The Isolate Caproiciproducens sp. 7D4C2 Produces n-Caproate at Mildly Acidic Conditions From Hexoses: Genome and rBOX Comparison With Related Strains and Chain-Elongating Bacteria

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.

Mixed Acid Fermentation of Carbohydrate-Rich Dairy Manure Hydrolysate

“…After Megasphaera ’s abundance in the DMB decreased, other lactic acid utilizing bacteria increased in abundance, such as the four ASVs assigned to Caproiciproducens ( Figure 5 ), a Firmicute in the Clostridia class ( Contreras-Dávila et al, 2020 ; Esquivel-Elizondo et al, 2021 ). Caproiciproducens has been shown to produce intracellular lactic acid and then use it to produce C4 and MCFAs when sugars are the initial substrate ( Esquivel-Elizondo et al, 2021 ) and is also proposed to be able to utilize lactic acid as an initial substrate ( Contreras-Dávila et al, 2020 ; Gao et al, 2021 ). In both reactors, ASVs assigned to another Clostridia genus, Clostridium sensu stricto 12 , began to accumulate after several days of reactor operation ( Figure 5 ).…”

“…For instance, given the isolation techniques that have been described for homofermentative S. inulinus ( Yu et al, 2011 ) and for MCFA producing Caproiciproducens spp. ( Esquivel-Elizondo et al, 2021 ), a synthetic microbiome of isolate representatives of these bacteria could theoretically recover all carbohydrate derived electron equivalents as intermediate lactic acid, which could then be chain-elongated to C6, producing CO 2 as a byproduct (based solely on the proposed metabolic pathways involved in these organisms).…”

Caproicibacterium lactatifermentans sp. nov., isolated from pit clay used for the production of Chinese strong aroma-type liquor

“…LBM19010’ and ‘ Caproicibacterium sp. JNU-WLY1368’ were assigned to the family Oscillospiraceae [3–9]. Caproicibacterium amylolyticum , a caproate-producing bacterium isolated from pit clay of Chinese strong aroma-type liquor fermentation system, was recently proposed to the novel genus Caproicibacterium , belonging to the family Oscillospiraceae in the class Clostridia.…”

Editorial: Microbial Chain Elongation- Close the Carbon Loop by Connecting-Communities

“…This is especially true for hydrolysates that contain xylose and glucose. The microbiome-member Caproiciproducens spp., for example, can utilize sugars for caproate or lactic acid production ( Esquivel-Elizondo et al, 2021 ). However, the network and redundancy analysis in Agler et al (2012b) and the redundancy analysis in Ingle et al make it more likely that lactic acid is the pertinent electron donor for chain elongation from hydrolysates, especially towards butyric acid production.…”