Studies on Biodegradation of Pyrene by Mycobacterium Frederiksbergense

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Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants produced via natural and anthropogenic sources, mainly generated during the incomplete combustion of solid and liquid fuels or derived from industrial activities. Due to high hydrophobicity and recalcitrant nature, they tend to contaminate soil and water and pose serious threat to receiving environment because of their mutagenicity and carcinogenicity. Of the various treatment methods for PAHs, biodegradation have been shown to be more successful, eco-friendly and cost effective one. In this study, Mycobacterium frederiksbergense, preliminarily known to degrade pyrene, was investigated of its potential to degrade this model PAH compound, in single and mixed substrate condition, employing three different biodegradation systems. In the first slurry phase degradation system containing pyrene as the sole substrate, the Mycobacterium showed a lag of 48 h in pyrene degradation in batch shake flask for an initial concentration of 50 mg l-1. Immediately following this lag period, pyrene was actively degraded with an observed rate of 19.86 mg l-1 d-1. However, in fermenter, pyrene was degraded without any lag phase with an over all rate of 6 mg l-1 d-1. To study biodegradation of pyrene in a ternary mixture along with naphthalene and anthracene in this slurry phase system, a 23 full factorial design of experiments was employed. Statistical analyses of the PAHs degradation rates by the culture performed in the form of analysis of variance (ANOVA) and studentDs t-test gave interpretation of main and interaction effects of the substrates.In surfactant aided system for biodegradation of pyrene, initially five synthetic chemical surfactants viz. TritonX-100, Tween 80, Tween 20, sodium dodecyl sulphate (SDS) and cetyl trimethyl ammonium bromide (CTAB) were evaluated of their molar solubilisation ratio for pyrene, which inferred superiority of Tween 80 over others. Also, a biosurfactant produced by an indigenous microbial culture isolated from the soil of a gasoline filling station, was comparatively evaluated of its efficiency to solubilise pyrene. The partially purified biosurfactant also showed emulsification activity, stability at different temperature, pH towards a range of solvents. However, the culture failed to biodegrade pyrene. Tween 80 aided pyrene biodegradation in batch shake flask indicated a prolonged lag phase, which was however absent in fermenter experiments with a maximum degradation rate at 25 mg l-1 initial pyrene concentration. Results of Tween 80 aided mixed PAHs biodegradation studies with pyrene, naphthalene and anthracene were also analysed in terms of their main and interaction effects on their biodegradation. To further evaluate the potential of the mycobacterium in biodegradation of pyrene, two liquid phase partitioning bioreactor (TPPB) system was developed. After an initial screening procedure, silicone oil was chosen as the non-aqueous phase liquid in the TPPB system. Further, results of hydrodynamic study in this TPPB system indicated the best operating conditions to be 1.5 vvm of aeration rate, 600 rpm agitation rate, with silicone oil fraction of 0.2. At this optimized sets of operating conditions in the TPPB system, biodegradation study carried out at different initial pyrene concentration in silicone oil (200-1000 mg l-1) gave very high pyrene degradat.
Supervisors: K. Pakshirajan and V. Venkata Dasu