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Kinetic assessment of antibiotic resistant bacteria inactivation by solar photo-Fenton in batch and continuous flow mode for wastewater reuse
Water Research 159 (2019) 184e191
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Kinetic assessment of antibiotic resistant bacteria inactivation by solar photo-Fenton in batch and continuous flow mode for wastewater reuse nez a, b, J.L. Casas Lo pez a, b, G. Rivas Iba n ~ ez a, c, B. Esteban García a, b, I. De la Obra Jime a , b, * nchez Pe rez J.A. Sa a b c
Solar Energy Research Centre (CIESOL), Joint Centre University of Almería-CIEMAT, 04120, Almería, Spain Department of Chemical Engineering, University of Almería, 04120, Almería, Spain Plataforma Solar de Almería, CIEMAT, 04200, Tabernas, Almería, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 February 2019 Received in revised form 28 April 2019 Accepted 30 April 2019 Available online 7 May 2019
The presence of antibiotic resistant bacteria in municipal wastewater treatment plants represents a real risk to human health. For the first time, this paper shows that the inactivation rate of cefotaxime resistant bacteria is the same as total bacteria when secondary effluents are treated by the solar photo-Fenton process. To obtain this result, an exhaustive and comparative kinetic study on the inactivation of both total and cefotaxime resistant bacteria (Total coliform, Escherichia coli and Enterococcus sp) was carried out, taking into account the effects of the main operation conditions, such as solar irradiance and iron concentration, and operation mode (batch and continuous). In all the operation conditions studied, no significant differences were found between the first order inactivation rate constants, ki, of total and cefotaxime resistant bacteria. Additionally, ki increased with solar irradiance and iron concentration. As for the effect of the operation mode, the main finding of this work is much quicker inactivation in continuous flow mode than in batch mode, pointing out its potential application at large scale. The best continuous operation condition to inactivate the bacteria to the detection limit (1 CFU mL1), was at 22.4 min of hydraulic residence time with 5 mg Fe2þ L1 and 30 mg H2O2$L1. This treatment time is approximately a third of that reported in batch mode. The efficiency, in terms of figure of merits, of the continuous flow operation was 2.7 m2 of solar collector area to reduce one log of E. coli concentration per m3 of treated water and per hour, in comparison with 2137 m2 calculated for batch operation under the same solar UVA irradiance, 30 W m2. This paper encourages research into continuous solar disinfection processes due to its enhanced efficiency with regard to the commonly used batch wise operation and shows that efficient removal of total bacteria ensures the removal of antibiotic resistant bacteria. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Wastewater disinfection Antibiotic resistant bacteria Disinfection kinetics Solar treatment Efficiency analysis
1. Introduction The reuse of treated wastewater for crop irrigation has become an increasingly recommended practice to address water scarcity in the EU and worldwide (Michael-Kordatou et al., 2018). However, the presence of the so-called “new contaminants” or “contaminants of emerging concern” in treated wastewater has led to increased concern about the potential direct and indirect effects on the environment and possible implications for human health
* Corresponding author. Department of Chemical Engineering, University of Almería, 04120, Almería, Spain. rez). E-mail address: [email protected] (J.A.S. Pe https://doi.org/10.1016/j.watres.2019.04.059 0043-1354/© 2019 Elsevier Ltd. All rights reserved.
(Kümmerer et al., 2016). In this regard, special attention has been paid to the contribution of treated wastewater concerning the emergence of antibiotic resistance in pathogenic bacteria, claimed to be “one of the major global threats to society” in the twenty first century by the World Health Organization (WHO, 2014). Municipal wastewater treatment plants (MWWTPs) are considered to be significant reservoirs of antibiotic resistant bacteria as they combine a high load of bacteria with a residual concentration of antibiotics (Fatta-kassinos, 2016). The biological treatments typically applied in MWWTPs are poorly effective in the complete disinfection of the wastewater, generating secondary effluents with about 109-1012 Colony Forming Units (CFU) per day and per inhabitant equivalent. Among these, at least 107e1010 CFU
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could have acquired antibiotic resistance (Rizzo et al., 2013a,b). Thus, the large amount of antibiotic resistant bacteria (ARB) discharged when treated wastewater is used for irrigation which may proliferate in soils or plants (Christou et al., 2017). Therefore, conventional MWWTPs should be upgraded with more efficient technologies such as tertiary treatments to provide enough protection for the potential risks related to water reuse, not only by meeting the current quality standards, but also by controlling the spread of ARB into the environment. In fact, currents standards in water reuse for irrigation purposes relies on the control of chemical and microbiological parameters like heavy metals or enteric pathogens (Escherichia coli and Enterococcus sp), but do not evaluate ARB levels (Fatta-kassinos, 2016). In this regard, the measurement of bacteria resistant to Cefotaxime (third-generation cephalosporin, which is on the WHO essential list of medicine) could be used as a reliable indicator of wastewater resistance levels as proposed by the Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) on the “Proposed EU minimum quality requirements for water reuse in agricultural irrigation and aquifer recharge”, a report prepared by the European Commission Joint Research Centre (SCHEER, 2017). Despite the most common tertiary treatments used as a disinfection step before secondary effluent reuse having proved to be efficient in microorganism inactivation (namely, ozonation, chlorination or UVC irradiation) (Xu et al., 2002; Rodriguez-Chueca et al., 2015; Rizzo et al., 2019), their efficacy for effective reduction of antibiotic resistance is not completely agreed upon (Luigi Rizzo et al., 2013; Yuan et al., 2015; Sousa et al., 2017; MichaelKordatou et al., 2018). Additionally, other drawbacks related to the generation of undesirable disinfection by-products through ozonation (Stalter et al., 2010) or chlorination (Watson et al., 2012) has forced research to seek out disinfection alternatives that are environmentally safer than conventional tertiary treatments, namely the solar photo-Fenton process. This Advanced Oxidation Process (AOP) has been found to be efficient, not only at degrading antibiotics, but also at inactivating microorganisms and ARB (Giannakis et al., 2018a) without increasing toxicity (Michael et al., 2012). It is based on the generation of highly oxidative and nonselective species, hydroxyl radicals (HO), that degrade the external membrane of bacteria, increasing its permeability and inducing internal reactions that eventually inactivate the microorganisms (Giannakis et al., 2018b). The generation of HO in the Fenton process relies on the repeated oxidation and reduction of iron (catalyst) by hydrogen peroxide. Moreover, in the presence of UVevis radiation, the HO production rate is increased due to the regeneration of Fe2þ by the photo-conversion of ferric iron to ferrous iron (Pignatello et al., 2006). Despite the most desirable pH for radical production being around 2.8e3.0, owing to the low solubility of Fe3þ at higher pH, many recent studies have confirmed the effectiveness of the photo-Fenton process for bacterial inactivation at neutral pH (Giannakis et al., 2016b). Nevertheless, to our knowledge, the industrial application of this solar process as a tertiary treatment for wastewater disinfection has not yet been developed, even though efforts have been directed towards making its application at large scale feasible via compound parabolic collectors (CPCs) (Rodríguez-Chueca et al., 2014) or low cost reactors such as raceway pond reactors (RPRs) (Esteban García et al., 2018). In particular, the latter has emerged as an attractive option, not only for the remarkable reduction in investment cost (RPR construction costs are 40 times cheaper than those for CPCs), but also for the larger volume of wastewater than can be treated per surface area (Carra et al., 2014). In fact, to bring closer to reality the solar photoFenton implementation, the large amount of secondary effluent generated in MWWTPs needs to be dealt with. With this in mind, the feasibility of the continuous flow operation of the solar photo-
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Fenton process in RPR for secondary effluent disinfection has recently been investigated with promising results (De la Obra et al., 2019). The objective of this work was to carry out an exhaustive and comparative kinetic study on the inactivation of both total and cefotaxime resistant bacteria by solar photo-Fenton at neutral pH in a wide range of operation conditions, taking into account different bacterial species (Total coliform, Escherichia coli and Enterococcus sp). To this end, the effects of solar irradiance and iron concentration as well as operation mode, batch vs. continuous, were analysed. 2. Materials and methods 2.1. Wastewater source Secondary effluent batches of 1 m3 were taken from the Municipal Wastewater Treatment Plant named “El Bobar” (Almeria, Southeastern Spain). A characterization of wastewater samples was carried out during the first three days of experimentation to assess their variability. The average values and standard deviation of the main physico-chemical parameters during the experimental period (from October 2017 to February 2019) are included as Supplementary Material in Table S1. All experiments were carried out at the natural pH of secondary effluent samples, around 7.3 ± 0.4 and remained constant during treatment time. 2.2. Experimental set-up Batch wise experiments were conducted in 5 cm-deep RPRs with initial hydrogen peroxide and iron concentrations of 50 and 20 mg L1, respectively. The effect of solar UVA irradiance on bacteria inactivation was evaluated at 12.85 ± 1.07 W m2, 30.38 ± 0.69 W m2 and 37.57 ± 0.55 W m2, corresponding to winter, spring and summer, respectively. These experiments have been partially reported by (De la Obra et al., 2019). In the present study, new results of cefotaxime resistant (CFX-R) bacteria inactivation are discussed. As for studying the influence of iron concentration on both total and CFX-R bacteria inactivation, iron concentration was varied (20, 10, 5 and 2.5 mg L1) while maintaining the initial H2O2 concentration at 50 mg L1 and the irradiance at 13 ± 1 W m2, corresponding to winter conditions. This experimental setup is shown in Fig. 1a. Temperature (LabJack EI1034) and pH (Crison 5335) were monitored with a LabJack U12 data acquisition card connected to a computer. UVA radiation was measured (W m2) with a horizontal global UV radiometer (Delta Ohm, LP UVA 02 AV), the spectral response range being 327e384 nm. Control experiments in batch mode were performed outdoors under the following conditions: i) Dark without any reagent to check the effect of the mechanical stress; ii) In the presence of sunlight (UVA) and iii) Sunlight with hydrogen peroxide (UVA/ H2O2). Mechanical stress had no effect on bacteria inactivation, while 2 and 2.5-log decrease were observed for TC and E. coli, respectively, after 120 min of treatment, and the synergic effect of UVA þ H2O2 caused 3-log inactivation after 200 min for TC and E. coli. Significant differences can be observed for Enterococcus sp., with no bacterial damage in any control assayed. These results are in concordance with those previously reported (Esteban García et al., 2018). Apart from that, the influence of hydraulic residence time (HRT) on total and CFX-R bacteria inactivation in continuous flow was studied at laboratory scale in a Sun Test CPS þ solar simulator at 30 W m2 of UVA irradiance and a temperature of 25 C using a Thermo Scientific NESLAB RTE-7 thermostatic bath. To
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2.3. Total and antibiotic resistant bacteria quantification Bovine liver catalase (Sigma-Aldrich, Spain) was added to the wastewater samples before microbiological analysis in order to ndez et al., 2012). remove residual hydrogen peroxide (García-Ferna The bacteria count in the samples collected during photo-Fenton experiments was carried out using the standard plated counting method through serial 10-fold dilutions with saline solution (0.8% NaCl) in selective media. To count antibiotic resistant bacteria in the samples, the selective media was enriched with 4 mg L1 of cefotaxime (CFX), a third-generation cephalosporin. This concentration was reported in the documented CLSI minimal inhibitory concentration (MIC) breakpoint levels for Enterobacteriaceae (Clinical and Laboratory Standards Institute, Wayne, PA, 2011). The enumeration of colonies was performed after one day of incubation for TC and E. coli in Chromocult-agar (Merk, Germany) and two days for Enterococcus sp. in Enterococcus-medium (Pronadisa, Spain) at 37 C in both total and CFX-R. For plate counting, 50-250-5001000 mL of sample were used, reaching the microbial detection limit (DL) of 1 CFU mL1. All procedures were carried out in duplicate for each sample. Regrowth in photo-Fenton treated water was checked by storing 200 mL of the sample in the dark at room temperature overnight. The stored samples were plated for colony counting as previously described. No regrowth was observed in any case. 2.4. Kinetic analysis Chick's first order kinetic equation was chosen to fit the decrease of total and CFX-R bacterial population, Equation (1)
log
Fig. 1. Experimental setups for wastewater disinfection by photo-Fenton: a) Raceway pond reactors operating in batch mode under natural sunlight. b) Stirred tank reactor operating in continuous flow mode under simulated solar light.
C ¼ ki $t C0
(1)
where C/C0 is the reduction in the bacterial concentration, ki is the disinfection rate constant in min1 and t is the photo-Fenton time in min. 2.5. Mass balances
this end, a 1 L-stirred PVC cylindrical reactor with 5 cm of liquid depth and 16 cm of internal diameter was selected to perform this study (Fig. 1b). To ensure perfect mixing, magnetic agitation was set to reach a mixing time of 2.4 s, a value much shorter than the photo-Fenton treatment time. Four input flows 33.3, 50, 100 and 200 mL min1 for the secondary effluent feed pump and two different input flows, 4.2 and 7.2 mL min1, for the H2O2 and Fe2þ stock solutions respectively, were established giving rise to four values of HRT, 22.4, 16.3, 9.0 and 4.7 min. The inlet iron and H2O2 concentrations were 5 mg L1 and 30 mg L1, respectively. The reactor start-up was carried out in batch mode for 2 h followed by the continuous supply of reactants and secondary effluent, giving rise to the continuous flow mode. Each HRT was maintained for 2 h to collect enough data once steady state was achieved. Additional control assays were carried out in continuous flow mode at 22.4 min of HRT. The effects of i) the mechanical stress, ii) UVA and iii) UVA þ H2O2 on TC, E. coli and Enterococcus sp. inactivation were evaluated. Mechanical stress due to magnetic stirring led to 1-log decrease for TC while not damage was observed for E. coli and Enterococcus sp. The exposure to UVA irradiance caused 1.5-log decrease in TC (1-log due to mechanical stress) and 1-log decrease in E. coli. In the case of Enterococcus sp. no bacterial damage was observed. The combined effect of UVA þ H2O2 did not cause any additional effect with regard to UVA for the three species studied.
For the kinetic studies, the mass balance of viable bacteria in the raceway pond reactor can be expressed as follows, Equation (2)
dCFU 1 ¼ ðC Cs Þ ki $Cs dt HRT i
(2)
where dCFU is the accumulation of bacterial communities in the dt 1 pilot-RPR during the solar photo-Fenton process, HRT is the dilution rate of the system in min1, ki is the bacterial inactivation rate constant (min1) and Ci and Cs are the inlet and outlet concentrations of microorganisms, respectively in CFU$mL1. 2.6. Process efficiency quantification The standard figures of merit suggested by IUPAC (Bolton et al., 2001) for the comparison and evaluation of advanced oxidation process (AOPs) can either be applied in terms of energy requirements, in the case of devices using artificial light, or in terms of area required, in the case of solar light applications, which is the case for the present study. In this regard, the figure of merit used was that intended for solar-driven systems, namely the collector area per order (Aco, m2$m3$order1). Aco is the collector area needed to inactivate 1log of microorganism per unit of volume over a time (t0) when “standardized” incident solar UV radiation was 30 W m2 and calculated for batch system (ABco) and continuous flow operation
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(ACco) following Equation (3) and Equation (4), respectively:
ABco ¼
ACco ¼
A$UV UV0 $t0 $V$0:4334$ki A$UV UV0 $t0 $Q $log CC0
(3)
(4)
where A (m2) is the collector area used, UV is the average of the incident solar UV radiation expressed in W$m2 and UV0 is “standardized” solar UVA radiation corresponding to 30 W m2 (typical solar UVA power on a perfect sunny Spring day around noon in Almería). V (m3) is the volume treated in batch operation and Q is the flow rate in m3$h1. C0 is the initial bacterial concentration and C is the bacterial concentration after 1 h of treatment. The average bacterial inactivation rate, ki , (h1) is obtained by a first order kinetic equation (Section 2.4). In addition, 0.4334 is the conversion factor between decimal and neperian logarithm. 2.7. Reagents and analytical methods Total and dissolved iron and hydrogen peroxide were measured by the standardized methods previously described by de la Obra et al. (De la Obra et al., 2019). The pH of iron stock solution was adjusted to 2.8 with sulphuric acid 0.1 M (95e98%, Panreac, Spain). To check the physico-chemical quality of secondary effluent batches, ion chromatography (Metrohm 881 Compact IC pro) and a TOC-V CSH/CSN analyser (SHIMADZU Corporation) were used for determining, by direct injection of filtered samples, anions and the dissolved carbon, organic and total inorganic carbon (TIC) concentrations, respectively. The chemical oxygen demand (COD) was quantified using a commercial kit (Hach LCK 314). Standard parameters such as pH and turbidity were measured using a portable pH-meter and turbidity-meter both acquired from Hanna, while the conductivity was determined by means of a conductivity-meter (Phywe). 3. Results and discussion 3.1. Influence of UV irradiance and iron concentration in batch mode operation on CFX-R bacteria inactivation The inactivation kinetics of ARB is studied in batch mode, since a simple first-order model is used to calculate the rate constants for comparison purposes. Fig. 2 shows the variation in the inactivation rate constants, ki, for total and CFX-R bacteria at three irradiance levels related to winter, spring and summer in Almería, Spain. In addition, the inactivation rate constants from Fenton control experiments were also included. In order to obtain accurate results and taking into account that the experiments were carried out in outdoor conditions, five replicates were carried out for 2 h at each irradiance level with initial iron and hydrogen peroxide concentrations of 20 and 50 mg L1, respectively, as reported to be the best reactant concentration for bacteria inactivation by solar photo mez et al., 2014). Significant difFenton in batch mode (Ortega-Go ferences in the initial bacterial concentration was found between total bacteria (2.9$104, 5.2$103 and 3.7$103 CFU mL1 for TC, E. coli and Enterococcus sp, respectively) and antibiotic resistant bacteria (1.7$102, 5.6$101 and 5.3$101 CFU mL1 for TC, E. coli and Enterococcus sp, respectively), in other words, around two orders of magnitude lower the CFX-R bacteria concentration with regard to the total. As per the total bacteria (TB), the highest inactivation rate constants were obtained for TC, while the lowest values were obtained for Enterococcus sp at each irradiance tested. These results
Fig. 2. Total and CFX-R bacteria inactivation rate constants by solar photo-Fenton in 5 cm-deep RPR in different seasons: winter (irradiance 12.85 ± 1.07 W m2), spring (irradiance 30.38 ± 0.69 W m2) and summer (irradiance 37.57 ± 0.55 W m2). 20 mg Fe2þ$L1 and 50 mg H2O2$L1.
corroborate previous studies where less bacterial damage and consequently a lower inactivation rate constant was observed in the case of Enterococcus sp than in the case of TC and E. coli (Esteban García et al., 2018). This can be explained due to the higher resistance shown by these gram-positive enteric bacteria (Enterococcus sp) because of the presence of a more resistant cell wall as reported elsewhere (Giannakis et al., 2016a). The highest bacterial inactivation rate constants were obtained with the highest irradiance in mez et al. (2016). Howsummer, in concordance with Ortega-Go ever, the irradiance values corresponding to spring and summer, 30.38 ± 0.69 and 37.57 ± 0.55 W m2, respectively, were not significantly different enough to justify the difference in the rate constant. Temperature also plays an important role in this inactimez et al., 2012) and, therefore, the difvation process (Ortega-Go ferences in temperature between spring and summer (24 ± 2 and 30 ± 1 C, respectively) made an important contribution to the total and CFX-R bacterial inactivation rates. Comparing the inactivation rate between total bacteria and CFX-R bacteria, no significant differences were observed. In both cases, inactivation rate constants very close to each other were obtained for the three microbiological groups studied. These results confirm those obtained by Giannakis et al. (2018a), who pointed out that antibiotic resistant bacteria do not need to be taken into account as a special factor in disinfection kinetics studies when the photo-Fenton process is applied. In addition, our study demonstrates that there are no differences between antibiotic resistant gram-positive and gram-negative bacteria (Enterococcus sp, TC and E. coli, respectively) since the inactivation rate constants were the same within confidence limits in all cases, corroborating once again, the aforementioned study. As for the effect of iron concentration on antibiotic resistant bacteria (CFX-R) inactivation, four iron concentrations (20, 10, 5 and 2.5 mg L1) were evaluated in triplicate with 50 mg L1 of hydrogen peroxide in winter (13 ± 1 W m2) (Fig. 3). The best iron concen mez et al., tration was 20 mg L1 as reported elsewhere (Ortega-Go 2014) and the linear increase of ki with Fe concentration shows the relevant effect of photo-Fenton on bacterial damage up to cellular inactivation. No significant differences between total and CFX-R bacteria inactivation constant were observed and the same slope in the ki vs. iron concentration linear relationship corroborates that the small differences shown in Fig. 3 are in the experimental error interval. To better compare total and CFX-R bacteria inactivation, a
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Fig. 3. Total and CFX-R bacteria inactivation rate constants by solar photo-Fenton in 5 cm-deep RPR at four iron concentrations (2.5, 5, 10 and 20 mg L1) and 13 ± 1 W m2. H2O2 concentration was 50 mg L1.
statistical analysis was carried out by pooling all data of ki obtained in batch mode, and representing ki for total bacteria (TB) vs. ki for CFX-R bacteria, Fig. 4. A statistically significant correlation between total and CFX-R bacteria inactivation with a confidence level of 95% was observed. The slope 1.003, demonstrates that the inactivation rate constants can be accepted as being the same for total and CFXR bacteria, the determination coefficient being 0.997. 3.2. Effect of HRT on CFX-R bacteria inactivation For scaling-up purposes, the solar photo-Fenton reactor must be operated in continuous flow mode, so it is necessary to study the effect of HRT on the inactivation of CFX-R bacteria. The study was carried out at four values of hydraulic residence time from 22.4 to 4.7 min working with the best iron (5 mg L1) and H2O2 (30 mg L1) concentrations previously reported for continuous flow disinfection of WWTP secondary effluents (De la Obra et al., 2019). The operation started in batch mode for 2 h and the continuous operation was then started by increasing the inflow rate every 2 h to verify the effect of the HRT reduction (Fig. 5). Bacterial inactivation
Fig. 4. Statistical analysis of the inactivation rate constants for total bacteria (TB) and cefotaxime resistant bacteria (CFX-R) obtained in batch mode by Fenton and photoFenton with different irradiance levels and iron concentrations. Experiments were carried out outdoors in a 5 cm-deep RPR.
Fig. 5. Concentration variation of total E. coli (closed squares) and CFX-R E. coli (open squares) by solar photo-Fenton as a function of hydraulic residence time: 22.4min ( ), 16.3 min ( ), 9 min ( ), 4.7 min ( ) and batch ( ). Hydrogen peroxide ( ) and total Fe concentrations ( ) are also presented. Experiments were carried out in a 5 cm-deep cylindrical reactor at 30 W m2 UVA irradiance.
below the DL was achieved after 70 min of treatment for total and CFX-R bacteria during batch mode operation. This result is consistent with that obtained in a previous study (Esteban García et al., 2018). In contrast, at continuous flow operation, significant differences were observed between 22.4 min and the other HRTs studied (16.3, 9.0 and 4.7 min). Although Fig. 5 only shows total and CFX-R E. coli inactivation, the results of total and CFX-R TC and Enterococcus sp are shown in Figures S1 and S2. In the case of 22.4 min, the system was able to reach bacterial inactivation under DL (1 CFU mL1) once steady state was achieved. However, in the cases of 16.3 min, 9.0 min and 4.7 min of HRT, the bacterial concentration increased up to values very close to the initial ones at steady state. It is important to remark that no regrowth was observed for any microbiological group studied in the collected samples during the experiments carried out at 22.4 min of HRT. Total and CFX-R E. coli inactivation rate constants were evaluated once steady state was achieved, showing rate constant values significantly higher (>143 ± 30 min1 for E. coli) in the case of 22.4 min of HRT when compared with 0.16 ± 0.01 min1, 0.18 ± 0.01 min1 and 0.30 ± 0.01 min1 in the case of 16.3, 9.0 and 4.7 min of HRT, respectively, Table 1. It is worth noting that when DL is achieved in steady state, there is uncertainty in the calculation of the kinetic constant due to the very high initial concentration and the unknown actual bacterial concentration below the DL, hence the reason for expressing ki > 143 ± 30 min1. Two features need to be highlighted from Table 1. First, the inactivation rate constants are much higher in continuous flow mode than in batch. In continuous flow operation, ferrous iron and hydrogen peroxide are continuously added to the reaction bulk and Fenton reaction continuously generates hydroxyl radicals and not only in the first few seconds of treatment as occurs in batch mode. This could explain a higher inactivation rate in continuous mode, so a Fenton assay in continuous operation was carried out with 20 mg L1 of iron and compared with continuous photo-Fenton (Table 2) to provide more insight on the explanation of this difference in inactivation rates. Both experiments were carried out with the highest iron concentration tested to enhance the Fenton effect. Although the Fenton inactivation rate constants in continuous mode were higher (one order of magnitude) than in batch, the differences between dark and illuminated operation in continuous mode were far higher. These results demonstrated the great influence of irradiance on the disinfection by solar photo-Fenton. As such, the explanation comes from the complex mechanisms of bacteria inactivation, as reported by Pulgarín and co-workers
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Table 1 Total and CFX-R bacteria inactivation rate constants by solar photo-Fenton at neutral pH in continuous flow mode as a function of HRT. Microbiological group
TC E. coli Enterococcus sp CFX-R TC CFX-R E. coli CFX-R Enterococcus sp
ki, min1 HRT ¼ 22.4 min
HRT ¼ 16.3 min
HRT ¼ 9.0 min
HRT ¼ 4.7 min
>629 ± 155 >143 ± 30 >43 ± 10 >8 ± 1 >5 ± 1 >3 ± 1
0.22 ± 0.03 0.16 ± 0.01 0.03 ± 0.01 0.15 ± 0.04 0.06 ± 0.01 0.01 ± 0.01
0.21 ± 0.03 0.18 ± 0.01 0.03 ± 0.01 0.16 ± 0.02 0.06 ± 0.01 0.02 þ 0.01
0.32 ± 0.03 0.30 ± 0.01 0.03 ± 0.01 0.21 ± 0.03 0.07 ± 0.01 0.03 ± 0.01
UVA irradiance ¼ 30 W m2. Temperature ¼ 25 C. Hydrogen peroxide and iron concentrations were 30 and 5 mg L1, respectively.
Table 2 Total and CFX-R bacteria inactivation rate constants by Fenton and solar photo-Fenton processes at neutral pH working at 22.4 min of HRT in continuous mode operation. Inactivation rate constant, ki, min1 Microbiological group
Fenton
photo-Fenton
TC E. coli Enterococcus sp
0.042 ± 0.001 0.033 ± 0.002 0.023 ± 0.002
>699 ± 108 >106 ± 23 >25 ± 3
CFX-R TC CFX-R E. coli CFX-R Enterococcus sp
0.031 ± 0.007 0.020 ± 0.008 0.018 ± 0.004
>14 ± 1 >4.3 ± 0.9 >3.1 ± 0.3
20 mg Fe2þ$L1 and 30 mg H2O2$L1. Photo-Fenton was carried out at 30 W m2 of UVA irradiance.
(Giannakis et al., 2016a). Photo-Fenton inactivation of bacterial cells is a synergic and additive effect of several factors such as radiation intensity, the oxidative action of hydrogen peroxide, the photoFenton reaction on cell membranes and the internal photoFenton reaction, giving rise to a multiple path oxidative destruction of vital cellular structures and molecules. In the case presented in this work, at neutral pH ferric iron formed from the reaction of H2O2, with freshly added ferrous iron, precipitates as ferric hymez et al., 2016). Iron hydroxides can produce droxides (Ortega-Go hydroxyl radicals through the heterogeneous photo-Fenton process (Ruales-Lonfat at el., 2015) and this precipitate can be adsorbed onto the cells, generating HO right at the membrane, favouring oxidative attack. In continuous flow, the dilution effect as the inlet stream enters the reactor, increases the likelihood of cell damage as opposed to initial batch conditions, when a much higher bacterial concentration is subjected to stressful conditions. Nonetheless, a minimum radiation energy requirement must be achieved, and energy dose must be taken into account. Following the procedure described by De la obra et al., (De la Obra et al., 2019), the cumulative radiation energy received per unit of treated water volume, QUVA, was calculated for the different HRTs, giving 0.80, 0.58, 0.32 and 0.17 kJ L1 for HRTs of 22.4, 16.3, 9.0 and 4.7 min, mez et al., respectively. As previously demonstrated (Ortega-Go 2016) the longer exposure time allows higher HO generation to be obtained, leading to higher bacterial inactivation being observed. A minimum of 1 kJ L1 has been reported to cause bacterial damage (De la Obra et al., 2019), a value similar to 0.80 kJ L1 obtained at 22.4 min HRT. The second feature in Table 1 is the apparent difference between inactivation rate constants for total and CFX-R bacteria when DL is reached in steady state. Unfortunately, the above-mentioned uncertainty in the calculation of ki from Eq. (2) with only two values of bacterial concentration, gives rise to a substantial impact of the inlet flow concentration, meaning the higher the initial concentration, the higher the rate constant. Accordingly, no conclusion can be drawn from these results and more research is needed for clarification. As for the HRTs when DL was not achieved, differences between rate constants for total and CFX-R bacteria were not significant.
3.3. Process efficiency analysis - figures of merit To evaluate the effect of the different factors tested in the previous sections on the scale up of the process, the figures of merit of the photo-Fenton process have been calculated as the collector area per order (Aco, m2$m3$order1) for batch system (ABco) and continuous flow operation (ACco) following Equations (3) and (4), respectively. The lower the collector area per order, the higher the efficiency of the process. Consequently, the determination of the collector area per order (ACO) for the operational modes (discontinuous and continuous), seasonal period (winter, spring and summer) and iron concentration used has been carried out. For that purpose, only the data for E. coli were used since a concentration limit is required by the Spanish legislation for that strain (RD 1620/2007). The results are shown in Table 3. The values obtained for discontinuous mode operation are two order of magnitude higher than for continuous mode. Among the three seasons, summer gives rise to the lowest ABco values. Regarding the effect of iron concentration in batch mode operation, the higher the iron concentration, the lower the area required and the higher the efficiency. In this respect, additional continuous conditions with lower HRT values (22.4, 16.3, 9 and 4.7 min) were tested in the solar simulator as shown in the Section 2.2. Under these experimental conditions, the figure of merit calculation is an extrapolation to the operation with natural sunlight in spring. In this case, only 22.4 min of HRT achieved the detection limit giving a value of ACco ¼ 2.7 square meters of solar collector area required to reduce in 1 h the amount of E. coli bacteria to one logarithmic unit (90% less) in one cubic meter of real secondary effluent as shown in Table 3. 4. Conclusions For the first time, the inactivation kinetics of total and cefotaxime resistant bacteria by solar photo-Fenton have been compared under very different treatment conditions, with no significant differences. The target of wastewater disinfection treatments can therefore be defined as total pathogen bacteria inactivation, as once
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Table 3 Effect of operation mode in different seasons on the estimated Aco values. Operational mode
Season
Irradiance (W m2)
[Fe] (mg$L1)
AC0 (m2$m3$order1)
Discontinuous
Winter Spring Summer Winter Winter Winter Winter Indoors Indoors Indoors Indoors
12.85 30.38 37.57 13.10 13.10 13.10 13.10 30 30 30 30
20 20 20 2.5 5 10 20 5 5 5 5
2009 ± 457 2137 ± 140 1063 ± 24 5977 ± 22 5287 ± 171 4104 ± 280 2311 ± 134 2.7 ± 0.3 11.9 ± 0.4 7.8 ± 0.3 4.4 ± 0.3
Continuous
HRT HRT HRT HRT
22.4 16.3 * 9* 4.7 *
* Detection limit was not achieved.
the detection limit has been achieved, ARB are also inactivated. The value obtained for the E. coli inactivation rate constant in continuous mode operation was around four orders of magnitude higher than in batch mode, with minimum energy dose (0.8 kJ L1 for HRT 22.4 min) to bring about disinfection below the detection limit (1 CFU mL1). For wastewater reuse in irrigation, these results of bacteria inactivation by solar photo-Fenton at neutral pH in continuous flow mode make this process suitable for large-scale application, since the treatment times are shorter than batch operation and reactor loading manoeuvres are avoided. Additionally, process efficiency has been evaluated in terms of figures of merit, the best condition being continuous flow operation, with 5 mg L1 of iron and 22.4 min of HRT giving the lowest value, 2.7 m2 m3$order1 of collector area per order and cubic meter, in contrast with 2137 m2 calculated for batch operation under the same solar UVA irradiance, 30 W m2. The operation of the solar photo-Fenton process in continuous flow mode opens up a new field of research as tertiary treatment because bacterial inactivation is much more efficient than in batch mode. However, more research is needed to elucidate the inactivation mechanisms and to develop process models in continuous flow. Acknowledgements This research has been supported by the Andalusian Regional Government (P12-RNM-1437) and the European Regional Development Fund (ERDF). I. de la Obra would like to acknowledge the Andalusian Regional Government for her grant. Ph.D. Gracia Rivas ~ ez wishes to thank MICINN for her Juan de la Cierva formation Ib an grant (FJCI-2017-34059). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2019.04.059. References Bolton, J.R., Bircher, K.G., Tumas, W., Tolman, C.A., 2001. Figures-of-merit for the technical development and application of advanced oxidation technologies for both electric- and solar-driven systems (IUPAC Technical Report). Pure Appl. Chem. 73, 627e637. https://doi.org/10.1351/pac200173040627. n Ferna ndez, F.G., Malato, S., S rez, J.A., Carra, I., Santos-Juanes, L., Acie anchez Pe 2014. New approach to solar photo-Fenton operation. Raceway ponds as tertiary treatment technology. J. Hazard Mater. 279, 322e329. https://doi.org/10. 1016/j.jhazmat.2014.07.010. Christou, A., Agüera, A., Bayona, J.M., Cytryn, E., Fotopoulos, V., Lambropoulou, D., €der, P., Fatta-Kassinos, D., 2017. The Manaia, C.M., Michael, C., Revitt, M., Schro potential implications of reclaimed wastewater reuse for irrigation on the agricultural environment: the knowns and unknowns of the fate of antibiotics and antibiotic resistant bacteria and resistance genes e a review. Water Res. 123, 448e467. https://doi.org/10.1016/j.watres.2017.07.004. CLSI, 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-
First Informational Supplement. CLSI document M100eS21. Clinical and Laboratory Standards Institute, Wayne, PA. De la Obra, I., Esteban, B., Rivas, G., Casas, J.L., Sanchez Perez, J.A., 2019. Continuous flow disinfection of WWTP secondary effluents by solar photo-Fenton at neutral pH in raceway pond reactors at pilot plant scale. Appl. Catal. B Environ. 247, 115e123. https://doi.org/10.1016/j.apcatb.2019.01.093. nchez Pe rez, J.A., 2018. Wild bacteria Esteban García, B., Rivas, G., Arzate, S., Sa inactivation in WWTP secondary effluents by solar photo-fenton at neutral pH in raceway pond reactors. Catal. Today 313, 72e78. https://doi.org/10.1016/j. cattod.2017.10.031. Fatta-kassinos, D., 2016. Wastewater Reuse and Current Challenges, the Handbook of Environmental Chemistry. Springer International Publishing, Cham. https:// doi.org/10.1007/978-3-319-23892-0. ndez, I., Polo-Lo pez, M.I., Oller, I., Fern n ~ ez, P., 2012. Bacteria García-Ferna andez-Iba and fungi inactivation using Fe3þ/sunlight, H2O2/sunlight and near neutral photo-Fenton: a comparative study. Appl. Catal. B Environ. 121e122, 20e29. https://doi.org/10.1016/j.apcatb.2012.03.012. pez, M.I., Spuhler, D., Sa nchez Pe rez, J.A., Fern n ~ ez, P., Giannakis, S., Polo Lo andez Iba Pulgarin, C., 2016a. Solar disinfection is an augmentable, in situ-generated photo-Fenton reactiondPart 1: a review of the mechanisms and the fundamental aspects of the process. Appl. Catal. B Environ. 199, 199e223. https://doi. org/10.1016/J.APCATB.2016.06.009. Giannakis, S., Ruales-Lonfat, C., Rtimi, S., Thabet, S., Cotton, P., Pulgarin, C., 2016b. Castles fall from inside: evidence for dominant internal photo-catalytic mechanisms during treatment of Saccharomyces cerevisiae by photo-Fenton at nearneutral pH. Appl. Catal. B Environ. 185, 150e162. https://doi.org/10.1016/J. APCATB.2015.12.016. Giannakis, S., Le, T.T.M., Entenza, J.M., Pulgarin, C., 2018a. Solar photo-Fenton disinfection of 11 antibiotic-resistant bacteria (ARB) and elimination of representative AR genes. Evidence that antibiotic resistance does not imply resistance to oxidative treatment. Water Res. 143, 334e345. https://doi.org/10.1016/ j.watres.2018.06.062. Giannakis, S., Voumard, M., Rtimi, S., Pulgarin, C., 2018b. Bacterial disinfection by the photo-Fenton process: extracellular oxidation or intracellular photo-catalysis? Appl. Catal. B Environ. 227, 285e295. https://doi.org/10.1016/j.apcatb. 2018.01.044. Kümmerer, K., Dionysiou, D.D., Fatta-Kassinos, D., 2016. Scope of the book wastewater reuse and current challenges. Handb. Environ. Chem. https://doi.org/10. 1007/698-2015-448. Michael, I., Hapeshi, E., Michael, C., Varela, A.R., Kyriakou, S., Manaia, C.M., FattaKassinos, D., 2012. Solar photo-Fenton process on the abatement of antibiotics at a pilot scale: degradation kinetics, ecotoxicity and phytotoxicity assessment and removal of antibiotic resistant enterococci. Water Res. 46, 5621e5634. https://doi.org/10.1016/j.watres.2012.07.049. Michael-Kordatou, I., Karaolia, P., Fatta-Kassinos, D., 2018. The role of operating parameters and oxidative damage mechanisms of advanced chemical oxidation processes in the combat against antibiotic-resistant bacteria and resistance genes present in urban wastewater. Water Res. 129, 208e230. https://doi.org/ 10.1016/j.watres.2017.10.007. mez, E., Fern n ~ ez, P., Ballesteros Martín, M.M., Polo-Lo pez, M.I., Ortega-Go andez-Iba nchez Pe rez, J.A., 2012. Water disinfection using photoEsteban García, B., Sa Fenton: effect of temperature on Enterococcus faecalis survival. Water Res. 46, 6154e6162. https://doi.org/10.1016/J.WATRES.2012.09.007. mez, E., Esteban, B., Ballesteros Martín, M.M., Ferna ndez Iba n ~ ez, P., Ortega-Go nchez Pe rez, J.A., 2014. Inactivation of natural enteric bacteria in real Sa municipal wastewater by solar photo-Fenton at neutral pH. Water Res. 63, 316e324. https://doi.org/10.1016/j.watres.2014.05.034. mez, E., Ballesteros Martín, M.M., Esteban García, B., Sa nchez Pe rez, J.A., Ortega-Go n ~ ez, P., 2016. Wastewater disinfection by neutral pH photoFern andez Iba Fenton: the role of solar radiation Appl. Catal. B Environ 181, 1e6. https://doi. org/10.1016/j.apcatb.2015.06.059. Pignatello, J.J., Oliveros, E., MacKay, A., 2006. Advanced oxidation processes for organic contaminant destruction based on the fenton reaction and related chemistry. Crit. Rev. Environ. Sci. Technol. 36, 1e84. https://doi.org/10.1080/
I. De la Obra Jimenez et al. / Water Research 159 (2019) 184e191 10643380500326564. 1620/2007, R.D., n.d. 7 de diciembre, por el que se establece el regimen juridico de la reutilizacion de las aguas depuradas. BOE no294 http:/www.boe.es/boe/dias/ 2003/02//21/pdfs/A07228-. Rizzo, L., Fiorentino, A., Anselmo, A., 2013a. Advanced treatment of urban wastewater by UV radiation: effect on antibiotics and antibiotic-resistant E. coli strains. Chemosphere 92, 171e176. https://doi.org/10.1016/j.chemosphere.2013. 03.021. Rizzo, L., Manaia, C., Merlin, C., Schwartz, T., Dagot, C., Ploy, M.C., Michael, I., FattaKassinos, D., 2013b. Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Sci. Total Environ. 447, 345e360. https://doi.org/10.1016/j.scitotenv.2013.01.032. rez, M., Ferna ndez-Iba n ~ ez, P., Rizzo, L., Agovino, T., Nahim-Granados, S., Castro-Alfe pez, M.I., 2019. Tertiary treatment of urban wastewater by solar and UVPolo-Lo C driven advanced oxidation with peracetic acid: effect on contaminants of emerging concern and antibiotic resistance. Water Res. 149, 272e281. https:// doi.org/10.1016/j.watres.2018.11.031. pez, M.I., Mosteo, R., Ormad, M.P., Ferna ndezRodríguez-Chueca, J., Polo-Lo n ~ ez, P., 2014. Disinfection of real and simulated urban wastewater effluents Iba using a mild solar photo-Fenton. Appl. Catal. B Environ. 150e151, 619e629. https://doi.org/10.1016/j.apcatb.2013.12.027. Rodriguez-Chueca, J., Ormad, M.P., Mosteo, R., Sarasa, J., Ovelleiro, J.L., 2015. Conventional and advanced oxidation processes used in disinfection of treated urban wastewater. Water Environ. Res. 87, 281e288. https://doi.org/10.2175/ 106143014X13987223590362.
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SCHEER, 2017. Scientific Committee on Health , Environmental and Emerging Risks Scientific Advice on Proposed EU Minimum Quality Requirements for Water Reuse in Agricultural Irrigation and Aquifer Recharge, vols. 1e24. Sousa, J.M., Macedo, G., Pedrosa, M., Becerra-Castro, C., Castro-Silva, S., Pereira, M.F.R., Silva, A.M.T., Nunes, O.C., Manaia, C.M., 2017. Ozonation and UV254nm radiation for the removal of microorganisms and antibiotic resistance genes from urban wastewater. J. Hazard Mater. 323, 434e441. https://doi. org/10.1016/j.jhazmat.2016.03.096. Stalter, D., Magdeburg, A., Oehlmann, J., 2010. Comparative toxicity assessment of ozone and activated carbon treated sewage effluents using an in vivo test battery. Water Res. 44, 2610e2620. https://doi.org/10.1016/j.watres.2010.01. 023. Watson, K., Shaw, G., Leusch, F.D.L., Knight, N.L., 2012. Chlorine disinfection byproducts in wastewater effluent: bioassay-based assessment of toxicological impact. Water Res. 46, 6069e6083. https://doi.org/10.1016/j.watres.2012.08. 026. WHO, 2014. Antimicrobial resistance: global health report on surveillance [WWW document]. Bull. World Health Organ. https://doi.org/10.1007/s13312-0140374-3. Xu, P., Janex, M.L., Savoye, P., Cockx, A., Lazarova, V., 2002. Wastewater disinfection by ozone: main parameters for process design. Water Res. https://doi.org/10. 1016/S0043-1354(01)00298-6. Yuan, Q. Bin, Guo, M.T., Yang, J., 2015. Fate of antibiotic resistant bacteria and genes during wastewater chlorination: implication for antibiotic resistance control. PLoS One 10, 1e11. https://doi.org/10.1371/journal.pone.0119403.
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Kinetic assessment of antibiotic resistant bacteria inactivation by solar photo-Fenton in batch and continuous flow mode for wastewater reuse nez a, b, J.L. Casas Lo pez a, b, G. Rivas Iba n ~ ez a, c, B. Esteban García a, b, I. De la Obra Jime a , b, * nchez Pe rez J.A. Sa a b c
Solar Energy Research Centre (CIESOL), Joint Centre University of Almería-CIEMAT, 04120, Almería, Spain Department of Chemical Engineering, University of Almería, 04120, Almería, Spain Plataforma Solar de Almería, CIEMAT, 04200, Tabernas, Almería, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 February 2019 Received in revised form 28 April 2019 Accepted 30 April 2019 Available online 7 May 2019
The presence of antibiotic resistant bacteria in municipal wastewater treatment plants represents a real risk to human health. For the first time, this paper shows that the inactivation rate of cefotaxime resistant bacteria is the same as total bacteria when secondary effluents are treated by the solar photo-Fenton process. To obtain this result, an exhaustive and comparative kinetic study on the inactivation of both total and cefotaxime resistant bacteria (Total coliform, Escherichia coli and Enterococcus sp) was carried out, taking into account the effects of the main operation conditions, such as solar irradiance and iron concentration, and operation mode (batch and continuous). In all the operation conditions studied, no significant differences were found between the first order inactivation rate constants, ki, of total and cefotaxime resistant bacteria. Additionally, ki increased with solar irradiance and iron concentration. As for the effect of the operation mode, the main finding of this work is much quicker inactivation in continuous flow mode than in batch mode, pointing out its potential application at large scale. The best continuous operation condition to inactivate the bacteria to the detection limit (1 CFU mL1), was at 22.4 min of hydraulic residence time with 5 mg Fe2þ L1 and 30 mg H2O2$L1. This treatment time is approximately a third of that reported in batch mode. The efficiency, in terms of figure of merits, of the continuous flow operation was 2.7 m2 of solar collector area to reduce one log of E. coli concentration per m3 of treated water and per hour, in comparison with 2137 m2 calculated for batch operation under the same solar UVA irradiance, 30 W m2. This paper encourages research into continuous solar disinfection processes due to its enhanced efficiency with regard to the commonly used batch wise operation and shows that efficient removal of total bacteria ensures the removal of antibiotic resistant bacteria. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Wastewater disinfection Antibiotic resistant bacteria Disinfection kinetics Solar treatment Efficiency analysis
1. Introduction The reuse of treated wastewater for crop irrigation has become an increasingly recommended practice to address water scarcity in the EU and worldwide (Michael-Kordatou et al., 2018). However, the presence of the so-called “new contaminants” or “contaminants of emerging concern” in treated wastewater has led to increased concern about the potential direct and indirect effects on the environment and possible implications for human health
* Corresponding author. Department of Chemical Engineering, University of Almería, 04120, Almería, Spain. rez). E-mail address: [email protected] (J.A.S. Pe https://doi.org/10.1016/j.watres.2019.04.059 0043-1354/© 2019 Elsevier Ltd. All rights reserved.
(Kümmerer et al., 2016). In this regard, special attention has been paid to the contribution of treated wastewater concerning the emergence of antibiotic resistance in pathogenic bacteria, claimed to be “one of the major global threats to society” in the twenty first century by the World Health Organization (WHO, 2014). Municipal wastewater treatment plants (MWWTPs) are considered to be significant reservoirs of antibiotic resistant bacteria as they combine a high load of bacteria with a residual concentration of antibiotics (Fatta-kassinos, 2016). The biological treatments typically applied in MWWTPs are poorly effective in the complete disinfection of the wastewater, generating secondary effluents with about 109-1012 Colony Forming Units (CFU) per day and per inhabitant equivalent. Among these, at least 107e1010 CFU
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could have acquired antibiotic resistance (Rizzo et al., 2013a,b). Thus, the large amount of antibiotic resistant bacteria (ARB) discharged when treated wastewater is used for irrigation which may proliferate in soils or plants (Christou et al., 2017). Therefore, conventional MWWTPs should be upgraded with more efficient technologies such as tertiary treatments to provide enough protection for the potential risks related to water reuse, not only by meeting the current quality standards, but also by controlling the spread of ARB into the environment. In fact, currents standards in water reuse for irrigation purposes relies on the control of chemical and microbiological parameters like heavy metals or enteric pathogens (Escherichia coli and Enterococcus sp), but do not evaluate ARB levels (Fatta-kassinos, 2016). In this regard, the measurement of bacteria resistant to Cefotaxime (third-generation cephalosporin, which is on the WHO essential list of medicine) could be used as a reliable indicator of wastewater resistance levels as proposed by the Scientific Committee on Health, Environmental and Emerging Risks (SCHEER) on the “Proposed EU minimum quality requirements for water reuse in agricultural irrigation and aquifer recharge”, a report prepared by the European Commission Joint Research Centre (SCHEER, 2017). Despite the most common tertiary treatments used as a disinfection step before secondary effluent reuse having proved to be efficient in microorganism inactivation (namely, ozonation, chlorination or UVC irradiation) (Xu et al., 2002; Rodriguez-Chueca et al., 2015; Rizzo et al., 2019), their efficacy for effective reduction of antibiotic resistance is not completely agreed upon (Luigi Rizzo et al., 2013; Yuan et al., 2015; Sousa et al., 2017; MichaelKordatou et al., 2018). Additionally, other drawbacks related to the generation of undesirable disinfection by-products through ozonation (Stalter et al., 2010) or chlorination (Watson et al., 2012) has forced research to seek out disinfection alternatives that are environmentally safer than conventional tertiary treatments, namely the solar photo-Fenton process. This Advanced Oxidation Process (AOP) has been found to be efficient, not only at degrading antibiotics, but also at inactivating microorganisms and ARB (Giannakis et al., 2018a) without increasing toxicity (Michael et al., 2012). It is based on the generation of highly oxidative and nonselective species, hydroxyl radicals (HO), that degrade the external membrane of bacteria, increasing its permeability and inducing internal reactions that eventually inactivate the microorganisms (Giannakis et al., 2018b). The generation of HO in the Fenton process relies on the repeated oxidation and reduction of iron (catalyst) by hydrogen peroxide. Moreover, in the presence of UVevis radiation, the HO production rate is increased due to the regeneration of Fe2þ by the photo-conversion of ferric iron to ferrous iron (Pignatello et al., 2006). Despite the most desirable pH for radical production being around 2.8e3.0, owing to the low solubility of Fe3þ at higher pH, many recent studies have confirmed the effectiveness of the photo-Fenton process for bacterial inactivation at neutral pH (Giannakis et al., 2016b). Nevertheless, to our knowledge, the industrial application of this solar process as a tertiary treatment for wastewater disinfection has not yet been developed, even though efforts have been directed towards making its application at large scale feasible via compound parabolic collectors (CPCs) (Rodríguez-Chueca et al., 2014) or low cost reactors such as raceway pond reactors (RPRs) (Esteban García et al., 2018). In particular, the latter has emerged as an attractive option, not only for the remarkable reduction in investment cost (RPR construction costs are 40 times cheaper than those for CPCs), but also for the larger volume of wastewater than can be treated per surface area (Carra et al., 2014). In fact, to bring closer to reality the solar photoFenton implementation, the large amount of secondary effluent generated in MWWTPs needs to be dealt with. With this in mind, the feasibility of the continuous flow operation of the solar photo-
185
Fenton process in RPR for secondary effluent disinfection has recently been investigated with promising results (De la Obra et al., 2019). The objective of this work was to carry out an exhaustive and comparative kinetic study on the inactivation of both total and cefotaxime resistant bacteria by solar photo-Fenton at neutral pH in a wide range of operation conditions, taking into account different bacterial species (Total coliform, Escherichia coli and Enterococcus sp). To this end, the effects of solar irradiance and iron concentration as well as operation mode, batch vs. continuous, were analysed. 2. Materials and methods 2.1. Wastewater source Secondary effluent batches of 1 m3 were taken from the Municipal Wastewater Treatment Plant named “El Bobar” (Almeria, Southeastern Spain). A characterization of wastewater samples was carried out during the first three days of experimentation to assess their variability. The average values and standard deviation of the main physico-chemical parameters during the experimental period (from October 2017 to February 2019) are included as Supplementary Material in Table S1. All experiments were carried out at the natural pH of secondary effluent samples, around 7.3 ± 0.4 and remained constant during treatment time. 2.2. Experimental set-up Batch wise experiments were conducted in 5 cm-deep RPRs with initial hydrogen peroxide and iron concentrations of 50 and 20 mg L1, respectively. The effect of solar UVA irradiance on bacteria inactivation was evaluated at 12.85 ± 1.07 W m2, 30.38 ± 0.69 W m2 and 37.57 ± 0.55 W m2, corresponding to winter, spring and summer, respectively. These experiments have been partially reported by (De la Obra et al., 2019). In the present study, new results of cefotaxime resistant (CFX-R) bacteria inactivation are discussed. As for studying the influence of iron concentration on both total and CFX-R bacteria inactivation, iron concentration was varied (20, 10, 5 and 2.5 mg L1) while maintaining the initial H2O2 concentration at 50 mg L1 and the irradiance at 13 ± 1 W m2, corresponding to winter conditions. This experimental setup is shown in Fig. 1a. Temperature (LabJack EI1034) and pH (Crison 5335) were monitored with a LabJack U12 data acquisition card connected to a computer. UVA radiation was measured (W m2) with a horizontal global UV radiometer (Delta Ohm, LP UVA 02 AV), the spectral response range being 327e384 nm. Control experiments in batch mode were performed outdoors under the following conditions: i) Dark without any reagent to check the effect of the mechanical stress; ii) In the presence of sunlight (UVA) and iii) Sunlight with hydrogen peroxide (UVA/ H2O2). Mechanical stress had no effect on bacteria inactivation, while 2 and 2.5-log decrease were observed for TC and E. coli, respectively, after 120 min of treatment, and the synergic effect of UVA þ H2O2 caused 3-log inactivation after 200 min for TC and E. coli. Significant differences can be observed for Enterococcus sp., with no bacterial damage in any control assayed. These results are in concordance with those previously reported (Esteban García et al., 2018). Apart from that, the influence of hydraulic residence time (HRT) on total and CFX-R bacteria inactivation in continuous flow was studied at laboratory scale in a Sun Test CPS þ solar simulator at 30 W m2 of UVA irradiance and a temperature of 25 C using a Thermo Scientific NESLAB RTE-7 thermostatic bath. To
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2.3. Total and antibiotic resistant bacteria quantification Bovine liver catalase (Sigma-Aldrich, Spain) was added to the wastewater samples before microbiological analysis in order to ndez et al., 2012). remove residual hydrogen peroxide (García-Ferna The bacteria count in the samples collected during photo-Fenton experiments was carried out using the standard plated counting method through serial 10-fold dilutions with saline solution (0.8% NaCl) in selective media. To count antibiotic resistant bacteria in the samples, the selective media was enriched with 4 mg L1 of cefotaxime (CFX), a third-generation cephalosporin. This concentration was reported in the documented CLSI minimal inhibitory concentration (MIC) breakpoint levels for Enterobacteriaceae (Clinical and Laboratory Standards Institute, Wayne, PA, 2011). The enumeration of colonies was performed after one day of incubation for TC and E. coli in Chromocult-agar (Merk, Germany) and two days for Enterococcus sp. in Enterococcus-medium (Pronadisa, Spain) at 37 C in both total and CFX-R. For plate counting, 50-250-5001000 mL of sample were used, reaching the microbial detection limit (DL) of 1 CFU mL1. All procedures were carried out in duplicate for each sample. Regrowth in photo-Fenton treated water was checked by storing 200 mL of the sample in the dark at room temperature overnight. The stored samples were plated for colony counting as previously described. No regrowth was observed in any case. 2.4. Kinetic analysis Chick's first order kinetic equation was chosen to fit the decrease of total and CFX-R bacterial population, Equation (1)
log
Fig. 1. Experimental setups for wastewater disinfection by photo-Fenton: a) Raceway pond reactors operating in batch mode under natural sunlight. b) Stirred tank reactor operating in continuous flow mode under simulated solar light.
C ¼ ki $t C0
(1)
where C/C0 is the reduction in the bacterial concentration, ki is the disinfection rate constant in min1 and t is the photo-Fenton time in min. 2.5. Mass balances
this end, a 1 L-stirred PVC cylindrical reactor with 5 cm of liquid depth and 16 cm of internal diameter was selected to perform this study (Fig. 1b). To ensure perfect mixing, magnetic agitation was set to reach a mixing time of 2.4 s, a value much shorter than the photo-Fenton treatment time. Four input flows 33.3, 50, 100 and 200 mL min1 for the secondary effluent feed pump and two different input flows, 4.2 and 7.2 mL min1, for the H2O2 and Fe2þ stock solutions respectively, were established giving rise to four values of HRT, 22.4, 16.3, 9.0 and 4.7 min. The inlet iron and H2O2 concentrations were 5 mg L1 and 30 mg L1, respectively. The reactor start-up was carried out in batch mode for 2 h followed by the continuous supply of reactants and secondary effluent, giving rise to the continuous flow mode. Each HRT was maintained for 2 h to collect enough data once steady state was achieved. Additional control assays were carried out in continuous flow mode at 22.4 min of HRT. The effects of i) the mechanical stress, ii) UVA and iii) UVA þ H2O2 on TC, E. coli and Enterococcus sp. inactivation were evaluated. Mechanical stress due to magnetic stirring led to 1-log decrease for TC while not damage was observed for E. coli and Enterococcus sp. The exposure to UVA irradiance caused 1.5-log decrease in TC (1-log due to mechanical stress) and 1-log decrease in E. coli. In the case of Enterococcus sp. no bacterial damage was observed. The combined effect of UVA þ H2O2 did not cause any additional effect with regard to UVA for the three species studied.
For the kinetic studies, the mass balance of viable bacteria in the raceway pond reactor can be expressed as follows, Equation (2)
dCFU 1 ¼ ðC Cs Þ ki $Cs dt HRT i
(2)
where dCFU is the accumulation of bacterial communities in the dt 1 pilot-RPR during the solar photo-Fenton process, HRT is the dilution rate of the system in min1, ki is the bacterial inactivation rate constant (min1) and Ci and Cs are the inlet and outlet concentrations of microorganisms, respectively in CFU$mL1. 2.6. Process efficiency quantification The standard figures of merit suggested by IUPAC (Bolton et al., 2001) for the comparison and evaluation of advanced oxidation process (AOPs) can either be applied in terms of energy requirements, in the case of devices using artificial light, or in terms of area required, in the case of solar light applications, which is the case for the present study. In this regard, the figure of merit used was that intended for solar-driven systems, namely the collector area per order (Aco, m2$m3$order1). Aco is the collector area needed to inactivate 1log of microorganism per unit of volume over a time (t0) when “standardized” incident solar UV radiation was 30 W m2 and calculated for batch system (ABco) and continuous flow operation
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187
(ACco) following Equation (3) and Equation (4), respectively:
ABco ¼
ACco ¼
A$UV UV0 $t0 $V$0:4334$ki A$UV UV0 $t0 $Q $log CC0
(3)
(4)
where A (m2) is the collector area used, UV is the average of the incident solar UV radiation expressed in W$m2 and UV0 is “standardized” solar UVA radiation corresponding to 30 W m2 (typical solar UVA power on a perfect sunny Spring day around noon in Almería). V (m3) is the volume treated in batch operation and Q is the flow rate in m3$h1. C0 is the initial bacterial concentration and C is the bacterial concentration after 1 h of treatment. The average bacterial inactivation rate, ki , (h1) is obtained by a first order kinetic equation (Section 2.4). In addition, 0.4334 is the conversion factor between decimal and neperian logarithm. 2.7. Reagents and analytical methods Total and dissolved iron and hydrogen peroxide were measured by the standardized methods previously described by de la Obra et al. (De la Obra et al., 2019). The pH of iron stock solution was adjusted to 2.8 with sulphuric acid 0.1 M (95e98%, Panreac, Spain). To check the physico-chemical quality of secondary effluent batches, ion chromatography (Metrohm 881 Compact IC pro) and a TOC-V CSH/CSN analyser (SHIMADZU Corporation) were used for determining, by direct injection of filtered samples, anions and the dissolved carbon, organic and total inorganic carbon (TIC) concentrations, respectively. The chemical oxygen demand (COD) was quantified using a commercial kit (Hach LCK 314). Standard parameters such as pH and turbidity were measured using a portable pH-meter and turbidity-meter both acquired from Hanna, while the conductivity was determined by means of a conductivity-meter (Phywe). 3. Results and discussion 3.1. Influence of UV irradiance and iron concentration in batch mode operation on CFX-R bacteria inactivation The inactivation kinetics of ARB is studied in batch mode, since a simple first-order model is used to calculate the rate constants for comparison purposes. Fig. 2 shows the variation in the inactivation rate constants, ki, for total and CFX-R bacteria at three irradiance levels related to winter, spring and summer in Almería, Spain. In addition, the inactivation rate constants from Fenton control experiments were also included. In order to obtain accurate results and taking into account that the experiments were carried out in outdoor conditions, five replicates were carried out for 2 h at each irradiance level with initial iron and hydrogen peroxide concentrations of 20 and 50 mg L1, respectively, as reported to be the best reactant concentration for bacteria inactivation by solar photo mez et al., 2014). Significant difFenton in batch mode (Ortega-Go ferences in the initial bacterial concentration was found between total bacteria (2.9$104, 5.2$103 and 3.7$103 CFU mL1 for TC, E. coli and Enterococcus sp, respectively) and antibiotic resistant bacteria (1.7$102, 5.6$101 and 5.3$101 CFU mL1 for TC, E. coli and Enterococcus sp, respectively), in other words, around two orders of magnitude lower the CFX-R bacteria concentration with regard to the total. As per the total bacteria (TB), the highest inactivation rate constants were obtained for TC, while the lowest values were obtained for Enterococcus sp at each irradiance tested. These results
Fig. 2. Total and CFX-R bacteria inactivation rate constants by solar photo-Fenton in 5 cm-deep RPR in different seasons: winter (irradiance 12.85 ± 1.07 W m2), spring (irradiance 30.38 ± 0.69 W m2) and summer (irradiance 37.57 ± 0.55 W m2). 20 mg Fe2þ$L1 and 50 mg H2O2$L1.
corroborate previous studies where less bacterial damage and consequently a lower inactivation rate constant was observed in the case of Enterococcus sp than in the case of TC and E. coli (Esteban García et al., 2018). This can be explained due to the higher resistance shown by these gram-positive enteric bacteria (Enterococcus sp) because of the presence of a more resistant cell wall as reported elsewhere (Giannakis et al., 2016a). The highest bacterial inactivation rate constants were obtained with the highest irradiance in mez et al. (2016). Howsummer, in concordance with Ortega-Go ever, the irradiance values corresponding to spring and summer, 30.38 ± 0.69 and 37.57 ± 0.55 W m2, respectively, were not significantly different enough to justify the difference in the rate constant. Temperature also plays an important role in this inactimez et al., 2012) and, therefore, the difvation process (Ortega-Go ferences in temperature between spring and summer (24 ± 2 and 30 ± 1 C, respectively) made an important contribution to the total and CFX-R bacterial inactivation rates. Comparing the inactivation rate between total bacteria and CFX-R bacteria, no significant differences were observed. In both cases, inactivation rate constants very close to each other were obtained for the three microbiological groups studied. These results confirm those obtained by Giannakis et al. (2018a), who pointed out that antibiotic resistant bacteria do not need to be taken into account as a special factor in disinfection kinetics studies when the photo-Fenton process is applied. In addition, our study demonstrates that there are no differences between antibiotic resistant gram-positive and gram-negative bacteria (Enterococcus sp, TC and E. coli, respectively) since the inactivation rate constants were the same within confidence limits in all cases, corroborating once again, the aforementioned study. As for the effect of iron concentration on antibiotic resistant bacteria (CFX-R) inactivation, four iron concentrations (20, 10, 5 and 2.5 mg L1) were evaluated in triplicate with 50 mg L1 of hydrogen peroxide in winter (13 ± 1 W m2) (Fig. 3). The best iron concen mez et al., tration was 20 mg L1 as reported elsewhere (Ortega-Go 2014) and the linear increase of ki with Fe concentration shows the relevant effect of photo-Fenton on bacterial damage up to cellular inactivation. No significant differences between total and CFX-R bacteria inactivation constant were observed and the same slope in the ki vs. iron concentration linear relationship corroborates that the small differences shown in Fig. 3 are in the experimental error interval. To better compare total and CFX-R bacteria inactivation, a
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Fig. 3. Total and CFX-R bacteria inactivation rate constants by solar photo-Fenton in 5 cm-deep RPR at four iron concentrations (2.5, 5, 10 and 20 mg L1) and 13 ± 1 W m2. H2O2 concentration was 50 mg L1.
statistical analysis was carried out by pooling all data of ki obtained in batch mode, and representing ki for total bacteria (TB) vs. ki for CFX-R bacteria, Fig. 4. A statistically significant correlation between total and CFX-R bacteria inactivation with a confidence level of 95% was observed. The slope 1.003, demonstrates that the inactivation rate constants can be accepted as being the same for total and CFXR bacteria, the determination coefficient being 0.997. 3.2. Effect of HRT on CFX-R bacteria inactivation For scaling-up purposes, the solar photo-Fenton reactor must be operated in continuous flow mode, so it is necessary to study the effect of HRT on the inactivation of CFX-R bacteria. The study was carried out at four values of hydraulic residence time from 22.4 to 4.7 min working with the best iron (5 mg L1) and H2O2 (30 mg L1) concentrations previously reported for continuous flow disinfection of WWTP secondary effluents (De la Obra et al., 2019). The operation started in batch mode for 2 h and the continuous operation was then started by increasing the inflow rate every 2 h to verify the effect of the HRT reduction (Fig. 5). Bacterial inactivation
Fig. 4. Statistical analysis of the inactivation rate constants for total bacteria (TB) and cefotaxime resistant bacteria (CFX-R) obtained in batch mode by Fenton and photoFenton with different irradiance levels and iron concentrations. Experiments were carried out outdoors in a 5 cm-deep RPR.
Fig. 5. Concentration variation of total E. coli (closed squares) and CFX-R E. coli (open squares) by solar photo-Fenton as a function of hydraulic residence time: 22.4min ( ), 16.3 min ( ), 9 min ( ), 4.7 min ( ) and batch ( ). Hydrogen peroxide ( ) and total Fe concentrations ( ) are also presented. Experiments were carried out in a 5 cm-deep cylindrical reactor at 30 W m2 UVA irradiance.
below the DL was achieved after 70 min of treatment for total and CFX-R bacteria during batch mode operation. This result is consistent with that obtained in a previous study (Esteban García et al., 2018). In contrast, at continuous flow operation, significant differences were observed between 22.4 min and the other HRTs studied (16.3, 9.0 and 4.7 min). Although Fig. 5 only shows total and CFX-R E. coli inactivation, the results of total and CFX-R TC and Enterococcus sp are shown in Figures S1 and S2. In the case of 22.4 min, the system was able to reach bacterial inactivation under DL (1 CFU mL1) once steady state was achieved. However, in the cases of 16.3 min, 9.0 min and 4.7 min of HRT, the bacterial concentration increased up to values very close to the initial ones at steady state. It is important to remark that no regrowth was observed for any microbiological group studied in the collected samples during the experiments carried out at 22.4 min of HRT. Total and CFX-R E. coli inactivation rate constants were evaluated once steady state was achieved, showing rate constant values significantly higher (>143 ± 30 min1 for E. coli) in the case of 22.4 min of HRT when compared with 0.16 ± 0.01 min1, 0.18 ± 0.01 min1 and 0.30 ± 0.01 min1 in the case of 16.3, 9.0 and 4.7 min of HRT, respectively, Table 1. It is worth noting that when DL is achieved in steady state, there is uncertainty in the calculation of the kinetic constant due to the very high initial concentration and the unknown actual bacterial concentration below the DL, hence the reason for expressing ki > 143 ± 30 min1. Two features need to be highlighted from Table 1. First, the inactivation rate constants are much higher in continuous flow mode than in batch. In continuous flow operation, ferrous iron and hydrogen peroxide are continuously added to the reaction bulk and Fenton reaction continuously generates hydroxyl radicals and not only in the first few seconds of treatment as occurs in batch mode. This could explain a higher inactivation rate in continuous mode, so a Fenton assay in continuous operation was carried out with 20 mg L1 of iron and compared with continuous photo-Fenton (Table 2) to provide more insight on the explanation of this difference in inactivation rates. Both experiments were carried out with the highest iron concentration tested to enhance the Fenton effect. Although the Fenton inactivation rate constants in continuous mode were higher (one order of magnitude) than in batch, the differences between dark and illuminated operation in continuous mode were far higher. These results demonstrated the great influence of irradiance on the disinfection by solar photo-Fenton. As such, the explanation comes from the complex mechanisms of bacteria inactivation, as reported by Pulgarín and co-workers
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Table 1 Total and CFX-R bacteria inactivation rate constants by solar photo-Fenton at neutral pH in continuous flow mode as a function of HRT. Microbiological group
TC E. coli Enterococcus sp CFX-R TC CFX-R E. coli CFX-R Enterococcus sp
ki, min1 HRT ¼ 22.4 min
HRT ¼ 16.3 min
HRT ¼ 9.0 min
HRT ¼ 4.7 min
>629 ± 155 >143 ± 30 >43 ± 10 >8 ± 1 >5 ± 1 >3 ± 1
0.22 ± 0.03 0.16 ± 0.01 0.03 ± 0.01 0.15 ± 0.04 0.06 ± 0.01 0.01 ± 0.01
0.21 ± 0.03 0.18 ± 0.01 0.03 ± 0.01 0.16 ± 0.02 0.06 ± 0.01 0.02 þ 0.01
0.32 ± 0.03 0.30 ± 0.01 0.03 ± 0.01 0.21 ± 0.03 0.07 ± 0.01 0.03 ± 0.01
UVA irradiance ¼ 30 W m2. Temperature ¼ 25 C. Hydrogen peroxide and iron concentrations were 30 and 5 mg L1, respectively.
Table 2 Total and CFX-R bacteria inactivation rate constants by Fenton and solar photo-Fenton processes at neutral pH working at 22.4 min of HRT in continuous mode operation. Inactivation rate constant, ki, min1 Microbiological group
Fenton
photo-Fenton
TC E. coli Enterococcus sp
0.042 ± 0.001 0.033 ± 0.002 0.023 ± 0.002
>699 ± 108 >106 ± 23 >25 ± 3
CFX-R TC CFX-R E. coli CFX-R Enterococcus sp
0.031 ± 0.007 0.020 ± 0.008 0.018 ± 0.004
>14 ± 1 >4.3 ± 0.9 >3.1 ± 0.3
20 mg Fe2þ$L1 and 30 mg H2O2$L1. Photo-Fenton was carried out at 30 W m2 of UVA irradiance.
(Giannakis et al., 2016a). Photo-Fenton inactivation of bacterial cells is a synergic and additive effect of several factors such as radiation intensity, the oxidative action of hydrogen peroxide, the photoFenton reaction on cell membranes and the internal photoFenton reaction, giving rise to a multiple path oxidative destruction of vital cellular structures and molecules. In the case presented in this work, at neutral pH ferric iron formed from the reaction of H2O2, with freshly added ferrous iron, precipitates as ferric hymez et al., 2016). Iron hydroxides can produce droxides (Ortega-Go hydroxyl radicals through the heterogeneous photo-Fenton process (Ruales-Lonfat at el., 2015) and this precipitate can be adsorbed onto the cells, generating HO right at the membrane, favouring oxidative attack. In continuous flow, the dilution effect as the inlet stream enters the reactor, increases the likelihood of cell damage as opposed to initial batch conditions, when a much higher bacterial concentration is subjected to stressful conditions. Nonetheless, a minimum radiation energy requirement must be achieved, and energy dose must be taken into account. Following the procedure described by De la obra et al., (De la Obra et al., 2019), the cumulative radiation energy received per unit of treated water volume, QUVA, was calculated for the different HRTs, giving 0.80, 0.58, 0.32 and 0.17 kJ L1 for HRTs of 22.4, 16.3, 9.0 and 4.7 min, mez et al., respectively. As previously demonstrated (Ortega-Go 2016) the longer exposure time allows higher HO generation to be obtained, leading to higher bacterial inactivation being observed. A minimum of 1 kJ L1 has been reported to cause bacterial damage (De la Obra et al., 2019), a value similar to 0.80 kJ L1 obtained at 22.4 min HRT. The second feature in Table 1 is the apparent difference between inactivation rate constants for total and CFX-R bacteria when DL is reached in steady state. Unfortunately, the above-mentioned uncertainty in the calculation of ki from Eq. (2) with only two values of bacterial concentration, gives rise to a substantial impact of the inlet flow concentration, meaning the higher the initial concentration, the higher the rate constant. Accordingly, no conclusion can be drawn from these results and more research is needed for clarification. As for the HRTs when DL was not achieved, differences between rate constants for total and CFX-R bacteria were not significant.
3.3. Process efficiency analysis - figures of merit To evaluate the effect of the different factors tested in the previous sections on the scale up of the process, the figures of merit of the photo-Fenton process have been calculated as the collector area per order (Aco, m2$m3$order1) for batch system (ABco) and continuous flow operation (ACco) following Equations (3) and (4), respectively. The lower the collector area per order, the higher the efficiency of the process. Consequently, the determination of the collector area per order (ACO) for the operational modes (discontinuous and continuous), seasonal period (winter, spring and summer) and iron concentration used has been carried out. For that purpose, only the data for E. coli were used since a concentration limit is required by the Spanish legislation for that strain (RD 1620/2007). The results are shown in Table 3. The values obtained for discontinuous mode operation are two order of magnitude higher than for continuous mode. Among the three seasons, summer gives rise to the lowest ABco values. Regarding the effect of iron concentration in batch mode operation, the higher the iron concentration, the lower the area required and the higher the efficiency. In this respect, additional continuous conditions with lower HRT values (22.4, 16.3, 9 and 4.7 min) were tested in the solar simulator as shown in the Section 2.2. Under these experimental conditions, the figure of merit calculation is an extrapolation to the operation with natural sunlight in spring. In this case, only 22.4 min of HRT achieved the detection limit giving a value of ACco ¼ 2.7 square meters of solar collector area required to reduce in 1 h the amount of E. coli bacteria to one logarithmic unit (90% less) in one cubic meter of real secondary effluent as shown in Table 3. 4. Conclusions For the first time, the inactivation kinetics of total and cefotaxime resistant bacteria by solar photo-Fenton have been compared under very different treatment conditions, with no significant differences. The target of wastewater disinfection treatments can therefore be defined as total pathogen bacteria inactivation, as once
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Table 3 Effect of operation mode in different seasons on the estimated Aco values. Operational mode
Season
Irradiance (W m2)
[Fe] (mg$L1)
AC0 (m2$m3$order1)
Discontinuous
Winter Spring Summer Winter Winter Winter Winter Indoors Indoors Indoors Indoors
12.85 30.38 37.57 13.10 13.10 13.10 13.10 30 30 30 30
20 20 20 2.5 5 10 20 5 5 5 5
2009 ± 457 2137 ± 140 1063 ± 24 5977 ± 22 5287 ± 171 4104 ± 280 2311 ± 134 2.7 ± 0.3 11.9 ± 0.4 7.8 ± 0.3 4.4 ± 0.3
Continuous
HRT HRT HRT HRT
22.4 16.3 * 9* 4.7 *
* Detection limit was not achieved.
the detection limit has been achieved, ARB are also inactivated. The value obtained for the E. coli inactivation rate constant in continuous mode operation was around four orders of magnitude higher than in batch mode, with minimum energy dose (0.8 kJ L1 for HRT 22.4 min) to bring about disinfection below the detection limit (1 CFU mL1). For wastewater reuse in irrigation, these results of bacteria inactivation by solar photo-Fenton at neutral pH in continuous flow mode make this process suitable for large-scale application, since the treatment times are shorter than batch operation and reactor loading manoeuvres are avoided. Additionally, process efficiency has been evaluated in terms of figures of merit, the best condition being continuous flow operation, with 5 mg L1 of iron and 22.4 min of HRT giving the lowest value, 2.7 m2 m3$order1 of collector area per order and cubic meter, in contrast with 2137 m2 calculated for batch operation under the same solar UVA irradiance, 30 W m2. The operation of the solar photo-Fenton process in continuous flow mode opens up a new field of research as tertiary treatment because bacterial inactivation is much more efficient than in batch mode. However, more research is needed to elucidate the inactivation mechanisms and to develop process models in continuous flow. Acknowledgements This research has been supported by the Andalusian Regional Government (P12-RNM-1437) and the European Regional Development Fund (ERDF). I. de la Obra would like to acknowledge the Andalusian Regional Government for her grant. Ph.D. Gracia Rivas ~ ez wishes to thank MICINN for her Juan de la Cierva formation Ib an grant (FJCI-2017-34059). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2019.04.059. References Bolton, J.R., Bircher, K.G., Tumas, W., Tolman, C.A., 2001. Figures-of-merit for the technical development and application of advanced oxidation technologies for both electric- and solar-driven systems (IUPAC Technical Report). Pure Appl. Chem. 73, 627e637. https://doi.org/10.1351/pac200173040627. n Ferna ndez, F.G., Malato, S., S rez, J.A., Carra, I., Santos-Juanes, L., Acie anchez Pe 2014. New approach to solar photo-Fenton operation. Raceway ponds as tertiary treatment technology. J. Hazard Mater. 279, 322e329. https://doi.org/10. 1016/j.jhazmat.2014.07.010. Christou, A., Agüera, A., Bayona, J.M., Cytryn, E., Fotopoulos, V., Lambropoulou, D., €der, P., Fatta-Kassinos, D., 2017. The Manaia, C.M., Michael, C., Revitt, M., Schro potential implications of reclaimed wastewater reuse for irrigation on the agricultural environment: the knowns and unknowns of the fate of antibiotics and antibiotic resistant bacteria and resistance genes e a review. Water Res. 123, 448e467. https://doi.org/10.1016/j.watres.2017.07.004. CLSI, 2011. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-
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