Survival and activity of genetically engineered degradative inocula in soil
In this study bioluminescence-marker systems were used to investigate the fate of two genetically engineered degradative bacteria in soil under different conditions. These bacteria were chromosomally lux-marked Alcaligenes eutrophus H850 Lr with luxAB genes which was able to degrade polychlorinated biphenyls (PCBs), and Pseudomonas stutzeri P16. P. stutzeri P16 was genetically marked with the luxAB tet cassette by insertional mutagenesis using mini Tn5-transposons and was characterised for lux gene stability, phenanthrene degradability and sensitivity to aldehyde. Insertion of lux gene into genome of P. stutzeri P16 provided a novel strain with lower maximum specific growth rate and less sensitivity to high concentrations of phenanthrene in liquid culture. Survival and activity of P. stutzeri P16 luxAB4 was further investigated in soil amended with low and high concentrations of phenanthrene. Initially high concentrations of phenanthrene had a less toxic effect on viable cell concentrations and luminescence activity of genetically engineered P. stutzeri P16 comparing with its wild type showing that lux-marking the above degradative strain made the marked strain more suitable for environmental application in heavily polluted sites. However, resistant cells to high concentration of phenanthrene were not able to survive longer than those which was inoculated to microcosms with lower concentrations of phenanthrene. This indicates that toxicity of the target compound and its concentration have to be considered before releasing the GEMs. Degradation of phenanthrene was enhanced in both sterile and non-sterile soil inoculated with P. stutzeri P16 luxAB4. This study could be used as model for further investigated of degradation of other PAHs such as fluoranthene, benz[a]anthracene, benzo[a]pyrene, by degradative bacteria and also monitoring the survival and activity of those bacteria in the environment using bioluminescence-marker systems.