Marine Waste to Nano-Wealth: Synthesis and Characterization of Chitosan Nanoparticles from Mollusca Shells with Antimicrobial Insights

Abstract

This study explores the transformation of molluscan shell waste from the Nellore coast of Andhra Pradesh into high-value chitosan nanoparticles (CNPs) using a green, biologically inspired approach. Chitin was extracted by enzymatic activity of A.niger, A. terrus and A. flavus used for deprotienization, Demineralisation and Bascillus sp used to obtain chitosan. The resulting chitosan was subjected to ionic gelation with sodium tripolyphosphate (TPP) to yield stable nanoparticles. These CNPs were thoroughly characterized using techniques including UV-Vis spectroscopy, FTIR, XRD, SEM-EDX, DLS, and zeta potential analyses. The average particle size was 112.4 ± 9.1 nm and the zeta potential measured +28.7 mV, suggesting good colloidal stability. Antimicrobial activity of the CNPs was systematically evaluated against a panel of bacterial and fungal pathogens, including Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus flavus. The nanoparticles demonstrated broad-spectrum efficacy with distinct zones of inhibition and low minimum inhibitory concentrations (MICs). Particularly notable was their potent bactericidal effect against Gram-positive S. aureus and significant antifungal activity against C. albicans. The antimicrobial mechanism is likely driven by electrostatic interactions with microbial membranes, leading to disruption of cellular integrity and leakage of intracellular contents. These results underscore the dual benefit of valorizing marine waste into eco-friendly nanomaterials and deploying them as effective antimicrobial agents. The study highlights the promising role of mollusca-derived chitosan nanoparticles in sustainable aquaculture management, water purification, and potential biomedical applications.

Introduction

1. Overview

Marine bio-waste, especially molluscan shells, is a rich source of chitin, a natural polymer that can be converted into chitosan—a biodegradable, biocompatible material with wide-ranging applications in biomedicine, agriculture, and food packaging. Transforming this waste into chitosan nanoparticles (CNPs) provides environmental and economic value, especially for antimicrobial use in aquaculture.

2. Source and Extraction of Chitin

• Shell Collection: Molluscan shells (e.g., Monacha cantiana, Lissachatina fulica, Murex trapa) were sourced from seafood waste in Nellore, Andhra Pradesh.

• Chemical Extraction: Involves deproteinization (NaOH), demineralization (HCl), and decolorization (ethanol). Optimized for temperature, pH, and concentration.

• Biological Extraction: Utilized fungi (Aspergillus niger, A. terrus, A. flavus) for eco-friendly deproteinization, demineralization, and decolorization. Resulted in:

  • Higher yield (514.32 ± 22.14 g vs. 474.66 ± 25.02 g chemically)

  • Better structural integrity and purity

3. Chitosan Production

• Biological Deacetylation: Bacillus species isolated from aquaculture sediments were screened for chitin deacetylase (CDA) activity.

• Optimization: Carbon and nitrogen sources, pH (5–8), and temperature (16–45°C) were optimized for maximum CDA activity.

• Enzyme Assays: Conducted to measure CDA activity and Degree of Deacetylation (DD).

4. Chitosan Nanoparticle (CNP) Synthesis

• Method: Ionic gelation using sodium tripolyphosphate (TPP).

• Process:

  • 0.1% chitosan in 1% acetic acid

  • TPP added dropwise under stirring

  • Centrifugation and washing to isolate nanoparticles

5. Characterization of CNPs

• Size & Charge: Dynamic Light Scattering (DLS) and Zeta Potential analysis showed nanoscale size and stable surface charge.

• Morphology: Scanning Electron Microscopy (SEM) revealed mostly spherical, smooth nanoparticles.

• Composition: EDX confirmed organic nature (C, N, O).

• Functional Groups: FTIR identified typical chitosan peaks (O–H, N–H, amide I/II, C–O–C).

• Crystallinity: XRD analysis showed reduced crystallinity, improving solubility and bioactivity.

6. Antimicrobial Activity

• Antibacterial Testing:

  • Well diffusion method used against E. coli, S. aureus, P. aeruginosa, B. subtilis.

  • Inhibition zones observed, increasing with CNP concentration.

• Antifungal Testing:

  • Poisoned food method used against Candida albicans, Aspergillus niger, Fusarium oxysporum.

  • Mycelial growth inhibition calculated.

7. MIC, MBC, and MFC Analysis

• MIC (Minimum Inhibitory Concentration):

  • Determined using broth microdilution.

  • Effective against both bacteria and fungi.

• MBC/MFC (Minimum Bactericidal/Fungicidal Concentration):

  • Determined via sub-culturing onto agar plates.

  • Confirmed complete microbial inhibition at defined concentrations.

8. Key Findings

• Chitosan nanoparticles from molluscan waste are effective antimicrobial agents, especially for aquaculture pathogens.

• Biological extraction methods are superior in terms of yield, environmental safety, and product quality.

• CNPs offer significant potential for eco-friendly solutions in managing antibiotic resistance in aquatic systems.

Conclusion

The current study successfully demonstrates the eco-friendly conversion of molluscan shell waste collected from the Nellore coast of Andhra Pradesh into high-value chitosan nanoparticles (CNPs) using a biologically inspired green synthesis approach. Chitin extracted from the shells underwent mild thermal deacetylation to produce chitosan, which was then crosslinked using sodium tripolyphosphate (TPP) via ionic gelation to yield stable nanoparticles. The synthesized CNPs exhibited favorable physicochemical properties, including an average particle size of 112.4 ± 9.1 nm and a positive zeta potential of +28.7 mV, indicating good colloidal stability. Characterization through UV-Vis, FTIR, XRD, SEM-EDX, DLS, and zeta potential confirmed the formation and structural integrity of the nanoparticles. The antimicrobial assessment revealed broad-spectrum efficacy of the CNPs against both bacterial and fungal pathogens. Notably, Staphylococcus aureus and Candida albicans showed significant susceptibility, suggesting the strong antimicrobial potential of the nanoparticles. The observed effects are likely driven by electrostatic interactions between the positively charged chitosan nanoparticles and negatively charged microbial membranes, resulting in cellular disruption and leakage of intracellular components. These findings emphasize the dual advantage of marine waste valorization and development of potent nanobiomaterials with biomedical and environmental relevance. Looking forward, the promising characteristics of these CNPs open new avenues for their application in aquaculture wastewater treatment. Aqua industry effluents often contain high levels of nutrients (phosphates, nitrates), organic matter, antibiotics, and microbial contaminants, which can lead to eutrophication and disease outbreaks. Chitosan nanoparticles, owing to their high surface area, cationic nature, and biocompatibility, offer a multifunctional solution by acting as both adsorbents and antimicrobial agents. Future research should focus on evaluating the adsorption efficiency of these nanoparticles for nutrient removal, optimizing their use in real aquaculture effluent systems, and integrating them into filtration or bioremediation platforms. Pilot-scale studies in aquafarms could further validate their impact on improving water quality, enhancing shrimp or fish health, and supporting sustainable aquaculture practices. The incorporation of probiotics or bioactive plant extracts along with CNPs may also enhance their functionality, making them valuable in the development of holistic wastewater treatment systems.

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Copyright © 2025 Samuel Sushma Angel, Gandhi N, Vijaya Ch. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.