Effects of the co-formulated antibiotics on sludge anaerobic digestion: transformation, microbial stress response, and community evolution

Abstract

Co-formulated antibiotics may exert more complex effects on sludge anaerobic digestion (AD) due to their synergistic mechanisms. However, studies on the transformation and effects of antibiotic combinations in AD systems remain limited. This study investigates the impacts of the representative co-formulated antibiotics sulfamethoxazole and trimethoprim (SMX/TMP) on sludge AD. Results demonstrated that both antibiotics interfered with key metabolic pathways, resulting in marked suppression of methane generation. Bliss Independence Model (BIM) analysis demonstrated that their joint effects were concentration-dependent, with synergistic inhibition occurred at lower doses and higher levels produced antagonistic interactions, likely reflecting metabolic saturation. Further experiments revealed co-exposure triggered excessive reactive oxygen species (ROS) accumulation, decreased key enzyme activity, cell activities. While partial biotransformation of SMX/TMP was observed, transformation products did not serve as carbon sources for methane enhancement. Partitioning assessment showed that both compounds were predominantly retained within extracellular polymeric substances (EPS), with notable fractions remaining in soluble EPS, indicating restricted cellular penetration and potential antibiotic residues in effluent. Microbial community analysis showed severe inhibition of hydrolytic and acidogenic microorganisms such as Syntrophomonas and Romboutsia, but significantly enhanced the abundance of antibiotic degrading functional microorganisms such as norank_f_Anaerolineaceae, which mean a potential degradation in AD system. Moreover, conventional methanogens decreased severely but Candidatus Methanofastidiosum displayed notable resilience, indicating a methane production feasibility in antibiotics stress AD system. These findings provide a comprehensive understanding of the concentration-dependent interaction, transformation, and partitioning behavior of co-occurring SMX/TMP, offering new mechanistic insights into their ecological risks and persistence in AD systems.

Graphical abstract

Keywords

Trimethoprim / Sulfamethoxazole / Waste activated sludge / Methane production / Anaerobic digestion

Highlight

	● TMP/SMX showed dose-dependent synergy at low levels and antagonism at high levels.
	● TMP and SMX induced oxidative stress and suppressed key enzyme activities in anaerobic digestion.
	● TMP accumulated intracellularly, whereas SMX bound extracellularly, leading to distinct inhibition mechanisms.
	● Partial antibiotic degradation occurred but did not contribute to methane production.

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