## 3 Results and Discussion
### Photometric Growth Inhibition Experiments
The growth rate as a ratio of test to control was significantly (_p_ < 0.05) lower in the acidified broth (0.37 and 0.20 for HCl and acetic acid, respectively) than in the standard broth (0.61). In the case of _E. coli_, elevated salt concentration increased the effectiveness of Co-cit, but the acids did not (Table 3). The fact that different compounds influence the efficacy of Co-cit against different target bacteria suggests that the increase in efficacy is due to increased physiological stress on the organism caused by the modified broth rather than any effect on the properties of Co-cit itself. The pH was unlikely to have affected the Co-cit at the values used in this experiment. The aqueous speciation of Co-cit at different pH values has been studied previously. Mononuclear Co-cit, with either one or two citrate ligands, is predominant in the pH of range 5-8, while dimers may form at higher pH values [28]. As an example of the apparent role of physiological stress, _E. coli_ is quite acid-tolerant but not salt-tolerant, whereas the reverse is true for _L. monocytogenes_. The sensitivity of _E. coli_ to Co-cit (in comparison to the determined sensitivity in standard nutrient broth) increased with the addition of salt but not by lowering the pH 5.5, whereas _L. monocytogenes_ sensitivity was increased by reduced pH but not by the addition of salt. However, _P. aeruginosa_ appears to be an exception to this trend. In four of the five modified broths, in which Co-cit showed significantly increased activity (5% NaCl was the exception), there was no evidence of inhibition caused by the modified broth alone. However, the fact that there was no significant decrease in growth rate in the modified broths does not exclude the possibility that an increased physiological burden on the bacteria would reduce their ability to withstand the effect of Co-cit. As previously mentioned, a large variation in Co-cit species composition would not be expected within the pH range used in the present experiments (5.5 to 7.3). Therefore, the species other than mononuclear Co-cit that could be responsible for the antimicrobial \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Modification of Nutrient broth**} & \multicolumn{4}{c}{**Ratio of Growth Parameters of _Pseudomonas aeruginosa_ in:**} \\ \cline{2-5}  & **Modified NB with Co-cit/Modified NB without Co-cit** & **Modified NB/Standard NB** \\ \hline  & **Max. Growth Rate** & **Lag Phase** & **Max. Growth Rate** & **Lag Phase** \\ \hline None (standard NB) & 0.51 * & 6.9 & - & - \\ Sorbate (10 g/L) & 0.69 *+ & 4.1 & 0.91 * & 1.8 \\ Benzoate (2 g/L) & 0.61 * & 6.9 & 0.84 * & 1.3 \\ Propionate (20 g/L) & 0.00 *+ & \textgreater{}15.0 \(\updownarrow\) & 1.07 & 1.6 \\ HCl (pH 5.5) & 0.00 *+ & \textgreater{}7.8 \(\updownarrow\) & 1.01 & 1.9 \\ Acetic acid (pH 5.5) & 0.04 *+ & \textgreater{}22.2 \(\updownarrow\) & 1.32 * & 6.6 \\ NaNO\({}_{2}\) (0.15 g/L) & 0.24 *+ & \textgreater{}5.7 \(\updownarrow\) & 1.06 & 1.1 \\ NaCl (50 g/L) & 0.00 *+ & \textgreater{}3.4 \(\updownarrow\) & 0.49 * & 1.8 \\ \hline \hline \end{tabular}  
* Significant (_p_ < 0.05) difference between the mean maximum growth rates in standard or modified broth with and without Co-cit (column 2) or between modified NB and standard NB (column 4). Statistical analysis was not performed on the lag phases.
* Significant (_p_ < 0.05) difference between the ratio of maximum growth rates with and without Co-cit in modified broth compared to that in unmodified nutrient broth (0.61). \(\updownarrow\) Some or all of the recorded lag phases in broths with Co-cit were >24 h.  
\end{table} 
Table 4: Relative maximum growth rates of _Pseudomonas aeruginosa_ in modified and standard nutrient broth (NB) with and without cobalt citrate [Co-cit (0.5 mM)]. activity are limited. It is possible that small amounts of free citrate are present in the solutions due to the dissociation of the ligands from the Co-cit complex.