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Immobilization of 15NH4 by cattle slurry decomposing in soil
Soil Biol. Biochem. Vol. 26. No. 6. pp. 743-745. 1994 Copyright 0 1994 Elsevier Science Ltd
003847 17(93)EOO23-F
Printed in Great Britain. All rights reserved 003%0717/94$7.00 + 0.00
IMMOBILIZATION OF “NH4 BY CATTLE DECOMPOSING IN SOIL
SLURRY
S. P. TREHAN Central Potato Research Station, Model Town P.O., Post Bag No. I, Jalandhar-144 003, India (Accepted
10 November
1993)
Summary-Almost half the lSN-labelled N added to a cattle slurry-soil were converted to an organic form in 9 days: 5% was fixed by clay minerals and ca 20% lost from the system. The ATP content of the slurry-soil mixture more than doubled in 9 days, suggesting that about 24% of the 15N converted to organic N at the end of 9 days was present in the form of lahelled microbial biomass.
INTRODUCTION Within a few days of the addition of cattle slurry to soil, numbers of bacteria and protozoa increase cu IO-fold (Opperman et al., 1989). Immobilization of 15NH, by cattle slurry occurs rapidly (Trehan and Wild, 1993), presumably because the added N is quickly assimilated by the microbial biomass. My aim was to follow the fate of “N-1abelled inorganic N added to cattle slurry and soil, measuring the quantity of labelled N converted to organic form, the quantity fixed by inorganic colloids of the soil, the amounts lost from the system and (using ATP as an index of biomass) the quantity present in living soil organisms. MATERIALSAND METHODS The soil used was a sandy loam, tentatively classified as a eutrochrept, from the Reading University Farm, Sonning. The sample (O-15 cm) was dried at 25°C to 20% water holding capacity (WHC), sieved (2 mm), and kept at the same water content for 1 week before imposing the experimental treatment. It had a pH of 6.0 (in 10 mM CaCl,) and contained 16% clay, 0.83% C and 0.09% N on a dry weight basis. The cattle slurry was collected from the floor of a dairy cattle shed at the University Farm, Sonning. Before use, it was kept for 4 days at 20°C when it was assumed that the urea contained in it had hydrolysed. The dry matter content at the time of use was 11.3%, containing, on a dry weight basis, 29.8%, 3.56% total N, 0.93% NH: and a trace of NOT-N. The cattle slurry was first supplemented with labelled ammonium sulphate to give a 15N enrichment of 13.79% atom excess of the NH:-N. It was then mixed thoroughly in a rotary mixer with the soil and sufficient water added to bring the soil to 50% WHC. The slurry addition contained 12.9mg dry
slurry gg ’ soil, supplying 140 pg NH:-N and 340 pg organic N g-i soil. Quantities (equivalent to 200 g oven dry soil) of the mixture were then transferred to conical flasks, each containing a vial of 4 M KOH to absorb CO,. Twelve flasks were connected via a manifold to an O2 cylinder so that, as the CO, was absorbed in the KOH, it was replaced by an equal volume of 02, thus keeping the atmosphere inside the flasks at constant 0, partial pressure (Clement and Williams, 1962). The treated soils were kept in the dark at 25 + 0.5”C for 1, 2, 3, 5, 7 or 9 days. After each period, two flasks were analysed for adenosine triphosphate (ATP), labelled N extracted by KCl, labelled N fixed by clay and labelled N present in organic matter. An additional 12 flasks containing a mixture (equivalent to 200 g oven dry soil) of soil, ammonium sulphate (140 pg N g-’ soil) and water were also incubated similarly to measure CO2 production without slurry present. ATP was measured in four replicate subsamples from each flask by the trichloroacetic acidphosphate-paraquat extraction procedure (Tate and Jenkinson, 1982) using a Beckman LS 1801 liquid scintillation spectrometer. Recovery of added ATP varied between 76 and 86%; ATP concentrations in Table 1 are corrected for incomplete recovery (Jenkinson and Oades, 1979). The distribution of labelled N was determined by first shaking 50 g moist soil with 125 ml 2 M KC1 and centrifuging. The extraction was repeated three more times and the combined extracts analysed for NH:-N plus NOT-N by distillation with magnesium oxide and Devarde’s alloy (Bremner, 1965a). The distillate was collected in 15 ml 5 mM H,SO, and titrated. The solution was acidified, evaporated to dryness in a vial and the i5N atom% was measured using a Micromass 602E mass spectrometer after converting ammonium to dinitrogen with lithium hypobromite (Pruden et al., 1985). The soil residue was dried at 70°C for 48 h and crushed (2 mm). Total N, which included
S. P. TREHAN
144
Table I. Distribution of N from cattle slurry incubated Mineral N
Incubation period (days)
(pk! g ‘) U&belled N&-N NO,-N 2 56 70 71 68 47 9 0.9
0
I 2 3 5 7 9 SES
II 5 6 II 21 46 19 I.1
(Kz g ‘)
I 4 4 4 5 5 5 0.2
I 24 26 30 36 43 48 0.6
136 85 82 75 58 30 2 0.7
0 I 2 5 16 36 55 0.5
RESULTS AND DISCUSSION
Production of COZ was stimulated much more by cattle slurry than by ammonium sulphate (Fig. 1). By 9 days 838 pg CO*-C gg’ soil had been produced from the treatment with cattle slurry compared with 116 pg CO,-C gg’ soil in the ammonium
sulphate treatment. represents 19% of another experiment, slurry C was evolved of the cattle slurry microorganisms.
4 -
n=
1
“0
2
-
I
I
I
4
6
8
I 10
Days
Fig. I. Carbon slurry (e)
ATP (LJg g ‘)
dioxide production after addition of cattle and ammonium sulphate (0) to soil.
99 81 81 81 82 81 79
0.41 0.86 0.80 0.92 0.92 0.81 I .09 0.03
The difference of 722 pg CO*-C the C added in the slurry. In not recorded here, 45% of the in 39 days as COZ. Clearly, part is an energy-rich substrate for
Transformations of labelled NH,-N There was some fixation of labelled N by clay, but much greater quantities were either immobilized or lost from the soil within 9 days (Table 1). Almost half the labelled NH:-N had nitrified in the same period. ATP measurements The ATP content of the soil increased from 0.41 pg gg’ soil at time zero to 1.09pg g-’ soil after 9 days (Table 1). Opperman (1989) showed that numbers of bacteria and protozoa in soil increased rapidly during the first 10 days after addition of cattle slurry and then decreased, fungi being unaffected. It is possible to make a rough estimate of the proportion of the labelled organic N that is held in living organisms from the ATP measurements in Table 1. Assume, following Table 1, that 0.68 pg ATP g-i soil is synthesized during the 9 day incubation (i.e. 1.094.41 pg ATP g-i soil) then calculating (Tate and Jenkinson, 1982) the biomass C content from the relationship, biomass
/
Recovery
Labelled NH,-N NO,-N
organic and clay-fixed N was determined by the Kjeldahl method (Bremner, 1965b). On a separate soil sample, non-exchangeable (clay-fixed) NH:-N was determined by the KOBr-KOH method (Silva and Bremner, 1966). For each constituent, ammonia was distilled into H,SO,, titrated, and the “N atom% was measured as described above. Clayfixed N was subtracted from total N to give soil organic N. The same calculation was made for labelled N. Evolved CO* was measured by transferring the KOH solution from the vials to beakers, adding excess BaCl, solution to precipitate carbonate and titrating the residual alkali with HCI using phenolphthalein as an indicator (adapted from Stotzky, 1965). All results are expressed on a oven-dry soil basis.
.
with soil
OrgdlliC N
Clay-fixed N (flcg &! ‘)
C = 171 (ATP content
of soil)
and assuming that the C:N ratio of soil microbial biomass is 6.7 (Shen et al., 1984). Then the extra 0.68 pg biomass ATP should correspond to 17.36 pg biomass N gg’ soil. Assume that this new biomass obtains all its labelled N from the mineral N pool of the soil (i.e. that there is no recycling of labelled organic N in the 9 days of the experiment) and that the labelled N fraction of this pool is the mean of its labelled N fraction at day 0 and day 9. From the data in Table 1 this mean labelled N fraction is (136/(2 + 11 + 136) + (2 + 55)/(9 + 79 + 2 + 55)/2 = 0.653. The quantity of labelled N in the newly synthesized biomass is then 0.653 x 17.35 = 11.3 mg labelled biomass N gg’ soil. This represents 23.6% of the labelled organic N present in the soil at the end of 9 days.
N immobilization Acknowledgements-The work was carried out in the Department of Soil Science, University of Reading, Reading, England. I thank Professor A. Wild for help during the course of the work. I also thank the Commonwealth Scholarship Commission for providing a scholarship and the Indian Council of Agricultural Research and the Director, Central Potato Research Institute, India, for granting study leave.
REFERENCES
Bremner J. M. (1965a) Inorganic forms of nitrogen. In Methods of Soil Analysis, Part 2 (C. A. Black, Ed.), 1179-1237. American Society of Agronomy, PP. Madison. Bremner J. M. (1965b) Total nitrogen. In Mefhoa!s of Soil Analysis, Part 2 (6. A. Black, Ed.), pp. 1149~1178. American Society of Agronomy, Madison. Clement C. R. and Williams T. E. (1962) An incubation technique for assessing the nitrogen status of soils newly ploughed from leys. Journal of Soil Science 13, 82-91. Jenkinson D. S. and Oades J. M. (1979) A method for measuring adenosine triphosphate in soil. Soil Biology & Biochemistry 11, 193-199.
by cattle slurry
745
Opperman M. H., Wood M. and Harris P. J. (1989) -Changes in microbial populations following the application of cattle slurry to soil at two temperatures. Soil Biology & Biochemistry 21, 263-268. Pruden G., Powlson D. S. and Jenkinson D. S. (1985) The measurement of 15N in soil and plant material. Fertilizer Research 6, 205-218. Shen S. M., Proden G. and Jenkinson D. S. (1984) Mineralization and immobilization of nitrogen in fumigated soil and the measurement of microbial soil nitrogen. Soil Biology & Biochemistry 16, 437444. Silva J. A. and Bremner J. M. (1966) Determination and isotope ratio analysis ofdifferent forms of nitrogen in soil: 5. Fixed ammonium. Soil Science Society America proceedings 30, 587-594. Stotzky G. (1965) Microbial respiration. In Mefhodc of Soil Analysis, Part 2 (C. A. Black, Ed.), pp. 1550-1572. American Society of Agronomy, Madison Tate, K. R. and Jenkinson D. S. (1982) Adenosine triphosphate measurement in soil: an improved method. Soil Biology & Biochemistry 14, 331-335. Trehan S. P. and Wild A. (1993) Effects of an organic manure on the transformations of ammonium nitrogen in planted and unplanted soil. Plant and Soil 151, 287-294.
003847 17(93)EOO23-F
Printed in Great Britain. All rights reserved 003%0717/94$7.00 + 0.00
IMMOBILIZATION OF “NH4 BY CATTLE DECOMPOSING IN SOIL
SLURRY
S. P. TREHAN Central Potato Research Station, Model Town P.O., Post Bag No. I, Jalandhar-144 003, India (Accepted
10 November
1993)
Summary-Almost half the lSN-labelled N added to a cattle slurry-soil were converted to an organic form in 9 days: 5% was fixed by clay minerals and ca 20% lost from the system. The ATP content of the slurry-soil mixture more than doubled in 9 days, suggesting that about 24% of the 15N converted to organic N at the end of 9 days was present in the form of lahelled microbial biomass.
INTRODUCTION Within a few days of the addition of cattle slurry to soil, numbers of bacteria and protozoa increase cu IO-fold (Opperman et al., 1989). Immobilization of 15NH, by cattle slurry occurs rapidly (Trehan and Wild, 1993), presumably because the added N is quickly assimilated by the microbial biomass. My aim was to follow the fate of “N-1abelled inorganic N added to cattle slurry and soil, measuring the quantity of labelled N converted to organic form, the quantity fixed by inorganic colloids of the soil, the amounts lost from the system and (using ATP as an index of biomass) the quantity present in living soil organisms. MATERIALSAND METHODS The soil used was a sandy loam, tentatively classified as a eutrochrept, from the Reading University Farm, Sonning. The sample (O-15 cm) was dried at 25°C to 20% water holding capacity (WHC), sieved (2 mm), and kept at the same water content for 1 week before imposing the experimental treatment. It had a pH of 6.0 (in 10 mM CaCl,) and contained 16% clay, 0.83% C and 0.09% N on a dry weight basis. The cattle slurry was collected from the floor of a dairy cattle shed at the University Farm, Sonning. Before use, it was kept for 4 days at 20°C when it was assumed that the urea contained in it had hydrolysed. The dry matter content at the time of use was 11.3%, containing, on a dry weight basis, 29.8%, 3.56% total N, 0.93% NH: and a trace of NOT-N. The cattle slurry was first supplemented with labelled ammonium sulphate to give a 15N enrichment of 13.79% atom excess of the NH:-N. It was then mixed thoroughly in a rotary mixer with the soil and sufficient water added to bring the soil to 50% WHC. The slurry addition contained 12.9mg dry
slurry gg ’ soil, supplying 140 pg NH:-N and 340 pg organic N g-i soil. Quantities (equivalent to 200 g oven dry soil) of the mixture were then transferred to conical flasks, each containing a vial of 4 M KOH to absorb CO,. Twelve flasks were connected via a manifold to an O2 cylinder so that, as the CO, was absorbed in the KOH, it was replaced by an equal volume of 02, thus keeping the atmosphere inside the flasks at constant 0, partial pressure (Clement and Williams, 1962). The treated soils were kept in the dark at 25 + 0.5”C for 1, 2, 3, 5, 7 or 9 days. After each period, two flasks were analysed for adenosine triphosphate (ATP), labelled N extracted by KCl, labelled N fixed by clay and labelled N present in organic matter. An additional 12 flasks containing a mixture (equivalent to 200 g oven dry soil) of soil, ammonium sulphate (140 pg N g-’ soil) and water were also incubated similarly to measure CO2 production without slurry present. ATP was measured in four replicate subsamples from each flask by the trichloroacetic acidphosphate-paraquat extraction procedure (Tate and Jenkinson, 1982) using a Beckman LS 1801 liquid scintillation spectrometer. Recovery of added ATP varied between 76 and 86%; ATP concentrations in Table 1 are corrected for incomplete recovery (Jenkinson and Oades, 1979). The distribution of labelled N was determined by first shaking 50 g moist soil with 125 ml 2 M KC1 and centrifuging. The extraction was repeated three more times and the combined extracts analysed for NH:-N plus NOT-N by distillation with magnesium oxide and Devarde’s alloy (Bremner, 1965a). The distillate was collected in 15 ml 5 mM H,SO, and titrated. The solution was acidified, evaporated to dryness in a vial and the i5N atom% was measured using a Micromass 602E mass spectrometer after converting ammonium to dinitrogen with lithium hypobromite (Pruden et al., 1985). The soil residue was dried at 70°C for 48 h and crushed (2 mm). Total N, which included
S. P. TREHAN
144
Table I. Distribution of N from cattle slurry incubated Mineral N
Incubation period (days)
(pk! g ‘) U&belled N&-N NO,-N 2 56 70 71 68 47 9 0.9
0
I 2 3 5 7 9 SES
II 5 6 II 21 46 19 I.1
(Kz g ‘)
I 4 4 4 5 5 5 0.2
I 24 26 30 36 43 48 0.6
136 85 82 75 58 30 2 0.7
0 I 2 5 16 36 55 0.5
RESULTS AND DISCUSSION
Production of COZ was stimulated much more by cattle slurry than by ammonium sulphate (Fig. 1). By 9 days 838 pg CO*-C gg’ soil had been produced from the treatment with cattle slurry compared with 116 pg CO,-C gg’ soil in the ammonium
sulphate treatment. represents 19% of another experiment, slurry C was evolved of the cattle slurry microorganisms.
4 -
n=
1
“0
2
-
I
I
I
4
6
8
I 10
Days
Fig. I. Carbon slurry (e)
ATP (LJg g ‘)
dioxide production after addition of cattle and ammonium sulphate (0) to soil.
99 81 81 81 82 81 79
0.41 0.86 0.80 0.92 0.92 0.81 I .09 0.03
The difference of 722 pg CO*-C the C added in the slurry. In not recorded here, 45% of the in 39 days as COZ. Clearly, part is an energy-rich substrate for
Transformations of labelled NH,-N There was some fixation of labelled N by clay, but much greater quantities were either immobilized or lost from the soil within 9 days (Table 1). Almost half the labelled NH:-N had nitrified in the same period. ATP measurements The ATP content of the soil increased from 0.41 pg gg’ soil at time zero to 1.09pg g-’ soil after 9 days (Table 1). Opperman (1989) showed that numbers of bacteria and protozoa in soil increased rapidly during the first 10 days after addition of cattle slurry and then decreased, fungi being unaffected. It is possible to make a rough estimate of the proportion of the labelled organic N that is held in living organisms from the ATP measurements in Table 1. Assume, following Table 1, that 0.68 pg ATP g-i soil is synthesized during the 9 day incubation (i.e. 1.094.41 pg ATP g-i soil) then calculating (Tate and Jenkinson, 1982) the biomass C content from the relationship, biomass
/
Recovery
Labelled NH,-N NO,-N
organic and clay-fixed N was determined by the Kjeldahl method (Bremner, 1965b). On a separate soil sample, non-exchangeable (clay-fixed) NH:-N was determined by the KOBr-KOH method (Silva and Bremner, 1966). For each constituent, ammonia was distilled into H,SO,, titrated, and the “N atom% was measured as described above. Clayfixed N was subtracted from total N to give soil organic N. The same calculation was made for labelled N. Evolved CO* was measured by transferring the KOH solution from the vials to beakers, adding excess BaCl, solution to precipitate carbonate and titrating the residual alkali with HCI using phenolphthalein as an indicator (adapted from Stotzky, 1965). All results are expressed on a oven-dry soil basis.
.
with soil
OrgdlliC N
Clay-fixed N (flcg &! ‘)
C = 171 (ATP content
of soil)
and assuming that the C:N ratio of soil microbial biomass is 6.7 (Shen et al., 1984). Then the extra 0.68 pg biomass ATP should correspond to 17.36 pg biomass N gg’ soil. Assume that this new biomass obtains all its labelled N from the mineral N pool of the soil (i.e. that there is no recycling of labelled organic N in the 9 days of the experiment) and that the labelled N fraction of this pool is the mean of its labelled N fraction at day 0 and day 9. From the data in Table 1 this mean labelled N fraction is (136/(2 + 11 + 136) + (2 + 55)/(9 + 79 + 2 + 55)/2 = 0.653. The quantity of labelled N in the newly synthesized biomass is then 0.653 x 17.35 = 11.3 mg labelled biomass N gg’ soil. This represents 23.6% of the labelled organic N present in the soil at the end of 9 days.
N immobilization Acknowledgements-The work was carried out in the Department of Soil Science, University of Reading, Reading, England. I thank Professor A. Wild for help during the course of the work. I also thank the Commonwealth Scholarship Commission for providing a scholarship and the Indian Council of Agricultural Research and the Director, Central Potato Research Institute, India, for granting study leave.
REFERENCES
Bremner J. M. (1965a) Inorganic forms of nitrogen. In Methods of Soil Analysis, Part 2 (C. A. Black, Ed.), 1179-1237. American Society of Agronomy, PP. Madison. Bremner J. M. (1965b) Total nitrogen. In Mefhoa!s of Soil Analysis, Part 2 (6. A. Black, Ed.), pp. 1149~1178. American Society of Agronomy, Madison. Clement C. R. and Williams T. E. (1962) An incubation technique for assessing the nitrogen status of soils newly ploughed from leys. Journal of Soil Science 13, 82-91. Jenkinson D. S. and Oades J. M. (1979) A method for measuring adenosine triphosphate in soil. Soil Biology & Biochemistry 11, 193-199.
by cattle slurry
745
Opperman M. H., Wood M. and Harris P. J. (1989) -Changes in microbial populations following the application of cattle slurry to soil at two temperatures. Soil Biology & Biochemistry 21, 263-268. Pruden G., Powlson D. S. and Jenkinson D. S. (1985) The measurement of 15N in soil and plant material. Fertilizer Research 6, 205-218. Shen S. M., Proden G. and Jenkinson D. S. (1984) Mineralization and immobilization of nitrogen in fumigated soil and the measurement of microbial soil nitrogen. Soil Biology & Biochemistry 16, 437444. Silva J. A. and Bremner J. M. (1966) Determination and isotope ratio analysis ofdifferent forms of nitrogen in soil: 5. Fixed ammonium. Soil Science Society America proceedings 30, 587-594. Stotzky G. (1965) Microbial respiration. In Mefhodc of Soil Analysis, Part 2 (C. A. Black, Ed.), pp. 1550-1572. American Society of Agronomy, Madison Tate, K. R. and Jenkinson D. S. (1982) Adenosine triphosphate measurement in soil: an improved method. Soil Biology & Biochemistry 14, 331-335. Trehan S. P. and Wild A. (1993) Effects of an organic manure on the transformations of ammonium nitrogen in planted and unplanted soil. Plant and Soil 151, 287-294.