Abstract
This study used 13C tracers and dynamic labeling to reveal metabolic features (nutrients requirements, pathway delineation and metabolite turnover rates) of Clostridium carboxidivorans P7, a model strain for industrial syngas fermentation, and its implication with bioreactor mass transfer. P7 shows poor activity for synthesizing amino acids (e.g., phenylalanine) and thus, needs rich medium for cell growth. The strain has multiple carbon fixation routes (Wood-Ljungdahl pathway, pyruvate:ferredoxin oxidoreductase reaction and anaplerotic pathways) and Re-citrate synthase (Ccar_06155) was a key enzyme in its tricarboxylic acid cycle (TCA) pathway. High fluxes were observed in P7’s Wood-Ljungdahl pathway, right branch of TCA cycle, pyruvate synthesis, and sugar phosphate pathways, but the cells anabolic pathways were strikingly slow. In bioreactor culture, when syngas flowrate increased from 1 to 10 mL/min, P7 strain produced same amount of total extracellular products (acids and alcohols) but high flowrate favored alcohol accumulation. This observation was due to the mass transfer limitation influencing energy metabolism (CO/H2 oxidation for cofactor generations) more prominently than carbon fixation. When syngas flowrate increased from 10 of 20 mL/min, the alcohol productivity was not improved and the labeling rate (~0.03 h−1) of key metabolite acetyl-CoA reached to P7 strain’s metabolism limitation regime.
Highlights
Biological utilization syngas such as CO2 and CO becomes an important research field due to cheap feedstock and the concerns of global warming
The information will inform us the influence of rich medium on labelling test and metabolic flux quantifications. This is because the complex carbon nutrients in yeast extract (YE) can incorporated into biomass, and interfere with a quantitative flux analysis
P7 barely grew in YE-free medium even carbon sources (glucose (GLU) or fructose (FRU)) were provided. This result was similar to previous report that P7 syngas fermentation in YE-free medium experienced a very long lag phase (5 days) with minimal cell density achieved over 600 hours[26]
Summary
Biological utilization syngas such as CO2 and CO becomes an important research field due to cheap feedstock and the concerns of global warming. In addition to mass transfer limitations, biotransformation of gaseous substrates into metabolites inside the cell can be another bottleneck in syngas fermentation. It is necessary to have a thorough investigation of the rate and routes of cell metabolism for conversion of acetyl-CoA into cascade metabolites during syngas fermentation. Isotope tracer technique has been used to determine the mass transport in bioreactors[13,14,15] This approach can investigate cell metabolism by analyzing isotopomer of both proteinogenic amino acids and fast turnover free metabolites[16]. The aim of this work is to elucidate functional pathway for cell growth and syngas conversion under different bioreactor mass transfer scenarios. Via 13C-fingerprinting of proteinogenic amino acid and fast-turnover metabolites, this study tracks cell adsorption of sugars or syngas (CO/CO2) from culture medium into its biosynthesis pathways[20]. The dynamic labeling/un-labelling of metabolites provides new insights into functions of individual central pathway under different mass transfer conditions
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