In last years, the occurrence of bacteria with the capacity of survive and multiply in the presence of the antibiotics specifically designed to kill them, the so-called Antibiotic-Resistant Bacteria (ARB) has become a global concern because of the potential threats they can pose to human health. Diseases produced by these ARB cannot be effectively treated with antibiotics, being responsible for more than 700,000 human deaths per year. ARB can transfer their antibiotic resistance genes (ARGs) from one bacterium to another, favouring the spread of antibiotic resistance in the environment. The World Health Organization (WHO) has considered ARB as one of the three main threats of the 21st century due to the large number of infections and deaths they cause worldwide each year.
One of the most concentrated sources of ARB are hospital effluents and, specifically, urine from immunocompromised patients where simultaneously coexist bacteria causing urinary tract infections with excreted pharmaceuticals. Currently, hospital urines are typically merged with other hospital effluents and then, discharged into the municipal sewer system and treated together with urban effluents in municipal wastewater treatment facilities (WWTPs). The inefficiency of biological treatments from WWTPs to treat hospital effluents leads the spread of ARB and ARGs in the environmental water sources, negatively affecting aquatic organisms and human health. This makes urine treatment a key objective for reducing the environmental and health impact of hospital effluents. For this reason, the priority of this project is to face the problem directly in the pollution source, within the framework of the regional and national projects "Electrochemical technologies for the treatment of hospital urines: reduction of environmental and health impact (SBPLY/17/180501/000396) and "Electrochemical technologies facing the challenge of hospital urine treatment (PID2019-110904RB-I00), respectively.
In this context, electrochemical oxidation (EO) has aroused great interest among disinfection processes since EO has been reported to attain complete disinfection in urban wastewaters thanks to the in-situ generation of disinfectant species from the electrooxidation of the ions naturally contained in the effluent treated. This PhD Thesis is focused on the removal of bacteria from hospital urines based on our previous works related to the disinfection of urban treated wastewater, facing the challenge of the reactor layout. Specifically, the main objective of the present PhD Thesis is to assess the technical feasibility of different electrochemical processes including electrolysis and photoelectrolysis, to reduce the environmental and health impact of ARB from hospital urines, avoiding the formation of undesired disinfection by-products. To meet this general objective, results presented in Chapter 5 have been divided in the following five different sections, which correspond to the five partial objectives of this work.
In section 5.1, a prospective study of pathogens causing Urinary Tract Infections (UTIs) was carried out within the period 2014 to 2018. This study was accomplished in collaboration with the University Hospital Complex of Albacete, Spain (CHUA) as model of sanitary facility. This allows to evaluate the occurrence and fate of ARB in a real environment as well as the problematic associated with ARB in a hospital complex. The study proved that E. coli was the most significant bacteria found in Positive Urines (PUs) and that gram-negative bacteria (E. coli, K. pneumoniae, P. aeruginosa and P. mirabilis) predominated over gram-positive bacteria (E. faecalis) and yeasts (C. albicans). K. pneumoniae showed the highest percentages of antibiotic resistance. However, the research carried out on the disinfection processes of ARB was mainly related to the removal of E. coli, which confirms that the importance given to K. pneumoniae as ARB from a sanitary viewpoint does not correspond to the research carried out on the disinfection processes. Hence, it was pointed out the need to search and develop novel technologies that allow to remove K. pneumoniae for decreasing the sanitary and environmental impact of the effluents infected with this bacterium.
In section 5.2, a preliminary study of the electrochemical technology as an alternative for the removal of bacteria in two different treatment scenarios (synthetic urban wastewater and synthetic hospital urines) was conducted using a parallel flow reactor with BDD anodes at 10 A m-2. This study proved that even the bacterial removal efficiency was lower in hospital urines (competitive oxidation reactions), the occurrence of hazardous disinfection by-products was avoided in this matrix since the production of hypochlorite and the subsequent formation of inorganic chloramines were favoured.
Subsequently, a preliminary evaluation of electrolysis for the reduction of the potential chemical risk of hospital urines was also conducted. Chloramphenicol (CAP) was selected as a model of antibiotic and the influence of anodic material (BDD and MMO) and current density (50, 25, 12.5 A m-2) was evaluated. This study concluded that the electrochemical oxidation with BDD anodes applying 12.5 A m-2 (8 Ah dm-3) allows to degrade CAP from hospital urines, increasing their biodegradability up to 40 % follows the Zahn-Wellens method. However, this study also pointed out that the electrical charges required for the removal of antibiotics were much higher than those required for disinfection (8 Ah dm-3 vs. < 0.15 Ah dm-3) and then, other parallel studies out of this thesis were carried out on this topic.
In this regard, the development of suitable disinfection technologies as a pre- treatment to remove K. pneumoniae from hospital urines was evaluated in section 5.3. The main objective of this section was to gain insight into the role of the electro- generated oxidants on the disinfection process. Firstly, the contribution of chloramines on the electrochemical disinfection process was evaluated testing two concepts of electrochemical cell design with MMO anodes: a conventional parallel flow reactor and a microfluidic flow-through reactor. The influence of current density (5-50 A m-2) was also studied, and results were compared with other simpler "urine" matrices and with chemical disinfection tests. Results showed that the disinfection process of K. pneumoniae from hospital urines relies on the current density and the reactor layouts. The presence of chlorides in hospital urines contributed to the generation of hypochlorite and chloramines. The formation of chloramines was enhanced using the flow-through reactor layout. Additionally, chemical disinfection tests proved a stronger bactericidal effect of hypochlorite in comparison with chloramines. However, chloramines played a key role in the disinfection process since they contribute not only as disinfectants but avoiding the generation of chlorine derived by-products.
Additionally, the contribution of ozone on the electrochemical disinfection process was also evaluated testing a PEM-electrolyzer (MIKROZON® cell) especially designed to produce ozone in low-conductivity water. The influence of current intensity and hospital urine composition was studied. Results showed that the MIKROZON® cell reached total disinfection from current intensities higher than 0.5 A. The combined effect of ozone and chlorine disinfectants attained higher disinfection rates than the values obtained when single ozone was the main disinfectant electrogenerated (urine without chlorides) at current intensities lower than 0.5 A. At values of 1.0 A, the disinfection rates were quite similar which revealed that large amounts of ozone were electrogenerated. The crystal violet assay showed that the combined effect of all disinfectants promoted higher cell damages, increasing the cell wall permeability. Ozone was proved to attack DNA to a greater extent from current intensities higher than 0.5 A. Finally, during the treatment of diluted urines at 1.0 A, higher disinfection rates were obtained due to the minimization of competitive reactions.
Electrochemical oxidation can promote the formation of hydroxyl radicals and disinfectant species from water electrolysis which may be activated by the irradiation of UV light, increasing the quantity of free radicals available for disinfection purposes. In section 5.4, the assessment of coupling of electrochemical oxidation with UV light was developed using a microfluidic flow-through reactor for the removal of K. pneumoniae in hospital urines. The influence of the current density (5-50 A m-2) and the anode material (BDD and MMO) was tested on the production of disinfectants not only of hypochlorite but also monochloramine, dichloramine and trichloramine. Results showed that UV disinfection could not reach the complete disinfection of K. pneumoniae. However, electrochemical oxidation with BDD and MMO anodes led to complete removal of ARB from urine when applying 50 A m-2. The disinfection rate was higher when working with MMO anodes since BDD anodes favoured competitive oxidation reactions between bacteria and the organics contained in urine. Finally, photoelectrolysis was proved to enhance single UV disinfection and electrolysis performances. A marked synergistic effect was found when UV disinfection was enhanced by electrolysis at 5 A m-2 with BDD and MMO anodes.
In section 5.5, the validation of electrochemical technology for the treatment of complex synthetic urine matrices (polymicrobial urines) was carried out. The polymicrobial hospital urines simulates real hospital urines since the pandemic caused by the coronavirus SARS-CoV-2 did not allow us to validate the electrochemical technology in a real environment due to the sanitary restrictions. Firstly, the simultaneous occurrence of more than one pathogen causing UTIs in real hospital urines was analysed based on data supplied by the Microbiology and Parasitology Service from CHUA. From this study, the most prevalent combinations of bacteria presented in real urines from hospitalized patients were: 1) E. faecalis and K. pneumoniae, 2) E. coli and E. faecalis, and 3) K. pneumoniae and E. coli. Subsequently, the microfluidic flow- through reactor and the MIKROZON® cell were evaluated for the abatement of ARB pairs working under the most suitable operating conditions reported in previous sections. Hence, the microfluidic flow-through reactor was tested with MMO anodes at 50 A m-2 and the MIKROZON® cell worked at 1 A. Results showed that the microfluidic flow-through reactor achieved removal rates between 5 and 6 logs after 180 min whereas the MIKROZON® cell reached the total disinfection (7 logs) after 60 min. Additionally, the denaturation of DNA and ARGs in polymicrobial hospital urines was also considered. While no noticeable changes in the ARGs concentration were observed with the flow-through reactor, the MIKROZON® cell reached a mean decrease in ARGs ranked as follows: blaKPC (4.18-logs) > blaTEM (3.96-logs) > ermB (3.23-logs), offering a mean depletion of 3.77-logs for all ARGs tested. Finally, Scanning Electron Microscope (SEM) images confirmed the complete disinfection attained using the MIKROZON® cell where severe damages were induced in the cell walls, resulting in the integrity loss of bacterial structures
