Medium-Chain Fatty Acids (MCFA) Production Through Anaerobic Fermentation Using Clostridium kluyveri: Effect of Ethanol and Acetate.

Medium-chain fatty acids (MCFA) are saturated monocarboxylic acids and can be used as antimicrobials, corrosion inhibitors, precursors in biodiesel, and bioplastic production. In the present study, MCFA production was evaluated with acetate and ethanol using the bacteria Clostridium kluyveri. Effects of substrate, electron donor, and methane inhibitor on MCFA production were evaluated. Bacteria successfully converted the ethanol and acetate to butyrate (C4), caproate (C6), and caprylate (C8) by chain elongation process. The highest concentrations of butyrate (4.6 g/l), caproate (3.2 g/l), and caprylate (0.5 g/l) were obtained under methane inhibition conditions than other conditions. The productions of butyrate and caproate were 1.6 and 1.48 times higher under methane inhibition conditions, respectively. Results denoted that the bacteria C. kluyveri can be used for conversion of acetate and ethanol into useful products like butyrate and caproate.

Predicting and experimental evaluating bio-electrochemical synthesis - A case study with Clostridium kluyveri.

Microbial electrosynthesis is a highly promising application of microbial electrochemical technologies for the sustainable production of organic compounds. At the same time a multitude of questions need to be answered and challenges to be met. Central for its further development is using appropriate electroactive microorganisms and their efficient extracellular electron transfer (EET) as well as wiring of the metabolism to EET. Among others, Clostridia are believed to represent electroactive microbes being highly promising for microbial electrosynthesis. We investigated the potential steps and challenges for the bio-electrochemical fermentation (electro-fermentation) of mid-chain organic acids using Clostridium kluyveri. Starting from a metabolic model the potential limitations of the metabolism as well as beneficial scenarios for electrochemical stimulation were identified and experimentally investigated. C. kluyveri was shown to not be able to exchange electrons with an electrode directly. Therefore, exogenous mediators (2-hydroxy-1,4-naphthoquinone, potassium ferrocyanide, neutral red, methyl viologen, methylene blue, and the macrocyclic cobalt hexaamine [Co(trans-diammac)]3+) were tested for their toxicity and electro-fermentations were performed in 1L bioreactors covering 38 biotic and 8 abiotic runs. When using C. kluyveri and mediators, maximum absolute current densities higher than the abiotic controls were detected for all runs. At the same time, no significant impact on the cell metabolism (product formation, carbon recovery, growth rate) was found. From this observation, we deduce general potential limitations of electro-fermentations with C. kluyveri and discuss strategies to successfully overcome them.

Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri.

Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.