STAGE II. Experimental models for the obtaining of materials based on raw chitosan

PERIOD: 01.01.2021-31.12.2021

OBJECTIVES

Act 2.1 – Optimization of the recipe for raw chitosan obtaining starting from chitin extracted from preconditioned crustaceans’ waste. Results: Functional model for the obtaining of raw chitosan.

2.1.1 –Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: The optimization of the recipe for obtaining raw chitosan starting from chitin extracted from preconditioned crustaceans’ waste

Act.2.2– The obtaining of MIP pearls based on raw chitosan (via experimental chitin) for antibiotics retention. Results: Experimental model for MIP pearls.

2.2.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: The obtaining of MIP pearls based on raw chitosan (via experimental chitin) for antibiotics retention.

Act.2.3– The synthesis of antibacterial hydrogels based on raw chitosan (via experimental chitin) and quaternary ammonium salts. Results: Experimental model of antibacterial hydrogels.

2.3.1 –Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: The synthesis of antibacterial hydrogels based on raw chitosan (via experimental chitin) and quaternary ammonium salts.

Act 2.4– The characterization of raw materials, intermediates and final materials. Results: 3 Characterization reports.

2.4.1 –Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: The physico-chemical characterization of raw materials, intermediates and final materials and quantification of the

antibiotic retention profile.

2.4.2 –Part – Partner (P1) – EDAS-EXIM SRL: The biochemical characterization of raw materials, intermediates and final materials and the quantification of the retention profile of pathogenic bacteria.

Act 2.5– Communication and dissemination of the results through national or international conferences, working visits at International Consortium Partners, national and international indexed web of science journals and competent national authorities, such as OSIM, for the protection of intellectual property. Results: Report about dissemination, communication and travel.

2.5.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Communication and dissemination of the results and the protection of intellectual property.

2.5.2 –Part-– Partner (P1) – EDAS-EXIM SRL: Communication and dissemination of the results and the protection of intellectual property.

SUMMARY

               The main objective of the research was the optimization of raw chitosan and raw chitosan-based materials obtaining, starting from shrimp carcasses, and the monitorization of effects on the final properties. Regarding this, the following secondary objectives have been set out:

OS1. The obtaining and optimization of the recipe for raw chitosan obtaining from chitin extracted from preconditioned crustaceans’ waste. Physico-chemical characterization of raw materials, intermediates and final materials.

OS2. The obtaining MIP pearls based on raw chitosan (via experimental chitin) for antibiotic retention. Physico-chemical characterization of raw materials, intermediates, final materials and quantification of the antibiotic retention profile.

OS3. The synthesis of antibacterial hydrogels based on raw chitosan (via experimental chitin) and quaternary ammonium salts. Physico-chemical and bacteriological characterization of raw materials, intermediates and final materials and the quantification of the retention profile of pathogenic bacteria.

OS1. The achievement of this objective involved the optimization of the obtaining process of raw chitosan from crustaceans’ carcasses. This phase ended with the obtaining of a chitosan with properties similar to those of commercial chitosan. However, some distinct features were also highlighted, which led to different results compared to the commercial one.

               The synthesis of chitosan from shrimp carcasses has been successfully performed. The FTIR spectra of the two types of chitosan synthesized in the laboratory (raw and commercial chitin) were compared with that of commercial chitosan. The samples showed all the characteristic bands of chitosan formation presented in the literature. XRD spectra confirmed the presence of calcium carbonate in raw chitosan, which is desirable in order to improve the mechanical properties of chitosan for future applications.

               The HPLC technique provided important information about the molecular weight of chitosan samples, so it can be seen that the molecular weight of chitosan obtained in the laboratory is higher than that of commercial chitosan. Raw chitosan has two different molecular masses, one of which is an order of magnitude larger than that of CC, thus explaining its weaker solubility.

               The deacetylation degree of chitosan was determined by two methods, titration and nuclear magnetic resonance; the obtained data highlighted a high deacetylation degree for both synthesized chitosan, compared to the commercial chitosan.

               In the next stage, studies will be continued to optimize the process of the obtaining raw chitosan from shrimp carcasses, in order to obtain a product with uniform molecular weight.

OS2. This objective involved the obtaining of raw chitosan-based MIP beads for antibiotic retention. As it was found in Stage I, the process of pearls

obtaining proved to be more complex than anticipated. Therefore, the obtaining of materials having antibiotic retention properties primarily involved obtaining cryogel granules with interpenetrated networks consisting of chitosan and biocellulose.

               The study provided information about hybrid cryogels obtaining with applications in the retention of antibiotics from wastewater. Both natural polymers and natural clays were used. Most part of the research has focused on the obtaining of hybrid cryogels based on chitosan-biocellulose and natural clays with superabsorbent properties for antibiotic retention.

               The study of the swelling degrees showed that cryogels had relatively high swelling degrees, which confirmed a very high swelling capacity of these hybrid materials. UV-Vis analysis highlighted the superabsorbent profiles of cryogels for the retention of antibiotics, especially penicillin G. In case of Sulfadiazine Argentic, the possibility of using cyclodextrins to improve water solubility will be

analyzed.

               Thermal analysis also showed the successful incorporation of kaolin, while highlighting stability differences between samples prepared using either commercial chitosan or laboratory synthesized chitosan. Scanning electron microscopy confirmed the incorporation of the clay into the Chitosan-Biocellulose polymer matrix, while also providing information on the porosity of the samples.

               It is noteworthy that the use of chitosan synthesized from crustaceans’ carcasses has led to materials with improved properties in comparison to those based on commercial chitosan. The next step involves testing the newly cryogel recipes in terms of tetracycline and vancomycin retention while optimizing the obtaining method (using molecular imprinting) in order to increase the specificity/selectivity for a particular antibiotic class.

O3. This objective was accomplished by synthesizing new hydrogels with antibacterial effect based on raw chitosan, respectively chitosan synthesized

from crustaceans’ carcasses with polycationic interpenetrated network of quaternary ammonium groups. At the end of this phase, hydrogels with interpenetrated networks were obtained, being found to show improved mechanical strength in comparison to those prepared within the previous stage.

               Characteristic bands for both synthetic polyelectrolyte and chitosan were observed in FTIR spectroscopic analyzes. The thermal degradation behavior of the synthesized IPN hydrogels showed a positive influence on the thermal stability with the increase of the concentration of synthetic polyelectrolyte.

               In the study on the swelling degree, it was observed the influence of the type of chitosan, aside from the obvious influence of the concentration of synthetic monomer used. The maximum swelling degree was 15704% for commercial chitosan and 12823% for raw chitosan. Also, compared to the results obtained in the previous stage regarding the hydrogels synthesized with lower amounts of crosslinker, in this case a better mechanical strength

was found during use/swelling. Hydrogels have maintained their integrity for over 24 hours vs. 6 hours for those obtained in the previous stage.

Bacteriological tests reflected the potential of the synthesized materials, especially raw chitosan-based hydrogel, synthesized from shrimp carcasses, to destroy both coliforms and clostridia by 51% and 53%, respectively. There was also an increase in the bacteriological effect when increasing the amount of polyelectrolyte in hydrogels, which proves a synergistic effect of the two types of interpenetrated polymers. In the next stage, the synthesis process of hydrogels will be optimized in order to increase the bactericidal effect, but at the same time to maintain the mechanical stability of the hydrogels.

DISSEMINATION

During Stage II / 2021, an online workshop was organized with 14 participants from the Partner Organizations (INCDCP-ICECHIM, EDAS-EXIM. SRL, NIBIO, University of Coimbra and BrINOVA) entitled: “Wastewater Purification using biopolymer-based materials” Workshop and Meeting on project progress in the frame of BLUEBIO Cofund 2019 Project BIOSHELL, Location: On-line, 1 OCTOBER 2021. During the workshop the following works were presented:

1.             Presentation of Andreea Miron “Synthesis and characterization of chitosan from commercial chitin and from shrimp wastes” (INCDCP- ICECHIM Bucharest, Romania) (5 min)

2.             Presentation of Marinela Dumitru “Synthesis and characterization of chitosan-based gels for the retention of penicillin” (INCDCP- ICECHIM Bucharest, Romania) (5 min)

3.             Presentation of Iulia Neblea “Synthesis and characterization of chitosan-based gels for bacteria inactivation and retention” (INCDCP- ICECHIM Bucharest, Romania) (5 min)

4.             Presentation of Dr. Andreea Olaru “Evaluation of bactericid effect of new materials on waste water samples” (EDAS-EXIM. SRL, Bucharest, Romania) (15 min)

5.  Presentation of Dr. Lisa Paruch “Development of Antibiotic Resistant Genes (ARGs) markers and screening examination of pathogens & ARGs in wastewater” (Norwegian Institute for Bioeconomy Research-NIBIO, Oslo-Aas, Norway) (15 min)

6.             Presentation of Prof. Artur Valente “Chitosan-based gels for metal and antibiotic adsorption” (University of Coimbra, Coimbra, Portugal) (15 min)

In the implementation plan of stage II, the publication of two ISI papers was foreseen. In this regard, the paper submitted for publication last year was published in September and another ISI indexed article developed in authorship with EDAS-EXIM and NIBIO project partners was submitted and published. Another paper was submitted in October and is now under review.

1.        L. Paruch, A.M. Paruch, T.-V. Iordache, A.G. Olaru, A. Sarbu, Mitigating antibiotic resistance genes in wastewater by sequential treatment of

novel nanomaterials, Polymers, Special Issue: “Hybrid Polymer Materials for Water Purification and Wastewater Treatment”, 13(10), 1593; 2021, https://doi.org/10.3390/polym13101593 WOS:000655148100001, Published in 2021

2.        A.M.Gavrila, S. Nedelcu-Flor, A. Sarbu, T. Sandu, A. Olaru, G. Hubca, D. Donescu, T.V. Iordache, Synthesis and properties of organosilica particles with quaternary ammonium bearings as bacteriostatic interfaces, Scientific Bulletin of UPB Series B, vol 3, 2021, Trimis in 2020 si , Published in 2021.

3.        M. V. Dumitru, T. Sandu, A. Ghebaur, S. A. Gârea, T. V. Iordache, A. L. Ciurlică, I. E. Neblea, B. Trică, H. Iovu, A. Sârbu, Organically Modified

Montmorillonite as pH Versatile Carriers for Delivery of 5-Aminosalicylic Acid , Applied Surface Science, Under review from October 2021.

In the work plan of the project, scientific communications (two) were considered for the dissemination of the results obtained. The communications made are the following:

1.      Dumitru Marinela-Victoria, Sandu Teodor, Sarbu Andrei, Miron Andreea, Coman Alina Elena, Botez Razvan Edward, Duldner Monica, Iovu Horia, Iordache Tanta Verona, Hybrid cryogels with advanced adsorbent properties for sulfadiazine, Bucharest Polymer Conference (BPC), 2nd Edition, 9-11 June 2021, Bucharest, Romania (short oral com.)

2.      Marinela-Victoria DUMITRU, Teodor SANDU, Iulia Elena NEBLEA, Anita-Laura CHIRIAC, Ionut Cristian RADU, Horia IOVU, Andrei SARBU, Tanta Verona IORDACHE, Hybrid Cryogels with Advanced Adsorbent Properties for Penicillin, Simpozion international Prioritatile Chimiei pentru o Dezvoltare Durabila PRIOCHEM – editia XVII, 27- 29 Octombrie 2020, Bucuresti, Romania. Poster

3.      Andreea MIRON, Cesar FILHO, Radu FIERASCU, Iulia NEBLEA, Anita-Laura CHIRIAC, Andrei SARBU, Horia IOVU, Tanta Verona IORDACHE, Calcium Carbonate Enriched-Chitosan Prepared from Shrimp Shell Waste, Simpozion international Prioritatile Chimiei pentru o Dezvoltare Durabila PRIOCHEM – editia XVII, 27- 29 Octombrie 2020, Bucuresti, Romania. Poster

4.      Iulia Elena NEBLEA, Andreea Gabriela OLARU, Anita-Laura CHIRIAC, Anamaria ZAHARIA, Razvan BOTEZ, Mircea TEODORESCU, Andrei SARBU, Tanta-Verona IORDACHE, Chitosan-Based Bactericidal Interpenetrated Hydrogels, Simpozion international Prioritatile Chimiei pentru

o Dezvoltare Durabila PRIOCHEM – editia XVII, 27- 29 Octombrie 2020, Bucuresti, Romania. Poster

               The innovative results obtained in this currently stage have made the object of two patent application meeting the purpose of protecting intellectual property rights, as follows:

1.      Patent application A/00651/27.10.2021: „Criogeluri hibride superadsorbante pe baza de polimeri naturali si argile silanizate si procedeu de obtinere a acestora”, Autori: Chiriac Anita-Laura, Iordache Tanta-Verona, Dumitru Marinela Victoria, Miron Andreea, Sandu Teodor, Sarbu Andrei, Gavrila Ana-Mihaela, Zaharia Anamaria.

2.      Patent application A/00686/16.11.2021: „Hidrogeluri bactericide cu retea interpenetrata pe baza de chitosan si procedeu de obtinerea al acestora”, Autori: Chiriac Anita-Laura, Iordache Tanta-Verona, Sarbu Andrei, Neblea Iulia Elena, Miron Andreea, Stoica Elena-Bianca, Gavrila Ana-Mihaela, Zaharia Anamaria, Olaru Andreea, Cosasu Dan.

Other results obtained within the project refer to the presentation of some doctoral scientific reports of the 3 PhD students Iulia Neblea, Marinela Dumitru and Andreea Miron, which summarizes the results obtained in Stages I and II of this project:

1. Report 1 PhD Iulia Neblea (December) with the title of the thesis: “New structures of micro and nano-gels based on natural and biocompatible polymers”. Doctoral coordinator: Prof. Dr. Eng. Mircea TEODORESCU.

2. Report 1 (June) and 2 (December) Doctorate Marinela Dumitru with the title of the thesis: “Valorization of chitosan from marine sources for obtaining water treatment agents”. Doctoral coordinator: Prof. Dr. Ing. Horia IOVU.

3. Report 1 (June) and 2 (December) Doctorate Andreea Miron with the title of the thesis: “Bio-sourced polymers with controlled properties”. Doctoral

coordinator: Prof. Dr. Ing. Horia IOVU.

STAGE III. Functional models for the obtaining of materials based on raw hitosan

PERIOD: 01.01.2022-31.12.2022

OBJECTIVES

Act 3.1 – Reproducibility of the method for obtaining crude chitosan starting from chitin extracted from preconditioned shellfish waste. Results: Laboratory technology for obtaining raw chitosan.

3.1.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Reproducibility of the method for obtaining crude chitosan starting from chitin extracted from preconditioned shellfish waste

Act 3.2 – Optimization of MIP bead networks based on raw chitosan (via experimental chitin) for antibiotic retention. Results: Functional Model MIP pearls.

3.2.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Optimization of MIP bead networks based on raw chitosan (via experimental chitin) for antibiotic retention

Act 3.3 – Optimization of synthesis networks of antibacterial hydrogels based on raw chitosan (experimental chitin) and quaternary ammonium salts. Results: Functional model antibacterial hydrogels.

3.3.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Optimization of synthesis networks of antibacterial hydrogels based on raw chitosan (experimental chitin) and quaternary ammonium salts

Act 3.4 – Characterization of raw materials, intermediates and final materials. Results: Characterization report.

3.4.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Characterization of raw materials, intermediates and final materials and validation of the retention profile of antibiotics from various contaminated solutions.

3.4.2 – Part – Partner (P1) – EDAS-EXIM SRL: Biochemical characterization of raw materials, intermediates and final materials and validation of the retention profile of pathogenic bacteria from wastewater.

Act.35.- Retention profile validation for antibiotics, heavy metals and pathogenic bacteria in a laboratory micro-pilot. Results: Demonstrator test report.

3.5.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Quantification of antibiotic retention profiles for chitosan-based materials tested in the EDAS laboratory micro-pilot.

3.5.2 – Part – Partner (P1) – EDAS-EXIM SRL: Quantification of the retention profiles of heavy metals and pathogenic bacteria for chitosan-based materials tested in a laboratory micro-pilot. Validation of their efficiency in the retention of antibiotics, heavy metals and pathogenic bacteria from contaminated samples

Act 3.6 – Communication and dissemination of results through national or international conferences, work visits to the International Consortium Partners and national or International web of science indexed journals. Results: Dissemination, communication and travel report

3.6.1 – Part-Coordinator (CO) – National Institute for Research & Development in Chemistry and Petrochemistry ICECHIM: Communication activities and dissemination of results.

3.6.2 – Part – Partner (P1) – EDAS-EXIM SRL: Communication activities and dissemination of results.

SUMMARY

The main objective of the research study was the development of experimental models for obtaining raw chitosan-based materials and their testing. In this respect, the methods of obtaining materials from chitin extracted from the preconditioned waste of crustaceans (according to Stage II) were optimized and the final properties of the materials obtained were re-evaluated. As in the previous stage, the reference materials were commercial chitosan and chitosan obtained from commercial chitin (according to the protocol established in Stage I). To fulfill this objective, the following secondary objectives were established:

OS1. Verification of the reproducibility of the method for obtaining raw chitosan starting from chitin extracted from the preconditioned waste of crustaceans (optimized method in stage II). Physico-chemical characterization of raw materials, intermediates and final materials.

OS2. Optimization of MIP synthesis methods based on raw chitosan (via experimental chitin) for antibiotic retention (the networks developed and tested, in Stages I and II, respectively). Physico-chemical characterization of raw materials, intermediates and final materials and determination of the retention profile of antibiotics from various contaminated solutions.

OS3. Optimizing the synthesis methods of antibacterial hydrogels based on raw chitosan (via experimental chitin) and quaternary ammonium salts (the networks developed and tested, in Stages I and II, respectively). Physico-chemical and bacteriological characterization of raw materials, intermediates and final materials and identification of the retention profile of pathogenic bacteria.

OS4. Testing the efficiency of the materials obtained in a laboratory micro-pilot. Quantification of retention profiles of antibiotics, heavy metals and pathogenic bacteria for chitosan-based materials.

OS1. This objective aimed to demonstrate the reproducibility of the process of obtaining raw chitosan from crustacean carcasses. The synthesis of chitosan starting from commercial chitin and from chitin obtained from waste shrimp carcasses were successfully obtained, showing similar degrees of deacetylation. The FTIR structural analysis showed all the characteristic bands of chitosan formation both in the CCH sample and in the SHC sample. The thermogravimetric analysis highlighted a similar thermal stability for the CCH and SHC chitosan samples, and the XPS analysis demonstrated the presence of calcium carbonate in the SHC chitosan sample, obtained from shrimp carcass waste. The SEM images revealed the presence of calcium carbonate in the structure and on the surface of the SHC chitosan sample, which was also confirmed by the XPS analysis. In conclusion, the SHC chitosan synthesized in Stage 3 presents reproducible characteristics with the one synthesized in Stage 2, which also reveals a good reproducibility of the proposed synthesis methods. The results obtained in this stage led to the development of a laboratory technology for obtaining chitosan, by excluding the demineralization stage of chitin.

OS2. Objective 2 was represented by the optimization of the synthesis process for MIP materials based on raw chitosan for antibiotic retention. The optimization of the synthesis procedure was aimed at improving the stability of cryogels at pH variation and improving the adsorption capacity for Penicillin G and Tetracycline. Several series of cryogels were obtained: cryogels based on chitosan or chitosan with APV, with silicate or modified kaolin content; cryogels based on chitosan and gelatin. The UV-Vis analysis allowed the determination of the retention capacity of the cryogels for two types of antibiotics (PG and Tetracycline). Following this study, it was demonstrated that samples based on CC as well as those based on SHC have superabsorbent properties, especially at basic pH. The FTIR spectra confirmed the obtaining of the desired cryogels by the appearance of specific polymer bands, as well as the successful incorporation of modified kaolin and, respectively, silica into the chitosan matrix. From the study of the degrees of swelling, it was proven that the recipes of the hybrid cryogels were optimized and improved, and the cryogels based on chitosan-gelatin seem to be an option to consider for solutions with a lower pH. Scanning electron microscopy revealed a foliar supermacroporous structure of the cryogels, which explains their superabsorbent character. In conclusion, the structure of the cryogels obtained in Stage II was improved by adding gelatin (for high performance at low pH) and adding modified clay/silicate to improve the stability and retention capacity of antibiotics at basic pH.

OS3. In the the frame for this objective, the optimization of the synthesis method of new hydrogels with antibacterial effect based on chitosan synthesized from crustacean carcasses with polycationic interpenetrated network of quaternary ammonium groups was followed. For this purpose, a macro-crosslinker, PEGDA700, was additionally used to improve the stability of the hydrogels over time. In the study of the degree of swelling, the influence of the crosslinker concentration and the influence of the monomer concentration were observed. The FTIR analyzes highlighted the presence of

bands characteristic of chitosan and of the VBTAC monomer. The thermal degradation behavior of the synthesized IPN hydrogels indicates two main degradation steps for the samples based on CC and SHC and three steps for those based on CHC. These results underline the fact that the use of CC and SHC leads to obtaining more homogeneous materials. Bacteriological tests reflected the potential of the synthesized materials, especially the SHC chitosan-based hydrogel. In this case, the tests were performed in triplicate using samples of used water from an industrial source containing Gram-negative, Colifomi, and Gram-positive bacteria, Clostridium Perfringens. A high capacity to retain bacteria was observed in the case of samples containing CCH and CC, but only in the case of Gram-negative bacteria. Regarding the samples containing SHC, a lower retention capacity was observed compared to the control sample, but the antibacterial action is manifested against both species of bacteria. In conclusion, the hydrogels with interpenetrated networks proved much more stable and with a higher bactericidal effect than the samples obtained in the previous stage.

OS4. The last proposed objective sought to demonstrate the effectiveness of SHC chitosan-based materials for the retention of bacteria, antibiotics and heavy metals, in dynamic conditions on a larger volume of contaminated water. For this purpose, the testing was carried out in a 500 mL micropilot with stirring of the EDAS Project Partner. The materials optimized in the third phase were tested in triplicate in order to obtain the retention profiles of bacteria, organic residues and heavy metals from industrial wastewater. The results demonstrated that the efficiency of SHC-based hydrogels can be increased by up to 30%, by stirring. In the next stage, the study on the retention of antibiotics in the micro-pilot of the cryogels optimized in the third phase will be finalized.

In this stage, trips were made to Valcea and Sinaia for the communication of some results within the organized conferences. Part of the scientific communications as well as the meeting with the Project partners were organized online. 4 scientific papers and an editorial were published in ISI indexed journals. Also, 5 Research Reports were submitted as part of some doctoral theses, with the theme closely related to that of the project.

Because the project partner BrINOVA decided to leave the consortium (following delays in signing the contract by 1 year) and due to the fact that the Univ. Coimbra signed the contract 1 year late, the joint activities, such as the testing in a micro-pilot laboratory of the retention capacity of antibiotics and heavy metals from the materials obtained both by ICECHIM and by the Univ. Coimbra were partially realized (only for a series of materials obtained by ICECHIM). Testing of the second series prepared by ICECHIM (cryogels) and of the series of samples prepared by Univ. Coimbra was postponed by 2 months (for the first two months of 2023). In conclusion, considering the small deviation from the general plan, not imputable to the coordinator, the degree of fulfillment of the proposed objectives was achieved in a proportion of 100% in relation to the expected results for this stage.

DISEMINATION

As part of stage III/2022, an online meeting was organized with participants from the 4 remaining Partner Organizations (INCDCP-ICECHIM, EDAS-EXIM. ​​SRL, NIBIO, University of Coimbra). BrINOVA did not sign the contract with the Contracting Authority and decided to leave the consortium. During the meeting, aspects related to the takeover of BrINOVA activities were disscused, which were taken over by the Univ. Coimbra and INCDCP-ICECHIM. For

these reasons and because of the fact that Univ. Coimbra signed the contract 1 year late, the joint activities, such as the testing in a micro-pilot laboratory of the retention capacity of antibiotics and heavy metals from the materials obtained both by ICECHIM and by the Univ. Coimbra were partially realized (only for a series of materials obtained by ICECHIM). Testing of the second series prepared by ICECHIM and the series of samples prepared by Univ. Coimbra was postponed by 2 months (for the first two months of 2023). In order to successfully fulfill all the common objectives of the project, it was also

decided to extend the duration of the project by at least 5 months (until August 31, 2023), in order to carry out all the activities proposed in the project (which include biodegradability tests of raw materials and obtained materials). The Addendum will be signed in January 2023.

In the implementation plan of stage III, the publication and communication of scientific works was foreseen. In this respect, the work sent last year for publication was published in January 2022, and 2 more articles and a review, indexed ISI, were also published during this stage. Also, the project and its results were discussed in an editorial. Two patent applications used as the background of the project were presented at 2 innovation fairs. The trips to Valcea, Sinaia, Iasi and Cluj for the communication of the results were supported from complementary funds, while the rest of the conferences were held online or organized by ICECHIM, for which only participation fees were paid.

Published articles:

1. Marinela Victoria Dumitru, Teodor Sandu, Ana Lorena Ciurlica, Iulia Elena Neblea, Bogdan Trica, Adi Ghebaur, Sorina Alexandra Garea, Horia Iovu, Andrei Sarbu, Tanța Verona Iordache, Organically modified montmorillonite as pH versatile carriers for delivery of 5-aminosalicylic acid, Applied Clay Science 218 (2022) 106415, https://doi.org/10.1016/j.clay.2022.106415

2. Iulia Elena Neblea, Ana‑Mihaela Gavrila, Tanta‐Verona Iordache, Anamaria Zaharia, Paul Octavian Stanescu, Ionut‑Cristian Radu, Sabina Georgiana

Burlacu, Georgeta Neagu, Anita‑Laura Chiriac, Andrei Sarbu, Interpenetrating networks of bacterial cellulose and poly (ethylene glycol) diacrylate as potential cephalexin carriers in wound therapy, Journal of Polymer Research (2022) 29:406, https://doi.org/10.1007/s10965-022-03250-9

3. Miron, A.; Sarbu, A.; Zaharia, A.; Sandu, T.; Iovu, H.; Fierascu, R.C.; Neagu, A.-L.; Chiriac, A.-L.; Iordache, T.-V. A Top-Down Procedure for Synthesizing Calcium Carbonate-Enriched Chitosan from Shrimp Shell Wastes. Gels 2022, 8, 742. https://doi.org/10.3390/gels8110742

4. Sandu, T.; Sârbu, A.; Caprarescu, S.; Stoica, E.-B.; Iordache, T.-V.; Chiriac, A.-L. Polymer Membranes as Innovative Means of Quality Restoring for Wastewater Bearing Heavy Metals. Membranes 2022, 12, 1179. https://doi.org/10.3390/membranes12121179 

5. Andrei Sarbu, Editorial, Special Issue on the Applications of Molecularly Imprinted Films: Appl. Sci. 2022, Volume 12, Issue 17, 8533 (FI= 2,838) https://doi.org/10.3390/app12178533

Scientific Communications:

1. Ana-Lorena Neagu, Bianca-Elena Stoica, Ana-Mihaela Gavrila, Tanta Verona Iordache, Sorin Dolana, Andrei Sarbu, Teodor Sandu, Horia Iovu, Catalin Zaharia, Petru Epure, Detection of Lipopolysaccharides from Multi-Drug Resistant Bacteria using Modified Plastic Screen-Printed Carbon Electrodes, Simpozion International Prioritatile Chimiei pentru o Dezvoltare Durabila PRIOCHEM – editia XVIII, 26- 28 Octombrie 2022, Bucuresti, Romania. Oral Com

2.Elena-Bianca Stoica, Sorin-Viorel Dolana, Tanţa-Verona Iordache, Anamaria Zaharia, Anita-Laura Chiriac, Teodor Sandu, Andrei Sârbu, Ana-Mihaela Gavrilă, Ephedrine Hydrochloride Detection Based on MIP Particles/Conductive Carbon Paste Modified Electrode, Simpozion international Prioritatile Chimiei pentru o Dezvoltare Durabila PRIOCHEM – editia XVIII, 26- 28 Octombrie 2022, Bucuresti, Romania. Oral Com

3.Andreea Miron, Tanta-Verona Iordache, Sorin-Viorel Dolana, Marinela Dumitru, Ana-Mihaela Gavrila, Anamaria Zaharia, Horia Iovu, Anita-Laura (Radu) Chiriac, Novel Nanocomposites Based on Mesoporous Titania/Acrylonitrile Obtained by Host-Guest Method, Applications of Chemistry in Nanosciences and Biomaterials Engineering NanoBioMat 2022 – Summer Edition, 22-24 June 2022, virtual. Poster

4.Iulia Elena Neblea, Tanța-Verona Iordache, Anamaria Zaharia, Andreea Olaru, Andreea Miron, Mircea Teodorescu, Andrei Sarbu, Anita-Laura Chiriac, Biopolymer-based interpenetrated hydrogels for wastewater treatment, Applications of Chemistry in Nanosciences and Biomaterials Engineering NanoBioMat 2022 – Summer Edition, 22-24 June 2022, virtual. Poster

5.Marinela Victoria Dumitru, Tanta-Verona Iordache, Miron Andreea, Teodor Sandu, Sorin Viorel Dolana, Horia Iovu, Andrei Sarbu, Anita-Laura Chiriac, Molecularly imprinted supermacroporous cryogels for penicillin G adsorption, Applications of Chemistry in Nanosciences and Biomaterials Engineering NanoBioMat 2022 – Summer Edition, 22-24 June 2022, virtual. Poster

6.Iulia Elena Neblea, Anamaria Zaharia, Andreea Olaru, Mircea Teodorescu, Tanța-Verona Iordache, Elena-Bianca Stoica, Teodor Sandu, Andreea Miron, Andrei Sarbu, Anita-Laura Chiriac, New Innovative Biopolymer-Based Interpenetrated Hydrogels with Potential Antibacterial Activity, 22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022. Oral Com

7. Marinela Victoria Dumitru, Tanta-Verona Iordache, Iulia Elena Neblea, Elena Bianca Stoica, Teodor Sandu, Andrei Sarbu, Ana-Mihaela Gavrila, Anita Laura Chiriac, Molecularly Imprinted Supermacroporous Cryogels for Penicillin G Adsorption, Conferinta Nationala de Chimie, Calimanesti-Caciulata Valcea, 4-7 oct. 2022. Oral Com

Other results obtained within the project refer to the presentation of doctoral scientific reports by the 3 doctoral students Iulia Neblea, Marinela

Dumitru and Andreea Miron, which summarize the results obtained in Stage III of the project:

1. PhD Raport 2 Iulia Neblea (June). Thesis title: „Noi structuri de micro și nano-geluri pe bază de polimeri naturali și biocompatibili”. PhD Mentor: Prof. Dr. Ing. Mircea TEODORESCU.

2. PhD Raport 3 (June) si 4 (December) Marinela Dumitru. Thesis title: „Valorificarea chitosanului din surse marine pentru obținerea de agenți de tratare a apelor”. PhD Mentor: Prof. Dr. Ing. Horia IOVU.

3. PhD Raport 3 (June) si 4 (December) Andreea Miron. Thesis title: „Polimeri biosursați cu proprietăți dirijate”. PhD Mentor: Prof. Dr. Ing. Horia IOVU.

Participation in innovation fairs with patent applications used as a background for the BIOSHELL project (medalized and awarded with various distinctions) and settled from institutional projects:

1. Iordache Tanţa Verona; Chiriac Anita Laura; Zaharia Anamaria; Sârbu Andrei; Sîrbu Carmen Eugenia; Gavrilă Ana Mihaela; Sandu Teodor; Stoica Elena Bianca; Cojocaru Crina Thea; Botez Răzvan Edward; Miron Andreea; Apostol Steluţa, GEOTEXTILE COMPOSITE FOR ENVIRONMENT PROTECTION AND PROCESS FOR PREPARING THE SAME, RO201900846A·2019-12-03, E U R O I N V E N T 14th European Exhibition of Creativity and Innovation Iasi, Romania, 26-28 May 2022 (Siver Medal, Exhibition Diploma)

2.Iordache Tanţa Verona, Radu Anita Laura, Sârbu Andrei Zaharia Anamaria, Gavrilă Ana Mihaela, Sandu Teodor, Apostol Steluţa, Stoica Elena Bianca, PARTICULE DE POLIMERI IMPRENTATI MOLECULAR PE SUPORT ANORGANIC SI PROCEDEU DE OBTINERE A ACESTORA, RO134369A2/2020-08-28, PRO INVENT-XX, 26-28 October 2022, Cluj-Napoca (PRO INVENT Medal and Excellence Diploma, Diploma from USAMV Bucuresti, Diploma from Justin Capra Association).

STAGE IV. Refurbishment of raw chitosan-based materials and development

of laboratory technologies for their preparation

PERIOD: 01.01.2023-31.08.2023

OBJECTIVES

Act 4.1 – Refurbishment of raw chitosan-based materials and their testing. Results: Reuse report.

4.1.1 – Part – Coordinator (CO) – National Research and Development Institute for Chemistry and Petrochemistry – ICECHIM Bucharest: Testing some methodologies for reconditioning chitosan-based materials and re-evaluating the efficiency of chitosan-based materials.

4.1.2 – Partner (P1) – EDAS-EXIM SRL: Re-evaluation of the effectiveness of chitosan-based materials for the retention of heavy metals and pathogenic bacteria.

Act 4.2 – Development of laboratory technologies for the preparation of MIP pearls and chitosan-based antibacterial hydrogels. Results: Laboratory

technology for obtaining MIP pearls and laboratory technology for obtaining antibacterial hydrogels.

4.2.1 – Part – Coordinator (CO) – National Research and Development Institute for Chemistry and Petrochemistry – ICECHIM Bucharest: Development of laboratory technologies for the preparation of MIP pearls and chitosan-based antibacterial hydrogels.

Act 4.3 – Communicating and disseminating the results through a final workshop organized by the Project Coordinator and through national or international web of science indexed journals. Results: Dissemination, communication and travel report.

4.3.1 – Part – Coordinator (CO) – National Research and Development Institute for Chemistry and Petrochemistry – ICECHIM Bucharest: Communication activities and dissemination of results.

4.3.2 – Part – Partner (P1) – EDAS-EXIM SRL: Communication activities and dissemination of results.

SUMMARY

The main objective of the research study was the reconditioning of raw chitosan-based materials and the development of laboratory technologies for their preparation. In this sense, the chitin materials extracted from the preconditioned crustacean waste (according to Stage II) were tested for reconditioning and re-evaluation for the retention of heavy metals and pathogenic bacteria. Following the establishment of the optimal results of reconditioning and re-evaluation of the retention, the protocols for obtaining the materials were optimized and, based on the demonstration of their reproducibility, the laboratory technologies for the preparation of MIP pearls and antibacterial hydrogels were developed. As in the previous stage, the reference materials were commercial chitosan and chitosan obtained from commercial chitin (according to the protocol established in Stage I). To fulfill this objective, the following secondary objectives were established:

OS1. Testing some methodologies for reconditioning chitosan-based materials and re-evaluating the effectiveness of chitosan-based materials for the retention of heavy metals and pathogenic bacteria. In order to recondition chitosan-based materials, a purification process similar to the purification step applied after the synthesis of hydrogels was used, following the same principle of the diffusion of harmful compounds retained in the 3D polymer network.

Following the analysis carried out by the project partner (EDAS-EXIM), chitosan-based samples (cryogels – for the retention of antibiotics and modified chitosan – Parter Univ. Coimbra for the retention of heavy metals), very good results were obtained for lead (33-58% reduction) and cadmium (90% reduction), as well as very good results for reducing total phosphorus. For the reconditioned materials based on chitosan (semi-IPN), a decrease in bactericidal efficiency of approximately 30% (total Coliforms and Clostridia) was observed, compared to the initial set analyzed in Stage 3. After determining the bacteriological and physico-chemical indicators, the samples of reconditioned material based on chitosan were prepared and shipped to Norway for determining the retention of pathogens and ARGs from wastewater.

OS2. Development of the laboratory technology for the preparation of chitosan-based MIP pearls using the optimized method for MIP synthesis from raw chitosan (via experimental chitin) for the retention of antibiotics (recipes developed and tested in Stage III).

This objective had as its main purpose the testing of samples at an industrial level on waste water in order to determine the ability of cryogels to retain antibiotics. As in the previous stages, the cryogel recipes were studied and improved to have a much higher antibiotic retention capacity. In this stage, the best samples were chosen and later tested. For this purpose, two samples were chosen based on chitosan prepared from shrimp carcasses (SHC), one with a high content of modified kaolin and one with a high content of organosilane. The testing was carried out on wastewater containing Penicillin G, because in the previous stages the best results were obtained for this antibiotic. The cryogels obtained were characterized both from a physical and physico-chemical point of view using appropriate techniques and equipment. Also, for the two series of cryogels, a common laboratory technology was created, which is supported by the patent application registered in the previous stages.

OS3. The development of laboratory technology for the synthesis of antibacterial hydrogels based on chitosan using the optimized synthesis methods

for antibacterial hydrogels based on raw chitosan (via experimental chitin) and quaternary ammonium salts (the developed and tested networks, in Stages I and II, respectively ).

Within this objective, the establishment of the laboratory technology for obtaining IPN antibacterial hydrogels based on chitosan and quaternary ammonium salts was considered. Thus, the reproducibility of the synthesis of IPN antibacterial hydrogels was taken into account, following the determination of the degree of swelling at equilibrium, the determination of the monomer conversion, as well as the specificity and reproducibility of the final antibacterial properties. The obtained IPN hydrogels were characterized both from a physical and physico-chemical point of view using appropriate techniques and equipment. Also, for the optimal series of IPN hydrogels, a laboratory technology was created, which is supported by the patent application registered in the previous stages.

In conclusion, the degree of fulfillment of the proposed objectives was achieved in a proportion of 100%, referring to the expected results for the final stage.

DISEMINATION

During stage IV/2023, a final online workshop was organized with participants from the remaining 4 Partner Organizations (INCDCP-ICECHIM, EDAS-EXIM. SRL, NIBIO, University of Coimbra). The workshop was titled “Biodegradable Cleaning Materials for Effective Wastewater Treatment”. During the meeting, the results obtained in the project up to that moment were presented, but there were also discussions about the implementation of the activities from the work plan in order to fulfill all the objectives and the provided indicators, as well as future perspectives. 4 oral presentations were given, as follows:

1. Dr. Ing. Anita-Laura Chiriac “Biopolymers-based materials for antibiotics elimination and bacteria inactivation and retention” (INCDCP- ICECHIM Bucharest, Romania) (15 min)

2. Dr. Andreea Olaru “Quantification of pathogenic bacteria and heavy metals retention for chitosan-based materials” (EDAS-EXIM. SRL, Bucharest, Romania) (15 min)

3. Dr. Lisa Paruch “Pathogens and ARGs removal from wastewater using developed semi-IPN hydrogels” (Norwegian Institute for Bioeconomy Research-NIBIO, Oslo-Aas, Norway) (15 min)

4. Prof. Artur Valente “Sorption of metal ions and drugs onto chitosan-based polymers” (University of Coimbra, Coimbra, Portugal) (15 min)

Because one of the partners signed the financing contract 1 year later, and one of the partners withdrew from the project, itnwas also decided to extend the duration of the project by 5 months (until August 31, 2023) , in order to successfully fulfill all the common objectives of the project. The Addendum was signed in February 2023.

In the implementation plan of stage IV, the publication of the original results and the communication of scientific works were foreseen. In this sense, 3 ISI indexed articles were published during this stage. Dissemination was also carried out through a national conference with international participation, during which a poster communication was presented. The two patent applications registered within the project were presented at 2 international innovation fairs.

Published articles:

1.      Iulia E. Neblea, Anita-L Chiriac*, Anamaria Zaharia, Andrei Sarbu, Mircea Teodorescu, Andreea Miron Lisa Paruch, Adam M. Paruch, Andreea G. Olaru, Tanta-V. Iordache*, Introducin semi-interpenetrating networks of chitosan and ammonium-quaternary polymers fo the effective removal of waterborne pathogens from wastewaters. Polymers 2023 15(5), 1091; https://doi.org/10.3390/polym15051091

2.       Marinela Victoria Dumitru Sandu Teodor, Andreea Miron, Anamaria Zaharia, Ionut Cristian Radu, Ana-Mihael Gavrila, Andrei Sarbu, Horia Iovu, Anita-Laura Chiriac*, Tanța Veron Iordache*, Hybrid cryogels with superabsorbent properties as promisin materials for penicillin G retention. Gels 2023,

9, 443. https://doi.org/10.3390/gels9060443 

3.       Lisa Paruch, Adam M. Paruch, Iulia Elena Neblea, Tanta-Verona Iordache, Andreea G. Olaru, Anita-L. Chiriac, Andrei Sarbu, Effective removal of antibiotic resistance genes from wastewater using marine waste-derived novel nanocomposites. Environmental Technology & Innovation 2023, 32, 103320; https://doi.org/10.1016/j.eti.2023.103320

Scientific Communications: Marinela-Victoria Dumitru, Andreea Miron, Teodor Sandu, Horia Iovu, Ana-Lorena Neagu (Ciurlica), Sorin Dolana, Anamaria Zaharia, Anita-Laura Chiriac, Tanta-Verona Iordache, Cryogels based on chitosan for antibiotics retention, NANOBIOMAT-2023, 28-30 June 2023, Poster

In order to valorize crustacean carcass waste, 3 new types of materials were obtained (chitosan enriched with minerals, prepared from shrimp carcasses, MIP cryogels for retaining antibiotics and bactericidal semi-interpenetrated hydrogels). Based on the optimization of the synthesis recipes of these materials, 3 laboratory technologies were developed, 2 of which were based on patent applications submitted to OSIM:

1. Laboratory technology for obtaining raw chitosan (described in Stage III)

2. Laboratory technology for obtaining MIP cryogels (described in Stage IV)

3. Laboratory technology for obtaining bactericidal hydrogels (described in Stage IV)

Other results obtained within the project refer to the presentation of doctoral scientific reports by 2 doctoral students Marinela Dumitru and Andreea Miron, which summarize the results obtained in Stage IV of the project:

1.     PhD Raport 5 (June) Marinela Dumitru. Thesis title: „Valorificarea chitosanului din surse marine pentru obținerea de agenți de tratare a apelor”. PhD Mentor: Prof. Dr. Ing. Horia IOVU.

2.     PhD Raport 5 (June) Andreea Miron. Thesis title: „Polimeri biosursați cu proprietăți dirijate”. PhD Mentor: Prof. Dr. Ing. Horia IOVU.

Also, part of the results of this Stage were obtained following two research internships (supported by complementary funds – ERASMUS), at the Project Partner Univ. Coimbra, by PhD students Andreea Miron and Marinela Dumitru. The two internships were carried out between March 1 and May 31, 2023 and coordinated by Prof. Luisa Duraes and Prof. Artur Valente (BIOSHELL Project Manager and, respectively, the key person from the Portuguese partner).

Participation in innovation fairs, settled from institutional projects, with the two patent applications submitted within the BIOSHELL project and published in BOPI at the beginning of 2023. The two CBIs were awarded medals and awards with various distinctions as follows:

1.      Chiriac Anita-Laura, Iordache Tanta-Verona, Dumitru Marinela Victoria, Miron Andreea, Sandu Teodor, Sarbu Andrei, Gavrila Ana-Mihaela, Zaharia Anamaria, „Superabsorbent hybrid cryogels based on natural polymers and silanized clays and process for preparing the same”, A/00651/27.10.2021, Japan Design, Idea&Invention Expo (JDIE 2023) International Invention Exhibition, Tokyo, Japonia, 6-10 July 2023 (Medalie Argint)

2.     Elena Iulia Neblea, Anita-Laura Chiriac, Tanța-Verona Iordache, Andrei Sârbu, Andreea Miron, Ana-Mihaela Gavrilă, Anamaria Zaharia, Elena Bianca Stoica, Andreea Olaru, Dan Cosașu, “Bactericidal hydrogels with interpenetrated network based on chitosan and process for their obtainment”, A 2021 00686 /16.11.2021, Fan Expo Canada 2023, Toronto, Canada, 24-27 August 2023.