Authors: Schmidt IK1, Reinsch S2, Christiansen CT3
Reviewers: Verbruggen E4, Berauer, B5
Measurement unit: mg C, N, or P g-1 dry soil or g-1 SOM; Measurement scale: plot; Equipment costs: none; Running costs: €; Installation effort: low; Maintenance effort: medium; Knowledge: medium; Measurement mode: manual
The soil microbial biomass constitutes 1–3% of the total soil organic carbon (C) content. Soil microbes also store a substantial amount of nutrients, nitrogen (N), and phosphorus (P) as the C:N and C:P ratios in microbes are relatively low compared to plant nutrient ratios (Schmidt et al., 2002; Zechmeister-Boltenstern et al., 2015). For example, microbial biomass P was about one-third of total soil P in an arctic heath (Jonasson et al., 1996, 1999), and on a global scale, estimates suggest that soil microbial N and P pools are equal to plant nutrient pools – despite plants storing far more C relative to microbes (Whitman et al., 1998; Bar-On et. al., 2018). Hence, even small fluctuations in microbial biomass may be crucial for mobilisation (release) and immobilisation (microbial uptake) rates of N and P in nutrient-deficient soils, i.e. where N and/or P limit plant growth (Smith & Paul, 1990; Schmidt et al., 2002). Consequently, microbial biomass pools are highly important components of an ecosystem’s carbon and nutrient storage potential and the pools are important to take into account when evaluating responses to experimental climate change or other drivers of global change.
2.2.1.1 What and how to measure?
Gold standard
Microbial biomass in soil can be quantified by the chloroform-fumigation extraction (CFE; Brookes et al., 1985b, also see Table 2.2.1.1). First, roots and stones (> 2 mm diameter) are removed from the fresh, homogenised soil samples. Then, one set of soil samples (minimum 10 g) is extracted for soluble C (dissolved organic carbon; DOC), N (total dissolved nitrogen; TDN), and P (PO4). Sorting out roots by hand is preferred as sieving may release substantial amounts of nutrients and if the sorted soil is stored, the microbes may utilise the released C and nutrients.
Simultaneously, another set of soil samples (minimum 10 g) are vacuum-incubated and treated with ethanol-free chloroform fumes for 24 h at room temperature (~ 20 ᵒC) in the dark (Jenkinson & Powlson, 1976; Tate et al., 1988). This fumigation step is performed using a desiccator and a pump placed in a well-aerated fume-hood. The chloroform treatment kills and lyses the living soil microorganisms (Brookes et al., 1985a, 1985b), releasing microbial C, N, and P contents into the soil solution (Vance et al., 1987). As chloroform is not only toxic for microbes, special care has to be taken when handling the chloroform as well as chloroform-treated soils. After 24 hours, the fumigated soil samples are extracted similarly to the non-fumigated samples. Microbial C, N, and P are commonly reported in mg C, N, and P per grams of dry soil or per grams of soil organic matter (SOM) content. Thus, a third soil subsample is needed for fresh to dry weight conversion – followed by loss on ignition combustion if SOM content is desired.
Microbial biomass C, N, and P is calculated as the difference in C, N, or P contents in pair-wise extracts of unfumigated and fumigated soil samples, for example:
Microbial biomass C (mg C per g dry soil) = Cfumigated soil – Cunfumigated soil
Table 2.2.1.1 Stepwise guide to microbial biomass measurements. The first row describes how soil samples are processed in the laboratory prior to fumigation. The second row is a guide to chloroform fumigation of soil samples. Another set of soil samples are extracted for initial content of inorganic and dissolved organic nutrients (third row), followed by a procedure to stop fumigation (fourth row). Rows five and six describe subsequent analytical analyses of the fumigated and unfumigated extractants, and the necessary calculations, respectively.
| Step 1 | Step 2 | Step 3 | Step 4 | |
| Soil samples | Sort out roots by hand | Weigh three sets of samples for an un-fumigated sample, a fumigated sample and for fresh:dry weight conversion | Place the fresh soil in an oven and dry at e.g. 55 °C until constant weight | Weigh the dry soil and calculate water content so all measures can be expressed on a g dry weight soil basis |
| Start fumigation | Use aluminium containers (alu-candle holder) or crucibles as chloroform will dissolve plastic | Place soil samples in dessicator – line bottom of desiccator with wet napkins to avoid soil drying | Clean chloroform for stabilising ethanol. Pass 10–30 ml through Al2O3 into a beaker.
USE FUME HOOD! |
Place ChCl3 beaker in dessicator and close the lid. Start the vacuum pump and continue until chloroform boils. Close the valve AND then turn off the pump. |
| Unfumigated sample | Extract soil sample in appropriate extractant (see details below) when the fumigated soil samples are in the dessicator | Filter and store cold. Samples can be stored in the freezer if more than a few days will pass before analysis, which should be at the same time as the batch of fumigated samples | ||
| Stop fumigation | Open the valve of the desiccator after 24 h and let air enter very carefully | Open the desiccator and remove the wet napkins and the ChCl3 beaker with the remaining chloroform | Close the lid on the desiccator and flush repeatedly 3–4 times, using the pump to remove remaining chloroform fumes | Remove the dessicator lid once more and take out the fumigated soil samples. These are now ready for extraction – same procedure for the unfumigated samples |
| Laboratory analyses | Measurement of DOC in extracts of fumigated and unfumigated samples for microbial C assessment | Measurement of TDN in extracts of fumigated and unfumigated samples for assessment of microbial N | Measurement of PO4-P in extracts of fumigated and unfumigated samples for assessment of microbial P | |
| Calculation | Microbial biomass C, N, and P are calculated as the difference in C, N, or P contents in pair-wise extracts of unfumigated and fumigated soil samples | Note that all extractions must be corrected for the soil sample specific dilution associated with its gravimetric soil moisture content. |
According to the literature, fumigated and unfumigated soil samples have most often been extracted with 0.5 M K2SO4, which is suitable for obtaining C and N. However, using a lower concentration of K2SO4 (0.05 M) or even extraction in pure water are highly recommended alternatives (see Special cases for details; Nordin et al., 2004). The extraction is followed by measurements of DOC, inorganic N, TDN, and PO4 on extracts of both fumigated and non-fumigated samples.
In dry soils (with a water content below 30% of field-capacity), the water content can be adjusted by adding a known amount of pure water to the samples right before the on-set of fumigation. Alternatively, when dealing with waterlogged soils, a modified chloroform-addition method has been developed (Witt et al., 2000). Here, chloroform is added directly into each inundated soil sample, and then samples are incubated for 24 h and extracted as described above.
Handling of soil samples for microbial C, N and P content
Microbial biomass C, N and P are usually measured in the top 10 or 30 cm of the soil where the microbial biomass and activity are highest. In the field, soil samples are placed in a cooling box at ca. 4 °C in a cooling room back in the laboratory. The samples should NOT be frozen or dried. The samples are stored as intact soil cores to minimize disturbance and microbial activity. The microorganisms are active also at low temperatures, so the samples should be processed within few days after sampling in the field following the steps in Table 2.2.1.1.
Where to start
THE CFE method has generally not changed much over the past 30 years, and details on the procedure are found in the classic method papers by Brookes et al. (1985b), Vance et al. (1987), and Tate et al. (1988). For current studies using the CFE method with slight modifications, see, for example, Christiansen et al. (2018).
2.2.1.2 Special cases, emerging issues, and challenges
Use of extractants
Historically, K2SO4 has been the preferred extractant used for microbial C and N extractions. However, 0.5 M K2SO4 is difficult to work with because i) the solution concentration is close to saturation, ii) possible salt precipitation occurs within sample vials when cooled or frozen, and iii) samples often leave salt deposits inside the instruments during analysis. Similar biomass pool sizes can be obtained when using low concentration 0.05 M K2SO4 or simple H2O extraction (Nordin et al., 2004). A test of extractability with 0.05 M K2SO4 or H2O shows no effect on microbial C, but the extractability of inorganic and dissolved organic N is approximately 20% lower with 0.05 M K2SO4 and 30% lower with water relative to 0.5 M K2SO4 (pers. obs.). If desired, this extractability difference can be adjusted for by using a lower KEN factor of 0.45 compared to 0.54 used by Brookes et al. (1985b), but see recommendations on extraction factors below.
NaHCO3 is generally used to extract PO4-P and microbial P in neutral to alkaline soils (Schmidt et al., 1999), while Bray-1 solution is used for PO4-P and microbial P in acidic soils (Bray & Kurtz, 1945; Wu et al., 2000); see Zederer et al. (2017) for details on microbial P determination.
Using extraction correction factors
Extractability of microbial C, N, and P varies across different soils (Joergensen, 1996; Joergensen & Mueller, 1996), microbial communities (Anderson & Domsch, 1978), and seasons (Schadt et al., 2003). CFE extraction efficiency is generally 40–60% of total microbial biomass, and extraction correction factors KEC, KEN, and KEP are often applied to adjust for the incomplete release of C, N, or P, respectively (Jonasson et al., 1996), for example, when absolute pool sizes are desired, such as when calculating total ecosystem pools or budgets. However, great care should be taken to apply a suitable correction factor – and it is relatively common not to use any correction factor at all. Based on the literature, general correction factors for microbial biomass C, N, and P are KEC = 0.45 (Wu et al., 1990; Joergensen, 1996), KEN = 0.54 (Brookes et al., 1985a), and KEP = 0.40 (Jonasson et al., 1996), respectively. Always specify whether correction factors were used in the calculations and what values were applied as correction.
Fatty acid analyses
The CFE method measures the total microbial biomass, i.e. combined bacterial and fungal C, N, and P. An ergosterol assay has been developed to quantify the fungal abundance in soil (Salmanowicz & Nylund, 1988; Nylund & Wallander, 1992). In contrast, fatty acid methyl esters (FAMEs) extracted from microbial cell walls are used to quantify the relative bacterial (PLFAs – phospholipid fatty acids) and fungal (mainly NLFAs – neutral-lipid fatty acids) abundance (Bligh & Dyer, 1959; Frostegård & Bååth, 1996). FAMEs are also used to broadly describe the microbial community composition. More advanced methods use sequences of genetic material (e.g. 16S and ITS regions of rRNA or DNA) of bacteria or fungi, respectively, to identify microbial community composition at a finer scale (Marsh, 1999), and qPCR can be used to quantify cell numbers which can then be used for biomass estimations (see 4.9 Soil microbial community composition). A comprehensive review of methods studying soil microbial diversity is published by Kirk et al. (2004). Note that there is generally a good agreement between biomass estimations based on CFE and PLFA methods (Schmidt et al., 2000).
CFE modifications relating to soil type
In dry soils with a water content below 30% of field capacity, the water content can be adjusted by adding a known amount of pure water to the samples immediately before the on-set of fumigation. Alternatively, when dealing with waterlogged soils, a modified chloroform-addition method has been developed (Witt et al., 2000). Here, liquid chloroform is added directly into each inundated soil sample, and then samples are incubated for 24 hours before extraction. For soils that are high in clay content, note that chloroform fumes can adsorb to clay minerals and potentially bias the microbial biomass C measurement – see Alessi et al. (2011) for uncertainties in biomass C estimates with the CFE.
Isotopic signatures of microbial C and N
Microbial C and N can be quantified using stable C and N isotopes. Stable isotope analysis allows for the quantification of C and N use by microbes under different climatic conditions in stable isotope labelling experiments (Nordin et al., 2004; Andresen et al., 2009, 2018; Reinsch et al., 2014) or continuous labelling experiments e.g. Free-Air CO2 Enrichment (FACE). Stable isotope analysis of soil extracts can also be used when C and N stabilisation in the microbial biomass is of interest (Thaysen et al., 2017). Isotopic values of soil microbial C and N can be determined after the CFE method. Unfumigated and fumigated soil samples are extracted in water (for later analyses of DOC and TDN) and then freeze-dried (Lipson & Monson, 1998). Extraction of soils with K2SO4 will leave salt deposits after freeze-drying and make packaging for stable isotope analysis difficult; K2SO4 deposits further ruin the separation columns used in isotope ratio mass spectrometry even at low salt concentrations. Thus, we recommend the use of water extraction so that solvent from the samples can be evaporated during freeze-drying without leaving a salty residue during stable isotope analysis of δ13C and δ15N (Nordin et al., 2004).
Prior to freeze-drying, pH in the extracts should be adjusted in extracts from non-acidic sites to 4.5 with HCl. The soil extracts are sublimated to dryness in a freeze-dryer. δ13C and 15N concentrations (in atom % compared to isotopic ratios of 13C/12C and 15N/14N) of soil microbes are calculated as the difference of 15N and 13C fumigated and unfumigated samples. However, it is difficult to collect all N and P after freeze drying so the isotopic analysis provides a C:N ratio of the microbial biomass and the excess 15N and 13C. To calculate the pool sizes, the unfumigated and fumigated extracts from the CFE analysis should be analysed for total organic C and TDN to provide the pool sizes.
2.2.1.3 References
Theory, significance, and large datasets and more on methods and existing protocols
The fumigation-extraction method has not changed significantly over the last decades, and details on the procedure are found in the classic method papers by Brookes et al. (1985b), Vance et al. (1987), and Tate et al. (1988). Further, key references will depend on the actual study in question. A key reference for PLFA in microbes is Frostegård & Bååth (1996).
All references
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Anderson, J. P. E., & Domsch, K. H. (1978). Mineralization of bacteria and fungi in chloroform-fumigated soils. Soil Biology and Biochemistry, 10, 207-213.
Andresen, L. C., Michelsen, A., Jonasson, S., Beier, C., & Ambus, P. (2009). Glycine uptake in heath plants and soil microbes responds to elevated temperature, CO2 and drought. Acta Oecologica, 35(6), 786-796.
Andresen, L. C., Domínguez, M. T., Reinsch, S., Smith, A., Schmidt, I. K., Ambus, P., … Tietema, A. (2018). Isotopic methods for non-destructive assessment of carbon dynamics in shrublands under long-term climate change manipulation. Methods in Ecology and Evolution, 9(4), 866-880.
Bar-On, Y. M., Phillips, R. & Milo, R. (2018). The biomass distribution on Earth. Proceedings of the National Academy of Sciences USA, 115(25) 6506-6511.
Bligh, E. G., & Dyer, W. J., (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917.
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Authors: Schmidt IK1, Reinsch S2, Christiansen CT3
Reviewers: Verbruggen E4, Berauer, B5
Affiliations
1 Department of Geosciences and Natural Resource Management, University of Copenhagen, Frederiksberg, Denmark
2 Centre for Ecology and Hydrology, Environment Centre Wales, Bangor, UK
3 NORCE Norwegian Research Centre and Bjerknes Centre for Climate Research, Bergen, Norway
4 Centre of Excellence PLECO (Plants and Ecosystems), Biology Department, University of Antwerp, Wilrijk, Belgium
5 Department of Disturbance Ecology, University of Bayreuth, Bayreuth, Germany