Trametes versicolor immobilized on rotating biological contactors as alternative biological treatment for the removal of emerging concern micropollutants

Water Research 170 (2020) 115313

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Trametes versicolor immobilized on rotating biological contactors as alternative biological treatment for the removal of emerging concern micropollutants
A. Cruz del Alamo, M.I. Pariente, F. Martínez, R. Molina*
stoles, Madrid, Spain Department of Chemical and Environmental Technology. ESCET. Rey Juan Carlos University, Mo

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 August 2019 Received in revised form 12 November 2019 Accepted 14 November 2019 Available online 19 November 2019

White rot fungi have been studied for the removal of micropollutants of emerging concern from wastewater during the last decade. However, several issues need to be overcome for its plausible implementation at full-scale installations such as the addition of supplementary substrates, the partial re-inoculation of fresh fungi or the use of extended hydraulic retention times. This work proposes the immobilization of Trametes versicolor on rotating biological contactors at bench scale (flowrates of 10 L/ d and reactor capacity of 10 L) for the treatment of different urban wastewater. This type of bioreactor achieved remarkable reductions of the total organic carbon loading of the wastewater (70e75%) in a wide range of C:N and C:P ratios with limited addition of supplementary substrates, non-refreshment of the fungal biomass and only 1-day of hydraulic retention. The addition of gallic acid as quinone-like mediator and quelated iron and manganese complexes increased the removal of pharmaceutical micropollutants mediated by the so-called advanced bio-oxidation process. The immobilization of Trametes versicolor on rotating biological contactors also showed a remarkable stabilization of the fungi during the continuous treatment of different urban wastewater under non-sterile conditions. Thus, this system is a sound alternative for biological urban wastewater treatment with pharmaceutical removal because overcome all the problems usually associated with the water treatment technologies based on white rot fungi that makes difficult the scaling-up of the process and its implementation in full scale wastewater treatment plants. © 2019 Elsevier Ltd. All rights reserved.

Keywords: White rot fungi Rotating biological contactors Advanced bio-oxidation process Continuous operation Urban wastewater

1. Introduction Pharmaceutical Compounds (PhACs) are considered a group of emerging concern micropollutants for the environment and human health due to their capacity of bioaccumulation, toxicity and
and Desrosiers, 2014). Municipal effluents are persistence (Sauve one of the main sources of PhACs in aquatic environment due to the limited efficiency of wastewater treatment plants (WWTPs) to remove them (Verlicchi et al., 2013). Nowadays, the development of performing sustainable wastewater facilities within the removal of emerging concern micropollutants, including PhACs (Commission Implementing Decision (EU) 2018/840), has emerged as a critical duty in the environmental research field (Evgenidou et al., 2015). White rot fungi (WRF) are basidiomycetes that can degrade the

* Corresponding author. E-mail address: [email protected] (R. Molina). https://doi.org/10.1016/j.watres.2019.115313 0043-1354/© 2019 Elsevier Ltd. All rights reserved.

lignin component of complex lignocellulose substrates. WRF are such a robust organisms with a high tolerance to toxic environments and they withstand high temperatures in a wide range of pH (Moore et al., 2011). The ability of the lignin-degrading WRF is related to their unspecific oxidative enzymatic system with ligninmodifiers extra-cellular enzymes, especially oxidases and peroxidases, and intra-cellular enzymatic complexes such as cytochrome P450 (Liang et al., 2012; Syed and Yadav, 2012). This ability of the fungi to oxidize and even mineralize aromatic ring-containing compounds make them applicable to other aromatic pollutants
n-Herna
ndez et al., 2017). (Olico In addition, the production of hydrogen peroxide in ligninolytic fungal cultures has been proven by different mechanisms based on peroxide-generating enzymes such as glucose or glyoxal oxidases (Daniel et al., 1994; Zhao and Janse, 1996). The ubiquitous formation of hydrogen peroxide upon WRF systems enables the generation of hydroxyl radicals through Fenton-like reactions by quelated iron complexes of the medium ((Krueger et al., 2016) This process has

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been suggested to be site-specific in nature, with production of hydroxyl radicals occurring in the fungi cell wall close to lignin and cellulose (Backa et al., 1993). Several works have also demonstrated that the production of hydroxyl radicals can be enhanced by addition of quinone-like mediators. These compounds favor quinone redox cycles driven by intracellular quinone reductase and extracellular lignin-modifying enzymes to produce hydrogen
mez-Toribio et al., 2009; Guille
n et al., 2000). The nonperoxide (Go specificity and high potential oxidation of hydroxyl radicals makes them very effective for the degradation of non-biodegradable pollutants. Thus, WRF are considered a potential alternative for the removal of micropollutants of emerging concern such as PhACs.
(Christoforidis et al., 2018; del Alamo et al., 2018; Marco-Urrea et al., 2009; Palli et al., 2017; Wen et al., 2011). Previous works of WRF-based systems have been focused on the removal of PhACs in malt-extract synthetic mediums (optimum for fungal growth) in order to explore their degradation pathways and by-products (Nguyen et al., 2013; Yang et al., 2013a). Others have been focused on the treatment of real effluents coming from urban
et al., 2013; Badia-Fabregat et al., 2017), hospital (Cruz-Morato
et al., 2014; Mir-Tutusaus et al., 2017) and veterinary (Cruz-Morato (Badia-Fabregat et al., 2016) depuration facilities. In case of real wastewater streams, the treatments were performed with fungal processes in which the wastewater was supplemented with a considerable amount of substrates. In these works, the removal of the organic loading and nutrients was not deeply studied under non-sterile conditions. Recently, a critical review of the limiting drawbacks of WRF to be considered a real wastewater treatment alternative for the removal of organics micropollutants has been reported (Mir-Tutusaus et al., 2018a). The addition of supplementary carbon and nitrogen sources readily biodegradable during operation is one of the limitations of
et al., 2013). Glucose WRF (Badia-Fabregat et al., 2015; Cruz-Morato and ammonium tartrate has been the preferred compounds in most of studies (Badia-Fabregat et al., 2015; Jelic et al., 2012; Zhang and Geißen, 2012). This is a serious limitation for up-scaling the process, due to the cost of these substrates when a large volume of wastewater is treated. Other important limitation is the bacterial contamination of the biomass fixed in the reactor under non-sterile conditions. These non-inoculated microorganisms exert the competition for substrates leading to the loss and destabilization of fungal biomass, and consequently, reducing the removal of micropollutants. The coexistence of the fungus and autochthonous microorganisms from wastewater, both native fungal and bacteria communities, has been recently studied by Badia-Fabregat et al., 2016, 2017. Several strategies have been proposed to promote the WRF growth over bacteria. They include the control of the C:N ratio, the periodical biomass renewal or the immobilization of the fungal biomass. However, none all of them has been completely satisfactory. Some works have studied the treatment of simulated wastewater with model micropollutants in optimal C:N conditions under sterile conditions at lab-scale (Malachova et al., 2013; Novotný et al., 2012). However, the C:N ratio is depending on the wastewater nature and the addition of supplementary sources to achieve the optimal composition is not feasible from an economical point of view. The option of partial biomass renovation requires an external system for cultivation and growth of fresh WRF that should be periodically inoculated in the reactor. It usually implies a significant withdrawal of WRF biomass from the reactor and replacement by
nquez et al., 2006). fresh WRF (Badia-Fabregat et al., 2016; Bla Finally, the immobilization of the fungal biomass into carriers/ supports allows the decoupling of hydraulic retention time (HRT) and the sludge retention time (SRT), producing the wash-out of the suspended bacteria microorganisms. This is a good strategy, but a minimum HRT of 2 days was necessary for an effective removal of


nquez et al., 2008; Hai et al., 2009). The micropollutants (Bla immobilization of fungal biomass also reduces the problems of WRF growth in dispersed mycelium on the reactor surfaces (wall or stirrers). Thus, different solutions have been studied, such as the
nquez et al., 2006; Borra s growth of fungus in form of pellets (Bla et al., 2008; Espinosa-Ortiz et al., 2016) or the immobilization on inert (polyurethane) or non-inert (wooden chips and wooden
n et al., residues) carriers (Li et al., 2016; Liang et al., 2012; Tora 2017). Fixed bed reactors, fluidized bed reactors and membrane biological reactors (MBR) were the technologies usually found in literature in these cases (Hai et al., 2013; Jelic et al., 2012; Nguyen
n et al., 2017; et al., 2013; Rodarte-Morales et al., 2012; Tora Zhang et al., 2017). Interestingly, the immobilization of fungi into carriers has shown an enhancement of the efficiency of the fungal treatment, especially in non-sterile conditions (Li et al., 2015). In order to overcome the current limitations of fungal biological treatments, this work deals with the use of rotating biological contactors (RBCs) for the immobilization of Trametes versicolor as well-known WRF. The performance of the process was assessed for the removal of the carbon and nutrients of two real urban wastewater after a primary treatment. The addition of a quinone-like mediator and quelated iron and manganese complexes was studied with the purpose to enhance the potential capacity of fungi for the removal of pharmaceutical micropollutants through the socalled advanced bio-oxidation process via the production of nonselective oxidizing hydroxyl radicals. The continuous treatment of real wastewaters was performed with non-addition of extra supplementary biodegradable sources and non-renovation of fungal biomass at reduced HRT of 1 day. Finally, the prevalence of fungi after the prolonged continuous treatment of the different wastewaters under non-sterile conditions was also analysed. 2. Material and methods 2.1. Fungus strain The strain T. versicolor (CECT, 20817) was collected from the
n Espan ~ ola de Cultivos Tipo (CECT)” and maintained by “Coleccio sub-culturing on 2% malt extract agar slants (pH 4.5) at 25
C. Blended mycelial mass was produced according to a procedure
previously described (Vasiliadou et al., 2016; del Alamo et al., 2018) and used as fungal inoculum to the rotating biological contactors (RBCs). 2.2. Immobilization of T. versicolor on rotating biological contactors (RBCs)
n S.L. (Huesca, The RBCs were provided by ACAI Depuracio Spain). The bioreactor made of AISI 304 stainless steel is equipped with five rotating discs (30 cm - diameter) made of polypropylene, which provide a total surface area of 0.71 m2. The working volume was set to 10 L with the immersion of ca. 40% of the discs into the wastewater during operation. To ensure an appropriate aeration of the reactor, the set of the discs was mechanically rotated at 12 rpm (Di Palma and Verdone, 2009). The bioreactor inoculated with the blended mycelial fungal inoculum was initially operated in fedbatch mode for 30 days (start-up period) until obtaining a homogeneous biofilm immobilized over the discs (Fig. S1). During this stage, the reactor was operated in draw-fill cycles (Vasiliadou et al., 2016) using a malt extract solution as specific fungal growth medium with ca. 3 g/L of TOC concentration and pH of 4.5. Once 50% of the initial TOC was consumed, 1 L of the reactor’s medium was replaced with fresh malt extract (10% volume exchange) in order to restore again a TOC concentration of 3 g/L and to start a new drawfill cycle. After this start-up period, the malt extract was substituted


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by a synthetic urban wastewater with a composition as described
elsewhere (del Alamo et al., 2018). The overall TOC loading and pH were maintained at 3 g/L and 4.5, respectively. The bioreactor was operated with the synthetic urban wastewater (acclimation stage) for 20 days. The excess of organic content and pH (4.5) in both stages was used in order to maximize the production of extracellular polysaccharides (EPS) that promotes the adhesion of T. versicolor over the discs surface (Nimtz et al., 2008; Tavares et al., 2005). Finally, the bioreactor was operated under continuous mode for the treatment of real urban wastewater effluents coming from two different wastewater treatment plants (WWTPs). 2.3. Wastewater samples and operation conditions of fungal biological treatment Different wastewater samples were collected from: i) demo pilot
stoles Campus, plant located in Rey Juan Carlos University, Mo Madrid (Spain) designed to 1800 equivalent inhabitants providing a capacity of 360 m3/d (DPP-URJC); and ii) wastewater treatment plant at Toledo (Spain) of 65,000 equivalent inhabitants currently working with 8800 m3/d (WWTP-Toledo). Several samples of both DPP-URJC and WWTP-Toledo facilities were used for the study of different operation conditions. Table 1 summarizes the characterization data of the wastewater samples and the operating conditions of RBCs for the treatment of the different real urban wastewater effluents. All the wastewater samples, except DPP-URJC-1, were treated under advanced bio-oxidation (ABO) conditions, that comprises the addition of a lignin derived quinone-type mediator (gallic acid, 85 mg/L) and complexed metal sources (Fe2(C2O4)3$6H2O, 145 mg/ L; and Mn(NO3)2$4H2O, 20 mg/L) as promoters of quinone-redox cycles and Fenton reactions. Concentrations of these chemicals
mez-Toribio et al., 2009; were taken from previous works (Go
Christoforidis et al., 2018; del Alamo et al., 2018). The loadings in the continuous influents of the RBCs for the treatment of all wastewaters were 0.85, 1.12 and 0.14 g/d of gallic acid, iron oxalate and manganese nitrate, respectively. The inlet streams were adjusted to a pH of 4.5 in order to inhibit the bacteria cellular transport leading to the predominance of fungi activity in the bioreactor (Jo et al., 2010; Mir-Tutusaus et al., 2018a). The hydraulic retention time (HRT) was fixed at 1 day for all the wastewater samples (minimum value reported in literature for continuous reactors based on white rot fungi (Mir-Tutusaus et al., 2018a), except for DPP-URJC-3 sample that was decreased at 0.5 day. 2.4. Characterization techniques and analytical methods Samples were dairy withdrawn from inlet and outlet streams of the RBCs to monitor the performance of the fungal biological

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treatments in terms of the macroscopic parameters shown in Table 1. Additionally samples taken each 4 days were used for determination of the removal of pharmaceutical micropollutants. Total organic carbon (TOC) was estimated using a combustion/nondispersive infrared gas analyser model TOC-V Shimadzu. 3Ammonium-nitrogen (NeNHþ 4 ), phosphate-phosphorous (P-PO4 ), total suspended solids (TSS) and volatile suspended solids (VSS) were determined following the standardized APHA methods (Rice et al., 2012). Dissolved oxygen (DO) and pH were periodically measured by CellOx 325 and a-Sentix 81 probes from WTW-Xylem, respectively. Remnant gallic acid was measured by High Performance Liquid Chromatography (HPLC) in a Varian Prostar equipped with a Phenomenex C18 column (3  150 mm) and a UVeVis detector at 254 nm. A mixture of methanol (49.5%), ultrapure water (49.5%) and glacial acetic acid (1%) at pH of 2e2.5 was used as mobile phase at 0.15 mL/min. LCeMS quality methanol and glacial acetic acid were obtained from Sigma-Aldrich. The amounts of Fe and Mn dissolved in the effluent were measured by ICP-AES analysis collected in a Varian Vista AX Pro-720ES spectrometer. The pharmaceutical micropollutants were analysed after solid phase extraction (SPE, Afonso-Olivares et al. (2017)) by ultra-highperformance liquid chromatography - tandem mass spectrometry (UHPLC-ESI-MS/MS) using a vortex electrospray ionization interface (Bruker UHPLC/MSMS EVOQ™ QUBE). Further details of the analytical method are included in the supplementary information. Following this method, 23 PhACs of 7 different therapeutic groups were identified and quantified. All the samples were analysed by triplicate. The detection and quantification limits (LOD and LOQ, respectively) of the method for each PhACs are shown in Table S1. 2.5. Assessment of fungal/bacteria activity for communities of the biofilm of RBCs Several bioassays were performed in order to estimate the presence of fungi and bacteria communities in the biofilm of the RBCs as consequence of the continuous treatment of wastewaters under non-sterile conditions. These tests were performed batchwise in 160 mL bottles using 100 mL of a synthetic wastewater adjusted to pH of 4.5 or 7 with diluted solutions of NaOH or H2SO4. The synthetic wastewater was prepared as described elsewhere
(del Alamo et al., 2018), but substituting the organic carbon sources by sodium acetate as readily biodegradable substrate. The bottles were inoculated with 200 mg dry-based VSS/L of biomass collected from the biofilm formed over the RBCs after the biological treatments of DPP-URJC and WWTP-Toledo wastewaters. Additionally, Gram- and Gram þ bactericides (ampicillin, 4 mg/L, and tetracycline, 128 mg/L) or fungicide (nystatin, 200 mg/L) were added and maintained for 24 h under continuous stirring at 25
C to inhibit the bacterial or fungal activity. The minimal inhibitory concentration of

Table 1 Characterization of wastewater samples and operation conditions of the RBCs.

Sampling time (2017) TOC (mg/L) NeNHþ 4 (mg/L) P-PO34 (mg/L) TOC/NeNHþ 4 TOC/P-PO34 TSS (g/L) VSS (g/L) Feed Flow (L/d) HRT (d) Addition of ABO mediators Treatment duration (d)

DPP-URJC-1

DPP-URJC-2

DPP-URJC-3

WWTP-Toledo-1

WWTP-Toledo-2

March 146 ± 20 118 ± 10 12 ± 3 1.2 12.2 0.083 ± 0.03 0.076 ± 0.03 10 1 NO 65

April 115 ± 29 175 ± 21 14 ± 3 0.6 8.2 0.093 ± 0.03 0.091 ± 0.02 10 1 YES 32

June 143 ± 33 28 ± 9 10 ± 1 5.1 14.3 0.13 ± 0.06 0.11 ± 0.05 20 0.5 YES 30

June 76 ± 18 29 ± 5 13 ± 4 2.6 5.9 0.31 ± 0.14 0.2 ± 0.1 10 1 YES 22

July 116 ± 3 41 ± 7 2.3 ± 0.2 2.8 50.4 0.7 ± 0.14 0.45 ± 0.1 10 1 YES

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each chemical was set according to previous studies (ArayaCloutier et al., 2017; Han et al., 2012; Mawabo et al., 2015). Finally, 250 mg/L of TOC as sodium acetate was added to the medium to start the bioassays. Further bioassays in absence of bactericides and fungicide were also carried out with the purpose of comparison. The bottles were incubated under controlled temperature (25
C) during 4 days at 300 rpm in absence of ABO promoters.

3. Results and discussion 3.1. Influence of ABO mediators on the fungal activity of T. versicolor Initially, the effect of adding advanced bio-oxidation (ABO) mediators was studied with DPP-URJC-1 and DPP-URJC-2 samples. The RBCs were operated with the DPP-URJC-1 wastewater in absence of oxidizing mediators for 65 days (fungal treatment, FungT), and then the ABO mediators were added to the inlet DPP-URJC-2 wastewater for further 32 days (advanced bio-oxidation treatment, ABO-T). The immersed area and rotation speed of RBCs as well as the HRT were kept constant. Fig. 1aeb shows the performance of 3RBCs in terms of TOC, NeNHþ 4 and P-PO4 removals and elimination of pharmaceutical micropollutants for both systems. The pH and DO in the effluents hardly changed for the fungal and advanced biooxidation treatments with values of 6.52 ± 0.39 and 4.39 ± 0.29 mg/ 3L, respectively. Confidence intervals of TOC, NeNHþ 4 and P-PO4 removals were calculated according to the t-distribution at 95% for

3Fig. 1. Removal of (a) TOC, NeNHþ 4 and P-PO4 and (b) PhACs for fungal and advanced bio-oxidation treatments of wastewaters from demo pilot plant at Rey Juan Carlos university.

30 (Fung-T) and 15 (ABO-T) samples. The fungal treatment showed remarkable removals of TOC (70 ± 8%) and phosphates (37 ± 29%). In contrast, the ammonium removal was practically negligible. The addition of ABO mediators showed similar TOC reductions and the increase of NeNHþ 4 and PPO34 removals, but these variations are not statistically significant. These results prove an active and stable performance of the biomass immobilized on RBCs for the treatment of a real wastewater with a low C:N ratio (between 1.2 and 0.6, too far from the theoretical optimum range for WRF of 7e10) operating without additional loadings of biodegradable carbon and nitrogen extra sources as usually employed in other works (Mir-Tutusaus et al., 2016; Tor
an et al., 2017). The potential capacity of reducing the TOC has been hardly studied in literature, but is a crucial point in the development of a real wastewater treatment process based on T. versicolor (Palli et al., 2017). The low ammonium removal seems to be related to the limited nitrogen uptake of the WRF (Cruz
et al., 2013; Mir-Tutusaus et al., 2018a). Moreover, NHþ Morato 4 can be released by ammonification of urea, with partial assimilation of nitrogen and release of the ammonium in excess to the medium (Geisseler et al., 2010). The balance between the uptake and release of ammonium is influenced by the nutritional status of the fungal community (starvation or nitrogen excess), the C:N ratio of the population and the quantity and chemical composition of dissolved organic matter. Typically, fungi release nitrogen as ammonium for a C:N ratio lower than 30:1 (Hodge et al., 2000). Thus, it is expected that the ammonification process happened considering the low C:N ratios of the urban wastewater used in this work, in particular DPPURJC wastewater (see Table 1). It must be also pointed out that the amount of gallic acid was almost completely consumed (98%) with a very low concentration (<2 mg/L) in the outlet effluent. Likewise, the concentrations of Fe and Mn from the quelated metal promoters were lower than 1 mg/L and ca. 8 mg/L, respectively. Thus, the use of the ABO promoters as well as derivative by-products did not show any sign of toxic effect on the biological treatment. In terms of the PhACs removal, only 9 out of the 23 PhACS analysed in this work (Table S2) were detected in samples of DPPURJC-1 and DPP-URJC-2 (Fig. 1b): 3 antibiotics (Amoxicillin, AMX, metronidazole, MDZ and sulfamethoxazole, SMX), 2 psychiatric drugs (carbamazepine, CPZ and caffeine, CFN), 1 analgesic (4Acetamidoantipyrine, 4-AAA), 1 lipid regulator (gemfibrozil, GFZ), 1 chemical diuretic (hydrochlorothiazide, HCT) and 1 contrast agent (iohexol, IHX). CFN and AMX, that the last one was recently included in the watch list of priority substances modified by the Commission Implementing Decision (EU) 2018/840, were the PhACs detected in the highest concentration in DPP-URJC-1 and DPP-URJC-2 samples. The fungal treatment (Fung-T) showed moderate removals with values below 40% for 7 PhACs. Only caffeine achieved an elimination higher than 80% but this compound is considered easily removed by other biological treatments (Ibrahim et al., 2014). The functional groups of the chemical structure as well as the hydrophilic/hydrophobic behaviour of the PhACs strongly affect their removal (Yang et al., 2013b). In general, PhACs containing electron donating functional groups such as amine (eNH2), hydroxyl (eOH) or alkoxy (eOR) groups are more susceptible to be biologically degraded by the oxidative catabolism of fungi. On the contrary, PhACs containing strong electron-acceptor functional groups, such as CZP containing azepine, an amide (eCONR2) group are majorly degraded by extracellular lignin modifying enzymes, but with lower removal degrees (Marco-Urrea et al., 2009; RodríguezRodríguez et al., 2010). On the other hand, hydrophobicity plays a critical role in biosorption of micropollutants in the WRF, enhancing its removal from the aqueous phase. Hydrophobic micropollutants (log Kow > 3.2),


A. Cruz del Alamo et al. / Water Research 170 (2020) 115313


ment et al., 2017). such as GFZ are easily removed by WRF (Grandcle However, elimination of hydrophilic compounds (log Kow < 3.2) is highly dependent on the presence of electron-donor and/or
ment et al., 2017; Yang et al., 2013b). The acceptor groups (Grandcle presence of the strong electron acceptor groups of amide and iodine in the IHX, prevails over its electron donating hydroxyl groups, hindering their biological oxidation. Moreover, the pH of the aqueous medium is also an important factor. Hydrophilic compounds with pKa < pHmedium are expected to be as ionic forms, promoting its solubility in the aqueous phase and making more difficult their elimination. It seems the case of 4-AAA, AMX, MDZ, SMX and HCT. For example, HCT possess electron withdrawing groups (sulphonamide and chlorine) and amine donating group, being the biodegradability comparable to that obtained for SMX, which accounts for the same opposing groups (sulphonamide and amide, see Table S2). The negative removals of IHX, AMX and 4-AAA are in agreement with previous observations (Collado et al., 2014) due to concentrations in the effluent higher than in the influent. This fact has been reported very often for recalcitrant compounds to biological oxidation like CPZ, DCF or AMX (Palli et al., 2019) attributing these results to different factors: i) the conversion of conjugated metabolites to their parent compound through enzymatic processes; ii) sudden release of pharmaceuticals from sludge; iii) sampling variations due to long hydraulic retention times and iv) the limited analytical capabilities for low concentrations. The promotion of highly oxidizing hydroxyl radicals by ABO mediators increased the elimination of 4-AAA, AMX and IHX up to 23, 34 and 77%, respectively. Significant increases were also accomplished for HCT and MDZ (both around 50%) and GFZ and SMX (82 and 89%, respectively). Likewise, it must be noted that these results were obtained under continuous operation of RBCs with a HRT of only 1 day and non-supplementary addition of extra biodegradable carbon and nitrogen sources, which have been very influencing factors in the performance of alternative bioreactors using T. versicolor, such as fixed bed columns or fluidized bed reactors, for the treatment of wastewaters (Bl
anquez et al., 2008; Hai
n et al., 2017). et al., 2009; Palli et al., 2017; Tora 3.2. Effect of HRT on the performance of advanced bio-oxidation treatment on RBCs (ABO-T RBCs) The immobilization of T. versicolor on the surface of the RBC’s discs makes possible decoupling the hydraulic retention time and cellular retention time. HRTs ranging from 1 to 3 days have been reported for fungal treatments. Lower HRTs usually limit the performance of the treatment, decreasing the removal of micropollutants. In order to consider the potential capacity of T.versicolor immobilized over RBCs as alternative to biological treatment to be implemented in a conventional WWTP, a HRT of 0.5 days was studied for the treatment of DPP-URJC-3 wastewater. Fig. 2aeb shows the performance of ABO-T on RBCs operating with a HRT of 1 day (DPP-URJC-2) and 0.5 days (DPP-URJC-3). As expected, the TOC removal decreased when reducing the HRT. A less significant reduction was observed for the phosphate content. In the case of ammonium, the removal was improved from ca. 10e20%. This is a consequence of a limited ammonification of the nitrogen-containing organic compounds, leading to less ammonium concentration by the nitrogen uptake of the microorganisms of the biological treatment. Moreover, it must be pointed out that the ammonium nitrogen content of DPP-URJC-3 is much lower than that found by DPP-URJC-2. Nevertheless, the most important influence of decreasing the HRT was obtained in the PhACs removal efficiency (Fig. 2b, Table S2). It should be pointed out that DPP-URJC-2 and DPP-URJC3 samples were taken from the same wastewater treatment plant

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Fig. 2. Influence of the hydraulic retention time on the performance of ABO-T on RBCs 3for the removal of: (a) TOC, NeNHþ 4 and P-PO4 and (b) PhACs.

but at different periods of time. Consequently, concentration of PhACs varied in their composition. Additionally, diclofenac (nonsteroidal anti-inflammatory drug, DCF) was detected during operation with DPP-URJC-3 instead of AMX (see Table S2). An extremely decrease in the PhACs removal was observed working at HRT of 0.5 days. GFZ and IHX removal efficiency decayed from 82 to 77% to 70 and 60%, respectively, whereas HCT and MDZ decreased from ca. 50 to 25 and 20%, respectively. Finally, 4-AAA, CFN, CZP and SMX were not eliminated, showing even negative removal as consequence of their biorefractory behaviour to be degraded and the reasons previously mentioned for the treatment of DPP-URJC-1. DCF also showed a removal of only 10%, probably due to the presence of nondonating electrons groups (carboxylic and chloride) that make it less willing for both oxidative catabolism and advanced biooxidation mediated by hydroxyl radicals attack (Mazzafera, 2002; Tang and Huang, 1996; Yan et al., 2017). Thus, it can be concluded that the decrease of the HRT from 1 to 0.5 day dramatically reduced the efficiency of PhACs removal on the rotating biological contactors. 3.3. Influence of wastewater composition on the biological performance of fungal RBCs The validation of the ABO-T on RBCs for a plausible implementation in a conventional wastewater treatment plant requires its evaluation for several types of wastewater. In this context, two wastewater samples (WWTP-Toledo-1 and WWTP-Toledo-2) taken

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at different periods of time from a WWTP in the north of Toledo (Spain) and a wastewater stream from the demo wastewater pilot plant at Rey Juan Carlos University (DPP-URJC-2) were compared. All the wastewaters were collected from the outlet streams of primary treatment in the depuration facilities. The WWTP-Toledo-1 and WWTP-Toledo-2 wastewater samples showed similar C:N ratios and they have higher C:N ratio than DPP-URJC-2 wastewater samples (ca. 3:1 vs 1:1.5, respectively). However, the C:P ratios of WWTP-Toledo-1 and WWTP-Toledo-2 streams are different (6:1 and 50:1, respectively), whereas the C:P ratio of DPP-URJC-2 (ca. 8:1) is quite similar to that of the WWTP-Toledo-1 (Table 1). Concerning the pharmaceutical micropollutants, AMX was not detected in WWTP-Toledo 1 and 2, IHX was not detected in WWTPToledo-2 and DCF and N,N-Diethyl-meta-toluamide (an insect repellent, DEET) were found only in the WWTP-Toledo-2 sample (Table S2). The variation of C:P composition of WWTP-Toledo-1 to WWTPToledo-2 wastewater did not modify the efficiency of the biological treatment during operation (macronutrients and PhACs removals). Thus, a low of phosphate content of the wastewater does not limit the performance of the advanced bio-oxidation fungal treatment (see Table S3). The higher C:N ratio of WWTP-Toledo-2 respect to DPP-URJC-2 wastewater samples promoted a slight 3improvement of the TOC, NeNHþ 4 , P-PO4 removals (Fig. 3a). This fact is probably due to the best performance of biological fungal systems with higher C:N ratios. A high C:N ratio mimics the optimal conditions of white rot fungi for degradation of lignin in natural lignocellulosic substrates, which increases the production of lignin-

~ A¥th, ~ modifying enzymes (Elgueta et al., 2016; Rousk and BA¥ 2007). Typically, it is reported in literature an optimum C:N ratio ranging from 7 to 10 (Jo et al., 2010; Mir-Tutusaus et al., 2018b). Moreover, a high C:N ratio ensures the fungal growth and maintenance under non-sterile conditions over bacterial populations. Regarding the effect of C:P content, there is not information reported about its influence on fungal biological systems. The elimination of PhACs (Fig. 3b, Table S2) showed similar removals for 4-AAA, CFN, CPZ and GFZ in both wastewater samples (WWTP-Toledo-2 and DPP-URJC-2). In turn, the removal of antibiotics (MDZ and SMX) slightly decreased for the WWTP-Toledo-2 wastewater. Thus, it seems that the slight improvement of biolog3ical performance in terms of the TOC, NeNHþ 4 and P-PO4 removals is not related to a higher efficiency of the removal of pharmaceuticals. However, it was seen that the total content of pharmaceuticals in the WWTP-Toledo-2 wastewater (ca. 59 mg/L) is considerable higher than in the DPP-URJC-2 wastewater (ca. 32 mg/ L). Taking into account these data, the efficiency of the treatment for the removal of pharmaceuticals could be affected by its loading rate, as it can be deduced from the overall efficiency of all the pharmaceuticals detected (59% for DPP-URJC-2 versus 46% for WWTP-Toledo-2). Likewise, it must be pointed out the remarkable tolerance of the fungal treatment for the range of the pharmaceutical loadings of these two wastewaters, from 317 mg/d (DPP-URJC2) to 590 mg/d (WWTP-Toledo-2). All the results related to the total concentration and overall removal efficiencies can be found in Table S4. 3.4. Assessment of the prevalence of fungal or bacterial communities in the biofilm of RBCs after treatment of DPP-URJC and WWTP-Toledo wastewaters

3Fig. 3. Removal of (a) TOC, NeNHþ 4 and P-PO4 and (b) PhACs for the ABO-T on RBCs of two different wastewaters from demo pilot plant at Rey Juan Carlos University (DPPURJC-2) and wastewaters treatment plant at Toledo (WWTP-Toledo-2).

As it has been mentioned, the treatment of non-sterile wastewater under continuous operation can promote the proliferation of bacterial colonies, which compete for the assimilable substrates of the wastewater. This fact can produce the prevalence of dominant bacterial colonies, which would reduce the efficiency of the fungal advanced bio-oxidation process for the removal of pharmaceutical micropollutants. In order to evaluate the prevalence of fungal or bacterial communities in the RBCs, several bioassays with bactericides, fungicide and none of them were performed using as inoculums the sludge of the biofilm of the RBCs discs after the treatment of DPP-URJC-2 and WWTP-Toledo-2 wastewaters. The consumption of sodium in terms of the TOC content was used as indirect measurement of the prevalence of fungal or bacterial communities in the RBCs. Fig. 4a and b shows the results of the activity tests after 4 days at pH 4.5 and 7, respectively. These pH values were chosen according to the pH of the inlet and outlet streams of the RBCs during treatment. The profiles of the TOC removal for all bioassays along the time are shown in Figs. S3 and S4. At pH 4.5 (Fig. 4a), the WWTP-Toledo-2 inoculum was more active than the DPP-URJC-2 one. In presence of the fungicide, the WWTP-Toledo-2 inoculum achieved a TOC removal of ca. 25%. In turn, a negligible TOC removal was observed by the DPP-URJC-2 inoculum. The bioassays using bactericides showed similar TOC removals than that obtained by the inoculums alone, even with a slight increase for the DPP-URJC-2 inoculum. These results clearly point to a prevalence of fungi in the DPP-URJC-2 and WWTPToledo-2 inoculums. At pH 7 (Fig. 4b), the DPP-URJC-2 inoculum was less active in absence of fungicide and bactericides in comparison to the results at pH 4.5. This is a consequence of a more favourable pH for bacterial communities and the decelerated metabolism of fungal communities. Likewise, the activity of bacteria when using the fungicide increased up to ca. 37% and 12% for


A. Cruz del Alamo et al. / Water Research 170 (2020) 115313

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DPP-URJC-2, a higher efficiency for PhACs removal was achieved, denoted by the major elimination of antibiotics. The higher proportion of fungal biomass will increase the production of nonselective oxidizing hydroxyl radicals through the advanced biooxidation process enhancing the removal of more bio-refractory micropollutants (Christoforidis et al., 2018). 4. Conclusions

Fig. 4. Fungal and bacterial activity of inoculums taken from biofilm of RBCs treating DPP-URJC-2 and WWTP-Toledo-2 wastewaters after 4 days of incubation at: a) pH 4.5 and b) pH 7.

the WWTP-Toledo-2 and DPP-URJC-2 inoculums, respectively. Nevertheless, the activity of fungi in bioassays performed with bactericides was much higher than those obtained by bacteria in those with the fungicide. This confirms the prevalence of fungal communities on the biofilm of RBCs. However, the development of bacterial communities during the treatment of both wastewaters is also evident, being more important for the inoculum of the WWTPToledo-2 treatment. Moreover, it is observed that this inoculum displayed a slight decrease of activity for the bactericides bioassays as compared to those performed with the inoculum alone. These results indicates that the higher proportion of bacteria microorganisms for this inoculum reduces the activity of both fungal and bacterial communities, probably due to the competitive substrate consumption. On the other hand, the increase of the bacterial proportion in the inoculum of the WWTP-Toledo-2 could be related to the higher C:N ratio of this wastewater, three times higher than the DPP-URJC-2 effluent. Summarizing and looking at the results obtained at pH 7, real pH of operation of the RBCs, the bacterial communities will be contributing to the biological performance of the RBCs. Moreover, the increasing competition of bacteria for the substrate could lead to loss of fungal biomass and the destabilization of fungal enzymes
(del Alamo et al., 2018; Hai et al., 2013). Additionally, the decrease in the fungi proportion would produce a reduction of the efficiency of PhACs’ removal (Badia-Fabregat et al., 2017). Thus, as the RBCs are less colonized by bacterial communities during the treatment of

The efficiency of T.versicolor as White Rot Fungi immobilized on rotating biological contactors (RBCs) for the treatment of urban wastewater of different depuration facilities has been demonstrated. The presence of advanced bio-oxidation promoters, a lignin-derived mediator and metal complexes with redox activity, enhanced the performance of fungal biological treatment for the removal of pharmaceutical micropollutants, increasing significantly the elimination of 4-AAA, AMX, IHX and SMX among others. Remarkable removals of the organic content (ca. 70e75% of TOC) were achieved with organic loadings between 32 and 60 mg TOC/h, and C:N and C:P ratios ranging from 0.6 to 2.8 and 6 to 50, respectively. These results were accomplished operating with nonaddition of supplementary biodegradable glucose source, nonfungal biomass refreshment, non-external aeration and 1 day of HRT, which are the main limitations of fungal biological treatments for plausible implementation in WWTPs. It was observed the proliferation of bacteria in the biofilm of RBCs as consequence of the continuous treatment of real wastewater under non sterile conditions. However, the fungal communities dominate the performance of the removal of the organic loading. The fungal treatment based on the immobilization of T.versicolor on RBCs (10 L/d in a reactor of 10 L capacity) is considered an effective technology for depuration of real urban wastewaters including the removal of pharmaceutical micropollutants, although further steps must be done in order to achieve a better stabilization of the fungal biomass and higher removals of nitrogen as nutrient. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors wish to thank to “Comunidad de Madrid” and European Structural Funds for their financial support to the REMTAVARES-CM project (S2013/MAE-2716 and S2018/EMT-4341). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2019.115313. References 
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