## 3 Results and Discussion  
The susceptibility to acidic environments was assessed in four human pathogenic bacteria, _E. coli, K. pneumoniae, Acinetobacter_ sp., and _S. aureus_[15, 16]. The experimental conditions assayed were 0.5-pH gradients between 3.0 and 7.0, obtained using either organic (acetic acid-AA, and formic acid-FA) or inorganic (hydrochloric acid-HA, and sulphuric acid-SA) acids. The selected bacteria were cultured in the pH-adjusted MHB mediums, in 96-well microplates, and their ability to grow was verified by adding INT [17]. Reduction of INT to a red-coloured product indicates bacterial growth. Figure 1 summarizes the results obtained, showing the ability of the selected bacteria to grow under the different pH conditions assayed. A subculture of each well where bacterial growth did not occur and the first condition in which there was significant growth (indicated by an intense pink colour) was then made on MHA plates, with neutral pH, to determine the bactericidal versus the bacteriostatic effect of the different pH media (Figure 2). Under bacteriostatic conditions, cell metabolism is largely affected, but cells survive, although are not able to divide. In this case, no reduction of the INT is observed in the 96-well plates, but isolated colonies will be formed in the subcultured agar plates, although in a small number since no bacterial growth occurred during the incubation time in the well. Table 1 consolidates the results from Figures 1 and 2 and highlights the bactericidal pH conditions (blue boxes) and bacteriostatic (white boxes) for each bacterium. Remain pH conditions do not affect bacterial growth (pink boxes). Overall, the results obtained reveal that bacteria's ability to cope with pH can vary significantly depending on the species, being _Acinetobacter_ sp. the most susceptible, only surviving at pH 5.0 or above, while _K. pneumoniae_ was the most resistant bacteria, tolerating pH 3.5, although under a bacteriostatic effect (Figure 2 and Table 1). More importantly, the type of acid (organic or inorganic) used to obtain the acidic environments in which bacterial cells were challenged, also dictates bacterial behaviour. As can be easily verified in Figures 1 and 2, although the selected bacteria can grow at least above pH 5.5, irrespectively of the acids involved in the media acidification, they are much more tolerant to inorganic (strong) acids (HA and SA) than organic (weak) acids (AA and FA, although to a lesser extension in to _Acinetobacter_ sp.). This observation is certainly related to the nature of the acids used. Strong acids, such as the HA and SA, completely dissociate in solution into CI- or SO\({}_{4}\)2- and H+, respectively, which cannot diffuse inside the cell due to the low ion permeability of the membrane [2; 18; 19]. On the other hand, weak acids like AA and FA, when in solution, exist substantially in the undissociated state, CH\({}_{3}\)COOH or CHOOH, respectively, which are relatively small and uncharged molecules that can easily diffuse through the cell membrane (using mechanisms like permeases or porins) [7; 19]. Figure 1: Differences between Gram-positive and Gram-negative bacterial growth in pH-adjusted MHB medium, assessed with the iodonitrotetrazolium chloride (INT) assay) [17]. Wells where bacterial growth occurred show a pink colour. The pH gradients shown were obtained with the addition of organic (AA and FA) and inorganic (HA and SA) acids. For simplification, a representative well for each bacteria species is shown out of the triplicates done in the same assay. Each assay was repeated twice on different days. The conditions above pH 5.5 did not affect bacterial growth and for simplification, they are not shown. Legend: AA-acetic acid, FA–formic acid, HA–hydrochloric acid, SA–sulphuric acid. However, once in the cytosol, and due to its higher pH, the undissociated acids easily dissociate, producing CH3 COO\({}^{-}\), CHOO\({}^{-}\), and H\({}^{+}\).