Since the Great Oxidation Event 2.4 billion years ago, microorganisms have evolved sophisticated responses to oxidative stress. These ancient adaptations remain relevant in modern engineered systems, particularly in conventional activated sludge (CAS) processes, which serve as significant reservoirs of antibiotic resistance genes (ARGs). While ROS-induced stress responses are known to promote ARG enrichment/emergence in pure cultures, their impact on ARG dynamics in wastewater treatment processes remains unexplored. Shotgun-metagenomics analysis of two hospital wastewater treatment plants showed that only 35-53 % of hospital effluent resistome was retained in final effluent. Despite this reduction, approximately 29-36 % of ARGs in CAS showed higher abundance than upstream stages, of which 20-22 % emerged de novo. Beta-lactamases and efflux pumps constituted nearly 47-53 % of these enriched ARGs. These ARGs exhibited significant correlations (p < 0.05) with ROS stress response genes (oxyR, soxR, sodAB, katG and ahpCF). The CAS resistome determined 58-75 % of the effluent ARG profiles, indicating treatment processes outweigh influent composition in shaping final resistome. Proof-of-concept batch reactor experiments confirmed increased ROS and ARG levels under high dissolved oxygen (8 mg/L) compared to low oxygen (2 mg/L) concentrations. Untargeted metaproteomics revealed higher expression of resistant proteins (e.g., OXA-184, OXA-576, PME-1, RpoB2, Tet(W/32/O)) under elevated ROS levels. Our findings demonstrate that CAS processes actively shape effluent resistome through ROS-mediated selection, indicating that treatment processes, rather than initial wastewater composition, determine final ARG profiles. This study indicates that the emergence of ARGs needs to be considered as an integral aspect of wastewater treatment design and operation to prevent antibiotic resistance dissemination.

As the global transition toward circular wastewater treatment intensifies, extracellular polymeric substances (EPS) have emerged as valuable targets for resource recovery. Although most related efforts have focused on aerobic granular sludge, conventional activated sludge systems, which account for most global wastewater treatment, remain underexploited. Building on the established link between oxidative stress and EPS biosynthesis in pure strains, it is proposed that strategically manipulating oxygen exposure patterns to intensify oxidative stress in activated sludge microbial communities could enhance EPS production. To test this, this study applied continuous oxygen perturbation under aerobic exposure to intensify oxidative stress. Compared to a stable oxygen condition simulating typical wastewater aeration, the perturbation considerably enhanced EPS yield to 74.4 mg/L/day, a 90.5 % increase over the stable condition (39.0 mg/L/day). To validate the role of oxidative stress in EPS enhancement, intermittent anoxic phases were introduced into the perturbation pattern to relieve oxidative stress, causing the EPS-enhancing effect to disappear, with yield dropping to 9.8 mg/L/day. Mechanistically, intensified oxidative stress under aerobic continuous perturbation was primarily driven by elevated reducing substrates for non-respiratory flavoenzymes, exemplified by glutamate synthase, glutathione reductase, and dihydrolipoamide dehydrogenase, which are prone to generate H₂O₂ as an unintended metabolic byproduct. Among the multiple microbial groups contributing to H₂O₂ production, Methylophilaceae, Comamonadaceae, and Rhodobacteraceae were distinguished by simultaneously exhibiting upregulation of EPS biosynthesis proteins, suggesting that taxa within these families collectively mediated both H₂O₂ production and EPS enhancement. By modulating aeration, this study offers a chemical-free, controllable strategy for enhancing EPS production within conventional activated sludge systems.

Climate change threatens freshwater ecosystems, potentially intensifying cyanobacterial blooms and antibiotic resistance. We investigated these risks in Cosseys Reservoir, New Zealand, using short-term warming simulations (22 °C, 24 °C, and 27 °C) with additional oxidative stress treatments. A metagenomic analysis revealed significant community shifts under warming. The cyanobacterial abundance increased from 6.11% to 20.53% at 24 °C, with Microcystaceae and Nostocaceae proliferating considerably. The microcystin synthesis gene (mcy) cluster showed a strong association with cyanobacterial abundance. Cyanobacteria exhibited enhanced nutrient acquisition (pstS gene) and an upregulated nitrogen metabolism under warming. Concurrently, antibiotic resistance genes (ARGs) increased, particularly multidrug resistance genes (50.82% of total ARGs). A co-association network analysis identified the key antibiotic-resistant bacteria (e.g., Streptococcus pneumoniae and Acinetobacter baylyi) and ARGs (e.g., acrB, MexK, rpoB2, and bacA) central to resistance dissemination under warming conditions. Oxidative stress exacerbated both cyanobacterial growth and ARGs’ proliferation, especially efflux pump genes (e.g., acrB, adeJ, ceoB, emrB, MexK, and muxB). This study demonstrated that even modest warming (2–5 °C) could promote both toxic cyanobacteria and antibiotic resistance. These findings underscore the synergistic effects of temperature and oxidative stress posed by climate change on water quality and public health, emphasizing the need for targeted management strategies in freshwater ecosystems. Future research should focus on long-term impacts and potential mitigation measures.

Dynamic oxygen fluctuations in activated sludge were investigated to enhance valuable biochemical production during wastewater treatment. Batch experiments compared constant aeration with rapid cycling between oxygen-rich and oxygen-poor states. Fluctuating oxygen concentrations (0-2 mg/L) significantly increased production of valuable biochemicals compared to constant oxygen concentration (2 mg/L). Continuous oxygen perturbations increased free amino acids by 35.7 ± 7.6 % and free fatty acids by 76.4 ± 13.0 %, while intermittent perturbations with anoxic periods enhanced free amino acids by 42.4 ± 8.1 % and free fatty acids by 39.3 ± 7.7 %. Fourteen standard amino acids showed significant increases, and most fatty acids had carbon chain lengths between C12-C22. Mechanistically, oxygen perturbations activated FNR and ArcA regulons, resulting in lower relative abundances of TCA cycle enzymes and higher abundances of amino acid and fatty acid biosynthetic enzymes. These findings demonstrate that controlled oxygen fluctuations in wastewater treatment can enhance the biochemical value of activated sludge with minimal process modifications, facilitating resource recovery.

Nitrous oxide (N ₂ O) emissions from wastewater treatment plants, with a warming potential 298 times that of CO ₂ , pose a significant challenge to lowering their carbon footprint. Current mitigation strategies focus on limiting N ₂ O formation during nitrification and denitrification but overlook microbial reduction mechanisms. This study examines the potential for enhancing nitrous oxide reductase (NosZ) activity to reduce N ₂ O to N ₂ . We hypothesize that strategic oxygen manipulation can enhance N ₂ O destruction by continuous NosZ expression and enable NosZ activation in microorganisms with superior NosZ capabilities. We assess microbial community function and metabolic regulation using metagenomics and metaproteomics to clarify the effect of intermittent aeration regimes on N ₂ O emission. Intermittent aeration with periodic anoxic exposure significantly reduced N ₂ O emissions with 71% nitrogen removal by enhancing the metabolic activity of Hyphomicrobium . NosZ activity increased by 4- to 6.5-fold after system adaptation to oxygen modulations, compared to continuous oxic-anoxic cycling without the anoxic phase. The latter resulted in increased N ₂ O emissions due to suppressed NosZ activity and higher N ₂ O production from Methylobacillus , which uses nitric oxide as an alternative electron acceptor. Our finding that strategic oxygen manipulation can energize N ₂ O destruction lays the foundation for developing next-generation wastewater treatment technologies for mitigating N ₂ O emissions.