Characterization of a limonene-degrading methanogenic enrichment culture

In this study, a limonene-degrading methanogenic enrichment culture was analyzed, revealing new insights into the predatory microbial interactions and potential limonene degradation pathways. The enrichment culture, established in 1999 using activated sludge obtained from a wastewater treatment plant, sustains a complex microbial community despite being supplied only with limonene as a carbon source.
Using long-read PacBio sequencing, a metagenome-assembled genome assigned to the
Syntrophobacteraceae family was identified as responsible for the initial step of limonene
degradation under methanogenic conditions. Within the genome of the Syntrophobacteraceae MAG, two operons were identified, with metatranscriptomic and metaproteomic data verifying the transcription of the genes and the presence of the proteins in the metaproteome. The initial activation of limonene is catalyzed by an enzyme annotated as benzylsuccinate synthase, which forms a new phylogenetic branch within the known and characterized fumarate-adding enzymes and was therefore named limonenylsuccinate synthase. Limonenylsuccinate synthase is possibly the first enzyme detected for monoterpene degradation in a methanogenic environment.
With 32 MAGs assembled from the metagenome, the microbial community in the enrichment culture is a complex syntrophic community. Most members of the community can be assigned to syntrophic bacteria, methanogenic archaea, or fermenters, who work together to degrade limonene. However, there is a predator thriving in the culture. Candidatus Velamenicoccus archaeovorus, a member of the candidate phylum Omnitrophota, that was visualized using CARD-FISH in combination with high-resolution microscopy and was found attached to the methanogen Methanothrix soehngenii and other microorganisms within the enrichment culture. Candidatus Velamenicoccus archaeovorus transcribes genes that allow it to attach to its prey, lyse the cells, and consume their biomass, showing similarities to the mechanisms of other predatory bacteria. Furthermore, a group I intron was detected in the predator’s 23S rRNA gene
and could be visualized using CARD-FISH in its prey cells, showing the transfer of mobile
genetic elements between prey and host.
The predation process introduces a new trophic level into the microbial community as it results in the release of necromass. The necromass, consisting of carbohydrates, lipids, and proteins from dead cells, is expected to be fermented by other members of the community, such as Anaerolineaceae and Lentimicrobium, closing the microbial loop and facilitating further syntrophic interactions. The complex interplay between syntrophic bacteria, methanogens, and the predatory bacterium illustrates the intricate dynamics within this anaerobic system. The discovery of the novel limonenylsuccinate synthase enzyme and the predatory role of Candidatus Velamenicoccus archaeovorus highlights the metabolic complexity of microbial biomass turnover in energy-limited ecosystems.