## 3 Results
### Metabolite Profiling  
A chemical derivatization procedure was chosen in order to detect the main amino and non-amino organic acids and their precursors in the central carbon metabolism and fatty acid biosynthesis. The overall list of identified metabolites is presented in Table 2.  
We have observed that both growth conditions (_i.e._, dilution rates) and the genetic characteristics of _E. coli_ strains (_i.e._, presence/absence of the _rel_A gene deletion) induced significant alterations in the metabolite profiles of bacterial cultures, though the number of metabolites that had their levels significantly different depending on the dilution rate was slightly higher than when comparing _E. coli_ strains (16 and 14 metabolites, respectively). Still, nine metabolites were commonly altered in both experimental conditions, indicating that metabolic states of cultures were profoundly affected in both cases. Almost 50% of the total metabolites were detected at significantly different levels in the mutant strain compared to the wild-type (Figure 1), meaning that, at the same steady state conditions, at least half of the detected metabolites presented significant differences in their abundances when comparing the two cultures. This suggests that enzymatic activities involving these metabolites are somehow influenced by this single mutation, leading to alterations in their levels. For instance, significant changes in amino and fatty acids levels, in particular tetradecanoate (_ttdca_, n-C14:0), pentadecanoate (_pdca_, n-C15:0), 10,13-dimethyltetradecanoate (_1013 mlt_), octadecanoate (_ocdca_, n-C18:0), isoleucine (_ile_), threonine (_thr_), aspartate (_asp_) and glutamate (_glu_) were observed. Other metabolites that revealed interesting differences include N-acetyl-L-glutamate (_acglu_), lysine (_lys_), malate (_mal_), alpha-ketoglutarate (_akg_), itaconate (_itcon_) and malonate (_ma_); that were uniquely detected in the _E. coli_ W3110 culture at a dilution rate of 0.1 h\({}^{-1}\) (see Figure S1). Although these were not retrieved as statistically significant in the [PERSON]'s test, since they were not detected in any other samples, they may contribute to the differentiation between the metabolic behavior of W3110 and _ \begin{table}
\begin{tabular}{l l l l} \hline _TCA intermediaries_ & _Faty acids_ & _Amino acids_ & _Others_ \\ \hline alpha-ketoglutate & Hexanoate (hxa, n-C6:0) & Aspartate (asp) & Benzoate* (bnz) \\ (akg) & Octanoate (octa, n-C8:0) & Isoleucine (ile) & NADP(H) \\ _cis_-Aconitate (acon-C) & Decanoate (dca, n-C10:0) & Lysine (lys) & Nicotinate (nac) \\ Citrate (cit) & Tetradecanoate (ttdca, n-C14:0) & Threonine (thr) & Phosphoenolpyruvate (pep) \\ Fumarate (fum) & 10,13-Dimethyltetradecanoate (1013 mlt) & Alanine (ala) & 5-oxo-D-proline* (pyrglu) \\ Malate (mal) & Pentadecanoate (pdca, n-C15:0) & Leucine (leu) & Malonate* (ma) \\ Succinate (succ) & 14-Methylpentadecanoate (14 mpdca) & Valine (val) & Itaonate* (itcon) \\ Octadecanoate (ocdca, n-C18:0) & Glycine (gly) & Lactate (lac) \\ Octadeconate (ocdcea, n-C18:1) & Serine (ser) & \\
9-_cis_,12-_cis_-Octadecadiene (ocdcin, n-C18:2) & Glutamate (glu) & \\ C18:2) & Proline (pro) & \\  & & Phenylalanine (phe) \\  & & (_2S_)-2-isopropylmalate \\  & & (3c3 hmp) \\  & & N-Acetyl-L-glutamate (acglu) \\ \hline \end{tabular}  
* Metabolites unknown to be synthesized by _E. coli_  
\end{table} 
Table 2: List of the intracellular metabolites identified after GC-MS analysis. cultures. These metabolites are essentially associated with amino acid biosynthetic activities or metabolic regulation, like the itaconate (_itcon_) and malonate (_ma_), known to be enzymatic inhibitors of the isocitrate lyase, an enzyme associated with the glyoxylate cycle. Besides differences in metabolite levels, we have paid attention to the changes in metabolite profiles produced by each _E. coli_ strain at different dilution rates.