A 17β-Estradiol-utilizing Bacterium, Sphingomonas Strain KC8: Part I

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Environ. Sci. Technol. 2010, 44, 4943–4950

A 17β-Estradiol-utilizing Bacterium, Sphingomonas Strain KC8: Part I Characterization and Abundance in Wastewater Treatment Plants HYUNGKEUN ROH AND KUNG-HUI CHU* Zachry Department of Civil Engineering, Texas A&M University, College Station, TX77843-3136

Received January 18, 2010. Revised manuscript received May 13, 2010. Accepted May 21, 2010.

A 17β-estradiol-utilizing bacterium, Sphingomonas strain KC8, was characterized in terms of its utilization kinetics toward 17β-estradiol, estrone, and testosterone. The maximum specific substrate utilization rates (qm) are 0.37, 0.50, and 0.17 mgsubstrate/mg-protein/day for 17β-estradiol, estrone, and testosterone, respectively. The half-velocity constants (Ks) are 1.9 mg/L for 17β-estradiol, 2.7 mg/L for estrone, and 2.4 mg/L for testosterone. Strain KC8 can grow on testosterone, glucose, sodium succinate, and sodium acetate, but not on phenol. Also, strain KC8 cannot degrade two common wastewater micropollutants, bisphenol A (a plasticizer) and triclosan (an antimicrobial agent). Unlike Novosphingobium sp. ARI-1 (a known estrogen-degrader) that would lose its degradation ability toward estrone after growing on a nutrient-rich estrogen-free medium for 7 days, strain KC8 was still able to degrade both 17β-estradiol and estrone after growing on the same medium for 15 days. Strains KC8 and ARI-1 were molecularly detected in activated sludge of municipal wastewater treatment plants (WWTPs) operating under solid retention times of 2-30 days. The concentrations of strain KC8 were 2-3 orders higher than those of strain ARI-1 in the WWTPs, suggesting that strain KC8 is ubiquitous in WWTPs and might play an important role in estrogen removal.

Introduction Exposure to estrogens (as low as in the range of ng/L) in the environment has been known to cause adverse effects on the reproductive system of aquatic wildlife (1-3). Estrogens (such as estrone, 17β-estradiol, and estriol) are reproductive steroidal hormones (4) that are produced naturally to modulate important biological function in humans and animals. After metabolized in the body, both conjugated and unconjugated estrogens are excreted in urine that enters wastewater treatment plants (WWTPs). Conventional WWTPs are designed to remove macropollutants (common organics) not micropollutants like estrogens. Unremoved estrogens are continuously released into the environment via effluent discharge. Not surprisingly, estrogens are detected in effluent (92% similar) (AY771798, AY771797, AY771794, X94101, AJ009706, AJ292601, DQ177493, and AY509242) with KC8 16S rRNA gene sequence (DQ066438). Similarly, primers (ARI-1f and ARI-1r) and TaqMan probe (ARI-1 Taq) were designed to target the 16S rRNA gene sequence (AB070237) of ARI-1 strain. Twenty-eight closely related bacterial 16S rRNA gene sequences (EU127294, DQ985055, AB219359, AY690709, AB177883, AJ416411, AJ303009, AB025012-4, U20756, U20773-4, AJ001051, DQ840049, EF628247, EF421434, EF044233, AB023290, AJ009707, AJ000920, AJ746092, AJ746094, AB110635, AF411072, AB362778, AB220123, AB220125) were used for the design. The specificity of designed primers and probes were checked using the Basic Local Alignment Search Tool (BLAST) of GenBank. The primers (KC8f and KC8r) and probe (KC8 Taq) had at least three total mismatches when comparing with more than 100 bacterial 16S rRNA gene sequences and the eight closely related bacterial 16S rRNA gene sequences mentioned above (accessed GenBank on 9/29/2005). Therefore, the designed primers and probe can discriminate 16S rRNA gene sequences of strain KC8 from closely related strains. Similarly, the primers (ARI-1f and ARI-1r) and probe (ARI-1 Taq) had at least three total mismatches after comparing with bacterial 16S rRNA gene sequences, based on the available sequences deposited in the GenBank (accessed on 3/13/2008). DNA Extraction and Real-time PCR Assays. FastDNA SPIN kit and FastDNA SPIN kit for soil (MP Biomedicals, LLC, Solon, OH) were used to extract genomic DNA of each

TABLE 1. Prevalence of Three Known Estrogen-Degrading Bacteria and amoA Gene in Selected WWTPs WWTPs

WWTP No. 1

WWTP No. 2

treatment capacity (MGD2)b SRT (d) MLVSS (g/L) BOD5 removal (%) ammonia removal (%)

9.5 7-8 2.2 97-98 95

75 10-12 1.5 97- 99 99

total bacteria

8.0 × 109 (2.2 × 109) 7.2 × 104 (7.2 × 104) 6.3 × 103 (6.3 × 103) 1.5 × 105 (1.5 × 105) 1.7 × 104 (8.4 × 103)

1.4 × 1010 (4.0 × 109) 2.8 × 105 (2.8 × 105) 2.1 × 102 (2.1 × 102) 1.9 × 105 (1.9 × 105) 2.1 × 104 (8.4 × 103)

WWTP No. 3 1st stage

operating parameters

strain KC8 real-time PCR assay, no. copies/mL (cells/mL)a

ARI-1 AOB amoA

2nd stage 200

2 1.4

20-30 1.1 91- 99 84 - 99

1.7 × 1010 (4.8 × 109) 2.8 × 104 (2.8 × 104) 5.2 × 102 (5.2 × 102) 7.4 × 105 (7.4 × 105) 1.5 × 104 (7.6 × 103)

6.5 × 109 (1.8 × 109) 5.5 × 104 (5.5 × 104) 2.7 × 102 (2.7 × 102) 4.8 × 105 (4.8 × 105) 1.6 × 104 (7.9 × 103)

a cells/mL ) (no. copies/mL)/(gene copy number/cell). Gene copy number/cell is assumed to be 3.6 (total 16S rRNA), 1.0 (strains KC8, ARI-1, and AOB), and 2 (amoA gene) (28). b MGD ) million gallons per day.

FIGURE 1. Degradation of testosterone by strain KC8 (A). Utilization of testosterone by strain KC8 in nitrate mineral salts medium (B). The bars represent the ranges of duplicate experiments. bacterium (pure) and activated sludge samples (mixed), respectively. Extraction procedure was followed as described by Yu et al. (24, 25). DNA concentrations were determined

using a Hoefer DyNa Quant 300 Fluorometer (Pharmacia Biotech, San Francisco, CA). Extracted DNAs were stored at -20 °C until use. VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Strain KC8 utilized glucose, sodium succinate, and sodium acetate, but not phenol, as a sole carbon source. The bars indicate the ranges of duplicate data points.

TABLE 2. Substrate Utilization Kinetic Parameters of Strain KC8a substrate parameters

17β-estradiol

estrone

q (mg-substrate/mg-protein/day) qm(mg-BODL/mg -protein/day)c Ks(mg-substrate/L) Ks(mg-BODL/L)c Y (g-VSS/g-BODL)c,e doubling time(hr)

0.37 ( 0. 02 1.00 ( 0.05 1.9 ( 0.2 5.1 ( 0.5 0.22d 27d

0.50 ( 0.02 1.35 ( 0.05 2.7 ( 0.3 7.3 ( 0.8 -

sodium sodium typical value for testos-terone glucose succinate acetate activated sludge processb 0.17 ( 0.01 0.48 ( 0.03 2.4 ( 0.4 7.0 ( 1.2 0.24 61

0.05 20

0.04 24

0.42 29

52-70e >10 0.42-0.49 -

a qm) maximum specific substrate utilization rate. Ks ) half velocity constants. Y ) yield coefficient. - ) Not available. ( ) The range of duplicate experiments. b Data from Rittmann and McCarty (27). c Based on theoretical oxygen demand, where 1 mg 17β-estradiol/L ) 2.7 mg BODL/L; 1 mg- testosterone/L ) 2.8 mg BODL/L; 1 mg glucose/L ) 1.1 mg BODL /L; 1 mg- sodium succinate/L ) 0.6 mg BODL/L; and 1 mg sodium acetate/L ) 0.5 mg BODL/L. d Data from Yu et al. (17). e The reported qm values range from 20-27 mg BODL/ mg VSS/day. For comparison, the unit of these values was converted to mg BODL/ mg protein/day. The conversion was done by using VSS (g) ) 2.6 × protein (g) as measured in the laboratory.

Real-time PCR assays were conducted to quantify total bacteria population, strains KC8 and ARI-1, AOB, and amoA gene. PCR mixture (25 µL of total volume) containing 600 nM forward and reverse primers (SI Table S1), 500 nM of probe, 12.5 µL of Taq Mastermix (or SYBR Green I for amoA gene), and 2-50 ng of DNA template, was used. The PCR amplification protocol was listed in SI Table S1. The PCR amplification reactions were performed using a Bio-Rad iQ5Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA). Details of standard curve development are available in the SI. The DNA concentrations in samples were determined by comparing to those of standard curves containing known DNA concentrations. Chemical Analysis. Estrogen concentrations in liquid samples (containing both cells and growth medium) were determined by GC/MS analysis as described by Yu et al. (24) (see SI for details). The detection limits for 17β-estradiol and estrone were 5 µg/L. Testosterone in samples was extracted with the same volume of ethyl ether, resuspended with pyridine, and then derivatized with BSTFA with 1% TMCS (trimethylchlorosilane). The derivatized samples were injected into GC/MS system using SIM mode. The primary ion, m/z 432, was selected for testosterone quantification. The detection limit for testosterone was 50 µg/L. Concentrations of bisphenol A and triclosan were determined by GC/ 4946

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MS analysis as described by Roh et al. (23) (see SI for details). The detection limits for triclosan and bisphenol A were 50 and 10 µg/L, respectively. The cellular proteins in the samples were released by using a sonicator (Branson Sonifier-150) at a power of 50 W for 10 s (26). The treatment was repeated for three times with cooling the samples on ice for 45 s between the treatments. The released protein content was then determined using a BCA protein assay kit. Determination of Monod Kinetic Parameters. Monod kinetics,q ) (qm · S)/(Ks + S), was used to describe utilization kinetics for estrogens and testosterone. The experimental data obtained from kinetic tests were plotted as specific substrate utilization rates (q, mass of substrate/mass of cell protein/time) against substrate concentrations (S, mass/ volume). The specific substrate utilization rates were obtained by dividing the initial substrate utilization rates over biomass added. The initial substrate utilization rates were determined by dividing the mass difference of substrate over 2 h of incubation period. No biomass changes were observed over 2 h. The maximum rate of specific substrate utilization (qm, mass of substrate/mass of cell protein/time) and the halfvelocity constant (Ks, mass of substrate/volume) were determined through curve fitting by using Sigmaplot 8.0 (SPSS Inc.) (27).

FIGURE 3. Monod degradation kinetic of 17β-estradiol (A), estrone (B), and testosterone (C) by strain KC8. The changes of biomass concentrations were negligible within 2 h (less than 5%). Solid symbols represent experimental data and the dash line represents fitted Monod kinetic curve. The bars indicate the ranges of duplicate data points.

Results Ability to Utilize and/or Degrade Wastewater Macro- and Micropollutants. Results of substrate degradation tests showed that strain KC8 can degrade testosterone, but not triclosan or bisphenol A. The inability of strain KC8 to degrade triclosan or bisphenol A might be due to toxicity of these compounds. It might be particularly true for triclosan, since it is an antimicrobial agent. However, the toxicity aspect was

not further examined in this study. Approximately 95% of added testosterone was degraded within 1 day (Figure 1A). Strain KC8 could also grow on testosterone. After 25 days of incubation, the biomass of strain KC8 increased 12 times, from 4 to 48 mg protein/L (Figure 1B). Assuming all the degraded testosterone was used for microbial growth and microbial decay was not considered, the average value of the observed yields was 0.26 mg protein/mg testosterone and VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Effects of nutrient-rich medium on 17β-estradiol degradation ability of strains KC8 and ARI-1. After growing in complex nutrient (R2A) medium without 17β-estradiol for 15 days, the resting cells of strain KC8 still can degrade17β-estradiol and estrone but at much slower rates (A). 17β-estradiol was rapidly degraded by the resting cells of strain KC8 when pregrown in R2A medium containing 17β-estradiol (B) (17). After growing in R2A medium without 17β-estradiol for 7 d, the resting cells of strain ARI-1 could only degrade 17β -estradiol, but not estrone (C) (19). Similarly, 17β-estradiol was rapidly degraded by the resting cells of strain ARI-1 that were grown in R2A medium containing 17β-estradiol (D) (19). Solid squares represent 17β-estradiol. Open squares represent estrone. Open circles represent 17β-estradiol in controls. The bars represent the ranges of duplicate data points. the doubling time was estimated to be 61 h. Strain KC8 was able to utilize all organic substrates tested to support its growth, except for phenol. The biomass of strain KC8 (expressed as protein content) increased from 9 to 41 mg/L for glucose in 3 days; from 9 to 38 mg/L for sodium succinate in 2.5 days; and from 11 to 47 mg/L for sodium acetate in 5 days (Figure 2). Assuming each substrate was completely depleted when reached maximum optical densities, the observed average yields ranged from 0.04 to 0.42 mg VSS/mg BODL (VSS (g) ) 2.6 × protein (g) was used. The conversion relationship was determined in the laboratory). The doubling times were estimated ranging from 20 to 29 h when using different substrates (Table 2). Estrogen/Testosterone Utilization Kinetic Parameters. The Monod equation described estrogens and testosterone degradation kinetics well (Figure 3). The maximum specific substrate utilization rate (qm) were estimated to be 0.37, 0.50, and 0.17 mg substrate/mg protein/day for 17β-estradiol, estrone, and testosterone, respectively. The estimated half velocity constants (Ks) are 1.9 mg 17β-estradiol/L, 2.7 mg estrone/L, and 2.4 mg testosterone/L (Table 2). Changes of cellular protein content in the vials were negligible (less than 5%) during the kinetic experiments. Effects of Complex Nutrients on Estrogen Degradability. After grown on R2A agar plates without 17β-estradiol for 15 days (i.e., subculturing to a new R2A agar plates every 3 days for five times), strain KC8 still retained its ability to degrade 17β-estradiol and estrone (Figure 4A). Approximately 62% of initial 17β-estradiol was degraded in 5 days. Meanwhile, estrone concentrations increased in the first 30 h and then decreased to nondetectable level after 5 days. The results 4948

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were different from those obtained from the strain KC8 grown with a complex nutrient medium containing 17β-estradiol (Figure 4B), where 17β-estradiol was rapidly degraded to near zero within 24 h (17). These results were different from those obtained from strain ARI-1, which lost its estrogen degradability after grown in the nutrient-rich medium without estrogen (Figures 4C and D) (19). Validation of Real-Time PCR Assays. Real-time PCR assay for strain KC8 was validated using strain KC8, a mixed culture of an in-house lab-scale bioreactor operating with 5 days of SRT, and thirteen other estrogen degraders (strains KC1 through KC7 and KC9 through KC14). A linear relationship (r2 ) 0.998) was observed between the Ct values and the amounts of DNA of strain KC8 in the templates (ranging from 0.2 ng to 100 ng) (SI Figure S1). Similarly, a linear relationship (r2 ) 0.999) was observed for the mixed culture in the templates (ranging 0.5-150 ng) (SI Figure S1). No signals (Ct > 40 cycles) were detected when the assay was challenged against all thirteen other 17β-estradiol-degrading bacteria. Validation of real-time PCR assay for ARI-1 was conducted similarly using known amounts of DNA of strain ARI-1. A linear range (r2 ) 0.994) for templates containing 0.25-250 ng of DNA of strain ARI-1 was observed. For DNA of the mixed culture, the linear range was from 0.25 to 250 ng (r2 ) 0.993) (SI Figure S2). Again, no signals (Ct > 40 cycles) were detected when the real-time PCR assay for ARI-1 was challenged against eight KC strains (KC1 through KC2 and KC9 through KC14). Prevalence of Estrogen Degraders in WWTPs. The quantities of three known estrogen degraders in WWTP samples were quantified using real-time PCR assays. In

addition, the quantity of amoA gene in the activated sludge samples was measured, because amoA gene is coded for ammonia monooxygenase enzyme which is responsible for ammonia oxidation by AOB. Despite the differences in operating conditions and process configurations among three WWTPs, the quantity of strain KC8 was not much different (ranging from 2.8 × 104 to 2.8 × 105 copies/mL). However, much lower gene copies of ARI-1, ranging from 2.1 × 102 to 6.3 × 103 copies/mL, were observed in all WWTP samples. The concentrations of AOB were between 7.6 × 104 and 3.7 × 105 copies/mL. The amoA gene copy numbers were measured from 1.5 × 104 to 2.1 × 104 copies/mL (Table 1).

Discussion In this study, the 17β-estradiol-utilizing bacterium Sphingomonas strain KC8 was characterized from many different aspects. One important finding of this study was that strain KC8 could degrade and further utilize testosterone as a growth substrate. Such an ability of strain KC8 is favorable, since strain KC8 would remove testosterone and estrogens (17βestradiol and estrone) simultaneously from wastewater. The utilization kinetics of estrogens and testosterone by strain KC8 is one of important factors to evaluate the potential of using strain KC8 for effective estrogen and testosterone removal in biological systems. For easy comparison, the units of utilization kinetic parameters were converted to BODL basis (see Table 2). The measured qm values (0.5-1.4 mg BODL/mg-protein/day) are also smaller than the typical value for heterotrophs in activated sludge (reported as 20-27 mg BODL/mg VSS/day (27) or 52-70 mg BODL/mg protein/day as shown in Table 1). Assuming strain KC8 uses 17β-estradiol as a sole carbon source, based on these kinetic values and the small growth yield of strain KC8 (0.23 mg protein/mg 17β-estradiol) (17), an impractically long solid retention time (SRT) will be required for completely mixed activated sludge systems to remove estrogens down to ng/L levels. Additionally, one should also note that application of these kinetic data to predict estrogen degradation at very low concentrations in wastewater might be limited, in part due to the high estrogen concentrations used in the kinetic tests, the presence of other organics that can be also degraded by strain KC8, and many other unknown estrogen degraders in wastewater. The need of the long SRTs might be overcome if strain KC8 can grow rapidly on common organic constituents (macro-pollutants like glucose and other organics) in wastewater. In addition to be able to grow rapidly on common wastewater organics, the strain also has to retain its degradation ability toward estrogenic compounds. To investigate this aspect, strain KC8 was examined for its ability to grow on glucose, sodium succinate, or sodium acetate. Glucose is a representative carbohydrate in wastewater, with a typical influent concentration 104-fold higher than the concentration of estrogens in wastewater. Sodium succinate and sodium acetate are two major compounds in TCA (tricarboxylic acid) cycle responsible for generating energy and carbon sources in many microorganisms. As shown in Figure 2 and Table 2, strain KC8 can utilize these three organics with short doubling times ranging from 20 to 29 h, suggesting that strain KC8 is very likely to thrive in existing activated sludge systems. This hypothesis was supported by the detection of strain KC8 in the surveyed WWTPs (Table 1). Based on the estrogen degradation and growth kinetics of this study, a minimum SRT (θ min ) 1/µmax ) 1/(qmax · yield)) of 12 days would be required when strain KC8 uses 17β-estradiol as a sole carbon source and microbial decay was ignored. The highest ratio of strain KC8 to total microbial population (0.003%) was observed in WWTP No. 2 which has 10-12 days of SRT (Table 1). Another way to achieve a long solid retention time is to employ an attached growth system. However, this aspect

has not been examined in this study. Future studies are needed to examine whether a biofilm system would permit greater accumulation of strain KC8 for enhance estrogen removal. Effective removal of estrone might be a key to reduce total estrogenicity in wastewater (17), as many field studies reported elevated estrone in treated wastewater and not many known estrogen-degrading isolates can degrade estrone effectively. To our knowledge, both strains ARI-1 and KC8 could degrade 17β-estradiol and estrone rapidly when grew on complex nutrients with 17β-estradiol ((Figure 4B and D), but only strain KC8 still retained its degradation ability toward 17β-estradiol and estrone (Figure 4A). Furthermore, the concentrations of strain ARI-1 were much lower than the concentrations of AOB and strain KC8 in three WWTPs, suggesting that strain ARI-1 might not be important for estrogen removal in engineered bioreactors. These results implied that strain KC8 would play an important role on degrading estrogen into nonestrogenic metabolites/end product even in wastewater with low, and/or fluctuating concentrations of estrogens. However, more studies are needed to examine whether strain KC8 can degrade low level of estrogens in the presence of various wastewater organics, or whether the strain would preferentially degrade “easy” substrates (like glucose and acetate) over the estrogens.

Acknowledgments We thank Dr. Chang-Ping Yu for his assistance in the experiment.

Supporting Information Available Detail experimental methods for (i) degradation tests of common wastewater organics, (ii) chemical analysis, (iii) results of the validation of real-time PCR assays for strain KC8 and strain ARI-1, and (iv) protocols of real-time-PCR assays. This material is available free of charge via the Internet at http://pubs.acs.org.

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