This demonstration project evaluated long-term performance of a two-tiered biobarrier system previously used at the 22 Area of Marine Corps Base (MCB) Camp Pendleton to treat methyl tert-butyl ether (MTBE) in groundwater. The system used pure oxygen injection/sparging to create a two-tiered biobarrier approach. The project objectives were two-fold: (1) evaluated the current microbial activity supporting natural attenuation of MTBE using a combination of conventional contaminant concentration data and geochemistry trend analyses and advanced molecular biological tools (MBTs), including metaproteomics and metagenomics, and (2) assessed the long-term impact of the biobarrier system on formation permeability.

In addition to evaluating data collected using conventional monitoring techniques, this project applied metagenomics and metaproteomics to improve the understanding of long-term impacts of the remedy on biodegradation at the site. Use of these advanced MBTs for quantification and detection of biomarkers, especially deoxyribonucleic acid (DNA) and peptides (protein fragments) in environmental samples has been rapidly expanding over the last few decades.

Technology Description

Unlike conventional MBTs, such as quantitative polymerase chain reaction (qPCR) or microarrays, metagenomics provides insight into gene sequence information for whole communities. Metagenomic sequencing of environmental samples provides a comprehensive picture of all bacterial and archaeal sequences within a sample, not just those microorganisms targeted with qPCR assays. Providing a larger snapshot of microbial community composition not only allows detection of microorganisms related to the degradation of a specific chemical, but also has a potential to link composition of microbial consortia and geochemical characteristics of the site.

Metaproteomics provides the most direct measure of microbial activity through detection of proteins of interest, providing information on molecular processes used by microorganisms. It can identify proteins encoded by genes in the metagenome and give a snapshot of community metabolic activities at the moment of sampling. The characterization of a proteome can be accomplished by interpreting mass-spectrometry-based peptide sequencing using data derived from 16S ribosomal ribonucleic acid (rRNA) gene sequencing. In environmental metaproteomics, a predicted protein database constructed from metagenomic information of the exact same sample is required to assign peptide sequence information to proteins from which the peptides were derived.

Metagenomic and metaproteomics are cutting-edge environmental microbiological tools that are rapidly developing. With the availability of metagenomic sequences and the increasing number of complete individual bacterial and archaeal genome sequences, it is now possible to apply postgenomic techniques (particularly proteomics) to complex microbial communities. Combined, these powerful tools provide a capability to reveal the presence of specific proteins within the microbial community to provide direct evidence of specific pathways involved in the degradation of contaminants.

Demonstration Results

The treatment system, consisting of a set of two biosparging biobarriers, was installed in 2004 at Marine Corps Base (MCB) Camp Pendleton, San Diego, California. Each barrier was comprised of a number of sparging wells used to inject oxygen into the aquifer. During operation of these biobarriers (from 2004 through 2010), MTBE concentrations in groundwater declined significantly such that only dilute levels of MTBE (i.e., 5 micrograms per liter [µg/L] to 40 µg/L) remained. In 2010 and 2012, regulatory agencies agreed to discontinue operation of the mid-plume and leading-edge biobarrier, respectively. However, since low-levels of MTBE still existed at the site that exceeded the State of California’s secondary maximum contaminant level (MCL) for MTBE (5 µg/L), the site was transitioned to MNA after shutdown of the biobarriers. 

This project showed that metageonomics and metaproteomics have the potential to provide robust lines of evidence that degradation of contaminants of concern (COCs) at a site continue to occur after an active remedy has been applied. At project completion, these techniques serve to augment conventional data, but may not be able to replace and/or reduce the frequency of application of conventional techniques at this time. The cost for these analyses for this demonstration were $350 and $1,800 per sample for the metagenomics and metaproteomic analyses, respectively, based on analysis of a batch of 7 samples. Cost for metagenomic analysis is not anticipated to decrease as the quantity of samples increases; however, the cost for the proteomic analysis will decrease as the number of samples increases. For instance, had 50 samples been analyzed during this demonstration, the resulting cost per sample would have been $750. 

Implementation Issues

The advanced MBTs and conventional groundwater analyses (contaminant concentration and geochemistry) used in this project are commonly employed for these types of assessments and the implementation issues are well understood. The general concerns of DoD site managers and other end users likely include the following: (1) regulatory acceptance; (2) insufficient confidence in results and access to specialized laboratories; and (3) technology cost compared to other more conventional monitoring options.