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Biodegradation of nonylphenol polyethoxylates under sulfate-reducing conditions
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Biodegradation of nonylphenol polyethoxylates under sulfate-reducing conditions Jian Lua,b , Qiang Jina , Yiliang Hea,⁎, Jun Wua,b , Juan Zhaoa a
School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China School of Resources & Environmental Engineering, Hefei University of Technology, Hefei 230009, PR China
b
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Article history:
Biodegradation behavior of nonylphenol polyethoxylates (NPEOs) under sulfate-reducing
Received 11 July 2007
conditions was investigated. The results showed that NPEOs were readily degraded under
Received in revised form
sulfate-reducing conditions. These compounds were degraded via sequential removal of
7 December 2007
ethoxyl units to nonylphenol (NP) without forming carboxylated intermediates under sulfate-
Accepted 1 January 2008
reducing conditions. The biodegradation of NPEOs under sulfate-reducing conditions was not inhibited even at very high initial concentrations of NPEOs. The maximum removal rate
Keywords:
increased about 1.3 μM d− 1 for each 10 μmol increase in initial concentration. The decrease in
Biodegradation
temperature caused a sharp decrease in the removal efficiency of NPEOs. The temperature
Nonylphenol polyethoxylates
coefficient (Ф) for the biodegradation of NPEOs under sulfate-reducing conditions was 0.008.
Environmental behavior
Severe accumulation of NP and short-chain NPEOs occurred when most NPEOs were removed
Sulfate
and this accumulation led to an increase in the estrogenic activity. The highest estrogenic
Estrogenic intermediate
activity appeared on day 21 when the total concentration of these metabolites reached its top
Terminal electron acceptor
(18.03 ± 4.73 μM). NP could inhibit the biodegradation of NPEOs under sulfate-reducing conditions only at relatively high concentration. These findings are of major environmental importance in terms of the environmental behavior of NPEO contaminants in natural environment. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Nonylphenol polyethoxylates (NPEOs) are by far the most commonly used nonionic surfactants, encompassing more than 80% of the world market (Warhurst, 1995). These compounds are discharged into environment and primarily degraded to more persistent breakdown products such as nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), and nonylphenol diethoxylate (NP2EO) (Ahel et al., 1994). Studies showed that these breakdown products had estrogenic activities (Jobling and Sumpter, 1993; Renner, 1997; Lin and Janz, 2006; Hashimoto et al., 2007). Since the wide occurrence of these harmful metabolites has been reported (Ying et al., 2002), the biodegradation behavior of NPEOs in environment has raised public concern. Biodegradation of NPEOs under aerobic conditions is well documented. However, reports on the biodegradation of NPEOs
under anaerobic conditions are quite rare (Thiele et al., 1997). Many studies showed that the anaerobic breakdown products of NPEOs (NP and short-chain NPEOs) could persist in diverse anaerobic environments such as aquatic sediments, digestor sludge and landfill sludge (Shang et al., 1999; Ejlertsson et al., 1999). Therefore, studies on the anaerobic biodegradation behavior of NPEOs under different anaerobic conditions are considered important. Microbial sulfate reduction is important in both carbon and electron flow in many natural environments. Bacteria can oxide a number of organic contaminants under sulfatereducing conditions. Studies showed that several compounds including benzene, naphthalene, toluene, and alkanes could be degraded under sulfate-reducing conditions (Coates et al., 1996; Kazumi et al., 1997; Heider et al., 1999). In this study, a sulfate-reducing culture enriched from the anaerobic digestor
⁎ Corresponding author. Tel.: +86 21 5474 5634; fax: +86 21 5474 0825. E-mail addresses: [email protected] (J. Lu), [email protected] (Y. He). 0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.01.003
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sludge of a typical sewage treatment plant (STP) was used to investigate the biodegradation of NPEOs under sulfate-reducing conditions. The objective of this study was to obtain initial information on the biodegradation behavior of NPEOs under sulfate-reducing conditions.
2.
Materials and methods
2.1.
Chemicals and reagents
A nonionic surfactant (Igepal CO-630), which has an average of nine ethoxyl units, was obtained from Sigma-Aldrich (St. Louis, MO, USA). According to the manufacturer's information, the number of average molecular weight (Mn) is 617. Analytical standards of NP (technical grade), NP1EO, NP2EO, nonylphenoxyacetic acid (NP1EC), and nonylphenoxyethoxyacetic acid (NP2EC) were obtained from Dr. Ehrenstorfer (Augsburg, Germany). The chemical structures of NPEOs and their typical breakdown products can be found in Renner (1997) and Di Corcia et al. (1998). High-performance liquid chromatography (HPLC) grade methanol, ethyl acetate, and gas chromatography (GC) resolve grade methylene chloride were purchased from Tedia (Fairfield, OH, USA). Acetyl chloride (purity of ≥ 98.5%) was obtained from Alfa-Aesar (Ward Hill, MA, USA). Chrysene-d12, the internal standard, was purchased from Supelco (Bellefonte, PA, USA). High-purity Milli-Q water was produced in a Milli-Q Plus system (Millipore, USA). Yeast extract was obtained from Oxoid (Hampshire, England). All other reagents used were of reagent grade.
2.2.
Microorganisms and media
A sulfate-reducing culture was enriched from the anaerobic digestor sludge of Minhang STP, Shanghai, China. Strict anaerobic microbial techniques were used throughout the experiment manipulations as previously described (Lu et al., 2007). Each liter of medium contained 2.5 g NaHCO3, 1.5 g NH4Cl, 0.1 g MgCl2 · 6H2O, 0.1 g CaCl2, 0.6 g KH2PO4, and 1.0 g yeast extract. The medium contained sulfate at a final concentration of 30 mM. Relatively high concentration of the sulfate was used to ensure the development of sulfate-reducing conditions. The medium was boiled for 20 min, and allowed to cool under a stream of oxygen-free nitrogen. After cooling, carbonate (30 mM), resazurin (0.001 g L− 1), FeCl2 · 4H2O (0.015 g L− 1), and Na2S · 9H2O (0.35 g L− 1) were added. Differences in the media pH might lead to different NPEO removal efficiencies. Therefore, media pH was adjusted to 7 using 1 M HCl to avoid disturbance of media pH on the NPEO biodegradation. Serum bottles were capped with rubber stoppers and the head space of the bottles was N2:CO2 (70:30, v/v). Enrichment was carried out in a 150 mL serum bottle containing 100 mL sediment-medium mixture (1: 2, v/v) and performed at 30 °C and 120 rpm in darkness for one month. The commercial surfactant (Igepal CO-630) was added to the slurry to a final concentration of 100 mg L− 1 (162.1 μM). To avoid the disturbance of the remaining NPEO contaminants in the enrichment medium, preincubation was carried out in a 150 mL serum bottle containing 50 mL active culture and 50 mL fresh medium for a week without adding NPEOs.
2.3.
Biodegradation assays
Biodegradation tests were carried out in 150 mL serum bottles. 90 mL fresh medium and 10 mL culture inoculum was added to each bottle (MLVSS 1.0 g L− 1). The incubations were performed at 120 rpm in darkness. Sterile controls were autoclaved three times on consecutive days before the biodegradation tests were initiated. At each sampling point the cultures were rigorously shaken and sampled with sterile syringes flushed with N2:CO2 (70:30, v/v). For the study on the stiochiometry of the primary biodegradation of NPEOs coupled to sulfate reduction, yeast extract was omitted from the medium. The head space of each bottle was oxygen-free nitrogen. Background controls were performed without NPEOs.
2.4.
Analytical methods
Samples were prepared as previously described (Lu et al., 2007) before being analyzed. Briefly, culture medium was withdrawn and acidified to pH 2 before extraction. The acidified liquid medium was then extracted by ethyl acetate and methylene chloride. The extracts were mixed together, dried by adding anhydrous sodium sulfate, and subjected to rotary evaporation. The dried sample was redissolved in 3 mL methanol. For NP, NP1EO and NP2EO, 1 mL methanol solution was blown to dryness by a N2 gas stream and then redissolved in 10 μL methylene chloride before gas chromatography-mass spectrometry (GC-MS) analysis. In case of short-chain nonylphenol polyethoxycarboxylates (NPECs) and carboxyalkylphenol polyethoxycarboxylates (CAPECs), 1 mL methanol solution was blown to dryness and then derivatization was performed. The propylated ester products were redissolved in 10 μL methylene chloride before injection. For the concentration of remaining total NPEOs, 0.5 mL methanol solution was blown to dryness, redissolved in 100 μL mixture of methanol/ water (50:50, v/v), and ready for HPLC analysis. The remaining solution was blown to dryness and then redissolved in a mixture of methanol/water (80:20, v/v) containing 1 mM sodium acetate for liquid chromatography-mass spectrometry (LC-MS) analysis. HPLC analysis, GC-MS analysis, and LC-MS analysis were performed as previously described (Lu et al., 2007) to monitor the biodegradation behavior of NPEOs under sulfate-reducing conditions. Briefly, a simple HPLC analysis was performed on a Shimadzu LC-2010AHT HPLC instrument to monitor the changes in the total concentration (in mole) of NPEOs. The injection volume was 10 μL. The chromatographic separation was carried out isocratically in the reversed-phase mode with a Cosmosil trimethylsilyl (TMS) C1 column (250 mm × 4.6 mm i.d. particle packing size 5 μm, Nacalai Tesque, Japan). The mobile phase used was a mixture of methanol/water (80:20, v/v) and the flow-rate was maintained at 0.8 ml/min. A GC-MS instrument (GC-MS-QP2010; Shimadzu) with a DB-5 fused-silica capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness) was used to determine low molecular biodegradation products (NP, short-chain NPEOs, short-chain NPECs and CAPECs, and unknown low molecular intermediates) for reliable identification. Helium was the carrier gas and injection volume was 1 μL. The solvent delay was set to 3 min. The identification of NP,
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NP1EO, and NP2EO was confirmed by comparing the GC retention times and mass spectra acquired from samples against spectra acquired from authentic standards. The identification of NP1EC and NP2EC was confirmed by matching the GC retention times and mass spectra of their propylated standards. The identification of the short-chain CAPECs was performed according to the characteristic ions given by Montgomery-Brown et al. (2003). The identification of other metabolites was performed by instrumental library searches applying the NIST (National Institute of Standards and Technology) mass spectral database. The quantification of typical intermediates was performed directly after intermediates identification. Chromatograms were registered using selective ion monitoring (SIM) of the main characteristic fragment ions of target compounds to greatly enhance the sensitivity. Ion (m/z) sets for chysene-d12 (internal standard), NP, NP1EO, and NP2EO were 240 + 120 + 236, 135 + 149, 179 + 193, and 223 + 237 respectively. The response factors for NP, NP1EO, and NP2EO were 0.163, 0.154, 0.605 (relative to the internal standard), respectively. Mass-spectrometric analyses were performed with a Hewlett Packard Series 1100 LC-MS with electrospray ionization and a quadrupole mass analyzer to monitor the biodegradation behavior of individual oligomers and the formation of long-chain NPECs. The analysis was operated in flow injection analysis (FIA) mode with an injection volume of 10 μL and the mobile phase was a mixture of methanol/water (80:20, v/v) containing 1 mM sodium acetate. Full scan positive ionization mode was performed to identify these intermediates. NPEOs with the ethoxyl chain number from 1 to 17 were identified by confirming the characteristic pattern showing the [M + Na]+ ions in the positive mode with m/z values from 287 ([NP1EO + Na+] ion) to 991 ([NP17EO + Na+] ion), increasing by 44. Long-chain NPECs were identified by confirming the characteristic pattern showing the [M + Na]+ ions in the positive mode with m/z values from 389 ([NP3EC + Na+] ion) to 1005 ([NP17EC + Na+] ion), increasing by 44. Electrospray mass was also used to monitor changes in the molecular weight distributions of NPEO samples because standards of long-chain NPEOs were not available. Changes in the relative abundances of each sodium-cationized NPEO molecules ([M + Na+] ions) were monitored during biodegradation period to investigate changes in the concentration distribution of oligomers of NPEOs. Mass calibration was performed using [NP10EO + Na+] ion at the beginning of the experiment whose relative abundance was registered as 100%. Sulfate was measured by a MIC ion chromatograph (Metohm, Switzerland).
2.5.
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relative estrogenic potencies of NP, NP1EO, and NP2EO were 6.25E-05, 6.14E-06, and 1.69E-06, respectively.
3.
Results and discussion
3.1. Biodegradation of NPEOs under sulfate-reducing conditions The biodegradation of NPEOs under sulfate-reducing conditions is shown in Fig. 1. NPEOs were rapidly removed under sulfate-reducing conditions. The maximum biodegradation rate was 34.85 ± 0.94 μM d− 1 and more than 80% of NPEOs were removed within seven days. The biodegradation rate sharply decreased when most NPEOs were removed. To determine if the biodegradation of NPEOs was associated with sulfate reduction, biodegradation test with molybdate (30 mM), a known inhibitor of sulfate reduction, was performed. The addition of molybdate resulted in immediate inhibition of NPEOs biodegradation, suggesting that sulfate reduction was necessary for NPEOs biodegradation. In order to further determine whether NPEOs biodegradation was coupled to sulfate reduction, the study on stoichiometry of NPEOs biodegradation was performed following the method described by Lu et al. (2007). Predicted values based on stoichiometric equation were compared to those measured in NPEO-degrading enrichment. The study on stoichiometry of NPEOs biodegradation under sulfate-reducing conditions was just performed for three days to avoid the disturbance of the further biodegradation of NP, which was formed during the biodegradation period of NPEOs. In the stoichiometric equation, it is assumed that the ethoxyl chain of NPnEO (n means the number of the repeating ether unit) is completely mineralized to CO2 as follows: − NPn EO þ 0:5Hþ þ 1:25SO2− 4 ¼ NPn − 1 EO þ 2:0HCO3 þ 1:25H2 S þ 1:5H2 O
ð1Þ
Based on Eq. (1), 1.25 mol SO2− 4 will be reduced when 1 mol ethoxyl chains are removed. The measured amount of SO2− 4 was 78 ± 8% of the theoretically expected amount, suggesting
Data calculation
The maximum biodegradation rate of NPEOs was determined from the time course of NPEOs disappearance, using points in the linear portion of graphs that released substrate concentration to time. Estradiol equivalents were calculated (calEEQ) by multiplying the molar concentration of each estrogenic intermediate (NP, NP1EO, or NP2EO) with its relative potency and adding up the values for these typical estrogenic intermediates following the method described by Vermeirssen et al. (2005) and Lu et al. (2007). According to a recent study with high sensitive analysis method (Kuruto-Niwa et al., 2005), the
Fig. 1 – Biodegradation of NPEOs under sulfate-reducing conditions. The incubation was performed at 25 °C. Initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). Error bars represent ±one standard deviation for three replicates.
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to the NP. Similar biodegradation pathway has been observed under Fe(III)-reducing conditions (Lu et al., 2007) and other anaerobic conditions (Renner, 1997; Ejlertsson et al., 1999). Previous studies showed that carboxylated intermediates were the main breakdown products under aerobic conditions (Ahel et al., 1994; Montgomery-Brown et al., 2003) while NP and short-chain NPEOs were the main breakdown products under anaerobic conditions (Ahel et al., 1994). It seems that the biodegradation pathway of NPEOs can be changed in the presence of different terminal electron acceptors. However, similar biodegradation pathway has been observed under diverse reducing conditions. Further study is needed to elucidate the influence of diverse terminal electron acceptors on the biodegradation behavior of NPEOs.
3.3. Effect of the initial concentration on the biodegradation of NPEOs under sulfate-reducing conditions
Fig. 2 – Changes in ion peak distribution of the NPEOs samples during the biodegradation period. The initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). The incubation was performed at 25 °C. The results shown are representative of triplicate incubations. that the primary biodegradation of NPEOs under sulfatereducing conditions was coupled to sulfate reduction.
3.2. Typical biodegradation intermediates and biodegradation pathway of NPEOs under sulfate-reducing conditions The low molecular breakdown products were analyzed by GC-MS for reliable identification. NP and short-chain NPEOs (NP1EO and NP2EO) were identified as biodegradation intermediates while low molecular carboxylated metabolites such as short-chain NPECs were not detected. Moreover, the typical electrospray mass spectra of NPEO samples collected at different incubation times showed that only oligomers of NPEOs were detected while long-chain NPECs were not formed during the whole biodegradation period. Fig. 2 summarized the histogram of the ion peak distribution of each biodegraded sample observed by electrospray mass. As the figure showed, stepwise ethoxyl chain shortening process occurred. The ion peak distributions gradually shifted to lower numbers of polymerization with increase of the incubation time. The rapid decrease in the relative peak abundance of long-chain NPEOs suggested that long-chain NPEOs were rapidly removed. Two different terminal oxidative biodegradation models for the ethoxyl chain scission of alkylphenol polyethoxylates (APEOs) have been reported by Sato et al. (2001) and Jonkers et al. (2001), respectively. Alkylphenol polyethoxycarboxylates (APECs) are the typical biodegradation intermediates in these oxidative models. Since NPECs were not detected, the terminal oxidative models may be not the case for the biodegradation of NPEOs under sulfate-reducing conditions. Stepwise ethoxyl chain shortening process from long-chain NPEOs to shorter chain polyethoxylates without forming related NPECs was observed in our experiment, suggesting that NPEOs were biodegraded through a non-oxidative pathway, through which NPEOs were degraded via sequential removal of ethoxyl units
Substrate concentration often has great influence on the reaction rate. The relationship between substrate concentration and specific removal rate is usually studied in bioremediation design. The specific NPEO-removing rate could not be measured in our experiment because of the consecutive change in the biomass with the elapse of incubation time. Therefore, the maximum removal rate was used to replace the specific removal rate in the study on the relationship between initial concentration of NPEOs and biodegradation rate. Maximum removal rate as a function of initial concentration was plotted in Fig. 3. Clearly, there is a linear relationship between initial concentration and the maximum removal rate (r2 = 0.988, P b 0.05). Increase in the initial concentration resulted in increased removal rate. The maximum removal rate increased about 1.3 μM d− 1 for each 10 μmol increase in initial concentration. High initial concentration might have possible surfactant toxicity which would subsequently inhibit the biodegradation of NPEOs. In general, the rate increases in direct proportion to the substrate concentration only at very low substrate concentrations. However, this linear relationship maintained
Fig. 3 – Maximum removal rate as a function of the initial concentration of Igepal CO-630 (r2 =0.988). Initial concentrations of Igepal CO-630 were 10 mg L− 1 (16.21 μM), 50 mg L− 1 (81.05 μM), 100 mg L− 1 (162.1 μM), 300 mg L− 1 (486.3 μM), and 500 mg L− 1 (810.5 μM). The incubation was performed at 25 °C. The results shown are representative of triplicate incubations.
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even at relatively high initial concentration, suggesting that the toxic effect had not occurred at very high initial concentration (500 mg L− 1). Since concentrations of NPEOs contaminants in natural environments are usually very low (Thiele et al., 1997), the toxic effect on the biodegradation of NPEOs under sulfate-reducing conditions can be negligible in most natural environments.
3.4. Effect of temperature on the biodegradation of NPEOs under sulfate-reducing conditions To investigate the effect of temperature within the usual mesophilic operational range on the biodegradation of NPEOs, incubations were performed at 15 °C, 25 °C, and 35 °C. Fig. 4 shows that temperature has great influence on the biodegradation of NPEOs under sulfate-reducing conditions. The decrease in temperature caused a delay in the start of the rapid removal of NPEOs. A significant lag phase was observed in the treatment at 15 °C while no lag phase was observed in the treatment at 35 °C. The removal efficiency of the total NPEOs also depended on the temperature. The decrease in temperature led to a rapid decline in the removal efficiency of NPEOs. In anaerobic treatment, the slow growth rate of microorganisms most critical to the biodegradation process makes temperature all the more important for bioremediation design. Based on Arrhenius equation, a simple equation (Rittmann and McCarty, 2001) which was widely used was developed to describe the relationship between reaction rate constant and temperature: k2 ¼ k1 eUðT2 T1 Þ :
ð2Þ
Where Ф is the temperature coefficient, k1 and k2 are the biodegradation rate constants at temperature T1 and T2, respectively. Clearly, kinetic parameters at different temperature are needed to describe the relationship between the anaerobic biodegradation and temperature in detail. However, it seems that study on the kinetics of the biodegradation of NPEOs may be difficult because commercial NPEOs are complex mixtures of isomers and oligomers. Moreover, the biodegradation kinetics analysis of total NPEOs was disturbed by the accumulation of persistent short-chain NPEOs in our experiment because these
Fig. 4 – Biodegradation of NPEOs at different temperatures. Initial concentration of Igepal CO-630 was 10 mg L− 1 (16.21 μM). The results shown are representative of triplicate incubations.
Fig. 5 – Changes in the concentration of estrogenic metabolites (A) and calculated estrogenic activity (B) during the biodegradation period. Initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). The incubation was performed at 25 °C. The results shown are representative of triplicate incubations. compounds are more persistent than their parent compounds. To avoid this disturbance caused by the accumulation of shortchain NPEOs, the biodegradation data collected in later phase when severe accumulation of short-chain NPEOs occurred were not used for kinetics analysis. As Fig. 4 shows, biodegradation data collected in early phase fit well with first-order kinetics (r2 =0.947–0.969, Pb 0.05). Therefore, the biodegradation constants (k) of total NPEOs at different temperatures were used to calculate the temperature coefficient (Ф) according to Eq. (2). The result showed that the temperature coefficient (Ф) for the biodegradation of NPEOs under sulfate-reducing conditions was 0.008.
3.5. Evolution of the typical estrogenic intermediates during biodegradation period NP, NP1EO, and NP2EO are weakly estrogenic (Jobling and Sumpter, 1993; Renner, 1997). Therefore, their evolution during the biodegradation period was monitored. Changes in the concentration of these estrogenic compounds during the
Fig. 6 – Removal of the total NPEOs in the presence of NP. Initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). The incubation was performed at 25 °C. Error bars represent ± one standard deviation for three replicates.
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biodegradation period were shown in Fig. 5A. The accumulation of NP, NP1EO, and NP2EO coincided with the rapid removal of total NPEOs. The concentration of NP2EO reached its top on day 7 which was much earlier than those of NP1EO and NP. The concentrations of NP1EO and NP reached their tops on day 21 when most NPEOs were removed. Since these compounds are estrogenic, their accumulation led to a significant increase in the estrogenic activity during the biodegradation period. The highest estrogenic activity appeared on day 21when the total concentration of these metabolites reached its top (18.03 ± 4.73 μM) (Fig. 5B). Similar phenomenon was observed in the biodegradation of NPEOs under Fe(III)-reducing conditions (Lu et al., 2007). However, it must be pointed out that the accumulation of these estrogenic intermediates was greatly alleviated under Fe (III)-reducing conditions. According to their molecular structures, these compounds are highly reduced organic molecules. These compounds have no highly electron-seeking groups such as carbonyls. In general, such compounds can be thermodynamically readily oxidized in the presence of terminal electron acceptor with relatively high redox potential (Heider et al., 1999). Therefore, the accumulation of these compounds was alleviated in the presence of terminal electron acceptor with relatively high redox potential.
3.6. Effect of typical biodegradation intermediate on the biodegradation of NPEOs under sulfate-reducing conditions NP, the most common toxic and estrogenic intermediates of NPEOs, was added into the culture medium to investigate the effect of typical biodegradation intermediate on the biodegradation of NPEOs under sulfate-reducing conditions (Fig. 6). The result showed that NP could inhibit the biodegradation of NPEOs under sulfate-reducing conditions. The maximum removal rate was sharply decreased from 34.90 ± 1.13 μM d− 1 to 16.18 ± 0.40 μM d− 1 when NP was added at high concentration (1000 μM). However, this inhibition effect became obvious only when NP was added at relatively high concentration. Since NP in natural environment usually maintains a low level (Thiele et al., 1997; Ying et al., 2002), this inhibit effect on the biodegradation of NPEOs under sulfate-reducing conditions can be negligible in most natural environments.
4.
Conclusions
Results reported in this paper demonstrated that NPEOs were readily biodegraded under sulfate-reducing conditions. NPEOs were degraded via sequential removal of ethoxyl units to NP without forming carboxylated intermediates during the biodegradation period. This ethoxyl chain removal process was coupled to sulfate reduction. Both temperature and the initial concentration played important roles in the removal efficiency of NPEOs. The decrease in temperature caused a sharp decrease in the removal efficiency of NPEOs. Increase in the initial concentration resulted in enhanced removal rate. The accumulation of NP and short-chain NPEOs led to a significant increase in the estrogenic activity during the biodegradation period. NP, the typical intermediate of NPEOs, could inhibit the anaerobic biodegradation of NPEOs only at high concentra-
tion. Since microbial sulfate reduction is an important process in environment, these findings have significant environmental implications in terms of the removal of NPEOs contaminants in environment.
Acknowledgments This work is financially supported by the National Key Basic Research Program (Grant No. 2007CB815603) and National Foundation of Science of China (Grant No. 50478019). The authors would like to thank Ping Tao (Instrumental Analysis Center of Shanghai Jiao Tong University) for making LC-MS facilities available to us. The authors also wish to thank the anonymous reviewers for their reading of the manuscript, and for their suggestions and critical comments.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v
Biodegradation of nonylphenol polyethoxylates under sulfate-reducing conditions Jian Lua,b , Qiang Jina , Yiliang Hea,⁎, Jun Wua,b , Juan Zhaoa a
School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China School of Resources & Environmental Engineering, Hefei University of Technology, Hefei 230009, PR China
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Article history:
Biodegradation behavior of nonylphenol polyethoxylates (NPEOs) under sulfate-reducing
Received 11 July 2007
conditions was investigated. The results showed that NPEOs were readily degraded under
Received in revised form
sulfate-reducing conditions. These compounds were degraded via sequential removal of
7 December 2007
ethoxyl units to nonylphenol (NP) without forming carboxylated intermediates under sulfate-
Accepted 1 January 2008
reducing conditions. The biodegradation of NPEOs under sulfate-reducing conditions was not inhibited even at very high initial concentrations of NPEOs. The maximum removal rate
Keywords:
increased about 1.3 μM d− 1 for each 10 μmol increase in initial concentration. The decrease in
Biodegradation
temperature caused a sharp decrease in the removal efficiency of NPEOs. The temperature
Nonylphenol polyethoxylates
coefficient (Ф) for the biodegradation of NPEOs under sulfate-reducing conditions was 0.008.
Environmental behavior
Severe accumulation of NP and short-chain NPEOs occurred when most NPEOs were removed
Sulfate
and this accumulation led to an increase in the estrogenic activity. The highest estrogenic
Estrogenic intermediate
activity appeared on day 21 when the total concentration of these metabolites reached its top
Terminal electron acceptor
(18.03 ± 4.73 μM). NP could inhibit the biodegradation of NPEOs under sulfate-reducing conditions only at relatively high concentration. These findings are of major environmental importance in terms of the environmental behavior of NPEO contaminants in natural environment. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Nonylphenol polyethoxylates (NPEOs) are by far the most commonly used nonionic surfactants, encompassing more than 80% of the world market (Warhurst, 1995). These compounds are discharged into environment and primarily degraded to more persistent breakdown products such as nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), and nonylphenol diethoxylate (NP2EO) (Ahel et al., 1994). Studies showed that these breakdown products had estrogenic activities (Jobling and Sumpter, 1993; Renner, 1997; Lin and Janz, 2006; Hashimoto et al., 2007). Since the wide occurrence of these harmful metabolites has been reported (Ying et al., 2002), the biodegradation behavior of NPEOs in environment has raised public concern. Biodegradation of NPEOs under aerobic conditions is well documented. However, reports on the biodegradation of NPEOs
under anaerobic conditions are quite rare (Thiele et al., 1997). Many studies showed that the anaerobic breakdown products of NPEOs (NP and short-chain NPEOs) could persist in diverse anaerobic environments such as aquatic sediments, digestor sludge and landfill sludge (Shang et al., 1999; Ejlertsson et al., 1999). Therefore, studies on the anaerobic biodegradation behavior of NPEOs under different anaerobic conditions are considered important. Microbial sulfate reduction is important in both carbon and electron flow in many natural environments. Bacteria can oxide a number of organic contaminants under sulfatereducing conditions. Studies showed that several compounds including benzene, naphthalene, toluene, and alkanes could be degraded under sulfate-reducing conditions (Coates et al., 1996; Kazumi et al., 1997; Heider et al., 1999). In this study, a sulfate-reducing culture enriched from the anaerobic digestor
⁎ Corresponding author. Tel.: +86 21 5474 5634; fax: +86 21 5474 0825. E-mail addresses: [email protected] (J. Lu), [email protected] (Y. He). 0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.01.003
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sludge of a typical sewage treatment plant (STP) was used to investigate the biodegradation of NPEOs under sulfate-reducing conditions. The objective of this study was to obtain initial information on the biodegradation behavior of NPEOs under sulfate-reducing conditions.
2.
Materials and methods
2.1.
Chemicals and reagents
A nonionic surfactant (Igepal CO-630), which has an average of nine ethoxyl units, was obtained from Sigma-Aldrich (St. Louis, MO, USA). According to the manufacturer's information, the number of average molecular weight (Mn) is 617. Analytical standards of NP (technical grade), NP1EO, NP2EO, nonylphenoxyacetic acid (NP1EC), and nonylphenoxyethoxyacetic acid (NP2EC) were obtained from Dr. Ehrenstorfer (Augsburg, Germany). The chemical structures of NPEOs and their typical breakdown products can be found in Renner (1997) and Di Corcia et al. (1998). High-performance liquid chromatography (HPLC) grade methanol, ethyl acetate, and gas chromatography (GC) resolve grade methylene chloride were purchased from Tedia (Fairfield, OH, USA). Acetyl chloride (purity of ≥ 98.5%) was obtained from Alfa-Aesar (Ward Hill, MA, USA). Chrysene-d12, the internal standard, was purchased from Supelco (Bellefonte, PA, USA). High-purity Milli-Q water was produced in a Milli-Q Plus system (Millipore, USA). Yeast extract was obtained from Oxoid (Hampshire, England). All other reagents used were of reagent grade.
2.2.
Microorganisms and media
A sulfate-reducing culture was enriched from the anaerobic digestor sludge of Minhang STP, Shanghai, China. Strict anaerobic microbial techniques were used throughout the experiment manipulations as previously described (Lu et al., 2007). Each liter of medium contained 2.5 g NaHCO3, 1.5 g NH4Cl, 0.1 g MgCl2 · 6H2O, 0.1 g CaCl2, 0.6 g KH2PO4, and 1.0 g yeast extract. The medium contained sulfate at a final concentration of 30 mM. Relatively high concentration of the sulfate was used to ensure the development of sulfate-reducing conditions. The medium was boiled for 20 min, and allowed to cool under a stream of oxygen-free nitrogen. After cooling, carbonate (30 mM), resazurin (0.001 g L− 1), FeCl2 · 4H2O (0.015 g L− 1), and Na2S · 9H2O (0.35 g L− 1) were added. Differences in the media pH might lead to different NPEO removal efficiencies. Therefore, media pH was adjusted to 7 using 1 M HCl to avoid disturbance of media pH on the NPEO biodegradation. Serum bottles were capped with rubber stoppers and the head space of the bottles was N2:CO2 (70:30, v/v). Enrichment was carried out in a 150 mL serum bottle containing 100 mL sediment-medium mixture (1: 2, v/v) and performed at 30 °C and 120 rpm in darkness for one month. The commercial surfactant (Igepal CO-630) was added to the slurry to a final concentration of 100 mg L− 1 (162.1 μM). To avoid the disturbance of the remaining NPEO contaminants in the enrichment medium, preincubation was carried out in a 150 mL serum bottle containing 50 mL active culture and 50 mL fresh medium for a week without adding NPEOs.
2.3.
Biodegradation assays
Biodegradation tests were carried out in 150 mL serum bottles. 90 mL fresh medium and 10 mL culture inoculum was added to each bottle (MLVSS 1.0 g L− 1). The incubations were performed at 120 rpm in darkness. Sterile controls were autoclaved three times on consecutive days before the biodegradation tests were initiated. At each sampling point the cultures were rigorously shaken and sampled with sterile syringes flushed with N2:CO2 (70:30, v/v). For the study on the stiochiometry of the primary biodegradation of NPEOs coupled to sulfate reduction, yeast extract was omitted from the medium. The head space of each bottle was oxygen-free nitrogen. Background controls were performed without NPEOs.
2.4.
Analytical methods
Samples were prepared as previously described (Lu et al., 2007) before being analyzed. Briefly, culture medium was withdrawn and acidified to pH 2 before extraction. The acidified liquid medium was then extracted by ethyl acetate and methylene chloride. The extracts were mixed together, dried by adding anhydrous sodium sulfate, and subjected to rotary evaporation. The dried sample was redissolved in 3 mL methanol. For NP, NP1EO and NP2EO, 1 mL methanol solution was blown to dryness by a N2 gas stream and then redissolved in 10 μL methylene chloride before gas chromatography-mass spectrometry (GC-MS) analysis. In case of short-chain nonylphenol polyethoxycarboxylates (NPECs) and carboxyalkylphenol polyethoxycarboxylates (CAPECs), 1 mL methanol solution was blown to dryness and then derivatization was performed. The propylated ester products were redissolved in 10 μL methylene chloride before injection. For the concentration of remaining total NPEOs, 0.5 mL methanol solution was blown to dryness, redissolved in 100 μL mixture of methanol/ water (50:50, v/v), and ready for HPLC analysis. The remaining solution was blown to dryness and then redissolved in a mixture of methanol/water (80:20, v/v) containing 1 mM sodium acetate for liquid chromatography-mass spectrometry (LC-MS) analysis. HPLC analysis, GC-MS analysis, and LC-MS analysis were performed as previously described (Lu et al., 2007) to monitor the biodegradation behavior of NPEOs under sulfate-reducing conditions. Briefly, a simple HPLC analysis was performed on a Shimadzu LC-2010AHT HPLC instrument to monitor the changes in the total concentration (in mole) of NPEOs. The injection volume was 10 μL. The chromatographic separation was carried out isocratically in the reversed-phase mode with a Cosmosil trimethylsilyl (TMS) C1 column (250 mm × 4.6 mm i.d. particle packing size 5 μm, Nacalai Tesque, Japan). The mobile phase used was a mixture of methanol/water (80:20, v/v) and the flow-rate was maintained at 0.8 ml/min. A GC-MS instrument (GC-MS-QP2010; Shimadzu) with a DB-5 fused-silica capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness) was used to determine low molecular biodegradation products (NP, short-chain NPEOs, short-chain NPECs and CAPECs, and unknown low molecular intermediates) for reliable identification. Helium was the carrier gas and injection volume was 1 μL. The solvent delay was set to 3 min. The identification of NP,
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NP1EO, and NP2EO was confirmed by comparing the GC retention times and mass spectra acquired from samples against spectra acquired from authentic standards. The identification of NP1EC and NP2EC was confirmed by matching the GC retention times and mass spectra of their propylated standards. The identification of the short-chain CAPECs was performed according to the characteristic ions given by Montgomery-Brown et al. (2003). The identification of other metabolites was performed by instrumental library searches applying the NIST (National Institute of Standards and Technology) mass spectral database. The quantification of typical intermediates was performed directly after intermediates identification. Chromatograms were registered using selective ion monitoring (SIM) of the main characteristic fragment ions of target compounds to greatly enhance the sensitivity. Ion (m/z) sets for chysene-d12 (internal standard), NP, NP1EO, and NP2EO were 240 + 120 + 236, 135 + 149, 179 + 193, and 223 + 237 respectively. The response factors for NP, NP1EO, and NP2EO were 0.163, 0.154, 0.605 (relative to the internal standard), respectively. Mass-spectrometric analyses were performed with a Hewlett Packard Series 1100 LC-MS with electrospray ionization and a quadrupole mass analyzer to monitor the biodegradation behavior of individual oligomers and the formation of long-chain NPECs. The analysis was operated in flow injection analysis (FIA) mode with an injection volume of 10 μL and the mobile phase was a mixture of methanol/water (80:20, v/v) containing 1 mM sodium acetate. Full scan positive ionization mode was performed to identify these intermediates. NPEOs with the ethoxyl chain number from 1 to 17 were identified by confirming the characteristic pattern showing the [M + Na]+ ions in the positive mode with m/z values from 287 ([NP1EO + Na+] ion) to 991 ([NP17EO + Na+] ion), increasing by 44. Long-chain NPECs were identified by confirming the characteristic pattern showing the [M + Na]+ ions in the positive mode with m/z values from 389 ([NP3EC + Na+] ion) to 1005 ([NP17EC + Na+] ion), increasing by 44. Electrospray mass was also used to monitor changes in the molecular weight distributions of NPEO samples because standards of long-chain NPEOs were not available. Changes in the relative abundances of each sodium-cationized NPEO molecules ([M + Na+] ions) were monitored during biodegradation period to investigate changes in the concentration distribution of oligomers of NPEOs. Mass calibration was performed using [NP10EO + Na+] ion at the beginning of the experiment whose relative abundance was registered as 100%. Sulfate was measured by a MIC ion chromatograph (Metohm, Switzerland).
2.5.
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relative estrogenic potencies of NP, NP1EO, and NP2EO were 6.25E-05, 6.14E-06, and 1.69E-06, respectively.
3.
Results and discussion
3.1. Biodegradation of NPEOs under sulfate-reducing conditions The biodegradation of NPEOs under sulfate-reducing conditions is shown in Fig. 1. NPEOs were rapidly removed under sulfate-reducing conditions. The maximum biodegradation rate was 34.85 ± 0.94 μM d− 1 and more than 80% of NPEOs were removed within seven days. The biodegradation rate sharply decreased when most NPEOs were removed. To determine if the biodegradation of NPEOs was associated with sulfate reduction, biodegradation test with molybdate (30 mM), a known inhibitor of sulfate reduction, was performed. The addition of molybdate resulted in immediate inhibition of NPEOs biodegradation, suggesting that sulfate reduction was necessary for NPEOs biodegradation. In order to further determine whether NPEOs biodegradation was coupled to sulfate reduction, the study on stoichiometry of NPEOs biodegradation was performed following the method described by Lu et al. (2007). Predicted values based on stoichiometric equation were compared to those measured in NPEO-degrading enrichment. The study on stoichiometry of NPEOs biodegradation under sulfate-reducing conditions was just performed for three days to avoid the disturbance of the further biodegradation of NP, which was formed during the biodegradation period of NPEOs. In the stoichiometric equation, it is assumed that the ethoxyl chain of NPnEO (n means the number of the repeating ether unit) is completely mineralized to CO2 as follows: − NPn EO þ 0:5Hþ þ 1:25SO2− 4 ¼ NPn − 1 EO þ 2:0HCO3 þ 1:25H2 S þ 1:5H2 O
ð1Þ
Based on Eq. (1), 1.25 mol SO2− 4 will be reduced when 1 mol ethoxyl chains are removed. The measured amount of SO2− 4 was 78 ± 8% of the theoretically expected amount, suggesting
Data calculation
The maximum biodegradation rate of NPEOs was determined from the time course of NPEOs disappearance, using points in the linear portion of graphs that released substrate concentration to time. Estradiol equivalents were calculated (calEEQ) by multiplying the molar concentration of each estrogenic intermediate (NP, NP1EO, or NP2EO) with its relative potency and adding up the values for these typical estrogenic intermediates following the method described by Vermeirssen et al. (2005) and Lu et al. (2007). According to a recent study with high sensitive analysis method (Kuruto-Niwa et al., 2005), the
Fig. 1 – Biodegradation of NPEOs under sulfate-reducing conditions. The incubation was performed at 25 °C. Initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). Error bars represent ±one standard deviation for three replicates.
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to the NP. Similar biodegradation pathway has been observed under Fe(III)-reducing conditions (Lu et al., 2007) and other anaerobic conditions (Renner, 1997; Ejlertsson et al., 1999). Previous studies showed that carboxylated intermediates were the main breakdown products under aerobic conditions (Ahel et al., 1994; Montgomery-Brown et al., 2003) while NP and short-chain NPEOs were the main breakdown products under anaerobic conditions (Ahel et al., 1994). It seems that the biodegradation pathway of NPEOs can be changed in the presence of different terminal electron acceptors. However, similar biodegradation pathway has been observed under diverse reducing conditions. Further study is needed to elucidate the influence of diverse terminal electron acceptors on the biodegradation behavior of NPEOs.
3.3. Effect of the initial concentration on the biodegradation of NPEOs under sulfate-reducing conditions
Fig. 2 – Changes in ion peak distribution of the NPEOs samples during the biodegradation period. The initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). The incubation was performed at 25 °C. The results shown are representative of triplicate incubations. that the primary biodegradation of NPEOs under sulfatereducing conditions was coupled to sulfate reduction.
3.2. Typical biodegradation intermediates and biodegradation pathway of NPEOs under sulfate-reducing conditions The low molecular breakdown products were analyzed by GC-MS for reliable identification. NP and short-chain NPEOs (NP1EO and NP2EO) were identified as biodegradation intermediates while low molecular carboxylated metabolites such as short-chain NPECs were not detected. Moreover, the typical electrospray mass spectra of NPEO samples collected at different incubation times showed that only oligomers of NPEOs were detected while long-chain NPECs were not formed during the whole biodegradation period. Fig. 2 summarized the histogram of the ion peak distribution of each biodegraded sample observed by electrospray mass. As the figure showed, stepwise ethoxyl chain shortening process occurred. The ion peak distributions gradually shifted to lower numbers of polymerization with increase of the incubation time. The rapid decrease in the relative peak abundance of long-chain NPEOs suggested that long-chain NPEOs were rapidly removed. Two different terminal oxidative biodegradation models for the ethoxyl chain scission of alkylphenol polyethoxylates (APEOs) have been reported by Sato et al. (2001) and Jonkers et al. (2001), respectively. Alkylphenol polyethoxycarboxylates (APECs) are the typical biodegradation intermediates in these oxidative models. Since NPECs were not detected, the terminal oxidative models may be not the case for the biodegradation of NPEOs under sulfate-reducing conditions. Stepwise ethoxyl chain shortening process from long-chain NPEOs to shorter chain polyethoxylates without forming related NPECs was observed in our experiment, suggesting that NPEOs were biodegraded through a non-oxidative pathway, through which NPEOs were degraded via sequential removal of ethoxyl units
Substrate concentration often has great influence on the reaction rate. The relationship between substrate concentration and specific removal rate is usually studied in bioremediation design. The specific NPEO-removing rate could not be measured in our experiment because of the consecutive change in the biomass with the elapse of incubation time. Therefore, the maximum removal rate was used to replace the specific removal rate in the study on the relationship between initial concentration of NPEOs and biodegradation rate. Maximum removal rate as a function of initial concentration was plotted in Fig. 3. Clearly, there is a linear relationship between initial concentration and the maximum removal rate (r2 = 0.988, P b 0.05). Increase in the initial concentration resulted in increased removal rate. The maximum removal rate increased about 1.3 μM d− 1 for each 10 μmol increase in initial concentration. High initial concentration might have possible surfactant toxicity which would subsequently inhibit the biodegradation of NPEOs. In general, the rate increases in direct proportion to the substrate concentration only at very low substrate concentrations. However, this linear relationship maintained
Fig. 3 – Maximum removal rate as a function of the initial concentration of Igepal CO-630 (r2 =0.988). Initial concentrations of Igepal CO-630 were 10 mg L− 1 (16.21 μM), 50 mg L− 1 (81.05 μM), 100 mg L− 1 (162.1 μM), 300 mg L− 1 (486.3 μM), and 500 mg L− 1 (810.5 μM). The incubation was performed at 25 °C. The results shown are representative of triplicate incubations.
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even at relatively high initial concentration, suggesting that the toxic effect had not occurred at very high initial concentration (500 mg L− 1). Since concentrations of NPEOs contaminants in natural environments are usually very low (Thiele et al., 1997), the toxic effect on the biodegradation of NPEOs under sulfate-reducing conditions can be negligible in most natural environments.
3.4. Effect of temperature on the biodegradation of NPEOs under sulfate-reducing conditions To investigate the effect of temperature within the usual mesophilic operational range on the biodegradation of NPEOs, incubations were performed at 15 °C, 25 °C, and 35 °C. Fig. 4 shows that temperature has great influence on the biodegradation of NPEOs under sulfate-reducing conditions. The decrease in temperature caused a delay in the start of the rapid removal of NPEOs. A significant lag phase was observed in the treatment at 15 °C while no lag phase was observed in the treatment at 35 °C. The removal efficiency of the total NPEOs also depended on the temperature. The decrease in temperature led to a rapid decline in the removal efficiency of NPEOs. In anaerobic treatment, the slow growth rate of microorganisms most critical to the biodegradation process makes temperature all the more important for bioremediation design. Based on Arrhenius equation, a simple equation (Rittmann and McCarty, 2001) which was widely used was developed to describe the relationship between reaction rate constant and temperature: k2 ¼ k1 eUðT2 T1 Þ :
ð2Þ
Where Ф is the temperature coefficient, k1 and k2 are the biodegradation rate constants at temperature T1 and T2, respectively. Clearly, kinetic parameters at different temperature are needed to describe the relationship between the anaerobic biodegradation and temperature in detail. However, it seems that study on the kinetics of the biodegradation of NPEOs may be difficult because commercial NPEOs are complex mixtures of isomers and oligomers. Moreover, the biodegradation kinetics analysis of total NPEOs was disturbed by the accumulation of persistent short-chain NPEOs in our experiment because these
Fig. 4 – Biodegradation of NPEOs at different temperatures. Initial concentration of Igepal CO-630 was 10 mg L− 1 (16.21 μM). The results shown are representative of triplicate incubations.
Fig. 5 – Changes in the concentration of estrogenic metabolites (A) and calculated estrogenic activity (B) during the biodegradation period. Initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). The incubation was performed at 25 °C. The results shown are representative of triplicate incubations. compounds are more persistent than their parent compounds. To avoid this disturbance caused by the accumulation of shortchain NPEOs, the biodegradation data collected in later phase when severe accumulation of short-chain NPEOs occurred were not used for kinetics analysis. As Fig. 4 shows, biodegradation data collected in early phase fit well with first-order kinetics (r2 =0.947–0.969, Pb 0.05). Therefore, the biodegradation constants (k) of total NPEOs at different temperatures were used to calculate the temperature coefficient (Ф) according to Eq. (2). The result showed that the temperature coefficient (Ф) for the biodegradation of NPEOs under sulfate-reducing conditions was 0.008.
3.5. Evolution of the typical estrogenic intermediates during biodegradation period NP, NP1EO, and NP2EO are weakly estrogenic (Jobling and Sumpter, 1993; Renner, 1997). Therefore, their evolution during the biodegradation period was monitored. Changes in the concentration of these estrogenic compounds during the
Fig. 6 – Removal of the total NPEOs in the presence of NP. Initial concentration of Igepal CO-630 was 100 mg L− 1 (162.1 μM). The incubation was performed at 25 °C. Error bars represent ± one standard deviation for three replicates.
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biodegradation period were shown in Fig. 5A. The accumulation of NP, NP1EO, and NP2EO coincided with the rapid removal of total NPEOs. The concentration of NP2EO reached its top on day 7 which was much earlier than those of NP1EO and NP. The concentrations of NP1EO and NP reached their tops on day 21 when most NPEOs were removed. Since these compounds are estrogenic, their accumulation led to a significant increase in the estrogenic activity during the biodegradation period. The highest estrogenic activity appeared on day 21when the total concentration of these metabolites reached its top (18.03 ± 4.73 μM) (Fig. 5B). Similar phenomenon was observed in the biodegradation of NPEOs under Fe(III)-reducing conditions (Lu et al., 2007). However, it must be pointed out that the accumulation of these estrogenic intermediates was greatly alleviated under Fe (III)-reducing conditions. According to their molecular structures, these compounds are highly reduced organic molecules. These compounds have no highly electron-seeking groups such as carbonyls. In general, such compounds can be thermodynamically readily oxidized in the presence of terminal electron acceptor with relatively high redox potential (Heider et al., 1999). Therefore, the accumulation of these compounds was alleviated in the presence of terminal electron acceptor with relatively high redox potential.
3.6. Effect of typical biodegradation intermediate on the biodegradation of NPEOs under sulfate-reducing conditions NP, the most common toxic and estrogenic intermediates of NPEOs, was added into the culture medium to investigate the effect of typical biodegradation intermediate on the biodegradation of NPEOs under sulfate-reducing conditions (Fig. 6). The result showed that NP could inhibit the biodegradation of NPEOs under sulfate-reducing conditions. The maximum removal rate was sharply decreased from 34.90 ± 1.13 μM d− 1 to 16.18 ± 0.40 μM d− 1 when NP was added at high concentration (1000 μM). However, this inhibition effect became obvious only when NP was added at relatively high concentration. Since NP in natural environment usually maintains a low level (Thiele et al., 1997; Ying et al., 2002), this inhibit effect on the biodegradation of NPEOs under sulfate-reducing conditions can be negligible in most natural environments.
4.
Conclusions
Results reported in this paper demonstrated that NPEOs were readily biodegraded under sulfate-reducing conditions. NPEOs were degraded via sequential removal of ethoxyl units to NP without forming carboxylated intermediates during the biodegradation period. This ethoxyl chain removal process was coupled to sulfate reduction. Both temperature and the initial concentration played important roles in the removal efficiency of NPEOs. The decrease in temperature caused a sharp decrease in the removal efficiency of NPEOs. Increase in the initial concentration resulted in enhanced removal rate. The accumulation of NP and short-chain NPEOs led to a significant increase in the estrogenic activity during the biodegradation period. NP, the typical intermediate of NPEOs, could inhibit the anaerobic biodegradation of NPEOs only at high concentra-
tion. Since microbial sulfate reduction is an important process in environment, these findings have significant environmental implications in terms of the removal of NPEOs contaminants in environment.
Acknowledgments This work is financially supported by the National Key Basic Research Program (Grant No. 2007CB815603) and National Foundation of Science of China (Grant No. 50478019). The authors would like to thank Ping Tao (Instrumental Analysis Center of Shanghai Jiao Tong University) for making LC-MS facilities available to us. The authors also wish to thank the anonymous reviewers for their reading of the manuscript, and for their suggestions and critical comments.
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