EGA60 Application

Determining Soil Microbial Biomass and Respiration Rate with the

EGA60 Multi-sample Analyser

Background

The International Organisation for Standardization (an independent NGO1) produced

‘International Standard 14240-1’ in 2002, relating specifically to the method of determining soil
microbial biomass. The Standard was prepared by Technical Committee ISO/TC 190, Soil quality,
Subcommittee SC 4, Biological methods1.

ISO 14240 is entitled ‘Soil quality — Determination of soil microbial biomass’ and comprises two
parts. Part 1 describes the ‘Substrate-Induced Respiration Method’, (known as SIR) 1.
This guiding document will detail the SIR method and its application in respect to the EGA60.

Definitions

The ISO defines the following terms:

Soil microbial biomass: The mass of intact microbial cells in a given soil.

Respiration is the recommended measurement method, as only intact microbial cells are
quantified. When carbon (or nitrogen) analysis is used, gases emitted as a result of dead cell
material are also detected.

Soil respiration rate: The volume of carbon dioxide released per unit mass of soil per unit time.

The purpose of measuring soil microbial biomass is manifold; to assess soil fertility and its
maintenance, to assess soil microbial ability to degrade organic materials (Knapp et al., 19834) and
to assess the effects of added materials on the natural microbial population.

The forerunner to the EGA60 (ADC BioScientific Ltd.) was the world renowned ADC225 MK3 Infra-
Red Gas Analyzer and WA161 multiplexer. The ADC225 was used by Heinemeyer et al. (1989)2 in
performing measurements to establish the Substrate Induced Respiration Method (SIR). Their
method set ‘Part 1’ of the ISO standard 14240 of measuring soil microbial respiration worldwide.

The SIR method was first published by Anderson and Domsch in 1978. The method is based on
measuring the “maximum initial respiratory response” of a soil sample after adding a substrate
such as glucose to it2. The EGA60 SIR method uses the soil micro-organisms’ physiological
response in the form of CO2 production, to the addition of this substrate, to quantify the microbial
activity in the soil.
SIR Method using the EGA60 system

1. The soil to be tested is generally sieved to remove any unwanted, large material and to
allow for even distribution of the substrate to be used.
2. The substrate now needs to be mixed with the soil. This can be in liquid form or in solid
form. If you are using solid form, mix with talc to aid mixing within the soil6. If soil is dry
add sufficient water so that the soil just holds together when firmly squeezed into a ball in
the hand.
3. Set up your EGA60 sample columns (using the quick start guide supplied upon purchase) in
an area where ambient temperature is stable at 22oC5. Place the samples into soil columns
and replace the sealing cap in the top of the column.
4. Incubate a soil sample mixed with the minimum volume of substrate for a predetermined
period of time. With your quick start guide and manual to help you, configure the EGA60
to record the CO2 analysis or C’an values (ppm) produced by each soil sample over a set
period of time.
5. Maximum initial respiration rate is determined by adding glucose to soil in a series of
increasing concentrations until respiration rate reaches a maximum. Active biomass (the
living soil component) is estimated from this maximum rate of respiration.1
6. Several re-runs of the experiment should be made with varying amounts of substrate to
determine the optimum concentration of substrate required to give the greatest increase
in respiration rate. At least five samples are recommended to test the glucose
concentration (one sample rack of 5 columns)1. Generally, soils with a low organic content
require a larger volume of substrate than those with a high organic content 8.
7. Repeat the experiment 2 or 3 times to confirm the maximum values of CO2 generated.
8. Once your experiment is complete, simply transfer the SD card to your computer/laptop
and save the comma delineated file as an Excel workbook.
9. Calculate; firstly, the maximum initial rate of respiration6 from the C’an data.
10. Secondly, the maximum initial rate of respiration must be converted into a rate of
respiration per hour.
11. The respiration rate per hour can now be used to estimate microbial carbon using
calculations such as that suggested by Anderson and Domsch 5:
Anderson and Domsch (1978) calculated the microbial biomass in soil incubated at 22oC using the
following formula5:

X = 40R + 0.37

This equation uses; the (pre-measured) concentration of soil microbial carbon, X, in mg/kg1, and
the rate of CO2 evolution, R, in ml/kg/hour1, measured with the EGA60.

The factor 40 is the correlation between respiration rate and soil microbial biomass as measured
by the fumigation and incubation method (see annex A of reference [5] in ISO 142409:1)1.
The SIR method5 provides a quick and relatively easy estimate of living biomass within the soil.
The substrate used is not limited to glucose, and substrate choice should be made with
consideration of the soil micro-organisms present as well as the type of soil being measured.
Anderson and Domsch, however, found that glucose produced the best results5.

Measurement of the CO2 evolution rate, immediately after addition of the substrate, provides an
accurate estimate of soil microbial carbon biomass6.

Substrate induced respiration has also been used in combination with selective microbial
inhibitors, such as cycloheximide or chlortetracycline, which can be used to subtract specific types
of micro-organisms from results observed7. This method can be conducted fairly quickly and
results can be gained in 1-3 hours. Results obtained by SIR correlate very well with other methods
of biomass estimation8.

Whilst many of the ADC225 systems are still in use worldwide today, the new EGA60, or multi-
sample soil respiration system comprises an IRGA and multiplexer in a smaller, equally versatile
instrument:

A 25 channel EGA60 system (showing 10 of the attached soil columns)

Following the same principal as the ADC2250, the EGA60 can be used to measure the evolution of
CO2 from several soil samples at regular time intervals (every hour for at least 6 hours, was the
method used to set ISO 14240)1 to calculate a rate of respiration.
From ADC2250 to EGA60

The EGA60 was designed to build on the functionality and versatility of the ADC2250 system,
which was used in pioneering studies such as Heinemeyer et al., (1989) and Subke et al., (2004) to
process a large amount of data from up to 24 samples, measured hourly. Both systems allow
constant purging of samples with ambient air. This prevents diffusion of CO2 from samples into a
traditional flask head-space, and also rapidly creates equilibrium between CO2 in the soil (solution)
and atmosphere. Therefore, any CO2 that is present in the system and not produced by soil
microbiota is greatly reduced (Heinemeyer et al., 1989).

The EGA60 has a dedicated ‘zero reference’ channel linked to a soda lime chemical column. This
provides automatic and regular calibration of the system with CO2 stripped air.

Using the EGA60 to obtain rate of formation of CO2:

The method of determining rate of CO2 formation within a soil sample using an InfraRed Gas
Analyser in a flow-through system, as found in ISO 16072:2002(E)3, uses the analyser (the ADC225
IRGA) to measure:

1. The difference between the CO2 concentration (in µL/L) of reference air entering soil
columns and that of air leaving the column.

2. The actual flow rate of air through each column, measured in ml /min, as converted from
the EGA60 flow data in µmol s-1.

3. Mass of each soil sample (moist) - measured before the soil sample is inserted into the
sample column.

The EGA measures the flow rate in µmol s-1. Should you wish to convert your flow data to ml min-1
the conversion factor will depend on the temperature of the soil sample and ambient air;

At 22oC and atmospheric pressure of 1027mb:

Multiply the µmol s-1 value by 1.46 (x 1.46) to give ml min-1.

References:
1. International Standard ISO 14240-1, 20-Jan-1998 BSDS, First Edition 1997-01-15, Reference
Number ISO 14240-1: 1997 (E). A copy of the International Standard can be purchased by
visiting the ISO website:
https://www.iso.org/obp/ui/#iso:std:iso:14240:-1:ed-1:v1:en:sec:foreword

2. Heinemeyer, O., Insam, H., Kaiser, E.A. and Walenzik, G. (1989) Soil microbial biomass and
respiration measurements: An automated technique based on infra-red gas analysis Plant
and Soil 116, 191-195.

3. International Standard ISO 16072:2002(E) Soil Quality – Laboratory methods for

determination of microbial soil respiration, First Edition 2002-12-15, Reference number ISO
16072:2002(E).

4. Knapp, E.B., Elliott, L.F., Campbell, G.S. (1983) Microbial respiration and growth during the
decomposition of wheat straw. Soil Biology and Biochemistry, 15, 5, 319-323.

5. Anderson, J.P.E and Domsch, K.H. (1978): A physiological method for the quantitative
measurement of microbial biomeasured in soils. Soil Biology and Biochemistry 10, 215-221

6. Sparling, G. P. (1995). The substrate induced respiration method, pp. 397–404. In K. Alef
and P. Nannipieri
Please(ed.), Methods
do contact in applied
us with soil microbiology
any queries, and biochemistry. Academic
feedback or comments:
Press, London, United Kingdom.

7. Michael H. Beare, Constance L. Neely, David C. Coleman, William L. Hargrove A substrate-

induced respiration (SIR) method for measurement of fungal and bacterial biomass on
plant residues. Soil Biol Biochem 22:585-594.

8. Horwath, W. R. and Paul, E. A. Microbial Biomass. pp. 760–763. In R. W. Weaver et al. (ed.),
Methods of soil analysis, Part 2. Microbiological and Biochemical Properties. SSSA Book
Series No. 5. SSSA, Madison, Wisconsin.

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