Bactericidal hybrid surfaces against Gram-negative and Gram-positive pathogenic bacteria: Smart Tools for Wastewater Purification

The final step of this project was to test/evaluate the pathogen inhibition potency of the developed nanostructural materials working in tandem (T) pattern. The fundamental study outcomes obtained from the previous efforts were promising and indicated that the film-based format, i.e. Molecularly Imprinted Polymer (MIP) and Non-Imprinted Polymer (NIP), together with functionalized kaolin (K) based microparticles exhibited individually remarkable antagonistic effect against both gram positive and negative pathogens at different levels. This is an essential and vital knowledge to enable the downstream study towards an extended investigation on the tandem effect of integration both formats into treatment trials. Efficiency evaluation on the first tandem trial: The first tandem testing samples were prepared in October 2019 including the control samples which were the raw wastewaters (the same type used for the entire project period) and the treated samples (24 hours incubating) using MIP/NIP films and kaolin based microparticles in tandem (MIP-T and NIP-T), all samples were prepared in triplicate. The frozen filters yielded from the ultrafiltration of all the wastewater samples were delivered to NIBIO and subjected to microbial genomic DNA extraction. The DNA concentration and quality were verified using Qubit 4.0 fluorometer. Probe quantitative real-time PCR (qPCR) was performed using the extracted genomic DNA to detect the targeted pathogens implementing the previously developed genetic markers. The archived results from the first tandem trial indicated a promising overall pathogen removal efficiency. Both MIP-T and NIP-T could attain 100% removal of Salmonella typhimurium, while MIP exhibited slightly higher efficacy than NIP in terms of clearance of Enterococcus faecalis (81% vs. 55%) and Clostridium perfringens (97% vs. 66%). In dealing with Campylobacter jejuni, MIP-T managed to eliminate 70% of pathogen but no clearance was detected in NIP-T treatment. In terms of Shiga-toxin producing E.coli (STEC), MIP-T and NIP-T were observed to exert similar performance in terms of removing the three key toxin encoded genes, i.e. 69% vs. 66% for stx1; 71% vs. 76% for stx2 and 53% vs. 49% for eae gene. The overall results from the first tandem experiment evidenced that integration of MIP and functionalized kaolin derived microparticles in tandem could significantly reduce the load of pathogens from the studied wastewater. Efficiency evaluation on the second tandem trial: In order to verify the promising results achieved in the first tandem trial, we designed and performed the second trial with the same experimental setup in Jan. - Mar. 2020. Upon receiving the frozen filters in NIBIO, we processed the fresh samples as we performed previously. qPCR was carried out to detect and quantify the target pathogen in water samples. We obtained very similar results as compared to the first tandem trial. MIP-T and NIP-T could remove about 100% of Salmonella typhimurium. MIP-T indicated slightly better performance than NIP-T on eliminating Enterococcus faecalis (99% vs. 97%); Clostridium perfringens (100% vs. 91%) and Campylobacter jejuni (100% vs. 79%). Concerning STEC toxic genes, again both displayed similar efficacies, i.e. 47% vs.48% for stx1; 57% vs. 50% for stx2 and 58% vs. 32% for eae. The second tandem trial obtained similar removal effects as detected in the first trial. The pathogen elimination efficacies achieved using MIP/NIP and functionalized kaolin microparticles in tandem are robust and reproducible. Concluding remark: The ultimate objective of this project was to develop novel materials engineered by nanotechnology for wastewater treatment, with a particular focus on pathogens removal. These innovative materials consist of lipopolysaccharide (LPS) molecularly imprinted films and kaolin microparticles modified with quaternary ammonium salts (QAS) groups. The overall efficiencies of LPS-MIP/NIP and kaolin-vinyltrimethoxysilane-vinylbenzyl trimethylammonium chloride (K-VTMS-VBTAC) used either individually or in tandem have been assessed and confirmed with high potency against target pathogens using genetic markers developed at NIBIO. These novel materials and application platform possess considerable potential for further development with regard to enhancing substantially the treatment capabilities and efficiency in mitigating pathogenic burden in wastewater, which constitutes the serious public and environmental health threats.

Prosjektet har generert originale resultater og produkter med store potensielle effekter i og utover prosjektperioden, og med flere nyttbare applikasjoner, inkludert kommersialisering. Dette er hovedsakelig relatert til utvikling og konstruksjon av den nye bio-tanken med bakteriedrepende overflater mot patogene gram-negative bakterier (GNB) og gram-positive bakterier (GPB) under behandling av avløpsvann. Et aktivt engasjement fra interessenter i prosjektet er avgjørende for effektive tverrsektorielle sammenhenger, overføringer og kommunikasjon med akademia, industri, frivillige organisasjoner, beslutningstakere og sluttbrukere. Koblingene mellom forskere, interessenter, industri og sluttbrukere sikrer kommunikasjon med beslutningstakere til hvem prosjektresultatene gir nyttig informasjon om innovative tiltak, nye teknologier og originale produkter utviklet for å forhindre negative effekter på folkehelse samt miljøet (spesielt akvatisk).

TANDEM project promotes originality and innovativeness through the conceptual developments, feasible designs and novel implementations, which include original aspects concerning (i) the synthesis methods beyond the state of art for obtaining two new types of bactericidal surfaces for Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB), respectively, (ii) the know-how solutions for re-conditioning of proposed bactericidal surfaces, (iii) the new knowledge of the two bactericidal surfaces transposed into building blocks (named smart tools) for prototyping a bio-tank, and (iv) the maximized tandem effort of these smart tools for developing more efficient wastewater purification strategies. All these original approaches will further convey to qualitative and quantitative results as follows: (i) novel synthesis technologies for bactericidal hybrid surfaces, (ii) know-how for designing and constructing the tandem bio-tank, and (iii) sustainable purification technology for domestic wastewater and source-separated grey- and black water. The on-set of the project is Technology Readiness Level (TRL) 3 and targets TRL 6. Each participant (from CO-P1 to P3) is entering the chain value at a different position, corresponding to the expertise of each Partner. The plateau from TRL 3 to TRL 6 is straightforward, being covered by well-defined interdisciplinary and complementary activities developed by all participants. The project consortium is well-balanced having aside two renowned RT&D entities from Romania and Norway that will guaranty the success for developing bactericidal hybrid surfaces (for GNB and GPB) and their subsequent transposition into building blocks (the smart tools). In addition, the consortium involves one medium enterprise from Romania entrusted with prototyping a bio-tank using these smart tools and demonstration of efficiency for the emerging wastewater purification technology in relevant and real environments.