Impact of pH on citric acid antimicrobial activity against Gram-negative bacteria

The antimicrobial activity of citric acid (CA) is often evaluated without pH adjustment or control and its impact on micro-organisms is better understood in acidic conditions. However, the biocidal action of the fully ionized CA molecule, predominantly available at higher pH, has not been previously investigated. The objective of this study was to evaluate the antimicrobial effect of high (10%) and low (1%) concentrations of CA, each adjusted over a wide range of pH values (4·5, 6·5 and 9·5) relative to the controls exposed to corresponding pH levels alone (no CA). The viability and morphology of Escherichia coli and Klebsiella aerogenes were evaluated using a culture-based enumeration assay in parallel with direct SEM imaging. Overall, the highest membrane damage and loss in viability were achieved with 10% CA at pH 9·5, which yielded at least 4·6 log₁₀ CFU per ml (P < 0·001) reductions in both organisms. Insight into the superior efficacy of CA at high pH is proposed based on zeta potential measurements which reveal a more negatively charged bacterial surface at higher pH. This pH-dependent increase in surface charge may have rendered the cells potentially more sensitive towards chelants such as CA^3- that interact with membrane-stabilizing divalent metals.