Bioremediation of polynitrated aromatic compounds: plants and microbes put up a fight

Bioremediation of polynitrated aromatic compounds: plants and microbes put up a fight Juan L Ramos, M Mar Gonza´lez-Pe´rez, Antonio Caballero and Pieter van Dillewijn Industrialization and the quest for a more comfortable lifestyle have led to increasing amounts of pollution in the environment. To address this problem, several biotechnological applications aimed at removing this pollution have been investigated. Among these pollutants are xenobiotic compounds such as polynitroaromatic compounds — recalcitrant chemicals that are degraded slowly. Whereas 2,4,6-trinitrophenol (TNP) can be mineralized and converted into carbon dioxide, nitrite and water, 2,4,6-trinitrotoluene (TNT) is more recalcitrant — although several microbes can use it as a nitrogen source. The most effective in situ biotreatments for TNT are the use of bioslurry (which can be preceded by an abiotic step) and phytoremediation. Phytoremediation can be enhanced by using transgenic plants alone or together with microbes. Addresses Estacio´n Experimental del Zaidı´n, Consejo Superior de Investigaciones Cientı´ficas, Apdo Correos 419, E-18008 Granada, Spain Corresponding author: Ramos, Juan L ([email protected])

Current Opinion in Biotechnology 2005, 16:275–281 This review comes from a themed issue on Environmental biotechnology Edited by Gerben J Zylstra and Jerome J Kukor

Nitroaromatic compounds are true xenobiotics. Although nitroaromatics with few substituents such as mononitrotoluenes, nitrobenzoates and nitrophenols, are easily degraded by ubiquitous microbes, polysubstituted nitroaromatic compounds are more difficult to degrade [1]. Furthermore, when microbes able to deal with these chemicals are isolated they degrade them at a very slow rate. This slow rate of degradation can result from the intrinsic toxicity of the compounds, as well as from their limited solubility. Vast sites worldwide are contaminated with 2,4,6trinitrotoluene (TNT) and 2-4-6-trinitrophenol (TNP), because of the large-scale manufacture of these compounds. Bioremediation is an attractive means for decontaminating these sites. Several microorganisms have recently been isolated as able to use TNT as the sole nitrogen source; however, very few cases of mineralization have been reported [2,3]. Yet, several species of Actinomicetales have been found to mineralize TNP [4–6,7,8,9]. These findings have prompted the search for enzymes capable of ‘putting up a fight’ against polynitrated aromatic compounds such as TNT and TNP. In this review we have analyzed recent advances in the removal of polynitroaromatic chemicals by microbes, plants and plant–microbe associations.

Available online 8th April 2005 0958-1669/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.03.010

Introduction Since the end of the nineteenth century humans have developed a series of technologies that have brought increasing comfort to industrialized societies. A side effect of the development of new materials during this industrialization has been the generation of wastes, with their consequent impact on the biosphere. When wastes are degraded at a slower rate than their appearance in the biosphere they accumulate and become what we call a pollutant, constituting a burden to the environment. Among these wastes, xenobiotic compounds, which are chemicals with structures or substituents that are rarely found in natural products, represent a serious problem as they are seldom mineralized by living organisms and are often toxic. www.sciencedirect.com

Enzymes involved in TNT and TNP degradation Although there are currently no known pathways for the complete catabolism of TNT [2], this compound can be transformed into diverse products in a wide range of microorganisms by a variety of enzymes. Increased interest has been bestowed on the enzymes involved in TNT degradation: the best known of these enzymes are flavonitroreductases belonging to the b/a old yellow enzyme (OYE) family, such as XenA and XenB in Pseudomonas, pentaerythritol tetranitrate (PETN) reductase in Enterobacter cloacae, NemA in Escherichia coli, morphinone reductase in Pseudomonas putida M10, and OYE in Saccharomyces cerevisie [10,11,12–16]. The OYE family of enzymes attacks TNT using either one or both of two possible pathways (Figure 1): the sequential reduction of the nitro groups to hydroxylamine and amine derivatives (which can be catalyzed by any of the six enzymes mentioned above) or the reduction of the aromatic ring by hydride addition, with the subsequent Current Opinion in Biotechnology 2005, 16:275–281

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Figure 1

CH3 NO2

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Initial TNT (compound 1) degradation pathways. A: Reduction of the aromatic ring by OYE-type enzymes via a Meisenheimer complex (compound 2) to unresolved products with the release of nitrite. B: Common reduction pathway of a nitro group to a hydroxylamine derivative (compound 3). This product could be further reduced to amino derivatives (compound 4) or transformed to as yet unknown products, releasing ammonium in the process.

release of nitrite (catalyzed only by XenB, NemA and PETN reductase). The mechanism of hydride addition to the nitroarene aromatic ring has been studied with the PETN reductase of E. cloacae by Khan and coworkers [11,12]. They showed that in the reductive half-reaction of PETN reductase NADPH binds to form an enzyme–NADPH charge transfer intermediate before hydride transfer from the nicotinamide coenzyme to flavin mononucleotide. Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from the formation of its hydride–Meisenheimer complex. This is consistent with a mechanism involving a direct nucleophilic attack by hydride from the flavin N5 atom on the electrondeficient aromatic nucleus of the substrate. This was further corroborated when the crystal structures of complexes of the oxidized enzyme bound to TNT were solved [12]. The hydride–Meisenheimer complex then breaks down to form alternative products. The chemical identities of these products are uncertain, but have been studied in reactions catalyzed by PETN reductase of E. cloacae and XenB from P. fluorescens [15]. It is believed that nitrite is released in this process. The utilization of nitrite by microbes requires further reduction to ammonium. In agreement with this observation, several microbes that use TNT as a nitrogen source transitorily accumulate nitrite in the culture medium, which upon induction of nitrite reductase can be used as a nitrogen source [17]. Current Opinion in Biotechnology 2005, 16:275–281

All of the enzymes described above, as well as many other nitroreductases, are able to carry out a different reaction: the reduction of one or more nitro groups of TNT to its hydroxylamine moiety. These derivatives tend to release ammonium from the aromatic ring through an unknown mechanism that could involve a Bamberger-like rearrangement [18]. The E. coli NfsA and NfsB flavin proteins, which use NADPH and NADH, respectively, as a source of reducing equivalents, and PnrA in P. putida are among the nitroreductases that carry out this type of reaction [19–23]. These enzymes reduce TNT in addition to a variety of structurally diverse nitroaromatic compounds, including nitrofurans and nitroimidazoles [19–21]. Ammonium released from the TNT ring can be used as a nitrogen source by P. putida and E. coli via the glutamine synthetase/glutamate synthase pathway [17]. Watrous et al. [24] identified a new enzyme, an Fe-only hydrogenase, involved in TNT metabolism in the strict anaerobe Clostridium acetobotulinicum. This enzyme catalyzes the H2-dependent reduction of the nitro group of TNT to the corresponding hydroxylamine in an acidogenic environment. This finding is relevant not only because it expands the range of enzymes capable of attacking TNT, but because it also allows us to envisage biotreatments using microbes that colonize different niches. TNP, also known as picric acid, is structurally similar to TNT and can be mineralized. Several bacteria belonging www.sciencedirect.com

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Figure 2

(a)

orfA

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Gene cluster and early reactions in 2,4,6-trinitrophenol (TNP) metabolism. (a) The npd gene cluster of R. opacus HL PM-1 showing the proteins that have been functionally identified: npdC, hydride transferase I (HTI); npdF, hydrolase; npdR, regulator; npdG, NADPH-dependent F420 reductase (NDFR); npdH, tautomerase; npdI, hydride transferase II (HTII). (b) The degradation pathway of TNP: 1, TNP; 2, H -TNP; 3a, aci-nitro form of 2H–-TNP; 3b, nitro form of 2H -TNP; 4, dinitrophenol (DNP); 5, H -DNP; 6, 2,4-dinitrocyclohexanone (2,4-DNCH); 7, 4,6-dinitrohexanoate (4,6-DNH). (Figure modified from [5].).

to the genera Rhodococcus and Nocardioides grow aerobically on TNP or dinitrophenol and utilize these compounds as their sole nitrogen, carbon and energy sources [4–6,7,8,25,26]. The genes and enzymes involved in TNP degradation have been characterized [5,7,9,27]. The TNP degradation genes of Rhodococcus opacus HL PM-1 were identified using a messenger RNA differential display system [9]. Subsequently, the genes that encode the earlier steps of TNP degradation were located in a cluster (Figure 2). The npdI gene encodes hydride transferase II (HTII), which transfers the first hydride to TNP; npdC encodes hydride transferase I (HTI), which catalyzes the second hydride transfer giving rise to 2H -TNP (Figure 2). Both enzymes require an NADPH-dependent F420 reductase (NDFR) encoded by npdG to supply the hydride ions in the form of F420–H2. The npdH gene encodes a tautowww.sciencedirect.com

merase that catalyzes a proton shift between the acid– nitro and the nitro forms of the dihydride–Meisenheimer complex of TNP, whereas npdF encodes a hydrolase that converts 2,4-dinitrocyclohexanone (2,4-DNCH) into 4,6dinitrohexanoate (4,6-DNH) [5]. HTI, HTII and NDFR have low activity with TNT and can produce 2H -TNT. The tautomerase is able to convert 2H -TNT to different tautomeric forms, but the nitrite-releasing enzyme cannot apparently denitrate the complex and thus 2H TNT accumulates as a dead-end product in R. opacus. The TNP degradation gene cluster (Figure 2) also contains a transcriptional regulator, NpdR. In an npdR deletion mutant, high activity was found for HTII and HTI irrespective of whether they had been previously induced [7]. Furthermore, in the wild-type background NpdR represses the expression of npdI and npdC in R. opacus HL PM-1, as confirmed by in vitro gel shift assays. Current Opinion in Biotechnology 2005, 16:275–281

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Biological treatments of TNT Biological field treatments of TNT include composting, bioslurry processes and phytoremediation [26,28–31]. Composting and bioslurry treatments involve cometabolic processes that are based on the feasibility of the reduction of the nitro groups of TNT by microorganisms [30]. The final outcome of these processes is that hydroxylamine and amine groups on the nitroarene ring react with quinones and carbonyl groups of the humic fraction of the soil, giving rise to immobilized TNT derivatives that are not bioavailable and thus exhibit decreased toxicity [30]. In composting, the soil is mixed with alternative degradable organic material that is used by the microorganisms in the soil to transform TNT [29]. The best conditions for composting are produced in windrow composting, using alternate anaerobic and aerobic phases. The first anoxic step leads to the rapid reduction of TNT and condensation of the amine derivatives to the humic material. In the second phase, the products from the anaerobic treatment are metabolized by soil microbes to non-toxic products.

ques requires an understanding of how the plant interacts with TNT and how much of the contaminant is tolerated. Plant tolerance to TNT depends on the species, the growth stage of the plant, TNT bioavailability, and soil characteristics [28,39,41–43]. For instance, germinating seeds, seedlings or mature plants of the same species tolerate different concentrations of TNT. When TNT is bioavailable at higher levels, for example under aqueous conditions, the plant tends to tolerate lower concentrations of the xenobiotic than in soils where bioavailability is more restricted. In general terms, aquatic and wetland plants show growth inhibition and chlorosis (loss of chlorophyll) at concentrations ranging from 1–5 mg TNT/L, whereas in soil most plants can withstand between 50 mg and 100 mg TNT/kg and some even up to 1600 mg TNT/ kg soil. However, the mechanisms of TNT toxicity remain unknown.

Bioslurry processes involve the mixture of contaminated material with water and nutrients [26]. Bioslurry treatment is faster than composting and a high rate of TNT reduction can be achieved. A field-scale process, called sequential anaerobic bioremediation, uses as combination of anaerobic and aerobic steps. Using this technique with 14 C-labelled TNT no 14CO2 release was detected, but 15 N nuclear magnetic resonance results indicated that TNT reduction products were eventually incorporated as part of the soil material [29,32–36]. Recently, Weiss and co-workers [35] have studied the cleavage of 15N–C bound in soil. In addition to the production of 15NH4+ and 15NO2 , they also detected 15N2O. This finding suggested the possible slow metabolism of the immobilized products. In both types of treatment, extensive monitoring and a final evaluation of remediation efficiency are necessary for environmental safety considerations [37].

Plants seem to deal with TNT as though they were a ‘green liver’, whereby the contaminant is detoxified via the chemical transformation of TNT, conjugated to plant metabolites, and sequestered within plant tissues or polymers rather than being mineralized to carbon dioxide and nitrogen [28,40]. TNT can thus be transformed by reductive and oxidative processes in the plant. A wide range of reduction derivatives (e.g. 2-amino-4,6-dinitrotoluene, 4amino-2,6-dinitrotoluene, 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene) have been found in several plant species [42]. Most of the amino-dinitrotoluenes remain in the roots where their concentrations usually exceed those of TNT, whereas the TNT derivatives accumulate to a lesser extent in the stem and leaves. In the aquatic plant Myriophyllum aquaticum, TNT oxidation products have been found (e.g. 2amino-4,6-dinitrobenzoate, 2-N-acetoxyamino-4,6-dinitrobenzaldehyde, 2,4-dinitro-6-hydroxybenzyl alcohol and 2,4-dinitro-6-hydroxytoluene). The last two of these products may be the result of ring hydroxylation together with the removal of a nitro group. This is of interest because derivatives with fewer nitro groups are more susceptible to microbial degradation [44].

Because of the limitations of the processes described above, Schrader and Hess [38] proposed a combined system consisting of a fast abiotic pre-treatment with high concentrations of hydroxyl radicals followed by a bioslurry treatment to degrade the products from the first step. An advantage of this procedure is that TNT products, instead of being immobilized, are mostly (97%) mineralized. Nonetheless, the abiotic step could lead to a drastic reduction in the microbial populations, which may affect the second step in some cases.

TNT metabolites are subsequently conjugated with plant-derived glucose, malonate or glutathione. The nature of the intermediates changes with time into unknown non-extractable bound material, suggesting that conjugation acts as an intermediate step between transformation and sequestration. Sens et al. [45] reported for wheat that 43% of TNT and its derivatives were found in the cytoplasm, whereas the remaining 57% was present in the cell-wall fraction. Of the TNT in this fraction, 27% was associated to lignin.

Phytoremediation, in which plants are used to remove TNT from the soil, is considered a cost-effective alternative to the biological systems mentioned above [28,39,40]. Implementation of phytoremediation techni-

Transgenic plants bearing bacterial nitroreductases have been shown to exhibit increased tolerance to TNT (Figure 3). French et al. [46] and Hannink et al. [40] obtained transgenic tobacco plants that expressed the

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Figure 3

TNT. Their results support the green liver hypothesis, as among the enzymes encoded by upregulated genes were P450 cytochromes, which oxidize many xenobiotics and might have a role in TNT metabolism. Moreover, other upregulated proteins included glutathione S-transferases, which may be involved in the conjugation of these transformation products. The authors also observed increased expression of the genes involved in the plant response to oxidative stress. Induction of these genes was also observed in response to TNT in microarrays covering about 30% of the Chlamydomonas reinhardii genome [48], which suggests that at least part of the toxicity of TNT is derived from oxidative stress.

Conclusions and future perspectives The biochemical mechanisms underlying the different modes of attack on TNT are of great interest. Very little, if anything, is known about the reactions that lead to the removal of the nitro groups by denitrases, and knowledge acquisition is essential for the different reductive processes in the aromatic ring, reduction of the carbon skeleton or nitro groups. Further characterization of denitrases and their interactions with TNT should shed light on these interesting reaction patterns. Traditional methods to detect TNT and TNP are based on chemical analyses; however, alternative techniques based on immunochemical reactions (immunosensors) or living organisms (biosensors) are being developed. Immunosensors of TNT based on anti-TNT monoclonal antibodies provide considerable sensitivity and selectivity for the development of field-portable systems [49]. Biosensors can be engineered by combining regulators that recognize TNT and promoters fused to luciferase (lux) genes.

Transgenic aspen hybrid exhibiting high resistance to TNT in soil with this xenobiotic. (Figure courtesy of P van Dillewijn and colleagues).

PETN reductase gene (onr) or the nitroreductase gene (nfs) from E. cloacae, respectively. In wild-type plants, growth was inhibited at 0.025 mM TNT, whereas onrtobacco lines germinated and grew normally at 0.05 mM TNT. Although these transgenic plants failed to grow in media containing 0.5 mM TNT, nfs lines germinated well at this concentration and removed TNT from hydroponic media faster than wild-type plants. Recent work by Ekman et al. [47] described differential gene expression in Arabidopdsis roots in the presence of www.sciencedirect.com

Although research efforts regarding issues related to mass balance in the case of TNT are desirable, new metagenomic approaches will aid the identification of new enzymes with activity against nitroaromatic compounds. These methods require a host strain free of enzymatic activities against TNT. Such hosts are now available in our laboratory and will be useful in exploring a variety of niches to find new enzymes, which in addition to their applications in the removal of explosives may be of potential pharmaceutical interest. Some concerns remain that need to be addressed to make the phytoremediation of TNT more effective. The toxicity of sequestered TNT derivatives and the recalcitrance of these compounds in the environment remain unknown. Another major concern is bioavailability: if the plants do not come into contact with the contaminant, because it is tightly bound to the soil, their potential for phytoremediation will be restricted. It is likely that combined treatments with microbes and plants will overcome the limitations of phytoremediation [50]. Current Opinion in Biotechnology 2005, 16:275–281

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Update A number of recent articles have investigated the phytoremediation of TNT. Among these, one article used tobacco cell suspension cultures and the identification of conjugates of sugars with hydroxylaminodinitrotoluene isomers to reveal a role for glycosyltransferases in TNT phytoremediation processes [51]. In a very different study, rotation of salicaceae and conifer trees was proposed to be used for efficient phytoremediation [52]. Robertson and Jjemba [53] reported that a bacterial consortium established in soils polluted with TNT was able to mineralize almost 50% of 14C-TNT, and identified a strain of the genus Enterobacter as one of the key microbes in the mineralization of TNT. This report shows promising advances in the removal of this pollutant. Among microbes, a marine yeast was found to be capable of removing nitro groups from TNT and yielded 2,4-dinitrotoluene in a process in which a hydride-Meisenheimer complex was identified as a potential intermediate [54].

Acknowledgements Work in the authors’ laboratory was supported by a grant from the European Commission (MADOX, QLRT-2001-00345) and Ministerio de Medio Ambiente (Ref. 059/2004/3). We thank Carmen Lorente for secretarial assistance and Karen Shashok for improving the use of English in the manuscript.

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Current Opinion in Biotechnology 2005, 16:275–281