Measuring soil microbial biomass using an automated ...

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Soil Biology & Biochemistry xxx (2010) 1e4

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Measuring soil microbial biomass using an automated procedure Rainer Georg Joergensen a, *, Jinshui Wu b, Philip C. Brookes c a

Department of Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany Institute of Sub-Tropical Agriculture, Changsha, PR China c Sustainable Soils and Grassland Systems, Rothamsted Research, Harpenden, Herts., AL5 2JQ, UK b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 September 2010 Accepted 20 September 2010 Available online xxx

Here we outline the development of the first automated procedure for measuring soil microbial biomass carbon (biomass C) by FumigationeExtraction (FE) and on which this Citation Classic is based. The method (and its later variations) has been used widely since it was published. It gives essentially the same results as the FumigationeIncubation (FI) method on which it was based and which it has now largely replaced. Analysis of the then current data clearly showed that the calibration value to convert extracted organic C to biomass C (kEC ¼ 0.45) using FE is still valid and that there was no need for a change. We also review some of the previous discussions about the method and outline future prospects for microbial biomass measurements in soil microbial ecology. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Biomass carbon measurements FumigationeExtraction FumigationeIncubation

1. Introduction The paper by Wu et al. (1990) is actually the fourth of a set of linked papers identified as Citation Classics in Soil Biology & Biochemistry (the first three being Vance et al., 1987; Brookes et al., 1982, 1985 e see Jenkinson et al., 2004). They are all based upon measuring the soil microbial biomass by, what was then, a comparatively new technique, termed FumigationeExtraction (FE). This rapidly replaced the older Fumigation-Incubation (FI) method introduced by Jenkinson (1966) and later developed by Jenkinson and Powlson (1976). Both methods share a common treatment, viz. fumigation of moist soil with chloroform, in order to lyse the microbial cells, followed by fumigant removal. At this stage the methods diverge. The FI method uses a 10-day incubation period (usually at 40e50% water holding capacity and 25 C) during which the killed cells are mineralised by surviving or inoculated microorganisms to CO2, which is trapped and measured. Appropriate calibration involves the subtraction of CO2 evolved from a ‘control’ non-fumigated soil to account for mineralization of non-biomass soil organic matter. There is a further correction, usually termed kC, based on the fact that about 45% of the killed biomass C is evolved as CO2eC (i.e. kC ¼ 0.45). Finally, an estimate of the initial soil microbial biomass C (Bc) is calculated from: Bc ¼ [(CO2eC evolved from CHCl3 fumigated soil in 10 d) (CO2eC evolved from non-fumigated soil in 10 d)]/kC.

* Corresponding author. Tel.: þ49 5542 98 1591; fax: þ49 5542981596. E-mail address: [email protected] (R.G. Joergensen).

In contrast, the FE method demands the immediate extraction of cell components after fumigation using simple salt solutions (e.g. 0.5 M K2SO4 for biomass C). A control value is also assessed, analogous to that of FI and based on correcting for the non-biomass soil organic C extracted. A final correction, based on the incomplete extraction of the killed biomass by the fumigant, is then made, using an analogous conversion value to that in FI and termed kEC; this is also 0.45 as proposed by Wu et al. (1990). Jenkinson et al. (2004) pointed out that the development of FE made the use of FI obsolete. The FE method certainly has several advantages over FI. It can be used, not only in virtually all soils but also for river sediments (Klaus et al., 1998), litter layers (Scholle et al., 1992), peat (Brake et al., 1999), and compost samples (Gattinger et al., 2004). Furthermore, a huge range of sample sizes can be analysed, depending on the homogeneity of the soil. Virtually all elements contained in the microbial cell, including C (Vance et al., 1987), N (Brookes et al., 1985), P (Brookes et al., 1982), S (Saggar et al., 1981; Wu et al., 1994), and metals, such as K, Na, Mg, Mn, Zn, Cu, and Ni (Khan et al., 2009) can be measured by using different extractants. The actual flux of elements through the microbial biomass can be determined by using radioactive isotopes such as 14C (Wu et al., 1993), 32P (Chen and He, 2004), 33P (Kouno et al., 2002; Achat et al., 2010) and 35S (Wu et al., 1994) and Q3 increasingly stable isotopes such as 13C (Ryan and Aravena, 1994) and 15N (Ocio et al., 1991) or 13C and 15N (Dijkstra et al., 2006; Schenck zu Schweinsberg-Mickan et al., 2010; Zareitalabad et al., 2010). The colorimetric measurement of ninhydrin-reactive N enables the estimation of microbial biomass without combustion or the need for expensive equipment (Amato and Ladd, 1988; Jorgensen and Brookes, 1990).

0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.09.024

Please cite this article in press as: Joergensen, R.G., et al., Measuring soil microbial biomass using an automated procedure, Soil Biology & Biochemistry (2010), doi:10.1016/j.soilbio.2010.09.024

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R.G. Joergensen et al. / Soil Biology & Biochemistry xxx (2010) 1e4

Several drawbacks of the FI method, such as the lengthy incubation period, the restriction to near-neutral soils, the lack of validity of the measurements in the presence of fresh decomposing organic matter (e.g. Martens, 1995), as well as the increasing demand for reliable microbial biomass estimates in a wide range of soils, all resulted in the need for some methodological improvement. This was resolved by a very able young American PhD student, Eric Vance, who worked for 2 years as a Fulbright Scholar at Rothamsted with David Jenkinson and Phil Brookes. Eric quickly saw the potential for an extraction-based method for biomass C, and, seemingly in no time, had produced some very convincing data. The paper by Vance et al. (1987) describing the new FE method, was published soon after. This has the honour of being the most highly cited paper in Soil Biology & Biochemistry to date. The success of the paper of Wu et al. (1990) built on that of Vance et al. (1987) and added some important methodological improvements, focussing on two main aspects: (1) automated measurement of C in K2SO4 soil extracts; and (2) the establishment of a conversion value from CHCl3 labile C to biomass C. There is one major methodological problem associated with measuring biomass C by the original FE method. This is that the organic C in the soil extracts was determined originally by wet digestion with K2Cr2O7 followed by back-titration with ferrous ammonium sulphate. The problem, common to all back-titration methods, is that the C is determined from the small difference between the volume of the original K2Cr2O7 solution and the often relatively large amount of K2Cr2O7, remaining unoxidised after digestion. In soils with reasonably large biomass C contents this was no problem but in low biomass soils the difference can be within the experimental error. A great deal of care and technical skill was needed to accurately measure biomass C in such soils. Here the problem rested until two enthusiastic young scientists, Jinshui Wu, a PhD student from China and Rainer Georg Joergensen, a Post-Doctoral scientist from Germany, joined our group. Rainer was aware of methods for measuring C in diluted solutions by automated analysis and we soon found local companies who were anxious to sell us their instruments. We finally settled on a Dohrmann DC-80 (Rosemount-Dohrmann, Santa Clara, USA), which measured CO2 by infra-red detection after a wet UV-persulphate oxidation procedure, and that machine gave us many years of service. Between it and our researchers, aided by an undergraduate student from Germany, Birgit Pommerening and advice from Rémi Chaussod from INRA Dijon, France, we soon arrived at a reliable method for measuring biomass C by automated liquid digestion (Wu et al., 1990). Later developments included the use of furnacetype auto analysers (e.g. Dimatoc 100, Dimatec, Essen) which give essentially the same results. The Wu et al. (1990) paper revealed two very useful results. Firstly, there was a highly significant linear correlation (r ¼ 0.99) between K2SO4-extractable C measured by dichromate digestion and that recorded by automated UV-persulphate oxidation; this correlation held for both fumigated and non-fumigated soil extracts. Overall, there was 19.44% more organic C measured by UV-persulphate than by dichromate oxidation. Secondly, there was a highly significant 1:1 linear correlation (r ¼ 0.99) between biomass C measured by both methods. Thus, it is easy to convert data from one method to another. In particular, it means that both methods concurred in that 45% of biomass C was either mineralised to CO2eC in 10 d with FI or extracted with FE after 24 h fumigation. This is considered in more detail below. 2. Calibration The conversion of measured data into microbial biomass values is an important step for assessing the flux of energy and elements

Table 1 Mean kEC values obtained by FumigationeIncubation (FI) and other methods for converting C released by CHCl3 fumigation and extraction to microbial biomass C. Method

kEC

Range

Number of soils

Indirect by FI (Wu et al., 1990) Indirect by FI, other authors (Joergensen, 1996) Indirect by FI (Beck et al., 1997) Indirect by other methodsb (Joergensen, 1996) Direct (Joergensen, 1996) Direct (Chen and He, 2002)

0.45 0.47a

0.39e0.67 0.23e0.98

20 176

0.42 0.48a

0.16e0.66

20 90

0.34a 0.31

0.15e0.40 0.27e0.35

21 8

All data

0.46a

0.15e0.98

335

a b

Weighted mean. Substrate-induced respiration, ATP, direct microscopy.

(Jenkinson and Ladd, 1981). Biomass values serve as an internal control by relating microbial growth and CO2 evolution data, and they help to prove the soundness of the values in comparison with other data. Microbial biomass data are also useful because most models, for the turnover of organic matter in soil, e.g. RothC (Coleman and Jenkinson, 1996; Cerri et al., 2007) and DAISY (Müller et al., 2006), contain at least one biomass compartment. Agreement between the amount of biomass predicted by a particular model and that actually measured provides a useful test of the adequacy of the model. In addition, the comparison of treatments is not affected by the conversion values. Similarly, in a literature review on the microbial biomass in tropical soils from 26 countries (Joergensen, 2010), 75.3% of the data were obtained by FE (Vance et al., 1987), 18.0% by FI (Jenkinson and Powlson, 1976), 4.9% by substrate-induced respiration (Anderson and Domsch, 1978), 1.3% by ATP (Jenkinson and Oades, 1979) and 0.5% by direct microscopy (Babiuk and Paul, 1970). It was possible to pool all these data due to the existence of appropriate values for converting the measured values into contents of microbial biomass C. Apart from the above mentioned references, several studies were carried out to calibrate the kEC value directly by addition of unlabelled and labelled microorganisms and by in situ or other methods. 2.1. Direct calibration of the conversion values Only one experiment has been published where the calibration of the kEC value was done by the addition of non-labelled, cultivated organisms (Tate et al., 1988). However, these cultivated organisms contain an unknown fraction of dead microbial material or spores (which are not attacked by CHCl3 fumigation) and would lower the kEC value (Tate et al., 1988). The calibration of the kEC value with in situ labelling of the soil microbial biomass has been achieved by the addition of 14C-labelled glucose (e.g. Voroney and Paul,1984; Sparling and West, 1988; Sparling et al., 1990; Bremer and van Kessel, 1990), followed by the FE method. This procedure requires that: (1) part of the non-mineralised and non-extractable 14C glucose will, after a certain time, be completely incorporated into the soil microbial biomass; (2) only a negligible part will have been transformed into non-biomass microbial residues; and (3) the extractability of the young 14C-labelled biomass is representative of the total microbial biomass. All assumptions may be wrong! The incorporation of glucose into the cytoplasm is not necessarily immediately followed by its metabolism, especially in a situation of N-deficiency (Bremer and van Kessel, 1990; Joergensen and Raubuch, 2002). Young cells contain more easily soluble components in the cytoplasm than older cells (Bremer and van Kessel, 1990). During the rapid glucoseinduced growth, a significant percentage of the microbial biomass is


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transferred immediately to non-biomass microbial residues (Chander and Joergensen, 2001; Engelking et al., 2007). Consequently, all direct calibration approaches lead to markedly lower conversion values than the indirect approaches (Table 1) and, consequently, are not recommended. 2.2. Indirect calibration of the conversion values As the FI method was most intensively calibrated in the early seventies (Jenkinson and Powlson, 1976; Jenkinson et al., 1976) it was usually the basis for most subsequent studies, although other methods, such as ATP measurements, substrate-induced respiration, and direct microscopy have also been used (Table 1). However, the use of the FI method for calibrating the FE method is hampered by a variety of methodological problems, especially the quantity of CO2 evolved from the fumigated soil compared to that in the non-fumigated control during the incubation (Jenkinson, 1988; Wu et al., 1990; Kemmitt et al., 2008). An excessive respiration rate measured in non-fumigated control samples lowers the biomass C values of the FI method and increases, consequently, the kEC values (Joergensen, 1996). The variance of the kEC values obtained by the FI method may be affected by the incubation temperature, the age structure of microbial community, the ratio fungal to bacterial biomass, but also by the pre-treatment of the soils, such as sampling, intensity of sieving, length of pre-incubation period, or the moisture adjustment. For these reasons, the mean kEC values obtained by the different authors differed more than those between the different forms of land use (Joergensen, 1996). 3. Conclusions, future needs and research questions Despite the obvious complexity involved in obtaining a true kEC for each biomass C for each soil and each situation, the extensive calibration data (Table 1) clearly reveals that the conversion value proposed by Wu et al. (1990) can be recommended for general use. Indeed, further calibration studies do not seem necessary, considering all of the other assumptions and errors, which are inherent in the fumigation methods. The same applies to the use of specific conversion values for individual soils or variable conversion values over seasons (Ross, 1990) or with increasing soil depth (Dictor et al., 1998), considering the uncertainties of the methods used for calibrating. However, it is certainly more appropriate to use a nonperfect conversion value than not to use one at all, as fumigation never renders 100% of the microbial biomass extractable (Oberson et al., 2001; Marschner et al., 2002; Bünemann et al., 2004). The FE method is simple to use and leads to meaningful and clear results, enabling all interested scientists to use it in experimental work in soil microbial ecology. However, some results obtained by the FE method have still not been fully considered by those using classical microbiological tools or modern molecular techniques e the seasonal fluctuations of the microbial biomass is an example. These variations are relatively small (at least in Northern Europe) if obtained by the FE method, and are often masked by a strong spatial variability of C input and soil properties (Nieder et al., 2008). Another example is the land use and its management, which has been repeatedly demonstrated to have strong impacts on biomass C obtained by the FE method (AcostaMartínez et al., 2004, 2010; Allen et al., 2008). In contrast, PLFA and molecular genetic tools often emphasise that seasonal variations of microbial communities override the effects of land-use management (Waldrop and Firestone, 2006; Toyota and Kuninaga, 2006; Hamer et al., 2008). Steel corers have even been disinfected with methanol between different samplings (Moore et al., 2010), as an immediate response of the autochthonous microbial community has been expected to those colonising from the inner corer surface.

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