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Review
. 2025 Jan 27;13(1):26.
doi: 10.1186/s40168-024-02009-2.

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

Chunxiao Wang  1 , Yu Yang  1 , Xiaoqing Xu  1 , Dou Wang  1 , Xianghui Shi  1 , Lei Liu  1 , Yu Deng  1   2 , Liguan Li  1   3 , Tong Zhang  4   5   6   7
Affiliations
Review

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

Chunxiao Wang et al. Microbiome. .

Abstract

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. Video Abstract.

Keywords: Absolute quantification; Cellular internal standard; Environmental analytical microbiology; Microbiome; Standardization.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: The manuscript does not report data collected from humans and animals. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Absolute abundance of environmental microbiome, including total microbial loads across different habitats using diverse AQ methods (a) (data source: [, –26]); pathogens and pathogenic antibiotic-resistant bacteria (PARB) density in water samples and wastewater treatment works (WWTPs) (b) (data source: [1, 23, 27]); and ARG concentrations in water samples and WWTPs using IS-based AQ methods (c) (data source: [1, 6, 27])
Fig. 2
Fig. 2
Absolute quantification (AQ) methods and critical issues to be addressed
Fig. 3
Fig. 3
Influencing factors and technical considerations for a reporting checklist in IS-based AQ methods
Fig. 4
Fig. 4
Applications using absolute quantitative microbiome in environmental analytical microbiology
See this image and copyright information in PMC

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