Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation

Abstract

Background: The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures.

Results: Here, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n-caproic acid, which were up to 40 mM day^-1 (4.64 g L^-1 day^-1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n-caproic acid production. In addition, n-caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions.

Conclusions: Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.

Keywords: Carboxylate platform; Carboxylic acids; Chain elongation; Syngas fermentation; n-Caproic acid; n-Caprylic acid.

Fig. 1

Simplified pathways showing the coupling of syngas fermentation, relying on the Wood–Ljungdahl pathway, and biological chain elongation, relying on reverse β-oxidation. The red and green box represents the boundary of the microorganism performing the pathway. More details on both pathways can be found in References [8] and [42]

Fig. 2

Bioreactor setup for fermentation with in-line product extraction (pertraction). The bioreactor broth is sent through a hollow-fiber unit (cell guard) to obtain a cell-free effluent and broth for the pertraction system

Fig. 3

Three separate batch experiments (1, 2, and 3) were carried out. a Growth (as OD) was highest for the standard DSMZ52 (DMSZ) medium and the Mock medium syngas fermentation effluent with added growth factors, but no yeast extract. b Production of n-caproic acid was the highest in the standard DSMZ52 medium, but comparable results were obtained with syngas fermentation effluent with additions. Data represent the mean (n = 3), error bars indicate the standard deviation

Fig. 4

Production rates of n-butyric acid (gray dotted line), n-caproic acid (gray line), and n-caprylic acid (black line) during continuous-mode operation of the bioreactor with pertraction (BP, top, operating pH 6) and without pertraction (BNP, bottom, operating pH 7). The moving average (n = 7) for the n-caproic acid concentration is provided. The seven operating phases (Table 2) are marked with vertical lines