Noah Fierer
updated: 4/10/2003
Soil Microbial Biomass Determination
Basic Idea:
The quantity of microbial biomass in a given soil sample is inherently difficult to
measure. To some degree, any measurement of microbial biomass is relative – different
methodologies or variations in methodologies will yield microbial biomass estimates that
are not directly comparable. For this reason, if you want to measure microbial biomass –
choose a method and stick with it exactly. Here, I’ll discuss 2 methods to measure
microbial biomass: substrate induced respiration (SIR) and simultaneous chloroform
fumigation-extraction (sCFE), a modification of the traditional chlorform-fumigation
extraction method.
SIR estimates microbial biomass based on the short-term respiration rates (CO2
production) when soil is given an excess supply of labile C. The higher the respiration
rate, the more microbial biomass in a given soil. The SIR method is fairly
straightforward, consistent, and reasonably quick, however, the biomass estimates are
relative – the method will not give direct estimates of microbial biomass C (or N) in soil.
sCFE measures microbial biomass based on the amount of microbial C or N
extractable from soil after treatment with chloroform, a cell membrane disruptor. There is
a lot of confusion about general chloroform fumigation-extraction techniques, and, (in
my opinion) the technique is often carried out in a manner that makes cross-comparison
of biomass estimates difficult. For these reasons, I have included a lengthy treatise on the
technique (see pages 2 – 4) along with a full description of my modifications to the
traditional chloroform-fumigation extraction method. The sCFE technique combines the
soil extraction and chloroform exposure steps, increasing the simplicity and efficacy of
the normal fumigation-extraction method. The modified method yields estimates of
microbial C and N that are well correlated with those obtained by the normal fumigation-
extraction method across a range of soil types. The simultaneous chloroform slurry-
extraction method is particularly useful for studying soils with a range of porosities
and/or water contents where differences in chloroform diffusion rates through soils may
affect biomass extraction efficiencies when using the normal fumigation-extraction
method.
SIR:
• • Weigh 5- 10g soil (fresh wt.) of soil into a 50mL centrifuge tubes
equipped with gas-tight lids that have rubber septa for gas sampling.
• • Add 10mL yeast solution to each tube. Yeast solution should deliver
20mg yeast/g dry soil. (3g autolyzed yeast extract to 250mL ddH2O). Other
SIR protocols use glucose but I’ve found that yeast gives higher respiration
rates and the goal of the method is to maximize CO2 production.
• • Cap tubes and shake horizontally in 200C room for the duration of the 4
h assay. 10 –20 minutes after sealing the tubes measure the initial (the T0 time
point) headspace CO2 concentrations. This is the TO time point. Measure
headspace CO2 concentrations two more times, approx. 2h and 4h after the T0
time point.
• • Measure headspace CO2 concentrations by injecting 5mL lab air into
tube, pull out 5mL air from tube and measure cumulative CO2 concentrations
on an IRGA equipped for static CO2 analysis. Make sure to record the time
that the gas sample was taken.
• • Calculate the slope of the line relating CO2 concentrations to time. The
average respiration rate (µg C-CO2/gsoil/h) over the 4 h incubation period is an
index of the SIR-responsive microbial biomass. Calculate an r2 value for the
line describing CO2 concentrations over time to make sure the relationship is
roughly linear.
• • This technique is modified from that described in: West and Sparling.
1986. Journal of Microbiological Methods. 5: 177-189.
sCFE:
Why modify the traditional CFE method?
The chloroform fumigation-extraction (CFE) method that is “traditionally” used
to measure microbial biomass in soil involves fumigating soils with chloroform for 24 h
to 4 days to lyse microbial cells. At the end of the fumigation, both the fumigated sample
and an unfumigated “control” sample are extracted with a salt solution. Extractable
microbial biomass C or N is then measured as the difference in the amount of C or N
extracted from the fumigated and the control samples.
There are several potential sources of error when estimating soil microbial
biomass C and N with this method. One problem is that the fumigation-extraction
technique relies on the gaseous diffusion of chloroform through soil, so fumigation
efficiencies can be reduced in soils that are wet or have low porosities. This may pose
particular problems when estimating annual variation in the size of the microbial biomass
pool where seasonal differences in soil water content may dramatically alter extraction
efficiencies.
Another possible problem is the potential for microbial activity or enzymes to
cause changes in extractable C and N during the relatively long fumigation period. One
assumption of the method is that the extractable C and N levels in the unfumigated
“control” samples are the same as those in the fumigated sample at the end of the
fumigation, minus the “flush” of microbial biomass C and N released by the chloroform.
However, chloroform fumigation does not stop all microbial or enzymatic activity and a
substantial portion of the microbial biomass may survive fumigation. We have measured
respiration rates during a 24 h fumigation period that are 64% and 90% of the respiration
rates measured in the equivalent unfumigated controls. Significant mineralization of soil
organic matter, or added substrate, during the fumigation period may cause changes in
non-biomass extractable C and N levels that would not be reflected in the unfumigated
“control”. This may be particularly evident in short-term soil labeling experiments with
14
C, 15N, or 3H labeled substrates where a prolonged fumigation period may lead to a
significant disparity between the unfumigated and fumigated samples.
The solution?
We addressed both of these problems by combining the chloroform-exposure and
extraction steps while reducing the amount of time samples are exposed to chloroform.
Treating the soils with chloroform in a slurry extract reduces any effects of chloroform
diffusivity on fumigation efficiencies and allows the control and fumigated (chloroform-
exposed) subsamples to be extracted simultaneously. Shortening the chloroform
exposure period reduces the chance that extractable C and N levels will change
substantially during chloroform exposure.
The traditional CFE and sCFE methods gave very comparable estimates for
microbial biomass C and N over a wide range of soil types (see Figure 1 below). On
average, the sCSE method rendered approximately 30% less biomass C and N extractable
than the traditional CFE method. The two techniques had similar levels of variability
between sample replicates in extractable microbial biomass C and N (average coefficients
of variation = 0.25% for C and 0.48% for N). The lower recovery of biomass C and N
with the simultaneous CSE method is not surprising considering that chloroform
exposure times were only 4 h compared to 24 h with the traditional CFE method. Further
description of the method and a comparison of the sCFE method to the traditional CFE
method is provided in Appendix 1 of my Ph.D. thesis (Fierer. 2003. Stress Ecology and
the Dynamics of Microbial Communities and Processes in Soil. University of California,
226 pages).
Method Description:
• • For each soil sample, weigh two soil subsamples (chloroform-exposed
and control) of 3 to 10 g each into 70 mL glass tubes (Pyrex No. 9825).
• • Add 40 mL of 0.5 M K2SO4 to each of the glass tubes containing the soil
subsamples. To one subsample add 0.5 mL of EtOH-free chloroform. Seal
both the chloroform-exposed and the control samples with chloroform
resistant screw caps and shake simultaneously at approximately 150 rev min-1
for 4 h.
• • After the shaking period, let the tubes settle for 10 minutes. Decant the
top 20-30 mL of the soil extracts and gravity filter through Whatman No. 1
paper folded into a glass funnel. Collect the filtrate in 50mL centrifuge tubes.
Filtering only the top portion of the soil extract reduces chloroform
contamination in the filtered extract since the liquid chloroform settles to the
bottom of the tube.
• • Immediately bubble the filtered extracts vigorously with air for 20-30
min., the most effective method for removing chloroform from extracts. We
use house air bubbled through long spinal tap needles. Bubble both the
chloroform-exposed and control extracts.
• • You need a blank for both the control and chloroform exposed extracts.
(40mL of 0.5M K2SO4 with and without chloroform). Treat the blanks in the
same manner as the samples. In general, the K2SO4 blanks with chloroform
added should have levels of dissolved carbon only 25% greater than the very
low C concentrations (1-2 ppm C) in the blanks without chloroform.
• • Analyze the control and chloroform-exposed extracts for total dissolved
C and N using a persulfate digestion technique. Dissolved C and N
concentrations in the K2SO4 blanks (no soil added) with and without
chloroform added are subtracted from the extract concentrations of the
chloroform-exposed and the control samples, respectively. Extractable
microbial biomass C and N are calculated as the difference in total dissolved
C and N (both organic and inorganic) between the chloroform-exposed
subsample and the corresponding control subsample. Express results as µg
chloroform extractable biomass C/g dry soil. In order to calculate total
microbial biomass C or N (extractable + non-extractable biomass) you need to
use a conversion factor. In general, 20 – 40% of the total biomass is extracted
by this technique but the percentage extractable will vary between different
soils. A further discussion of conversion factors is available in Tate et al.
1988 (Soil Biology & Biochemistry 20:329-335) and Dictor et al. 1998 (Soil
Biology & Biochemistry 30:119-127).
