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. 2022 Feb 17;8:7. doi: 10.1038/s41522-022-00267-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — High-sulfate (Nance, N-L) and low-sulfate (Flowers Goodale, FG-L; Terret, T-L) CBM wells were sampled utilizing a Subsurface Environmental Sampler (SES) to retrieve in situ CBM samples of the attached fraction under environmentally relevant conditions. Following retrieval, samples were either prepared for BONCAT incubation (purple track) or shotgun metagenomics (blue track). For shotgun metagenomics, DNA was extracted and sequenced. BONCAT incubations with the synthetic amino acid tracer, HPG, lasted 24 h followed by cell removal from coal, click chemistry, staining, and cell sorting of BONCAT+ and BONCAT-Total (SYTO stained) cell fractions, DNA extraction and amplified metagenomic sequencing. The No HPG control was used to determine sorting gates for BONCAT+ events.