Community Structure and Microbial Associations in Sediment-Free Methanotrophic Enrichment Cultures from a Marine Methane Seep
Abstract
Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b ("Candidatus Methanocomedenaceae") and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses.
Additional Information
© 2022 American Society for Microbiology. Received: 10 November 2021. Accepted: 27 April 2022. Published online: 23 May 2022. We thank Grayson Chadwick for assistance with fluorescence-activated aggregate sorting, Connor Skennerton for assistance with data management and bioinformatics, and Ranjani Murali for genome phylogeny of SRB. We thank Igor Antoshechkin and Vijaya Kumar for metagenomic library preparation and sequencing at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at California Institute of Technology. We also acknowledge Peter Brewer and members of the shipboard party (Kat Dawson and Ally Pasulka), crew, and pilots of the 2013 research expedition on the R/V Western Flyer, owned and operated by the Monterey Bay Aquarium Research Institute (MBARI). We thank the editor and anonymous reviewers for their constructive comments on the manuscript. This study is supported by funding from the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under award number DE-SC0016469 and the NSF-supported Center for Dark Energy Biosphere Investigations (C-DEBI). Cell aggregate sorting and sequencing were supported by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, under contract no. DE-AC02-05CH11231. D.R.S. was additionally supported by the Netherlands Organisation for Scientific Research, Rubicon award 019.153LW.039. V.J.O. is a CIFAR fellow in the Earth 4D program. This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed or represents that its use would not infringe privately owned rights. Reference here to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. We declare we have no competing interests. Data availability. The 16S rRNA gene amplicon sequences, shotgun metagenome sequences, and metagenome-assembled genomes from the sediment-free enrichment cultures have been deposited in the National Center for Biotechnology Information (NCBI) under BioProject accession no. PRJNA758896. The raw sequences for flow sorting controls, sorted single cells, and aggregates from the sediment-free enrichments have been deposited in the Joint Genome Institute (JGI) Genomes Online Database under study ID Gs0133461.Attached Files
Supplemental Material - aem-02109-21-s0001.pdf
Supplemental Material - aem-02109-21-s0003.xlsx
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Supplemental Material - aem-02109-21-s0006.xlsx
Supplemental Material - aem02109-21-s0002.xlsx
Updated - aem-02109-21-s0005.xlsx
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Additional details
- PMCID
- PMC9195934
- Eprint ID
- 115726
- Resolver ID
- CaltechAUTHORS:20220721-8119000
- Department of Energy (DOE)
- DE-SC0016469
- Center for Dark Energy Biosphere investigations (C-DEBI)
- Department of Energy (DOE)
- DE-AC02-05CH11231
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
- 019.153LW.039
- Canadian Institute for Advanced Research (CIFAR)
- Created
-
2022-07-22Created from EPrint's datestamp field
- Updated
-
2023-10-06Created from EPrint's last_modified field
- Caltech groups
- Millard and Muriel Jacobs Genetics and Genomics Laboratory, Division of Geological and Planetary Sciences, Division of Biology and Biological Engineering