Fulvic acid belongs to a family of organic materials known as humic substances, the dark, chemically complex residues left behind when microorganisms decompose plant and animal matter. Humic substances are conventionally divided into three fractions according to their solubility: humic acid, which dissolves in alkaline solutions but precipitates when the pH drops below 2; humin, which is insoluble under all conditions; and fulvic acid, the fraction that remains soluble across the entire pH range. This solubility gives fulvic acid unusual versatility, and its molecular architecture is studded with reactive functional groups, including carboxyl, phenolic, quinone, carbonyl, hydroxyl, and amine moieties, many of which have documented biological activity. The exact structure of fulvic acid remains incompletely understood, but capillary electrophoresis and mass spectrometry studies suggest it should be viewed not as a single high-molecular-weight compound but as an assembly of smaller molecules that associate in solution.

The research team collected cow dung from cattle farms in village Chanesar Chachar, Taluka Pano Aqil, District Sukkur, dried it for five to six days at room temperature, and ground it into a fine powder. Extraction followed the protocol of the International Humic Substances Society: the powdered manure was mixed with 80 milliliters of 5 percent sodium hydroxide solution, shaken for four hours, and left to stand for twelve hours. After dilution with deionized water and centrifugation at 3000 revolutions per minute, hydrochloric acid was added to bring the pH down to between 1 and 2, precipitating the humic acid fraction and leaving fulvic acid in the supernatant, which was then collected and dried for analysis. The method is deliberately simple, relying on cheap reagents and standard laboratory equipment, which is precisely what makes the approach attractive for resource-limited settings.

One of the study’s most technically interesting observations concerns extraction efficiency. When 25 grams of dung powder were processed per liter of 0.5 N sodium hydroxide, the yield was 2.5 grams, or 10 percent of the starting mass. Doubling the raw material to 50 grams produced 4 grams, an 8 percent yield, and 100 grams yielded just 6 grams, or 6 percent. The percentage yield therefore falls as the solid loading rises, an inverse relationship the authors attribute to reduced solvent accessibility and less efficient mass transfer at higher concentrations, possibly compounded by saturation effects in the alkaline solubilization process. For anyone hoping to scale up production, the lesson is counterintuitive but clear: less material per batch extracts more efficiently, a trade-off between throughput and yield that will need optimization before any industrial process could be contemplated.

Characterization of the extracted material relied on three complementary analytical techniques. Ultraviolet-visible spectroscopy showed a maximum absorption at 316 nanometers, closely matching a commercial fulvic acid standard from Aldrich, indicating similar chromophoric structures arising from pi-to-pi-star transitions in aromatic and conjugated systems. Fourier transform infrared spectroscopy then mapped the functional group chemistry: a broad band between 3400 and 2900 reciprocal centimeters corresponding to O-H and N-H stretching, a sharp peak near 2929 from aliphatic C-H stretching of methyl and methylene groups, bands between 1598 and 1507 assigned to aromatic carbon-carbon stretching and quinone and conjugated carbonyl contributions, a peak at 1420 reflecting carboxylate asymmetric stretching and phenolic C-O stretching, and a strong absorption at 1034 from C-O stretching of alcohols and polysaccharide structures. The spectrum of the extracted material closely paralleled that of the standard, confirming successful isolation of humic-type functional groups.

X-ray diffraction completed the structural picture. The diffraction pattern displayed a broad background hump between 15 and 35 degrees two-theta, the signature of a largely amorphous material, superimposed with several sharp crystalline peaks. The most intense peak appeared at approximately 44.71 degrees with a d-spacing of 2.0276 angstroms, and a second prominent peak at 31.74 degrees with a d-spacing of 2.8195 angstroms. The combination of diffuse scattering and discrete reflections indicates a semi-crystalline structure in which small crystalline domains are embedded in a dominant amorphous matrix, with minimal impurities, consistent with the successful isolation of a heterogeneous natural organic mixture rather than a single crystalline compound.

The antimicrobial testing followed classical microbiological methodology. Reference strains of E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and Salmonella Typhi (ATCC 14028) were cultured on Mueller-Hinton agar, and inocula were standardized to the 0.5 McFarland turbidity standard. Fulvic acid was dissolved in dimethyl sulfoxide, with the solvent held at or below 1 percent by volume and a solvent-only control included to rule out false positives. In the agar well diffusion assay, the dose-response relationship was unambiguous. At 0.1 grams per 5 milliliters, no inhibition zones appeared against any of the three organisms. At 0.2 grams per 5 milliliters, zones of 11, 10, and 9 millimeters emerged for E. coli, P. aeruginosa, and S. Typhi respectively. Raising the concentration to 0.4 grams per 5 milliliters expanded the zones to 12, 11, and 11 millimeters, and at 0.8 grams per 5 milliliters the zones reached 14, 15, and 12 millimeters. The maximum concentration tested, 1.6 grams per 5 milliliters, produced the largest zones: 16 millimeters against E. coli, 15 against P. aeruginosa, and 14 against S. Typhi.

Quantitative susceptibility testing sharpened the hierarchy. Using a two-fold dilution series from 1000 down to 31.25 micrograms per milliliter, the team determined minimum inhibitory concentrations ranging from 250 to 1000 micrograms per milliliter and minimum bactericidal concentrations from 500 to 2000 micrograms per milliliter. E. coli proved the most susceptible, inhibited at 250 micrograms per milliliter, followed by P. aeruginosa at 500, while Salmonella Typhi required a full 1000 micrograms per milliliter. The authors connect this graded susceptibility to differences in cell envelope architecture: P. aeruginosa deploys a famously impermeable outer membrane and active efflux pumps, while E. coli and S. Typhi present different membrane compositions and permeability characteristics. Mechanistically, the antimicrobial action is attributed to fulvic acid’s phenolic, carboxylic, and quinone functional groups, which may disrupt microbial membranes, chelate essential metal ions, and generate redox-mediated oxidative stress, mechanisms well documented for structurally related phytophenolic compounds.

The authors are careful to frame these results as preliminary, and the caveats deserve emphasis. The activity was demonstrated only against planktonic, free-floating bacteria in vitro, using agar diffusion and broth dilution assays. No mechanistic studies of bacterial interaction pathways, no cytotoxicity profiling on mammalian cell lines, no stability or bioavailability assessments, and no tests against clinically resistant strains or biofilm models were performed. The dataset, as the researchers themselves state, is insufficient to support direct application in biomedical, agricultural, or environmental systems, and any translational implications must be considered tentative. What the study does establish is a proof of concept: cow dung is a viable, low-cost raw material for fulvic acid extraction, the isolated product carries the structural fingerprints of authentic fulvic acid, and it exerts measurable, concentration-dependent antibacterial activity against three major Gram-negative pathogens.

Even within those limits, the work lands at a compelling moment. Humic substances are already used in agriculture as soil conditioners and fertilizers, added to animal feed to improve growth and immunity, and investigated as anti-inflammatory, antitumor, and wound-healing agents. If subsequent research confirms that fulvic acid’s antimicrobial effects survive purification, formulation, and contact with living systems, a substance that farmers currently discard, or that poses a disposal and sanitation burden in much of the developing world, could be converted into a feedstock for natural antimicrobials. The road from a petri dish in Khairpur to a clinic or a feed additive is long, and the required in vivo studies, toxicity evaluations, and resistance testing will take years. But the underlying idea, that the answer to drug-resistant bacteria might be composting at the edge of the village, is exactly the kind of cross-disciplinary leap that the antimicrobial resistance crisis demands, and this study offers the first careful, spectroscopically verified evidence that the leap is worth taking.

Subject of Research: Antimicrobial activity of fulvic acid extracted from cow dung against Gram-negative bacteria

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

Article References: Chachar, Z. A., Dharejo, S. A., & Pirzada, T. (2026). Antimicrobial activity of fulvic acid extracted from bovine dung against selected gram-negative pathogens: a preliminary in vitro assessment. Discover Chemistry, 3(1), Article 548. https://doi.org/10.1007/s44371-026-00935-8

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00935-8

Keywords: fulvic acid, cow dung, antimicrobial resistance, Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhi, humic substances, FTIR spectroscopy, X-ray diffraction, minimum inhibitory concentration, natural antimicrobials