QUANTIFICATION OF ENHANCED BIOREDUCTION OF CR(VI) USING 13C-PLFA MONITORING COUPLED WITH ADVANCED MICROBIOLOGICAL TECHNIQUES

Cr(VI) is a widespread groundwater contaminant. Under appropriate geochemical conditions, it can be biologically reduced to Cr(III), a much less toxic/mobile form of Cr. To stimulate Cr(VI) bioreduction in groundwater at the Hanford 100H field site, 18 kg of HRC® was injected into the aquifer. HRC® consists of polylactate esterified to a glycerol backbone that slowly releases lactic acid, providing a carbon source to support microbial activity. 10 g of ¹³C-labeled polylactate was added to the HRC® giving it a δ¹³C value of ~40‰ (vs. an unlabeled value of -15‰). The response of the subsurface microbial communities to the HRC® injection is being monitored in water samples collected from 4 depth intervals in the injection well and in an extraction well located ~5 m down-gradient of the injection well. Standard geochemical parameters (e.g., dissolved O₂, E_h, anion chemistry) and the δ¹³C values of dissolved inorganic carbon (DIC), dissolved organic carbon, and phospholipid fatty acid (PLFA) extracts of biomass in the samples are being analyzed. Cell counts, PLFA and 16s rDNA analyses are also being monitored.

Microbial populations increased from background levels of <10⁴ cells/ml to 2x10⁷ cells/ml 13-17 days after the injection of the HRC®. 16S rDNA microarray analysis showed enrichment of specific bacterial populations (e.g., Pseudomonas, Acidovorax, Thauera, Flavobacteria, Desulfovibrio) and archael (only non-methanogenic Crenarchaeotes) populations. This corresponds with the onset of reducing conditions, disappearance of NO₃, decrease in SO₄ and removal of Cr(VI) from the groundwater. At the same time, the δ¹³C of DIC increased from -15‰ to greater than 50‰, then began to decrease. The δ¹³C values of PLFA peaks common to many organisms showed a similar pattern of ¹³C-enrichment, but reached values (>200‰) much higher the bulk δ¹³C of the labeled HRC®, reflecting the faster dissolution rate for the ¹³C-labeled polylactate relative to the HRC®. After the δ¹³C of the PLFA peaked, it quickly returned to background values (-15‰) indicating rapid turnover of biomass in the system. Several PLFA peaks specific to organisms known to metabolize glycerol (e.g., Flavobacteria) increased, but did not show the same increase in δ¹³C. These data are being used to model changes in biological activity resulting from HRC® injection.