Enrichment and characterization of steroid-degrading microbes for targeted removal of steroid hormone micropollutants in small-scale wastewater treatment solutions

In remote areas, wastewater treatment typically occurs directly at the source in decentralized systems, which often work well in removing biochemical oxygen demand, nitrogen, and phosphorus, but not micropollutants like pharmaceuticals and steroid hormones. Microorganisms capable of degrading such micropollutants could be used in bioengineered systems to increase micropollutant removal. In this study, bacteria were enriched from municipal wastewater in flow-through reactors supplemented with testosterone (TE), androsta-1,4-dien-3,17-dion (ADD), 17β-estradiol (E2), or 17α-ethinylestradiol (EE2). The removal of TE and ADD was stable over multiple transfers, and several androgen-degrading <i>Comamonas</i> strains were isolated. In contrast, the removal of E2 and EE2 was considerably slower, and no estrogen-degrading strains were isolated, suggesting that estrogens were removed mainly by adsorption and precipitation rather than by biological activity. The system's complexity was then increased to approach real-life conditions by increasing reactor size, adding lactate and acetate as alternative carbon sources, and/or sterilized real wastewater and supplementing a hormone mix. Sequencing-based community analysis confirmed <i>Comamonas</i> as the main androgen degrader in these reactors but did not identify any known estrogen degraders. Alternative carbon, but not real wastewater, reduced hormone removal rates. These results show that (i) efficient androgen-degrading bacteria can be readily isolated using flow-through reactors and low hormone concentrations, while isolation of estrogen degraders is more difficult, (ii) androgen-degrading <i>Comamonas</i> strains are good candidates for bioaugmentation of small-scale water treatment solutions, and (iii) alternative carbon sources might hinder steroid degradation. This provides a basis to improve steroid removal from wastewater by bioaugmentation to prevent hormone discharge into water bodies.IMPORTANCESteroid hormones are endocrine-disrupting compounds exhibiting adverse effects on humans and the environment even at very low concentrations. Their removal during water treatment is often insufficient, and novel methods are required to increase hormone micropollutant removal from wastewater. This study enriched and isolated steroid hormone-degrading microorganisms in a flow-through system and tested their usability for bioaugmentation of small-scale water treatment solutions. While removal of estrogen hormones was found to be mainly abiotic, efficient biological androgen degradation was achieved. The combined results show that flow-through reactors, along with appropriate surface materials, are suitable for isolating biofilm-forming and hormone-degrading bacteria and that these microbes can be used for bioaugmentation to remove low concentrations of steroid hormones from wastewater. Based on these results, recommendations for future enrichment studies for bioaugmentation are formulated, and potential pitfalls are discussed.