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

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.


Introduction
Over the last few decades, pesticide usage has shown a sustained growing tendency as a result of the exponential increase of the human population that brings along the need for intensifying food production. In this context, herbicides act as a key component of modern global agricultural systems (Bilal et al., 2019). Diuron [3-(3,4dichlorophenyl)-1,1-dimethylurea], a phenylurea herbicide, is used extensively to control weeds throughout the world, not only in agriculture but also in urban and industrial scenarios (Liu, 2014). This herbicide is stable to hydrolysis at neutral pH (pH 5-9) and is generally persistent in soil with a half-live of 320-330 days, and thus, can To reduce diuron risks and protect water ecosystems, the European Commission has included this herbicide in the priority hazardous substance list (Directive 2013/39/EU) and established environmental quality standards (EQS) in surface waters. Due to its potential effects, diuron is also one of the 92 substances that are currently being assessed by the European Chemicals Agency (ECHA) as a potential endocrine disruptor.
Despite worldwide efforts to control its environmental presence, diuron is still frequently detected in surface and ground waters (Kaonga et (Singh and Singh, 2014). Although different reactors based on WRF have been successfully designed for the degradation of various xenobiotics, their implementation at full-scale still needs to overcome some problems, like bacterial contamination (Mir-Tutusaus et al., 2018). In comparison with other reactors such as stirred tank reactor and fluidized bed reactor, trickle-bed reactors (TBR) offer multiple advantages, such as lower operational costs, greater operational flexibility, and higher sustainability (Luo et al., 2014). Furthermore, the immobilization of T. versicolor on wood has been proven to be advantageous to maintain fungal activity and has been successfully used to remove pharmaceuticals in hospital wastewater (Torán et al., 2017).
The purpose of the present study was: i) to screen out one WRF species that could effectively degrade diuron, ii) explore the enzymatic system involved in the degradation, and iii) evaluate the potential transformation products (TPs) formed during the process.
A further objective was to estimate the operational variables of a TBR immobilized with the selected fungus for diuron removal, operated under non-sterile conditions, to improve reactor performance and overcome the current limitations for its application in real scenarios. The defined medium used in the degradation batch experiments in Erlenmeyer flask consisted of (per liter) 8 g glucose, 3.3 g ammonium tartrate, 1.68 g dimethyl succinate, 10 mL micronutrients, and 100 mL macronutrients (Kirk et al., 1978). pH was adjusted to 4.5.

Fungal selection for diuron removal
To select the best fungal species for diuron removal, degradation experiments were performed in 250 mL Erlenmeyer flasks containing 50 mL of defined medium fortified with diuron at a final concentration of 10 mg L -1 . Briefly, pellets of each fungus were transferred into flasks as inoculum, thereby achieving a concentration of approximately 3.4 g dry weight (DW) L -1 . Then the cultures were incubated at 25 °C under continuous orbital-shaking (135 rpm) for 7 days. To obviate the influence of photodegradation, the incubation were performed in the dark. Abiotic (uninoculated) and heat-killed culture (121 °C for 30 min) were used as controls. Each set was tested in triplicate. Aliquots were taken at specific time intervals during incubation to measure diuron and glucose concentrations.

Identification of diuron transformation products
Experiments were conducted in 500 mL Erlenmeyer flasks containing 100 mL of fresh define medium. In brief, after transferring 2.6 g DW L -1 of T. versicolor pellets, cultures were incubated for 7 days under the same conditions described in section 2.3.
Diuron was added at a concentration of 1 mg L -1 . Abiotic control containing only diuron was also prepared. Each experiment was conducted in triplicate. At selected times, 4 mL of the culture was withdrawn and centrifuged (17, 700 × g, 4 min) at room temperature. Then, 1.5 mL of supernatant was transferred into a 2-mL amber vial containing 75 μL of diuron-d6 solution at a concentration of 10 μg mL -1 and kept in the dark at -20 °C until analysis. Simultaneously, laccase activity was determined during incubation.
Also, another degradation experiment using 3,4-DCA as substrate (10 mg L -1 ) was performed in parallel, to explore the degradation ability of T. versicolor towards the main metabolite formed by diuron and elucidate the biochemical pathway of diuron degradation. In these experiments, 3.2 g DW L -1 of biomass was employed and all the other conditions were identical to those used in diuron degradation experiments (section 2.3). Samples were taken at selected times and filtered as mentioned above before the analysis of 3,4-DCA residues.

Diuron removal in fungal TBR under non-sterile condition
As shown in Figure 1, a cylindrical glass TBR (Ø 3 cm, h 25 cm) with a working volume of 160 cm 3 was set up. Specifically, 60 g (29 g DW) of autoclaved wet pine wood chips (2 cm × 1 cm × 0.5 cm) were inoculated with blended mycelial suspension and incubated statically for 9 days as described by Torán et al. (2017). Tap water (adjusted to pH 4.5) was fortified with diuron to a final concentration of 10 mg L -1 , and fed into the reservoir bottle from where it was pumped continuously to the top of the reactor through an external recirculation loop. The hydraulic retention time (HRT) corresponding to the total aqueous volume in the system divided by the influent flow rate (Fin) was established as 3 days. Humid air was introduced from the top, and the pH was maintained at 4.5 in the reservoir by adding either 1 M HCl or 1 M NaOH, and mixing with a magnetic stirrer.

Figure 1 Schematic representation of the trickle-bed reactor set-up
Response surface methodology (RSM) was applied for assessing the factors that influence diuron removal performance. Given the configuration of this particular system and its variable parameters, recycling ratio (RR), defined as the ratio between recirculating flow rate (Fr) and influent flow rate (Fin), and the ratio of influent flow rate to the working volume of the reactor (Fin/VR) were set as independent factors.
Correspondingly, the response analyzed was diuron removal at steady state, i.e., after 9 days of continuous treatment. For each experiment, the TBR was newly set up with freshly colonized wood chips. Design Expert V8.06 (Stat-Ease, Inc., MN, USA) was used to design the experiment. A factorial experimental design with 2 factors at 3 levels was employed, as described in Table 1

Biomass quantification
Biomass was determined by the dry weight of pellets. It was obtained through filtration of the culture, followed by washing with sterile distilled water and drying at 105 °C to constant weight.

Glucose concentrations
Samples were filtrated through a nylon membrane (0.45 μm) and then measured with a biochemistry analyzer (2700 select, Yellow Springs Instrument, USA).

Laccase activity
Laccase activity was measured through the oxidation of 2,6-dymethoxyphenol (DMP) by the enzyme as described elsewhere (Wariishi et al., 1992). Activity units per liter (AU L -1 ) are defined as the amount of DMP in μM which is oxidized per minute. The molar extinction coefficient of DMP was 24.8 mM -1 cm -1 .

Evaluation and identification of TPs
Analysis of TPs was performed using an ultra-high performance liquid chromatography (

HRMS data processing
The acquired LC-HRMS data were processed using Compound Discover 3.1 software (Thermo Fisher Scientific). Briefly, experimental samples collected at different times were compared, considering samples collected at t=0 as the control. m/z features in the different samples were aligned and deconvoluted using 2 min as maximum retention time shift and 5 ppm of mass tolerance. Then, they were grouped and assigned to a specific molecular ion, for which an elemental composition was predicted. In parallel, a search by formula or mass was performed in various MS libraries and compound databases (ChemSpider, mzCloud, mzVault) for the assignment of a potential compound identity. This workflow produced a list of peaksthat was manually revised to identify real TPs, i.e., those peaks that were only present in samples collected after 2 and/or 7 days of degradation, and absent at time 0. Once identified, the molecular structures proposed by the software were evaluated according to the elemental composition of the molecular and fragment ions, fragment rationalization (assisted by fragment ion search scoring), and isotopic patterns.

Data analysis
The mean and standard deviation (SD) of data were calculated and subjected to analysis of variance (ANOVA). Statistical significance was determined using SPSS V22.0.

Role of laccase and cytochrome P450 inhibitor in the degradation of diuron
The activity of the cytochrome P450 enzymatic system in diuron degradation was evaluated in vivo experiments after the addition of the cytochrome P450 inhibitor 1aminobenzotriazole. The presence of the cytochrome P450 inhibitor in the culture hindered diuron degradation as compared to the inhibitor-free culture system, although some removal occurred initially, which was probably attributed to sorption processes ( Figure 3). This finding is in agreement with previous reports that showed that the addition of 1-aminobenzotriazole significantly inhibited diuron degradation by P.
chrysosporium and G. lucidum, as well as the metabolites produced in the process

TPs generated during degradation of diuron by T. versicolor
Main TPs generated during the degradation of diuron by T. versicolor and detected using LC-HRMS are shown in Table 4. In total five ions were identified as TPs; however, logical tentative structures could be only proposed for three of them with an  (Figure 4). TP248 is believed to be formed after carbon hydroxylation at the tertiary amine moiety. This reaction may be mediated by the cytochrome P450 system, according to the enzymatic exploration results and to a previous study on the detoxification of chlortoluron by the grass weed A. myosuroides (Hall et al., 1995). This is the first report of the formation of TP248 during microbial degradation of diuron.  3,4-DCA has been widely reported to be a diuron TP that could be generated after 3 the amide bond hydrolysis of DCPU; however, it was not detected in the experimental 4 culture media in this study. Since 3,4-DCA is much more toxic than diuron, the ability 5 of T. versicolor to remove this TP was also evaluated. A degradation experiment using Based on our results and previous findings, a pathway of diuron degradation by T. 21 versicolor is proposed in Figure 6; however, further investigation is still needed to 22 understand the entire metabolic pathway.  effect. The lack of fit was non-significant, which evidences the good correlation 68 between the experimental factors or predictors and the response variable. Besides, Table   69 5 shows the agreement between the experimental data and those predicted by the model.