The objective of this project was to demonstrate the efficacy of cryogenic core technology as an integrated approach to sample collection and handling as a tool for monitoring subsurface remediation. This included the development of an in situ cryogenic coring system, the evaluation of molecular biological tool (MBT) analysis in conjunction with that system, and the demonstration that the cryogenic sampling protocols were robust and ready for application by the remediation user community.

The performance of the cryogenic coring system was evaluated for both sample collection and sample preparation. The effects of sample collection and handling were evaluated using “benchmark” samples consisting of both laboratory-generated frozen cores samples and cryogenically sampled cores taken from a well-controlled physical model environment. These 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).

The cryogenic coring system allowed samples to be frozen in situ. Once brought to the surface, the frozen cores were packed in dry ice and shipped to the laboratory for further processing and analysis. The approach prevented redistribution of fluids during sample recovery and shipping, and because the cores were frozen in situ, there is little loss of solid material during retrieval to ground surface. The data indicated that the vertical distribution of DNA within the core can be measured at the centimeter scale, providing unprecedented characterization of subsurface biogeochemical interfaces.

Additionally, this project did not observe any significant degradation due to freezing or storage for a suite of genes and gene transcripts, including short-lived messenger RNA (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 ribosomal DNA (rDNA). Furthermore, freezing and storage for 5 months at -80ºC did not affect the microbial community composition of samples from the model aquifer. Of even greater significance was that freezing and storage did not affect the relative abundance of 16S ribosomal RNA (rRNA) phylotypes, since in vivo rRNA content is often correlated with cellular growth rate. Thus, it was concluded that cryogenic preservation and storage of intact sediment samples can be used for accurate molecular characterization of microbial populations.

Following the characterization of the effects of whole-sample freezing, quantitative PCR was used to profile the abundance of genes associated with Toluene degradation across a contaminant plume in a model aquifer. This approach was important because profiles generated from sediment samples better reflect in situ contaminant degradation. Experiments using Nitrate to stimulate anaerobic Toluene biodegradation revealed that bacteria containing the gene for benzylsuccinate synthase (bssA), although present, were not actively degrading Toluene in groundwater lacking Nitrate. Results were consistent with the hypothesis that organisms, and their genes, detected in groundwater may be artifacts of microbial transport from upstream where chemical conditions favored their growth. The transcription of bssA genes from denitrifying Toluene degraders was induced by Toluene, but only in the presence of Nitrate. Transcript abundance dropped rapidly following the removal of either Toluene or Nitrate. The drop in bssA transcripts pursuant to the removal of Toluene could be described by an exponential decay function with a rate constant of 0.44 hr^-1 and a half-life of 1.6 hrs.

Interestingly, bssA transcripts never disappeared completely, but rather appeared to be constitutively transcribed in the absence of inducers. A significant implication of this was that the detection of transcripts may not be sufficient as evidence of contaminant degradation. Instead, an integrated approach combining functional gene abundance and gene transcript analysis is recommended. This approach circumvents the complications associated with interpreting pore water gene abundance data that arise from microbial transport, thus making reliable assessments possible from water samples, as opposed to sediment samples. It also avoids the possibility of mistakenly associating basal-level gene expression with actively degrading microbial populations.

This project demonstrated that labile biomolecules can be preserved in aquifer solids if those solids are collected cryogenically. This result will make it possible to analyze aquifer materials for biomolecules that serve as indicators of activity (e.g., mRNA of targeted genes), a significant advancement over the current approach that relies on DNA of functional genes, which, while providing direct confirmation of the presence of specific types of organisms, does not provide direct evidence of their activities.

As with most research projects, this one yielded unexpected benefits. In this case, the improved ability to collect labile molecules led directly to improvements in diagnosis of in situ activity. Specifically, the physical model studies showed that ratios of mRNA to DNA for functional genes could be used to identify areas where bacterial populations were and were not actively degrading contaminants. Of perhaps equal importance, the approach also allowed researchers to identify where degradation activities were not taking place, even though DNA analyses indicated that those organisms were present (i.e., under conditions where DNA-only analysis would have given a false positive result).

The cryogenic sampling technique developed in this project builds on current practice for core sampling and was designed to be easily integrated into routine field sampling. As a consequence, it is now possible to collect intact aquifer samples containing both solids and pore water. This approach couples well with quantitative PCR methods, which are the basis for molecular-tool based approaches for groundwater remediation.