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BIOTEK2021-Bioteknologi for verdiskaping

ERA-NET: Recycling crustaceans shell wastes for developing biodegradable wastewater cleaning composites (BIOSHELL)

Awarded: NOK 4.7 mill.

This project periodical report updates the major work delivered by NIBIO research group since 01.12.2022. The earlier developed semi-IPN hydrogels have demonstrated great pathogens and ARGs removal effects in wastewater (WW). As a continuous effort of novel nanomaterial development, new materials were produced for further testing. These variants are similar as the format of the earlier products, such as chitosan from commercial source (CC); chitosan derived from commercial chitin (CCH); chitosan prepared from raw marine residues, e.g., shrimp shells, (SHC). However, a new component, i.e., PEGDA 700 (a hydrophilic biopolymer) was added to all new hydrogels. The variants of each product series are also reflecting the amount balance between vinyl benzyl trimethylammonium chloride (VBTAC) and PEGDA. WW samples after being treated by these new materials were sent to NIBIO for molecular assessments. The treated WW samples as well as the raw/original WW were undergone DNA extraction to acquire microbial genomic DNA (gDNA) using Qiagen PowerWater kit. After quality control and quantity measurement, gDNA was used for qPCR pathogen analyses using developed marker genes. Among all examined samples, Campylobacter jejuni and Legionella pneumophila were not detected. Overall, all tested series displayed highly variable pathogen removal rates, ranging from 16% up to 90%, most effectively against Enterococcus faecalis (Ent) (51-78%), Clostridium perfringens (CP) (33-90%), and Salmonella typhimurium (STM) (23-74%). Among them, all series with high PEGDA and low VBTAC (V0.5 series) exhibited better results than those with low PEGDA and high VBTAC (IPN series). Specifically, CC-V0.5 performed the best on removal of Ent, CP and STM, while SHC-0.5 represented the second best one. Notably, in case of Shigella spp., CC-IPN showed comparable activity as V0.5 series at 45-50% reduction efficiency. For Shiga-toxin producing E. coli (STEC) removal, all variants of both series generated similar activity, removing STEC by 40-50%. In addition to pathogen examination, content of antibiotic resistance genes (ARGs) was also analyzed on the extracted WW gDNA. Consistent with previous finding, vanA and mecA were not detected in all tested samples. Similar to the activity trend identified for pathogen examination, V0.5 series exhibited notably higher ARG mitigation effects than IPN series, particularly towards intI1 (55-69%), blaCTXm (56-62%), ermB (57-75%), qrnS (37-78%), sul1(60-68%) and tetO (46-66%). Interestingly, towards multidrug resistance genes, such as efflux pump encoding genes, IPN series performed distinctly better on reducing qacE1 (64-100%) and qacA/B (52-100%). This is a sort of surprising finding of which the underlying mechanisms warrant further investigation and elaboration. In summary, the overall molecular assessment indicated that the developed new hydrogels derived from marine waste (SHC series) can exert comparable/equally good pathogen and ARGs removal effectiveness as CC (commercial one) and CCH (derived from commercial chitin), especially once higher amount of PEGDA700 is present in the formula. Considering relatively high price of VBTAC, using more PEGDA700 instead is rather cost-effective. The new developed nanocomposites are primarily intended for metal retention. It takes great advantages of superior biodegradability, biocompatibility, hydrophilicity inherited by PEGDA. The formed 3D network structural hydrogels can contribute to the adsorption of metal ions and other substances, thus facilitating pollutants removal from WW. By combining with bioactivity proven VBTAC, based on our recent investigation, the new nanomaterials could exert substantial pathogens and ARGs retention & removal as well. To this end, the new type of hydrogels generated from marine wastes strengthened by the input of PEGDA and VBTAC proved to be effective against pathogens and ARGs in WW, in addition to their exhibited metal removal effects, for instance, towards Cu, Ni and Cd (examined and analyzed by other project partners).

After 3 years collaborative endeavor of all project's partners regardless of COVID impact, BIOSHELL has achieved the major goals as outlined/anticipated in the proposal. The key outcomes are reflected as such: (i) successful development of chitosan extraction from marine wastes derived from fishery and seafood production; (ii) successful generation of novel chitosan-based nanocomposites for pathogen and ARGs removal from wastewater; (iii) Establishment of genetic marker-based molecular diagnostic platform for reliable and rapid assessment of treatment effectiveness. The achieved project results have been effectively and intentionaly disseminated to broad audiences through e.g. open access scientific journals as such: 1. Paruch, L., Paruch, A.M., Neblea, I.E, Iordache, T.-V, Olaru, A.G., Chiriac, A.-L, Sarbu, A. 2023. Effective removal of antibiotic resistance genes from wastewater using marine waste-derived novel nanocomposites, Environmental Technology & Innovation, 32,103320. https://doi.org/10.1016/j.eti.2023.103320. 2. Neblea, I.E., Chiriac, A.-L., Zaharia, A., Sarbu, A., Teodorescu, M., Miron, A., Paruch, L., Paruch, A.M., Olaru, A.G., Iordache, T.-V. 2023. Introducing semi-interpenetrating networks of chitosan and ammonium-quaternary polymers for the effective removal of waterborne pathogens from wastewaters. Polymers, 15(5), 1091. https://doi.org/10.3390/polym15051091 3. Paruch, L., Paruch A.M. 2022. An overview of microbial source tracking using host-specific genetic markers to identify origins of fecal contamination in different water environments. Water, 14(11), 1809. https://doi.org/10.3390/w14111809 4. Paruch, L. 2022. Molecular Diagnostic Tools Applied for Assessing Microbial Water Quality. Int. J. Environ. Res. Public Health,19, 5128. https://doi.org/10.3390/ijerph19095128 5. Paruch L, Paruch A.M. 2022. Molecular Identification of Infectious Enteropathogens in Faeces of Healthy Horses. Microbiol Insights. 15,11786361221089005. http://doi.org/10.1177/11786361221089005 6. 7. Paruch L., Paruch A.M., Iordache T-V, Olaru A.G., Sarbu A. 2021. Mitigating antibiotic resistance genes in wastewater by sequential treatment with novel nanomaterials. Polymers, 13(10), 1593. https://doi.org/10.3390/polym13101593 The obtained data have been also reached out to broader audience through national and international conferences, symposiums as oral presentation and posters (by combining the results from different partners). In terms of IPR protection, several national (in Romania) patents have been filed/in application. Since the projects outcomes are innovative and appealing in technology aspect, the methodology and novel nanoproducts have great potential in post-project period to attract interest for technology upgrading/transfer in industry (e.g. novel nanoproducts fabrication using massive marine wastes) and wastewater treatment sectors (e.g. integrating the developed nanomaterials into the biological purificaiton filter media).

Wastes from agriculture and fishery cause harmful effects on the environment and implicitly on humans. Yet, many of these wastes can be recycled. One of the current global issues refers to minimizing waste production, effective wastewater treatment, biosafe food production, and reducing hazards from the exposure to pathogens. Most of the threatening microorganisms, especially Emerging Pathogens (EPs), derive from wastewater. Moreover, antibiotics residues present in wastewater lead bacterial pathogens to develop Antibiotic Resistance Genes (ARGs). In addition, heavy metals are among the most harmful non-microbial pollutants due to their toxicity to humans. The BIOSHELL project aims at synergistically solving economic, environmental and health problems caused by agricultural and food industry wastes. The project focuses on utilizing the wastes from seafood preparation, such as crustacean carcasses, in the development of innovative and efficient inorganic-organic functionalized hydrogel nanocomposites, suitable to facilitate the sustainable wastewater purification technologies for heavy metals retention, antibiotics elimination, EPs and ARGs removal. Functional biopolymer-based hydrogels starting from valorized crustacean's shell wastes will be developed both for the metal and antibiotics retention in waters as well as for anti-bacterial treatment. These competitive materials will be Ion Imprinted Polymers (IIPs) or Molecularly Imprinted Polymers (MIPs). They will benefit from new synthesis methodologies applied for chelating the chitosan nanocomposites and for the chemical grafting of the bactericidal hybrid surfaces. The development of new approaches for the valorization of crustacean wastes, by the new functionalized bio-hydrogels, will improve water purification and wastewater treatment in general, but particularly for decentralised locations/on-site systems. The regeneration of new bio-based agents shall also be targeted.

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Funding scheme:

BIOTEK2021-Bioteknologi for verdiskaping

Thematic Areas and Topics

BioteknologiMedisinsk bioteknologiAntimikrobiell resistensHavbrukDelportefølje KvalitetGrunnforskningHavbrukAnnen havbruksrelevant forskningPolitikk- og forvaltningsområderMiljø, klima og naturforvaltningPolitikk- og forvaltningsområderDelportefølje Et velfungerende forskningssystemMarinHavbrukLTP3 Klima, polar og miljøBioteknologiIndustriell bioteknologiLTP3 Et kunnskapsintensivt næringsliv i hele landetResponsible Research & InnovationAnvendt forskningNaturmangfold og miljøLTP3 Muliggjørende og industrielle teknologierResponsible Research & InnovationRRI Utviklings- og prosessorienteringBioteknologiMarin bioteknologiPortefølje Klima og miljøLTP3 Fagmiljøer og talenterBioteknologiBransjer og næringerFiskeri og havbrukNaturmangfold og miljøTerrestrisk forurensning inkl. miljøgifterBioøkonomiØvrig bioøkonomiCo-Funded/ERA-NETPortefølje Banebrytende forskningKlimarelevant forskningInternasjonaliseringMarinMarin bioteknologiLTP3 Hav og kystInternasjonaliseringInternasjonalt samarbeid om utlysningBioøkonomiMarinLTP3 Nano-, bioteknologi og teknologikonvergensBransjer og næringerPortefølje ForskningssystemetCo-Funded/ERA-NETERA-NET Cofund H2020Portefølje Mat og bioressurserDelportefølje InternasjonaliseringPortefølje Muliggjørende teknologierInternasjonaliseringInternasjonalt prosjektsamarbeidLTP3 Bioøkonomi og forvaltningLTP3 Høy kvalitet og tilgjengelighetLTP3 Klima, miljø og energiLTP3 Marine bioressurser og havforvaltningLTP3 Rettede internasjonaliseringstiltakLTP3 Styrket konkurransekraft og innovasjonsevne