CET Volume 86 DOI: 10.3303/CET2186143 Paper Received: 25 October 2020; Revised: 30 January 2021; Accepted: 3 May 2021 Please cite this article as: Aranciaga Pajuelo R.B., Vargas Lopez J.P., Castaneda Olivera C., Jave Nakayo J.L., Benites Alfaro E.G., Cabrera Carranza C.F., 2021, Inactivation of Antibiotic Resistant Bacteria in Hospital Wastewater by Tio₂/h₂o₂ Photocatalysis, Chemical Engineering Transactions, 86, 853-858 DOI:10.3303/CET2186143 CHEMICAL ENGINEERING TRANSACTIONS VOL. 86, 2021 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Sauro Pierucci, Jiří Jaromír Klemeš Copyright © 2021, AIDIC Servizi S.r.l. ISBN 978-88-95608-84-6; ISSN 2283-9216 Inactivation of Antibiotic Resistant Bacteria in Hospital Wastewater by TiO₂/H₂O₂ Photocatalysis Raúl B. Aranciaga Pajueloa, Jonathan P. Vargas Lópeza, Carlos A. Castañeda Oliveraa,*, Jorge L. Jave Nakayoa,b, Elmer G. Benites Alfaroa, Carlos F. Cabrera Carranzaa,b a Universidad César Vallejo, Campus Los Olivos, Lima, Peru b Universidad Nacional Mayor de San Marcos, Lima, Perú caralcaso@gmail.com Contamination of wastewater by drug-resistant bacteria is an emerging problem because it can have a direct effect on human health and the environment. Therefore, this research determined the photocatalytic efficiency of TiO2 and H2O2 in the inactivation of Escherichia coli (E. coli) and intestinal Enterococci (intestinal E.) bacteria. The tests were carried out in two photoreactors of 12 L each, achieving a percentage of efficiency of 75.90% and 93.01% in the inactivation of Escherichia coli and intestinal Enterococci, respectively, with a dose of 500 mg/L of TiO2 and 100 mg/L of H2O2. In addition, a higher percentage of efficiency in the inactivation of intestinal Enterococci was evidenced with respect to Escherichia coli, and the chemical and physical parameters of the wastewater improved with photocatalytic treatment, reaching values below the Maximum Allowable Values for non-domestic wastewater discharges. Finally, the results obtained showed that photocatalysis as a wastewater treatment method for the inactivation of antibiotic-resistant bacteria is efficient and could be used as an alternative to treat hospital wastewater. 1. Introduction The contamination of wastewater by Escherichia coli and intestinal Enterococci resistant to antibiotics is due to the indiscriminate use of antibiotics which have endowed resistance towards them, causing a negative environmental impact (Proia et al., 2018). According to the WHO (2018), resistance to antibiotics by bacteria is one of the biggest problems for public health. On the other hand, wastewater from hospital and urban areas, as well as wastewater treatment plants are points of generation of resistance to antibiotics (Kordatou, Karaolia and Kassinos, 2018). Different studies proved that advanced oxidation processes, specifically photocatalytic processes, can inactivate the bacterial reproductive capacity or eliminate them as a consequence of the process (Giannakis et al., 2018; Ttofa et al., 2019). The main function of photocatalysis in the inactivation of E. coli and intestinal Enterococci, is to eliminate and/or inhibit the reproductive properties of these bacteria, through photocatalytic reactions induced by the absorption of photons of light which can be from a natural or artificial source. In this photocatalytic reaction, the energy of the photons is absorbed by an electron from the valency band, which is promoted to the conduction band of the semiconductor, generating reactive oxygen species such as -OH radicals, which degrade organic compounds in a non-selective way, thereby microbial growth is inhibited damaging the cell membrane or breaking DNA chains (Moreira et al., 2018; Ameta et al., 2018). Previous investigations used TiO2 and H2O2 as photocatalysts under strong ultraviolet radiation, demonstrating an inactivation level greater than 80%, obtaining regrowth levels below the samples without treatment, after treatment by photocatalysis (Guo et al., 2017). An important aspect related to the treatment is the residual persistence of the disinfection effect after the treatment because the damaged bacteria can regrow under suitable conditions (Fiorentino et al., 2015). In Peru, hospital wastewater is drained directly without prior treatment, taking into account D.S N°. 021-2009-Vivienda, which indicates the Maximum Admissible Standards for physical and chemical parameters, but not microbiological parameters. 853 The use of photocatalysis for the degradation and / or inactivation of bacteria resistant to antibiotics, appears as a promising solution, and with the aim of achieving lower operating costs in the treatment of hospital wastewater, and to prevent the dissemination of these bacteria in the environment (Zhou et al., 2020; Vaiano et al., 2017). That is why the present research is based on the principles of microbial inactivation by photocatalysis using TiO2 and H2O2 with the main objective of determining the efficiency of photocatalytic treatment in the inactivation of E. coli and intestinal E. resistant to antibiotics in wastewater from the Naval Medical Center. 2. Materials and methods 2.1 Sample collection The samples came from the Naval Medical Center, Callao - Peru, collecting two samples of 1100 mL of water in the sewer network (272416 E and 8665953 N), to identify and quantify E. coli and intestinal E., as well as to analyze the physical parameters and starting chemicals. Likewise, the two photoreactors were filled with 12 L of wastewater each. 2.2 Photoreactor Construction The photoreactor is made of four transparent acrylic tubes, pipes, and a 12 L container attached to a 0.5 hp water transport pump, which assembled a closed circuit ensuring the homogenization of the photocatalysts with the wastewater to be treated. As shown in Figure 1, we are based on (Blanco, 2002). Figure 1: Photoreactor 2.3 Antibiotic Resistance Analysis The antibiotic resistance test according to Hudzicki, (2009) was applied. For this, Mueller Hinton agar and discs impregnated with antibiotics (penicillin, ampicillin and ciprofloxacin) were used, and the inhibition halos were measured using a vernier. Table 1 shows the level of sensitive, intermediate sensitive or resistant to the antibiotics. Table 1. Inhibition halos 2.4 Photocatalysis process For the photocatalysis process, 250 and 500 mg/L of TiO2 were used with 50 and 100 mg/L of H2O2, with experimentation times of 90 and 180 minutes to determine the best inactivation time of E. coli and intestinal E. and the effect on physical and chemical parameters. 2.4.1 Quantification of Escherichia coli and intestinal Enterococci The methodology proposed by the standard methods for the examination of water and wastewater was followed, (Baird, Eaton and Rice, 2017). For E. coli, 1.981 g of McConkey agar was prepared in 40 mL of distilled water and sterilized in an autoclave for one hour. Then, the solution was poured into Petri dishes (20 mL), then the microorganisms were inoculated and incubated at 37 °C for 24 hours. Finally, it was calculated from the colony forming units (CFU/100mL), using the following Equation 1. Antibiotic Disk content (µg) Diameter of the inhibition halo in mm (Escherichia coli) Diameter of the inhibition halo in mm (Intestinal Enterococci) Resistant Intermediate Sensitive Resistant Intermediate Sensitive Penicillin 10 ≤ 28 --- ≥ 29 ≤ 14 --- ≥ 15 Ampicillin 10 ≤ 13 14 -16 ≥ 17 ≤ 16 --- ≥ 17 Ciprofloxacin 5 ≤ 15 16 - 20 ≥ 21 ≤ 15 16 -20 ≥ 21 854 /100 = . ( ) ∗ (1) C.e = Colonies enumerated For intestinal Enterococci, 100 mL of Azide Dextrose (AD) broth culture medium was used, put in 10 fermentation tubes and verified pH in the range of 7.2 ± 0.2. 10 mL of the residual water sample was inoculated in each of the tubes with the AD broth and incubated at 35 ° C for 48 hours. Check if there was turbidity, if this is the case, continue with the analysis, but if the sample is not discarded. Transfer the presumptive tubes to Petri dishes with bile esculin agar; previously verify that the pH is between 7.1 ± 0.2; incubate at 35 ° C for 24 hours. The blackish colonies with brown halos are transferred to brain heart infusion (BHI) broth with 6.5% NaCl and incubated at 35°C for 48 hours. Finally, calculate the most probable number (MPN), Equation 2. /100 = 100 ∗ 10 (2) v = Sample volume of the lowest selected dilution 2.4.2 Physical and Chemical Analysis For the physical and chemical analysis, the methodology proposed by the standard methods for the examination of water and wastewater (Baird, Eaton and Rice, 2017) was used. The parameters analysed were pH, Oils and Fats (O and F), Biological Oxygen Demand (BOD5), Chemical Oxygen Demand (COD), Total Suspended Solids (TSS) and Temperature (T°), which were compared with the Maximum Permissible Values (MPV) established by the Ministerio de Vivienda Construcción y Saneamiento (2009) in in Supreme Decree No. 021-2009-VIVIENDA. These average values are: pH (6 - 9), O and F (100 mg/L), BOD5 (500 mg/L), COD (1000 mg/L), TSS (500 mg/L) and T° (<35 ° C). 2.5 Inactivation efficiency To determine the percentage of efficiency, the concentrations of the microorganisms before and after the treatment were considered. For this, Equation 3 was used, in which the initial and final data of E. coli and intestinal E. will be taken, in order to calculate the efficiency (Valencia et al., 2012). All bacterial inactivation tests were performed in triplicate. . . . ∗ 100 (3) Where: Initial C.: Initial concentration and Final C.: Final concentration 3. Results and discussion 3.1 Efficiency level in the inactivation of Escherichia coli and intestinal Enterococci The level of efficiency in the inactivation of Escherichia coli and intestinal Enterococci for each dose is shown in Figure 2. Figure 2: Percentage of inactivation efficiency: D1: 250 and 50 mg/L of TiO2 and H2O2, respectively; D2: 500 and 100 mg/L of TiO2 and H2O2, respectively; T1: 90 minutes; T2: 180 minutes D1T1 D1T2 D2T1 D2T2 0 20 40 60 80 100 E ff ic ie n cy ( % ) Dose Escherichia coli intestinal Enterococci 855 As shown in Figure 2, for dose D1 at time T1, no efficacy was shown for both bacteria in all three tests, it could be associated with low solar radiation during experimentation, which did not lead the photocatalytic reaction inactivate the E. coli and intestinal E. (Sillanpää, 2020), as well as the morphological conditions of each drug-resistant bacteria, which can endow them with a certain resistance to treatment (Serna et al., 2019; Sharma et al., 2016), but as the reaction time T2 increased in all three tests, a percentage of 16.1% and 31% is appreciated for E. coli and intestinal E. respectively. Likewise, for the D2 dose after a T1 reaction time in all three tests, it had an efficiency level of 63.3% and 33% for E. coli and intestinal E., respectively, but by increasing the T2 reaction time, the highest percentage of efficiency was gotten in both cases reaching 75.9% and 93.01% for E. coli and intestinal E., respectively, in all three tests. These results coincide with previous works on the inactivation of drug-resistant bacteria by TiO2 and H2O2, showing that the treatment has a good performance (Maniakova et al., 2020). For all the doses worked, the inactivation of E. coli and intestinal E. had an increasing trend, and the highest inactivation efficiency was achieved with the D2 dose at time T2, being higher for intestinal E. bacteria. This difference may be due to the fact that E. coli has a more resistant structure that does not allow the exchange of solutes with the medium (Giannakis et al., 2018). 3.1.2. Minimum time and effective dose The minimum time and effective dose were evaluated as a function of the decrease in bacterial concentration of Escherichia coli and intestinal Enterococci, as shown in Figure 3. Figure 3. Behaviour of the microorganisms during treatment: a) E. coli (5,40 E+8 CFU/100mL - Control) and b) intestinal Enterococci (2,40 E+06 MPN/100 mL - Control) From Figure 3 it is observed that the inactivation of the bacteria occurs mainly in time T1 when applying the D2 dose, determining that at a dose of 500 mg/L of TiO2 and 100 mg/L of H2O2 with a reaction time of 180 minutes are the minimum parameters to initiate bacterial inactivation for both Escherichia coli and intestinal Enterococci. In comparison, Pantoja et al. (2015) achieved total inactivation of E. coli after 20 min of photocatalysis treatment (UV-C/TiO2/SiO2). This difference in inactivation values is due to the type of wastewater and treatment used (Tiwari, Drogui and Tyagi, 2020). In addition, in Figure 3 it was also observed that the inactivation level initially increased by adding more TiO2 and H2O2, based on the fact that increasing the dose of photocatalysts increased the number of reactive oxygen species such as hydroxyl (-OH) radicals, which facilitated bacterial inactivation (Mun et al., 2016). However, it is known that by exceeding doses of photocatalysts greater than 1 g/L, the turbidity of the wastewater to be treated would increase and consequently inhibit the photocatalytic effect of TiO2 and H2O2, this is because the photons of light from ultraviolet rays, would not properly affect the particles of photocatalysts, reducing the efficiency of inactivation (White, 2002). 3.1.3. Sensitivity level The level of sensitivity of E. coli and intestinal E. during treatment was determined. According to Figure 2, it was possible to analyse that intestinal E. are more sensitive with regard to E. coli after photocatalytic 856 treatment, showing the highest percentage of efficiency in their inactivation, reaching values above 90%, being this bacterium considered the most sensitive to treatment. Comparing to the researches of Giannakis et al. (2018), Serna et al. (2019) and Ttofa et al. (2019), they obtained an efficiency percentage greater than 90% for E. coli, which worked under pH levels of 5.8 and the samples came from the biological treatment of a wastewater treatment plant (WWTP). 3.1.4. Evaluation of the physical and chemical parameters with the Maximum Permissible Values For the evaluation of the physical and chemical parameters, the pre- and post-test results of each parameter studied were considered. These values were compared with the maximum permissible values (MPV) established by the Peruvian Ministry of Housing, Construction and Sanitation. See Table 2. Table 2: Results of the physical and chemical analyses Parameter Unit Pre Test Post Test MPV pH BOD5 COD TSS O and F 1 -14 mg/L mg/L mg/L mg/L 6.26 286 344 270 109 7.28 241 316 266 68 6 – 9 500 1000 500 100 Temperature °C 21,1 21 < 35 As shown in Table 2, the photocatalytic treatment had a positive effect on the physical and chemical parameters, since the post-test results did not exceed the Maximum Permissible Values (Ministerio de Vivienda, Construcción y Saneamiento, 2009), but instead decreased compared to the results before treatment, determining that the treatment in question improves the parameters established by the standard. It should be noted that the values of Oils and Fats had a significant decrease, taking into account that the photocatalytic reaction triggers oxide reduction reactions, degrading the organic compounds present in the wastewater (Ameta et al., 2018; Vaiano et al., 2016). 4. 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