Novel multi-kingdom consortium for the production of antimicrobial caproic acid

Microbial fermentation systems can be designed to produce specific short chain carboxylic acids. The strong antimicrobial caproic acid can be produced by strains of the strictly anaerobe Clostridium kluyveri by chain elongation using acetate and ethanol as electron donor. We hypothesized that multi-kingdom microbial consortium composed of C. kluyveri, the heterofermentative Limosilactobacillus reuteri and the yeast Saccharomyces cerevisiae leads to the production of caproic acid in presence of glucose and fructose.

Fermentations were performed in 400 ml bioreactors at 37°C and pH 6.8. We carried out first fed batch fermentation consisting of a bacterial consortium (BC) of L. reuteri and C. kluyveri with external addition of ethanol, and afterwards batch fermentations with a multi-kingdom consortium (MKC) of L. reuteri, C. kluyveri and S. cerevisiae. Substrate consumption and metabolite production were quantified by HPLC-RI, UHPLC-DAD and confirmed by 1H NMR, and the cells growth was monitored by qPCR. The minimum inhibitory dilution fold 50 % (MIDF50) value of the fermentates was tested against Salmonella enterica subsp. enterica CCM 4420. Fermentations were started with L. reuteri or L. reuteri with S. cerevisiae to initiate acetate and ethanol production. C. kluyveri was inoculated 24 h later in both BC and MKC.

During 12 days of fermentation, only L. reuteri increased in cell counts by 1.5±0.7 and 0.7±0.1 log cells/ml in BC and MKC, respectively. Acetic acid levels were highest (about 12-18 mM) between days 2-5. In the same period, the concentration of butyric acid increased, and after 5-6 days of fermentation caproic acid was detected. After 12 days, 25.3± 0.1 and 31.2±11.0 mM of caproic acid were produced by BC and MKC, respectively. In addition to caproic acid, the fermentates contained lactic acid (116-108 mM), butyric acid (4-6.8 mM), propionic acid (4.1-5.1 mM) and ethanol (9.7-10.2 mM). Fermentates produced by BC and MKC showed high inhibitory effects against S. enterica.

We designed a self-contained process to produce fermentates containing antimicrobial caproic acid by a novel multi-kingdom microbe consortium with potential in food preservation.