Linking meta-omics and biogeochemistry approaches in soils has remained challenging. This study evaluates the use of an internal RNA extraction standard and its potential for making quantitative estimates of a given microbial community size (biomass) in soil metatranscriptomics. We evaluate commonly used laboratory protocols for RNA processing, metatranscriptomic sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Metatranscriptomic profiles from soil samples were generated using two library preparation protocols and prepared in triplicates. RNA extracted from pure cultures of Saccharolobus solfataricus was added to the samples as an internal nucleic acid extraction standard (NAEstd). RNA reads originating from NAEstd were identified with a 99.9% accuracy. A remarkable replication consistency between triplicates was seen (average Bray-Curtis dissimilarity 0.03 ± 0.02), in addition to a clear library preparation bias. Nevertheless, the between-sample pattern was not affected by library type. Estimates of 16S rRNA transcript abundance derived from qRT-PCR experiments, NAEstd and a previously published quantification method of metatranscriptomics (hereafter qMeTra) were compared with microbial biomass carbon (MBC) and nitrogen (MBN) extracts. The derived biomass estimates differed by orders of magnitude. While most estimates were significantly correlated with each other, no correlation was observed between NAEstd and MBC extracts. We discuss how simultaneous changes in community size and the soils nucleic acid retention strength might hamper accurate biomass estimation. Adding NAEstd has the potential to shed important light on nucleic acid retention in the substance matrix (e.g., soil) during extraction.

Agricultural University of Iceland Hvanneyri Iceland

Archaea Biology and Ecogenomics Unit Department of Functional and Evolutionary Ecology University of Vienna Vienna Austria

Department of Arctic and Marine Biology UiT The Arctic University of Norway Tromsø Norway

Department of Bacterial Physiology University Greifswald Greifswald Germany

Department of Microbiology and Ecosystem Science University Vienna Vienna Austria

Institute of Bioinformatics University Medicine Greifswald Greifswald Germany

Institute of Mathematics and Computer Science University Greifswald Greifswald Germany

Institute of Soil Biology and Biogeochemistry Biology Centre CAS České Budějovice Czech Republic

Interfaculty Institute of Genetics and Functional Genomics University Greifswald Greifswald Germany

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Adl, S. M., A. Habura, and Y. Eglit. 2014. “Amplification Primers of SSU rDNA for Soil Protists.” Soil Biology and Biochemistry 69: 328–342. https://doi.org/10.1016/j.soilbio.2013.10.024.

Alteio, L. V., J. Séneca, A. Canarini, et al. 2021. “A Critical Perspective on Interpreting Amplicon Sequencing Data in Soil Ecological Research.” Soil Biology and Biochemistry 160: 108357. https://doi.org/10.1016/j.soilbio.2021.108357.

Anthony, M. A., S. F. Bender, and M. G. A. van der Heijden. 2023. “Enumerating Soil Biodiversity.” Proceedings of the National Academy of Sciences of the United States of America 120, no. 33: e2304663120. https://doi.org/10.1073/pnas.2304663120.

Apprill, A., S. Mcnally, R. Parsons, and L. Weber. 2015. “Minor Revision to V4 Region SSU rRNA 806R Gene Primer Greatly Increases Detection of SAR11 Bacterioplankton.” Aquatic Microbial Ecology 75, no. 2: 129–137. https://doi.org/10.3354/ame01753.

Bastida, F., D. J. Eldridge, C. García, G. Kenny Png, R. D. Bardgett, and M. Delgado‐Baquerizo. 2021. “Soil Microbial Diversity–Biomass Relationships Are Driven by Soil Carbon Content Across Global Biomes.” ISME Journal 15, no. 7: 2081–2091. https://doi.org/10.1038/s41396‐021‐00906‐0.

Bölscher, T., C. Vogel, F. K. Olagoke, et al. 2024. “Beyond Growth: The Significance of Non‐Growth Anabolism for Microbial Carbon‐Use Efficiency in the Light of Soil Carbon Stabilisation.” Soil Biology and Biochemistry 193: 109400. https://doi.org/10.1016/j.soilbio.2024.109400.

Bonato Asato, A. E., C. Wirth, N. Eisenhauer, and J. Hines. 2023. “On the Phenology of Soil Organisms: Current Knowledge and Future Steps.” Ecology and Evolution 13, no. 4: e10022. https://doi.org/10.1002/ece3.10022.

Bradford, M. A., W. R. Wieder, G. B. Bonan, N. Fierer, P. A. Raymond, and T. W. Crowther. 2016. “Managing Uncertainty in Soil Carbon Feedbacks to Climate Change.” Nature Climate Change 6, no. 8: 751–758. https://doi.org/10.1038/nclimate3071.

Bratbak, G., and I. Dundas. 1984. “Bacterial Dry Matter Content and Biomass Estimations.” Applied and Environmental Microbiology 48, no. 4: 755–757.

Bushnell, B. 2015. “BBmap.” Joint Genome Institute. Accessed January 2021. https://sourceforge.net/projects/bbmap/.

Cleaves, H. J., C. M. Jonsson, C. L. Jonsson, D. A. Sverjensky, and R. M. Hazen. 2010. “Adsorption of Nucleic Acid Components on Rutile (TiO2) Surfaces.” Astrobiology 10, no. 3: 311–323. https://doi.org/10.1089/ast.2009.0397.

Coclet, C., P. O. Sorensen, U. Karaoz, et al. 2023. “Virus Diversity and Activity Is Driven by Snowmelt and Host Dynamics in a High‐Altitude Watershed Soil Ecosystem.” Microbiome 11, no. 1: 237. https://doi.org/10.1186/s40168‐023‐01666‐z.

Conant, R. T., M. G. Ryan, G. I. Ågren, et al. 2011. “Temperature and Soil Organic Matter Decomposition Rates—Synthesis of Current Knowledge and a Way Forward.” Global Change Biology 17, no. 11: 3392–3404. https://doi.org/10.1111/j.1365‐2486.2011.02496.x.

Crowther, T. W., K. E. O. Todd‐Brown, C. W. Rowe, et al. 2016. “Quantifying Global Soil Carbon Losses in Response to Warming.” Nature 540, no. 7631: 104–108. https://doi.org/10.1038/nature20150.

Dahl, M. B., A. Söllinger, P. Sigurðsson, et al. 2023. “Long‐Term Warming‐Induced Trophic Downgrading in the Soil Microbial Food Web.” Soil Biology and Biochemistry 181: 109044. https://doi.org/10.1016/j.soilbio.2023.109044.

de Mendiburu, F. 2023. “agricolae: Statistical Procedures for Agricultural Research.” R Package Version 1.3‐7. https://CRAN.R‐project.org/package=agricolae.

Filser, J., J. H. Faber, A. V. Tiunov, et al. 2016. “Soil Fauna: Key to New Carbon Models.” Soil 2, no. 4: 565–582. https://doi.org/10.5194/soil‐2‐565‐2016.

Gagelidze, N. A., L. L. Amiranashvili, T. A. Sadunishvili, G. I. Kvesitadze, T. F. Urushadze, and T. O. Kvrivishvili. 2018. “Bacterial Composition of Different Types of Soils of Georgia.” Annals of Agrarian Science 16, no. 1: 17–21.

Gann, E. R., Y. Kang, S. T. Dyhrman, C. J. Gobler, and S. W. Wilhelm. 2021. “Metatranscriptome Library Preparation Influences Analyses of Viral Community Activity During a Brown Tide Bloom.” Frontiers in Microbiology 12: 664189. https://doi.org/10.3389/fmicb.2021.664189.

Geisen, S., A. T. Tveit, I. M. Clark, et al. 2015. “Metatranscriptomic Census of Active Protists in Soils.” ISME Journal 9, no. 10: 2178–2190. https://doi.org/10.1038/ismej.2015.30.

Gifford, S. M., S. Sharma, J. M. Rinta‐Kanto, and M. A. Moran. 2011. “Quantitative Analysis of a Deeply Sequenced Marine Microbial Metatranscriptome.” ISME Journal 5, no. 3: 461–472. https://doi.org/10.1038/ismej.2010.141.

Gruber‐Vodicka, H. R., B. K. B. Seah, and E. Pruesse. 2020. “phyloFlash: Rapid Small‐Subunit rRNA Profiling and Targeted Assembly From Metagenomes.” mSystems 5, no. 5: e00920‐20. https://doi.org/10.1128/mSystems.00920‐20.

Hadziavdic, K., K. Lekang, A. Lanzen, I. Jonassen, E. M. Thompson, and C. Troedsson. 2014. “Characterization of the 18s rRNA Gene for Designing Universal Eukaryote Specific Primers.” PLoS One 9, no. 2: e87624. https://doi.org/10.1371/journal.pone.0087624.

Joergensen, R. G. 1996. “The Fumigation‐Extraction Method to Estimate Soil Microbial Biomass: Calibration of the K‐ EC Value.” Soil Biology and Biochemistry 28, no. 1: 25–31.

Jonasson, S., A. Michelsen, I. K. Schmidt, E. V. Nielsen, and T. V. Callaghan. 1996. “Microbial Biomass C, N and P in Two Arctic Soils and Responses to Addition of NPK Fertilizer and Sugar: Implications for Plant Nutrient Uptake.” Oecologia 106, no. 4: 507–515. https://doi.org/10.1007/BF00329709.

Kim, B. H., and G. M. Gadd. 2008. “Bacterial Physiology and Metabolism.”

Kopylova, E., L. Noé, and H. Touzet. 2012. “SortMeRNA: Fast and Accurate Filtering of Ribosomal RNAs in Metatranscriptomic Data.” Bioinformatics 28, no. 24: 3211–3217. https://doi.org/10.1093/bioinformatics/bts611.

Kramer, S., D. Dibbern, J. Moll, et al. 2016. “Resource Partitioning Between Bacteria, Fungi, and Protists in the Detritusphere of an Agricultural Soil.” Frontiers in Microbiology 7, no. SEP: 1–12. https://doi.org/10.3389/fmicb.2016.01524.

Lanzén, A., S. L. Jørgensen, D. H. Huson, et al. 2012. “CREST – Classification Resources for Environmental Sequence Tags.” PLoS One 7, no. 11: e49334. https://doi.org/10.1371/journal.pone.0049334.

Loferer‐Krößbacher, M., J. Klima, and R. Psenner. 1998. “Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis.” Applied and Environmental Microbiology 64, no. 2: 688–694. https://doi.org/10.1128/AEM.64.2.688‐694.1998.

Matamouros, S., T. Gensch, M. Cerff, et al. 2023. “Growth‐Rate Dependency of Ribosome Abundance and Translation Elongation Rate in Corynebacterium glutamicum Differs From That in Escherichia coli.” Nature Communications 14, no. 1: 5611. https://doi.org/10.1038/s41467‐023‐41176‐y.

Metting, F. B. (Ed.). 1993. Soil Microbial Ecology. Marcel Dekker. Topley and Wilson's Microbiology and Microbial Infections, Guy R. Knudsen, Bacteriology of Soils and Plants, p. 2.

Milo, R., and R. Phillips. 2015. “Cell Biology by Numbers.”

Moran, M. A., B. Satinsky, S. M. Gifford, et al. 2012. “Sizing Up Metatranscriptomics.” ISME Journal 7, no. 2: 237–243. https://doi.org/10.1038/ismej.2012.94.

Naylor, D., N. Sadler, A. Bhattacharjee, et al. 2020. “Soil Microbiomes Under Climate Change and Implications for Carbon Cycling.” Annual Review of Environment and Resources 45, no. 1: 29–59. https://doi.org/10.1146/annurev‐environ‐012320‐082720.

Neidhardt, F. C., J. L. Ingraham, and M. Schaechter. 1990. Physiology of the Bacterial Cell: A Molecular Approach, 442–462. Sinauer Associates Inc.

Neidhardt, F. C., and H. E. Umbarger. 1996. “Chemical composition of Escherichia coli.” In: EcoSal—Escherichia coli and Salmonella: Cellular and Molecular Biology, Böck A, Curtiss III R, Kaper JB, et al (eds). 2nd edn. ASM Press.

Nielsen, U. N., E. Ayres, D. H. Wall, and R. D. Bardgett. 2011. “Soil Biodiversity and Carbon Cycling: A Review and Synthesis of Studies Examining Diversity – Function Relationships.” European Journal of Soil Science 62: 105–116. https://doi.org/10.1111/j.1365‐2389.2010.01314.x.

Novinscak, A., and M. Filion. 2011. “Effect of Soil Clay Content on RNA Isolation and on Detection and Quantification of Bacterial Gene Transcripts in Soil by Quantitative Reverse Transcription‐PCR.” Applied and Environmental Microbiology 77: 6249–6252. https://doi.org/10.1128/AEM.00055‐11.

Oksanen, J., G. Blanchet, M. Friendly, R. Kindt, P. Legendre, and D. McGlinn. 2021. “Vegan: Community Ecology Package R Version 2.6‐2.” https://CRAN.R‐project.org/package=vegan.

Parada, A. E., D. M. Needham, and J. A. Fuhrman. 2016. “Every Base Matters: Assessing Small Subunit rRNA Primers for Marine Microbiomes With Mock Communities, Time Series and Global Field Samples.” Environmental Microbiology 18, no. 5: 1403–1414. https://doi.org/10.1111/1462‐2920.13023.

Paulin, M. M., M. H. Nicolaisen, C. S. Jacobsen, A. L. Gimsing, J. Sørensen, and J. Bælum. 2013. “Improving Griffith's Protocol for Co‐Extraction of Microbial DNA and RNA in Adsorptive Soils.” Soil Biology and Biochemistry 63: 37–49. https://doi.org/10.1016/j.soilbio.2013.02.007.

Peng, J., C.‐E. Wegner, Q. Bei, P. Liu, and W. Liesack. 2018. “Metatranscriptomics Reveals a Differential Temperature Effect on the Structural and Functional Organization of the Anaerobic Food Web in Rice Field Soil.” Microbiome 6, no. 1: 169. https://doi.org/10.1186/s40168‐018‐0546‐9.

Peng, J., X. Zhou, C. Rensing, W. Liesack, and Y.‐G. Zhu. 2024. “Soil Microbial Ecology Through the Lens of Metatranscriptomics.” Soil Ecology Letters 6, no. 3: 230217. https://doi.org/10.1007/s42832‐023‐0217‐z.

Petters, S., V. Groß, A. Söllinger, et al. 2021. “The Soil Microbial Food Web Revisited: Predatory Myxobacteria as Keystone Taxa?” ISME Journal 15, no. 9: 2665–2675. https://doi.org/10.1038/s41396‐021‐00958‐2.

Piwosz, K., T. Shabarova, J. Tomasch, et al. 2018. “Determining Lineage‐Specific Bacterial Growth Curves With a Novel Approach Based on Amplicon Reads Normalization Using Internal Standard (ARNIS).” ISME Journal 12, no. 11: 2640–2654. https://doi.org/10.1038/s41396‐018‐0213‐y.

Portillo, M. C., J. W. Leff, C. L. Lauber, and N. Fierer. 2013. “Cell Size Distributions of Soil Bacterial and Archaeal Taxa.” Applied and Environmental Microbiology 79, no. 24: 7610–7617. https://doi.org/10.1128/AEM.02710‐13.

Poursalavati, A., J. V. Javaran, I. Laforest‐Lapointe, and L. M. Fall. 2023. “Soil Metatranscriptomics: An Improved RNA Extraction Method Toward Functional Analysis Using Nanopore Direct RNA Sequencing.” Phytobiomes Journal 7: 42–54. https://doi.org/10.1094/PBIOMES‐12‐22‐0108‐TA.

R Core Team. 2024. “R: A Language and Environment for Statistical Computing.” R Foundation for Statistical Computing, Vienna, Austria. https://www.R‐project.org/.

Schostag, M., A. Priemé, S. Jacquiod, J. Russel, F. Ekelund, and C. S. Jacobsen. 2019. “Bacterial and Protozoan Dynamics Upon Thawing and Freezing of an Active Layer Permafrost Soil.” ISME Journal 13: 1345–1359. https://doi.org/10.1038/s41396‐019‐0351‐x.

Shi, H., Y. Zhou, E. Jia, M. Pan, Y. Bai, and Q. Ge. 2021. “Bias in RNA‐Seq Library Preparation: Current Challenges and Solutions.” BioMed Research International 2021: 1–11. https://doi.org/10.1155/2021/6647597.

Sigurdsson, B. D., N. I. W. Leblans, S. Dauwe, et al. 2016. “Geothermal Ecosystems as Natural Climate Change Experiments: The ForHot Research Site in Iceland as a Case Study.” Icelandic Agricultural Sciences 29, no. 1: 53–71. https://doi.org/10.16886/IAS.2016.05.

Söllinger, A., L. S. Ahlers, M. B. Dahl, et al. 2024. “Microorganisms in Subarctic Soils Are Depleted of Ribosomes Under Short‐, Medium‐, and Long‐Term Warming.” ISME Journal 18: wrae081. https://doi.org/10.1093/ismejo/wrae081.

Söllinger, A., J. Séneca, M. Borg Dahl, et al. 2022. “Down‐Regulation of the Bacterial Protein Biosynthesis Machinery in Response to Weeks, Years, and Decades of Soil Warming.” Science Advances 8, no. 12: eabm3230. https://doi.org/10.1126/sciadv.abm3230.

Söllinger, A., A. T. Tveit, M. Poulsen, et al. 2018. “Holistic Assessment of Rumen Microbiome Dynamics Through Quantitative Metatranscriptomics Reveals Multifunctional Redundancy During Key Steps of Anaerobic Feed Degradation.” mSystems 3, no. 4: 1–19.

Starr, E. P., E. E. Nuccio, J. Pett‐Ridge, J. F. Banfield, and M. K. Firestone. 2019. “Metatranscriptomic Reconstruction Reveals RNA Viruses With the Potential to Shape Carbon Cycling in Soil.” Proceedings of the National Academy of Sciences of the United States of America 116, no. 51: 25900–25908. https://doi.org/10.1073/pnas.1908291116.

Täumer, J., S. Marhan, V. Groß, et al. 2022. “Linking Transcriptional Dynamics of CH4‐Cycling Grassland Soil Microbiomes to Seasonal Gas Fluxes.” ISME Journal 16, no. 7: 1788–1797. https://doi.org/10.1038/s41396‐022‐01229‐4.

Thompson, L. R., J. G. Sanders, D. McDonald, et al. 2017. “A Communal Catalogue Reveals Earth's Multiscale Microbial Diversity.” Nature 551, no. November: 457–463. https://doi.org/10.1038/nature24621.

Thorn, C. E., C. Bergesch, A. Joyce, et al. 2019. “A Robust, Cost‐Effective Method for DNA, RNA and Protein Co‐Extraction From Soil, Other Complex Microbiomes and Pure Cultures.” Molecular Ecology Resources 19, no. 2: 439–455. https://doi.org/10.1111/1755‐0998.12979.

Tkacz, A., M. Hortala, and P. S. Poole. 2018. “Absolute Quantitation of Microbiota Abundance in Environmental Samples.” Microbiome 6: 110. https://doi.org/10.1186/s40168‐018‐0491‐7.

Torsvik, V., L. Øvreås, and T. F. Thingstad. 2002. “Prokaryotic Diversity—Magnitude, Dynamics, and Controlling Factors.” Science 296, no. 5570: 1064–1066. https://doi.org/10.1126/science.1071698.

Tveit, A. T., T. Urich, P. Frenzel, and M. M. Svenning. 2015. “Metabolic and Trophic Interactions Modulate Methane Production by Arctic Peat Microbiota in Response to Warming.” Proceedings of the National Academy of Sciences of the United States of America 112, no. 19: E2507–E2516. https://doi.org/10.1073/pnas.1420797112.

Tveit, A. T., T. Urich, and M. M. Svenning. 2014. “Metatranscriptomic Analysis of Arctic Peat Soil Microbiota.” Applied and Environmental Microbiology 80, no. 18: 5761–5772. https://doi.org/10.1128/AEM.01030‐14.

Urich, T., A. Lanzén, J. Qi, D. H. Huson, C. Schleper, and S. C. Schuster. 2008. “Simultaneous Assessment of Soil Microbial Community Structure and Function Through Analysis of the Meta‐Transcriptome.” PLoS One 3, no. 6: e2527. https://doi.org/10.1371/journal.pone.0002527.

Vaulot, D., S. Geisen, F. Mahé, and D. Bass. 2022. “pr2‐Primers: An 18S rRNA Primer Database for Protists.” Molecular Ecology Resources 22, no. 1: 168–179. https://doi.org/10.1111/1755‐0998.13465.

Verbrigghe, N., K. Meeran, M. Bahn, et al. 2022. “Long‐Term Warming Reduced Microbial Biomass but Increased Recent Plant‐Derived C in Microbes of a Subarctic Grassland.” Soil Biology and Biochemistry 167: 108590. https://doi.org/10.1016/j.soilbio.2022.108590.

Wang, H., G. Jurasinski, J. Täumer, et al. 2023. “Linking Transcriptional Dynamics of Peat Microbiomes to Methane Fluxes During a Summer Drought in Two Rewetted Fens.” Environmental Science & Technology 57, no. 12: 5089–5101. https://doi.org/10.1021/acs.est.2c07461.

Wang, Y., M. Hayatsu, and T. Fujii. 2012. “Extraction of Bacterial RNA From Soil: Challenges and Solutions.” Microbes and Environments 27, no. 2: 111–121. https://doi.org/10.1264/jsme2.ME11304.

Wasimuddin, K. Schlaeppi, F. Ronchi, S. L. Leib, M. Erb, and A. Ramette. 2020. “Evaluation of Primer Pairs for Microbiome Profiling From Soils to Humans Within the One Health Framework.” Molecular Ecology Resources 20, no. 6: 1558–1571. https://doi.org/10.1111/1755‐0998.13215.

Wickham, H. 2007. “Reshaping Data With the Reshape Package.” Journal of Statistical Software 21, no. 12: 1–20. http://www.jstatsoft.org/v21/i12/.

Wickham, H. 2016. ggplot2: Elegant Graphics for Data Analysis. Springer‐Verlag New York. ISBN 978‐3‐319‐24277‐4. https://ggplot2.tidyverse.org.

Wimmer, E., I. A. Zink, L. H. Hodgskiss, M. Kerou, and C. Schleper. 2024. “The Type III‐B CRISPR‐Cas System Affects Energy Metabolism and Adaptation in the Archaeon Saccharolobus solfataricus.” Preprint at bioRxiv 2024.09.02.610847. https://doi.org/10.1101/2024.09.02.610847.

Yilmaz, P., R. Kottmann, E. Pruesse, C. Quast, and F. O. Glöckner. 2011. “Analysis of 23S rRNA Genes in Metagenomes—A Case Study From the Global Ocean Sampling Expedition.” Systematic and Applied Microbiology 34, no. 6: 462–469. https://doi.org/10.1016/j.syapm.2011.04.005.

Yilmaz, P., L. W. Parfrey, P. Yarza, et al. 2014. “The SILVA and “All‐Species Living Tree Project (LTP)” TAXONOMIC FRAMEWORKS.” Nucleic Acids Research 42, no. 1: D643–D648. https://doi.org/10.1093/nar/gkt1209.

Zhang, M., L. Zhang, S. Huang, et al. 2022. “Assessment of Spike‐AMP and qPCR‐AMP in Soil Microbiota Quantitative Research.” Soil Biology and Biochemistry 166: 108570. https://doi.org/10.1016/j.soilbio.2022.108570.

Zillig, W., K. O. Stetter, S. Wunderl, W. Schulz, H. Priess, and I. Scholz. 1980. “The Sulfolobus‐“Caldariella” Group: Taxonomy on the Basis of the Structure of DNA‐Dependent RNA Polymerases.” Archives of Microbiology 125: 259–269. https://doi.org/10.1007/BF00446886.