Spatial and Temporal Evaluation of Dechlorinating Populations During Crab Shell Enhanced Bioremediation
Open Access
- Author:
- McElhoe, Jennifer A
- Graduate Program:
- Environmental Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 07, 2011
- Committee Members:
- Rachel Alice Brennan, Dissertation Advisor/Co-Advisor
Rachel Alice Brennan, Committee Chair/Co-Chair
William D Burgos, Committee Member
John Michael Regan, Committee Member
Maryann Victoria Bruns, Committee Member - Keywords:
- Crab Shell
Bioremediation
Trichloroethene
Chitin
qPCR
FISH - Abstract:
- Due to its dense, hydrophobic, and toxic nature, the restoration of trichloroethene (TCE) contaminated aquifers presents a significant challenge to both government and industry. Remediation of contaminated sites typically falls into two categories: the traditional pump and treat, which is costly and generally inefficient at removing dense non-aqueous phase liquids (DNAPLs) like TCE; and bioremediation with native or bioaugmented halorespiring organisms. Hydraulic fracturing (“fracking”) of substrates and proppants into TCE contaminanted soils is a developing, but understudied, practice of stimulating in situ bioremediation. Laboratory studies combining chemical and molecular analyses to evaluate reductive dechlorination stimulated through the addition of a crab shell and sand mixture (substrate used in fracking processes) may lend insight into the assessment and monitoring of contaminated field locations. This study evaluates the spatial and temporal distribution of dechlorinating populations during crab shell enhanced bioremediation. Laboratory tests were conducted using a commercially available mixed dechlorinating culture (Bio-Dechlor INOCULUM) containing Dehalococcoides species, capable of complete dechlorination of TCE to ethene, and crab shell (SC-20, SC-40, or SC-80) as a slowly fermentable substrate. Semi-continuous flow columns tests were conducted to evaluate the ability of different grades of commercially available crab shell to enhance degradation of chlorinated solvents at various concentrations. Continuous flow column tests were conducted to further assess the distribution of dechlorinating populations throughout the course of anaerobic reductive dechlorination. In the semi-continuous flow column study, the SC-20 grade of crab shell provided the greatest variety of fermentation products, supported the greatest ethene production, and maintained a more stable pH than the other grades of crab shell for all test parameters. The less processed, less costly SC-20 has a higher proportion of protein which contributed to the increased variety of fermentation products, supplying an increased source of reducing equivalents and supporting greater conversion of TCE to ethene. A circum-neutral pH is also important for the efficient conversion of TCE to ethene, and the natural buffering capacity of the CaCO3 in the SC-20 maintained pH in the columns within the optimal range for dechlorination. Due to the findings of this study, all subsequent tests were conducted using the SC-20 grade of crab shell. In the continuous flow column tests, Dehalococcoides cell numbers were observed to increase from <1% of the total bacterial population in the influent end of the columns to 9-40% in the second half of the columns. The individual reductive dehalogenase (RDase) genes only accounted for a portion of the total Dehalococcoides 16S rRNA gene copies suggesting that there may be unidentified RDases in the BDI inoculum. T-RFLP fingerprinting analysis showed the greatest richness and diversity in communities associated with the solid-phase column materials in the beginning of the experiment (<~10 pore volumes) and in communities associated with pore water at the end of the experiment, suggesting Statistical analysis of the T-RFLP data indicated that neither the solid-phase nor the pore water sampling effectively captured all the dominant populations in the continuous flow columns. This has implications for field assessments of subsurface microbiology which are traditionally conducted through groundwater sampling. Fluorescent in situ hybridization (FISH) processing of intact biofilm extracted from the columns showed archaeal cells in close proximity to Dehalococcoides cells, indicating that competition, if it exists, is not creating spatial stratification, and suggests the possibility of a synergistic relationship. The close proximity of potentially competing hydrogenotrophs also indicates that the crab shell substrate is capable of supporting multiple hydrogen utilizing microbes.