Noninvasive quantitative measurement of bacterial growth in porous media under unsaturated-flow conditions.

Glucose-dependent growth of the luxCDABE reporter bacterium Pseudomonas fluorescens HK44 was monitored noninvasively in quartz sand under unsaturated-flow conditions within a 45- by 56- by 1-cm two-dimensional light transmission chamber. The spatial and temporal development of growth were mapped daily over 7 days by quantifying salicylate-induced bioluminescence. A nonlinear model relating the rate of increase in light emission after salicylate exposure to microbial density successfully predicted growth over 4 orders of magnitude (r(2) = 0.95). Total model-predicted growth agreed with growth calculated from the mass balance of the system by using previously established growth parameters of HK44 (predicted, 1.2 x 10(12) cells; calculated, 1.7 x 10(12) cells). Colonization expanded in all directions from the inoculation region, including upward migration against the liquid flow. Both the daily rate of expansion of the colonized zone and the population density of the first day's growth in each newly colonized region remained relatively constant throughout the experiment. Nonetheless, substantial growth continued to occur on subsequent days in the older regions of the colonized zone. The proportion of daily potential growth that remained within the chamber declined progressively between days 2 and 7 (from 97 to 13%). A densely populated, anoxic region developed in the interior of the colonized zone even though the sand was unsaturated and fresh growth medium continued to flow through the colonized zone. These data illustrate the potential of a light transmission chamber, bioluminescent bacteria, and sensitive digital camera technology to noninvasively study real-time hydrology-microbiology interactions associated with unsaturated flow in porous media.

A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media.

A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm(3) H(2)O/cm(3) of sand, respectively), and at cell densities ranging between 1 x 10(6) and 8.5 x 10(8) cells/ml. Before O(2) availability became a limiting factor, the rate of light emission (L) increased with the square of time (t) and linearly with increasing cell density (c). A nonlinear model was developed that contains a "rate of increase in light emission" constant, B', which is determined directly from the slope of a plot of radical L/c against t. The model predicted the behavior of lux induction in HK44 under a variety of conditions. Similar B' values were determined [49.0-57.6 x 10(-10) light units/(cell min(2))] for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand. Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B'=61.6+/-3.2, log phase cells plus glucose, B'=63.2+/-7.2). The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media.

A modification to the Bouwer and Rice method of slug-test analysis for large-diameter, hand-dug wells.

The Bouwer and Rice method of estimating the saturated hydraulic conductivity (Ks) from slug-test data was evaluated for geometries typical of hand-dug wells. A two-dimensional, radially symmetric and variably saturated, ground water transport model was used to simulate well recovery given a range of well and aquifer geometries and unsaturated soil properties, the latter in terms of the van Genuchten parameters. The standard Bouwer and Rice method, when applied to the modeled recharge rates, underestimated Ks by factors ranging from 1.3 to 5.6, depending on the well geometry and the soil type. The Bouwer and Rice analytical solution was modified to better explain the recovery rates as predicted by the numerical model, which revealed a significant dependence on the unsaturated soil for the shallow and wide geometries that are typical of traditional wells. The modification introduces a new parameter to the Bouwer and Rice analysis that is a measure of soil capillarity which improves the accuracy of Ks estimates by tenfold for the geometries tested.