New Isolated Shrimp (Litopenaeus setiferus) Chitosan-Based Films Loaded with Fly Ash for Antibacterial Evaluation

New three fabricated chitosan (CS) loaded with fly ash (FA) films were developed in this study. The shell waste of white shrimp was used as a precursor for the isolation of chitin and converted into chitosan by carrying out a deacetylation process. The formation of chitosan was conducted by various preparation steps deproteinization, demineralization, and deacetylation. The degree of deacetylation was found to be 95.2%. The obtained chitosan was used to prepare three different chitosan loaded-fly ash films. The prepared films contained various fly ash: chitosan ratios (2:1, FA-CSF1), (1:1, FA-CSF2), and (1:2, FA-CSF3). The obtained films were characterized using FTIR, XRD, and SEM. The micrograph images of the formed films showed spherical particles with an average size of 10 µm. The surface area, adsorption-desorption properties, thermal stability, and water/fat binding features of the fabricated chitosan films were studied. The results revealed that the prepared films displayed typical BET graphs with surface areas ranging from 2.436 m2 g−1 to 8.490 m2 g−1. The fabricated FA-CSF films also showed high thermal stability at temperatures up to 284.9 °C and excellent water/fat binding capacities. The antibacterial potential of the designed films was screened against E. coli (Gram-negative) and B. cereus (Gram-positive) bacterial strains. The tested solution of CS (1%) exhibited inhibition zones for E. coli and B. cereus as 18.51 mm and 14.81 mm, respectively, while in FA solution (1%), the inhibition zones were found to be 10.16 mm, and 13.57 mm, respectively. The results encourage and open up the new and promising areas of research for applying chitosan extracted from waste materials in biological applications.


Introduction
Litopenaeus setiferus (White shrimp) has become one of the most significant aquaculture shrimps in the world. It has a wide range of salt tolerance, quick growth, and other features appropriate for intensive aquaculture [1]. Shrimps are small marine crustaceans that are widely consumed as shellfish. Because of their high vitamin and mineral content, such as copper, calcium, zinc, iron, and phosphorus, as well as their high amino acid and protein content, they constitute a typically nutritious meal [2]. The biochemical composition of shrimps is highly influenced by various factors including, species, size, age, natural diet, feed composition, and environmental factors [3]. Shrimps have a seasonal shift in their fatty acid profile, cholesterol, and total carotenoid concentration [4,5]. Their physicochemical, proximate composition, microbiological, and sensory qualities, on the other hand, have received little attention in relation to their origin and treatment [6].
Chitin is the world's second most prevalent polysaccharide after cellulose and represents the main component in the exoskeleton of arthropods such as insects, and crustaceans. It is present in green algae, fungi's cell walls, insect and spider cuticles, and in the crustacean's exoskeletons [7]. The leftover residues from crustacean (shrimp, crab, lobster, and prawn) processing factories are the principal source of chitin on an industrial scale. The majority of crustacean shells are composed of chitin (15-40%), protein (20-40%), calcium setiferus) shells, sunflower oil, and olive oil was purchased from the local markets in Riyadh, Saudi Arabia.

Bacterial Strains and Maintaining Culture
Two bacterial strains, Bacillus cereus (B. cereus, Gram-positive, ARCC14579) and Escherichia coli (E. coli, Gram-negative, ATCC25966) were obtained from the Microbiology Department, College of Science, King Saud University, Saudi Arabia. The test strains were cultured in Mueller-Hinton broth (Sigma-Aldrich, Hamburg, Germany) and incubated at 37 • C overnight in a Thermo Scientific TM MaxQ TM 4000 Benchtop Orbital Shakers. Using a UV spectrophotometer Thermo Scientific TM Orion AquaMate 8000, the optical density (OD) was optimized to 0.1 at λ = 625 nm.

Isolation of Chitin from White Shrimp Shells
White shrimp shells were used to isolate chitin. The separation process was conducted in four conventional steps including demineralization, deproteinization, decolorization, and deacetylation using the previously standard reported method [42]. Briefly, the shells of shrimps were scraped from the loose tissues, collected, and washed thoroughly with tap water to eliminate any contaminants. The cleaned shells were air-dried and blended into small pieces of less than 20 mesh. The deproteinization step was carried out using 100 g of the homogenized shells with 1.0 mol L −1 sodium hydroxide at 100 • C for 8 h. The demineralization step was performed by treating the same quantity of homogenized shells with 1.0 mol L −1 of hydrochloric acid (1500 mL). The pH of the resulting solid material was neutralized using distilled water. The isolated chitin was neutralized using distilled water. The isolation process was performed in triplicates. The isolated chitin was then rinsed using ethanol followed by acetone to eliminate any remaining contaminants. The purified chitin was dried to a consistent weight in a vacuum oven for 2 h at 50 • C. The deacetylation step to convert chitin to chitosan was generally conducted by treating the chitin sample with concentrated (40-50%, w/v) sodium hydroxide at 100 • C for 30 min. The resulting chitosan was filtered, oven-dried, and stored in a dissector for further experiments (Scheme 1). partment, College of Science, King Saud University, Saudi Arabia. The test strains cultured in Mueller-Hinton broth (Sigma-Aldrich, Hamburg, Germany) and incuba 37 °C overnight in a Thermo Scientific TM MaxQ TM 4000 Benchtop Orbital Shakers. Us UV spectrophotometer Thermo Scientific TM Orion AquaMate 8000, the optical de (OD) was optimized to 0.1 at λ = 625 nm.

Isolation of Chitin from White Shrimp Shells
White shrimp shells were used to isolate chitin. The separation process was ducted in four conventional steps including demineralization, deproteinization, de ization, and deacetylation using the previously standard reported method [42]. Br the shells of shrimps were scraped from the loose tissues, collected, and washed oughly with tap water to eliminate any contaminants. The cleaned shells were airand blended into small pieces of less than 20 mesh. The deproteinization step was ca out using 100 g of the homogenized shells with 1.0 mol L −1 sodium hydroxide at 1 for 8 h. The demineralization step was performed by treating the same quantity of ho enized shells with 1.0 mol L −1 of hydrochloric acid (1500 mL). The pH of the resulting material was neutralized using distilled water. The isolated chitin was neutralized distilled water. The isolation process was performed in triplicates. The isolated chiti then rinsed using ethanol followed by acetone to eliminate any remaining contamin The purified chitin was dried to a consistent weight in a vacuum oven for 2 h at 50 °C deacetylation step to convert chitin to chitosan was generally conducted by treatin chitin sample with concentrated (40-50%, w/v) sodium hydroxide at 100 °C for 30 The resulting chitosan was filtered, oven-dried, and stored in a dissector for furth periments (Scheme 1). Scheme 1. Schematic illustration described the isolation steps of chitin from white shrimp shel the production of chitosan by deacetylation of the isolated chitin.

Fabrication of Chitosan-Based Films Loaded with Fly Ash
Chitosan-based films loaded with fly ash were prepared with different ratios Scheme 1. Schematic illustration described the isolation steps of chitin from white shrimp shells and the production of chitosan by deacetylation of the isolated chitin.

Fabrication of Chitosan-Based Films Loaded with Fly Ash
Chitosan-based films loaded with fly ash were prepared with different ratios of FA and CS. Three different chitosan-loaded fly ash (FA-CS) films were fabricated using approximately 0.5 g of chitosan dissolved in 1% (v/v) acetic acid under agitation for 5 min, then 1.0 g of fly ash was added to prepare FA: CS concentration ratio of 2:1 (FA-CSF1), 0.5 g of fly ash was added to 0.5 g CS to prepare 1:1 (FA-CSF2), and 0.5 FA: 1.0 g CS was used to prepare 1:2 (FA-CSF3) films, respectively. The suspended mixtures were poured into a rounded flat container and incubated overnight at 50 • C [43].

Characterization of the Fabricated FA-CS Films
The fabricated chitosan-loaded fly ash films were characterized using various spectroscopic and microscopic techniques. A Thermo Nicolet Avatar-360 ESP FTIR spectrophotometer (Thermo Scientific™, Waltham, MA, USA) was used in a wavenumber range of 4000-400 cm −1 to scan and determine the functional groups present in the as-prepared films. X-ray diffraction (XRD) Bruker D5005 diffractometer (Bruker™, Billerica, MA, USA) was applied using Cu Kα radiation (45 kV, 30 mA), 2θ = 5-65 • at a scan rate of 4 min −1 , and at temperature of 25 • C to detect the crystallinity of the films [44]. A scanning electron microscope (SEM JEOL JSM-6300, Billerica, MA, USA) was used to determine the morphology differences between the fabricated films. The samples were gold-sputtered prior to examination. The electron beam was with 10 kV energy and magnifications of ×2000 [45].

Thermogravimetric Analysis
The thermal properties of the fabricated chitosan films were studied. Thermogravimetric analysis (TGA) was utilized to measure the thermal stability and to determine the dedradation temperature of dried film powder, using a VersaTherm© Thermogravimetric Analyzer (Cahn Scientific, Irvine, CA, USA). The mass of each sample was ranged from 5 to 6 mg. The temperature was optimized from 25 to 800 • C with a heating rate (10 • C/min) and maintained in isotherm for 5 min under a nitrogen gas condition with a flow rate of 50 mL min −1 . The mass of the sample was continuously recorded as a function of temperature. The degradation percentage and temperatures were estimated in triplicates from the percentage weight loss first derivative graph (DTGA) against temperature [46].

Brunauer-Emmett-Teller Surface-Area Analysis
The surface area and porosity of the fabricated chitosan (CSF), as well as chitosan loaded with fly-ash (FA-CSF) films, were evaluated using the Brunauer-Emmett-Teller (BET) method. A Micromeritics Gemini 2360 surface area analyzer (Norcross, GA, USA) was used to carry out the measurements. The chitosan films were degassed at 25 • C before testing. The adsorption-desorption of nitrogen gas molecules were carried out based on the standard pressure range (0.05 < P/Po < 0.3) and the occupied area by nitrogen molecule 16.2 Å 2 at 77.35 K.

Water and Oil Binding Capacity
The water and fat binding capacity of the fabricated chitosan films were studied by weighing a centrifuge tube of 15 mL containing 50 mg of each sample. The samples were dispersed by adding 4.0 mL of distilled water and vortexing for 1 min at 3400 rpm. The last step was repeated by adding approximately 4.0 mL of olive oil and sunflower oil. The ingredients were left at room temperature for 1 h. After that, the samples were centrifuged for 30 min at 6000 rpm. The tubes were weighed after the supernatant was discarded. Triplicates of each sample were examined [47].

Degree of Deacetylation Determination
The degree of deacetylation was measured by dissolving 0.5 g dried CS in 20 mL of 0.1 mol L −1 hydrochloric acid and 25 mL deionized water under continual stirring for 30 min. Stirring was continued for another 30 min after adding another 25 mL deionized water. The solution was titrated with a 0.1 mol L −1 sodium hydroxide solution using an automated burette once the chitosan was completely dissolved (0.01 mL accuracy). The degree of chitosan deacetylation (DD) was estimated using the formula: where m expressed the weight of the sample (0.5 g CS), V1 and V2 represent volumes of NaOH (0.1 mol L −1 ) corresponding to the deflection points. The values 2.03 and 0.0042 expressed the coefficient resulting from the molecular weight of chitin monomer unit and that resulting from Chitin-chitosan molecular weight difference.

Antibacterial Activity
The antibacterial potential of the FA, CS, and the fabricated (FA-CSF) films was studied against two bacterial strains B. cereus and E. coli using the agar diffusion method. The Mueller-Hinton agar plates were inoculated with the two bacterial strains (100 µL of density 1 × 10 6 CFU/mL) and incubated for 24 h at 37 • C. Prior to the inclusion of bacteria, the test samples were sterilized in a biosafety cabinet that was dosed with UV radiation for 1 h. In the shaking incubator, sterilized samples were cultured with bacterial strains overnight at 37 • C. The experiment was done in triplicates, and the inhibition zones were measured in millimeters (mm) by measuring the diameter of the zone with no bacterial growth [48].

Statistical Analysis
The data were presented in the form of means and standard deviations (SD). To determine statistical significance, ANOVA test using Excel of Microsoft Office V 10.0.0 was used to estimate the variance, followed by the student's t-test. The significance level was set at a p-value of < 0.05.

Results and Discussion
The isolation process of chitin from shells of white shrimp waste can be performed in many stages. The two critical steps were the deproteinization and demineralization. In this study, the removal of protein was carried out using sodium hydroxide prior to the demineralization to control the formation of foam during the treatment process and hence, could optimize the yield of chitin [49]. The demineralization step was successfully accomplished by treating the shrimp waste with 1.0 mol L −1 hydrochloric acid solution to dissolve calcium carbonate and remove other organic matters. The obtained residue was chitin. Meanwhile, chitosan is the deacetylated product of chitin. Therefore, the deacetylation degree of the isolated chitin to obtain chitosan was studied and the results revealed a high deacetylation degree with a value 95.2%.

Determination of Degree of Deacetylation
A simple titration method was used to determine the degree of deacetylation of the isolated chitosan. The titration analysis was based on the titration of chitosan solution dissolved in 0.1 mol L −1 hydrochloric acid and titrated with 0.1 mol L −1 of sodium hydroxide and from the two inflection points (V1 and V2) corresponding to the acid consumed by the amine groups. The calculated deacetylation value was found to be 95.2%, which is consistent with the results reported in previous studies [50].

Characterization of the Fabricated FA-CS Films
FTIR analysis was used to characterize the functional groups of the prepared chitosanbased films loaded with fly ash (Figure 1). It was observed that all samples showed a strong band at about 3427 cm −1 corresponding to OH stretching vibration and asymmetric stretching vibration of primary amine (-NH 2 ). The symmetric and asymmetric C-H stretching vibration peaks of CSF were recorded at 2926 and 2858 cm −1 , respectively. In all samples, a significant band at 1626 cm −1 was observed corresponding to residual N-H bending vibration group obtained from primary amine [51], and bending O-H vibration group suggesting the presence of molecular water [36]. These bands are typical of polysaccharides, which are found not only in CS but also in Xylan, carrageenan, and glucan. A band at about 1080 cm −1 was found in the Chitosan-loaded fly ash samples (FA-CSF1, FA-CSF2, and FA-CSF3) due to Si-O-Si or Si-O-Al asymmetric stretching vibration. In addition to this, at approximately 598 cm −1 , a band appears in FA-CSF1, FA-CSF2, and FA-CSF3 suggesting Si-O-Al vibration [52][53][54][55].

Characterization of the Fabricated FA-CS Films
FTIR analysis was used to characterize the functional groups of the prepared c tosan-based films loaded with fly ash (Figure 1). It was observed that all samples show a strong band at about 3427 cm −1 corresponding to OH stretching vibration and asymm ric stretching vibration of primary amine (-NH2). The symmetric and asymmetric C stretching vibration peaks of CSF were recorded at 2926 and 2858 cm −1 , respectively. In samples, a significant band at 1626 cm −1 was observed corresponding to residual N bending vibration group obtained from primary amine [51], and bending O-H vibrat group suggesting the presence of molecular water [36]. These bands are typical of po saccharides, which are found not only in CS but also in Xylan, carrageenan, and gluc A band at about 1080 cm −1 was found in the Chitosan-loaded fly ash samples (FA-CS FA-CSF2, and FA-CSF3) due to Si-O-Si or Si-O-Al asymmetric stretching vibration. In dition to this, at approximately 598 cm −1 , a band appears in FA-CSF1, FA-CSF2, and F CSF3 suggesting Si-O-Al vibration [52][53][54][55]. The XRD analysis was used to study the crystalline phases of FA, CSM, FA-CS FA-CSF2, and FA-CSF3. The XRD pattern of the FA showed two main crystalline pha Quartz and Mullite. The large hump at 2θ at 25-30° indicated the amorphous phases the fly ash [56]. The early studies revealed that the Al2O3, SiO2, and Fe2O3 are the m oxides present in the FA and represent approximately 70% of the total mass (Figure 2 The XRD pattern of CS-based film displayed three main 2θ position peaks at 10°, 20° a 31° indicating the presence of the crystalline phase of the chitosan [57]. The XRD patte of the FA-CSF1, FA-CSF2, and FA-CSF3 films showed the presence of the crystall phases of chitosan and FA indicating well loading of FA on the chitosan surface (Fig  2b-d).
The surface morphology and effect of fly ash loading on the chitosan-based fil microspheres with smaller ones enveloped or minerals within these spheres called plerospheres [58]. Furthermore, the smoothness of the microsphere and the clearer surfaces were evident after combining chitosan with fly ash, suggesting the incorporation of the CS with FA (Figure 3a-c).  The surface morphology and effect of fly ash loading on the chitosan-based films CSF, FA-CSF1, FA-CSF2, and FA-CSF3 were studied by SEM. Microspheres comprise the majority of FA particles. FA particles were found in two different spherical forms (Figure 3). It is possible to detect hollow spheres (cenospheres) and thin-walled hollow microspheres with smaller ones enveloped or minerals within these spheres called plerospheres [58]. Furthermore, the smoothness of the microsphere and the clearer surfaces were evident after combining chitosan with fly ash, suggesting the incorporation of the CS with FA (Figure 3a-c).

Thermogravimetric Analysis
TGA analysis allows continuous monitoring of the mass of the samples as a function of temperature and time, while an inert gas atmosphere was passed over the samples. A dynamic TGA was performed for CSF, FA-CSF1, FA-CSF2, and FA-CSF3. The results showed that in the FA-CSF1, fly ash loaded chitosan-based films with a ratio of 2:1, an initial weight loss of 0.21%, followed by 26.65% with a decomposition temperature of 66.2 • C, and 276 • C (Figure 4a). Moreover, in the FA-CSF2, fly ash loaded chitosan-based films with a ratio of 1:1, an initial weight loss of 0.13%, followed by 0.45%, and 19.7% with a decomposition temperature of 61.2 • C, 177.2 • C, and 284.9 • C (Figure 4b). Additionally, FA-CSF3, fly ash loaded chitosan-based films with a ratio of 1:2, an initial weight loss of 2.66%, followed by 5.90%, and 26.64% with a decomposition temperature of 61.2 • C, 177.2 • C, and 284.9 • C (Figure 4c). Finally, the CSF, chitosan-based films, an initial weight loss of 7.05%, followed by 10.49%, and 43.44% with a decomposition temperature of 82.5 • C, 170.6 • C, and 281.1 • C (Figure 4d). The outcomes revealed that all samples showed decomposition temperatures that have matched those obtained by chitosan-based films [59,60].

Thermogravimetric Analysis
TGA analysis allows continuous monitoring of the mass of the samples as a function of temperature and time, while an inert gas atmosphere was passed over the samples. A dynamic TGA was performed for CSF, FA-CSF1, FA-CSF2, and FA-CSF3. The results showed that in the FA-CSF1, fly ash loaded chitosan-based films with a ratio of 2:1, an initial weight loss of 0.21%, followed by 26.65% with a decomposition temperature of 66.2 °C, and 276 °C (Figure 4a). Moreover, in the FA-CSF2, fly ash loaded chitosan-based films with a ratio of 1:1, an initial weight loss of 0.13%, followed by 0.45%, and 19.7% with a decomposition temperature of 61.2 °C, 177.2 °C, and 284.9 °C (Figure 4b). Additionally, FA-CSF3, fly ash loaded chitosan-based films with a ratio of 1:2, an initial weight loss of 2.66%, followed by 5.90%, and 26.64% with a decomposition temperature of 61.2 °C, 177.2 °C , and 284.9 °C (Figure 4c). Finally, the CSF, chitosan-based films, an initial weight loss of 7.05%, followed by 10.49%, and 43.44% with a decomposition temperature of 82.5 °C, 170.6 °C, and 281.1 °C (Figure 4d). The outcomes revealed that all samples showed decomposition temperatures that have matched those obtained by chitosan-based films [59,60].

Brunauer-Emmett-Teller (BET) Surface-Area Analysis
The surface area and adsorption-desorption properties of the fabricated CSF, FA-CSF1, FA-CSF2, and FA-CSF3 films were investigated using BET analysis. The results showed that typical-BET isotherm graphs were obtained. At low pressure (P/Po ˂ 0.8) rel-

Water and Oil Binding Capacity
Water and fat binding capacities using sunflower oil and olive oil for the CSF, FA-CSF1, FA-CSF2, and FA-CSF3 were evaluated, and the results were summarized in Table 1. The results showed that the loading of the chitosan-based films with fly ash increased the WBC ranging from 305.24% to 1725.24%. These could be attributed to the dissimilarity in the chitosan crystallinity, number of salt-forming functional groups, and the residual protein content after isolation. Meanwhile, OBC is one of the most crucial properties of chitosan that estimates its usefulness for various applications. Previous studies [61] addressed that the FBC is directly influenced by the deproteinization and demineralization steps. The FBC was performed for all CS films using sunflower oil and olive oil. The results showed that increasing the FA concentration in relation to the decrease in CS and the OBC of CSF in olive oil was decreased from 753.52% to 584.99%, whereas increasing the FA concentration increased the OBC in sunflower oil. The WBC and OBC of the chitosan-based film were consistent with the previously reported results [62].

Antibacterial Activity
Chitosan was known for its wide range of antibacterial activity against a broad range of microorganisms. Many mechanisms have been proposed for the action of chitosan on the bacterial cell, but the most meaningful mechanism is the polymer backbone charged groups and their ionic interactions with the constituents of the bacterial cell wall leading to the hydrolysis of the peptidoglycans in the bacterial cell wall, allowing the leakage of intracellular [63].
The antibacterial activity of CS (1%), FA (1%) solutions, FA-CSF1, FA-CSF2, and FACSF3 films displayed varying degrees of sensitivity against E. coli and B. cereus. Collectively, both bacterial microorganisms were sensitive to the tested samples. The results showed that the fabricated films exhibited potent antibacterial activities against E. coli and B. cereus, followed by CS and FA. The strongest antibacterial effect of FA-CSF films was towards E. coli. The results showed inhibition zones (12, 15 and 22 mm), (10, 11 and 20 mm) for FA-CSF1, FA-CSF2, and FACSF3 against E. coli and B. cereus, respectively ( Figure 6A). Meanwhile, the CS and FA displayed antibacterial activity against both types of bacterial cells [36]. The CS (1%) solution showed a bacterial inhibition zone diameter larger than FA on both E. coli and B. cereus ( Figure 6B). The CS inhibition zones on E. coli and B. cereus were 18.51 mm and 14.81 mm, respectively, while in FA the inhibition zones were 10.16 mm, and 13.57 mm, respectively [64].
Furthermore, the bacterial growth % and bacterial growth reduction % were determined. The bacterial growth % was compared with the bacterial cell growth without the addition of any sample (control) where the growth % was considered 100%. The results revealed that the growth % was the lowest with the CSF. The % growth rate was 30.56%, 54.62%, 68.52%, and 79.28% with the E. coli, and 14.04%, 27.77%, 44.29%, and 60.13% with the B. cereus, using CSF, FA-CSF3, FA-CSF2, and FA-CSF1, respectively (Figure 7a). The results showed that by decreasing the fly ash-loaded concentration with CS, bacterial growth was inhibited and decreased. This was also visualized in Figure 7b by representing the samples by subtracting the bacterial growth % from 100. It was reported that purified FA reduced the bacterial growth of E. coli by 59.89%. However, the FA reduced the growth of the tested bacteria by only 4.95%.
showed that the fabricated films exhibited potent antibacterial activities against E. coli and B. cereus, followed by CS and FA. The strongest antibacterial effect of FA-CSF films was towards E. coli. The results showed inhibition zones (12, 15 and 22 mm), (10, 11 and 20 mm) for FA-CSF1, FA-CSF2, and FACSF3 against E. coli and B. cereus, respectively ( Figure  6A). Meanwhile, the CS and FA displayed antibacterial activity against both types of bacterial cells [36]. The CS (1%) solution showed a bacterial inhibition zone diameter larger than FA on both E. coli and B. cereus ( Figure 6B). The CS inhibition zones on E. coli and B. cereus were 18.51 mm and 14.81 mm, respectively, while in FA the inhibition zones were 10.16 mm, and 13.57 mm, respectively [64]. Furthermore, the bacterial growth % and bacterial growth reduction % were determined. The bacterial growth % was compared with the bacterial cell growth without the   (Figure 7a). The results showed that by decreasing the fly ash-loaded concentration with CS, bacterial growth was inhibited and decreased. This was also visualized in Figure 7b by representing the samples by subtracting the bacterial growth % from 100. It was reported that purified FA reduced the bacterial growth of E. coli by 59.89%. However, the FA reduced the growth of the tested bacteria by only 4.95%.

Conclusions
The present study describes the isolation process of chitin from the shells of white shrimp (Litopenaeus setiferus) waste. The isolation procedure was conducted using different steps including, deproteinization, demineralization, and deacetylation to obtain chitosan. The deacetylation degree of the isolated chitin to obtain chitosan was studied and the results revealed a high deacetylation degree with a value 95.2%. The isolated chitosan was loaded with different percentages of fly ash and used to fabricate three FA-CSF films. The physical and chemical properties of the fabricated chitosan films were studied using various spectroscopic and microscopic techniques such as FTIR, XRD, and SEM. Moreover, the thermal stability, the adsorption-desorption, surface area, and the water/oil binding features of the fabricated films were also studied. The results revealed high stability of the fabricated films with excellent water/oil binding capacities. The antibacterial potential of 1% CS and FA solutions and the fabricated FA-CSF films were screened against two bacterial strains E. coli and B. cereus. The fabricated FA-CSF3 showed the largest zone of inhibition against E. coli stains compared to B. cereus and others. The outcomes of the current study open up a promising view in the future to exploit the isolated chitosan from shrimp waste to be applied in various biomedical applications.