## 4 Discussion  
The growth rate-dependent regulation of the metabolism is fundamental to fine-tune the fueling and biosynthetic reactions in such a way that cells can rapidly adapt to the existing environmental conditions. Typically, the cellular metabolism increases with the growth rate to promote biomass formation in a more efficient way, as demonstrated by biomass yields in chemostat cultures (Table 1), _i.e._, increased biomass yields were observed at higher dilution rates. However, it has been shown that at reduced dilution rates (e.g., 0.05 and 0.1 h\({}^{-1}\)), metabolism is not directly related to the growth rate, as cell growth becomes limited by cell-carbon supply [1]. As a result, the non-linearity observed in most metabolic profiles (Figure 2) must be an effect of the selected growth conditions that are inherently dependent on the energy-efficient use of the carbon substrate for biomass production. In this study, the majority of intracellular metabolite levels had a maximum at a dilution rate of 0.1 h\({}^{-1}\), decreasing below and above this dilution rate. This was previously suggested to be associated with the extremely low residual glucose concentrations in glucose-limited cultures that triggers a series of cellular responses to adapt growth to these nutritional conditions [1, 23]. According to [PERSON] _et al._[24], at a dilution rate of 0.1 h\({}^{-1}\), large flux variations are verified in the metabolic network, in particular at the oxaloacetate node where two anaperotic reactions converge. The carbon flux through the glyoxylate cycle (_i.e._, an anaperotic pathway that converts isocitrate to succinate or to malate via glyoxylate) is maximum at this dilution rate and decreases at higher dilution rates [1, 25, 26]. It was proposed [24, 26, 27] that at nutrient starvation conditions the cAMP-mediated catabolite repression of enzymes in the glyoxylate cycle is limited and the activity of the competing enzyme, _i.e._, the isocitrate dehydrogenase, is decreased. As such, it is believed that anaperotic reactions are stimulated in hungry _E. coli_ cells and, at higher dilution rates, are restrained as a consequence of the increasing glucose concentrations and catabolite repression [28]. Clearly, depending on culture conditions, metabolic flux distributions can differ considerably, reflecting the variable efficiency of carbon utilization either for biomass formation or starvation responses. Besides the aforementioned activities, a general increase in the central metabolism seems to occur at a dilution rate of 0.1 h\({}^{-1}\), at which most metabolites reached its maximum levels. It is generally accepted that under steady state conditions an increase in metabolite levels would correspond to an increase in metabolic activities, since metabolism is fully balanced and no accumulation of intracellular metabolites is expected to occur due to a tight coupling of the anabolism and catabolism [29, 30]. In this work, metabolic profiles of chemostat cultures of two _E. coli_ strains (W3110 and the isogenic \(\Delta\)_rel_A mutant) were determined by GC-MS analysis to explore the effects of different growth rate conditions on the _E. coli_ metabolism, as well as to verify the involvement of RelA under such conditions. It has been proposed that under low growth, the RelA-dependent stringent control of many cellular activities is promoted, including some key metabolic activities [8, 31, 32, 33, 34, 35]. Yet, little is known about the RelA-dependent ppGpp control over the _E. coli_ metabolism and its influence on central metabolic activities. Our results show that metabolite pools were strongly affected by the _rel_A gene mutation as well as by the dilution rate. Though it was expected that metabolite levels would be altered with the dilution rate, due to the capacity of cells to alter their metabolism to cope with new growth conditions, the effect of the introduction of the single gene mutation (\(\Delta\)_rel_A) was more difficult to predict.