# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
Data are expressed as fold induction relative to the untreated control (artificial water) and were normalized to the 16r RNA gene. Dotted lines indicate expression differences between treatments. production through direct interaction with the cell surface or dissolved organic compounds. The presence of additional organic and inorganic pollutants could have further intensified these effects, for example, by forming corrosion layers around nanoparticles or promoting their aggregation, altering their toxicity [45; 46; 47]. Concurrently, _ctaD_ expression was moderately increased, suggesting disturbances in the electron transport chain and elevated energy requirements resulting from stress conditions. These effects likely promoted the activation of alternative respiratory pathways, such as those involving cytochrome o ubiquinol oxidase (cyo) or cytochrome bd oxidase, which can serve as terminal oxidases under stress conditions, bypassing the cytochrome c oxidase pathway encoded by _ctaD_[48; 49]. These alternative oxidases, characterized by high oxygen affinity, may help maintain redox balance and support cellular respiration when cytochrome c oxidase activity is compromised, ensuring sufficient ATP production for defence and repair systems. Thus, the presence of Al2O3 NPs in raw wastewater appears to elicit both immediate antioxidant responses and adaptive shifts in energy metabolism [45]. As biological treatment progressed, the transcriptional responses of _katE_, _alpC_, and _ctaD_ in _P. putida_ gradually declined, reflecting environmental detoxification and reduced bioavailability of nanoparticle fractions. Physicochemical data likewise showed a decrease in dissolved Al3+ concentrations over time [31], potentially reducing cellular stress. In the control group, the observed decay in bacterial growth likely resulted from the depletion of bioavailable organic compounds and the accumulation of inhibitory metabolites in the wastewater matrix, which could limit metabolic activity and growth over time, even in the absence of nanoparticles or bulk material. These factors collectively contributed to the observed reduction in transcriptional activity and growth across all experimental conditions. Nevertheless, _katE_ and _alpC_ remained upregulated relative to the control, possibly due to residual nanoparticle activity or delayed effects from prior exposure. Interestingly, _ctaD_ expression was initially low but increased in treated wastewater at later time points, possibly indicating the compensatory upregulation of energy pathways in response to membrane dysfunction, altered electron flow, or increased ATP demand. Similar adaptive mechanisms, including upregulation of cytochrome oxidase genes and alternative respiratory pathways, have been reported in _P. putida_ responding to redox and oxidative stress induced by environmental stressors [44]. According to data from the literature, biological wastewater treatment generally reduces the intensity of transcriptional responses, aligning with environmental detoxification and a decline in nanoparticle bioavailability [20]. However, persistent gene upregulation may result from residual nanoparticle activity, secondary stress effects, or the presence of other micropollutants. Even in treated effluents, trace levels of heavy metals, nanoparticles, and pharmaceutical residues may remain [20; 31; 50; 51; 52]. Chronic exposure to such compounds can disrupt membrane potential, induce oxidative stress, and damage lipid structures. These effects increase the energy demand of cells, potentially activating _ctaD_ and other respiratory genes. Furthermore, under conditions of limited oxygen availability--common in sludge environments--bacteria often upregulate high-affinity cytochrome oxidases such as cytochrome c oxidase to optimize oxygen usage [53; 54]. Organic (e.g., humic acids) and inorganic substances present in wastewater can also influence nanoparticle transformation and behavior.