Substrate-induced Respiration (SIR) Substrate-induced respiration (SIR) is the measurement of soil respiration in the presence of an added substrate such as glucose, glutamic acid, mannitol and amino acids. The SIR method uses the soil organism’s physiological respiration reactions to substrate addition, such as CO2 production or O2 consumption, as a means of quantifying microbial activities in soils. This method was developed by Anderson and Domsch in 1978 to provide a quick estimate of living microorganism carbon biomass in soils. The substrates that can be utilized are not limited to the aforementioned substrates; substrate choice should reflect the target organisms and type of soil being tested. Though other substrates can be used to produce measurable respiration Anderson and Domsch found that glucose produced the greatest results. Measurement of the levels of production and consumption can be measured immediately following the addition of the substrate to provide an estimate of soil microbial carbon biomass (Sparling). Substrate-induced respiration has also been used in conjunction with solutions designed to act as selective inhibitors, such as cycloheximide or chlortetracycline, to limit the microbial communities being observed (Williams and Rice). This method can be conducted fairly quickly and has an analysis time of approximately 1-3 hours, depending on the apparatus used to measure respiration. The results obtained by the SIR method also seem to correlate well with other established biomass estimation procedures (Horwath and Paul).
Steps to conducting an SIR analysis
1)The soils to be tested using the SIR analysis are generally prepared by sieving to remove living plants and other unwanted organic materials. These materials must be removed in order to prevent the input and output of photosynthesis, like CO2 absorption and O2 production, from affecting accurate measurement of microbial respiration. The sieving of the soils to be tested also allows for an even distribution of added substrate which in turn produces a more dispersed release of the produced CO2 (Sparling).
2)The chosen substrate needs to be added to the soil sample. This can be done in a liquid form in which the substrate is diluted in water or dry form in which case it is suggested that the substrate be mixed with talc to aid in achieving an even integration with the soil sample (Sparling).
3)Place the soil sample in a gas-tight container. The sample size is dependent on the soil sample being tested, soils low in organic matter require larger sample sizes and soils that are high in organic matter can be done with smaller samples. Several trials should be run using different substrate additions as to determine the minimum amount of substrate needed to produce the greatest rate of respiration (Horwath and Paul).
4)Incubate the soil sample (mixed with the minimal substrate level) and measure the rate of respiration over a predetermined time. There are many ways in which soil microbial respiration can be measured and the rate should be measured at its greatest initial respiration level (Sparling).
5)The measured greatest initial rate of respiration must now be used to calculate the rate of respiration per hour. The rate per hour can be used to determine an estimate of microbial carbon. This can be done by either using a previously published factor or by using a calibration curve between SIR response and microbial carbon that you produced prior to conducting the experiment (Sparling).
Gas Measurement Devices
Soil microbial analysis via SIR is not restricted to the utilization of a single apparatus or method, rather there are several in which one can use. Examples of apparatuses and methods used in this endeavor include, but are not limited to; Gas chromatography, the Warburg device, infrared gas analyzers, and the Wosthoff CO2 analyzer (Sparling).
Gas Chromatography: Gas chromatography is a quick and very sensitive technique that can be used in the separation and measurement of many gases. This method utilizes a carrier gas (usually helium) and temperature to control gas separation producing peaks that can be measured to quantitatively determine productivity (Pavia). Due to the speed at which the gas chromatographer separates out the gases it is limited to being used with relatively minute soil samples (Sparling). Click here to learn more about Gas Chromatography
Warburg Device: The Warburg device is essentially a differential respirometer that can be used to measure CO2 output. This apparatus in its simplest form consists of a specialized flask containing some form of alkali, usually KOH, and a sample compartment for the soil. The CO2 produced through microbial respiration is absorbed and trapped in the alkali solution and can be quantitated via titration (Atlas and Bartha). Click here to learn more about the Warburg Apparatus
Infrared Gas Analysis: The infrared gas analysis is a relatively complex computer controlled system. The device consists of several independent sample lines allowing for analysis of different flow rates. Each sample line contains a gas pump, humidifier and flow regulation valves that direct the air flow past an infrared gas analyzer which can measure CO2 concentrations (Alef). Click here to learn more about Infrared Gas Analysis
Wosthoff CO2 analyzer: The Wosthoff CO2 analyzer uses a NaOH solution that reacts with CO2 resulting in a measurable changes in electrolytic conductivity. These conductivity changes are directly proportional to the concentration of CO2 being produced from the sample, therefore quantitative analysis is obtained quickly. This device is also capable of measuring CO2 production from four different samples at the same time (Sparling).
Calculation of Biomass Carbon
Anderson and Domsch calculated the SIR determined microbial carbon biomass of samples incubated at 22°C by using the following equation (Horwath and Paul).
x = 40.04y + 0.37 where x = total microbial carbon biomass and y = greatest initial rate of CO2 respiration
(mL of CO2 per g-1 dry weight soil)
References
Sparling, G. P. (1995). The substrate induced respiration method, pp. 397–404.
In K. Alef and P. Nannipieri (ed.), Methods in applied soil microbiology and
biochemistry. Academic Press, London, United Kingdom
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
Pavia, Donald L. et al (2007). Introduction to Organic Laboratory Techniques: A Microscale Approach (4th Ed.). Thomson Brooks/Cole, Independence, Kentucky. pp. 797-817
Alef, K. (1995). The infrared gas analysis, p. 218–219
In K. Alef and P. Nannipieri (ed.), Methods in applied soil microbiology and
biochemistry. Academic Press, London, United Kingdom
Atlas, R. and Bartha, R. (1998). Microbial Ecology: Fundamentals and Applications (4th Ed.). Benjamin/Cummings Science Publishing, Menlo Park, California. pp. 258-260
Williams, M. A. and Rice, C. W. (2007). Seven years of enhanced water availability influences the physiological, structural, and functional attributes of a soil microbial community. Appl. Soil Ecol. 35, 535-545
Substrate-induced respiration (SIR) is the measurement of soil respiration in the presence of an added substrate such as glucose, glutamic acid, mannitol and amino acids. The SIR method uses the soil organism’s physiological respiration reactions to substrate addition, such as CO2 production or O2 consumption, as a means of quantifying microbial activities in soils. This method was developed by Anderson and Domsch in 1978 to provide a quick estimate of living microorganism carbon biomass in soils. The substrates that can be utilized are not limited to the aforementioned substrates; substrate choice should reflect the target organisms and type of soil being tested. Though other substrates can be used to produce measurable respiration Anderson and Domsch found that glucose produced the greatest results. Measurement of the levels of production and consumption can be measured immediately following the addition of the substrate to provide an estimate of soil microbial carbon biomass (Sparling). Substrate-induced respiration has also been used in conjunction with solutions designed to act as selective inhibitors, such as cycloheximide or chlortetracycline, to limit the microbial communities being observed (Williams and Rice). This method can be conducted fairly quickly and has an analysis time of approximately 1-3 hours, depending on the apparatus used to measure respiration. The results obtained by the SIR method also seem to correlate well with other established biomass estimation procedures (Horwath and Paul).
Steps to conducting an SIR analysis
1) The soils to be tested using the SIR analysis are generally prepared by sieving to remove living plants and other unwanted organic materials. These materials must be removed in order to prevent the input and output of photosynthesis, like CO2 absorption and O2 production, from affecting accurate measurement of microbial respiration. The sieving of the soils to be tested also allows for an even distribution of added substrate which in turn produces a more dispersed release of the produced CO2 (Sparling).
2) The chosen substrate needs to be added to the soil sample. This can be done in a liquid form in which the substrate is diluted in water or dry form in which case it is suggested that the substrate be mixed with talc to aid in achieving an even integration with the soil sample (Sparling).
3) Place the soil sample in a gas-tight container. The sample size is dependent on the soil sample being tested, soils low in organic matter require larger sample sizes and soils that are high in organic matter can be done with smaller samples. Several trials should be run using different substrate additions as to determine the minimum amount of substrate needed to produce the greatest rate of respiration (Horwath and Paul).
4) Incubate the soil sample (mixed with the minimal substrate level) and measure the rate of respiration over a predetermined time. There are many ways in which soil microbial respiration can be measured and the rate should be measured at its greatest initial respiration level (Sparling).
5) The measured greatest initial rate of respiration must now be used to calculate the rate of respiration per hour. The rate per hour can be used to determine an estimate of microbial carbon. This can be done by either using a previously published factor or by using a calibration curve between SIR response and microbial carbon that you produced prior to conducting the experiment (Sparling).
Gas Measurement Devices
Soil microbial analysis via SIR is not restricted to the utilization of a single apparatus or method, rather there are several in which one can use. Examples of apparatuses and methods used in this endeavor include, but are not limited to; Gas chromatography, the Warburg device, infrared gas analyzers, and the Wosthoff CO2 analyzer (Sparling).
Gas Chromatography:
Gas chromatography is a quick and very sensitive technique that can be used in the separation and measurement of many gases. This method utilizes a carrier gas (usually helium) and temperature to control gas separation producing peaks that can be measured to quantitatively determine productivity (Pavia). Due to the speed at which the gas chromatographer separates out the gases it is limited to being used with relatively minute soil samples (Sparling). Click here to learn more about Gas Chromatography
Warburg Device:
The Warburg device is essentially a differential respirometer that can be used to measure CO2 output. This apparatus in its simplest form consists of a specialized flask containing some form of alkali, usually KOH, and a sample compartment for the soil. The CO2 produced through microbial respiration is absorbed and trapped in the alkali solution and can be quantitated via titration (Atlas and Bartha). Click here to learn more about the Warburg Apparatus
Infrared Gas Analysis:
The infrared gas analysis is a relatively complex computer controlled system. The device consists of several independent sample lines allowing for analysis of different flow rates. Each sample line contains a gas pump, humidifier and flow regulation valves that direct the air flow past an infrared gas analyzer which can measure CO2 concentrations (Alef). Click here to learn more about Infrared Gas Analysis
Wosthoff CO2 analyzer:
The Wosthoff CO2 analyzer uses a NaOH solution that reacts with CO2 resulting in a measurable changes in electrolytic conductivity. These conductivity changes are directly proportional to the concentration of CO2 being produced from the sample, therefore quantitative analysis is obtained quickly. This device is also capable of measuring CO2 production from four different samples at the same time (Sparling).
Calculation of Biomass Carbon
Anderson and Domsch calculated the SIR determined microbial carbon biomass of samples incubated at 22°C by using the following equation (Horwath and Paul).
x = 40.04y + 0.37
where
x = total microbial carbon biomass
and
y = greatest initial rate of CO2 respiration
(mL of CO2 per g-1 dry weight soil)
References
Sparling, G. P. (1995). The substrate induced respiration method, pp. 397–404.
In K. Alef and P. Nannipieri (ed.), Methods in applied soil microbiology and
biochemistry. Academic Press, London, United Kingdom
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
Pavia, Donald L. et al (2007). Introduction to Organic Laboratory Techniques: A Microscale Approach (4th Ed.). Thomson Brooks/Cole, Independence, Kentucky. pp. 797-817
Alef, K. (1995). The infrared gas analysis, p. 218–219
In K. Alef and P. Nannipieri (ed.), Methods in applied soil microbiology and
biochemistry. Academic Press, London, United Kingdom
Atlas, R. and Bartha, R. (1998). Microbial Ecology: Fundamentals and Applications (4th Ed.). Benjamin/Cummings Science Publishing, Menlo Park, California. pp. 258-260
Williams, M. A. and Rice, C. W. (2007). Seven years of enhanced water availability influences the physiological, structural, and functional attributes of a soil microbial community. Appl. Soil Ecol. 35, 535-545