AUTHOR=Esquivel-Elizondo Sofia , Bağcı Caner , Temovska Monika , Jeon Byoung Seung , Bessarab Irina , Williams Rohan B. H. , Huson Daniel H. , Angenent Largus T. 

TITLE=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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.594524

DOI=10.3389/fmicb.2020.594524

ISSN=1664-302X

ABSTRACT=<p>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, <italic>n</italic>-butyrate, <italic>n</italic>-caproate, biomass, and H<sub>2</sub>/CO<sub>2</sub> from hexoses. With only fructose as substrate (pH 5.5), the maximum <italic>n</italic>-caproate specificity (i.e., products <italic>per</italic> other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and <italic>n</italic>-butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and <italic>n</italic>-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing <italic>n</italic>-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 <italic>n</italic>-caproic acid was ∼0.3 mM), the <italic>n</italic>-caproate selectivity (i.e., product <italic>per</italic> 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 <italic>Caproicibacter fermentans</italic> (99.5%) and <italic>Caproiciproducens galactitolivorans</italic> (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, <italic>C. fermentans</italic>, and <italic>C. galactitolivorans</italic> belong to the same genus of <italic>Caproiciproducens</italic>. 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 <italic>Megasphaera</italic> spp. In conclusion, <italic>Caproiciproducens</italic> spp., comprising strain 7D4C2, <italic>C. fermentans</italic>, <italic>C. galactitolivorans</italic>, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. <italic>Caproiciproducens</italic> sp. 7D4C2 (DSM 110548) was studied here to understand <italic>n</italic>-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into <italic>n</italic>-caproate.</p>