Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Increased Biotransformation Efficiency of Chunk-TNT-Contaminated Soil Using Acetone Pretreatments

Particulate, or chunk 2,4,6-trinitrotoluene (TNT), in soil was found to be recalcitrant to composting down to particle sizes of approximately 2 mm. Evidence for the colonization of TNT surfaces was obtained, but no pitting or otherwise preferential solid TNT solubilization was observed. Acetone pretreatments were used to make the chunk-TNT-contaminated soil more amenable to bioremediation. A pretreatment of acetone slurrying to dissolve and redisperse solid TNT in soil before applying remedial treatments was developed. The well-described treatment of composting was subsequently applied to native and acetone-pretreated contaminated soils. Acetone-pretreated soil responded to composting significantly better than untreated soil. After evaporating off the acetone used as pretreatment, composting microcosms held at 55°C showed sporadic removal from 3000 ppm to 300 ppm TNT in 24 days for untreated soil, while pretreated soil demonstrated conclusive removal from 3000 ppm to 18.1 ppm TNT in 6 days. Separate results indicated that residual acetone from pretreatment without subsequent evaporation was found to delay, but not otherwise inhibit, the compost's ability to degrade TNT. Community level physiological profile testing of 13-day-old composts, with pretreatment and residual acetone, suggests that three significantly different microbiological compost communities were equally adept at degrading the repartitioned TNT. The superior removal rates and efficiencies in the acetone-pretreated systems are likely to be due to the increased availability of TNT to the necessary microflora.     

Bioremediation of Soil Contaminated with Explosives at the Naval Weapons Station Yorktown

The explosives TNT, HMX, and RDX are integral components of many munitions. The wastes from the manufacture and the use of these and other explosives has resulted in substantial contamination of water and soil. White rot fungi have been proposed for use in the bioremediation of contaminated soil and water. Strains of Phanerochaete chrysosporium and Pleurotus ostreatus adapted to grow on high concentrations of TNT were studied with regard to their ability to degrade TNT in liquid cultures. Both strains were able to cause extensive degradation of TNT. Field bioremediation studies using P. ostreatus were performed on site at the Yorktown Naval Weapons Station Yorktown (Yorktown, VA). In two plots, 6 cubic yards of soil contaminated with TNT, HMX, and RDX were blended with 3 cubic yards of a substrate mixture containing nutrients that promote the growth of fungi. In soil amended with growth substrate and P. ostreatus, concentrations of TNT, HMX and RDX were reduced from 194.0±50, 61±20 mg/kg and 118.0±30 to 3±4, 18±7 and 5±3?mg/kg, respectively, during a 62-day incubation period. Interestingly, in soil that was amended with this substrate mixture, but not with P. ostreatus, the concentrations of TNT, HMX, and RDX were also reduced substantially from 283±100, 67±20, and 144±50?mg/kg to 10±10, 34±20, and 12±10?mg/kg, respectively, during the same period. Thus, it appears that addition of amendments that enhance the growth and activity of indigenous microorganisms was sufficient to promote extensive degradation of these compounds in soil.     

Microbial Transformation of 2,4,6-Trinitrotoluene in Aerobic Soil Columns

2,4,6-Trinitrotoluene (TNT)-contaminated soil material of a former TNT production plant was percolated aerobically in soil columns. Nineteen days of percolation with a potassium phosphate buffer supplemented with glucose or glucose plus ammonium sulfate caused an over 90% decline in the amount of extractable nitroaromatics in soils containing 70 to 2,100 mg of TNT per kg (dry weight). In the percolation solution, a complete elimination of TNT was achieved. Mutagenicity and soil toxicity were significantly reduced by the percolation process. 4-N-Acetylamino-2-amino-6-nitrotoluene was generated in soil and percolation fluid as a labile TNT metabolite.     

Treatment of munitions in soils using phytoslurries

Phytoremediation is an established technology for the treatment of explosives in water and soil. This study investigated the possibility of using slurried plants (or phytoslurries) to treat explosives (TNT and RDX). The degradation of TNT in solution using intact and slurried parrotfeather (Myriophyllum aquaticum), spinach (Spinicia oleracea), and mustard greens (Brassica juncea) was evaluated. Phytoslurries of parrotfeather and spinach removed the TNT faster than the intact plant. Conversely, the removal rate constants for slurried and intact mustard greens were about the same. A study using pressurized heating to destroy enzymatic activity in the phytoslurries was also conducted to compare removal from released plant chemicals to adsorptive removal. Aqueous phase removal of TNT by autoclaved spinach phytoslurry was compared with nonautoclaved spinach phytoslurry. The autoclaved phytoslurry did remove TNT, but not as completely as nonautoclaved slurry. This suggests that some removal is due to adsorption, but not all. Phytoslurries of mustard greens and parrotfeather had higher RDX removal rates compared with intact plant removal, but the rates for parrotfeather in either case were relatively low. Phytoslurries of spinach had relatively modest increases in RDX removal rates compared with intact plant. Studies were then conducted with phytoslurry/soil mixtures at two scales: 60 ml and 1.5 l. In both cases, phytoslurries of mustard greens and spinach removed TNT and RDX at higher levels than control slurries.     

Trinitrotoluene removal in a soil slurry and soil box systems by an oil-degrading mixed bacterial culture

Bioremediation of trinitrotoluene (TNT)-contaminated soil has proven difficult due to the low bioavailability of the contaminant and its resistance to biocatalytic attack, causing slow rates of biodegradation. We have previously described a mixed bacterial culture acclimated and maintained on crude oil-containing medium that is capable of high rates of TNT biotransformation activity with low production of metabolites. We investigated the ability of this culture to bioremediate TNT-spiked soil and artificially weathered soil slurry systems, as well as a soil box system. The culture was able to remove up to 302 ppm (mg/l) of TNT within 24 h in a spiked-soil slurry system, which is among the highest rates of TNT removal reported to date. The toxicity of artificially weathered TNT-spiked soil to Vibrio fischeri decreased over a period of 39 h from a 15-min EC₅₀ of 15.7 to 32.5 ppm. Preliminary results of a soil box system, in which no agitation was used, showed similar TNT removal to the soil slurry system, with 100 ppm TNT being removed within 24 h.     

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.     

Fate of explosives and their metabolites in bioslurrytreatment processes

Microcosm tests simulating bioslurry reactors with 40% soil content, containing high concentrations of TNT and/or RDX, and spiked with either [14C]-TNT or [14C]-RDX were conducted to investigate the fate of explosives and their metabolites in bioslurry treatment processes. RDX is recalcitrant to indigenous microorganisms in soil and activated sludge under aerobic conditions. However, soil indigenous microorganisms alone were able to mineralize 15% of RDX to CO2 under anaerobic condition, and supplementation of municipal anaerobic sludge as an exogenous source of microorganisms significantly enhanced the RDX mineralization to 60%. RDX mineralizing activity of microorganisms in soil and sludge was significantly inhibited by the presence of TNT. TNT mineralization was poor (< 2%) and was not markedly improved by the supplement of aerobic or anaerobic sludge. Partitioning studies of [14C]-TNT in the microcosms revealed that the removal of TNT during the bioslurry process was due mainly to the transformation of TNT and irreversible binding of TNT metabolites onto soil matrix. In the case of RDX under anaerobic conditions, a significant portion (35%) of original radioactivity was also incorporated into the biomass and bound to the soil matrix.     

Non-Ligninolytic TNT Mineralization in Contaminated Soil by Phanerochaete chrysosporium

The explosive 2,4,6-trinitrotoluene (TNT) is widely used and results in widespread soil contamination. The white-rot fungus Phanerochaete chrysosporium has been shown to degrade TNT, using the peroxidase enzyme. In this study, we report peroxidase-independent degradation of TNT by non-ligninolytic P. chrysosporium. Significant disappearance of TNT from highly contaminated soil using P. chrysosporium has been observed. Soil highly contaminated with TNT (2270 ppm [10 mM]) was diluted to 100 ppm (0.44 mM) with malt extract medium. Pregrown (48 hours) mycelial pellets of P. chrysosporium were added in 100 mL malt extract medium and incubated in Gledhill flasks. Analysis by high-performance liquid chromatography (HPLC) was conducted on soil extracts at specific time points to estimate the disappearance of TNT from contaminated soil incubated with P. chrysosporium. When the pregrown mycelial pellets were added, TNT disappeared within 48 hours. The dissolved concentration of 2-amino-4,6-dinitrotoluene (2Am-DNT) increased up to the third day, then declined before its final disappearance by day 10. Results show that the pregrown mycelial pellets of P. chrysosporium mineralized up to 17.3±6.3% [¹⁴C]-TNT within 30 days.     

Biodegradation of nitro-explosives

Environmental contamination by nitro compounds is associated principally with the explosives industry. However, global production and use of explosives is unavoidable. The presently widely used nitro-explosives are TNT (Trinitrotoluene), RDX (Royal Demolition Explosive) and HMX (High Melting Explosive). Nevertheless, the problems of these nitro-explosives are almost parallel due to their similarities of production processes, abundance of nitro-explosives and resembling chemical structures. The nitro-explosives per se as well as their environmental transformation products are toxic, showing symptoms as methaemoglobinaemia, kidney trouble, jaundice etc. Hence their removal/degradation from soil/water is essential. Aerobic and anaerobic degradation of TNT and RDX have been reported, while for HMX anaerobic or anoxic degradation have been described in many studies. A multisystem involvement using plants in remediation is gaining importance. Thus the information about degradation of nitro-explosives is available in jigsaw pieces which needs to be arranged and lacunae filled to get concrete degradative schemes so that environmental pollution from nitro-explosives can be dealt with more successfully at a macroscale. An overview of the reports on nitro-explosives degradation, future outlook and studies done by us are presented in this review.     

Lime Treatment of Explosives-Contaminated Soil from Munitions Plants and Firing Ranges

Microcosms were prepared using soils from munitions plants and active firing ranges and treated with hydrated lime. The presence of particulate explosives and co-contaminants, and the concentration of soil total organic carbon (TOC) on the alkaline hydrolysis reaction were studied. Trinitrobenzene (TNB) and dinitrobenzene (DNB) were sensitive to alkaline hydrolysis under these experimental conditions. The TNT metabolites, 2A- and 4A-DNT, were also removed, although more slowly than the parent compound, and the reaction required a higher pH (>12). RDX retention in the soil was proportional to the TOC content. The degradation intermediates of the alkaline hydrolysis reaction partitioned in the soil matrix in a manner similar to the parent. Solid particles of explosives are also degraded by alkaline hydrolysis. RDX and HMX exhibited 74 and 57% removal, respectively, in 21 days. TNT, as whole and broken grains, showed 83 and 99.9% removal in 21 days, respectively. The propellants, 2,4- and 2,6-DNT, were insensitive to alkaline hydrolysis. Alkaline hydrolysis is an inexpensive and effective means of reducing the varied explosives contamination.     

Ligninolytic and Nonligninolytic Mineralization of Trinitrotoluene by Several White Rot Basidiomycetes

The explosive 2,4,6-trinitrotoluene (TNT) is considered a toxic environmental pollutant that contaminates the soil and ground water. The white rot fungus Phanerochaete chrysosporium is well known for the degradation of TNT under ligninolytic condition. Very few, if any, studies have been done using other white rot fungi. In this study four fungal species, namely, P. chrysosporium, Kuehneromyces mutabilis, Hypholoma fasciculare, and Phlebia radiata, were used to investigate TNT degradation. All fungi were grown under ligninolytic (low-nitrogen) and nonligninolytic (high-nitrogen) conditions containing 25 parts per million (ppm) (0.11 mM) of TNT. Analysis by high-performance liquid chromatography (HPLC) showed biotransformation of TNT under both conditions. Complete degradation occurred under ligninolytic conditions (peroxidase enzymes were present) by P. chrysosporium and P. radiata. A nitrite release assay at 6 days indicated the denitrifying abilities of all the tested varieties of white rot fungi. For both ligninolytic and non-ligninolytic conditions, mass-balance studies showed biotransformation of 0.5 μ Ci ¹⁴C-labeled TNT with pregrown mycelial pellets of all fungal species, in which 5% to 15% of the TNT was converted to CO₂. These studies show that TNT may be degraded by several other species of white rot fungi and provided additional information on the biodegradation of nitroaromatic compounds in the environment.     

Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions

Summary Composting was investigated as a bioremediation technology for clean-up of sediments contaminated with explosives and propellants. Two field demonstrations were conducted, the first using 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and N-methyl-N,2,4,6-tetranitroaniline (tetryl) contaminated sediment, and the second using nitrocellulose (NC) contaminated soil. Tests were conducted in thermophilic and mesophilic aerated static piles. Extractable TNT was reduced from 11840 mg/kg to 3 mg/kg, and NC from 13090 mg/kg to 16 mg/kg under thermophilic conditions. Under mesophilic conditions, TNT was reduced from 11 190 mg/kg to 50 mg/kg. The thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT, 17.3 and 30.1 days for RDX, and 22.8 and 42.0 days for HMX, respectively. Known nitroaromatic transformation products increased in concentration over the first several weeks of the test period, but decreased to low concentrations thereafter.     

Long-Term Explosive Contamination in Soil: Effects on Soil Microbial Community and Bioremediation

Explosive contamination in soil is a great concern for environmental health. Following 50 years of munitions manufacturing and loading, soils from two different sites contained ≥ 6,435 mg 2,4,6-trinitrotoluene (TNT), 2,933 mg hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,135 mg octahydrol-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) kg^{? 1} soil. Extractable nitrate-N was as high as 315 and ammonium-N reached 150 mg N kg^{? 1} soil. Water leachates in the highly contaminated soils showed near saturation levels of TNT and RDX, suggesting great risk to water quality. The long-term contamination resulted in undetectable fungal populations and as low as 180 bacterial colony forming units (CFU) g^–1 soil. In the most severely contaminated soil, dehydrogenase activity was undetectable and microbial biomass carbon was very low (< 3.4 mg C _mic kg^–1 soil). The diminished biological activity was a consequence of long-term contamination because short-term (14 d) contamination of TNT at up to 5000 mg TNT kg^–1 soil did not cause a decline in the culturable bacterial population. Natural attenuation may not be a feasible remediation strategy in soils with long-term contamination by high concentrations of explosives.     

Effect of plant extract on the degradation of nitroaromatic compounds by soil microorganisms

Remediation of soils contaminated by nitroaromatic compounds and nitramines, i.e. explosives, is known as very important, complicated, and rapidly developing area of biotechnology. A search for optimal growth conditions for soil bacteria is of a great importance in order to isolate various xenobiotic degraders. Bacteria consortium A43 was isolated from soils contaminated with explosives. In the presence of carbohydrate and plant extract, an addition of TNT to the solidified minimal medium stimulated the growth of the tested bacteria, as compared to other bacteria consortium isolated from the same soils. Reducing sugars as carbohydrates, and cabbage leaf extract as a plant extract were used in these experiments. Cultivation of the A43 in liquid medium of the same content showed that addition of cabbage leaf extract alone to medium is much more efficient for TNT degradation by growing biomass as compared to addition of carbohydrate alone.     

Growth of a sitka spruce plantation: Spatial distribution and seasonal fluctuations of lengths,weights and carbohydrate concentrations of fine roots

Summary As part of an investigation into the primary production of a forest the activity of fine roots was estimated by taking weekly soil cores from 24 May to 27 September in the 11 year of growth of a plantation of Picea sitchensis. Distinct maxima were found in 1) starch and soluble carbohydrate concentration in the root, mid-June, 2) root weight/soil volume, early July, and 3) root length/soil volume, late July with a second maximum in early September. However, root concentrations in the soil were the same at the end as at the start of the period and it is suggested that the fine root system of the forest had reached a dynamic equilibrium.The growth of the fine root system, from mid-May to late July is described as a continuous process; there is no indication that root activity ceases during the period of shoot elongation.Two populations of fine roots were found in the forest. In the surface horizons of the soil roots classified with a diameter < 0.5 mm have a greater mean diameter, more root tips per unit length and are present in greater concentrations than in the peat and mineral soil below. Higher concentrations of root were found both in regions of soil which had been disturbed by preplanting cultivation and in regions close to the tree trunk. re]19760304     

Anaerobic/Aerobic Composting of Soil Contaminated with 2,4,6-Trinitrotoluene

A loam soil from Pennsylvania without a history of exposure to explosives was incubated with 5 g kg^-1 of ¹⁵N-labeled 2,4,6-trinitrotoluene (TNT) and 200 μCi kg^-1 of ¹⁴C-TNT for 3 days and then amended with compost at a 1:2 soil to compost ratio. The compost was prepared by mixing 40% alfalfa hay, 40% grass hay, 10% spent mushroom compost, and 10% municipal biosolids. The mixture of soil and compost was inoculated with methanogens from cattle manure, amended with glucose and starch, and incubated for 37 days under anaerobic conditions. The anaerobic incubation was followed by 26 days of forced aerobic incubation. At the end of the aerobic phase, most of the radioactivity was associated with organic matter; only 8.7% could be extracted with water and methanol, but no TNT was present in the extracts as determined by high-performance liquid chromatography. The unextractable radioactivity was associated with humic acid (40.0±1.0%), fulvic acid (14.3±1.4%), and humin (28.2±0.5%). Radioactive materials associated with humic acid and humin were analyzed by solid-state ¹⁵N-nuclear magnetic resonance (NMR) spectrometry. The NMR spectra indicated that nitro groups of TNT had been reduced to amino groups thatwere subsequently involved in the formation of covalent bonds with soil organic matter.     

Plant processes important for the transformation and degradation of explosives contaminants

Environmental contamination by explosives is a worldwide problem. Of the 20 energetic compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are the most powerful and commonly used. Nitroamines are toxic and considered as possible carcinogens. The toxicity and persistence of nitroamines requires that their fate in the environment be understood and that contaminated soil and groundwater be remediated. This study, written as a minireview, provides further insights for plant processes important for the transformation and degradation of explosives. Plants metabolize TNT and the distribution of the transformation products, conjugates, and bound residues appears to be consistent with the green liver model concept. Metabolism of TNT in plants occurs by reduction as well as by oxidation. Reduction probably plays an important role in the tolerance of plants towards TNT, and, therefore a high nitroreductase capacity may serve as a biochemical criterion for the selection of plant species to remediate TNT. Because the activities and the inducibilities of the oxidative enzymes are far lower than of nitroreductase, reducing processes may predominate. However, oxidation may initiate the route to conjugation and sequestration leading ultimately to detoxification of TNT, and, therefore, particularly the oxidative pathway deserves more study. It is possible that plants metabolize RDX also according to the green liver concept. In the case of plant metabolism of HMX, a conclusion regarding compliance with the green liver concept was not reached due to the limited number of available data.     

Effects of a soil fulvic acid on the growth and nutrient content of cucumber (Cucumis sativus) plants

Summary Cucumber (Cucumis sativus) plants were grown in Hoagland solution to which 20 to 2000 ppm of a soil fulvic acid (FA) were added. The addition of 100 to 300 ppm of FA produced highly significant increases in the growth and development of above and below ground plant parts, in the uptake of nutrient elements (N, P, K, Ca, Mg, Cu, Fe and Zn), and in the formation of numbers of flowers per plant. Effects of adding 500 and more ppm of FA were less beneficial.