COFUND-BLUEBIO-BIOSHELL“Recycling crustaceans shell

wastes for developing biodegradable wastewater cleaning composites – BIOSHELL”

Project Partners and Institutions:

Project Director:

CS I. Dr. Eng. RADU Anita-Laura, Advanced Polymer Materials and Polymer Recycling Group, Polymers Department,

Coordinator:

National Research & Development Institute for Chemistry and Petrochemistry-ICECHIM, Bucharest, Romania (www.icechim.ro)

Project Responsible:

CS III. Dr. biochim. Andreea Gabriela OLARU, Director of the R&D Dept. and Quality Dept., S.C. EDAS-EXIM S.R.L.

Prof. Dr. Artur Jose Monteiro VALENTE, University of Coimbra, Faculty of Sciences and Technology – Department of Chemistry,

Dr. Alexandre Cabral CRAVEIRO, Brinova Bioquimica Lda.,

Prof.Dr.eng. Lisa PARUCH, Professor at Environment and Natural Resources Division,

Partners:·        

  • S.C. EDAS-EXIM.SRL, Bucharest, Romania, http://www.edas.ro·        
  • University of Coimbra, Faculty of Science and Technology, Coimbra,
  • Portugal, http://www.uc.pt·  Brinova Bioquimica Lda., Évora, Portugalia, http://www.brinova.eu·        
  • Norwegian Institute of Bioeconomy Research –NIBIO, Division of Environment and Natural Resources As, Norway, http://www.nibio.no

National Funding Agencies

Unitatea executiva pentru finantarea invatamantului superior, a cercetarii, dezvoltarii  si inovarii (UEFISCDI): https://www.uefiscdi.ro/

UEFISCDI and CE: 250 000 €

Co-financing amount from EDAS-EXIM. SRL 30.000 €

Fundação para a Ciência e a Tecnologia (FCT): http://www.fct.pt/

FTC and CE: 99.431 €

Co-financing amount from University of Coimbra: 88.122 €

Co-financing amount from BRINOVA Lda: 84.000 €

Duration of project: 36 months (start date: 10 April 2020)

Abstract

Wastes from agriculture and fishery cause harmful effects on the environment and implicitly on humans. But, many of these wastes can berecycled. 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) derivefrom 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. BIOSHELL aims atsynergistically solving economic, environmental and health problems. The project focuses on utilizing the wastes from sea foodpreparation such as crustacean carcasses in the development of innovative and efficient inorganic-organic functionalized hydrogelnanocomposites, suitable to facilitate the sustainable wastewater purification technologies about heavy metals retention, antibioticselimination, EPs and ARGs removal.

Objective

Functional biopolymer-based hydrogels starting from valorized crustacean’s shell wasteswill be developed both for the metal and antibiotics retention in waters as well as for anti-bacterial treatment. These competitive materialswill be ion imprinted polymers (IIPs) or molecularly imprinted polymers (MIPs). They will benefit from new synthesis methodologies appliedfor chelating the chitosan nanocomposites and for the chemical grafting of the bactericidal hybrid surfaces. The development of newapproaches for the valorization of crustacean wastes, by the new functionalized biohydrogels, will improve the on-site wastewater treatmentin EU. The regeneration of new bio-based agents is also targeted.

Estimated Results

Consortium Results:

(i) 6 scientific ISI papers;

(ii) 2 patent applications;

(iii) 6 communications at prestigious Symposia ;

(iv) 3 organized workshops;

(v) 1 participation to Invention Salons;

(vi) project website.


STAGE I. Studies for obtaining raw chitosan and chitosan-based materials

PERIOD: 10.04.2020-31.12.2020

OBJECTIVES

Act 1.1 – Obtaining chitosan starting from commercial chitin and obtaining crude chitosan starting from chitin extracted from preconditioned waste of crustaceans. Results: Experimental model for obtaining chitosan.

Act 1.2– Studies on obtaining MIP pearls based on chitosan (via commercial chitin) for the treatment of antibiotics. Results: Concept for the MIP pearls.

Act 1.3 – Studies on the synthesis of antibacterial hydrogels based on chitosan (via commercial chitin) and quaternary ammonium salts. Results: Concept for the antibacterial hydrogel.

Act 1.4 – Characterization of raw materials, intermediates and final materials. Results: 3 Characterization reports.

Act 1.5 – Communicating the existing results of the project partners by organizing a kick-off meeting at the headquarters of the Project Coordinator. Results: Report of dissemination, communication and travel.

SUMMARY

The main objective of the research activity was to perform studies of obtaining crude chitosan and chitosan-based materials, starting from commercial chitin, and tracking the effects on the final properties. In this respect, the following secondary objectives have been established:

OS1. Obtaining chitosan from commercial chitin and obtaining crude chitosan from chitin extracted from preconditioned shellfish waste. Physico-chemical

characterization of raw materials, intermediates and final materials.

OS2. Studies on obtaining MIP beads based on chitosan (via commercial chitin) for antibiotic retention. Physico-chemical characterization of raw materials, intermediates and final materials.

OS3. Studies regarding the synthesis of antibacterial hydrogels based on chitosan (via commercial chitin) and quaternary ammonium salts. Physico-chemical and

bacteriological  characterization of raw materials, intermediates and final materials.

OS1. In order to obtain chitosan from commercial chitin, two synthesis methods were used in which the percentage of NaOH was varied. To obtain the chitosan, the deacetylation process involved the homogenization of chitin with NaOH in a 250 mL round-bottomed flask. The suspension was refluxed under stirring and the obtained chitosan was washed until the pH of the filtered product was neutral. Laboratory synthesized chitosan samples were compared with commercial chitosan (Ccom) of medium molecular weight, in which case some similarities were found in both FTIR and TGA analysis. In fact, the results were compared with a sample of commercial chitin that was used as raw material in the synthesis of chitosan.

However, the Micro-CT images and the degrees of deacetylation obtained from the NMR analysis for the synthesized chitosan were different compared to the commercial chitosan sample. The chitosan synthesized in the laboratory indicated a degree of deacetylation of over 86% compared to the commercial one of 75% and the

degree of compaction of the synthesized chitosan was lower, the proof being the Micro-CT analysis which indicated towards a material with a more increased intrinsic porosity than that of the commercial reference.

OS2. The study on obtaining MIP pearls based on chitosan allowed to obtain information regarding the production of hybrid pearls or granules with applications in the retention of antibiotics from wastewaters. In this regard, molecularly imprinted cryogels based on chitosan and biocellulose were obtained for penicillin retention. Thus, molecularly imprinted and non-imprinted cryogels based on chitosan were obtained (4 series of NIP/MIP pairs).

The study of swelling degrees showed that all cryogels adsorb a lot of water, but also the fact that some of them fragment due to low mechanical strength (gels being physically crosslinked with ammonium acid carbonate). UV-Vis analysis allowed to highlight the washing/extraction degree of penicillin for the obtained cryogels. In fact, it also proved that the MIP sample re-adsorbed 5.24 more specifically penicillin than the reference cryogel NIP.

Thermal analysis also showed that imprinting took place for all MIP series and highlighted differences in stability between the samples obtained with commercial chitosan vs the one synthesized in the laboratory. In the next stage, the method of obtaining cryogels will be optimized in order to

obtain more stable materials from a mechanical point of view.

OS3. Obtaining new polymer networks with interpenetrated structure consisted in the polymerization of a monomer, generator of quaternized ammonium groups, in the presence of chitosan (commercial or synthesized). Hydrogels based on commercial/synthesized chitosan were characterized in terms of swelling degree. The results showed that the type of chitosan influenced the degree of swelling for each hydrogel. For the hydrogels obtained using a smaller amount of crosslinker, differences were observed between the values of maximum swelling degrees. This showed that the use of a higher concentration of crosslinker led to much more rigid polymer networks which prevented the absorption of a larger amount of water.

Regarding the commercial/synthesized chitosan-based hydrogels, FTIR spectra showed the characteristic bands for chitosan. TGA/DTG analysis confirmed the presence of chitosan in the final structure of polymer networks.

Bactericidal tests reflected the potential of the synthesized materials, especially the chitosan-based hydrogel synthesized in the laboratory, to destroy both coliforms and clostridia in proportions of 83% and 69%, respectively.

DISSEMINATION

During stage 1/2020, a project kick-off meeting was organized with the physical participation of the project partners on 03.03.2020.

The Project Director participated in the BLUEBIO projects kick-off meeting (Online kick-off of the BlueBio ERA-NET COFUND 9 June 2020) and in a networking event (Connectivity among Blue Bioeconomy Cofund projects 20 November 2020).

An ISI indexed article was published and one was submitted for publication as follows:

1.Teodor Sandu, Maria Luiza Jecu, Iuliana Raut, Mariana Calin, Elvira Alexandrescu, Tanta Verona Iordache, Marinela Victoria Dumitru, Andrei Sarbu, Hybrid Beads Bearing Immobilized Bacteria As Advanced Means For The Removal Of Acid Blue 93 Dye, Materiale Plastice (Mater. Plast.),Year 2020, Volume 57, Issue 3.


2.Ana-Mihaela Gavrilă, Simona Nedelcu-Flor, Andrei Sârbu, Teodor Sandu, Andreea Gabriela Olaru, Gheorghe Hubca, Dan Donescu, Tanța-Verona Iordache, Synthesis And Properties Of Organosilica Particles With Quaternary Ammonium Bearings As Bacteriostatic Interfaces, Trimis spre publicare U.P.B. Sci. Bull., Series.

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.