# Bielectroanalytical Detection of Lactic Acid Bacteria
## 3 Results and Discussion
For a higher inoculum size of 10,000 CFU, double that reported for the _E. coli_ SHSP mutant, the detection time is around three hours vs. a six-hour detection time for 5000 CFU of wildtype _E. coli_ and 14 h for the fermentative strain [13]. The detection time achieved here for 10,000 CFU _E. faecalis_ resembles respiratory behavior. The genome of _E. faecalis_ is reportedly deficient in heme, but supplementation of this growth factor and or menaquinone induces respiratory behavior in _E. faecalis_ by activating the redox center of cytochrome bd and providing a quinone pool to transfer electrons from NADH\({}^{+}\) to terminal reductases. Respiratory behavior is defined in this sense by observations of increased vitality or biomass production accompanied by less acidification of the medium from lactate accumulation [25]. Upon analyzing the growth yields of _E. faecalis_ in bench top experiments, we observed that the optical density (OD\({}_{600}\)) was always higher in aerobic conditions than it was in anaerobic ones (Figure 3A). This phenomenon was reported previously [26] and it is suggestive of respiratory behavior and could arise from residual heme or quinoids in the undefined medium that we used as was reported for the Todd Hewitt broth r by [PERSON] and [PERSON] (2008). Further analysis shows that the addition of heme, menaquinone, or both does not alter this trend, although menaquinone does give a slight boost in growth, observed by an increase in OD in anaerobic conditions, suggesting a role in managing reactive oxygen species (Figure 2A). A comparative analysis of Lactobacillus electron trans  
Figure 2: (**A**) Calibration curve for _E. faecalis_ detection showing detection time (minutes) with inoculum size as log CFU mL\({}^{-1}\) (**r**\({}^{2}\) = 0.96). (**B**) Chronoamperometry curves of corresponding inoculum sizes and from which calibration curve is derived. The dotted line corresponds to the thresholdof detection.. Mean values are shown, error bars correspond to the standard deviation of three replicates and are represented by bars (**A**) or by the shaded area (**B**). port chain stimulation by heme and menaquinone by [PERSON] et al. (2009) [25] showed similar behavior in _E. faecalis_, i.e., that _E. faecalis_ growth is stimulated by menaquinone and, to greater extent, by the addition of both heme and menaquinone. However, the difference in pH between 'fermentative' and'respiratory' growth ranged only between 0.2 pH units (5.62-5.82) [25], although growth increased by 38%. Under equivalent conditions, we observed a quantitatively lower increase in growth (OD\({}_{600}\)) of around 14% and the pH to be similarly stable (Figure 3B). We observed a pH with a delta (\(\Delta_{\text{pH}}\)) of only 0.13 pH units but an overall moderately lower pH of 5.14 and 5.27 between the unsupplemented control and supplemented conditions, respectively. The \(\Delta_{\text{pH}}\) between fermentative LAB and the supplemented treatment thathad been induced to respire was previously reported to be greater what we have observed here, and the observed \(\Delta_{\text{pH}}\) is usually close to a single pH unit; for example, the \(\Delta_{\text{pH}}\) between a heme and menaquinone-stimulated _Streptococcus enterica_ culture compared to an unsupplemented control was 1.07, with the final pH being 4.42 and 5.49 for fermentative and respiratory conditions, respectively [25]. Taken together, although _E. faecalis_ exhibits less respiratory stimulation upon the addition of heme in BHI medium relative to other LAB, it is clear that it does derive some growth benefits from medium supplementation with respiratory components. Despite the small difference in pH between respiratory and fermentative growth in _E. faecalis_, [PERSON] (2009) still concluded that _E. faecalis_ is both heme and menaquinone stimulated [25]. Concluding that _E. faecalis_ OG1 RF will grow vigorously and that its response to additive respiratory components is minimal, we proceeded to examine the quantitative extent of _E.