Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SEstained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10 5 CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

Bacterial transport experiments were conducted using intact sediment cores collected from sites on the Delmarva Peninsula near South Oyster, VA, to delineate the relative importance of physical and chemical heterogeneity in controlling transport of an adhesion-deficient bacterial strain. Electron microscopy revealed that the sediments consisted of quartz and feldspar with a variable amount of clay and iron and aluminum hydroxide coatings on the grains. A nonmotile, gram-negative indigenous groundwater strain, designated as Comamonas sp. DA001, was injected into the cores along with a conservative tracer bromide (Br). DA001 cells were 1.2 x 0.6 microm in size with a hydrophilic surface and a slightly negative surface charge. Bacterial breakthrough preceded that of Br. This differential advection phenomenon can be accounted for by reduction of the effective porosity for the bacteria relative to Br. The distribution of cells remaining in the core as determined by scintillation counting and phosphor imaging techniques was variable, ranging from nearly uniform concentrations throughout the core to exponentially decreasing concentrations with distance from the point of injection. The fraction of bacterial retention in the core was positively correlated with the abundance of the metal hydroxides and negatively correlated with grain size. Because grain size was inversely correlated with the abundance of the metal hydroxide coatings, it was necessary to separate the effects of grain size and mineralogy. The fraction of the bacterial retention accounting for the effect of grain size, the collision efficiency, exhibited no correlation with the abundance of the metal hydroxides, indicating that the bacterial retention was primarily controlled by grain size. Reasons for the lack of influence of mineralogy on bacterial transport include (i) the slightly negatively charged bacterial surfaces; (ii) an insufficient heterogeneity of sediment surface properties; and (iii) the masking of the positive charge of the metal hydroxide surfaces by adsorbed organic carbon (up to 1180 ppm). This study demonstrates that the laboratory-based bacterial transport experiments are effective in delineating physical versus chemical controlling factors and provide an important link to field-based bacterial transport studies.

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Abstract-The effects of 2,4,6-trinitrotoluene (TNT) and other munitions compounds on indigenous microbial communities in several soils were examined. Culturable heterotrophs, concentrations of phospholipid fatty acid (PLFA), and basal respiration rates exhibited slight negative correlations with high TNT and 1,3,5-trinitrobenzene (TNB) levels. Heat-shock-resistant culturable heterotrophs, percentage of gram-positive soil isolates, mole percent of branched PLFA, and 10Me18:0 (tuberculostearic acid) were observed to be significantly lower in highly contaminated soils. Total soil nitrogen levels were positively correlated with high TNT and TNB concentrations, whereas total soil carbon exhibited no significant correlation with either compound. Multivariate analysis of PLFA data resulted in distinct separation of soils with respect to their degree of contamination, with specific signature PLFAs for gram-positive bacteria, fungi, and protozoa being negatively associated with high contaminant levels. Apparent concentrations of TNT resulting in 50% reductions in indicators of gram-positive populations were much higher than values from pure culture experiments, possibly as a result of low bioavailability due to sorption onto clay and soil organic matter. Few effects of other munitions compounds were observed. Closer examination of a highly contaminated soil revealed that the number of culturable heterotrophs growing on 0.3% molasses plates decreased by 50% when 67 g TNT/ml was added to the medium; a 99% decrease was observed for soil contaminated with less than 20 g TNT/g. Highly contaminated soil harbored a greater number of organisms that were able to grow on plates amended with greater than 10 g TNT/ml. Gram-positive isolates from both soils demonstrated marked growth inhibition when greater than 8-16 g TNT/ml was present in the culture medium. These results indicate that chronic exposure to munitions compounds can dramatically alter soil microbial communities.

A systematic evaluation of the ability of different bacterial genera to transform 2,4,6-trinitrotoluene (TNT), and grow in its presence, was conducted. Aerobic Gram-negative organisms degraded TNT and evidenced net consumption of reduced metabolites when cultured in molasses medium. Some Gram-negative isolates transformed all the initial TNT to undetectable metabolites, with no adsorption of TNT or metabolites to cells. Growth and TNT transformation capacity of Gram-positive bacteria both exhibited 50% reductions in the presence of approximately 10 microg TNT ml-1. Most non-sporeforming Gram-positive organisms incubated in molasses media amended with 80 microg TNT ml-1 became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 microg TNT ml-1, indicating that TNT sensitivity is linked to metabolic activity. These results indicate that the microbial ecology of soil may be severely impacted by TNT contamination.

The effects of 2,4,6-trinitrotoluene (TNT) and other munitions compounds on indigenous microbial communities in several soils were examined. Culturable heterotrophs, concentrations of phospholipid fatty acid (PLFA), and basal respiration rates exhibited slight negative correlations with high TNT and 1,3,5-trinitrobenzene (TNB) levels. Heat-shock-resistant culturable heterotrophs, percentage of gram-positive soil isolates, mole percent of branched PLFA, and 10Me18:0 (tuberculostearic acid) were observed to be significantly lower in highly contaminated soils. Total soil nitrogen levels were positively correlated with high TNT and TNB concentrations, whereas total soil carbon exhibited no significant correlation with either compound. Multivariate analysis of PLFA data resulted in distinct separation of soils with respect to their degree of contamination, with specific signature PLFAs for gram-positive bacteria, fungi, and protozoa being negatively associated with high contaminant levels. Apparent concentrations of TNT resulting in 50% reductions in indicators of gram-positive populations were much higher than values from pure culture experiments, possibly as a result of low bioavailability due to sorption onto clay and soil organic matter. Few effects of other munitions compounds were observed. Closer examination of a highly contaminated soil revealed that the number of culturable heterotrophs growing on 0.3% molasses plates decreased by 50% when 67 g TNT/ml was added to the medium; a 99% decrease was observed for soil contaminated with less than 20 g TNT/g. Highly contaminated soil harbored a greater number of organisms that were able to grow on plates amended with greater than 10 g TNT/ml. Gram-positive isolates from both soils demonstrated marked growth inhibition when greater than 8-16 g TNT/ml was present in the culture medium. These results indicate that chronic exposure to munitions compounds can dramatically alter soil microbial communities.

Metam sodium (MS) is a widely used soil fumigant with broad biocidal activity. The impact of MS on soil microbial community structure and function is largely unknown. We investigated the effects of fumigation with field‐relevant doses of MS in soil microcosms using traditional heterotrophic activity measures (C and N mineralization), a substrate‐utilization assay, and fatty acid analysis. Our results suggest that fumigation with MS results in persistent changes (at least 18 wk) in heterotrophic activity and fatty acid composition of the microbial biomass. Lack of recovery of heterotrophic activity and changes in fatty acid composition after fumigation suggest that MS has the potential to alter important microbially mediated functions such as nutrient cycling and pollutant degradation. Preliminary fatty acid data suggest that actinomycetes and other gram‐positive organisms may recover preferentially after fumigation with MS. Although community fatty acid analysis shows promise as a screening tool in soil microbial toxicity studies, more detailed information can be obtained from analysis of phospholipid fatty acid (PLFA) and other specific lipid fractions.

Significant challenges remain in developing reliable techniques to monitor in situ biodegradation. Stable carbon and oxygen isotope analyses of the contaminants, products of degradation, and electron acceptor(s) may provide robust means for monitoring the occurrence, pathways, and rates of intrinsic or enhanced in situ biodegradation. Results of a laboratory study using diesel fuel and a mixed microbial culture show that combined stable carbon isotope analyses of carbon dioxide and stable oxygen isotope analyses of molecular oxygen allow monitoring of the occurrence and pathways of degradation. The first-order rate constants for contaminant degradation (about -0.04 day -1 ) obtained from oxygen and contaminant concentrations are in excellent agreement with those obtained from isotopic data for oxygen (-0.04 to -0.05 day -1 ), indicating that oxygen isotope analyses of molecular oxygen can be used for quantifying the rate of contaminant degradation. Based on our results and a review of the published literature on oxygen isotope systematics of molecular oxygen and other common electron acceptors (nitrate and sulfate), it is suggested that combined carbon and oxygen isotope analyses of carbon dioxide and the electron acceptors provide effective tools for monitoring intrinsic and enhanced in situ biodegradation of fuel or chlorinated hydrocarbons under aerobic and anaerobic conditions.