Exploring the potential of seaweed derivatives for the development of biodegradable plastics: a comparative study

Citation: Wan Yahaya, W.A.; Azman, N.A.M.; Adam, F.; Subramaniam, S.D.; Abd Hamid, K.H.; Almajano, M.P. Exploring the Potential of Seaweed Derivatives for the Development of Biodegradable Plastics: A Comparative Study. Polymers 2023,15, 2884. https:// doi.org/10.3390/polym15132884 Academic Editor: Dimitrios Bikiaris Received: 29 March 2023 Revised: 16 June 2023 Accepted: 27 June 2023 Published: 29 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). polymers Article Exploring the Potential of Seaweed Derivatives for the Development of Biodegradable Plastics: A Comparative Study Wan Amnin Wan Yahaya 1, Nurul Aini Mohd Azman 1,2,* , Fatmawati Adam 1,2, Sarmilaah Dewi Subramaniam 1, Khadijah Husna Abd Hamid 1and Maria Pilar Almajano 3,* 1 Faculty of Chemical and Process Engineering Technology, Universiti Malaysia Pahang, Lebuhraya Persiaran Tun Khalil Yaakob, Gambang 26300, Pahang, Malaysia; [email protected] (W.A.W.Y.) 2Centre for Research in Advanced Fluid and Processes, Lebuhraya Persiaran Tun Khalil Yaakob, Gambang 26300, Pahang, Malaysia 3 Chemical Engineering Department (DEQ), Escola Tècnica Superior d’Enginyeria Industrial de Barcelona (ETSEIB), Universitat Politècnica de Catalunya (UPC), Av, Diagonal 647, 08028 Barcelona, Spain *Correspondence: [email protected] (N.A.M.A.); m.pilar[email protected] (M.P.A.) Abstract: Biodegradable films made from biopolymer materials have the potential to replace conventional plastics, which can reduce waste disposal problems. This study aims to explore the potential of different seaweed derivate films consisting of 2% (w/w) of kappaphycus alverezi (KA), kappa carrageenan (KC), refined carrageenan (RC) and semi-refined carrageenan (SRC) as bio-based materials with 0.9% (w/w) glycerol (G), and reinforced with different concentrations of cellulose nanofibers (CNFs) derived from palm waste. A characterization of the glycerol-plasticized seaweed derivatives containing 0, 5, 10, and 15% (v/w) cellulose nanofiber is carried out. The CNFs were studied based on their mechanical, physical and thermal properties including mechanical properties, thickness, moisture content, opacity, water solubility, water vapor permeability and thermal stability. The hydrogen bonding was determined using the DFT calculation generated by Gauss view software version 9.6. The KA + G + 10%CNF film exhibited a surface with slight cracks, roughness, and larger lumps and dents, resulting in inferior mechanical properties (18.50 Mpa), making it unsuitable for biofilm production. The KC + G + 10%CNF film exhibited mechanical properties 24.97 Mpa and water vapor permeability of 1.42311 × 10 −11 g s −1 m −1 Pa −1 . The RC/G/10%CNF film displayed the highest TS (48.23 MPa) and water vapor permeability (1.4168 × 10 −11 g s −1 m −1 Pa −1 ), but it also had higher solubility in water (66%). In contrast, the SRC + G + 10%CNF film demonstrated excellent mechanical properties (45.98 MPa), low water solubility (42.59%), low water vapor permeability (1.3719 × 10 −11 g s −1 m −1 Pa −1 ), and a high decomposition temperature (250.62 ◦ C) compared to KA, KC and RC. These attributes develop films suitable for various applications, including food packaging with enhanced properties and stability. Keywords: seaweed derivatives; plasticizers; cellulose nanofibers; biopolymer; Gaussian 1. Introduction Petroleum-based polymers such as polyethylene, polypropylene, and polystyrene, are widely used in the manufacture of commercial packaging materials such as plastic bags, bottles, and food containers [ 1 ]. Non-degradable materials often end up in landfills, oceans and other natural environments. It can take hundreds or even thousands of years for these materials to degrade, resulting in continued pollution of the environment and harm to wildlife. Therefore, biomass-based packaging materials derived from renewable resources, agricultural wastes, and agro-industrial byproducts have received attention due to their advantages such as low price, easy availability, and environmental friendliness compared to synthetic products [ 2 ]. Of the biodegradable polymers, polysaccharides derived from natural monomers offer the greatest opportunity to become alternative packaging materials due to their potential biodegradability and environmental compatibility [ 3 ]. Among Polymers 2023,15, 2884. https://doi.org/10.3390/polym15132884 https://www.mdpi.com/journal/polymers Polymers 2023,15, 2884 2 of 18 biopolymers, seaweed-based biopolymers such as alginate, agar, and carrageenan have received great interest due to their good barrier properties and mechanical properties. Seaweed derivatives are mostly used in the food industry as thickeners, gelling agents and stabilizers in concentrations ranging from 0.005% to 2.0% (w/w). Seaweed derivatives, such as Kappaphycus alvarezii (KA), kappa carrageenan (KC), refined-carrageenan (RC), and semi-refined-carrageenan (SRC), are used as biopolymers in the production of biodegradable films. These seaweeds are known for their high content of carrageenan, a polysaccharide that forms a gel-like substance when dissolved in water. KA, also known by the trade name cotonii, is a class of Rhodophyceae [ 4 ]. This species of algae occurs in reddish, yellowish and green colors, depending on the concentration of phycoerythrin pigment. It is easy to cultivate and grows rapidly, with an increase of almost 4.5% daily. KA is the main industrial source of κ -carrageenan [ 4 , 5 ], as this polysaccharide accounts for up to 37 wt% of the alga, on a dry weight basis. Due to the properties of κ - carrageenan, such as thickening, gelling, stabilization and emulsification, it has a variety of applications, such as a thickening agent for milk-based desserts. In general, the KA consists on average of 50.8% carbohydrates, 3.3% lipids, 3.3% proteins, 12.4% sulfated groups, 15.6% ash and 3.0% insoluble aromatics [ 6 , 7 ]. Meanwhile, semi-refined carrageenan (SRC) is the end product of KC processing, which involves alkali treatment to remove the carrageenan. Remaining components, such as cellulosic materials, can be removed using additional processes such filtration and purification, resulting in refined carrageenan (RC) [8]. SRC’s global pricing is often only two-thirds that of traditional RC because there are fewer processing stages involved. The cosmetics and pharmaceutical industries employ refined carrageenan extensively, whereas SRC is mostly used in other applications, such food packaging, that do not require high refinement. However, they face some problems due to their natural hydrophilicity, which frequently needs to be modified by grafting/blending with other polymers or by adding fillers to improve their competitiveness with standard polymers [9,10]. Oil palm biomass (OPB), which is made up of empty fruit bunches (EFBs), was abundantly generated in Malaysia and can be used as a source of cellulose for the production of nanocellulose where only 10% is used, and it is frequently left inexhaustible. These OPBs offer enormous resources for the translation into value-added products based on scientific results [ 11 ]. Nanocellulose is produced by breaking down cellulose fibers into nano-sized particles, which can be used to reinforce polymer composites and improve their mechanical properties. The extraction process of nanocellulose involves the removal of hemicellulose and lignin from wood pulp, followed by mechanical or chemical treatment to obtain the desired particle size (<100 nm). Dai et al. [ 12 ] has demonstrated the flexibility of hydroxypropyl guar/cellulose-nanofibrils films using a casting technique. In comparison to the control film, the composite films have better mechanical properties and higher oxygen and water vapor barriers. The manufacturing technology of biodegradable films involves mixing the biopolymers with other components, such as glycerol and nanocellulose, to form a composite material. The composite is then molded into thin films using techniques such as extrusion or casting. The economic market value of biodegradable films made from seaweed derivatives and nanocellulose is growing rapidly, as consumers and manufacturers become more aware of the environmental impact of conventional plastics. Grand View Research’s analysis estimates that the global market for biodegradable plastics was worth USD 3.39 billion in 2020 and is projected to increase at a CAGR of 14.5% from 2021 to 2028. The increasing demand for sustainable packaging solutions, particularly in the food and beverage industry, is driving the growth of the biodegradable plastics market. Therefore, further studies were conducted by adding nano-fillers as reinforcing agents to meet the requirements of packaging applications. This study aims to explore the potential of these four comparison materials from seaweed derivatives such as KA (pure seaweed without the extraction process), KC (pure carrageenan), RC and SRC with natural plasticizers from glycerol, reinforced with different concentrations of cellulose nanofibers (0,5,10 and 15% v/v). The films produced were characterized based on mechanical, physical, and thermal Polymers 2023,15, 2884 3 of 18 properties and its morphology to develop a potential bio-nanocomposite film. Overall, this work has significant implications for the development of sustainable materials that can help reduce plastic waste and mitigate its environmental impact. 2. Materials and Methods 2.1. Materials KA, KC, RC and SRC range < 200 µ m powder size were obtained from TACARA Sdn., Bhd., and cellulose nanofiber (CNF) was purchased from UPM Biomass Centre, Malaysia. Glycerol (99%), hydrochloric acid (HCl), potassium chloride (KCl), and potassium oxide (KOH) were supplied by Sigma-Aldrich, Gillingham, England. 2.2. Preparation of Kappaphycus Alvarezii (KA) The dried seaweed was pre-treated by removing visible foreign matters such as sand and stones. The seaweeds were further washed with running deionized water for 5 min to reduce salt content. Then, the preheated seaweed was dried in an oven at 60 ◦ C until a constant weight, to fully remove the moisture content. The preheated seaweeds were grounded for 400 rpm using a Retsch ball mill grinder and sieved as powder <200 µm. 2.3. Extraction of κappa Carrageenan (KC) The extraction of κ appa-carrageenan was prepared according to Manuhara et al. [ 13 ]. The dried seaweed was washed with tap water and soaked in water for 24 h. After soaking, it were cut and crushed with a blender to prepare an algal slurry. Then the slurry was mixed with water and conditioned in an alkaline solution, which was heated at 90 ◦ C for 2 h with constant stirring. After extraction, the residue was separated from the viscous filtrate. It was coagulated with a KCl solution for 15 min and stirred, and then the mixture was filtered to separate the water and carrageenan gel. The gel was completely soaked in 96% alcohol for one hour with continuous stirring. It was separated from the alcohol and water by filtration. Finally, it was dried at 70 ◦C for 24 h and ground into powder. 2.4. Extraction of Refined Carrageenan (RC) Kappaphycus alvarezii was submerged in 3 L of water for 24 h after being rinsed with flowing water. Then, the algae were cut with scissors and ground with a blender to produce the algal pulp. The water and pulp were then combined in a ratio of 1:80 (v/v). Ca(OH) 2 solution was used to condition the mixture into an alkaline state (pH 9). Then, the extraction was carried out by heating at 90 ◦ C for two hours while stirring continuously. Following extraction, solid algal waste was isolated from the filtrate. The filtrate was then warmed at 60 ◦ C for 30 min after being neutralized with a 1% HCl solution to a pH of 7. The mixture was filtered to separate the carrageenan gel and water after the filtrate had been coagulated with KCl solution (1.5%, 2.5%, or 3.5%) and KCl solution in a 1:1 ratio for 15 min. After that, the carrageenan gel was thoroughly dissolved in 96% alcohol and swirled constantly for an hour. Filtration was used to separate the carrageenan gel from the alcohol and water. The carrageenan was milled into an 80-mesh size after being dried in a cabinet drier at 70 ◦C for 24 h [6]. 2.5. Extraction of Semi-Refined Carregeenan (SRC) Semi-refined carrageenan was prepared according to the Normah and Nazarifah method [ 14 ], with slight modifications. Kappaphycus alvarezii were washed under running tap water to remove residues, sand and salt. Then, 200 g of algae were extracted in 2.5 L of hot alkaline solution containing about 150 g of potassium hydroxide solution with an alkaline pH of 13 at a temperature of 70 ◦ C for 2 h. Then, the algae were neutralized by soaking in water for 4 h followed by overnight soaking. The neutralized algae were dried overnight at a temperature of 50 ◦ C. Finally, the dried SRC were ground and stored in a desiccator for further analysis. Polymers 2023,15, 2884 4 of 18 2.6. Preparation of Bio-Nanocomposite Films The seaweed derivative powder was mix in distilled water (2% w/w, based on the dry weight) and heated to a temperature of 80 ◦ C with constant stirring. Then, the plasticizer glycerol (0.9% v/v) was included at 80 ◦ C under magnetic stirring. The temperature was maintained at 800 rpm for 10 min with continuous stirring to achieve gelation. After the addition of glycerol, the film solution was heated to 90 ◦ C and the temperature of the film solution was maintained at 90 ◦ C for ± 1 min. Then, the cellulose suspension was added at different concentrations (0, 5, 10, and 15% v/v). The control film was prepared without the addition of plasticizers. A total of 100 mL of the film-forming solution (FFS) was poured onto a polyacrylic casting plate (16 cm × 16 cm × 0.3 cm) and dried for 1 day at a temperature of 40 ± 2 ◦ C. The dried film was eventually removed from the plates. Table 1 shows the different concentrations of the film sample. Table 1. The different concentrations of film sample. Sample Seaweed Derivatives Glycerol Cellulose Nanofiber KA KA 0 0 KA + G KA 0.9 0 KA + G + 5%CNF KA 0.9 5 KA + G + 10%CNF KA 0.9 10 KA + G + 15%CNF KA 0.9 15 KC KC 0 0 KC + G KC 0.9 0 KC + G + 5%CNF KC 0.9 5 KC + G + 10%CNF KC 0.9 10 KC + G + 15%CNF KC 0.9 15 RC RC 0 0 RC + G RC 0.9 0 RC + G + 5%CNF RC 0.9 5 RC + G + 10%CNF RC 0.9 10 RC + G + 15%CNF RC 0.9 15 SRC SRC 0 0 SRC + G SRC 0.9 0 SRC + G + 5%CNF SRC 0.9 5 SRC + G + 10%CNF SRC 0.9 10 SRC + G + 15%CNF SRC 0.9 15 KA: kappaphacus Alvarezii KC: Kappa carrageenan RC: refined carrageenan SRC: semi-refined carrageenan. 2.7. Tensile Strength (TS) and Elongation at Break (EAB) The TS and EAB of the films were evaluated using a testing machine (AG-Xplus Series, Shimadzu, Japan). Film samples were cut into 1.5 cm × 10 cm. Then, the samples were tested using the testing machine and measured with a deformation rate of 50 mm/min [ 15 ]. The tested film were equilibrated in desiccators at 25 ◦ C and 50% relative humidity for 48 h. The TS and EAB of the film samples were measured according to the standard method ASTM D882-12. The TS and EAB value of the films were calculated using the following equation: Ts(Mpa)=Fmax ∅(1) where F max is the maximum load and Φ is the cross-sectional area of the film. The EAB of the films will be calculated using the following equation: EAB(%)=∆l lo ×100 (2) where ∆l is the film extension and l0is the initial length of the film sample. Polymers 2023,15, 2884 5 of 18 2.8. Thickness Measurement A hand-held digital micrometer (Mitutoyo Co., Tokyo, Japan) was used to measure the thickness of the film samples. The mean values of five measurements were calculated at random positions of the film samples. The mean value of thickness was used for the opacity calculation and mechanical properties [16]. 2.9. Opacity Measurement The opacity of the film was measured by using a spectrophotometer. The film was cut into 3 cm × 0.3 cm and put into the cuvette, according to the ASTM D523-08 method proposed by Shojaee-Aliabadi et al. [ 17 ] The opacity was examined at 600 nm using UV–Vis. The opacity was calculated based on the following equation: Opacity =Abs600 b(3) where Abs 600 is the value of absorbance at 600 nm and bis the thickness of film (mm). 2.10. Solubility in Water The water solubility (WS) of the film samples was evaluated using the Farhan and Hani method. The film samples were sliced uniformly (2 cm × 2 cm) and dried in a laboratory oven at 100 ◦ C for 24 h. To ascertain their initial dry weight, they were weighed to the closest 0.0001 g. Then, the films were placed in 50 mL screw-capped centrifuge tubes with 30 mL of distilled water and put into a water bath with continuous stirring at 25 ◦ C for 24 h. Undissolved films were then filtered using Whatman No. 1 filter paper and dried at 100 ◦C for 24 h to ascertain their final dry weight. Each sample was measured three times. The WS (%) was performed as a percentage, using the following equation: WS(%)=WO−Wf WO ×100 (4) where W O is the initial dried weight of the film and W f is the final weight of the dried, undissolved film. 2.11. Moisture Content The moisture content (MC) of the films was measured according to the method studied by Nur Fatin Nazurah and Nur Hanani [ 18 ]. The moisture contents of the film samples were determined by measuring the weight loss of the films (2 cm × 2 cm) before and after drying in a laboratory oven at 100 ◦ C for 24 h. The measurement was performed in triplicate. MC was examined in percentage according to the equation below: MC(%)=MCwet −MCdry MCwet ×100 (5) where W is the weight of the film sample. 2.12. FT-IR Spectroscopy Interactions of the different constituents within the Kappaphycus alvarezii seaweed, kappa-carrageenan, refined carrageenan and semi-refined carrageenan-based films incorporated with 10% of CNF film were obtained using a Fourier-transform infrared spectrometer (Thermo Scientific Nicolet iS5 FT-IR Spectrometer, Massachusetts, USA). The IR spectrum was measured over the wave number range from 4000 to 650 cm −1 , using OMNIC software. 2.13. Thermal Properties The TA Q500 thermogravimetric analyzer was used to analyze the thermal stability of the samples using the TGA method. A continuous heating rate of 10 ◦ C per minute was used for the measurements. Under a nitrogen atmosphere, the temperature was varied Polymers 2023,15, 2884 6 of 18 from 30 ◦ C to 600 ◦ C [ 19 ]. The derivation of the TGA curves was used to calculate the decomposition temperatures (DTG). 2.14. Scanning Electron Microscopy A small layer of gold was applied to the film specimens before they were placed on aluminum stubs with double-sided tape. A scanning electron microscope (Jeol, model JSM-5800, Tokyo, Japan) operating at 5 kV and a magnification of 1000 kx was used for morphological observations on the surface of the films (which were broken under liquid nitrogen before imaging). 2.15. Water Vapor Permeability According to Nur Hanani, Roos, and Kerry (2012), the film sample was firmly placed over the cup’s rim and each crucible was filled with 6 mL of distilled water. To prevent leaks, a vacuum seal grease was employed. The crucibles were put into a desiccator with silica gel beads as a desiccant (50 ± 5% RH and 23 ± 2 ◦ C). Over the course of eight hours, the weight differences were tracked at one-hour intervals, and the WVP was determined using the following [20]: WVP =∆w.l A.t. P (6) where ∆ w is the weight difference (g), l is the film thickness (m), A is the exposed area of the film (m 2 ), t is the time elapsed, and P is the partial pressure difference of water vapor across the film (Pa). 2.16. Quantum Mechanic Simulation The chemical interaction between carrageenan, glycerol, and cellulose nano-fiber was simulated using Gaussian 09W molecular dynamics software. The Pubchem database was used to obtain the molecular structures. To save costs on computation and simulation time, the chemical structures of carrageenan and cellulose nanofiber were shortened. The carrageenan structure was reduced to a single molecular unit, whereas the cellulose nanofiber structure was reduced to two units. DFT calculations were used to optimize the geometries of all molecules using Becke’s three parameters in conjunction with the correlation functions of Lee, Yang, and Parr (B3LYP) with a 6-31G (d,p) basis set. The molecular electronic surface potentials (MESPs) of the carrageenan, glycerol, and cellulose nanofibers were calculated using geometry optimization. The interaction energy was calculated using the generated energy of the self-consistent field (SCF), as shown below: Interaction energy =ESCFcomplex −(ESCFcarrageenan +ESCFglycerol +ESCFcn f )(7) 3. Results 3.1. Mechanical Properties of Films TS, EAB and Young’s modulus are commonly used to determine the breaking strength of the packaging materials. The value of TS indicates the resistance of the film at maximum tensile load, while the value of EAB (%) indicates the maximum allowable elongation of the film [ 21 ]. Meanwhile, Young’s modulus indicates the elasticity that characterizes the stiffness or flexibility of the material. Table 2shows the TS (MPa), EAB (%) and young modulus value of all treated samples. Table 2. Result of mechanical strength of films. Sample Mechanical Properties TS (Mpa) EAB (%) Young Modulus KA 11.36 ±1.22 a1.99 ±1.03 a5.71 KA + G 15.02 ±1.64 b5.54 ±2.09 b2.71 Polymers 2023,15, 2884 7 of 18 Table 2. Cont. Sample Mechanical Properties TS (Mpa) EAB (%) Young Modulus KA + G + 5%CNF 16.79 ±0.86 c5.96 ±1.64 c2.82 KA + G + 10%CNF 18.50 ±0.53 d6.58 ±1.12 d2.81 KA + G + 15%CNF 20.21 ±1.08 e9.40 ±1.39 e2.15 KC 46.87 ±1.1 a0.97 ±0.03 a48.23 KC + G 18.31 ±0.04 b22.32 ±0.13 b0.82 KC + G + 5%CNF 24.22 ±0.16 c20.27 ±0.20 c1.19 KC + G + 10%CNF 24.97 ±2.83 d15.22 ±1.39 d1.64 KC + G + 15%CNF 22.08 ±1.40 e13.45 ±0.99 e1.64 RC 59.03 ±0.48 a2.65 ±0.13 a22.27 RC + G 40.63 ±0.52 b18.46 ±0.16 b2.20 RC + G + 5%CNF 42.90 ±6.98 c11.10 ±1.73 c3.86 RC + G + 10%CNF 48.23 ±2.54 d8.22 ±1.68 d5.87 RC + G + 15%CNF 41.39 ±7.34 e12.86 ±2.71 e3.22 SRC 50.83 ±1.52 a1.02 ±0.13 a49.83 SRC + G 36.08 ±1.79 b15.82 ±1.06 b2.28 SRC + G + 5%CNF 39.63 ±0.95 c23.56 ±3.88 c1.68 SRC + G + 10%CNF 45.98 ±0.57 d19.18 ±0.78 d2.39 SRC + G + 15%CNF 26.72 ±2.28 e20.28 ±3.42 e1.32 The mean and standard deviation of the values are shown. Differing letters in the same column denote substantially different values (p< 0.05). The addition of glycerol 0.9% as a plasticizer significantly changes the value of TS and EAB% for all samples (p< 0.05) compared to the unplasticized film, decreasing the brittleness of the sample and increasing the elasticity of the film. According to Balqis et al., these phenomena are due to the increased spatial distance between the polymer chains, located between the polymer molecules, due to the plasticizers [ 18 ]. On the other hand, EAB was found to be inversely correlated with TS. Plasticizers such as glycerol increase the mobility of the polymer chains, resulting in more stretchable and flexible films, which increases the EAB and decreases the TS. Further improvements were achieved by adding CNF as a filler. This led to positive results, as the mechanical properties of the film improved. This indicates that hydrogen bonds are formed between CNF and carrageenan, which increases the cross-linking between polymer chains and CNF fills the voids created by plasticizers, which eventually decreases the movement of chains. This finding was supported by Bagheri et al. (2019) in their studies, where the addition of CNF to whey gluten improved its mechanical properties [ 22 ]. On the other hand, for kappa-carrageenan (KC) and semi-refined carrageenan (SRC), the TS decreases after the addition of 15% CNF. This is due to the inhomogeneity of the biopolymers and the CNF composite film, where the CNF agglomerates and is incompatible with the biopolymer matrix. The agglomeration of the fibers affects the result, because the structure of the polymer chains is different [ 23 ]. According to Zare et al. 2016, agglomeration is attributed to direct mutual attraction between nanoparticles through van der Waals forces or chemical bonding [ 24 ]. A similar finding was observed by Sogut et al., in that the aggregation potential of CNF particles occurs once a sufficient concentration is reached in the film solution [ 25 ]. Apart from this, Xu et al. observed that CNFs reinforced with chitosan improved the mechanical properties of the film from 2% to 10%, resulting in a significant increase in elongation at the break, due to better adhesion of the nanosized cellulose to the matrix of the biopolymers [ 26 ]. Meanwhile, KA with 15% CNF increased in TS but with a lower EAB compared to other samples, due to its properties which make the KA film easy to break and prevent it forming good mechanical properties. Biopolymer films with a suitable plasticizer concentration can be obtained with good mechanical properties in the range of 10–100 Mpa. Moreover, the reinforced CNF film is in the range of 10–100 Mpa, which shows good mechanical characteristics that can be used for the film development [ 27 ]. In addition, the modulus Polymers 2023,15, 2884 8 of 18 of elasticity provides valuable information about the ability of the films to resist external forces and maintain their structural integrity. A higher modulus of elasticity indicates a stiffer material with lower elasticity, while a lower modulus of elasticity indicates a more flexible and elastic film [ 28 ]. Among the control samples, KA, KC, RC and SRC, SRC had a relatively high Initial Young’s modulus of 49.83. However, with the addition of glycerol, the Young’s modulus decreased significantly to 2.71, 0.82, 2.20 and 2.28, respectively, indicating increased flexibility. The addition of CNF to KA + G films had different effects on the elastic modulus. While KA + 5%CNF slightly increased the elastic modulus to 2.82, KA + 10%G and KA + 15%G decreased it to 2.81 and 2.15, respectively, indicating increased flexibility with increasing CNF content. In contrast, KC-reinforced CNF was affected by the further addition of 5%, 10%,and15% CNF, remaining low at 1.19, 1.64, and 1.64, respectively. The elastic modulus of RC reinforced with 5%, 10%,and 15% CNF resulted in higher values of elasticity. The highest initial elastic modulus was found for the SRC samples (49.83), while SRC + G films was slightly decreased by the addition of CNF, indicating higher flexibility. 3.2. Physical Properties of Films The thickness of packaging films is one of the most important factors in product protection. The gas permeability of the film can be affected by the thickness of the film. As the thickness increases, the gas permeability of the film can also increase. Table 3shows the effect of glycerol and different concentrations of cellulose nanofibers (CNF) on the thickness of seaweed derivative films. Table 3. Result of physical properties of films. Sample Physical Properties Thickness (mm) Opacity Water Solubility (%) Moisture Content (%) KA 0.060 ±0.00 4.50 ±0.40 b60.00 ±4.60 b1.64 ±0.27 b KA + G 0.060 ±0.00 3.86 ±0.26 b67.92 ±0.30 c39.23 ±0.33 c KA + G + 5%CNF 0.060 ±0.00 5.63 ±0.50 c57.01 ±4.02 d30.18 ±1.13 d KA + G + 10%CNF 0.060 ±0.00 7.32 ±0.25 c47.22 ±3.48 e28.85 ±0.12 d KA + G + 15%CNF 0.060 ±0.00 7.29 ±0.18 c43.92 ±0.39 f27.31 ±0.39 e KC 0.040 ±0.00 9.98 ±1.10 a60.52 ±5.40 a1.98 ±0.07 a KC + G 0.092 ±0.01 b1.90 ±0.12 b80.00 ±6.72 a35.83 ±0.16 b KC + G + 5%CNF 0.092 ±0.01 b3.09 ±0.25 b55.45 ±1.36 b30.27 ±1.05 c KC + G + 10%CNF 0.092 ±0.01 c3.92 ±0.03 c53.26 ±0.66 c30.12 ±0.08 d KC + G + 15%CNF 0.100 ±0.00 3.93 ±0.02 c53.01 ±5.92 d29.62 ±0.29 d RC 0.020 ±0.00 3.65 ±0.48 c82.00 ±0.57 e8.48 ±0.36 e RC + G 0.024 ±0.01 c2.68 ±0.30 c88.03 ±7.89 f35.61 ±0.16 e RC + G + 5%CNF 0.040 ±0.00 3.51 ±0.29 a68.18 ±0.81 a29.08 ±0.54 a RC + G + 10%CNF 0.040 ±0.00 6.94 ±1.38 b66.71 ±3.68 a29.06 ±0.95 b RC + G + 15%CNF 0.040 ±0.00 7.27 ±0.78 b66.00 ±0.54 b26.24 ±0.41 c SRC 0.068 ±0.01 c10.88 ±0.5 c66.69 ±2.09 c1.50 ±0.08 d SRC + G 0.072 ±0.01 c7.91 ±0.44 c93.19 ±6.18 d31.85 ±0.48 d SRC + G + 5%CNF 0.072 ±0.01 c8.62 ±0.02 c53.04 ±2.59 e29.36 ±0.39 e SRC + G + 10%CNF 0.080 ±0.00 8.68 ±0.17 c42.59 ±7.78 a28.84 ±0.71 e SRC + G + 15%CNF 0.088 ±0.01 c8.85 ±0.85 a40.00 ±2.86 a27.35 ±0.29 a The mean and standard deviation of the values are shown. Differing letters in the same column denote substantially different values (p< 0.05). The thickness of all biopolymer film increased significantly after the addition of glycerol and CNF (p> 0.05), except for Kappaphycus Alvarezi (KA). This is due to the plasticizer molecules in the film matrix increasing the interstitial space between the polymer chains in the film matrix, resulting in an increase in thickness [ 16 ]. According to Ili Balqis et al. (2017), the thickness of the film increased because glycerol tends to absorb more moisture than an unplasticized film. Therefore, these films swelled to a greater extent, increasing their thickness. In addition, the thickness increased when CNF was added, as the solid content Polymers 2023,15, 2884 9 of 18 of the resulting film increased, with CNF serving as a filler for the space created by the plasticizers [29]. The film transparencies are shown in Table 3. The lower opacity values determined good film transparency, making its properties more attractive and clearer. The KC/G control film shows the highest transparency value, while the SRC control shows the highest opacity value. For all four types of seaweed derivative films, the initial opacity value is higher (without plasticizer), but the opacity value decreases when glycerol is added, and it increases when CNF is added, depending on the concentration. According to Farhan et al., the presence of glycerol reduces the intermolecular interactions between the polymer chains while increasing the space between them, allowing light transmission into the biopolymer film and resulting in great transparency. Because of the strong contact between CNF and the biopolymer matrix, CNF functions as a filler in the plasticized biopolymer matrix, resulting in lesser light scattering and increased light transmittance in the films. [ 2 ]. Meanwhile, SRC has the highest value for opacity, which is due to the presence of the cellulose that was present in the original algae. Therefore, it gives a turbid solution compared to RC and KC, which give a clear solution, while KA (the original alga) gives a clear solution compared to SRC, due to some impurities in the SRC derivatives [30]. Film solubility serves as an indicator of the quality of films used as packaging materials, including their integrity, water resistance, and biodegradability. In certain applications, it may be necessary for films to be water-insoluble in order to enhance product integrity and water resistance. As demonstrated in Table 3, adding plasticizers and CNF considerably increased the films’ solubility (p> 0.05). The result shows that the enhancement of CNF in the seaweed-based film significantly decreased the film solubility compared to the control films, which almost completely dissolved during the analysis as a result of the hydrophilicity of carrageenan. Film solubility increased when a plasticizer was introduced into the biopolymer matrix, compared to the unplasticized film. This is due to the nature of the plasticizer itself, which has hydrophilic properties [ 16 , 29 ]. Previous studies have shown that in the case of CNF, the primary factor contributing to a decrease in water solubility is the filling of voids between biopolymer chains by CNF, which reduces the mobility of these chains and consequently slows down the diffusion rate of water molecules [ 22 ]. Cellulose nanofibers (CNFs) improve the compactness of the biopolymer structure and increase resistance to water molecule permeability. This implies a stronger interaction between the biopolymer chains and CNFs, which is facilitated by better dispersion of the nanoparticles within the polymer matrix [ 31 ]. Another explanation for the decrease in water solubility with increasing CNF concentration, as illustrated in morphology image, could be the large molecular size and low solubility of CNFs in water [2]. Moisture content in the films can be measured by determining the percentage of moisture loss. Table 3indicates a significant increase in percent moisture content (MC) in film plasticized with glycerol as compared to control films (without plasticizer) (p< 0.05). The percent moisture content for all control films (non-plasticized) shows the lowest value of MC compared to the other samples. It shows a drastic change when glycerol is added, which could be due to the hydrophilic nature of glycerol, where a higher number of hydroxyl groups (OH) are present in the plasticizer, leading to an increase in MC ( Ili Balqis et al., 2017) [32] . Increasing hydrophilic plasticizers’ concentration leads to a reorganization of the polysaccharide network, free volume, and an increase in segmental movements, which makes it easier for water molecules to diffuse and results in a greater moisture content of the film. [33]. 3.3. FT-IR Spectroscopy FT-IR spectroscopy presented information about the chemical composition and specific functional groups of the carrageenan-based films for this study. Figures 1and 2show the spectra of the absorption bands between 650 cm −1 and 3600 cm −1 for KA-, KC-, RC- and SRC-based films incorporated with 10% of CNF. Polymers 2023,15, 2884 16 of 18 Funding: Thisresearchwasfunded bytheMinistryofHigherEducationNo. FFRGS/1/2021/TK0/UMP/ 02/51 (University Reference RDU210149) and Universiti Malaysia Pahang under grant (PGRS 210368). Institutional Review Board Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The Ministry of Higher Education supported this work through the Fundamental Research Grant Scheme (FRGS) under No. FFRGS/1/2021/TK0/UMP/02/51 (University Reference RDU210149). In addition, the research was financially supported by Universiti Malaysia Pahang under grant PGRS 210368. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ferreira, L.F.; Figueiredo, L.P.; Martins, M.A.; Luvizaro, L.B.; de Blara, B.R.B.; Oliveira, C.R.d.; Júnior, M.G.; Tonoli, G.H.D.; Dias, M.V. Active coatings of thermoplastic starch and chitosan with alpha-tocopherol/bentonite for special green coffee beans. Int. J. Biol. Macromol. 2021,170, 810–819. 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