Measurement of microbial biomass C and N is indirect and thus is an approximation of actual total biomass. The biomass is based on the mineralization of a fixed proportion of the cellular C and N, following the death of the soil biota after incubation of a soil sample with chloroform (CHCl3). Biomass is then determined by the measurement of the products of cell lysis following death. Essentially, a fresh soil sample is subsampled for (1) immediate extraction in K2SO4 (‘unfumigated’), and (2) fumigation with chloroform and then extraction in K2SO4 (‘fumigated’)
50 ml centrifuge tubes for extractions
15-ml centrifuge or 20-ml scintillation tubes for filtrate storage
Scoop for ~5 g soil
0.5M K2SO4 (50 ml per sample, plus blanks)
Whatman #1 filter paper, 18.5 cm diameter (or to match funnels)
Chloroform (ethanol-free, either amylene-stabilized or distilled)
Aluminum weigh boats
Carboy
Balance (2-place)
Repipettor with 1L bottle
Funnels and funnel racks
Orbital shaker
Acid bath
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Acid wash funnels, carboy, storage tubes. Store funnels in acid washed bin covered with foil. Or for very-short term, store upside-down on clean paper towels and cover with paper towels.
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Make 0.5M K2SO4 (molarity is 174.2592 g/mol) in acid-washed carboy. Make enough for all samples, blanks, and standards in your entire experiment. For 1L, add 87.1 g of K2SO4 to 800 ml of distilled water, mix to dissolve, add distilled water to bring up to 1L, mix again.
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Label tubes for extraction and filtrate collection. Record tube+cap weights. Use repipettor (calibrated with graduated cylinder) to fill extraction cups with 25ml 0.5M K2SO4 and record tube+cap+ K2SO4 weights. Store tubes in refrigerator if not using immediately.
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Number and weigh aluminum weigh boats. Record weights.
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Label 15-20 ml tubes for filtrate storage.
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Collect soil (e.g., by coring) and place in plastic bag. Mix thoroughly (make sure the mixing time is the same for all samples).
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Scoop the following from each soil sample:
a. ~5 g of homogenized fresh soil into the pre-weighed aluminum weigh dish; record wet soil + tin weight; place in 105°C oven
b. ~5 g of homogenized fresh soil sample into 50-ml tube pre-filled with 25 ml 0.5M K2SO4; record soil + tube + cap + K2SO4 weight
c. ~5 g of homogenized fresh soil sample into 50 ml tube (for chloroform); record soil + tube + cap weight
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Place tubes with soil and K2SO4 in shaker racks and shake for one hour (record start and stop times). Make sure tubes are horizontal or deeply angled to get thorough mixing. Place funnels in funnel racks, add filter paper. After 1 hour, filter extract through the leached filter paper into acid-washed, pre-labeled storage tubes. Freeze immediately at -20C.
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While the unfumigated extracts are shaking, begin the fumigation. Place tubes with soil in hood and add 2-ml of chloroform using a designated pipettor with filter tip. Chloroform can be added on a cotton ball, or pipetted directly onto soil (if the latter, allow extra time for evaporation/venting in step 5). Cap tightly and cover cap with parafilm. Allow to sit for 24 hours.
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After 24 hours, remove caps and allow tubes to vent in hood for 30 min. Then add 25 ml 0.5M K2SO4; record soil + tube + cap + K2SO4 weight. Place on shaker for 1 hour, filter, and freeze as above.
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For soil in tins, dry in oven for 48 hrs at 105°C or until sample reaches constant weight. Remove from oven and place in desiccator for 20 min to cool. Record dry+tin weight. Calculate % soil moisture and gravimetric soil water content (see Gravimetric Soil Moisture Protocol).
Use the microplate protocols for quantifying C and N, or use the Apollo 9000 TOC/TN to measure the quantities of C and N in your samples. Alternatively, samples can be further digested for total nitrogen using the persulfate method.
Extractable microbial biomass C and N are reported as micrograms per gram of dry soil.
Correct microplate or TOC ug ml-1 for blanks (subtract). Then calculate
ug g-1 = (ug ml-1 *ml extractant) / g dry soil.
Dry soil is calculated as g dry soil = (g fresh wt soil – (g fresh wt soil * % soil moisture/100))
Subtract unfumigated from fumigated.
Convert the chloroform-labile pool to microbial biomass N by dividing by 0.54 and to microbial biomass C by dividing by 0.45 (soil-specific, but these are usually good estimates). Also see spreadsheet templates for these calculations.
K2SO4 extracts from the unfumigated portion can also be used to measure NH4+ and NO3-
Beck T, Joergensen RG, Kandeler E, Makeschin F, Nuss E, Oberholzer HR, Scheu S. 1997. An inter- laboratory comparison of ten different ways of measuring soil microbial biomass C. Soil Biology and
Biochemistry 29:1023-1032 Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: A rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology and Biochemistry 17:837-842
Joergensen RG. 1995. The fumigation extraction method. Pp 376-382 in (K Alef & P Nannipieri, Eds.). Methods in Applied Soil Microbiology and Biochemistry. Academic Press, London.
Joergensen RG. 1996. The fumigation-extraction method to estimate soil microbial biomass: calibration of the kEC factor. Soil Biology and Biochemistry 28: 25-31.
Scott-Denton LE, Rosenstiel TN, Monson RK (2006) Differential controls by climate and substrate over the heterotrophic and rhizospheric components of soil respiration. Global Change Biology 12:205-216
Sparling GP, West AW (1988) A direct extraction method to estimate soil microbial carbon: Calibration in situ using microbial respiration and carbon-14 labelled cells. Soil Biology and Biochemistry 20:337-344
Vance ED, Brookes PC, Jenkinson DS (1987) An Extraction Method for Measuring Soil Microbial Biomass Carbon. Soil Biology and Biochemistry 19:703-708
Wu J, Joergensen RG, Pommerening B, Chaussod R, Brookes PC. 1990. Measurement of soil microbial biomass C by fumigation-extraction – an automated procedure. Soil Biology and Biochemistry 22: 1167-1169.