Inspiration of combined additive on the yield of humic and fulvic acids and the formation of functional groups to mitigate heavy metal bioremediation.

Current humic substances (HSs) production for bioremediation suffers from low yields and inconsistent functionality. The present work examined humic acid (HA) and fulvic acid (FA) extraction from compost treated with (1) molasses alone (CK), (2) Pseudomonas aeruginosa (T1), (3) Bacillus firmus (T2), and (4) a synergistic combination of molasses and both microbes (T3). The humic substances (HSs) were extracted using the standard alkaline extraction followed by the acid precipitation method. HSs were characterized over a period of 75 days using inductively coupled plasma mass spectrometry (ICP-MS) for heavy metals, Fourier-transform infrared spectrophotometer (FTIR), UV-Vis spectrophotometer, and X-ray photoelectron spectroscopy (XPS) for functional groups, along with elemental analysis. Results demonstrated that T3 significantly enhanced HA yield (4.76 g/kg) with optimal C/N (1.06) and E4/E6 (4.33) ratios, while T1 yielded the highest FA (2.18 g/kg) by day 60. In the HA fraction, T3 significantly reduced HM concentrations of Cd by 73%, Zn by 68%, and Fe by 45% in HA fractions, while revealing Cu and Mn bioavailability, providing a novel insight for targeted remediation indicating enhanced stabilization. Functional group engineering was validated by XPS, which indicated that T3 is uniquely enriched with redox-active groups, specifically carbonyl (C = O, 531.99 eV) and hydroxyl (C-OH, 532.96 eV). These groups facilitate the binding of nitrogen/sulfur species (amide: 400.24 eV; thiol: 163.45 eV), thereby enhancing bioremediation processes. Elemental analysis revealed enriched carbon (HA: 55.04%; FA: 56.24%) and oxygen (HA: 31.87%; FA: 31.73%), alongside elevated nitrogen, sulfur, and hydrogen. The T3 synergy demonstrates immediate applicability in the rehabilitation of contaminated soil.