Nanoenabled strategies to reduce the presence of contaminants of emerging concern in aquatic environment

Contaminants of emerging concern such as antibiotics, pathogens and antimicrobial resistant bacteria in water bodies associated to intensive fish and inland animal farming, represent a great threat to the environment and human health. The aim of the AMROCE project was to reduce antibiotic pollution and spread of resistant bacteria in the entire water cycle through a platform of novel antibiotic-free antimicrobial products. AMROCE worked with development of antimicrobial/antibiofilm fish cage nets and wastewater filtration membranes through polymer and surface nano-engineering. Marine-derived antimicrobial agents and antibiofilm enzymes were nano-formulated as alternative to antibiotics for fish and animal feed supplement. Human and environmental nano-safety during the manufacturing and use of the novel nanotechnology-embedded products have been evaluated to anticipate nano-safety issues. Different marine raw materials were explored by SINTEF Ocean to find and describe new lipids and antimicrobial proteins which can be used for the antimicrobial bio-based material in different formulations. SINTEF Ocean produced several fractions and ingredients from various marine raw materials included fish (salmon) rest raw materials, seaweeds (Alaria Esculenta, Saccharina latissima), jellyfish (Aurelia aurita, Periphylla periphylla), and low trophic crustaceans (gammarids). The project explored drying, fractionation, and hydrolysis as potential technological processes. In collaboration with UPC (Universitat Politècnica de Catalunya), the project coordinator from Spain), several rounds of bioactivity evaluations were conducted. Fractions from helmet jellyfish (Periphylla periphylla) showed the best antimicrobial properties, while hydrolysates from salmon bones were defined as ingredients with the best antioxidant potential. SINTEF Ocean also focused on extracting lipids and peptides from various microalgae species. Fatty acid fractions with higher amounts of omega 3 fatty acids (EPA or DHA) demonstrate antimicrobial properties. As a result, SINTEF Ocean cultivated different microalgae species with high potential for producing substantial amounts of EPA or DHA. Microalgae Nannochloropsis oceanica produced the highest amount of lipids, while the highest amount of polyunsaturated fatty acids (EPA or DHA) was produced by Chlorella vulgaris. Optimal conditions (algae type and harvest stage) for maximizing EPA and DHA production were established. erring oil showed antimicrobial. When processed into concentrated omega-3 fatty acid oils, fish oil undergoes hydrolysis, transforming fatty acids into ethyl ester form. The ethyl ester mixture is then fractionated into light (short-chain saturated fatty acids) and heavy (rich in long-chain omega-3 fatty acids) fractions. Typically, the light fraction is considered a by-product and used in low-value applications. However, AMROCE identified that the light fraction possesses good antimicrobial properties, presenting an opportunity for its use as an antimicrobial ingredient. In collaboration with the Bar-Ilan Institute of Nanotechnology & Advanced Materials (Israel), NTNU tested the efficiency of antifouling-coated nets. The coated (with different concentrations of antifouling coating) and uncoated net pen sections were placed in a controlled aquarium simulating the conditions of an aquaculture farm. Biofilters, enriched with microbiome from a sea-based farm, were added to the aquarium to evaluate the antifouling properties and biotoxicity of the coatings. Results showed that the uncoated net exhibited typical biofilm formation, with microbiome diversity matching that of the biofilter microbiome after three months. Biofilm began forming on the uncoated net after one week and grew steadily in both microbiome richness and visible biomass. Nets with a high and low concentration of antifouling coating were tested. The high-concentration coating exhibited significant biotoxicity, killing nearly all microorganisms in the biofilters and aquarium within a week. The low-concentration antifouling-coated net delayed biofilm attachment for 5-6 weeks, and after three months, some biofilm had formed, though significantly less than on the uncoated section. The antifouling experiment was conducted in collaboration with biotechnology students from NTNU, and the full thesis report is available in NTNU Open. To promote the project, share research outcomes, and exchange experiences, SINTEF Ocean, together with project partners, organized the workshop "Marine-derived bioactives and their nanoformulation into high-added-value products," held on 13 September 2023 in Trondheim. This event was a joint session connecting two projects, AMROCE and Tardis (Nano-enabled stimuli-responsive scaffolds for targeted antimicrobials delivery to treat Staphylococcus aureus infections and restore skin homeostasis), whose research areas complement each other.

The project fostered a strong multidisciplinary approach by integrating expertise from various scientific and technological fields. Collaboration between experts in chemistry, materials engineering, microbiology, nanobiotechnology, nanotoxicology, LCC/LC Aanalysis ensured the development of effective, antibiotic-free antimicrobial strategies to improve the animal and fish living conditions, reducing the use of antibiotics, the accumulation of CECs in the environment and the emergence of antimicrobial resistant (AMR). AMROCE key achievements include: i) the extraction of novel antimicrobial lipids and AMPs from marine sources, ii) the development of antibiotic-free, antibacterial, and antibiofilm nanoformulations that do not provoke AMR emergence, for use in fish feed supplements, i) the production of innovative CuO NP-based antimicrobial FCN and WFMable to reduce microbial contamination. Furthermore, comprehensive LCA and LCC analyses were performed to evaluate the sustainability and economic impacts of these innovations, alongside the delivery of critical reports on environmental and regulatory compliance. By combining technological innovation with regulatory insight and industry needs, AMROCE addressed major challenges related to AMR and environmental contamination, paving the way for safer and more sustainable practices in aquaculture and water treatment.

Contaminant of emerging concern (CECs) such as antibiotics, pathogens and antimicrobial resistant (AMR) bacteria in water bodies associated to intensive ?sh and inland animal farming, represent a great threat to the environment and human health. AMROCE aims at reducing antibiotic pollution and spread of AMR bacteria in the entire water cycle through a platform of novel antibiotic-free antimicrobial products. AMROCE will develop antimicrobial/antibio?lm ?sh cage nets and wastewater ?ltration membranes through polymer bulk and surface nano-engineering. Marine-derived antimicrobial agents and antibio?lm enzymes will be nano-formulated as alternative to antibiotics for ?sh and animal feed supplement. Human and environmental nanosafety during the manufacturing and use of the novel nanotechnology-embedded products will be continuously evaluated to anticipate nanosafety issues.