Heterophase Synthesis of Humic Substances at Low Substrate Concentrations and Flow-Through Conditions

Abstract

Abstract—Humic substances (HS) are heterogeneous and polydisperse compounds formed in soils, sediments and waters during the decay and transformation of organic residues (the process called humification). The role of extracellular free-radical condensation reactions (secondary synthesis) in the formation of HS is a subject of debate. Here we have studied the formation of HS from a mixture of monomers under the dynamic conditions and at low substrate concentrations in the presence of laccase from the fungusCoprinus comatusF-2940. Laccase was immobilized on kaolinite modified with aluminum hydroxide. We have investigated some properties of the enzyme, reactivity of phenolic acids and amino acids in the presence of laccase. The optimum of 2.6-dimethoxyphenol (2.6-DMP) oxidation by free laccase was at pH 6.0. Upon immobilization, it shifted to the acidic region (pH 4.4), the thermal stability decreased, however the kinetic parameters of 2.6-DMP oxidation remained unchanged. In terms of reactivity (% of substrate conversion by free laccase) the individual phenolic acids formed a series: caffeic (72) > ferulic (53) > gallic = syringic (43) > protocatechuic (5.5) > vanillic = p-hydroxybenzoic (0). In the mixture of phenolic acids, gallic acid was most efficiently oxidized (50%), while the other acids were oxidized in comparable amounts (13–17%). The conversion of phenolic acids increased in the presence of lysine. When a mixture of gallic, protocatechuic, syringic, ferulic acids (0.01 mM each) and lysine (0.02 mM) was passed through a flow-through microcolumn, immobilized laccase effectively oxidized the phenolic acids, the reaction products bound to the mineral phase, staining it dark. According to high performance liquid chromatography, the molecular weights of compounds extracted from the mineral phase did not exceed 900 Da, thus fulvic acid-like substances were formed. Results of the study suggest an important role of free-radical heterophase reactions in the formation of the molecular composition of the liquid phase and organo-mineral complexes.