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Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas europaea
ARTICLE IN PRESS
Water Research 38 (2004) 2323–2330
Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas europaea Jianghong Shi*, Saori Fujisawa, Satoshi Nakai, Masaaki Hosomi Department of Applied Chemistry, Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo 184-8588, Japan Received 24 September 2003; received in revised form 2 February 2004; accepted 17 February 2004
Abstract This report describes the uses of nitrifying activated sludge (NAS) and ammonia-oxidizing bacterium Nitrosomonas europaea to significantly degrade estrone (E1), 17b-estradiol (E2), estriol (E3), and 17a-ethynylestradiol (EE2). Using NAS, the degradation of estrogens obeyed first-order reaction kinetics with degradation rate constants of 0.056 h1 for E1, 1.3 h1 for E2, 0.030 h1 for E3, and 0.035 h1 for EE2, indicating that E2 was most easily degraded. Then, we confirmed that E2 was degraded via E1 by NAS. With/without the ammonia oxidation inhibitor, it was observed that ammonia-oxidizing bacteria in conjunction with other microorganisms in NAS degraded estrogens. Using N. europaea, the degradation of estrogens reasonably obeyed zero-order reaction kinetics, and no remarkable difference is present among the four estrogens degradation rates and it was found that E1 was not detected during E2 degradation period. We suggested that E2 was degraded to E1 in NAS could be caused by other heterotrophic bacteria, not by ammoniaoxidizing bacteria. r 2004 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Natural and synthetic estrogens; Nitrifying activated sludge; Nitrosomonas europaea; Products
1. Introduction There is growing concern about the persistence and degradation pathways of natural and synthetic estrogens in the environment. Natural estrogens, i.e., estrone (E1), 17b-estradiol (E2), and estriol (E3), are excreted by humans and animals through their urine principally as inactive polar conjugates such as glucuronides and sulphates [1]. The synthetic estrogen, 17a-ethynylestradiol (EE2), is a key ingredient in oral contraceptives and is mainly eliminated as conjugates in urine [1]. Many of these conjugates of natural and synthetic estrogens are cleaved to free estrogens through microbial processes before or during sewage treatment [2–4]. Ascenzo et al. *Corresponding author. Tel.: +81-42-388-7070; fax: +8142-381-4201. E-mail address: [email protected] (J. Shi).
have investigated the concentrations of both free and conjugated estrogens in the six STPs in Roman, indicating that E3-16-glucuronide, E2-3-glucuronide, and E2-3-sulfate surviving in the sewer system were completely removed by the STP treatment, while E1, E2, E3, E1-3-glucuronide, E1-3-sulfate, and E3-3-sulfate were detected in the effluents [5]. Both natural and synthetic estrogens have been detected in the ng L1 range in effluents [1,6–9], indicating that these estrogens are not completely removed during the treatment process. For example, Desbrow et al. have reported that E2 and E1 were detected in the ranges of 1– 50 ng L1 and 1–80 ng L1 in seven effluents from sewage treatment plants in Britain. In addition, EE2 was detected in the range of 0.2–7.0 ng L1 from the three sewage treatment plants [6]. Recently, a multitude of chemicals have shown to be endocrine disrupters that disturb the hormonal systems
0043-1354/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.02.022
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of aquatic organisms. Among these, natural and synthetic estrogens are already effective at the lower ng L1 level [10,11]. A laboratory study on the endocrine-disrupting potency of EE2 demonstrated that EE2 at low concentrations of 1–10 ng L1 caused estrogenic response in caged fish [11]. Since most endocrine disrupters such as bisphenol A and nonylphenol are weakly estrogenic, often with potencies three or more orders of magnitude less than that of E2, E1, or EE2, estrogens have been suggested as the major compounds responsible for endocrine disruption in sewage wastewater [6,12]. Due to the endocrine-disrupting potency of natural and synthetic estrogens, there has been an increasing interest in biodegradation of these estrogens by using microorganisms. Ternes et al. [4] showed that the activated sludge collected from a STP oxidized E2 of 1 mg L1 to E1 and E1 was then eliminated; however, EE2 of 1 mg L1 appeared to be mainly stable in contact with the activated sludge. Lee et al. [13] showed that E2 of 200 mg L1 has been almost quantitatively oxidized to E1 by sewage bacteria under both aerobic and anaerobic conditions. Michael et al. [14] and Colucci et al. [15] reported that E2, E1, and EE2 were rapidly biodegraded in agricultural soils. Ying et al. [16,17] investigated degradations of E2 and EE2 using aquifer materials, or a marine sediment and seawater under aerobic condition, indicating that E2 of 1 mg/g in the sediment was degraded quickly with a half-life of 2 or 4.4 d, while EE2 was degraded much more slowly with a half-life of 81 or >20 d. Jurgens et al. [18] showed that microorganisms in the river water sample were capable of transforming E2 to E1, and that E1 was then degraded at similar rates, but EE2 was much more resistant. Regarding EE2, Vader et al. [19] demonstrated that nitrifying activated sludge (NAS) could degrade EE2 at an initial concentration of 50 mg L1 within 6 d; a result allowing us to surmise that NAS may degrade natural estrogens including E1, E2, and E3. Further studies are necessary, however, to determine which estrogen can be degraded by NAS and which microorganism in NAS is responsible for degradation of the estrogens. In our previous work [20], we isolated an EE2-degrading microorganism Fusarium proliferatum strain HNS-1 that can use EE2 as the sole source of carbon to degrade 97% of EE2 at an initial concentration of 25 mg L1 at a first-order rate constant, 0.6 d1. This research shows that EE2 can be degraded by certain microorganisms. NAS containing a lot of nitrifying bacteria is used to oxidize ammonia to nitrite and nitrate. Nitrosomonas europaea in nitrogen removal systems, an obligate chemolithotrophic ammonia-oxidizing bacterium, is usually responsible for the oxidation of ammonia to nitrite, deriving its energy for growth exclusively from the oxidation of ammonia to nitrite. It is also known to be capable of oxidizing various hydrocarbon
compounds such as methane, methanol, phenol, and benzene, as well as halogenated hydrocarbons such as TCE [21,22]. Investigation of the extent in the biodegradation of estrogens by nitrifying bacteria is clearly of research interest. Accordingly, we decided to (i) investigate the biodegradability of natural and synthetic estrogens using NAS, (ii) determine the relationship between nitrifying activity of NAS and degradation rates of the estrogens, and (iii) investigate the biodegradability of natural and synthetic estrogens using the ammonia-oxidizing bacterium N. europaea.
2. Materials and Methods 2.1. Chemicals E1, E2, E3, and EE2 were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 2.2. Nitrifying activated sludge (NAS) NAS (MLSS 5400 mg L1) was obtained from a nitrification tank treating landfill leachate in Tokyo, Japan. NAS was pre-cultivated by a fill-and-draw operation with a 3-d cycle in a 2-L reactor at 30 C. At the start of pre-cultivation, 1.0 L of NAS and 1.0 L of mineral-salts medium A [(NH4)2SO4, 6.6 g L1; KH2PO4, 1.36 g L1; MgSO4 7H2O, 2.46 g L1; NaCl, 2.92 g L1; CaCO3, 1.0 g L1 and EDTA-Fe, 6 mg L1] [23] were mixed and aerated. In each cycle, half the supernatant was drawn after settling for 30 min, then fresh mineral salts medium A of the same volume was added. The pH in the reactor was controlled at 7.5–8.0 using 40 g L1 NaHCO3. 2.3. Batch experiments using nitrifying activated sludge Pre-cultivated NAS and sterilized mineral salts medium A were added to sterilized 200-mL Erlenmeyer flasks. The amount of NAS in the flasks was adjusted to obtain an MLSS concentration of 2700 mg L1. Each estrogen in ethanol solution was added to a flask to achieve a final concentration of 1.0 mg L1. Controls for these experiments were produced by sterilized precultivated NAS, sterilized mineral salts medium A, and estrogens. The initial pH was adjusted to 8.0 using 40 g L1 NaHCO3, the pH of the culture medium was then adjusted to about 7.5–8.0 using 40 g L1 NaHCO3 during cultivation. Flasks were cultivated at 30 C on a rotary shaker at 100 rpm. During cultivation estrogens and NHþ 4 -N concentrations were measured. All batch experiments were performed in triplicate.
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2.4. N. europaea Ammonia-oxidizing bacterium N. europaea (NCIMB 11850) was purchased from NCIMB Japan CO., LTD. Freeze-dried N. europaea was added to a predetermined medium of NCIMB [(NH4)2SO4, 235 mg L1; KH2PO4, 200 mg L1; CaCl2 2H2O, 40.0 mg L1; MgSO4 7H2O, 40.0 mg L1; FeSO4U7H2O, 0.5 mg L1; NaEDTA, 0.5 mg L1; and Phenol red, 0.5 mg L1] and then cultivated at 70 rpm at 30 C. The pH of the culture medium was adjusted to about 8.0 by 5% Na2CO3 during cultivation.
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lylthiourea were added into sterilized 200-mL Erlenmeyer flasks containing 40 mL sterilized mineral salts medium B and 0.4 mg L1 of estrogen. During the cultivation period, estrogens, NHþ 4 -N, NO2 -N, and NO-3 -N concentrations were measured. All inhibition experiments were performed in triplicate. 2.7. Analysis
In the estrogen-degradation experiment by using N. europaea, we used modified medium B [22] consisting of 10 mM (NH4)2SO4, 3 mM KH2PO4, 750 mM MgSO4, 200 mM CaCl2, 16 mM Fe-EDTA, 1 mM CuSO4 and 0.04 % (w/v) Na2CO3, being buffered with 43 mM NaH2PO4 and 4 mM K2HPO4 (pH 8.0). The estrogen in ethanol solution was added to a 200-mL Erlenmeyer flask to achieve a final concentration of 0.4 mg L1. Following solvent evaporation, 40 mL of sterilized modified medium B was dispensed into the flasks and 2 mL of pre-cultivated solution of N. europaea was inoculated into the flasks which were subsequently cultivated in a rotary shaker at 100 rpm and 30 C. During the cultivation period, estrogen and NHþ 4 -N concentrations, pH, and turbidity were measured. The controls for these experiments only contained sterilized modified medium B and estrogens. All batch experiments were performed in triplicate.
Samples for the measurement of estrogen concentrations were prepared by adding CH3CN to the remaining culture medium at a volume ratio of 1:1 to uniformly dissolve the estrogen. Following filtration (pore size 0.22 mm), the sample (100 mL) was subjected to a reversephase high-performance liquid chromatography (RPHPLC) using an ODS column (TSK-gel ODS 80Ts, Tosoh, Japan) and an electrochemical detector (1049 A, Hewlett-Packard) that detects estrogen by oxidizing its phenolic group. In the RP-HPLC analysis, elution was carried out by using 45% (E3 35%) v/v acetonitrile/ water at a flow rate of 1.0 mL/min. In a recovery experiment conducted beforehand (data not shown), we confirmed that more than 95% of estrogens adsorbed on sludge or microorganisms could be collected using acetonitrile extraction. NHþ 4 -N concentration was determined by the green-colored indophenol method using a spectrophotometer (UV-160, Shimadzu). NO-2 -N and NO-3 -N concentrations were measured using an ion chromatographic analyzer (IC 7000, Yakogawa Analytical Systems, Japan). The pH of the culture medium was measured with a pH meter, while turbidity was measured using a turbidity meter (MODEL T-2600D, Tokyodenshoku, Japan).
2.6. Inhibitor and inhibition experiment of ammonia oxidation
3. Results and Discussion
2.5. Batch experiments using n. europaea
Allylthiourea was used as the AMO (the action of ammonia monooxygenase) inhibitor [22] to assess estrogen degradation activities of NAS and N. europaea in the absence of ammonia oxidation. NAS was precipitated by centrifugation at 3000 rpm for 5 min and washed three times with a phosphate buffer (pH 8.0) in order to remove NHþ 4 -N, NO2 -N, and NO3 -N contained in it. The washed NAS suspension was then added into sterilized 200-mL Erlenmeyer flasks containing 40 mL sterilized mineral salts medium A at an initial MLSS of 700 mg L1, after which estrogen in ethanol solution was added to the flasks to achieve an initial concentration of 0.2 mg L1. Following addition of Allylthiourea (10 mg L1), the initial pH was adjusted to 8.0 with 40 g L1 NaHCO3, then the pH of the culture medium was adjusted to about 7.5–8.0 using 40 g L1 NaHCO3 during cultivation. These flasks were cultivated on a rotary shaker at 100 rpm and 30 C. Similarly, the pre-cultivated solution of N. europaea and Al-
3.1. Estrogen degradation using NAS Fig. 1 shows time variations in EE2, E1, E2, and E3 concentrations during the batch-experiment cultivation period of NAS at an initial concentration of 1 mg L1. The degradation of estrogens obeyed first-order reaction kinetics with degradation rate constants of 0.056 h1 for E1, 1.3 h1 for E2, 0.030 h1 for E3, and 0.035 h1 for EE2. These results confirm that NAS significantly degrades both synthetic and natural estrogens. Among the four estrogens, E2 was most easily degraded. Ternes et al. [4] reported that E2 at initial concentration of 1 mgL1 could be degraded using activated sludge over a period of 1–3 h. Here, NAS degraded 98% of E2 at 1 mg L1 within 2 h, which indicates that NAS also has excellent E2-degradation ability. Regarding EE2, Vader et al. [19] showed that 0.050 mg L1 of EE2 could be degraded using NAS within 6 d. We found a similar trend in the decrease of EE2 concentrations.
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1.4
Estrogens Conc. (mgL-1)
1.2 1 E1 with with NAS E1 i NAS E2 with with NAS E2 i NAS E3 with with NAS E3 i NAS EE2 with with NAS EE2 i NAS Control of of E1 E1 Control Control of E2 E2 Control r of Control of E3 E3 Control t of Control of EE2
0.8 0.6 0.4 0.2 0 0
24
48
72
96
Time (hour) Fig. 1. Changes in EE2, E1, E2 and E3 concentrations over NAS cultivation period.
3.2. Estrogen-degrading pathways and products using NAS As reported in the previous studies [4,13,18], E1 was produced by NAS during E2 degradation periods. Fig. 2 shows changes in E1 concentrations during E2 degradation by NAS, where E1 was detected and then degraded further. E1 produced from E2 degradation was confirmed using spiked tests in which E1 was added to the culture samples. Considering the amount of E1 produced from E2, we surmised whether or not E2 was consecutively degraded via E1 by NAS, and then calculated the concentration of E1 using a consecutive first-order reaction equation to verify other possible products of E2 degradation in addition to E1. The consecutive first-order reaction is CE2
kE2 -
kE1 CE1
E2 or E1 Conc. (mgL-1)
1 E2
0.8
Measured E1 Calculated l l E1
0.6 0.4 0.2 0 0
12
24
36
48
60
72
Time (hr)
Fig. 2. Prediction of E1 concentration during the E2-degradation period using NAS.
ð1Þ
- Products;
dCE2 =dt ¼ kE2 CE2 ;
ð2Þ
dCE1 =dt ¼ kE2 CE2 kE1 CE1 ;
ð3Þ 1
where kE2 is the E2 degradation rate constant (h ), kE1 the E1 degradation rate constant (h1), CE2 the E2 concentration [mg L1], and CE1 the E1 concentration [mg L1]. From Eqs. (2) and (3), the E1 concentration via E2 degradation by NAS can be calculated using CE10 k t CE1 1 ¼ e E1 ; ð4Þ ekE1 t ekE2 t þ CE20 1 k CE20 where CE20 is the initial E2 concentration (1.0 mg L1), CE10 the initial E1 concentration (0 m g L1), k= kE1XkE2, kE2 = 1.3 h1, and kE1 = 0.054 h1. Since
the E1 values calculated from Eq. (4) show a good agreement with the actual measured E1 values (Fig. 2), it is clear that E1 was only a transient by-product and consecutively degraded to other compounds by NAS. Fig. 3 shows chromatograms of the NAS-culture medium after E2 degradation at 0, 2.3, and 72 hr, where the peak at 3.72 min is considered to be an unknown degradation intermediate via E1 degradation. It should be noted that the unknown degradation intermediate of E1 was further degraded and vanished after 72 hr. Similar to the degradation of E1, the unknown degradation intermediate of EE2 and E3 at about 3.7 min was also further degraded and barely detected after 96 hr. These unknown intermediate products were
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þ þ Fig. 4. Changes in NHþ 4 -N, NH4 -N, and NH4 -N concentrations during EE2 degradation by NAS with/without inhibitor.
Fig. 3. Chromatogram obtained by HPLC–ECD analysis of NAS-culture medium in the degradation of E2.
Estrogens r Conc.. (mgL-1)
degraded by NAS and disappeared over time. The RPHPLC analysis showed the unknown intermediate products were eluted faster than the estrogens. Due to the elution characteristics of RP-HPLC and the detection mode of the electrochemical detector, we surmise that these products may be more polar compounds carrying a phenolic group. Furthermore, since the peaks areas of these products decreased, this suggests that the phenolic frames were cleaved during degradation by NAS.
0.3 EE2 EE2 + Inhibitor Inhibi r E1 E1+Inhibitor E2 E2 + Inhibitor i
0.25 0.2 0.15 0.1 0.05 0 0
3.3. Estrogen-degrading activity of nas with the inhibitor of ammonia oxidation Allylthiourea was used as the inhibitor of ammonia oxidation in estrogen-degradation experiments. Fig. 4 shows the time changes in NHþ 4 -N, NO2 -N, and NO3 -N concentrations during EE2 degradation by NAS with/ without allylthiourea. When only NAS and EE2 are added into mineral salts medium A, NHþ 4 -N gradually decreases during the estrogen degradation period of 96 h, NO-2 -N temporarily increases and then decreases, NO-3 -N increases via NO-2 -N oxidation, and the total nitrogen concentrations of NHþ 4 -N, NO2 -N, and NO3 N are nearly uniform. In contrast, when NAS, EE2, and allylthiourea are added into mineral salts medium A, NHþ 4 -N concentration shows minimal change, and neither NO-2 -N, nor NO-3 -N are generated during the EE2 degradation period, which indicates that allylthiourea completely inhibited NHþ 4 -N oxidation to NO-2 -N. In addition, time changes in NHþ 4 -N, NO2 -N, and NO-3 -N concentrations in E1 and E2 degradations by NAS with/without allylthiourea were similar to EE2
16
32 48 64 Time (hour)
80
96
Fig. 5. Changes in E1 or E2 and EE2 degradation by NAS with/without inhibitor.
degradation. Since ammonia-oxidizing bacteria like N. europaea use ammonia as their sole energy source to oxidize ammonia to nitrite, this strongly suggests that ammonia-oxidizing bacteria in NAS did not grow during the estrogen-degradation experiments in the presence of the inhibitor allylthiourea. Fig. 5 shows estrogens degradation results by NAS with/without inhibitor, where all the decreases in estrogens concentrations obeyed first-order reaction kinetics. At an initial estrogens concentration of 0.2 mg L1, the degradation rate constants were 0.036 h1 for E1, 0.60 h1 for E2, and 0.059 h1 for EE2 without the inhibitor, whereas the degradation rate constants were only 0.004 h1 for E1, 0.32 h1 for E2, and 0.0085 h1 for EE2 with the inhibitor. These results suggested that ammonia oxidation bacteria together with other microorganisms in NAS degraded the
ARTICLE IN PRESS estrogens. In addition, the inhibitor of ammonia oxidation allylthiourea did not change E1 production behavior during E2 degradation by NAS. 3.4. Estrogens degradation using N. europaea To confirm whether or not estrogens are biodegraded by ammonia-oxidizing bacteria, we investigated estrogen degradation abilities of one ammonia-oxidizing bacterium, N. europaea. Figs. 6 and 7 show time variations in estrogens and ammonia nitrogen, respectively. During a 187-h exposure of N. europaea to ammonium and the estrogen (E1, E2, E3, or EE2) at an initial concentration of 0.4 mg L1, the estrogen and NHþ 4 -N concentrations gradually decreased, while turbidity increased with time. The deceases both in estrogen concentrations and ammonia nitrogen consumption reasonably obey zeroorder reaction kinetics. No remarkable difference is present among the four estrogen degradation rates, with estrogen biodegradation rate constants of 0.0019 mg L1 h 1 for EE2, 0.0022 mg L1 h –1 for E1, 0.0020 mg L1 h –1 for E2, and 0.0016 mg L1 h –1 for E3. Corresponding ammonia nitrogen consumption rates 1 1 were 1.5 mgNHþ h for EE2, 1.5 mgNHþ 4 -N L 41 1 1 1 NL h for E1, 1.4 mgNHþ h for E2, 4 -N L 1 and 1.3 mg NHþ h 1 for E3. These results 4 -N L suggest that ammonia-oxidizing bacteria such as N. europaea can contribute to the estrogen degradations by NAS and that N. europaea is not only able to cometabolize various hydrocarbon compounds [21,22], but also able to degrade estrogens, E1, E2, E3 and EE2. 3.5. Estrogen degradation products using N. europaea Fig. 8 shows the HPLC-ECD chromatogram of EE2 culture medium after degradation by N. europaea starting at 120 hr, where a large peak at 4–7 min is
0.5
Estrogens Conc.. (mgL-1)
EE2 0.4
E2 E1
0.3
E3
Ammonia nitrogen i Conc. (mgL-1)
J. Shi et al. / Water Research 38 (2004) 2323–2330
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300
200 EE2 E2 E1 E3
100
0 0
4 40
80
1 120
1 160
200
Time (hour)
Fig. 7. Changes in NHþ 4 -N concentration by N. europaea.
Fig. 8. Chromatogram obtained by RP-HPLC-ECD analysis of N. europaea-culture medium containing remaining EE2 and its degradation products at 120 hr.
apparent, being considered an unknown degradation product of EE2. Corresponding results for E1, E2, and E3 also show an unknown product near 4–7 min (data not shown). Note that this peak gradually increases in area until the end of estrogen degradation, indicating that unknown degradation products are not further degraded. The unknown products of estrogen degradation were eluted faster than the estrogens, and due to the elution and detection modes, these degradation products may well be more polar compounds that have a phenolic group than estrogens. Future research is needed to determine whether or not these degradation products have endocrine-disrupting activity and to identify these degradation products
0.2
3.6. Estrogen degradation test using N. europaea with ammonia oxidation inhibitor
1 0.1 0 0
40
80
120
1 160
200
Time (hour) Fig. 6. Degradation of E1, E2, E3, and EE2 by N. europaea.
In N. europaea, ammonia is initially oxidized to hydroxylamine by ammonia monooxygenase (AMO), with allylthiourea being a well-known strong inhibitor of AMO at very low concentrations. Fig. 9 show changes in E2 concentrations during E2 degradation
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Accordingly, since generation of E1 from E2 appeared in nitrifying activated sludge and in conventional activated sludge [4], E2 degradation via E1 by NAS is considered to be caused by other heterotrophic bacteria and not by nitrifying bacterium such as N. europaea.
0.5
0.4 E2 Conc. (mgL-1)
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E2 0.3 E2+ N. europaea
4. Conclusions
E2+N. europaea { Inhibitor i i
0.2
*
0.1
0.0 0
50
100
150
200
*
Time (hour) Fig. 9. Changes in E2 degradation by N. europaea with / without inhibitor. *
by N. europaea with/without the inhibitor, where a concentration of 10 mg L1 Allylthiourea effectively inhibits E2 degradation and nitrification (data not shown) of N. europaea; a result suggesting that AMO plays some role in estrogen degradation.
*
3.7. Comparison of the estrogen-degraded characteristics of nitrifying activated sludge and N. europaea Both NAS and N. europaea degraded the four estrogens, yet differences occurred in estrogen degradation rates and degradation products. NAS degrades estrogens and their degradation intermediate, while N. europaea only degrades estrogens with no further degradation of their intermediate. This suggests that other microorganisms exist in NAS which are not ammonia-oxidizing bacteria, and that they are responsible for intermediate degradation. In addition, since estrogens were degraded by NAS with/without ammonia oxidation inhibitor, both ammonia-oxidizing bacteria and other unknown microorganisms could well exist in NAS and be responsible for its estrogen- degradation ability. As shown in Figs. 4 and 5, a concentration 10 mg L1 Allylthiourea effectively prevents nitrification of NAS, yet at this concentration most estrogen-degradation ability of NAS is not inhibited; i.e., allylthiourea is only an inhibitor of ammonia monooxygenase in ammonia-oxidizing bacteria and not an inhibitor of the enzyme secreted by other microorganisms in NAS, that enzyme most likely degraded the estrogens. The results showed that E1 was generated when NAS degraded E2, whereas E1 was not generated when N. europaea degraded E2. Obviously then, NAS and N. europaea exhibit differently in E2 degradation pathways.
*
Degradation of natural and synthetic estrogens was studied by using nitrifying activated sludge (NAS) and ammonia-oxidizing bacterium N. europaea. The results indicate that both NAS and N. europaea significantly degrade E1, E2, E3, and EE2. E2 was most easily degraded among the four estrogens and was consecutively degraded to other products via E1 by using NAS. While, no remarkable difference is present among the four estrogen degradation rates and E2 was not degraded to E1 by using N. europaea. In the presence of ammonia oxidation inhibitor, we confirmed ammonia-oxidizing bacteria in NAS did not grow. However, these results suggested that the other microorganisms except ammonia oxidation bacteria in NAS could degrade the four estrogens. N. europaea only degrades estrogens with no further degradation of their intermediate. This suggests that other microorganisms exist in NAS which are not ammonia-oxidizing bacteria, and that they are responsible for intermediate of estrogens. In further work, we would like to identify the estrogen degradation products and investigate the estrogenic activity during estrogen degradations using NAS or N. europaea.
Acknowledgements This research was supported by generous grants from the Nippon Life Insurance Foundation and Japan New Activated Sludge Technology. Additional support was provided by the 21st Century Center of Excellence Program (COE), Evolution and Survival of TechnologyBased Civilization, Tokyo University of Agriculture and Technology.
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yeast. T. J. of Japan Society on Water Environ 2001;24(11):750–6. Lee HB, Liu D. Degradation of -estradiol and its metabolites by sewage bacteria. Water, Air, Soil Pollution 2002;134:353–68. Colucci M, Bork H, Topp E. Persistence of estrogenic hormones in agricultural soils: 1. 17a-estradiol and estrone. J Environ Qua. 2001;30:2070–6. Colucci M, Topp E. Persistence of estrogenic hormones in agricultural soils: 2. 17a-ethynylestradiol. J. Environ Qua, 2001;30:2077–80. Ying Guang-Guo, Kookana Rai S, Peter Dillon. Sorption and degradation of selected five endocrine disrupting chemicals in aquifer material. Water Research 2003; 37:3785–91. Ying Guang-Guo, Kookana Rai S. Degradation of five selected endocrine-disrupting chemicals in seawater and marine sediment. Enviro Sci Technol 2003;37:1256–60. Jurgens M, Holthaus K, Johnson A, Smith J, Hetheridge M, Williams R. The potential for estradiol and ethinylestradiol degradation in English rivers. Environ Toxicology and Chemistry 2002;21(3):480–8. Vader JS, Ginkel CGvan, Stokman FM, Sperling GM, Jong Jde, Boer Wde, Graaf JSde, Most Mvander, Stokman PGW. Degradation of ethinyl estradiol by nitrifying activated sludge. Chemosphere 2000;41(8):1239–43. Shi JH, Suzuki Y, Lee B-D, Nakai S, Hosomi M. Isolation and characterization of the ethynylestradiol-biodegrading microorganism. Fusarium proliferatum strain HNS- Water Sci and Technol 2002;45:175–9. Hyman MR, Murton Ian B, Arp DJ. Interaction of ammonia monooxygenase from nitrosomonas europea with alkanes, alkenes and alkynes. Appl Environ Microbial 1988;54:3187–90. Rasche ME, Hyman MR, Arp DJ. Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia monooxygenase and substrate specificity. Appl and Environ Microb 1991;57(10):2986–94. The Meeting of Microbe Research Methods. Microbiological experiment methods. Japan: Koudan Publishing Company; 1984. p. 428.
Water Research 38 (2004) 2323–2330
Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas europaea Jianghong Shi*, Saori Fujisawa, Satoshi Nakai, Masaaki Hosomi Department of Applied Chemistry, Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka, Koganei, Tokyo 184-8588, Japan Received 24 September 2003; received in revised form 2 February 2004; accepted 17 February 2004
Abstract This report describes the uses of nitrifying activated sludge (NAS) and ammonia-oxidizing bacterium Nitrosomonas europaea to significantly degrade estrone (E1), 17b-estradiol (E2), estriol (E3), and 17a-ethynylestradiol (EE2). Using NAS, the degradation of estrogens obeyed first-order reaction kinetics with degradation rate constants of 0.056 h1 for E1, 1.3 h1 for E2, 0.030 h1 for E3, and 0.035 h1 for EE2, indicating that E2 was most easily degraded. Then, we confirmed that E2 was degraded via E1 by NAS. With/without the ammonia oxidation inhibitor, it was observed that ammonia-oxidizing bacteria in conjunction with other microorganisms in NAS degraded estrogens. Using N. europaea, the degradation of estrogens reasonably obeyed zero-order reaction kinetics, and no remarkable difference is present among the four estrogens degradation rates and it was found that E1 was not detected during E2 degradation period. We suggested that E2 was degraded to E1 in NAS could be caused by other heterotrophic bacteria, not by ammoniaoxidizing bacteria. r 2004 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Natural and synthetic estrogens; Nitrifying activated sludge; Nitrosomonas europaea; Products
1. Introduction There is growing concern about the persistence and degradation pathways of natural and synthetic estrogens in the environment. Natural estrogens, i.e., estrone (E1), 17b-estradiol (E2), and estriol (E3), are excreted by humans and animals through their urine principally as inactive polar conjugates such as glucuronides and sulphates [1]. The synthetic estrogen, 17a-ethynylestradiol (EE2), is a key ingredient in oral contraceptives and is mainly eliminated as conjugates in urine [1]. Many of these conjugates of natural and synthetic estrogens are cleaved to free estrogens through microbial processes before or during sewage treatment [2–4]. Ascenzo et al. *Corresponding author. Tel.: +81-42-388-7070; fax: +8142-381-4201. E-mail address: [email protected] (J. Shi).
have investigated the concentrations of both free and conjugated estrogens in the six STPs in Roman, indicating that E3-16-glucuronide, E2-3-glucuronide, and E2-3-sulfate surviving in the sewer system were completely removed by the STP treatment, while E1, E2, E3, E1-3-glucuronide, E1-3-sulfate, and E3-3-sulfate were detected in the effluents [5]. Both natural and synthetic estrogens have been detected in the ng L1 range in effluents [1,6–9], indicating that these estrogens are not completely removed during the treatment process. For example, Desbrow et al. have reported that E2 and E1 were detected in the ranges of 1– 50 ng L1 and 1–80 ng L1 in seven effluents from sewage treatment plants in Britain. In addition, EE2 was detected in the range of 0.2–7.0 ng L1 from the three sewage treatment plants [6]. Recently, a multitude of chemicals have shown to be endocrine disrupters that disturb the hormonal systems
0043-1354/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.02.022
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of aquatic organisms. Among these, natural and synthetic estrogens are already effective at the lower ng L1 level [10,11]. A laboratory study on the endocrine-disrupting potency of EE2 demonstrated that EE2 at low concentrations of 1–10 ng L1 caused estrogenic response in caged fish [11]. Since most endocrine disrupters such as bisphenol A and nonylphenol are weakly estrogenic, often with potencies three or more orders of magnitude less than that of E2, E1, or EE2, estrogens have been suggested as the major compounds responsible for endocrine disruption in sewage wastewater [6,12]. Due to the endocrine-disrupting potency of natural and synthetic estrogens, there has been an increasing interest in biodegradation of these estrogens by using microorganisms. Ternes et al. [4] showed that the activated sludge collected from a STP oxidized E2 of 1 mg L1 to E1 and E1 was then eliminated; however, EE2 of 1 mg L1 appeared to be mainly stable in contact with the activated sludge. Lee et al. [13] showed that E2 of 200 mg L1 has been almost quantitatively oxidized to E1 by sewage bacteria under both aerobic and anaerobic conditions. Michael et al. [14] and Colucci et al. [15] reported that E2, E1, and EE2 were rapidly biodegraded in agricultural soils. Ying et al. [16,17] investigated degradations of E2 and EE2 using aquifer materials, or a marine sediment and seawater under aerobic condition, indicating that E2 of 1 mg/g in the sediment was degraded quickly with a half-life of 2 or 4.4 d, while EE2 was degraded much more slowly with a half-life of 81 or >20 d. Jurgens et al. [18] showed that microorganisms in the river water sample were capable of transforming E2 to E1, and that E1 was then degraded at similar rates, but EE2 was much more resistant. Regarding EE2, Vader et al. [19] demonstrated that nitrifying activated sludge (NAS) could degrade EE2 at an initial concentration of 50 mg L1 within 6 d; a result allowing us to surmise that NAS may degrade natural estrogens including E1, E2, and E3. Further studies are necessary, however, to determine which estrogen can be degraded by NAS and which microorganism in NAS is responsible for degradation of the estrogens. In our previous work [20], we isolated an EE2-degrading microorganism Fusarium proliferatum strain HNS-1 that can use EE2 as the sole source of carbon to degrade 97% of EE2 at an initial concentration of 25 mg L1 at a first-order rate constant, 0.6 d1. This research shows that EE2 can be degraded by certain microorganisms. NAS containing a lot of nitrifying bacteria is used to oxidize ammonia to nitrite and nitrate. Nitrosomonas europaea in nitrogen removal systems, an obligate chemolithotrophic ammonia-oxidizing bacterium, is usually responsible for the oxidation of ammonia to nitrite, deriving its energy for growth exclusively from the oxidation of ammonia to nitrite. It is also known to be capable of oxidizing various hydrocarbon
compounds such as methane, methanol, phenol, and benzene, as well as halogenated hydrocarbons such as TCE [21,22]. Investigation of the extent in the biodegradation of estrogens by nitrifying bacteria is clearly of research interest. Accordingly, we decided to (i) investigate the biodegradability of natural and synthetic estrogens using NAS, (ii) determine the relationship between nitrifying activity of NAS and degradation rates of the estrogens, and (iii) investigate the biodegradability of natural and synthetic estrogens using the ammonia-oxidizing bacterium N. europaea.
2. Materials and Methods 2.1. Chemicals E1, E2, E3, and EE2 were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 2.2. Nitrifying activated sludge (NAS) NAS (MLSS 5400 mg L1) was obtained from a nitrification tank treating landfill leachate in Tokyo, Japan. NAS was pre-cultivated by a fill-and-draw operation with a 3-d cycle in a 2-L reactor at 30 C. At the start of pre-cultivation, 1.0 L of NAS and 1.0 L of mineral-salts medium A [(NH4)2SO4, 6.6 g L1; KH2PO4, 1.36 g L1; MgSO4 7H2O, 2.46 g L1; NaCl, 2.92 g L1; CaCO3, 1.0 g L1 and EDTA-Fe, 6 mg L1] [23] were mixed and aerated. In each cycle, half the supernatant was drawn after settling for 30 min, then fresh mineral salts medium A of the same volume was added. The pH in the reactor was controlled at 7.5–8.0 using 40 g L1 NaHCO3. 2.3. Batch experiments using nitrifying activated sludge Pre-cultivated NAS and sterilized mineral salts medium A were added to sterilized 200-mL Erlenmeyer flasks. The amount of NAS in the flasks was adjusted to obtain an MLSS concentration of 2700 mg L1. Each estrogen in ethanol solution was added to a flask to achieve a final concentration of 1.0 mg L1. Controls for these experiments were produced by sterilized precultivated NAS, sterilized mineral salts medium A, and estrogens. The initial pH was adjusted to 8.0 using 40 g L1 NaHCO3, the pH of the culture medium was then adjusted to about 7.5–8.0 using 40 g L1 NaHCO3 during cultivation. Flasks were cultivated at 30 C on a rotary shaker at 100 rpm. During cultivation estrogens and NHþ 4 -N concentrations were measured. All batch experiments were performed in triplicate.
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2.4. N. europaea Ammonia-oxidizing bacterium N. europaea (NCIMB 11850) was purchased from NCIMB Japan CO., LTD. Freeze-dried N. europaea was added to a predetermined medium of NCIMB [(NH4)2SO4, 235 mg L1; KH2PO4, 200 mg L1; CaCl2 2H2O, 40.0 mg L1; MgSO4 7H2O, 40.0 mg L1; FeSO4U7H2O, 0.5 mg L1; NaEDTA, 0.5 mg L1; and Phenol red, 0.5 mg L1] and then cultivated at 70 rpm at 30 C. The pH of the culture medium was adjusted to about 8.0 by 5% Na2CO3 during cultivation.
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lylthiourea were added into sterilized 200-mL Erlenmeyer flasks containing 40 mL sterilized mineral salts medium B and 0.4 mg L1 of estrogen. During the cultivation period, estrogens, NHþ 4 -N, NO2 -N, and NO-3 -N concentrations were measured. All inhibition experiments were performed in triplicate. 2.7. Analysis
In the estrogen-degradation experiment by using N. europaea, we used modified medium B [22] consisting of 10 mM (NH4)2SO4, 3 mM KH2PO4, 750 mM MgSO4, 200 mM CaCl2, 16 mM Fe-EDTA, 1 mM CuSO4 and 0.04 % (w/v) Na2CO3, being buffered with 43 mM NaH2PO4 and 4 mM K2HPO4 (pH 8.0). The estrogen in ethanol solution was added to a 200-mL Erlenmeyer flask to achieve a final concentration of 0.4 mg L1. Following solvent evaporation, 40 mL of sterilized modified medium B was dispensed into the flasks and 2 mL of pre-cultivated solution of N. europaea was inoculated into the flasks which were subsequently cultivated in a rotary shaker at 100 rpm and 30 C. During the cultivation period, estrogen and NHþ 4 -N concentrations, pH, and turbidity were measured. The controls for these experiments only contained sterilized modified medium B and estrogens. All batch experiments were performed in triplicate.
Samples for the measurement of estrogen concentrations were prepared by adding CH3CN to the remaining culture medium at a volume ratio of 1:1 to uniformly dissolve the estrogen. Following filtration (pore size 0.22 mm), the sample (100 mL) was subjected to a reversephase high-performance liquid chromatography (RPHPLC) using an ODS column (TSK-gel ODS 80Ts, Tosoh, Japan) and an electrochemical detector (1049 A, Hewlett-Packard) that detects estrogen by oxidizing its phenolic group. In the RP-HPLC analysis, elution was carried out by using 45% (E3 35%) v/v acetonitrile/ water at a flow rate of 1.0 mL/min. In a recovery experiment conducted beforehand (data not shown), we confirmed that more than 95% of estrogens adsorbed on sludge or microorganisms could be collected using acetonitrile extraction. NHþ 4 -N concentration was determined by the green-colored indophenol method using a spectrophotometer (UV-160, Shimadzu). NO-2 -N and NO-3 -N concentrations were measured using an ion chromatographic analyzer (IC 7000, Yakogawa Analytical Systems, Japan). The pH of the culture medium was measured with a pH meter, while turbidity was measured using a turbidity meter (MODEL T-2600D, Tokyodenshoku, Japan).
2.6. Inhibitor and inhibition experiment of ammonia oxidation
3. Results and Discussion
2.5. Batch experiments using n. europaea
Allylthiourea was used as the AMO (the action of ammonia monooxygenase) inhibitor [22] to assess estrogen degradation activities of NAS and N. europaea in the absence of ammonia oxidation. NAS was precipitated by centrifugation at 3000 rpm for 5 min and washed three times with a phosphate buffer (pH 8.0) in order to remove NHþ 4 -N, NO2 -N, and NO3 -N contained in it. The washed NAS suspension was then added into sterilized 200-mL Erlenmeyer flasks containing 40 mL sterilized mineral salts medium A at an initial MLSS of 700 mg L1, after which estrogen in ethanol solution was added to the flasks to achieve an initial concentration of 0.2 mg L1. Following addition of Allylthiourea (10 mg L1), the initial pH was adjusted to 8.0 with 40 g L1 NaHCO3, then the pH of the culture medium was adjusted to about 7.5–8.0 using 40 g L1 NaHCO3 during cultivation. These flasks were cultivated on a rotary shaker at 100 rpm and 30 C. Similarly, the pre-cultivated solution of N. europaea and Al-
3.1. Estrogen degradation using NAS Fig. 1 shows time variations in EE2, E1, E2, and E3 concentrations during the batch-experiment cultivation period of NAS at an initial concentration of 1 mg L1. The degradation of estrogens obeyed first-order reaction kinetics with degradation rate constants of 0.056 h1 for E1, 1.3 h1 for E2, 0.030 h1 for E3, and 0.035 h1 for EE2. These results confirm that NAS significantly degrades both synthetic and natural estrogens. Among the four estrogens, E2 was most easily degraded. Ternes et al. [4] reported that E2 at initial concentration of 1 mgL1 could be degraded using activated sludge over a period of 1–3 h. Here, NAS degraded 98% of E2 at 1 mg L1 within 2 h, which indicates that NAS also has excellent E2-degradation ability. Regarding EE2, Vader et al. [19] showed that 0.050 mg L1 of EE2 could be degraded using NAS within 6 d. We found a similar trend in the decrease of EE2 concentrations.
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1.4
Estrogens Conc. (mgL-1)
1.2 1 E1 with with NAS E1 i NAS E2 with with NAS E2 i NAS E3 with with NAS E3 i NAS EE2 with with NAS EE2 i NAS Control of of E1 E1 Control Control of E2 E2 Control r of Control of E3 E3 Control t of Control of EE2
0.8 0.6 0.4 0.2 0 0
24
48
72
96
Time (hour) Fig. 1. Changes in EE2, E1, E2 and E3 concentrations over NAS cultivation period.
3.2. Estrogen-degrading pathways and products using NAS As reported in the previous studies [4,13,18], E1 was produced by NAS during E2 degradation periods. Fig. 2 shows changes in E1 concentrations during E2 degradation by NAS, where E1 was detected and then degraded further. E1 produced from E2 degradation was confirmed using spiked tests in which E1 was added to the culture samples. Considering the amount of E1 produced from E2, we surmised whether or not E2 was consecutively degraded via E1 by NAS, and then calculated the concentration of E1 using a consecutive first-order reaction equation to verify other possible products of E2 degradation in addition to E1. The consecutive first-order reaction is CE2
kE2 -
kE1 CE1
E2 or E1 Conc. (mgL-1)
1 E2
0.8
Measured E1 Calculated l l E1
0.6 0.4 0.2 0 0
12
24
36
48
60
72
Time (hr)
Fig. 2. Prediction of E1 concentration during the E2-degradation period using NAS.
ð1Þ
- Products;
dCE2 =dt ¼ kE2 CE2 ;
ð2Þ
dCE1 =dt ¼ kE2 CE2 kE1 CE1 ;
ð3Þ 1
where kE2 is the E2 degradation rate constant (h ), kE1 the E1 degradation rate constant (h1), CE2 the E2 concentration [mg L1], and CE1 the E1 concentration [mg L1]. From Eqs. (2) and (3), the E1 concentration via E2 degradation by NAS can be calculated using CE10 k t CE1 1 ¼ e E1 ; ð4Þ ekE1 t ekE2 t þ CE20 1 k CE20 where CE20 is the initial E2 concentration (1.0 mg L1), CE10 the initial E1 concentration (0 m g L1), k= kE1XkE2, kE2 = 1.3 h1, and kE1 = 0.054 h1. Since
the E1 values calculated from Eq. (4) show a good agreement with the actual measured E1 values (Fig. 2), it is clear that E1 was only a transient by-product and consecutively degraded to other compounds by NAS. Fig. 3 shows chromatograms of the NAS-culture medium after E2 degradation at 0, 2.3, and 72 hr, where the peak at 3.72 min is considered to be an unknown degradation intermediate via E1 degradation. It should be noted that the unknown degradation intermediate of E1 was further degraded and vanished after 72 hr. Similar to the degradation of E1, the unknown degradation intermediate of EE2 and E3 at about 3.7 min was also further degraded and barely detected after 96 hr. These unknown intermediate products were
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þ þ Fig. 4. Changes in NHþ 4 -N, NH4 -N, and NH4 -N concentrations during EE2 degradation by NAS with/without inhibitor.
Fig. 3. Chromatogram obtained by HPLC–ECD analysis of NAS-culture medium in the degradation of E2.
Estrogens r Conc.. (mgL-1)
degraded by NAS and disappeared over time. The RPHPLC analysis showed the unknown intermediate products were eluted faster than the estrogens. Due to the elution characteristics of RP-HPLC and the detection mode of the electrochemical detector, we surmise that these products may be more polar compounds carrying a phenolic group. Furthermore, since the peaks areas of these products decreased, this suggests that the phenolic frames were cleaved during degradation by NAS.
0.3 EE2 EE2 + Inhibitor Inhibi r E1 E1+Inhibitor E2 E2 + Inhibitor i
0.25 0.2 0.15 0.1 0.05 0 0
3.3. Estrogen-degrading activity of nas with the inhibitor of ammonia oxidation Allylthiourea was used as the inhibitor of ammonia oxidation in estrogen-degradation experiments. Fig. 4 shows the time changes in NHþ 4 -N, NO2 -N, and NO3 -N concentrations during EE2 degradation by NAS with/ without allylthiourea. When only NAS and EE2 are added into mineral salts medium A, NHþ 4 -N gradually decreases during the estrogen degradation period of 96 h, NO-2 -N temporarily increases and then decreases, NO-3 -N increases via NO-2 -N oxidation, and the total nitrogen concentrations of NHþ 4 -N, NO2 -N, and NO3 N are nearly uniform. In contrast, when NAS, EE2, and allylthiourea are added into mineral salts medium A, NHþ 4 -N concentration shows minimal change, and neither NO-2 -N, nor NO-3 -N are generated during the EE2 degradation period, which indicates that allylthiourea completely inhibited NHþ 4 -N oxidation to NO-2 -N. In addition, time changes in NHþ 4 -N, NO2 -N, and NO-3 -N concentrations in E1 and E2 degradations by NAS with/without allylthiourea were similar to EE2
16
32 48 64 Time (hour)
80
96
Fig. 5. Changes in E1 or E2 and EE2 degradation by NAS with/without inhibitor.
degradation. Since ammonia-oxidizing bacteria like N. europaea use ammonia as their sole energy source to oxidize ammonia to nitrite, this strongly suggests that ammonia-oxidizing bacteria in NAS did not grow during the estrogen-degradation experiments in the presence of the inhibitor allylthiourea. Fig. 5 shows estrogens degradation results by NAS with/without inhibitor, where all the decreases in estrogens concentrations obeyed first-order reaction kinetics. At an initial estrogens concentration of 0.2 mg L1, the degradation rate constants were 0.036 h1 for E1, 0.60 h1 for E2, and 0.059 h1 for EE2 without the inhibitor, whereas the degradation rate constants were only 0.004 h1 for E1, 0.32 h1 for E2, and 0.0085 h1 for EE2 with the inhibitor. These results suggested that ammonia oxidation bacteria together with other microorganisms in NAS degraded the
ARTICLE IN PRESS estrogens. In addition, the inhibitor of ammonia oxidation allylthiourea did not change E1 production behavior during E2 degradation by NAS. 3.4. Estrogens degradation using N. europaea To confirm whether or not estrogens are biodegraded by ammonia-oxidizing bacteria, we investigated estrogen degradation abilities of one ammonia-oxidizing bacterium, N. europaea. Figs. 6 and 7 show time variations in estrogens and ammonia nitrogen, respectively. During a 187-h exposure of N. europaea to ammonium and the estrogen (E1, E2, E3, or EE2) at an initial concentration of 0.4 mg L1, the estrogen and NHþ 4 -N concentrations gradually decreased, while turbidity increased with time. The deceases both in estrogen concentrations and ammonia nitrogen consumption reasonably obey zeroorder reaction kinetics. No remarkable difference is present among the four estrogen degradation rates, with estrogen biodegradation rate constants of 0.0019 mg L1 h 1 for EE2, 0.0022 mg L1 h –1 for E1, 0.0020 mg L1 h –1 for E2, and 0.0016 mg L1 h –1 for E3. Corresponding ammonia nitrogen consumption rates 1 1 were 1.5 mgNHþ h for EE2, 1.5 mgNHþ 4 -N L 41 1 1 1 NL h for E1, 1.4 mgNHþ h for E2, 4 -N L 1 and 1.3 mg NHþ h 1 for E3. These results 4 -N L suggest that ammonia-oxidizing bacteria such as N. europaea can contribute to the estrogen degradations by NAS and that N. europaea is not only able to cometabolize various hydrocarbon compounds [21,22], but also able to degrade estrogens, E1, E2, E3 and EE2. 3.5. Estrogen degradation products using N. europaea Fig. 8 shows the HPLC-ECD chromatogram of EE2 culture medium after degradation by N. europaea starting at 120 hr, where a large peak at 4–7 min is
0.5
Estrogens Conc.. (mgL-1)
EE2 0.4
E2 E1
0.3
E3
Ammonia nitrogen i Conc. (mgL-1)
J. Shi et al. / Water Research 38 (2004) 2323–2330
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300
200 EE2 E2 E1 E3
100
0 0
4 40
80
1 120
1 160
200
Time (hour)
Fig. 7. Changes in NHþ 4 -N concentration by N. europaea.
Fig. 8. Chromatogram obtained by RP-HPLC-ECD analysis of N. europaea-culture medium containing remaining EE2 and its degradation products at 120 hr.
apparent, being considered an unknown degradation product of EE2. Corresponding results for E1, E2, and E3 also show an unknown product near 4–7 min (data not shown). Note that this peak gradually increases in area until the end of estrogen degradation, indicating that unknown degradation products are not further degraded. The unknown products of estrogen degradation were eluted faster than the estrogens, and due to the elution and detection modes, these degradation products may well be more polar compounds that have a phenolic group than estrogens. Future research is needed to determine whether or not these degradation products have endocrine-disrupting activity and to identify these degradation products
0.2
3.6. Estrogen degradation test using N. europaea with ammonia oxidation inhibitor
1 0.1 0 0
40
80
120
1 160
200
Time (hour) Fig. 6. Degradation of E1, E2, E3, and EE2 by N. europaea.
In N. europaea, ammonia is initially oxidized to hydroxylamine by ammonia monooxygenase (AMO), with allylthiourea being a well-known strong inhibitor of AMO at very low concentrations. Fig. 9 show changes in E2 concentrations during E2 degradation
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Accordingly, since generation of E1 from E2 appeared in nitrifying activated sludge and in conventional activated sludge [4], E2 degradation via E1 by NAS is considered to be caused by other heterotrophic bacteria and not by nitrifying bacterium such as N. europaea.
0.5
0.4 E2 Conc. (mgL-1)
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E2 0.3 E2+ N. europaea
4. Conclusions
E2+N. europaea { Inhibitor i i
0.2
*
0.1
0.0 0
50
100
150
200
*
Time (hour) Fig. 9. Changes in E2 degradation by N. europaea with / without inhibitor. *
by N. europaea with/without the inhibitor, where a concentration of 10 mg L1 Allylthiourea effectively inhibits E2 degradation and nitrification (data not shown) of N. europaea; a result suggesting that AMO plays some role in estrogen degradation.
*
3.7. Comparison of the estrogen-degraded characteristics of nitrifying activated sludge and N. europaea Both NAS and N. europaea degraded the four estrogens, yet differences occurred in estrogen degradation rates and degradation products. NAS degrades estrogens and their degradation intermediate, while N. europaea only degrades estrogens with no further degradation of their intermediate. This suggests that other microorganisms exist in NAS which are not ammonia-oxidizing bacteria, and that they are responsible for intermediate degradation. In addition, since estrogens were degraded by NAS with/without ammonia oxidation inhibitor, both ammonia-oxidizing bacteria and other unknown microorganisms could well exist in NAS and be responsible for its estrogen- degradation ability. As shown in Figs. 4 and 5, a concentration 10 mg L1 Allylthiourea effectively prevents nitrification of NAS, yet at this concentration most estrogen-degradation ability of NAS is not inhibited; i.e., allylthiourea is only an inhibitor of ammonia monooxygenase in ammonia-oxidizing bacteria and not an inhibitor of the enzyme secreted by other microorganisms in NAS, that enzyme most likely degraded the estrogens. The results showed that E1 was generated when NAS degraded E2, whereas E1 was not generated when N. europaea degraded E2. Obviously then, NAS and N. europaea exhibit differently in E2 degradation pathways.
*
Degradation of natural and synthetic estrogens was studied by using nitrifying activated sludge (NAS) and ammonia-oxidizing bacterium N. europaea. The results indicate that both NAS and N. europaea significantly degrade E1, E2, E3, and EE2. E2 was most easily degraded among the four estrogens and was consecutively degraded to other products via E1 by using NAS. While, no remarkable difference is present among the four estrogen degradation rates and E2 was not degraded to E1 by using N. europaea. In the presence of ammonia oxidation inhibitor, we confirmed ammonia-oxidizing bacteria in NAS did not grow. However, these results suggested that the other microorganisms except ammonia oxidation bacteria in NAS could degrade the four estrogens. N. europaea only degrades estrogens with no further degradation of their intermediate. This suggests that other microorganisms exist in NAS which are not ammonia-oxidizing bacteria, and that they are responsible for intermediate of estrogens. In further work, we would like to identify the estrogen degradation products and investigate the estrogenic activity during estrogen degradations using NAS or N. europaea.
Acknowledgements This research was supported by generous grants from the Nippon Life Insurance Foundation and Japan New Activated Sludge Technology. Additional support was provided by the 21st Century Center of Excellence Program (COE), Evolution and Survival of TechnologyBased Civilization, Tokyo University of Agriculture and Technology.
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