Soil Biology & Biochemistry 36 (2004) 57 www.elsevier.

com/locate/soilbio

Citation classics

Measuring soil microbial biomass

David S. Jenkinson*, Philip C. Brookes, David S. Powlson
Agriculture and Environent Division, Rothamsted Research, Harpenden, Hertforshire AL5 2JQ, UK

1. Introduction Methods papers are much quoted, either because people want to use them, or because people think them wrong. Our three highly cited papers have attracted attention for both reasons. The rst (Vance et al., 1987) describes a method for measuring the amount of biomass carbon (C) in soil, the second (Brookes et al., 1985b), a parallel method for biomass nitrogen (N) and the third (Brookes et al., 1982) for biomass phosphorus (P). In all three we treat the soil microbial biomass as a whole, just as one might choose to study a forest as an entity, although well aware that it is made up of different kinds of tree.

the corresponding unfumigated soil and this CO2 was also more heavily labelled. From this and other experiments it was concluded that the extra CO2 released by the fumigated soil came from heavily labelled organisms formed during the decomposition of the plant material (Jenkinson, 1966). These were killed during the fumigation phase and then decomposed during the incubation phase. Stormer had indeed been right.

3. The fumigation/incubation method for measuring microbial biomass Our rst attempts to measure the quantity of microbial biomass in soil (Jenkinson, 1966; Jenkinson and Powlson, 1976b) were based on the size of the ush of respiration that follows fumigation with chloroformthe fumigation/incubation (FI) method. Microbial biomass was given by F=kC ; where the ush F is given by (CO2 C evolved on incubating fumigated soil for 10 days, minus CO2 C evolved in 10 days by an unfumigated control soil). The constant kC represented the fraction of killed biomass C converted to CO2 during a 10 day incubation period: it was set at 0.5, based on incubations with a small selection of organisms (two yeasts, two fungi, an actinomycete, seven bacteria and a soil invertebrate), all added seperately to soil (Jenkinson, 1976). Later, following a survey of the literature (Jenkinson and Ladd, 1981), it was modied to 0.45. This method has been widely used, despite its disadvantagesthe time needed to obtain a result, and more importantly, its failure with strongly acid soils and in soils containing large amounts of recently-added substrate. There has also been some controversy (Voroney and Paul, 1984) about the need for a control incubationan issue that still smolders on (Franzluebbers et al., 1999). Although we stand by our view that a control is essential (Wu et al., 1996), we now view the FI method as obsolete.

2. Background All three papers arose from work done almost 40 years ago on the effects of chloroform fumigation on soil respiration (Jenkinson, 1966). Long before that, fumigation had been known to enhance soil respiration for a day or twofor a review of the early literature see Jenkinson and Powlson (1976a). Stormer (1908) had postulated that the ush of decomposition that follows fumigation is caused by the decomposition of killed organisms by the survivors, but his explanation was displaced by fancier theories, for example that fumigation disrupted the balance between different components of the soil microbial population. In the early 1960s one of us was examining the distribution of label in a soil that had been incubated in the eld for a year with plant material uniformly labelled with 14C (Jenkinson, 1966). The soil was exposed to pure chloroform vapour and, after removal of the vapour, inoculated with a small amount of unfumigated soil and incubated in the laboratory. For a few days, the fumigated soil evolved CO2 much more rapidly than
* Corresponding author. E-mail address: davidsjenkinson@tiscali.co.uk (D.S. Jenkinson). 0038-0717/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2003.10.002

D.S. Jenkinson et al. / Soil Biology & Biochemistry 36 (2004) 57

4. Fumigation/extraction Phil Brookes joined the biomass group at Rothamsted in 1978, at a time when David Powlson was becoming more and more involved with eld experiments using 15N labelled fertilizers. Phil was interested in soil P and decided to see if he could measure soil microbial P from the difference between the quantities of P extracted by the Olsen bicarbonate reagent from fumigated and unfumigated soil. The paper on measuring P in the soil microbial biomass (Brookes et al., 1982) appeared in the same issue of Soil Biology & Biochemistry as a completely independent paper by Hedley and Stewart (1982), describing an almost identical procedure. Brookes et al. (1982) was the rst of our fumigation/extraction papers. By the early 1980s we had become increasingly dissatised with the FI method for microbial C. We knew that chloroform disrupts cell membranes, allowing the contents to leak out. We also knew that the extractability of non-living soil organic matter was little, if at all, affected by chloroform fumigation. Was the incubation really necessary, or could we measure the quantities of C or N released directly, immediately after fumigation? The arrival of three talented young students gave us the opportunity to re-examine the 1976 work and see if we could devise something better. Andrea Landman and Jan Kragt, then students at the Agricultural University, Wageningen, The Netherlands, joined us in a detailed study of the release of ammonium and organic N during fumigation, leading to the papers describing the fumigation/extraction method for measuring microbial N (Brookes et al., 1985a,b). Gordon Pruden, that most meticulous of analysts, was a co-author of the second of these papers and the whole group were saddened by his untimely death in 1984. Eric Vance, at the time a PhD student at the University of Missouri, USA (and now at the National Council for Air and Stream Improvement, North Carolina, USA) came to Rothamsted with a Fulbright Scholarship. One of the papers produced during his stay (Vance et al., 1987) describes the fumigation/extraction procedure for measuring soil microbial biomass. This procedure can be used in conditions where the original FI method fails: in acid soils; in soils recently amended with fresh substrate; (Ocio and Brookes, 1990) and in waterlogged (paddy) soils (Inubushi et al., 1991). Some of the biomass measurements in Vance et al. (1987) paper were based on data taken directly from an earlier publication (Powlson and Jenkinson, 1976). Had we not been obsessed with incubation techniques in 1976, we had all the information needed to develop the fumigation/ extraction procedure a decade earlier. A little later (and quite independently), the New Zealand group led by Kevin Tate described a very similar procedure for measuring biomass C by fumigation/extraction (Tate et al., 1988).

5. Criticisms and weaknesses of the fumigation/extraction techniques Argument is part of science and our fumigation/extraction papers have been criticized on three main grounds. The rst is our choice of a 24 h exposure to chloroform vapour. Enzymic activity continues during fumigation, with extractable N continuing to increase for at least 5 days, at rates that are not quite the same in different soils (Brookes et al., 1985a). However, 5 days is too long to wait if you want a rapid biomass assay, so we settled for 24 h as a reasonable compromise. The second criticism is that chloroform solublises more than just biomass C, N or P, by rendering other, non-living, fractions of the soil organic matter extractible. We have never been able to entirely exclude this possibilityalthough we did show that the quantity of non-biomass C thus solubilised (if any) was tiny compared to that from killed biomass. However, if treatments such as heating or irradiation are used to kill the biomass, signicant quantities of non-biomass C are solubilised (Jenkinson, 1966; Powlson and Jenkinson, 1976). Another cause of trouble is the use of unpuried chloroform. The literature contains instances where chloroform fumigation causes enormous ushes of CO2, usually accompanied by the disappearance of inorganic N. This problem can condently be attributed to the failure to remove ethanol, added to commercial chloroform as stabilizer. By far the most serious criticism of all three papers is directed towards the way we related the quantity of material solubilized by chloroform to the size of the original biomass, i.e. how we set the values of kEC ; kEN and kEP : The values we now use are all ultimately derived from measurements made on a small selection of organisms added to soil and fumigated. These organisms, nearly all grown on nutrient-rich media, may not be good models for the mostly starving and diverse soil population. Several authors have measured kEC by a totally different method. A soluble 14C-labelled substrate such as glucose is added to soil, allowed to decompose for a day or two, and fumigated. The 14C rendered extractable by fumigation, expressed as a fraction of the insoluble 14C remaining in the soil at the time of fumigation, gives kEC : Dictor et al. (1998) discuss this approach and give a brief review of the relevant literature. The difculty lies in the assumption that all of the insoluble 14C is present as live microbial biomass, with none stabilized in non-living structures such as insoluble polysaccharide gums, making the values of kEC thus obtained too small. Neither the addition of externally grown organisms, nor the cultivation of organisms from a pulse of labelled substrate in-situ will give more than rough values for the fraction of the soil microbial biomass C rendered extractable by chloroform.

D.S. Jenkinson et al. / Soil Biology & Biochemistry 36 (2004) 57

6. Do we need a different factor for every soil? Scientists are often classied as lumpers or splitters. Splitters put each new observation in a class of its own. We are rmly in the lumper camp: there are similarities between the biomass in different soils, for example, in ATP content (about 11 mmol ATP g21 biomass C: Contin et al., 2001) or in the way biovolume is distributed between small and large organisms in different soils (Vance et al., 1991). This gives us condence that the same values of kEC ; kEN or kEP can be applied to different aerobic surface soils without major error. Of course it is not necessary to have a kEC value to make valid comparisons between soilsbut a value is essential if, for example, you wish to know just how much biomass is formed from a given quantity of substrate. We feel it is time for an up-to-date review of the literature on kEC ; kEN and kEP : In the meanwhile we use 0.45 for kEC ; 0.45 for kEN and 0.40 for kEP : Better analytical methods are now available for measuring extractable C and N than we had in the 1980s. The 0.5 M K2SO4 extractant we used is too concentrated for many of the modern automated methods and more dilute solutions can be usedprovided the concentration is sufcient to keep the soil occulated and expanding clays closed.

7. Retrospect In the 1980s the comparative freedom long enjoyed by Rothamsted scientists to choose their own research topics was coming to an end and the ponderous new system of grant applications, commissioned research and interdisciplinary collaboration directed from above was beginning to make itself felt. In many ways, these three papers represent the last owering of the old way of doing research. We saw the opportunity, did the work and wrote the papers.

References
Brookes, P.C., Kragt, J.F., Powlson, D.S., Jenkinson, D.S., 1985a. Chloroform fumigation and the release of soil nitrogen: the effects of fumigation time and temperature. Soil Biology & Biochemistry 17, 831835. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985b. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method for measuring microbial biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837 842. Brookes, P.C., Powlson, D.S., Jenkinson, D.S., 1982. Measurement of microbial biomass phosphorus in soil. Soil Biology & Biochemistry 14, 319329.

Contin, M., Todd, A., Brookes, P.C., 2001. The ATP concentration of the soil microbial biomass. Soil Biology & Biochemistry 33, 701 704. Dictor, M.-C., Tessier, L., Soulas, G., 1998. Reassessment of the KEC coefcient of the fumigationextraction method in a soil prole. Soil Biology & Biochemistry 30, 119 127. Franzluebbers, A.J., Haney, R.L., Hons, F.M., 1999. Relationships of chloroform-incubation to soil organic matter pools. Soil Biology & Biochemistry 31, 395405. Hedley, M.J., Stewart, J.W.B., 1982. Method to measure microbial biomass phosphorus in soils. Soil Biology & Biochemistry 14, 377 385. Inubushi, K., Brookes, P.C., Jenkinson, D.S., 1991. Measurements of soil microbial biomass C, N and ninhydrin-N in anaerobic and aerobic soils by the fumigation extraction method. Soil Biology & Biochemistry 23, 737 742. Jenkinson, D.S., 1966. Studies on the decomposition of plant material in soil. II. Partial sterilization of soil and the soil biomass. Journal of Soil Science 17, 280302. Jenkinson, D.S., 1976. The effects of biocidal treatments on metabolism in soil. IV. The decomposition of fumigated organisms in soil. Soil Biology & Biochemistry 8, 203208. Jenkinson, D.S., Powlson, D.S., 1976a. The effects of biocidal treatments on metabolism in soil. I. Fumigation with chloroform. Soil Biology & Biochemistry 8, 167 177. Jenkinson, D.S., Powlson, D.S., 1976b. The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biology & Biochemistry 8, 209213. Jenkinson, D.S., Ladd, J.N., 1981. Microbial biomass in soil: measurement and turnover. In: Paul, E.A., Ladd, J.N. (Eds.), Soil Biochemistry, vol 5. Dekker, New York, pp. 415 471. Ocio, J.A., Brookes, P.C., 1990. An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the characterization of the biomass that develops. Soil Biology & Biochemistry 22, 685694. Powlson, D.S., Jenkinson, D.S., 1976. The effects of biocidal treatments on metabolism in soil. II. Gamma irradiation, autoclaving, air-drying and fumigation. Soil Biology & Biochemistry 8, 179 188. Stormer, K., 1908. Uber die Wirkung des Schwefelkohlenstoffs und ahnlicher Stoffe auf den Boden. Zentralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten II 20, 282 286. Tate, K.R., Ross, D.J., Feltham, C.W., 1988. A direct extraction method to estimate soil microbial biomass C: effects of experimental variables and some different calibration procedures. Soil Biology & Biochemistry 20, 329 335. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1991. Conrmation of a relationship between the size of non-hyphal organisms and their contribution to soil biovolume. Soil Biology & Biochemistry 23, 10971098. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology & Biochemistry 19, 703707. Voroney, R.P., Paul, E.A., 1984. Determination of kC and kN in situ for calibration of the chloroform fumigationincubation method. Soil Biology & Biochemistry 16, 914. Wu, J., Brookes, P.C., Jenkinson, D.S., 1996. Evidence for the use of a control in the fumigation incubation method for measuring microbial biomass carbon in soil. Soil Biology & Biochemistry 28, 511518.