Background: The carboxylate platform is a promising technology for substituting petrochemicals in the provision of specific platform chemicals and liquid fuels. It includes the chain elongation process that exploits reverse β-oxidation to elongate short-chain fatty acids and forms the more valuable medium-chain variants. The pH value influences this process through multiple mechanisms and is central to effective product formation. Its influence on the microbiome dynamics was investigated during anaerobic fermentation of maize silage by combining flow cytometric short interval monitoring, cell sorting and 16S rRNA gene amplicon sequencing.
Results: Caproate and caprylate titres of up to 6.12 g L-1 and 1.83 g L-1, respectively, were achieved in a continuous stirred-tank reactor operated for 241 days. Caproate production was optimal at pH 5.5 and connected to lactate-based chain elongation, while caprylate production was optimal at pH 6.25 and linked to ethanol utilisation. Flow cytometry recorded 31 sub-communities with cell abundances varying over 89 time points. It revealed a highly dynamic community, whereas the sequencing analysis displayed a mostly unchanged core community. Eight key sub-communities were linked to caproate or caprylate production (rS > | ± 0.7|). Amongst other insights, sorting and subsequently sequencing these sub-communities revealed the central role of Bifidobacterium and Olsenella, two genera of lactic acid bacteria that drove chain elongation by providing additional lactate, serving as electron donor.
Conclusions: High-titre medium-chain fatty acid production in a well-established reactor design is possible using complex substrate without the addition of external electron donors. This will greatly ease scaling and profitable implementation of the process. The pH value influenced the substrate utilisation and product spectrum by shaping the microbial community. Flow cytometric single cell analysis enabled fast, short interval analysis of this community and was coupled with 16S rRNA gene amplicon sequencing to reveal the major role of lactate-producing bacteria.
Keywords: 16S rRNA gene sequencing; Anaerobic fermentation; Caproic acid; Caprylic acid; Flow cytometry; MCFA; Microbial chain elongation; Microbial community; Process monitoring; Single cell analytics.