Abstract
Bulk production of medium-chain carboxylates (MCCs) with 6–12 carbon atoms is of great interest to biotechnology. Open cultures (e.g., reactor microbiomes) have been utilized to generate MCCs in bioreactors. When in-line MCC extraction and prevention of product inhibition is required, the bioreactors have been operated at mildly acidic pH (5.0–5.5). However, model chain-elongating bacteria grow optimally at neutral pH values. Here, we isolated a chain-elongating bacterium (strain 7D4C2) that grows at mildly acidic pH. We studied its metabolism and compared its whole genome and the reverse β-oxidation (rBOX) genes to other bacteria. Strain 7D4C2 produces lactate, acetate, n-butyrate, n-caproate, biomass, and H2/CO2 from hexoses. With only fructose as substrate (pH 5.5), the maximum n-caproate specificity (i.e., products per other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and n-butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and n-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing n-caproate specificity from ∼92% at mildly acidic pH (pH 4.5-5.0) to ∼24% at alkaline pH (pH 9.0). Moreover, when carboxylates were extracted from the broth (undissociated n-caproic acid was ∼0.3 mM), the n-caproate selectivity (i.e., product per substrate fed) was 42.6 ± 19.0% higher compared to 7D4C2 cultures without extraction. Based on the 16S rRNA gene sequence, strain 7D4C2 is most closely related to the isolates Caproicibacter fermentans (99.5%) and Caproiciproducens galactitolivorans (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, C. fermentans, and C. galactitolivorans belong to the same genus of Caproiciproducens. Their rBOX genes are conserved and located next to each other, forming a gene cluster, which is different than for other chain-elongating bacteria such as Megasphaera spp. In conclusion, Caproiciproducens spp., comprising strain 7D4C2, C. fermentans, C. galactitolivorans, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. Caproiciproducens sp. 7D4C2 (DSM 110548) was studied here to understand n-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into n-caproate.
Highlights
Medium-chain carboxylates (MCCs, 6–12 carbon atoms) are precursors to liquid fuels (Levy et al, 1981)
We revived the sample with ethanol (40 mM), acetate (4 mM), n-caproate (4 mM), and n-caprylate (4 mM) in basal medium that was buffered with 91.5 mM MES and supplemented with 0.05% w/v yeast extract and vitamins (Supplementary Figure 1)
To identify phenotypic differences between strain 7D4C2, C. fermentans, and C. galactitolivorans, we studied the carbohydrate utilization of strain 7D4C2 using the AN MicroPlateTM from Biolog (Hayward, CA, United States) (Supplementary Figure 10) and we compared the results to those reported for C. fermentans (Flaiz et al, 2020) and C. galactitolivorans (Kim et al, 2015)
Summary
Medium-chain carboxylates (MCCs, 6–12 carbon atoms) are precursors to liquid fuels (Levy et al, 1981). MCCs are much easier to separate from the culture broth compared to short-chain carboxylates (SCCs, 2–5 carbon atoms) due to their hydrophobic carbon chains (Levy et al, 1981; Xu et al, 2015; Angenent et al, 2016) Besides their use for fuel production, MCCs are feedstocks in the chemical, pharmaceutical, food, and agricultural industries for the manufacture of a wide variety of products (Levy et al, 1981; Kenealy et al, 1995; Desbois, 2012; Harvey and Meylemans, 2014). N-caproate, which is a 6-carbon MCC (here referred to as the total of dissociated and undissociated forms), is toxic to microbes at pH values near its pKa (Agler et al, 2012a; Ge et al, 2015)
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