BUT HELP is on the way. A joint research project by the Center for Agroforestry and Department of Veterinary Pathobiology at the University of Missouri has finished preliminary testing on a novel and cheap method to apply pollution-eating enzymes to these contaminants.
Initial studies show that the process works in groundwater. Lab testing has just begun to determine how well the delivery system works in soil. The project is headed by Chung-Ho Lin, a research assistant professor of bioremediation and phytoremediation in the School of Natural Resources’ Center for Agroforestry, and George Stewart, the McKee Professor of Microbial Pathogenesis and chair of the Department of Veterinary Pathobiology.
Lin and Stewart are building on known qualities of these enzymes to degrade the nitrogen-rich explosive compounds left over from TNT, HMX and RDX manufacture. Lin says the challenge in using enzymes effectively is that they are fragile and don’t stay put in the environment. The enzymes are easily inactivated or washed away by rain before they can finish degrading the pollutants.
Lin and Stewart’s trick is to use bacterial endospores as a vehicle to deliver the enzymes to the pollution, thus allowing these “tethered” enzymes to persist there over time. The soil bacterium Bacillus thuringiensis, a spore-forming organism that has been widely utilized as a safe biocontrol agent against insect pests, forms the basis of this bioremediation system.
“Nature has designed these spores to be relatively immobile in soil, to allow the bacteria to persist in these environments,” Stewart says. “We have engineered B. thuringiensis to express enzymes tethered to the surface of the spores. The spore normally has a protein called BclA on its surface. Using the BclA attachment signals, we developed a method to trick the bacterium into attaching a foreign protein instead of BclA.”
Stewart says the expressed enzyme is engineered to be present in substantial quantities over the entire spore surface. Tethering an enzyme to the spore surface extends the time the enzyme is active. “Thus, high levels of stabilized enzymes are present on these spore particles,” he says.
In this system, the spores function as particles with attached enzymes. Because the spores need to remain as spores, rather than germinate to their bacterial cell form, the spores are killed prior to use. This effectively turns the spores into inert particles with enzymes attached. Because the spores are engineered to display the enzymes, no expensive protein purification steps are involved, greatly reducing costs.
“Inactivation of the spores produces nonviable particles which remain in the spore form,” says Stewart. “This biocatalytic system thus alleviates the major disadvantages of using free enzymes for breakdown of environmental pollutants. The spore platforms do not break down easily, as they are naturally resistant to harmful environments, such as acidic soil, heat and desiccation.”
And there are other benefits. “In addition to their potential use in soil environments, the spores can be immobilized on filters in a bioreactor, which can then be used to remove pollutants from groundwater environments,” Stewart says.
The predominant enzymes involved in biodegradation of explosives include nitroreductases from TNT and Cytochrome P450 reductase from RDX. These enzymes were first isolated from the microbial degraders such as Pseudomonas putida and Rhodococcus rhodochrous.
The goal, the researchers say, is to use the nitroreductases and Cytochrome P450 reductase to replace the expensive and environmentally destructive remediation techniques currently in use, chief among them incineration, composting and detonation. These typically cost from $100,000 to $1 million per acre.
The compounds Lin and Stewart are targeting are mostly remnants of munitions manufactured decades ago, including the nitrogen-rich explosive compounds left over from TNT (2,4,6-trinitrotoluene), HMX (high-melting explosive or Octogen) and RDX (royal demolition explosive or cyclonite). All are potentially detrimental to human health.
The Environmental Protection Agency has determined, for example, that TNT and RDX are possible human carcinogens. TNT exposures may also increase the risk of anemia, abnormal liver function and skin irritations. HMX has been linked to neurobehavioral effects in laboratory animals, while chronic exposure, the EPA says, is associated with liver and kidney damage.
Such risks may be compounded by these toxins’ staying power. “TNT and RDX are usually recalcitrant in the environment, meaning that they have a long residence time and are hard to decompose naturally,” says Lin.
Other leftover weapons components have also been implicated. Exposure to the propellant perchlorate may cause metabolic disorders of the thyroid. White phosphorus, used in incendiary munitions, can trigger reproductive disorders, along with potentially fatal liver, heart and kidney damage.
Most of the contaminated U.S. sites that could be cleansed by Lin and Stewart’s process are World War II-era munitions plants. During their operation, they generated industrial wastes that were disposed of using accepted practices of the times, which included on-site dumping, burial and open burning. Manufacturing processes and disposal caused contamination of soil and groundwater.
The Department of Defense has identified 2,307 sites that are potentially contaminated with military munitions. Of these, 25 were located in Missouri, 62 in Kansas, 29 in Nebraska, and 35 in Illinois. The state with the largest number of sites is California, with 376 known areas of contamination.
In all, according to the Government Accounting Office, more than 15 million acres in the United States are contaminated with unexploded ordnance, discarded military munitions and munitions constituents such as propellants and other chemicals.
Much of this land has been or will be converted to non-military uses such as farming, recreational or residential and commercial development. The Weldon Springs Ordnance Works, located about 30 miles west of St. Louis, is typical of these sites.
Opened just prior to America’s entry into WWII, during the height of the conflict the facility employed close to 5,000 people and produced millions of pounds of TNT and DNT. As with other plants of the era, contaminated wastes from the site were summarily dumped, buried and burned.
Although most pollutants from the 17,000-acre Ordnance Works site have since been cleaned up using current state-of-the-art methods, remediated areas, according to the EPA, will need to be monitored for the “foreseeable future.” Among such areas, the agency says, are “seven unlined lagoons where TNT wastewater was stored, TNT production lines, two DNT production lines, drainage ditches below TNT production lines, and eight areas where explosive wastes were burned.”
Lin has been studying ways to more efficiently remediate similar sites for more than a decade. When he first came to MU, he tested ways to use Missouri native grasses to absorb and degrade the nitrogen-rich explosive compounds in soil left over from TNT and RDX manufacture. He then considered introducing living microorganisms into this system to further enhance the breakdown of the compounds, but these microorganisms stopped utilizing munitions residue as food sources when they found other nitrogen sources in the soils. Lin’s interest in using enzymes started when he learned of previous studies showing that certain enzymes — if in place long enough — could reduce the presence of nitrites by 66 to 76 percent.
Lin and Stewart, along with Brian Thompson and Hsin-Yeh Hsieh, postdoctoral fellows in their lab, hit upon the idea of using bacterial spores as the enzyme platform during studies to characterize spore development. The mechanism for attachment of proteins to the spore surface was identified and characterized by Thompson and Stewart. Lin, Thompson and Hsieh, meanwhile, characterized the enzymes responsible for detoxifying the pollutants.
“Thus, the collaboration involving the MU Center for Agroforestry, the College of Veterinary Medicine and the Bond Life Sciences Center was born,” Stewart says. Lin and Stewart are encouraged by the initial research findings. The spore-enzyme system, they say, will degrade the pollution 20 to 50 times more effectively than applying the enzymes to the pollution alone. In an early test, TNT compounds began to be significantly degraded within 10 minutes.
“Our results,” Lin says, “suggested that more than 30 percent of TNT in water was degraded by the constructed spore-enzyme system within the first two hours” — an outcome that, if sustained, could go a long way toward finally bringing to a close a toxic legacy leftover from a heroic struggle.