Investigation of a microbial consortium able to degrade selected emerging pollutants with endocrine-disrupting capabilities from wastewater in Bloemfontein

Abstract

Emerging pollutants (EPs) have become a growing environmental concern due to their persistence, ubiquity, and potential to cause adverse ecological and human health effects. Among these, endocrine-disrupting chemicals (EDCs) are of particular interest because of their ability to interfere with hormonal systems. Endocrine-disrupting chemicals (EDCs) such as atrazine (ATZ), bisphenol A (BPA), 17α-ethinylestradiol (EE2), and triclosan (TCS) are known to be persistent in the environment and their presence is of global concern. These compounds are often detected in the final effluent of wastewater, indicating the incomplete removal of EDCs. Some wastewater treatment plants (WWTPs) utilise wastewater stabilisation ponds (WSPs) that use physical and biological processes to remove EPs. This research aimed to address the identified knowledge gap by investigating the occurrence and persistence of selected EDCs in a wastewater stabilisation system, particularly in two maturation ponds, in Bloemfontein, South Africa, and to determine their biodegradation by the indigenous microorganism consortium. The concentrations of ATZ, BPA, EE2 and TCS in the inflow and outflow of two wastewater maturation ponds (the inflow and the outflow ponds) were quantified by LC-MS/MS. Microbial composition was characterised using next generation sequencing (NGS). The inhibitory effects of these EDCs on microbial community were also investigated by determining the minimum inhibitory concentrations (MIC). Laboratory enrichment experiments were conducted in mineral salts medium (MSM) inoculated with pond water, with each EDC supplemented at 10 mg/L, incubated at 26°C, and shaken for 35 days. Degradation was monitored over 35 days by LC-MS/MS using peak area (AUC) as a proxy for relative concentration, and microbial growth was tracked through optical density (OD) and colony-forming units (CFU). All four target EDCs were detected in the maturation ponds at variable concentrations. The concentrations of EE2 in the inflow pond ranged from 0.0057 μg/mL to 125.0 μg/mL. Physicochemical variables revealed that pH and TDS influenced the microbial communities in these ponds. Microbial diversity investigated by 16S rRNA sequencing showed the presence of Proteobacteria, the most predominant phylum across the inflow and outflow samples. At the genus level, Flavobacterium and Microcystis were predominant, followed by Dechloromonas and Pseudomonas across the inflow and outflow pond samples. ITS analysis revealed unidentified phyla that accounted for 68.85% of all ASVs, followed by Ascomycota that occurred across the inflow and outflow samples. The inhibitory effects of EDCs on the microbial growth in the wastewater maturation ponds under study were evident. MIC assay results indicated that microbial growth was largely unaffected at lower concentrations (0.1 - 1 μg/mL), with inhibition becoming evident at higher doses (10 - 100 μg/mL). ATZ showed the lowest toxicity, sustaining microbial growth even at 100 μg/mL. BPA exhibited moderate inhibition, with growth declining significantly at 100 μg/mL. EE2 induced marked inhibition between 10 - 100 μg/mL, while TCS demonstrated the highest toxicity, suppressing microbial growth even at concentrations as low as 1 - 10 μg/mL. Maximum optical density (OD600nm) and colony forming units (CFU/mL) of LogOD600nm 0.387, and 1.23 x 107 CFU/mL were observed by Day 35. Microbial growth data confirmed active proliferation, suggesting a potential biological contribution to the reduction in the concentrations of EDCs. Study showed 90% signal reduction within 14 days for EE2 and BPA, while TCS showed only limited removal (66%) relative to controls. A spike in TCS control flasks on Day 35 and an increase in the experimental flasks suggested analytical challenges. A possible interference in MSM matrix with ionisation and recovery of TCS was possible. This study demonstrated that the maturation ponds in the wastewater stabilisation system acted as reservoirs for diverse EDCs and harboured microbial communities with biodegradative potential. The integration of field monitoring, microbial characterisation, and laboratory biodegradation assays highlighted both the promise and challenges of EDC bioremediation. While BPA and EE2 were readily degraded, ATZ remained recalcitrant, and TCS posed both analytical and biological barriers to removal. These findings underscore the importance of coupling environmental assessments with laboratory experiments to fully understand EDC. Future work should refine detection methods and media component interference for triclosan detection, investigate metabolic pathways, and explore the role of microbial consortia in enhancing pollutant removal.