Using classical tax-onomic methods, the researchers were not able to differentially assign the isolate to either the B. thuringiensis or B. cereus species. The main objec-tives of this project are to develop molecular tools that can phylogenetically position GTN-degrading bacilli and to develop economical detoxification technologies. We have attempted the first route with mixed success. There are many degradation products and some may be strongly bound to the organic matrix of the wetland . This report considers the alternative approach using radio labelled 14C TNT. We used the same system of for other wetland experiments. A series of 1.5 L, replicated wetlands microcosms were set up using dead fragmented plant matter and a small soil inoculum. The microcosms were initially set up with nitrate to stimulate dentrifying bacteria. It is likely that bacteria that degrade nitrate will also be able to degrade TNT. The system was considered ready for TNT degradation when the nitrate concentration had fallen. The microcosms are anoxic, sparged with nitrogen gas and are slow flow-through systems. Incubations with added [U-14C] TNT were used to investigate mass balance and determine metabolite production including 14CO2. Any CO2 from the reaction solution was trapped in a series of three bottles containing 12-mL of scintillation cocktail. At the end of the experiment, the solution was acidified to pH 1 using concentration H2SO4 to volatilize any CO2 that remained in the solution. 14C in the final solution phase was also captured by scintillation cock-tail,blueberry plants in pots and then analyzed using a Beckman LS 6000 SC Scintillation Counter .
The projected costs of Department of Defense site restoration using existing technologies are staggering: the estimated cleanup cost is at least $24.5 billion for the 7,313 identified U.S. sites . The pollutants at these sites include chlorinated hydrocarbons, metals, petroleum products, explosives, mixed waste and other organics. There is clearly a need for new cost-effective treatment technologies. Bio-remediation, the use of microorganisms to detoxify hazardous waste, promises to provide economical and ecologically sound clean-up strategies. Tropical environments may have a large number of niches where unique microorganisms might have developed that can biodegrade organic contaminants and bio-transform heavy metals. This study is focused on the screening of new microorganisms in water samples from several sites in Puerto Rico and their potential use as agents for the transformation of wastes such as heavy metals, nitroaromatic compounds and other organic contaminants. The sites to be sampled include San Juan Bay in northern Puerto Rico and Yabucoa Bay to the southeast. An experiment has been designed that will use barrels holding soil contaminated with diesel fuel. The basic design is shown in Figure 1. Warm, humidified air will be pumped into the soil barrels. The warm, humidified air is meant to accelerate rates of bio-degradation within the barrels. A total of nine barrels will be used. Three barrels will have diesel contamination in the soil but will only be exposed to ambient dry air in order to provide a base line for monitored bio-degradation parameters. The diesel fuel will be mixed into the sand at a concentration of about 1,000 ppm. The sand, with a grain size of about 1 mm, will be spread out on a plastic liner. Diesel fuel will be dropped onto the sand while the sand is stirred to obtain a homogeneously contaminated sand. Then the contaminated sand will be put into the barrels.
The other three barrels with diesel-contaminated soil will be vented with warm, humidified air. The last three barrels will have a nitrogen-based fertilizer added to the diesel-contaminated soil to ensure that there is not a lack of nitrogen acting as a limiting factor to the rate of bio-degradation that can occur. Bio-degradation parameters will be monitored using the sampling ports as shown in Figure 1. Material and supplies needed for the project have been ordered and delivered to the project site, the University of Texas at El Paso Solar Pond facility. The barrels are currently assembled at the project site. The experiment started Jan. 10, 2000, with a six-month monitoring phase. Analysis and interpretation of results will start shortly after the first monitoring data are obtained.Most Department of Defense sites are contaminated with polycyclic aromatic hydrocarbons . PAHs contaminate military property as a result of open burning, incomplete fossil fuel combustion and accidental or deliberate hydrocarbon spills. PAHs rank among the most important environmental contaminants because of their persistence in the environment and their carcinogenic and mutagenic properties. Bio-remediation has been demonstrated to remove PAHs from contaminated soil. However there is evidence that the PAHs are not always mineralized during bio-remediation, but are transformed to stable intermediates that may remain in the soil after treatment. The identification of these intermediates is incomplete. Our task, the bio-degradation of PAHs, is an investigation of how bacteria transform high molecular weight PAHs in both laboratory culture and in the field. Our hypothe-sis is that we can use metabolite turnover to measure intrinsic PAH degradation. The scientific goals of the project are to develop a greater understanding of how bacteria that are able to grow on light PAHs can also oxidize heavier PAHs, such as pyrene and benz[a]pyrene. The technology or application goals of the project are to develop tools that allow us to study PAH degradation and PAH metabolite turnover in contaminated soils.
If we can link metabolite turnover and PAH degradation, we believe we can make a strong argument for an intrinsic degradation procedure based on monitoring metabolite turnover. The application of intrinsic remediation to fuel hydrocarbon contamination has proven a very cost effective approach to site clean-up and it would be beneficial if this technology could be applied to PAHs as well.A method for the extraction and analysis of PAH metabolites has been developed. This method uses an acetone extraction followed by a fluoracil column clean-up procedure that produces a methylene chloride fraction for GC analysis and an ethyl acetate fraction for HPLC analysis. This procedure was tested against known phenanthrene metabolites and found to be effective for their analysis in soil. Our current results suggest that this method is selective for carboxylic acids and excludes many alcohols. An HPLC method with fluorescence detection is used to selectively ana-lyze polynuclear compounds. Metabolite production was studied in a pure, well-characterized culture . As the bacteria grow on phenanthrene , we see a transitory accumulation of a metabolite. This metabolite is present only during active PAH turnover. Trinitrotoluene is chiefly used in military operations because it is a high explosive that is unaffected by shock or abrasion. TNT is also used in certain types of film processing. The compound has been manufactured and used for over a century with concomitant accumulation of TNT waste and unexploded ordinance. Although TNT is flammable, it is readily soluble in water and has accumulated in the groundwater of many military sites. In addition, TNT can also be found in small pellets,drainage plant pots which have become coated and are quite resistant to water. As with many hazardous wastes, TNT can be excavated from soil and incinerated, but costs are high and air pollution can be a problem. Thus, less costly alternatives are needed to remove TNT from a large number of sites covering a large area of the United States and elsewhere. It would prevent much trouble if the removal of TNT occurred on-site, since this would avoid shipping large volumes of contaminated soil or water on public highways. In the last two decades, wetlands have shown great promise for treatment of wastes, and continue to do so. In particular, there are now several large treatment wetlands for nitrate. These have operated for several years and are part of the water treatment system for several large public agencies. A number of investigators have already made important findings showing that TNT and other explosives could be taken up and transformed by living plants. Some of the plants tested may be amenable to full-scale wet-lands treatment systems. Wetlands are characterized as reducing systems but treatment wetlands are quite different from natural wetlands. In particular, treatment wetlands use plants grown in the wetland to provide carbon and habitat for bacteria that carry out the desired process. The soil underlying the dead leaf litter layer and the live plants play little role in the degradation or immobilization processes. It is not that these two components are not active, but that the flow rate and reaction rate in the leaf litter is engineered to dwarf that elsewhere.The main objective of this project is to study the ability of desert plants to adsorb toxic heavy metal ions from contaminated waters, thus reducing the threat to the public’s health. Metals such as lead , copper , nickel and chromium have been deter-mined to cause acute health effects in humans as well as ecological damage.
However, many desert plants have been found to survive in heavy metal contaminated soils. This resistance may be in part due to the development of chemical binding sites found on the exposed desert plant’s cell walls. Therefore, nature could hold the answer to removal of heavy metal ions from aqueous solutions. By utilizing the plant tissues that have evolved a natural affinity for heavy metal ions, we may be able to develop a method to remove these contaminants in a low-cost and highly effective manner. However, before a method can be developed to remove the heavy metal ions using desert plant tissues, the binding mechanism must be better understood.At the present time we are performing experiments with the desert plant Solanum ealeagnifolium . The plants were collected, dried and ground in preparation for the various metal binding studies. Batch laboratory methods are being employed to characterize the metal binding abilities of the desert plant. In addition, chemical modification experiments have been utilized to further study the metal binding mechanism of Solanum ealeagnifolium with the different metal ions being studied. Metal analysis for the batch experimentation has been performed utilizing flame atomic absorption spectroscopy and graphite atomic absorption spectroscopy with Zeeman background correction. In addition, samples have been analyzed utilizing synchrotron X-ray Absorption Spectroscopy at Stanford Synchrotron Radiation Laboratories at Stanford University in California. The XAS data are currently being examined to determine the X-ray Absorption Near Edge Structure of bound metals such as Cu and Cr. By using the computer software, Fourier transformation of the spectra can be performed, which provides the Extended X-ray Absorption Fine Structure of the metal bound on the biomass. These data can then be com-pared with model compounds to delineate the nearest neighbor atom bound to the metal ion on the biomass. The XAS data along with the chemical modification will help us pin down the actual chemical functional groups responsible for binding of the metal ions and help to find the most effective method to remove metal ions from contaminated waters. Thus far, we have determined that Solanum ealeagnifolium is able to bind appreciable amounts of metals tested. Figure 1 shows the percent of metal boundat various pHs. It is clear from Figure 1 that the binding of these metals is pH-dependent, with maximum binding occurring at or near pH 5.0. Since carboxyl lig-ands are known to have pKa values in the 3-4 region, we believe that carboxylic acids found on the cell walls may be involved in the binding of these metal ions. However, the binding of Cr did not follow the same trend. This indicates that Cr may be binding through a different mechanism. In order to better understand how carboxyl ligands on the cell walls of Solanum ealeagnifolium are involved in the metal binding process, we chemically modified the plant tissues and performed batch experiments with modified biomass. The results of the esterification and hydrolyzation capacity experiments are shown in Table 1. From the table, it is clear that the esterification of the cell walls of Solanum ealeagnifolium blocked the majority of the binding for these metals. These data support the finding from the pH profile, which indicates that carboxyl ligands are involved in the metal binding process. In addition, it can be seen form the table that by creating additional carboxyl ligands, there is an increase in the binding of these metals, which further supports that carboxyl groups found on the cell walls of Solanum ealeagni-folium are involved in the binding of these metal ions.