Abstract
Functional profiling of anaerobic digestion microbiomes is routinely performed against KEGG or MetaCyc, whose paid commercial licences block fee-for-service analysis for biogas operators, the setting where the results have the most immediate operational value. We present digestome, a curated marker-gene panel for anaerobic digestion built exclusively on sources that are free for commercial use (NCBIfam, public domain; Pfam, CC0; Rhea, CC BY), together with a scorer that reports pathway completeness, branch capability and, for hydrolysis, whether the enzyme is built for export. Benchmarked against 1,401 metagenome-assembled genomes from 134 anaerobic digesters, the panel assigned no methanogenesis route to any of 1,361 non-methanogens, and every acetoclastic call fell within
Methanosarcina
or
Methanothrix
. The result held for 3,043 species representatives from the Genome Taxonomy Database, spanning 203 phyla, which are built differently from binned metagenomes: the only background genomes given a route were two archaea carrying mcrA, and none of the 90 anaerobic methane and alkane oxidisers was given one. Specificity against non-methanogens does not show that a methanogen gets the right route: with a family-level check, 59 of 156 acetoclastic calls in that sample fell on methylotrophic genera that do not use acetate, and a lineage policy removed them, with 14 more left unconfirmed in unnamed genera. Testing whether a catalytic domain shares a polypeptide with an export module reduced the genomes credited with cellulolytic capacity from 597 to 50. A causal graph of the measurement shows that a profile reports the community together with three measurement choices (sampling, DNA extraction and sequencing depth, and reference genomes), which fixes what one profile can attribute and what a prospective validation would have to record.