Biodegradation of 2-chlorobenzoate by recombinant Burkholderia cepacia expressing Vitreoscilla hemoglobin under variable levels of oxygen availability.

Abstract

The influence of bacterial hemoglobin, VHb, on dechlorination and degradation of 2-chlorobenzoate (2-CBA) by recombinant Burkholderia sp. under variable oxygen availability with an initial dissolved oxygen concentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Ability to express VHb was provided to recombinant Burkholderia by transformation with the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded in cultures with 85% and 70% of total volume as headspace air in closed reactors by both wild type and recombinant Burkholderia. The recombinant cultures were able to dechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 degrees C compared to more than 120 hours for wild type cultures. The rate and extent of CBA degradation by recombinant cultures with 40% of total volume as headspace air was higher than those achieved by wild type cells at the end of the 168 hours of incubation period, 98 and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40% of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinant strains, whereas it was non-stoichiometric (molar ratio = 0.24) for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorination and degradation pathway for recombinant cells under hypoxic conditions. The degradation and dechlorination ability of both types of cells was also investigated in continuous reactor studies by varying the dilution rate under hypoxic conditions. Regarding potential of the recombinant strain for 2-CBA degradation in either open ecosystems or closed bioreactor bioremediation systems, the stability of the plasmid containing vgb in the recombinant cells was also studied; the plasmid was 100% stable at 0.025 h(-1) dilution rate (approximately 1.7 d hydraulic retention time), even after one month.