Mechanical, UV protection, and antibacterial properties of poly(vinyl alcohol)/marine mucilage biocomposite films

The mucilage that accumulated in the Sea of Marmara in 2021 turned into an environmental disaster that threatened not only marine life but also the shipping and tourism industries. However, because of the partially organic structure of marine mucilage, it has the potential to be used as an additive for polymer films. In this study, bioplastic films were prepared from PVA with a series of mucilage content from 0% to 50% by mass, and their properties were investigated. The FT‐IR spectra revealed a shift in hydroxyl peaks, suggesting an interaction between the mucilage and PVA. SEM images showed the formation of crystal forms at the higher mucilage content of 20%, indicating ionic saturation of the PVA matrix. EDX and XRD analyses revealed that the crystal was NaCl coming from the mucilage composition. The tensile strength of the neat PVA film was measured as 9.86 MPa and improved to 14.56 MPa with 5% mucilage content, which is attributed to the ionic cross‐linking. The tensile properties were preserved up to 20% of the mucilage content, and then sudden decrements were observed. The transmittance percentage (T%) of the neat PVA film in UV–Vis spectra (800–200 nm) was detected as 75% at 500 nm and decreased down to 39% with increased mucilage content up to 50%. Furthermore, a rising limit of linearity (LOL) of the spectrums was observed from 350 to 580 nm, with increasing mucilage content from 5% to 50%. The biofilms showed antibacterial activity against Escherichia coli and Staphylococcus aureus bacteria. Hence, a robust and biodegradable film having UV protection and antibacterial activity was developed from the marine mucilage bio‐waste.


| INTRODUCTION
The formation of mucilage, which can accumulate on the surface and through the water column of seas, begins with the extracellular organic substances released by marine microorganisms, particularly phytoplankton, in response to specific stressors or when they die.These organic substances, which generally degrade through time, may accumulate under certain environmental conditions, such as alterations in the temperature and oxygen regimes. 1,2he structure of mucilage is composed of proteins, carbohydrates, lipids, and DNA, as well as inorganics like silicon and aluminum, and ions like calcium. 3 In its sticky gel-like consistency, mucilage (also known as sea saliva) may contain many marine microorganisms such as viruses, phytoplankton, and bacteria. 3ome groups of phytoplankton, particularly diatoms, have been identified as the most prevalent causative organisms in mucilage samples from all over the world. 4ollowing research, it was discovered that dinoflagellate species also played an important role in the formation of mucilage. 4,5Some of the species thought to trigger mucilage formation have been reported as Gonyaulax fragilis and Cylindrotheca closterium, as well as Skeletonema costatum, Cyclotella sp., and Thalassiosira rotula, which were also detected in the mucilage used in this study. 1,4,5umerous environmental stresses affect phytoplankton. 6oth climate change and human activity contribute to the occurrence of extreme algal blooms.0][11] Mucilage was discovered for the first time in the North Adriatic Sea in 1729. 9Marine mucilage has been commonly observed in the Adriatic and Tyrrhenian Seas and less commonly in the Aegean and Marmara Seas, 12 sometimes causing significant environmental damages, including the death of nearly 90% of benthic organisms in 1991 in the Tyrrhenian Sea. 13 In 2021, the surface and water column of the Sea of Marmara was covered in mucilage for months, obstructing fishermen's nets, halting commercial shipping operations, suffocating marine life, and endangering tourism and the economy. 12,14As a result of the efforts of the Ministry of Environment, Urbanization, and Climate Change along with local municipalities, 11,000 metric tons of mucilage was collected from the surface of the Marmara Sea in the summer of 2021. 15However, agencies did not know how to dispose of this massive waste.We hypothesized that mucilage could be turned into a bioplastic material owing to its partially organic structure. 16ioplastics can be defined as sustainable alternative plastics derived from renewable natural resources such as plants, algae, bacteria, biomass, and biological wastes, 17,18 providing the same functions as petroleumderived plastics. 19The European Bioplastics Organization defines bioplastic as either biobased or biodegradable or both. 20Their non-toxicity, easier recycling, lesser use of fossil fuels in production, and sustainability make them superior to conventional plastics. 17,19,21arious materials with different properties are used in the production of bioplastics, such as corn, potato, wheat, wood, milk proteins, starch obtained from food, soy and feed plants, and other wastes consisting of biomass. 18,21,22owever, competition of biomass sources with feed and food is a major obstacle in using these bioplastic raw materials.In addition, these crops require large amounts of fertilizer, fertile soil, and irrigation water. 23acteria are another source for the production of bioplastics.Some of the bacterial species are used for polyhydroxyalkanoate (PHA) production.PHA is degraded by microorganisms and is produced from renewable resources such as waste from food and agricultural industries. 24However, in addition to these beneficial properties, the risk of contamination and the need for special conditions for bacterial cultures limit the production of bioplastics from bacteria. 22lgae are attracting attention as a sustainable alternative in bioplastic production to overcome the limitations of the above-mentioned resources and to ensure environmental safety.Advantages of algae include rapid growth, high biomass availability, short harvest time, non-competition with food, possibility to extract high-value metabolites, and their ability to be grown even in non-arable land. 17,18,21,257][28][29] In addition to the mentioned properties, it is also preferred in biodegradable film and bioplastic studies due to its superior film-forming capacity and hydrophilicity. 30,31On the other hand, PVA has high ultraviolet (UV) radiation transmittance, so it is not suitable for UV-protective material as used alone. 32Numerous studies have been conducted on the development of UV-protective PVA products in order to increase the UV-shielding ability of PVA.Modifying the properties of PVA-based materials through blending with specific fillers is a straightforward and efficient method.However, when combined with polymer matrices, the majority of conventional organic UV absorbers undergo photodegradation and migration.Some of them are also toxic despite their good UV-shielding performance. 32,33The UV protection property of a material is significant because ultraviolet radiation can cause harmful effects on human health, such as skin aging, DNA damage, and cancer.Moreover, UV light may cause photodegradation of pharmaceuticals and foods, altering their color and taste. 34Moreover, materials such as polymers were affected negatively by UV radiation as discoloration and deformation of mechanical properties.Therefore, UV-shielding capacity is an attractive feature for UV-protective films and coatings for biomedical, food, and optoelectronic products to provide a safe and healthy environment by preventing the harmful effects of UV radiation. 32his study aimed to investigate the performance of mucilage in bioplastics to be considered as a value-added material rather than its disposal.The produced bioplastic showed antibacterial activity against Escherichia coli and Staphylococcus aureus.In UV-Vis spectra, the transmittance of 0M-PVA film at 400 nm was 75%, while the transmittance of 20M-PVA film decreased to 40%.In 50M-PVA, the transmittance of the film decreased below 20% at 400 nm.Therefore, the films offered improved UV protection with the increasing percentage of mucilage.

| Materials
Mucilage was collected from the Mudanya shores of the Marmara Sea in Bursa, Turkey (40 23 0 01.4 00 N 28 51 0 32.2 00 E).The salt content of the dried mucilage was determined to be 57% by mass, as described in the next section.The rest of the content consists of carbohydrates, lipids, and proteins, as determined in our previous study. 35Additional materials for bioplastic production were polyvinyl alcohol (PVA) (Acros Organics, 95.5%-96.5% hydrolyzed, MW approx.85,000-124,000), glycerol (Carlo Erba Reagents, 30 Be, Vegetal origin) and acetic acid (Macron Fine Chemicals, glacial 99.5%).Ultrapure water was obtained from a Millipore-Direct-Q 3 UV device (Darmstadt, Germany).

| Preparation of mucilage powder
Mucilage was poured into tubes and then centrifuged for 15 min at 1462 g (Beckman-Coulter, Allegra X30R, USA).After the supernatant was discarded, mucilage was poured into glass petri plates and dried in an oven at 50 C. Dried mucilage was pulverized by a laboratory mortar (Figure 1).In order to determine the salt content, a certain amount of the dried mucilage was dissolved in distilled water, and the salinity was measured using an YSI multiparameter instrument (Ohio, USA).The salt content was determined to be 57% by mass.

| Preparation of mucilage bioplastic films
Mucilage-PVA bioplastics were prepared by the solvent casting method using different ratios of mucilage and PVA.Hence, separate solutions of 10% mucilage and 10% PVA were prepared in 10% acetic acid aqueous solution by mixing for approximately 1 h at 70-80 C using a magnetic stirrer.Solutions were then allowed to cool to room temperature.The polymer and mucilage solutions were mixed in a total volume of 10 mL with a certain amount of 10% mucilage and 10% PVA solutions.For instance, 50M-PVA was prepared by mixing 5 mL of 10% PVA and 5 mL of 10% mucilage solutions.As a plasticizer, 0.4 g of glycerol were added to each film.The prepared mixtures were poured into glass petri dishes, and the water was evaporated in a fume hood at room temperature for 2 days.Hence, the bioplastic films were prepared with final mucilage ratios of 5%, 10%, 15%, 20%, 25%, 40%, and 50%.The notations of the films were given by the mucilage content followed by M-PVA.For instance, the biofilm containing 5% mucilage was notated as 5M-PVA.The production of bioplastic films is schematically shown in Figure 2.

| Characterization of bioplastic films
Structural analysis of mucilage-PVA bioplastic films was performed using Fourier-Transform Infrared (FTIR) spectroscopy (Nicolet-iS50, Massachusetts, ABD).FTIR spectra were obtained with 16 scans in the range of 400-4000 cm À1 with a resolution of 4 cm À1 .
The thermal stability of the bioplastic films was investigated using the Ta/SDT650 thermal gravimetric analyzer (TGA) (New Castle, USA).Bioplastic films were cut into small pieces of 30-35 mg.TGA analyses were carried out at 10 C/min increments between 25 and 900 C under a nitrogen atmosphere.The % weight loss of bioplastic films was recorded at different temperatures.
The thermal properties of the bioplastic films and the variation of the percentage of crystallinity were determined by differential scanning calorimetry (DSC) (DSC25, TA Instruments, New Castle, USA) instrument.The weights of the samples were between 9 and 10 mg and examined at 10 C/min increments between 70 and 300 C under a nitrogen atmosphere.X-ray diffractograms, which give quantitative and qualitative phase information and crystalline structure 36 of the prepared bioplastics, were performed at a scanning speed of 2 C/min increments between 5 and 90 C to examine their crystal structures (Bruker AXS/Discovery D8, Massachusetts, USA).
Scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDS) were used to observe the morphology of the bioplastic films, the crystal structures within, and the distribution of the mucilage in the matrix (Carl Zeiss/Gemini 300, Oberkochen, Germany).Samples were coated with a thin layer of gold-palladium before testing.The acceleration voltage was kept at 5.0 kV.
UV-Visible absorbance spectra were obtained using a SHIMADZU-UV3800 UV-Vis-NIR spectrophotometer (Kyoto, Japan) to analyze the transmittance of the produced bioplastic films.Scans were collected in the 200-900 nm range at 0.5 nm intervals.Mucilage-free PVA film was taken as a reference for each sample.
Mechanical properties of bioplastic films were determined using a universal mechanical test instrument (SHIMADZU-AGS-X, Kyoto, Japan) according to ASTM D 882-02 (2002).In order to determine the mechanical properties, 1 Â 8 cm films were prepared with a sample preparation device (INSTRON-6054.000,Norwood, USA).The distance between the gauge length was 60 mm, and the crosshead speed was set as 500 mm/min.The tests were performed in triplicate, and the average of results is given with standard deviations.

| Antibacterial activity of bioplastic films
Antibacterial properties of raw mucilage and 10M-PVA bioplastic film were investigated by the disc diffusion method. 37Bacterial strains used in the disc diffusion test were Gram-positive S. aureus ATCC 25923 and Gramnegative E. coli ATCC 25922.For the antibacterial activity, 0.5 g of powdered mucilage was pressed into 1.6 cm diameter discs using 3 tons of force in a manual press machine (Manual MSE LP/M2S10, Turkey).The turbidity of liquid bacteria cultures was set to 0.5 McFarland. 38hen, liquid cultures were inoculated onto solid agar medium in petri dishes with the spread plate method, and the discs were placed on them.Petri dishes were incubated at 37 C for 24 h.To examine the antibacterial activity of the 10M-PVA bioplastic film, 1.6 cm diameter pieces were cut from the film.

| RESULTS AND DISCUSSION
3.1 | Structure of mucilage/PVA biofilms FTIR analyses were performed to understand the interactions between marine mucilage and PVA.The FTIR spectra of the marine mucilage sample, virgin PVA in powder form, and bioplastic films are shown in Figure 3A.In the mucilage spectrum, the bands at 1634 and 1567 cm À1 corresponded to the amide I and amide II protein structures. 39he peaks at 3240 and 3350 cm À1 were attributed to amine and hydroxyl groups of the proteins and carbohydrates in the mucilage composition. 40The band between 1024 and 1040 cm À1 belonged to the CO polysaccharide and carbohydrate groups. 39In the PVA spectrum, the strong and broad band between 3000 and 3600 cm À1 is due to the hydroxyl groups, and the weak bands observed between 2850 and 2960 cm À1 are due to the CH from alkyl groups.Furthermore, the bands at 1037 and 1091 cm À1 were attributed to the stretching of the CO bond.The bands at 1750 and 1735 cm À1 in the PVA spectrum reflect the C O and C O bond from the acetate group of PVA.The peaks between 1320 and 1420 cm À1 were attributed to the bending of C H of the polymer backbone. 41he spectra of bioplastic films were substantially similar to that of PVA because of overlapping with the mucilage peaks.However, as more mucilage was incorporated into the structure of the bioplastic film, the hydroxyl peak intensity increased, and the peak shifted, especially after 20% mucilage (Figure 3B).This shift is thought to be caused by intermolecular hydrogen bonding of the hydroxyl groups of mucilage and the PVA. 42Furthermore, ionic cross-linking, originating from the salts, may cause the shifting.

| Scanning electron microscopy (SEM)
The morphologies and the dispersion of the mucilage in the biofilms were investigated by SEM images.Figure 4 shows SEM images of the produced bioplastic films at 100Â and 5000Â magnifications.According to the SEM images, there was no additive in the film that did not contain mucilage (0-PVA), whereas the mucilage appeared as splinters in the bioplastic films containing 5% and 10% mucilage.While 5M-PVA and 10M-PVA had homogeneous mucilage distributions, 15M-PVA and higher had heterogeneous regions formed by crystalline particles.The crystal structures were exposed to EDS, and they were identified as NaCl salts (Figure S1).The absence of the crystals in films containing 5% and 10% mucilage was thought to be due to the dissolution of Na and Cl in the PVA matrix and being in ionic form.The SEM images of bioplastic films containing 15% or more mucilage showed salt crystals.In bioplastics containing 40% and 50% mucilage, salt crystals were observed to increase.Consequently, as the ratio of mucilage raised, so did the crystal content of the bioplastic films.The matrix reached ionic saturation at a certain mucilage ratio (15% mucilage content or higher), and excess ions formed heterogeneous regions in the structure in the form of salt crystals.

| Crystallinity of mucilage/PVA films
The XRD pattern of the films revealed the characteristic peaks of the semi-crystalline structure of PVA as a broad peak at 2θ = 10-15 and a strong peak at 2θ = 20 . 43The peak intensity at 2θ = 20 indicated that the polymer's crystalline state decreased as the mucilage ratio increased, and the peak disappeared at high mucilage contents (Figure 5).The XRD model of bioplastic films containing 5%, 10%, and 15% mucilage was identical to the PVA XRD model. 43The films having above 15% mucilage content showed new sharp peaks, which are attributed to the NaCl salt crystals.These new peaks gradually increased as the mucilage content increased, corresponding to the XRD pattern of NaCl reported by Palevicius et al. 44 These results proved the presence of NaCl in ionic from up to 15% mucilage content and in crystal form for the higher concentrations, supporting the SEM-EDS results.The NaCl crystal formation at over 15% content was ascribed to the saturation of the polymer matrix by the ions.
The crystallinity of the polymer was calculated using Equation (1) 45 from the XRD graphs (Figure 5).Crystallinity values of polymer used in bioplastic films were 41.20%, 38.18%, 37.72%, and 34.98% for 5M-PVA, 10M-PVA, 15M-PVA, and 20M-PVA, respectively.The crystallinity of the polymer decreased as the mucilage ratio increased due to the increase in salt concentration. 46n their study, Tretinnikov and Zagorskaya prepared PVA films with and without LiCl and examined their crystallinity.They observed that the addition of LiCl reduced the crystallinity.
where, X c is the crystallinity.A c is the crystalline area.A a is the amorphous area.

| Thermal properties of mucilage/PVA films
TGA thermograms corresponding to the samples are given in Figure 6.The mass loss below 100 C was due to the loss of absorbed moisture.Mucilage showed threestage degradation.The first decomposition took place between 200 and 500 C, corresponding to 10.5% weight loss, and in the second decomposition, the mass loss slightly continued until 750 C. The first and second degradations were due to the polysaccharide structure, similar to a study done with natural polysaccharides. 47The third decomposition, starting from 750 C, was due to the NaCl content. 48he mass loss of 0M-PVA occurred in three stages.The first stage of massive mass loss occurred between 180 and 220 C for 0M-PVA, corresponding to 11.12% F I G U R E 5 X-ray diffraction (XRD) graphs of bioplastic films.weight loss, while the second stage occurred between 250 and 310 C due to the loss of hydroxyl groups leaving the polyene structure.In the third stage, which occurred between 400 and 480 C, the polyene structures decomposed.The thermogram results of the 0M-PVA in this study are similar to a reported study by Devangamath et al. 49 Above 600 C, a weight loss was seen for 0M-PVA, and the residual was 8% at 900 C.However, mucilagecontaining biofilms exhibited a continuous weight loss above 500 C due to the mucilage.The increased rate of weight loss at about 800 C is ascribed to the NaCl content.Among the mucilage-containing biofilms, the residuals increased with increasing mucilage content.The films containing 5%, 10%, 15%, and 20% mucilage exhibited lesser residual since the salt content of these films was low.On the other hand, for the bioplastics containing 25%, 40%, and 50% mucilage, the amount of residue seen was higher than in the film without mucilage (0M-PVA) due to the high salt content.
Differential Scanning Calorimetry (DSC) was used to examine the impact of mucilage on the thermal characteristics of bioplastic films.Figure 7 illustrates the DSC thermograms of the samples.In the DSC curve of mucilage, the endothermic peak at 116.5 C was due to the water loss of polysaccharide content.In literature, similar endothermic peaks were reported for the mucilage from different sources.For instance, the peak value slightly varied based on the source. 50The other endothermic peaks that appeared at 148 and 200.8 C were attributed to the melting of the proteins in the composition. 51he glass transition temperature of the mucilage-free PVA film (0M-PVA) was 66 C, similar to the reported value by Patel et al., 43 who found the glass transition temperature of pure PVA as 66 C. The melting point of PVA was determined to be 199 C.
The T g values of the bioplastic films increased with the incorporation of mucilage.This increment was due to the ionic crosslinking, which restricted the free motion of the PVA chains.Similar results were reported by Lee et al. 52 for PVA and nitrate salt mixture.The melting points of 5M-PVA and 10M-PVA films were determined to be 188 and 181 C, respectively.The hydrogen bonding interaction between the functional groups of mucilage and PVA may be responsible for the decrease in melting temperatures.This established interaction may weaken the interaction between PVA chains, resulting in lower melting temperatures.For the rest of the bioplastics, the melting peak disappeared, indicating a crystalline structure, which agreed well with XRD results.For 50% M-PVA, the endothermic peaks above 175 C were due to the decomposition of the film.The TGA curve of this film supported this result as the initial degradation temperature of this film was determined to be 163 C.

| UV-visible spectroscopy
UV (ultraviolet) radiation causes discoloration and a loss of mechanical properties of sensitive materials such as polymers.In addition, UV radiation has been linked to skin aging, DNA damage, and cancer.Moreover, it leads to the photodegradation of pharmaceuticals and foods.Therefore, UV-protective films are used in food packaging and protective equipment.Thus, biomedical products were studied to restrain the detrimental impact of UV radiation and provide a safe and healthy environment.The UV region of the Sun's UV-Vis light contains wavelengths classified as UV-C (220-280 nm), UV-B (280-320 nm), and UV-A (320-400 nm). 53The absorption capability of the films as transmittance percentage (T%) in the range of 800-180 nm was tested.The results are given in Figure 8.
The 0M-PVA film exhibited a T% of around 75 with no change in the range between 800 and 220 nm.This result demonstrated that the PVA film showed the highest transmission in visible wavelengths of light.In the study by Abral et al., 54 it was shown that pure PVA film had a transmission of 70.3% at 400 nm, which was less than our PVA film.The initial T% of the bioplastic films containing mucilage at 800 nm decreased with increasing mucilage content.T% of the bioplastic films declined in the studied range.However, the declines became more obvious as the slope of the curve increased at a specific wavelength for each biopolymer.In other words, a limit of linearity (LOL) was detected for all the biofilms.LOL increases with increasing mucilage content (Table 1).For example, the LOL for 5M-PVA was 350 nm, while 580 nm for 50M-PVA.Similar behaviors were reported for cellulose-lignin films. 55While the transmittance of the 5M-PVA decreased below 400 nm, the wavelength at which the transmission began to decrease shifted to 600 nm as the mucilage ratio increased.At 400 nm (UV-A), the transmittance of the 20M-PVA and 25M-PVA films slightly decreased to 42.4% and 41.9%, respectively, while of the 40M-PVA and 50M-PVA films dropped to 25.9% and 18.04% respectively.The film containing 50% mucilage was UV-C protective because it showed no transmittance below 280 nm.In fact, it showed very low transmittance up to 400 nm, so it was acceptable as UV-A and UV-B protective material. 56In the study conducted by Andrés et al., 56 bio-oil was added to PVA film and UV protection was evaluated.At the highest concentration of bio-oil, they measured 85% transmittance at 750 nm, whereas our films provided superior protection with a 50% transmittance value at 50% mucilage.In another study conducted by Abral et al., 54 Uncaria gambir (UG) extract was added to improve the UV protection of PVA.They measured the transmittance at 750 and 400 nm as 72% and 10.2%, respectively, with the highest concentration of UG extract.In our study, 50M-PVA film showed higher protection with 50% transmittance at 750 nm and a similar protection value at 400 nm.These results indicated that the UV absorption of bioplastic films increased with increasing mucilage content and exhibited effective protection against UV light (Figure 8).
In order to study the UV protection performance of the biocomposite films further, the sun protection factor (SPF) values were calculated according to the literature. 57PF is considered a measure of UV-B shielding performance.With the increasing mucilage content, SPF values rise (Table 1).In order to see the effect of the mucilage content, the protection percentage, which is equal to 100 À (100/SPF), 55 was calculated.The results showed that, at low mucilage content, the increment rate of protection percentages is higher.For instance, when the mucilage content increases from 5% to 10%, the increment in protection percentage is about 20%.However, there was only a 2% change when the content increased from 40% to 50%.This result shows that the effect of chemical or physical properties of mucilage on UV protection is a more decisive factor than its concentration.

| Mechanical properties of mucilage/PVA films
The mechanical properties of the films were investigated by the tensile test, and tensile strength, elongation at break, and elastic modulus values were calculated.The results are given in Figure 9.The tensile strength value of neat PVA film (0M-PVA) was determined to be 9.86 MPa.A similar result was reported by Koteswararao et al. 58 as $10 MPa for the tensile strength of a solution cast neat PVA film, which had the same molecular weight as the one used in this study.The tensile strength of films composed of 5, 10, 15, and 20M-PVA were determined as 14.56, 11.90, 11.56, and 11.25 MPa, respectively (Figure 9B).Compared with the neat PVA film, the enhanced strength of these films was attributed to the ionic cross-linking of PVA chains by Na and Cl ions coming from the mucilage.Figure 9A depicts the ionic F I G U R E 8 Ultraviolet-visible (UV-Vis) spectra of bioplastic films.
T A B L E 1 Ultraviolet-visible (UV-Vis) spectra results of the biofilms.cross-linking between PVA and mucilage in the films.With further increasing the mucilage ratio, the tensile strength of the films was adversely affected because of the salt crystal formations, as previously shown by SEM and XRD results.The tensile strength was weakened by the heterogeneity of salt crystals and suddenly dropped to 5.59, 4.45, and 2.88 MPa for 25, 40, and 50M-PVA, respectively (Figure 9B).As a result, the strength of the films was enhanced by ionic cross-linking up to a critical mucilage content (20%), then worsened at the higher contents because of the salt crystal formation.

Sample
Tensile test results revealed that the inclusion of mucilage has a slightly negative impact on the elongation at the break of the bioplastic films (Figure 9C).In general, the elongation exhibits an opposite behavior with tensile strength. 59Our results corresponded to this relation.The ionic cross-linking led to a strong interaction between PVA chains and hardened the chains sliding over each other under tensile force, hence causing lower elongations.
The highest elastic modulus values of the bioplastic films were determined for 5M-PVA and 10M-PVA as 22.70 and 22.81 MPa, respectively.The elastic modulus of 15M-PVA and 20M-PVA were around 15 MPa, which was close to the pure PVA film (Figure 9D).At the above mucilage contents, the modulus values further decreased and were calculated as 5.63, 8.30, and 3.37 MPa for 25M-PVA, 40M-PVA, and 50M-PVA, respectively.These results suggested that the addition of a certain amount of the mucilage increased the elastic modulus up to a critical value as a consequence of the increasing strength by the ionic cross-linking.
Supplementary Figure S2 demonstrates the obtained stress-strain curves for all samples.With the addition of mucilage to the PVA bioplastic film, the stress-strain curves altered, and depending on the mucilage ratio, the curves with different behaviors were seen.The stressstrain curves clearly demonstrated that the tensile strength increased as the mucilage ratio reached 20%.
Moreover, among bioplastic films, 10M-PVA had greater flexibility with high tension.Therefore, this film would break at a slower and more consistent rate.The deterioration of the mechanical properties of the films containing 25%, 40%, and 50% mucilage was further supported by stress-strain curves.

| Antibacterial test results
Regarding the mechanical performance and the mucilage content, 10M-PVA was determined to be an optimum film.Hence, the antibacterial tests were performed with this bioplastic film.As seen in Figure 10, the 10M-PVA bioplastic films exhibited antibacterial activity against E. coli and S. aureus and generated an antibacterial zone surrounding the film (Table 2).It is well known from the literature that PVA does not have antibacterial activity.So, the antibacterial activity of 10M-PVA was attributed to the mucilage content.Therefore, the antibacterial activity of the mucilage was further investigated.
As seen in Figure 11, the mucilage discs showed antibacterial activity.The mucilage contained 57% salt by mass, and the activity may have stemmed from the salt content.The test was also performed with dialyzed mucilage to understand whether the antibacterial activity was due to the salt present in the mucilage.To do so, powdered mucilage was dialyzed against distilled water to remove salts, and 1% salt by mass was determined after dialysis.
Dialyzed (D +) mucilage discs did not exhibit antibacterial activity against E. coli and S. aureus, while nondialyzed (D À) mucilage discs exhibited antibacterial activity against E. coli and S. aureus (Figure 11).These results strongly suggest that the antibacterial activity of the biofilm originated from the salt content of the mucilage.Supportively, the antibacterial activity of NaCl is known from the literature. 60

| CONCLUSION
The marine mucilage that occurred in the Sea of Marmara in 2021 blanketed the sea column and its surface, resulting in various detrimental consequences.This catastrophe accelerated mucilage prevention and disposal studies significantly.In this study, the usage of mucilage bio-waste as an additive for plastics to gain functional properties was aimed.So, the marine mucilage and PVA-containing bioplastic films were effectively produced using the solution casting method.The prepared bioplastic films were characterized using FTIR, TGA, DSC, XRD, SEM-EDS, and UV-vis.The mechanical qualities and antibacterial activity of the films were also investigated.
From the FTIR spectrums, the interaction between the mucilage and PVA was concluded due to the shift of the hydroxyl peaks.SEM images revealed a homogenous distribution of the mucilage in the PVA matrix up to 20% mucilage content, and at the above mucilage contents, salt crystal formations were seen and attributed to the ionic saturation of the polymer matrix.The EDX analysis confirmed that the crystal structure was NaCl salt.It has been discovered that increasing the mucilage ratio in bioplastic films results in better UV-Vis protection.Furthermore, the mucilage content in PVA films increased the strength of the films up to 20% mucilage content and influenced the mechanical properties favorably.The enhancements in the mechanical properties were attributed to ionic cross-linking of PVA by Na + and Cl À ions.The antibacterial tests by the disc diffusion method revealed that the mucilage films had antibacterial activity due to the salt in the content.
Consequently, a biodegradable biocomposite film with good mechanical properties, UV protection capability, and antibacterial activity was developed from the marine mucilage bio-waste.Thus, mechanical, chemical, physical, and biological interactions of marine mucilage with polymers have been studied and elucidated in detail.Although it may be considered a restricted biomaterial, the characterization results of marine mucilage, which has a multicomponent structure containing inorganic and organic compounds, revealed in this study will provide valuable information about the properties of many other natural materials incorporated into polymers.

F
I G U R E 1 Schematic illustration of the collection and pulverization of mucilage.F I G U R E 2 Schematic illustration of the preparation of mucilage bioplastic films.

F
I G U R E 3 Fourier-transform infrared (FTIR) spectra of marine mucilage, virgin polyvinyl alcohol (PVA), and bioplastics (A) and the increasing of hydroxyl peak intensity and shifting of peak (B).

F I G U R E 6
Thermogravimetric analysis (TGA) of bioplastic films.F I G U R E 7 Differential scanning calorimetry (DSC) graphs of bioplastic films.

T A B L E 2 27 F I G U R E 1 1
The zone diameters (mm) formed by mucilage and bioplastic film.Antibacterial test results of dialyzed (D (+)) and non-dialyzed (D (À)) mucilage.