• Energy Research

Abstract Background Growth substrates, aerobic/anaerobic conditions, specific growth rate (μ) etc. strongly influence Escherichia coli cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA. Results We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor E. coli K-12 MG1655 flux and energy metabolism dynamic responses to change of μ and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing μ and carbon wasting to non-CO2 by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of E. coli pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on μ, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway. Conclusions It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.

Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C 1 waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen Clostridium autoethanogenum is being used as a cell factory in industrial-scale gas fermentation.

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h(-2) after initial stabilization of the cultures in chemostat at D = 0.2-0.3 h(-1). It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used-at an acceleration rate 0.003 h(-2) the quasi steady state growth was observed until mu (crit) = 0.59 h(-1), which is also the mu (max) value for the culture. Lower values of mu (crit) were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h(-1) was immediately disrupted after initiating acceleration at the highest acceleration rate studied-0.06 h(-2). Observation was made that differences [Delta(mu - D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h(-2) (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate-it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S (0) = 5 g L(-1) glucose instead of S (0) = 10 g L(-1). It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility-Delta(mu - D) was less than 5%, standard deviations of biomass yields per ATP produced (Y (ATP)), and biomass yields per glucose consumed (Y (XS)) were less than 15%.

Acetogens are promising cell factories for producing fuels and chemicals from waste feedstocks via gas fermentation, but quantitative characterization of carbon, energy, and redox metabolism is required to guide their rational metabolic engineering. Here, we explore acetogen gas fermentation using physiological, metabolomics, and transcriptomics data for Clostridium autoethanogenum steady-state chemostat cultures grown on syngas at various gas-liquid mass transfer rates. We observe that C. autoethanogenum shifts from acetate to ethanol production to maintain ATP homeostasis at higher biomass concentrations but reaches a limit at a molar acetate/ethanol ratio of ∼1. This regulatory mechanism eventually leads to depletion of the intracellular acetyl-CoA pool and collapse of metabolism. We accurately predict growth phenotypes using a genome-scale metabolic model. Modeling revealed that the methylene-THF reductase reaction was ferredoxin reducing. This work provides a reference dataset to advance the understanding and engineering of arguably the first carbon fixation pathway on Earth.