# Molecular Response of Bacteria Exposed to Wastewaters-Borne Nanoparticles
## 1 Introduction  
The growing presence of nanoparticles in wastewater has emerged as a significant environmental concern due to their expanding use across various industrial sectors. Engineered nanomaterials, including aluminum oxide nanoparticles (Al2O3 NPs), are increasingly applied in medicine [1, 2, 3], cosmetics [4], textiles [5], electronics and materials science [6, 7], water treatment [8, 9], and environmental remediation [10]. These nanoparticles are introduced into aquatic systems through industrial discharges and consumer products, raising critical concerns regarding their long-term ecological impact, particularly in the context of wastewater treatment. Due to their high surface reactivity, small size (<50 nm), and large specific surface area, Al2O3 NPs may interact strongly with biological systems and pose risks to both environmental and human health [11]. Studies have shown varied effects on bacteria, with _Escherichia coli_ exhibiting mild growth inhibition due to reactive oxygen species (ROS) generation, _Pseudomonas stutzeri_ showing oxidative stress responses without significant toxicity, and _Staphylococcus aureus_ experiencing cell wall perforation and membrane disruption [11, 12, 13, 14]. The increasing detection of Al\({}_{2}\)O\({}_{3}\)NPs in wastewater systems underscores the need for improved regulatory frameworks and advanced methods of toxicological assessment. A major limitation of conventional toxicity tests is their inability to elucidate the mechanisms of nanoparticle toxicity [15]. Standard assays, which typically focus on survival and reproduction, fail to detect subtle molecular effects or stress responses. Recent studies have highlighted the importance of molecular biomarkers, such as gene expression changes, as sensitive early indicators of nanoparticle-induced stress, revealing mechanisms like ROS-mediated oxidative stress and membrane damage [10; 11; 12; 13; 14; 16]. These molecular responses provide insight into how nanoparticles affect cellular processes, including oxidative stress and detoxification pathways. Despite progress in the field, the specific toxicological mechanisms of many nanomaterials remain poorly understood [17]. In aquatic organisms such as _Daphnia magna_, exposure to metal nanoparticles (e.g., copper, silver) has been shown to induce differential expression of genes involved in oxidative stress responses, immune regulation, and energy metabolism [18; 19; 20]. However, similar studies on microorganisms, especially bacteria, are still limited. Although bacteria play a crucial role in aquatic ecosystems and are key to biological wastewater treatment processes, their molecular responses to nanoparticles are underexplored. Existing studies primarily focus on general community-level effects, including oxidative stress, microbial diversity shifts, and the potential for horizontal gene transfer linked to antibiotic resistance [20; 21; 22; 23]. The specific responses of individual bacterial species to distinct types of nanomaterials, particularly metal-based or polymeric nanoparticles, remain insufficiently characterized [24; 25]. Such studies are crucial, as Al\({}_{2}\)O\({}_{3}\)NPs may disrupt bacterial cell membranes, generate ROS, or alter energy metabolism, potentially affecting wastewater treatment efficiency. The aim of this study was to evaluate the transcriptional response of _Pseudomonas putida_ to wastewater with aluminum oxide nanoparticles and their bulk counterparts, focusing on genes involved in oxidative stress and energy metabolism. _P. putida_ was selected as the model bacterium due to its ecological relevance in wastewater treatment systems, where it contributes to biodegradation and nutrient cycling [26; 27]. Its metabolic versatility, robustness, and well-characterized genetic background make it ideal for studying nanoparticle-induced stress responses, particularly through molecular techniques like RT-qPCR [28; 29].