Assessment of anaerobic toluene biodegradation activity by bssA transcript/gene ratios.

Benzylsuccinate synthase (bssA) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of bssA genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in bssA transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly, bssA transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples.

Effects of cryogenic preservation and storage on the molecular characteristics of microorganisms in sediments.

Sediment samples from a large physical-model aquifer and laboratory-generated samples were used to systematically assess the effects of whole-sample freezing on the integrity of biomolecules relevant to bioremediation. Impacts of freezing on DNA and RNA were assessed using quantitative polymerase chain reaction (PCR) as well as the community fingerprinting method, PCR single-strand conformation polymorphism (PCR-SSCP). We did not observe any significant degradation of a suite of genes and gene transcripts, including short-lived mRNA transcripts, from P. putida F1 or from B. subtilis JH642 in single-species samples, or from archaea in enrichment culture samples that also contained members of diverse bacterial phyla. Similarly, freezing did not change the relative abundance of dominant phylotypes in enrichment culture samples as measured by PCR-SSCP of bacterial 16S rDNA. Additionally, freezing and storage for 5 months at -80 °C did not affect the microbial community composition of samples from the model aquifer. Of even greater significance is that freezing and storage did not affect the relative abundance of 16S rRNA phylotypes, since in vivo rRNA content is often correlated with cellular growth rate. Thus, we conclude that cryogenic preservation and storage of intact sediment samples can be used for accurate molecular characterization of microbial populations and may facilitate high-resolution capture of biogeochemical interfaces important to bioremediation.

Detachment-influenced transport of an adhesion-deficient bacterial strain within water-reactive porous media.

Bacteria and carboxylate-modified microsphere transport experiments were performed in glass bead packed columns in order to examine the distribution of retained colloids on the sediment. Solution pH was allowed to vary from 6.0 to 9.4 across the length of the column (20 cm) in order to examine potential effects of solution chemistry on the retained profiles. Both the microspheres and the bacteria showed retained profiles that deviated strongly from log-linear behavior expected from a spatially invariant colloid deposition rate coefficient. Deviation for the microspheres was in the form of steeper-than-expected decreases in retained concentrations with distance from source. Deviation for the bacteria was in the form of maximum retained concentrations that were located down-gradient from the column inlet. Subsidiary experiments with varying elution times showed that detachment during elution moved the peak of mass of retained bacteria down-gradient of the column inlet; however, the disproportional translation of the peak of mass relative to elution time indicated that processes operating during injection produced the initial down-gradient translation of peak concentrations of retained cells.