Escherichia colimetabolism under short-term repetitive substrate dynamics: Adaptation and trade-offs

Abstract

7Background 8 Microbial metabolism is highly dependent on the environmental conditions. Especially, the 9 substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-10 scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen 11 availability (mixing limitations), which influence their metabolism and subsequently their 12 physiology. Earlier, single substrate pulse experiments were not able to explain the observed 13 physiological changes generated under large-scale industrial fermentation conditions. 14 Results 15In this study we applied a repetitive feast-famine regime in an aerobic Escherichia coli culture 16 in a time-scale of seconds. The regime was applied for several generations, allowing cells to 17 adapt to the (repetitive) dynamic environment. The observed response was highly reproducible 18 over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an 19 increase of the specific substrate and oxygen consumption (average) rates during the feast-20 famine regime, compared to a steady-state (chemostat) reference environment. The increased 21 rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, 22 this drop was not followed by increased by-product formation, pointing to the existence of 23 energy-spilling reactions and/or less effective ATP synthesis. During the feast-famine cycle, 24 the cells rapidly increased their uptake rate. Within 10 seconds after the beginning of the 25 feeding, the substrate uptake rate was higher (4.68 μmol/gCDW/s) than reported during batch 26 growth (3.3 μmol/gCDW/s). The high uptake led to an accumulation of several intracellular 27 metabolites, during the feast phase, accounting for up to 34 % of the carbon supplied. Although 28 the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, 29 3 suggesting well-controlled balance between ATP producing and ATP consuming reactions. The 30 role of inorganic polyphosphate as an energy buffer is discussed. 31 Conclusions 32The adaptation of the physiology and metabolism of Escherichia coli under substrate dynamics, 33 representative for large-scale fermenters, revealed the existence of several cellular mechanisms 34 coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling 35 processes resulted to be of great importance. These metabolic strategies consist a meaningful 36 step to further tackle reduced microbial performance, observed under large-scale cultivations. 37 38 Keywords 39 Escherichia coli; feast-famine; substrate dynamics; dynamic metabolic responses; energy 40 homeostasis 41 4 Introduction 42Microorganisms are widely used for the production of chemicals, ranging from small organic 43 acids to large proteins, including biopharmaceuticals, biochemicals and bulk biofuels [1][2][3]. In 44 order to meet the cost targets and demands, large-scale production cultivations are and will be 45 required [4]. ...

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