Armillaria sp. F022, a white-rot fungus isolated from a tropical rain forest in Samarinda, Indonesia, was used to biodegrade benzo[a]pyrene (BaP). Transformation of BaP, a 5-ring polycyclic aromatic hydrocarbon (PAH), by Armillaria sp. F022, which uses BaP as a source of carbon and energy, was investigated. However, biodegradation of BaP has been limited because of its bioavailability and toxicity. Five cosubstrates were selected as cometabolic carbon and energy sources. The results showed that Armillaria sp. F022 used BaP with and without cosubstrates. A 2.5-fold increase in degradation efficiency was achieved after addition of glucose. Meanwhile, the use of glucose as a cosubstrate could significantly stimulate laccase production compared with other cosubstrates and not using any cosubstrate. The metabolic pathway was elucidated by identifying metabolites, conducting biotransformation studies, and monitoring enzyme activities in cell-free extracts. The degradation mechanism was determined through the identification of several metabolites: benzo[a]pyrene-1,6-quinone, 1-hydroxy-2-benzoic acid, and benzoic acid.
In this study we sought to optimize recovery of fluorescent aromatic compounds (FACs) from the bile of African catfish (Clarias gariepinus) injected with 10mg/kg benzo[a]pyrene (BaP). Fractions of pooled bile were hydrolyzed, combined with ten volumes of methanol, ethanol, acetonitrile, or acetone, centrifuged and supernatants were analyzed by high-performance liquid chromatography with fluorescent detection (HPLC/FL). As well, to test whether FACs were being lost in solids from the centrifugation, pellets were resuspended, hydrolyzed and mixed with six volumes of the organic solvent that produced best FAC recovery from the supernatant, and subjected to HPLC/FL. Highest FAC concentrations were obtained with 2000μl and 1250μl acetone for supernatants and resuspended pellets respectively. FACs concentrations were negatively correlated with biliary protein content but were unaffected by addition of bovine serum albumin (BSA) followed by no incubation indicating that the presence of proteins in the biliary mixture does not simply interfere with detection of FACs. In another experiment, efficiency of acetone addition was compared to two different liquid-liquid extractions (L-LEs). Acetone additions provided significantly higher biliary FACs than the L-LE methods. The new two-stage bile preparation with acetone is an efficient, inexpensive and easily performed method.