Biological Activity of Humic Substances Is Related to Their Chemical Structure

To understand if the biological activity of humic substances may be related to their molecular weight or chemical structure, two humic substances, derived from an Ah horizon of uncultivated couch grass [Elytrigia repens (L.) Desv. ex Nevski] and an Ah horizon of forest soil, were extensively characterized by means of different spectroscopic techniques (diffuse reflectance infrared Fourier transform [DRIFT] and 1H nuclear magnetic resonance [NMR]). The two humic substances, each separated in fractions with low (3500 Da) relative molecular mass were compared for their effects on Pinus nigra J.F. Arnold callus. Growth of callus, the soluble sugar content, free amino acid pool, and the activities of the key enzymes involved in C and N metabolism were investigated. Callus was grown for a subculture period (28 d) on basal Murashige and Skoog medium plus humic matters with or without different hormones: indole‐3‐acetic acid, 2,4‐dichlorophenoxyacetic acid, or 6‐benzylaminopurine. The results of 1H‐NMR spectra and the DRIFT spectroscopy showed significant differences in the chemical composition between forest and grass humic substances. A large amount of aliphatic and H‐sugarlike component and an intense chemical shift of the β‐CH3 region in both grass humic fractions were observed, while high contents of betaine, organic acid, and COOH groups in both forest humic fractions were detected. A different biological activity between the grass and forest humic fractions was also observed. Thus, the different activity of the two humic substances used seems related to the diverse chemical composition rather than to different molecular weights.