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

BackgroundMicrobial metabolism is highly dependent on the environmental conditions. Especially, the substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen availability (mixing limitations), which influence their metabolism and subsequently their physiology. Earlier, single substrate pulse experiments were not able to explain the observed physiological changes generated under large-scale industrial fermentation conditions.ResultsIn this study we applied a repetitive feast-famine regime in an aerobic Escherichia coli culture in a time-scale of seconds. The regime was applied for several generations, allowing cells to adapt to the (repetitive) dynamic environment. The observed response was highly reproducible over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an increase of the specific substrate and oxygen consumption (average) rates during the feast-famine regime, compared to a steady-state (chemostat) reference environment. The increased rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, this drop was not followed by increased by-product formation, pointing to the existence of energy-spilling reactions and/or less effective ATP synthesis. During the feast-famine cycle, the cells rapidly increased their uptake rate. Within 10 seconds after the beginning of the feeding, the substrate uptake rate was higher (4.68 μmol/gCDW/s) than reported during batch growth (3.3 μmol/gCDW/s). The high uptake led to an accumulation of several intracellular metabolites, during the feast phase, accounting for up to 34 % of the carbon supplied. Although the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, suggesting well-controlled balance between ATP producing and ATP consuming reactions. The role of inorganic polyphosphate as an energy buffer is discussed.ConclusionsThe adaptation of the physiology and metabolism of Escherichia coli under substrate dynamics, representative for large-scale fermenters, revealed the existence of several cellular mechanisms coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling processes resulted to be of great importance. These metabolic strategies consist a meaningful step to further tackle reduced microbial performance, observed under large-scale cultivations.

Quantifying the Responses of Mixed Rumen Microbes to Excess Carbohydrate

ABSTRACTThe aim of this study was to determine if a mixed microbial community from the bovine rumen would respond to excess carbohydrate by accumulating reserve carbohydrate, energy spilling (dissipating excess ATP energy as heat), or both. Mixed microbes from the rumen were washed with N-free buffer and dosed with glucose. Total heat production was measured by calorimetry. Energy spilling was calculated as heat production not accounted by (i) endogenous metabolism (heat production before dosing glucose) and (ii) synthesis of reserve carbohydrate (heat from synthesis itself and reactions yielding ATP for it). For cells dosed with 5 mM glucose, synthesis of reserve carbohydrate and endogenous metabolism accounted for nearly all heat production (93.7%); no spilling was detected (P= 0.226). For cells dosed with 20 mM glucose, energy spilling was not detected immediately after dosing, but it became significant (P< 0.05) by approximately 30 min after dosing with glucose. Energy spilling accounted for as much as 38.7% of heat production in one incubation. Nearly all energy (97.9%) and carbon (99.9%) in glucose were recovered in reserve carbohydrate, fermentation acids, CO2, CH4, and heat. This full recovery indicates that products were measured completely and that spilling was not a methodological artifact. These results should aid future research aiming to mechanistically account for variation in energetic efficiency of mixed microbial communities.

Kinetic Analysis of Growth Rate, ATP, and Pigmentation Suggests an Energy-Spilling Function for the Pigment Prodigiosin of Serratia marcescens

ABSTRACTSerratia marcescensis a gram-negative environmental bacterium and opportunistic pathogen.S. marcescensexpresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes forS. marcescensduring cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.

Minimization of activated sludge production by chemically stimulated energy spilling

Minimization of excess sludge production in activated sludge processes has been pursued around the world in order to meet stringent environmental regulations on sludge treatment and disposal. To achieve this goal, physical, chemical, and biological approaches have been proposed. In this paper, a chemical compound, 3,3′,4′,5-tetrachlorosalicylanilide (TCS) was tested for enhancing microbial energy spilling of the sludgeso as to minimize its growth. In order to examine this, an exploratory study was conducted using both batch and continuous activated sludge cultures. Batch experiments with these two cultures were carried out at different initial concentrations of TCS. It has been confirmed that an addition of TCS is effective in reducing the production of both the sludge cultures, particularly the continuous culture where the observed growth yield was reduced by around 70%, when the initial TCS concentration was 0.8 ppm. Meanwhile, the substrate removal activity of this culture was found not to be affected at this TCS concentration. To further evaluate the TCS effect, a pure microbial culture of E. coli was employed. Batch experiment results with this culture implied that TCS might be able to reduce the cell density of E. coli drastically when an initial TCS concentration was greater than 0.12 ppm. It was also found that TCS was not toxic to this type of bacteria. Microscopic examinations with a 4′, 6-diamidino-2-phenylindole (DAPI) staining technique revealed that TCS neither affected the cell division nor altered the cell size of E. coli. However, both the cell ATP content and the cell dry weight were reduced significantly with the addition of TCS.