Continuous n-valerate formation from propionate and methanol in an anaerobic chain elongation open-culture bioreactor

Abstract

Background: Chain elongation forms a new platform technology for the circular production of biobased chemicals from renewable carbon and energy sources. This study aimed to develop a continuous methanol-based chain elongation process for the open-culture production of a new-generation biofuel precursor and potential platform chemical: n-valerate. Propionate was used as a substrate for chain elongation to n-valerate in an anaerobic open-culture bioreactor. In addition, the co-production of n- and iso-butyrate in addition to n-valerate via, respectively, acetate and propionate elongation was investigated.

Results: n-Valerate was produced during batch and continuous experiments with a pH in the range 5.5-5.8 and a hydraulic retention time of 95 h. Decreasing the pH from 5.8 to 5.5 caused an increase of the selectivity for n-valerate formation (from 58 up to 70 wt%) during methanol-based propionate elongation. n-Valerate and both n- and iso-butyrate were produced during simultaneous methanol-based elongation of propionate and acetate. Propionate was within the open-culture preferred over acetate as a substrate with 10-30% more consumption. Increasing the methanol concentration in the influent (from 250 to 400 mM) resulted in a higher productivity (from 45 to 58 mmol C/L/day), but a lower relative product selectivity (from 49 to 43 wt%) of n-valerate. The addition of acetate as a substrate did not change the average n-valerate productivities. Within the continuous bioreactor experiments, 6 to 17 wt% of formed products was methane. The microbial community during all steady-states in both methanol-based elongation bioreactors was dominated by species related to Clostridium luticellarii and Candidatus Methanogranum. C. luticellarii is the main candidate for n-valerate formation from methanol and propionate.

Conclusions: n-Valerate was for the first time proven to be produced from propionate and methanol by an open-culture bioreactor. Methanogenic activity can be inhibited by decreasing the pH, and the n-valerate productivity can be improved by increasing the methanol concentration. The developed process can be integrated with various biorefinery processes from thermochemical, (bio)electrochemical, photovoltaic and microbial technologies. The findings from this study form a useful tool to steer the process of biological production of chemicals from biomass and other carbon and energy sources.

Keywords: Biobased chemicals; Butyrate; Chain elongation; Methanol; Mixed-culture fermentation; Open-culture fermentation; Selective pressure; n-Valerate.

Fig. 1

Concentration profile (a) during a batch experiment with methanol and propionate with the initial pH 7 at 308 K. Initially (day 0–25), acetate is formed via CO₂ elongation with methanol concurrently with a pH drop. Propionate elongation to valerate starts slowly in the beginning; however, after 40 days when the pH is 5.8 it is the most prevalent metabolic activity. The total conversions at the end of the batch experiment are shown as well (b). The error bars represent the minimum and maximum values measured in the duplo experiments

Fig. 2

Volumetric productivities in time during continuous methanol-based propionate elongation in an anaerobic open-culture reactor at 309 K. The production of protons in mmol/day is also shown ( ). The vertical lines indicate the major changes in the setup: change of the hydraulic retention time (HRT) from 42 to 95 h ( ), pH change from 6.3 to 5.8 ( ) and pH change from 5.8 to 5.5 ( )

Fig. 3

Average volumetric production and consumption rates during the steady-states of phase III (day 78–90) (pH 5.8) and phase IV (day 111–120) (pH 5.5) of continuous methanol-based propionate elongation in an anaerobic open-culture reactor and during the steady-state of phase V (day 64–71) (methanol in influent 250 mM) and during the last days of phase VI (day 97–104) (methanol in influent 400 mM) of continuous methanol-based propionate and acetate elongation in an anaerobic open-culture reactor at 309 K. The error bars represent the minimum and maximum values measured during the phase. Table 2 shows an overview of the average concentrations, productivities and relative product selectivities of n-valerate during the same four phases. The carbon balances for the four steady-states were 88 ± 2, 93 ± 4, 88 ± 4 and 92 ± 3% from left to right, the electron balances were 92 ± 2, 98 ± 4, 87 ± 4 and 91 ± 3%, respectively. The balances can be found in Additional file 1: Figure S3

Fig. 4

Relative selectivity of the formed n-butyrate (n-C4), iso-butyrate (i-C4), n-valerate (n-C5), iso-valerate (i-C5), n-caproate (n-C6), carbon dioxide (CO₂) and methane (CH₄) of continuous methanol-based propionate and acetate elongation in an anaerobic open-culture reactor at 36 °C at the steady-state with 250 mM methanol in the influent (a) (day 64–71) and at the last days with 400 mM methanol in the influent (b) (day 111–120). The values are calculated based on the production rates in g/L/day; the total production rates were 1.37 ± 0.26 (a) and 2.12 ± 0.18 (b) g/L/day

Fig. 5

The hypothetically proposed mechanism for methanol-based propionate elongation to n-valerate [49]. Within the Wood–Ljungdahl pathway, one methanol is oxidized via the THF route to formate/CO, whilst another methanol is supplied to the ACS complex via a CH₃–[Co]-enzyme intermediate [45, 46]. The ACS complex then catalyzes the formation of acetyl-CoA. Depending on the intracellular potential, formate could either be directly utilized for the formation of CO (dotted line) [46], or alternatively CO formation would require the bifurcating hydrogenase as well as an Rnf complex to balance the redox compounds (dashed line) [45]. The formed acetyl-CoA is then likely used in a thiolase-driven condensation step with propionyl-CoA to form 3-ketopentanoyl-CoA, similar to the reverse beta-oxidation mechanism in C. kluyveri [6]. The two NADHs generated during the oxidation of methanol are subsequently used to reduce 3-ketopentanoyl-CoA to 3-hydroxypentanoyl-CoA and to reduce pent-2-enoyl-CoA to pentanoyl-CoA. Because the methanol-based chain elongation of propionate to n-valerate (Table 1R3) has a ΔG of − 106.1 kJ/reaction, an ATP yield of 1.5 ATP would be expected (106.1 kJ/~ 70 kJ/ATP [50] = 1.5 ATP). This suggests that additional energy would be gained via a proton/Na⁺ motive force (pmf) that is likely generated at the oxidation of CH₃-THF [50]. Potentially, additional bifurcation steps within the reverse beta-oxidation part might be necessary, depending on the intracellular redox potentials of the redox cofactors [50, 51]

Fig. 6

Schematic overview of the proposed main conversions that occurred during continuous methanol-based elongation of propionate (Pro) and simultaneous propionate and acetate (Pro&Ac) elongation at 309 K. The four scenarios represent the steady-states in the propionate elongation reactor at pH 5.8 (a) and pH 5.5 (b) and in the simultaneous propionate and acetate elongation reactor with 250 (c) and 400 (d) mM methanol in the reactor influent. The conversions with dashed arrows are proposed to maintain the electron balance. The main reactions are indicated with thicker arrows. For simplicity, only the productivities of the compounds with a value higher than 5 mmol/L/day are shown. The reaction equations and Gibbs free energy of the numbered conversions can be found in Additional file 1

Fig. 7

Schematic (a) and actual (b) setup of upflow anaerobic bioreactor used for continuous methanol-based chain elongation