Coupled Transport and Population Growth of Pseudomonas Putida Cells in the Vicinity of a Toluene Source Plume in a Saturated Sand-packed 2-D Microcosm

Nearly half of all Americans drinking water comes from groundwater, while it suffers a great potential threat from the leakage of hazardous substances from underground storage tanks. According to US EPA, there are more than 400,000 confirmed underground storage tank leaks nationwide. Bioremediation is the treatment that uses naturally occurring microorganisms to break down these hazardous substances into less toxic or nontoxic substances. In the past years, increasing amount of data have been reported regarding the enhancement of the contaminant biodegradation rate by the chemotaxis of the microorganisms to these pollutants due to their increased delivery to the contaminated site, the process of which is often hindered by the non-uniform distributions of subterranean hydraulic conductivity. Although mixing transverse to the flow path is suggested to be a primary limiting factor for biodegradation efficiency, other steps can also contribute to the hindrance of the bioremediation process, for instance, the first step for the bacteria to degrade the contaminants is their uptake into the cell body. In this study, we investigated the impact of the toluene uptake step on the bioremediation efficiency using Pseudomonas putida F1 wild type and knockout mutant strains (PpF1 wild type, PpF1 (ΔtodX), and PpF1 (ΔtodXΔcymDΔF1fadL)) whose toluene trans- membrane transport channel(s) were removed, yet their chemotaxis to toluene was preserved. To this end, a bench-scale 2-D microcosm system was used as the study platform mimicking features of the naturally occurring groundwater system to study bioremediation efficiency limiting factors under conditions closer to those in natural aquifers. The application of mathematical models that incorporate the cell growth rate during this process - which is dependent on the toluene uptake that provides the cells with the carbon and energy source for survival and proliferation – will further elucidate to what extent the uptake step contributes to the bioremediation process in comparison with other steps such as dispersion and chemotaxis. The results revealed that PpF1 (ΔtodX) that has reduced toluene transport capability, exhibited a lower percent recovery as compared with the wild type, implying less toluene consumption, and furthermore, exposure of the bacterial population to an increased toluene concentration (up to a certain level) increased the cell percent recovery. Furthermore, PpF1 (ΔtodXΔcymDΔF1fadL)) that had even lower toluene uptake capability due to additional removal of toluene transport related channels, showed even lower recovery from the 2-D microcosm under the same experimental conditions. A mathematical model was then set up to study in detail the transport processes of the species during the microcosm experiments, and it was confirmed the sensitiveness of the toluene degradation efficiency to bacterial growth- related parameters which was directly correlated with toluene uptake capability, regardless of the presence of mass transfer barrier. And this sensitiveness was reserved under different conditions, for example, in a region more distant to toluene source and at an increased flow rate which can occur at different geographic locations. This suggests that, in addition to mechanisms that enhance the delivery of the degrading agents to the contaminated site, transport membrane proteins of bacteria can also play a key role in bioremediation enhancement. Moreover, it suggests the potential of engineering bacteria toward higher contaminant permeability for enhanced bioremediation efficiencies.