A Clostridium Group IV Species Dominates and Suppresses a Mixed Culture Fermentation by Tolerance to Medium Chain Fatty Acids Products

Abstract

A microbial community is engaged in a complex economy of cooperation and competition for carbon and energy. In engineered systems such as anaerobic digestion and fermentation, these relationships are exploited for conversion of a broad range of substrates into products, such as biogas, ethanol, and carboxylic acids. Medium chain fatty acids (MCFAs), for example, hexanoic acid, are valuable, energy dense microbial fermentation products, however, MCFA tend to exhibit microbial toxicity to a broad range of microorganisms at low concentrations. Here, we operated continuous mixed population MCFA fermentations on biorefinery thin stillage to investigate the community response associated with the production and toxicity of MCFA. In this study, an uncultured species from the Clostridium group IV (related to Clostridium sp. BS-1) became enriched in two independent reactors that produced hexanoic acid (up to 8.1 g L^-1), octanoic acid (up to 3.2 g L^-1), and trace concentrations of decanoic acid. Decanoic acid is reported here for the first time as a possible product of a Clostridium group IV species. Other significant species in the community, Lactobacillus spp. and Acetobacterium sp., generate intermediates in MCFA production, and their collapse in relative abundance resulted in an overall production decrease. A strong correlation was present between the community composition and both the hexanoic acid concentration (p = 0.026) and total volatile fatty acid concentration (p = 0.003). MCFA suppressed species related to Clostridium sp. CPB-6 and Lactobacillus spp. to a greater extent than others. The proportion of the species related to Clostridium sp. BS-1 over Clostridium sp. CPB-6 had a strong correlation with the concentration of octanoic acid (p = 0.003). The dominance of this species and the increase in MCFA resulted in an overall toxic effect on the mixed community, most significantly on the Lactobacillus spp., which resulted in a decrease in total hexanoic acid concentration to 32 ± 2% below the steady-state average. As opposed to the current view of MCFA toxicity broadly leading to production collapse, this study demonstrates that varied tolerance to MCFA within the community can lead to the dominance of some species and the suppression of others, which can result in a decreased productivity of the fermentation.

Keywords: Clostridium; MCFA; carboxylic acids; fermentation; microbial community; toxicity.

Figure 1

(A,B) Concentration over time of total VFA, hexanoic acid, and octanoic acid in Reactor 1 (A) and Reactor 2 (B) fed from a common source of biorefinery stillage, including the detection of decanoic acid. The filled diamond represents the days at which decanoic acid was detected significantly above the feed baseline, with a blank diamond representing no significant detection. The top four most relatively abundant microorganisms over time in the two identical reactors, Reactor 1 (C) and Reactor 2 (D).

Figure 2

Redundancy analysis demonstrating the alignment of octanoic acid (Oct), heptanoic acid (Hep), and CE (chain elongation, i.e., concentration of volatile fatty acids greater than C5 chain length) in the opposite direction to the total VFA and hexanoic acid (Hex) concentration. Rx_Dy corresponds to Reactor x on day y.

Figure 3

Phylogenetic tree at species-level resolution showing some genetic heterogeneity among close relatives of the dominant sequence of Reactor 1 on day 34, referred to as Clostridium sp. BS-1_sec. The scale bar indicates 0.1 estimated changes per nucleotide. The alignment considers 974 bp of the phylogenetic gene maker16S rRNA. See Table S1 in Supplementary Material for more information.

Figure 4

(A) The proposed substrate pathway for the fermentation prior to the onset of MCFA toxicity, as observed days 14–21 in Reactor 1 and days 14–52 in Reactor 2. (B) The hypothetical impact of MCFA toxicity on the proposed substrate pathway, as observed from days 21 to 68 in Reactor 1. The dashed line borders represent stressed, low relative abundance species.