Journal of Water Process Engineering 55 (2023) 104105 Contents lists available at ScienceDirect Journal of Water Process Engineering journal homepage: www.elsevier.com/locate/jwpe Effect of dissolved oxygen on the degradation activity and consumption capacity of white-rot fungi Eduardo Beltrán-Flores, Shamim Tayar, Paqui Blánquez *, Montserrat Sarrà Departament d'Enginyeria Química Biològica i Ambiental, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: In recent decades, bioremediation using white rot fungi (WRF) has become an attractive alternative for the Pesticide removal of xenobiotics from water. However, WRF are aerobic microorganisms whose degradative capacity may Flame retardant be reduced when operating in oxygen-restricted environments. This work determines the limiting dissolved Oxygen limitation oxygen level of Trametes versicolor in terms of degradation of two target micropollutants: bentazon and tributyl Chemical oxygen demand removal White rot fungi phosphate. When the dissolved oxygen concentration was set below 15 % saturation (1.3 mg O2⋅L− 1), the results Bioremediation revealed a considerable decrease in degradation capacity and laccase synthesis. Hence, 15 % dissolved oxygen was established as a reference value of aerobic conditions to assess dissolved oxygen profiles in both a rotating drum bioreactor (RDB) and a fixed-bed bioreactor (FBB). Restrictive oxygen conditions were achieved after an operating period of 24 h in the RDB and an empty bed contact time of 44 min in the FBB. In addition, growth kinetics on Q. ilex wood and organic matter removal (in terms of COD) were studied, obtaining 0.059 mg ergosterol⋅g wood DW− 1⋅day− 1 and 16.23 mg O2⋅L− 1⋅h− 1, respectively. Therefore, T. versicolor has demonstrated a remarkable ability to assimilate complex carbon sources and a high micropollutant degradation activity, especially when operating in non-limiting oxygen regimes. 1. Introduction difficulties related to biomass maintenance, presents increasing evi­ dence of its potential application in micropollutant abatement. Partic­ Micropollutants in the aquatic environment have become a world­ ularly, white rot fungi (WRF) are basidiomycetes known for their ability wide issue of rising environmental concern. These compounds constitute to degrade a wide range of micropollutants through their powerful a diverse and expanding group of anthropogenic and natural pollutants enzyme system, which is composed of extracellular enzymes, such as that include pharmaceuticals, personal care products, steroid hormones, laccase, and the intracellular enzyme system known as cytochrome P450 industrial chemicals, pesticides and flame retardants, among others [1]. [5]. Among WRF, T. versicolor is considered one of the best candidates Even though these xenobiotics are generally detected at low concen­ because of its excellent degradation efficiency for many xenobiotics [6]. trations, many of them raise significant toxicological concerns in both Like other WRF, T. versicolor is an aerobic fungus whose growth and wildlife and human beings [2,3]. Therefore, remediation techniques metabolic activity have traditionally been considered to be affected by must be developed and applied to remove these toxic compounds from the dissolved oxygen (DO) concentration in the medium [7,8]. However, the environment. some investigations have shown that this fungus can operate under The most established micropollutant treatments are physical- anaerobic conditions, e.g. in the conversion of xylose to ethanol [9], or chemical, as they are considered relatively fast, simple and effective even degrade recalcitrant compounds such as industrial dyes [10]. Even methods against micropollutants. However, these techniques have though T. versicolor seems to degrade micropollutants to some extent important limitations, such as the formation of residues in sorption, under oxygen restricted conditions, this fungus may be oxygen-limited fouling in membrane technology, and high operational costs and the and hence its degradative activity may be reduced. In fact, a common generation of transformation products in advanced oxidation processes practice in the field of fungal bioremediation is to overlook the DO level [4]. In contrast, bioremediation is considered a sustainable and envi­ as long as the fungus exhibits some degradation activity. In any case, no ronmentally friendly technology that, despite possible technical study has quantitatively determined the lower level at which a WRF (e. * Corresponding author. E-mail address: paqui.blanquez@uab.cat (P. Blánquez). https://doi.org/10.1016/j.jwpe.2023.104105 Received 7 May 2023; Received in revised form 20 July 2023; Accepted 26 July 2023 Available online 2 August 2023 2214-7144/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 g., T. versicolor) is oxygen-limited for pollutant degradation. DO content Sigma Aldrich (Barcelona, Spain). High-performance liquid chroma­ is an essential parameter and must be properly monitored, otherwise tography (HPLC) grade acetonitrile, methanol and formic acid were oxygen may be under-supplied, reducing treatment performance, or supplied by Merck (Darmstadt, Germany). D(+)-Glucose was supplied over-supplied, increasing operating costs. by Acros Organics (New Jersey, USA). Ammonium tartrate was acquired Another key parameter in wastewater treatment is chemical oxygen from Scharlau (Barcelona, Spain). All chemicals were of high purity demand (COD). WRF generally degrade recalcitrant micropollutants via grade. co-metabolism, thus requiring an easily assimilated carbon supply for maintenance. In addition, micropollutant concentrations are too low to support metabolic requirements [11]. Nevertheless, some WRF, such as 2.3. Fungal strain and culture conditions Pleurotus ostreatus and Phanerochaete chrysosporium, have been reported to successfully assimilate intrinsic COD from high organic load waste­ T. versicolor ATCC 42530 was purchased from the American Type waters without the addition of supplementary nutrients [12,13]. In Culture Collection. T. versicolor was maintained by subculturing on agar contrast, T. versicolor has shown a doubtful ability to reduce COD plates of malt extract (2 % w/v) every 30 days at 25 ◦ C. T. versicolor [14,15], although there is still scarce evidence and many information mycelial suspension and pellets were prepared as previously described gaps to investigate. elsewhere [22]. T. versicolor immobilized on Q. ilex wood chips was Therefore, the aim of this study was to determine the DO concen­ prepared as described by Beltrán-Flores et al. [23]. tration at which T. versicolor was oxygen-limited in terms of pollutant degradation, where bentazon and tributyl phosphate (TBP) were used as target compounds. Two different types of chemicals, a pesticide (ben­ 2.4. Bioreactor set-ups and procedures tazon) and a flame retardant compound (TBP), were chosen to cross- sectionally study the effect of DO on the degradation of micro­ 2.4.1. Stirred tank bioreactor: biodegradation pollutants. Bentazon was chosen as the target pesticide because it has Maintenance medium used in the bioreactor was prepared (per litre) recently been detected in surface waters of the Ebro River Delta, is with 3 g glucose, 21 mg ammonium tartrate, 10 mL micronutrients and rapidly degraded by T. versicolor and has a low sorption propensity 100 mL macronutrients, as described in previous works [24]. The me­ [16–18]. Besides, bentazon has been associated with potential health dium was sterilized at 120 ◦ C for 30 min. Glucose and ammonium problems and is considered of particular concern according to the tartrate were supplied at the consumption rate, approximately 1 Directive 2008/105/EC on Environmental Quality Standards of the EC g⋅d− 1⋅L− 1 and 7 mg⋅d− 1⋅L− 1, respectively. The glucose and ammonium [19]. Meanwhile, TBP has been shown to pose toxicological risks in tartrate stock solutions were 0.3 g⋅mL− 1 and 3.2 mg⋅mL− 1, respectively. several organisms [20,21]. The limiting DO level was used as a reference Experiments were performed in a stirred tank bioreactor Applikon to assess whether limiting conditions were reached in two well- model ez-Control (Applikon Biotechnology, Netherland) of 2 L established fungal bioreactors, an FBB and an RDB. Thus, this study maximum useful volume, with 1.5 L operating volume. The pH was set allows to determine the aeration requirements in common fungal bio­ to 4.5 and controlled using 1 M HCl and NaOH. The temperature was reactors, a key information gap to date. In addition, this study analyses fixed at 25 ◦ C. DO levels were set at 5, 10, 15 and 30 %, depending on the T. versicolor kinetic growth and capacity to remove organic matter (in experiment, so that the aeration rate was automatically adjusted to terms of COD) under sterile conditions. maintain these percentages, keeping a constant agitation of 200 rpm. Experiments were conducted at different DO levels in order to evaluate 2. Materials and methods the effect of oxygen on the fungus degradation ability. The DO set-point was fixed between 5 and 30 % saturation. This operational range was 2.1. Agricultural wastewaters established based on the fact that most wastewater treatment plants (WWTP) work above 25 % saturation, which is considered as non‑oxy­ Agricultural wastewater (AW) was collected from an agricultural gen limited region [25]. Hence, a saturation of 30 % DO was initially drainage channel in the Llobregat River Basin in Gavà in December 2020 selected to work under non‑oxygen limiting conditions, at which and stored in the fridge at 4 ◦ C until use. Table 1 summarizes the AW T. versicolor was supposed to maintain its intrinsic degradation capacity physicochemical parameters. intact, while lower DO levels of 15 %, 10 % and 5 % were chosen to assess the effect of oxygen limitation on degradation activity. A total of 80 g L− 1 wet weight of pellets (equivalent to 3.5 g DW⋅L− 1) 2.2. Chemical and materials were inoculated to the reactor. In each cycle, a pulse of 10 ppm bentazon or 5 ppm TBP from their respective stock solutions (10 mg⋅mL− 1 in Bentazon, TBP and sodium acetate anhydrous were purchased from methanol) was injected into the bioreactor every 24 or 48 h, respec­ tively. A total of 3 cycles were performed for each DO level, which Table 1 means a total injection of 30 ppm bentazon and 15 ppm TBP. Separate Physicochemical characterization of the agricultural wastewater. Values are experiments were conducted for each DO level, i.e., with fresh medium means ± standard deviation for triplicate samples. and biomass. Samples of 2 mL withdrawn from the bioreactor were Parameter Agricultural wastewater (Llobregat River centrifuged at 15.000g during 1 min to separate the supernatant from Basin, 2020) the pellets. pH 8.06 ± 0.02 Conductivity (mS⋅cm− 1) 3.17 ± 0.01 Colour at 650 nm 0.10 ± 0.011 2.4.2. Stirred tank bioreactor: OUR estimation Chloride (mg Cl⋅L− 1) 653.7 ± 6.1 The reactor was set up as described in Section 2.4.1. In this case, 1 L Sulphate (mg S⋅L− 1) 86.3 ± 1.6 of maintenance medium (without micropollutants) was saturated above Nitrite (mg N⋅L− 1) 0.4 ± 0.1 50 % DO, when aeration was stopped. The specific oxygen uptake rate Nitrate (mg N⋅L− 1) 0 (sOUR) measures the rate at which oxygen is consumed by a microor­ Ammonia (mg N⋅L− 1) 0 TSS (mg⋅L− 1) 16 ± 2 ganism/consortium and is defined as the mass of oxygen consumed per COD (mg O2⋅L− 1) 44 ± 4 unit of fungal biomass and time (g O2⋅g biomassDW− 1⋅day− 1). The sOUR TOC (mg⋅L− 1) 16 ± 1 was obtained by dividing the value of the slope of the linear regression of Heterotrophic plate count 3.50⋅104 ± 1.50⋅104 the DO concentrations over time by the biomass amount. The operating (CFU⋅mL− 1) conditions were the same as in Section 2.4.1. 2 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 2.4.3. Stirred tank bioreactor: organic matter removal using an OXROB10 robust oxygen probe coupled to a FireSting-PRO (4 Regarding the study of the organic matter removal, the consumption channels) fiber-optical multi-analyte meter (Pyroscience, Germany). rate of glucose was compared with that of another less assimilable The reactor was fed with an upward stream of AW saturated at 100 % source of COD, sodium acetate. For this purpose, 1 L of maintenance DO at flow rates of 12, 23 and 40 mL⋅min− 1, equivalent to empty bed medium was treated but replacing glucose with an equivalent amount of contact times (EBCT) of 59, 31 and 18 min, respectively. The pH of the sodium acetate in terms of COD (2 g O2⋅L− 1). Thus, a pulse of acetate was AW was previously adjusted to 4.5. In this case, the sOUR was calculated added in the medium from a stock solution of 0.2 g⋅mL− 1. Once the for each reactor section according to Eqs. (1) and (2). acetate was almost consumed, a new acetate pulse was injected, [DOE − DOS ]⋅Q obtaining two complete cycles. In addition, a control experiment with sOUR = (1) Mbiomass ⋅1000 glucose was conducted. In these cases, the DO level was maintained at 30 % and the medium was not spiked with any micropollutant. Samples Xergo of 2 mL withdrawn from the bioreactor were centrifuged at 15.000g for Mbiomass = ⋅Mwood (2) RE− B 1 min to separate the supernatant from the pellets. The resulting su­ pernatant was used to measure glucose or acetate. The operating con­ where DOE and DOS are the DO concentrations at the inlet and outlet of ditions were the same as in Section 2.4.1. each section (mg O2⋅L− 1), respectively, Q the flow rate (L day− 1), Mbiomass the fungal biomass of each section (g DW), Xergo the ergosterol 2.4.4. Fixed-bed bioreactor content in the wood (mg ergosterol⋅g wood DW− 1), RE-B is the correla­ The FBR consisted of a cylindrical methacrylate tube (4.4 cm diam­ tion between ergosterol mass and fungal biomass of T. versicolor and eter and 62 cm height) assembled with 250 g DW of inoculated wood corresponds to 6.61 (mg ergosterol⋅g biomass DW− 1) [26], and Mwood (Fig. 1). DO measurements were performed by inserting the oxygen the wood mass in each section (g). probe through a series of holes, plugged with septums, which were made in the column. Thus, the DO was measured at 0, 7 (P-1), 19 (P-2), 34 (P- 2.4.5. Rotating drum bioreactor 3) and 49 (P-4) cm height (bed volume = 0.74 L, useful volume = 0.45 L) The RDB was constructed with a methacrylate tube (length: 45.0 cm Fig. 1. Representation of the FBB and DO measurement points. 3 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 × radius: 4.2 cm) supported on a polyvinylchloride gutter (length: 51.0 USA). The column temperature was 30 ◦ C, injection volume was 2 μL cm × radius: 7.0 cm). The wastewater was contained within the channel and the mobile phase consisted of 75 % methanol and 25 % ultrapure while the colonized wood chips were placed inside the tube. The tube water (v/v), which was pumped isocratically at 0.25 mL⋅min− 1 (total was provided with multiple 8 mm diameter holes to ensure adequate runtime: 14 min). The mass spectrometer was operated in the selected- contact between the biomass and the wastewater and it was covered ion monitoring (SIM) positive mode electrospray ionization (ESI+) and with a plastic mesh to prevent biomass loss. Approximately 30 % of the was set up to monitor the ions m/z 267.10 Da. Data in the tuning file biomass was submerged in the liquid phase, while the rest was in direct were selected for parameters which contains the lens voltage values, contact with air. The inner tube was connected to an electric motor Interface, Q-array and others. High purity nitrogen was used as nebu­ (Worm Gear Motor, model: 4632-370, 12 V) that rotates to alternate the lizing gas at 1.50 L/min and drying gas pressure was zero. The detection submerged biomass fraction. The working volume was approximately voltage was set at 1.5 kV. The other operation parameters for MS were 2.3 L. No additional substrate or nutrient was added to the reactor. optimized as follows: capillary voltage: 4.50 kV; heat block temperature: DO concentrations were analysed in a RDB that had been previously 200 ◦ C; CDL temperature: 250 ◦ C. used for AW treatment [27,28]. The feed tank was stirred during the entire operation, from which water (100 % DO) was pumped to one side 2.6. Acetate measurement of the reactor (HRT = 5 days). The water left the reactor from the other side by overflow. An external recirculation loop (4.7 L⋅day− 1) was The samples were filtered prior measurement with 0.22 μm pore-size required for pH adjustment, which was performed in a recirculation tank Millipore Millex-GV PVDF filters and, when required, diluted with ul­ (≈0.4 L). The pH was automatically controlled at 4.5 by adding either 1 trapure water to have concentration between the calibration ranges. M HCl or NaOH. A total of 545 g DW of inoculated wood was introduced Acetate was measured by HPLC (Ultimate 3000, Dionex Integrion HPLC, into the inner tube, which was rotated one and a half turns every 24 h. USA) calibrated to measure volatile fatty acids (VFA) in the range of 20 Aeration was initially provided through a diffuser located in the reactor to 1000 mg⋅L− 1 (acetate, propionate, formate, isobutyrate, valeric, until reaching oxygen saturation. Afterwards, aeration was turned off, butyrate and isovaleric). The separation was performed in an ICSep ICE- and the dynamic profile of DO uptake was monitored until equilibrium, CPREGEL 87H3 column (height: 150 mm, diameter: 7.8 mm, Trans­ which was reached after 30 h of operation. The oxygen level was genomic, Omaha, NE, USA) at 30 ◦ C working with a sulphuric acid (6 measured at 3 different points of the reactor: inlet, intermediate, and mM H2SO4) as mobile phase pumped at a constant flow rate of 0.5 outlet; and at two depths: 4 and 6 cm (reactor dimensions are length: 51 mL⋅min− 1. Total run time was 25 min. The HPLC was equipped with a cm × radius 7 cm), as shown in Fig. 2. refractive index detector (Waters 2410) with an IR of 1024 and an autosampler (Ultimate 3000 Autosampler). Under these conditions, the 2.5. Pesticide analysis in liquid phase retention time was approximately 18.31 min. Residual concentration of bentazon and TBP were analysed by LC, 2.7. Other analyses with a limit of detection of 0.5 ppm for both compounds. Liquid samples were initially filtered through Millipore Millex-GV PVDF filters (0.22 Laccase activity was determined through the absorbance changes μm). In the case of bentazon, the analysis was conducted by a Dionex induced by the oxidation of 2,6-dymetoxyphenol (DMP) and expressed Ultimate 3000 HPLC system with a UV detector operating at 254 nm. in activity units per litre (UA⋅L− 1), as described elsewhere [30]. Ergos­ The separation was performed in a C18 reversed-phase column (Phe­ terol content was used as an indicator to quantify fungal biomass and nomenex®, Kinetex® EVO C18 100, 5 μm, 4.6 mm 150 mm) with a was analysed by homogenizing and extracting inoculated wood in mobile phase of 0.01 % formic acid solution (v/v) (A) and acetonitrile triplicate based on a modified method previously described [31]. (B) at 30 ◦ C. The organic gradient used in chromatographic separation Briefly, 0.5 g of each homogenized sample was transferred to a test tube was: 35 % B from 0 min to 5 min, linear increase to 45 % B from 5 min to to be extracted with 1 mL cyclohexane and 3 mL KOH-methanol solution 15 min, restoration of initial conditions in 1 min and maintenance of (10 %, w/v) by 15 min sonication followed by heating at 70 ◦ C for 90 initial conditions for 2 min more (total runtime: 18 min) [16]. The min. After adding 1 mL of distilled water and 2 mL of cyclohexane, the flowrate was 0.9 mL⋅min− 1, and the sample injection volume was 40 μL. tube was vortexed for 30 s and then centrifuged at 3.500 rpm for 5 min. TBP chromatography study was adapted from a method previously The upper organic phase was recovered while the aqueous phase was reported in the literature [29]. This analysis was performed using a washed twice with 2 mL cyclohexane. The organic phases were pooled Prominence UFLC that was controlled by an LCMSsolution Chroma­ and evaporated to dryness with nitrogen. The residue was reconstituted tography Data System software, consisting of a SIL/20A autosampler in 1 mL methanol at 40 ◦ C for 15 min, vortexed for 30 s, transferred to and LC-20AD solvent-delivery system and equipped with a LCMS-2010A Eppendorf vials and centrifuged at 6000 rpm for 3 min. The resulting detector, all of them from Shimadzu (Japan). Chromatographic sepa­ solution was transferred to 2 mL amber vial for quantification by HPLC ration was achieved by an Acclaim Organic Acid (OA) column from (Ultimate 3000, Dionex, USA) equipped with a UV detector at 282 nm, Dionex, OA 5 μm, 120 Å, 4.0 × 150 mm (Thermo Scientific, Waltham, using a C18 reverse phase column (Phenomenex®, Kinetex® EVO C18 Fig. 2. Representation of the RDB and DO measurement points. 4 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 100 Å, 4.6 mm × 150 mm, 5 μm). Acetonitrile was isocratically supplied Supplementary Material. According to these results, the fungus was at 1 mL⋅min− 1 as eluent, obtaining a retention time of approximately found to be in an oxygen-limiting region when the DO was decreased 7.6 min. The injection volume was 40 μL and the oven temperature was below 15 %, i.e., 10 % and 5 %. In this region, the degradation capacity 35 ◦ C. was dramatically reduced by up to 84 %. In contrast, it cannot be clearly Conductivity was determined using a CRISON MicroCM 2100 con­ concluded whether the fungus is oxygen-limited when the DO was ductometer. An UNICAM 8625 UV/VIS was used to detect colour as restricted to 15 % since the biodegradation rate was only moderately absorbance at a wavelength of 650 nm. The commercial kits LCK114 or reduced for bentazon and remained high for TBP. At 30 % DO, LCK314m and LCH303 were used to quantify COD and ammonia con­ T. versicolor was even able to completely remove bentazon at 10 ppm centrations respectively (Hach Lange, Germany). Total suspended solids after 6 h and TBP at 5 ppm after 32 h. Note that the degradation profiles (TSSs) and volatile suspended solids (VSSs) were determined using changed slightly between cycles, which was mainly attributed to the standard procedures 2540 D and 2540 E respectively, and HPC was accumulation of metabolites and extracellular enzymes. This variation calculated according to standard method 9215 [32]. Chloride, nitrite could also be associated with changes in the morphology of the fungal and sulphate concentrations were quantified by ion chromatography pellets, although the stirring speed was relatively low (200 rpm) and using a Dionex ICS-2000. Glucose concentration was measured with a apparently no morphological alteration was observed on a macroscopic YSI model 2700 biochemical analyser (Yellow Spring Instruments & Co., scale. USA) after filtering the sample through a 0.22 μm Millipore Millex-GV The results of the degradation kinetics of bentazon are in line with PVDF filter. those reported previously [16], in which bentazon was eliminated in less than 3 days. Regarding TBP, no data have been found on its degradation 3. Results and discussion by fungi, but some bacteria such as Sphingomonas sp. have been able to degrade TBP with similar kinetics [35]. 3.1. Stirred tank bioreactor Furthermore, laccase activity was analysed as an indicator of fungal activity. This extracellular enzyme was only monitored for TBP, since a 3.1.1. Effect of dissolved oxygen on the biodegradation of two model recent study has shown that, although laccase is slightly involved in micropollutants bentazon degradation, the main mechanism of degradation is via cyto­ The relative remaining concentrations of the pollutants are shown in chrome P450 [16]. Fig. 4 shows the evolution of laccase activity for each Fig. 3. T. versicolor was only able to completely eliminate both micro­ DO value. Enzyme activity reached maximum values at 30 % DO, while pollutants in the first cycle studied when working at 30 % DO. This result it was substantially limited as DO decreased, especially below 15 %. indicates that the maximum performance of the fungus was accom­ These results show that the enzymatic activity of T. versicolor, and thus plished at the highest DO level. However, the degradation efficiencies its degradation potential, is inhibited when working near anoxic con­ obtained when working at 30 % and 15 % DO were clearly better than ditions. In this regard, Pinheiro et al. (2020) evaluated laccase produc­ those achieved under more restricted oxygen conditions in all cycles. In tion by T. versicolor in three different reactors, detecting higher activity this regard, Leisola (1983) and Li (2016) demonstrated that the presence values when aeration was incorporated into the system [36]. Therefore, of oxygen was a critical factor in the removal of lignin and carbamaz­ laccase production was found to be correlated to DO concentration epine respectively by the WRF P. chrysosporium [33,34]. Nevertheless, during the process, which is in good agreement with the results of the T. versicolor was able to partially degrade both compounds, bentazon present study. Additionally, a higher DO concentration may not only and TBP, even at a very low DO level of 5 %, which is in line with other lead to an increase in laccase production by T. versicolor, but could also research, in which the fungus remained metabolically active in anaer­ enhance TBP degradation efficiency, as oxygen is an essential electron obic environments [10], e.g., being able to bioconvert xylose to ethanol acceptor in certain laccase-mediated oxidation reactions [37]. without oxygen [9]. Bettin et al. (2020) also reported maximum laccase activity working This finding was further analysed by studying the biodegradation at 30 % DO for another WRF, Pleurotus sajor-caju PS-2001 [38]. When kinetic constants of bentazon and TBP, as shown in Table 2. The equa­ the DO concentration was higher than 30 % DO, i.e., more favourable for tions used for the determination of the kinetic constants are shown in the the fungus, instead of promoting laccase production, it stimulated Fig. 3. (A) Bentazon concentration profiles for three batches of 24 h and (B) TBP concentration profiles for three batches of 48 h at different DO levels. Initial concentrations are 10 ppm bentazon and 5 ppm TBP. 5 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 Table 2 First-order kinetic constants of bentazon and TBP biodegradation at four different DO levels (30, 15, 10 and 5 %) and three cycles. Average kinetic constants and degradation decline were calculated from the regression of the point cloud of all cycles. R2 is the correlation coefficient of the first-order kinetics. k (h− 1) Bentazon TBP DO (%) 30 15 10 5 30 15 10 5 Cycle 1 0.287 0.119 0.086 0.095 0.125 0.099 0.026 0.010 2 0.397 0.355 0.296 0.086 0.077 0.106 0.019 0.020 3 0.348 0.397 0.126 0.144 0.082 0.051 0.027 0.017 Average (point 0.335 ± 0.055 0.288 ± 0.150 0.164 ± 0.112 0.109 ± 0.031 0.096 ± 0.026 0.096 ± 0.030 0.025 ± 0.004 0.015 ± 0.005 cloud) (R2 = 0.914) (R2 = 0.789) (R2 = 0.767) (R2 = 0.798) (R2 = 0.753) (R2 = 0.847) (R2 = 0.898) (R2 = 0.790) Degradation 0 14.0 51.04 67.5 0 0 74.0 84.4 decline (%) Fig. 4. Time course profile of laccase activity for TBP degradation at different dissolved oxygen concentrations. biomass growth. In the present study, biomass growth was limited by the the Supplementary materials). Considering that the oxygen saturation use of a maintenance medium (1 g⋅L− 1 of glucose) instead of a growth (100 % DO) is 8.30 mg O2⋅L− 1 (at 25 ◦ C) and that the inoculated biomass medium (8 g ⋅L− 1 of glucose), remaining in all cases below 4.5 g DW⋅L− 1 was 3.50 g DW⋅L− 1 (at 25 ◦ C), a sOUR of 0.10 g O2⋅g biomass of fungal biomass at the end of the experiments (initial biomass con­ DW− 1⋅day− 1 was obtained. This consumption was predictably lower centration was 3.50 ± 0.40 g DW⋅L− 1). than that (1.39–2.05 g O2⋅g− 1⋅day− 1) obtained by Thiruchelvam and Therefore, the DO level was found to be a critical variable to control Ramsay (2007) since in the latter case a growth medium was used and, according to these results, at least 15 % DO is desirable to remain instead of a maintenance medium [40]. the maximum degradative potential of T. versicolor. Aeration represents the largest contribution to the costs of most WWTPs cost [39], thus 3.1.3. Organic matter removal reducing the oxygen content from 30 to 25 % (typically used in WWTP) WRF degrade micropollutants mainly by co-metabolism, as these to 15 % can result in large energy savings. This is the first study that fungi require a more abundant carbon source for their maintenance quantitatively determine the DO level at which T. versicolor, as an [41]. Carbon sources that have been traditionally used in experimental example of WRF, is oxygen-limited in a bioremediation process. studies as substrate for WRF include glucose, which is an easily assim­ ilable nutrient [42], straw and wood, which are natural substrates for 3.1.2. Specific oxygen uptake ratio (OUR) estimation these fungi [23]. These substrates are generally required because the A fundamental parameter for the design of any aerobic bioprocess is intrinsic organic matter in the water usually consists of complex com­ the sOUR. The sOUR can be estimated from the oxygen uptake of the pounds that are difficult to be assimilated by WRF. In fact, COD reduc­ fungus in a specific period. This study requires working under tion from wastewater by WRF is inconsistent and scarcely reported non‑oxygen limiting conditions. In this case, the DO level was set at [43,44]. In this section, the ability of T. versicolor to reduce COD under approximately 52 %. From the slope of its linear regression, the oxygen sterile and non‑oxygen-limited conditions was evaluated. For this pur­ consumption was found to be 3.00 % min− 1 approximately (Fig. S.1 of pose, acetate (sodium acetate) was added to the medium as a less 6 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 assimilable carbon source for T. versicolor, which is commonly used in progressively decreased throughout this last cycle to 1480 mg O2⋅L− 1 synthetic waters to simulate COD [45]. In this case, there are no pesti­ (52 % removal). These results contrast with other studies that have re­ cides nor any other organic compound in the medium and DO was set at ported the inability of T. versicolor to reduce COD. In this regard, Badia- 30 % to operate in the non‑oxygen limiting region, preventing these Fabregat et al. (2017) studied several fungal treatments under non- factors from influencing acetate consumption. sterile conditions in continuous operational mode with veterinary hos­ Fig. 5 shows the results of two degradation experiments using either pital wastewater and reverse osmosis concentrate with COD concen­ acetate and glucose (control) as COD sources. As expected, T. versicolor trations of 245–264 mg O2⋅L− 1 and 65 mg O2⋅L− 1 respectively [15]. In was able to completely consume glucose in a short period, obtaining a both cases, significant increases in COD concentration were reported, decreasing trend that was adjusted to a straight line (R ≈ 1.00) whose which were attributed to the possible production of metabolites. Anas­ slope was 83.28 mg O2⋅L− 1⋅h− 1. For 3.5 g pellets DW⋅L− 1, the glucose tasi et al. (2012) examined the treatment of textile wastewater by consumption rate was 15.86 mg O2⋅L− 1⋅g pellets DW− 1⋅h− 1. Interest­ different fungal strains and concluded that fungal treatment was more ingly, the fungus was also able to consume an equivalent amount of effective in decolourisation than in COD reduction [48]. In particular, acetate in terms of COD, although over a considerably longer period T. versicolor was the worst performing strain in terms of COD reduction, (approximately 130 versus 26 h). Once consumed, a second acetate considerably raising the initial level. Hu et al. (2021) also reported a pulse was added to verify whether the fungus was able to maintain its considerable increase in COD concentration during the treatment of metabolic activity over time in an additional cycle. In the second pulse, agricultural water by T. versicolor in a fluidized bed reactor [49]. In this T. versicolor was also able to almost completely degrade acetate for the case, glucose was used as substrate, thus the COD increase was attrib­ same period of time (130 h). The decreasing acetate trends of both cycles uted to the addition of antifoam (Tween 80), which could not be were fitted to straight lines, giving a slope of 16.23 mg O2⋅L− 1⋅h− 1 (R = consumed by T. versicolor. By comparing the results of the reported 0.96) for the first pulse, and 15.91 mg O2⋅L− 1⋅h− 1 (R = 0,90) for the studies with those obtained in the present work, it can be deduced that second pulse. For 3.5 g pellets DW⋅L− 1, the acetate consumption rates although the fungus is capable of consuming acetate, hence reducing were 3.09 and 3.03 mg O2⋅L− 1⋅g pellets DW− 1⋅h− 1 for the first and COD, the efficiency of the treatment can depend on other factors, such as second cycle, respectively. Therefore, the consumption rate of acetate the characteristics of the organic content, the matrix, the operating was approximately 5 times lower than that of glucose, which was conditions and the reactor type. attributed to an energetically less efficient assimilation of the former compound by this fungus [46,47]. Although acetate was found to be a 3.2. DO study in a fixed-bed bioreactor carbon source not as readily biodegradable as glucose by T. versicolor, the fungus was able to consume acetate for 264 h (11 days). The wood was inoculated with T. versicolor in a box under sterile In addition, soluble COD analyses were performed at the beginning conditions, reaching 0.395 ± 0.034 mg⋅g DW− 1 of ergosterol content and end of each cycle to ascertain whether the acetate was completely after 50 days of culture (Fig. S2 of the Supplementary materials). Fig. 6 mineralized or whether some transformation products (TPs) were shows the DO evolution over time in the FBB working in continuous formed. In the first cycle, soluble COD was reduced from approximately mode for different flow rates. The results showed that the DO con­ 2000 to 892 mg O2⋅L− 1. This result indicates that although T. versicolor sumption rate of the system varied over time until reaching the equi­ was able to completely remove acetate, some TPs that contribute to COD librium (maximum). This transition period was attributed to the were probably generated. Anyway, T. versicolor was found to be able to interaction between water and residual air during the column filling reduce COD by 55 % at the end of the first cycle. After spiking the stage. This evolution was hardly observed when working at a flow rate of medium with approximately 2 g⋅L− 1 of acetate at the beginning of the 40 mL⋅min− 1 (Fig. 6 (C)) as the column was rapidly filled up. second cycle, the soluble COD increased to 3072 mg O2⋅L− 1, which Fig. 7 shows two graphs that allow a better interpretation of the Fig. 5. Acetate (black circle) and glucose (blue circle) consumption profiles in terms of equivalent COD level over time. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 7 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 Fig. 6. Dissolved oxygen depletion profiles in the FBB at flow rates of 12 (a), 23 (b) and 40 (c) mL⋅min− 1. The reactor measurement points were located at 7 (P-1), 19 (P-2), 34 (P-3) and 49 (P-4) cm heights. sOUR (g O2·g biomass DW ·day ) -1 -1 Outgoing DO (%) Fig. 7. Correlations between outgoing dissolved oxygen and EBCT (a), and between sOUR and incoming dissolved oxygen. Dashed lines are linear (a) and expo­ nential (b) regressions. 8 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 results in the steady-state. In this case, the steady-state results in the FBB worked with discontinuous rotation showed better biomass mainte­ have been unified to form point cloud plots that allow a better corre­ nance when the rotation frequency was changed from 1.5 turns every 4 h lation between variables. Fig. 7 (A) shows that there is a strong corre­ to every 24 h [28]. Although operating under more static conditions lation (R2 = 0.73) between the EBCT and the DO level in the reactor. The could favour biomass immobilization on wood and thus increased higher the EBCT, the longer period the water remained inside the reactor treatment yield, the lack of agitation may cause the submerged biomass and thus, the more DO was assimilated by T. versicolor. This correlation to be oxygen limited. In this section, DO was measured at different indicates that an FBB with the characteristics described in the present points of the reactor (at the inlet, middle and outlet) to evaluate whether study operated under limiting DO concentrations (i.e., less than 15 % the fungus was under limiting conditions in the RDB (Fig. 8). DO, Section 3.1.1) from 43 min of EBCT onwards. These results show DO depletion profiles were fitted to first-order kinetics (Table 3). The that, even if the influent is fed at 100 % saturation, an FBB may require equations used for the determination of the kinetic constants are shown internal aeration in order to guarantee aerobic conditions for the fungus. in the Supplementary Material. The oxygen limiting conditions (15 %) The sOUR can be calculated from DO results, the level of ergosterol were reached at all studied points of the RDB before 30 h of operation. in the wood and the amount of wood in each reactor section [Eqs. (1) According to the kinetics, oxygen limiting conditions were reached for and (2)]. DO consumption was basically attributed to the activity of the first time in the RDB (intermediate point, 6 cm depth) after T. versicolor since the presence of other microorganisms was considered approximately 9.7 h; and at all studied points after 30 h. Furthermore, negligible. Different sOURs were obtained for each flow rate and reactor rotation performed after 24 h (1.5 turns) did not result in an appreciable section studied. As shown in Fig. 7 (B), an exponential correlation (R2 = increase in DO concentrations. These results indicate that if the RDB is 0.95) was found between the DO concentration and the sOURs of the operated under the same conditions for a period longer than 9.7 h system. Thus, the existence of different sOURs was mainly attributed to (conservative scenario) without additional aeration, the submerged the DO level measured in each reactor section. These results contrast fungal fraction can be oxygen limited. However, fungal biomass has with those obtained in the stirred-tank bioreactor (see Section 3.1.2). In proven to remain active and immobilized on wood in the RDB for a long the latter case, sOUR was considered constant in the non‑oxygen period of operation [28]. These results indicate that the alternation of limiting region. However, it should be noted that in the stirred-tank the submerged fraction may mitigate the effects of anoxic conditions and bioreactor there was a much better oxygen transfer than in the FBB. In contribute to maintaining the metabolic activity of the fungus. the FBB, low DO liquid interfaces, or even dead zones were probably Although DO concentrations were considerably reduced after 30 h in created around the fungus with diffusional and oxygen transfer limita­ the channel, it remained around 82 % in the recirculation tank. Other tions that, consequently, restricted oxygen availability to the fungus reported studies have also periodically monitored the DO concentration [50]. Therefore, delocalized oxygen supply along the column and/or in the recirculation tank of the RDB and it remained above 30 % working in more turbulent conditions should be considered to favour a throughout the treatment [23,52]. This higher DO level in the recircu­ better transfer and availability of oxygen to the fungus. lation tank compared to the channel may be due to aeration during liquid dripping in the recirculation tank, a larger liquid-air contact surface and stirring of the recirculation tank. Accordingly, measure­ 3.3. DO study in a rotating drum bioreactor ments were also conducted in the feed tank, obtaining an average value of 34.5 ± 4.1 % DO, but it is feasible to assume that the actual DO The RDB was inoculated with wood colonized by T. versicolor, which concentration at which the effluent entered the reactor was significantly was previously cultured for 24 days (0.24 mg ergosterol⋅g wood DW− 1). increased by dripping. The RDB is a reactor with a rotating mechanism that maintains aerobic DO concentrations varied as a function of depth and axial location, as conditions using two strategies: alternation of the submerged biomass demonstrated by fitting the data to a first-order kinetics of oxygen and aeration of the liquid medium by agitation. depletion (Table 3). The consumption curves were approximately fitted Consequently, high speeds and long periods of rotation promote the to first-order kinetics, from which the kinetic constants were calculated. aeration and thus the DO saturation in the system. In this regard, a DO decrease was faster at 6 cm than at 4 cm depth, probably due to constant and relatively high rotational speed (12 rpm) maintained high worse oxygen diffusion, and at the middle point than at the extremes of DO saturation (8.0–6.5 mg O2⋅L− 1) in a rotating biological contactor the RDB, which was attributed to a lower air-water contact surface. inoculated with T. versicolor immobilized on polypropylene discs [51]. These results indicate that the submerged biomass operates under In contrast, continuous rotation (6 rpm) has also been reported to pro­ oxygen limiting conditions from the first 30 h onwards (in some points duce fungus detachment from wood [52], which is probably attributed even earlier), and therefore, the incorporation of an additional aeration to the generation of shear forces that damage the mycelial structure or/ system in the RDB would be highly recommended to enhance the and to a better performance of the fungus in this substrate when working treatment performance. This improvement should be explored in future under static conditions. Regarding the latter, another recent study that Fig. 8. Dissolved oxygen depletion profiles measured at two depths (4 and 6 cm) at the inlet (A), intermediate zone (B) and outlet (C) of the RDB. Dashed lines are the curves described by the fitted first-order kinetics (T: theoretical). 9 E. Beltrán-Flores et al. Journal of Water Process Engineering 55 (2023) 104105 Table 3 Kinetics of dissolved oxygen consumption in the RDB. Inlet Intermediate Outlet Depth (cm) 4 6 4 6 4 6 Kinetic rate (h− 1) 6.09⋅10− 2 1.42⋅10− 1 7.93⋅10− 2 1.88⋅10− 1 7.41⋅10− 2 1.60⋅10− 1 R2 0.93 0.91 0.93 0.91 0.92 0.88 work on the RDB, along with the evaluation of operational costs, prior to [3] W.H. Leong, S.Y. Teh, M.M. Hossain, T. Nadarajaw, Z. Zabidi-Hussin, S.Y. Chin, K. S. Lai, S.H.E. Lim, Application, monitoring and adverse effects in pesticide use: the its eventual implementation at full-scale. importance of reinforcement of Good Agricultural Practices (GAPs), J. Environ. According to the results obtained in the stirred tank bioreactor, at Manag. 260 (2020) 1–12, https://doi.org/10.1016/j.jenvman.2019.109987. least 15 % DO is required to avoid limiting the degradative activity of [4] J. Benner, D.E. Helbling, H.P.E. Kohler, J. Wittebol, E. Kaiser, C. Prasse, T. T. versicolor. 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Based on this result, two well-studied re­ structural analysis of the polysaccharide secreted by Trametes (Coriolus) versicolor actors in fungal bioremediation, the fixed-bed and the rotating drum ATCC 200801, Appl. Microbiol. Biotechnol. 81 (2009) 827–837, https://doi.org/ 10.1007/s00253-008-1700-2. bioreactors, were found to be oxygen limited for an EBCT longer than 43 [9] K. Okamoto, A. Uchii, R. Kanawaku, H. Yanase, Bioconversion of xylose, hexoses min and an operational period longer than 9.7 h, respectively. In case and biomass to ethanol by a new isolate of the white rot basidiomycete Trametes these limits are exceeded, aeration should be added to ensure full aer­ versicolor, Springerplus 3 (2014) 1–9, https://doi.org/10.1186/2193-1801-3-121. [10] S.H. Chen, A.S. Yien Ting, Biodecolorization and biodegradation potential of obic conditions. 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Trametes versicolor immobilized on lignocellulosic support for the continuous treatments of hospital wastewater, Bioresour. Technol. 243 (2017) 966–974, Data availability https://doi.org/10.1016/j.biortech.2017.07.055. [15] M. Badia-Fabregat, D. Lucas, T. Tuomivirta, H. Fritze, T. Pennanen, S. Rodríguez- Mozaz, D. Barceló, G. Caminal, T. Vicent, Study of the effect of the bacterial and Data will be made available on request. fungal communities present in real wastewater effluents on the performance of fungal treatments, Sci. Total Environ. 579 (2017) 366–377, https://doi.org/ 10.1016/j.scitotenv.2016.11.088. Acknowledgements [16] M. García-Vara, K. Hu, C. Postigo, L. Olmo, G. Caminal, M. Sarrà, M. López de Alda, Remediation of bentazone contaminated water by Trametes versicolor: Project PID2019-103989RB-100 financed by MCIN/AEI/10.130 characterization, identification of transformation products, and implementation in a trickle-bed reactor under non-sterile conditions, J. Hazard. 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