Bharat Manna

Aquatic environments can maintain and express antibiotic resistance genes even without antibiotic pressure, but the conditions that govern their activity remain poorly understood. Redox oscillations are pervasive in natural and engineered waters and strongly alter microbial metabolism, yet whether their frequency shapes resistance expression is unknown. We reanalyzed paired metagenomes and metatranscriptomes from a published chemostat experiment in which a sulfidic stream community was cycled between oxic and anoxic conditions at high, medium and low frequency (HF, MF, LF), with strain-resolved population genomics of the dominant Pseudomonas lineage. Resistance genes were broadly distributed across treatments, whereas transcription concentrated under fast cycling, reaching a median 168 TPM under HF against 38 under LF although no antibiotic was present, so genetic potential and expression diverged. Fast cycling induced ROS-generating flavoproteins, antioxidant defenses and transcription-coupled DNA repair, and Pseudomonas supplied 99% of resistance-gene transcripts under HF, chiefly through its intrinsic efflux systems. Within the dominant Pseudomonas genome, however, the four resistance genes carried no sequence variants under HF, where coverage was deepest, and no fixed difference from the reference in any sample. Genome-wide selection remained purifying throughout (pooled pN/pS 0.72 under HF against 0.12 under LF, both below 1), and the relaxation trended toward redox and central metabolism. Redox frequency therefore raises resistance expression by selecting a metabolically versatile, high-efflux lineage, while the resistance sequences themselves stay conserved. Resistance activity in aquatic systems responds to an abiotic driver, which argues for weighing expression alongside gene presence in environmental surveillance. Abstract Figure: Highlights: Fast redox cycling raised ARG expression without any antibiotic added. High-frequency cycling induced ROS sources, antioxidant defense and DNA repair. Fast cycling swept one Pseudomonas lineage to community dominance. ARGs in the dominant Pseudomonas genome carried no variants under HF. Gene-based AMR surveillance would miss the rise in resistance expression.

Nitrous oxide (N₂O) emissions from biological wastewater treatment pose a disproportionate challenge to carbon neutrality, as small releases substantially elevate the greenhouse gas footprint. Conventional treatment systems operate with aerobic phases lasting hours, during which oxygen suppresses nitrous oxide reductase (NosZ) activity and allows dissolved N₂O to accumulate. Shortening these phases to the minute scale through dynamic feedback-controlled aeration could couple N₂O production and reduction, but whether frequent oxic-anoxic cycling enables NosZ reactivation during postoxic anoxia remains unclear. In 1 L lab-scale suspended-growth reactors, we compared oxygen-only pulsing without imposed anoxia (continuous perturbation, CP) and paired oxic-anoxic cycling (intermittent perturbation, IP). At an oxygen ceiling of 2 mg/L, CP increased the N₂O fraction of gaseous nitrogen (GN) to 0.25-0.74, whereas IP maintained a low N₂O-N/GN ratio of 0.02-0.24 while releasing 79% of nitrified ammonium-N as GN. After adaptation, dissolved N₂O consumption rates were 4-6.5 times higher under IP than CP. Metaproteomic evidence confirmed NosZ enrichment under IP, with Hyphomicrobium as a key contributor. These results establish minute-scale oxic-anoxic cycling as a key operational prerequisite for NosZ reactivation during postoxic anoxia, recoupling N₂O production and reduction, and providing a practical framework for reducing N₂O emissions through PLC-based aeration control without retrofitting.

The uncontrolled release of organic micropollutants (OMPs) from wastewater treatment plants highlights a failure to synergize co-metabolic and reactive oxygen species (ROS)-driven biodegradation pathways. Here, we demonstrate that engineered redox cycling—dynamic fluctuations between oxic and anoxic conditions—fundamentally reshapes microbial metabolism and OMP reaction networks to dramatically enhance removal. Using an integrated multi-omics approach centered on paired mass distance (PMD) reactomics, we show that redox cycling increased the removal of 32 diverse OMPs from a baseline of 32% up to 67%. This enhancement was driven by a complete restructuring of degradation mechanisms: cycling stimulated microbial amino acid and fatty acid metabolism by up to 42%, which coupled to controlled ROS production where oxidative pathways (+15.995 Da) accounted for 47% of transformations under intermittent aeration. We mapped distinct “enzymatic fingerprints,” with strong correlations (r > 0.7) linking Proteobacteria monooxygenases to oxidative reactions and Rhodobacteraceae dehydrogenases to reductive ones (+2.016 Da), revealing clear functional specialization. Ultimately, the redox regime dictates the entire OMP transformation network topology, shifting pathways from simple hydroxylation to complex, multi-step networks. This work provides a mechanistic framework establishing redox manipulation as a powerful strategy to synergistically activate degradation pathways, allowing existing infrastructure to meet stringent discharge regulations.

Climate warming presents a critical challenge to global ecosystems, with rising temperatures inducing the emergence of antibiotic resistance genes (ARGs) in aquatic environments. Given the natural correlation between rising temperatures and declining oxygen levels in aquatic systems, we hypothesized that decoupling temperature and oxygen stress would reveal regulatory mechanisms mitigating ARG expression under future warming scenarios. Through transcriptomic analysis of Escherichia coli under temperature upshift (TU), dissolved oxygen upshift (DOU), and dissolved oxygen downshift (DOD), we identified 101 distinct regulons with 8 showing significant regulation (|log₂FC| > 1.5). Key regulons ( cusR , mhpR , pdhR ) exhibited parallel regulation under TU and DOD but opposite patterns under DOU, revealing mechanistic links between temperature and oxygen stress through shared regulatory networks involving oxidative stress regulators ( oxyR - metR ) and aerobic respiration control ( arcA - betI ). Evolved strains from controlled temperature-oxygen fluctuations demonstrated sophisticated metabolic reprogramming, including enhanced carbohydrate metabolism, nucleotide biosynthesis, and putrescine degradation. These strains exhibited optimized energy metabolism through reduced downregulation of ATP synthase subunits and NADH dehydrogenase complex genes, preserving electron transport chain function under thermal stress. Simultaneously, they displayed strategic ROS management with reduced upregulation of peroxiredoxin ( ahpF ) and Cu/Zn-SOD ( sodC ), while reversing Fe-SOD ( sodB ) expression patterns. To validate these mechanisms, we conducted independent batch reactor experiments with two dissolved oxygen conditions (2-8 mg/L) using intermittent perturbation patterns. Non-targeted metaproteomic analysis revealed that lowered oxygen levels (2 vs. 8 mg/L) triggered comprehensive reconfiguration of antimicrobial resistance mechanisms, including downregulation of penicillin-binding proteins, alanine racemase, and cationic antimicrobial peptide resistance systems, while modulating two-component regulatory networks. Our findings reveal coordinated regulatory networks optimizing energy metabolism, ROS management, and antibiotic resistance, suggesting oxygen management strategies could mitigate climate warming effects on antibiotic resistance in aquatic environments.

Aquatic ecosystems are facing severe threats from climate change, with rising temperatures as a major driving force posing a significant challenge to their ecological balance. Microorganisms constitute the majority of biomass in the water ecosystems and mediate the flux of carbon, nitrogen, sulfate and other essential nutrients. Climate warming profoundly influences microbial communities by shaping their distribution and ecological roles in the ecosystem, and in turn microorganisms play a significant role in climate feedback. The Yellow River, China’s second-longest river, is crucial for agricultural irrigation and provides drinking water for millions, yet little is known about microbial community adaptations under future warming scenarios in the system. In this study, we analyzed a publicly available metagenomic dataset from Yellow River water samples that were previously subjected to temperature treatments of 23, 26, 29, 32, and 35 degrees Celsius. We employed metagenomic analysis to identify how gradually increasing temperatures affected microbial community profiles. The study identified 140 species tolerant to high temperatures, showing a significant increase in abundance with elevated temperatures. The elevated temperature stress impacted the network properties of microbial communities substantially. Certain temperature-tolerant species were identified as hubs in the network across five temperatures, with their number increasing with temperature. Deltaproteobacteria bacterium was present at all five temperatures, while Parcubacteria bacterium, Parvularcula sp ., Phenylobacterium sp ., Phycisphaeraceae bacterium , and Sphingobium xenophagum were only present at high temperatures of 32 °C and 35 °C. The percentage of taxa nodes connected to tolerant hubs in the overall network rose from 57.48% to 95.94%, indicating the growing importance of these tolerant hubs. The positive connections among these tolerant hubs also increased, with the number of positive edges rising from 1166 to 2811, indicating a potential collaborative relationship among these taxa in response to temperature stress. These findings suggest that tolerant hubs may initially respond to temperature stress and subsequently transfer this function to other species through cooperative interactions. Understanding these microbial dynamics is crucial for developing strategies to maintain freshwater ecosystem health amid climate change.

As the global transition toward circular wastewater treatment intensifies, extracellular polymeric substances (EPS) have emerged as valuable targets for resource recovery. Although most efforts have focused on aerobic granular sludge, conventional activated sludge systems, which account for most global wastewater treatment, remain underexploited for EPS recovery. Building on the established link between oxidative stress and EPS biosynthesis in pure strains, we propose that strategically manipulating oxygen exposure patterns to intensify oxidative stress in activated sludge microbial communities could enhance EPS production. To test this, we 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 H ₂ O ₂ as an unintended metabolic byproduct. Among the multiple microbial groups contributing to H ₂ O ₂ production, Methylophilaceae, Comamonadaceae , and Rhodobacteraceae were distinguished by simultaneously exhibiting upregulation of EPS biosynthesis proteins, suggesting that taxa within these families collectively mediated both H ₂ O ₂ production and EPS enhancement. By modulating aeration, this study offers a chemical-free, controllable strategy for enhancing EPS production within conventional activated sludge systems. Graphical