Perchlorate has been used by the Department of Defense (DoD) for more than 50 years as a component in munitions, pyrotechnics, propellants, explosives, mines, and rocket and missile fuels. It is toxic to humans and the environment and is highly persistent in drinking water. The perchlorate anion is particularly difficult to treat in water because it is highly soluble, adsorbs poorly to minerals or activated carbon, and moves freely in groundwater. A variety of perchlorate remediation technologies are currently commercially available. One of them, biological degradation, involves perchlorate reducing bacteria, which are widespread in the environment. PRBs have the ability to grow in either the presence or absence of air, provided proper nutrients are available in the environment. Both in situ and ex situ biological treatment systems have already been applied at full scale to treat perchlorate contamination. Implementation of novel techniques such as optimization and creation of microbial consortia using the Genetic Algorithm have great potential to improve the existing technology.  The objective of this project was to demonstrate that transformation of natural subsurface biofilms by free DNA encoding constitutively expressed genes for perchlorate degradation (reduction) can be employed to establish a process of engineered natural attenuation for in situ treatment of perchlorate in groundwater.

A novel in situ approach for the bioremediation of perchlorate contamination of groundwater was developed by way of a field-laboratory linked research effort. Genes encoding perchlorate and chlorite degradation were used to transform natural subsurface biofilms such that members of the biofilm microbial community attained the ability to reduce perchlorate and dismutate chlorite. First, a large collection of novel, extremophilic, perchlorate-reducing bacteria was generated and characterized to serve as a pool for cloning novel genes that encode biological perchlorate reduction ability. Those members of the strain collection that are most effective at perchlorate reduction were identified by optimizing perchlorate reduction by a subset of the strains using the methods of evolutionary computation. Associated perchlorate reductase and chlorite dismutase genes were cloned and sequenced. Plasmids were then constructed carrying these genes under the control of a constitutive promoter, and the genes were expressed in laboratory bacteria to characterize the kinetics of perchlorate reduction by the encoded proteins. Methods for growing natural subsurface biofilms on sterile basalt substrates were developed, and substrates were deployed in wells for colonization by natural aquifer bacteria.

State-of-the-art tools of genomics and proteomics were developed that allow for sensitive, real-time monitoring of processes that remove perchlorate from the environment, such as facilitated remediation and monitored natural attenuation. Specifically, the results demonstrated that the liquid chromatography/mass spectrometry (LC/MSⁿ)-based proteomic method is a promising tool for both identification and quantification of perchlorate and chlorate-reducing enzyme systems in pure and mixed cultures, including those derived from environmental samples. Signature peptides from chlorite dismutase, perchlorate reductase, and chlorate reductase were detected in all of the samples. Quantification results show that Dechlorosoma sp. KJ had the highest level of expression of the enzymes involved in perchlorate reduction among the microbial samples analyzed. For example, the concentration of chlorite dismutase in Dechlorosoma sp. KJ was found to be approximately 10 times and 4 times higher than in the environmental sample (BR-enrichment) and D. hortensis, respectively. Similarly, the concentrations of perchlorate reductase subunits A, B, and D also were higher in Dechlorosoma sp. KJ. The concentrations of perchlorate reducing enzymes were found to be higher in early-log phase as compared to the late-log growth phase in D. hortensis. This may be because the enzymes (CD and PR) are destroyed or inactivated in the late growth phase.

This project provided DoD with a new technique for in situ treatment of perchlorate-contaminated aquifers. Genetic algorithms and environmental proteomics could become tools in environmental microbiology for an efficient control of the functioning of natural and undefined microbial ecosystems. In the future, the proteomics-based technique developed could be used to obtain quantitative proteomics data from other perchlorate and chlorate-reducing microorganisms. The most efficient pure strain(s) or natural consortia, in terms of the expression levels and perchlorate reducing enzyme stability, might then be selected for use in perchlorate bioremediation process development. This research also will help develop proteomic biomarkers that can be used to measure the exposure of a given environment to perchlorate contamination and determine the current perchlorate degradation status within that environment.