# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
The formation of corrosion layers or particle aggregation alters metal bioavailability and interactions with bacterial cells [45], thereby modulating nanoparticle toxicity. These complex environmental factors can lead to persistent low-level stress, driving restructuring of bacterial metabolism, including enhanced ATP production pathways and the adaptation of electron transport systems [20; 31; 36; 55; 56]. The literature highlights that most studies on nanoparticle effects on bacteria have been conducted in simplified model systems using pure cultures and defined media. Researchbased on real wastewater samples remains limited. One notable exception is the study by [PERSON] et al. (2016), which showed that ZnONPs reduced microbial diversity and disrupted functional processes in activated sludge [57]. Similarly, [PERSON] et al. (2020) found that ZnONPs modulated the expression of nitrification genes (_amoA_, _luo_, _nirK_) in wastewater systems depending on oxygen levels and nanoparticle aggregation [58]. These findings emphasize the importance of considering both the physical form of the material (nano vs. bulk) and environmental context (raw vs. treated wastewater), as nanoparticle effects on microorganisms are strongly matrix-dependent. In summary, our data suggest that _katE_ and _alpC_ are sensitive biomarkers of acute oxidative stress induced by Al2O3 NPs in wastewater, while _ctaD_ reflects later-stage metabolic adaptations related to energy management in stressed bacterial cells. In untreated wastewater containing bulk Al2O3 (WW-Bulk-I), gene expression levels were also elevated relative to controls but lower than those observed in the nanoparticle treatments. The expression of _katE_ ranged between 3.0 and 3.5 (Figure 2), _alpC_ between 3.1 and 3.4 (Figure 3), and _ctaD_ between 1.5 and 1.8 (Figure 4). While a slight downward trend was observed over time, the response remained detectable across all time points. In the treated wastewater supplemented with bulk material (WW-Bulk-E), a further reduction in gene expression was observed. _KatE_ expression declined from \(\sim\)3.0 to \(\sim\)2.5 (Figure 2), _alpC_ from \(\sim\)3.1 to \(\sim\)2.2 (Figure 3), and _ctaD_ from \(\sim\)1.7 to \(\sim\)1.2 (Figure 4). Compared to untreated bulk samples, these findings reflect a diminished cellular response, likely due to the physical or chemical transformation of the material during wastewater treatment. In light of these observations, it is worth emphasizing that both in wastewater and in pristine suspensions, Al2O3 nanoparticles induced stronger transcriptional responses in _P. putida_ cells compared to their bulk counterparts. Under pristine conditions, the nanoparticles triggered significantly higher expression levels of genes associated with oxidative stress and energy metabolism (_katE_, _alpC_, _ctaD_), consistent with their enhanced reactivity, solubility, and bioavailability [39; 40]. In contrast, the bulk form elicited only a moderate gene expression response, which aligns with previous studies indicating its limited cellular penetration and weaker surface interactions [37]. [PERSON], in turn, showed that the exposure of _Shewanella oneidensis_ to functionalized gold nanoparticles induced _sodB_ expression, while the ligand alone had no such effect, highlighting the critical role of surface properties in stress induction. Stronger biological responses to nanoparticles have also been demonstrated in environmental studies [17]. [PERSON] et al. (2016) found that ZnO NPs significantly reduced bacterial taxonomic diversity in activated sludge, whereas bulk ZnO had no such effect, indicating lower interaction with microbial cells [57]. Similarly, [PERSON] et al. (2018) reported that _Bacillus subtilis_ exposed to ZnONPs exhibited a stringent response and significant changes in gene expression related to the TCA cycle and pentose phosphate pathway--key metabolic and redox processes [59]. These molecular effects were absent in the bulk treatment.