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

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, -butyrate, -caproate, biomass, and H/CO from hexoses. With only fructose as substrate (pH 5.5), the maximum -caproate specificity (i.e., products other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and -butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and -butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing -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 -caproic acid was ∼0.3 mM), the -caproate selectivity (i.e., product 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 (99.5%) and (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, , and belong to the same genus of . 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 spp. In conclusion, spp., comprising strain 7D4C2, , , and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. sp. 7D4C2 (DSM 110548) was studied here to understand -caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into -caproate.