Understanding bioremediation of contaminated groundwater: Application of a lux bioreporter to monitor in situ bacterial catabolism of naphthalene in saturated porous media

Abstract

One attractive technology for restoration of hydrocarbon-contaminated groundwater is in situ bioremediation, a process where the degradative capacity of biological systems, usually bacteria, is harnessed to facilitate clean-up of environmental pollutants. However, the successful implementation of in situ bioremediation is contingent upon understanding how physicochemical and microbial factors affect the formation and dynamics of microbially active regions, known as bioactive zones (BAZs), in porous media. In this study, a novel, laboratory-scale fiber optic detection system was developed and employed to monitor real-time, in situ BAZ formation and dynamics during naphthalene transport in saturated porous media. Biological activity was measured non-destructively by detecting in situ bioluminescence from Pseudomonas putida RB1353, a naphthalene degrading, lux reporter organism. The first investigation focused on examining the impact of temperature, pH and initial cell number on P. putida RB1353's peak luminescence and V max during naphthalene catabolism. Statistical analyses based on general linear models indicated that temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism. These results demonstrated that with careful characterization and standardization of measurement conditions, attainment of a reproducible luminescence response and an understanding of the response are feasible. The second investigation evaluated several potential limitations of the fiber optic detection system and the ability of the detection system to capture BAZ dynamics. The results indicated that the system is not adversely affected by biofilm formation on the optical fiber tips or by bioluminescence attenuation in the porous medium. Additionally, the utility of the detection system was demonstrated by effectively capturing the dynamics of in situ bacterial activity during naphthalene catabolism under changing physicochemical conditions. The third investigation employed the detection system to monitor real-time, in situ BAZ formation and dynamics during naphthalene transport in saturated porous media containing defined physicochemical and microbial heterogeneities. Despite successful transport of bacteria into sterile regions, BAZ formation was limited by local physicochemical conditions. Furthermore, bacterial transport against the advective flow enabled BAZ formation upgradient of inoculated regions. Ultimately, such investigations will improve the utility of in situ bioremediation by enhancing our understanding of BAZ dynamics in complex, heterogeneous systems.