The potential of caproate (hexanoate) production using Clostridium kluyveri syntrophic cocultures with Clostridium acetobutylicum or Clostridium saccharolyticum

Abstract

Caproate (hexanoate) and other medium-chain fatty acids are valuable platform chemicals produced by processes utilizing petroleum or plant oil. Clostridium kluyveri, growing on short chain alcohols (notably ethanol) and carboxylic acids (such as acetate) is noted for its ability to perform chain elongation to produce 4- to 8-carbon carboxylates. C. kluyveri has been studied in monoculture and coculture conditions, which lead to relatively modest carboxylate titers after long fermentation times. To assess the biosynthetic potential of C. kluyveri for caproate production from sugars through coculture fermentations, in the absence of monoculture data in the literature suitable for our coculture experiments, we first explored C. kluyveri monocultures. Some monocultures achieved caproate titers of 150 to over 200 mM in 40–50 h with a production rate of 7.9 mM/h. Based on that data, we then explored two novel, syntrophic coculture partners for producing caproate from sugars: Clostridium acetobutylicum and Clostridium saccharolyticum. Neither species has been cocultured with C. kluyveri before, and both demonstrate promising results. Our experiments of C. kluyveri monocultures and C. kluyveri—C. saccharolyticum cocultures demonstrate exceptionally high caproate titers (145–200 mM), fast production rates (3.25–8.1 mM/h), and short fermentation times (18–45 h). These results represent the most caproate produced by a C. kluyveri coculture in the shortest known fermentation time. We also explored the possibility of heterologous cell fusion between the coculture pairs similar to the results seen previously in our group with C. acetobutylicum and Clostridium ljungdahlii. Fusion events were observed only in the C. acetobutylicum—C. kluyveri coculture pair, and we offer an explanation for the lack of fusion between C. saccharolyticum and C. kluyveri. This work supports the promise of coculture biotechnology for sustainable production of caproate and other platform chemicals.

Species-Specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

“…In addition to cytoplasmic exchange studies, rRNA-FISH has several advantages over other methods as a tool for subpopulation tracking [Fig. [4][5][6]49)] as well as for identifying the fraction of actively translating (and thus actively metabolizing) cells, which, as we have shown here (Fig. 7-9) is very different for different species.…”

“…Work characterizing the metabolic consequences of C. acetobutylicum-C. kluyveri and a C. ljungdahlii - C. kluyveri co-cultures has been published largely focusing on the metabolic potential of the co-cultures (6, 11). Our 2022 paper provides the first evidence for cytoplasmic material exchange between C. acetobutylicum and C. kluyveri (6). We sought to explore the interspecies relationships, especially cytoplasmic exchange, between these organisms.…”

“…(6,11). Our 2022 paper provides the first evidence for cytoplasmic material exchange between C. acetobutylicum and C. kluyveri (6). We sought to explore the interspecies relationships, especially cytoplasmic exchange, between these organisms.…”

Environmental biotechnologies can make water pollutants part of the path to mitigating climate change

“…Carboxylic acids with longer chain lengths are easier to separate and also have higher economic value per carbon [51,53,54]. One trend to notice in the three examples is that chain elongation is a reduction process, which means that delivering an electron donor, such as H 2 (shown here) or an alcohol, can upgrade the chain length and value of the organic products [52,55,56].…”

Evaluating the feasibility of medium chain oleochemical synthesis using microbial chain elongation

“…While most chain elongators remain uncultivated, over 15 strains have been isolated and sequenced to date, with most new isolates being reported in the last 10 years (Candry & Ganigué, 2021). These isolates, particularly Clostridium kluyveri, have been used to establish synthetic co-cultures to convert gaseous or sugar-based feedstocks to MCFAs and their corresponding alcohols (Bäumler et al, 2022; Diender et al, 2016; Haas et al, 2018; Otten et al, 2022; R. Lynd et al, 2022) which could be expanded to more complex feedstocks by selecting lactate and/or ethanol-producing partners with improved hydrolytic capabilities.…”