Cellulose nanocrystal (CNC) has attracted significant attention due to its attractive properties. The aim of this research was to evaluate the properties of CNC/chitosan composite film with the addition of glutaraldehyde as crosslinker under different curing conditions. CNC has been successfully isolated from oil palm empty fruit bunch (EFB) using sulphuric acid hydrolysis preceded by alkaline deep eutectic solvent (DES) pre-treatment and bleaching. The processing parameters of acid hydrolysis were optimized using Box-Behnken Design. The results showed that the yield of CNC was 37.1%, under the optimal conditions of 60.0 wt% acid concentration at 46.1 °C for 58.5 minutes. Fourier transform infrared (FTIR) spectroscopy indicated the efficient elimination of unwanted impurities from the raw EFB fibers by pre-treatment. The average diameter and length of CNC were 6.78 ± 2.12 nm and 160.06 ± 32.58 nm, respectively. The raw EFB fibre, treated cellulose and CNC showed crystallinities of 38.7%, 51.2%, and 65.3%, respectively. The CNC had lower thermal stability, which was ascribed to the sulphate group present on the CNC surface. The addition of CNC and GA as crosslinker into chitosan composite films was studied. Crosslinking of chitosan composite films was carried out using different crosslinking methods (conventional heating and microwave curing). Tensile strength and modulus elasticity of chitosan composites were enhanced significantly by the addition of CNC and GA especially for microwave-cured samples. The changes in chemical interaction of the chitosan composite films were investigated by FTIR. The microwave-cured-GA-crosslinked chitosan composite films were more thermally-stable than non-crosslinked and conventionally-heated-GA-crosslinked chitosan composite films due to the formation of a more stable network structure between GA and chitosan matrix. Furthermore, the functional properties such as swelling, water solubility and water vapor permeability of composite films were reduced significantly by the addition of CNC and GA. However, the reduced antimicrobial efficacy of composite films against Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae was observed in crosslinked composite films. The crosslinked chitosan composite films were also found to have a lower degradation rate than the non-crosslinked composites. The degrading-bacteria were identified as Enterobacter kobei and E. roggenkampii whereas the degrading-fungi was Ophiocordyceps heteropoda.
