Determination of Microbial Biomass in Soil using Digital Droplet PCR (ddPCR)
April 24th, 2026
This study presents a rapid ddPCR method for quantifying soil microbial biomass and fungal-to-bacterial ratios. Although ddPCR-derived microbial biomass carbon shows only weak correlation with chloroform fumigation estimates, the method provides a sensitive, scalable molecular approach for assessing microbial abundance without additional soil sampling or the use of harsh chemicals.
Our study uses droplet digital PCR and whole genome sequencing to accurately quantify microbial biomass across various soil types. Conventional methods require large soil samples and harmful chemicals. ddPCR uses minimal soil for absolute gene and biomass carbon quantification.
To begin, assemble the reagents of the soil DNA extraction kit to isolate the DNA from soil microbes at 20 degrees Celsius room temperature. Spin the two milliliter bead-beating tube briefly and verify that the beads have settled at the bottom. Add 100 milligrams of soil, then 800 microliters of Solution CD1 and vortex briefly to mix.
Use a bead mill homogenizer at a speed of six meters per second for 40 seconds to lyse the sample thoroughly and centrifuge the bead-beating tube at 15, 000 g for one minute at room temperature to achieve mechanical cell lysis. Transfer the supernatant that may still contain soil particles to a clean two milliliter microcentrifuge tube. Add 200 microliters of Solution CD2.
Vortex for five seconds and centrifuge at 15, 000 g for one minute. Transfer up to 700 microliters of supernatant to a clean two milliliter microcentrifuge tube without disturbing the pellet. Add 600 microliters of Solution CD3 to the supernatant and vortex for five seconds.
Load 650 microliters of the mixture onto a spin column and centrifuge at 15, 000 g for one minute. Discard the flow-through and repeat the process for the remaining lysate. Next, place the spin column into a clean two milliliter collection tube.
Add 500 microliters of Solution EA to the spin column and centrifuge at 15, 000 g for one minute at room temperature. Discard the flow-through and place the spin column back into the same collection tube. Add 500 microliters of Solution C5 to the spin column and centrifuge as demonstrated previously.
After discarding the flow-through, place the spin column into a new two milliliter collection tube and centrifuge at 16, 000 g for two minutes at room temperature. Place the spin column in a new 1.5 milliliter elution tube. Add 50 to 100 microliters of the Solution C6 to the center of the filter membrane of the spin column.
Centrifuge at 15, 000 g for one minute. Store the tube containing the eluent at four degrees Celsius after discarding the spin column. Quantify extracted DNA using a fluorometer and a double-stranded DNA broad range assay kit following the manufacturer's instructions.
For analysis of bacteria and fungi, select specific 16S rRNA and 18S rRNA primers. After thawing, vortex all reagents on ice. Set up the Digital Droplet Polymerase Chain Reaction, or ddPCR.
Place the droplet generator cartridge, or DG8, into the cartridge holder. Add a total of 20 microliters of the reaction mixture into each of the sample wells in the cartridge. Then add 70 microliters of Droplet Generation Oil for Evagreen into the cartridge well labeled oil.
Place the cartridge into the droplet generator and cover it with a gasket. Transfer 40 microliters of the generated droplets to each well of a 96-well semi-skirted plate for PCR amplification. Seal the plate using a plate sealer preset to 180 degrees Celsius for five seconds.
Configure the thermal cycling profile and set the heated lid to 105 degrees Celsius. After completion of PCR, transfer the 96-well plate to the plate reader. Using QuantaSoft version 2.1, adjust the threshold of the amplitude to separate negative and positive droplets.
From each well, evaluate the parameters, like number of gene copies per nanogram of DNA, total gene copies per nanogram of extracted DNA, gene copies per gram dry weight of soil processed in the extraction, and cell count per gram dry weight of soil. Further, calculate the Microbial Biomass Carbon, or MBC, corresponding to bacteria and fungi per gram dry weight of soil per well. Triplicate analysis of soil DNA from five locations yielded average microbial DNA concentrations ranging from 32 nanograms to 4, 590 nanograms per gram of dry soil per sample.
Soil B contained the fewest 16S rRNA gene copies at an average of 8.8 times 10 to the power of seven and 18S rRNA gene copies at an average of 4.6 times 10 to the power of six per gram of dry soil. In contrast, Soil E had the highest counts of 16S and 18S rRNA gene copies with an average of 4.4 times 10 to the power of nine and 2.5 times 10 to the power of nine respectively per gram of dry soil, differing significantly from all other samples. While fungal copies remained similar across Soils A, B, C, and D, bacterial counts in Soil D differed significantly from both B and E.The highest microbial content was observed in Soil E, while the lowest microbial content was found in samples collected from Soil B differing significantly from A, D, and E.This protocol allows researchers to measure soil community dynamics and structure through microbial biomass and fungal to bacterial ratio.
The same template DNA used for droplet digital PCR can be used for DNA sequencing to enable microbial community and functional analysis.
Overview
This article presents an optimized workflow for quantifying bacterial and fungal abundance in soil using digital droplet PCR (ddPCR) targeting the 16S and 18S rRNA genes. The protocol enables absolute quantification of microbial gene copies, conversion to cell numbers, and estimation of microbial biomass carbon (MBC). The method is compared to conventional chloroform fumigation extraction and is demonstrated across soils from five globally distributed agricultural systems.
Key Study Components
Area of Science
- Soil microbiology
- Molecular ecology
- Environmental science
Background
- Soil is the largest terrestrial pool of organic carbon, but significant carbon loss has occurred over the past two centuries.
- Microbial biomass, especially fungi and bacteria, drives carbon and nutrient cycling in soils.
- Conventional methods for microbial biomass quantification have limitations, including high sample requirements and conversion uncertainties.
- Accurate, scalable, and sensitive methods are needed for soil health assessment.
Purpose of Study
- To develop and validate a ddPCR-based protocol for quantifying bacterial and fungal abundance in soil.
- To compare ddPCR-derived microbial biomass estimates with conventional chloroform fumigation methods.
- To assess the protocol's applicability across diverse soil types and agricultural systems.
Methods Used
- Soil DNA extraction using a bead-beating and spin column protocol.
- Quantification of extracted DNA with a fluorometer and broad range assay kit.
- ddPCR targeting 16S rRNA (bacteria) and 18S rRNA (fungi) genes with specific primers.
- Calculation of gene copies, cell counts, and microbial biomass carbon per gram of dry soil.
- Comparison of ddPCR results with chloroform fumigation extraction and whole genome sequencing.
Main Results
- ddPCR enabled high-resolution, absolute quantification of bacterial and fungal gene copies in soil samples.
- Microbial DNA concentrations ranged from 32 ng to 4,590 ng per gram of dry soil across samples.
- Soil E had the highest microbial content, while Soil B had the lowest.
- ddPCR provided broader and more sensitive estimates of fungal-to-bacterial (F:B) ratios than whole genome sequencing.
- ddPCR-derived MBC showed only a weak correlation with chloroform fumigation values.
Conclusions
- The ddPCR protocol offers a reproducible, scalable, and sensitive approach for quantifying soil microbial abundance and biomass.
- This method allows for improved assessment of ecological indicators such as the F:B ratio.
- ddPCR can be integrated with compositional analyses to advance soil health assessment and research.
