17β-Estradiol-Degrading Bacteria Isolated from ... - ACS Publications

Environ. Sci. Technol. 2007, 41, 486-492

17β-Estradiol-Degrading Bacteria Isolated from Activated Sludge CHANG-PING YU, HYUNGKEUN ROH, AND KUNG-HUI CHU* Zachry Department of Civil Engineering, Texas A&M University, College Station, Texas 77843-3136

Fourteen phylogenetically diverse 17β-estradiol-degrading bacteria (strains KC1-14) were isolated from activated sludge of a wastewater treatment plant. These isolates widely distributed among eight different generasAminobacter (strains KC6 and KC7), Brevundimonas (strain KC12), Escherichia (strain KC13), Flavobacterium (strain KC1), Microbacterium (strain KC5), Nocardioides (strain KC3), Rhodococcus (strain KC4), and Sphingomonas (strains KC8KC11 and KC14)sof three Phyla: Proteobacteria, Actinobacteria, and Bacteroidetes. All 14 isolates were capable of converting 17β-estradiol to estrone, but only three strains (strains KC6, KC7, and KC8) showed the ability to degrade estrone. Only strain KC8 could use 17β-estradiol as a sole carbon source. Based on the degree of estrogens being transformed and the estrogenicity of metabolites and/ or end products of estrogen degradation, three different degradation patterns (patterns A-C) were observed from degradation tests using resting cells. Eleven out of 14 isolates showed degradation pattern A, where 17β-estradiol was stoichiometrically converted to estrone. Estrone was confirmed to be a degradation product of 17β-estradiol; however, estrone was not further degraded during the course of experiments. Strains KC6 and KC7 exhibited degradation pattern B, where both17β-estradiol and estrone were degraded, with slower 17β-estradiol degradation rates than those observed in pattern A. Strain KC8 was the only strain exhibited degradation pattern C, where 17βestradiol and estrone were rapidly degraded within 3 days. No residual 17β-estradiol and estrone or estrogenic activity was detected after 5 days, suggesting that strain KC8 could degrade 17β-estradiol into nonestrogenic metabolites/end products. Strains KC6-8 exhibited nonspecific monooxygenase activity but not nonspecific dioxygenase activity. However, the relationship between nonspecific monooxygenase activity and its estrogen degradation ability was unclear.

Natural (17β-estradiol and estrone) and synthetic (17Rethynyl estradiol, a major ingredient in contraceptives) estrogens in treated wastewater are considered to contribute the most of the estrogenic activity (7, 8) since estrogens have 3 orders of magnitude higher estrogenic potencies than other identified EDCs in wastewater. Estrogens produced naturally by humans and animals or used for personal care are excreted in urine and feces as inactive polar conjugates. The conjugates can be converted back to unconjugated estrogens (active forms) by bacterial enzymes in the raw wastewater and during the wastewater treatment processes. Estrogens survived from the treatment processes are subsequently released into the environment through effluent. Thus, treated wastewater is considered one of the most likely estrogenic sources released into the environment. Numerous laboratory and field studies have been focused on the fate of estrogens in wastewater treatment plants in the past decades (see review (9) and refs 10-17). Removal of estrogens by WWTPs was observed at different degreessranging from 19 to 94% for estrone, 7692% for 17β-estradiol, and 83-87% for 17R-ethynyl estradiol (10). While estrogens were suggested to be mainly removed via biodegradation during wastewater treatment, little is known about the microorganisms responsible for estrogen degradation. Early studies have reported that several human intestinal bacteria and oral microorganisms are capable of converting estradiol to estrone and vice versa (18-20). Shi et al. (21) reported that biodegradation of estrogens by nitrified activated sludge and Nitrosomonas europaea as well as suggested that other heterotrophic bacteria might be involved in 17β-estradiol degradation. Recently, Fujii et al. (22) isolated the first 17β-estradiol-degrading bacterium, Novosphingobium species (ARI-1), from activated sludge. Lately, six estrogen-degrading isolates (four Rhodococcus strains, an Achromobacter strain, a Ralstonia strain) from activated sludge were reported by Yoshimoto et al. (23) and Weber et al. (24). In light of the recent success in the isolation of estrogen-degrading cultures from activated sludge, the wide removal range for different estrogens strongly suggested that estrogen-degrading cultures are widespread in activated sludge. We hypothesized that (i) many different types of estrogen-degrading bacteria in addition to Novosphingobium species, Achromobacter species, Ralstonia species, Rhodococcus species, and Nitrosomonas species are present in activated sludge and (ii) their different degradation abilities toward estrogens contribute to the wide range of estrogen removal observed by many field studies. This study reported 14 phylogenetically diverse 17β-estradiol-degrading cultures (strains KC1-14) isolated from activated sludge. Three distinctive degradation patterns toward 17β-estradiol and estrone were observed and discussed. Three isolates (strains KC6-8) that showed an ability to degrade estrone were further characterized.

Introduction

Materials and Methods

Endocrine disrupting compounds (EDCs), including estrogens, are chemicals of an increasing public concern because the exposure of EDCs has caused adverse health impacts on wildlife (1, 2). Many studies have suggested that estrogens in treated wastewater are responsible for male fish feminization and sexual disruption in many aquatic organisms (35). A recent study demonstrated that exposure to 17β-estradiol (>16 ng/L) affected the reproduction of marine male fish (6).

Chemicals. Three estrogens (17β-estradiol (>99% pure), 17Restradiol (>98% pure), and estrone (>98% pure)) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Due to low solubility of estrogens in water (∼3 mg/L at roomtemperature determined in our laboratory), stock solutions of estrogens were prepared in acetone (1000 mg of estrogen/L of acetone). Dimethylformamide (DMF) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were purchased from Pierce Biotechnology, Inc. (Rockford, IL). Sodium formate (ACS reagent grade) and glycerol (ultrapure) were purchased from MP Biomedicals, Inc. (Solon, OH). Tetrazotized o-

* Corresponding author phone: (979) 845-1403; fax: (979) 8621542; e-mail: [email protected]. 486

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10.1021/es060923f CCC: $37.00

 2007 American Chemical Society Published on Web 12/13/2006

dianisidine was purchased from Fluka Chemical Corp. (Ronkonkoma, NY). Naphthalene (99.6% pure) was obtained from Alfa Aesar (Ward Hill, MA). NADH was produced from Research Organics (Cleveland, OH). Dithiothreitol (DTT) was produced from Promega Corp. (Milwaukee, WI). Ferrous sulfate and indol (>99% pure) were purchased from Mallinckrodt Baker, Inc. (Philipsburg, NJ) and Sigma-Aldrich, Inc. (St. Louis, MO), respectively. Enrichment of Estrogen-Degrading Consortium. Estrogen-degrading consortium was enriched in a 500 mL gasflow through laboratory-scale bioreactor constructed from a 2-L flask, a rubber stopper with one inlet and one outlet, and a magnetic stirrer. Oxygen was supplied by house air bubbled at a rate of 120 mL/min. Activated sludge collected from the aeration basin of a municipal wastewater treatment plant in Knoxville, TN, was used as an inoculum (for detailed description of the plant see ref 25). The bioreactor was originally supplied with a mixture of equal concentrations of three estrogens (∼3 mg/L of each estrogen: 17R-estradiol, 17β-estradiol, and estrone) as sole carbon sources in nitrate mineral salts (NMS) medium (26). The acetone-free NMSestrogen medium was prepared as follows: Following the additions of the known amounts of three estrogen stock solutions into the NMS medium, the medium was heated to 80 °C (the boiling point of acetone is 59 °C) and purged with air at a flow rate of 120 mL/min for 30 min to removal remaining acetone in the medium. The acetone-free NMSestrogen growth medium was cooled to room temperature before use. The bioreactor was operated in batch-draw mode at the room temperature. Approximately every 20 days, 250 mL of cell suspension was discarded from the bioreactor and then replaced with the equal amount of acetone-free, NMSestrogen medium. The average solid retention time of the bioreactor was 40 days. Each cycle, the OD600 of cell suspension increased from 0.05 to 0.1. In the first cycle, complete degradation was observed for 17β-estradiol and 17R-estradiol but not for estrone. After the second cycle, enhanced and complete degradation of all three estrogens was observed within 3 days. Isolation of 17β-Estradiol-Degrading Cultures. After 6 months of operation, the bioreactor culture was used as a source for the isolation of 17β-estradiol-degrading cultures. One milliliter of the reactor culture was used to streak on NMS or R2A agar plates containing about 3 mg/L of 17βestradiol. 17β-Estradiol was the sole carbon source in NMS agar plates but not in R2A agar plates. The plates were incubated at 30 °C in the dark. Due to the poor growth of colonies on the NMS-estrogen agar plates, the isolation efforts were thus focused on the colonies growing on the R2Aestrogen agar plates. After numerous streaking, morphologically distinct colonies were selected and tested for their degradation ability toward 17β-estradiol and estrone. Isolates showing degradation abilities were further identified by using 16S rRNA gene sequences. DNA Extraction and Sequencing. Genomic DNA of each isolate was extracted using a FastDNA kit (Q-Biogene, Carlsbad, CA) in accordance with the manufacturer’s instructions. DNA concentrations were determined using a Hoefer DyNa Quant 200 Fluorometer (Pharmacia Biotech, San Francisco, CA). The extracted genomic DNA was used as a template for PCR amplification of 16S rRNA gene sequences using bacterial universal primers 8-27F (5′AGAGTTTGATCMTGGCTCAG-3′) and 1392-1407R (5′ACGGGCGGTGTGTACA-3′). Each PCR reaction was performed in a total volume of 25 µL, with Taq PCR Master Mix (QIAGEN Inc., Valencia, CA), 400 nM forward and reverse primers, and 1 µL of DNA templates. The PCR thermal cycle was 95 °C for 10 min, followed by 45 cycles of 95 °C for 45 s, 57 °C for 1 min, and 72 °C for 2 min. A final elongation step

of 72 °C for 7 min was included. The PCR products were excised from a 1.5% agarose gel in 1x TAE buffer, recovered, and purified with QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and were sequenced on Applied Biosystems 3100 DNA sequencer (Perkin-Elmer, Foster City, CA). Sequence Analysis and Construction of a Phylogenic Tree. Raw sequence data from both strands were assembled into full-length sequences using the Manipulate Sequences program (http://www.vivo.colostate.edu/molkit/manip/index.html). Closely related sequences were identified by comparing the partial 16S rRNA gene sequences with the sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) (27). The closest relatives identified from searches were included in further phylogenetic analysis. Sequence alignment and phylogenic relationships were completed with CLUSTAL X software. The online computer tool, Classifier of the Ribosomal Database-II project (http:// rdp.cme.msu.edu/index.jsp) (28), was used to assign 16S rRNA gene sequences to the taxonomical hierarchy. The accession numbers of the sequences in GenBank are from DQ066431 to DQ066444. Estrogen Degradation Tests. Experiments were designed to examine whether isolates were capable of (i) degrading different types of estrogens and (ii) converting estrogens to nonestrogenic compounds. 17β-Estradiol and estrone were used as two model compounds. The degradation tests were conducted in a series of 300-mL vials containing 100 mL of resting estrogen-degrading cells and an initial concentration of 3 mg/L of 17β-estradiol or estrone. All isolates, except for strains KC2 and KC8, were pregrown overnight in tryptic soy broth (TSB) medium containing 3 mg/L of 17β-estradiol before harvesting by centrifugation for experimental use. Due to the slow growth of strains KC2 and KC8, strains KC2 and KC8 were grown in the medium for 3 days and 2 days, respectively. The harvested cells were washed with 10 mM phosphate buffered saline (pH 7.0, containing 10 mM NaCl and 2.5 mM KCl) before resuspending in the acetone-free, NMS-estrogen growth medium to an optical density (OD600) of 1 (ranging from 400 to 850 mg/L of MLVSS depending on strain types). The vials were vigorously mixed by hand before collecting the liquid samples. The first samples were collected within 20 min after inoculation. The vials were incubated on a rotary shaker at 150 rpm at 30 °C. Liquid samples collected over time were analyzed for estrogen concentrations by GC/ MS analysis and for estrogenic activity by Yeast Estrogenic Screening (YES) assays. All degradation tests were performed in duplicate. Autoclave-killed controls were used. The loss of 17β-estradiol/or estrone in the controls was less than 10%. Growth Tests. Selected isolates capable of degrading both 17β-estradiol and estrone were further examined for their abilities to use 17β-estradiol as a sole carbon source. Sufficient 17β-estradiol in acetone-free NMS growth medium during the tests was provided and prepared as described below. A known amount of stock solution (containing 20 mg of 17βestradiol) was first added into a 300-mL flask followed by gentle purging with air to evaporate the acetone in the flask. After complete evaporation of acetone, 100 mL of NMS medium was added into the flask to dissolve estrogen. The flask was shaken at 150 rpm for 2 days before inoculated with isolates. The flasks were incubated at 30 °C at 150 rpm for 25 days. Negative controls containing only NMS medium and cells (no 17β-estradiol) were used. Samples were collected over time for 17β-estradiol analysis and protein content measurements. The protein contents were released from cells by using a sonicator (model Branson Sonifier-150) and were determined by using a bicinchoninic acid protein assay reagent kit purchased from Pierce Biotechnology, Inc. (Rockford, IL). Estrogen Analysis. Estrogen concentrations in liquid samples (containing cells and growth medium) were deterVOL. 41, NO. 2, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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mined by GC/MS analysis as described by Yu et al. (29). Briefly, the estrogens in liquid samples were extracted with ethyl ether, resuspended with DMF, and then derivatized with BSTFA. The derivatized samples were injected into a HP 6890 series gas chromatograph equipped with a DB-5MS capillary column (J&W Scientific; 30 m × 0.25 mm i.d.; 0.25 µm film thickness) and an HP 5973 mass selective detector. The analysis was performed in the SIM (selective ion monitoring) mode. The primary ions selected for quantification were m/z 416 for 17β-estradiol and m/z 342 for estrone. The column temperature was initially set at 240 °C for 2 min, then ramped at 10 °C /min to 300 °C, and held constant for 3 min. Helium was the carrier gas, and a flow rate of 1 mL/ min was used. The detection limits for 17β-estradiol and estrone were 5 µg/L. Yeast Estrogenic Screening (YES) Assay. The estrogenic activities of metabolites and/or end products of estrogens degradation by 17β-estradiol-degrading isolates were determined by yeast estrogenic screening (YES) assays. The YES assay is an in vitro, yeast-based reporter assay originally developed by Routledge et al. (30). A recombinant yeast strain, Saccharomyces cerevisiae, carrying a human estrogen receptor (ER R) and a reporter gene (Lac Z), was used to interact with environmental estrogens. When environmental estrogens bind to the receptor in the yeast, the yeast will then produce a red product. The absorbance of the red product is corresponding to the potency of estrogenic compounds in the samples. The YES assays were conducted as described by Layton et al. (13). Briefly, ether extracts were dried and resuspended in ethanol for YES assay. The samples were incubated with the cell-suspension of recombinant Saccharomyces cerevisiae strain for 3 days. The production of red product was then measured at absorbance at 540 nm. Positive controls and standard curves were constructed by using 2-fold serial dilutions of 17β-estradiol at the concentration of 3.1 µg/L. The detection limit for YES assay was 0.02 µg/L (0.09 nM), a well concentration corresponding to 0.4 µg/L in sample. Since the GC/MS detection limits for 17β-estradiol and estrone (5 µg/L) are much higher than that of YES assays (0.4 µg/L), samples with detectable estrogen concentrations by GC/MS were not further analyzed by YES assays. Therefore, only samples from estrogen degradation tests of strain KC8 were analyzed with YES assays. Nonspecific Monooxygenase Activity Assays. The activities of nonspecific monooxygenases in strains KC6-8 were examined by using a naphthalene oxidation assay as described by Chu and Alvarez-Cohen (31). Nonspecific monooxygenase can oxidize naphthalene into naphthol, which can interact with tetrazotized o-dianisidine to produce purple naphthol-diazo complex. The quantity of the purple complex can be determined by using a Hewlett-Packard G1130A UVvisible spectrophotometer at 530 nm. Negative controls without naphthalene were used. Nitrosomonas europaea, an ammonia-oxidizing bacterium, was used as positive controls. Nonspecific Dioxygenase Activity Assays. The activities of nonspecific dioxygenases in strains KC6-8 were determined by using an indole oxidation assay that was originally developed for determining activity of toluene dioxygenases (32). Toluene dioxygenase can oxidize indole into dark red colored, indoxyl. The production of indoxyl was monitored using a Hewlett-Packard G1130A UV-visible spectrophotometer at 400 nm. Experiments were conducted as described by Jenkins et al. (32). Reaction mixtures excluding indole were used for negative controls. An E. coli strain expressing naphthalene dioxygenase (E. coli TG1/pBS (Kan) NDO) (33) was used as positive controls for indole oxidation assays. 488

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Results Phylogenetic Diversity of 17β-Estradiol-Degrading Isolates. Fourteen 17β-estradiol-degrading bacteria, designated as strains KC1-14, were successfully isolated from the estrogendegrading bioreactor that was originally inoculated with activated sludge. The morphological properties of the 14 isolates can be seen in Table S1, Supporting Information. The analysis of 16S rRNA gene sequences (∼1300 bp) of these isolates revealed that they were phylogenetically diverse (Figure 1). These isolates widely distributed among eight different generasAminobacter, Brevundimonas, Escherichia, Flavobacterium, Microbacterium, Nocardioides, Rhodococcus, and Sphingomonassof three Phyla: Proteobacteria, Actinobacteria, and Bacteroidetes. Strains KC1-5 belonged to five different genera in Actinobacteria and Bacteroidetes. The rest of the isolates (strains KC6-14) clustered around R-proteobacteria, except for strain KC13 that belonged to γ-proteobacteria. No isolate belonged to β-proteobacteria. Many isolates showed high degrees of similarities to many known bacteria. For example, strains KC3, KC4, KC5, KC12, and KC13 showed >99% homology in 16S rRNA gene sequence of Nocardioides simplex, Rhodococcus rubber M2, Microbacteria testaceum, Brevundimmonas vesicularies, and Escherichia coli, respectively. Strains KC6 and KC7 are members in the genus Aminobacter containing strains known for degrading atrazine (34). Another five isolates, strains KC8, KC9, KC10, KC11, and KC14, were clustered to the genus Sphingomonas. Strain KC2 has a 91% homology to an uncultured bacterium clone MIZ31. Unlike other 13 isolates, strain KC2 showed no close relationship to any 16S rRNA gene sequences of known bacterial isolates deposited in the GenBank, suggesting that strain KC2 might be a novel species in the genus Flavobacterium. According to 16S rRNA analysis, all isolates are different from known estrogen-transforming bacteria (18-20) and hormone-degrading cultures, Novosphingobium sp ARI-1 (the first reported 17β-estradioldegrader (22)), species of Rhodococcus zopfii and Rhodococcus equi (estrogen-degrading bacteria (23)), one strain of Achromobacter and one strain of Ralstonia (24), and Comamonas testosteroni (a known testosterone-degrader (35)). Estrogen Degradation Patterns. Results of 17β-estradiol degradation tests indicated that all 14 isolates could convert 17β-estradiol to estrone within 7 days. However, only three strains (strains KC6-8) were capable of degrading estrone. Based on the extent of estrogen being transformed and the estrogenicity of metabolites and/or end products of estrogen degradation, three different degradation patterns (patterns A-C) were observed. These three degradation patterns A-C are described as follows: Degradation Pattern A. Isolates in this group were capable of degrading 17β-estradiol but not estrone within 7 days. One-to-one stoichiometric conversion of 17β-estradiol to estrone was observed, and the sum of molar concentration of 17β-estradiol and estrone remained unchanged for 7 days. Complete transformation of 17β-estradiol to estrone occurred rapidly within 1 day (pattern A1, Figure 2) or slowly within 7 days (pattern A2, Figure 2). However, as the concentrations of 17β-estradiol decreased, the concentrations of estrone increased and accumulated over the tested period, suggesting that estrone was a metabolite during 17β-estradiol transformation. Strains KC4, KC5, KC9, KC10, and KC11 exhibited degradation pattern A1 (data not shown). Strains KC1, KC2, KC3, KC12, KC13, and KC14 exhibited degradation pattern A2 (data not shown). No estrone was degraded during estrone degradation tests using these 11 isolates. The observation was consistent with the results from 17β-estradiol degradation that these isolates lacked the ability to degrade estrone. Degradation Pattern B. Unlike degradation pattern A, isolates in this group exhibited the ability to transform estrone

FIGURE 1. Phylogenetic distribution of estrogen-utilizing cultures isolated from enrichment cultures. Calculation of the phylogenetic tree was based on the neighbor-joining method with bootstrapping. The tree was rooted with the 16S rRNA gene sequence of Methanococcus thermolithotrophicus (belonging to the domain Archaea) as the outgroup. Since the 16S rRNA gene sequences of four 17β-estradioldegrading Rhodococcus strains (23) are not available from the GenBank, two closely related strains, Rhodococcus equi and Rodocuccus zopfii, are used. Values near the branches points are based on 100 bootstrap replications. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. within 7 days. In pattern B, the transformation of 17β-estradiol to estrone occurred much slower compared to those observed in pattern A. After the initial accumulation of estrone during 17β-estradiol degradation, the concentrations of estrone began to decline after day 5. Strains KC6 and KC7 showed this degradation trend (Figure 3). Their abilities to degrade estrone were consistent with results observed from the results of the 7-day estrone degradation tests. Approximately 21 ( 5% and 27 ( 4% of estrone were degraded by strains KC6 and KC7, respectively. Degradation Pattern C. Degradation of 17β-estradiol and estrone were much improved in pattern C. 17β-Estradiol was rapidly transformed into nonestrogenic compounds within 7 days. Only strain KC8 showed this degradation pattern (Figure 4). Interestingly, an increase of estrone concentration was observed in the first 20 min of 17β-estradiol degradation.

Within 24 h, 17β-estradiol was no longer detected, and estrone was measured at an average concentration of 48 µg/L. On day 3, only trace estrone was detected (14 µg/L). On day 5 and day 7, neither 17β-estradiol nor estrone was detected by GC/MS. No estrogenic activity was observed in day 5 and day 7 samples as determined by YES assays, suggesting the absence of estrogenic metabolites and/or end products from 17β-estradiol degradation. The ability of strain KC8 to degrade estrone was further confirmed through estrone degradation tests. Estrone was rapidly degraded by strain KC8 within 1 day (data not shown), consistent with those observed in pattern C of 17β-estradiol degradation. The overall results indicated that strain KC8 could rapidly degrade 17β-estradiol and estrone into nonestrogenic compounds. VOL. 41, NO. 2, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Estrogen degradation pattern A. 17β-Estradiol was completely converted to estrone that was later accumulated in liquid medium. Strain KC4 rapidly converted 17β-estradiol into estrone within 1 day (A, pattern A1). Strain KC14 converted 17βestradiol into estrone at a slower rate in 7 days (B, pattern A2). Solid diamonds (17β-estradiol) and open squares (estrone). The bars indicate the ranges of duplicate experiments. 17β-Estradiol as a Growth Substrate. After 25 days of incubation, the amended 17β-estradiol was depleted in flasks inoculated with strain KC8 (Figure 5). Protein contents in the flasks increased from 6 mg/L to 63 mg/L. The average growth yield was estimated to be 0.23 mg of protein/mg of 17β-estradiol. The doubling time of strain KC8 using 17βestradiol was estimated to be 27 h. However, no significant degradation of 17β-estradiol or increase in protein contents was observed for strains KC6 or KC7 over 36 days (data not shown), suggesting that strains KC6-7 were not able to use 17β-estradiol as a sole carbon source for growth. Nonspecific Oxygenase Activity in Strains KC6-8. Positive results of naphthalene oxidation assays were observed for all three strains, suggesting the presence of nonspecific monooxygenase enzymes. Negative results of indole oxidation assays were observed for strains KC6-8, implicating that these strains might not have nonspecific dioxygenase enzymes.

Discussion This study reported 14 phylogenetically diverse 17β-estradioldegrading bacteria isolated from activated sludge. None of these isolates were similar to these previously reported 17βestradiol-transforming bacteria nor to seven recently known estrogen-degrading strains isolated from activated sludge (one strain of Novosphingobium (22), one strain of Achromobacter, one strain of Ralstonia (24), and four strains of Rhodococcus (23). In two recent studies (22, 23, Yoshimoto, 2004 #3098), high concentrations of 17β-estradiol (0.1% w/v) were used for enrichment and isolation. In contrast, this study employed 10-fold lower concentrations of estrogen 490

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FIGURE 3. Estrogen degradation pattern B. 17β-Estradiol and estrone were degraded at slower rates, leading to reduction of total estrogenic activity and estrogen concentrations. Solid diamonds (17β-estradiol) and open squares (estrone). The bars indicate the ranges of duplicate experiments.

FIGURE 4. Estrogen degradation pattern C. 17β-Estradiol and estrone were degraded rapidly into none estrogenic compounds in 7 days. Solid diamonds (17β-estradiol) and open squares (estrone). The bars indicate the ranges of duplicate experiments. mixtures for enrichment and 30-fold lower concentrations of 17β-estradiol (∼3 mg/L) for isolation. Our experimental approaches might be responsible for the success in isolating 14 phylogenetically diverse 17β-estradiol-degrading bacteria. Since the ambient concentrations of estrogens in wastewater are even much lower than the concentrations used in this study, further studies are needed to address the prevalence of these isolates in various biological treatment processes as well as to determine the significance of these isolates in estrogen removal in situ. While the ability to transform 17β-estradiol to estrone was observed in all 14 isolates, the ability to degrade estrone was observed only in three strains (strains KC6-8). Along

FIGURE 5. Degradation of 17β-estradiol and growth of strain KC8 in NMS-estrogen medium. The growth was monitored by measuring the protein concentration of the cultures. Solid diamonds (17βestradiol), open diamonds (protein content, with 17β-estradiol), and open circles (protein content, without 17β-estradiol (negative controls)). The bars indicate the ranges of duplicate experiments. with the production and/or accumulation of estrone during 17β-estradiol degradation (Figures 2-4), three points could be made. First, the ability to oxidize the secondary alcohol on the C17 position of 17β-estradiol to ketone might be a common feature among these isolates. Second, estrone was a major metabolite during 17β-estradiol biodegradation. Third, the step to degrade estrone might be the rate-limiting step for converting estrogens (17β-estradiol and estrone) to nonestrogenic metabolites or end products. These suggestions were supported by many previous studies. For example, many field studies observed a good removal of 17β-estradiol but a less satisfactory removal of estrone by wastewater treatment plants (9, 10, 14, 36). In some cases, much higher concentrations of estrone were observed in effluent than in influent (9, 10). Laboratory batch tests using 14C-labeled estrogens also observed a better removal for 17β-estradiol than for estrone (13). Another important finding of this study was to elucidate three distinctive patterns of estrogen degradation (patterns A-C). One key aspect in evaluating their degradation abilities was to know whether these isolates can transform estrogens to nonestrogenic compounds, such that the overall estrogenic activity could be completely diminished. Since the potency of estrone is about 38-50% of that of 17β-estradiol according to YES assays (9, 37), the transformation of 17β-estardiol to estrone reduced the overall estrogenic activity of the tested samples. Accordingly, 11 isolates showing degradation pattern A reduced total estrogenicity but not total estrogen concentration (17β-estradiol + estrone), while strain KC8 showed degradation pattern C tranformed 17β-estradiol to nonestrogenic metabolites and/or end products (i.e., where the estrogenicity was completely diminished). The different degradation abilities of these isolates might be explained by two possible degradation mechanisms, nongrowth linked (cometabolic) and/or growth-linked (metabolic) reactions. Cometabolic reactionssnonbeneficial reactions catalyzed by existing enzymes that are designed specific for other purposessmight be responsible for estrogen degradation resulting in degradation pattern A. This hypothesis was supported by several lines of evidence observed in this study. Strains showing the ability to convert 17βestradiol to estrone in a rapid and stoichiometric matter (pattern A1) strongly suggested that degradation of 17βestradiol might be due to cometabolic reactions using enzyme(s) that was already present in these strains. The slower transformation of 17β-estradiol to estrone observed in degradation pattern A2 might be due to a weaker affinity of enzyme to 17β-estradiol. Furthermore, several attempts to grow these strains with 17β-estrodial as a sole carbon source were unsuccessful (data not shown).

These observations of a slight removal of estrone (2030% in 7 days) were insufficient to indicate which degradation mechanism was responsible for estrogen degradation in strains KC6 and KC7. A recent study has reported estrone was converted to 17R-estradiol under nitrate-reducing conditions (38). In our 17β-estradiol and estrone degradation tests, no 17R-estradiol was detected. Yet, more tests conducted with a longer experimental duration are needed to clearly illustrate the biodegradation of estrone. Nonetheless, the results of growth tests for strains KC6-7 suggested that estrogens were most likely degraded by strains KC6 and KC7 via cometabolic reactions. Growth-linked, metabolic reactionsscommonly involved in energy or carbon sources for microbial growthsmight explain the degradation pattern C shown by strain KC8. This hypothesis was supported by the results of growth tests. As shown in Figure 5, strain KC8 was capable of growing in the acetone-free NMS-estrogen medium (with 20 mg of 17βestradiol) for 2 weeks, with an average growth yield of 0.23 mg of protein/mg of 17β-estradiol and a doubling time of 27 h. The implication of the widespread ability to transform 17β-estradiol to estrone among 14 phylogenetically diverse isolates is important, since the total estrogenic activity is readily reduced. Interestingly, only three strains were found to degrade estrone. Since estrone still has a relatively high estrogenic potency (37) and higher average effluent concentrations (about 3.5 times higher than 17β-estradiol) (9, 10), our results suggested that effective removal of estrone in wastewater is probably the key to reduce total estrogenicity in the effluent. Three strains, strains KC6, KC7, and KC8, had exhibited degradation ability toward estrone and thus hold a great promise for completely mineralizing estrogens. The results of enzymatic characterization showed that strain KC8 exhibited nonspecific monooxygenase activity but not dioxygenase activity. Similar findings were also obtained for strains KC6 and KC7. Nonspecific monooxygenases are known to be responsible for metabolic and/or cometabolic reactions of a wide range of organics (39, 40). Shi et al. suggested that ammonia monooxygenase (a nonspecific monooxygenase enzyme produced by ammonia-oxidizing bacteria) might be responsible for estrogen degradation (21). In this study, it is likely that nonspecific monooxygenase enzymes were responsible for estrogen degradation by strain KC8. However, more experiments are needed to test this hypothesis. Several aspects, including the ability to use other available organics and other estrogenic compounds in wastewater, the pathways, enzymes, and kinetics of estrogen degradation, are currently under investigation in order to fully capitalize the degradation ability of these three strains for enhanced estrogen degradation.

Acknowledgments The authors would like to thank Mr. Hsion-Wen Kuo and Ms. Sara K. Ferrell for assistance in streaking, cloning, and sequencing. The authors also thank Dr. Thomas K. Wood, Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX for supplying the strain E. coli TG1/pBS (Kan) NDO.

Supporting Information Available Morphological properties of the 14 isolates (Table S1). This material is available free of charge via the Internet at http:// pubs.acs.org.

Literature Cited (1) Tyler, C. R.; Jobling, S.; Sumpter, J. P. Endocrine disruption in wildlife: a critical review of the evidence. Crit. Rew. Toxicol. 1998, 28, 319-361. VOL. 41, NO. 2, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Received for review April 17, 2006. Revised manuscript received October 20, 2006. Accepted October 31, 2006. ES060923F