Galacturonate Metabolism in Anaerobic Chemostat Enrichment Cultures: Combined Fermentation and Acetogenesis by the Dominant sp. nov. "Candidatus Galacturonibacter soehngenii".

Agricultural residues such as sugar beet pulp and citrus peel are rich in pectin, which contains galacturonic acid as a main monomer. Pectin-rich residues are underexploited as feedstocks for production of bulk chemicals or biofuels. The anaerobic, fermentative conversion of d-galacturonate in anaerobic chemostat enrichment cultures provides valuable information toward valorization of these pectin-rich feedstocks. Replicate anaerobic chemostat enrichments, with d-galacturonate as the sole limiting carbon source and inoculum from cow rumen content and rotting orange peels, yielded stable microbial communities, which were dominated by a novel Lachnospiraceae species, for which the name "Candidatus Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H2 as coproducts. The observed molar ratio of acetate and the combined amounts of H2 and formate deviated significantly from 1, which suggested that some of the hydrogen and CO2 formed during d-galacturonate fermentation was converted into acetate via the Wood-Ljungdahl acetogenesis pathway. Indeed, metagenomic analysis of the enrichment cultures indicated that the genome of "Candidatus G. soehngenii" encoded enzymes of the adapted Entner-Doudoroff pathway for d-galacturonate metabolism as well as enzymes of the Wood-Ljungdahl pathway. The simultaneous operation of these pathways may provide a selective advantage under d-galacturonate-limited conditions by enabling a higher specific ATP production rate and lower residual d-galacturonate concentration than would be possible with a strictly fermentative metabolism of this carbon and energy source.IMPORTANCE This study on d-galacturonate metabolism by open, mixed-culture enrichments under anaerobic, d-galacturonate-limited chemostat conditions shows a stable and efficient fermentation of d-galacturonate into acetate as the dominant organic fermentation product. This fermentation stoichiometry and population analyses provide a valuable baseline for interpretation of the conversion of pectin-rich agricultural feedstocks by mixed microbial cultures. Moreover, the results of this study provide a reference for studies on the microbial metabolism of d-galacturonate under different cultivation regimes.

Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems.

The Saprospiraceae family within the phylum Bacteroidota is commonly present and highly abundant in wastewater treatment plants (WWTPs) worldwide, but little is known about its role. In this study, we used MiDAS 4 global survey with samples from 30 countries to analyze the abundance and distribution of members of Saprospiraceae. Phylogenomics were used to delineate five new genera from a set of 31 high-quality metagenome-assembled genomes from Danish WWTPs. Newly designed probes for fluorescence in situ hybridization (FISH) revealed rod-shaped morphologies for all genera analyzed, including OLB8, present mostly inside the activated sludge flocs. The genomes revealed potential metabolic capabilities for the degradation of polysaccharides, proteins, and other complex molecules; partial denitrification; and storage of intracellular polymers (glycogen, polyphosphate, and polyhydroxyalkanoates). FISH in combination with Raman microspectroscopy confirmed the presence of intracellular glycogen in Candidatus Brachybacter, Candidatus Parvibacillus calidus (both from the former genus OLB8), and Candidatus Opimibacter, and the presence of polyhydroxyalkanoates in Candidatus Defluviibacterium haderslevense and Candidatus Vicinibacter. These results provide the first overview of the most abundant novel Saprospiraceae genera present in WWTPs across the world and their potential involvement in nutrient removal and the degradation of macromolecules.

"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of "Candidatus Galacturonibacter soehngenii," from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of "Ca. G. soehngenii" was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the "Ca. G. soehngenii" MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of "Ca. G. soehngenii" enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of "Ca. G. soehngenii."

A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway.

D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant Lactobacillus suebicus strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and in vitro product identification demonstrated that D-galacturonate metabolism in L. suebicus occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.