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Release of ninhydrin-reactive compounds during fumigation of soil to estimate microbial C and N
Sod Biol. Biochem. Vol. 25, No. 12, pp. 1803-1805,
1993
0038-0717/93
Printed in Great Britain. All rights reserved
$6.00 + 0.00
Copyright 0 1993 Pergamon Press Ltd
SHORT COMMUNICATION RELEASE OF NINHYDRIN-REACTIVE COMPOUNDS DURING FUMIGATION OF SOIL TO ESTIMATE MICROBIAL C AND N G. P. SPARLING,'* V.V.S.R.
GWTA~ and CHUNYA ZHU’
‘Soil Science and Plant Nutrition, Faculty of Agriculture, The University of Western Australia, Nedlands, WA 6009 and *Division of Plant Industry, CSIRO G.P.O. Box 1600, Canberra, ACT 2601, Australia (Accepted 9 June 1993)
Amato and Ladd (1988) showed that ninhydrin-positive compounds (NPC) accumulated in soil when under CHCl, fumigation, and that the release of NPC after a 10 day fumigation could be used to estimate soil microbial C. By calibration against microbial C and N estimated by fumigation-incubation methods, the relationship: microbial C = 21 x NPC release, was proposed, and a provisional relationship of microbial N = 3.1 x NPC release, was also suggested (Amato and Ladd, 1988). They noted that the release of NPC after 24 h was only some 60% of that obtained after fumigations for 10 days. However, Carter (1991) and Joergensen and Brookes (1990) used the more convenient 24 h fumigations in some Canadian and British soils and reported similar conversion factors (21 and 4.95) to those of Amato and Ladd (1988). We applied the modified ninhydrin method of Joergensen and Brookes (1990) to soils from Western Australia, but the suggested routine (24 h fumigation, extraction with 0.5 M K,SG,, modified reagents to measure NPC, and factors of 21 to calculate microbial C and 4.95 for microbial N), appeared to substantially underestimate biomass C and N, as judged by biomass estimates obtained using alternative techniques (Sparling and Zhu, 1993). Concurrent work on soils from New South Wales also suggested that the release of NPC was incomplete after 24 h. *Author for correspondence.
We report here on the pattern of release of NPC during fumigation of soils for up to 10 days and reexamine the suggested factors to calculate the microbial biomass C and N after 24 or 240 h (10 day) fumigations. The release of NPC and soluble-N during fumigation of five topsoils from the southern region of Western Australia (WA) and four from New South Wales (NSW) was followed (Table 1, Fig. 1). The soils were conditioned at 22°C for 7 days at 40 and 55% of moisture holding capacity, respectively, for the WA and NSW soils. The NPC content of the soils was then measured using the method of Joergensen and Brookes (1990), and the soluble-N content of WA soils measured using the method of Ross (1992). Fumigations were carried out for 24,48,72, 168 and 240 h, at 22°C using purified CHCl, (Jenkinson and Powlson, 1976). Microbial C and N were estimated by fumigationincubation; estimates obtained by the calculation methods of Jenkinson and Powlson (1976), Ayanaba et al. (1976) and Voroney and Paul (1984) were averaged to provide a ‘combined estimate’ as described earlier (Sparling and Zhu, 1993). In all cases the release of NPC after 24 h fumigation was very much less than after 186-240 h (Fig. 1). On average, 54% of the NPC in WA soils, and 56% of that in NSW soils, had been released after 24 h compared to 240 h (Table 1). The release of soluble-N by the WA soils was less affected by the duration of fumigation, with an average of 67% released after 24 h (Table 1). The NPC released after 24 and 240 h was regressed against the microbial C and N.
Table 1. Soil characteristics, total C, pH, microbial biomass C and N (rg g-l). and amounts of ninhydrin-N and total soluble-N (PgN g-‘) extracted after fumigation with CHCI, and the proportion (%) released after 24 h fumigation compared to 240 h, in soils from WA and NSW Microbial Land use
Ninhydrin-N
Soluble-N
State WA WA WA WA WA Mean of WA soils
C%
pH
C
N
24h
240h
%
24h
240h
%
Kalannie
Arable
0.86
Allandalc Merredin Wongan Beverley
Pasture Arable Arable Arable
3.70 1.00 0.41 1.10 1.43
4.7 5.3 5.7 5.0 5.2 5.2
90 328 238 113 114 177
12.7 50.2 34.6 26.2 30.4 30.8
3.7 8.6 5.3 1.8 2.2 4.3
4.2 15.1 11.4 5.9 4.5 8.2
88 57 46 31 49 54
12.7 26.5 11.7 4.7 4.8 12.1
14.6 46.0 16.1 10.0 6.8 18.7
87 58 73 47 71 67
NSW NSW NSW
Temora Harden 1 Harden 2
Arable Arable Arable
1.59 5.8
763 685 661
148 157 137
13.1 13.5 11.5
23.9 24.7 21.5
55 55 54
NT NT
NT NT
NT NT
NSW Mean of
Ginninderrs
NT
Arable
Site
1.78 5.9 1.09
5.5
469
109
9.8
16.4
61
NT NT
NT NT
1.5
5.9
644
138
12.0
21.6
56
NT
NT
1.54
6.2
NSW soils NT = not tested.
1803
NT NT
Short Communication
1804
lb.
la. WFSTERN AUSTRALIA
20
NEW SOUTH WALES
30
0
0 012
3 Fumigation
7
10
01
3
(days)
7
Fumigation
10
(days)
Fig. 1. Ninhydrin-positive compounds (PgN as leucine equivalents g-r soil) extracted in 0.5 MKr SO, from soils after fumigation with CHCI, for up to 240 h (a) WA soils: Wongan (0); Beverley (a); Kalannie (A); Allandale (A); Met&in (0); (b) NSW soils: Temora (A); Harden-B (0); Harden-C (0); Ginninderra (A). None of the intercepts differed significantly from zero, and the relationships in Table 2 were obtained. For the WA soils the relationships for 240 h (IO day) fumigations are almost identical to those obtained by Amato and Ladd (1988) and suggests their factors to calculate microbial C and N are also valid for these WA soil types. In contrast, the factors obtained for 24 h fumigations are almost double those obtained by Joergensen and Brookes (1990) and Carter (1991). The factors were also consistently higher in the NSW soils than the WA soils, which may possibly be related to differences in soil type and texture, but again, the factors for 24 h fumigations were almost double those for the 240 h fumigations. For both sets of soils, this was a direct consequence of the release of NPC after 24 h being almost half of that after 240 h. Gcio and Brookes (1990) also surmised that the release of NPC was incomplete after 24 h fumigation, but their suggested factors to calculate microbial C and N (31.2 and 4.95, respectively) are substantially lower than ours. The factors controlling the release of NPC are not fully understood, but enzyme activity is essential for the release of ninhydrin-reactive amino-N (Amato and Ladd, 1988). Enzyme activity during fumigation also affects the solubilization and forms of microbial N (Brookes er al., 1985a, b; Sparling and West, 1989). It seems possible that in our soils there was low enzyme activity and reaction times >24 h were required to allow the accumulation of NPC derived from the killed microbial biomass. The duration of fumigation also affected the release of total soluble N, but to a lesser extent than NPC. Even so, when calculating &-factors for the fumigation-extraction method it appears advisable to standardize the fumigation period. Enzyme activity is also important to release microbial P into extractable inorganic phosphate during the estimation of microbial biomass P (Brookes er a/., 1982;
Sparling and West, 1989). However, preliminary observations on the release of microbial P during fumigation of WA soils, have shown little further increase beyond 24 h fumigation. We consider that the ninhydrin method is a useful and reliable technique to estimate microbial C and N, but that the pattern of NPC release during fumigation of the soils should be checked and calibrated against estimates of microbial C and N. If this is not possible, and standard factors are to be applied, the longer fumigations of 7-10 days are to be preferred over 24 h in order to avoid underestimating the microbial biomass C and N. Acknowfedgemenrs-This study was completed while GPS was the Wheatgrower Senior Research Fellow under funding provided by the Grains Research and Develop ment Corp., and forms part of the research programme of the Cooperative Research Centre for Legumes in Mediterranean Agriculture. The work completed by WSRG forms part of the LWRRDC and Land and Water Care Programme, CSIRO. REFERENCES
Amato M. and Ladd J. N. (1988) An assay for microbial biomass based on ninhydrin-reactive nitrogen in extracts of fumigated soils. Soil Biology & Biochemistry 20, 107-l 14.
Ayanaba A., Tuckwell S. B. and Jenkinson D. S. (1976) The effects of clearing and cropping on the organic reserves and biomass of tropical forest soils. Soil Biology & Biochemistry
8, 519-525.
Brookes P. C., Powlson D. S. and Jenkinson D. S. (1982) Measurement of microbial biomass phosphorus in soil. Soil Biology & Biochemistry
14, 319-329.
Table 2. Factors to calculate microbial C and N from the rekasc of ninhydrin-positive wmoounds after 24 or 240 h fumiaations in soils from WA and NSW Western Australia
New South Wales
Fumigation
Microbial C
Microbial N
Microbial C
Microbial N
24h 240h
40.4 f 2.9. 21.4kO.56
6.47 f 1.06 3.49 f 0.41
64.8 f 13.5 29.8 f 5.16
11.89f 1.19 5.33 f 0.45
*Slope and standard error. Intercepts did not differ significantly from zero and all regressions were significant at P = eO.05.
Short Communication Brookes P. C., Kragt J. F., Powlson D. S. and Jenkinson D. S. (1985a) Chloroform fumigation and the release of soil nitrogen: the effects of fumigation time and temperature. Soil Biology & Biochemistry 17, 831-835. Brookes P. C., Landman A., Pruden G. and Jenkinson D. S. (1985b) Chloroform fumiaation and the release of . soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837-842. Carter M. R. (1991) Ninhydrin-reactive N released by the fumigation+xtraction method as a measure of microbial biomass under field conditions. Soil Biology & Biochemistry 23, 139-143. Jenkinson D. S. and Powlson D. S. (1976) The effects of biocidal treatments on metabolism in soil. V. A method to measure soil microbial biomass. Soil Biology & Biochemistry 8, 209-213. Joergensen R. G. and Brookes P. C. (1990) Ninhydrin-reactive nitrogen measurements of microbial biomass in 0.5 M K,SO, soil extracts. Soil Biology & Biochemistry 22, 1023-1027. I
1805
Gcio J. A. and Brookes P. C. (1990) An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the characterisation of the biomass that develops. Soil Biology _. & Biochemistry 22, 685-694. Ross D. J. (1992) Influence of sieve mesh size on estimates of microbial carbon and nitrogen by fumigationextraction procedures in soils under pasture. Soil Biology & Biochemirtry 24, 343-350. Sparling G. P. and West A. W. (1989) Importance of soil water content when estimating soil microbial C, N and P by the fumigation-extraction methods. Soil Biology & Biochemistry 21, 245-253. Sparling G. P. and Zhu C. (1993) Evaluation and calibration of biochemical methods to measure microbial biomass C and N in soils from Western Australia. Soil Biology & Biochemistry 25, 1795-1803. Voroney R. P. and Paul E. A. (1984) Determination of the kc and k, in situ for calibration of the chlorofonnfumigation-incubation method. Soil Biofogy & Biochemistry 16, 9-14.
1993
0038-0717/93
Printed in Great Britain. All rights reserved
$6.00 + 0.00
Copyright 0 1993 Pergamon Press Ltd
SHORT COMMUNICATION RELEASE OF NINHYDRIN-REACTIVE COMPOUNDS DURING FUMIGATION OF SOIL TO ESTIMATE MICROBIAL C AND N G. P. SPARLING,'* V.V.S.R.
GWTA~ and CHUNYA ZHU’
‘Soil Science and Plant Nutrition, Faculty of Agriculture, The University of Western Australia, Nedlands, WA 6009 and *Division of Plant Industry, CSIRO G.P.O. Box 1600, Canberra, ACT 2601, Australia (Accepted 9 June 1993)
Amato and Ladd (1988) showed that ninhydrin-positive compounds (NPC) accumulated in soil when under CHCl, fumigation, and that the release of NPC after a 10 day fumigation could be used to estimate soil microbial C. By calibration against microbial C and N estimated by fumigation-incubation methods, the relationship: microbial C = 21 x NPC release, was proposed, and a provisional relationship of microbial N = 3.1 x NPC release, was also suggested (Amato and Ladd, 1988). They noted that the release of NPC after 24 h was only some 60% of that obtained after fumigations for 10 days. However, Carter (1991) and Joergensen and Brookes (1990) used the more convenient 24 h fumigations in some Canadian and British soils and reported similar conversion factors (21 and 4.95) to those of Amato and Ladd (1988). We applied the modified ninhydrin method of Joergensen and Brookes (1990) to soils from Western Australia, but the suggested routine (24 h fumigation, extraction with 0.5 M K,SG,, modified reagents to measure NPC, and factors of 21 to calculate microbial C and 4.95 for microbial N), appeared to substantially underestimate biomass C and N, as judged by biomass estimates obtained using alternative techniques (Sparling and Zhu, 1993). Concurrent work on soils from New South Wales also suggested that the release of NPC was incomplete after 24 h. *Author for correspondence.
We report here on the pattern of release of NPC during fumigation of soils for up to 10 days and reexamine the suggested factors to calculate the microbial biomass C and N after 24 or 240 h (10 day) fumigations. The release of NPC and soluble-N during fumigation of five topsoils from the southern region of Western Australia (WA) and four from New South Wales (NSW) was followed (Table 1, Fig. 1). The soils were conditioned at 22°C for 7 days at 40 and 55% of moisture holding capacity, respectively, for the WA and NSW soils. The NPC content of the soils was then measured using the method of Joergensen and Brookes (1990), and the soluble-N content of WA soils measured using the method of Ross (1992). Fumigations were carried out for 24,48,72, 168 and 240 h, at 22°C using purified CHCl, (Jenkinson and Powlson, 1976). Microbial C and N were estimated by fumigationincubation; estimates obtained by the calculation methods of Jenkinson and Powlson (1976), Ayanaba et al. (1976) and Voroney and Paul (1984) were averaged to provide a ‘combined estimate’ as described earlier (Sparling and Zhu, 1993). In all cases the release of NPC after 24 h fumigation was very much less than after 186-240 h (Fig. 1). On average, 54% of the NPC in WA soils, and 56% of that in NSW soils, had been released after 24 h compared to 240 h (Table 1). The release of soluble-N by the WA soils was less affected by the duration of fumigation, with an average of 67% released after 24 h (Table 1). The NPC released after 24 and 240 h was regressed against the microbial C and N.
Table 1. Soil characteristics, total C, pH, microbial biomass C and N (rg g-l). and amounts of ninhydrin-N and total soluble-N (PgN g-‘) extracted after fumigation with CHCI, and the proportion (%) released after 24 h fumigation compared to 240 h, in soils from WA and NSW Microbial Land use
Ninhydrin-N
Soluble-N
State WA WA WA WA WA Mean of WA soils
C%
pH
C
N
24h
240h
%
24h
240h
%
Kalannie
Arable
0.86
Allandalc Merredin Wongan Beverley
Pasture Arable Arable Arable
3.70 1.00 0.41 1.10 1.43
4.7 5.3 5.7 5.0 5.2 5.2
90 328 238 113 114 177
12.7 50.2 34.6 26.2 30.4 30.8
3.7 8.6 5.3 1.8 2.2 4.3
4.2 15.1 11.4 5.9 4.5 8.2
88 57 46 31 49 54
12.7 26.5 11.7 4.7 4.8 12.1
14.6 46.0 16.1 10.0 6.8 18.7
87 58 73 47 71 67
NSW NSW NSW
Temora Harden 1 Harden 2
Arable Arable Arable
1.59 5.8
763 685 661
148 157 137
13.1 13.5 11.5
23.9 24.7 21.5
55 55 54
NT NT
NT NT
NT NT
NSW Mean of
Ginninderrs
NT
Arable
Site
1.78 5.9 1.09
5.5
469
109
9.8
16.4
61
NT NT
NT NT
1.5
5.9
644
138
12.0
21.6
56
NT
NT
1.54
6.2
NSW soils NT = not tested.
1803
NT NT
Short Communication
1804
lb.
la. WFSTERN AUSTRALIA
20
NEW SOUTH WALES
30
0
0 012
3 Fumigation
7
10
01
3
(days)
7
Fumigation
10
(days)
Fig. 1. Ninhydrin-positive compounds (PgN as leucine equivalents g-r soil) extracted in 0.5 MKr SO, from soils after fumigation with CHCI, for up to 240 h (a) WA soils: Wongan (0); Beverley (a); Kalannie (A); Allandale (A); Met&in (0); (b) NSW soils: Temora (A); Harden-B (0); Harden-C (0); Ginninderra (A). None of the intercepts differed significantly from zero, and the relationships in Table 2 were obtained. For the WA soils the relationships for 240 h (IO day) fumigations are almost identical to those obtained by Amato and Ladd (1988) and suggests their factors to calculate microbial C and N are also valid for these WA soil types. In contrast, the factors obtained for 24 h fumigations are almost double those obtained by Joergensen and Brookes (1990) and Carter (1991). The factors were also consistently higher in the NSW soils than the WA soils, which may possibly be related to differences in soil type and texture, but again, the factors for 24 h fumigations were almost double those for the 240 h fumigations. For both sets of soils, this was a direct consequence of the release of NPC after 24 h being almost half of that after 240 h. Gcio and Brookes (1990) also surmised that the release of NPC was incomplete after 24 h fumigation, but their suggested factors to calculate microbial C and N (31.2 and 4.95, respectively) are substantially lower than ours. The factors controlling the release of NPC are not fully understood, but enzyme activity is essential for the release of ninhydrin-reactive amino-N (Amato and Ladd, 1988). Enzyme activity during fumigation also affects the solubilization and forms of microbial N (Brookes er al., 1985a, b; Sparling and West, 1989). It seems possible that in our soils there was low enzyme activity and reaction times >24 h were required to allow the accumulation of NPC derived from the killed microbial biomass. The duration of fumigation also affected the release of total soluble N, but to a lesser extent than NPC. Even so, when calculating &-factors for the fumigation-extraction method it appears advisable to standardize the fumigation period. Enzyme activity is also important to release microbial P into extractable inorganic phosphate during the estimation of microbial biomass P (Brookes er a/., 1982;
Sparling and West, 1989). However, preliminary observations on the release of microbial P during fumigation of WA soils, have shown little further increase beyond 24 h fumigation. We consider that the ninhydrin method is a useful and reliable technique to estimate microbial C and N, but that the pattern of NPC release during fumigation of the soils should be checked and calibrated against estimates of microbial C and N. If this is not possible, and standard factors are to be applied, the longer fumigations of 7-10 days are to be preferred over 24 h in order to avoid underestimating the microbial biomass C and N. Acknowfedgemenrs-This study was completed while GPS was the Wheatgrower Senior Research Fellow under funding provided by the Grains Research and Develop ment Corp., and forms part of the research programme of the Cooperative Research Centre for Legumes in Mediterranean Agriculture. The work completed by WSRG forms part of the LWRRDC and Land and Water Care Programme, CSIRO. REFERENCES
Amato M. and Ladd J. N. (1988) An assay for microbial biomass based on ninhydrin-reactive nitrogen in extracts of fumigated soils. Soil Biology & Biochemistry 20, 107-l 14.
Ayanaba A., Tuckwell S. B. and Jenkinson D. S. (1976) The effects of clearing and cropping on the organic reserves and biomass of tropical forest soils. Soil Biology & Biochemistry
8, 519-525.
Brookes P. C., Powlson D. S. and Jenkinson D. S. (1982) Measurement of microbial biomass phosphorus in soil. Soil Biology & Biochemistry
14, 319-329.
Table 2. Factors to calculate microbial C and N from the rekasc of ninhydrin-positive wmoounds after 24 or 240 h fumiaations in soils from WA and NSW Western Australia
New South Wales
Fumigation
Microbial C
Microbial N
Microbial C
Microbial N
24h 240h
40.4 f 2.9. 21.4kO.56
6.47 f 1.06 3.49 f 0.41
64.8 f 13.5 29.8 f 5.16
11.89f 1.19 5.33 f 0.45
*Slope and standard error. Intercepts did not differ significantly from zero and all regressions were significant at P = eO.05.
Short Communication Brookes P. C., Kragt J. F., Powlson D. S. and Jenkinson D. S. (1985a) Chloroform fumigation and the release of soil nitrogen: the effects of fumigation time and temperature. Soil Biology & Biochemistry 17, 831-835. Brookes P. C., Landman A., Pruden G. and Jenkinson D. S. (1985b) Chloroform fumiaation and the release of . soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837-842. Carter M. R. (1991) Ninhydrin-reactive N released by the fumigation+xtraction method as a measure of microbial biomass under field conditions. Soil Biology & Biochemistry 23, 139-143. Jenkinson D. S. and Powlson D. S. (1976) The effects of biocidal treatments on metabolism in soil. V. A method to measure soil microbial biomass. Soil Biology & Biochemistry 8, 209-213. Joergensen R. G. and Brookes P. C. (1990) Ninhydrin-reactive nitrogen measurements of microbial biomass in 0.5 M K,SO, soil extracts. Soil Biology & Biochemistry 22, 1023-1027. I
1805
Gcio J. A. and Brookes P. C. (1990) An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the characterisation of the biomass that develops. Soil Biology _. & Biochemistry 22, 685-694. Ross D. J. (1992) Influence of sieve mesh size on estimates of microbial carbon and nitrogen by fumigationextraction procedures in soils under pasture. Soil Biology & Biochemirtry 24, 343-350. Sparling G. P. and West A. W. (1989) Importance of soil water content when estimating soil microbial C, N and P by the fumigation-extraction methods. Soil Biology & Biochemistry 21, 245-253. Sparling G. P. and Zhu C. (1993) Evaluation and calibration of biochemical methods to measure microbial biomass C and N in soils from Western Australia. Soil Biology & Biochemistry 25, 1795-1803. Voroney R. P. and Paul E. A. (1984) Determination of the kc and k, in situ for calibration of the chlorofonnfumigation-incubation method. Soil Biofogy & Biochemistry 16, 9-14.