Control of redox potential in a novel continuous bioelectrochemical system led to remarkable metabolic and energetic responses of Clostridium pasteurianum grown on glycerol

Abstract

Background: Electro-fermentation (EF) is an emerging tool for bioprocess intensification. Benefits are especially expected for bioprocesses in which the cells are enabled to exchange electrons with electrode surfaces directly. It has also been demonstrated that the use of electrical energy in BES can increase bioprocess performance by indirect secondary effects. In this case, the electricity is used to alter process parameters and indirectly activate desired pathways. In many bioprocesses, oxidation-reduction potential (ORP) is a crucial process parameter. While C. pasteurianum fermentation of glycerol has been shown to be significantly influenced electrochemically, the underlying mechanisms are not clear. To this end, we developed a system for the electrochemical control of ORP in continuous culture to quantitatively study the effects of ORP alteration on C. pasteurianum by metabolic flux analysis (MFA), targeted metabolomics, sensitivity and regulation analysis.

Results: In the ORP range of -462 mV to -250 mV, the developed algorithm enabled a stable anodic electrochemical control of ORP at desired set-points and a fixed dilution rate of 0.1 h^-1. An overall increase of 57% in the molar yield for 1,3-propanediol was observed by an ORP increase from -462 to -250 mV. MFA suggests that C. pasteurianum possesses and uses cellular energy generation mechanisms in addition to substrate-level phosphorylation. The sensitivity analysis showed that ORP exerted its strongest impact on the reaction of pyruvate-ferredoxin-oxidoreductase. The regulation analysis revealed that this influence is mainly of a direct nature. Hence, the observed metabolic shifts are primarily caused by direct inhibition of the enzyme upon electrochemical production of oxygen. A similar effect was observed for the enzyme pyruvate-formate-lyase at elevated ORP levels.

Conclusions: The results show that electrochemical ORP alteration is a suitable tool to steer the metabolism of C. pasteurianum and increase product yield for 1,3-propanediol in continuous culture. The approach might also be useful for application with further anaerobic or anoxic bioprocesses. However, to maximize the technique's efficiency, it is essential to understand the chemistry behind the ORP change and how the microbial system responds to it by transmitted or direct effects.

Keywords: BES; Clostridium pasteurianum; Continuous fermentation; ORP; Redox metabolism; Regulation analysis; Symbolic metabolic control analysis.

Fig. 1

Different strategies for ORP control in fermentation processes. a Addition of liquid reducing/oxidizing agents. b Sparging with reducing/oxidizing gases (control of aeration rate and agitation). c Application of electrical energy in a BES. WE = working electrode; CE = counter electrode

Fig. 2

Online ORP values (in mV) during the continuous electrochemically ORP-controlled cultivation of C. pasteurianum in a BES at D = 0.1 h⁻¹. Captures indicate the following: Number of steady state (I-IV); set-points for the electrochemical ORP control (in mV) / applied current (in mA) / pulsing time (in ms). A detailed description of the control mechanism is given in the text. The electrochemical pulsing was interrupted by 100 ms without current application. Black blocks indicate time points of fast-sampling and changing of controller set-points

Fig. 3

Results of the metabolic flux analysis during the continuous and ORP-controlled cultivation of C. pasteurianum in Biebl medium with glycerol (36 g L⁻¹ feed concentration and D = 0.1 h⁻¹). For better visualization, data points obtained from the flux analysis were connected by linear curves. For all graphs: y-axis gives the reaction rate in mmol g⁻¹ h⁻¹ and x-axis ORP in mV. For simplicity, only NADH, ATP, Ferredoxin (Fd_red), and protons are shown as cofactors. Dashed lines indicate measured reaction rates at steady state. Solid lines indicate calculated rates

Fig. 4

Molar NADH/NAD ratio, adenylate energy charge (AEC), and intracellular concentrations of glyceralaldehyde-3-phosphate (G-3-P), pyruvate, butyryl-CoA and acetyl-CoA during the continuous and ORP controlled cultivation of C. pasteurianum in Biebl medium with glycerol (36 g L⁻¹ feed concentration and D = 0.1 h⁻¹). Small letters above the blue curve denote that the data point belongs to a statistically significantly (α = 0.05) differing group. When no letters are shown in the subpanels, data did not differ significantly, as tested by ANOVA

Fig. 5

Sensitivity analysis of intracellular reactions in response to electrochemically-controlled ORP changes during the continuous cultivation of C. pasteurianum grown on glycerol. Δq₁: -462 mV → -416 mV; Δq₂: -416 mV → -337 mV; Δq₃: -337 mV → -250 mV. If no value is shown, the reaction rate was zero at the new state and normalization is not possible

Fig. 6

Reaction network used for symbolic MCA and regulation analysis. G3P = glyceralaldeyde-3-phosphate

Fig. 7

Visualization of regulation analysis in response to electrochemically-controlled ORP changes during the continuous cultivation (D = 0.1 h⁻¹) of C. pasteurianum grown on glycerol. a) Δq₁: − 462 mV → -− 16 mV; b) Δq₂: − 416 mV → -− 37 mV; c) Δq₃: − 337 mV → -− 250 mV. Green links indicate an activation/positive response of the flux or metabolite, and red links indicate an inhibition/negative response. Starting from the center node (Δq_i), the first edges in both directions indicates the negative and positive response of a flux or intermediate (sum of negative and positive partial integrated responses). The second respective links towards the outer vertices shows through which flux the response was transmitted (equals partial integrated response). Thickness of the links correspond to the relative strength/value of the (partial) integrated responses. Missing of fluxes or intermediates in one scenario means that they were not affected by the external parameter change