Concentration-dependent response of estrone-degrading bacterial community in activated sludge analyzed by microautoradiography-fluorescence in situ hybridization

Inefficient removal of estrone (E1) in wastewater treatment plants (WWTPs) causes feminizing effects in male aquatic creatures. As E1 is mainly removed by biodegradation, investigation of E1 degradation is important to determine better removal strategies. Using microautoradiography-fluorescence in situ hybridization (MAR-FISH), we demonstrated that the structures of [3H]E1-incorporating bacterial communities were different at different E1 concentrations applied to activated sludge. At 200 μg/L E1, almost all [3H]E1-incorporating cells were associated with either Betaproteobacteria or Gammaproteobacteria (60% and 40% of MAR (+) cells, respectively). The proportion of Betaproteobacteria and Gammaproteobacteria in the total number of [3H]E1-incorporating cells decreased as the concentration of E1 decreased. In contrast, the proportion of Alphaproteobacteria in the total number of [3H]E1-incorporating cells increased as the concentrations of E1 decreased. At the lowest applied concentration (540 ng/L), almost all the [3H]E1-incorporating cells were Alphaproteobacteria (96%). The results of MAR-FISH applied to sludge samples collected from various plant locations and activated sludge processes, and during different seasons also demonstrated the high contribution of Alphaproteobacteria to the entire E1-degrading bacterial community (50.4 ± 11% of the total number of [3H]E1-incorporating cells) at 1 μg/L E1. Since the E1 concentration in domestic wastewater is at sub-μg/L levels, the key E1 degraders in activated sludge of domestic WWTPs are probably be Alphaproteobacteria. All [3H]E1-incorporating Alphaproteobacteria were hybridized with probe ALF968. Few MAR (+) cells were Sphingomonadales. An E1-degrading bacterial community at low E1 concentration appeared to consist of diverse bacterial groups of Alphaproteobacteria. This study suggested that substrate concentration is an essential factor for revealing E1-degrading bacteria in complex communities.