## 3 Results and Discussion
These forms, being charged, cannot easily diffuse out from the cell, and consequently accumulate intracellularly [7; 19]. This proton accumulation reduces the cytosolic pH to dramatic values that deregulate the metabolic machinery of the cell [7]. Therefore, the bactericidal or bacteriostatic ability of an acid (organic or inorganic) will depend primarily on the concentration of its undissociated form, which is determined by the dissociation constant, pKa. This indicates the pH at which the ratio of dissociated to undissociated forms is 50:50. For that reason, AA and FA, with a pKa of 4.8 and 3.8, respectively, are more efficient in inhibiting bacterial growth than HA and SA, whose pKa are \(-6.3\) and \(-3.0\), respectively. Remarkably, the results obtained not only show differences between organic and inorganic acids but also slight variations inside each group (AA/FA or HA/SA). In a simplistic approach, this would be a predictable result for the weak acids used, which originate relatively similar molecules, CH3 COO\({}^{-}\) or CHOO\({}^{-}\) (AA and FA respectively), and eventually to a larger extent for the strong acids used. In this case, the pKa (\(-6.3\) and \(-3.0\)) stoichiometry of the dissociation reactions (one proton versus two protons) and size of the anions generated (CI\({}^{-}\) and SO4\({}^{2-}\)), respectively,  
Figure 2: Effect of different acidic environments on bacterial viability, evaluated by subculturing in neutral pH MHA medium after growth in pH adjusted MHB medium. For simplification, only a representative plate for each combination of bacteria/condition out of the triplicate assays performed on two different days is presented. Legend: AA–acetic acid, FA–formic acid, HA–hydrochloric acid, SA–sulphuric acid. are significantly different. This would be additional evidence supporting the mechanisms of diffusion of weak acids into the bacterial cytoplasm followed by dissociation and internal medium acidification. There are, however, additional considerations that certainly play a role in the results obtained, as the structure of the cell wall of the bacteria studied. Gram positive (_S. aureus_) and Gram negative (_E. coli_, _K. pneumoniae_, and _Acinetobacter_ sp.) present significant differences in what concerns to the complexity and composition of their cell walls, for instance in terms of polysaccharides and peptidoglycans proportions and transporters. Furthermore, other metabolic features must be considered in the explanation of the higher toxicity of weak acids, at least in certain bacteria. Acetic acid, for instance, has an important role in _E. coli_ metabolism and can be used both as a substrate or product (acetate switch), while formate is only a product [20]. A deeper discussion of these and other mechanisms affecting the response of different bacteria to acidic conditions can be found elsewhere [11; 13; 14; 20; 21; 22]. A simplified diagram representing the differences in the mechanism of cytosolic acidification of bacteria is presented in Figure 3.  
As referred to above, the bacteria studied in this work have been selected by their involvement in human pathogenic situations [15; 16], but these are different bacteria in phylogenetic terms. _S. aureus_ is the only Gram-positive bacteria assayed in this work and the most divergent of all, while _K. pneumoniae_ and _E. coli_ are the two bacteria that are phylogenetically more similar. Moreover, all these bacteria grow normally in different niches and pH environments. This has consequences in the mechanisms each specie developed to cope with acidic environments and the organization of the cell membrane is certainly the main factor contributing to this differentiation. _Acinetobacter_ sp. is the least tolerant bacteria to acidic environments. Moreover, it is the species that exhibits fewer variations to the acidic conditions according to the type of acid (organic or inorganic) used  
\begin{table}
\begin{tabular}{c  
to obtain the pH-adjusted media (Table 1). Although it is a Gram-negative bacterium, such as _E. coli_ and _K.