## 2 Results and Discussion
### Investigation of Discriminating Features
Additionally, Ag\({}^{+}\) can be generated during the oxidation of nanoparticles in aqueous environment under acidic conditions, therefore promoting the release of silver ions under aerobic conditions [55]. Ag\({}^{+}\) released in the vicinity of the bacteria can lead to the disruption of cell wall and membrane, thus configuring as a AgNPs-associatedbiocide mechanism [56]. Observed elevated levels of succinate may be linked to the hampering of cellular respiration: silver ions were found inhibiting the oxidation of glucose and causing decrease in the levels of this intermediary [57]. Furthermore, Ag\({}^{+}\) seemed to impair transport of ions in bacterial cell, resulting in an outflow of succinate. Such mechanism is possibly connected with the inhibition of NADP-linked glutamate dehydrogenase [58]. Specifically, at C2, an augmentation in the responses of the following VOCs was verified: 2,6,7-trimethyldodecane (in _E. coli_ and _P. mirabilis_), butanetioic acid (in _E. coli_ and _E. faecalis_) and 2-nonenal (in _E. coli_). 2-Nonenal may be yielded during the oxidative breakdown of palmitoleic acid [59]. Regarding the appearing of _p_-xylene, aromatic compounds are reported to be synthetized by the shikimate pathway--a biochemical route responsible for production of aromatic amino acids [60]. It was found that silver ions displace copper ions operating in enzymatic processes as those related to the shikimate pathway, including themselves in the active sites of involved enzymes [61]. Therefore, the enzymatic generation of differential aromatic products may be related to such imbalance. However, connected with this factor, unbound copper results in the deficit of cuproenzymes, which serve as cellular antioxidative defenses, thus allowing ROS to accumulate in the periplasmic space [61]. C3 caused the occurrence of only few volatiles, such as 2-undecenal (_E. coli_ and _K. pneumoniae_) and 2-decenal (_E. faecalis_). Like 2-undecenal, 2-decenal can also be generated by the oxidation of lipids from plasma membrane, particularly through the \(\alpha\) scission of oleic acid after interaction with ROS. At C2 and C3, continuous increasing of compounds such as decanal (for _E. coli_), nonanoic acid (for _P. mirabilis_) and 2-undecenal (for _E. faecalis_) was observed. Nonanoic acid, despite possibly being formed by fatty acids biosynthesis pathway, can also be released through \(\alpha\) scission of oleic acid after oxidized in the position C-10. Besides the disappearing of several VOCs previously generated by the cultures, increasing concentrations of agent resulted in elevated responses of compounds potentially related to cell membrane damage. Indeed, plasma membrane is described as the main target of AgNPs. Further effects associated with this phenomenon were loss of potassium ions from the intracellular space and decreased production of energy (ATP) [62]. The profile of straight-chain saturated and mono-unsaturated fatty acids for both Gram-negative and Gram-positive bacteria appear to be rather similar, mainly consisting of palmitic (C16:0), oleic (C18:1), palmitoleic (C16:1) and stearic (C18:0) acids [63]. Due to this, the correlation made between some of the discriminating features as products of lipid oxidative cleavage appear to be reasonable, once these involve oleic and palmitoleic acids (principal unsaturated lipids of bacteria membrane) as main substrates. A part of the aforementioned compounds has in common their reported behavior as bioactive volatiles, i.e., VOCs which have a role in bacteria adaptation to an environment and inter-species signaling [39]. Therefore, bio-AgNPs may elicit the production of biologically active VOCs, enabling bacteria to better adapt to the modified medium. However, VOCs with such properties are generally present at lower concentrations of nanoparticles (C1, or even C2), evidencing the predominance of deleterious effects of nanoparticles over the ability of bacteria to customize their metabolism according to the environment.