Phytoremediation of TNT and RDX Shree Nath Singh and Shweta Mishra 1 Introduction Besides other organic contaminants, soil contamination by explosives also poses a serious environmental concern. Explosive compounds are released into the environment during manufacturing, handling and disposal operations at military sites to contaminate surface and ground waters, soils and sediments (Sunahara et al. 2009) In addition, aquatic environments are also contaminated with unexploded ordnance (UXO) and dumped ammunition wastes (Darrach et al. 1998; Rodacy et al. 2000; Dave 2003; Ek et al. 2006). Explosive compounds are heterocyclic nitramines and mostly nitro derivatives of benzene, toluene and phenol. They can be classified into two groups i.e. primary and secondary, based on their susceptibility to initiation when exposed to stimuli, such as heat, shock, friction etc. Primary explosives are highly susceptible to initiation and hence, often used to ignite secondary explosives, such as TNT (2,4,6trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazinane), HMX (1,3,5,7-tetranitro1,3,5,7-tetrazocane), and tetryl (N-methyl-N,2,4,6-tetranitroaniline).They exhibit low bioaccumulative potential due to weak hydrophobicity (Lotufo et al. 2009). Only a few natural nitroaromatic compounds, such as chloramphenicol, nitropyoluteorin, oxypyrrolnitrin and phidolopin are known to date. Apart from them, no other natural nitoaromatic compounds are available which show recalcitrance to biological degradation. These are carcinogenic and mutagenic in nature and also cause a disease to human, known as pancytopenia as a result of bone marrow failure and also have harmful effects on the liver (Amdur et al. 1991). The most common contaminants, found around active military firing ranges, are TNT, RDX and HMX. TNT and RDX are also priority pollutants in the list of United States Environmental Protection Agency (USEPA 2004). The distribution of these contaminants in sub-surface environments occurs through the dissolution S. N. Singh (&)
S. Mishra CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India e-mail: drsn06@gmail.com S. N. Singh (ed.), Biological Remediation of Explosive Residues, Environmental Science and Engineering, DOI: 10.1007/978-3-319-01083-0_16, Ó Springer International Publishing Switzerland 2014 371 372 S. N. Singh and S. Mishra of mixed solid-phase energetic residues, which are found to be spread around military ranges after detonation events. The dissolution of the residues occurs through the direct impact of precipitation events and by flowing surface runoff, or by percolating soil pore water. TNT, first time synthesized in 1863, was initially used in the dye industry before becoming in the 20th century, the main conventional explosive used by military forces worldwide. TNT is obstinate to oxygenolytic transformation due to mutual steric and electrophilic effects of multiple nitro substitutions on the aromatic nucleus (Esteve-Núñez et al. 2001; Preuss and Rieger 1995). On the other hand, RDX, which was formerly used as a rat poison, is today considered a carcinogen by the EPA (Binks et al. 1995; Lachance et al. 1999). RDX has low aqueous solubility (*40 mg/l) (Talmage et al. 1999). However, once dissolved, RDX can migrate with groundwater to pollute down gradient aquifers. A lifetime health advisory of 2 lg/l of TNT in drinking water and a water-quality limit of 105 lg/l of RDX have been recommended (Etnier 1989; Ross and Hartley 1990). Even though, many reports have suggested that this compound can be readily biodegraded, but RDX persists in the sub-surface environments for a longer time (Meyers et al. 2007). 2 Physical and Chemical Properties of RDX and TNT The uptake and transformation of energetic substances, such as TNT and RDX, by plants are regulated by both their physical and chemical properties (Table 1). TNT is a nitroaromatic compound and chemically known as 1-methyl-2,4,6-trinitrobenzene and commonly known as tolite. TNT is a highly reactive energetic compound, as three nitro functional groups are attached to an aromatic ring. It can undergo oxidation and reduction in both aerobic and anaerobic conditions (Hawari et al. 2000). But due to the presence of aromatic ring, TNT is resistant to electrophilic attack and hence rarely metabolized (Spain 1995). RDX - a hetrocyclic nitramine also known as hexagen, hexolite, trinitrohexahydrotriazine and cyclotrimethylenetrinitramine, is a major component of military explosives, such as Composition B (Comp B) and Composition 4 (C4) (Hewitt et al. 2007). Since RDX is fairly soluble, it does not get easily sorbed to soil particles, and hence, more transportable in the environment as compared to TNT. Octanol water partition coefficient is an important factor for the uptake of compounds by the plants from the soil and also for their movement through the membrane of the roots (Yoon et al. 2005). Many studies have reported that hydrophilic compounds, having log KOW less than 1.8, are not able to penetrate the lipid-rich membrane of roots, while hydrophobic compounds with log KOW greater than 3.8, will be easily taken up into the roots, but not translocated to the shoots (Yoon et al. 2005). The major difference between these two explosive compounds is the logarithm of their soil organic carbon–water coefficient (KOC). Since log KOC of TNT is over a hundred fold greater than RDX, it is strongly adsorbed to the Phytoremediation of TNT and RDX 373 Table 1 Physical and chemical properties of RDX and TNT (USEPA 2011a) Properties RDX TNT State at room temperature Molecular weight (g/mol) Water solubility (mg/l) Octanol-water partition coefficient log (Kow) Soil organic carbon water coefficient log (Koc) Vapour pressure at 25 °C (mm Hg) Henry’s law constant (atmm3/Mol) Molecular structure White crystalline solid 222 42 (at 20 °C) 0.87 1.80 Yellow, odorless solid 227 130 (at 25 °C) 1.6 300 4.0 9 10-9 1.99 9 10-4 1.96 9 10-11 (at 25 °C) 4.57 9 10-7 (at 20 °C) other organic matter present in the soil and gets immobilized, whereas RDX mainly moves deeply through the soil to the groundwater (Kalderis et al. 2011). 3 Phytoremediation of Ammunition Wastes Traditional treatments for the remediation of the toxic ammunition wastes (e.g., open burning and open detonation, adsorption onto activated carbon, photooxidation, etc.) are very costly and also deteriorate the environment. In many cases, it was found practically infeasible. Therefore, an inexpensive and environmentfriendly treatment was developed which is based on either microorganisms or plants or their combination. Phytoremediation is an attractive technology which uses green plants for the partial degradation of explosive compounds present in the soil and water. It was developed a few decades ago based on our knowledge that plants are capable of metabolizing toxic pesticides. It utilizes a variety of biological and physical characteristics of plants to aid in site remediation. Different classes of organisms, such as bacteria, fungi and plants, have been reported for the biotransformation of TNT, RDX, and HMX. The transformation occurs through a sequential reduction of the nitro groups to form toxic aromatic amino derivatives which are further transformed. Transformation is based on the ‘‘Green Liver’’ model which 374 S. N. Singh and S. Mishra describes the fate of organic contaminants within the plant tissues (Sandermann 1994; Burken and Schnoor 1997; Salt et al. 1998; Hannink et al. 2002). Phytoremediation encompasses several different technologies (1) phytoextraction involves bioconcentrating contaminants in the harvestable zones of the plant; (2) phytostabilization allays the bioavailability of contaminants by binding them to plant tissues; (3) phytodegradation degrades toxic compounds by the enzyme systems of the plants and plant-associated microorganisms and (4) phytovolatilization volatilizes the contaminants by the plants. Many plants, such as poplar trees, reed grass and agronomic plants have been reported to take up RDX and TNT (Sikora et al. 1997; Price et al. 2002; Best et al. 2004; Vila et al. 2007a) and concentrate them mainly in new growth (Seth-Smith et al. 2002). Harvey et al. (1991) also studied the uptake, translocation and transformation of RDX by plants, but the results were found quite different from that of TNT (Adrian et al. 2003). Besides, maize (Zea mays L.) and broad beans (Vicia faba L.) are also able to remove TNT from the soils (Van Dillewijn et al. 2007). Plants have developed the ability to take up the chemicals from the vapor, liquid and solid phases, but the movement of the organics within the plant usually occurs in solution. The uptake efficiency of the plants generally depends on following factors, such as pH, pKa, soil water, organic content, water partition coefficients (log Kow) and plant physiology (MacFarlane et al. McFarlane et al. 1990). Only chemicals, having log octanol: water partition coefficients (log Kow) between 0.5 and 3.0, are taken up by the plants. Among nitroaromatic explosives, nitrotoluene has log Kow  2:37, while 2,4-DNT possesses log Kow  1:98 (Briggs et al. 1982). Besides, water solubility and uptake of the contaminants can be enhanced by the use of both synthetic surfactant (Triton X-100) and naturally produced biosurfactants (rhamnolipids) (Salt et al. 1998). Once the explosive compounds have entered the plant tissues, they can be metabolized, stored (often in the root system) or volatilized. The biological degradation of contaminants can also be enhanced by another process of phytoremediation, known as rhizodegradation, in which, a symbiotic relationship between plants and microorganisms exists. Bacteria and fungi increase their activity in the rhizosphere of plants (Susarla et al. 2002) and result in the reduced toxicity and reduced nutrient deficiency in both bacteria and plants (Wenzel 2009). An increased removal of TNT has been also observed from an active rhizospheric zone of the prairie grass (Wolfe et al. 1994). The plant secretes some sugars, alcohols and acids which encourage the growth of rhizospheric bacteria around the root system (Schnoor et al. 1995). The bacteria humidify the organics and secrete the degradative enzymes, such as peroxidases and thereby, augment the degradation of contaminants (Dec and Bollag 1994). Besides, a few enzymes, such as nitroreductases, laccases and peroxidases, have been reported to be involved in the phytodegradation of nitroaromatic compounds (Schnoor et al. 1995). In addition, another process, known as rhizofiltration, also plays an Phytoremediation of TNT and RDX 375 important role in the remediation by mediating absorption and adsorption of the contaminants to the roots of the plant. Thus, plant root system plays an active role in the remediation of explosive contaminants (Salt et al. 1998). 4 ‘‘Green Liver’’ Model The uptake and transformation of energetic substances in plants is driven by simple diffusion and degradative enzymes. The assimilation of organics in plants is essential for the contact between plant cell enzymes and organic contaminants. The ‘‘Green Liver’’ model illustrates the process of transformation of explosive contaminants in plants, once they are taken up from the soil. According to early studies, plants deal with organic explosives, such as RDX and TNT in three phases as depicted in Fig. 1 (Van Dillewijn et al. 2008; Rylott and Bruce 2009): Phase I (transformation)—The contaminant is metabolized into a more soluble and less toxic intermediate products by several reactions, such as oxidation, reduction, or hydrolysis. The oxidative metabolism of explosive compounds is generally mediated by cytochrome P450 mono-oxygenase in plants. Infact, hydrophobic pollutants are emulsified to make them highly reactive electrophilic compounds for conjugation. In plants, cytochrome P450 forms a largest group of plant protein which plays an important role in degradation of explosives (Morant et al. 2003). Phase II (conjugation)—In conjugation between organic contaminant and endogenous hydrophilic molecules, such as D-glucose, glutathione, or amino acids, soluble or insoluble substances are produced to be subsequently sequestered in different cellular compartments of the plant for storage (Yoon et al. 2005; Schnoor et al. 2006). Conjugation also enhances metabolic activity which is further catalyzed by glycosyl-, malonyl-, and glutathione S-transferases. Phase III (compartmentation)—The soluble contaminants and breakdown products are sequestered into vacuoles or cell wall from the cytosol of the plants via ATP-binding, ABC transporters and multi-drug resistant proteins which play an important role in sequestration or compartmentation to reduce their toxicity and finally, the insoluble compounds are stored into the cell wall (Yoon et al. 2005). 5 Biotransformation 5.1 TNT Transformation by Plants Periwinkle (Catharanthus roseus) and parrot feather (Myriophyllum aquaticum) are two plants involved in the transformation of TNT. In this process, two main metabolites i.e. 2-amino-4,6-dinitrotoluene (2-ADNT)) and 4-amino-2,6-dinitrotoluene (4-ADNT), are formed as primary reduction products during the 376 S. N. Singh and S. Mishra Organic Compound in Environment Uptake Transport Transport, Xylem Flow Organic Compound in Xylem or Leaf Organic Compound in Root Tissue Transformation Reactions Metabolite in Plant Conjugation Conjugate Bound or Soluble Sequestration Compartmentalized Conjugated Compound Fig. 1 Green liver model for the metabolism of xenobiotics in plants (Burken et al. 2000) degradation of TNT by plants (Palazzo and Leggett 1986; Thompson et al. 1998; Bhadra et al. 1999). The formation of diaminotoluenes (2,4-diamino-6-nitrotoluene and 4,6-diamino-2-nitrotoluene) and azoxy compounds was also observed under strong reducing conditions and by the condensation of hydroxylamines, respectively (Pavlostathis et al. 1998; Sens et al. 1999; Thompson et al. 1998). TNT transformation pathway has been proposed by Rylott and Bruce (2009) as reflected in Fig. 2. However, Bhadra et al. (1999) studied the oxidation of TNT by plants and identified six oxidized metabolites, such as 2-amino-4,6-dinitrobenzoic acid, 2,4- dinitro-6-hydroxy-benzyl alcohol, 2-N-acetoxyamino-4,6 dinitrobenzaldehyde, 2,4-dinitro-6-hydroxytoluene, and two binuclear metabolites from azoxytetranitro toluenes during oxidative transformation of TNT (Fig. 3). The oxidized metabolites were detected in parrot feather (Myriophyllum aquaticum) during degradation of TNT (Subramanian 2004). Besides, they also concluded that oxidation of TNT could occur before the reductive transformation in plants. The reductive transformation of TNT has also been reported in non-axenic culture and aquatic plant systems where nitro groups of TNT undergo reduction with the formation of 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) and 4- hydroxylamino-2,6-dinitrotoluene (4HADNT) (Pavlostathis et al. 1998; Wang Phytoremediation of TNT and RDX 377 Vacuole Fig. 2 Proposed TNT degradation mechanism in plants (Rylott and Bruce 2009) RDX MNX DNX Light-mediated Breakdown CH2O CH3OH CO2 Fig. 3 RDX degradation mechanism in plants (Rylott and Bruce 2009) 378 S. N. Singh and S. Mishra et al. 2003). These hydroxylamines were also observed in the axenic hairy roots of Catharanthus and axenic Arabidopsis seedlings (Subramanian 2004; Subramanian et al. 2005). According to Subramanian and Shanks (2003) and Wang et al. (2003), hydroxylamines are the first transformation products which form other metabolites by undergoing reduction, oxidation, conjugation, and polymerization process. After transformation, the products of the TNT move to another phase called conjugation and are sequestered in the plant cells. Bhadra et al. (1999) reported that monoamines were the precursors to the conjugates. They have characterized four conjugates of TNT metabolites having a 6-carbon moiety in Catharanthus roseus and Myriophyllum aquaticum and found that out of four conjugates, two were similar to 2-ADNT and others were similar to 4-ADNT in molecular structures. Similarly, Vila et al. (2005) have also reported conjugates of TNT metabolites formed by conjugation of glucose on the hydroxylamine group of either 2HADNT or 4HADNT and also various other diglycoside conjugates with gentiobioside or sophoroside including monoglycosides by tobacco cell cultures. The conjugation of plant sugars with monoamines and hydroxylamines was also observed by Subramanian (2004) and Subramanian et al. (2005). 5.2 RDX Transformation by Plants The degradation of RDX was studied by Van Aken et al. (2004) in poplar tissue cultures and crude extracts of leaves (Fig. 4). In plant cells, during transformation process, RDX undergoes reduction and the metabolites produced were identified as hexahydro-1-nitroso-1, 3-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) (Fig. 5). Subsequently, MNX and DNX were transformed to formaldehyde and methanol, both in crude extracts and in intact cultures in the presence of light. In the final step, light-independent mineralization of one-carbon metabolites by intact plant cultures was observed, but not reported in crude extracts. Some of transformed products may be assimilated by the plants. In plants, enzymes mediate conjugation of formaldehyde to form S-formyl-glutathione (Just and Schnoor 2004). After complete mineralization of RDX by plants, small quantities of CO2 are produced to be re-assimilated in the photosynthesis process (Van Aken et al. 2004). 6 Phytoremediation 6.1 Phytoremediation of TNT The uptake and fate of energetic substances in plant system were found different for nitroaromatic and nitramine explosives. The degradation of TNT is largely found in the plant roots, where TNT remains due to its high biochemical activity of Phytoremediation of TNT and RDX TNT 379 4HA26DNT 2HA46DNT 4A26DNT 2A46DNT Fig. 4 TNT and its major metabolites RDX MNX MDNA DNX BHNA TNX NDAB Fig. 5 RDX and its major metabolites aromatic nitro group of TNT. It forms oxidative couplings on roots and hence, little or no translocation to leaves and stems occurs as examined by phosphor imager autoradiography (Schneider et al. 1996; Brentner et al. 2010). The most observed transformation process in the case of TNT is the aerobic reduction by the plants (Burken et al. 2000) and the most commonly observed reduction products formed in plants were monoaminated TNT metabolites (2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene). However, only a few plants have the potential to translocate TNT to leaves (Schneider et al. 1996). Hughes et al. (1997) also reported partial degradation and formation of metabolites in the aqueous medium by hairy root cultures of axenically grown Catharanthus roseus and Myriophyllum aquaticum plants. This observation was also endorsed by Bhadra et al. (1999) who reported 25 ppm of TNT degradation 380 S. N. Singh and S. Mishra within a few weeks by the hairy root cultures of Catharanthus roseus. Similarly, Pavlostathis et al. (1998) observed 100 % removal of TNT with the concentration up to 49 lM by Myriophyllum spicatum (Eurasian watermilfoil). However, smooth bromegrass (Bromus inermis L.), grown in a sterilized environment, was found to remove and/or break down TNT into less toxic by-products. Nelson (2001) reported that hydroponic cultures of sago pondweed (Potamogeton pectinatus L.) were able to dissipate TNT from water at a faster rate (below HPLC detection limits within 48–96 h) as compared to non-cultured plants, where only 37–56 % of the added TNT was lost. It was also observed during the experiment that when TNT was applied in successive doses (once every 4 days), sago pondweed was able to tolerate up to 0.5 mg/l TNT. However, a concentration of TNT 60 mg/l did not influence tuber germination of sago pondweed. Bae et al. (2004) observed that Indian mallow (Abuliton avicennae) removed 76.8 % of TNT from the soil, while 31.6 % was recovered in the soil as ADNTs and 0.2 % as TNT and ADNTs in the shoots and roots, respectively. However, in unplanted column, 51.9 % of the TNT was mineralised in the soil and 37.3 % was recovered as ADNTs. Working on degradation of TNT through plants, such as, Phragmites australis, Juncus glaucus, Carex gracillis and Typha latifolia, Vanek et al. (2006) observed a maximum of 90 % of TNT transformation within 10 days of cultivation. Among four plants, the most potential degrader was found to be Phragmites australis which transformed about 90 % of TNT within 10 days and 4-amino-2,6-dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT) were the first stable products formed during the degradation process. Similarly, Lee et al. (2007) worked on four plant species i.e. barnyard grass (Echinochloa crusgalli), sunflower (Helianthus annuus), Indian mallow (Abutilon avicennae) and Indian jointvetch (Aeschynomene indica), for the remediation of TNT contaminated soil and observed that all the four species had a high potential to remove TNT and its metabolites, regardless of whether the culture was grown single or mixed. The concentrations of TNT and its metabolites, 2-amino-4,6-dinitrotoluene (2-ADNT)) and 4-amino-2,6 dinitrotoluene (4-ADNT) were found very high in H. annuus, A. indica and A. avicennae except Echinochloa crusgalli. Ouyang et al. (2007) observed 25 % of the TNT removal from the soil by the poplar tree in 90 days by the use of UTCSP model (dynamic model for Uptake and Translocation of Contaminants from a Soil–Plant ecosystem). They also monitored a diurnal variation pattern in the uptake of TNT by roots and observed that TNT uptake was enhanced during the day and decreased during the night, most likely due to changes in leaf water transpiration as result of diurnal variations in xylem water potentials. Earlier also, Ouyang et al. (2005) made similar observation using CTSPAC model (mathematical model for coupled transport of water, solutes, and heat in the soil–plant-atmosphere continuum) on TNT removal from contaminated sandy soil by a single poplar (Populus fastigiata) tree. According to CTSPAC model, no TNT was found in the stem and leaves and only about 1 % of total TNT was observed in the roots due to rapid biodegradation and transformation of TNT into its intermediate products. About 13 % of the soil TNT was removed by root Phytoremediation of TNT and RDX 381 uptake of the poplar tree. Brentner et al. (2010) also investigated the localization of RDX and TNT in the plant tissues of Populus deltoids x nigra DN34 (poplar) and Panicum vigratum, Alamo (switchgrass) by the use of phosphor imager autoradiography. They observed that in both plants, TNT and/or TNT-metabolites remained predominantly in the root tissues, while RDX and/or RDX metabolites were readily translocated to leaf tissues. Makris et al. (2007) studied the uptake of 40 mg TNT/l for 8 days by vetiver grass in a hydroponic system and found that in aqueous medium, the concentration of TNT reached to method detection limit (1 mg/l) within 8 days, indicating vetiver high affinity for TNT with no visible toxicity. Das et al. (2010) also reported that vetiver grass potentially removed TNT when treated together with different TNT (0–100 mg/kg) and urea (0, 125, 350 and 1,000 mg/kg) concentrations. In the presence of urea, the removal rate of TNT was found as high as 91 %, indicating fast translocation of TNT from root to shoot. Major TNT metabolites, such as 2-ADNT, 4-ADNT and 1,3,5-TNB were detected in the plant tissues. In addition, Chekol et al. (2002) reported reed canary grass (Phalaris arundinacea L.) and switch grass (Panicum virgatum L.) as the most effective plant species which enhanced TNT transformation. About 77 and 73 % transformations of TNT (100 mg/kg) were observed by switch grass and reed canary grass, respectively. Jiamjitrpanich et al. (2012) discovered a new technology known as nanophytoremediation which is a combination between phytoremediation and nanoscale zero valent iron (nZVI) for removal of trinitrotoluene (TNT) from contaminated soil. In this study, a hyperaccumulator plant purple guinea grass (Panicum maximum) was used for nano-phytoremediation in soil with the TNT/ nZVI ratio of 1/10 (100 mg/kg initial TNT concentration) and observed a complete TNT remediation within 60 days. 6.2 Phytoremediation of RDX RDX is fairly soluble and mobile in the environment, as it does not bind well to organic or soil fractions. Therefore, it is readily translocated to shoots and leaves of the plants after its uptake as compared to TNT (Thompson et al. 1999). About 70 % of RDX was accumulated in the aerial parts of the plant. Photolytic transformation, that occurs in the aerial parts of the plant, is the primary mechanism of transformation during the degradation of RDX (Just and Schnoor 2004). Photolysis occurs mainly when water containing organic contaminants is taken up by plants and is released into the air through their leaves. Phytophotolysis phenomenon for the mineralization of RDX in poplar plant tissues was also observed by Van Aken et al. (2004). According to them, the mineralization of RDX occurs in three steps (1) a light independent reduction of RDX to MNX (hexahydro-1-nitroso-3,5dinitro-1,3,5-triazine) and DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine) by plant cells, (2) a plant/light mediated breakdown of RDX, MNX, or DNX into 382 S. N. Singh and S. Mishra metabolites (formaldehyde and methanol), (3) a light-independent mineralization of metabolites to CO2. The uptake of RDX by plants generally occurs, because log Kow of RDX is 0.87 (Burken and Schnoor 1997; Talmage et al. 1999). Brentner et al. (2010) monitored the translocation and transformation of RDX in the plant leaves by phosphor imager autoradiography. Thompson et al. (1999) studied plant uptake of RDX from both soil and hydroponic system and reported that due to decreased bioavailability of RDX in soil, the RDX uptake was slower than in the hydroponic system. About 71 % of RDX was taken up by hybrid poplar from the hydroponic system in 7 days. Similar observation was also made earlier by Harvey et al. (1991). They reported that less than 16 % of RDX was taken up by bush beans (Phaseolus vulgaris) from soil after 60 days, while 60 % was removed from the hydroponic system by the same plant after 7 days. It was also observed that the transformation and translocation of RDX were different from its co-contaminant TNT (Adrian et al. 2003). Thus, various studies have confirmed the uptake of energetic substances, such as RDX and TNT, by many plant species i.e. terrestrial, agronomic and wetland (Price et al. 2002; Vila et al. 2007a, b). Many agricultural crops such as lettuce (Lacutca sativa), tomato (Lycopersicon esculentum), corn (Zea mays), and cyperus (Cyperus esculentus) also play an important role in the removal of RDX through accumulation (Larson 1997). Bhadra et al. (2001) studied the uptake and transformation of 8 mg/L RDX by two plants i.e. Catharanthus roseus hairy root cultures and whole Myriophyllum aquaticum (parrot feather) and found that both plants have a high potential to remove RDX from the hydroponic system. They also pointed out that C. roseus had an intrinsic capability for removal of RDX. Similar observation for RDX removal by C. roseus and production of bound metabolites, was also made by Hughes et al. (1997). The formation of polar metabolites and bound residues of RDX metabolites was also observed during transformation of RDX (Hannink et al. 2002; Just and Schnoor 2004). Besides, mineralization of RDX was also reported by Van Aken et al. (2004). The production of mononitroso and dinitroso transformation products in the plant tissues during RDX degradation by plants has also been reported by other workers (Vila et al. 2007a; Reynolds et al. 2006; Larson et al. 1999). Thus, phytoaccumulation is the main process involved in the phytoremediation of RDX, as more than 90 % of soluble residues of RDX were detected as the parent compound (Price et al. 2002; Hannink et al. 2002; Vila et al. 2007a, b). In contrast to this hypothesis, some workers have suggested that during phytoremediation of RDX, phytodegradation may also act as a possible technology for its removal. Panicum maximum was reported as an effective species for the removal of RDX in Hawaii by Paquin et al. (2004). Lamichhane et al. (2012) observed that in the presence of molasses, the phytoremediation of RDX by guinea grass (Panicum maximum) was enhanced which resulted in RDX disappearance mainly in the root zone. Phytoremediation of TNT and RDX 383 6.3 Enzymes Phytodegradation (phytotransformation) is a mechanism by which plants tissue degrades contaminant by plant enzymes or enzyme co-factors (Susarla et al. 2002). For example, nitroreductase and laccase are a few enzymes reportedly involved in the breakdown of TNT and the metabolites are incorporated into new plant materials (Schnoor et al. 1995). The reduction of nitro groups of nitroaromatic compounds generally occurs through a group of enzymes known as nitroreductases (Bryant and DeLuca 1991). These enzymes use flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups and nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as reducing agents to catalyse the reduction of nitro-substituted compounds (Bryant et al. 1981; Bryant and DeLuca 1991). According to Schnoor et al. (1995), Myriophyllum aquaticum, an aquatic plant, produces a nitroreductase enzyme which mediates the partial degradation of TNT (128 ppm) within one week in the flooded soil. Transgenic plants are found to be more efficient degrader of explosive compounds than the wild type. These plants minimize the phytotoxic effects by expressing the bacterial genes which are reported to be involved in the degradation of TNT and RDX (Rylott and Bruce 2009; Van Aken 2009). For example, when pentaerythritol tetranitrate (PETN) reductase gene from Enterobacter cloacae strain PB2 was expressed in transgenic tobacco, an improved tolerance of transgenic tobacco to TNT was reported. Similarly, when bacterial nitroreductase gene (nfsI) was expressed in transgenic tobacco, the degradation of TNT was found much faster than the control plants (Hannink et al. 2001) as shown in Fig. 6. Van Dillewijn et al. (2008) had also reported that when Pseudomonas strain containing nitroreductase gene (pnrA) was expressed in poplar plant, it resulted in increased uptake of TNT from both water and soil. Gandia-Herrero et al. (2008) also observed overexpression of many important enzymes in plants in response to nitroaromatic compounds through microarray and other gene expression assay. According to them, phytoremediation of TNT can be improved not only by upregulating genes involved in the nitroreductase step, but also in the conjugation step. During microarray analysis, an over-expression of two uridine glycosyl transferases from Arabidopsis has resulted in both conjugate production and TNT detoxification. Similarly, oxophytodienoate reductases (OPRs) and glutathione-S-transferases (GSTs) were also upregulated in A. thaliana and Populus trichocarpa, respectively, in response to TNT exposure (Mezzari et al. 2005). Gene expression analysis in poplar trees exposed to TNT has clearly shown that genes for glutathione S-transferases may be mainly responsible for detoxification of TNT in the plants (Brentner et al. 2008). Rylott et al. (2006) reported that Rhodococcus rhodochrous 11Y had xplA gene (CYP177) encoding an enzyme, known as flavodoxin-cytochrome P450 which played a central role in the biodegradation of RDX. The tolerance and removal of RDX from soil was found ten times higher when xplA gene was expressed in the 384 S. N. Singh and S. Mishra Nitroreductase 2 nitroso dinitrotoluene (NODNT) hydroxylamino dinitrotoluene (HADNT) aminodinitrotoluene (ADNT) O Pentaerythritol tetranitrate reductase hydride-Meissenheimer complex (H- - TNT) dihydride-Meissenheimer complex (2H- - TNT) Fig. 6 Proposed transformation pathway of TNT by pentaerythritol tetranitrate reductases and nitroreductase (Williams et al. 2001) transgenic Arabidopsis plant as compared to non-transgenic plant. Jackson et al. (2007) also reported that the transformation of RDX by plants could be increased by 30 folds through the co-expression xplA and xplB (a flavodoxin reductase) in transgenic plants as compared to xplA alone. Similarly, the co-expression of nfsI and xplA genes in Poplar plants increased the removal of both TNT and RDX. 6.4 Rhizodegradation In phytoremediation, the degradation of pollutants is mainly mediated by rhizospheric microbes, a process called rhizodegradation. Rhizosphere is a zone between roots and soil which can be characterized by low redox potentials, abundant energy and nutrients, low pH, and high microbial activities due to root activities. The rhizoremediation is generally mediated by three step processes i.e. (a) sequestration or immobilization or retention of toxicants within a confined area, (b) removal of contaminants from the soil/waste water, and (c) destruction/degradation of organic pollutants by plant-microbial association. The contaminated soil is mainly treated by these three strategies either individually or in combination with each other. The microbes found in the plant roots are also involved in xenobiotic metabolism. Both bacteria and fungi present in the rhizosphere show catabolic activity mediated by the enzymes involved in the degradation process. Organic chemicals, released from both living and dead roots, used to modulate enzyme activity. It was observed that neither a single plant or microbe worked extremely well in immobilization, removal and destruction properties, nor a single Phytoremediation of TNT and RDX 385 species had shown faster degradation of organic contaminants. Hence, the contaminated soil may be successfully treated by a combination of plant species with appropriate remediation properties, aided by rhizospheric communities (bacteria and fungi) which are active against the specific contaminants present in the soil. The microbes in the rhizosphere have been also observed to possess plant growth stimulating properties (Campbell and Greaves 1990). They may fix nitrogen, synthesize siderophores and phytohormones, like auxins and cytokinins, and solubilize soil minerals for the plant growth (Glick 2003). The beneficial microorganisms in the rhizosphere are closely attached to the plant roots and are also known as plant growth promoting bacteria (PGPR). These microbes play an important role in recycling of plant nutrients, maintenance of soil structure, detoxification of noxious chemicals, and control of plant pests (Mackova et al. 2006; Rajkumar et al. 2009, 2010). Among the rhizospheric microorganisms, the Plant Growth Promoting Rhizobacteria (PGPR) and Arbuscular Mycorrhizal Fungi (AMF) have gained prominence all over the world to treat the contaminated soil (Ma et al. 2011). The microbes in the rhizosphere utilize root exudates containing sugars, organic acids and amino acids as source of nutrient and energy (Vancura and Hovadik 1965). The growth and activity of microbes in the rhizosphere can be increased by specific plant species which target the biodegradation of explosive contaminants. The rhizospheric microbes also reduce the plant toxicity of explosives through increased biodegradation process of explosives. Several studies have reported that grass species harbour a large population of bacteria in their vigorous root system which are found suitable for rhizodegradation. This process can be enhanced by increasing aeration of the soil by the plant roots, which penetrate the soil with highly developed fine roots and also by enhancing the contact of colonized bacteria with the organic pollutants (Kuiper et al. 2004). Yang (2010), during his experiment on rhizodegradation of TNT and RDX by two selected Missouri native grasses i.e. eastern gamma grass (EG, Tripsacum dactyloides) and switchgrass (SW, Panicum virgatum L.), observed that when 14C-spiked RDX and TNT rhizosphere soils were incubated for 8 weeks, both grasses were able to stimulate the rhizodegradation of RDX, TNT and its metabolites. More than 13 % of applied RDX was converted into CO2 as compared to 5 % as observed in the control. Eastern gamma grass was found more effective in augmenting RDX rhizodegradation than switch grass. But in case of TNT, more than 95 % of applied TNT was degraded in the first 7 days, but less than 2 % TNT was transformed into CO2 and six major degradation metabolites were identified. In contrast to RDX degradation, switchgrass appeared to be more effective for degrading TNT than eastern gamma grass. The degradation of explosive compound can also be enhanced by inoculating bacteria in the rhizosphere soil. The bacteria can be acclimatized in the rhizosphere by inoculating bacteria with coating seeds in the rhizospheric zone. Pseudomonas spp., are predominant plant growth promoting bacteria found in the rhizosphere. Other than bacteria, fungi are also reported to colonize plant roots, and increase the plant uptake of nutrients. The synergistic interaction between microbes and plants enhances the feasibility of the application of phytoremediation 386 S. N. Singh and S. Mishra technology on a large scale at relatively high explosive concentrations. Yang (2010) reported that when 14C-TNT spiked rhizospheric soils of both eastern gamma grass and switch grass were inoculated with Pseudomonas putida KT2440 and incubated for 8 weeks, more than 90 % TNT disappeared in the first 7 days and less than 1.2 % TNT was mineralized into harmless CO2. It was also observed that TNT degradation followed a second order kinetics of degradation in soils. Although P. putida KT2440 suppressed mineralization, more non-extractable residues were detected in the soil. Overall, switchgrass with P. putida KT2440 acted to possess the best capacity to degrade TNT in soil. 7 Conclusions Recently, phytoremediation has gained importance as an eco-friendly and selfsustaining technology against the conventional technologies available. It has got many advantages which include in situ applicability, low costs, no need for specific equipment and no introduction of new chemical substances into the environment to deal with various environmental toxicants. Over the years, there has been a substantial increase in our knowledge of the mechanisms involved in the uptake, transport, and detoxification of pollutants by plants and their associated microbes. However, phytoremediation efficiency has been not fully explored due to lack of our knowledge about basic plant processes and plant microbe interactions. Besides, there is a need for more phytoremediation field studies to demonstrate its effectiveness for enhanced acceptance by the public. Several researches are still ongoing for the development of plant-based remediation technology to make it a commercially viable industry. However, some key technical hurdles have to be overcome to make it a commercially viable technology. These include identifying more species with high remediation potentials, optimizing phytoremediation processes, such as appropriate plant selection and agronomic practices, understanding more about how plants uptake, translocate, and metabolize contaminants, identifying genes responsible for uptake and/or degradation for transfer to appropriate high-biomass plants, decreasing the length of time needed for phytoremediation to work, devising appropriate methods for contaminated biomass disposal, particularly for heavy metals and radionuclides that do not degrade to harmless substances, and protecting wildlife from feeding on plants used for remediation. Although, plants have the ability to remove explosives from the environment, but the processes for removal and transformation of TNT, RDX and HMX are different from the metals and regulated by enzymes and other several factors. Their products are conjugated and stored largely in vacuoles and cell walls of plants. Transgenic plants are specifically designed with bacterial genes to have a higher phytoremediation capacity than wild-type plants. Transgenic lines are more resistant to the toxic effects of RDX and TNT, take up higher quantities of explosives and more effectively degrade these substances. But, research on the use Phytoremediation of TNT and RDX 387 of transgenic plant lines in phytoremediation is still in the laboratory phase. The next phase would include experiments in the greenhouse and field trials to test their efficacy in explosive transformation and mineralization. No doubt, transgenic studies are very encouraging for the future of phytoremediation technology. The enhanced metabolism of GTN and TNT, as demonstrated in transgenic tobacco, indicates that the introduction of PETN reductase and the bacterial nitroreductase into grasses or fast-growing deep-rooted trees, such as poplars, more suitable for phytoremediation purposes, could significantly increase explosive removal in the field application. Besides, use of specific plant promoters to direct transgene expression to specific plant tissues is of great potential interest in enhancing the phytoremediation properties of plants. Application of new genomic technologies will provide an invaluable help in the identification of genes which enable explosives tolerance and their regulatory systems. The identification of enzymes mediating the detoxification systems will provide new targets for future rounds of genetic engineering which may provide robust plants for degradation of explosive compounds. 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