Curr Microbiol (2011) 63:450–457 DOI 10.1007/s00284-011-0001-7
Recent Advances in the Biodegradation of Chlorothalonil Guangli Wang • Bin Liang • Feng Li Shunpeng Li
•
Received: 28 January 2011 / Accepted: 15 August 2011 / Published online: 31 August 2011 Ó Springer Science+Business Media, LLC 2011
Abstract Chlorothalonil (TPN; 2,4,5,6-tetrachloroiso phthalonitrile) has been widely used as a broad-spectrum chlorinated aromatic fungicide and its application resulted in global pollution commonly detected in the diverse ecosystems. Recently, microbial degradation of TPN has been studied extensively as an effective and environmental-friendly method to reduce TPN residue levels in the environment. This review summarizes the current knowledge of recent developments in the biodegradation of TPN. Diverse pure culture strains capable of degrading TPN were widely distributed among Proteobacteria and several metabolic pathways of TPN biotransformation were discovered. The two key genes (glutathione S-transferase and chlorothalonil hydrolytic dehalogenase coding gene) responsible for the conversion of TPN and recent findings for future practical bioremediation of TPN-contaminated ecosystem are also discussed.
Guangli Wang and Bin Liang contributed equally to this study. G. Wang B. Liang S. Li (&) Department of Microbiology, Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, People’s Republic of China e-mail:
[email protected] G. Wang F. Li (&) Anhui Key Laboratory of Plant Resources and Biology, College of Life Sciences, Huaibei Normal University, Huaibei 235000, Anhui, People’s Republic of China e-mail:
[email protected] B. Liang State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, Heilongjiang, People’s Republic of China
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Introduction Chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) belongs to the group of halogenated benzonitriles which is one of the most widely used fungicides in the world. It was invented in 1963 by the Diamond Alkali Co. and was first registered for use in the US in 1966. TPN has become the second most widely used agricultural fungicide in the US, with five million kilograms applied annually [11]. In China, the annual production of TPN exceeds 8,000 tons [25]. Although the exact mechanism is not fully understood [14], it is believed that TPN works by contacting with target fungi, inhibiting glutathione-related enzymes in cell respiration [3]. TPN is highly toxic to fish, birds, and aquatic invertebrates [6, 12] and can cause human dermatitis, severe eye, skin irritation, and gastrointestinal problems [13]. It is considered as a probable human carcinogen by US Environmental Protection Agency. The half-life of TPN is 1–2 months [48] but may remain in soil for 100 days [47], or even 1 year after repeated application [29]. Owing to its widespread use and persistence in soil, TPN is commonly detected in diverse ecosystems such as vegetables and fruits [52, 56], soil and water [7, 11], groundwater [55], greenhouse air [20], and the area as remote as the surface micro-layer and fog of the Chukchi Arctic ecosystem [8]. Bioremediation of TPN pollution is recognized as a cost-effective and reliable method. Therefore, there is a need to study the microbiological degradation of TPN, both for application and action mode. This review presents an overview of recent advances in the microbiological degradation of TPN involving pure culture strains and soil microbial consortia capable of degrading TPN on metabolic pathways of TPN biodegradation, bioremediation of TPN in soils and the key genes
G. Wang et al.: Biodegradation of Chlorothalonil
responsible for the TPN biotransformation as well as perspectives of the bioremediation of TPN-contaminated ecosystem.
Pure Culture Strains Capable of Biodegrading TPN Among the TPN-biodegrading pure culture bacterial strains that have been isolated in the past decades, Flavobacterium NL0-1 and A0-6 [17], unidentified bacterium TBI [29], and Ochrobactrum lupini strain TP-D1 [42] were reported to removal TPN without providing other carbon sources (Table 1). In recent experiment, sixteen strains (designated CTN-1 to CTN-16) capable of degrading high concentration of TPN (50 mg l-1) have been isolated that exhibit the same metabolic pathway transforming TPN to 2,5,6-trichloro4-hydroxybenzene-1,3-dicarbonitrile (TPN-OH) in the absence of a carbon source, although they cannot use TPN for growth [25, 26, 49, 50]. These isolates belonged to eight different genera (Ochrobactrum, Shinella, Caulobacter, Rhizobium, Bordetella, Pseudomonas, Pseudoxanthomonas, and Lysobacter) in different classes of Proteobacteria [26] (Table 1). Seven TPN-degrading strains, belonged to Pseudomonas, Achromobacter, Ochrobactrum, Ralstonia, and Lysobacter, were isolated from another TPN-contaminated environment [36]. Soil microorganisms capable of co-metabolizing low concentrations of CTN (B0.5 mg l-1) in the presence of other carbon sources (glucose, acetate or 1/10 diluted nutrient broth), such as Bacillus cereus NS1 [58], Azomonas sp. A40-2 [17–19], Moraxella sp. A0-1 [17, 18], Acinetobacter calcoaceticus IFO 12552 [18], Agrobacterium radiobacter IFO 13532 [18], and other diverse TPNdegraders were compiled Table 1. Among these TPN cometabolizing strains, 21 strains could form metabolite Methylthiotrichloroisopathalonitrile (MT) during TPN (0.5 mg l-1) biotransformation in the 1/10 diluted nutrient broth [18]. In addition, 14 strains placed in the phylogenetic group of Bacillales (Firmicutes), Actinobacteria, and Flavobacteria (Bacteroidetes). 77.8% of TPN-degrading strains were affiliated to various classes of Proteobacteria which indicated that TPN-degrading bacteria are widely distributed among Proteobacteria in TPN-contaminated or uncontaminated ecosystems.
Metabolic Pathways of TPN Biodegradation in Microorganisms Mechanisms for microbial attack on TPN have been described in the literatures. Until now about 20 kinds of TPN metabolites have been detected during TPN biotransformation by pure culture strains or soil microbial
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consortia [7, 18, 19, 21–23, 25, 26, 29, 34–39, 42, 49]. There are five main metabolic pathways to biodegrade TPN. One mechanism is to displace 4-chlorine atom with a hydroxyl group (via hydrolytic dechlorination) to generate TPN-OH [18, 23, 29, 34, 36, 38, 39, 42]. We observed that the 16 strains isolated recently exhibited this hydrolytic dechlorination pathway and form 4-OH-TPN from TPN biodegradation [25, 26, 49]. The second metabolic pathway involved the oxidation/hydration of cyano group to corresponding amides, thiazoles, and acidic groups [7, 23, 34, 35, 37, 38, 42]. The third pathway involves the biodegradation of TPN by the glutathione-dependent glutathione S-transferase (GST), such as in O. anthropi SH35B [21, 22]. The fourth pathway suggests that the 4-chlorine atom of TPN could be substituted by a methylthio group to generate 2,5,6-trichloro-4-(methylthio)isophthalonitrile [18, 19, 34], or by a methoxy group to generate 2,5,6trichloro-4-(methoxy)isophthalonitrile [39]. In the fifth pathway the chlorine atoms of TPN could be substituted with hydrogen atoms by a reductive dechlorination process [18, 23, 34, 39]. The related metabolic pathways of TPN biodegradation are shown in detail in Fig. 1. Among those metabolites, TPN-OH is probably the primary metabolite resulting from TPN degradation often found in diverse ecosystems [1, 20, 33, 34, 37, 38].
Two Key Genes Responsible for the TPN Biodegradation Currently, various metabolic pathways involved in the biodegradation of TPN have been reported. However, only two genes responsible for the TPN dechlorination have been cloned from different TPN-degraders [21, 22, 26, 49]. O. anthropi SH35B degrades TPN by GST [21, 22], which can utilize the glutathione (GSH) to replace the three chlorine atoms of TPN with glutathione thiol molecules by GST catalysis (Fig. 1). In fact, the GSTlike enzyme working as a dehalogenase has been reported for the dechlorination of the chlorinated compounds dichloromethane [24] and pentachlorophenol [4, 15]. Interestingly, in Ochrobactrum sp. CTN-11, even though the cloned GST-like gene has 88% similarity with that from O. anthropi SH35B, it is not functionally expressed in the presence of glutathione. This indicates that TPN was not reductively dechlorinated by thiolytic substitution catalyzed by GST in strain CTN-11 [25]. Subsequently, it was confirmed that dehalogenation of TPN in strain CTN-11 was due to hydrolytic action. The different degrading mechanisms of TPN between Ochrobactrum sp. CTN-11 and O. anthropi SH35B indicated the metabolic diversity of TPN in the same genus [25, 26].
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G. Wang et al.: Biodegradation of Chlorothalonil
Table 1 The summary of microorganisms capable of biodegrading TPN Microorganisms
Metabolites of TPN biodegradation
Phylogenetic group
References
Unidentified strain TB1
(14)
ND
[29]
Unidentified strain Y1
ND
ND
[57]
Achromobacter sp. RB-16
(14)
b-Proteobacteria
[36]
Acinetobacter calcoaceticus IFO 12552
(12)
c-Proteobacteria
[18]
Agrobacterium radiobacter IFO 13532
(12)
a-Proteobacteria
[18]
Alcaligenes faecalis IFO 13111
(12)
b-Proteobacteria
[18]
Azomonas sp. A40-2
(12); (14) and (9), (10) or (11)
c-Proteobacteria
[17–19]
Azomonas sp. I40-1 and N40-1
ND
c-Proteobacteria
[17]
Bordetella sp.CTN-10 and CTN-16
(14)
b-Proteobacteria
[26]
Bacillus cereus IFO 15305 and NS1
ND
Bacillales
[18, 58]
Bacillus megaterium ATCC 13368
ND
Bacillales
[18]
Bacillus subtilis DB101
(12)
Bacillales
[18]
Bacillus sp. B75
ND
Bacillales
[18]
Caulobacter sp.CTN-14
(14)
a-Proteobacteria
[26]
Corynebacterium sp. MM22
(12)
Actinobacteridae
[18]
Flavobacterium sp. A0-6a
(14)
Flavobacteria
[17, 18]
Flavobacterium sp. NL0-1a
ND
Flavobacteria
[17, 18]
Klebsiella pneumoniae ATCC 13906
(12)
c-Proteobacteria
[18]
Lysobacter sp. RB-38
(14)
c-Proteobacteria
[36]
Lysobacter ruishenii CTN-1T
(14)
c-Proteobacteria
[26, 50]
Micrococcus luteus IFO 3333
Trace amount of (12)
Actinobacteridae
[18]
Moraxella sp. A0-1 and A0-5
(12)
c-Proteobacteria
[17, 18]
Moraxella sp. A40-3
ND
c-Proteobacteria
[17]
Nocardia asteroids IFO 3384
ND
Actinobacteridae
[18]
Ochrobactrum sp. CTN-11 and RB-28
(14)
a-Proteobacteria
[25, 26, 36]
Ochrobactrum anthropi SH35B
Biotransformation by GSTb
a-Proteobacteria
[21, 22]
Ochrobactrum lupini TP-D1
(14), (16), (18), (22)
a-Proteobacteria
[42]
Pseudoxanthomonas sp. CTN-8 and CTN-9
(14)
c-Proteobacteria
[26]
Pseudomonas sp. A0-3, N40-1 and I40-1
(12)
c-Proteobacteria
[18]
Pseudomonas sp. A0-2
ND
c-Proteobacteria
[17]
Pseudomonas fluorescens IFO 12180
(12)
c-Proteobacteria
[18]
Pseudomonas putida IFO 14164
(12), (14)
c-Proteobacteria
[18]
Pseudomonas stutzeri IFO 3773, IFO 12510 and IFO 13596
(12)
c-Proteobacteria
[18]
Pseudomonas stutzeri A6
ND
c-Proteobacteria
[18]
Pseudomonas vesicularis IFO 12165
ND
c-Proteobacteria
[18]
Pseudomonas sp. CTN-2, CTN-3, CTN-5, CTN-6, CTN-7, RB-4, RB-5, and RB-9
(14)
c-Proteobacteria
[26, 36]
Ralstonia sp. RB-29
(14)
b-Proteobacteria
[36]
Rhizobium sp. CTN-4 and CTN-15
(14)
a-Proteobacteria
[26]
Rhodococcus coprophilus NCIB 11211
ND
Actinobacteridae
[18]
Rhodococcus globerulus IFO 14531
Trace amount of (12)
Actinobacteridae
[18]
Rhodococcus rhodochrous IFO 11277
ND
Actinobacteridae
[18]
Shinella sp. CTN-12 and CTN-13
(14)
a-Proteobacteria
[26]
Sphingomonas sp. K-1400 and K-1402
ND
a-Proteobacteria
[18]
Staphylococcus aureus IFO 12732
ND
Bacillales
[18]
Xanthomonas maltophilia A21
ND
c-Proteobacteria
[18]
a
TPN as the carbon and energy source during the TPN biodegradation
b
TPN was rapidly transformed by the GST in the presence of glutathione and mono-, di-, and triglutathione conjugates of TPN by the GST catalysis were identified The numbers of brackets are the same as those in Fig. 1, ND not determined
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453
Fig. 1 Proposed metabolic pathways of TPN metabolism in microorganisms. Dashed line indicated that the metabolites were not identified. Symbols of ‘‘()’’ denote as follows: (1) isophthalonitrile (IPN); (2) 5-Clisophthalonitrile, (3) 4-Cl-isophthalonitrile, (4) 4,6-C12-IPN, (5) 2,4C12-IPN, (6) 2,5-C12-IPN, (7) 4,5-C12-IPN, (8) 2,5,6-C13-4-(OCH3)IPN, (9) 4,5,6-C13-IPN, (10) 2,4,5-C13-IPN, (11) 2,4,6-C13-IPN, (12) methylthiotrichloroisophthalonitrile, (13) 2,4,5,6-C14-IPN (TPN), (14) 2,5,6-C13-4-(OH)-IPN, (15) 1-carbamoyl-3-cyano-4-hydroxy-2,5,6-
trichlorobenzene, (16) methyl 2,5,6-trichloro-3-cyano-4-methoxybenzoate, (17) 3-cyano-2,4,5,6-tetrachlorobenzamide, (18) methyl 3-cyano-2,4,5,6-tetrachlorobenzoate, (19) 4-(glutathion-S-yl)-2,5, 6-trichloroisophtalonitril, (20) 4,5,6-tri-(glutathion-S-yl)-2-chloroisophtalonitril, (21) 4,6-bis(glutathion-S-yl)-2,5-dichloroisophtalonitril, (22) 3-methoxycarbonyl-2,4,5,6-tetrachlorobenzeneacetamide, (23) 3-cyano-2,4,5,6-tetrachlobenzoamide, (24): 3-carbamyl-2,4,5-trichlorobenzoic acid
A novel chlorothalonil hydrolytic dehalogenase gene (chd) was cloned from Pseudomonas sp. CTN-3 by shortgun method [49]. Chd (36 kDa) contains a putative
conserved domain of the metallo-b-lactamase superfamily and shows the highest identity with several metallohydrolases (24–29%) [49]. Until now, two hydrolytic
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dehalogenases for halogenated aromatics have been reported: the 4-chlorobenzoate dehalogenase complex and Chd. The hydrolytic dehalogenation activity of the 4-chlorobenzoate dehalogenase complex is comprised of three separate enzymes, 4-chlorobenzoate-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoylCoA thioesterase and needs CoA and ATP. On the contrary, the Chd enzyme required no other cofactors for hydrolytic dehalogenation [40, 41, 49]. Site-directed mutagenesis of Chd revealed that histidines 128 and 157, serine 126, aspartates 45, 130 and 184, and tryptophan 241 were essential for the dehalogenase activity [49]. The comparison of the chd gene of strain CTN-3 and that amplified from other 23 TPN-degraders were found to be highly conserved ([98%) [26, 36].
Persistence of TPN and Bioremediation of TPN in Soils The persistence of TPN residues and its effects on soil microorganisms after single application have been investigated. The half-life of TPN in soil varies from 1 to 12 months [29, 47, 48], depending on the soil type [16, 31, 43]. TPN residues have been shown to potentially influence soil bacterial and fungal populations [28, 43], soil enzymes activities and the total number of soil microorganisms [44, 45]. Soil microorganisms and enzymes were only affected by the first one or two applications of TPN at a field relevant application rate. Additional applications of TPN at the same rate did not inhibit the overall population densities of soil microorganisms or soil enzymatic functions [57]. A silty clay loam soil was treated with TPN at three different dosages (2.5, 25, and 250 mg/kg). All dosages of TPN significantly decreased fungal biomass as estimated by soil ergosterol content [32]. TPN significantly altered soil microbial activity. However, changes in soil microbial biomass were only observed in soil treated with higher dosages of TPN, which can reduce actinomycetes population, suggesting that application of TPN decreased the ratio of Gram-positives to Gram-negatives [32]. Research on 14C-labeled TPN dissipation in three acid Brazilian soils revealed that biodegradation and soil binding are main reasons for fast TPN dissipation instead of mineralization by TPN-degrading microorganisms [35]. Most soil-bound 14C residues were formed in the first day, but aging also contributed to the formation of less reversible forms of TPN-soil complexes. The 3-carbamyl-2,4,5trichlorobenzoic acid was the most abundant metabolite formed from TPN breakdown in these soils [35]. The anaerobic TPN biodegradation is strongly affected by the properties of the four paddy soils. Soils associating with rich total carbon, repeated TPN application, and neutral pH have shown the high capacity to biodegrade TPN [51]. The
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G. Wang et al.: Biodegradation of Chlorothalonil
higher the initial TPN concentration the lower TPN removal efficiency will be. In addition, anaerobic TPN biodegradation was accompanied by methane generation and a decrease of the oxidation–reduction potential [51]. Liang and collaborators found that the inoculation of Ochrobactrum sp. CTN-11 cells in sterile soil and nonsterile soil resulted in a quicker degradation of TPN and that 50 mg kg-1 TPN were degraded to a non-detectable level in both sterile and non-sterile soils in 3 days [25]. Shi et al. found that O. lupini TP-D1 could degrade 95.0% of TPN (50 mg kg-1) after 3-day incubation and 99.7% after 7 days in autoclaved soil [42]. However, the degradation rate of TPN was significantly decreased [29] or suppressed [17, 47] after repeated application in field experiments. The rod-shaped bacterial strain Y1 was isolated from the soil 21-day after the fourth treatment with TPN; this strain degraded 67.9% of TPN (10 mg l-1) in mineral salts medium after 14 days inoculation [57], indicating that some indigenous microorganisms have adapted to TPN after its repeated applications. Moreover, Carlo-Rojas et al. [5] reported that 56–95% of 0.432–1.298 lg/g TPN was depleted in biologically active soil microcosms after 25-day incubation as compared to a 37% loss of TPN in a sterile soil. Mori et al. [27, 28] also reported that the degradation of TPN in a manure amended soil was faster than that in non-manure soil, suggesting that the addition of other organic carbon sources enhanced microbial degradation of TPN. In soils receiving farmyard manure, the fungal contribution to TPN biodegradation was higher than the bacterial contribution. On the other hand, the bacterial contribution was higher in the soils without farmyard manure [27]. The TPN dissipation rate increased with an increase in water content of up to 60% water holding capacity [39]. However, at 100% water holding capacity, where anaerobic conditions dominate, the dissipation was very slow [39]. According to the radio-labeled study [38, 39], TPN degradation in virgin soil started by substitution of chlorine with a hydroxyl group, dechlorination and hydrolysis of the nitrile group. These results suggest that TPN-degrading bacteria and fungi could be successfully used for the removal of TPN from contaminated soils but the efficiency depends to some extent on the presence and absence of selective organic nutrients. These results would influence the design of future studies on the bioremediation of TPN. Motonaga et al. [30] reported TPN-OH to have a suppressive effect on the degradation of TPN. Therefore, although the theoretical toxicity of TPN in Bacillus subtilis [10] is approximately 50 times greater than that of the metabolite TPN-OH, the actual toxicity of TPN-OH appears to be not lower than that of TPN. Studies indicate that TPN-OH is more persistent in soil and has lower adsorption coefficient than the parent compound. The
G. Wang et al.: Biodegradation of Chlorothalonil
persistence and bioavailability of the metabolite appear to lead to suppression of TPN degradation after repeated application [30]. In addition, through analyzing the toxic effects of TPN and TPN-OH to the growth of P. putida KT2440 and Escherichia coli DH5a, it was found that 1 mM TPN had a more serious toxic effect on the growth of both strains than the same concentration of TPN-OH [26]. Chu et al. [9] discovered that chlorpyrifos degradation in the soil tested was not significantly altered by the addition of TPN, but the inhibitory effect of chlorpyrifos on soil microorganisms was largely increased by its combination with TPN. This result indicated that the combination of pesticide residues might have a more toxic effect on soil microorganisms. Recent studies have shown that TPN can significantly reduce the dissipation of herbicides such as metolachlor in soil. In addition, TPN seems to affect the activity of soil GST, a common pesticide detoxification process in soil [54]. One possible explanation for metolachlor dissipation kinetics is a build-up of the TPN intermediate (TPN-OH) which restricted soil microbial activity and depleted GST from TPN detoxification [53]. In a recent experiment, GST-like genes were cloned from Ochrobactrum sp. CTN-11 but, though it showed high sequence similarity to that from O. anthropi SH35B [25], it was not functionally expressed in the presence of glutathione. Subsequently, the chd gene from strain CTN-11 was cloned which can transform TPN to TPN-OH. So previous results are consistent with these findings, suggesting TPN-OH might inhibit GST activity of soil microbial community when facing pressure by TPN co-contaminated.
Discussions The use of microorganisms as an efficient, feasible, and cost-effective biotechnological approach for bioremediation has received increasing global attention. Chlorine removal from chlorinated compounds reduces both the recalcitrance to biodegradation and the risk of forming toxic intermediates during subsequent metabolic steps. As a result, the key reaction for microbial detoxification of chlorinated compounds is dechlorination [46]. In soil or other diverse ecosystem, the main metabolite of TPN is TPN-OH [1, 20, 33, 34, 37, 38], which is not ideal for the bioremediation purpose as TPN-OH is not only persistent in soil by binding to soil particles but also it is more readily dissolved in water leading to secondary pollution [30]. TPN-OH has been considered ‘‘slightly toxic’’ to fish and invertebrates by the US Environmental Protection Agency and photo-degradation of TPN-OH in aqueous solution suggested that it could be rapidly degraded to small aliphatic acids by sunlight [2]. So far, no pure culture with the
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ability to biodegrade TPN-OH or mineralize TPN has been isolated. Therefore, development of new bioremediation strategies for TPN-pollution, especially by enrichment and isolation of new strains or stable microbial communities with the ability to mineralize TPN or to biodegrade TPNOH, cloning the reductive dechlorination gene with the aim of avoiding the accumulation of TPN-OH and development the efficient biotechnology for the TPN-OH removal in the soil ecosystem will be significant goals for the thorough elimination of TPN contamination in the environment. Acknowledgments This study was supported by grants from the Chinese National Natural Science Foundation (31100083), the major project from the Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences (2010hzszdzx001), and the National Undergraduate Innovative Test Program (101030717).
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