## 4 Discussion
Differences observed in biomass yields have originally pointed to distinct metabolic behaviorsbetween the two strains, _i.e.,_ biomass yields were higher in the \(\Delta\)_rel_A mutant cultures and were not linearly-dependent on the growth rate at lower dilution rates (0.05 and 0.1 h\({}^{-1}\)). Additionally, metabolomics analysis revealed that approximately 50% of the whole set of metabolites detected in this study presented significant changes between the _E. coli_ W3110 and the \(\Delta\)_rel_A mutant cultures (Figure 1). Most of these differences consisted in altered levels of amino acids and fatty acids indicating that the RelA-dependent ppGpp control of metabolic activities involving these metabolites might be affected. This seems to be the case of fatty acids like octadecanoate (_ocdca_), tetradecanoate (_ttdca_), pentadecanoate (_pdca_) and 10,13-dimethyltetradecanoate (_1013 mlt_), that presented maximum levels at a dilution rate of 0.05 h\({}^{-1}\) in the _E. coli_ W3110 culture. Other examples include metabolites that were uniquely detected in the _E. coli_ W3110 culture at a dilution rate of 0.1 h\({}^{-1}\): \(N\)-acetyl-L-glutamate (_acglu_), lysine (_lys_), malate (_mal_), alpha-ketoglutarate (_akg_), itaconate (_itcon_) and malonate (_ma_) (see Figure S1). It seems that, at this particular dilution rate, the behaviour and regulation of metabolic activities associated (directly or indirectly) with those metabolites might be dependent on the activity of the RelA enzyme and, as a result, these metabolites could not be detected in the \(\Delta\)_rel_A cultures, at least at traceable amounts. One of the most interesting phenotypic traits of the \(\Delta\)_rel_A mutant strain is the reduced accumulation of acetate if compared to the control strain (0.02 and 0.34 g\(\cdot\)L\({}^{-1}\), respectively). Acetate was only detected in cultures at a dilution rate of 0.2 h\({}^{-1}\), but differences between the two cultures reveal that the mutation influences the metabolic overflow metabolism. The overflow metabolism has an impact on biomass yields, as observed in our study, _i.e.,_ the biomass yields of the mutant and wild-type cultures were 0.67 and 0.55 g of biomass per g of glucose, respectively, and may lead to growth arrest if the accumulation of by-products, such as acetate, reaches toxic levels. The acetate overflow metabolism has been recently investigated [36, 37] and researchers found that acetate overflow results from the unbalanced synthesis and scavenging activities that are controlled by different mechanisms, including the CRP-cAMP-dependent catabolic repression. Under higher dilution rates (e.g., 0.2 h\({}^{-1}\)), the CRP-cAMP-dependent catabolic repression augments the overflow metabolism through the down-regulation of the acetyl-CoA synthetase that scavenges acetate. We hypothesize that this mutant is less responsive to this phenomenon and thus, acetate accumulation is reduced. Besides these differences, it was found that some metabolite profiles correlate poorly when comparing _E. coli_ W3110 and \(\Delta\)_rel_A cultures at different dilution rates. This was mainly observed in fatty acids (octadecanoate (_ocdca_), tetradecanoate (_ttdca_), pentadecanoate (_pdca_) and 10,13-dimethyltetradecanoate (_1013 mlt_)) that have also shown largest differences in the Mack-Skillings test for the strain factor (p-values of 0.0002) and threonine (_thr_), lactate (_lac_) and succinate (_succ_) profiles, which presented the lowest correlation coefficients (r \(<\) 0.6). This suggests that _E. coli_A_ mutant cells are unable to maintain a close-to-wild-type behavior of the central carbon metabolism that may lead to important imbalances in metabolic functions. It has been described that fatty acid biosynthetic genes are stringently controlled by ppGpp [38, 39] and under nutrient-limiting conditions bacterial cells tend to adjust their cell wall composition [35, 40, 41].