Fiona Jordan

Analysis of artifacts suggests DGGE should not be used for quantitative diversity analysis.

PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with α-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real time PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis.

Biodegradation during contaminant transport in porous media: 4. Impact of microbial lag and bacterial cell growth.

Miscible-displacement experiments were conducted to examine the impact of microbial lag and bacterial cell growth on the transport of salicylate, a model hydrocarbon compound. The impacts of these processes were examined separately, as well as jointly, to determine their relative effects on biodegradation dynamics. For each experiment, a column was packed with porous medium that was first inoculated with bacteria that contained the NAH plasmid encoding genes for the degradation of naphthalene and salicylate, and then subjected to a step input of salicylate solution. The transport behavior of salicylate was non-steady for all cases examined, and was clearly influenced by a delay (lag) in the onset of biodegradation. This microbial lag, which was consistent with the results of batch experiments, is attributed to the induction and synthesis of the enzymes required for biodegradation of salicylate. The effect of microbial lag on salicylate transport was eliminated by exposing the column to two successive pulses of salicylate, thereby allowing the cells to acclimate to the carbon source during the first pulse. Elimination of microbial lag effects allowed the impact of bacterial growth on salicylate transport to be quantified, which was accomplished by determining a cell mass balance. Conversely, the impact of microbial lag was further investigated by performing a similar double-pulse experiment under no-growth conditions. Significant cell elution was observed and quantified for all conditions/systems. The results of these experiments allowed us to differentiate the effects associated with microbial lag and growth, two coupled processes whose impacts on the biodegradation and transport of contaminants can be difficult to distinguish.

Effective removal of microbial contamination from harvested rainwater using a simple point of use filtration and UV-disinfection device

There is a growing interest in the utilisation of harvested rainwater as an alternative water source. This study summarises an evaluation of the water quality obtained for privately-owned cisterns sampled in Tucson, AZ and the feasibility to obtain high-quality water from harvested rainwater by fitting cisterns with Point of Use (POU) filtration and UV disinfection devices. Of the 11 cisterns sampled, 30–50% tested positive for E. coli, 100% contained Enterococci and a few tested positive for lead at concentrations above the MCL. All physicochemical and microbiological constituents analysed were present at higher numbers in the summer than in the winter. The POU devices sufficiently cleared the harvested rainwater of total coliforms, E. coli, and Enterococci but performed marginally with respects to improving turbidity and heterotrophic bacteria.

Ethanol Addition for Enhancing Denitrification at the Uranium Mill Tailing Site in Monument Valley, AZ

Past mining and processing of uranium ore at a former uranium mining site near Monument Valley, AZ has resulted in nitrate contamination of groundwater. The objective of this study was to investigate the potential of ethanol addition for enhancing the reduction of nitrate in groundwater. The results of two pilot-scale field tests showed that the concentration of nitrate decreased, while the concentration of nitrous oxide (a product of denitrification) increased. In addition, changes in aqueous concentrations of sulfate, iron, and manganese indicated that the ethanol amendment caused a change in prevailing redox conditions. The results of compound-specific stable isotope analysis for nitrate–nitrogen indicated that the nitrate concentration reductions were biologically mediated. Denitrification rate coefficients estimated for the pilot tests were approximately 50 times larger than resident-condition (non-enhanced) values obtained from prior characterization studies conducted at the site. The nitrate concentrations in the injection zone have remained at levels three orders of magnitude below the initial values for many months, indicating that the ethanol amendments had a long-term impact on the local subsurface environment.

Biodegradation during contaminant transport in porous media: 8. The influence of microbial system variability on transport behavior and parameter determination

[1] The impact of microbial system variability on the biodegradation and transport behavior of a model solute, salicylate, was investigated with a series of miscible displacement experiments. Four systems of increasing complexity were employed: a sterilized, well-sorted sand inoculated with a single bacterial isolate, a sterilized soil inoculated with the same isolate, and two soils, each of which contained an indigenous multiple-population community of bacteria. The experiments were conducted in replicate (three or four experiments per set) and with paired controls. The biodegradation and transport behavior of salicylate exhibited a small degree of variability among the replicates for the two inoculated systems and a relatively large degree of variability for the two indigenous systems. The greater variability observed for the two indigenous systems is attributed primarily to greater variability of microbial system properties, such as initial cell density, metabolic status, and community composition. Values for maximum specific growth rate coefficient, mean lag time, and lag time variance were determined by model calibration to the measured breakthrough curves and compared to values obtained from batch experiments. Reasonable correspondence was observed between the two sets of values for both the inoculated and indigenous systems. The maximum specific growth rate coefficient exhibited a relatively small degree of uncertainty for all four systems, whereas greater uncertainty was associated with the lag time mean and variance. The variability in calibrated parameters among each set of replicate experiments was significantly greater than the uncertainty associated with the individual experiment calibrations and the measured input parameters. These results illustrate that variability inherent to natural microbial systems can cause variability in transport behavior even under controlled laboratory conditions and concomitantly enhance the uncertainty of biokinetic parameters obtained from laboratory studies.

Development of an agar lift-DNA/DNA hybridization technique for use in visualization of the spatial distribution of Eubacteria on soil surfaces

While microbial growth is well-understood in pure culture systems, less is known about growth in intact soil systems. The objective of this work was to develop a technique to allow visualization of the two-dimensional spatial distribution of bacterial growth on a homogenous soil surface. This technique is a two-step process wherein an agar lift is taken and analyzed using a universal gene probe. An agar lift is comprised of a thin layer of soil that is removed from a soil surface using an agar slab. The agar is incubated to allow for microbial growth, after which, colonies are transferred to a membrane for conventional bacterial colony DNA/DNA hybridization analysis. In this study, a eubacterial specific probe was used to demonstrate that growing bacterial populations on soil surfaces could be visualized. Results show that microbial growth and distribution was nonuniform across the soil surface. Spot supplementation of the soil with benzoate or glucose resulted in a localized microbial growth response. Since only growing colonies are detected, this technique should facilitate a greater understanding of the microbial distribution and its response to substrate addition in more heterogenous soil systems.