The Role o Glycerol in the Degradation of Dibenzothiophene by Burkholderia sp. C3.

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent, ubiquitous and carcinogenic chemicals found in the environment. PAHs can be formed anthropogenically and naturally. Dibenzothiophene (DBT) is a sulfur-containing PAH typically used as a model chemical to study biodegradation of PAHs and bioremediation. Biostimulation using more readily metabolized substrates can increase the biodegradation rate of PAHs, but the molecular mechanisms are not well understood. In this thesis, the effects of using glycerol as a co-substrate were investigated during DBT biodegradation by Burkholderia sp. C3 with respect to (i) DBT biodegradation kinetics, (ii) bacterial growth, (iii) proteome profiling, (iv) secretion of the biosurfactant rhamnolipid (RL), (v) RL biosynthesis inhibition, (vi) RL characterization and (vii) polyhydroxyalkanoates (PHA) vesicle formation. The results indicated that glycerol enhanced DBT biodegradation and supported bacterial growth, a phenomenon known as cometabolism. Optimized glycerol to DBT molar ratios increased DBT biodegradation rate constants up to 18-fold and enhanced DBT cometabolism by 25-30% at day 1 relative to DBT alone. At day 7 DBT (0.5 mM or 100 ppm) was completely biodegraded at a 100:1 glycerol to DBT molar ratio. Such increases were associated with nearly tripled amounts of RL secreted. RL analysis indicated biosynthesis, secretion and relevance of RLs to the enhanced DBT cometabolism. RLs reduced the surface tension of cultures with a DBT and glycerol mixture from 60-65 to 48-51 mN/m. RL biosynthesis inhibition with 2-bromooctanoic acid at 5 mM decreased DBT biodegradation rate comparable to that with DBT alone. Production of PHA vesicles were induced with glycerol and inhibited with 2 bromoalkanoic acids. RLs and PHA vesicles shared the same lipid precursor. All proteins involved in dTDP-rhamnose sugar precursor and R-3-hydroxydecanoyl-CoA lipid precursor for RL biosynthesis were accumulated under the conditions tested. RhlABC proteins mediate the final steps in RL formation. The work presented in this dissertation showed that glycerol induces DBT cometabolism at a dose dependent manner over time, which positively associates with bacterial growth, PHA lipid granule formation and RL biosynthesis and secretion. This is the first description of anabolic pathways involved in RL biosynthesis and bromooctanoic acid inhibitory mechanism in a Burkholderia species.