Environmental Science and Pollution Research

https://doi.org/10.1007/s11356-017-1053-4

ADVANCED OXIDATION PROCESSES FOR WATER/WASTEWATER TREATMENT

Removal of pharmaceutical compounds from urban wastewater by an

advanced bio-oxidation process based on fungi Trametes versicolor
immobilized in a continuous RBC system
Ana Cruz del Álamo 1 & María Isabel Pariente 1 & Ioanna Vasiliadou 1 & Beatriz Padrino 1 & Daniel Puyol 1 & Raúl Molina 1 &
Fernando Martínez 1

Received: 14 August 2017 / Accepted: 13 December 2017

# Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract
Conventional wastewater treatment plants (WWTPs) are not able to remove completely some emerging contaminants, such as
residual pharmaceutical compounds (PCs) with potential ecotoxicity to water bodies. An advanced bio-oxidation process
(ABOP) using white-rot fungi (WRF) has been proposed as alternative biological treatment for degradation of non-
biodegradable compounds. A synthetic and real wastewater spiked with 12 PCs at 50 μg L−1 was treated by means of ABOP
based on WRF in a rotating biological contactor (RBC) at 1 day of hydraulic retention time (HRT). The ABOP achieved a
remarkable biological performance in terms of TOC removal and reduction of N-NH4+ and P-PO43− nutrients. Likewise, 5 of the
12 PCs were eliminated with removal efficiencies ranging from 80 to 95%, whereas 6 of 12 PCs were eliminated with removal
values ranging from 50 to 70%. The anaerobic digestion of the fungal sludge generated upon the treatment was also evaluated,
obtaining a methane yield of 250 mL CH4 gVS−1. These results evidenced that the proposed ABOP is a promising alternative for
the sustainable wastewater treatment of urban effluents, combining advanced oxidation with biological operation for the removal
of emerging PCs and energy recovery.

Keywords White-rot fungi . Rotatingbiological contactor . Pharmaceutical compounds . Bio-methanogenic potential tests . Urban
wastewater

Introduction in conventional wastewater treatment plants (WWTPs) and

their poor biodegradability, which has negative and bio-
In recent years, pharmaceutical compounds (PCs) have re- accumulative effects on the environment (Zenker et al. 2014;
ceived an increasing attention from the scientific community Archer et al. 2017).
due to their presence in different water environmental bodies Among the technologies used in wastewater treatment, ad-
(Valcárcel et al. 2012; Mendoza et al. 2016). This constant vanced oxidation processes (AOPs) are well-known as pow-
detection of PCs is a consequence of their ineffective removal erful technologies for the degradation of chemical pollutants,
in particular those refractories to biological processes (Oller
et al. 2011). AOPs are technologies based on the production of
Responsible editor: Vítor Pais Vilar highly reactive species such as hydroxyl radicals (HO·). This
Electronic supplementary material The online version of this article radical is a non-selective and highly oxidative agent. To date,
(https://doi.org/10.1007/s11356-017-1053-4) contains supplementary the production of HO· is based on chemical, photochemical,
material, which is available to authorized users. or electrochemical techniques (Pignatello et al. 2006).
However, it has been recently reported that HO· radicals can
* Raúl Molina be also produced in biological systems through a BFenton-like
[email protected]
procedure^ without external addition of hydrogen peroxide as
oxidant (Marco-Urrea et al. 2009; Wei et al. 2010). This prom-
1
Department of Chemical and Energy Tech., Chemical and ising system, termed as advanced bio-oxidation process
Environmental Tech., Mechanical Tech., and Analytical Chemistry, (ABOP), has been successfully applied for the degradation
ESCET, Universidad Rey Juan Carlos, 28933 Móstoles, Madrid,
Spain of some specific pharmaceuticals (Marco-Urrea et al. 2010).
 Environ Sci Pollut Res

This ABOP is based on the incubation of white-rot fungi Materials and methods
(WRF) with lignin-derived quinones and chelated ferric ions
(Gómez-Toribio et al. 2009). Under these conditions, fungi are Fungus and chemicals
able to promote the conversion of the quinone into hydroqui-
none (DBQH2) by an intracellular quinone reductase and the The strain T. versicolor (CECT 20817) was collected from the
oxidation of DBQH2 to semiquinone radicals (DBQ·−) in the BColección Española de Cultivos Tipo (CECT)^ and main-
extracellular medium by any of the lignin-modifying enzymes tained by sub-culturing on 2% malt extract agar slants
of the WRF (laccase and peroxidases). Then, Fenton’s reagent (pH 4.5) at 25 °C. Subcultures were routinely prepared every
(Fe2+/Fe3+/H2O2) is formed by DBQ·− autoxidation catalyzed 15 days. Mycelial mass was produced according to a proce-
by Fe3+, in which Fe2+ and superoxide anion radicals (O2·−) dure previously described by Vasiliadou et al. (2016). The
are generated (reactions 1 and 2), followed by O 2 · − blended mycelial suspensions were used as fungal inoculum
dismutation (reaction 3). to the rotating biological contactor.
The pharmaceutical compounds were acquired from
DBQ•− þ Fe3þ →DBQ þ Fe2þ ð1Þ Sigma-Aldrich (Spain). Twelve pharmaceutical compounds
from different therapeutic groups were selected (Table S1):
Fe2þ þ O2 →Fe3þ þ O2 •− ð2Þ 4-AAA, antipyrine; CFA, clofibric acid; ATN, atenolol;
O2 •− þ HO2 • þ Hþ →O2 þ H2 O2 ð3Þ CFN, caffeine; CZP, carbamazepine; DCF, diclofenac; GFZ,
gemfibrozil; HCT, hydrochlorothiazide; IBP, ibuprofen; RNT,
This quinone redox cycle mechanism applied to WRF in- ranitidine; SMX, sulfamethoxazole; and SPD, sulpiride.
creases the range of environmental pollutants susceptible to be These compounds were chosen due to their occurrence and
biologically degraded due to the high oxidation power and potential toxicity in the effluent of WWTPs (Al Aukidy et al.
low substrate specificity of the oxidizing species generated. 2012; Kay et al. 2017). Malt extract (CAS 8002-48-0), sac-
The investigation of this biotechnology has been scarcely ex- charide, ammonium tartrate, sodium phosphate dibasic anhy-
plored for the degradation of emerging chemical pollutants drous, sodium phosphate monobasic monohydrate, Fe3+-oxa-
and the identification of the key steps to improve its efficiency late hexahydrated, Mn2+-nitrate monohydrated, and gallic ac-
as well as the degradation rate of pollutants appears as a stim- id were also purchased from Sigma-Aldrich (Spain). LC–MS
ulating challenge. quality methanol and the reagents used to adjust the pH were
To date, the works based on WRF as ABOP for the re- also obtained from Sigma-Aldrich.
moval of PCs have been performed at lab-scale in batch-wise
to treat sterilized simulated wastewaters spiked with PCs for Wastewater characterization
concentrations in the range of mg L−1 to μg L−1 (Marco-
Urrea et al. 2009, 2010). More recently, a real hospital waste- Two different wastewater streams (synthetic and real urban
water effluent under non-sterile conditions was studied in a wastewater effluents) were considered in this study. The syn-
batch aerated fluidized bed bioreactor with supplementary thetic urban wastewater (SUWW) was prepared according to
additions of glucose and ammonium tartrate during the treat- an adapted procedure described by Marco-Urrea et al. (2009).
ment (Cruz-Morató et al. 2014; Badia-Fabregat et al. 2016). This SUWW contained 0.6 g L−1 saccharide, 37 mg L−1 of
Considering these previous works, the main challenge of this ammonium tartrate, and 10 and 100 mL/L of micro- and
technology is the treatment of non-sterile effluents under macro-nutrient solutions, respectively. The macro-nutrient so-
continuous operation, with a realistic hydraulic retention lution contained 0.02 g L−1 KH2PO4, 0.005 g L−1 MgSO4·
time (HRT), for application in urban wastewater treatment 7H2O, and 10 mg L−1CaCl2 in distilled water, while the
plants, with non-addition of supplementary carbon and ni- micro-nutrient solution was consisted of minerals and vita-
trogen sources. The aim of the present work was to assess the mins. The real urban wastewater (RUWW) was collected after
viability of Trametes versicolor as WRF immobilized on the primary treatment of the pilot-scale WWTP located at
rotating biological contactor (RBC) for the continuous treat- Universidad Rey Juan Carlos at Móstoles, Madrid (Spain).
ment of a real effluent of primary treatment of WWTP. The Table S2 shows main characteristics of the SUWW and
advanced bio-oxidation of PCs was assessed with a HRT of RUWW.
1 day and non-supplementary loadings of nutrients. RBC
offers a large stability and low energy and maintenance de- Continuous ABOP in the RBC system
mands (Hassard et al. 2015). The feasibility of waste fungal
biomass as a substrate of anaerobic digestion (AD) for bio- The rotating biological contactor at lab-scale was provided by
gas production was also evaluated to achieve a sustainable ACAI Depuracion S.L. (Huesca, Spain). The bioreactor is
process, self-sufficient in energy, and minimizing waste made of AISI 304 stainless steel and has a total volume of
generation. 10 L with five disks. The diameter of each disk is 30 cm, and
Environ Sci Pollut Res

the total surface area for biomass attachment is 0.71 m2, being electrospray ionization interface as described by Vasiliadou
around 40% of each disk submerged in water. The disk rota- et al. (2013). Prior to the analysis, the samples were filtered
tional speed was 12 rpm (Pariente et al. 2013). Figure 1 shows through a durapore hydrophilic filter (PVDF, 0.65 μm) and
a schematic representation of the experimental bioreactor extracted using a polymeric cartridge (Oasis HLB SPE,
setup. 200 mg, from Waters). The solid-phase extraction (SPE) fol-
The RBC system was inoculated with blended mycelial lows a protocol with five steps: (i) conditioning of the car-
inoculum and initially operated in fed-batch mode for 45 days tridges with LC-MS methanol, (ii) equilibration of the car-
until obtaining a homogeneous biofilm immobilized over the tridges with ultrapure water, (iii) loading of the sample at
disks (start-up stage). During this step, the reactor was oper- constant flow rate, (iv) washing of the sorbent bed with a
ated in draw-fill cycles using a malt extract solution as specific solution of 5% LC-MS methanol in ultra-pure water, and (v)
fungal growth medium with ca. 3 g L−1 in terms of TOC drying under vacuum and elution with LC-MS methanol
concentration. Once the TOC was consumed, 1 L of the reac- (Afonso-Olivares et al. 2017).
tor’s medium was replaced with fresh malt extract (10% vol-
ume exchange) in order to restore again a TOC concentration Biochemical methane potential tests
of 3 g L−1 and start a new cycle. After that, the RBC system
was continuously operated with a HRT of 1 day for 15 days BMP tests were performed at 37 °C following a standardized
feeding an enriched SUWW (8 g L−1 saccharide, 498 mg L−1 assay described previously by Angelidaki et al. (2009). Ratios
ammonium tartrate, and 10 and 100 mL L−1 of the micro and of substrate/inoculum from 0.5 to 2 (1:2 to 2:1) w/w were
macronutrient solution, respectively) in absence of PCs (accli- studied in triplicate. The substrate of the bioassay was the
mation stage). Finally, the RBC was operated with the same residual fungal sludge settled from the outlet RBC effluent
HRT feeding the SUWW and RUWW for 120 and 40 days, in the treatment of RUWW. As inoculum, an anaerobic sludge
respectively. In this part of the study, the SUWW and RUWW collected from a full-scale anaerobic reactor located at Arroyo
were spiked with 8 and 12 pharmaceutical compounds at del Soto WWTP in Móstoles (Madrid, Spain) was used.
50 μg L−1 each (treatment stage), respectively. Likewise, as Biogas production was periodically measured for 34 days by
promoters of advanced bio-oxidation, metallic species in form water column displacement method using a Boyle-Mariotte
of Fe3+-oxalate (145 mg L−1) and Mn2+-nitrate (20 mg L−1) apparatus. The biogas composition (methane, carbon dioxide,
and quinone-like compounds as gallic acid (85 mg L−1) were and hydrogen) was analyzed using a gas chromatograph
added to both urban wastewaters. The concentrations of metal (7820A GC-TCD, Agilent Technologies, CA, USA).
sources and gallic acid were taken from the previous work of Methane yield was calculated as accumulated net methane
advanced bio-oxidation process performed by Gómez-Toribio volume divided by the amount of fungal biomass (mLCH4
et al. (2009). The inlet streams were adjusted to a pH of 4.5 as gVS−1) added initially to each bioassay (Segura et al. 2016).
acidification inhibits the bacteria cellular transport leading to
predominance of fungi activity in biological process, which
could be critical in case of non-sterilized RUWW (Woo-Sik Jo Results and discussion
et al. 2010). The HRT time of RBC was maintained at 1 day in
order to be more comparable to the HRT of biological second- The performance of white-rot fungi T. versicolor for advanced
ary treatments of urban WWTP (Vasiliadou et al. 2013). bio-oxidation of emerging pharmaceutical compounds at
Samples were periodically withdrawn from the inlet and outlet RBC in SUWW and RUWW was studied in terms of the
streams of RBC (Fig. 1, streams 2 and 3, respectively) for the following: (i) uptake of the carbon substrate and nutrients
treatment stage of SUWW and RUWW. (N-NH4+ and P-PO43−), (ii) the potential removal of pharma-
ceutical compounds, and (iii) evaluation of the biochemical
Analytical methods methane potential of residual fungal sludge for anaerobic
digestion.
Total organic carbon (TOC) was estimated using a
combustion/non-dispersive infrared gas analyzer model Performance of ABOP for carbon and nutrient
TOC-V Shimadzu. Chemical oxygen demand (COD), ammo- removals
nia nitrogen concentration, phosphate concentration, total
suspended solids (TSS), and volatile suspended solids (VSS) The SUWW and RUWW showed TOC/N/P ratios of 32:4:1
were determined following the standardized APHA methods and 69:8:1, respectively. Figures 2 and 3 depict the results of
(APHA 2005). pH was measured using a pH/ion-meter TOC and N-NH4+ of the inlet and outlet streams for the con-
(Metrohm781, Herisau/Switzerland). The pharmaceutical tinuous operation of RBC using a HRT of 1 day with SUWW
compounds were quantified by a LC-MS/MS triple quadru- from day 1 to day 120 and RUWW from day 121 to day 160.
pole mass spectrometer (Varian 325-MS) using vortex The inlet wastewater was acidified at pH 4.5 and spiked with
 Environ Sci Pollut Res

Fig. 1 Experimental setup: (1) RUWW, (2) feed flow with pH adjustment and spiked PCs, (3) treated wastewater from the RBC system, (4) clarified
water, and (5) residual sludge for valorization and final disposal

PCs at 50 μg L−1. The P-PO43− was not continuously moni- conditions, as it has been discussed in other studies demon-
tored along the operation time as TOC or N-NH4+, and only strating the release of nitrogen under these conditions (Zhang
data of days 149, 151, 153, and 154 of the treatment for and Geiβen 2012; Cruz-Morató et al. 2013; Badia-Fabregat
RUWW were analyzed (Table S3). et al. 2017). The growth of indigenous microbial of the
The results of TOC and N-NH4+ removals for the treatment RUWW in the RBC and the competition of these microorgan-
of both SUWW and RUWW evidenced a good performance of isms with fungus under the non-sterilized conditions are prob-
the fungal biomass. The TOC removal increased from 68 ± 25 ably giving a lower availability of carbon and nutrients for the
to 82 ± 12% for the RUWW. This improvement could be re- fungal population, producing the autolysis of the fungal
lated to the partial contribution of the bacterial activity of the mycelia and consequently, the release of nitrogen in the me-
real wastewater in spite of the partial bacterial inhibition in- dium. The comparison of the performance of white-rot fungi
duced by the reduction of pH to 4.5 (Yang et al. 2013a). as advanced bio-oxidation process in continuous RBC with
However, the nitrogen removal decreased from 73 ± 22 to conventional biological treatments in WWTPs evidenced that
61 ± 21% after ca. 140 days once the SUWW was shifted to the TOC removals (from 68 ± 25 to 82 ± 12%) were in the
RUWW and this wastewater was fed for 20 days. The increase range of the values of conventional activated sludge in
in the N-NH4+ concentration in the effluent of the RBC could WWTPs (Stensel et al. 2002). However, the range of removals
be related to the autolysis of fungal mycelia under starvation for N-NH4+ (from 73 ± 22 to 61 ± 21%) and P-PO43− (70 ±
5%, results only for the treatment of RUWW) were higher
RBC-influent RBC-effluent
than those of conventional activated sludge according to the
6000 results reported by six WWTPs (45 ± 24 and 65 ± 20% for N-
SUWW stage RUWW stage
NH4+ and P-PO43−, respectively) (Rodriguez-Garcia et al.
5000 2011). Therefore, the use of white-rot fungi as advanced bio-
oxidation process seems to be a good alternative for the con-
4000 tinuous treatment in RBC of non-sterile urban wastewater
TOC (mg d )
-1

acidified at 4.5 without additional substrates for fungi growth

3000 and 1 day of HRT, achieving remarkable removals of the TOC
and also of N-NH4+ and P-PO43− nutrients.
2000

1000
Performance of ABOP for emerging pharmaceutical
compounds
0
0 20 40 60 80 100 120 140 160 The efficiency of advanced bio-oxidation of T. versicolor was
Time (days) initially evaluated for the removal of eight spiked pharmaceu-
Fig. 2 Profiles of the TOC loadings for the SUWW and RUWW tical compounds at 50 μg L−1 each in the synthetic SUWW
treatment by ABOP (SMX, DCF, CFA, 4-AAA, CFN, GFZ, HCT, and CZP).
Environ Sci Pollut Res

Fig. 3 Profiles of the N-NH4+ RBC-influent RBC-effluent

100 600
loadings for the SUWW and SUWW stage RUWW stage
RUWW treatment by ABOP 90
500
80

N-NH4 (mg d ) SUWW

N-NH4 (mg d ) RUWW

70
400
60

-1

-1
50 300

+
40

30 200

20
100
10

0 0
0 20 40 60 80 100 120 140 160
Time (days)

Figure 4a shows the removal efficiencies of PCs for the con- CZP in an air pulsed fluidized bioreactor operated in continu-
tinuous operation of RBC with SUWW during 120 days and ous mode with a HRT of 3 days but only for the degradation of
1 day of HRT. The results correspond to the minimum, max- this compound as model pollutant. On the other hand, CZP
imum, and average removal percentage of 11 samples taken was not removed in experiments performed with sterilized and
during the treatment in the RBC (days 1, 4, 20, 31, 49, 57, 72, non-sterilized urban wastewater in the work reported by Cruz-
74, 81, 108, and 114, see Table S4). Taking into account the Morató et al. (2013).
average removals of these samples, efficiencies ranging from A more exhaustive evaluation of removal of pharmaceuti-
27% (SMX) to 53% (GFZ) were obtained for all the studied cal compounds was performed with the RUWW expanding
compounds, except for CFN that ca. 77% was achieved. the PCs to 12 target compounds spiked at 50 μg L−1 each in
However, a considerable dispersion of the results during the the RUWW. Figure 4b shows the removal efficiencies of PCs
120 days on operation of RBC is noteworthy, especially for for the continuous operation of RBC during 40 days (from day
CZP, HCT, and CFN. Nevertheless, these results of the ABOP 121 to day 160) and a HRT of 1 day. The results correspond to
with T. versicolor are similar or slightly better than others the minimum, maximum, and average removal efficiencies of
already published with this type of WRF. Thus, Vasiliadou 7 samples taken during the RBC operation (days 121, 125,
et al. (2016) reported removal values lower than 30% for 129, 136, 139, 153, and 156, see Table S5). Interestingly, the
ATN and lower than 20% for HCT, DCF, 4-AAA, and CZP removal efficiencies of PCs for the treatment of RUWW were
in discontinuous bioassays for 7 days of incubation under slightly higher than those obtained in the SUWW for the stud-
sterilized conditions using a synthetic defined medium. Jelic ied compounds. This increase is probably due to the additional
et al. (2012) also reported a removal efficiency of 54% for microbial activity of the indigenous microorganisms of the

Fig. 4 Intervals of removal of pharmaceutical compounds for the treatment of a SUWW and b RUWW by ABOP (red dot: average value)
 Environ Sci Pollut Res

real wastewater, which could grow with fungi during the con- achieved in WWTPs, 55% for 4-AAA and around 30% for
tinuous treatment in the RBC at non-sterilized conditions, in RNT. In the case of GFZ, the release of the parent compound
spite of its inhibition at reduced pH (4.5) (Badia-Fabregat from glucuronides or other conjugated metabolites during the
et al. 2016, 2017). This fact is likely the reason of the en- biological process produces negative removal values in con-
hanced removal capacity in the treatment of the RUWW as ventional activated sludge, only one WWTP reported values
compared to the SUWW. Likewise, the removal efficiency of in the range of 74–82%, being the rest of them lower than 50%
the ABOP for the RUWW was classified into three different or even lower with negative removals in some cases. HCT and
groups depending on their average removal values: moderate CFA displayed similar removal efficiencies in the ABOP and
removal (40–60%), significant removal (60–80%), and high the biological treatments of WWTPs with values ranging from
removal (80–100%). According to the results displayed in Fig. 40 to 61% (Rosal et al. 2010; Verlicchi et al. 2013). Finally,
4b, HCT, DCF, CFA, and CZP showed a moderate removal; PCs that are usually removed in low extension in WWTPs
GFZ, 4-AAA, and RNT significant removals, and SMX, ATN, such as DCF, lower than 30%, or CZP, with a wider range
CFN, IBP; and SPD high removal efficiencies. between negative values to almost 90%, but usually values
Previous works of air pulsed fluidized bioreactors using lower than also 30%, underwent higher removals for ABOP
T. versicolor for removal of pharmaceuticals in urban and with average values of 56 and 60% for DCF and CZP,
hospital wastewater with HRT of 3 or 2 days in some cases respectively.
(Mir-Tutusaus et al. 2017; Badia-Fabregat et al. 2017) re- The bio-refractory behavior of CZP for white-rot fungi has
vealed removal efficiencies ranging from 32 to 78% for been attributed to the presence of electron-acceptor functional
SMX, 14 to 67% for ATN, 20 to 96% for RNT, and 80 to groups (amide, azepine) in its molecular formula. Yang et al.
85% for IBP. These values are slightly lower than those ob- (2013b) have reported that PCs with electron-acceptor func-
tained in this work for the treatment of a real effluent coming tional groups are more difficult to remove by WRF than PCs
from a primary treatment of a pilot WWTP using RBC with with electron-donor functional groups. However, the pharma-
1 day of HRT. Moreover, it must be noted that the treatment of ceutical compounds usually have both type of functional
RUWW in the RBC was performed with non-addition of sup- groups, and then, its behavior is not easy to predict. Thus,
plementary nutrients (glucose and ammonia) and non-partial IBP with a carboxyl (electron-acceptor) and a methyl
renovation of fungal biomass unlike the previous mentioned (electron-donor) groups and SMX with a sulfonamide
works for urban and hospital wastewaters (Mir-Tutusaus et al. (electron-acceptor) and an amine (electron-donor) groups
2017; Badia-Fabregat et al. 2017). achieved high removal degrees (95 and 87%, respectively).
On the other hand, the efficiency of the ABOP in the RBC However, DCF with an electron-donor (amine) and various
was compared to the results of conventional biological treat- electron-acceptor groups (carboxylic and chlorine) only
ments of WWTPs. The pharmaceutical removal efficiencies of achieved 56%. Therefore, other factors are affecting the re-
eight WWTPs used for this study are summarized in Table S6 moval efficiency of PCs in biological processes. In this way,
(Martin et al. 2012; Verlicchi et al. 2013; Bollmann et al. the hydrophobicity of the PCs can promote its affinity to the
2016; Han-Tran and Yew-Hoong Gin 2017; Thiebault et al. solid phase of fungal biomass, becoming a complementary
2017). As can be seen from the data of WWTPs, the removal removal mechanism for hydrophobic compounds like the
efficiency significantly varies from one to other WWTP, main- cases of compounds with log KOW > 3.2. Only two of the
ly due to the different characteristics of the inlet wastewater studied pharmaceuticals possess log K OW > 3.2 (see
and operating conditions (HRT, SRT, and type of biological Table S1), GFZ and IBP, which achieved removals of 66
process). In any case, IBP, CFN, and ATN showed remarkable and 95%, respectively. In these cases, the removal by bio-
removals for the biological treatments of WWTPS. IBP and adsorption could be feasible or at least an important contribu-
CFN with ranges from 80 to 100% and ATN slightly lower tion. Additionally, hydrophilic compounds with pKa >
between 65 and 96%. The values of ABOP were around 90% pHmedium could be also adsorbed on the fungal biomass as
for IBP and CFN and 86% for ATN. In the case of SPD, the higher affinity is developed by non-ionic molecules. These
ABOP achieved a higher removal (95%, average removal val- could be the cases of HCT, CZP, SMX, RNT, CFN, ATN,
ue) whereas removals from 0 to 30% or 9 to 73 for the best and SPD. Hydrophilic compounds with pKa < pHmedium are
conditions were achieved for the biological treatments of expected to be as ionic forms promoting its dissolution in the
WWTP (Bollmann et al. 2016; Han-Tran and Yew-Hoong aqueous phase, such as DCF, 4-AAA, and CFA (Yang et al.
Gin 2017). The ABOP also showed a higher SMX removal 2013b; Grandclément et al. 2017). Hydrophilic PCs with
than biological treatments of WWTPs, with ca. 86% for pKa > pHmedium reached significant (60–80%) or high (80–
ABOP and much lower values of 17 or 52% in the case of 100%) removal yields, which can be associated to partial re-
activated sludge with nitrification/denitrification processes. moval by sorption processes of the fungal biomass. However,
The ABOP also accomplished removals between 60 and there have been published several works that discard signifi-
80% for GFZ, 4-AAA, and RNT which were higher than those cant contributions of sorption processes on the removal of PC
Environ Sci Pollut Res

compounds. Thus, Vasiliadou et al. (2016) reported a negligi- 1:2 1:1 2:1

ble contribution in the removal of GFZ, IBP, and other PCs 250
such as DCF, SMX, and SPD when using T. versicolor. A
more exhaustive and recent work focused on the role of sorp- 200

Yield (mLCH4 gvs )

-1
tion processes with different fungi, including T. versicolor, has
also reported values accounting for ca. 7% as mean value for 4 150
PCs that includes carbamazepine, diclofenac, iopromide, and
venlafaxine (Lucas et al. 2018). 100

Biochemical methane potential of fungal sludge 50

Figure 5 shows the cumulative methane production in the 0

BMP assays at different substrate to inoculum ratios. The 0 5 10 15 20 25 30 35

1:1 and 1:2 ratios achieved high methane yield (250 and Time (days)

225 mL CH4 gVS−1, respectively), and the biogas production Fig. 5 Time course of methane yield production for BMP tests
is almost completed after 20 days. The lower methane yield
for the substrate to inoculum ratio of 2:1 can be explained by fact seems to be also related to the enhancement of the ABOP
the accumulation of volatile fatty acid produced in anaerobic for the removal of pharmaceuticals in the treatment of the real
digestion of high concentration of organic substrate easily RUWW as compared to the SUWW. The ABOP operated in
fermentable in anaerobic digestion. It produces drop in pH, the RBC for the treatment of the real RUWW with 1 day of
which can inhibit the growth of methanogens, lowering the HRT, non-supplementary loads of extra substrates and no par-
methane production (Bouallagui et al. 2005; Ye et al. 2013). tial fungal replacement, showed higher removal efficiencies of
To date, there are only very few studies based on anaerobic pharmaceutical compounds than other fungal bioreactors re-
digestion of white-rot fungi sludge. Hom-Diaz et al. (2016) ported in literature and conventional biological processes of
reported a methane yield of 367 mL CH4 gVS−1 with a sub- WWTPs. Thus, 11 of the studied 12 studied PCs were elimi-
strate to inoculum ratio of 1:2, although these results were nated with removal efficiencies ranging from 50 to 95%.
obtained with fungal sludge as substrate grown in batch- Among them, compounds such as diclofenac or carbamaze-
wise with a very high organic matter concentration, far from pine, commonly characterized as refractory compounds for
real operation conditions of an urban WWTP. biological treatments, were removed with efficiencies of 56
As compared to sludge from conventional WWTPs as or- and 61%, respectively. Biochemical methane potential of re-
ganic substrates, the methane yield of fungal sludge is rela- sidual fungal sludge of the continuous RBC produced a meth-
tively higher than the results reported in previous studies. ane yield of 250 mLCH4 gVS−1 in anaerobic digestion process-
Activated sludge collected from a municipal WWTP achieved es. Therefore, the use of T. versicolor as WRF in RBC is
a methane yield of 192 mL CH4 gVS−1 and a sewage sludge considered a sustainable advanced biological treatment with
from a treatment facility obtained a value of 165 mL CH4 potential application for emerging PC removal and feasible
gVS−1(Park et al. 2016). Thus, these results show that valori- energy recovery by anaerobic digestion.
zation of fungal waste sludge through anaerobic digestion
could be an interesting alternative for a partial recovery of Acknowledgements The authors gratefully acknowledge the financial
support of Regional Government of Madrid provided through project
energy for the operation of wastewater treatment plant.
REMTAVARES (S2013/MAE-2716) and the European Social Fund and
Spanish Ministry of Economy and Competitiveness (MINECO) in the
frame of the collaborative international consortium WATERJPI2013-
Conclusions MOTREM of the Water Challenges for a Changing World Joint
Programming Initiative (Water JPI) Pilot Call.

The advanced bio-oxidation (ABOP) based on the immobili-

zation of T. versicolor as WRF over RBC was assessed in the
continuous treatment of a synthetic urban (SUWW) and real
urban (RUWW) wastewater. The biological performance of References
ABOP in terms of the TOC removal increased for the treat-
ment of RUWW. However, this performance decreased in Afonso-Olivares C, Čadková T, Sosa-Ferrera Z, Santana-Rodríguez JJ,
terms of the N-NH4+ elimination, which seems to indicate Nováková L (2017) Simplified solid-phase extraction procedure
combined with liquid chromatography tandem–mass spectrometry
signs of autolysis of fungal mycelia under starvation condi- for multiresidue assessment of pharmaceutical compounds in envi-
tions, probably due to the coexistence of fungal and indige- ronmental liquid samples. J Chromatogr 1487:54–63. https://doi.
nous microbial of the RUWW at non-sterile conditions. This org/10.1016/j.chroma.2017.01.059
 Environ Sci Pollut Res

Al Aukidy M, Verlicchi P, Jelic A, Petrovic M, Barceló D (2012) review. Process Saf Environ 94:285–306. https://doi.org/10.1016/j.
Monitoring release of pharmaceutical compounds: occurrence and psep.2014.07.003
environmental risk assessment of two WWTP effluents and their Hom-Diaz A, Baldi F, Blánquez P, Lombardi L, Martín-González L,
receiving bodies in the Po Valley, Italy. Sci Total Environ 438:15– Vicent T (2016) Exhausted fungal biomass as a feedstock for in-
25. https://doi.org/10.1016/j.scitotenv.2012.08.061 creasing methane production during the anaerobic digestion of or-
Angelidaki I, Alves M, Bolzonella D, Borzacconi L, Campos JL, Guwy ganic wastes. Waste Biomass Valori 7(2):307–315. https://doi.org/
AJ, Kalyuzhnyi S, Jenicek P, Van Lier JB (2009) Defining the 10.1007/s12649-015-9450-8
biomethane potential (BMP) of solid organic wastes and energy Jelic A, Cruz-Morató C, Marcos-Urrea E, Sarrà M, Pérez S, Vicent T,
crops: a proposed protocol for batch assays. Water Sci Technol Petrovic M, Barceló D (2012) Degradation of carbamazepine by
59(5):927–934. https://doi.org/10.2166/wst.2009.040 Trametes versicolor in an air pulsed fluidized bed bioreactor and
APHA (2005) Standard methods for the examination of water and waste- identification of intermediates. Water Res 46:955–964. https://doi.
water. American Public Health Association, Washington DC org/10.1016/j.watres.2011.11.063
Archer E, Petrie E, Zasprzyk-Horderm B, Wofaardt GG (2017) The fate Kay P, Hughes SR, Ault JR, Ashcroft AE, Brown LE (2017) Widespread,
of pharmaceuticals and personal care products (PPCPs), endocrine routine occurrence of pharmaceuticals in sewage effluent, combined
disrupting contaminants (EDCs), metabolites and illicit drugs in a sewer overflows and receiving waters. Environ Pollut 220(Pt B):
WWTW and environmental waters. Chemosphere 174:437–446. 1447–1455. https://doi.org/10.1016/j.envpol.2016.10.087
https://doi.org/10.1016/j.chemosphere.2017.01.101 Lucas D, Castellet-Rovira F, Villagrasa M, Badia-Fabregat M, Barceló D,
Badia-Fabregat M, Lucas D, Alcina Pereira M, Alves M, Pennanen T, Vicent T, Caminal G, Sarrá M, Rodriguez-Mozaz S (2018) The role
Fritze H, Rodríguez-Mozaz S, Barcelò D, Vicent T, Caminal G of sorption processes in the removal of pharmaceuticals by fungal
(2016) Continuous fungal treatment of non-sterile veterinary hospi- treatment of wastewater. Sci Total Environ 610-611:1147–1153.
tal effluent: pharmaceuticals removal and microbial community as- https://doi.org/10.1016/j.scitotenv.2017.08.118
sessment. Appl Microbiol Biotechnol 100(5):2401–2415. https:// Marco-Urrea E, Pérez-Trujillo M, Vicent T, Caminal G (2009) Ability of
doi.org/10.1007/s00253-015-7105-0 white-rot fungi to remove selected pharmaceuticals and identifica-
Badia-Fabregat M, Lucas D, Tuomivirta T, Fritze H, Pennanen T, tion of degradation products of ibuprofen by Trametes versicolor.
Rodríguez-Mozaz S, Barcelò D, Caminal G, Vicent T (2017) Chemosphere 74(6):765–772. https://doi.org/10.1016/j.
Study of the effect of the bacterial and fungal communities present chemosphere.2008.10.040
in real wastewater effluents on the performance of fungal treatments. Marco-Urrea E, Radjenović J, Caminal G, Petrović M, Vicent T, Barceló
Sci Total Environ 570:366–377. https://doi.org/10.1016/j.scitotenv. D (2010) Oxidation of atenolol, propranolol, carbamazepine and
2016.11.088 clofibric acid by a biological Fenton-like system mediated by the
Bollmann AF, Seitz W, Prasse C, Lucke T, Schulz W, Ternes T (2016) white-rot fungus Trametes versicolor. Water Res 44(2):521–532.
Occurrence and fate of amisulpride, sulpiride, and lamotrigine in https://doi.org/10.1016/j.watres.2009.09.049
municipal wastewater treatment plants with biological treatment Martin J, Camacho-Muñoz D, Santos JL, Aparicio I, Alonso E (2012)
and ozonation. J Hazar Mater 320:204–215. https://doi.org/10. Occurrence of pharmaceutical compounds in wastewater and sludge
1016/j.jhazmat.2016.08.022 from wastewater treatment plants: removal and ecotoxicological im-
Bouallagui H, Touhami Y, Ben Cheikh R, Hamdi M (2005) Bioreactor pact of wastewater discharges and sludge disposal. J Hazard Mater
performance in anaerobic digestion of fruit and vegetable wastes. 239-240:40–47. https://doi.org/10.1016/j.jhazmat.2012.04.068
Process Biochem 40(3-4):989–995. https://doi.org/10.1016/j. Mendoza A, Zonja B, Mastroianni N, Negreira N, López de Alda M,
procbio.2004.03.007 Pérez S, Barcelò D, Gil A, Valcárcel Y (2016) Drugs of abuse,
Cruz-Morató C, Ferrando-Climent L, Rodriguez-Mozaz S, Barcelò D, cytostatic drugs and iodinated contrast media in tap water from the
Marco-Urrea E, Vicent T, Sarrà M (2013) Degradation of pharma- Madrid region (central Spain):a case study to analyse their occur-
ceuticals in non-sterile urban wastewater by Trametes versicolor in a rence and human health risk characterization. Environ Int 86:107–
fluidized bed bioreactor. Water Res 47(14):5200–5210. https://doi. 118. https://doi.org/10.1016/j.envint.2015.11.001
org/10.1016/j.watres.2013.06.007 Mir-Tutusaus JA, Parladé E, Llorca M, Villagrasa A, Barceló D,
Cruz-Morató C, Lucas D, Llorca M, Rodríguez-Mozaz S, Gorga M, Rodriguez-Mozaz S, Martinez-Alonso M, Gaju N, Caminal G,
Petrovic M, Barcelò D, Vicent T, Sarrà M, Marco-Urrea E (2014) Sarrà M (2017) Pharmaceuticals removal and microbial community
Hospital wastewater treatment by fungal bioreactor: removal effi- assesment in a continuous fungal treatment of non-sterile real hos-
ciency for pharmaceuticals and endocrine disruptor compounds. Sci pital wastewater after coagulation-flocculation pretreatment. Water
Total Environ 493:365–376. https://doi.org/10.1016/j.scitotenv. Res 116:65–75. https://doi.org/10.1016/j.watres.2017.03.005
2014.05.117 Oller I, Malato S, Sánchez-Pérez JA (2011) Combination of advanced
Gómez-Toribio V, García-Martín AB, Martínez MJ, Martínez AT, Guillén oxidation processes and biological treatments for wastewater decon-
F (2009) Enhancing the production of hydroxyl radicals by tamination: a review. Sci Total Environ 409(20):4141–4166. https://
Pleurotus eryngii via Quinone redox cycling for pollutant. Appl doi.org/10.1016/j.scitotenv.2010.08.061
Environ Microbiol 75(12):3954–3962. https://doi.org/10.1128/ Pariente MI, Siles JA, Molina R, Botas JA, Melero JA, Martinez F (2013)
AEM.02138-08 Treatment of an agrochemical wastewater by integration of hetero-
Grandclément C, Seyssiecq I, Piram A, Wong-Wah-Chung P, Vanot G, geneous catalytic wet hydrogen peroxide oxidation and rotating bi-
Tiliacos N, Roche N, Doumenq P (2017) From the conventional ological contactors. Chem Eng J 226:409–415. https://doi.org/10.
biological wastewater treatment to hybrid processes, the evaluation 1016/j.cej.2013.04.081
of organic micropollutant removal: a review. Water Res 111:297– Park KY, Jang HM, Park MR, Lee K, Kim D, Kim YM (2016)
317. https://doi.org/10.1016/j.watres.2017.01.005 Combination of different substrates to improve anaerobic digestion
Han-Tran N, Yew-Hoong Gin K (2017) Occurrence and removal of phar- of sewage sludge in a wastewater treatment plant. Int Biodeterior
maceuticals, hormones, personal care products, and endocrine dis- Biodegradation 109:73–77. https://doi.org/10.1016/j.ibiod.2016.01.
rupters in a full-scale water reclamation plant. Sci Total Environ 006
599-600:1503–1516. https://doi.org/10.1016/j.scitotenv.2017.05. Pignatello JJ, Oliveros E, MacKay A (2006) Advanced oxidation pro-
097 cesses for organic contaminant destruction based on the Fenton re-
Hassard F, Biddle J, Cartmell E, Jefferson B, Tyrrel S, Stephenson T action and related chemistry. Crit Rev Environ Sci Tech 36(1):1–84.
(2015) Rotating biological contactors for wastewater treatment – a https://doi.org/10.1080/10643380500326564
Environ Sci Pollut Res

Rodriguez-Garcia G, Molinos-Senante M, Hospido A, Hernández- Verlicchi P, Galleti A, Petrovic M, Barcelò D, Al Aukidy M, Zambello E
Sancho F, Moreira MT, Feijoo G (2011) Environmental and eco- (2013) Removal of selected pharmaceuticals from domestic waste-
nomic profile of six typologies of wastewater treatment plants. water in an activated sludge system followed by a horizontal sub-
Water Res 45(18):5997–6010. https://doi.org/10.1016/j.watres. surface flow bed — analysis of their respective contributions. Sci
2011.08.053 Total Environ 454:411–425. https://doi.org/10.1016/j.scitotenv.
Rosal R, Rodriguez A, Perdigon-Melon JA, Petre A, Garcia-Calvo E, 2013.03.044
Gomez MJ, Agüera A, Fernandez-Alba AR (2010) Occurrence of Wei D, Houtman CJ, Kapich AN, Hunt CG, Cullen D, Hammel KE
emerging pollutants in urban wastewater and their removal through (2010) Laccase and its role in production of extracellular reactive
biological treatment followed by ozonation. Water Res 44(2):578– oxygen species during wood decay by the Brown rot basidiomycete
588. https://doi.org/10.1016/j.watres.2009.07.004 Postia placenta. Appl Environ Microbiol 76(7):2091–2097. https://
Segura Y, Puyol D, Ballesteros L, Martínez F, Melero JA (2016) doi.org/10.1128/AEM.02929-09
Wastewater sludges pretreated by different oxidation systems at mild Woo-Sik Jo MJK, Seong-Yong C, Young-Bok Y, Soon-Ja S, Hee-Young
conditions to promote the biogas formation in anaerobic processes. J (2010) Culture conditions for mycelial growth of Coriolus
Environ Sci Pollut Res Int 23(23):24393–24401. https://doi.org/10. versicolor. Mycobiology 38(3):195–202. https://doi.org/10.4489/
1007/s11356-016-7535-y MYCO.2010.38.3.195
Tchobanoglous G, Stensel HD, Tsuchihashi R, Burton FL (2002) Yang S, Hai FI, Nghiem LD, Nguyen LN, Roddick F, Price WE (2013a)
Wastewater engineering: treatment and reuse, Metcalf and Eddy Removal of bisphenol a and diclofenac by a novel fungal membrane
Inc, 4th edn. McGraw-Hill Education, New York bioreactor operated under non-sterile conditions. Int Biodeterior
Thiebault T, Boussafir M, Le Milbeau C (2017) Occurrence and removal Biodegradation 85:483–490. https://doi.org/10.1016/j.ibiod.2013.
efficiency of pharmaceuticals in an urban wastewater treatment plant: 03.012
mass balance, fate and consumption assessment. J Environ Chem Yang S, Hai FI, Nghiem LD, Price WE, Roddick F, Moreira MT, Magram
Eng 5(3):2894–2902. https://doi.org/10.1016/j.jece.2017.05.039 SF (2013b) Understanding the factors controlling the removal of
Valcárcel Y, Martínez F, Gónzalez-Alonso S, Segura Y, Catalá M, Molina trace organic contaminants by white-rot fungi and their lignin mod-
R, Montero-Rubio JC, Mastroianni N, López de Alda M, Postigo C, ifying enzymes: a critical review. Bioresour Technol 141:97–108.
Barcelò D (2012) Drugs of abuse in surface and tap waters of the https://doi.org/10.1016/j.biortech.2013.01.173
Tagus River basin: heterogeneous photo-Fenton process is effective
Ye J, Li D, Sun Y, Wang G, Yuan Z, Zhen F, Wang Y (2013) Improved
in their degradation. Environ Int 41:35–43. https://doi.org/10.1016/j.
biogas production from rice straw by co-digestion with kitchen
envint.2011.12.006
waste and pig manure. Waste Manag 33(12):2653–2658. https://
Vasiliadou IA, Molina R, Martínez F, Melero JA (2013) Biological re-
doi.org/10.1016/j.wasman.2013.05.014
moval of pharmaceutical and personal care products by a mixed
microbial culture: sorption, desorption and biodegradation. Zenker A, Cicero MR, Prestinaci F, Bottoni P, Carere M (2014)
Biochem Eng J 81:108–119. https://doi.org/10.1016/j.bej.2013.10. Bioaccumulation and biomagnification potential of pharmaceuticals
010 with a focus to the aquatic environment. J Environ Manag 133:378–
Vasiliadou IA, Sánchez-Vázquez R, Molina R, Martínez F, Melero JA, 387. https://doi.org/10.1016/j.jenvman.2013.12.017
Bautista LF, Iglesias J, Morales G (2016) Biological removal of Zhang Y, Geiβen S-V (2012) Elimination of carbamazepine in a non-
pharmaceutical compounds using white-rot fungi with concomitant sterile fungal bioreactor. Bioresour Technol 112:221–227. https://
FAME production of the residual biomass. J Environ Manag 180: doi.org/10.1016/j.biortech.2012.02.073
228–237. https://doi.org/10.1016/j.jenvman.2016.05.035