The research addresses the environmental concerns associated with fossil-based plastic food packaging by exploring the use of bio-based alternatives. The study focuses on coccolithophore microalgae, a sustainable resource containing both calcium carbonate (CaCO3) and bioactive compounds. The hypothesis is that incorporating coccolithophore microalgae into starch-based films will improve the films' properties, making them suitable for active and sustainable food packaging applications. This research is significant because it explores a novel material source for bioplastics, aiming to reduce reliance on non-renewable resources and improve food packaging's sustainability and functionality. Current biodegradable film development often utilizes polysaccharides like starch, but these frequently lack sufficient mechanical and barrier properties. The addition of fillers is a common strategy to enhance these characteristics. Previous studies have shown improvements using inorganic fillers like CaCO3 or organic fillers like cellulose fibers, but using microalgae biomass has yielded mixed results, often decreasing tensile strength. The unique composition of coccolithophores, combining CaCO3 and bioactive compounds, provides a potentially superior filler material. The study aims to determine the effectiveness of this novel approach and to characterize the resulting films' properties, advancing the development of active and sustainable food packaging.

The literature review covers existing research on sustainable food packaging, focusing on bioplastics and the use of starch-based films. It highlights challenges associated with starch films' mechanical and barrier properties and discusses strategies to improve them through the addition of fillers. Several studies are cited demonstrating the use of both organic and inorganic fillers, including calcium carbonate (CaCO3), cellulose fibers, and other microalgae species. While microalgae have been explored as an additive in starch-based films, the results have been inconsistent. Some studies demonstrated a decrease in tensile strength, while others noted improvement in barrier properties. Existing literature lacks investigations into the use of coccolithophore microalgae in this context. The review establishes the need for research into the unique properties of coccolithophores, including their bioactive compounds and CaCO3 content, as potential sources for creating sustainable and functional food packaging materials. The distinctive chemical composition and structure of coccolithophores, characterized by the presence of coccoliths, are also highlighted as potential advantages for material science applications.

The study employed a comprehensive methodology to evaluate the use of coccolithophore microalgae as fillers in starch-based films. Two species of coccolithophores, Emiliania huxleyi (EHUX) and Chrysotila pseudoroscoffensis (CP), were cultured and their biomass characterized by elemental analysis. Potato starch-based films were prepared using a solvent casting technique, incorporating different weight percentages (2.5, 5, 10, and 20% w/w) of EHUX, CP, and commercial CaCO3 for comparison. The resulting films were characterized using a variety of techniques. CIELab color parameters were measured to assess color changes. Thermogravimetric analysis (TGA) determined thermal stability. Mechanical properties, including Young's modulus, tensile strength, and elongation at break, were measured using a texture analyzer. Water contact angle measurements determined hydrophobicity. Film thickness and moisture content were also determined. Water vapor permeability (WVP) was assessed using a standardized method. Finally, the antioxidant activity of the films was evaluated using the ABTS radical inhibition assay. Statistical analysis, using F-tests and Student's t-tests, determined significant differences between film types. SEM microscopy imaged the microalgae and film surface morphology. The specific culture conditions for both EHUX and CP were described, including nutrient media, temperature, light intensity, and aeration. The detailed procedures for film production, including sieving, gelatinization, degassing, and casting, were provided. All measurements were performed in triplicate to ensure reproducibility. The detailed steps for each characterization technique were also described, including specific equipment and protocols used. The statistical analysis ensured that the observed differences were statistically significant and not due to random variation.

The key findings demonstrate that the incorporation of coccolithophore microalgae biomass into starch-based films significantly alters their properties. The incorporation of both E. huxleyi and C. pseudoroscoffensis, resulted in green-yellowish films. Films incorporating commercial CaCO3 showed a whitish appearance. The addition of either microalgae or commercial CaCO3 resulted in a reduction in Young's modulus, indicating a significant decrease in film rigidity. The incorporation of coccolithophores caused a more pronounced reduction in Young's modulus compared to the use of commercial CaCO3, which was most evident at the 20% filler concentration. The tensile strength was also significantly reduced with increasing microalgae concentration. Elongation at break showed a similar trend, decreasing with increasing microalgae concentration, indicating a decrease in film extensibility. The microalgae significantly improved the hydrophobicity of the films, with water contact angles exceeding 90° for films containing microalgae, especially on the top surface. This contrasts with the control films, which exhibited hydrophilic behavior. The addition of microalgae significantly enhanced the antioxidant activity of the films, as measured by ABTS radical inhibition. The antioxidant activity increased with increasing microalgae concentration, reaching 60.4% inhibition for films with 20% EHUX. This is significantly higher than the antioxidant activity observed in films with commercial CaCO3, where the inhibition remained low. The water vapor permeability (WVP) of the films was generally not significantly affected by filler addition, with a few exceptions. In particular, the use of CaCO3 at 2.5% and EHUX at 5% caused a significant decrease in WVP, while CP at 10% increased WVP. SEM micrographs confirmed the presence of intact coccoliths within the starch matrix, although some agglomeration was observed, particularly at higher filler concentrations. The protein content of EHUX and CP were consistent with previous findings reported in the literature.

The findings address the research question by demonstrating the potential of coccolithophore microalgae as viable fillers in starch-based films for food packaging applications. The significant reduction in film rigidity alongside the increase in hydrophobicity and antioxidant activity are notable achievements. The enhanced antioxidant activity is particularly important for food preservation, as it can help to extend the shelf-life of packaged goods by preventing oxidation. These improvements outweigh the decrease in tensile strength, indicating potential for specific applications requiring enhanced hydrophobicity and antioxidant properties. The results highlight the benefits of using microalgae over commercial CaCO3. The increased hydrophobicity likely results from the presence of hydrophobic compounds in the microalgae biomass such as lipids. This improved water resistance could enhance the films’ effectiveness as packaging materials. The study's limitations regarding the potential agglomeration of microalgae at higher concentrations and the need for further investigation into long-term stability and barrier properties warrant further research. Future studies should focus on optimizing the dispersion of microalgae within the starch matrix and exploring different processing methods to improve mechanical properties. The successful incorporation of microalgae biomass into bioplastics could contribute to a more sustainable and functional food packaging industry.

This study successfully demonstrated the potential of coccolithophore microalgae as fillers in starch-based films for creating sustainable and active food packaging. Both E. huxleyi and C. pseudoroscoffensis produced films with desirable properties, notably enhanced hydrophobicity and antioxidant activity. The results suggest that the bioactive compounds within the microalgae contribute significantly to these improvements. Future research could focus on optimizing film production to improve mechanical properties and exploring different types of microalgae and processing techniques. Scaling up production and conducting real-world food packaging trials will be crucial to determine the viability of this approach.

The study acknowledges some limitations, including the observation of microalgae agglomeration at higher concentrations. This agglomeration could potentially affect film properties and should be addressed in future research through optimization of the film-making process or pre-treatment of the microalgae. Further research is needed to thoroughly investigate the long-term stability of the films under various storage conditions, including assessing the maintenance of hydrophobicity and antioxidant activity. Additionally, a more in-depth analysis of the films' gas barrier properties is required to fully evaluate their suitability for different food packaging applications. The present study only focused on the short-term antioxidant activity; therefore, long-term stability assays under real-world conditions would provide more comprehensive insights.