Abstract
Limonene is a natural monoterpene omnipresent in human environments. It enters wastewater and is also metabolised in methanogenic digesters. A stable limonene-degrading methanogenic enrichment culture was investigated by metagenomic, metatranscriptomic and metaproteomic data sets to characterise the microbial community and identify the limonene degradation pathway. Thirty-two metagenome-assembled genomes revealed a complex community of bacteria and methanogenic archaea dominated by Candidatus Velamenicoccus archaeovorus as the top predator, contributing two-thirds of the reads in the metagenome. The presence of several fermenting bacteria (Anaerolineaceae, Aminidesulfovibrio, Smithellaceae, Lentimicrobium) indicated the recycling of necromass in a microbial loop. Only one hydrocarbon-activating enzyme system was expressed, a member of the alkyl- and arylsuccinate synthase family which is a glycine radical enzyme that adds fumarate to hydrocarbons. The limonenylsuccinate synthase gene encodes a modified substrate binding pocket with two smaller amino acids, suggesting an adaptation for the larger structure of limonene. The limonenylsuccinate synthase operon and a ring cleavage operon, as well as genes for the final syntrophic fermentation to acetate, hydrogen and formate were encoded in a Syntrophobacteraceae genome. Almost all genes for this degradation pathway were highly transcribed and expressed, demonstrating a catalytic role for glycine radical enzymes in methanogenic systems degrading limonene.