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Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids.

ABSTRACT: Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate. The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60 % of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150 % increase in ethanol production.

SUBMITTER: Benito-Vaquerizo S

PROVIDER: S-EPMC7658664 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids.

Benito-Vaquerizo Sara S Diender Martijn M Parera Olm Ivette I Martins Dos Santos Vitor A P VAP Schaap Peter J PJ Sousa Diana Z DZ Suarez-Diez Maria M

Computational and structural biotechnology journal 20201016

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcoh ...[more]

PMID: 33240469

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Project description:BackgroundThe 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.ResultsHere, 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.ConclusionsClostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.

Project description:A microbial community is engaged in a complex economy of cooperation and competition for carbon and energy. In engineered systems such as anaerobic digestion and fermentation, these relationships are exploited for conversion of a broad range of substrates into products, such as biogas, ethanol, and carboxylic acids. Medium chain fatty acids (MCFAs), for example, hexanoic acid, are valuable, energy dense microbial fermentation products, however, MCFA tend to exhibit microbial toxicity to a broad range of microorganisms at low concentrations. Here, we operated continuous mixed population MCFA fermentations on biorefinery thin stillage to investigate the community response associated with the production and toxicity of MCFA. In this study, an uncultured species from the Clostridium group IV (related to Clostridium sp. BS-1) became enriched in two independent reactors that produced hexanoic acid (up to 8.1 g L-1), octanoic acid (up to 3.2 g L-1), and trace concentrations of decanoic acid. Decanoic acid is reported here for the first time as a possible product of a Clostridium group IV species. Other significant species in the community, Lactobacillus spp. and Acetobacterium sp., generate intermediates in MCFA production, and their collapse in relative abundance resulted in an overall production decrease. A strong correlation was present between the community composition and both the hexanoic acid concentration (p = 0.026) and total volatile fatty acid concentration (p = 0.003). MCFA suppressed species related to Clostridium sp. CPB-6 and Lactobacillus spp. to a greater extent than others. The proportion of the species related to Clostridium sp. BS-1 over Clostridium sp. CPB-6 had a strong correlation with the concentration of octanoic acid (p = 0.003). The dominance of this species and the increase in MCFA resulted in an overall toxic effect on the mixed community, most significantly on the Lactobacillus spp., which resulted in a decrease in total hexanoic acid concentration to 32 ± 2% below the steady-state average. As opposed to the current view of MCFA toxicity broadly leading to production collapse, this study demonstrates that varied tolerance to MCFA within the community can lead to the dominance of some species and the suppression of others, which can result in a decreased productivity of the fermentation.

Project description:The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO2-containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter PcooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHAMCL), enhanced gene expression through the PcooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Plac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHAMCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition.Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.

Dataset Information

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids.

Publications

Modeling a co-culture of <i>Clostridium autoethanogenum</i> and <i>Clostridium kluyveri</i> to increase syngas conversion to medium-chain fatty-acids.

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