Use of a Highly Specialized Biocatalyst to Produce Lactate or Biohydrogen and Butyrate from Agro-Industrial Resources in a Dual-Phase Dark Fermentation

This study aimed at investigating the feasibility of using a highly specialized bacterial inoculum harboring lactic acid bacteria (LAB) and lactate-oxidizing, hydrogen-producing bacteria (LO-HPB) to produce either lactate or biohydrogen and butyrate from several agro-industrial resources via dual-phase dark fermentation. The feedstocks were fruit–vegetable waste, cheese whey, coffee wastewater, tequila vinasse, and maize processing wastewater, and were tested in both mono- and co-fermentation. The results obtained indicated that the biocatalyst used was able to perform a dual-phase lactate fermentation, producing high lactate (13.1–36.4 g/L), biohydrogen (0.2–7.5 NL H2/Lfeedstock, equivalent to 0.3–1.7 mol H2/mol hexose), and butyrate (3.3–13.9 g/L) with all the tested feedstocks. A series of self-fermentation tests were also performed with crude cheese whey and fruit–vegetable waste for comparison purposes. Compared to inoculum-aided fermentations, the self-fermentation exhibited a reduced bioconversion efficiency. Short-length 16S rRNA gene sequencing analysis showed that LO-HPB was the dominant microbial group (86.0%) in the biocatalyst, followed by acetic acid bacteria (5.8%) and LAB (5.7%). As expected, the molecular analysis also showed significant differences in the microbial community structure of the biocatalyst and those that evolved from self-fermentation. Besides lactate fermentation and oxidation, the biocatalyst also assisted the bi-phasic lactate fermentation via oxygen consumption, and apparently, via substrate hydrolysis. Overall, this study can lay the foundation for robust inoculum development, which is of special significance in the field of dark fermentation, and proposes an innovative bioprocess for agro-industrial valorization through a trade-off approach, tailoring the metabolic pathway to the target product(s).