This study assessed cryogenic preservation for molecular characterization of microorganisms in sediment using samples from a toluene-contaminated model aquifer. qPCR, RT-qPCR, and PCR-SSCP showed no significant differences between frozen and unfrozen sediments, validating cryogenic coring for accurate subsurface microbial analysis. Functional gene profiling revealed distinct sediment-attached versus suspended toluene-degrading communities, with sediment data better reflecting groundwater chemistry. Functional gene abundance and transcripts alone proved unreliable indicators of biodegradation due to basal expression. An integrated approach using the ratio of transcripts to gene copies is proposed as a more accurate measure of in-situ metabolic activity, requiring system-specific activity thresholds.
