Degradation of Selected PAH’s By Laccase-Mediator System in Soil

This study investigated the degradation of selected PAHs using laccase produced through cocultivation of Trichoderma
\nviride (EXF8977) and Penicillium chrysogenum (EXF1818) on solid state fermentation and its mediator system.
\nThe main objective of this work was to evaluate the ability of the laccase-mediator systems to degrade PAHs in soil, as
\nwell as the effects of natural and synthetic mediators on the laccase activity during the process of PAHs degradation.
\nThe laccase production was optimized in packed-bed bioreactor, by testing different airflow (0.05 L min-1
\n, 0.1 L min-1
\nand 0.2 L min-1
\n), temperature (RT, 30 and 40 °C) and time (2 and 3 weeks) on kiwi peels as substrate using single fungi
\nspecies and cocultivation. Extracellular laccase activity was measured spectrophotometrically using 0.5 mM 2,2'-azino-bis(3-
\nethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate on 420 nm and enzyme activity was expressed in units per
\nmillilitre (U/mL).
\nBatch degradation experiments were carried out in 100 mL Erlenmeyer flask on soil pH 5, containing PAH mixture
\n(fluorene, anthracene, pyrene, chrysene and benzo(a)pyrene) from 50 till 600 ppm in dark at 20, 30 and 40 °C. In order to
\nobtain optimum laccase degradation conditions different concentration (0.1 mM, 0.5 mM and 1 mM) of synthetic (ABTS)
\nand natural mediators (p-coumaric acid and ferulic acid) were studied. Samples were collected after 0, 3 and 5 incubation
\ndays and the compounds were measured by UHPLC. All the assays including controls were performed in triplicate.
\nThereafter column packed bed reactor was set up to evaluate the degradation with continue flow (0.015 mL/min) of crude
\nlaccase enzyme with 1 mM ABTS, p-coumaric acid and ferulic acid for 25 days, by taking samples each 5 days and evaluating
\ndegradation ratio and the remaining concentration of laccase in samples.
\nThe selected fungi were able to produce 2.95 U/mL of laccase during SSF in Erlenmeyer flask, however this amount
\nincreased with continuous airflow. Highest values were obtained at aeration rates 0.2 L min-1
\n, with a production of 6.32
\nU/mL, followed by 0.1 and 0.05 L min -1 with productions of 5.45 and 3.79 U/mL, respectively.
\nData showed laccase extracted from cocultivation of Penicillium chrysogenum and Trichoderma viride on kiwi peels
\nwith an appropriate mediator promoted the degradation of fluorene, anthracene, pyrene, chrysene and benzo(a)pyrene in
\nconcentration from 50 ppm till 600 ppm. Reaction system reached highest degradation at 3 days in case of fluorene, chrysene
\nand benzo(a)pyrene, but with anthracene and pyrene maximum degradation rate was observed between 3 and 5 days. The
\noptimizing mediator for fluorene and pyrene was p-coumaric acid, with removal of 81% and 98%, respectively. For
\nanthracene, the best mediator was ABTS with 42% degradation, and laccase with ferulic acid achieved highest degradation
\nrate with chrysene, 61%, and benzo(a)pyrene, 5.4%.
\nHowever, in packet bed reactor with continuous flow of laccase with mediator at 0.015 mL/min, for 25 days at RT (26-
\n28 °C), ABTS was found to be the optimum mediator for fluorene, anthracene, chrysene and benzo(a)pyrene, with
\ndegradation reaching 74.8 %, 71.9%, 81.8% and 96%, respectively. Around 72.2 % of phenanthrene was degraded with
\nferulic acid as mediator, and 99.9% degradation of pyrene was reached with p-coumaric acid. Less than 60% degradation
\nrate in 25 days was observed for phenanthrene and anthracene with p-coumaric acid as mediator.
\nThe results achieved in the present research demonstrated the utility of laccase from SSF production and its use in
\nbioremediation in soil, especially to reduce the concentration of PAH. However, more research must be done in real
\nconditions.

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