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Biotransformation of 2,4-dinitrotoluene by obligate marine Shewanella marisflavi EP1 under anaerobic conditions
Bioresource Technology 180 (2015) 200–206
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Biotransformation of 2,4-dinitrotoluene by obligate marine Shewanella marisflavi EP1 under anaerobic conditions Jiexun Huang a, Guojing Ning a, Feili Li a, G. Daniel Sheng b,⇑ a b
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
h i g h l i g h t s Reduction of 2,4-DNT by S. marisflavi EP1 was a process of microbial respiration. 200 lM 2,4-DNT was completely reduced by growing cells in 24 h. 2,4-DNT was reduced in a wide range of NaCl concentration (2–8%). Flavins enhanced the reduction of 2,4-DNT by EP1. Effective transformation occurred at a low temperature of 4 °C.
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Article history: Received 30 October 2014 Received in revised form 30 December 2014 Accepted 31 December 2014 Available online 8 January 2015 Keywords: Shewanella Dinitrotoluene Biotransformation Anaerobic reduction
a b s t r a c t Anaerobic transformation of 2,4-DNT by obligate marine Shewanella marisflavi EP1 was investigated. The cell growth of EP1 was proportional to the total amount of 2,4-DNT reduced. The eventual transformation product was 2,4-diaminotoluene, via 2-amino-4-nitrotoluene and 4-amino-2-nitrotoluene as intermediates. The presence of Cu2+, dicumarol, metyrapone and flavins intensively influenced the reduction activity of 2,4-DNT, suggesting that dehydrogenease, menaquinone, cytochromes and flavins are essentially involved in electron transport process for 2,4-DNT reduction. These results indicate that biotransformation of 2,4-DNT by EP1 is a form of microbial anaerobic respiration. Furthermore, EP1 was capable of transforming 2,4-DNT at relatively alkaline range of pH (7–9), and at a wide range of temperature (4–40 °C) and salinity (2–8% NaCl concentration). Our findings not only deepen our understanding of the environmental fate of 2,4-DNT, but also provide an extension to the application of shewanellae in the site bioremediation and/or wastewater treatment. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction 2,4-Dinitrotoluene (2,4-DNT) is widely used as a chemical intermediate in the manufacturing of polyurethane, dyes, explosives, and pesticides (Snellix et al., 2002). With improper disposal practices, the release of DNTs has resulted in the contamination of groundwater, soil and sediment adjacent to many manufacturing sites (Bradley et al., 1994). 2,4-DNT has posed a significant environmental concern due to its toxicity to humans (EPA, 2008). Removal of DNT from the contaminated sites is therefore imperative. The typical treatment of 2,4-DNT contaminated wastewater is activated carbon adsorption and followed by incineration of the exhausted carbon (Vanderloop et al., 1999). Although effective, this ⇑ Corresponding author. Tel.: +86 21 65989857; fax: +86 21 65983869. E-mail address: [email protected] (G.D. Sheng). http://dx.doi.org/10.1016/j.biortech.2014.12.108 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
procedure is expensive due to the high carbon requirements and poses air quality concern introduced by the incineration (Snellix et al., 2002). Alternative biological treatment of 2,4-DNT contamination was therefore given more attentions by many investigators. Due to the strong electrophilic character of nitro substituents, 2,4DNT is quite recalcitrant to microbial attack by the oxidative pathway (Cheng et al., 1998). With industrial applications of the aerobic biodegradation of 2,4-DNT, it is difficult to achieve compliance with EPA discharge limits (Sponza and Atalay, 2003). Under anaerobic conditions, however, 2,4-DNT could be reduced to some readily degradable compounds, which could be easily degraded by subsequential aerobic biological processes (Sponza and Atalay, 2003; Wang et al., 2011). Although several mixed or pure microbial cultures were found able to function in those anaerobic processes in laboratory experiments (Cheng et al., 1998; Vanderloop et al., 1999; Hughes et al., 1999; Noguera and Freedman, 1996; Shin et al., 2005), little is
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known about the in situ bioremediation of DNT by the indigenous microorganisms in nature. A recent report provided an insight into the impact of 2,4-DNT on the indigenous bacterial community in the marine sediments from two unexploded ordnance sites (Yang et al., 2009). Phylogenetic analysis found that the presence of 2,4-DNT led to enrichment of Shewanella species. The results indicated that bacteria within Shewanella genus might play a large role in the in situ transformation of 2,4-DNT in marine sediments. Species of Shewanella can be grouped into two clusters based on the phylogenetic analysis of 16S rDNA or gyrB gene sequence, as well as their phenotypic properties (Zhao et al., 2010). Cluster I Shewanella usually isolated form marine sediments are obligate marine members required Na+ for growth and more tolerant to higher salinity as compared to the other Shewanella from freshwater environments included in cluster II. The ocean and their sediments are the final sink for contaminants from anthropic activities, including 2,4-DNT pollution. Two cluster II Shewanella, Shewanella putrefaciens CN32 (Luan et al., 2010) and Shewanella oneidensis MR-1 (Cai et al., 2012), were previously reported the reduction of nitrobenzene. However, none of cluster II Shewanella was found in the undersea unexploded ordnance contaminated site, but dominant with obligate marine Shewanella (Zhao et al., 2010). Moreover, these non-obligate marine Shewanella reduced nitrobenzene under low or non-saline conditions, which mismatch the presence of large amounts of salts in many industrial effluents. Although DNT reduction by obligate marine Shewanella has been mentioned in the previous study (Zhao et al., 2010), little was known about the reduction mechanism and the effects of environmental factors on the DNT transformation by obligate marine Shewanella. In this study, Shewanella marisflavi EP1, an obligate marine Shewanella, which was recently isolated from marine sediments was shown to possess the ability to anaerobically reduce 2,4-DNT and grow coupled to this reduction. 2. Methods 2.1. Chemicals and stock solutions 2,4-Dinitrotoluene (2,4-DNT, 99%), 2-amino-4-nitrotoluene (2A4NT, 98%), 4-amino-2-nitrotoluene (4A2NT, 98%) and 2,4-diaminotoluene (2,4-DAT, 98%) were all purchased from Aladdin. Dicumarol and metyrapone were purchased from Sigma–Aldrich. Solvents for high performance liquid chromatography were HPLC-grade and obtained from Tedia (USA). All other chemicals obtained from Sangon Biotech (Shanghai), are analytical grade or higher. A stock solution of 2,4-DNT (5.0 g L 1) was prepared in methanol in brown glass. Stock solutions of sodium lactate (2.0 M), sodium fumarate (0.45 M) and flavins (riboflavin and flavin mononucleotide, 1:1, 250 lM) were prepared in milli-Q water and then injected into empty autoclaved serum vials using syringes with 0.22 lm pore-sized filters (millipore). All stock solutions were stored at 4 °C. 2.2. Bacterial strain, media and cultivation S. marisflavi EP1 (CCTCC M 209016) was previously obtained from marine sediments in Xiamen, China (Huang et al., 2010). All incubations were done at 30 °C. The EP1 growth and 2,4-DNT reduction studies were performed in a basal medium (BM) containing (per liter): K2HPO4, 1.3 g; KH2PO4, 0.45 g; (NH4)2SO4, 1.19 g; NaCl, 20.0 g; MgSO4, 0.25 g; CaCl2, 0.05 g; L-arginine HCl, 0.02 g, L-glutamic acid, 0.02 g; L-serine, 0.02 g; and Wolfe’s minerals of 100 ml. The initial pH of the medium was adjusted to 7.0. BM was boiled up for 20 min and allowed to cool, then flushed with N2
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(99.999%) to remove oxygen before autoclaving in sealed serum bottles. Lactate was added as electron donor throughout this study. To obtain inoculums, cells of EP1 were maintained in anoxic BM amended with 20 mM lactate and 40 mM fumarate as the electron donor and acceptor, respectively. Cells were collected after centrifugation at 3000 rpm for 10 min and then resuspended in sterile BM. 2.3. Biotransformation of 2,4-DNT To study the biotransformation of 2,4-DNT, standard anaerobic techniques (Miller and Wolin, 1974) for the culturing of EP1 were used throughout. Cells were inoculated into 50-ml BM in serum bottles containing 10 mM lactate with 2,4-DNT serving as electron acceptor. The initial concentration of cells was 1.3 105 to 2.5 105 CFU/ml unless noted otherwise. All treatments were incubated statically at 30 °C in dark. To monitor the reactions, a 1-ml incubation mix was withdrawn from each serum bottle using a sterile syringe and immediately centrifuged at 6000 rpm for 90 s using a mini Spinner (Eppendorf). The supernatant was used for subsequent analysis. Effect of flavins or respiratory inhibitors on the biotransformation of 2,4-DNT by EP1 was further investigated. Different concentration of flavins or respiratory inhibitors were spiked into sterile 50-ml serum vials containing BM, 2,4-DNT, lactate and cells suspension. Concentrations of 2,4-DNT and its metabolites were also monitored by periodically sampling and analysis. 2.4. Effect of environmental factors on the 2,4-DNT reduction The precultured cells of strain EP1 were washed and resuspended in anoxic BM, then inoculated into the experimental systems containing 50 ml anoxic BM and 10 mM lactate as electron donors under different environmental factors. Salinity of culture medium was controlled by adding different concentrations of NaCl (ranging from 0% to 10%). The initial pH of medium was adjusted to the range of 6.0–10.0 by using 1 M HCl or NaOH. The temperature range for EP1 growth using 2,4-DNT as electron acceptor was examined at 4 °C, 15 °C, 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C. The tests of 2,4-DNT tolerance by EP1 were set up with the concentration of 2,4-DNT at 50 lM, 100 lM, 200 lM, 300 lM, and 400 lM. 2.5. Analytical methods 2,4-DNT and its metabolites were measured via high performance liquid chromatography (Waters e2695) with a C18 column (Sun Fire, 5 lm, 4.6 250 mm) operated with UV detection. The mobile phase composed of water–methanol for the separation of 2,4-DNT, 4A2NT, and 2A4NT, was programmed as follows: 50% (v/v) methanol at 0 min, 70% (v/v) methanol at 11 min, and isotropic from 11 min to 13 min. For 2,4-DAT, an acetonitrile–phosphate buffer (20 mM, pH 7.0, 30/70, v/v) was used as the eluant at 1.0 mL min 1. All the analyses were conducted at room temperature and the sample volumes injected were 10 lL. Cell growth was determined by absorbance at 600 nm or plate counting on LB medium. 3. Results and discussion 3.1. Biotransformation of 2,4-DNT coupled to the anaerobic growth of EP1 Previous studies demonstrated that Shewanella possess a remarkable respiratory versatility allowing them to utilize a diversity of final electron acceptors (Hau and Gralnick, 2007). However,
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the respiratory versatility of one given Shewanella strain could be variable. Shewanella halifaxensis could rapidly reduce hexahydro1,3,5-trinitro-1,3,5-triazine (RDX), but could not reduce Fe(III) (Zhao et al., 2006). In contrast, S. oneidensis MR-1 could effectively reduce Fe(III), but reduce RDX at extremely low rates (Luan et al., 2010). Although nitrobenzene reduction has been performed by non-obligate marine Shewanella, little was known about DNT reduction by obligate marine members. S. marisflavi EP1 isolated from marine sediments fell into cluster I Shewanella based on 16S rDNA sequence analysis (Huang et al., 2010). Na+ requirement for growth and tolerance at high salinity make EP1 more suitable for in-situ remediation of DNT contamination in marine sediments. Thus, it was necessary to study the biotransformation of 2,4-DNT by obligate marine strain EP1. As the results shown in Fig. 1, different treatments and controls were set in a defined medium under anaerobic conditions. EP1 was able to completely reduce 2,4-DNT of 100 lM in less than 24 h using lactate as electron donor. However, lactate was not able to transform 2,4-DNT in the absence of EP1. Reduction of 2,4-DNT was also not observed in the controls inoculated with heat-killed (95 °C, 30 min) cells or in the absence of lactate. When S. marisflavi EP1 was inoculated to the defined medium with lactate as the sole electron donor and different concentrations of 2,4-DNT as electron acceptor, cell growth was monitored over time. Fig. 2 shows the correlation of anaerobic growth of EP1 with the reduction of 2,4DNT. No growth occurred in the absence of 2,4-DNT. A maximal cell density of 6.9 107 CFU/ml was obtained when 200 lM 2,4DNT was completely reduced in the defined medium. The cell yield was evidently proportional to the total amount of 2,4-DNT reduced (R2 = 0.9653). These results demonstrated that the transformation of 2,4-DNT was a microbial process performed by S. marisflavi EP1, and 2,4-DNT was utilized as the terminal electron acceptor. Given that there is a strong preference for lactate as carbon source in Shewanella (Nealson and Scott, 2005), lactate was chosen as the electron donor throughout this study. Generally, microorganisms metabolize organic compounds to produce energy supporting growth via fermentation or respiration. As lactate is the terminal product generated by the fermentative microorganisms in nature, use of lactate to reduce 2,4-DNT by EP1 excluded the possibility of fermentation. Actually, shewanellae have been found nonfermentative (Hau and Gralnick, 2007). In the defined medium, 2,4-DNT was the only available electron acceptor considering that the biomass yield was proportional to the reduced 2,4-DNT. Our
Fig. 1. Anaerobic biotransformation of 2,4-DNT by S. marisflavi EP1 with lactate as electron donors. Error bars represent one standard deviation calculated from triplicates.
Fig. 2. Anaerobic growth of S. marisflavi EP1 coupled to the reduction of different concentrations of 2,4-DNT. No growth was observed in the absence of 2,4-DNT. Error bars represent one standard deviation calculated from replicates.
studies undoubtedly indicate that EP1 oxidize lactate to release electrons to reduce 2,4-DNT as electron acceptor via respiration process that supports anaerobic growth. 3.2. Intermediates and products of 2,4-DNT transformation Tests were conducted in the defined medium using growing cells of EP1, the intermediates and products of 2,4-DNT transformation was analyzed by HPLC. The initial reduction of 2,4-DNT had a high rate, two intermediates produced soon and a terminal product commenced to be detected after 24 h (Fig. 3). The retention times of the two intermediates in the chromatography were 9.431 min and 10.343 min, respectively. The peak of the product appeared in chromatography at 4.459 min. Through comparing the retention times and full wavelength UV spectrograms, the intermediates and product well matched with the analytical standards of 2-amino-4-nitrotoluene (2A4NT), 4-amino-2-nitrotoluene (4A2NT) and 2,4-diaminotoluene (2,4-DAT), confirming that the disappearance of 2,4-DNT was accompanied with concurrent formation of 2A4NT and 4A2NT. Both intermediates were further reduced to the eventual product, 2,4-DAT.
Fig. 3. Temporal concentrations of metabolites during the transformation of 2,4DNT by S. marisflavi EP1. Error bars represent one standard deviation calculated from triplicates.
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Of the two aminonitrotoluene (ANT) isomers, 4A2NT was generated faster than 2A4NT. After 24 h of incubation, all of the DNT was biotransformed, with 4A2NT and 2A4NT accounting for 60% and 34% of the original DNT, respectively. The preferential formation of 4A2NT could be explained by the fact that the para nitro group is more open and better solvated than the ortho nitro group due to the shielding effect of methyl group on the ortho nitro group (Oh and Chiu, 2009). Similar reaction pattern has been observed in other microbial 2,4-DNT transformation (Bradley et al., 1994; Noguera and Freedman, 1996). In contrast, anaerobic transformation of 2,4-DNT by Clostridium acetobutylicum favored formation of dihydroxylaminotoluenes with a limited reduction to ANT (Hughes et al., 1999). The different profiles of DNT reduction might be derived from the different terminal reductases in the microorganisms. 3.3. Experiments about the respiratory electron transport chain To further understand the metabolic mechanism of 2,4-DNT reduction by EP1, some factors capable of affecting the electron transport chain were investigated. Cu2+ ions and dicumarol, two kinds of respiratory inhibitors, are thought to inhibit the membrane-impermeable dehydrogenas (Fernandez et al., 1989) and electron transport of menaquinone (MK) (Arnold et al., 1986) in bacteria, respectively. The respiration of 2,4-DNT by EP1 was almost completely inhibited by 10 lM Cu2+ (Fig 4a) or 50 lM dicumarol (Fig 4b), suggesting that lactate dehydrogenease and MK are essential components of electron transport chain. On the contrary, although 2,4-DNT reduction was greatly repressed by cytochrome P450 inhibitor metyrapone (Williams et al., 2004), it was not completely inhibited with even up to 1000 lM of metyrapone (Fig 4c). Since Shewanella species have a robust cytochrome pool (Fredrickson et al., 2008), this versatility may be exploited by EP1 cells in which the disabled cytochrome P450 can be functionally replaced by other cytochromes. In addition, the effects of various concentration of flavins on 2,4-DNT transformation by EP1 were evaluated. As shown in Fig. 5, flavins demonstrated a stimulating effect on 2,4-DNT transformation. The enhancing effects increased with the increase of flavins concentrations up to 7.5 lM. With addition of 2.5 lM flavins, the transformation efficiency increased 2.5-fold after 6 h incubation, compared with that obtained in the absence of flavins. This result was in agreement with the previous observations that the addition of flavins could also enhance the reduction of carbon electrode (Marsili et al., 2008). Using electron transport chain inhibitors or promoters is proven one of the effective methods to study the respiratory chain components. The current experiments indicated that electron transport chain for anaerobic transformation of 2,4-DNT by EP1 might include the components such as dehydrogenases, menaquinone, cytochromes, and maybe flavins. This profile matched well with the extracellular respiratory pathway exploited by S. oneidensis MR-1 (Brutinel and Gralnick, 2012), the most intensively studied member in Shewanella genus. Based on these similarities, a hypothetical respiratory mode is proposed for EP1. In this mode, the MK pool accepts the electrons produced from the lactate dehydrogenase, and transfer these electrons to a series of cytochromes, termed Mtr pathway through which electrons eventually move to the cell surface. Then the extracellular flavins accept electrons from cell surface and move to the terminal electron acceptors by diffusion. Alternatively, electrons at the cell surface can transfer to electron acceptors by direct contact considering that 2,4-DNT reduction occurred in the absence of flavins in the present experiments. However, there is still the possibility that flavins can be synthesized and secreted by EP1 itself, as flavins have been identified as redox-active mediator secreted by a number of Shewanella
Fig. 4. Effect of respiration inhibitors on 2.4-DNT reduction by S. marisflavi EP1. Reduction of 2,4-DNT were measured in the presence of different concentrations of Cu2+ (a), dicumarol (b), and metyrapone (c). Error bars represent one standard deviation calculated from triplicates.
species (von Canstein et al., 2008). However, a recent study found that intracellular reactions were involved in nitrobenzene reduction in S. oneidensis MR-1 (Cai et al. 2012), which makes this mechanism more complex. The exact mechanism of anaerobic respiration of 2,4-DNT in EP1 remains unclear and needs further investigations. 3.4. Effects of environmental factors on the 2,4-DNT transformation The transformation of 2,4-DNT by S. marisflavi EP1 occurred over a pH range from 6.0 to 10.0 (Fig. 6a). Maximum activity of
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Fig. 5. Effect of flavins on the transformation of 2,4-DNT by S. marisflavi EP1.
EP1 presented at pH 8.0 under the experimental conditions. The absence of 2,4-DNT transformation in the treatment at pH 10.0 indicated that 2,4-DNT reduction at pH 8.0 was a microbial process catalyzed by EP1 rather than a chemically spontaneous process under alkaline conditions. Fig. 6b shows EP1 could transform 2,4-DNT at the low temperature of 4 °C, although only 20% of 2,4-DNT were reduced within 24 h. Increasing temperature
enhanced the activity of 2,4-DNT transformation by EP1 up to 40 °C. However, EP1 cells were heat-inactivated at 45 °C. As shown in Fig. 6c, 2,4-DNT could be reduced by EP1 in the presence of various concentration of NaCl (2–8%). Compared to the absence of 2,4DNT transformation observed under non-saline conditions, adding certain amounts of NaCl was in favor of 2,4-DNT transformation by EP1. At optimum salinity with 2% NaCl, 100 lM 2,4-DNT was almost completely transformed around 12 h. A small amount of 2,4-DNT reduced was detected at the salinity of 8% NaCl. Fig 6d showed the percent of 2,4-DNT reduced by EP1 under various initial concentrations of DNT. After 12 h, initial 50 lM or 100 lM 2,4DNT could be completely transformed, but 200 lM, 300 lM and 400 lM of initial concentration remained 20%, 80%, and 90% unreduced, respectively. By calculation of the average removal rate within 9 h, the optimum removal activity of EP1 occurred at initial concentration of 200 lM 2,4-DNT, increasing 30% compared to that at 100 lM or 300 lM 2,4-DNT (Inset in Fig. 6d). These results were consistent with the typical traits of the shewanellae, such as slight alkaline optimal pH for growth (Venkateswaran et al., 1999; Yoon et al., 2004), the capacity to thrive at low temperature (Hau and Gralnick, 2007), and somewhat halophilic (Yoon et al., 2004). Meanwhile, the salinity tolerance of EP1 was different from that of the most intensively studied S. oneidensis MR-1 (Venkateswaran et al., 1999). The former was not able to thrive with 0% NaCl, similar to S. halifaxensis which required at least 1% NaCl for growth (Zhao et al., 2006). EP1 was able to completely reduce 100 lM 2,4-DNT at 4 °C within 96 h
Fig. 6. Biotransformation of 2,4-DNT by S. marisflavi EP1 under different pH (a), temperature (b), salinity (c), and initial concentrations of 2,4-DNT (d). Inset in (b) shows the complete transformation of 100 lM 2,4-DNT at 4 °C. Inset in (d) shows the average removal rate calculated from transformation data under different initial concentrations of 2,4-DNT within the early 9 h.
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J. Huang et al. / Bioresource Technology 180 (2015) 200–206 Table 1 Comparison of 2,4-DNT transformation with other reports.
a
Catalyst
Electron donor
Electron acceptor
pH
Salinitya
Initial concentration (lM)
Efficiency
Refs.
n-Hexane soot Poultry litter biochar Microbial community Clostridium acetobutylicum Pseudomonas putida OU83 Arthrobacter sp. K1 Pseudomonas aeruginosa S. putrefaciens CN32 S. oneidensis MR-1 S. marisflavi EP1
Dithiothreitol Dithiothreitol Ethanol Saccharides – 2,4-DNT Ethanol Lactate Lactate Lactate
2,4-DNT 2,4-DNT 2,4-DNT 2,4-DNT Oxygen Oxygen 2,4-DNT Nitrobenzene 2,4-DNT Anaerobic
7.4 7.0 7.2 7.0 7.5 7.0 6.5–7.8 6.8 7.0 7.0–9.0
0% 0% – 0% 0% 0.5% 0% 0% 0% 2%
178 100 88 549 400 477 240 149 90 200
100%/7 d 75%/10 d 100%/120 h 100%/1 h 98%/48 h 66%/10 d 100%/13 d 100%/24 h 100%/24 h 100%/24 h
Oh and Chiu (2009) Oh et al. (2013) Cheng et al. (1998) Hughes et al. (1999) Walia et al. (2002) Küce et al. (2014) Noguera and Freedman (1996) Luan et al. (2010) Huang et al. (2013) This study
Salinity: NaCl concentration (wt/vol.%).
(Inset in Fig. 6b). It is actually in accordance with most Shewanella strains that demonstrate the capacity to grow at low temperature, but their temperature optimum for growth is beyond 16 °C (Hau and Gralnick, 2007). 3.5. Comparison with other reported 2,4-DNT transformations Recent years, many remediation approaches were developed to mitigate the DNT contamination (Table 1). In these approaches, catalysts could be biotic or abiotic and reactions could be under anaerobic or aerobic conditions. Comparison of 2,4-DNT transformation by S. marisflavi EP1 with the reactions in other reports indicates that only C. acetobutylicum presented higher reducing activity than EP1. C. acetobutylicum was capable of transforming 100% 2,4DNT (549 lM) in 1 h (Hughes et al., 1999). Such high activity might be attributed to the spiking of 2,4-DNT in reaction system during the log-growth phase of C. acetobutylicum when the cultures gave a very high density of cells. However, in this study transformation of 2,4-DNT by EP1 commenced at the time of inoculation when the reaction system gave comparatively lower cell density. Other three bacterial pure cultures, Pseudomonas putida OU83 (Walia et al., 2002), Arthrobacter sp. K1 (Küce et al., 2014) and Pseudomonas aeruginosa (Noguera and Freedman, 1996) could transform 98% 2,4-DNT (400 lM) in 48 h, 66% 2,4-DNT (477 lM) in 10 days, and 100% 2,4-DNT (240 lM) in 13 days, respectively. Recently, black carbon-mediated reduction of 2,4-DNT has received much attentions, but the reduction catalyzed by n-hexane soot (Oh and Chiu, 2009) and poultry litter biochar (Oh et al., 2013) remained low activities compared with that catalyzed by EP1. Two non-obligate marine Shewanella were also compared in Table 1. S. putrefaciens CN32 could reduce 100% nitrobenzene (149 lM) in 24 h, albeit at a high resting cell density of 1 108 cell ml 1 (Luan et al., 2010). By contrast, the growing cells of EP1 only reached 6.9 107 CFU/ ml after complete reduction of 200 lM 2,4-DNT. While S. oneidensis MR-1 performed the optimum reduction of 2,4-DNT at 0% salinity. 3.6. Environmental significance In nature, redox states of contaminants influence their environmental fate. For example, oxidation states of inorganic elements, such as uranium and chromium, change their solubility and mobility in water and sediments (Fredrickson et al., 2008). Some redoxsensitive organic chemicals, such as NACs, their biodegradability under aerobic conditions largely depends on the oxidation states (Snellix et al., 2002). Reduction of these contaminants to change their redox states via microbial respiration offer the potential to effectively clean up the contaminated environments. The shewanellae, as a group of dissimilatory metal-reducing bacteria, are regarded as appropriate candidates for application in bioremediation of these contaminants because of their outstanding capacity
to respire a broad range of electron acceptors. More than 20 electron acceptors that are found to be respired by Shewanella so far include toxic organic compounds and inorganic elements (Hau and Gralnick, 2007). And the list increases continually. In this study, we demonstrated that 2,4-DNT reduction by S. marisflavi EP1 was a process of respiration. Furthermore, we also illustrated that trinitrotoluene and a dinitroaniline pre-emergence herbicide pendimethalin could be reduced and transformed by S. marisflavi EP1 and S. oneidensis MR-1 (data not shown). This respiration form may expand the range of electron acceptors utilized by the shewanellae to include enormous NACs. It is well known that members of Shewanella genus are wildly distributed in marine and freshwater environments, especially thrived in redox-stratified environments where nitroaromatic compounds are readily and strongly sorbed (Yang et al., 2009). Based on this, our finding is expected to not only deepen our understanding on the environmental fate and ecotoxicity of 2,4-DNT, in addition to other nitroaromatic pollutants, but also suggest a potential site bioremediation and/or wastewater treatment approach. Addition of electron donors preferred by the shewanellae may stimulate the reduction of 2,4-DNT, and facilitate its completely mineralized and detoxified in environments. Moreover, as industrial effluents frequently have an elevated salinity and NOCs-contaminated sites in natural environments undergo a comparatively low temperature such as sea sediments (Yang et al., 2009), a high-performance activity of DNT transformation by the shewanellae at high salinity and low temperature significantly extend their industrial and environmental applications.
4. Conclusions The biotransformation of 2,4-DNT by S. marisflavi EP1 under anaerobic conditions is a process of respiration in which EP1 can utilize lactate as electron donor for dissimilatory reducing 2,4DNT. The terminal product of 2,4-DNT transformation is 2,4-diaminotoluene via temporary formation of two intermediates, 2-amino4-nitrotoluene and 4-amino-2-nitrotoluene. Due to the toxicity of amino-derivatives, however, further studies will be necessarily required to generate ecologically safe remediation products. Furthermore, rapid and effective reduction of 2,4-DNT conducted by EP1 at relatively wide range of pH, temperature and salinity, suggest a potential strategy for the cleanup of nitroaromatic contaminants in natural environments.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21437004, 21307112).
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Nealson, K.H., Scott, J., 2005. Ecophysiology of the genus Shewanella. In: Dworkin, M. (Ed.), The Prokaryotes. Springer-Verlag, New York. Noguera, D.R., Freedman, D.L., 1996. Reduction and acetylation of 2,4dinitrotoluene by a Pseudomonas aeruginosa strain. Appl. Environ. Microbiol. 62, 2257–2263. Oh, S.Y., Chiu, P.C., 2009. Graphite- and soot-mediated reduction of 2,4dinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine. Environ. Sci. Technol. 43, 6383–6388. Oh, S.Y., Son, J.G., Chiu, P.C., 2013. Biochar-mediated reductive transformation of nitro herbicides and explosives. Environ. Toxicol. Chem. 32, 501–508. Shin, K.H., Lim, Y., Ahn, J.H., Khil, J., Cha, C.J., Hur, H.G., 2005. Anaerobic biotransformation of dinitrotoluene isomers by Lactococcus lactis subsp. lactis strain 27 isolated from earthworm intestine. Chemosphere 61, 30–39. Snellix, Z., Nepovím, A., Taghavi, S., Vangronsveld, J., Vanek, T., van der Lelie, D., 2002. Biological remediation of explosives and related nitroaromatic compounds. Environ. Sci. Pollut. Res. 9, 48–61. Sponza, D.T., Atalay, H., 2003. Treatability of 2,4-dinitrotoluene in anaerobic/aerobic sequential process. J. Environ. Sci. Health 38, 1529–1548. Vanderloop, S.L., Suidan, M.T., Moteleb, M.A., Maloney, S.W., 1999. Biotransformation of 2,4-dinitrotoluene under different electron acceptor conditions. Water Res. 33, 1287–1295. von Canstein, H., Ogawa, J., Shimizu, S., Lloyd, J.R., 2008. Secretion of flavins by Shewanella species and their role in extracellular electron transfer. Appl. Environ. Microbiol. 74, 615–623. Wang, Z.Y., Ye, Z.F., Zhang, M.H., 2011. Bioremediation of 2,4-dinitrotoluene (2,4DNT) in immobilized micro-organism biological filter. J. Appl. Microbiol. 110, 1476–1484. Walia, S.K., Ali-Sadat, S., Brar, R., Chaudhry, G.R., 2002. Identification and mutagenicity of dinitrotoluene metabolites produced by strain Pseudomonas putida OU83. Pestic. Biochem. Physiol. 73, 131–139. Williams, P.A., Cosme, J., Vinkovic, D.M., Ward, A., Angove, H.C., Day, P.J., Vonrhein, C., Tickle, I.J., Jhoti, H., 2004. Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science 305, 683–686. Venkateswaran, K., Moser, D.P., Dollhopf, M.E., Lies, D.P., Saffarini, D.A., MacGregor, B.J., Ringelberg, D.B., White, D.C., Nishijima, M., Sano, H., Burghardt, J., Stackebrandt, E., Nealson, K.H., 1999. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. Int. J. Syst. Bacteriol. 49, 705–724. Yang, H., Zhao, J.S., Hawari, J., 2009. Effect of 2,4-dinitrotoluene on the anaerobic bacterial community in marine sediment. J. Appl. Microbiol. 107, 1799–1808. Yoon, J.-H., Yeo, S.-H., Kim, I.G., Oh, T.K., 2004. Shewanella marisflavi sp. nov. and Shewanella aquimarina sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea. Int. J. Syst. Evol. Microbiol. 54, 2347–2352. Zhao, J.-S., Deng, Y., Manno, D., Hawari, J., 2010. Shewanella spp. genomic evolution for a cold marine lifestyle and in-situ explosive biodegradation. PLoS ONE 5 (2), e9109. http://dx.doi.org/10.1371/journal.pone.0009109. Zhao, J.-S., Manno, D., Leggiadro, C., O’Neil, D., Hawari, J., 2006. Shewanella halifaxensis sp. nov., a novel obligately respiratory and denitrifying psychrophile. Int. J. Syst. Evol. Microbiol. 56, 205–212.
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Biotransformation of 2,4-dinitrotoluene by obligate marine Shewanella marisflavi EP1 under anaerobic conditions Jiexun Huang a, Guojing Ning a, Feili Li a, G. Daniel Sheng b,⇑ a b
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
h i g h l i g h t s Reduction of 2,4-DNT by S. marisflavi EP1 was a process of microbial respiration. 200 lM 2,4-DNT was completely reduced by growing cells in 24 h. 2,4-DNT was reduced in a wide range of NaCl concentration (2–8%). Flavins enhanced the reduction of 2,4-DNT by EP1. Effective transformation occurred at a low temperature of 4 °C.
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Article history: Received 30 October 2014 Received in revised form 30 December 2014 Accepted 31 December 2014 Available online 8 January 2015 Keywords: Shewanella Dinitrotoluene Biotransformation Anaerobic reduction
a b s t r a c t Anaerobic transformation of 2,4-DNT by obligate marine Shewanella marisflavi EP1 was investigated. The cell growth of EP1 was proportional to the total amount of 2,4-DNT reduced. The eventual transformation product was 2,4-diaminotoluene, via 2-amino-4-nitrotoluene and 4-amino-2-nitrotoluene as intermediates. The presence of Cu2+, dicumarol, metyrapone and flavins intensively influenced the reduction activity of 2,4-DNT, suggesting that dehydrogenease, menaquinone, cytochromes and flavins are essentially involved in electron transport process for 2,4-DNT reduction. These results indicate that biotransformation of 2,4-DNT by EP1 is a form of microbial anaerobic respiration. Furthermore, EP1 was capable of transforming 2,4-DNT at relatively alkaline range of pH (7–9), and at a wide range of temperature (4–40 °C) and salinity (2–8% NaCl concentration). Our findings not only deepen our understanding of the environmental fate of 2,4-DNT, but also provide an extension to the application of shewanellae in the site bioremediation and/or wastewater treatment. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction 2,4-Dinitrotoluene (2,4-DNT) is widely used as a chemical intermediate in the manufacturing of polyurethane, dyes, explosives, and pesticides (Snellix et al., 2002). With improper disposal practices, the release of DNTs has resulted in the contamination of groundwater, soil and sediment adjacent to many manufacturing sites (Bradley et al., 1994). 2,4-DNT has posed a significant environmental concern due to its toxicity to humans (EPA, 2008). Removal of DNT from the contaminated sites is therefore imperative. The typical treatment of 2,4-DNT contaminated wastewater is activated carbon adsorption and followed by incineration of the exhausted carbon (Vanderloop et al., 1999). Although effective, this ⇑ Corresponding author. Tel.: +86 21 65989857; fax: +86 21 65983869. E-mail address: [email protected] (G.D. Sheng). http://dx.doi.org/10.1016/j.biortech.2014.12.108 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
procedure is expensive due to the high carbon requirements and poses air quality concern introduced by the incineration (Snellix et al., 2002). Alternative biological treatment of 2,4-DNT contamination was therefore given more attentions by many investigators. Due to the strong electrophilic character of nitro substituents, 2,4DNT is quite recalcitrant to microbial attack by the oxidative pathway (Cheng et al., 1998). With industrial applications of the aerobic biodegradation of 2,4-DNT, it is difficult to achieve compliance with EPA discharge limits (Sponza and Atalay, 2003). Under anaerobic conditions, however, 2,4-DNT could be reduced to some readily degradable compounds, which could be easily degraded by subsequential aerobic biological processes (Sponza and Atalay, 2003; Wang et al., 2011). Although several mixed or pure microbial cultures were found able to function in those anaerobic processes in laboratory experiments (Cheng et al., 1998; Vanderloop et al., 1999; Hughes et al., 1999; Noguera and Freedman, 1996; Shin et al., 2005), little is
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known about the in situ bioremediation of DNT by the indigenous microorganisms in nature. A recent report provided an insight into the impact of 2,4-DNT on the indigenous bacterial community in the marine sediments from two unexploded ordnance sites (Yang et al., 2009). Phylogenetic analysis found that the presence of 2,4-DNT led to enrichment of Shewanella species. The results indicated that bacteria within Shewanella genus might play a large role in the in situ transformation of 2,4-DNT in marine sediments. Species of Shewanella can be grouped into two clusters based on the phylogenetic analysis of 16S rDNA or gyrB gene sequence, as well as their phenotypic properties (Zhao et al., 2010). Cluster I Shewanella usually isolated form marine sediments are obligate marine members required Na+ for growth and more tolerant to higher salinity as compared to the other Shewanella from freshwater environments included in cluster II. The ocean and their sediments are the final sink for contaminants from anthropic activities, including 2,4-DNT pollution. Two cluster II Shewanella, Shewanella putrefaciens CN32 (Luan et al., 2010) and Shewanella oneidensis MR-1 (Cai et al., 2012), were previously reported the reduction of nitrobenzene. However, none of cluster II Shewanella was found in the undersea unexploded ordnance contaminated site, but dominant with obligate marine Shewanella (Zhao et al., 2010). Moreover, these non-obligate marine Shewanella reduced nitrobenzene under low or non-saline conditions, which mismatch the presence of large amounts of salts in many industrial effluents. Although DNT reduction by obligate marine Shewanella has been mentioned in the previous study (Zhao et al., 2010), little was known about the reduction mechanism and the effects of environmental factors on the DNT transformation by obligate marine Shewanella. In this study, Shewanella marisflavi EP1, an obligate marine Shewanella, which was recently isolated from marine sediments was shown to possess the ability to anaerobically reduce 2,4-DNT and grow coupled to this reduction. 2. Methods 2.1. Chemicals and stock solutions 2,4-Dinitrotoluene (2,4-DNT, 99%), 2-amino-4-nitrotoluene (2A4NT, 98%), 4-amino-2-nitrotoluene (4A2NT, 98%) and 2,4-diaminotoluene (2,4-DAT, 98%) were all purchased from Aladdin. Dicumarol and metyrapone were purchased from Sigma–Aldrich. Solvents for high performance liquid chromatography were HPLC-grade and obtained from Tedia (USA). All other chemicals obtained from Sangon Biotech (Shanghai), are analytical grade or higher. A stock solution of 2,4-DNT (5.0 g L 1) was prepared in methanol in brown glass. Stock solutions of sodium lactate (2.0 M), sodium fumarate (0.45 M) and flavins (riboflavin and flavin mononucleotide, 1:1, 250 lM) were prepared in milli-Q water and then injected into empty autoclaved serum vials using syringes with 0.22 lm pore-sized filters (millipore). All stock solutions were stored at 4 °C. 2.2. Bacterial strain, media and cultivation S. marisflavi EP1 (CCTCC M 209016) was previously obtained from marine sediments in Xiamen, China (Huang et al., 2010). All incubations were done at 30 °C. The EP1 growth and 2,4-DNT reduction studies were performed in a basal medium (BM) containing (per liter): K2HPO4, 1.3 g; KH2PO4, 0.45 g; (NH4)2SO4, 1.19 g; NaCl, 20.0 g; MgSO4, 0.25 g; CaCl2, 0.05 g; L-arginine HCl, 0.02 g, L-glutamic acid, 0.02 g; L-serine, 0.02 g; and Wolfe’s minerals of 100 ml. The initial pH of the medium was adjusted to 7.0. BM was boiled up for 20 min and allowed to cool, then flushed with N2
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(99.999%) to remove oxygen before autoclaving in sealed serum bottles. Lactate was added as electron donor throughout this study. To obtain inoculums, cells of EP1 were maintained in anoxic BM amended with 20 mM lactate and 40 mM fumarate as the electron donor and acceptor, respectively. Cells were collected after centrifugation at 3000 rpm for 10 min and then resuspended in sterile BM. 2.3. Biotransformation of 2,4-DNT To study the biotransformation of 2,4-DNT, standard anaerobic techniques (Miller and Wolin, 1974) for the culturing of EP1 were used throughout. Cells were inoculated into 50-ml BM in serum bottles containing 10 mM lactate with 2,4-DNT serving as electron acceptor. The initial concentration of cells was 1.3 105 to 2.5 105 CFU/ml unless noted otherwise. All treatments were incubated statically at 30 °C in dark. To monitor the reactions, a 1-ml incubation mix was withdrawn from each serum bottle using a sterile syringe and immediately centrifuged at 6000 rpm for 90 s using a mini Spinner (Eppendorf). The supernatant was used for subsequent analysis. Effect of flavins or respiratory inhibitors on the biotransformation of 2,4-DNT by EP1 was further investigated. Different concentration of flavins or respiratory inhibitors were spiked into sterile 50-ml serum vials containing BM, 2,4-DNT, lactate and cells suspension. Concentrations of 2,4-DNT and its metabolites were also monitored by periodically sampling and analysis. 2.4. Effect of environmental factors on the 2,4-DNT reduction The precultured cells of strain EP1 were washed and resuspended in anoxic BM, then inoculated into the experimental systems containing 50 ml anoxic BM and 10 mM lactate as electron donors under different environmental factors. Salinity of culture medium was controlled by adding different concentrations of NaCl (ranging from 0% to 10%). The initial pH of medium was adjusted to the range of 6.0–10.0 by using 1 M HCl or NaOH. The temperature range for EP1 growth using 2,4-DNT as electron acceptor was examined at 4 °C, 15 °C, 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C. The tests of 2,4-DNT tolerance by EP1 were set up with the concentration of 2,4-DNT at 50 lM, 100 lM, 200 lM, 300 lM, and 400 lM. 2.5. Analytical methods 2,4-DNT and its metabolites were measured via high performance liquid chromatography (Waters e2695) with a C18 column (Sun Fire, 5 lm, 4.6 250 mm) operated with UV detection. The mobile phase composed of water–methanol for the separation of 2,4-DNT, 4A2NT, and 2A4NT, was programmed as follows: 50% (v/v) methanol at 0 min, 70% (v/v) methanol at 11 min, and isotropic from 11 min to 13 min. For 2,4-DAT, an acetonitrile–phosphate buffer (20 mM, pH 7.0, 30/70, v/v) was used as the eluant at 1.0 mL min 1. All the analyses were conducted at room temperature and the sample volumes injected were 10 lL. Cell growth was determined by absorbance at 600 nm or plate counting on LB medium. 3. Results and discussion 3.1. Biotransformation of 2,4-DNT coupled to the anaerobic growth of EP1 Previous studies demonstrated that Shewanella possess a remarkable respiratory versatility allowing them to utilize a diversity of final electron acceptors (Hau and Gralnick, 2007). However,
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the respiratory versatility of one given Shewanella strain could be variable. Shewanella halifaxensis could rapidly reduce hexahydro1,3,5-trinitro-1,3,5-triazine (RDX), but could not reduce Fe(III) (Zhao et al., 2006). In contrast, S. oneidensis MR-1 could effectively reduce Fe(III), but reduce RDX at extremely low rates (Luan et al., 2010). Although nitrobenzene reduction has been performed by non-obligate marine Shewanella, little was known about DNT reduction by obligate marine members. S. marisflavi EP1 isolated from marine sediments fell into cluster I Shewanella based on 16S rDNA sequence analysis (Huang et al., 2010). Na+ requirement for growth and tolerance at high salinity make EP1 more suitable for in-situ remediation of DNT contamination in marine sediments. Thus, it was necessary to study the biotransformation of 2,4-DNT by obligate marine strain EP1. As the results shown in Fig. 1, different treatments and controls were set in a defined medium under anaerobic conditions. EP1 was able to completely reduce 2,4-DNT of 100 lM in less than 24 h using lactate as electron donor. However, lactate was not able to transform 2,4-DNT in the absence of EP1. Reduction of 2,4-DNT was also not observed in the controls inoculated with heat-killed (95 °C, 30 min) cells or in the absence of lactate. When S. marisflavi EP1 was inoculated to the defined medium with lactate as the sole electron donor and different concentrations of 2,4-DNT as electron acceptor, cell growth was monitored over time. Fig. 2 shows the correlation of anaerobic growth of EP1 with the reduction of 2,4DNT. No growth occurred in the absence of 2,4-DNT. A maximal cell density of 6.9 107 CFU/ml was obtained when 200 lM 2,4DNT was completely reduced in the defined medium. The cell yield was evidently proportional to the total amount of 2,4-DNT reduced (R2 = 0.9653). These results demonstrated that the transformation of 2,4-DNT was a microbial process performed by S. marisflavi EP1, and 2,4-DNT was utilized as the terminal electron acceptor. Given that there is a strong preference for lactate as carbon source in Shewanella (Nealson and Scott, 2005), lactate was chosen as the electron donor throughout this study. Generally, microorganisms metabolize organic compounds to produce energy supporting growth via fermentation or respiration. As lactate is the terminal product generated by the fermentative microorganisms in nature, use of lactate to reduce 2,4-DNT by EP1 excluded the possibility of fermentation. Actually, shewanellae have been found nonfermentative (Hau and Gralnick, 2007). In the defined medium, 2,4-DNT was the only available electron acceptor considering that the biomass yield was proportional to the reduced 2,4-DNT. Our
Fig. 1. Anaerobic biotransformation of 2,4-DNT by S. marisflavi EP1 with lactate as electron donors. Error bars represent one standard deviation calculated from triplicates.
Fig. 2. Anaerobic growth of S. marisflavi EP1 coupled to the reduction of different concentrations of 2,4-DNT. No growth was observed in the absence of 2,4-DNT. Error bars represent one standard deviation calculated from replicates.
studies undoubtedly indicate that EP1 oxidize lactate to release electrons to reduce 2,4-DNT as electron acceptor via respiration process that supports anaerobic growth. 3.2. Intermediates and products of 2,4-DNT transformation Tests were conducted in the defined medium using growing cells of EP1, the intermediates and products of 2,4-DNT transformation was analyzed by HPLC. The initial reduction of 2,4-DNT had a high rate, two intermediates produced soon and a terminal product commenced to be detected after 24 h (Fig. 3). The retention times of the two intermediates in the chromatography were 9.431 min and 10.343 min, respectively. The peak of the product appeared in chromatography at 4.459 min. Through comparing the retention times and full wavelength UV spectrograms, the intermediates and product well matched with the analytical standards of 2-amino-4-nitrotoluene (2A4NT), 4-amino-2-nitrotoluene (4A2NT) and 2,4-diaminotoluene (2,4-DAT), confirming that the disappearance of 2,4-DNT was accompanied with concurrent formation of 2A4NT and 4A2NT. Both intermediates were further reduced to the eventual product, 2,4-DAT.
Fig. 3. Temporal concentrations of metabolites during the transformation of 2,4DNT by S. marisflavi EP1. Error bars represent one standard deviation calculated from triplicates.
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Of the two aminonitrotoluene (ANT) isomers, 4A2NT was generated faster than 2A4NT. After 24 h of incubation, all of the DNT was biotransformed, with 4A2NT and 2A4NT accounting for 60% and 34% of the original DNT, respectively. The preferential formation of 4A2NT could be explained by the fact that the para nitro group is more open and better solvated than the ortho nitro group due to the shielding effect of methyl group on the ortho nitro group (Oh and Chiu, 2009). Similar reaction pattern has been observed in other microbial 2,4-DNT transformation (Bradley et al., 1994; Noguera and Freedman, 1996). In contrast, anaerobic transformation of 2,4-DNT by Clostridium acetobutylicum favored formation of dihydroxylaminotoluenes with a limited reduction to ANT (Hughes et al., 1999). The different profiles of DNT reduction might be derived from the different terminal reductases in the microorganisms. 3.3. Experiments about the respiratory electron transport chain To further understand the metabolic mechanism of 2,4-DNT reduction by EP1, some factors capable of affecting the electron transport chain were investigated. Cu2+ ions and dicumarol, two kinds of respiratory inhibitors, are thought to inhibit the membrane-impermeable dehydrogenas (Fernandez et al., 1989) and electron transport of menaquinone (MK) (Arnold et al., 1986) in bacteria, respectively. The respiration of 2,4-DNT by EP1 was almost completely inhibited by 10 lM Cu2+ (Fig 4a) or 50 lM dicumarol (Fig 4b), suggesting that lactate dehydrogenease and MK are essential components of electron transport chain. On the contrary, although 2,4-DNT reduction was greatly repressed by cytochrome P450 inhibitor metyrapone (Williams et al., 2004), it was not completely inhibited with even up to 1000 lM of metyrapone (Fig 4c). Since Shewanella species have a robust cytochrome pool (Fredrickson et al., 2008), this versatility may be exploited by EP1 cells in which the disabled cytochrome P450 can be functionally replaced by other cytochromes. In addition, the effects of various concentration of flavins on 2,4-DNT transformation by EP1 were evaluated. As shown in Fig. 5, flavins demonstrated a stimulating effect on 2,4-DNT transformation. The enhancing effects increased with the increase of flavins concentrations up to 7.5 lM. With addition of 2.5 lM flavins, the transformation efficiency increased 2.5-fold after 6 h incubation, compared with that obtained in the absence of flavins. This result was in agreement with the previous observations that the addition of flavins could also enhance the reduction of carbon electrode (Marsili et al., 2008). Using electron transport chain inhibitors or promoters is proven one of the effective methods to study the respiratory chain components. The current experiments indicated that electron transport chain for anaerobic transformation of 2,4-DNT by EP1 might include the components such as dehydrogenases, menaquinone, cytochromes, and maybe flavins. This profile matched well with the extracellular respiratory pathway exploited by S. oneidensis MR-1 (Brutinel and Gralnick, 2012), the most intensively studied member in Shewanella genus. Based on these similarities, a hypothetical respiratory mode is proposed for EP1. In this mode, the MK pool accepts the electrons produced from the lactate dehydrogenase, and transfer these electrons to a series of cytochromes, termed Mtr pathway through which electrons eventually move to the cell surface. Then the extracellular flavins accept electrons from cell surface and move to the terminal electron acceptors by diffusion. Alternatively, electrons at the cell surface can transfer to electron acceptors by direct contact considering that 2,4-DNT reduction occurred in the absence of flavins in the present experiments. However, there is still the possibility that flavins can be synthesized and secreted by EP1 itself, as flavins have been identified as redox-active mediator secreted by a number of Shewanella
Fig. 4. Effect of respiration inhibitors on 2.4-DNT reduction by S. marisflavi EP1. Reduction of 2,4-DNT were measured in the presence of different concentrations of Cu2+ (a), dicumarol (b), and metyrapone (c). Error bars represent one standard deviation calculated from triplicates.
species (von Canstein et al., 2008). However, a recent study found that intracellular reactions were involved in nitrobenzene reduction in S. oneidensis MR-1 (Cai et al. 2012), which makes this mechanism more complex. The exact mechanism of anaerobic respiration of 2,4-DNT in EP1 remains unclear and needs further investigations. 3.4. Effects of environmental factors on the 2,4-DNT transformation The transformation of 2,4-DNT by S. marisflavi EP1 occurred over a pH range from 6.0 to 10.0 (Fig. 6a). Maximum activity of
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Fig. 5. Effect of flavins on the transformation of 2,4-DNT by S. marisflavi EP1.
EP1 presented at pH 8.0 under the experimental conditions. The absence of 2,4-DNT transformation in the treatment at pH 10.0 indicated that 2,4-DNT reduction at pH 8.0 was a microbial process catalyzed by EP1 rather than a chemically spontaneous process under alkaline conditions. Fig. 6b shows EP1 could transform 2,4-DNT at the low temperature of 4 °C, although only 20% of 2,4-DNT were reduced within 24 h. Increasing temperature
enhanced the activity of 2,4-DNT transformation by EP1 up to 40 °C. However, EP1 cells were heat-inactivated at 45 °C. As shown in Fig. 6c, 2,4-DNT could be reduced by EP1 in the presence of various concentration of NaCl (2–8%). Compared to the absence of 2,4DNT transformation observed under non-saline conditions, adding certain amounts of NaCl was in favor of 2,4-DNT transformation by EP1. At optimum salinity with 2% NaCl, 100 lM 2,4-DNT was almost completely transformed around 12 h. A small amount of 2,4-DNT reduced was detected at the salinity of 8% NaCl. Fig 6d showed the percent of 2,4-DNT reduced by EP1 under various initial concentrations of DNT. After 12 h, initial 50 lM or 100 lM 2,4DNT could be completely transformed, but 200 lM, 300 lM and 400 lM of initial concentration remained 20%, 80%, and 90% unreduced, respectively. By calculation of the average removal rate within 9 h, the optimum removal activity of EP1 occurred at initial concentration of 200 lM 2,4-DNT, increasing 30% compared to that at 100 lM or 300 lM 2,4-DNT (Inset in Fig. 6d). These results were consistent with the typical traits of the shewanellae, such as slight alkaline optimal pH for growth (Venkateswaran et al., 1999; Yoon et al., 2004), the capacity to thrive at low temperature (Hau and Gralnick, 2007), and somewhat halophilic (Yoon et al., 2004). Meanwhile, the salinity tolerance of EP1 was different from that of the most intensively studied S. oneidensis MR-1 (Venkateswaran et al., 1999). The former was not able to thrive with 0% NaCl, similar to S. halifaxensis which required at least 1% NaCl for growth (Zhao et al., 2006). EP1 was able to completely reduce 100 lM 2,4-DNT at 4 °C within 96 h
Fig. 6. Biotransformation of 2,4-DNT by S. marisflavi EP1 under different pH (a), temperature (b), salinity (c), and initial concentrations of 2,4-DNT (d). Inset in (b) shows the complete transformation of 100 lM 2,4-DNT at 4 °C. Inset in (d) shows the average removal rate calculated from transformation data under different initial concentrations of 2,4-DNT within the early 9 h.
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J. Huang et al. / Bioresource Technology 180 (2015) 200–206 Table 1 Comparison of 2,4-DNT transformation with other reports.
a
Catalyst
Electron donor
Electron acceptor
pH
Salinitya
Initial concentration (lM)
Efficiency
Refs.
n-Hexane soot Poultry litter biochar Microbial community Clostridium acetobutylicum Pseudomonas putida OU83 Arthrobacter sp. K1 Pseudomonas aeruginosa S. putrefaciens CN32 S. oneidensis MR-1 S. marisflavi EP1
Dithiothreitol Dithiothreitol Ethanol Saccharides – 2,4-DNT Ethanol Lactate Lactate Lactate
2,4-DNT 2,4-DNT 2,4-DNT 2,4-DNT Oxygen Oxygen 2,4-DNT Nitrobenzene 2,4-DNT Anaerobic
7.4 7.0 7.2 7.0 7.5 7.0 6.5–7.8 6.8 7.0 7.0–9.0
0% 0% – 0% 0% 0.5% 0% 0% 0% 2%
178 100 88 549 400 477 240 149 90 200
100%/7 d 75%/10 d 100%/120 h 100%/1 h 98%/48 h 66%/10 d 100%/13 d 100%/24 h 100%/24 h 100%/24 h
Oh and Chiu (2009) Oh et al. (2013) Cheng et al. (1998) Hughes et al. (1999) Walia et al. (2002) Küce et al. (2014) Noguera and Freedman (1996) Luan et al. (2010) Huang et al. (2013) This study
Salinity: NaCl concentration (wt/vol.%).
(Inset in Fig. 6b). It is actually in accordance with most Shewanella strains that demonstrate the capacity to grow at low temperature, but their temperature optimum for growth is beyond 16 °C (Hau and Gralnick, 2007). 3.5. Comparison with other reported 2,4-DNT transformations Recent years, many remediation approaches were developed to mitigate the DNT contamination (Table 1). In these approaches, catalysts could be biotic or abiotic and reactions could be under anaerobic or aerobic conditions. Comparison of 2,4-DNT transformation by S. marisflavi EP1 with the reactions in other reports indicates that only C. acetobutylicum presented higher reducing activity than EP1. C. acetobutylicum was capable of transforming 100% 2,4DNT (549 lM) in 1 h (Hughes et al., 1999). Such high activity might be attributed to the spiking of 2,4-DNT in reaction system during the log-growth phase of C. acetobutylicum when the cultures gave a very high density of cells. However, in this study transformation of 2,4-DNT by EP1 commenced at the time of inoculation when the reaction system gave comparatively lower cell density. Other three bacterial pure cultures, Pseudomonas putida OU83 (Walia et al., 2002), Arthrobacter sp. K1 (Küce et al., 2014) and Pseudomonas aeruginosa (Noguera and Freedman, 1996) could transform 98% 2,4-DNT (400 lM) in 48 h, 66% 2,4-DNT (477 lM) in 10 days, and 100% 2,4-DNT (240 lM) in 13 days, respectively. Recently, black carbon-mediated reduction of 2,4-DNT has received much attentions, but the reduction catalyzed by n-hexane soot (Oh and Chiu, 2009) and poultry litter biochar (Oh et al., 2013) remained low activities compared with that catalyzed by EP1. Two non-obligate marine Shewanella were also compared in Table 1. S. putrefaciens CN32 could reduce 100% nitrobenzene (149 lM) in 24 h, albeit at a high resting cell density of 1 108 cell ml 1 (Luan et al., 2010). By contrast, the growing cells of EP1 only reached 6.9 107 CFU/ ml after complete reduction of 200 lM 2,4-DNT. While S. oneidensis MR-1 performed the optimum reduction of 2,4-DNT at 0% salinity. 3.6. Environmental significance In nature, redox states of contaminants influence their environmental fate. For example, oxidation states of inorganic elements, such as uranium and chromium, change their solubility and mobility in water and sediments (Fredrickson et al., 2008). Some redoxsensitive organic chemicals, such as NACs, their biodegradability under aerobic conditions largely depends on the oxidation states (Snellix et al., 2002). Reduction of these contaminants to change their redox states via microbial respiration offer the potential to effectively clean up the contaminated environments. The shewanellae, as a group of dissimilatory metal-reducing bacteria, are regarded as appropriate candidates for application in bioremediation of these contaminants because of their outstanding capacity
to respire a broad range of electron acceptors. More than 20 electron acceptors that are found to be respired by Shewanella so far include toxic organic compounds and inorganic elements (Hau and Gralnick, 2007). And the list increases continually. In this study, we demonstrated that 2,4-DNT reduction by S. marisflavi EP1 was a process of respiration. Furthermore, we also illustrated that trinitrotoluene and a dinitroaniline pre-emergence herbicide pendimethalin could be reduced and transformed by S. marisflavi EP1 and S. oneidensis MR-1 (data not shown). This respiration form may expand the range of electron acceptors utilized by the shewanellae to include enormous NACs. It is well known that members of Shewanella genus are wildly distributed in marine and freshwater environments, especially thrived in redox-stratified environments where nitroaromatic compounds are readily and strongly sorbed (Yang et al., 2009). Based on this, our finding is expected to not only deepen our understanding on the environmental fate and ecotoxicity of 2,4-DNT, in addition to other nitroaromatic pollutants, but also suggest a potential site bioremediation and/or wastewater treatment approach. Addition of electron donors preferred by the shewanellae may stimulate the reduction of 2,4-DNT, and facilitate its completely mineralized and detoxified in environments. Moreover, as industrial effluents frequently have an elevated salinity and NOCs-contaminated sites in natural environments undergo a comparatively low temperature such as sea sediments (Yang et al., 2009), a high-performance activity of DNT transformation by the shewanellae at high salinity and low temperature significantly extend their industrial and environmental applications.
4. Conclusions The biotransformation of 2,4-DNT by S. marisflavi EP1 under anaerobic conditions is a process of respiration in which EP1 can utilize lactate as electron donor for dissimilatory reducing 2,4DNT. The terminal product of 2,4-DNT transformation is 2,4-diaminotoluene via temporary formation of two intermediates, 2-amino4-nitrotoluene and 4-amino-2-nitrotoluene. Due to the toxicity of amino-derivatives, however, further studies will be necessarily required to generate ecologically safe remediation products. Furthermore, rapid and effective reduction of 2,4-DNT conducted by EP1 at relatively wide range of pH, temperature and salinity, suggest a potential strategy for the cleanup of nitroaromatic contaminants in natural environments.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21437004, 21307112).
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