successfully designed for the degradation of various xenobiotics, their implementation at full-scale 36 still needs to overcome some problems, like bacterial contamination (Mir-Tutusaus et al., 2018). In 37 comparison with other reactors such as stirred tank reactor and fluidized bed reactor, trickle-bed 38 reactors (TBR) offer multiple advantages, such as lower operational costs, greater operational 39 flexibility, and higher sustainability (Luo et al., 2014). Furthermore, the immobilization of T. 40 versicolor on wood has been proven to be advantageous to maintain fungal activity and has been 41 successfully used to remove pharmaceuticals in hospital wastewater (Torán et al., 2017). 42 The purpose of the present study was: i) to screen out one WRF species that could effectively 43 degrade diuron, ii) explore the enzymatic system involved in the degradation, and iii) evaluate the 44 potential transformation products (TPs) formed during the process. A further objective was to 6 45 estimate the operational variables of a TBR immobilized with the selected fungus for diuron 46 removal, operated under non-sterile conditions, to improve reactor performance and overcome the 47 current limitations for its application in real scenarios. 48 49 2. Materials and methods 50 2.1. Microorganisms and media 51 Trametes versicolor ATCC 42530 was acquired from American Type Culture Collection, 52 Gymnopilus luteofolius FBCC 466 and Stropharia rugosoannulata FBCC 475 were obtained from 53 Fungal Biotechnology Culture Collection (FBCC) of the University of Helsinki (Finland), and 54 Pleurotus ostreatus was isolated from a fruiting body collected from rotting wood (Palli et al., 2014) 55 and preserved in our laboratory. Fungal strains were maintained by subculturing every 30 days on 56 2% (w/v) malt extract plates (pH 4.5) at 25 °C. Blended mycelial suspensions and pellets were 57 prepared using malt extract medium (pH 4.5) as previously described (Blánquez et al., 2004). 58 The defined medium used in the degradation batch experiments in Erlenmeyer regime consisted 59 of (per liter) 8 g glucose, 3.3 g ammonium tartrate, 1.68 g dimethyl succinate, 10 mL micronutrients, 60 and 100 mL macronutrients (Kirk et al., 1978). pH was adjusted to 4.5. 61 62 2.2. Chemicals and reagents 63 Diuron (purity, ≥ 98%), 3,4-DCA (98%), laccase mediators violuric acid monohydrate (VA, ≥ 64 97%) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS, 98%), 65 cytochrome P450 inhibitor 1-aminobenzotriazole (ABT, purity, 98%), and commercial laccase 66 purified from T. versicolor (20 AU mg– 1) were purchased from Sigma-Aldrich (Barcelona, Spain). 7 67 Diuron-d6, used as a surrogate standard in chemical analyses, was purchased from Toronto Research 68 Chemicals (Ontario, Canada). Hydrated 1-hydroxybenzotriazole (HOBT, ≥ 98%) was obtained from 69 Fluka (Barcelona, Spain). Chromatographic grade acetonitrile was purchased from Carlo Erba 70 Reagents S.A.S (Barcelona, Spain). HPLC-grade methanol, and formic acid (>98%) used as a 71 mobile phase modifier, were obtained from Merck (Darmstadt, Germany). Stock solutions (5 mg 72 mL– 1) of diuron and 3,4-DCA to be used in in vivo and in vitro degradation experiments were 73 prepared by appropriate dilution of the substances in ethanol and stored at – 20 °C until use. 74 75 2.3. Fungal selection for diuron removal 76 To select the best fungal species for diuron removal, degradation experiments were performed 77 in 250 mL Erlenmeyer flasks containing 50 mL of defined medium fortified with diuron at a final 78 concentration of 10 mg L– 1. Briefly, pellets of each fungus were transferred into flasks as inoculum, 79 thereby achieving a concentration of approximately 3.4 g dry weight (DW) L– 1. Then the cultures 80 were incubated at 25 °C under continuous orbital-shaking (135 rpm) for 7 days. To obviate the 81 influence of photodegradation, the incubation were performed in the dark. Abiotic (uninoculated) 82 and heat-killed culture (121 °C for 30 min) were used as controls. Each set was tested in triplicate. 83 Aliquots were taken at specific time intervals during incubation to measure diuron and glucose 84 concentrations. 85 86 2.4. Evaluation of the enzymatic system involved in diuron degradation 87 T. versicolor was selected for subsequent experiments based on its decomposition efficiency. To 88 evaluate the enzymatic system involved in diuron degradation, in vitro experiments with T. 8 89 versicolor laccase and in vivo experiments with an inhibitor of the cytochrome P450 system were 90 conducted. 91 Laccase-mediated degradation experiments were performed in 250 mL Erlenmeyer flasks 92 containing 50 mL laccase-sodium malonate dibasic monohydrate solution (250 mM, pH 4.5) at a 93 final enzyme activity of 500 AU L– 1. Diuron concentration was set to 10 mg L– 1. Different laccase- 94 mediators including VA, ABTS, and HOBT were added individually to a final concentration of 1 95 mM (Marco-Urrea et al., 2009) for evaluation of their effects on diuron degradation. Abiotic (no 96 laccase) and flasks without the addition of mediators were also prepared as controls. The flasks were 97 incubated for 3 days on an orbital shaker (135 rpm) at 25 °C in the dark and each experimental 98 condition was conducted in triplicate. At selected times, 1 mL sample aliquot was withdrawn from 99 each flask and mixed with 100 μL of 1 M HCl to stop the reaction. Then, it was filtered through a 100 Millipore Millex-GV PVDF 0.22 μm membrane before the analysis of diuron. 101 For evaluating the activity of cytochrome P450, the cytochrome P450 inhibitor ABT (dissolved 102 in ethanol) was added to in vivo cultures at 5 mM final concentration (Marco-Urrea et al., 2009). 103 Briefly, experiments in triplicate were carried out in 100 mL Erlenmeyer flasks containing 25 mL 104 of defined medium spiked with diuron at a final concentration of 10 mg L– 1. After inoculating 105 pellets (3.2 g DW L– 1), cultures were incubated for 7 days at 25 °C in the dark under continuous 106 orbital-shaking (135 rpm). Experimental control was run in parallel in the absence of ABT. Samples 107 were taken at selected times and filtered as mentioned above before the analysis of diuron residues. 108 109 2.5 Identification of diuron transformation products 110 Experiments were conducted in 500 mL Erlenmeyer flasks containing 100 mL of fresh define 9 111 medium. In brief, after transferring 2.6 g DW L– 1 of T. versicolor pellets, cultures were incubated 112 for 7 days under the same conditions described in section 2.3. Diuron was added at a concentration 113 of 1 mg L– 1. Abiotic control containing only diuron was also prepared. Each experiment was 114 conducted in triplicate. At selected times, 4 mL of the culture was withdrawn and centrifuged (17, 115 700 × g, 4 min) at room temperature. Then, 1.5 mL of supernatant was transferred into a 2-mL amber 116 vial containing 75 μL of diuron-d6 solution at a concentration of 10 μg mL– 1 and kept in the dark at 117 – 20 °C until analysis. Simultaneously, laccase activity was determined during incubation. 118 Also, another degradation experiment using 3,4-DCA as substrate (10 mg L– 1) was performed in 119 parallel, to explore the degradation ability of T. versicolor towards the main metabolite formed by 120 diuron and elucidate the biochemical pathway of diuron degradation. In these experiments, 3.2 g 121 DW L– 1 of biomass was employed and all the other conditions were identical to those used in diuron 122 degradation experiments (section 2.3). Samples were taken at selected times and filtered as 123 mentioned above before the analysis of 3,4-DCA residues. 124 125 2.6. Diuron removal in fungal TBR under non-sterile condition 126 As shown in Figure 1, a cylindrical glass TBR (Ø 3 cm, h 25 cm) with a working volume of 160 127 cm3 was set up. Specifically, 60 g (29 g DW) of autoclaved wet pine wood chips (2 cm × 1 cm × 128 0.5 cm) were inoculated with blended mycelial suspension and incubated statically for 9 days as 129 described by Torán et al. (2017). Tap water (adjusted to pH 4.5) was fortified with diuron to a final 130 concentration of 10 mg L– 1, and fed into the reservoir bottle from where it was pumped continuously 131 to the top of the reactor through an external recirculation loop. The hydraulic retention time (HRT) 132 corresponding to the total aqueous volume in the system divided by the influent flow rate (Fin) was 10 133 established as 3 days. Humid air was introduced from the top, and the pH was maintained at 4.5 in 134 the reservoir by adding either 1 M HCl or 1 M NaOH, and mixing with a magnetic stirrer. Air Parameters: Height: 25 cm Internal diameter: 3 cm Working volume: 160 cm3 Porosity: 60% Effluent Influent 135 Stirrer 136 Figure 1 Schematic representation of the trickle-bed reactor set-up 137 138 Response surface methodology (RSM) was applied for assessing the factors that influence diuron 139 removal performance. Given the configuration of this particular system and its variable parameters, 140 recycling ratio (RR), defined as the ratio between recirculating flow rate (Fr) and influent flow rate 141 (Fin), and the ratio of influent flow rate to the working volume of the reactor (Fin/VR) were set as 142 independent factors. Correspondingly, the response analyzed was diuron removal at steady state, 143 i.e., after 9 days of continuous treatment. For each experiment, the TBR was newly set up with 144 freshly colonized wood chips. Design Expert V8.06 (Stat-Ease, Inc., MN, USA) was used to design 145 the experiment. A factorial experimental design with 2 factors at 3 levels was employed, as 146 described in Table 1. This translates into 12 experimental runs including 4 replicates. 11 147 148 149 Table 1 Factors and levels for response surface methodology Levels Factors Codes –1 0 1 Recycling ratio (RR) X1 200 500 800 Fin/VR (h– 1) X2 0.42 1.04 1.67 150 151 2.7. Analytical methods 152 2.7.1. Biomass quantification 153 Biomass was determined by the dry weight of pellets. It was obtained through filtration of the 154 culture, followed by washing with sterile distilled water and drying at 105 °C to constant weight. 155 156 2.7.2. Glucose concentrations 157 Samples were filtrated through a nylon membrane (0.45 μm) and then measured with a 158 biochemistry analyzer (2700 select, Yellow Springs Instrument, USA). 159 160 2.7.3. Laccase activity 161 Laccase activity was measured through the oxidation of 2,6-dymethoxyphenol (DMP) by the 162 enzyme as described elsewhere (Wariishi et al., 1992). Activity units per liter (AU L– 1) are defined 163 as the amount of DMP in μM which is oxidized per minute. The molar extinction coefficient of 164 DMP was 24.8 mM– 1 cm– 1. 165 166 2.7.4. Diuron and 3,4-DCA concentrations 167 Diuron and 3,4-DCA residual concentrations were determined using high-performance liquid 12 168 chromatography (HPLC, Ultimate 3000, Dionex, USA) and UV detection. Briefly, 1 mL of culture- 169 aliquot was withdrawn and filtered (0.22 μm, PVDF) before HPLC analysis. HPLC-UV analysis 170 was performed using a C18 reversed-phase column (Phenomenex®, Kinetex® EVO C18 100 Å, 4.6 171 mm × 150 mm, 5 μm) at 30 ºC and a mobile phase consisting of acetonitrile and water (40:60, v/v) 172 at a constant flow rate of 0.9 mL min– 1. Analytes were detected using a wavelength of 252 nm. The 173 sample injection volume was 40 µL. 174 175 2.7.5. Evaluation and identification of TPs 176 Analysis of TPs was performed using an ultra-high performance liquid chromatography (UHPLC) 177 system Acquity (Waters, Milford, MA, USA) coupled to a hybrid quadrupole-Orbitrap mass 178 spectrometer Q Exactive (Thermo Fisher Scientific, San Jose, CA, USA), equipped with a heated- 179 electrospray ionization source (HESI). Chromatographic separation was achieved with a Purospher® 180 STAR RP-18 endcapped Hibar® HR (150 × 2.1 mm, 2 µm) column (Merck, Darmstadt, Germany) 181 and a linear gradient of the organic constituent of the mobile phase. The mobile phase employed 182 under positive ionization mode (HESI+) consisted of (A) water and (B) methanol, both containing 183 0.1% of formic acid (flow rate of 0.2 mL min– 1), whereas under negative ionization mode (HESI–) 184 a mobile phase of (A) water and (B) acetonitrile (flow rate of 0.3 mL min– 1) was used. The organic 185 gradient applied was: 0–1 min, 5% B; 3 min, 20% B; 6 min, 80% B; 7 min, 100% B; 9 min, 100% 186 B; 9.5 min, 5% B; 14 min, 5% B. The injection volume was 10 µL. 187 The specific conditions used in the HESI interface in both polarity modes were: spray voltage, 188 3.0 kV; sheath gas flow rate, 40 arbitrary units; auxiliary gas, 10 arbitrary units; capillary 189 temperature, 350 ºC, and vaporizer temperature, 400 ºC. Nitrogen (>99.98%) was employed as 13 190 sheath, auxiliary and sweep gas. Accurate mass detection was conducted in data-dependent 191 acquisition (DDA) mode. First, a full MS scan was done within the m/z range 70-1,000 at 70,000 192 full width at half maximum (FWHM) resolution(at m/z 200). Then, data-dependent MS/MS scan 193 events (17,500 FWHM resolution at m/z 200) were recorded for the five most intense ions (>10e5) 194 detected in each scan, with a normalized collision energy of 40%. Data acquisition was controlled 195 by Xcalibur 2.2 software (Thermo Fisher Scientific). 196 197 2.7.6. HRMS data processing 198 The acquired LC-HRMS data were processed using Compound Discover 3.1 software (Thermo 199 Fisher Scientific). Briefly, experimental samples collected at different times were compared, 200 considering samples collected at t=0 as the control. m/z features in the different samples were 201 aligned and deconvoluted using 2 min as maximum retention time shift and 5 ppm of mass tolerance. 202 Then, they were grouped and assigned to a specific molecular ion, for which an elemental 203 composition was predicted. In parallel, a search by formula or mass was performed in various MS 204 libraries and compound databases (ChemSpider, mzCloud, mzVault) for the assignment of a 205 potential compound identity. This workflow produced a list of peaksthat was manually revised to 206 identify real TPs, i.e., those peaks that were only present in samples collected after 2 and/or 7 days 207 of degradation, and absent at time 0. Once identified, the molecular structures proposed by the 208 software were evaluated according to the elemental composition of the molecular and fragment ions, 209 fragment rationalization (assisted by fragment ion search scoring), and isotopic patterns. 210 211 2.7.7. Data analysis 14 212 The mean and standard deviation (SD) of data were calculated and subjected to analysis of 213 variance (ANOVA). Statistical significance was determined using SPSS V22.0. 214 3. Results and discussion 215 3.1. Degradation of diuron by different white-rot fungi 216 Firstly, the ability of different white-rot fungi to degrade diuron was evaluated. As shown in 217 Figure 2, diuron in abiotic controls showed high chemical stability after 7 days of incubation. This 218 means that any removal observed in the experimental treatment must be exclusively attributed to 219 fungal sorption and biodegradation mechanisms. The four tested microorganisms demonstrated 220 different diuron elimination efficiencies, ranging from 18% to 83%. Although there were small 221 differences, the variation patterns of diuron concentration in the different heat-killed fungal cultures 222 were essentially similar, showing an initial fast drop due to sorption onto the biomass, after which 223 concentration remains stable. Thus, sorption contributed to diuron elimination to some extent. This 224 is in agreement with data reported by Lucas et al. (2018) in similar WRF elimination experiments 225 conducted with pharmaceuticals. On average, diuron adsorption observed in this study (16%) was 226 more than double than that observed for other xenobiotics with similar or higher hydrophobicity 227 than diuron (Log Kow 2.68), i.e., venlafaxine (Log Kow 3.28) and carbamazepine (2.45) (Lucas et 228 al., 2018). Thus, additional physical-chemical characteristics may affect the adsorption of the 229 compound onto the biomass (e.g, pKa, or water solubility). 15 T. versicolor G. luteofolius 1.2 1.2 1.0 1.0 0.8 0.8 0.6 C/C0 0.6 C/C0 0.4 0.4 0.2 0.2 0.0 0.0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (d) Time (d) S. rugosoannulata P. ostreatus 1.2 1.2 1.0 1.0 0.8 0.8 0.6 C/C0 0.6 C/C0 0.4 0.4 0.2 0.2 0.0 0.0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Time (d) Time (d) 230 231 Figure 2 Time-course degradation of diuron by different fungi. C represents the residual 232 concentration of diuron in the sample (mg L– 1), and C0 corresponds to the initial concentration of 233 diuron in the sample (mg L– 1); Blue lines with filled squares, experimental; red lines with empty 234 triangles, killed control; green lines with filled circles, abiotic. Average values of three replicates 235 with the corresponding standard deviation are shown. 236 237 As for glucose, its concentration plunged to almost zero after 7 days of incubation in T. versicolor, 238 G. luteofolius, and S. rugosoannulata cultures (Supplementary Information (SI), Table S1). 239 Although the capability of P. ostreatus in degrading different xenobiotics such as plastic, 240 organochlorine pesticides, and polycyclic aromatic hydrocarbons has been well documented 241 (Bhattacharya et al., 2014; da Luz et al., 2013; Purnomo et al., 2010; Purnomo et al., 2017), an 242 exceptional scenario was found in diuron degradation, as it presented a much lower consumption of 243 glucose than the other investigated fungi, with 4.85 g L– 1 glucose remaining in solution at the end 244 of the experiment. This is indicative of a lower metabolism of this fungal strain as compared to the 245 others, which results in nearly no biodegradation (as shown in Figure 2). A similar observation was 16 246 reported regarding the degradation of venlafaxine by this species in an analogous medium (Llorca 247 et al., 2019), in spite that it showed excellent performance for the removal of other pharmaceuticals, 248 namely, diclofenac, ketoprofen, and atenolol, in sterile hospital wastewater (Palli et al., 2017). 249 Hence, further research is needed to address the mechanisms behind this selective performance. 250 This is the first study that confirms the potential of S. rugosoannulata to degrade diuron, thus 251 enriching the diuron-degrading microbe pool. Considering the better performance in diuron 252 degradation by T. versicolor compared to the other investigated WRF species, it was selected for 253 further research. 254 255 3.2. Role of laccase and cytochrome P450 inhibitor in the degradation of diuron 256 Several in vitro experiments were carried out to investigate whether laccase and laccase-mediator 257 systems are involved in diuron degradation by T. versicolor. Laccase activity was found to reach a 258 maximum level of 27.68 AU L– 1 during diuron degradation (TP formation) experiments. Moreover, 259 results of in vitro degradation experiments showed that diuron concentrations remained constant 260 throughout the incubation period and were similar to those observed in the abiotic control (Table S2 261 in SI), despite substantial laccase activity (500 AU L– 1) was introduced. These results indicate that 262 laccase is not involved in the first step of diuron degradation. Nonetheless, an opposite conclusion 263 was drawn in a previous study (Coelho-Moreira et al., 2018), in which diuron was successfully 264 depleted in the presence of crude laccase from G. lucidum, and improved yields were obtained after 265 adding synthetic mediators, namely, ABTS and acetylacetone. Furthermore, Coelho-Moreira et al. 266 (2018) reported that diuron acted as a laccase inducer. The genetic difference among genera and 267 species of WRF could explain the discrepancy of the results obtained. Likewise, the effect of 17 268 different redox potentials between the laccase enzyme and the monophenolic substrate may be a key 269 factor (Glazunova et al., 2018). In this regard, it is also worthy to point out that low levels of Mn 270 peroxidase were detected in culture filtrate from G. lucidum (Coelho-Moreira et al., 2018). 271 272 The activity of the cytochrome P450 enzymatic system in diuron degradation was evaluated in 273 vivo experiments after the addition of the cytochrome P450 inhibitor 1-aminobenzotriazole. The 274 presence of the cytochrome P450 inhibitor in the culture hindered diuron degradation as compared 275 to the inhibitor-free culture system, although some removal occurred initially, which was probably 276 attributed to sorption processes (Figure 3). This finding is in agreement with previous reports that 277 showed that the addition of 1-aminobenzotriazole significantly inhibited diuron degradation by P. 278 chrysosporium and G. lucidum, as well as the metabolites produced in the process (Coelho-Moreira 279 et al., 2013, 2018). 1.0 0.8 0.6 C/C0 0.4 0.2 0.0 0 1 2 3 4 5 6 7 8 Time (d) 280 281 Figure 3 Effect of the cytochrome P450 inhibitor 1-aminobenzotriazole on the degradation of 282 diuron by T. versicolor. C represents the residual concentration of diuron in the sample (mg L– 1), 283 and C0 corresponds to the initial concentration of diuron in the sample (mg L– 1); Filled squares, 284 inhibitor-free; empty circles, inhibitor. Average values of three replicates with the corresponding 285 standard deviation are shown. 18 286 3.3. TPs generated during degradation of diuron by T. versicolor 287 Main TPs generated during the degradation of diuron by T. versicolor and detected using LC- 288 HRMS are shown in Table 2. In total five ions were identified as TPs; however, logical tentative 289 structures could be only proposed for three of them with an identification confidence level of 3 (CL3) 290 according to Schymanski’s scale (Schymanski et al., 2014). The remaining two TPs were appointed 291 as “unequivocal molecular formulae” (identification confidence level of 4), as no sufficient evidence 292 existed to propose possible structures. The CL3 TPs were TP248 3-(3,4-dichlorophenyl)-1- 293 hydroxymethyl-1-methylurea (DCPHMU), TP218 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), 294 and TP204 3,4-dichlorophenylurea (DCPU). TP218 (DCPMU) and TP204 (DCPU) have been 295 previously reported as aerobic degradation by-products of diuron regardless of the organism 296 (bacteria or fungi) used for its degradation. They are formed after successive N-demethylations 297 reactions of diuron (Badawi et al., 2009; Coelho-Moreira et al., 2013, 2018; Ellegaard-Jensen et al., 298 2014; Sørensen et al., 2008). Demethylation of diuron may occur intracellularly, as diuron and these 299 demethylated metabolites were found to be present in mycelial extracts of G. lucidum (Coelho- 300 Moreira et al., 2018). This is also in agreement with the results obtained in the laccase experiments 301 conducted in this study. All identified TPs remained in solution after 7 days of treatment; however, 302 TP204 (DCPU), TP137 and TP195 were the ones that presented an increasing trend by the end of 303 the experiment. Thus, TP248 (DCPHMU) and TP218 (DCPMU) can be considered as intermediate 304 byproducts that further degrade in contact with the fungus (Figure S1 in SI). TP248 is believed to 305 be formed after carbon hydroxylation at the tertiary amine moiety. This reaction may be mediated 306 by the cytochrome P450 system, according to the enzymatic exploration results and to a previous 307 study on the detoxification of chlortoluron by the grass weed A. myosuroides (Hall et al., 1995). 19 308 This is the first report of the formation of TP248 during microbial degradation of diuron. 20 309 Table 2 Transformation products formed during diuron degradation by T. versicolor Full Scan MS/MS tR HESI Suspect identity Number Δm Δ Chemical structure (min) mode m/z Formula RDB m/z Formula RDB (Confidence level) (ppm) (ppm) H Cl N N OH TP248 6.7 – 247.0030 C9H9O2N2Cl2 5.5 -2.1 159.9708 C6H4NCl2 4.5 – 4.6 DCPHMU (CL3) O Cl H H Cl N N TP218 8.6 + 219.0093 C8H9Cl2ON2 4.5 3.3 161.9876 C6H6Cl2N 3.5 3.8 DCPMU (CL3) O Cl 161.9877 C6H6Cl2N 3.5 3.4 H Cl N NH 2 TP204 8.5 + 204.9937 C7H6Cl2N2O 4.5 3.5 159.9728 C6H4Cl2N 4.5 8.2 DCPU (CL3) O 92.0503 C6H6N 4.5 8.5 Cl TP137 2.9 + 138.0556 C7H8O2N 4.5 4.4 110.0608 C6H8ON 3.5 7.1 n/a (CL4) 178.0508 C9H8O3N 6.5 3.6 TP195 4.3 + 196.0612 C9H10O4N 5.5 4.0 n/a (CL4) 150.0556 C8H8O2N 5.5 4.5 310 tR: chromatographic retention time, HESI, heated-electrospray ionization; Δm, mass measurement error; RDB, ring and double-bound equivalents 21 311 3,4-DCA has been widely reported to be a diuron TP that could be generated after the amide bond 312 hydrolysis of DCPU; however, it was not detected in the experimental culture media in this study. 313 Since 3,4-DCA is much more toxic than diuron, the ability of T. versicolor to remove this TP was 314 also evaluated. A degradation experiment using 3,4-DCA as substrate was conducted. T. versicolor 315 showed an exceptional ability to degrade 3,4-DCA Initial 3,4-DCA concentrations (10 mg L– 1) were 316 degraded in less than 24 h (Figure 4). This may explain why 3,4-DCA was not identified as a diuron 317 TP in this study. The biodegradation of 3,4-DCA under aerobic conditions has been extensively 318 described, and it could be reduced to a dechlorination step followed by ring-cleavage (Giacomazzi 319 and Cochet, 2004; Tasca and Fletcher, 2018). 1.2 1.0 0.8 0.6 C/C0 0.4 0.2 0.0 0 1 2 3 4 5 6 7 8 Time (d) 320 321 Figure 4 Time-course degradation of 3,4-DCA by T. versicolor. C represents the residual 322 concentration of 3,4-DCA in the sample (mg L– 1), and C0 corresponds to the initial concentration 323 of 3,4-DCA in the sample (mg L– 1); Blue lines with filled squares, experimental; red lines with 324 empty triangles, killed control; green lines with filled circles, abiotic. Average values of three 325 replicates with the corresponding standard deviation are shown. 326 327 Based on our results and previous findings, a pathway of diuron degradation by T. versicolor is 328 proposed in Figure 5; however, further investigation is still needed to understand the entire 329 metabolic pathway. 22 H Cl N N O Cl Diuron H Cl N N OH O Cl DCPHMU H H Cl N N O Cl DCPMU H Cl N NH2 O Cl DCPU Cl NH2 Cl 330 3,4-DCA 331 Figure 5 Proposed pathway of diuron degradation by T. versicolor. Full lines, the reactions 332 observed in this study; dashed lines, the reaction described in the literature 333 334 3.4. Influence of operational variables on diuron removal using TBR under non-sterile condition 335 TBR performance is difficult to describe using a mathematical model due to the various physical 336 and biological processes involved in it. On one side, the contribution of the wood is not limited to 337 the immobilization of the biomass. It also plays an important role in pollutant removal due to 23 338 adsorption, which occurs mainly in the early stages of a continuous treatment process. On the other 339 side, wood is a nutrient source that promotes fungal growth. The water is distributed from the bottom 340 to the top of the TBR and flows through a bed with a remarkably high porosity (60%), which is 341 mainly occupied by air. Thus, the removal yield will depend mainly on the contact time between the 342 immobilized biomass and the liquid phase. The biomass is directly related to the mass of the wood, 343 referred to as the working volume of the bioreactor (VR) (Fig. 1). Since the working volume (VR) 344 and the HRT were fixed, the factors amenable to modification were narrowed down to the 345 recirculation flow rate (Fr), the influent flow rate (Fin), and the water volume in the reservoir tank 346 (Vr). The number of times that the water is recirculated through the bioreactor is designated as RR 347 (Fr/Fin). The HRT, defined as the ratio between the volume of water in the system (i.e., the volume 348 of water in the reservoir tank (Vr)) and the Fin, was fixed to 3 days according to previous experiences 349 (Torán et al., 2017). The ratio between Fin and VR stands for the pollutant load to the system. 350 Therefore, the factors RR (X1) and Fin/VR (X2) were selected for studying each effect, as well as 351 their interaction on the removal of diuron. According to the factorial experimental design with 3 352 levels for each of these two factors, 12 trials were performed using tap water fortified with diuron, 353 and each experimental result along with model predicted values are given in Table 3. 354 355 Table 3 Central Composite Design matrix showing actual and predicted values for diuron removal Response Factors in coded value Run (Diuron removal, %) X1 X2 Actual Predicted 1 0 –1 74.4 74.1 2 1 –1 67.3 66.9 3 1 0 44.7 44.1 4 0 0 56.7 56.1 5 –1 0 64.6 68.2 6 –1 –1 83.0 81.2 24 7 1 1 26.5 21.3 8 –1 1 62.6 55.2 9 0 0 29.5 38.2 10 –1 –1 84.0 81.2 11 –1 –1 82.0 81.2 12 –1 –1 78.0 81.2 356 357 In each trial, the packing material was renovated to avoid fungus aging and adverse effect of 358 saturated wood on adsorption. The response of each run was the mean removal value obtained 359 during the steady state, which was achieved from day 9 to day 18 of continuous operation. Based 360 on the fitness of the data obtained, the following linear regression model for diuron removal in the 361 TBR system is proposed: Y = 56.61 – 11.98X1 – 17.72X2 – 5.05X1 X2 (1) 362 To further verify the goodness of the proposed model for diuron removal, analysis of variance 363 (ANOVA) was applied (Table S3 in SI). The results obtained indicate that the proposed model is 364 significant with a confidence level of 95% (F = 58.54, p < 0.0001), which also addressed its stability. 365 The adjusted R2 was 0.9401, indicating that 94 % of the data variance could be explained by the 366 studied factors (with a level of confidence of 95%). The two factors and the interaction between 367 them were also significant to predict diuron removal (p < 0.05) on which the predictor Fin/VR 368 demonstrate highest effect. The lack of fit was non-significant, which evidences the good correlation 369 between the experimental factors or predictors and the response variable. Besides, Table 3 shows 370 the agreement between the experimental data and those predicted by the model. Figure 6 illustrates 371 that the decrease in RR and Fin/VR enhanced the removal of diuron. Therefore, it is clear to infer 372 here the improvement of yield that can be obtained by setting those two factors at the lowest possible 373 levels, having in mind the following condition as VR is constant. 25 ∆Fr ∆Fin < (2) Fr Fin Diuron removal (%) Fin/VR (h– 1) RR 374 375 Figure 6 Response surface showing the effects of RR and Fin/VR on diuron removal 376 377 The results evidenced, as expected, that the lower the volumetric load (Fin/VR) the higher the 378 pollutant removal, but also that lower recirculating rates entail better removal yields. The RR is 379 related to the times that the water is passed through the bioreactor. In this regard, according to our 380 results, and contrary to expected, low RR values result in better performance than high RR values. 381 This could be explained by the fact that the highest contact time between the immobilized fungus 382 and the pollutant may contribute considerably to increase the removal efficiency, which is also 383 consistent with the fact that the cytochrome P450 system was the one found to be involved in diuron 384 degradation instead of extracellular enzymes. High RR requires high recirculating flow rates 385 promoting a coalescing effect between water drops, which increases the crossing velocity through 386 the column and consequently results in a reduction of the contact time with the fungus. However, a 387 lower value of diuron removal (72% vs 81.2%) was obtained in an additional experiment using RR 26 388 of 200 and optimal Fin/VR. This result indicates that better removal could be achieved by enlarging 389 the experimental domain to the corner where the optimum is. In summary, to design an effective 390 TBR is important to ensure a high contact time between the water and the fungus. This will depend 391 on the operational parameters that further influence the water crossing velocity through the 392 bioreactor. 393 394 4. Conclusions 395 Of the four fungi species investigated, T. versicolor was found to be the one that most efficiently 396 degraded both diuron and its main byproduct 3,4-DCA. The cytochrome P450 enzymatic system 397 was found to be the one catalyzing diuron removal. Up to five TPs were formed during the process, 398 and structures could be tentatively proposed for three of them. One intermediate TP, namely 399 DCPHMU, was detected for the first time, providing a better understanding of the occurring 400 demethylation process. The effect of operational variables on diuron removal from water using a 401 trickle-bed reactor was also evaluated. The contact time between the immobilized fungus and the 402 water played an important role in diuron removal. This represents a pioneering step forward to 403 overcome key barriers to upscale this type of bioreactor and apply this technology to the 404 bioremediation for real diuron-contaminated waters since the TBR tested in this study was operated 405 under non-sterile conditions. 406 407 Acknowledgment 408 This work has been supported by the Spanish Ministry of Economy and Competitiveness State 409 Research Agency (CTM2016-75587-C2-1-R and CTM2016-75587-C2-2-R) and co-financed by the 27 410 European Union through the European Regional Development Fund (ERDF) and the Horizon 2020 411 research and innovation WATERPROTECT project (727450). This work was partly supported by 412 the Generalitat de Catalunya (Consolidate Research Group 2017-SGR-14) and the Ministry of 413 Science and Innovation (Project CEX2018-000794-S). The Department of Chemical, Biological and 414 Environmental Engineering of the Universitat Autònoma de Barcelona is a member of the Xarxa de 415 Referència en Biotecnologia de la Generalitat de Catalunya. K. Hu acknowledges the financial 416 support from the Chinese Scholarship Council. 417 418 Conflict of interest 419 We declare that no conflict of interest exists in the submission of this manuscript. 420 421 References 422 Azubuike, C.C., Chikere, C.B., Okpokwasili, G.C., 2016. Bioremediation techniques-classification 423 based on site of application: principles, advantages, limitations and prospects. World J. Microb. 424 Biot. 32, 180. https://doi.org/10.1007/s11274-016-2137-x. 425 Badawi N., Rønhede, S., Olsson, S., Kragelund, B.B., Johnsen, A.H., Jacobsen, O.S, Aamand, J., 426 2009. 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Chem. 267, 23688-23695. 34 Supplementary information Fungal bioremediation of diuron-contaminated waters: evaluation of its degradation and the effect of amendable factors on its removal in a trickle-bed reactor under non-sterile conditions Kaidi Hu1, Josefina Torán1, Ester López-García2, Maria Vittoria Barbieri2, Cristina Postigo2, Miren López de Alda2, Gloria Caminal3, Montserrat Sarrà1*, Paqui Blánquez1 1Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain 2Water, Environmental and Food Chemistry Unit (ENFOCHEM), Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain 3Institut de Química Avançada de Catalunya (IQAC), CSIC. Jordi Girona 18-26, 08034 Barcelona, Spain *Corresponding author: Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel: + 34- 34935812789; E-mail: montserrat.sarra@uab.cat 35 Table S1 Time-course of glucose concentrations in the experimental culture media (g L– 1) Incubation time (d) Fungus 0 1 3 7 T. versicolor 7.00 ± 0.13 5.35 ± 0.16 1.83 ± 0.40 < 0.01 G. luteofolius 6.63 ± 0.10 4.77 ± 0.02 0.54 ± 0.29 < 0.01 S. rugosoannulata 7.02 ± 0.13 4.34 ± 0.02 < 0.01 ND P. ostreatus 7.01 ± 0.12 6.21 ± 0.09 5.67 ± 0.05 4.85 ± 0.10 Note: Each value of glucose concentration represents the mean of triplicate measurements ± SD. ND: no detected 36 Table S2 Effect of laccase on diuron degradation in the presence and absence of mediators Diuron (mg L– 1) Time (h) Abiotic control Laccase VA ABTS HOBT 0 10.01 ± 0.41 9.69 ± 0.91 9.02 ± 0.31 9.58 ± 0.72 9.41 ± 0.29 8 9.83 ± 0.38 9.74 ± 0.59 8.96 ± 0.47 10.35 ± 0.21 9.25 ± 0.71 24 9.45 ± 0.75 9.40 ± 0.60 9.69 ± 0.49 9.66 ± 0.35 10.60 ± 0.83 72 9.32 ± 1.10 9.01 ± 0.89 9.54 ± 1.10 10.74 ± 0.16 9.13 ± 1.47 Note: Each value of laccase concentration represents the mean of triplicate measurements ± SD. VA, violuric acid monohydrate; ABTS, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt; HOBT, hydrated 1-hydroxybenzotriazole 37 Table S3 Analysis of variance of the linear regression model proposed Source Sum of squares Degrees of freedom Mean square F p Model 4, 214.66 3 1, 404.89 58.54 < 0.0001* X1 1, 028.52 1 1, 028.52 42.86 0.0002* X2 2, 248.02 1 2, 248.02 93.68 < 0.0001* X1X2 134.73 1 134.73 5.61 0.0454* Residual 191.98 8 24.00 Lack of fit 171.23 5 34.25 4.95 0.1091 Pure error 20.75 3 6.92 Total 4, 406.64 11 *Significant (p < 0.05) R2 = 0.9564 R2adj = 0.9401 38 Figure S1. Evolution of the TPs identified during T. versicolor-mediated degradation of diuron 39