Metabolomic and kinetic investigations on the electricity-aided production of butanol by Clostridium pasteurianum strains

Abstract

In this contribution, we studied the effect of electro-fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3-5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro-fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms.

Keywords: BES; Clostridium pasteurianum; butanol production; electro‐fermentation; redox metabolism.

FIGURE 1

Main metabolic pathways of glycerol fermentation in C. pasteurianum. 1,3‐PDO, 1,3‐propanediol; Fd/FdH, oxidized/reduced ferredoxin

FIGURE 2

Measured dry cell weight (DCW) (A), extracellular concentrations of 1,3‐propanediol (PDO) (B), butanol (C), ethanol (D), glycerol (E) and total amount of consumed glycerol (F) during the fed‐batch cultivation of C. pasteurianum in Biebl medium. Initial glycerol concentration was 25 g L^–1. Feed started after 8 h with 3 g L h^–1, reduced after 20 h to 1 g L h^–1. Application of −400 mA current after 8 h in the BES cultivations

FIGURE 3

Extracellular concentrations of lactate (A), formate (B), acetate (C), and butyrate (D) during the fed‐batch cultivation of C. pasteurianum in Biebl medium. Initial glycerol concentration was 25 g L^–1. Feed start after 8 h with 3 g L h^–1, reduced after 20 h to 1 g L h^–1. Application of −400 mA current after 8 h in the BES cultivations

FIGURE 4

Estimated cell‐specific rate of glycerol uptake (A, B) and butanol production (C, D) of C. pasteurianum cells during fed‐batch and electricity‐aided fed‐batch cultivation. PDO production is only shown for the R525 strain (E), since no PDO production was observed for the dhaB mutant strain. F: online measured H₂/CO₂ ratio from all four conducted cultivations

FIGURE 5

Intracellular NADH/NAD ratio (A, B) and adenylate energy charge (C, D) of C. pasteurianum cells during fed‐batch and electricity‐aided fed‐batch cultivation. Errors indicate the standard deviation of concentrations obtained from three separate metabolite extractions. When no error bar is stated, the average of two samples is shown

FIGURE 6

Intracellular concentrations of pyruvate (A, B), acetyl‐CoA (C, D), and butyryl‐CoA (E, F) in C. pasteurianum cells during fed‐batch and electricity‐aided fed‐batch cultivation. Errors indicate the standard deviation of concentrations obtained from three separate metabolite extractions. When no error bar is stated, the average of two samples is shown