A novel P450-initiated biphasic process for sustainable biodegradation of benzo[a]pyrene in soil under nutrient-sufficient conditions by the white rot fungus Phanerochaete chrysosporium

Abstract

High molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) such as benzo[a]pyrene (BaP) are resistant to biodegradation in soil. Conventionally, white rot fungus Phanerochaete chrysosporium has been investigated for HMW-PAH degradation in soil primarily using nutrient-deficient (ligninolytic) conditions, albeit with limited and non-sustainable biodegradation outcomes. In this study, we report development of an alternative novel biphasic process initiated under nutrient-sufficient (non-ligninolytic) culture conditions, by employing an advanced experimental design strategy. During the initial nutrient-sufficient non-ligninolytic phase (16 days), the process showed upregulation (3.6- and 22.3-fold, respectively) of two key PAH-oxidizing P450 monooxygenases pc2 (CYP63A2) and pah4 (CYP5136A3) and formation of typical P450-hydroxylated metabolite. This along with abrogation (84.9%) of BaP degradation activity in response to a P450-specific inhibitor implied key role of these monooxygenases. The subsequent phase triggered on continued incubation (to 25 days) switched the process from non-ligninolytic to ligninolytic resulting in a significantly higher net degradation (91.6% as against 67.4% in the control nutrient-limited set) of BaP with concomitant de novo ligninolytic enzyme expression making it a biphasic process yielding improved sustainable bioremediation of PAH-contaminated soil. To our knowledge this is the first report on development of such biphasic process for bioremediation application of a white rot fungus.

Keywords: Genetic algorithm; Non-ligninolytic; P450; PAH biodegradation; Response surface methodology; Transcript analysis in soil.

Fig. 1

Overall design of the optimization strategy for the developed Biphasic process for BaP degradation in soil.

Fig. 2

Response surface plots depicting influence of parameter interactions on the biodegradation of BaP by P. chrysosporium in the soil treatment process. (a) Carbon and nitrogen interaction; (b) carbon and pH interaction; (c) carbon and sawdust interaction; (d) pH and sawdust interaction; (e) nitrogen and pH interaction; (f) nitrogen and sawdust interaction. In addition to the variables considered in the individual plots, hold values were 4% (w/v) carbon, 25 mM nitrogen, 4.5 initial pH, and 5% (w/w) sawdust.

Fig. 3

Response surface plots depicting influence of parameter interactions on the concentration of the P450 monooxygenase metabolite 3OH-BaP generated by P. chrysosporium in the soil treatment. (a) Carbon and nitrogen interaction; (b) carbon and pH interaction; (c) carbon and sawdust interaction; (d) nitrogen and pH interaction; (e) nitrogen and sawdust interaction; (f) sawdust and pH interaction. In addition to the variables considered in the individual plots, hold values were 4% (w/v) carbon, 25 mM nitrogen, 4.5 initial pH, and 5% (w/w) sawdust.