The ability to enzymatically immobilize TNT biotransformation metabolites onto humic monomers was investigated using new laccase producing fungal isolates, namely: Trametes modesta BT2368, Trametes versicolor BT2570, Trametes hirsuta BT2566, T. hirsuta BT2133 and Sclerotium rolfsii CBS 350.80. Degradation of TNT by these organisms was confirmed using HPLC and it was shown to lead predominantly to biotransformation. Aminodinitrotoluenes (4-aminodinitrotoluene (4-AMDNT) and 2-aminodinitrotoluene (2-AMDNT)) were identified as the major biotransformation products. T. hirsuta BT2566 cultures supplemented with 220 mu M TNT produced the highest amounts of 4-AMDNT and 2-AMDNT up to a concentration of 104 and 52 mu M after 96 h of incubation, respectively. Incorporation of 200 mM of humic monomers especially ferulic acid, guaiacol and catechol during the biotransformation of TNT drastically reduced the accumulation of aminodinitrotoluenes to below 30%. In the presence of a purified laccase from T. hirsuta complete immobilization of 2-hydroxylaminodinitrotoluenes (2-HADNT) and 4-hydroxylaminodintrotoluenes (4-HADNT) by the different humic monomers was achieved. Ferulic acid was the most effective humic monomer in immobilizing TNT metabolites both in in vitro and culture experiments. (c) 2006 Elsevier Inc. All rights reserved.