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Release of microbial cell N during chloroform fumigation
SoilBiol. Biochem. Vol. 27,No. 9,pp. 1235-1236, 1995 Copyright d 1995 Elsevier Science Ltd
Pergamon
003&0717(95)00023-2
Printed
in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00
SHORT COMMUNICATION RELEASE OF MICROBIAL CELL N DURING CHLOROFORM FUMIGATION L. G. GREENFIELD Department
of Plant and Microbial
Sciences,
University
of Canterbury,
Christchurch,
New Zealand
(Accepted 4 February 1995)
Soil microbial biomass (MB) methods are constantly being refined and evaluated so that greater reliability and concordance between the results from different MB methods can be achieved (Jenkinson, 1988; Sparling and Zhu, 1993). The fumigation-incubation (FI) and fumigation+xtraction (FE) methods both involvs: a 24 h fumigation treatment. In FI, kc- and k,-factors refer to the fraction of the killed biomass mineralized when the fumigated soil is incubated under standard conditions. :[n FE, k,,- and k,,-factors refer to the fraction of the killed biomass rendered extractable to K,SO, solution by fumigation. Although k-factors for C and N have been obtained using pure microbial cells (Jenkinson, 1988) for use with the: FI method, there has been little work reported on this aspect for the FE method. Tate et al. (1988), using four fungal and two bacterial species, determined a mean k,,-factor of 0.20 using the FE method, which is considerably less than the &-factor of 0.48 estimated from the FI method. Here I report on the release of N from pure microbial cultures subjected to FE. Cultures were grown in basal media (Schimel ef al., 1989), harvested after IO days growth at 18°C by centrifugation and washed 3 times with isotonic saline phosphate buffer. Fungal mycelia were blotted to form a moist pad of even thickness. Bacterial cells were gently stirred in 5 ml water. Duplicate cores (I cm dia, fungi) or volumes (0.5 ml, bacteria) were then used for each of the following procedures: (a) oven-dried, (lO5”C, 24 h) to determine dry weights; (b) freeze-dried and N contents determined by Kjeldahl analysis; and (c) subjected to FE. Typically, 100 mg (dry wt basis) of fresh cells were placed into tared 50ml glaslr centrifuge tubes and fumigated and extracted as described by Br&kes et al. (19856). In this method. samules are fumigated with ethanol-free CHCI, for 24 h at’ 25”d and then extracted with 0.5 M K,SO, for 30min. The supernatants were acidified and evaporated for N determination (Kjeldahl digestion using Hg catalyst and a salt-to-acid ratio of 0.6). In some cases, following the initial K,SO, extraction, fresh K,SO,, was added to the previously-fumigated and extracted cell residues, along with 0.1 ml of CHCI, to prevent microbial growth and to serve as fumigant. Tubes were tightly capped and stood for a further 7d at 18°C with occasional hand shaking, after which time they were centrifuged as before. Supernatants were analysed for N, and cell residues washed with water before oven drying to determine mass loss. In some cases, FE was performed on freeze-dried, whole or ground samples. Freshly-harvested cells (from the same batch used earlier) extracted with 0.5 M K,SO, with no prior CHCI, treatment served as controls, and in all cases (except dry samples) ~6% of total cell N was found to be K$O,soluble. These controls were further extracted with fresh
K,SO, (7 d at 18°C) in the presence of CHCI, to compare with earlier samples previously fumigated (24 h), extracted (30 min) and re-extracted for 7 d. Mean results are presented in Table I; the variation between duplicates was always 18%. Allowing for controls (column l), it can be seen that after 24 h fumigation and 30 min extraction, considerably more N was recovered from whole fungal than bacterial cells (column 5). Insufficient results (columns 3 and 7) do not allow this observation to be confidently extended to cell mass (column 7, even assuming C = 50% soluble matter). However from Table 4, column 8 in Tate et al. (l988), more cell C was extractable from fungi (mean = 23%) than bacteria (mean = lS%), as a consequence of fumigation. Schimel et al. (1989) found that i 5% of the C and N of a Pseudomonas sp. and Aspergillus Jaws was K,SO,-soluble after a 24 h fumigation. Given the species similarities in Schimel et al. (1989) and my study it is difficult to reconcile the divergent results. Large amounts of N and mass became soluble when controls (columns I and 3), which registered little initial soluble N or mass, were stood for 7 d in the presence of CHCI, (columns 2 and 4). The amounts recovered approached the concentrations found in equivalent but fumigated samples (cf. columns 2 and 6, and 4 and 8); this suggests that considerable amounts of potentiallymineralizable N-rich cytoplasmic material remain within dead cells when the FE method of Brookes et al. (1985b) was used. Several authors have commented on the increase in total soluble N or ninhydrin-positive compounds when fumigation of soil was prolonged for up to IOd (Brookes et al., 1985a; Amato and Ladd, 1988; Ross and Tate, 1993; Sparling and Zhu, 1993). The use of freeze-dried, including ground cells, resulted in larger amounts of N and mass appearing in K$O, extracts, particularly in control samples, e.g. B. subfilis and F. solani (column 1). The mean &,-value for all organisms in Table I (excepting dried) was 0.28 + 0. I6 (SD), which could reflect the fact that only five bacterial and three fungal species were studied. The k,,-value would reflect the ratio of fungal-to-bacterial biomass in a particular soil (Anderson and Domsch, 1978). The present -/+,-value is lower than the value of 0.45 obtained bv Jenkinson (1988) but given the variability, kO.16, arobnd the mean’k,,-;alue of 0.28, it is probably similar to the value of 0.38 obtained by Sparling and Zhu (1993) for West Australian soils. Tate et al. (1988) obtained a mean k,,-factor of 0.20 for microbial cells using the FE method, but suggested that the age of the cells, growth and harvest conditions used could affect &-values. They also pointed out the need for more research into the effect of such 1235
1236
Short Communication Table
I. Cell N (U of initial) and cell mass (% of initial)
solubilized
in K,SO, after CHCI, fumigation
Conwol Soluble Species
Experimenral
Soluble mass
N
Soluble N
Soluble mass
%N
CT)
J)
G)
c:)
G)
(i)
G)
G)
9.62 9.71 9.71
3 2 18
ND 39 ND
ND 31 ND 48 ND ND ND ND
20 21 26 ND 18 25 I6 26
ND 49 ND ND 44 ND 38 ND
ND ND 29 ND 20 ND I9 37
ND 37 ND ND ND ND ND ND
ND 32 26 ND ND
49 56 45 53 55
70 74 63 ND ND
ND ND 22 ND 42
ND ND 35 ND ND
Bacteria
B. licheniformis B. suh~ilis B. subri/is* B. subrilist B. circus P. gingeri P. ,puore.w% P. puores(P.s *
Fungi A. frarus A. niger
F. solani F. .solani* F. soiani:
9.06
30
75
IO.31 IO.68 II.60 II.60
3 5 2 I6
ND ND 36 ND
ND ND IO ND 4 ND 5 I2
3.92 4.05 5.24 5.24 5.24
6 2 3 I ND
63 15 61 ND ND
ND ND ND ND ND
A = material
released after 30 min KiSO, extraction; B = material released after a further 7 d K,SO, extraction of the residue from treatment A. *Freeze-dried; tfreeze-dried Sigma Chemical Co. product, other cells were recent soil isolates; #freeze-dried and ground control values not subtracted. ND = not determined.
basic variables as extraction time and shaking conditions on subsequent k,,-factors. Such comments apply equally to k,,-factors. Davidson et al. (1989) pointed out that insufficient data exists concerning the effects of the duration of fumigation and soil moisture variability in FE that uncertainties still remain in conversion of N-flush to MB values. Jenkinson (1988), in discussing weaknesses in calculating biomass N, also cited the variability of the N content of microbial cells grown in vitro. In conclusion, the fact that considerably less N is extractable from bacterial than fungal cells in FE suggests that MB values based on /c-factors derived by FE of individual microbial cell species may be underestimates. However, the reliability of any &,-value would depend on the initial calibration and this has often entailed using microbial N results determined from mineral N-flush data. With this approach, Ross (1992) found a k,,-value of 0.35 k 0.05 for several pasture soils. My findings need to be confirmed using other microbial cells but the work reported here shows that there are still fundamental gaps in our understanding of many of the factors and processes which affect MB values and results obtained using currentlyavailable methods are probably more reliable as relative rather than absolute measures (Ross and Tate, 1993).
Acknowledgements-1 am most grateful to the anonymous reviewer, Professor D. S. Jenkinson, Drs M. Beare, D. J. Ross, G. P. Sparling and D. Wardle for helpful discussion and comments on the manuscript.
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 & Biochemisfry 20, 107-I 14. Anderson, J. P. E. and Domsch K. H. (1978) Mineralization
of bacteria and fungi in chloroform fumigated soils. Soil Bioloav & Biochemistrv 10, 207-213. Brooked-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 fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837-842. Davidson E. A., Eckert R. W., Hart S. C. and Firestone M. K. (1989) Direct extraction of microbial biomass nitrogen‘ from forest and grassland soils of California. Soil Biology & Biochemistry 21, 773-778. Jenkinson D. S. (1988) Determination of microbial biomass carbon and nitrogen in soil. In Aduunces in Nitrogen Cycling in Agricultural Ecosystems (J. R. Wilson, Ed.), pp. 368-386. Commonwealth Agricultural Bureau International, Wallingford. Ross D. J. (1992) Influence of sieve mesh on estimates of microbial carbon and nitrogen by fumigationextraction procedures in soils under pasture. Soil Biology & Biochemistry 24, 343-350. Ross D. J. and Tate K. R. (1993) Microbial C and N in litter and soil of a southern beech (Nothofagus) forest: comparison of measurement procedures. Soil Biology & Biochemistry 25, 467-475. Schimel J. P., Scott W. J. and Killham K. (1989) Changes in cytoplasmic carbon and nitrogen pools in a soil bacterium and a fungus in response to salt stress. Applied and Environmental Microbiology 55, 1635-1637. 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, 1793-1801. Tate K. R., Ross D. J. and Feltham C. W. (1988) A direct extraction method to estimate soil microbial C: effects of experimental variables and some different calibration procedures. Soil Biology & Biochemistry 20, 329-335.
Pergamon
003&0717(95)00023-2
Printed
in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00
SHORT COMMUNICATION RELEASE OF MICROBIAL CELL N DURING CHLOROFORM FUMIGATION L. G. GREENFIELD Department
of Plant and Microbial
Sciences,
University
of Canterbury,
Christchurch,
New Zealand
(Accepted 4 February 1995)
Soil microbial biomass (MB) methods are constantly being refined and evaluated so that greater reliability and concordance between the results from different MB methods can be achieved (Jenkinson, 1988; Sparling and Zhu, 1993). The fumigation-incubation (FI) and fumigation+xtraction (FE) methods both involvs: a 24 h fumigation treatment. In FI, kc- and k,-factors refer to the fraction of the killed biomass mineralized when the fumigated soil is incubated under standard conditions. :[n FE, k,,- and k,,-factors refer to the fraction of the killed biomass rendered extractable to K,SO, solution by fumigation. Although k-factors for C and N have been obtained using pure microbial cells (Jenkinson, 1988) for use with the: FI method, there has been little work reported on this aspect for the FE method. Tate et al. (1988), using four fungal and two bacterial species, determined a mean k,,-factor of 0.20 using the FE method, which is considerably less than the &-factor of 0.48 estimated from the FI method. Here I report on the release of N from pure microbial cultures subjected to FE. Cultures were grown in basal media (Schimel ef al., 1989), harvested after IO days growth at 18°C by centrifugation and washed 3 times with isotonic saline phosphate buffer. Fungal mycelia were blotted to form a moist pad of even thickness. Bacterial cells were gently stirred in 5 ml water. Duplicate cores (I cm dia, fungi) or volumes (0.5 ml, bacteria) were then used for each of the following procedures: (a) oven-dried, (lO5”C, 24 h) to determine dry weights; (b) freeze-dried and N contents determined by Kjeldahl analysis; and (c) subjected to FE. Typically, 100 mg (dry wt basis) of fresh cells were placed into tared 50ml glaslr centrifuge tubes and fumigated and extracted as described by Br&kes et al. (19856). In this method. samules are fumigated with ethanol-free CHCI, for 24 h at’ 25”d and then extracted with 0.5 M K,SO, for 30min. The supernatants were acidified and evaporated for N determination (Kjeldahl digestion using Hg catalyst and a salt-to-acid ratio of 0.6). In some cases, following the initial K,SO, extraction, fresh K,SO,, was added to the previously-fumigated and extracted cell residues, along with 0.1 ml of CHCI, to prevent microbial growth and to serve as fumigant. Tubes were tightly capped and stood for a further 7d at 18°C with occasional hand shaking, after which time they were centrifuged as before. Supernatants were analysed for N, and cell residues washed with water before oven drying to determine mass loss. In some cases, FE was performed on freeze-dried, whole or ground samples. Freshly-harvested cells (from the same batch used earlier) extracted with 0.5 M K,SO, with no prior CHCI, treatment served as controls, and in all cases (except dry samples) ~6% of total cell N was found to be K$O,soluble. These controls were further extracted with fresh
K,SO, (7 d at 18°C) in the presence of CHCI, to compare with earlier samples previously fumigated (24 h), extracted (30 min) and re-extracted for 7 d. Mean results are presented in Table I; the variation between duplicates was always 18%. Allowing for controls (column l), it can be seen that after 24 h fumigation and 30 min extraction, considerably more N was recovered from whole fungal than bacterial cells (column 5). Insufficient results (columns 3 and 7) do not allow this observation to be confidently extended to cell mass (column 7, even assuming C = 50% soluble matter). However from Table 4, column 8 in Tate et al. (l988), more cell C was extractable from fungi (mean = 23%) than bacteria (mean = lS%), as a consequence of fumigation. Schimel et al. (1989) found that i 5% of the C and N of a Pseudomonas sp. and Aspergillus Jaws was K,SO,-soluble after a 24 h fumigation. Given the species similarities in Schimel et al. (1989) and my study it is difficult to reconcile the divergent results. Large amounts of N and mass became soluble when controls (columns I and 3), which registered little initial soluble N or mass, were stood for 7 d in the presence of CHCI, (columns 2 and 4). The amounts recovered approached the concentrations found in equivalent but fumigated samples (cf. columns 2 and 6, and 4 and 8); this suggests that considerable amounts of potentiallymineralizable N-rich cytoplasmic material remain within dead cells when the FE method of Brookes et al. (1985b) was used. Several authors have commented on the increase in total soluble N or ninhydrin-positive compounds when fumigation of soil was prolonged for up to IOd (Brookes et al., 1985a; Amato and Ladd, 1988; Ross and Tate, 1993; Sparling and Zhu, 1993). The use of freeze-dried, including ground cells, resulted in larger amounts of N and mass appearing in K$O, extracts, particularly in control samples, e.g. B. subfilis and F. solani (column 1). The mean &,-value for all organisms in Table I (excepting dried) was 0.28 + 0. I6 (SD), which could reflect the fact that only five bacterial and three fungal species were studied. The k,,-value would reflect the ratio of fungal-to-bacterial biomass in a particular soil (Anderson and Domsch, 1978). The present -/+,-value is lower than the value of 0.45 obtained bv Jenkinson (1988) but given the variability, kO.16, arobnd the mean’k,,-;alue of 0.28, it is probably similar to the value of 0.38 obtained by Sparling and Zhu (1993) for West Australian soils. Tate et al. (1988) obtained a mean k,,-factor of 0.20 for microbial cells using the FE method, but suggested that the age of the cells, growth and harvest conditions used could affect &-values. They also pointed out the need for more research into the effect of such 1235
1236
Short Communication Table
I. Cell N (U of initial) and cell mass (% of initial)
solubilized
in K,SO, after CHCI, fumigation
Conwol Soluble Species
Experimenral
Soluble mass
N
Soluble N
Soluble mass
%N
CT)
J)
G)
c:)
G)
(i)
G)
G)
9.62 9.71 9.71
3 2 18
ND 39 ND
ND 31 ND 48 ND ND ND ND
20 21 26 ND 18 25 I6 26
ND 49 ND ND 44 ND 38 ND
ND ND 29 ND 20 ND I9 37
ND 37 ND ND ND ND ND ND
ND 32 26 ND ND
49 56 45 53 55
70 74 63 ND ND
ND ND 22 ND 42
ND ND 35 ND ND
Bacteria
B. licheniformis B. suh~ilis B. subri/is* B. subrilist B. circus P. gingeri P. ,puore.w% P. puores(P.s *
Fungi A. frarus A. niger
F. solani F. .solani* F. soiani:
9.06
30
75
IO.31 IO.68 II.60 II.60
3 5 2 I6
ND ND 36 ND
ND ND IO ND 4 ND 5 I2
3.92 4.05 5.24 5.24 5.24
6 2 3 I ND
63 15 61 ND ND
ND ND ND ND ND
A = material
released after 30 min KiSO, extraction; B = material released after a further 7 d K,SO, extraction of the residue from treatment A. *Freeze-dried; tfreeze-dried Sigma Chemical Co. product, other cells were recent soil isolates; #freeze-dried and ground control values not subtracted. ND = not determined.
basic variables as extraction time and shaking conditions on subsequent k,,-factors. Such comments apply equally to k,,-factors. Davidson et al. (1989) pointed out that insufficient data exists concerning the effects of the duration of fumigation and soil moisture variability in FE that uncertainties still remain in conversion of N-flush to MB values. Jenkinson (1988), in discussing weaknesses in calculating biomass N, also cited the variability of the N content of microbial cells grown in vitro. In conclusion, the fact that considerably less N is extractable from bacterial than fungal cells in FE suggests that MB values based on /c-factors derived by FE of individual microbial cell species may be underestimates. However, the reliability of any &,-value would depend on the initial calibration and this has often entailed using microbial N results determined from mineral N-flush data. With this approach, Ross (1992) found a k,,-value of 0.35 k 0.05 for several pasture soils. My findings need to be confirmed using other microbial cells but the work reported here shows that there are still fundamental gaps in our understanding of many of the factors and processes which affect MB values and results obtained using currentlyavailable methods are probably more reliable as relative rather than absolute measures (Ross and Tate, 1993).
Acknowledgements-1 am most grateful to the anonymous reviewer, Professor D. S. Jenkinson, Drs M. Beare, D. J. Ross, G. P. Sparling and D. Wardle for helpful discussion and comments on the manuscript.
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 & Biochemisfry 20, 107-I 14. Anderson, J. P. E. and Domsch K. H. (1978) Mineralization
of bacteria and fungi in chloroform fumigated soils. Soil Bioloav & Biochemistrv 10, 207-213. Brooked-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 fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837-842. Davidson E. A., Eckert R. W., Hart S. C. and Firestone M. K. (1989) Direct extraction of microbial biomass nitrogen‘ from forest and grassland soils of California. Soil Biology & Biochemistry 21, 773-778. Jenkinson D. S. (1988) Determination of microbial biomass carbon and nitrogen in soil. In Aduunces in Nitrogen Cycling in Agricultural Ecosystems (J. R. Wilson, Ed.), pp. 368-386. Commonwealth Agricultural Bureau International, Wallingford. Ross D. J. (1992) Influence of sieve mesh on estimates of microbial carbon and nitrogen by fumigationextraction procedures in soils under pasture. Soil Biology & Biochemistry 24, 343-350. Ross D. J. and Tate K. R. (1993) Microbial C and N in litter and soil of a southern beech (Nothofagus) forest: comparison of measurement procedures. Soil Biology & Biochemistry 25, 467-475. Schimel J. P., Scott W. J. and Killham K. (1989) Changes in cytoplasmic carbon and nitrogen pools in a soil bacterium and a fungus in response to salt stress. Applied and Environmental Microbiology 55, 1635-1637. 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, 1793-1801. Tate K. R., Ross D. J. and Feltham C. W. (1988) A direct extraction method to estimate soil microbial C: effects of experimental variables and some different calibration procedures. Soil Biology & Biochemistry 20, 329-335.