A Narrow pH Range Supports Butanol, Hexanol, and Octanol Production from Syngas in a Continuous Co-culture of Clostridium ljungdahlii and Clostridium kluyveri with In-Line Product Extraction

Abstract

Carboxydotrophic bacteria (CTB) have received attention due to their ability to synthesize commodity chemicals from producer gas and synthesis gas (syngas). CTB have an important advantage of a high product selectivity compared to chemical catalysts. However, the product spectrum of wild-type CTB is narrow. Our objective was to investigate whether a strategy of combining two wild-type bacterial strains into a single, continuously fed bioprocessing step would be promising to broaden the product spectrum. Here, we have operated a syngas-fermentation process with Clostridium ljungdahlii and Clostridium kluyveri with in-line product extraction through gas stripping and product condensing within the syngas recirculation line. The main products from C. ljungdahlii fermentation at a pH of 6.0 were ethanol and acetate at net volumetric production rates of 65.5 and 431 mmol C·L−1·d−1, respectively. An estimated 2/3 of total ethanol produced was utilized by C. kluyveri to chain elongate with the reverse β-oxidation pathway, resulting in n-butyrate and n-caproate at net rates of 129 and 70 mmol C·L−1·d−1, respectively. C. ljungdahlii likely reduced the produced carboxylates to their corresponding alcohols with the reductive power from syngas. This resulted in the longer-chain alcohols n-butanol, n-hexanol, and n-octanol at net volumetric production rates of 39.2, 31.7, and 0.045 mmol C·L−1·d−1, respectively. The continuous production of the longer-chain alcohols occurred only within a narrow pH spectrum of 5.7–6.4 due to the pH discrepancy between the two strains. Regardless whether other wild-type strains could overcome this pH discrepancy, the specificity (mol carbon in product per mol carbon in all other liquid products) for each longer-chain alcohol may never be high in a single bioprocessing step. This, because two bioprocesses compete for intermediates (i.e., carboxylates): (1) chain elongation; and (2) biological reduction. This innate competition resulted in a mixture of n-butanol and n-hexanol with traces of n-octanol.

Impact of carbon monoxide partial pressures on methanogenesis and medium chain fatty acids production during ethanol fermentation

“…CO metabolites are involved in chain elongation reactions. Microbial elongation of acetate (2 carbons) to propionate (3 carbons), butyrate (4 carbons), and MCFA using H 2 :CO 2 and/or ethanol as electron and carbon donors has been demonstrated (Diender, Stams, & Sousa, ; Grootscholten, Kinsky dal Borgo, Hamelers, & Buisman, ; Richter, Molitor, Diender, Sousa, & Angenent, ; Steinbusch et al, ). The stoichiometry of these elongation reactions and the corresponding ΔG°′ values (kJ/e − equivalent) are summarized in Table S2.…”

“…Production of caproate and alcohols from syngas (CO with H 2 and CO 2 ) was recently demonstrated by a syngas‐enriched mixed culture (Ganigué, Sánchez‐Paredes, Bañeras, & Colprim, ), and in pure culture studies (Gildemyn et al, ). In addition, co‐culture experiments have shown that CO can be converted to ethanol and then to MCFA in a controlled environment (Diender et al, ; Richter et al, ). However, the environments required for each step are not ideal for the opposite microbial partner, posing large challenges to further optimization and scale‐up with the co‐culture process (Diender et al, ; Richter et al, ).…”

“…In addition, co‐culture experiments have shown that CO can be converted to ethanol and then to MCFA in a controlled environment (Diender et al, ; Richter et al, ). However, the environments required for each step are not ideal for the opposite microbial partner, posing large challenges to further optimization and scale‐up with the co‐culture process (Diender et al, ; Richter et al, ). Besides identification or use of Clostridium spp.…”

Upgrading syngas fermentation effluent using Clostridium kluyveri in a continuous fermentation

“…Diender et al and Richter et al have recently shown that a direct conversion of syngas to n -caproic acid with a co-culture of a carboxydotrophic bacterium and C. kluyveri is also possible [51, 57]. However, they found that a pH discrepancy exists between the two members in the co-culture, and that n -caproic acid was further reduced to n -hexanol, resulting in a low specificity for either product.…”

Diversity, Function, and Application of Clostridium in Chinese Strong Flavor Baijiu Ecosystem: A Review

“…Interspecies complementary metabolism occurs whereby the products of one Clostridium are the substrates of another Clostridium and this is very prevalent in Clostridium present within the SFB ecosystem (Figure ). For example, C. ljungdahlii was able to cometabolize CO 2 and H 2 to acetate and ethanol, which can be used for the production of butyrate and caproic acid by C. kluyveri (Richter, Molitor, Diender, Sousa, & Angenent, ). Another synergistic interaction is the interspecies hydrogen transfer (Thauer, Kaster, Seedorf, Buckel, & Hedderich, ), as the existence of many hydrogen producing Clostridium bacteria and methanogenic archaea in the SFB ecosystem (Ding et al., , , ; Hu et al., ; Li et al., ; Luo et al., ; Tao et al., ; Wu, Ding, Huang, & Zhou, ; Zheng et al., ).…”