Microbial Diversity Analysis of Soil

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Importance of Microbial Diversity in Soil


The microbial community in soil comprises a diverse assortment of microbial strains. These microorganisms play crucial roles in sustaining and managing soil functions within both natural and managed ecosystems, owing to their active participation in critical processes such as soil structure formation, toxin removal, organic matter decomposition, and nutrient cycling of carbon, nitrogen, phosphorus, and sulfur. Furthermore, microbes are vital in promoting plant growth, inhibiting soil-borne plant diseases, and fostering vegetation development. Shifts in vegetation can, therefore, occur as a result of these microbial contributions. Consequently, the diversity of microorganisms in soil habitats has garnered increasing interest as a potential indicator of ecosystem health.

Methods for soil microbial diversity analysis

Soil microbial diversity analysis methods include:

Traditional Cultivation Method: This method estimates the quantity of specific microorganisms in a sample through the use of particular mediums and conditions. Its advantages are its quick and intuitive features, while its limitation lies in the requirement for specific cultivation environments. Moreover, many microorganisms' cultivation conditions remain unknown, and the process may become contaminated with miscellaneous bacteria.

Phospholipid Fatty Acids (PLFA) Method: This technique labels microbial communities by detecting phospholipid fatty acids in cell membranes, bypassing the need for microbial cultivation. However, it can only reflect the community of active microbes.

Biolog ECO Technique: This approach characterizes microbial physiological traits and community features by leveraging microorganisms' differential utilization of various carbon sources.

High Throughput Sequencing: This surveying method can detect microbial population distribution within samples and is commonly applied for diversity studies of bacteria and archaea.

Fluorescent Quantitative Polymerase Chain Reaction (PCR): This is employed for the quantitative analysis of specific microbial populations.

GeoChip Functional Gene Microarray: Aimed at detecting the functional genes of soil microorganisms, this method infers ecological roles and functions.

Accelerate Research and Practice in Soil Microbiology

Traditionally, the diversity of soil microorganisms has been analyzed through viable and cultivable methods, Community level physiological profiling (CLPP), and flow cytometry techniques. However, the limitations of these methods have been a persistent hindrance to further exploration. We propose the use of PCR, new-generation sequencing (NGS), and long-read sequencing methods (PacBio SMRT sequencing and Nanopore sequencing) for identifying, quantifying, and characterizing both culturable and unculturable microbes present in soil habitats. These technologies offer more accurate methodologies for measuring microbial diversity and abundance and for exploring microbial functionality.

What Can We Do?

  • 1. Identification of soil microflora
  • 2. Quantitation of soil microorganisms
  • 3. Study the evolutionary relationship of soil microorganisms
  • 4. Study the relationship between soil microbiome and the environment
  • Note: Our service are for research use only, not for disease diagnosis or treatment.

Detectable Objects

  • DNA samples of soil microbiome from diverse soil types can be tested.

Detectable Microorganisms

  • Viruses, fungi, bacteria, archaea, mycoplasma, etc.

Technical Platforms

  • We are equipped with Illumina Hiseq/Miseq, PacBio SMRT systems, and Nanopore systems for 16S/18S/ITS sequencing and metagenomics, PCR-DGGE (PCR-denaturing gradient gel electrophoresis), real-time qPCR, clone library, and other analytical platforms.

Bioinformatics Analysis

OTU Clustering Distribution-Based OTU-Calling
Rarefaction Curve
Shannon-Wiener Curve
Rank Abundance Curve
Diversity Index
OTU-Based Analysis Heatmap
Principal Components Analysis (PCA)
Hierarchical Clustering
Comparative Analysis RDA/CCA
Network Analysis
Functional analysis BLASTX, KEGG, eggNOG, CAZy, CARD, ARDB etc.
Phylogenetic Analysis (Un)Weighted Unifrac
Phylogenetic Trees


High-throughput sequencing analysis process.Figure 1. High-throughput sequencing analysis process.

PCR-DGGE analysis process.Figure 2. PCR-DGGE analysis process.

Sample Requirement

  • Metagenome Sequencing: DNA≥500 ng, Minimum Quantity≥20 ng, concentration≥5 ng/µL,
    16S/18S/ITS Sequencing: DNA≥100 ng, Minimum Quantity≥10 ng, concentration≥1 ng/µL, and OD260/280 = 1.8-2.0.
  • Ensure that the DNA is intact and not degraded.
  • Avoid repeated freezing and thawing cycles.
  • Dry ice or ice packs should be used for sample submission.


  1. Dubey A, Malla M A, Kumar A. Role of next-generation sequencing (NGS) in understanding the microbial diversity//Molecular genetics and genomics tools in biodiversity conservation. Singapore: Springer Nature Singapore, 2022: 307-328.
  2. Dodd, J.C., Boddington, C.L., Rodriguez, A., et al. Mycelium of arbuscular mycorrhizal fungi (AMF) from different genera: form, function and detection. Plant Soil, 2000, 226, 131 – 151.
  3. O'Donnell, A.G., Seasman, M., Macrae, A., et al. Plants and fertilisers as drivers of change in microbial community structure and function in soils. Plant Soil, 2001, 232, 135 – 145.
  4. Kirk J L, Beaudette L A, Hart M, et al. Methods of studying soil microbial diversity. Journal of microbiological methods, 2004, 58(2): 169-188.

1. How do you test the microbial content of soil?

The methodologies for evaluating microbial content in soil primarily encompass bacterial plate count method, fluorescence staining, biomass estimation, molecular biology-based techniques, and Phospholipid Fatty Acid (PLFA) analysis, amongst others.

The fluorescence staining procedure employs fluorescent dyes such as 4',6-diamidino-2-phenylindole (DAPI) to stain microbial cells within soil samples, followed by utilizing a fluorescence microscope to observe and enumerate the cells.Biomass Determination, an apt method to appraise microbial quantities, estimates these amounts by measuring constituents such as carbon, protein, or amino acid content within soil samples.

Molecular Biology Techniques, encompassing Polymerase Chain Reaction (PCR), Real-Time PCR, DNA microarray, and gene sequencing, provide an analytical avenue to unveil the structure and abundance of microbial communities in soil. These techniques involve the direct extraction of DNA or RNA from the soil samples.

Phospholipid Fatty Acid, a key component of microbial cell membranes, serves as another estimator of microbial numbers in the aforementioned strategies.

2. What affects soil microbial diversity?

The biodiversity of soil microorganisms is modulated by various factors, spanning across biological, chemical, and physical domains.

The type and density of vegetation wield significant influence on the structure and diversity of soil microbial communities. Different types of vegetation introduce varying types and quantities of organic matter, thereby impacting the ecological niches and abundance of microorganisms. Moreover, plant root exudates, a crucial nutritional source for soil microbial growth and metabolism, can attract, promote or inhibit the growth of specific microorganisms.Besides, soil inhabitants such as earthworms, ants, and various soil microarthropods modulate microbial diversity by reshaping soil physical properties and the rate of organic matter decomposition.

Microbial communities exhibit a broad spectrum of pH adaptability, thus, fluctuations in soil pH levels can reshape community composition and abundance. The content of organic matter in the soil directly steers the microorganisms' ecological niches and nutrient sources. For instance, high organic matter content often leads to increased microbial diversity. Nutrient contents in the soil, such as nitrogen, phosphorus, and potassium, can affect microbial growth and metabolic activities, thereby influencing microbial diversity.

Lastly, soil texture determines soil pore structure and aeration. Adequate moisture facilitates microbial growth and reproduction, whereas excess or limited water content might constrain the ecological functionality and diversity of microorganisms.

3. How does microbial diversity change with soil depth?

Soil depth represents a fundamental determinant steering the structure of microbial communities in agricultural soils. The deep soil strata serve as pivotal zones for soil formation and carbon sequestration.

Soils at varying depths exhibit disparate physical, chemical, and biological characteristics — including organic matter content, temperature, root exudates, and microbial interactions — which intrinsically influence the composition and diversity of the resident microbial communities. More often than not, the bacterial abundance, species richness, and diversity in deeper soil horizons are found to be lesser than those in the surface soils.

4. Why soil microbe diversity is important to building rich soil?

Microorganisms play a multitude of crucial roles in soil, significantly impacting the ecosystem's function and health. A diversity of beneficial microbes, such as nitrogen-fixing bacteria or phosphate-dissolving bacteria, inhabit the soil microbiome. These microorganisms assist plants in absorbing and utilizing nutrients in the soil, for instance, by converting atmospheric nitrogen into a form accessible for plant utilization or facilitating the absorbability of phosphorus in soils via mineral solubilization. Hence, the biodiversity of these beneficial microbes can enhance plant growth and development, thereby augmenting soil fertility.

Soil microorganisms specialize in the decomposition of organic matter and plant residue, fostering organic matter breakdown and humus formation in the soil. These processes result in the release of essential nutrients such as nitrogen, phosphorus, and potassium, providing vital nutritional resources for plant growth. Certain soil microorganisms, funguses and bacteria for instance, can excrete colloidal substances that promote the amalgamation of soil particles, thus aiding in the formation of favorable soil structure. An optimal soil structure promotes soil aeration and moisture content, which is beneficial for the growth of plant root systems and nutrient absorption.

Amid the diversity of the soil microbiome, some strains are beneficial for their ability to inhibit soil pathogenic microbes. They suppress the quantity of pathogens in soil via various mechanisms, including competition for nutritional resources, antibiotic secretion, and the production of antimicrobial substances.

Therefore, in summary, microorganisms embody an array of critical roles in soil, playing a crucial part in maintaining the soil ecosystem's functionality and health.

Reduction of microbial diversity in grassland soil is driven by long-term climate warming
Journal: Scientific reports
Impact factor: 28.3
Published: June 13, 2022


Recognizing the extensive negative impacts that loss of biodiversity, precipitated by climate change, can have on ecosystem functioning and the quality and quantity of its eco-services, we turn to explore a facet of biodiversity that warrants deeper understanding - soil microbial diversity. Although previous research has illuminated the way changes in climate, specifically warming, can impact aspects such as respiration feedback responses, decomposition, biomass, community composition and succession, effects on temporal scale, and complexity and stability of networks, scant data exists when it comes to in-situ, repeat empirical observation in the field over an extensive timeline. This has resulted in a considerable gap in our knowledge about the mechanism of how warming affects subterranean microbial diversity. To narrow this gap, we embarked on a seven-year, in-situ experiment, designed to unveil how climate change - encapsulated in increased warming, alterations in precipitation, and mowing - and the interaction thereof, impact plant prairie soil bacteria, fungi and protozoa community diversity and, inherently, ecosystem functionality.


soil microbial biomass

  • Soil phospholipid fatty acids (PLFA)

DNA extraction

  • Sodium dodecyl sulfate (SDS)-based cell lysis

Data analyses

  • Picante R package
  • Linear mixed-effects models
  • Structural equation modelling (SEM)


1. Effects of warming on soil microbial diversity

Compared to rainfall regulation and mowing, warming exerts a significant primary impact on soil and plant variables by increasing the temperature and reducing soil moisture, concurrently lowering soil pH and augmenting NO3-. While mowing has a significantly negative effect on plant biomass, it positively impacts plant richness (Fig.2).

Effects of experimental treatments on soil and plant variables by linear mixed-effects models (LMMs).Fig. 2 Effects of experimental treatments on soil and plant variables by linear mixed-effects models (LMMs).

In this study, we consider two dimensions of microbial community diversity: taxonomic diversity (species richness and their relative abundance) and phylogenetic diversity. Warming has a significant negative effect on taxonomic diversity, phylogenetic diversity, and biomass across three distinct types of microbial groups (Figs. 3, 4). There were no significant feedback responses in microbial diversity to interactive effects among multiple factors, with the only exception being a positive response of fungi and protozoa to the synergistic defoliation effect accompanying warming. Therefore, the key findings suggest that warming has a dominant effect on soil bacteria, fungi, and protozoan diversity. The likely reason is that under warming conditions, changes in microbial diversity are primarily driven by alteration in the soil microenvironment and geochemistry aspects (such as soil pH, soil moisture, and soil temperature), which are significantly influenced by warming.

Yearly differences of bacterial (a), fungal (b), and protistan (c) richness between warmed and unwarmed samples.Fig. 3 Yearly differences of bacterial (a), fungal (b), and protistan (c) richness between warmed and unwarmed samples.

Effects of Different treatments on soil bacterial, fungal, and protist diversity indices, lineage diversity, and biomassFigure 4. Effects of Different treatments on soil bacterial, fungal, and protist diversity indices, lineage diversity, and biomass

Warming exert differential effects on the microbial diversity of various lineages (Fig. 5). Within bacteria, an increase in temperature elicits a noticeable reduction in both the abundance and relative abundance of most bacterial phyla. Only the abundance and relative abundance of Actinobacteria, as well as the relative abundance of Firmicutes and Bacillus, manifest a significant positive feedback. A possible explanation for this may be their ability to form spores, which helps them withstand desiccation stress and therefore becomes more adaptive to relatively dry soils. In regard to fungi and protozoa, warming also markedly imposes negative impact on their taxonomic diversity and phylogenetic diversity, except for an evident positive feedback on both abundance and phylogenetic diversity of the Conosa in protozoans.

Effects of experimental warming on different microbial taxa.Fig. 5. Effects of experimental warming on different microbial taxa.

2. The Mechanisms of Microbial Diversity Reduction

At the inception of this study, we hypothesized that a warming climate would diminish microbial diversity by altering environmental conditions and biotic interactions. Our findings, drawing on a 2021 study by our research team, demonstrated that sustained warming heightened the complexity and convolutedness of the microbial species network. This further elucidates that global warming fosters competitive relations amongst microorganisms, eventually leading to the extinction of less competitive species and subsequent reduction in microbial diversity.

Warming acts as a decisive filtering factor in our research, positively selecting for microorganisms related to spore formation and negatively selecting for those unrelated to this process. This aligns synergistically with previous research findings. Consequently, to unveil the driving mechanisms behind the warming-induced decline of microbial diversity in this study, we employed Structural Equation Modeling to analyze the direct and indirect effects of environmental drivers on microbial diversity, as illustrated in Figure 6.

This model accounted for over half of the interpretation in microbial diversity, therefore leading to the conclusion that the environmental filtering effect, by influencing microbial activities and interactions, serves as the primary driving factor in the decline of microbial diversity.

Environmental drivers of microbial diversity.Fig.6. Environmental drivers of microbial diversity.

3. The Relationship Between Microbial Diversity and Ecosystem Functioning"

Beyond the domain of solely documenting warming-reduced microbial diversity, the current study bridges ecological functional process, coaxing an understanding of how alterations in microbial diversity due to climate change could, in turn, influence the ecological functional process. Observations indicate that warming downgrades various ecological functions such as total microbial biomass, bacterial biomass, primary productivity, and ecosystem respiration (refer Figure 7). Additionally, a significant positive correlation was found between microbial diversity and the ecological functional process (as depicted in Figure 6).

Effects of experimental warming on different ecosystem functions.Fig. 7 Effects of experimental warming on different ecosystem functions.


The impacts of global warming, acting as a decisive filtering factor, instigates aridity stress. Subsequently, it manipulates microbial activity and interplay, instigating a decline in microbial diversity and concomitantly rendering the associated ecosystem functions more vulnerable. Notably, the effects of increased temperature vary immensely across different microbial taxa. For instance, beneficial fungi within an ecosystem, Arbuscular Mycorrhizal Fungi (AMF), tend to decline due to warming, hence compromising the associated ecosystem functions under a future warming climate. Furthermore, it appears that the soil moisture reduction largely mediates the microbial diversity response to warming. Thus, in global drylands, encompassing arid, semi-arid, and dry sub-humid ecosystems, the impact of global warming on microbial diversity loss might be more severe. Consequently, investigating further whether the decline in microbial diversity due to warming and its corresponding driving mechanisms apply to other ecosystems is warranted.


  1. Wu L, Zhang Y, Guo X, et al. Reduction of microbial diversity in grassland soil is driven by long-term climate warming. Nature Microbiology, 2022, 7(7): 1054-1062.

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