Noves tècniques per al control i desinfecció de virus contaminants d’aigua

[cat] Arrel de la necessitat de garantir la disponibilitat d’aigua en un context d’escassetat i assegurar-ne un bon ús que no suposi un risc microbiològic per la població, aquesta Tesi Doctoral ha estat centrada en l’optimització, el desenvolupament i la validació de noves tècniques pel control de virus contaminants d’aigua. Per una banda, s’han optimitzat i desenvolupat mètodes ràpids, sensibles, metodològicament senzills i adaptables per a la concentració i detecció in situ de virus en diferents contextos; i per una altra, s’ha avaluat l’aplicabilitat i eficiència de desinfecció dels sistemes electroquímics d’oxidació avançada com a tractaments sostenibles per a la reutilització d’aigües. Pel que respecta a mètodes de concentració d’aigua per a la detecció de virus, en primer lloc s’ha adaptat el mètode de floculació orgànica amb llet descremada perquè sigui aplicable a contextos de baixa renda donat el seu baix cost econòmic, o a situacions d’emergència humanitària en què no es disposi d’un laboratori equipat, a més l’equipament s’ha adaptat perquè no es requereixi de corrent elèctrica. Aquest mètode, ha estat validat en dos contextos reals, un camp de refugiats ubicat a Banghi (República Centreafricana) i a la zona de Pedernales (Equador) afectada per un terratrèmol. El mètode i protocol desenvolupats, van demostrar ser metodològicament senzills per tal de ser duts a terme per usuaris no experts, demostrant ser un mètode eficient detectant contaminació viral en les mostres d’aigua testades, podent ser una eina útil en la prevenció de brots vírics en els països amb menys recursos, els quals són els més afectats per les malalties de transmissió hídrica donades les limitacions existents de sanejament de l’aigua. La resta de mètodes de concentració avaluats, estan basats en l’ús d’ultrafiltres per a la detecció de virus en aigua. Per una banda, s’ha caracteritzat un mètode ràpid per a l’anàlisi de grans volums d’aigua en aigües superficials i aigua de mar, aplicant la nova tecnologia d’elució d’escuma humida amb la que es redueix el temps de processat de la mostra així com la simplificació metodològica del mètode. A més, s’ha adaptat perquè sigui realitzable en el mateix punt de presa de la mostra, sent validat en dos contextos diferents, un on es va realitzar la concentració completa de mostres in situ, i l’altre on es va realitzar la filtració i posterior enviament del filtre al laboratori per a la seva anàlisi, demostrant la versatilitat del mètode. Finalment, el mètode es va optimitzar amb un segon pas de concentració utilitzant un dispositiu d'ultrafiltració centrífuga (Centricon® Plus-70) per tal d’augmentar-ne la sensibilitat. El mètode ha estat testat mitjançant diverses tècniques, permetent la detecció de virus mitjançant mètodes tan d’infectivitat com moleculars, incloent la detecció per qPCR i tècniques de seqüenciació massiva, com són l’enriquiment per dianes i posterior seqüenciació massiva d’amplicons. Amb l’arribada de la pandèmia de la COVID-19, i arrel de l’escassetat de subministraments de material necessaris per a l’anàlisi del SARS-CoV-2 en aigües residuals, es van caracteritzar dos mètodes ràpids de concentració d’aigües residuals per la detecció de virus embolcallats. Així doncs, es va validar un dispositiu d'ultrafiltració centrífuga (Centricon® Plus-70) i la pipeta concentradora automatitzada CP-Select™, com a mètodes ràpids, i el cas de la pipeta concentradora, factible per a treballar en vitrines de bioseguretat. Validant així, mètodes d’anàlisi d’aigües residuals perquè puguin ser implementats en laboratoris d’anàlisi rutinaris sense requerir de personal altament especialitzat. En la segona part de la tesi, s’ha avaluat l’aplicabilitat i eficiència de desinfecció dels sistemes electroquímics d’oxidació avançada com a tractaments sostenibles per a la reutilització d’aigües, testant dos tipus de materials, un ànode de Ruteni i un ànode de BDD en matrius naturals. Els assajos realitzats, han estat centrats en la inactivació de microorganismes patògens amb un focus especial en els virus, així com en l’avaluació de la idoneïtat dels microorganismes índex utilitzats clàssicament en els tractaments de desinfecció, la determinació dels subproductes tòxics i els requeriments energètics del sistema. Amb tot, s’ha demostrat que els sistemes electroquímics poden ser aplicats com a pas final de desinfecció d’aigües naturals, encara que els microorganismes índex han de ser revisats, donada la major sensibilitat de bacteris i bacteriòfags, en relació a altres microorganismes, però demostrant ser efectius per tal d’assolir un logaritme de reducció de virus humans amb baixos requeriments energètics, tenint potencial per ser sistemes descentralitzats de depuració d’aigües, tot i que han de ser adaptats per evitar la formació de subproductes tòxics.[eng] Given the need to guarantee the availability of water in a context of scarcity and ensure a good use that does not pose a microbiological risk to the population, this Thesis has been focused on developing and validating new techniques for the control of viruses contaminants of water. Firstly, fast, sensitive, methodologically simple, and adaptable methods have been developed for their application in situ for the detection of viruses in different contexts; and secondly, the applicability and disinfection efficiency of advanced oxidation electrochemical systems have been evaluated as sustainable treatments for water reuse. Regarding the development of water concentration methods for the detection of viruses, in the first place the skimmed milk organic flocculation method has been adapted so that it is applicable to low-income situations given its low economic cost, or to situations of a humanitarian emergency in which there is no equipped laboratory, in addition, the equipment has been adapted so that no electricity is required. This method has been validated in two real contexts, a refugee camp located in Banghi (Central African Republic) and in Pedernales (Ecuador) affected by an earthquake. The method and protocol developed proved to be methodologically simple to be carried out by non-expert users, proving to be an efficient method for detecting viral contamination in the tested matrices, being a useful tool in the prevention of viral outbreaks in countries with fewer resources, which are the most affected by water-borne diseases given the lesser sanitation of the water sources. The rest of the concentration methods evaluated are based on the use of ultrafilters for the detection of viruses in water. Firstly, a fast method has been characterized for the analysis of large volume water samples in surface water and seawater, applying the new wet foam elution technology with which the processing time of the sample is reduced as well as the methodological simplification of the method. In addition, it has been adapted so that it can be carried out at the same sampling point, being validated in two different contexts, one where the complete concentration of samples was carried out in situ, and the other where the filtration was carried out and later sending the filter to the laboratory for analysis, demonstrating the versatility of the method. Finally, the method was optimized with a second concentration step using a centrifugal ultrafiltration device (Centricon® Plus-70) to increase sensitivity. The method has been tested using various techniques, allowing the detection of viruses using infectivity and molecular techniques, including detection by qPCR and new generation sequencing techniques, such as target enrichment and amplicon deep sequencing. With the advent of the COVID-19 pandemic, and due to the shortage of material supplies necessary for the analysis of SARS-CoV-2 in wastewater, two rapid methods of wastewater concentration were characterized for the detection of enveloped viruses. Thus, a centrifugal ultrafiltration device (Centricon® Plus-70) and the CP-Select™ automated concentrator pipette were validated as fast methods, methodologically simple and in the case of the concentrator pipette, allowing to work under biosafety conditions. Thus, wastewater analysis methods have been validated, so they can be implemented in routine analysis laboratories without requiring highly specialized personnel. In the second part of the thesis, the applicability and disinfection efficiency of advanced oxidation electrochemical systems have been evaluated as sustaining treatments for water reuse, testing two types of materials, a Ruthenium anode, and an anode of BDD in natural matrices. The disinfection trials carried out have been focused on the inactivation of bacteria, viruses, bacterial spores and protozoa, as well as on the evaluation of the suitability of the index microorganisms classically used in disinfection treatments, the determination of toxic by-products and energy requirements of the system. The main results of these studies have shown that electrochemical systems can be applied as a final step in the disinfection of natural waters, although the index microorganisms must be reviewed, given the greater sensitivity of bacteria and bacteriophages, but proving to be effective to achieve a one logarithm reduction of virus with low energy requirements, having the potential to be implemented as decentralized water purification systems, although they must be adapted to avoid the formation of toxic by-products

Amphiphilic ionic complexes of hyaluronic acid with organophosphonium compounds and their antimicrobial activity

Amphiphilic ionic complexes of hyaluronic acid and alkyltrimethylphosphonium soaps with alkyl chains containing even numbers of carbons from 12 to 22 have been produced. The complexes have a nearly stoichiometric composition, are non-water soluble, and are stable to heat up to temperatures above 200¿°C. These complexes are amphiphilic and able to adopt a biphasic structure with the paraffinic and polysaccharide phases ordered arranged with a periodicity ranging between 3 and 5¿nm depending on n. The paraffinic phase in complexes with n¿=¿18 was crystallized and showed melting at temperatures between 58 and 70¿°C depending on the n value. The complexes decomposed upon incubation in water under physiological conditions, and undergone extensive biodegradation by the action of hyaluronidases. Biocide assays carried out in both solid and liquid media demonstrated a high antimicrobial activity of the complexes against Gram-positive S. aureus but moderate against Gram-negative E. coli and C. albicans fungiPostprint (updated version

NGS techniques reveal a high diversity of RNA viral pathogens and papillomaviruses in fresh produce and irrigation water

Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples

Exploring the diversity of coronavirus in sewage during COVID-19 pandemic: Don't miss the forest for the trees

Coronavirus; Secuenciación de nueva generación; Viroma de aguas residualesCoronavirus; Seqüenciació de nova generació; Viroma d'aigües residualsCoronavirus; Next generation sequencing; Sewage viromeIn the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater.This study was partially supported by the Ministry of Science, Innovation and Universities (AGL2017-86797-C2-1-R) through the University of Barcelona and the Direcció General de Recerca i Innovació en Salut (DGRIS) Catalan Health Ministry Generalitat de Catalunya through Vall d'Hebron Research Institute (VHIR). Sílvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona

Sono-enzymatically embedded antibacterial silver-lignin nanoparticles on cork filter material for water disinfection

Providing clean drinking water is a great challenge worldwide, especially for low-income countries where the access to safe water is limited. During the last decade, new biotechnological approaches have been explored to improve water management. Among them, the use of antimicrobial nanoparticles for designing innovative centralized and decentralized (point-of-use) water treatment systems for microbial decontamination has received considerable attention. Herein, antimicrobial lignin capped silver nanoparticles (AgLNP) were embedded on residual cork pieces using high-intensity ultrasound coupled with laccase-mediated grafting to obtain biofunctionalized nanomaterial. The developed AgLNP-coated cork proved to be highly efficient to drastically reduce the number of viable Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus in liquid medium. Additionally, the coated-cork was characterized using FTIR-ATR spectroscopy and SEM imaging, and further used as a filter bed in a point-of-use device for water disinfection. The constructed water filtering system significantly reduced the amount of viable E. coli and resistant Bacillus cereus spores from filtered water operating at increasing residence times of 1, 4, 6, 16, 24, and 48 h. Therefore, the presented results prove that the obtained cork-based antimicrobial nanocomposite material could be used as a filtering medium for the development of water filtration system to control pathogen dissemination.Peer ReviewedPostprint (published version

Effect of aeration on nitrogen removal-associated microbial community in an innovative vertical cork-based constructed wetland for winery wastewater treatment

The wine industry produces large quantities of wastewater that often contains high levels of organic matter and nutrients, like nitrogen and phosphorous. In particular, nitrogen pollution can be harmful, even at low levels, since infants are vulnerable to nitrates in drinking water, and excess nitrogen can harm the health of soils and waterways. Herein, an innovative compact, modular, and mobile treatment wetland (TW) using cork by product as the only granular media was used to treat the final effluents from the Codorniu winery (Sant Sadurní d'Anoia, Spain). The TW was operated during two 5-month periods with or without intermittent induced aeration (40 min cycles, 90 L/min air flow rate). Nitrate (N-NO3) and total nitrogen (TN) removal were higher (52.8% and 46.8%, respectively, p < 0.05) during TW operation under aerated-condition. Additionally, qPCR analysis of 16S rRNA, nirS and nosZ genes revealed that intermittent induced aeration facilitates N-NO3 reduction by the stimulation of denitrifying bacteria in the TW biofilm (11.4% increase in nirS copies number/g cork sample, p < 0.05) as well as increasing the number of heterotrophic bacteria adhered to cork (25.5% increase in 16S rRNA copies number/g cork sample, p < 0.05). Moreover, SEM images demonstrated the suitability of cork as a resistant filter media for TW after long-term system operation (1.5 years). In conclusion, our results suggest that aeration improved ni- trogen compounds removal compared to the non-aerated period, without affecting phosphorous elimination. Additionally, residual cork is presented here in a circular bioeconomy view, as a suitable filling media to treat winery wastewater that can provide additional carbon source to increase C/N rate stimulating denitrification, as well as a reliable organic substrate for biomass growth.To LIFE Programme, the EU's financial instrument supporting environmental, nature conservation, and climate action projects throughout the EU that supports LIFE ECORKWASTE 14 ENV/ES/460.Peer ReviewedPostprint (published version

Exploring the diversity of coronavirus in sewage during COVID-19 pandemic: Don't miss the forest for the trees.

In the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater

Exploring the diversity of coronavirus in sewage during COVID-19 pandemic: Don't miss the forest for the trees

In the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater.This study was partially supported by the Ministry of Science, Innovation and Universities (AGL2017-86797-C2-1-R) through the University of Barcelona and the Direcció General de Recerca i Innovació en Salut (DGRIS) Catalan Health Ministry Generalitat de Catalunya through Vall d'Hebron Research Institute (VHIR). Sílvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona.Peer reviewe

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.The authors wish to thank the staff from all the WWTPs monitored for their help and technical support during the sampling campaigns. The authors acknowledge the funding received from the ACA and the ASPCAT from the Catalan Government (Generalitat de Catalunya). ICRA authors acknowledge the funding provided by the Generalitat de Catalunya through the Consolidated Research Group grants ICRA-ENV 2017 SGR 1124 and ICRA-TiA 2017 SGR 1318. ICRA researchers also thank the funding from the CERCA program of the Catalan Government.Peer reviewe