Chitosan is formed from chitin deacetylation, but its insolubility remains challenging for industrial applications. An alternative would be employing Ionic Liquids (ILs) as a potential green solvent to dissolve chitosan. Hence, this research aims to study the optimum conditions of chitosan-[BMIM]Cl dissolution using Response Surface Methodology (RSM) and evaluate the ecotoxicity of chitosan-[BMIM]Cl mixture against Gram-positive and Gram-negative bacteria. Chitosan was obtained from heterogenous N-deacetylation of chitin using 50% sodium hydroxide solution at 100°C for 2.5 h. Chitosan dissolution in [BMIM]Cl was optimised using Central Composite Design (CCD) via RSM based on three independent factors: temperature, initial chitosan loading and dissolution time. Ecotoxicity of chitosan-[BMIM]Cl was evaluated using broth microdilution test against Escherichia coli and Staphylococcus aureus. Chitosan with a degree of deacetylation (DD) of 83.42% was obtained after three successive alkali treatments. Fourier Transform Infrared Spectroscopy (FTIR) revealed the presence of free hydroxyl groups, additional amino groups, and reduced C=O and C-H stretch intensity, indicating successful chitin deacetylation. The regression model for chitosan dissolution in [BMIM]Cl was significant (p < 0.05) with a non-significant lack of fit (p > 0.05). The optimised conditions to dissolve chitosan in [BMIM]Cl was 130°C, 1 wt. % and 72 h with a mean relative error of 1.78% and RMSE of 5.0496 wt. %. The toxicity of 10 wt. % chitosan-[BMIM]Cl mixture was “relatively harmless” (EC₅₀ > 1000 mg/L) with an EC₅₀ value of 3.1 wt. % for Escherichia coli and 3.2 wt. % for Staphylococcus aureus.

• Ahing, F. A., & Wid, N. (2016). Optimization of shrimp shell waste deacetylation for chitosan production. International Journal of Advanced and Applied Sciences, 3(10), 31-36. https://doi.org/10.21833/ijaas.2016.10.006

• Ahyat, N. M., Mohamad, F., Ahmad, A., & Azmi, A. A. (2017). Chitin and chitosan extraction from Portunus pelagicus. Malaysian Journal of Analytical Sciences, 21(4), 770-777. https://doi.org/10.17576/mjas-2017-2104-02

• Akakuru, O. U., Louis, H., Amos, P. I., Akakuru, O., Nosike, E. I., & Ogulewe, E. F. (2018). The chemistry of chitin and chitosan justifying their nanomedical utilities. Biochemistry & Pharmacology, 7, Article 1000241. https://doi.org/10.4172/2167-0501.1000241

• Ali, M. H., & Abustan, I. (2014). A new novel index for evaluating model performance. Journal of Natural Resources and Development, 4, 1-9. https://doi.org/10.5027/jnrd.v4i0.01

• Aranaz, I., Acosta, N., Civera, C., Elorza, B., Mingo, J., Castro, C., Gandia, M. D. I. L., & Caballero, A. H. (2018). Cosmetics and cosmeceutical applications of chitin, chitosan and their derivatives. Polymers, 10(2), Article 213. https://doi.org/10.3390/polym10020213

• Behera, S. K., Meena, H., Chakraborty, S., & Meikap, B. C. (2018). Application of response surface methodology (RSM) for optimization of leaching parameters for ash reduction from low-grade coal. International Journal of Mining Science and Technology, 28, 621-629. https://doi.org/10.1016/j.ijmst.2018.04.014

• Borkowski, A., Ławniczak, L., Cłapa, T., Narożna, D., Selwet, M., Pęziak, D., Markiewicz, B., & Chrzanowski, L. (2016). Different antibacterial activity of novel theophylline-based ionic liquids - Growth kinetic and cytotoxicity studies. Ecotoxicology and Environmental Safety, 130, 54-64. https://doi.org/10.1016/j.ecoenv.2016.04.004

• Brugnerotto, J., Lizardi, J., Goycoolea, F. M., Argüelles-Monal, W., Desbrières, J., & Rinaudo, M. (2001). An infrared investigation in relation with chitin and chitosan characterization. Polymer, 42(8), 3569-3580. https://doi.org/10.1016/S0032-3861(00)00713-8

• Bubalo, M., Radošević, K., Redovniković, L., Slivac, I., & Srček, V. (2017). Toxicity mechanisms of ionic liquids. Arhiv za Higijenu Rada I Toksikologiju, 68(3), 171-179. https://doi.org/10.1515/aiht-2017-68-2979

• Chen, Q., Xu, A., Li, Z., Wang, J., & Zhang, S. (2011). Influence of anionic structure on the dissolution of chitosan in 1-butyl-3-methylimidazolium-based ionic liquids. Green Chemistry, 13(12), 3446-3452. https://doi.org/10.1039/C1GC15703E

• Chowdhury, Z., Zain, S. M., Hamid, S. B. A., & Khalid, K. (2014). Catalytic role of ionic liquids for dissolution and degradation of biomacromolecules. Bioresources, 9(1), 1787-1823. https://doi.org/10.15376/BIORES.9.1.1787-1823

• Claros, M., Graber, T. A., Brito, I., Albanez, J., & Gavin, J. A. (2010). Synthesis and thermal properties of two new dicationic ionic liquids. Journal of the Chilean Chemical Society, 55(3), 396-398. https://doi.org/10.4067/S0717-97072010000300027

• Csonka, L. N. (1989). Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews, 53, 121-147. https://doi.org/10.1128/mr.53.1.121-147.1989

• Czechowska-Biskup, R., Jarosińska, D., Rokita, B., Ulański, P., & Rosiak, J. (2012). Determination of degree of deacetylation of chitosan - Comparison of methods. Progress on Chemistry and Application of Chitin and its Derivatives, 2012, 5-20.

• da Costa Lopes, A. M., Lins, R. M. G., Rebelo, R. A., & Łukasik, R. M. (2018). Biorefinery approach for lignocellulosic biomass valorisation with acidic ionic liquid. Green Chemistry, 20(17), 4043-4057. https://doi.org/10.1039/C8GC01763H

• Dennis, G., Harrison, W., Agnes., K., & Erastus, G. (2016). Effect of biological control antagonists adsorbed on chitosan immobilized silica nanocomposite on Ralstonia solanacearum and growth of tomato seedlings. Advances in Research, 6(1), 1-23. https://doi.org/10.9734/AIR/2016/22742

• Docherty, K. M. (2005). Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids. Green Chemistry, 7, 185-189. https://doi.org/10.1039/B419172B

• Egorova, K. S., & Ananikov, V. P. (2014). Toxicity of ionic liquids: Eco(cyto)activity as complicated, but unavoidable parameter for task-specific optimization. Chemistry-Sustainability-Energy-Materials, 7(2), 336-360. https://doi.org/10.1002/cssc.201300459

• El Knidri, H., El Khalfaouy, R., Laajeb, A., & Lahsini, A. (2016). Eco-friendly extraction and characterization of chitin and chitosan from the shrimp shell waste via microwave irradiation. Process Safety and Environmental Protection, 104, 395-405. https://doi.org/10.1016/j.psep.2016.09.020

• Elieh-Ali-Komi, D., & Hamblin, M. R. (2016). Chitin and chitosan: Production and application of versatile biomedical nanomaterials. International Journal of Advanced Research, 4(3), 411-427. https://pubmed.ncbi.nlm.nih.gov/27819009

• Fatima, B. (2020). Quantitative analysis by IR: Determination of chitin/chitosan DD. In M. Khan (Ed.), Modern Spectroscopic Techniques and Applications (pp. 1-24). IntechOpen. https://doi.org/10.5772/intechopen.89708

• Forsyth, S. A., MacFarlane, D. R., Thomsom, R. J., & Itzstein, M. V. (2002). Rapid, clean and mild o-acetylation of alcohols and carbohydrates in an ionic liquid. Chemical Communications, 7(7), 714-715. https://doi.org/10.1039/b200306f

• Ghanem, O. B., Mutalib, M. I. A., El-Harbawi, M., Gonfa, G., Chong, F. K., Alitheen, N. B. M., & Lévêque, J. M. (2015). Effect of imidazolium-based ionic liquids on bacterial growth inhibition investigated via experimental and QSAR modelling studies. Journal of Hazardous Materials, 297, 198-206. https://doi.org/10.1016/j.jhazmat.2015.04.082

• Gschwend, F. J. V., Chambon, C. L., Biedka, M., Brandt-Talbot, A., Fennell, P. S., & Hallett, J. P. (2019). Quantitative glucose release from softwood after pretreatment with low-cost ionic liquids. Green Chemistry, 21, 692-703. https://doi.org/10.1039/C8GC02155D

• IEA Bioenergy. (2020). Annual Report 2019 IEA Bioenergy. https://www.ieabioenergy.com/wp-content/uploads/2020/05/IEA-Bioenergy-Annual-Report-2019.pdf

• Islam, S., Arnold, L., & Padhye, R. (2015). Comparison and characterization of regenerated chitosan from 1-butyl-3-methylimidazolium chloride and chitosan from crab shells. BioMed Research International, 2015, Article 874316. https://doi.org/10.1155/2015/874316

• Kusuma, H. S., Agasi, H., & Darmokoesoemo, H. (2015). Effectiveness inhibition of fermentation legen using chitosan nanoparticles. Journal of Molecular and Genetic Medicine, 9(3), Article 1000173. https://doi.org/10.4172/1747-0862.1000173

• Lei, Z., Chen, B., Koo, Y. M., & MacFarlane, D. R. (2017). Introduction: Ionic liquids. Chemical Reviews, 117(10), 6633-6635. https://doi.org/10.1021/acs.chemrev.7b00246

• Liu, L., Wang, Y., Xie, H., Zhang, B., & Zhang, B. (2022). Enhancing the antioxidant ability of Momordica grosvenorii saponin to resist gastrointestinal stresses via microcapsules of sodium alginate and chitosan and its application in beverage. Beverages, 8(4), Article 70. https://doi.org/10.3390/beverages8040070

• Ma, Q., Gao, X., Bi, Z., Han, Q., Tu, L., Yang, Y., Shen, Y., & Wang, M. (2019). Dissolution and deacetylation of chitin in ionic liquid tetrabutylammonium hydroxide and its cascade reaction in enzyme treatment for chitin recycling. Carbohydrate Polymers, 230, Article 115605. https://doi.org/10.1016/j.carbpol.2019.115605

• Manzanares, P. (2020). The role of biorefining research in the development of a modern bioeconomy. Acta Innovations, 37, 47-56. https://doi.org.10.32933/ActaInnovations.37.4

• Matute, A. I. R., Cardelle-Cobas, A., García-Bermejo, A. B., Montilla, A., Olano, A., & Corzo, N. (2013). Synthesis, characterization and functional properties of galactosylated derivatives of chitosan through amide formation. Food Hydrocolloids, 33, 245-255. https://doi.org/10.1016/j.foodhyd.2013.03.016

• Megaw, J., Busetti, A., & Gilmore, B. F. (2013). Isolation and characterization of 1-alkyl-3-methylimidazolium chloride ionic liquid-tolerant and biodegrading marine bacteria. PLoS One, 8(4), Article e60806. https://doi.org/10.1371/journal.pone.0060806

• Mester, P., Wagner, M., & Rossmanith, P. (2015). Antimicrobial effects of short chained imidazolium-based ionic liquids - Influence of anion chaotropicity. Ecotoxicology and Environmental Safety, 111, 96-101. https://doi.org/10.1016/j.ecoenv.2014.08.032

• Morin-Crini, N., Lichtfouse, E., Torri, G., & Crini, G. (2019). Applications of chitosan in food, pharmaceuticals, medicine, cosmetics, agriculture, textiles, pulp and paper, biotechnology, and environmental chemistry. Environmental Chemistry Letters, 17, 1667-1692. https://doi.org/10.1006/s10311-019-00904-x

• Naseeruteen, F., Hamid, N. S. A., Suah, F. B. M., Ngah, W. S. W., & Mehamod, F. S. (2018). Adsorption of malachite green from aqueous solution by using novel chitosan ionic liquid beads. International Journal of Biological Macromolecules, 107, 1270-1277. https://doi.org/10.1016/j.ijbiomac.2017.09.111

• Palpandi, C., Shanmugam, V., & Shanmugam, A. (2009). Extraction of chitin and chitosan from shell and operculum of mangrove gastropod Nerita (Dostia) crepidularia Lamarck. International Journal of Medical Sciences, 1(5), 198-205.

• Passino, D. R. M., & Smith, S. B. (1987). Acute bioassays and hazard evaluation of representative contaminants detected in great lakes fish. Environmental Toxicology and Chemistry, 6(11), 901-907. https://doi.org/10.1002/etc.5620061111

• Peric, B., Sierra, J., Martí, E., Cruañas, R., Garau, M. A., Arning, J., Bottin-Weber, U., & Stolte, S. (2013). (Eco)toxicity and biodegradability of selected protic and aprotic ionic liquids. Journal of Hazardous Materials, 261, 99-105. https://doi.org/10.1016/j.jhazmat.2013.06.070

• Poerio, A., Girardet, T., Petit, C., Eleutot, S., Jehl, J., Arab-Tehrany, E., Mano, J. F., & Cleymand, F. (2021). Comparison of the physicochemical properties of chitin extracted from Cicada orni sloughs harvested in three different years and characterization of the resulting chitosan. Applied Sciences, 11, Article 11278. https://doi.org/10.3390/app112311278

• Puspawati, N. M., & Simpen, I. N. (2010). Optimasi deasetilasi khitin dari kulit udang dan cangkang kepiting limbah restoran seafood menjadi khitosan melalui konsentrasi NaOH [Optimization of deacetylation of chitin from seafood restaurant waste shrimp skin and crab shell into chitosan through NaOH concentration]. Jurnal Kimia, 4, 79-90. https://ojs.unud.ac.id/index.php/jchem/article/view/2760

• Qi, L., Xu, Z., Jiang, X., Hu, C., & Zou, X. (2004). Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate Research, 339(16), 2693-2700. https://doi.org/10.1016/j.carres.2004.09.007

• Rahim, A. H. A., Yunus, N. M., Man, Z., Sarwono, A., Hamzah, W. S. W., & Wilfred, C. (2018). Ultrasonic assisted dissolution of bamboo biomass using ether-functionalized ionic liquid. AIP Conference Proceedings, 2016, Article 020010. https://doi.org/10.1063/1.5055412

• Rinaudo, M. (2006). Chitin and chitosan: Properties and application. Progress in Polymer Science, 31(7), 603-632. https://doi.org/10.1016/j.progpolymsci.2006.06.001

• Rodríguez, H. (2021). Ionic liquids in the pretreatment of lignocellulosic biomass. Acta Innovations, 38, 23-26. https://doi.org/10..32933/ActaInnovations.38.3

• Roller, S., & Covill, N. (1999). The antifungal properties of chitosan in laboratory media and apple juice. International Journal of Food Microbiology, 47(1-2), 67-77. https://doi.org/10.1016/S0168-1605(99)00006-9

• Romanazzi, G., Gabler, F. M., Margosan, D., Mackey, B. E., & Smilnick, J. L. (2009). Effect of chitosan dissolved in different acids on its ability to control postharvest gray mold of table grape. Phytopathology, 99(9), 1028-1036. https://doi.org/10.1094/PHYTO-99-9-1028

• Rosatella, A. A., Branco, L. C., & Afonso, C. A. M. (2009). Studies on dissolution of carbohydrates in ionic liquids and extraction from aqueous phase. Green Chemistry, 11, 1406-1413. https://doi.org/10.1039/b900678h

• Rumenagan, I., Suryanto, E., Modaso, R., Wullur, S., Tallei, T., & Limbong, D. (2014). Structural characteristics of chitin and chitosan isolated from the biomass of cultivated rotifer, Brachionus rotundiformis. International Journal of Fisheries and Aquatic Sciences, 3, 12-18.

• Santos, E., Rodríguez-Fernández, E., Casado-Coterillo, C., & Irabien, A. (2016). Hybrid ionic liquid-chitosan membranes for CO2 separation: Mechanical and thermal behavior. International Journal of Chemical Reactor Engineering, 14(3), 713-718. https://doi.org/10.1515/ijcre-2014-0109

• Schmitz, C., Auza, L. G., Koberidze, D., Rache, S., Fischer, R., & Bortesi, L. (2019). Conversion of chitin to defined chitosan oligomers: current status and future prospects. Marine Drugs, 17(8), Article 452. https://doi.org/10.3390/md17080452

• Sivapragasam, M., Jaganathan, J. R., Levêque, J., Moniruzzaman, M., & Mutalib, M. I. A. (2019). Microbial biocompatibility of phosphonium- and ammonium-based ionic liquids. Journal of Molecular Liquids, 273, 107-115. https://doi.org/10.1016/j.molliq.2018.10.022

• Sivapragasam, M., Moniruzzaman, M., & Goto, M. (2020). An overview on the toxicological properties of ionic liquids towards microorganisms. Biotechnology Journal, 15(4), Article e1900073. https://doi.org/10.1002/biot.201900073

• Stepnowski, P., Składanowski, A. C., Ludwiczak, A., & Laczyńska, E. (2004). Evaluating the cytotoxicity of ionic liquids using human cell line HeLa. Human & Experimental Toxicology, 23, 513-517. https://doi.org/10.1191/0960327104ht480oa

• Sun, N., Rahman, M., Qin, Y., Maxim, M. L., Rodríguez, H., & Rogers, R. D. (2009). Complete dissolution and partial delignification of wood in the ionic liquid 1-ethyl-3-methylimidazolium acetate. Green Chemistry, 11, 646-655. https://doi.org/10.1039/B822702K

• Sun, X., Tian, Q., Xue, Z., Zhang, Y., & Mu, T. (2014). The dissolution behaviour of chitosan in acetate-based ionic liquids and their interactions: From experimental evidence to density functional theory analysis. RSC Advances, 4, 30282-30291. https://doi.org/10.1039/c4ra02594f

• Sun, X., Xue, Z., & Mu, T. (2014). Precipitation of chitosan from ionic liquid solution by the compressed CO2 anti-solvent method. Green Chemistry, 16, 2102-2106. https://doi.org/10.1039/C3GC42166J

• Swatloski, R. P., Spear, S. K., Holbrey, J. D., & Rogers, R. D. (2002). Dissolution of cellulose with ionic liquids. Journal of the American Chemical Society, 124(18), 4974-4975. https://doi.org/10.1021/ja025790m

• Tamzi, N. N., Faisal, M., Sultana, T., & Ghosh, S. K. (2020). Extraction and properties evaluation of chitin and chitosan prepared from different crustacean waste. Bangladesh Journal of Veterinary and Animal Sciences, 8(2), 69-76.

• Tan, H. T., & Lee, K. (2012). Understanding the impact of ionic liquid pretreatment on biomass and enzymatic hydrolysis. Chemical Engineering Journal, 183, 448-458. https://doi.org/10.1016/j.cej.2011.12.086

• Thomas, P. A., & Marvey, B. B. (2016). Room temperature ionic liquids as green solvent alternatives in the metathesis of oleochemical feedstocks. Molecules, 21(2), Article 184. https://doi.org/10.3390/molecules21020184

• Wang, W. T., Zhu, J., Wang, X. L., Huang, Y., & Wang, Y. Z. (2010). Dissolution behavior of chitin in ionic liquids. Journal of Macromolecular Science, Part B, 49(3), 528-541. https://doi.org/10.1080/00222341003595634

• Wang, X., Li, H., Cao, Y., & Tang, Q. (2011). Cellulose extraction from wood chip in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Bioresource Technology, 102, 7959-7965. https://doi.org/10.1016/j.biortech.2011.05.064

• Weyhing-Zerrer, N., Gundolf, T., Kalb, R., Oßmer, R., Rossmanith, P., & Mester, P. (2017). Predictability of ionic liquid toxicity from a SAR study on different systematic levels of pathogenic bacteria. Ecotoxicology and Environmental Safety, 139, 394-403. https://doi.org/10.1016/j/ecoenv.2017.01.055

• Wu, J., Zhang, J., Zhang, H., He, J., Ren, Q., & Guo, M. (2004). Homogenous acetylation of cellulose in a new ionic liquid. Biomacromolecules, 5(2), 266-268. https://doi.org/10.1021/bm034398d

• Wu, Y., Sasaki, T., Irie, S., & Sakurai, K. (2008). A novel biomass-ionic liquid platform for the utilization of native chitin. Polymer, 49, 2321-2327. https://doi.org/10.1016/j.polymer.2008.03.027

• Xie, H., Zhang, S., & Li, S. (2006). Chitin and chitosan dissolved in ionic liquids as reversible sorbents of CO2. Green Chemistry, 8(7), 630-633. https://doi.org/10.1039/B517297G

• Xu, B., Li, Q., Zhuang, L., Wang, Q., Li, C., Wang, G., Xie, F., & Halley, P. J. (2016). Dissolution and regeneration behavior of chitosan in 3-methyl-1-(ethylacetyl)imidazolium chloride. Fibers and Polymer, 17(11), 1741-1748. https://doi.org/10.1007/s12221-016-6747-6

• Yang, X., Qian, C., Li, Y., & Li, T. (2016). Dissolution and resourcfulization of biopolymers in ionic liquids. Reactive and Functional Polymers, 100, 181-190. https://doi.org/10.1016/j.reactfunctpolym.2016.01.017

• Zakrzewska, M. E., Bogel-Łukasik, E., & Bogel-Łukasik, R. (2010). Solubility of carbohydrates in ionic liquids. Energy Fuels, 24, 737-745. https://doi.org/10.1021/ef901215m

• Zhang, C., Shao, Y., Zhu, L., Wang, J., Wang, J., & Guo Y. (2017). Acute toxicity, biochemical toxicity and genotoxicity caused by 1-butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium tetrafluoroborate in zebrafish (Danio rerio) livers. Environmental Toxicology and Pharmacology, 51, 131-137. https://doi.org.10.1016/j-etap.2017.02.018

• Zhang, H., Wu, J., Zhang, J., & He., J. (2005). 1-allyl-3-methylimidazolium chloride room temperature ionic liquid: A new and powerful nonderivatizing solvent for cellulose. Macromolecules, 38(20), 8272-8277. https://doi.org/10.1021/ma0505676

• Zhang, M., Zhang, F., Li, C., An, H., Wan, T., & Zhang, P. (2022). Application of chitosan and its derivative polymers in clinical medicine and agriculture. Polymers, 14, Article 958. https://doi.org/10.3390/polym14050958

• Zhang, Y., Liu, Y, Chu, Z., Shi, L., & Jin, W. (2013). Amperometric glucose biosensor based on direct assembly of prussian blue film with ionic liquid-chitosan matrix assisted enzyme immobilization. Sensors and Actuators B: Chemical, 176, 978-984. https://doi.org/10.1016/j.snb.2012.09.080

• Zhao, D., Yu, S., Sun, B., Gao, S., Guo, S., & Zhao, K. (2018). Biomedical applications of chitosan and its derivative nanoparticles. Polymers, 10(4), Article 462. https://doi.org/10.3390/polym10040462

• Zhu, S., Yu, P., Lei, M., Tong, Y., Zheng, L., Zhang, R., Ji, J., Chen, Q., & Wu, Y. (2013). Investigation of the toxicity of the ionic liquid 1-butyl-3-methylimidazolium chloride to Saccharomyces cerevisiae AY93161 for lignocellulosic ethanol production. Polish Journal of Chemical Technology, 15(2), 94-98. https://doi.org/10.2478/pjct-2013-0029

• Zhuang, L., Zhong, F., Qin, M., Sun, Y., Tan, X., Zhang, H., Kong, M., Hu, K., & Wang, G. (2020). Theoretical and experimental studies of ionic liquid-urea mixtures on chitosan dissolution: Effect of cationic structure. Journal of Molecular Liquids, 317, Article 113918. https://doi.org/10.1016/j.molliq.2020.113918