## 2 Results and Discussion
### Differences in VOC Profiles Composition
Some  
Figure 4: (**a**) Venn diagram displaying the number of compounds coincident between the studied untreated bacterial strains. Networks presenting the connections between the bacterial strains (squares) and respective emitted compounds (circles) for VOC profiles regarding (**b**) untreated cultures and those supplemented with bio-AgNPs at concentrations (**c**) 12.5, (**d**) 25 and (**e**) 50 ug mL\({}^{-1}\). EC = _E. coli_, EF = _E. faecalis_, KP = _K. pneumoniae_, PM = _P. mirabilis_. steps of the pathways responsible for generating volatile metabolites may not be active in all of the studied bacteria. Moreover, originally displayed enzymatic activity can differ depending on the investigated organism. When comparing the profiles generated by unstressed bacteria and those acquired using the greatest concentration of agent (50 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\)), it is observed a reduction of 44, 52, 67 and 80% in the number of VOCs previously detected for _E. coli_, _E. faecalis_, _K. pneumoniae_ and _P. mirabilis_ (consecutively). The most abrupt decrease in volatiles number occurred for nanoparticles incremented at concentration of 25 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\) for _K. pneumoniae_ and _P. mirabilis_, and 50 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\) for the remaining species. Such observations suggest that _E. coli_ and _E. faecalis_ display greater resistance to bio-AgNPs supplementation, in terms of preservation of their original metabolism. In general terms, the increase in the number of detected VOCs can be associated to the elicitation of molecular mechanisms induced by the introduced agent. These can possibly include the upregulation of certain pathways in order to compensate bacterial functions which were impaired, or to generate products with specific biological roles. VOCs generated after treatment can also be byproducts of chemical reactions with cell components promoted by nanoparticles. A reduction in the number of produced volatiles can be simply due to the inhibition of biochemical processes, or a consequence of metabolic shifts intending to adapt cell survival to the new environment. Figure 5b expresses the changes in the proportion of compounds used in each of the performed assays, according to their respective chemical groups. In general, the contribution of hydrocarbons to the profiles from cultures processed with lower concentrations of bio-AgNPs tends to increase. This indicates that, at the MIC level, the occurred metabolic alterations encompassed the augmented production of this class of compounds. The biosynthesis of hydrocarbons is documented in several species of microorganisms, being mainly associated to the fatty acid metabolism. Precursor molecules of acetyl-CoA (or structural derivatives) are elongated with malonate units, the decarboxylation of the formed products can generate alkanes and alkenes (both straight and branched chain, depending on the employed starter unit) [18; 28]. The composition of hydrocarbons emitted by microorganisms appears to be related to their physiological groups and the conditions  
Figure 5: (**a**) Plot displaying the number of compounds detected in each of the performed experiments, according with functional group—the color scale refers to the range from lower (dark blue) to greater (red) incidence of VOCs; (**b**) bar plot presenting the proportion of VOCs detected referring to the respective chemical classes. EC = _E. coli_, EF = _E. faecalis_, KP = _K. pneumoniae_, PM = _P. mirabilis_. provided for their growth [29]. Still, there is a lack of knowledge regarding the possible biological/physiological roles of biogenic hydrocarbons. It is believed that produced hydrocarbons may serve as source of carbon and energy for the cell. Above that, these compounds likely develop functions in regulation of membrane permeability [29]. Overall, the proportion of classes such as aldehydes (for all species), VNCs (in _E. coli_, _E. faecalis_ and _K. pneumoniae_), VSCs (in _E. faecalis_, _K. pneumoniae_ and _P. mirabilis_) and ethers (in _E. coli_, _E.