Journal of Environmental Management 293 (2021) 112859 Contents lists available at ScienceDirect Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman Comparison between two reactors using Trametes versicolor for agricultural wastewater treatment under non-sterile condition in sequencing batch mode Kaidi Hu a, Montserrat Sarrà a, *, Gloria Caminal b a Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain b Institut de Química Avançada de Catalunya (IQAC), CSIC. Jordi Girona 18-26, 08034, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Agricultural wastewater is a major source of herbicides, which pose environmental and health concerns owing to Micropollutants their substantial use and poor elimination rate in conventional wastewater treatment plants. White-rot fungi are Herbicides versatile in degrading xenobiotics; however, the key problem encountered with their application in actual sce­ Trickle-bed reactor narios is competition with indigenous microorganisms, mainly bacteria. To address this barrier, two different Fluidized-bed reactor Residence time distribution strategies were implemented in the present study. One strategy was to set up a trickle bed with Trametes versicolor Fungal bioremediation immobilized on pine wood, and another strategy was to employ a T. versicolor-pelleted, fluidized-bed reactor to remove diuron and bentazon from actual wastewater under non-sterile conditions. The residence time in the trickle bed was estimated using three methodologies. With 10 batches of a 3-day cycle operation, although the trickle-bed reactor possessed a shorter contact time (8.5 h per cycle) and lower laccase activity compared with those of the fluidized-bed reactor, it demonstrated a higher removal yield and lower bacterial counts. In addition, the utilization of pine wood as a carrier obviously reduced the cost since no additional nutrients were required. Hence, after evaluating all advantages and limitations of both bioreactors, for the purpose of treating over the long term and scaling up, a trickle-bed reactor is the preferred choice. 1. Introduction agricultural crops, such as fruit, cotton, and sugar cane (Liu, 2014), while bentazon [3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2, In recent decades, diverse micropollutants that demonstrate persis­ 2-dioxide] is a thiadiazine herbicide and used largely postemergence tence, partly because of their high volumes of production and con­ in rice cultivation areas for the control of broadleaf weeds as well as sumption resulting from anthropogenic activities and partly because of sedges (Gillespie et al., 2011). Due to either recalcitrance (Giacomazzi their poor elimination rates in conventional wastewater treatment and Cochet, 2004; Liu, 2014) or extended use (López-Piñeiro et al., plants (WWTPs), have represented a severe global concern, and these 2017), both pollutants are ubiquitous in different water bodies (Barbieri micropollutants include pesticides, pharmaceuticals, personal care et al., 2020a; Barbieri et al., 2021; Vieira et al., 2016; Wan et al., 2020) products, steroid hormones, industrial chemicals and many other through leaching or runoff (Giacomazzi and Cochet, 2004; Hua et al., emerging compounds (Luo et al., 2014). Among them, pesticides are 2009; López-Piñeiro et al., 2017), and accumulating evidence shows that considered the main trigger of environmental deterioration, although they pose multifaceted threats towards non-target organisms and eco­ their use addresses the increasing food demand and population explo­ systems (Giacomazzi and Cochet, 2004; Liu, 2014; Macedo et al., 2008; sion (Meftaul et al., 2020; Rani et al., 2020). Turcant et al., 2003). Hence, the development of techniques to address As a key component of modern global agricultural systems, herbicide this concern is strongly encouraged and imperative. sales account for the largest proportion of agrochemicals and currently In comparison with physical and chemical approaches, bioremedia­ for more than 40% of their consumption (Carvalho, 2017). Diuron [3-(3, tion is regarded as a low-cost, eco-friendly, and efficient alternative 4-dichlorophenyl)-1,1-dimethylurea], one type of phenylurea herbicide, technique for the abatement of a broad range of pollutants (Azubuike is used extensively to control pre- and postemergence weeds for many et al., 2016). In particular, as a result of a battery of well-developed * Corresponding author. E-mail address: montserrat.sarra@uab.cat (M. Sarrà). https://doi.org/10.1016/j.jenvman.2021.112859 Received 9 February 2021; Received in revised form 4 May 2021; Accepted 19 May 2021 Available online 24 May 2021 0301-4797/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). K. Hu et al. Journal of Environmental Management 293 (2021) 112859 Fig. 1. Schematic representations of the trickle-bed reactor (A) and the fluidized-bed reactor (B) setup. ligninolytic enzymes, white-rot fungi (WRF) are effective at degrading purchased from Sigma-Aldrich (Barcelona, Spain). The commercial various recalcitrant contaminants (Mir-Tutusaus et al., 2018). The herbicide KAOS-B (bentazon, 48%) was obtained from SAPEC AGRO application of fungal reactors, especially fungal pelleted reactors, in (Barcelona, Spain). D (+)-Glucose was purchased from Acros Organics wastewater treatment has been well documented (Espinosa-Ortiz et al., (New Jersey, USA). Ammonium chloride was obtained from Scharlau 2016). Our group has focused on air-pulsed fluidized-bed reactors (Barcelona, Spain). Chromatographic grade acetonitrile was purchased (FBRs) using pellets of Trametes versicolor for years, and good results from Carlo Erba Reagents S.A. S (Barcelona, Spain). Formic acid (purity, have been obtained for different xenobiotics under sterile conditions ≥ 98%) was used as a mobile phase modifier, and ferric chloride was (Blánquez et al., 2007; Jelic et al., 2012). However, some issues obtained from Merck (Darmstadt, Germany). PROSEDIM CS 209 was appeared under non-sterile conditions, including bacterial contamina­ acquired from Degrémont Iberia (Bilbao, Spain). Stock solutions (10 mg tion and biomass ageing (Mir-Tutusaus et al., 2018). Although several mL– 1) of diuron and bentazon to be used for wastewater fortification solutions have been proposed for long-term application, such as partial were prepared by appropriate dilution of the substances in ethanol and biomass renovation, pH adjustment or coagulation-flocculation pre­ stored in the dark at 4 ◦ C until use. treatment (Mir-Tutusaus et al., 2017), economically, FBR is still an expensive option. Notably, a trickle-bed reactor (TBR) with T. versicolor 2.3. Agricultural wastewater immobilized on pine wood was set up by Hu et al. (2020), and it could successfully remove diuron from tap water. However, a comparison of Agricultural wastewater (AW) was collected from irrigation channels the performances of these two reactors in the scenario of in Gavà, within the Llobregat River Basin (Catalonia, Spain), but during pesticide-contaminated wastewater treatment is still unknown. two different periods. The wastewater used for the TBR (AW I) was To address this gap, the objective of the present work was to set up a collected in February 2019, whereas that for the FBR (AW II) was ob­ trickle-bed reactor and fluidized-bed reactor using T. versicolor as inoc­ tained in August 2020. They were stored at 4 ◦ C until use. In the case of ulum for agricultural wastewater treatment under non-sterile conditions AW II, a coagulation-flocculation pretreatment was applied to reduce and then to compare their performances and practicality from different algal biomass. Coagulant ferric chloride and flocculant PROSEDIM CS perspectives. It is hoped that this work can provide perspectives for 209 were employed in this process, during which 2 min of coagulation at scaling-up approaches and long-term processes. 200 rpm, 15 min of flocculation at 20 rpm and 30 min of settling were performed. Specifically, AW II was pretreated with 40 mg L– 1 coagulant 2. Materials and methods and 2 mg L– 1 flocculant. The characteristics of the pretreated AW II and AW I are given in Table S1 of the supplementary material (SM). 2.1. Microorganism and medium 2.4. Bioreactor setup T. versicolor ATCC 42530 was acquired from the American Type Culture Collection and maintained by subculturing every 30 days on 2% 2.4.1. Trickle-bed reactor (w/v) malt extract Petri dishes at 25 ◦ C. Blended mycelial suspensions As shown in Fig. 1A, a cylindrical methacrylate TBR (Ø 8.3 cm, H 59 and pellets were prepared using malt extract as described by Blánquez cm) with an approximate working volume of 2.5 L and a porosity of 60% et al. (2004). was set up. Essentially, 1 L of AW I fortified with diuron and bentazon to For the TBR experiment, the packing material was prepared by a final concentration of 10 mg L– 1 for both pollutants was loaded into inoculating a blended mycelial suspension into autoclaved pine wood the packing bed from the top of the reactor through a rotary distributor (Pinus sp.) chips as reported by Torán et al. (2017), followed by 4 weeks and then collected by the reservoir tank placed at the bottom. The of incubation at 25 ◦ C. collected water was mixed by a magnetic stirrer, and its pH was main­ tained at 4.5 by adding either 1 M HCl or 1 M NaOH. An external 2.2. Chemicals and regents bottom-to-top recirculation loop (flow rate 70 mL min– 1) was provided. Simultaneously, an identical reactor filled with non-colonized chips was Diuron (purity, ≥ 98%) and 2,6-dymetoxyphenol (DMP, 99%) were operated in parallel to assess the removal contribution from 2 K. Hu et al. Journal of Environmental Management 293 (2021) 112859 lignocellulosic supporting material. Multiple runs were implemented in sequencing batch mode at room temperature, and a 3-day cycle was adopted. Samples were taken from the tank after each batch to measure laccase, pesticide concentration, heterotrophic plate counts (HPCs) and chemical oxygen demand (COD), and then, the wastewater was totally replenished. 2.4.2. Fluidized-bed reactor Pellets were transferred into a glass air-pulsed fluidized reactor, achieving the same biomass in terms of dry weight (DW) as that in the TBR. This reactor was designed with a cylindrical vertical centre body connected with a diametrically wider head (Fig. 1B). The useful volume of this FBR was 1.5 L, and it carried 1 L of AW II spiked with diuron and bentazon. The concentration was set to 10 mg L– 1 for each herbicide. The reactor was operated in sequencing batch mode with a 3-day cycle at 25 ◦ C. The pH was controlled at a constant value of 4.5 by the addition of 1 M HCl or 1 M NaOH through several ports at the top. Air was introduced from the bottom using an electrovalve through which the fluidized condition (1 s air pulse every 4 s) in the centre body was sus­ tained. The aeration rate was approximately 0.8 L min– 1. Nutrients including glucose and NH4Cl were fed with a molar C/N ratio of 7.5 based on the glucose consumption rate of T. versicolor (Mir-Tutusaus et al., 2017). At the end of each batch, samples were withdrawn for laccase, pesticide concentration, HPCs, COD and glucose analyses, and then, pellets were harvested and reinoculated to replenished wastewater. 2.5. Residence time of the TBR Taking into account that the contact time between the immobilized fungi and the water has been suggested to play a vital role in the regime of a TBR (Hu et al., 2020), residence time determination is important for the employed reactor. In this work, the residence time was determined through three different techniques: (1) Volumetric quantification. After the wood was soaked with water, the pump was switched on for a certain time, and all the introduced distilled water was collected at the outlet. The time corresponding to collecting a 10 mL aliquot was recorded. (2) Pulse tracer injection. After injecting 3 mL of 3 M KCl at the top of the reactor, the effluent was collected every 15 s, and the conductivity was measured. (3) Step tracer input. The influent was changed to a KCl so­ lution (46 mM) at a certain point, and then, the samples were collected and analysed analogously to the pulse input strategy. A flow rate of 70 mL min– 1 was employed in all experiments, and each kind of test was conducted in duplicate. 2.6. Analytical procedures 2.6.1. Laccase After filtration through a 0.22 μm hydrophilic polypropylene syringe filter (Scharlau, Barcelona, Spain), laccase activity was measured through the oxidation of DMP by the enzyme as described elsewhere (Wariishi et al., 1992). Activity units per L (AU L– 1) were defined as the amount of DMP in μM that was oxidized in 1 min. The molar extinction coefficient of DMP was 24.8 mM– 1 cm– 1. 2.6.2. Pesticide residues Samples were filtered using a Millipore Millex-GV unit equipped with a polyvinylidene difluoride (PVDF) membrane (0.22 μm) prior to Fig. 2. Responses to TBR residence time determination using different tech­ analysis. The residual diuron and bentazon concentrations were deter­ niques. a. volumetric quantification. b. pulse tracer injection. c. step tracer mined through an HPLC (Ultimate 3000, Dionex, USA) equipped with a input. Empty circles, input volume; filled squares, output volume; filled triangles, UV detector. Chromatographic analysis was achieved with a mobile tracer concentration at outlet. phase consisting of 0.01% (v/v) formic acid in water (A) and acetonitrile (B) at a flow rate of 0.9 mL min– 1 and a C18 reversed-phase column (Phenomenex®, Kinetex® EVO C18 100 Å, 4.6 mm × 150 mm, 5 μm) set at 30 ◦ C. A gradient elution programme was performed as follows: the percentage of B was 35% from 0 min to 0.5 min, then linearly increased 3 K. Hu et al. Journal of Environmental Management 293 (2021) 112859 Table 1 Average residence time of the TBR determined through different techniques. Technique Residence time (s) Volumetric quantification 102 ± 2 Pulse tracer injection 173 ± 1 Step tracer input 259 ± 6 Note: Means and standard deviation of duplicates are shown. to 45% from 0.51 min to 12 min, followed by decreasing to 35% in 1 min and maintaining 35% for 2 min. The injection volume was 40 μL. The detection wavelength was set at 254 nm, and a limit of detection of 0.5 mg L– 1 was accomplished by using this analysis condition. 2.6.3. Biomass Biomass in terms of pellets was determined by the dry weight, ob­ tained after filtrating the culture and drying the residue at 105 ◦ C to a constant weight. With regard to the TBR, biomass was quantified by measuring ergosterol as previously described by Rodríguez-Rodríguez et al. (2010). However, the HPLC analysis method was slightly modified since a C18 reversed-phase column (Phenomenex®, Kinetex® EVO C18 100 Å, 4.6 mm × 150 mm, 5 μm) was adopted in the present study. The isocratic eluent was switched to acetonitrile (100%), and the retention time was 7.593 min. 2.6.4. Other analyses The glucose concentration was measured using a biochemistry ana­ lyser (2700 select, Yellow Springs Instrument, USA) after filtrating the sample with a Millipore Millex-GV PVDF syringe filter (0.22 μm). The absorbance at 650 nm was determined by a UNICAM 8625 UV/ VIS spectrometer, and the conductivity was monitored by a CRISON MicroCM 2100 conductometer. The total suspended solids (TSSs) and volatile suspended solids (VSSs) were measured according to the stan­ dard methods 2540 D and 2540 E (Baird et al., 2017), respectively. The total organic carbon (TOC) was determined using an Analytik Jena multi N/C 2100 S/1 analyser. The HPC results were reported as the logarithm of colony-forming units (CFU) per mL [lg (CFU mL– 1)] using the spread-plate method with a plate count agar (PCA) following the stan­ dard method 9215 (Baird et al., 2017). The N–NH+ Fig. 3. Profiles of pesticides removal by the two different fungal reactors in 4 concentration and the COD were analysed by using the commercial kits LCK 303 and LCK sequencing batch mode under non-sterile conditions. a. diuron; b. bentazon. Gray columns, trickle-bed reactor; medium columns, fluidized-bed reactor. 314 or LCK 114 or LCK 014 (Hach Lange, Germany). Chloride, sulfate, nitrite and nitrate anions were quantified by a Dionex ICS-2000 inor­ ganic chromatograph equipped with a Dionex IonPac AS18-HC column average time required for the liquid to pass through the reactor, namely, (250 mm × 4 mm), which was eluted at 1 mL min– 1 with a 13 mM KOH the residence time. However, this characteristic changed to significantly aqueous solution. higher values when pulse input and step input were used (Table 1) since the conductivity proportional responses were not symmetric and the 2.6.5. Data analysis curves contained long tails (Fig. 2b and c). Similar profiles were ob­ The degradation percentage was calculated using the following tained by Sá and Boaventura (2001) using siliceous granular material to equation: support the biomass in the TBR. A reasonable explanation could be that part of the packing bed was relatively stagnant due to the irregular shape Removal percentage(%) = M0 − M × 100% of the wood chips, for which even a rotary distributor had been provided M0 at the top of the reactor. The main water flow went downwards through preferred routes, while a small amount of tracer was retained in the where M0 corresponds to the initial amount of contaminant (mg), and M reactor and left very slowly. Therefore, it can be concluded that the TBR represents the residual amount of contaminant (mg). employed in this work did not perform as an ideal plug-flow reactor. However, if the relatively stagnant volume (tails) was ignored, as 3. Results and discussion mentioned by Levenspiel (2002), 102 s corresponds to the time at which half of the mass of 80% of the tracer injection (active volume) was 3.1. TBR residence time collected in the scenario of pulse input and to the moment when half of the volume of flow in the rector was substituted by tracer solution in the To determine the residence time of the TBR, three different strategies scenario a step input. Hence, we consider that among the residence were adopted in this study, and the responses and calculated results are times, 102 s was the most representative residence time. Given that an presented in Fig. 2 and Table 1. For the volumetric quantification operation recirculation rate of 300 (recirculation flow rate at 70 mL technique shown in Fig. 2a, the flow rate of the outlet became constant min– 1) was applied in the present study, the total contact time was and equalled that of the inlet after 102 s, which can be assumed to be the 4 K. Hu et al. Journal of Environmental Management 293 (2021) 112859 Table 2 HPC and COD of the agricultural wastewater throughout the sequencing batch treatment by the two different fungal reactors. Batch Microbial counts [Log (CFU) mL –1] COD (mg L ) –1 TBR FBR TBR FBR 1 4.44 8.23 671 1172 2 2.90 8.90 590 1188 3 0 8.76 521 1706 4 3.90 8.52 438 2107 5 6.00 8.24 382 1695 6 6.10 7.85 318 1815 7 6.13 7.20 280 1696 8 5.99 7.29 288 2097 9 5.92 7.79 208 2565 10 5.87 7.95 248 2611 Note: TBR, trickle-bed reactor; FBR, fluidized-bed reactor. Fig. 4. Profile of laccase activity during operation. Filled squares, trickle-bed 3.3. Comparison between the trickle-bed reactor and air-pulsed fluidized- reactor; empty circles, fluidized-bed reactor. bed reactor actually 8.5 h for each batch. Clearly, the TBR demonstrated better results than the FBR based on the contaminant removal yield, as mentioned above, but this evidence is not strong enough to determine which fungal reactor is an ideal option. 3.2. Pesticide removal performance in the two different reactors Therefore, attention was also paid to bioactivity persistence in the two systems. Laccase activity was monitored during operation because it Several pieces of colonized wood chips were used before operation to partly reflects fungal activity (Mir-Tutusaus et al., 2017), and the results measure the initial biomass in the TBR, and the ergosterol concentration are shown in Fig. 4. An enzymatic activity peak of 47 AU L– 1 was was approximately 0.044 ± 0.002 mg per g of dry wood. Considering achieved in the TBR at the end of first batch, probably resulting from that 367 g of dry chips was introduced and the ergosterol content of the accumulation within the static incubation, whereas this peak occurred selected strain corresponded to 6.61 mg gDW– 1 of fungal biomass in second running period of the FBR, at 31 AU L– 1. Then, although (Rodríguez-Rodríguez et al., 2010), we determined that the total laccase activity displayed a decreasing pattern in both setups and biomass of the TBR was approximately 2.45 g DW. Hence, an equivalent eventually remained at a low constant level, its variation was obviously pelleted inoculum was transferred into the FBR for operation. slower in the FBR than in the TBR. The primary reasons behind this As shown in Fig. 3, considerable removals of diuron and bentazon observation could be the different nutrient conditions in the reactors and occurred in the TBR, yielding 69.38% and 47.21% removal on average, the possibility that extracellular laccase was adsorbed onto the wood respectively; in contrast, the FBR was less effective, at 29.79% and (Arora and Gill, 2000; Li et al., 2018). Furthermore, adequate oxygen 37.22% removal, respectively. However, the elimination performance transfer can be guaranteed by employing air-pulse conditions, while air was similar between the two reactors at the beginning of operation, and supply should be included in future work using TBRs. this performance was even better for bentazon in the FBR than in the In addition, the HPC and COD of the wastewater after treatment by TBR. This result could be explained by the fact that although both sets the trickle-bed reactor and fluidized-bed reactor are presented in employed a 3-day cycle, the contact time between the fungi and Table 2. A dramatic increase in the bacterial amount was observed in the pollutant was quite low in the case of the TBR based on the above FBR compared that in the raw water. This result could be explained by empirical calculations (8.5 h per cycle). Therefore, the contribution of the continuous feeding of glucose [1200 mg glucose (gDW d) – 1] for the cytochrome P450 system of T. versicolor to the selected pesticide maintenance (Mir-Tutusaus et al., 2017), and the supplemented glucose degradation was restricted due to the configuration of the TBR, and this was almost fully consumed in each batch (final concentration < 0.1 g L – particular intracellular enzyme is largely involved in diuron and ben­ 1 ). With respect to the TBR, although the microbial growth also accel­ tazon degradation (García-Vara et al., 2020; Hu et al., 2020). On the erated after 4 batches, it was largely hindered initially, especially during other hand, bearing in mind that the lignocellulosic-supporting material the 3rd batch, appearing as having no microbial count. In addition, in the TBR, namely, pine wood chips, played an indispensable role in the bacterial contamination remained at a lower level in the TBR than in the treatment owing to its adsorption since the results from the control FBR, indicating that utilization of pine wood as a supporting material is reactor showed that 83.22% of diuron was removed and 28.02% of a successful strategy to limit bacterial growth, and this represents a bentazon was removed; these values were 25.75% and 8.47%, respec­ bottleneck in fungal reactor application regimes (Mir-Tutusaus et al., tively, in regard to pellets in an Erlenmeyer test (García-Vara et al., 2018). At the same time, the total COD increased in the two treatments 2020; Hu et al., 2020). Such apparent differences between the two compared with that in the initial wastewater. A common reason may be pesticides could be ascribed to their different hydrophobicities accord­ that the spiked pesticides were not completely addressed. This result ing to the Pesticide Properties Database (https://sitem.herts.ac.uk/aer could also be explained by the elution of wood particles from the packed u/ppdb/en/index.htm). However, regardless, the presence of lignocel­ bed and/or wood rotting by T. versicolor in the TBR, whereas the lulosic carriers enhances and maintains the capability of reactors to contribution was from the addition of antifoam (Tween 80) in the FBR. eliminate target pesticides from water bodies, which is consistent with Fortunately, foam formation was not a concern for the trickle-bed the results of a more recently published study where a great extent of reactor treatment since it is somewhat an open-scale system. pharmaceuticals (73.3%) were adsorbed by the bed that was mainly Regardless, future efforts need to focus economic costs while moving composed of rice husks (Tormo-Budowski et al., 2021). This evidence to a scaling-up process. Apart from preparing mycelial suspensions, further indicates that these organic packing materials play a vital role in there is an extra process before starting fluidized-bed reactor treatments wastewater treatment using a TBR. Additionally, it is worth noting that a compared to starting trickle-bed reactors, that is, pellet formation. decreasing trend in the removal yield was observed in both reactors, Although a low-cost procedure using a defined medium instead of a malt probably ascribed to the fungal ageing behaviour and biomass washing extract to culture fungi was developed to obtain pellets and can satisfy a out. 5 K. Hu et al. Journal of Environmental Management 293 (2021) 112859 Table 3 Declaration of competing interest Advantages and limitations of the two reactor configurations for pesticides contaminated wastewater treatment in sequencing batch mode under non-sterile The authors declare that they have no known competing financial conditions. interests or personal relationships that could have appeared to influence Reactor Advantages Limitations the work reported in this paper. type Trickle-bed ● Simple construction and great ● Short contact time Acknowledgement operational flexibility ● Low mass transfer ● Retain biomass in the reactor ● Poor oxygen supply This work has been supported by the Spanish Ministry of Economy ● Hinder bacterial growth Risk of clogging in long and Competitiveness State Research Agency (CTM2016-75587-C2-1-R) ● ● Low-cost and high term operation sustainability ● Lack of homogeneity and co-financed by the European Union through the European Regional ● No nutrients required ● High pumping energy Development Fund (ERDF). This work was partly supported by the ● Nature conditions emulated Generalitat de Catalunya (Consolidate Research Group 2017-SGR- Fluidized- ● High homogeneity ● Required pellet preparation 0014). K. Hu acknowledges the financial support from Chinese Schol­ bed ● Adequate cellular retention process time ● Continuous nutrient supply arship Council. ● Efficient mass transfer ● Higher bacterial competition Appendix A. Supplementary data ● Foam formation ● Pellet break-up or Supplementary data to this article can be found online at https://doi. aggregation ● Complicated separation of org/10.1016/j.jenvman.2021.112859. biomass ● Biomass renovation for References long term operation Arora, D.S., Gill, P.K., 2000. Laccase production by some white rot fungi under different nutritional conditions. Bioresour. Technol. 73, 283–285. large demand (Borràs et al., 2008), other raw materials still bring cost Azubuike, C.C., Chikere, C.B., Okpokwasili, G.C., 2016. 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Improved of heating energy resulting from sterilization during biomass prepara­ fully automated method for the determination of medium to highly polar pesticides tion is reasonably higher than the mechanical energy that was necessary in surface and groundwater and application in two distinct agriculture-impacted for recirculation with a pump. In addition, other problems related to areas. Sci. Total Environ. 745, 140650. Barbieri, M.V., Peris, A., Postigo, C., Moya-Garcés, A., Monllor-Alcaraz, L.S., Rambla- pellet break-up appeared during operation, resulting in biomass loss Alegre, M., Eljarrat, E., López de Alda, M., 2021. Evaluation of the occurrence and because of adherence to the bioreactor wall and difficulties in separating fate of pesticides in a typical Mediterranean delta ecosystem (EBRO RIVER delta) biomass from the liquid phase during emptying. In contrast, the TBR is a and risk assessment for aquatic organisms. Environ. Pollut. 115813. relatively more robust configuration. 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