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Degradation and mineralization of DDT by the ectomycorrhizal fungi, Xerocomus chrysenteron
Chemosphere 92 (2013) 760–764
Contents lists available at SciVerse ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Degradation and mineralization of DDT by the ectomycorrhizal fungi, Xerocomus chrysenteron Yi Huang, Jie Wang ⇑ College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
h i g h l i g h t s The mineralization ability of ECMF on DDT was investigated for the first time. We are the first to prove DDT could be mineralized by Xerocomus chrysenteron. X. chrysenteron showed much more powerful DDT tolerance than other fungi in similar studies.
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Article history: Received 17 December 2012 Received in revised form 13 March 2013 Accepted 1 April 2013 Available online 4 May 2013 Keywords: Ectomycorrhizal fungi Xerocomus chrysenteron Mineralization DDT
a b s t r a c t One strain of ectomycorrhizal fungi, Xerocomus chrysenteron, had been investigated for its ability to degrade 1,1,1-trichloro-2,2-bis(4-chlorophe-nyl) ethane (DDT) by measuring unlabeled DDT and identifying its metabolites, and determining the mineralization of [13C]DDT in pure cultures. After 45 d incubation, about 55% of the added DDT disappeared from the culture system, less than 5% remained in the nutrient solution, and about 44% was retained in the mycelium. Inoculation with mycelium enhanced the degradation of DDT in soil, and alleviated enrichment of DDT in plants. The metabolites identified by gas chromatography–mass spectrometry were 1,1-dichloro-2,2-bis(4-chlorophenyl) ethane (DDD), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), and 4,40 -dichlorobenzophenone (DBP). There were significant differences in the d13C of released CO2 between [13C]DDT and DDT cultures, which indicated X. chrysenteron was able to mineralize DDT to CO2. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Even though 1,1,1-trichloro-2,2-bis(4-chlorophe-nyl) ethane (DDT) was banned for use in China for almost thirty years, high concentrations of DDT in the soils and sediments of agricultural regions are still being observed in recent years (Jaward et al., 2005; Chiu et al., 2006; Lin et al., 2009). Moreover, DDT is still being widely used for controlling malaria in Africa because of its cost effectiveness (Kinuthia et al., 2010). Exposure to DDT results in increased risk of cancer and endocrine disruption, and it has been proved that DDT is linked to liver damage and hepatocarcinogenesis in animals, even in human beings (Rossi et al., 1983; Person et al., 2012). Therefore, considering the potential negative effects, it is necessary to address the environmental persistence of this insecticide and to look for effective methods of remediation. Due to its extremely slowly degradation in natural environments, enhancement of the degradation or mineralization process of DDT by microorganisms has been gaining popularity in the last decades and a range of bacteria and white rot fungi have been ⇑ Corresponding author. Tel./fax: +86 10 62757867. E-mail address: [email protected] (J. Wang). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.04.002
demonstrated to enhance the degradation process in both pure cultures and natural soils (Xie et al., 2011; Purnomo et al., 2008, 2010, 2011). But the processes of DDT degradation by microbes often stop before ring cleavage, and the final degradation produces such as DDD, DDE, and DBP still have one or two stable benzene rings which still have negative impacts on environment and human health. However, quite a few researchers has dominated that white rot fungi is capable of mineralizing DDT (Bumpus and Aust, 1987; Purnomo et al., 2008). Ectomycorrhizal fungi (ECMF), as a species of heterotrophic fungi, were approved to have the abilities of enhancing the tolerance of host plants, and alleviating environmental impacts such as excessive heavy metal, nutrient exhaustion and high salinity (Bumpus and Aust, 1987; Kamanavalli and Ninnekar, 2004; Huang et al., 2007; Wang et al., 2011). Moreover, we have demonstrated that ectomycorrhizal fungi could degrade DDT through a similar pathway found in white rot fungi in previous research (Huang et al., 2007). Four ectomycorrhizal fungi were used and four oxidative enzymes, belonging to the ligninase system and being relevant to DDT degradation, and the metabolites were investigated. The results showed the same enzyme activities with Phanerochaete chrysosporium (Bumpus and Aust, 1987), a typical strain of white rot
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fungi, and that the remaining DDT degraded to DDD and DBP. Therefore we were interested in whether the ectomycorrhizal fungi have the ability to mineralize DDT to carbon dioxide. Thus, the purpose of this study is to investigate the possibility of mineralization DDT by selected mycorrhizal fungi Xerocomus chrysenteron and explore the pathway.
same way, but alcohol was used to take place of acetone, and the temperature was 114 °C, then CO2 was sequestrated because it was condensed to dry ice immediately. After 20 min collection, the U-tube was melted at the capillaries. White solidified CO2 could be seen when the test tube was removed from the cold trap.
2. Materials and methods
2.4. Determination of DDT and its metabolites
2.1. Degradation of DDT by X. chrysenteron
The mycelia and its liquid media were separated by filter papers. The mycelia were put into a mortar, ground for 5 min with 15 mL hexane. The suspension was centrifuged at 10 000 rpm for 10 min and the supernatant was collect. The nutrient medium was also extracted with normal hexane in a 25 mL separating funnel 3 times (5 mL for each), and then the extraction solutions were combined for each culture and dehydrated through 1 cm thick anhydrous sodium sulfate. The hexane was then removed by evaporation under nitrogen. The hexane extracts were then dissolved in 2 mL hexane, and ready for analysis of the DDT’s fate. Soil samples (around the roots) were ground, dehydrated with equal weight of anhydrous sodium sulfate, wrapped by filter paper (extracted for 24 h before using to eliminated potential contamination), extracted by 80 mL hexane and acetone mixture (1:1, v/v) using Soxhlet for 16 h at 56 °C. Then the solution was evaporated to approximately 1 mL by using a rotary evaporator, and then dissolved in 2 mL hexane for analysis. A gas chromatographic analysis of hexane extracts was performed with an Agilent 6890 N GC equipped with a 5973I MSD, using a DB5-MS narrow bore column (30 m 0.25 mm ID, HP). The oven temperature was programmed at 50 °C for 1 min, followed by a linear increase of 15 °C min1 to 200 °C, holding at 200 °C for 1 min, followed by a linear increase of 10 °C min1 to 280 °C, holding at 280 °C for 5 min. The injector temperature was 250 °C and the detector temperature was 280 °C. Helium was used as the carried gas.
The ECMF, X. chrysenteron, was isolated from a conifer forest in Beijing Western Hills, and identified by Beijing Forest University. And then sterilized (121 °C for 20 min) modified Kottke medium (Kottke et al., 1987) supplied with 80 mg L1 DDT was divided into 20 mL liquid cultures (triangular flasks) and each culture was inoculated with three agar pieces with active X. chrysenteron mycelium. The liquid cultures were incubated in a shaker at 25 °C and rotated at 150 rpm for 45 d. Cultivations without inoculation served as blank control under the same conditions as above. 2.2. Degradation of artificial DDT-contaminated soil Surface soil was collected from Weiming lake in Peking University, and passed a 2 mm nylon sieve and sterilized (121 °C, 20 min). The physicochemical properties of the soil were: pH 7.5 (1:2.5); total organic carbon (TOC) content 16.68 g kg1; total nitrogen (TN) 0.94 g kg1; total phosphorus (TP) 2.67 g kg1; and no detective DDT contamination. The pine seeds (Pinus tabulaeformis) were provided by the Beijing Municipal Bureau of Landscape and Forestry. Details of growth of seedlings and mycelium were in Supplementary materials. After 4 weeks growth, the roots of P. tabulaeformis seedlings were dipped into a mycelium suapension of X. chrysenteron, then the plants were planted into 15 cm diameter pots with 500 g sterilized soil. The cultivars were kept in the growth chamber under a light regime of 13 h light (with a light intensity of 8000 lx at 25 °C) and 11 h darkness (at 18 °C), and watered with 200 mL of 1/10 Kottke solution. Symbiosis was formed after 4 weeks, and the plants with inoculation rate more than 95% were used for the following experiments (Wang et al., 2011). The pines were planted into the plastic pots with 500 g DDT-contaminated soil. There were three DDT concentration gradients, 2.5, 5, and 10 mg kg1. The cultivars were kept in the growth chamber (13 h light at 25 °C, 11 h dark at 18 °C), and watered with 200 mL of 1/10 Kottke solution for 50 d.
2.5. [13C] measurement and calculation The relative abundance of [13C]CO2 was measured by SIRMS (Stable Isotope Ratio Mass Spectrometry, Laboratory for Stable Isotope Geochemistry, Institute of Geology and Geophysics), and the results were expressed in d13C relative to the standards in the conventional d per mil notation as follows: d13C = [(13C/12C)sample/ (13C/12C)standard 1] 1000‰ (Kayler et al., 2011)
2.3. Mineralization of [13C]DDT [13C]DDT was purchased from the Cambridge Isotope Laboratory (purity > 99%). Each large test tube (Supplemental Fig. 1) containing 160 mL sterilized modified Kottke medium was inoculated with six agar pieces with active X. chrysenteron mycelium. [13C]DDT was added into the tubes until the final DDT concentration was 10 mg L1, corresponding to 10 mg L1 unlabeled DDT as the blank control. Each test tube was plugged with a rubber stopper and sealed with the membrane, all treatments were carried out in triplicate and incubated at 25 °C for 36 d. By flushing with pure O2 (>99.99%, boiling point 183 °C), the evolved [13C]CO2 was collected every 9 d. All gaseous substances released were bubbled through two sequential U-tubes which were places in two cold traps (Supplemental Fig. 2). The first trap was prepared by pouring liquid nitrogen into acetone until the solid–liquid ratio reached 1:1, and the temperature was finally reduced to 50 °C, so water vapor and other impurities were first removed from the whole gas. The second trap was made in the
3. Results 3.1. DDT removal by X. chrysenteron mycelium Inoculated with mycelia of X. chrysenteron, DDT concentration decreased rapidly in liquid culture, while its concentration remained relatively constant in the non-mycorrhizal nutrient solution control (Fig. 1 and Table 1). On day 12, only 19% of the added DDT was detected in the culture solution. Since the biomass of X. chrysenteron mycelium was too small to measure at the beginning of the experiment, DDT concentrations in mycelium were measured after two weeks of the cultivation. After 45 d culture, only about 0.10 mg DDT was found in the medium and 0.69 mg in the fungal mycelium, which means that about 51% of the added DDT was removed from the cultivation system, about 44% was retained in the mycelium, and less than 5% remained in the liquid medium.
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were measured every 9 d. The results were shown in Table 3, and just like our expectation, the results of d13C in the labeled group were larger than the unlabeled group all over the time, which reached a peak on day 18, and then decreased. On the contrary, the d13C of the unlabeled group showed little change during the 36 d. Moreover, the d13C of the labeled group in day 9 and day 18 were 6.130‰ and 17.726‰ respectively, which were both significantly different with the unlabeled group. There was no significant difference between labeled and unlabeled treatments on day 27 and day 36.
1.6 1.4
DDT Concentration (mg)
1.2 1 0.8 0.6
4. Discussions
0.4
Many studies have confirmed that numbers of ectomycorrhizal fungi (ECMFs) had the ability to degrade POPs (Merharg and Cairnet, 2000). In this study, cultured in liquid media supplied with DDT for 45 d, 93% of DDT disappeared from the culture medium, and about 50% of it was found in the mycelium of X. chrysenteron (Fig. 1 and Table 1). Moreover, inoculation with mycelium in the soil, there were less DDT and more DDD in the inoculated treatments than in the non-inoculated treatments, which meant that inoculation enhanced the degradation of DDT in the soil (Table 2). These results, the disappearance of DDT and the identification of its metabolites, demonstrate that X. chrysenteron own the ability to degrade DDT. The GC/MS analysis illustrated three metabolic products of DDT degradation by X. chrysenteron. This result is similar with a study of Purnomo et al. (2008) showing that Gloeophyllum trabeum, a strain of brown rot fungi, produced DDE, DDD, and DBP from DDT degradation. In the liquid cultures, there was no DBP found in the nutrient solutions, moreover, in the DDT contaminated soil, no DBP could be detected in the soils and plants under the treatments of non-inoculation by X. chrysenteron. These indicate that the production of DBP during the DDT degradation could only happen in the mycelium of X. chrysenteron. The DDD concentrations in liquid cultures and soil cultures were highest than DDE and DBP, suggesting that DDT is the main metabolic product of DDT degradation by X. chrysenteron, which is similar with the research of Bumpus and Aust (1987) on DDT degradation by white rot fungi, P. chrysosporium. According to other researches in white rot fungi and brown rot fungi (Bumpus and Aust, 1987; Purnomo et al., 2008; Xiao et al., 2011), DDD and DDE were converted to DBP by reductive dechlorination and hydroxylation, which is to say that DBP is the metabolic product of DDE and DDD. However, whether DDT could be degraded to DBP is still unknown. It was generally considered that, the metabolites of DDT, DDD and DDE had little activity and may be the terminal metabolites, which means the further degradation of DDD and DDE was very difficult, and was the key step for DDT degradation or mineralization. According to Purnomo et al. researches (2008, 2010), less than 0.25% of the initial [14C]DDT was mineralized to [14C]CO2 by G. trabeum. For the low mineralization rates, it is unlikely that the fungi can mineralize DDT. Bumpus and Aust (1987) reported that the mineralization of [14C]DDT by G. trabeum was less than 0.1% in nitrogen-deficient or -sufficient cultures. In our study, as expected, intensive mineralization occurred. There were significant differences between [13C]CO2 release in labeled and unlabeled
0.2 0 0
5
10
15
20
25
30
35
40
45
Time (day) Fig. 1. DDT degradation curves of pure liquid cultures (non-mycelium control N, mycelium j and culture solution ; error bar means the SD).
3.2. DDT removal by X. chrysenteron in artificial DDT contaminated soil The concentrations of DDT in the soil and plants were shown in Table 2. No matter what the initial DDT concentrations were, the concentrations in soil were much lower under the condition of inoculation. After 50 d incubation, the removal rates of inoculation controls were 63.12%, 78.92% and 56.81% respectively, which were significantly higher that the non-inoculation treatments. Meanwhile, DDT concentrations in the roots and shoots decreased after being inoculated, which proved inoculation with X. chrysenteron could enhance the pesticide tolerance of plants. 3.3. Identification of metabolic products To confirm the degradation of DDT by X. chrysenteron and identify metabolic products, nutrient solution and mycelium in liquid cultures, and soil, roots and shoots in inoculation systems were extracted by hexane, and the extractions were analyzed with GC/MS by comparing retention times and mass spectra of their compounds with guide samples. The GC/MS analysis revealed three metabolites with retention times at 17.56, 15.11 and 16.75 min, which were identified as 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 4,40 -dichlorobenzophenone (DBP), and 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) respectively (Fig. 2). Analysis of the nutrient solution extractions also revealed the presence of DDE and DDD, but DBP was not detected. Moreover, DBP could not been found under the condition of non-inoculation, regardless of its positions. 3.4. Mineralization of DDT by X. chrysenteron mycelium [13C] labeled DDT was added into the culture media with mycelium of X. chrysenteron for 36 d. The CO2 samples in the U-tubes
Table 1 Degradation of DDT and its metabolic products by X. chrysenteron in liquid cultures during a 45-d inoculation period. Nutrient solution
X. chrysenteron
Mycelium
DDT (mg)
DDD (mg)
DDE (ug)
DBP (ug)
DDT (mg)
DDD (mg)
DDE (ug)
DBP (ug)
0.104 ± 0.038
0.004 ± 0.001
0.774 ± 0.091
–
0.694 ± 0.093
0.038 ± 0.004
1.693 ± 0.342
1.852 ± 0.178
‘‘–’’, Not detectable by GC/MS, date points represent means and standard deviation (n = 3)
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Fig. 2. Gas chromatogram of extraction from mycelium of X. chrysenteron after 15 d cultures.
Table 2 Residue of DDT and its metabolic products in soil and plants after 50 d incubation by X. chrysenteron. Initial DDT (mg kg1)
2.5
5.0
10.0
Inoculation
Non inoculation
Inoculation
Non inoculation
Inoculation
Non inoculation
Soil (mg kg1)
DDT DDD DDE DBP
0.922 ± .002de 0.413 ± 0.005 0.101 ± 0.000 0.011 ± 0.000
1.349 ± 0.023d 0.223 ± 0.008 0.093 ± 0.009 –
1.054 ± .052de 0.521 ± 0.035 0.117 ± 0.003 0.009 ± 0.002
2.446 ± 0.114c 0.273 ± 0.022 0.102 ± 0.006 –
4.319 ± 0.291b 0.464 ± 0.002 0.128 ± 0.001 0.015 ± 0.001
9.706 ± 0.692a 0.596 ± 0.061 0.119 ± 0.003 –
Roots (mg kg1)
DDT DDD DDE DBP
1.700 ± 0.023d 1.199 ± 0.134 0.336 ± 0.004 0.013 ± 0.007
2.576 ± 0.056d 1.258 ± 0.004 0.473 ± 0.002 –
2.815 ± 0.153d 2.571 ± 0.070 1.052 ± 0.003 0.095 ± 0.047
9.878 ± 0.215b 2.759 ± 0.003 0.810 ± 0.010 –
6.476 ± 0.168c 3.124 ± 0.031 1.742 ± 0.021 0.178 ± 0.035
13.853 ± 0.862a 3.501 ± 0.023 1.138 ± 0.032 -
Shoots (mg kg1)
DDT DDD DDE DBP
0.150 ± 0.015c 0.089 ± 0.002 – 0.126 ± 0.003
0.457 ± 0.102c 0.050 ± 0.004 – –
0.569 ± 0.096 0.170 ± 0.026 – 0.232 ± 0.006
4.274 ± 0.201a 0.023 ± 0.000 – –
1.874 ± 0.231b 0.781 ± 0.056 – 0.500 ± 0.052
5.496 ± 0.412a 0.558 ± 0.053 – –
‘‘–’’, Not detectable by GC/MS; data points represent means and standard deviation (n = 3); different small letters indicated significant (p < 0.05) difference between different treatments according to one-way ANOVA.
treatments on day 9 and day 18, which proved [13C]DDT was translated to [13C]CO2 through mineralization during this period. All these results illustrated a possibility for intensive degradation of DDT, which was the first demonstration of DDT mineralization by the ECMF, X. chrysenteron. Furthermore, compared to other ectomycorrhizal fungi and brown rot fungi used in previous studies, X. chrysenteron performed better at withstanding the stress of DDT (Supplemental Table 1). In our study, based on a semi-inhibiting concentration experiment (data not show), we set 80 mg L1 as the initial concentration of DDT in the liquid medium, which was much higher than that added in most previous biodegradation experiments, nevertheless, good results were obtained. Therefore, despite the heavy DDT stress, X. chrysenteron removed most of DDT by bioadsorption and biodegradation. Above all, not only do these results reveal the ability of X. chrysenteron to biodegrade DDT, but also
Table 3 Mineralization of DDT by X. chrysenteron during 36 d incubation. Time (d) 13
d C (‰)
9 18 27 36
[13C]DDT treatments a
6.130 ± 0.512 17.726 ± 1.235a 10.719 ± 1.021a 10.799 ± 0.985a
DDT treatments 9.507 ± 0.874b 9.940 ± 1.362b 10.839 ± .2142a 12.756 ± 1.962a
Small letters a and b indicated significant (p < 0.05) difference between different treatments according to one-way ANOVA.
provide a potential model strain for further research and application in soil bioremediation. Combined with our previous studies, and on the basis of the identification of the metabolic, the DDT degradation pathway by X. chrysenteron is proposed as shown in Fig. 3. This species of
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Fig. 3. Proposed pathway for DDT degradation in X. chrysenteron.
ectomycorrhizal fungi initially degrade DDT to form DDD and DDE by dechlorination of the ethane group. DDD or DDE is further degradation by sequential steps involving dechlorination and hydroxylation to DBP. All these metabolic products would be then mineralized to CO2 by X. chrysenteron and released into the air. This pathway is similar with the proposed pathways in white rot fungi and brown rot fungi, but yet, has some differences. DDD is the main metabolic product of DDT degradation by X. chrysenteron, which is the same with white rot fungi, and different with brown rot fungi, however, X. chrysenteron produces DDE just like brown rot fungi, which white rot fungi do not produce. According to previous studies, ectomycorrhizal fungi possibly have the same ligninase system with white rot fungi, it is potential that ectomycorrhizal fungi also use an enzymatic system similar with brown rot fungi. However, the general scheme presented in Fig. 3 may not be the sole pathway for DDT degradation in ectomycorrhizal fungi, the identities of additional metabolites remain to be determined and isotope labelling techniques would be need to trace DDT transformation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.04.002. References Bumpus, J.A., Aust, S.D., 1987. Biodegradation of DDT [1,1,1-trichloro-2,2-bis(4chlorophenyl) ethane] by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 53, 2001–2008. Chiu, S.W., Ho, K.M., Chan, S.S., So, O.M., Lai, K.H., 2006. Characterization of contamination in and toxicities of a shipyard area in Hong Kong. Environ. Pollut. 142, 512–520. Huang, Y., Zhao, X., Luan, S.J., 2007. Uptake and biodegradation of DDT by 4 ectomycorrhizal fungi. Sci. Total. Environ. 385, 235–241.
Jaward, F.M., Zhang, G., Nam, J.J., Sweetman, A.J., Obbard, J.P., Kobara, Y., Jones, K.C., 2005. Passive air sampling of polychlorinated biphenyls, organochlorine compounds, and polybrominated diphenyl ethers across Asia. Environ. Sci. Technol. 39, 8638–8645. Kamanavalli, C.M., Ninnekar, H.Z., 2004. Biodegradation of DDT by a Pseudomonas species. Curr. Mocrobiol. 2004 (48), 10–13. Kayler, Z.E., Kaiser, M., Gessler, A., Ellerbrock, H.R., Sommer, M., 2011. Application of d13C and d15N isotopic signatures of organic matter fractions sequentially separated from adjacent arable and forest soils to identify carbon stabilization mechanisms. Biogeosciences 8, 2895–2906. Kinuthia, M., Hamadi, I.B., Anne, W.M., Ciira, K., Muniru, K.T., 2010. Degradation of dichlorodiphenyltrichloroethane (DDT) by bacterial isolates from cultivated and uncultivated soil. Afr. J. Microbiol. Res. 4, 185–196. Kottke, I., Guttenberger, M., Hammpp, R., Oberwinkler, G., 1987. An in vitro method for establishing mycorrhizae on coniferous tree seedlings. Trees 1, 191–194. Lin, T., Hu, Z.H., Zhang, G., Li, X.D., Xu, W.H., Tang, J.H., Li, J., 2009. Levels and mass burden of DDTs in sediments from fishing harbors: the importance of DDT containing antifouling paint to the coastal environment of China. Environ. Sci. Technol. 43, 8083–8088. Merharg, A.A., Cairnet, J.W.G., 2000. Ecotomycorrhizaa-extending the capabilities of rhizosphere remediation. Soil. Biol. Biochem. 32, 1475–1484. Person, E.C., Graubard, B.I., Evans, A.A., London, W.T., Weber, J.P., Leblanc, A., Chen, G., Lin, W.Y., McGlynn, K.A., 2012. Dichlorodiphenyltrichloroethane and risk of hepatocellular carcinoma. Int. J. Cancer. 131, 2078–2084. Purnomo, A.S., Kamei, I., Kondo, R., 2008. Degradation of 1,1,1-trichloro-2,2-bis(4chlorophenyl) ethane (DDT) by brown-rot Fungi. J. Biosci. Bioeng. 105, 614–621. Purnomo, A.S., Koyama, F., Mori, T., Kondo, R., 2010. DDT degradation potential of cattle manure compost. Chemosphere 80, 619–624. Purnomo, A.S., Mori, T., Kamei, I., Kondo, R., 2011. Basic studies and applications on bioremediation of DDT: a review. Int. Biodeter. Biodegra. 65, 921–930. Rossi, L., Barbieri, O., Sanguineti, M., Cabral, J.R., Bruzzi, P., Santi, L., 1983. Carcinogenicity study with technical-grade dichlorodiphenyltrichloroethane and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene in hamsters. Cancer Res. 43, 776–781. Wang, J., Huang, Y., Jiang, X.Y., 2011. Influence of ectomycorrhizal fungi on absorption and balance of essential elements of Pinus tabulaeformis seedlings in saline soil. Pedosphere 21, 400–406. Xiao, P.F., Mori, T., Kamei, I., Kondo, R., 2011. A novel metabolic pathway for biodegradation of DDT by the white rot fungi, Phlebia lindtneri and Phlebia brevispora. Biodegradation 22, 859–867. Xie, H., Zhu, L.S., Xu, Q.F., Wang, J., Liu, W., Jiang, J.H., Meng, Y., 2011. Isolation and degradation ability of the DDT-degrading bacterial strain KK. Environ. Earth. Sci. 62, 93–99.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Degradation and mineralization of DDT by the ectomycorrhizal fungi, Xerocomus chrysenteron Yi Huang, Jie Wang ⇑ College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
h i g h l i g h t s The mineralization ability of ECMF on DDT was investigated for the first time. We are the first to prove DDT could be mineralized by Xerocomus chrysenteron. X. chrysenteron showed much more powerful DDT tolerance than other fungi in similar studies.
a r t i c l e
i n f o
Article history: Received 17 December 2012 Received in revised form 13 March 2013 Accepted 1 April 2013 Available online 4 May 2013 Keywords: Ectomycorrhizal fungi Xerocomus chrysenteron Mineralization DDT
a b s t r a c t One strain of ectomycorrhizal fungi, Xerocomus chrysenteron, had been investigated for its ability to degrade 1,1,1-trichloro-2,2-bis(4-chlorophe-nyl) ethane (DDT) by measuring unlabeled DDT and identifying its metabolites, and determining the mineralization of [13C]DDT in pure cultures. After 45 d incubation, about 55% of the added DDT disappeared from the culture system, less than 5% remained in the nutrient solution, and about 44% was retained in the mycelium. Inoculation with mycelium enhanced the degradation of DDT in soil, and alleviated enrichment of DDT in plants. The metabolites identified by gas chromatography–mass spectrometry were 1,1-dichloro-2,2-bis(4-chlorophenyl) ethane (DDD), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), and 4,40 -dichlorobenzophenone (DBP). There were significant differences in the d13C of released CO2 between [13C]DDT and DDT cultures, which indicated X. chrysenteron was able to mineralize DDT to CO2. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Even though 1,1,1-trichloro-2,2-bis(4-chlorophe-nyl) ethane (DDT) was banned for use in China for almost thirty years, high concentrations of DDT in the soils and sediments of agricultural regions are still being observed in recent years (Jaward et al., 2005; Chiu et al., 2006; Lin et al., 2009). Moreover, DDT is still being widely used for controlling malaria in Africa because of its cost effectiveness (Kinuthia et al., 2010). Exposure to DDT results in increased risk of cancer and endocrine disruption, and it has been proved that DDT is linked to liver damage and hepatocarcinogenesis in animals, even in human beings (Rossi et al., 1983; Person et al., 2012). Therefore, considering the potential negative effects, it is necessary to address the environmental persistence of this insecticide and to look for effective methods of remediation. Due to its extremely slowly degradation in natural environments, enhancement of the degradation or mineralization process of DDT by microorganisms has been gaining popularity in the last decades and a range of bacteria and white rot fungi have been ⇑ Corresponding author. Tel./fax: +86 10 62757867. E-mail address: [email protected] (J. Wang). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.04.002
demonstrated to enhance the degradation process in both pure cultures and natural soils (Xie et al., 2011; Purnomo et al., 2008, 2010, 2011). But the processes of DDT degradation by microbes often stop before ring cleavage, and the final degradation produces such as DDD, DDE, and DBP still have one or two stable benzene rings which still have negative impacts on environment and human health. However, quite a few researchers has dominated that white rot fungi is capable of mineralizing DDT (Bumpus and Aust, 1987; Purnomo et al., 2008). Ectomycorrhizal fungi (ECMF), as a species of heterotrophic fungi, were approved to have the abilities of enhancing the tolerance of host plants, and alleviating environmental impacts such as excessive heavy metal, nutrient exhaustion and high salinity (Bumpus and Aust, 1987; Kamanavalli and Ninnekar, 2004; Huang et al., 2007; Wang et al., 2011). Moreover, we have demonstrated that ectomycorrhizal fungi could degrade DDT through a similar pathway found in white rot fungi in previous research (Huang et al., 2007). Four ectomycorrhizal fungi were used and four oxidative enzymes, belonging to the ligninase system and being relevant to DDT degradation, and the metabolites were investigated. The results showed the same enzyme activities with Phanerochaete chrysosporium (Bumpus and Aust, 1987), a typical strain of white rot
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fungi, and that the remaining DDT degraded to DDD and DBP. Therefore we were interested in whether the ectomycorrhizal fungi have the ability to mineralize DDT to carbon dioxide. Thus, the purpose of this study is to investigate the possibility of mineralization DDT by selected mycorrhizal fungi Xerocomus chrysenteron and explore the pathway.
same way, but alcohol was used to take place of acetone, and the temperature was 114 °C, then CO2 was sequestrated because it was condensed to dry ice immediately. After 20 min collection, the U-tube was melted at the capillaries. White solidified CO2 could be seen when the test tube was removed from the cold trap.
2. Materials and methods
2.4. Determination of DDT and its metabolites
2.1. Degradation of DDT by X. chrysenteron
The mycelia and its liquid media were separated by filter papers. The mycelia were put into a mortar, ground for 5 min with 15 mL hexane. The suspension was centrifuged at 10 000 rpm for 10 min and the supernatant was collect. The nutrient medium was also extracted with normal hexane in a 25 mL separating funnel 3 times (5 mL for each), and then the extraction solutions were combined for each culture and dehydrated through 1 cm thick anhydrous sodium sulfate. The hexane was then removed by evaporation under nitrogen. The hexane extracts were then dissolved in 2 mL hexane, and ready for analysis of the DDT’s fate. Soil samples (around the roots) were ground, dehydrated with equal weight of anhydrous sodium sulfate, wrapped by filter paper (extracted for 24 h before using to eliminated potential contamination), extracted by 80 mL hexane and acetone mixture (1:1, v/v) using Soxhlet for 16 h at 56 °C. Then the solution was evaporated to approximately 1 mL by using a rotary evaporator, and then dissolved in 2 mL hexane for analysis. A gas chromatographic analysis of hexane extracts was performed with an Agilent 6890 N GC equipped with a 5973I MSD, using a DB5-MS narrow bore column (30 m 0.25 mm ID, HP). The oven temperature was programmed at 50 °C for 1 min, followed by a linear increase of 15 °C min1 to 200 °C, holding at 200 °C for 1 min, followed by a linear increase of 10 °C min1 to 280 °C, holding at 280 °C for 5 min. The injector temperature was 250 °C and the detector temperature was 280 °C. Helium was used as the carried gas.
The ECMF, X. chrysenteron, was isolated from a conifer forest in Beijing Western Hills, and identified by Beijing Forest University. And then sterilized (121 °C for 20 min) modified Kottke medium (Kottke et al., 1987) supplied with 80 mg L1 DDT was divided into 20 mL liquid cultures (triangular flasks) and each culture was inoculated with three agar pieces with active X. chrysenteron mycelium. The liquid cultures were incubated in a shaker at 25 °C and rotated at 150 rpm for 45 d. Cultivations without inoculation served as blank control under the same conditions as above. 2.2. Degradation of artificial DDT-contaminated soil Surface soil was collected from Weiming lake in Peking University, and passed a 2 mm nylon sieve and sterilized (121 °C, 20 min). The physicochemical properties of the soil were: pH 7.5 (1:2.5); total organic carbon (TOC) content 16.68 g kg1; total nitrogen (TN) 0.94 g kg1; total phosphorus (TP) 2.67 g kg1; and no detective DDT contamination. The pine seeds (Pinus tabulaeformis) were provided by the Beijing Municipal Bureau of Landscape and Forestry. Details of growth of seedlings and mycelium were in Supplementary materials. After 4 weeks growth, the roots of P. tabulaeformis seedlings were dipped into a mycelium suapension of X. chrysenteron, then the plants were planted into 15 cm diameter pots with 500 g sterilized soil. The cultivars were kept in the growth chamber under a light regime of 13 h light (with a light intensity of 8000 lx at 25 °C) and 11 h darkness (at 18 °C), and watered with 200 mL of 1/10 Kottke solution. Symbiosis was formed after 4 weeks, and the plants with inoculation rate more than 95% were used for the following experiments (Wang et al., 2011). The pines were planted into the plastic pots with 500 g DDT-contaminated soil. There were three DDT concentration gradients, 2.5, 5, and 10 mg kg1. The cultivars were kept in the growth chamber (13 h light at 25 °C, 11 h dark at 18 °C), and watered with 200 mL of 1/10 Kottke solution for 50 d.
2.5. [13C] measurement and calculation The relative abundance of [13C]CO2 was measured by SIRMS (Stable Isotope Ratio Mass Spectrometry, Laboratory for Stable Isotope Geochemistry, Institute of Geology and Geophysics), and the results were expressed in d13C relative to the standards in the conventional d per mil notation as follows: d13C = [(13C/12C)sample/ (13C/12C)standard 1] 1000‰ (Kayler et al., 2011)
2.3. Mineralization of [13C]DDT [13C]DDT was purchased from the Cambridge Isotope Laboratory (purity > 99%). Each large test tube (Supplemental Fig. 1) containing 160 mL sterilized modified Kottke medium was inoculated with six agar pieces with active X. chrysenteron mycelium. [13C]DDT was added into the tubes until the final DDT concentration was 10 mg L1, corresponding to 10 mg L1 unlabeled DDT as the blank control. Each test tube was plugged with a rubber stopper and sealed with the membrane, all treatments were carried out in triplicate and incubated at 25 °C for 36 d. By flushing with pure O2 (>99.99%, boiling point 183 °C), the evolved [13C]CO2 was collected every 9 d. All gaseous substances released were bubbled through two sequential U-tubes which were places in two cold traps (Supplemental Fig. 2). The first trap was prepared by pouring liquid nitrogen into acetone until the solid–liquid ratio reached 1:1, and the temperature was finally reduced to 50 °C, so water vapor and other impurities were first removed from the whole gas. The second trap was made in the
3. Results 3.1. DDT removal by X. chrysenteron mycelium Inoculated with mycelia of X. chrysenteron, DDT concentration decreased rapidly in liquid culture, while its concentration remained relatively constant in the non-mycorrhizal nutrient solution control (Fig. 1 and Table 1). On day 12, only 19% of the added DDT was detected in the culture solution. Since the biomass of X. chrysenteron mycelium was too small to measure at the beginning of the experiment, DDT concentrations in mycelium were measured after two weeks of the cultivation. After 45 d culture, only about 0.10 mg DDT was found in the medium and 0.69 mg in the fungal mycelium, which means that about 51% of the added DDT was removed from the cultivation system, about 44% was retained in the mycelium, and less than 5% remained in the liquid medium.
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were measured every 9 d. The results were shown in Table 3, and just like our expectation, the results of d13C in the labeled group were larger than the unlabeled group all over the time, which reached a peak on day 18, and then decreased. On the contrary, the d13C of the unlabeled group showed little change during the 36 d. Moreover, the d13C of the labeled group in day 9 and day 18 were 6.130‰ and 17.726‰ respectively, which were both significantly different with the unlabeled group. There was no significant difference between labeled and unlabeled treatments on day 27 and day 36.
1.6 1.4
DDT Concentration (mg)
1.2 1 0.8 0.6
4. Discussions
0.4
Many studies have confirmed that numbers of ectomycorrhizal fungi (ECMFs) had the ability to degrade POPs (Merharg and Cairnet, 2000). In this study, cultured in liquid media supplied with DDT for 45 d, 93% of DDT disappeared from the culture medium, and about 50% of it was found in the mycelium of X. chrysenteron (Fig. 1 and Table 1). Moreover, inoculation with mycelium in the soil, there were less DDT and more DDD in the inoculated treatments than in the non-inoculated treatments, which meant that inoculation enhanced the degradation of DDT in the soil (Table 2). These results, the disappearance of DDT and the identification of its metabolites, demonstrate that X. chrysenteron own the ability to degrade DDT. The GC/MS analysis illustrated three metabolic products of DDT degradation by X. chrysenteron. This result is similar with a study of Purnomo et al. (2008) showing that Gloeophyllum trabeum, a strain of brown rot fungi, produced DDE, DDD, and DBP from DDT degradation. In the liquid cultures, there was no DBP found in the nutrient solutions, moreover, in the DDT contaminated soil, no DBP could be detected in the soils and plants under the treatments of non-inoculation by X. chrysenteron. These indicate that the production of DBP during the DDT degradation could only happen in the mycelium of X. chrysenteron. The DDD concentrations in liquid cultures and soil cultures were highest than DDE and DBP, suggesting that DDT is the main metabolic product of DDT degradation by X. chrysenteron, which is similar with the research of Bumpus and Aust (1987) on DDT degradation by white rot fungi, P. chrysosporium. According to other researches in white rot fungi and brown rot fungi (Bumpus and Aust, 1987; Purnomo et al., 2008; Xiao et al., 2011), DDD and DDE were converted to DBP by reductive dechlorination and hydroxylation, which is to say that DBP is the metabolic product of DDE and DDD. However, whether DDT could be degraded to DBP is still unknown. It was generally considered that, the metabolites of DDT, DDD and DDE had little activity and may be the terminal metabolites, which means the further degradation of DDD and DDE was very difficult, and was the key step for DDT degradation or mineralization. According to Purnomo et al. researches (2008, 2010), less than 0.25% of the initial [14C]DDT was mineralized to [14C]CO2 by G. trabeum. For the low mineralization rates, it is unlikely that the fungi can mineralize DDT. Bumpus and Aust (1987) reported that the mineralization of [14C]DDT by G. trabeum was less than 0.1% in nitrogen-deficient or -sufficient cultures. In our study, as expected, intensive mineralization occurred. There were significant differences between [13C]CO2 release in labeled and unlabeled
0.2 0 0
5
10
15
20
25
30
35
40
45
Time (day) Fig. 1. DDT degradation curves of pure liquid cultures (non-mycelium control N, mycelium j and culture solution ; error bar means the SD).
3.2. DDT removal by X. chrysenteron in artificial DDT contaminated soil The concentrations of DDT in the soil and plants were shown in Table 2. No matter what the initial DDT concentrations were, the concentrations in soil were much lower under the condition of inoculation. After 50 d incubation, the removal rates of inoculation controls were 63.12%, 78.92% and 56.81% respectively, which were significantly higher that the non-inoculation treatments. Meanwhile, DDT concentrations in the roots and shoots decreased after being inoculated, which proved inoculation with X. chrysenteron could enhance the pesticide tolerance of plants. 3.3. Identification of metabolic products To confirm the degradation of DDT by X. chrysenteron and identify metabolic products, nutrient solution and mycelium in liquid cultures, and soil, roots and shoots in inoculation systems were extracted by hexane, and the extractions were analyzed with GC/MS by comparing retention times and mass spectra of their compounds with guide samples. The GC/MS analysis revealed three metabolites with retention times at 17.56, 15.11 and 16.75 min, which were identified as 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 4,40 -dichlorobenzophenone (DBP), and 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) respectively (Fig. 2). Analysis of the nutrient solution extractions also revealed the presence of DDE and DDD, but DBP was not detected. Moreover, DBP could not been found under the condition of non-inoculation, regardless of its positions. 3.4. Mineralization of DDT by X. chrysenteron mycelium [13C] labeled DDT was added into the culture media with mycelium of X. chrysenteron for 36 d. The CO2 samples in the U-tubes
Table 1 Degradation of DDT and its metabolic products by X. chrysenteron in liquid cultures during a 45-d inoculation period. Nutrient solution
X. chrysenteron
Mycelium
DDT (mg)
DDD (mg)
DDE (ug)
DBP (ug)
DDT (mg)
DDD (mg)
DDE (ug)
DBP (ug)
0.104 ± 0.038
0.004 ± 0.001
0.774 ± 0.091
–
0.694 ± 0.093
0.038 ± 0.004
1.693 ± 0.342
1.852 ± 0.178
‘‘–’’, Not detectable by GC/MS, date points represent means and standard deviation (n = 3)
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Fig. 2. Gas chromatogram of extraction from mycelium of X. chrysenteron after 15 d cultures.
Table 2 Residue of DDT and its metabolic products in soil and plants after 50 d incubation by X. chrysenteron. Initial DDT (mg kg1)
2.5
5.0
10.0
Inoculation
Non inoculation
Inoculation
Non inoculation
Inoculation
Non inoculation
Soil (mg kg1)
DDT DDD DDE DBP
0.922 ± .002de 0.413 ± 0.005 0.101 ± 0.000 0.011 ± 0.000
1.349 ± 0.023d 0.223 ± 0.008 0.093 ± 0.009 –
1.054 ± .052de 0.521 ± 0.035 0.117 ± 0.003 0.009 ± 0.002
2.446 ± 0.114c 0.273 ± 0.022 0.102 ± 0.006 –
4.319 ± 0.291b 0.464 ± 0.002 0.128 ± 0.001 0.015 ± 0.001
9.706 ± 0.692a 0.596 ± 0.061 0.119 ± 0.003 –
Roots (mg kg1)
DDT DDD DDE DBP
1.700 ± 0.023d 1.199 ± 0.134 0.336 ± 0.004 0.013 ± 0.007
2.576 ± 0.056d 1.258 ± 0.004 0.473 ± 0.002 –
2.815 ± 0.153d 2.571 ± 0.070 1.052 ± 0.003 0.095 ± 0.047
9.878 ± 0.215b 2.759 ± 0.003 0.810 ± 0.010 –
6.476 ± 0.168c 3.124 ± 0.031 1.742 ± 0.021 0.178 ± 0.035
13.853 ± 0.862a 3.501 ± 0.023 1.138 ± 0.032 -
Shoots (mg kg1)
DDT DDD DDE DBP
0.150 ± 0.015c 0.089 ± 0.002 – 0.126 ± 0.003
0.457 ± 0.102c 0.050 ± 0.004 – –
0.569 ± 0.096 0.170 ± 0.026 – 0.232 ± 0.006
4.274 ± 0.201a 0.023 ± 0.000 – –
1.874 ± 0.231b 0.781 ± 0.056 – 0.500 ± 0.052
5.496 ± 0.412a 0.558 ± 0.053 – –
‘‘–’’, Not detectable by GC/MS; data points represent means and standard deviation (n = 3); different small letters indicated significant (p < 0.05) difference between different treatments according to one-way ANOVA.
treatments on day 9 and day 18, which proved [13C]DDT was translated to [13C]CO2 through mineralization during this period. All these results illustrated a possibility for intensive degradation of DDT, which was the first demonstration of DDT mineralization by the ECMF, X. chrysenteron. Furthermore, compared to other ectomycorrhizal fungi and brown rot fungi used in previous studies, X. chrysenteron performed better at withstanding the stress of DDT (Supplemental Table 1). In our study, based on a semi-inhibiting concentration experiment (data not show), we set 80 mg L1 as the initial concentration of DDT in the liquid medium, which was much higher than that added in most previous biodegradation experiments, nevertheless, good results were obtained. Therefore, despite the heavy DDT stress, X. chrysenteron removed most of DDT by bioadsorption and biodegradation. Above all, not only do these results reveal the ability of X. chrysenteron to biodegrade DDT, but also
Table 3 Mineralization of DDT by X. chrysenteron during 36 d incubation. Time (d) 13
d C (‰)
9 18 27 36
[13C]DDT treatments a
6.130 ± 0.512 17.726 ± 1.235a 10.719 ± 1.021a 10.799 ± 0.985a
DDT treatments 9.507 ± 0.874b 9.940 ± 1.362b 10.839 ± .2142a 12.756 ± 1.962a
Small letters a and b indicated significant (p < 0.05) difference between different treatments according to one-way ANOVA.
provide a potential model strain for further research and application in soil bioremediation. Combined with our previous studies, and on the basis of the identification of the metabolic, the DDT degradation pathway by X. chrysenteron is proposed as shown in Fig. 3. This species of
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Fig. 3. Proposed pathway for DDT degradation in X. chrysenteron.
ectomycorrhizal fungi initially degrade DDT to form DDD and DDE by dechlorination of the ethane group. DDD or DDE is further degradation by sequential steps involving dechlorination and hydroxylation to DBP. All these metabolic products would be then mineralized to CO2 by X. chrysenteron and released into the air. This pathway is similar with the proposed pathways in white rot fungi and brown rot fungi, but yet, has some differences. DDD is the main metabolic product of DDT degradation by X. chrysenteron, which is the same with white rot fungi, and different with brown rot fungi, however, X. chrysenteron produces DDE just like brown rot fungi, which white rot fungi do not produce. According to previous studies, ectomycorrhizal fungi possibly have the same ligninase system with white rot fungi, it is potential that ectomycorrhizal fungi also use an enzymatic system similar with brown rot fungi. However, the general scheme presented in Fig. 3 may not be the sole pathway for DDT degradation in ectomycorrhizal fungi, the identities of additional metabolites remain to be determined and isotope labelling techniques would be need to trace DDT transformation. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.04.002. References Bumpus, J.A., Aust, S.D., 1987. Biodegradation of DDT [1,1,1-trichloro-2,2-bis(4chlorophenyl) ethane] by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 53, 2001–2008. Chiu, S.W., Ho, K.M., Chan, S.S., So, O.M., Lai, K.H., 2006. Characterization of contamination in and toxicities of a shipyard area in Hong Kong. Environ. Pollut. 142, 512–520. Huang, Y., Zhao, X., Luan, S.J., 2007. Uptake and biodegradation of DDT by 4 ectomycorrhizal fungi. Sci. Total. Environ. 385, 235–241.
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