Development and Performance Evaluation of a UV-C Coupled Recirculation System for Enhancing Cistern Water Microbiological Quality

Development and Performance Evaluation of a UV-C Coupled Recirculation System for Enhancing Cistern Water Microbiological Quality | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Performance Evaluation of a UV-C Coupled Recirculation System for Enhancing Cistern Water Microbiological Quality Jardielen Chaves Sousa, Adelaide Sampaio Oliveira, Dionéia Evangelista Cesar, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7359132/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In semi-arid regions, the absence of adequate water treatment for human consumption frequently gives rise to intestinal disorders and, in more severe cases, severe microbial infections. Within the context of the semi-arid region, many households depend on cistern water for their potable needs. This project aimed to develop and use a system composed of a UV-C radiation-emitting lamp coupled to a water recirculation pump to ensure the bacteriological safety of cistern water. The recirculation system was operated for 6, 12, and 24 hours, and tests were conducted using environmental samples or with E. coli ATCC 25922. The results demonstrated that the system could achieve 100% inhibition of the growth of total coliforms and thermotolerant coliforms after 6 hours of operation. The low-cost system has the potential to function as a social technology, thereby ensuring water security in semi-arid regions where there is no public water distribution system. Potable water semiarid public policies environmental sanitation sustainable development goals Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Rainwater harvesting is common in many parts of the world [ 1 ]. One way to store rainwater is through the use of cisterns, which provide water to families that do not have access to public supply systems [ 2 , 3 ]. Although it is considered a sustainable alternative to ensure water security, the consumption of water stored in cisterns can pose health risks to the population that consumes it [ 4 ]. Cisterns are usually low-cost structures built close to houses, where rainwater that falls on the roof is channeled and directed into the reservoir [ 5 ]. The conditions for collecting and storing water can favor the growth of pathogenic microorganisms [ 6 ], and health control and education are needed to prevent diseases resulting from microbiological contamination of water in cisterns. Diarrhea is one of the diseases caused by microorganisms in water consumed without adequate treatment. It is estimated that diarrhea caused 1.17 million deaths worldwide in 2021 [ 7 ], making it one of the leading causes of death among people of all ages. Taking into account estimates for children under 5 years of age, diarrhea is the fifth leading cause of death worldwide, with 446,000 deaths in 2016 [ 8 ]. Bacteria, protozoa and viruses, represented by Escherichia coli , Giardia spp . and Rotavirus A, respectively, are microorganisms that cause infections [ 9 – 11 ] leading to diarrhea in children in the Brazilian semi-arid region. Research by Hamilton et al. [ 4 ] shows that rainwater collected from roofs and stored in tanks is contaminated with E. coli in between 24 and 92% of samples. The use of chemical compounds to disinfect water is common, but their potential risks to human health are known. Ultraviolet C (UV-C) radiation has a known antimicrobial effect when used to reduce the density of microorganisms in water [ 12 ] and air [ 13 ]. UV radiation emitted by low-power lamps has a low potential to form harmful by-products [ 12 ]. UV-C radiation with a spectrum between 200 and 280 nm inactivates microorganisms by damaging their nucleic acids. The wavelength range of 250–270 nm is strongly absorbed by nucleic acids, making it highly effective for inducing cellular lethality or viral inactivation [ 14 ]. This is due to the peak absorption of DNA occurring near 260 nm [ 15 ]. Most of the lethal effects of UV radiation on biological systems are attributed to photochemical changes in pyrimidine residues [ 16 ]. There are several studies in the literature demonstrating the success of using UV-C radiation to control microbial growth on surfaces [ 17 ], in air [ 18 , 19 ], and in water [ 20 , 21 ]. In terms of water disinfection, UV-C radiation is effective against several pathogenic species, including E. coli [ 22 , 23 ]. The use of devices emitting radiation in the UV-C range can reduce the microbial density and consequently improve the quality of cistern water, making it potable from a microbiological point of view. The present study aimed to assemble a water recirculation system consisting of a water pump coupled to a device containing a UV-C lamp and to evaluate its efficiency in inactivating total and thermotolerant coliforms, including E. coli , in cistern water. 2. Material and Methods 2.1. Structure and Operation of the Water Recirculation System Each recirculation unit was assembled to contain a 20-litre PVC barrel, a submersible water recirculation pump with a capacity of 150 L h- 1 , and a device including a 15W Osram® UVC tubular fluorescent lamp with peak emission at 254 nm and 7.800 cd of light intensity. Water flows unidirectionally between the surface of the lamp and a PVC tube into which it is inserted, with a maximum distance of 1 cm from the lamp when it enters the device, which has enough volume for 180 mL of water. The recirculation pump, located inside the drum, was connected to the device containing the UV-C lamp through a silicone tube, with the other end of the device containing another tube that supplied water directly to the barrel (Fig. 1 ). 2.2. Study site and sampling Water samples were collected from concrete cisterns used to store rainwater at five sites in the interior of the states of Ceará and Piauí, Brazil (Fig. 2 ). Approximately 45 liters of water were collected at each sampling time and immediately sent for experimental set-up and physicochemical analysis. 2.2.1. Physicochemical analysis The collected samples were analysed for pH, chloride concentration, electrical conductivity, turbidity, total hardness, total alkalinity, and calcium content. The determination of pH, electrical conductivity, and turbidity was carried out using a LUCA-210 pH meter (Lucadema), an AT-255 microprocessor-based conductivity meter (Alfakit), and a 2020we portable turbidity meter (LaMotte), respectively. The remaining parameters were quantified according to the methods described in the Standard Methods for the Examination of Water and Wastewater [ 24 ]. 2.2.2. Carrying out experiments with recirculation Before the experiments, the entire system was disinfected with 0.1% (v/v) sodium hypochlorite. For each experiment, a set of 4 tanks was used, including a "control" tank in which the water was recirculated but without activation of the UV-C lamp (NoUV treatment) and another 3 in which the ultraviolet radiation was kept on to obtain samples in periods of 6h, 12h and 24h of continuous operation (UV treatments). Each barrel received 10 L of water from the same cistern, and the water was circulated at 150 L h − 1 . The barrels were kept closed, and the laboratory was dark throughout the experiment. After each interval, water samples were taken in triplicate and then subjected to microbiological analysis. 2.2.3. Microbiological analysis 2.2.3.1. Cultivable heterotrophic bacteria Aliquots of 1.0 mL were used to perform the pour plate technique on R2A agar (Himedia®) containing 0.64% fluconazole (to inhibit fungal colony growth). Cultures were performed in triplicate using samples from three of the five cisterns evaluated. Plates were incubated at 30 ºC for 48 hours and the colonies obtained were counted. 2.2.3.2. Total and thermotolerant coliforms Total and thermotolerant coliforms were determined according to the method described by the American Public Health Association [ 24 ]. Presumptive tests were performed using tubes containing lactose broth (Himedia®). Confirmatory tests were performed using tubes containing Brilliant Green broth 2% (Himedia®) and EC broth (Himedia®) to detect total and thermotolerant coliforms, respectively. Analyses were performed using the most probable number method (MPN.100 mL − 1 ) with Durham tubes inverted in the medium to detect gas production. As a control (time 0 h), microbiological analyses were performed on the water from the cisterns as soon as it arrived at the laboratory. 2.2.3.3. Typical E. coli cells Aliquots of 100 mL of water were vacuum filtered through 0.45 µm pore filter membranes designed to retain bacterial cells. The membranes were transferred to Petri dishes containing eosin-methylene blue agar (Himedia®). Plates were incubated at 37ºC for 24 hours, followed by colony counting. Analyses were performed in triplicate for each time the system was irradiated with UV-C radiation. 2.3. Assays with Escherichia coli ATCC 25922 2.3.1. Determination of the growth curve for E. coli ATCC 25922 First, E. coli ATCC 25922 was activated in TSA medium (Himedia®) with incubation at 37ºC for 24 h. After obtaining isolated colonies, one was inoculated into 50 mL of nutrient broth (Himedia®) in a 125 mL Erlenmeyer flask to obtain a pre-inoculum. The incubation was carried out with shaking at 37ºC for 24 h. The absorbance was measured in a spectrophotometer at 600 nm, and the pre-inoculum was added to an Erlenmeyer flask containing 400 mL of nutrient broth (Himedia®) in a volume sufficient to obtain an OD600 of 0.05. Incubation was carried out at 37ºC with shaking for 24 h. Every two hours, a 3 mL aliquot of the culture was taken to determine the absorbance at 600 nm to determine the bacterial growth curve. 2.3.2. Preparation of mesocosms with E. coli ATCC 25922 After preparation and sterilization of 40 L of 0.1% peptone water, each barrel received 10 L. At the same time, E. coli ATCC 25922 cells were grown as described in 2.3.1 to log phase, then washed and centrifuged at 6,000 rpm for 5 min. The supernatant was discarded, and the sedimented cells were resuspended in sterile 0.9% saline using a vortex mixer. This procedure was repeated two more times. The resuspended contents were assayed for absorbance at 600 nm, and 1 mL of the saline solution containing cells with an OD600 of 0.05 was added to each tube. The experiment was carried out according to the methodology described in point 2.2.2. To obtain the representative sample at time 0h (control), the system was left to run for 10 min without UV-C irradiation to allow homogenization of the cells. Aliquots were then taken for analysis. 2.3.3. Microbiological analysis 2.3.3.1. Thermotolerant coliforms The most probable number (MPN) was determined following the procedures outlined in section 2.2.3.2, with E. coli cells serving as the sole representatives of thermotolerant coliforms in the experiment. 2.3.3.2. Prokaryotic density Samples were fixed in 2% formalin and stored for later filtration through 0.22 µm polycarbonate membrane filters. Fluorescence in situ hybridization (FISH) was used to quantify E. coli ATCC 25922 cells using the ENT183 probe [ 25 ]. The hybridization procedure was performed according to Del`Duca et al. [ 26 ], using 20% formamide and NaCl concentrations for hybridization and washing, respectively. A generic probe was used as a negative control. The probes were labeled with Cy3 fluorochrome. Prokaryotic density was determined by evaluating ten random fields using an Olympus BX60 epifluorescence microscope (Olympus, Japan) equipped with Chroma U-N41007, U-MWU2, and U-MWG2 filters. Total prokaryotic density was determined by staining with 4',6-diamidino-2-phenylindole - DAPI [ 27 ]. 2.4. Statistical analysis Statistical analyses were conducted using SigmaPlot 12.0 software. Data distribution was assessed for normality using the Shapiro-Wilk test. Subsequently, comparisons were performed using Dunnett’s test or t-test at a significance level of 5%. 3. Results and discussion 3.1. Physicochemical parameters The analysis of the physicochemical data revealed that only one of the water samples did not present the basic pH value, with an average value of 8.3 ± 0.86. Concerning the conductivity data, a variation was observed from 15.5 µS cm- 1 to 159.8 µS cm − 1 . The turbidity values ranged from 0.2 to 1.16 NTU, while the chlorides occurred at an average of 9.6 ± 4.17 mg L − 1 . The alkalinity and hardness data indicate that the water stored in the evaluated cisterns is primarily influenced by calcium compounds [ 28 ] as basic components, likely resulting from the gradual release of calcium from the cement used in the construction of the cisterns. During the evaluation process, the Guideline for Drinking Water Quality [ 29 ] was utilized to establish limits for conductivity, chlorides, and turbidity, with maximum values of 2,000 µS cm − 1 , 250 mg L − 1, and 5.0 NTU, respectively, and a pH range between 6.5 and 8.5. Accordingly, the World Health Organization asserts that the water in the cisterns exhibits adequate physical-chemical quality for human consumption. When considering UV-C radiation for water disinfection, turbidity is recognized in the literature as one of the physical-chemical parameters that cause the most significant interference. Research has demonstrated that elevated turbidity levels can substantially compromise the efficacy of UV disinfection systems. Nourmoradi et al. [ 30 ] performed experiments using a UV reactor and observed that increasing turbidity from 1 to 5 NTU resulted in a 0.2 to 0.5 log reduction in microbial inactivation. Wu and Doan [ 31 ] observed a similar decline in disinfection efficiency as turbidity increased within the same range. 3.2. Evaluation of the effectiveness of the system for cistern water After evaluating the density of heterotrophic bacteria, the average values showed a significant variation between the system operating when UV radiation was applied and when compared to the control (system operating without UV radiation) (Fig. 3 ). Considering the density of heterotrophic bacteria present in the water immediately after sampling, according to the Dunnett's test at 5% probability, only the 6-hour treatment in the recirculation system promoted a significant reduction in the density of these microorganisms, causing an average reduction of 99.6%, considering the average data of the water from the cisterns evaluated (Fig. 3 a). When the system is operated without UV radiation, there is a tendency for the bacterial density to increase. This is probably due to the movement of the water, which increases the availability of oxygen and promotes metabolic activity and population growth of heterotrophic bacterial groups [ 32 ]. The experiments were conducted under conditions of darkness to prevent photoreactivation, a process that has been demonstrated to reverse specific types of ultraviolet (UV) damage [ 33 ]. However, it is important to note that bacterial DNA damage repair mechanisms function through various pathways that do not depend on light. These mechanisms are crucial for maintaining genomic integrity in response to diverse DNA lesions caused by environmental factors. Key repair pathways include direct reversal, base excision repair, nucleotide excision repair, and double-strand break repair, each employing distinct proteins and processes to address specific types of damage [ 34 , 35 ]. This may provide a rationale for the increase in the number of heterotrophic bacteria quantified after 12 hours and 24 hours of system operation. The data for total and thermotolerant coliforms shows that the recirculation system combined with the ultraviolet radiation was effective across all exposure times tested (Table 2 ). In all the treatments, for all cisterns, no bacterial growth was observed at any dilution level tested using the multiple-tube technique. Despite the lack of a consensus microorganism that can be universally established as an indicator of microbiological water quality, certain microbial groups are widely used for this purpose, with total coliforms and fecal coliforms being the most commonly used [ 36 , 37 ]. In the present study, a significant reduction in the concentration of the microorganisms analyzed was observed in all the time intervals evaluated, demonstrating that even in the shortest time of exposure to the equipment (6h), the water presented a satisfactory microbiological quality, in according to Guideline for Drinking Water Quality [ 29 ]. Table 1 Physicochemical parameters evaluated in the water from the cisterns used in the present study Cistern 1 2 3 4 5 pH 6.85 9.12 8.74 8.49 8.33 Conductivity (µS cm − 1 ) 15.53 20.77 70.26 159.83 157.73 Turbidity (NTU) 1.17 0.9 0.17 0.2 0.7 Chlorides (mg L − 1 de Cl − ) 9.12 3.61 8.56 14.54 12.49 Alkalinity (mg L − 1 de CaCO 3 ) 13.27 27.52 58.93 74.2 50.94 Total Hardness (mg L − 1 de CaCO 3 ) 6.86 14.21 56.68 58.96 45.57 Partial Hardness (mg L − 1 de Ca 2+ ) 4.74 14.37 54.23 54.72 43.45 Table 2 Density of total coliforms and thermotolerant coliforms (MPN.mL − 1 ) in aliquots of water obtained from cisterns (Control) and after 6h, 12h and 24h of the same water kept in a closed recirculation system coupled to a type C ultraviolet radiation emitting system. 0h (Control) 6h 12h 24h Total coliforms (MPN.mL − 1 ) Cistern 1 46 < 0,3 < 0,3 < 0,3 Cistern 2 0,36 < 0,3 < 0,3 < 0,3 Cistern 3 11 < 0,3 < 0,3 < 0,3 Cistern 4 2,3 < 0,3 < 0,3 < 0,3 Cistern 5 46 < 0,3 < 0,3 < 0,3 Thermotolerant coliforms (MPN.mL − 1 ) Cistern 1 0,36 < 0,3 < 0,3 < 0,3 Cistern 2 0,36 < 0,3 < 0,3 < 0,3 Cistern 3 1,1 < 0,3 < 0,3 < 0,3 Cistern 4 2,3 < 0,3 < 0,3 < 0,3 Cistern 5 15 < 0,3 < 0,3 < 0,3 Adhikari et al. [ 38 ] indicate that turbidity can interfere with the effectiveness of ultraviolet radiation in inactivating E. coli in water. According to the results obtained in the present study, the range between 0.2 and 1.17 NTU was not large enough to interfere with the effectiveness of the radiation applied to reduce total and thermotolerant coliforms. This is evidenced by the fact that the results are the same for all system recirculation times, regardless of the turbidity level of the samples. Concerning the analysis of typical E. coli cells performed employing the filter membrane technique, the results demonstrated the effectiveness of the system in inactivating the cells for all times evaluated (Table 3 ). Table 3 Density of typical E. coli cells (cells.100 mL − 1 ) estimated from the membrane technique in aliquots of water obtained from cisterns (Control) and after 6h, 12h, and 24h from the same water kept in a closed recirculation system coupled to a type C ultraviolet radiation emitting system Typical E. coli cells (cells.100 mL − 1 ) 0h (Control) 6h 12h 24h Cistern 1 0 0 0 0 Cistern 2 0.33 0 0 0 Cistern 3 1 0 0 0 Cistern 4 0 0 0 0 Cistern 5 1.67 0 0 0 3.3. System evaluation with E. coli ATCC 25922 After contaminating 0.1% peptone water with E. coli ATCC 25922 and introducing it into the recirculation systems—both with and without UV-C radiation—the initial quantification of thermotolerant coliforms was conducted by measuring the E. coli density, which was found to be 2.4 x 10 3 MPN mL − 1 (Fig. 4 ). Following the initiation of system operation, a statistically significant variation was observed between the treatments containing or not ultraviolet radiation (UV x NoUV). During the 6-hour operating period of the system with ultraviolet radiation, no positive tubes for thermotolerant coliform growth were detected. In contrast, the quantification of thermotolerant coliforms reached 7.5 x 10⁴ MPN mL⁻¹ when the system operated without ultraviolet radiation. A similar outcome was observed in the 12-hour UV treatment, while the 12-hour NoUV treatment resulted in data points exceeding 1.1 x 10 6 MPN.mL − 1 . After 24 hours of operation, the NoUV system maintained a thermotolerant coliform density above 1.1 x 10⁶ MPN.mL⁻¹, whereas the UV system effectively controlled these coliforms, with a quantified density of 83.6 MPN mL⁻¹. The lack of microbial growth in the tests conducted for 6-hour and 12-hour operating times confirms the system's effectiveness in inactivating total and thermotolerant coliforms, using E. coli as the indicator microorganism. After 12 hours of system operation without UV-C radiation, an increase in the density of thermotolerant coliforms (represented by E. coli ) was observed, although the exact magnitude of this increase could not be accurately quantified. This finding suggests that UV-C radiation may have been the factor responsible for regulating the population under study. The quantification of 83.6 MPN.mL − 1 of thermotolerant coliforms after 24 hours of UV-C treatment may be attributable to the heightened sensitivity of E. coli population to radiation during the exponential growth phase. However, after repeated exposure cycles, genomic adaptation can lead to increased resistance to ultraviolet radiation (Selveshwari et al., 2021). Moreover, Maghsoodi et al. [ 40 ] demonstrated that E. coli strains can repair DNA damage even in the absence of light, independent of photoreactivation, enabling some cells to remain viable post-repair despite dark conditions. Furthermore, Recacha et al. [ 41 ] identified several light-independent repair genes in the genome of strain ATCC 25922, including recA , recB , lexA , uvrA and uvrB . The expression of these and other genes involved in the response to UV-C-induced damage explains the detection of cells 48 hours after treatment in the recirculation system. By direct counting using DAPI, the average density of prokaryotic cells was initially quantified at 0.34 x 10 6 cells.mL − 1 . Similarly, by fluorescent in situ hybridization using the ENT183 probe, which allows the quantification of members of the Enterobacteriaceae family, we attempted to quantify the population of E. coli ATCC 25922 cells introduced into the system, resulting in an initial density of 0.14 x 10 6 cells.mL − 1 . As shown in Fig. 5 , during the first 6 hours of system operation, there is a significant difference in the density of both prokaryotic cells and Enterobacteriaceae representatives, according to the t-test at 5% probability. Only for prokaryotic cells the difference does not occur for the 12-hour period. The direct counting data, both for total prokaryotic cells and for Enterobacteriaceae, confirm the effectiveness of the system. The efficacy is even more evident if we look specifically at the initial time (0h) of the 6h UV treatment (Fig. 6 ). In this case, a significant reduction is observed for both prokaryotic cells and representatives of Enterobacteriaceae. After 6 hours of operation of the recirculation system with UV-C, there is a reduction of prokaryotic cells and Enterobacteriaceae to 0.13 x 10 6 cells.mL − 1 and 0.03 x 10 6 cells.mL − 1 , respectively. This represents a percentage of 60.77% for prokaryotic cells and 76.21% for Enterobacteriaceae. Considering that the system was disinfected and E. coli ATCC 25922 cells were added as a representative of Enterobacteriaceae, a 76.21% reduction in E. coli density in the system after 6 hours of recirculation is indicated. Comparing the quantification of thermotolerant coliforms with that of prokaryotic cells and Enterobacteriaceae under UV irradiation, a variation in the magnitude of the data is observed. While for thermotolerant coliforms there is an increase only during the 24h treatment and even then with a most probable number of 83.6 cells.mL − 1 , the microbial density by direct count indicates a greater growth over time. With 12h recirculation, even with the presence of ultraviolet radiation, there was an increase in the total number of prokaryotic cells from 0.34 to 2.16 x 10 6 cells.mL − 1 , an increase of 6.35x. Considering the 24-hour period, the increase was 19-fold, as the density increased to 6.47 x 10 6 cells.mL − 1 . The same occurred for Enterobacteriaceae cells, which increased 2.85x (0.4 x 10 6 cells.mL − 1 ) and 47.85x (6.7 x 10 6 cells.mL − 1 ) for the 12h and 24h times, respectively. These variations indicate that although the system is functional for a recirculation time of 6h, times longer than this do not contribute to an improvement in the microbiological quality of the water, but rather result in an increase in both cultivable and non-cultivable microbial populations. The differences between cultivation data and direct microbial enumeration data (Fig. 7 ) indicate that the abiotic conditions of the recirculation system maintain a large proportion of cells in the viable but non-culturable (VBNC) state. Considering the reductions in microbial densities observed between the initial time point and after 6 hours of exposure to UV-C radiation in the recirculation system, which demonstrated higher disinfection efficiency, several notable trends were identified. A 2.5-fold decrease was detected in the total prokaryotic cell density, as determined by DAPI staining. The density of Enterobacteriaceae, represented by E. coli ATCC 25922 cells introduced as contaminants, showed a 4.2-fold reduction based on the FISH method using the Ent183 probe. Additionally, there was at least an 800-fold decrease in the density of thermotolerant coliforms, also represented by ATCC 25922, as quantification yielded values below 3.0 MPN.mL⁻¹. After 24 hours, the reappearance of coliforms detected by the Most Probable Number (MPN) method indicated that a portion of the bacterial population either survived UV-C exposure or repaired DNA damage. This suggests that some cells remained in a viable but non-culturable (VBNC) state. The observed discrepancy between the reduction in cultivable cells and the total cell counts, which include non-culturable cells, under identical experimental conditions supports this hypothesis, indicating a potential induction of the VBNC state by the recirculation system. The discrepancy between cultivation-based and direct cell counting methods may lead to an underestimation of microbiological risk in water samples intended for human consumption [ 42 ]. Moreover, the use of specific probes in the FISH technique - such as ENT183, employed in this study to quantify E. coli - enhances the accuracy of detecting target groups, even when these bacteria are unable to form colonies on culture media [ 26 ]. As demonstrated by Oliver [ 43 ], exclusive reliance on cultivation-independent methods may overlook the persistence of microorganisms in the viable but nonculturable (VBNC) state. Therefore, the integration of multiple analytical approaches is essential for accurately evaluating the true effectiveness of disinfection systems, such as the one examined in this study. UV radiation induces a VBNC state in E. coli , characterized by a loss of culturability while retaining metabolic activity [ 44 , 45 ]. Recently, the use of ultraviolet radiation for water disinfection has been reported as a contributing factor in maintaining E. coli in the VBNC state [ 46 ]. The presence of VBNC E. coli in treated water can lead to an underestimation of health risks associated with drinking water [ 47 ], as these cells are capable of producing toxins while in the VBNC state. Some lineages, however, remain avirulent during this state but can regain virulence upon resuscitation [ 48 ]. 4. Conclusions Based on these findings, a 6-hour recirculation time is the most advantageous option for water disinfection. It reduces time and energy consumption, effectively controls heterotrophic bacteria, total and thermotolerant coliforms, and lowers the risk of UV-C-resistant mutant development, as well as the growth of culturable and viable but non-culturable (VBNC) cells. The disinfection technology developed and presented in this study offers a practical solution for ensuring microbiologically safe access to cistern water, directly enhancing community health autonomy and water security. This initiative aligns with multiple Sustainable Development Goals, particularly SDG 6 – Clean Water and Sanitation, and SDG 3 – Good Health and Well-being, by promoting resilient and sustainable access to safe water under conditions of environmental vulnerability. Declarations Acknowledgments This work was supported by the Ceará Foundation for Scientific and Technological Development Support (Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – FUNCAP), which provided financial resources (Project Grant No. BP5-00197-00078.01.00/22) for its development. We also thank Me. Cassiano Ricardo de Souza for providing the georeferenced map. Data Availability No datasets were generated during the current study Author Contribution J.C.S. Methodology, Formal analysis, Investigation, Writing - Original Draft. A.S.O. Methodology, Formal analysis, Investigation, Writing - Original Draft. D.E.C. Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft. C.P.S. Methodology, Formal analysis, Writing - Original Draft. E.M.R. Conceptualization, Validation, Investigation, Data Curation, Supervision, Project administration, Funding acquisition, Writing - Review & Editing. Conflict of interest The authors report there are no competing interests to declare. References Alim MA, Rahman A, Tao Z, Samali B, Khan MM, Shirin S (2020) Suitability of Roof Harvested Rainwater for Potential Potable Water Production: A Scoping Review. 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FEMS Microbiol Rev 34(4):415–425. https://doi.org/10.1111/j.1574-6976.2009.00200.x Zhang S, Guo L, Yang K, Zhang Y, Ye C, Chen S, Yu X, Huang WE, Cui L (2018) Induction of Escherichia Coli Into a VBNC State by Continuous-Flow UVC and Subsequent Changes in Metabolic Activity at the Single-Cell Level. Front Microbiol 9:2243. https://doi.org/10.3389/fmicb.2018.02243 Xiao Y, Wang J, Sun P, Ding T, Li J, Deng Y (2025) Formation and Resuscitation of Viable but Non-Culturable (VBNC) Yeast in the Food Industry: A Review. Int J Food Microbiol 426:110901. https://doi.org/10.1016/j.ijfoodmicro.2024.110901 Zhu L, Shuai X, Xu L, Sun Y, Lin Z, Zhou Z, Meng L, Chen H (2022) Mechanisms Underlying the Effect of Chlorination and UV Disinfection on VBNC State Escherichia Coli Isolated from Hospital Wastewater. J Hazard Mater 423:127228. https://doi.org/10.1016/j.jhazmat.2021.127228 Gehr R (2015) Comment on UV Disinfection Induces a Vbnc State in Escherichia Coli and Pseudomonas Aeruginosa . Environ Sci Technol 49(12):7501–7501. https://doi.org/10.1021/acs.est.5b00769 Pienaar JA, Singh A, Barnard TG (2016) The Viable but Non-Culturable State in Pathogenic Escherichia Coli : A General Review. Afr J Lab Med 5(1):9. https://doi.org/10.4102/ajlm.v5i1.368 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7359132","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501798520,"identity":"bb153516-cc89-48de-8331-52b422032d1f","order_by":0,"name":"Jardielen Chaves Sousa","email":"","orcid":"","institution":"Instituto Federal de Educação","correspondingAuthor":false,"prefix":"","firstName":"Jardielen","middleName":"Chaves","lastName":"Sousa","suffix":""},{"id":501798521,"identity":"e6640849-2a33-4ba0-b4f1-77c93d325cc0","order_by":1,"name":"Adelaide Sampaio Oliveira","email":"","orcid":"","institution":"Instituto Federal de Educação","correspondingAuthor":false,"prefix":"","firstName":"Adelaide","middleName":"Sampaio","lastName":"Oliveira","suffix":""},{"id":501798522,"identity":"5e75e69e-1ef9-422c-89ec-bd8e7215e926","order_by":2,"name":"Dionéia Evangelista Cesar","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora, - Juiz de Fora/MG","correspondingAuthor":false,"prefix":"","firstName":"Dionéia","middleName":"Evangelista","lastName":"Cesar","suffix":""},{"id":501798523,"identity":"7e44f39b-07f2-4c47-bd29-18a2fa153ac3","order_by":3,"name":"Camila Portela Silva","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora, - Juiz de Fora/MG","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Portela","lastName":"Silva","suffix":""},{"id":501798524,"identity":"095e12ba-96a2-430e-b551-d17982256c29","order_by":4,"name":"Edmo Montes Rodrigues","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYFCCBBiD+QADYwOUDWfg0SLBwMCWQLIWHgPitPCzZyc+ulFxp46f/8w3iZ87bOQY2A8f3cC44x5OLZI9bzcb55x5JiE5I3ebZO+ZNGMGnrS0G4xninFqMbiRu006t+2whMEN3m0SvG2HExskeMxuMLYl4NRiD9Nif/7MM8m/xGgxkIDZwpDDJk2ULRJnwH45LDnjRpqxtWxbmjEbyC+JZ3Br4W/P3fg4p+IwP3//4Yc337bZyPGzHz524+MO3FqQAYsEiGQDEcRpAKaYD0QqHAWjYBSMghEGACDpVxwKvumWAAAAAElFTkSuQmCC","orcid":"","institution":"Instituto Federal de Educação","correspondingAuthor":true,"prefix":"","firstName":"Edmo","middleName":"Montes","lastName":"Rodrigues","suffix":""}],"badges":[],"createdAt":"2025-08-12 21:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7359132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7359132/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89463106,"identity":"7147310d-cbb6-4d87-8fba-30100ba68035","added_by":"auto","created_at":"2025-08-20 08:09:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":646543,"visible":true,"origin":"","legend":"\u003cp\u003eWater recirculation system with type C ultraviolet radiation emitting device in (a) front view and (b) front section, with indications of the constituent parts. 1. Barrel; 2. Silicone hose for water outlet; 3. Silicone hose for water return; 4. Device containing UV-C radiation emitting lamp; 5. Water to be recirculated; 6. Recirculation pump; 7. PVC pipe; 8. UV-C radiation emitting lamp; 9. Lamp ballast; 10. Water inlet hose connector; 11. Water outlet hose connector.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/d2d5627e66f2c571ebcdb7ca.png"},{"id":89463104,"identity":"4da8317f-2f8e-4c90-b892-af5c1629165e","added_by":"auto","created_at":"2025-08-20 08:09:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":887996,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical localization of (a) Ceará, Brazil; and (b) geographical distribution of cisterns sampling sites.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/01b6794c2969aa5cf8792b0c.png"},{"id":89464147,"identity":"8ec98b7d-7627-47a6-824a-140a51f62d35","added_by":"auto","created_at":"2025-08-20 08:17:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":761904,"visible":true,"origin":"","legend":"\u003cp\u003eDensity of total heterotrophic bacteria considering the (a) mean value of the densities evaluated for water from three cisterns, whose statistical analyses demonstrated that for all the evaluated times, they were statistically different, comparing the treatments with exposure (UV) and without exposure (NoUV) to ultraviolet radiation. Considering each cistern as an experimental unit, both for (b) cistern 1, (c) cistern 2, and (d) cistern 3, the density values ​​were statistically different at the same time, considering the UV and NoUV treatments.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/5cb263b2b399d6c14640544b.png"},{"id":89463107,"identity":"389bf8d4-ec97-4f59-8062-80ce083edc7d","added_by":"auto","created_at":"2025-08-20 08:09:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":273630,"visible":true,"origin":"","legend":"\u003cp\u003eDensities of thermotolerant coliforms determined in water recirculation systems with ultraviolet radiation (UV) and without radiation (NoUV) at different operating times after contamination with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. The values at each point on the graph refer to the density data (MPN.mL\u003csup\u003e-1\u003c/sup\u003e) of thermotolerant coliforms.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/f358fe3d4e2d68678c9cb486.png"},{"id":89465283,"identity":"38a78ad1-9434-44ea-91e2-2281b082f5bc","added_by":"auto","created_at":"2025-08-20 08:25:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":283073,"visible":true,"origin":"","legend":"\u003cp\u003eDensity of (a) prokaryotic cells and (b) Enterobacteriaceae cells for experiments conducted in the presence (UV) and absence (NoUV) of ultraviolet radiation. Asterisks indicate significant difference for the same time according to the t-test at 5% probability.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/cb58a52ce6f791d11e04113c.png"},{"id":89463109,"identity":"9f040758-5500-4398-844d-acd47251dbe1","added_by":"auto","created_at":"2025-08-20 08:09:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":172136,"visible":true,"origin":"","legend":"\u003cp\u003eDensity of prokaryotic and Enterobacteriaceae cells at the beginning of the experiment (0h) and after 6h of recirculation with ultraviolet radiation. There is a statistically significant difference according to t-test at 5% probability in cell densities between the two time points for both prokaryotic cells and Enterobacteriaceae cells.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/e82726482c82b8b4ff53f3e2.png"},{"id":89463113,"identity":"13fde072-93ba-49e4-9701-55a188cf8352","added_by":"auto","created_at":"2025-08-20 08:09:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":423152,"visible":true,"origin":"","legend":"\u003cp\u003eDensity of cultivable and non-cultivable microbial cells after contamination with E. coli ATCC 25922 cells at the initial time and different treatment times in the water recirculation system with exposure to UV-C radiation quantified using three different methods.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/69d88c827ecf46d1141a8b86.png"},{"id":89466970,"identity":"c3d2eaec-1c68-4925-ac92-abe6ec382dfb","added_by":"auto","created_at":"2025-08-20 08:41:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4205301,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7359132/v1/fae1c312-8054-49cb-ac43-5835e3a38d34.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Performance Evaluation of a UV-C Coupled Recirculation System for Enhancing Cistern Water Microbiological Quality","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRainwater harvesting is common in many parts of the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One way to store rainwater is through the use of cisterns, which provide water to families that do not have access to public supply systems [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although it is considered a sustainable alternative to ensure water security, the consumption of water stored in cisterns can pose health risks to the population that consumes it [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCisterns are usually low-cost structures built close to houses, where rainwater that falls on the roof is channeled and directed into the reservoir [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The conditions for collecting and storing water can favor the growth of pathogenic microorganisms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and health control and education are needed to prevent diseases resulting from microbiological contamination of water in cisterns. Diarrhea is one of the diseases caused by microorganisms in water consumed without adequate treatment. It is estimated that diarrhea caused 1.17\u0026nbsp;million deaths worldwide in 2021 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], making it one of the leading causes of death among people of all ages. Taking into account estimates for children under 5 years of age, diarrhea is the fifth leading cause of death worldwide, with 446,000 deaths in 2016 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBacteria, protozoa and viruses, represented by \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eGiardia spp\u003c/em\u003e. and Rotavirus A, respectively, are microorganisms that cause infections [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] leading to diarrhea in children in the Brazilian semi-arid region. Research by Hamilton et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] shows that rainwater collected from roofs and stored in tanks is contaminated with \u003cem\u003eE. coli\u003c/em\u003e in between 24 and 92% of samples. The use of chemical compounds to disinfect water is common, but their potential risks to human health are known.\u003c/p\u003e\u003cp\u003eUltraviolet C (UV-C) radiation has a known antimicrobial effect when used to reduce the density of microorganisms in water [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and air [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. UV radiation emitted by low-power lamps has a low potential to form harmful by-products [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. UV-C radiation with a spectrum between 200 and 280 nm inactivates microorganisms by damaging their nucleic acids. The wavelength range of 250\u0026ndash;270 nm is strongly absorbed by nucleic acids, making it highly effective for inducing cellular lethality or viral inactivation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This is due to the peak absorption of DNA occurring near 260 nm [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Most of the lethal effects of UV radiation on biological systems are attributed to photochemical changes in pyrimidine residues [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThere are several studies in the literature demonstrating the success of using UV-C radiation to control microbial growth on surfaces [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], in air [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and in water [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In terms of water disinfection, UV-C radiation is effective against several pathogenic species, including \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe use of devices emitting radiation in the UV-C range can reduce the microbial density and consequently improve the quality of cistern water, making it potable from a microbiological point of view. The present study aimed to assemble a water recirculation system consisting of a water pump coupled to a device containing a UV-C lamp and to evaluate its efficiency in inactivating total and thermotolerant coliforms, including \u003cem\u003eE. coli\u003c/em\u003e, in cistern water.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Structure and Operation of the Water Recirculation System\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEach recirculation unit was assembled to contain a 20-litre PVC barrel, a submersible water recirculation pump with a capacity of 150 L h-\u003csup\u003e1\u003c/sup\u003e, and a device including a 15W Osram\u0026reg; UVC tubular fluorescent lamp with peak emission at 254 nm and 7.800 cd of light intensity. Water flows unidirectionally between the surface of the lamp and a PVC tube into which it is inserted, with a maximum distance of 1 cm from the lamp when it enters the device, which has enough volume for 180 mL of water. The recirculation pump, located inside the drum, was connected to the device containing the UV-C lamp through a silicone tube, with the other end of the device containing another tube that supplied water directly to the barrel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Study site and sampling\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWater samples were collected from concrete cisterns used to store rainwater at five sites in the interior of the states of Cear\u0026aacute; and Piau\u0026iacute;, Brazil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Approximately 45 liters of water were collected at each sampling time and immediately sent for experimental set-up and physicochemical analysis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Physicochemical analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe collected samples were analysed for pH, chloride concentration, electrical conductivity, turbidity, total hardness, total alkalinity, and calcium content. The determination of pH, electrical conductivity, and turbidity was carried out using a LUCA-210 pH meter (Lucadema), an AT-255 microprocessor-based conductivity meter (Alfakit), and a 2020we portable turbidity meter (LaMotte), respectively. The remaining parameters were quantified according to the methods described in the Standard Methods for the Examination of Water and Wastewater [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Carrying out experiments with recirculation\u003c/h2\u003e\u003cp\u003eBefore the experiments, the entire system was disinfected with 0.1% (v/v) sodium hypochlorite. For each experiment, a set of 4 tanks was used, including a \"control\" tank in which the water was recirculated but without activation of the UV-C lamp (NoUV treatment) and another 3 in which the ultraviolet radiation was kept on to obtain samples in periods of 6h, 12h and 24h of continuous operation (UV treatments). Each barrel received 10 L of water from the same cistern, and the water was circulated at 150 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The barrels were kept closed, and the laboratory was dark throughout the experiment. After each interval, water samples were taken in triplicate and then subjected to microbiological analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Microbiological analysis\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section4\"\u003e\u003ch2\u003e2.2.3.1. Cultivable heterotrophic bacteria\u003c/h2\u003e\u003cp\u003eAliquots of 1.0 mL were used to perform the pour plate technique on R2A agar (Himedia\u0026reg;) containing 0.64% fluconazole (to inhibit fungal colony growth). Cultures were performed in triplicate using samples from three of the five cisterns evaluated. Plates were incubated at 30 \u0026ordm;C for 48 hours and the colonies obtained were counted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section4\"\u003e\u003ch2\u003e2.2.3.2. Total and thermotolerant coliforms\u003c/h2\u003e\u003cp\u003eTotal and thermotolerant coliforms were determined according to the method described by the American Public Health Association [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Presumptive tests were performed using tubes containing lactose broth (Himedia\u0026reg;). Confirmatory tests were performed using tubes containing Brilliant Green broth 2% (Himedia\u0026reg;) and EC broth (Himedia\u0026reg;) to detect total and thermotolerant coliforms, respectively. Analyses were performed using the most probable number method (MPN.100 mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with Durham tubes inverted in the medium to detect gas production. As a control (time 0 h), microbiological analyses were performed on the water from the cisterns as soon as it arrived at the laboratory.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section4\"\u003e\u003ch2\u003e2.2.3.3. Typical \u003cem\u003eE. coli\u003c/em\u003e cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAliquots of 100 mL of water were vacuum filtered through 0.45 \u0026micro;m pore filter membranes designed to retain bacterial cells. The membranes were transferred to Petri dishes containing eosin-methylene blue agar (Himedia\u0026reg;). Plates were incubated at 37\u0026ordm;C for 24 hours, followed by colony counting. Analyses were performed in triplicate for each time the system was irradiated with UV-C radiation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Assays with \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922\u003c/h2\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Determination of the growth curve for \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/h2\u003e\u003cp\u003eFirst, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was activated in TSA medium (Himedia\u0026reg;) with incubation at 37\u0026ordm;C for 24 h. After obtaining isolated colonies, one was inoculated into 50 mL of nutrient broth (Himedia\u0026reg;) in a 125 mL Erlenmeyer flask to obtain a pre-inoculum. The incubation was carried out with shaking at 37\u0026ordm;C for 24 h. The absorbance was measured in a spectrophotometer at 600 nm, and the pre-inoculum was added to an Erlenmeyer flask containing 400 mL of nutrient broth (Himedia\u0026reg;) in a volume sufficient to obtain an OD600 of 0.05. Incubation was carried out at 37\u0026ordm;C with shaking for 24 h. Every two hours, a 3 mL aliquot of the culture was taken to determine the absorbance at 600 nm to determine the bacterial growth curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Preparation of mesocosms with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter preparation and sterilization of 40 L of 0.1% peptone water, each barrel received 10 L. At the same time, \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells were grown as described in 2.3.1 to log phase, then washed and centrifuged at 6,000 rpm for 5 min. The supernatant was discarded, and the sedimented cells were resuspended in sterile 0.9% saline using a vortex mixer. This procedure was repeated two more times. The resuspended contents were assayed for absorbance at 600 nm, and 1 mL of the saline solution containing cells with an OD600 of 0.05 was added to each tube. The experiment was carried out according to the methodology described in point 2.2.2. To obtain the representative sample at time 0h (control), the system was left to run for 10 min without UV-C irradiation to allow homogenization of the cells. Aliquots were then taken for analysis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. Microbiological analysis\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.1. Thermotolerant coliforms\u003c/h2\u003e\u003cp\u003eThe most probable number (MPN) was determined following the procedures outlined in section 2.2.3.2, with \u003cem\u003eE. coli\u003c/em\u003e cells serving as the sole representatives of thermotolerant coliforms in the experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.2. Prokaryotic density\u003c/h2\u003e\u003cp\u003eSamples were fixed in 2% formalin and stored for later filtration through 0.22 \u0026micro;m polycarbonate membrane filters. Fluorescence in situ hybridization (FISH) was used to quantify \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells using the ENT183 probe [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The hybridization procedure was performed according to Del`Duca et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], using 20% formamide and NaCl concentrations for hybridization and washing, respectively. A generic probe was used as a negative control. The probes were labeled with Cy3 fluorochrome. Prokaryotic density was determined by evaluating ten random fields using an Olympus BX60 epifluorescence microscope (Olympus, Japan) equipped with Chroma U-N41007, U-MWU2, and U-MWG2 filters. Total prokaryotic density was determined by staining with 4',6-diamidino-2-phenylindole - DAPI [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eStatistical analyses were conducted using SigmaPlot 12.0 software. Data distribution was assessed for normality using the Shapiro-Wilk test. Subsequently, comparisons were performed using Dunnett\u0026rsquo;s test or t-test at a significance level of 5%.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Physicochemical parameters\u003c/h2\u003e\u003cp\u003eThe analysis of the physicochemical data revealed that only one of the water samples did not present the basic pH value, with an average value of 8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86. Concerning the conductivity data, a variation was observed from 15.5 \u0026micro;S cm-\u003csup\u003e1\u003c/sup\u003e to 159.8 \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The turbidity values ranged from 0.2 to 1.16 NTU, while the chlorides occurred at an average of 9.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The alkalinity and hardness data indicate that the water stored in the evaluated cisterns is primarily influenced by calcium compounds [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] as basic components, likely resulting from the gradual release of calcium from the cement used in the construction of the cisterns.\u003c/p\u003e\u003cp\u003eDuring the evaluation process, the Guideline for Drinking Water Quality [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] was utilized to establish limits for conductivity, chlorides, and turbidity, with maximum values of 2,000 \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 250 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e and 5.0 NTU, respectively, and a pH range between 6.5 and 8.5. Accordingly, the World Health Organization asserts that the water in the cisterns exhibits adequate physical-chemical quality for human consumption.\u003c/p\u003e\u003cp\u003eWhen considering UV-C radiation for water disinfection, turbidity is recognized in the literature as one of the physical-chemical parameters that cause the most significant interference. Research has demonstrated that elevated turbidity levels can substantially compromise the efficacy of UV disinfection systems. Nourmoradi et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] performed experiments using a UV reactor and observed that increasing turbidity from 1 to 5 NTU resulted in a 0.2 to 0.5 log reduction in microbial inactivation. Wu and Doan [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] observed a similar decline in disinfection efficiency as turbidity increased within the same range.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Evaluation of the effectiveness of the system for cistern water\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter evaluating the density of heterotrophic bacteria, the average values showed a significant variation between the system operating when UV radiation was applied and when compared to the control (system operating without UV radiation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Considering the density of heterotrophic bacteria present in the water immediately after sampling, according to the Dunnett's test at 5% probability, only the 6-hour treatment in the recirculation system promoted a significant reduction in the density of these microorganisms, causing an average reduction of 99.6%, considering the average data of the water from the cisterns evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). When the system is operated without UV radiation, there is a tendency for the bacterial density to increase. This is probably due to the movement of the water, which increases the availability of oxygen and promotes metabolic activity and population growth of heterotrophic bacterial groups [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe experiments were conducted under conditions of darkness to prevent photoreactivation, a process that has been demonstrated to reverse specific types of ultraviolet (UV) damage [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, it is important to note that bacterial DNA damage repair mechanisms function through various pathways that do not depend on light. These mechanisms are crucial for maintaining genomic integrity in response to diverse DNA lesions caused by environmental factors. Key repair pathways include direct reversal, base excision repair, nucleotide excision repair, and double-strand break repair, each employing distinct proteins and processes to address specific types of damage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This may provide a rationale for the increase in the number of heterotrophic bacteria quantified after 12 hours and 24 hours of system operation.\u003c/p\u003e\u003cp\u003eThe data for total and thermotolerant coliforms shows that the recirculation system combined with the ultraviolet radiation was effective across all exposure times tested (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In all the treatments, for all cisterns, no bacterial growth was observed at any dilution level tested using the multiple-tube technique. Despite the lack of a consensus microorganism that can be universally established as an indicator of microbiological water quality, certain microbial groups are widely used for this purpose, with total coliforms and fecal coliforms being the most commonly used [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, a significant reduction in the concentration of the microorganisms analyzed was observed in all the time intervals evaluated, demonstrating that even in the shortest time of exposure to the equipment (6h), the water presented a satisfactory microbiological quality, in according to Guideline for Drinking Water Quality [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysicochemical parameters evaluated in the water from the cisterns used in the present study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eCistern\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e8.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eConductivity (\u0026micro;S cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e159.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e157.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTurbidity (NTU)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eChlorides (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ede Cl\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e12.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAlkalinity (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ede CaCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e74.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e50.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal Hardness (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ede CaCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e58.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e45.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePartial Hardness (mg L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ede Ca\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e54.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e43.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDensity of total coliforms and thermotolerant coliforms (MPN.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in aliquots of water obtained from cisterns (Control) and after 6h, 12h and 24h of the same water kept in a closed recirculation system coupled to a type C ultraviolet radiation emitting system.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0h (Control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e24h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eTotal coliforms (MPN.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eThermotolerant coliforms (MPN.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0,36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCistern 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAdhikari et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] indicate that turbidity can interfere with the effectiveness of ultraviolet radiation in inactivating \u003cem\u003eE. coli\u003c/em\u003e in water. According to the results obtained in the present study, the range between 0.2 and 1.17 NTU was not large enough to interfere with the effectiveness of the radiation applied to reduce total and thermotolerant coliforms. This is evidenced by the fact that the results are the same for all system recirculation times, regardless of the turbidity level of the samples.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eConcerning the analysis of typical \u003cem\u003eE. coli\u003c/em\u003e cells performed employing the filter membrane technique, the results demonstrated the effectiveness of the system in inactivating the cells for all times evaluated (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDensity of typical \u003cem\u003eE. coli\u003c/em\u003e cells (cells.100 mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) estimated from the membrane technique in aliquots of water obtained from cisterns (Control) and after 6h, 12h, and 24h from the same water kept in a closed recirculation system coupled to a type C ultraviolet radiation emitting system\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eTypical \u003cem\u003eE. coli\u003c/em\u003e cells (cells.100 mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0h (Control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCistern 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCistern 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCistern 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCistern 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCistern 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3. System evaluation with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/h2\u003e\u003cp\u003eAfter contaminating 0.1% peptone water with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and introducing it into the recirculation systems\u0026mdash;both with and without UV-C radiation\u0026mdash;the initial quantification of thermotolerant coliforms was conducted by measuring the \u003cem\u003eE. coli\u003c/em\u003e density, which was found to be 2.4 x 10\u003csup\u003e3\u003c/sup\u003e MPN mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Following the initiation of system operation, a statistically significant variation was observed between the treatments containing or not ultraviolet radiation (UV x NoUV). During the 6-hour operating period of the system with ultraviolet radiation, no positive tubes for thermotolerant coliform growth were detected. In contrast, the quantification of thermotolerant coliforms reached 7.5 x 10⁴ MPN mL⁻\u0026sup1; when the system operated without ultraviolet radiation. A similar outcome was observed in the 12-hour UV treatment, while the 12-hour NoUV treatment resulted in data points exceeding 1.1 x 10\u003csup\u003e6\u003c/sup\u003e MPN.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After 24 hours of operation, the NoUV system maintained a thermotolerant coliform density above 1.1 x 10⁶ MPN.mL⁻\u0026sup1;, whereas the UV system effectively controlled these coliforms, with a quantified density of 83.6 MPN mL⁻\u0026sup1;.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe lack of microbial growth in the tests conducted for 6-hour and 12-hour operating times confirms the system's effectiveness in inactivating total and thermotolerant coliforms, using \u003cem\u003eE. coli\u003c/em\u003e as the indicator microorganism. After 12 hours of system operation without UV-C radiation, an increase in the density of thermotolerant coliforms (represented by \u003cem\u003eE. coli\u003c/em\u003e) was observed, although the exact magnitude of this increase could not be accurately quantified. This finding suggests that UV-C radiation may have been the factor responsible for regulating the population under study. The quantification of 83.6 MPN.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of thermotolerant coliforms after 24 hours of UV-C treatment may be attributable to the heightened sensitivity of \u003cem\u003eE. coli\u003c/em\u003e population to radiation during the exponential growth phase. However, after repeated exposure cycles, genomic adaptation can lead to increased resistance to ultraviolet radiation (Selveshwari et al., 2021). Moreover, Maghsoodi et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] demonstrated that \u003cem\u003eE. coli\u003c/em\u003e strains can repair DNA damage even in the absence of light, independent of photoreactivation, enabling some cells to remain viable post-repair despite dark conditions. Furthermore, Recacha et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] identified several light-independent repair genes in the genome of strain ATCC 25922, including \u003cem\u003erecA\u003c/em\u003e, \u003cem\u003erecB\u003c/em\u003e, \u003cem\u003elexA\u003c/em\u003e, \u003cem\u003euvrA\u003c/em\u003e and \u003cem\u003euvrB\u003c/em\u003e. The expression of these and other genes involved in the response to UV-C-induced damage explains the detection of cells 48 hours after treatment in the recirculation system.\u003c/p\u003e\u003cp\u003eBy direct counting using DAPI, the average density of prokaryotic cells was initially quantified at 0.34 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Similarly, by fluorescent in situ hybridization using the ENT183 probe, which allows the quantification of members of the Enterobacteriaceae family, we attempted to quantify the population of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells introduced into the system, resulting in an initial density of 0.14 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, during the first 6 hours of system operation, there is a significant difference in the density of both prokaryotic cells and Enterobacteriaceae representatives, according to the t-test at 5% probability. Only for prokaryotic cells the difference does not occur for the 12-hour period. The direct counting data, both for total prokaryotic cells and for Enterobacteriaceae, confirm the effectiveness of the system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe efficacy is even more evident if we look specifically at the initial time (0h) of the 6h UV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In this case, a significant reduction is observed for both prokaryotic cells and representatives of Enterobacteriaceae. After 6 hours of operation of the recirculation system with UV-C, there is a reduction of prokaryotic cells and Enterobacteriaceae to 0.13 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.03 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. This represents a percentage of 60.77% for prokaryotic cells and 76.21% for Enterobacteriaceae. Considering that the system was disinfected and \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells were added as a representative of Enterobacteriaceae, a 76.21% reduction in E. coli density in the system after 6 hours of recirculation is indicated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComparing the quantification of thermotolerant coliforms with that of prokaryotic cells and Enterobacteriaceae under UV irradiation, a variation in the magnitude of the data is observed. While for thermotolerant coliforms there is an increase only during the 24h treatment and even then with a most probable number of 83.6 cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the microbial density by direct count indicates a greater growth over time. With 12h recirculation, even with the presence of ultraviolet radiation, there was an increase in the total number of prokaryotic cells from 0.34 to 2.16 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an increase of 6.35x. Considering the 24-hour period, the increase was 19-fold, as the density increased to 6.47 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The same occurred for Enterobacteriaceae cells, which increased 2.85x (0.4 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 47.85x (6.7 x 10\u003csup\u003e6\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for the 12h and 24h times, respectively.\u003c/p\u003e\u003cp\u003eThese variations indicate that although the system is functional for a recirculation time of 6h, times longer than this do not contribute to an improvement in the microbiological quality of the water, but rather result in an increase in both cultivable and non-cultivable microbial populations. The differences between cultivation data and direct microbial enumeration data (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) indicate that the abiotic conditions of the recirculation system maintain a large proportion of cells in the viable but non-culturable (VBNC) state.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsidering the reductions in microbial densities observed between the initial time point and after 6 hours of exposure to UV-C radiation in the recirculation system, which demonstrated higher disinfection efficiency, several notable trends were identified. A 2.5-fold decrease was detected in the total prokaryotic cell density, as determined by DAPI staining. The density of Enterobacteriaceae, represented by \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 cells introduced as contaminants, showed a 4.2-fold reduction based on the FISH method using the Ent183 probe. Additionally, there was at least an 800-fold decrease in the density of thermotolerant coliforms, also represented by ATCC 25922, as quantification yielded values below 3.0 MPN.mL⁻\u0026sup1;. After 24 hours, the reappearance of coliforms detected by the Most Probable Number (MPN) method indicated that a portion of the bacterial population either survived UV-C exposure or repaired DNA damage. This suggests that some cells remained in a viable but non-culturable (VBNC) state. The observed discrepancy between the reduction in cultivable cells and the total cell counts, which include non-culturable cells, under identical experimental conditions supports this hypothesis, indicating a potential induction of the VBNC state by the recirculation system.\u003c/p\u003e\u003cp\u003eThe discrepancy between cultivation-based and direct cell counting methods may lead to an underestimation of microbiological risk in water samples intended for human consumption [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, the use of specific probes in the FISH technique - such as ENT183, employed in this study to quantify \u003cem\u003eE. coli\u003c/em\u003e - enhances the accuracy of detecting target groups, even when these bacteria are unable to form colonies on culture media [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As demonstrated by Oliver [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], exclusive reliance on cultivation-independent methods may overlook the persistence of microorganisms in the viable but nonculturable (VBNC) state. Therefore, the integration of multiple analytical approaches is essential for accurately evaluating the true effectiveness of disinfection systems, such as the one examined in this study.\u003c/p\u003e\u003cp\u003eUV radiation induces a VBNC state in \u003cem\u003eE. coli\u003c/em\u003e, characterized by a loss of culturability while retaining metabolic activity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Recently, the use of ultraviolet radiation for water disinfection has been reported as a contributing factor in maintaining \u003cem\u003eE. coli\u003c/em\u003e in the VBNC state [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The presence of VBNC \u003cem\u003eE. coli\u003c/em\u003e in treated water can lead to an underestimation of health risks associated with drinking water [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], as these cells are capable of producing toxins while in the VBNC state. Some lineages, however, remain avirulent during this state but can regain virulence upon resuscitation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eBased on these findings, a 6-hour recirculation time is the most advantageous option for water disinfection. It reduces time and energy consumption, effectively controls heterotrophic bacteria, total and thermotolerant coliforms, and lowers the risk of UV-C-resistant mutant development, as well as the growth of culturable and viable but non-culturable (VBNC) cells. The disinfection technology developed and presented in this study offers a practical solution for ensuring microbiologically safe access to cistern water, directly enhancing community health autonomy and water security. This initiative aligns with multiple Sustainable Development Goals, particularly SDG 6 \u0026ndash; Clean Water and Sanitation, and SDG 3 \u0026ndash; Good Health and Well-being, by promoting resilient and sustainable access to safe water under conditions of environmental vulnerability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Cear\u0026aacute; Foundation for Scientific and Technological Development Support (Funda\u0026ccedil;\u0026atilde;o Cearense de Apoio ao Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico \u0026ndash; FUNCAP), which provided financial resources (Project Grant No. BP5-00197-00078.01.00/22) for its development. We also thank Me. Cassiano Ricardo de Souza for providing the georeferenced map.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo datasets were generated during the current study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.C.S. Methodology, Formal analysis, Investigation, Writing - Original Draft. A.S.O. Methodology, Formal analysis, Investigation, Writing - Original Draft. D.E.C. Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft. C.P.S. Methodology, Formal analysis, Writing - Original Draft. E.M.R. Conceptualization, Validation, Investigation, Data Curation, \u0026nbsp;Supervision, Project administration, Funding acquisition, Writing - Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report there are no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlim MA, Rahman A, Tao Z, Samali B, Khan MM, Shirin S (2020) Suitability of Roof Harvested Rainwater for Potential Potable Water Production: A Scoping Review. 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Afr J Lab Med 5(1):9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4102/ajlm.v5i1.368\u003c/span\u003e\u003cspan address=\"10.4102/ajlm.v5i1.368\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Potable water, semiarid, public policies, environmental sanitation, sustainable development goals","lastPublishedDoi":"10.21203/rs.3.rs-7359132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7359132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn semi-arid regions, the absence of adequate water treatment for human consumption frequently gives rise to intestinal disorders and, in more severe cases, severe microbial infections. Within the context of the semi-arid region, many households depend on cistern water for their potable needs. This project aimed to develop and use a system composed of a UV-C radiation-emitting lamp coupled to a water recirculation pump to ensure the bacteriological safety of cistern water. The recirculation system was operated for 6, 12, and 24 hours, and tests were conducted using environmental samples or with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922. The results demonstrated that the system could achieve 100% inhibition of the growth of total coliforms and thermotolerant coliforms after 6 hours of operation. The low-cost system has the potential to function as a social technology, thereby ensuring water security in semi-arid regions where there is no public water distribution system.\u003c/p\u003e","manuscriptTitle":"Development and Performance Evaluation of a UV-C Coupled Recirculation System for Enhancing Cistern Water Microbiological Quality","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 08:09:29","doi":"10.21203/rs.3.rs-7359132/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a2b4798-34eb-4789-9518-09269f90a386","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-20T08:09:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 08:09:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7359132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7359132","identity":"rs-7359132","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}