Potent antimicrobial and antibiofilm activity of citric acid coated magnetite nanoparticles for leather preservationDownload PDF Download PDF ArticleOpen accessPublished: 31 July 2025Amina Hayat1,Asma Irshad1,Uzair Ishtiaq2,3,Qudsia Mushtaq4,Alexis Spalletta5,Patrick Martin5,Rabbia Jawad6 &…Tahira Batool7 Scientific Reports volume 15, Article number: 27889 (2025) Cite this articleSubjectsBiotechnologyChemistryEnvironmental sciencesMicrobiologyNanoscience and technologyAbstractThe leather industry is a key contributor to the country’s economy but faces serious concerns about surface protection from microbial contamination. Various chemical methods are being applied to leather surface processing but they often release topic compounds dangerous for human body. Nanoparticles endowed with antimicrobial properties are proved to be an efficient approach for leather protection. The current study provides eco-friendly approach for synthesis and characterization of citric acid-coated magnetite nanoparticles, examining their potential antimicrobial agent within the leather industry. Magnetite nanoparticles (Fe3O4) were synthesized via aqueous co-precipitation method, subsequently functionalized with citric acid and characterized through UV-visible spectroscopy, FTIR, SEM-EDAX, and XRD. The antimicrobial activity against pathogenic bacteria and fungi was evaluated through agar well-diffusion method, minimum inhibitory concentration (MIC), and biofilm inhibition. All the results were statistically calculated through one-way ANOVA. UV-visible spectroscopy showed peak for Fe3O4 NPs at 280 nm while for Fe3O4@CA at 310 nm. The FTIR spectrum showed various distinct peaks at 3211.48, 1579.70, 1409.96, 1344.38, 1018.41, and 675.09 cm−1 and SEM-EDAX revealed semi-spherical morphology of nanoparticles with average particle size 40 nm. The XRD graph showed peaks at 2Ɵ of 27.2o, 35.7o, 47.1o, 57.0o and 60.8o which intimated to the crystal plane of (220), (311), (400), (511) and (440), respectively. The distinct zones of inhibition were observed against these pathogenic strains i.e. and Escherichia coli (ATCC 15597) (27 ± 0.9 mm), followed by Aspergillus niger (23 ± 0.2 mm) and Staphylococcus aureus (ATCC 25923) (22 ± 0.7 mm). Results of MIC i.e. 0.3 mg/mL for bacterial strains and 0.625 mg/mL for fungal strains were the least concentration of inhibition while biofilm inhibition with no visible growth in Fe3O4@CA containing samples, revealed the excellent antimicrobial potential of Fe3O4@CA nanoparticles. These findings suggest an effective method for synthesizing Fe3O4@CA nanoparticles, whose antimicrobial properties will be advantageous for protecting leather material from various microbial contaminations.IntroductionLeather industry is a keystone for the global economy as it significantly contributes to various sectors including fashion, furniture, bags and automotive engineering1. Despite economic importance, the safe and eco-friendly leather production and protection remains a pressing concern. Leather processing has various stages, which causes contamination issues, for environment and leather consumers, comprising different factors i.e. harmful chemical residues, pollutant absorption, and microbial proliferation and penetration2. Hence these problems imposed substantial challenges in the quality and durability of leather and leather-based accessories3. Moreover, after leather processing, improper disposal of excessive waste material from leather factories introduces environmental hazards due to integration of toxic chemicals and reagents from leather manufacturing into biological life cycle4. Despite all these artificial chemical-based treatments, pathogenic microbial strains are still a serious challenge for leather industry as various fungal and bacterial strains i.e. Aspergillus niger and Escherichia coli, respectively, continuously grow on the leather surface leads to reduced quality and life span of leather products5. Addressing these issues, need an innovative, sustainable and eco-friendly approach, which aligns with the global requirements in order to reduce microbial pollution as well as human health hazards. In this regard, nanotechnology has played remarkable role in the field of scientific research where wide range of nanoparticles, with their excellent and unique physio-chemical properties6, are extensively used to treat leather and leather based-products7,8.Recent advancements in nanotechnology have led to the manufacturing of multifunctional nanomaterials with wide range of applications in biomedical and environmental sectors. For example, waste water treatment through reduced porphyrin conjugated graphene oxide nanocomposite9, degradation of methylene blue10 and microbial contamination11 in waste water through reduced graphene oxide by using its photocatalytic activity12. Similarly, surface modification of nanoparticles can improve their antimicrobial potential and helps to treat various pathogenic resistant microbial strains. A study has explained the antifungal and antibacterial nature of titanium dioxide (TiO2) doped zinc oxide (ZnO) nanoparticles to protect leather surface and provides a better quality and durable leather products13. They have reported significant antimicrobial effects of TiO2 doped ZnO NPs which highlighted the importance of nanotechnology in leather industry13. Among various nanomaterials, magnetite (Fe3O4) nanoparticles have gained tremendous attention due to their exclusive physio-chemical and magnetic properties, high surface-to-volume ratio, and biologically compatible nature14,15. Based on these properties, Fe3O4 nanoparticles are widely used in water purification16, pollutant remediation17, and biomedical treatments18. The nature of nanoparticles to be manipulated through surface modifications make them particularly attractive for targeted applications19. Fe3O4 nanoparticles exhibited excellent antibacterial potential against broad range of pathogenic microorganisms by producing excessive amount of reactive oxygen species (ROS)20,21. These ROS enters into the bacterial cell where they penetrate into the cellular organelles, damage protein functioning, destruct enzymes molecules and inhibit nucleic acid proliferation and replication as shown in the Fig. 1. By incorporating these mechanisms, Fe3O4 nanoparticles create pores in the cell membrane of bacteria and ultimately leads to the cell death22.Fig. 1(Self-Drawn on bio render (Online)): Antimicrobial Potential mechanism of citric acid coated magnetite nanoparticles by inhibiting DNA proliferation and replication, protein denaturation and cell membrane destruction.Full size imageLiterature studies have reported limitations regarding chemical-based methods i.e. high cost, usage of toxic chemicals, and extra time consumption. These methods also prevented nanoparticles to be used for biological applications. Hence, for human use, the synthesis of nanoparticles through organic molecules is gaining importance due to better quality nanoparticles with reduce cytotoxicity. In this context, the functionalization of nanoparticles with biocompatible and eco-friendly agents, i.e. citric acid, further enhances their potential activities. Citric acid, a natural organic acid, have tremendous bioactive compounds with excellent antibacterial properties which can improves the dispersibility, stability, and functional properties of various nanoparticles23,24. Selenium nanoparticles have synthesized in the presence of citric acid using a green synthesis approach with excellent antibacterial, antifungal, antioxidant and anticoagulant potential activities25. Organic compounds are crucial to synthesize high-quality and biocompatible nanoparticles, hence offering a sustainable and bio-friendly alternative to traditional chemical and physical methods. Rich in varied bioactive components including flavonoids, polyphenols, and alkaloids, organic compounds serve as natural capping, stabilizing, and reducing agents during nanoparticle synthesis. This green synthesis approach has eradicated the use of toxic chemicals in comparison to conventional chemical methods.While use of citric acid, to synthesize nanoparticles, has been expansively employed in various applications, its use in leather processing represents a novel and propitious frontier. Incorporating green-synthesized nanoparticles into leather processing can provides an innovative approach to leather industries to revolutionize traditional methods with clean, safe, and efficient substitute to traditional chemical treatments in order to get better quality and durable leather products. This study has focused on the synthesis, characterization and biological applications of Fe3O4 nanoparticles coated with citric acid encountering adverse effects of chemical residues and microbial growth on leather surface. The structural, chemical, functional and antimicrobial properties of Fe3O4 nanoparticles have investigated in addressing leather contamination challenges. This work aims to provide a comprehensive background for the excellent antimicrobial potential of Fe3O4 nanoparticles against resistive microorganisms hence providing better leather processing, contributing to both industrial innovation and environmental conservation.Materials and methodsThe leather samples were randomly collected from various leather industries in Sialkot, Pakistan. The microorganisms i.e. Staphylococcus aureus, Escherichia coli and Aspergillus Niger were isolated from collected leather samples. The citrus lemon was purchased from local market in Lahore, Pakistan.ChemicalsAll the chemicals and reagents of analytical grade were used without further purification. Iron (II) chloride tetrahydrate (FeCl2. 4H2O > 99% purity), Iron (III) chloride hexahydrate (FeCl3. 6H2O > 99% purity), ammonia (NH4OH), and sodium hydroxide were purchased from Sigma Aldrich while citric acid was obtained from Merck. De-ionized water was used as a solvent.Isolation and incubation of fungal strainThe obtained leather samples were placed inside the desiccator as a source of tropical chamber at temperature 28 °C. Continuous sprinkling of autoclaved water was applied to maintain humidity required for the optimal growth of fungal strains. This was kept for 7 days and cultured on Potato Dextrose Agar (PDA) in the presence of 10 mg of streptomycin and 1 mL of 10% tartaric acid and incubated at 28 °C for two days. This protocol was followed by the guidelines of Asma Irshad et al., 202413. The isolated fungus was purified on yeast peptone dextrose agar, identified through Olympus CX23 microscope and characterized by Fungal Bank of University of the Punjab, Lahore, Pakistan.Synthesis of citric acid coated Fe 3O 4nanoparticlesThe magnetite nanoparticles were prepared through chemical co-precipitation method by reacting different concentration of ferrous chloride and ferric chloride i.e. 1 molar concentration of Fe2+ (FeCl2. 4H2O) and 2 molar concentration of Fe3+ (FeCl3. 6H2O), respectively. The mixture was homogenized by adding 25% ammonia (NH4OH) dropwise, with continuous stirring for 10 min at 80 °C. The pH was maintained at 8 to 12 and black precipitates were obtained, washed with de-ionized water and subjected to magnetic field. The protocol was followed by the guidelines of26 with slight modifications. The purified magnetic nanoparticles were dried, ground to fine powder and further processed for citric acid coating. For this purpose, 1 g citric acid and 1 g nanoparticles with 2 mL water were dissolved, stirred for 30 min at 90 °C and reduced the temperature as coating process was went to completion. The citric acid coated magnetite nanoparticles (Fe3O4@CA) were washed with de-ionized water, dried and ground to fine powder.Characterization of citric acid coated magnetite nanoparticlesUV-Vis spectroscopyThe concentration of synthesized nanoparticles was determined through UV-Vis spectroscopy. For this, solution of Fe3O4@CA nanoparticles was prepared in de-ionized water and UV-Vis analysis was done with the instrument UV-Vis spectrophotometer Shimadzu UV-1900i (Shimadzu Corporation, Kyoto, Japan). The spectrum was obtained in the wavelength ranges from 200 to 500 nm by keeping de-ionized water as blank13.Fourier transform infrared (FT-IR) spectroscopyThe functional groups and chemical bonds present between citric acid components and magnetite nanoparticles were investigated through FT-IR in dry air at room temperature (26 °C). The spectrum for Fe3O4@CA NPs was obtained in the presence of potassium bromide pellet by using Fourier transform infrared spectroscopy type ThermoFisher-Scientific Nicolet iS50 (ThermoFisher Scientific, USA) in the wavenumber ranges from 400 to 4000 cm−127.Scanning electron Microscopy-Energy dispersive X-Ray (SEM-EDX)The surface morphology and size distribution of Fe3O4@CA NPs was determined via scanning electron microscopy by using Nova NanoSEM 450 analyzer (ThermoFisher Scientific, USA), and Image J software was used to get the size of nanoparticles. Furthermore, SEM associated EDX profile was obtained to determine the elemental composition of Fe3O4@CA nanoparticles27.X-Ray diffraction (XRD)The crystalline structure and phase purity of Fe3O4@CA nanoparticles were determined by using Diffractometer-Bruker AXS D8 Advance (Bruker Corporation, Karlsruhe, Germany) using copper anticathode (Cu K = 1.5406 Å) under maintained constant conditions over a 2θ range from 10° to 80° for the step of 0.0101/minute13. The obtained diffractograms have been analyzed via system, based on the data sheets ASTM (American Society for Testing and Materials) with interplanar spacing (d) 2θ recorded software.The average crystalline size of Fe3O4@CA nanoparticles from FWHM (full width at half maximum) was calculated using Scherrer Eq. 1 given below;$${\rm D = K\lambda/\beta\:cos\:\theta}$$(1), where D is the average crystalline size, K is constant (shape factor, commonly 0.9), λ is the wavelength (1.5406 Å) of X-rays, β is the full width at half maximum (FWHM) of diffraction peak, and θ is the Bragg angle.Antimicrobial potential of citric acid coated magnetite nanoparticlesAgar well-diffusion methodAntimicrobial activities of Fe3O4@CA nanoparticles were estimated by agar well diffusion method against bacterial strains i.e. gram-positive strain Staphylococcus aureus, gram-negative strain Escherichia coli and fungal strain fungal strain Aspergillus niger. Potato dextrose and Luria Bertani agar were used to cultured fungal and bacterial strains, respectively. Wells were formed using cork-borer, each filling 100 µl of 1 mg/mL Fe3O4@CA nanoparticles solution. Streptomycin was used as a positive control for bacterial species while Fluconazole for fungal species by keeping water as a negative control. Plates were incubated at 37 °C (bacteria) or 28°C fungi for 24–48 h13,28.Minimum inhibitory concentration (MIC)The antimicrobial efficacy of Fe3O4@CA nanoparticles was also investigated through standard broth dilution method with reference to minimum inhibitory concentration (MIC). The MIC is the lowest nanoparticle concentration, which has ability to inhibit microbial growth. Serial two-fold dilutions of Fe3O4@CA nanoparticles were prepared in the concentrations 5, 2.5, 1.25, 0.625, and 0.312 mg/mL. The microbial concentration was arranged to 108 CFU/mL (0.5 McFarland’s standard) and both control and experimental samples were incubated at 37 °C for 24 h. Control samples were Streptomycin (for bacteria) and Fluconazole (for fungus)28.Anti-biofilm potentialThe crystal violet dye assay was performed to determine the biofilm inhibition potential of Fe3O4@CA nanoparticles. The CV dye has ability to binds with the extracellular polymeric substances of bacterial cells. The total biofilm mass formation was measured through this assay. For this, bacterial and fungal cultures were prepared and incubated at 37 °C for 48 h. Streptomycin and Fluconazole were used as reference for bacteria and fungi, respectively. The MIC Fe3O4@CA nanoparticles were added in the test sample tubes and after incubation, this material was discarded, tubes were stained with 2% CV dye, and washed with phosphate buffer saline (PBS). Then test-tube material was dissolved in 30% glacial acetic acid and absorbance was measured at 570 nm to obtain biofilm inhibition activity13.Statistical analysisThe obtained values were calculated through Minitab Statistical Software version 22.1 and results were expressed as mean ± SD. The p