Formatted Title
Identifying the Microorganisms Involved in Carbon Uptake from Trichloroethene under Different Incubation Conditions
Background/Objectives
The overall rationale is to provide knowledge to enhance the aerobic remediation of the groundwater contaminant, trichloroethene (TCE), for oxic sites. Although anaerobic bioremediation has been successful for many chlorinated solvent sites, it has significant limitations. For example, it is unlikely to be employed at large oxic sites because of the requirement for highly reducing conditions and the associated cost of driving such large sites anaerobic. Also, the accumulation of the known human carcinogen, vinyl chloride, from the dechlorination process represents a significant risk, if complete dechlorination does not occur. Aerobic contaminant biodegradation has the added advantage that biodegradation can occur to low contaminant concentrations. The aim is to determine which microorganisms are associated with the most sustainable rates of co-metabolic biodegradation of TCE in environmental samples and this will be achieved by examining the fate of carbon from TCE using stable isotope probing (SIP).
Approach/Activities
Sediments collected from two contaminated sites and one agricultural soil were used in microcosm studies to examine the impact of different substrate amendments on TCE biodegradation rates. For each soil or sediment, four microcosms were amended with 13C labelled TCE, four were amended with unlabeled TCE and three remained as abiotic controls. The study involved two different substrates previously associated with TCE removal, propane and methane. TCE removal rates are currently being monitored and when approximately 75% of TCE is degraded, nucleic acids will be extracted from the microcosms. The extracted DNA will be subject to ultracentrifugation and fractionation to separate the 13C labeled DNA from the background DNA. The fractions will then be subject to 16S rRNA gene amplicon sequencing. Additionally, the phylotypes associated with a group of key functional genes (propane monooxygenase, toluene monooxygenase, methane monooxygenase and ammonia monooxygenase) will be determined using a predictive approach.
Results/Lessons Learned
The removal of TCE is ongoing and is expected to be completed within a short time frame. Following this, DNA will be extracted and SIP sequencing data will be analyzed using Mothur, PICRUSt2 and RStudio. The molecular analysis is expected to be completed quickly, as these are commonly used methods within our research group. The data generated on the key functional genes and active microorganisms will be used to design qPCR assays to test groundwater and sediment samples to determine if these microorganisms are present and active across contaminated sites.