A new direct microscopy based method for evaluating in-situ bioremediation

A new epifluorescent microscopy based method using 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) and 5-(4,6-dichlorotriazinyl) aminofluoroscein (DTAF) was developed for quantifying total microbial biomass and evaluating levels of microbial activity. CTC is a tetrazolium dye that forms fluorescent intracellular formazan when biologically reduced by components of the electron transport system and/or dehydrogenases of metabolically active bacteria. DTAF is a fluorescein-based fluorochrome that selectively stains bacterial cell walls thereby enabling quantification of total bacterial biomass. CTC can be used in conjunction with DTAF to provide the optical resolution necessary to differentiate metabolically active cells from inactive cells in microbial populations associated with subsurface soils. The CTC/DTAF staining method has been shown to be effective for quantifying the metabolic activity of not only aerobic bacteria, but also diverse groups of anaerobic bacteria. This method allows for the rapid quantification of total and active bacterial numbers in complex soil samples without enrichment or cell elution. In this study, CTC/DTAF staining was applied to evaluate in-situ microbial activity in petroleum hydrocarbon contaminated subsurface soils from Sites 3 and 13 at Alameda Point, CA. At each site, subsurface microbial activity at two locations within contaminated plumes were examined and compared to activity at two geologically similar but uncontaminated background locations. Significant bacterial populations were detected in all soils examined, and the biomass estimates were several orders of magnitude higher than those obtained by conventional culture-based techniques. Both the total bacterial concentrations and the numbers of active bacteria in soils from contaminated areas were substantially higher than those observed in soils from background locations. Additionally, the percentages of metabolically active bacteria in the contaminated areas were consistently higher than those detected in background areas, suggesting that the enhanced microbial activity was due to microbial contaminant degradation. Although conventional heterotrophic plate counts failed to show significant microbial activity at either of the sites, soil gas carbon dioxide and methane measurements confirmed that hydrocarbon contaminant degradation was occurring in both areas. The CTC/DTAF staining protocol proved to be a rapid, reliable, and inexpensive method to evaluate the progress of in-situ bioremediation.