Project Description

Abstract

The island municipality of Puerto Rico, Vieques, remains contaminated from decades of military activity with the high-energy compound RDX (hexahydro-1,3,5-trinitroso-1,3, 5-triazine), posing severe risks to human health and local ecosystems. To address this issue, we engineered a genetic circuit in Escherichia coli for RDX biodegradation and biocontainment. The system integrates two modules: (1) a biodegradation module containing an RDX-inducible riboswitch that regulates xplA and xplB, encoding cytochrome P450 and its flavodoxin reductase responsible for degrading RDX into formaldehyde and nitrite, and (2) a killswitch module that detects these byproducts to trigger colicin-mediated cell lysis. The circuit will be implemented in a four-stage Serial Bioreactor System designed to promote bacterial growth, induce degradation, treat byproducts, and ensure safe environmental discharge. Our system provides a controlled and sustainable synthetic biology approach for mitigating RDX contamination and supporting environmental restoration in Vieques.

Our Problem

During World War II, global demand for explosives like TNT, ERW, and PETN ignited a dramatic search in manufacturing [1]. But among them, one explosive stood out for its immense power and stability: Royal Demolition Explosive (RDX). RDX is a secondary high explosive that is widely used by the military in shells, bombs, and demolition charges [2]. Large-scale production of RDX began in the United States in 1943, where it was often blended with oils, waxes, and other explosives to create effective military munitions. However, due to manufacturing impurities and environmental degradation products, RDX remains a major source of explosive contamination at active and former U.S. military sites [3]. Currently, there are 31 out of 1,699 hazardous waste sites contaminated by RDX catalogued as Superfund National Priorities List by the EPA [4]. Some of the countries most affected by this contamination include Israel, United States, Germany, and the Anones Lagoon in Vieques, Puerto Rico.

Although Puerto Rico is known for its rich culture, vibrant music, and deep connection with the land and sea, during the 1940s it was used as a military training site. For a span of 60 years, Vieques’ Municipality was used to test live ammunition in two-thirds of the island, leaving multiple unexploded artillery in terrestrial and marine environments [5]. The tests were conducted 180 days per year, eight miles away from 9,000 to 14,000 of Vieques civilians [6]. This practice left behind an array of contamination of heavy metals and organic compounds, including a vast quantity of RDX [5]. Furthermore, the Anones Lagoon, the greatest contamination site in Vieques, is directly connected to the Caribbean Sea, meaning the water’s current can transport the contaminants to open water.

Acute exposure to RDX has been associated with neurotoxic effects like seizures, nausea, and loss of consciousness [7]. Chronic exposure studies have suggested liver, kidney, and reproductive effects, and the compound has been classified with “suggestive evidence of carcinogenic potential” [8].

In Vieques, Puerto Rico, the ATSDR evaluated soil, water, and air samples and concluded that measured concentrations at that time did not exceed health-based guidelines, while noting the importance of continued monitoring [9]. Later analyses have identified energetic compounds and metals in marine organisms, such as lobsters and snails, pointing to potential bioaccumulation in aquatic ecosystems [10].

Epidemiological assessments reported differences in cancer incidence between Vieques and the rest of Puerto Rico during certain periods, highlighting the need for more research on possible exposure–health links [6].

Source: Cary, T. J., Rylott, E. L., Zhang, L., Routsong, R. M., Palazzo, A. J., Strand, S. E., & Bruce, N. C. (2021, May 3). Field trial demonstrating phytoremediation of the military explosive RDX by XPLA/XPLB-expressing switchgrass. Nature News. https://www.nature.com/articles/s41587-021-00909-4

Why RDX?

High-energy compounds like RDX are persistent and toxic contaminants that have been detected in soil, groundwater, and even air near military testing and training sites. The stability of these types of molecules makes them resistant to natural degradation processes, therefore making the exposure to RDX linked to harmful effects on both ecosystems and human health, including neurological disorders and carcinogenic risks. These concerns contribute to the need of developing effective remediation strategies.

Attempts at addressing RDX contamination have faced significant challenges. Traditional methods such as incineration, soil excavation, or pump-and-treat systems are expensive, energy-intensive, and often generate secondary environmental impacts. Biological methods could be useful, however they frequently depend on specialized microorganisms that are difficult to cultivate or maintain in natural environments. Such limitations reveal the need for an accessible and environmentally friendly solution that can be simulated in real-world conditions.

Synthetic biology as an emerging field offers an innovative way to apply engineering principles to biology, enabling the creation of modular devices that perform specific functions.Previous research has demonstrated that the enzymes XplA and XplB are capable of degrading RDX into less harmful products such as nitrite and formaldehyde. Although these metabolites remain toxic, their formation enhances the overall detoxification process, as they can be more readily captured or further transformed into non-toxic end products. Leaning upon these findings, our project proposes the development of R-DetoX 2.0, a genetic device that integrates detection and degradation functions into a single system.

By combining an activation mechanism called a riboswitch specific to RDX with degradation enzymes and a fluorescent reporter, R-DetoX 2.0 serves as a dual-purpose prototype that not only identifies the presence of RDX but also actively aims to break it down. This strategy emphasizes accessibility, efficiency, and environmental safety, offering a potential breakthrough for remediation efforts.

In choosing this project, we aim to integrate existing bioremediation attempts and practical applications. A successful system could provide a sustainable, cost-effective tool to clean up contaminated sites worldwide, demonstrating how synthetic biology can transform persistent environmental problems into opportunities for innovative solutions.