Antimicrobial activity of fulvic acid extracted from bovine dung against selected gram-negative pathogens: a preliminary in vitro assessment

Abstract This study investigates the extraction, physicochemical characterization, and antimicrobial activity of fulvic acid (FA) derived from cow dung as a low-cost natural resource. Fulvic acid was extracted through alkaline treatment using 0.5 N sodium hydroxide, and the extraction efficiency was evaluated at varying raw material loadings. An inverse relationship between cow dung quantity and percentage yield was observed, with the maximum yield (10%) obtained from 25 g of cow dung, followed by 8% from 50 g to 6% from 100 g per liter of NaOH solution, indicating enhanced extraction efficiency at lower solid concentrations. Structural characterization was performed using Fourier transform infrared (FTIR) spectroscopy, which revealed characteristic functional groups of fulvic acid, including broad O–H/N–H stretching vibrations (3300 cm⁻¹), aliphatic C–H stretching (2900 cm⁻¹), aromatic and carbonyl functional groups (1500–1624 cm⁻¹), and C–OH stretching vibrations (1000–1026 cm⁻¹). X-ray diffraction (XRD) analysis indicated a semi-crystalline structure with minimal impurities, confirming the successful isolation of fulvic acid. The antimicrobial activity of the extracted FA was evaluated against Gram-negative bacterial strains, including Escherichia coli , Pseudomonas aeruginosa , and Salmonella Typhi , using the agar well diffusion and broth microdilution methods. The extract exhibited concentration-dependent antibacterial activity, with inhibition zones ranging from 0 to 16 mm , where the maximum inhibition (16 mm) was observed against E. coli at 1.6 g/5 mL, while no inhibition was detected at 0.1 g/5 mL. The minimum inhibitory concentration (MIC) values ranged from 250 to 1000 µg/mL , whereas the minimum bactericidal concentration (MBC) values ranged from 500 to 2000 µg/mL , indicating that E. coli was the most susceptible organism, followed by Pseudomonas aeruginosa and Salmonella Typhi . These findings suggest that fulvic acid derived from cow dung possesses promising antibacterial properties against selected Gram-negative pathogens and may serve as a potential natural antimicrobial agent.