Degradation of 17β-Estradiol by a Gram-Negative Bacterium Isolated from Activated Sludge in a Sewage Treatment Plant in Tokyo, Japan

Abstract

A 17β-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new Novosphingobium species. Gas chromatography-mass spectrometry and ¹H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.

There has been increasing concern recently over the potential of sewage treatment plant (STP) effluent to cause estrogenic effects on aquatic fauna. Widespread sexual disruption, or so-called feminization, is thought to be due to environmental contaminants in the effluent and has been observed for riverine fish in several developed nations, including the United Kingdom, the United States, and Japan (3, 4, 7, 8, 10, 13). While there still remains some room for discussion about the chemicals that cause this phenomenon, natural estrogens entering the environment through the excretions of humans, domestic or farm animals, and wildlife are thought to be the most likely suspects, because they induce biological effects at environmentally relevant concentrations, on the order of nanograms per liter (12, 18, 21). Among the natural estrogens, 17β-estradiol (E2) is the most potent and is found ubiquitously in many water systems (2, 9, 11, 14, 24, 28). Therefore, it is thought that E2 is responsible for the majority of the estrogenic effects found for STP effluent.

Although the fate and behavior of E2 after excretion, during processing in STPs, or following discharge into rivers are not fully understood, there is some evidence supporting the importance of this problem. The efficiency of removal of natural estrogens, including E2, during STP processing is estimated to be 50 to 90%, depending on the facility and the location (2, 14). However, this removal is thought to be mainly due to adsorption of E2 into activated sludge or other factors independent of microbial degradation, because it has been suggested previously that natural steroidal estrogens, including E2, are poorly degraded during STP processing (17, 19). Furthermore, the proportion of steroidal estrogens remaining in the effluent is still capable of producing estrogenic effects (18). It has also been reported previously that conjugated E2 (an estrogenically inactive form) excreted from humans and animals is converted back into free E2 (the active form) before or during passage through the STP, suggesting that microbes in the process can cleave the conjugates (16). These facts, considered together with the increasing public consciousness regarding environmental preservation and the views of scientists (19, 26), make it clear that the degradation capabilities and removal efficiencies of STPs need to be improved further. Moreover, recent overpopulation in urban areas may make the feminization problem more serious. These prospects led us to search for microorganisms with strong E2-degrading activity.

To find microbes with E2-degrading activity, we collected 11 samples, including 4 soil and 3 water samples from natural environments and 2 wastewater and 2 activated sludge samples from STPs in Tokyo and Yokohama, Japan. Yeast nitrogen base without amino acids (YNB; pH 7.0 at 25°C; Difco Laboratories) with added E2 (Wako Pure Chemical Industries, Osaka, Japan) was used as the minimal medium (E2-YNB medium). YNB consists of (NH₄)₂SO₄ as a nitrogen source, other salts (KH₂PO₄, MgSO₄, NaCl, and CaCl₂), trace metals, and a very small amount of vitamins. No other carbon sources are present in YNB, and thus, E2 is considered to be nearly the sole carbon source in the E2-YNB medium. For cultivation, 100 μl of each sample was inoculated directly into E2-YNB (30 mg/30 ml), but sediment samples (10 g) were suspended with 10 ml of YNB before inoculation. For the assay of E2-degrading activity, the samples were cultured at 25°C while being rotated at 150 rpm. After cultivation, each culture was mixed with 120 ml of methanol followed by filtration through a 0.2-μm-pore-size Millex LG13 filter (Millipore Corporation, Bedford, Mass.). The filtrates were directly subjected to a reverse-phase high-pressure liquid chromatography (HPLC) system with a Mightysil RP-18GP octadecyl silica column (150 by 4.6 mm [inside diameter]; Kanto Chemical, Tokyo, Japan) for monitoring UV absorption at 210 nm. As the mobile phase (flow rate, 1.0 ml/min), a mixture of 80% methanol-20% water was used. In consequence of the assay, only one sample from activated sludge in an STP showed E2-degrading activity. In this sample, 60% of E2 was degraded in 14 days (data not shown). Then, the sample was subcultured three times and streaked on nutrient agar (Eiken Chemical, Tokyo, Japan) for isolation of E2-degrading microbes. After a 1-week incubation at 25°C, tiny whitish brown colonies emerged on the agar. A single colony was randomly picked and named strain ARI-1.

Subsequently, the E2-degrading activity of ARI-1 pure culture was examined under the same assay conditions as described above. A single colony of ARI-1 from a nutrient agar plate was inoculated into fresh E2-YNB medium and cultivated at 25°C while being rotated at 150 rpm. Figure 1 shows a typical time course for E2 degradation by ARI-1. ARI-1 steadily degraded 30, 10, and 5 mg of E2 in 30 ml of medium. Plate counts on nutrient agar showed that the cell density in 25-day-old ARI-1 pure culture (10 mg of E2/30 ml) was about 0.5 × 10⁹ CFU/ml, while it was 0.8 × 10³ CFU/ml in 0-day-old culture, suggesting that ARI-1 utilizes E2 as a carbon source. In general, E2 is found in natural environments in parts-per-trillion (nanograms-per-liter) concentrations (12, 18, 21). Thus, it is important to know whether ARI-1 can degrade E2 at the environmentally relevant concentration. However, we could not obtain definitive data for this question because we could not measure E2 below parts-per-billion (micrograms-per-liter) concentrations under the conditions that we used for HPLC. A more sensitive analytical method is needed to address this point.

FIG. 1.

Typical time courses for the degradation of E2 by the strain ARI-1. Degradation of 30, 10, or 5 mg of E2 in 30 ml of medium is indicated by open circles, concentric circles, or solid circles, respectively.

Since E2-YNB is a so-called minimal medium, we tried cultivation of ARI-1 in E2-YNB (10 mg/30 ml) supplemented with yeast extract (0.5% [wt/vol], a commonly used concentration) or glucose (0.05% [wt/vol], a molarity equal to that of E2) as a conutrient to accelerate the degradation of E2. However, the degradation activity was not accelerated by these nutrients (data not shown), though they are more easily assimilated by most bacteria and have sufficient concentrations to take the place of E2 as another carbon source in our culture conditions. Although various other culture conditions have to be examined before we can reach a definitive conclusion, ARI-1 seems to assimilate E2 as a preferred carbon source.

We examined whether ARI-1 could degrade other estrogens, including estron (E1), estriol (E3), and ethynylestradiol (EE2). A single colony of ARI-1 was inoculated into E1-YNB (10 mg/30 ml), E3-YNB (10 mg/30 ml), or EE2-YNB (10 mg/30 ml) medium and cultivated. The experimental conditions used in the E2 degradation assay were adopted. ARI-1 was found to degrade E1 and E3, but not EE2. About 40% of E1 was degraded in 20 days, while E3 was degraded almost completely within 10 days (data not shown).

Although UV absorption at 210 nm was used for monitoring in the HPLC analysis of the culture media, we did not find any aromatic compounds other than E2. Therefore, gas chromatography-mass spectrometry (GC-MS) was used to identify metabolites derived from E2 degradation. ARI-1 was cultivated in E2-YNB (10 mg/30 ml), and 0-, 5-, 10-, 15-, 20-, 25-, and 30-day-old cultures were extracted with hexane or chloroform. These solvents were used for the extraction because they have different octanol-water partition coefficients (3.29 and 1.52, respectively), which are often used as an index of hydrophobicity. The extracts were analyzed with a Varian-3800 gas chromatograph-mass spectrometer. The conditions were as follows: column, BPX-5 capillary column (25 m by 0.22 mm [inside diameter]) purchased from SGE (Melbourne, Victoria, Australia); injection volume, 3 μl; carrier gas, helium (1.0 ml/min); temperature gradient, 50°C for 3.4 min, 50 to 370°C at 10°C/min, and 370°C for 10 min. The ionization energy for electron impact ionization was 70 eV. However, we could not find any metabolites in extracts of 0- through 30-day-old cultures. Figure 2 shows total ion chromatograms of the chloroform extracts. During cultivation, only a decrease of the E2 peak was observed, as shown.

FIG. 2.

Total ion chromatograms of the chloroform extracts of 0 (A)- and 30 (B)-day-old cultures. The E2 peaks are indicated by arrowheads.

Many aromatic compounds, like phenol, benzene, or toluene, are toxic to aquatic organisms. Furthermore, some have recently been found to be estrogenic. Thus, it is very important to know whether the aromatic part of E2 is degraded by ARI-1. To examine this, chloroform extracts of 0- and 30-day-old cultures were dissolved in CD₃OD and analyzed by a nuclear magnetic resonance (NMR) spectrometer (¹H NMR). The ¹H-NMR spectra were obtained with a JEOL JNM-EX400 NMR spectrometer at 400 MHz on sample solutions in 5-mm-diameter tubes. The signal for the protons of both the aromatic and nonaromatic parts of E2 was found to have disappeared almost completely in the 30-day-old culture (Fig. 3). These data are consistent with those obtained from GC-MS (Fig. 2) and strongly suggest that E2 is degraded by ARI-1 to compounds with very low molecular mass (for instance, CO₂) or simple organic acids which may not be extractable with the conditions used. In summary, data from GC-MS and NMR consistently suggested that no toxic or accumulative metabolites of E2 were produced from the degradation pathway.

FIG. 3.

NMR spectra of the chloroform extracts of 0 (A)- and 30 (B)-day-old cultures. Each numbered proton signal in panel A corresponds to a position in the structure of E2 as shown in panel C. The asterisk indicates signals of methylene protons (positions 6, 7, 11, 12, 15, and 16 in E2) and methine protons (positions 8, 9, and 14 in E2). Signals at 3.3 and 4.9 ppm are derived from the solvent (CD₃OD and HDO, respectively).

The phylogenic analysis of ARI-1 was also carried out. Gram staining and optical microscopic study revealed that ARI-1 is a gram-negative and oval-shaped bacterium. The nearly complete 16S ribosomal DNA (rDNA) (1,417 bases) of the bacterial strain ARI-1 was amplified by PCR, using a universal primer set corresponding to positions 8 to 27 (forward primer) and 1492 to 1510 (reverse primer) of the Escherichia coli numbering system (27). We used the PCR operating conditions described by Suzuki and Yamasato (23). Direct sequencing of the amplified DNA fragments was carried out as described by Satomi et al. (20). A homology search using the BLAST algorithm (1) and all known sequence data in the GenBank, EMBL, and DDBJ databases was conducted to determine the similarities of the 16S rDNA sequences of ARI-1 and strains of other species. As a result of the BLAST search, it was found that 16S rDNA of ARI-1 was highly homologous to those of several Novosphingobium spp. (N. subterraneum, 97%; N. aromaticivorans, 96%; N. stygium, 95%; and N. capsulatum, 95%) and some Sphingomonas sp. strains (Sphingomonas sp. strain MT1, 95%; strain A28241, 95%; and strain MBIC4193, 95%), suggesting that ARI-1 is a member of the genus Novosphingobium. Novosphingobium (25) is one of the recently proposed genera which were previously included in the genus Sphingomonas (29). The genus Novosphingobium is well known for including many species that can assimilate biodegradation-resistant compounds. For instance, N. subarcticum can assimilate tetrachlorophenol (15); N. aromaticivorans was shown elsewhere to degrade several aromatic compounds including toluene and naphthalene (5); and N. stygium and N. subterraneum can degrade fluorene, biphenyl, and dibenzothiophene (6). That ARI-1 is a species of Novosphingobium supports its role as a bioremediator of E2. However, the similarity of the 16S rDNA of ARI-1 to that of its neighbors was at most 97% (data not shown), suggesting that ARI-1 may be a species which has not previously been reported (22). A more detailed analysis is in progress to ascertain whether ARI-1 is a novel species in this new genus, and the results will be reported soon.

In this study, we isolated the E2-degrading bacterium ARI-1, possibly a new member of the genus Novosphingobium, from activated sludge in an STP in Tokyo, Japan. ARI-1 degraded E2 steadily, and GC-MS and ¹H-NMR data (Fig. 2 and 3) suggested that no accumulative toxic metabolites of E2 were produced, supporting the practicality of this strain for the preservation of aquatic environments via treatment of sewers. However, there are several hurdles to be overcome before the use of ARI-1 for bioremediation will be practical. The most important challenge will be the enhancement of the E2-degrading activity of ARI-1, so that the time course of the degradation can be shortened and the treatment of E2-contaminated wastewater can be made efficient.

Nucleotide sequence accession number.

The 16S rDNA sequence of ARI-1 has been deposited in the DDBJ database under accession no. AB070237.