PNNL

Abstract

Historical unplanned releases at the UPR-100-N-17 166-N Diesel Oil Supply Line Leak waste site located within the 100-NR-1 OU resulted in diesel fuel contamination of the unsaturated vadose zone and site groundwater. Enhanced in situ bioremediation of the deep vadose zone was the final remedy decision for the 100-NR-1 Operable Unit (OU) and the full-scale bioventing demo began in 2012. For this remedy, oxygen is actively supplied to the deep vadose zone to stimulate aerobic hydrocarbon degrading microorganisms to metabolize petroleum contamination. Degradation rate calculations based on vadose zone respirometry measurements, however, have indicated declining activity over recent years, though the precise reason for this apparent decline has not been determined. In this investigation 100-NR-1 OU sediment cores were analyzed for total microbial biomass, as well as enumeration of diesel and BTEX degrading bacteria to determine if these microbial populations have declined relative to previous measurements taken in 2009. The main findings from this investigation are as follows. • Classical microbiological techniques and DNA based molecular analyses demonstrate that 100-NR-1 OU sediments maintain a persistent microbial community that ranges from 103 to 105 cells / gram of 100-NR-1 OU sediment between locations and with depth. • Most Probable Number (MPN) plating assays showed that 100-NR-1 OU sediments maintain nearly equivalent population densities of both aerobic and anaerobic bacteria and fungi. All should be considered as viable pathways for in situ diesel degradation, not just aerobic bacteria. • Diesel and BTEX degrading populations have persisted; biomass estimations as determined by MPN assays were comparable to a previous report in 2009 indicating stable population size. • Theoretical calculations of biomass based on DNA extraction yields suggest higher levels of total microbial biomass should be present in the 100-NR-1 OU sediments than is being fully captured by the specific approaches used for this investigation (i.e., MPN assays and specific enumeration of bacteria). These results provide clear evidence that 100-NR-1 OU sediments maintain microbiological potential for in situ degradation of diesel fuel, though the apparent cause for declining degradation activity is not known. In Section 4.0, recommendations are offered which could provide for a more complete analysis of the microbial community and diesel degradation specifically to enable a more definitive understanding of degradation activity constraints in situ.