Self-Synchronized Oscillatory Metabolism of Clostridium pasteurianum in Continuous Culture

Abstract

By monitoring the real-time gas production (CO2 and H2) and redox potential at high sampling frequency in continuous culture of Clostridium pasteurianum on glycerol as sole carbohydrate, the self-synchronized oscillatory metabolism was revealed and studied. The oscillations in CO2 and H2 production were in sync with each other and with both redox potential and glycerol in the continuous stirred tank reactor (CSTR). There is strong evidence that the mechanism for this is in the regulation of the oxidative pathway of glycerol metabolism, including glycolysis, and points toward complex, concerted cycles of enzyme inhibition and activation by pathway intermediates and/or redox equivalents. The importance of understanding such an “oscillatory metabolism” is for developing a stable and highly productive industrial fermentation process for butanol production, as unstable oscillations are unproductive. It is shown that the oscillatory metabolism can be eradicated and reinstated and that the period of oscillations can be altered by modification of the operating parameters. Synchronized oscillatory metabolism impacted the product profile such that it lowered the selectivity for butanol and increased the selectivity for ethanol. This elucidates a possible cause for the variability in the product profile of C. pasteurianum that has been reported in many previous studies.

Metabolic Oscillation Phenomena in Clostridia Species—A Review

“…Such oscillation forms a challenge in the establishment of a true steady state, hindering more efficient production, and needs to be understood to be mitigated. Figure 1 compares the different timescales of the oscillatory behaviours of two different Clostridia species, both reported in 2020 [32,33]. The oscillation in gas consumption by Clostridium autoethanogenum follows a 60-100 h period [33], while the gas production of Clostridium pasteurianum [32] oscillates every 2 h. In both cases, the metabolites and cell concentrations are also affected by the oscillation.…”

“…Figure 1 compares the different timescales of the oscillatory behaviours of two different Clostridia species, both reported in 2020 [32,33]. The oscillation in gas consumption by Clostridium autoethanogenum follows a 60-100 h period [33], while the gas production of Clostridium pasteurianum [32] oscillates every 2 h. In both cases, the metabolites and cell concentrations are also affected by the oscillation. In the case of C. pasteurianum [32], this could only be shown through carefully timed sampling at the peaks and valleys of the online signals obtained from off-gas and redox probes (glycerol and butanol data shown in Figure 1).…”

“…The oscillation in gas consumption by Clostridium autoethanogenum follows a 60-100 h period [33], while the gas production of Clostridium pasteurianum [32] oscillates every 2 h. In both cases, the metabolites and cell concentrations are also affected by the oscillation. In the case of C. pasteurianum [32], this could only be shown through carefully timed sampling at the peaks and valleys of the online signals obtained from off-gas and redox probes (glycerol and butanol data shown in Figure 1). The oscillation would have likely evaded detection based on off-line analysis only, and butanol and glycerol data would have simply appeared as "noisy", had the sampling time not been synchronized with the oscillation rate.…”

Metabolism, morphology and transcriptome analysis of oscillatory behavior of Clostridium butyricum during long-term continuous fermentation for 1,3-propanediol production

“…Possible reasons for the oscillation could be pyruvate metabolism disorder [ 28 ] and/or toxicity of intermediate 3-HPA [ 27 ]. Recently, another 1,3-PDO producer, Clostridium pasteurianum exhibited spontaneous oscillation in continuous fermentation, probably caused by toxicity of byproduct butanol [ 26 ] and/or relative to concerted cycles of inhibition and activation of enzymes for glycerol oxidation [ 25 ]. Exploration of the microbial oscillation not only helps us to better understand the internal metabolic regulation of microbial cells, but also contributes to the process stability in industrial application.…”

“…Intracellular feedback regulation, on the other hand, includes cell cycle (rhythmic phenomena for higher animal/cell division for microorganism), induction/repression of enzyme and allosteric control of enzyme activity. For microorganism particularly, reasons for the oscillatory behavior are complex, which are associated with type of microorganisms and culture condition, and one species of microorganism could exist several potential mechanisms [25][26][27][28].…”