Fourteen phylogenetically diverse 17beta-estradiol-degrading bacteria (strains KC1-14) were isolated from activated sludge of a wastewater treatment plant. These isolates widely distributed among eight different genera--Aminobacter (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 KC8-KC11 and KC14)--of three Phyla: Proteobacteria, Actinobacteria, and Bacteroidetes. All 14 isolates were capable of converting 17beta-estradiol to estrone, but only three strains (strains KC6, KC7, and KC8) showed the ability to degrade estrone. Only strain KC8 could use 17beta-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 17beta-estradiol was stoichiometrically converted to estrone. Estrone was confirmed to be a degradation product of 17beta-estradiol; however, estrone was not further degraded during the course of experiments. Strains KC6 and KC7 exhibited degradation pattern B, where both 17beta-estradiol and estrone were degraded, with slower 17beta-estradiol degradation rates than those observed in pattern A. Strain KC8 was the only strain exhibited degradation pattern C, where 17beta-estradiol and estrone were rapidly degraded within 3 days. No residual 17beta-estradiol and estrone or estrogenic activity was detected after 5 days, suggesting that strain KC8 could degrade 17beta-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.

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This study reported the application of 15N-stable isotope probing (SIP) to identify active hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-utilizing microorganisms in groundwater microcosms. Fifteen 16S rRNA gene sequences were derived from the 15N-DNA fraction (contributed from active microorganisms capable of using RDX as a nitrogen source) of microcosms receiving cheese whey. The 16S rRNA gene sequences belonged to Actinobacteria (two clones), alpha-Proteobacteria (seven clones), and gamma-Proteobacteria (six clones). Except for five sequences with high similarity to two known RDX degraders (Enterobacter cloacae and Pseudomonas fluorescens I-C), our results suggested that phylogenetically diverse microorganisms were capable of using RDX as a nitrogen source. Six sequences of the xplA gene (a known RDX-degrading catabolic gene) were detected from the 15N-DNA fraction. The xplA gene sequences were 96-99% similar to the xplA gene of Rhodococcus sp. DN22(a known RDX utilizer), suggesting that other RDX utilizers might contain xplA-like genes. Twenty-five 16S rRNA gene sequences recovered from the unenriched, RDX-contaminated groundwater clustered differently from those obtained from the 15N-DNA fraction of the cheese-whey-amended microcosm. Our results suggested that active RDX utilizers can be stimulated by nutrient source additions even if they are present at low densities, and that use of 15N-SIP can identifythese functional members of the microbial community.

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A 17beta-estradiol-utilizing bacterium, Sphingomonas strain KC8, was characterized in terms of its utilization kinetics toward 17beta-estradiol, estrone, and testosterone. The maximum specific substrate utilization rates (q(m)) are 0.37, 0.50, and 0.17 mg-substrate/mg-protein/day for 17beta-estradiol, estrone, and testosterone, respectively. The half-velocity constants (K(s)) are 1.9 mg/L for 17beta-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 17beta-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.