Production of high-concentration n-caproic acid from lactate through fermentation using a newly isolated Ruminococcaceae bacterium CPB6

Abstract

Background: n-Caproic acid (CA), as a medium-chain carboxylic acid, is a valuable chemical feedstock for various industrial applications. The fermentative production of CA from renewable carbon sources has attracted a lot of attentions. Lactate is a significant intermediate waste in the anaerobic breakdown of carbohydrates that comprise 18-70% of the chemical oxygen demand (COD) in municipal and some industrial wastewaters. Recently, researchers (including our own group) reported the CA production using lactate as electron donor with newly identified microbiome systems. However, within such processes, it was hard to determine whether the CA production was completed by a single strain or by the co-metabolism of different microorganisms.

Results: Here, we report the CA production using lactate as electron donor using the strain CPB6, which we isolated from a microbiome for CA production as described previously. Strain CPB6 is affiliated with Clostridium cluster IV of the family of Ruminococcaceae based on 16S rRNA gene sequence analysis. The strain prefers acidic initial pH condition (pH 5.0-6.5), and the temperature ranging from 30 to 40 °C for CA production. In a fed-batch fermentation with non-sterilized lactate-containing organic wastewater as feedstock, strain CPB6 produced 16.6 g/L CA (from 45.1 g/L lactate) with a maximum productivity of 5.29 g/L/day. Enzyme assays with crude cell extract showed that CPB6 can metabolize acetate and butyryl-CoA to produce n-butyric acid, and acetate/n-butyrate and caproyl-CoA to produce CA, respectively.

Conclusion: This study demonstrated that high concentration of CA production can be obtained by a newly isolated pure culture CPB6. This strain can be employed as a powerful workhorse for high-efficient CA recovery from lactate-containing waste streams. Our preliminary investigation suggested that the CA production from lactate in strain CPB6 might be via the chain elongation pathway of the reverse β-oxidation; the detailed mechanism, however, warrants further investigation using various molecular microbiology techniques.

Keywords: Caproic acid; Chain elongation; Hexanoate; Lactate; Organic waste; Ruminococcaceae bacterium.

Fig. 1

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences. The numbers at the nodes indicate the level of bootstrap values (1000 replications, >50%). Bar 0.02 indicates substitutions per nucleotide position

Fig. 2

Caproic acid (CA) formation from lactate by strain CPB6 in a fed-batch fermentation

Fig. 3

Effect of pH on caproic acid (CA) production from lactate: lactate consumption (a), CA production (b), acetate consumption (c), and butyrate accumulation (d)

Fig. 4

Effect of temperature on caproic acid (CA) production from lactate: lactate consumption (a), CA production (b), acetate consumption (c), and butyrate accumulation (d)

Fig. 5

Caproic acid (CA) recovery from lactate-containing wastewater by strain CPB6 under non-sterilized conditions. The fermentation was started using the mixed wastewater as described in the “Methods” section. When the wastewater was depleted, additional wastewater was added to support the further CA production

Fig. 6

Caproic acid (CA) production in strain CPB6 using different substrates: a lactate; b lactate and acetate; c lactate and butyrate

Fig. 7

Proposed metabolic pathways for butyrate and caproic acid (CA) formation in strain CPB6. This was extended and modified from previous models for CA production [2, 5] with the combination of lactate oxidation and chain elongation. Pathways were also included here indicating that CA could be formed from condensation of either acetate and caproyl-CoA or butyrate and caproyl-CoA