The quest for environmental analytical microbiology: Absolute quantitative microbiome using cellular internal standards

Background: High-throughput sequencing has revolutionized environmental microbiome research, providing both quantitative and qualitative insights into nucleic acid targets in the environment. The resulting microbial composition (community structure) data are essential for environmental analytical microbiology, enabling characterization of community dynamics and assessing microbial pollutants for the development of intervention strategies. However, the relative abundances derived from sequencing impede comparisons across samples and studies. 

Results: This review systematically summarizes various absolute quantification (AQ) methods and their applications to obtain the absolute abundance of microbial cells and genetic elements. By critically comparing the strengths and limitations of AQ methods, we advocate the use of cellular internal standard-based high-throughput sequencing as an appropriate AQ approach for studying environmental microbiome originated from samples of complex matrices and high heterogeneity. To minimize ambiguity and facilitate cross-study comparisons, we outline essential reporting elements for technical considerations, and provide a checklist as a reference for environmental microbiome research. 

Conclusions: In summary, we propose absolute microbiome quantification using cellular internal standards for environmental analytical microbiology, and we anticipate that this approach will greatly benefit future studies. Copyright ©  2025 The Author(s).