# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 2 Materials and Methods
### RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
The observed differences are primarily attributed to higher surface reactivity and ion release potential of nanoparticles, which can generate oxidative stress and facilitate stronger interaction with cell membranes [37]. Our results support this mechanism, showing more pronounced Al3+ ion release from both pristine and wastewater-associated nanoparticles than from the bulk form [20; 31]. Moreover, the larger specific surface area of nanoparticles promotes adsorption to bacterial membranes and potential internalization, which may further amplify cellular responses [36]. Together, these findings indicate that nanoform Al2O3 represents a distinct environmental risk compared to bulk material, and this distinction should be considered in risk assessment, particularly in complex systems such as wastewater. In this study, we conducted an interspecies comparative analysis to explore whether genes involved in similar cellular functions exhibit comparable transcriptional patternsin response to Al2O3 NPs. Specifically, we examined the expression of homologous genes related to oxidative stress (_katE/cat_, _alpC/gst_) and energy metabolism (_ctaD/nadh_) in two taxonomically distant organisms: the bacterium _P. putida_ and the crustacean _D. magna_ (Figure 5). These species represent different biological domains--prokaryotic and eukaryotic--and occupy distinct ecological roles, with _P. putida_ being a model organism for microbial communities in wastewater treatment systems and _D. magna_ serving as a widely accepted indicator species in aquatic ecotoxicology. This comparative approach was intended to better understand nanoparticle-induced toxicity mechanisms and to assess the potential for identifying universal molecular stress markers. Gene expression data for _D. magna_ were obtained from our previously published study using the same exposure conditions and target genes ([[https://doi.org/10.1016/j.dwt.2025.101000](https://doi.org/10.1016/j.dwt.2025.101000)]([https://doi.org/10.1016/j.dwt.2025.101000](https://doi.org/10.1016/j.dwt.2025.101000))) [20]. A comparison of genes with homologous function expression in _P. putida_ and _D. magna_ revealed consistent transcriptional response patterns across three functional groups: xenobiotic detoxification (_katE/cat_), oxidative stress response (_alpC/gst_), and energy metabolism (_ctaD/nadh_) (Figure 5). In response to pristine Al2O3 nanoparticles, both organisms showed strong upregulation of detoxification (_katE_, _cat_) and oxidative stress genes (_alpC_, _gst_), with peak expression occurring at different time points. For energy metabolism genes, contrasting responses were observed: _ctaD_ expression increased in _P. putida_, while _nadh_ expression decreased in _D. magna_ (Figure 5). Under exposure to untreated wastewater containing Al2O3 NPs, expression profiles became more aligned between species. The genes _katE/cat_ and _alpC/gst_ remained moderately upregulated throughout the exposure period, while _ctaD_ and _nadh_ both showed a gradual decrease (Figure 5). A similar pattern was observed in treated wastewater: transcriptional activity of detoxification and oxidative stress genes was sustained, whereas energy metabolism-related genes remained downregulated in both organisms (Figure 5). The comparison of genes with homologous function expression in _P. putida_ and _D. magna_ revealed consistent transcriptional response patterns across three functional groups: xenobiotic detoxification (_katE/cat_), oxidative stress response (_alpC/gst_), and energy metabolism (_ctaD/nadh_) (Figure 5). In response to pristine Al2O3 nanoparticles, both organisms showed strong upregulation of detoxification (_katE_, _cat_) and oxidative stress genes (_alpC_, _gst_), with peak expression occurring at different time points. For energy metabolism genes, contrasting responses were observed: _ctaD_ expression increased in  
Figure 5: Time-course expression of genes with homologous function involved in oxidative stress (_katE/cat_, _alpC/gst_) and energy metabolism (_ctaD/nadh_) in _P. putida_ and _D.