Biol Fertil Soils (2000) 30 : 510–519 Q Springer-Verlag 2000 ORIGINAL PAPER Christian Witt 7 John L. Gaunt 7 Catherine C. Galicia Johannes C.G. Ottow 7 Heinz-Ulrich Neue A rapid chloroform-fumigation extraction method for measuring soil microbial biomass carbon and nitrogen in flooded rice soils Received: 1 June 1999 Dedicated to Prof. K. Vlassak on the occassion of his 65th birthday C. Witt (Y) 7 C.C. Galicia International Rice Research Institute (IRRI), Soil and Water Sciences Division, MCPO Box 3127, 1271 Makati City, The Philippines e-mail: c.witt6cgiar.org J.L. Gaunt IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, UK J.C.G. Ottow Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Senckenbergstrasse 3, D-35390 Giessen, Germany H.-U. Neue UfZ-Center for Environmental Research, Department of Soil Sciences, Hallesche Strasse 44, D-06246 Bad Lauchstaedt, Germany Abstract A chloroform-fumigation extraction method with fumigation at atmospheric pressure (CFAP, with- out vacuum) was developed for measuring microbial biomass C (C BIO ) and N (N BIO ) in water-saturated rice soils. The method was tested in a series of laboratory experiments and compared with the standard chloro- form-fumigation extraction (CFE, with vacuum). For both methods, there was little interference from living rice roots or changing soil water content (0.44–0.55 kg kg –1 wet soil). A comparison of the two techniques showed a highly significant correlation for both C BIO and N BIO (P~0.001) suggesting that the simple and rapid CFAP is a reliable alternative to the CFE. It ap- peared, however, that a small and relatively constant fraction of well-protected microbial biomass may only be lysed during fumigation under vacuum. Determina- tions of microbial C and N were highly reproducible for both methods, but neither fumigation technique gener- ated N BIO values which were positively correlated with C BIO . The range of observed microbial C:N ratios of 4–15 was unexpectedly wide for anaerobic soil condi- tions. Evidence that this was related to inconsistencies in the release, degradation, and extractability of N BIO rather than C BIO came from the observation that in- creasing the fumigation time from 4 h to 48 h signifi- cantly increased N BIO but not C BIO . The release pattern of C BIO indicated that the standard fumigation time of 24 h is applicable to water-saturated rice soils. To cor- rect for the incomplete recovery of C BIO , we suggest applying the k C factor of 2.64, commonly used for aero- bic soils (Vance et al. 1987), but caution is required when correcting N BIO data. Until differences in fumiga- tion efficiencies among CFE and CFAP are confirmed for a wider range of rice soils, we suggest applying the same correction factor for both methods. Key words Microbial biomass 7 Fumigation extraction method 7 Flooded soil 7 Lowland rice 7 Microbial C : N ratio Introduction There is a lack of information about the size and dy- namics of the labile nutrient pool of soil microbial bio- mass (SMB) and the microbially mediated nutrient supply to irrigated lowland rice (Gaunt et al. 1995; Cassman et al. 1998). This is, in part, due to a lack of suitable methods for direct quantification of the SMB in water-saturated soils (Inubushi et al. 1989; Inubushi et al. 1991). One of the most successful, rapid and relia- ble methods for a direct quantification of the SMB in aerobic soils is the chloroform-fumigation extraction (CFE). Soils are fumigated with chloroform vapour in- ducing a lysis of microbial cell membranes and, thus, the subsequent release of microbial constituents which can be extracted and quantified, e.g. as microbial C (Vance et al. 1987) or N (Brookes et al. 1985a). While efficient lysis of microbial cells in aerobic soils can eas- ily be achieved with chloroform vapour, Inubushi et al. (1991) proposed the direct addition of chloroform to anaerobic soil in order to overcome limitations in the fumigation efficiency when exposing water-saturated