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Short-term effects of tillage on mineralization of nitrogen and carbon in soil
Soil Biology & Biochemistry 35 (2003) 979–986 www.elsevier.com/locate/soilbio
Short-term effects of tillage on mineralization of nitrogen and carbon in soil H.L. Kristensena,b,*, K. Deboszc,d, G.W. McCartye b
a Department of Terrestrial Ecology, National Environmental Research Institute, DK-8600 Silkeborg, Denmark Department of Horticulture, Danish Institute of Agricultural Sciences, P.O. Box 102, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark c Department of Crop Physiology and Soil Science, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark d Supertrae A/S, DK-7362 Hampen, Denmark e USDA-ARS Environmental Chemistry Laboratory, Beltsville, MD 20705, USA
Received 4 May 2001; received in revised form 28 February 2003; accepted 14 March 2003
Abstract Tillage is known to decrease soil organic nitrogen (N) and carbon (C) pools with negative consequences for soil quality. This decrease is thought partly to be caused by exposure of protected organic matter to microbial degradation by the disturbance of soil structure. Little is known, however, about the short-term effects of tillage on mineralization of N and C, and microbial activity. We studied the short-term effects of two types of tillage (conventional plough- and a non-inverting-tillage) on mineralization and microbial N and C pools in a sandy loam under organic plough-tillage management. The release of active and protected (inactive) N by tillage was further studied in the laboratory by use of 15N labelling of the active pool of soil N followed by simulation of tillage by sieving through a 2 mm sieve. Results showed that the two types of tillage as well as the simulation of tillage had very few effects on mineralization and microbial pools. The simulation of tillage caused, however, a small release of N from a pool which was otherwise protected against microbial degradation. The use of soil crushing for disruption of larger macroaggregates (.425 mm) and chloroform fumigation for perturbation of the microbial biomass increased the release from both active and protected N pools. The relative contribution from the protected N pool was, however, similar in the three treatments (22 –27%), thus the pools subjected to mineralization were characterised by similar degree of protection. On the basis of isotopic composition the pools of N mineralised were indistinguishable. This suggests that the released N originated from the same pool, that is the soil microbial biomass. The study points to the microbial pool as the main source of labile N which may be released by tillage, and thus to its importance for sustained soil fertility in agricultural systems. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Carbon; Microbial biomass; Mineralization; Nitrogen; Physical protection; Organic matter; Tillage
1. Introduction There is considerable interest in the effects of tillage on soil quality. The effects are especially important in organic farming where nutrients are made available to plants solely by microbial decomposition of added or in situ organic matter because use of inorganic fertilisers is not allowed. Studies of ecosystems under long-term management involving plough-tillage and no-tillage practices as well as undisturbed native soils have demonstrated that tillage may * Corresponding author. Address: Department of Horticulture, Danish Institute of Agricultural Sciences, P.O. Box 102, Kirstinebjergvej 10, Aarslev DK-5792, Denmark. Tel.: þ45-6390-4133; fax: þ 45-6390-4394. E-mail address: [email protected] (H.L. Kristensen).
cause a substantial decrease of soil organic matter content and mineralization of nitrogen and carbon (e.g. Elliott, 1986; Beare et al., 1994a; McCarty et al., 1995; Six et al., 1999). Many studies of such long-term changes have focused on soil structure and the properties of soil aggregates. This work has formed the basis of a conceptual model for aggregate hierarchy (Tisdall and Oades, 1982). The model describes how primary mineral particles are bound together with bacterial, fungal, and plant debris into microaggregates. The microaggregates are bound together into macroaggregates by transient binding mechanisms such as microbial- and plant-derived polysaccharides. Roots and fungal hyphae are thought to act as temporary binding agents as they enmesh microaggregates into macroaggregates (Tisdall and Oades, 1982; Elliott, 1986; Oades, 1993;
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0038-0717(03)00159-7
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Jastrow et al., 1998; Six et al., 1999). The microaggregates, micropores as well as water stable macroaggregates are thought to protect a pool of organic matter from microbial degradation (Rovira and Greacen, 1957; Hassink, 1992; Beare et al., 1994a; Franzluebbers and Arshad, 1997). Part of this pool may be released by disturbance of soil structure by tillage and to some extent cause the differences in organic matter content seen for example between no-tillage and plough-tillage soils. The main effect of tillage on release of protected pools may be an increased turnover of macroaggregates and thereby a slower rate of microaggregate formation and organic matter stabilization (Six et al., 1999). Most of the studies of effects of soil structure disturbance on soils have used methods involving sieving, drying and fractionation of soil samples. Further they have focused on long term changes of physical, chemical and biological properties of soil aggregates or whole-soil samples. Less is known about the short-term response of microbial processes such as nitrogen mineralization by disturbance of intact soil samples (Rice et al., 1987; Franzluebbers, 1999), and few have focused on the effects of soil disturbance on soils which were disturbed regularly, for example plough-tillage soils (Nordmeyer and Richter, 1985; Calderon et al., 2000, 2001). Despite the regular disturbance of soil structure plough-tillage soils are found to contain a considerable amount of macro- and microaggregates (Beare et al., 1994b), and Jastrow (1996) shows that macroaggregate formation may take place relatively quickly after disturbance. To understand the cause of low organic matter and mineralizable N and C pools in plough-tillage soils it is important to improve our knowledge about the mechanisms that act on plough-tillage soils at each tillage event. A few studies have tried to relate N release to the mechanisms of protection of organic matter from microbial turnover (Grace et al., 1993; Hassink, 1992; Franzluebbers, 1999). Kristensen et al. (2000) suggested a new approach for laboratory studies of release of N from active or protected (inactive) N pools after disturbance of soil structure. This approach related mineralization of N to microbial activity by labelling the active microbial pool in intact samples with 15 N. The release of N from active or protected pools could consequently be studied by disturbance of soil structure and calculations based on mineralization of 15N and 14N from both intact and disturbed samples. The mechanisms for protection of N were studied by use of different methods for physical or biological disturbance of the intact soil samples. For example protected N was released by perturbation of the microbial biomass by chloroform fumigation of intact samples. This release was interpreted as coming from a biological protected N pool, which for example, may be protected from degradation inside fungal hyphae with low activity. The method used by Kristensen et al. (2000), however, should be combined with field experiments. This would show if the detailed laboratory study gives
meaningful results for short-term effects of tillage on mineralization of organic matter. The purpose of this study was to investigate the shortterm effects of soil structure disturbance by tillage on N and C mineralization in a plough-tillage soil. To better understand the origin of released N, mineralization from active and protected pools of soil organic N was related to mechanisms of protection such as protection inside macroaggregates and the microbial biomass. The approach involved a field experiment with two types of tillage and disturbance of soil structure in a laboratory experiment.
2. Materials and methods 2.1. Field site and experiment The field experiment was conducted in autumn 1998 on Rugballega˚rd Experimental Station, Denmark (098470 E, 558520 N) on sandy loam developed on diluvial clay, sand and gravel with 13% clay, 13% silt, 38% fine sand, and 33% coarse sand (Munkholm et al., 2001). An in situ analysis of field moist soil after performance of the two tillage types found that large macroaggregates were distributed with 19, 17 and 65% in size classes of , 2, 2– 4 and . 4 mm, respectively (Per Schjønning, unpublished data). The organic matter content was 3%, C/N 11 and pHCaCl2 5.8. The experimental field was converted to organic farming practices in 1995 and was under conventional plough-tillage to a depth of 20 cm. The last time plough-tillage was performed was in the autumn 1997 before initiation of this study. After harvest and removal of the preceding crop of oat (Avena sativa L.), treatments of non-inverting deep loosening tillage (NIT) and conventional plough-tillage (PT) were performed to prepare the soil for sowing of winter wheat. The tillage treatments were applied in a randomised block design with three replicate blocks of field plots (10 £ 40 m) each containing three replicate sampling sites as a block effect was expected. The tillage treatment of NIT was performed with a tillage implement composed of subsoiler tines combined with a rotovator and a drill. The depth of subsoil loosening was 35 cm. The PT treatment included mouldboard ploughing to a depth of 20 cm, followed by secondary tillage and drilling in one pass by a combined implement. Details on the tillage treatments are given in Munkholm et al. (2001). The tillage was performed on 30 Sept. and 1 Oct. 1998. The soil was sampled at three occasions: before tillage (23 Sept. 1998), one day after tillage (2 Oct. 1998), and twenty days after tillage (20 Oct. 1998). Intact as well as composite bulk soil samples were taken from 8 – 12 cm depth from the soil surface at each sampling site. The 8– 12 cm depth was chosen because the sample would approximately be the same soil before and after tillage with both tillage types. No differences were found for bulk density between treatments after performance of the two
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tillage types (Munkholm et al., 2001). The soil sampling was done by excavation of soil to make an even soil surface in the wanted sampling depth. Then stainless steel tubes (6 cm diameter, 4 cm length) were hammered into the soil and dug out. The intact samples in the sampling tubes were brought to the laboratory where they remained in the tubes throughout the experiment. The tubes were covered by a special flange during hammering and all handling of the samples were done carefully to minimize disturbance of soil structure. The average gravimetric soil water content at the 8 –12 cm depth was 16.8% of soil dry weight at 23 Sept.; 18.4% at 2 Oct.; and 23.3% at 20 Oct. 1998. 2.2. Mineralization and microbial biomass in the field experiment Net N and C mineralization in intact samples were determined according to Debosz et al. (1996). This involved immediate extraction of six replicate intact samples (two 2 from each block) for analysis of NHþ 4 and NO3 content as well as aerobic incubation of six other replicate samples (still in tubes and intact) for 14 days at 20 8C. Incubation was performed in 1 l bottles sealed with septa for gas sampling for CO2 analysis and containing a beaker with 20 ml of water for humidity control. Gas sampling was conducted from the bottles including blanks without soil samples biweekly during incubation after which the soil samples were extracted 2 for analysis of NHþ 4 and NO3 content. After each gas sampling, incubation bottles were purged with moistened air. Microbial biomass N and C content were determined on a composite soil sample from each treatment and block using a modification of the fumigation-extraction procedure with kC ¼ 0:45 and kN ¼ 0:54 (Vance et al., 1987; Kaiser et al., 1992). Soil from each sample was fumigated with ethanolfree CHCl3 for 18 h at 25 8C. The fumigated soil samples together with unfumigated samples were extracted in 0.5 M K2SO4 for total C and N analysis. 2.3. Nitrogen-15 labelling in the disturbance experiment The laboratory study of simulated tillage effects on protected and active N release involved incubation of a total of 63 intact soil samples taken from the nine sampling sites (three in each block) in the field experiment before tillage (23 Sept. 1998). In the laboratory the intact samples were left uncovered overnight for adjustment of water content by evaporation (avg. loss of 1.9 ml/sample) to allow injection of a 15N labelling solution without altering soil moisture. Each sample was then cut to an approximate dry weight of 125 g (3.0 –3.7 cm length) by removal of soil from the top end of the sampling tube. The dry weight obtained was based on bulk density measurements of three extra samples collected from each block. The active N pool in the intact samples was labelled with 15N. This was done by injection of a solution containing (15NH4)2SO4 (5 mg N g21 dry weight soil, 98% 15N) and glucose (150 mg C g21 dry
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weight soil) followed by incubation for 6 days at 20 8C. The incubation allowed incorporation of the label into the active soil N pool participating in mineralization – immobilization turnover within this period (Kristensen et al., 2000). The injections were performed in series of nine injections of the labelling solution in each sample by use of a 1-ml hypodermic syringe fitted with a needle (0.5 mm o.d., 30 mm length). The depth of injection was 2.5 –3.0 cm and the needle was pulled out evenly as the solution was dispensed. A total of 1.8 ml of solution was injected into each sample giving a final soil water content of 15.7% of soil dry weight. The treated soil samples were placed in random order together with wet paper towel into three plastic boxes (36 L) and incubated for 6 days at 20 8C. The boxes were opened for aeration every third day. 2.4. Incubation series in the disturbance experiment After 15N labelling of the active organic N pool, one sample from each of the 9 replicate sampling sites was extracted for analysis of initial content of inorganic 15N and 14 N. The remaining replicates were subjected to different treatments of disturbance of soil structure/soil microbial biomass or left intact. The treatments were: (a) left intact; (b) sieving through a 2-mm mesh followed by repacking to original bulk density to simulate tillage; (c) crushing to pass a 425-mm mesh to disrupt larger macroaggregates followed by repacking to original bulk density; (d) left intact followed by incubation in acetylene atmosphere; (e) chloroform fumigation of intact samples for 24 h to perturb microbial biomass followed by incubation in acetylene atmosphere. All samples were then incubated for 14 days at 20 8C with wet paper towels for moisture control to allow for mineralization of soil organic N. The samples from (a), (b) and (c) were incubated in 36 l boxes with aeration every third day as described above for the labelling procedure. The samples from (d) and (e) were incubated in a vacuum desiccator which had acetylene injected through a septum to a concentration of 1% of the atmosphere. The desiccator was opened for aeration followed by injection of acetylene every third day. The incubation with acetylene was to inhibit nitrification activity in the intact samples as chloroform fumigation is known to inhibit nitrification in soil. The accumulation of NHþ 4 as end product of mineralization in the soil may stimulate NHþ 4 immobilization and thus influence net mineralization of soil organic N. 2.5. Sample analysis After termination of the net N and C mineralization incubation and the disturbance experiment, each sample was sieved (mesh size 2 mm), mixed, and subsampled for 2 analysis of the soil inorganic N pool. Soil NHþ 4 and NO3 were extracted in 1 M KCl, the extracts were centrifuged and the supernatant was analysed by standard colorimetric methods. Net N mineralization was calculated as
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the difference between final and initial inorganic N contents of samples. Gas samples (1 ml) were withdrawn from incubation bottles and the CO2 content was determined by gas chromatography with thermal conductivity detection (Microlab, Denmark). The C mineralization was calculated as the difference between CO2 –C measured in bottles containing samples and in blanks. The K2SO4 soil extracts of microbial biomass C and N pools were analysed for total organic C content by automated ultraviolet persulphate oxidation using a Dohrmann DC-180 carbon analyser. Total organic N in the extracts was determined by the Kjeldahl method, on a Tecator total N-analyser. The KCl extracts from the disturbance experiment were prepared for analysis of isotopic content of inorganic N þ (NO2 3 þ NH4 ) by use of the diffusion method by Brooks et al. (1989) slightly modified according to Rackwitz (unpublished data) where Eppendorf cups instead of stainless steel wire were used for placement of the acidified filter disk during diffusion. Analysis of 15N in diffused samples as well as in dried and ground soil samples was performed using an isotope mass spectrometer (Europa Scientific Ltd.) interfaced with an automated N –C analyser. The analysis by the mass spectrometer also provided measures of total N and C content in dried soil samples. The average soil water contents in the samples were 15.4 and 15.0% of dry weight in the intact and sieved/crushed treatments, respectively, at the end of the disturbance experiment. At this point recovery of 15N was found to be 87 – 94% of the amount measured after the labelling incubation.
Ax ¼ atom percent 15N in the active N pool assumed to be the atom percent 15N measured in the mineralized NO2 3 pool in each intact soil sample Y ¼ amount of protected N mineralized to NO2 3 in each disturbed soil sample Ay ¼ atom percent 15N in the protected N pool (assumed to be natural abundance 0.366%) Amin ¼ atom percent 15N in the mineralized NO2 3 pool in each disturbed soil sample. In the field experiment, effects of tillage treatment and sampling date on soil C and N pools and mineralization rates were examined by analysis of variance using a general linear mixed model with subsamples nested within treatments. No block effects were found. Significant interaction was found between tillage system and sampling dates. Accordingly, a repeated-measures model was constructed including tillage and sampling dates as fixed factors taking into account the different variances from sampling dates and the covariances between sampling dates. Statistical significance of differences in release of N pools between treatments in the laboratory experiment were tested by analysis of variance (F-test) with subsamples nested within treatments. The test was followed by pairwise comparisons by Tukey’s studentized range test (Proc GLM). All statistical tests were performed by use of the SAS system for data analysis (SAS Institute Inc., Cary, NC, USA). Statistical significant differences between results were considered at p , 0:05:
2.6. Data analysis
3. Results
The contributions from active and protected pools of soil organic N to the mineral N pool formed during incubation of the disturbed soil samples were calculated according to Kristensen et al. (2000). The calculation was based on measurements of the dilution of 15N label by 14N in the pool of mineralized N in disturbed relative to intact samples. The 15 N and 14N already present in the samples after the labelling incubation and prior to the performance of disturbance treatments was accounted for by subtraction of the amounts found in the intact samples extracted at this point. The principle of isotope dilution (Jansson, 1958) can be expressed in the following equation:
3.1. The field experiment
Amin ¼ ðAx X þ Ay YÞ=ðX þ YÞ
ð1Þ
Mineral N pools and net N mineralization showed no significant differences between tillage systems in the soil from 8 –12 cm depth at the two sampling dates after tillage (Table 1). The level of the mineral N pool increased in both Table 1 2 Effect of tillage system on soil inorganic N content (NHþ 4 þ NO3 ) and net N mineralization for three sampling dates. Numbers in parentheses are standard errors ðn ¼ 6Þ: Different letters behind means denote significant differences between tillage treatments and between sampling dates Tillage
After harvest, 23 Sept. 1998
Post-tillage 2 Oct. 1998
20 Oct. 1998
which can be solved for both X and Y when: Z ¼XþY
ð2Þ
where Z ¼ amount of N mineralized to NO2 3 in each disturbed soil sample X ¼ amount of active N mineralized to NO2 3 in each disturbed soil sample
Inorganic N content (mg N g21 dry weight soil) PTa 7.5 (0.4)b 13.2 (1.0)a NITb 10.8 (1.0)a
5.4 (1.0)b 5.6 (1.0)b
Net N mineralization (mg N g21 dry weight soil day21) PTa 0.3 (0.1)b 0.3 (0.1)b NITb 0.3 (0.1)b
0.6 (0.1)a 0.7 (0.1)a
a b
PT: plough-tillage. NIT: non-inverting tillage.
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Table 2 Effect of tillage system on CO2 production for three sampling dates. Numbers in parentheses are standard errors ðn ¼ 6Þ: Different letters behind means denote significant differences between tillage treatments and between sampling dates for each incubation period Tillage
After harvest
Post-tillage
23 Sept. 1998
2 Oct. 1998
Day 0–4
Day 4–10
CO2 production (mg C g21 dry weight soil day21) PTa 5.6 (1.3)b 4.4 (0.6)c NIT a
Day 0– 4
Day 4 –13
Day 0–6
Day 6– 15
7.2 (1.8)ab 9.0 (0.8)a
4.5 (0.7)c 5.3 (0.7)c
6.4 (1.1)ab 8.4 (1.1)a
3.0 (1.0)c 4.0 (1.0)c
Abbreviation as in Table 1.
tillage systems immediately after tillage and decreased after 3 weeks. Net N mineralization was at the same level before and the day after tillage. Three weeks later the rate increased to approximately double the rate just after tillage. The CO2 production during the first 4 days of incubation was higher the day after NIT tillage than before (Table 2). Three weeks after NIT tillage the CO2 production during the first 6 days of incubation was also elevated. Soil tillage had no effect on microbial biomass C and N content in the 8– 12 cm depth (Table 3). 3.2. The disturbance experiment In general a substantial amount of inorganic 15N and 14N was present in the samples after labelling the active biomass, and the pools increased in all treatments during the next 14 days of incubation (Table 4). In incubations without chloroform or acetylene treatment, NHþ 4 pools were found to be small and stayed more or less at the same level (app. 0.5 mg N kg21) during the experimental period. Where chloroform and/or acetylene were applied the NO2 3 pool remained at the same level (7 – 8 mg N kg21) during the incubation which indicated an effective inhibition of nitrification during the experiment (results not shown). The results presented in Table 4 were used for calculation of contributions of active and protected N pools to mineral N release as shown in Fig. 1. In the laboratory experiment Table 3 Effect of tillage system on microbial biomass C and N content for three sampling dates. Numbers in parentheses are standard errors ðn ¼ 3Þ: Different letters behind means denote significant differences between tillage treatments and between sampling dates Tillage
After harvest, 23 Sept. 1998
Table 4 þ Total pools of inorganic 15N and 14þ15N(NO2 3 þ NH4 ) used for calculation of net mineralization of protected and active N during incubation of soil samples in the disturbance experiment. Numbers in parentheses are standard errors ðn ¼ 9Þ: Different letters behind means denote significant differences between disturbance treatments Treatment
15
N (mg g21 dry weight soil)
N (mg g21 dry weight soil)
Start Intact Sieved Crushed
0.82 (0.13)a 0.97 (0.14)a 0.97 (0.13)a 1.26 (0.14)ab
9.24 (1.54)a 13.03 (3.51)a 14.17 (2.17)a 23.86 (4.00)a
Start þ acetylene Intact þ acetylene Intact þ chloroform fumigation þ acetylene
0.71 (0.11)a 0.82 (0.14)a 1.91 (0.11)b
8.86 (2.37)a 11.43 (2.17)a 47.82 (4.86)b
14þ15
20 Oct. 1998
Microbial biomass C (mg g21 dry weight soil) PTa 197 (11)a 185 (9)a NIT 199 (9)a
181 (17)a 200 (17)a
Microbial biomass N (mg g21 dry weight soil) PT 37 (2)a 36 (1)a NIT 40 (1)a
39 (4)a 39 (4)a
Abbreviation as in Table 1.
sieving through 2 mm mesh size was used to simulate soil structure disturbance by tillage and this treatment did not significantly influence net N mineralization (Table 4, Fig. 1). The rates found in intact and sieved samples were similar to the rates of net mineralization measured just before and after tillage in the field experiment (0.3 –0.4 mg N g21 day21). Any differences between the results obtained from the field and the laboratory studies were, however, not tested statistically due to major differences in experimental conditions (e.g. field or lab conditions, 15N/glucose injection). The total mineral N release caused by soil sieving was 4.9 mg N g21 of which 1.1 mg N g21 was released as protected N and constituted 22% of net N mineralization (Fig. 1). This amount was, however, not significantly different from zero. The more extreme physical disturbance caused by soil crushing to pass a 425 mm mesh increased net mineralization by a factor three to 14.6 mg N g21. The contribution from protected N pools amounted to 3.8 mg N g21 and made up 26% of total net N mineralization after crushing. The intact cores that had been incubated in acetylene atmosphere had a net N mineralization rate at the same level as those incubated without acetylene (Fig. 1). Perturbation of the microbial biomass by chloroform fumigation increased net mineralization by a factor of 12 amounting to 39.0 mg N g21 compared to
Post-tillage 2 Oct. 1998
a
20 Oct. 1998
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Fig. 1. The amount of inorganic N released from active (black columns) or protected (grey columns) N pools during incubation of soil samples in which soil structure or the microbial biomass had been disturbed. The soil samples were Intact; Sieved (2 mm mesh size); Crushed (.425 mm); Intact þ C2H2 (incubated in acetylene atmosphere) or Chloroform þ C2H2 (fumigated with chloroform and incubated in acetylene atmosphere). Bars indicate standard errors ðn ¼ 9Þ:
the intact þ acetylene treatment. Protected N release was increased to 10.5 mg N g21 and made up 27% of net mineralization. The percentage of protected N contributions was not significantly different between the sieved, crushed, and chloroform fumigated treatments (Fig. 1).
4. Discussion 4.1. Effect of tillage on N and C mineralization and microbial pools Net N mineralization rates were not significantly influenced by NIT and PT treatments in the field or by simulation of tillage by soil sieving in the laboratory experiment (Table 1, Fig. 1). This result is comparable to that of laboratory studies that found that net N mineralization was not influenced by soil structure disturbance in PT soils (Beare et al., 1994a; Calderon et al., 2000; Kristensen et al., 2000). In the present study the NIT soil was comparable to a PT soil as the NIT treatment was performed for the first time after several years of conventional ploughtillage management. A few laboratory studies have reported a stimulating effect on net N mineralization rates of soil structure disturbance by mixing of PT soil (Nordmeyer and Richter, 1985; Stenger et al., 1995). By contrast, stimulating
effects are generally observed in no-tillage and grassland soils when disturbed by mixing or sieving (Hassink, 1992; Grace et al., 1993; Kristensen et al., 2000); although decrease of net N mineralization has also been reported (Franzluebbers, 1999). The increase in CO2 production in the NIT soil after tillage indicated release of a small amount of readily available C (Table 2). Others have instead found a decrease in CO2 production after tillage or tillage simulation (Petersen and Klug, 1994; Calderon et al., 2000; 2001). Any effects on CO2 mineralization did not, however, influence the pools of microbial C biomass. Both the NIT and PT treatments did not significantly affect the microbial N pool (Table 3). Also Jenkinson (1988) and Calderon et al. (2000) have reported minimal effects of soil structure disturbance by sieving on microbial pools in plough-tillage soils, whereas Calderon et al. (2001) found short-term effects of tillage on the microbial N pool and microbial community structure. 4.2. Protected N release by simulation of tillage The lack of effects of soil structure disturbance on net N mineralization rates found both in the field and laboratory experiments indicates that disturbance of soil structure by sieving through 2 mm mesh and by the two field tillage
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treatments were comparable with regard to effects on net N mineralization. The lack of effects, however, forms only a weak basis for judging the general usefulness of sieving through 2 mm mesh for simulation of tillage as sieving also can be said to simulate undisturbed conditions as the treatments gave similar results. The protected N release of 22% for total mineral N in the soil with sieving treatment was comparable to results obtained from a PT silt loam by use of the same 15N labelling methodology and sieving through 2 mm mesh (Kristensen et al., 2000). In the silt loam the kinetics of N release were studied and all protected N was found to be released within 3 weeks after sieving. Within the first 2 weeks, 92% of this was released amounting to 3.4 mg N g 21. With a total mineral N release of 15.2 mg N g21 during the 2 weeks the protected N release made up 23% of the total. Thus similar amounts of protected N relative to total mineral N were released as in the present study even though rates of net mineralization and microbial N pools were three times higher in the silt loam. For comparison, the protected N release was 33% after sieving when the silt loam was under no-tillage management which indicated a higher protecting ability compared to the PT soils (Kristensen et al., 2000). 4.3. Protected N release by soil crushing Protected N release was increased by the crushing treatment. This suggests that the larger macroaggregates and other matter (. 425 mm) that were disrupted by the treatment contained a pool of N which was released by net mineralization. The pool released by crushing had, however, the same relative contribution from protected N as the N released by sieving. The matter that was crushed seemed therefore to have the same protecting ability as the wholesoil when disturbed by the sieving treatment. This is surprising as the crushing treatment was a more extreme disturbance event than tillage as also indicated by the increase of net N mineralization due to crushing. Also Franzluebbers and Arshad (1997) found crushing of smaller aggregates (0.25 – 1.00 mm) to release more protected C than crushing of larger macroaggregates (1.0 –5.6 mm) in a PT soil. Macroaggregates in PT soils have, however, been found to have relatively low stability (Beare et al., 1994b). Furthermore results by Six et al. (1999) have indicated that the turnover of macroaggregates in PT soils is high due to the continuous disruption of soil structure by tillage. This may decrease the formation of microaggregates inside macroaggregates and thereby decrease the stabilization of organic matter in the soil (Six et al., 1999). If such an effect have caused a low degree of protection of organic matter in the PT soil, it may explain why the crushing treatment did not release relatively more protected N compared to that of sieving.
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4.4. Origin of protected N release Comparison of results obtained from the sieving and crushing treatments may point in another direction for interpretation when also considering the results from the microbial biomass perturbation treatment by chloroform fumigation. In this treatment the release of N by net mineralization was further increased and again the amount of released protected N was equivalent to that of the sieved and crushed treatments when calculated on a relative basis to total N release. This suggests that N released by the three treatments may have originated from the same pool of organic N. The chloroform fumigation treatment followed by incubation for mineralization is used widely as a method for release of microbial biomass N (e.g. Franzluebbers et al., 1995). If the N released by soil structure disturbance originated from the same pool of organic N involved in the release caused by biomass perturbation, then this suggests that N mineralization flush associated with tillage originates from the microbial biomass N pool. This is supported by findings of Kristensen et al. (2000) who studied the kinetics of N release over a 5 week period after soil structure disturbance. Their results showed that any stimulating effect of sieving on net N mineralization was seen already within the first week after sieving. This indicates that the released N originated from a readily available N pool, which could be the microbial biomass. Similarly Gupta and Germida (1988) suggested that microbial biomass constitutes the primary source of organic matter released by tillage. Other physical and chemical disturbance treatments like soil crushing, drying– rewetting and freeze –thaw cycles may also cause a short-term release of organic N or C by mineralization, typically within less than 10 days (Kieft et al., 1987; DeLuca et al., 1992; Franzluebbers and Arshad, 1997; Franzluebbers, 1999). These studies also suggested that the release mainly originated from the microbial biomass in the soil. The three treatments in the present study: sieving, crushing and chloroform fumigation may possibly be regarded as three degrees of treatments to perturb the soil organic N pool representing a low, intermediate, and high degree of disturbance, respectively. It is possible that the treatments also represent three degrees of disturbance of the microbial biomass, being the main contributor to short-term mineral N release.
5. Conclusions Short-term effects of plough- and non-inverting tillage on net N and C mineralization and microbial pools were few in plough-tillage soil. The use of soil sieving through 2 mm mesh for simulation of soil structure disturbance by tillage gave similar results as obtained by tillage in the field. Despite the lack of effects of tillage on net N mineralization, simulation of tillage was found to cause a small release of N
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from an organic pool which was previously protected from microbial degradation. The use of soil crushing for disruption of larger macroaggregates (. 425 mm) and chloroform fumigation for perturbation of the microbial biomass were more extreme treatments for disturbance of the soil organic N pool, and caused an increased release of both active and protected pools. The pools released by the three treatments were characterized by similar degree of protection. This suggests that the mineral N released by the treatments originated from the same organic N pool in the soil, that is the soil microbial biomass. Thus the microbial biomass seemed to contain a significant pool of protected (inactive) N. Aggregates did not give more protection against microbial degradation than the whole-soil when under plough-tillage management.
Acknowledgements This work was supported by the Danish Research Centre for Organic Farming and the OECD Cooperative Research Programme: Biological Resource Management for Sustainable Agriculture Systems. We thank Dr K. Kristensen for ˚ en and statistical support and Anette Clausen, Karen A Karen K. Jakobsen for skilful technical assistance.
References Beare, M.H., Cabrera, M.L., Hendrix, P.F., Coleman, D.C., 1994a. Aggregate-protected and unprotected organic matter pools in conventional- and no-tillage soils. Soil Science Society of America Journal 58, 787– 795. Beare, M.H., Hendrix, P.F., Coleman, D.C., 1994b. Water-stable aggregates and organic matter fractions in conventional- and no-tillage soils. Soil Science Society of America Journal 58, 777–786. Brooks, P.D., Stark, J.M., McInteer, B.B., Preston, T., 1989. Diffusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society of America Journal 53, 1707–1711. Calderon, F.J., Jackson, L.E., Scow, K.M., Rolston, D.E., 2000. Microbial responses to simulated tillage in cultivated and uncultivated soils. Soil Biology and Biochemistry 32, 1547–1559. Calderon, F.J., Jackson, L.E., Scow, K.M., Rolston, D.E., 2001. Short-term dynamics of nitrogen, microbial activity, and phospholipid fatty acids after tillage. Soil Science Society of America Journal 65, 118–126. Debosz, K., Schjønning, P., Simmelsgaard, S.E., 1996. N mineralization in undisturbed soil. In: Van Cleemput, O., Hofman, G., Vermoesen, A. (Eds.), Progress in Nitrogen Cycling Studies, Kluwer, Dordrecht, pp. 37–40. DeLuca, T.H., Keeney, D.R., McCarty, G.W., 1992. Effect of freeze–thaw events on mineralization of soil nitrogen. Biology and Fertility of Soils 14, 116 –120. Elliott, E.T., 1986. Aggregate structure and carbon, nitrogen, and phosphorus in native and cultivated soils. Soil Science Society of America Journal 50, 627–633. Franzluebbers, A.J., 1999. Potential C and N mineralization and microbial biomass from intact and increasingly disturbed soils of varying texture. Soil Biology and Biochemistry 31, 1083– 1090. Franzluebbers, A.J., Arshad, M.A., 1997. Soil microbial biomass and mineralizable carbon of water-stable aggregates. Soil Science Society of America Journal 61, 1090–1097.
Franzluebbers, A.J., Hons, F.M., Zuberer, D.A., 1995. Soil organic carbon, microbial biomass, and mineralizable carbon and nitrogen in sorghum. Soil Science Society of America Journal 59, 460– 466. Grace, P.R., MacRae, I.C., Myers, R.J.K., 1993. Temporal changes in microbial biomass and N mineralization under simulated field cultivation. Soil Biology and Biochemistry 25, 1745–1753. Gupta, V.V.S.R., Germida, J.J., 1988. Distribution of microbial biomass and its activity in different soil aggregate size classes as affected by cultivation. Soil Biology and Biochemistry 20, 777–786. Hassink, J., 1992. Effects of soil texture and structure on carbon and nitrogen mineralization in grassland soils. Biology and Fertility of Soils 14, 126–134. Jansson, S.L., 1958. Tracer studies on nitrogen transformations in soil with special attention to mineralisation–immobilization relationships. Kungliga Lantbruksho¨gskolans Annaler 24, 100– 361. Jastrow, J.D., 1996. Soil aggregate formation and the accrual of particulate and mineral-associated organic matter. Soil Biology and Biochemistry 28, 665–676. Jastrow, J.D., Miller, R.M., Lussenhop, J., 1998. Contributions of interacting biological mechanisms to soil aggregate stabilization in restored prairie. Soil Biology and Biochemistry 30, 905 –916. Jenkinson, D.S., 1988. Determination of microbial biomass carbon and nitrogen in soil. In: Wilson, J.R., (Ed.), Advances in nitrogen cycling in agricultural ecosystems, CAB International, pp. 368 –386. Kaiser, E.A., Mueller, T., Joergensen, R.G., Insam, H., Heinemeyer, O., 1992. Evaluation of methods to estimate the soil microbial biomass and the relationship with soil texture and organic matter. Soil Biology and Biochemistry 24, 675–683. Kieft, T.L., Soroker, E., Firestone, M.K., 1987. Microbial biomass response to a rapid increase in water potential when dry soil is wetted. Soil Biology and Biochemistry 19, 119–126. Kristensen, H.L., McCarty, G.W., Meisinger, J.J., 2000. Effects of soil structure disturbance on mineralization of organic soil N. Soil Science Society of America Journal 64, 371 –378. McCarty, G.W., Meisinger, J.J., Jenniskens, F.M.M., 1995. Relationships between total-N, biomass-N and active-N in soil under different tillage and N fertilizer treatments. Soil Biology and Biochemistry 27, 1245–1250. Munkholm, L.J., Schjønning, P., Rasmussen, K.J., 2001. Non-inversion tillage effects on soil mechanical properties of a humid sandy loam. Soil and Tillage Research 62, 1–14. Nordmeyer, H., Richter, J., 1985. Incubation experiments on nitrogen mineralization in loess and sandy soils. Plant and Soil 83, 433–445. Oades, J.M., 1993. The role of biology in the formation, stabilization and degradation of soil structure. Geoderma 56, 377 –400. Petersen, S.O., Klug, M.J., 1994. Effects of sieving, storage, and incubation temperature on the phospholipid fatty acid profile of a soil microbial community. Applied and Environmental Microbiology 60, 2421– 2430. Rice, C.W., Grove, J.H., Smith, M.S., 1987. Estimating soil net nitrogen mineralization as affected by tillage and soil drainage due to topographic position. Canadian Journal of Soil Science 67, 513 –520. Rovira, A.D., Greacen, E.L., 1957. The effect of aggregate disruption on the activity of microorganisms in the soil. Australian Journal of Agricultural Research 8, 659 –673. Six, J., Elliott, E.T., Paustian, K., 1999. Aggregate and soil organic matter dynamics under conventional and no-tillage systems. Soil Science Society of America Journal 63, 1350–1358. Stenger, R., Priesack, E., Beese, F., 1995. Rates of net nitrogen mineralization in disturbed and undisturbed soils. Plant and Soil 171, 323 –332. Tisdall, J.M., Oades, J.M., 1982. Organic matter and water-stable aggregates in soils. Journal of Soil Science 33, 141–163. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. Microbial biomass measurements in forest soils: the use of chloroform fumigationincubation method in strongly acid soils. Soil Biology and Biochemistry 19, 697–702.
Short-term effects of tillage on mineralization of nitrogen and carbon in soil H.L. Kristensena,b,*, K. Deboszc,d, G.W. McCartye b
a Department of Terrestrial Ecology, National Environmental Research Institute, DK-8600 Silkeborg, Denmark Department of Horticulture, Danish Institute of Agricultural Sciences, P.O. Box 102, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark c Department of Crop Physiology and Soil Science, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark d Supertrae A/S, DK-7362 Hampen, Denmark e USDA-ARS Environmental Chemistry Laboratory, Beltsville, MD 20705, USA
Received 4 May 2001; received in revised form 28 February 2003; accepted 14 March 2003
Abstract Tillage is known to decrease soil organic nitrogen (N) and carbon (C) pools with negative consequences for soil quality. This decrease is thought partly to be caused by exposure of protected organic matter to microbial degradation by the disturbance of soil structure. Little is known, however, about the short-term effects of tillage on mineralization of N and C, and microbial activity. We studied the short-term effects of two types of tillage (conventional plough- and a non-inverting-tillage) on mineralization and microbial N and C pools in a sandy loam under organic plough-tillage management. The release of active and protected (inactive) N by tillage was further studied in the laboratory by use of 15N labelling of the active pool of soil N followed by simulation of tillage by sieving through a 2 mm sieve. Results showed that the two types of tillage as well as the simulation of tillage had very few effects on mineralization and microbial pools. The simulation of tillage caused, however, a small release of N from a pool which was otherwise protected against microbial degradation. The use of soil crushing for disruption of larger macroaggregates (.425 mm) and chloroform fumigation for perturbation of the microbial biomass increased the release from both active and protected N pools. The relative contribution from the protected N pool was, however, similar in the three treatments (22 –27%), thus the pools subjected to mineralization were characterised by similar degree of protection. On the basis of isotopic composition the pools of N mineralised were indistinguishable. This suggests that the released N originated from the same pool, that is the soil microbial biomass. The study points to the microbial pool as the main source of labile N which may be released by tillage, and thus to its importance for sustained soil fertility in agricultural systems. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Carbon; Microbial biomass; Mineralization; Nitrogen; Physical protection; Organic matter; Tillage
1. Introduction There is considerable interest in the effects of tillage on soil quality. The effects are especially important in organic farming where nutrients are made available to plants solely by microbial decomposition of added or in situ organic matter because use of inorganic fertilisers is not allowed. Studies of ecosystems under long-term management involving plough-tillage and no-tillage practices as well as undisturbed native soils have demonstrated that tillage may * Corresponding author. Address: Department of Horticulture, Danish Institute of Agricultural Sciences, P.O. Box 102, Kirstinebjergvej 10, Aarslev DK-5792, Denmark. Tel.: þ45-6390-4133; fax: þ 45-6390-4394. E-mail address: [email protected] (H.L. Kristensen).
cause a substantial decrease of soil organic matter content and mineralization of nitrogen and carbon (e.g. Elliott, 1986; Beare et al., 1994a; McCarty et al., 1995; Six et al., 1999). Many studies of such long-term changes have focused on soil structure and the properties of soil aggregates. This work has formed the basis of a conceptual model for aggregate hierarchy (Tisdall and Oades, 1982). The model describes how primary mineral particles are bound together with bacterial, fungal, and plant debris into microaggregates. The microaggregates are bound together into macroaggregates by transient binding mechanisms such as microbial- and plant-derived polysaccharides. Roots and fungal hyphae are thought to act as temporary binding agents as they enmesh microaggregates into macroaggregates (Tisdall and Oades, 1982; Elliott, 1986; Oades, 1993;
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0038-0717(03)00159-7
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Jastrow et al., 1998; Six et al., 1999). The microaggregates, micropores as well as water stable macroaggregates are thought to protect a pool of organic matter from microbial degradation (Rovira and Greacen, 1957; Hassink, 1992; Beare et al., 1994a; Franzluebbers and Arshad, 1997). Part of this pool may be released by disturbance of soil structure by tillage and to some extent cause the differences in organic matter content seen for example between no-tillage and plough-tillage soils. The main effect of tillage on release of protected pools may be an increased turnover of macroaggregates and thereby a slower rate of microaggregate formation and organic matter stabilization (Six et al., 1999). Most of the studies of effects of soil structure disturbance on soils have used methods involving sieving, drying and fractionation of soil samples. Further they have focused on long term changes of physical, chemical and biological properties of soil aggregates or whole-soil samples. Less is known about the short-term response of microbial processes such as nitrogen mineralization by disturbance of intact soil samples (Rice et al., 1987; Franzluebbers, 1999), and few have focused on the effects of soil disturbance on soils which were disturbed regularly, for example plough-tillage soils (Nordmeyer and Richter, 1985; Calderon et al., 2000, 2001). Despite the regular disturbance of soil structure plough-tillage soils are found to contain a considerable amount of macro- and microaggregates (Beare et al., 1994b), and Jastrow (1996) shows that macroaggregate formation may take place relatively quickly after disturbance. To understand the cause of low organic matter and mineralizable N and C pools in plough-tillage soils it is important to improve our knowledge about the mechanisms that act on plough-tillage soils at each tillage event. A few studies have tried to relate N release to the mechanisms of protection of organic matter from microbial turnover (Grace et al., 1993; Hassink, 1992; Franzluebbers, 1999). Kristensen et al. (2000) suggested a new approach for laboratory studies of release of N from active or protected (inactive) N pools after disturbance of soil structure. This approach related mineralization of N to microbial activity by labelling the active microbial pool in intact samples with 15 N. The release of N from active or protected pools could consequently be studied by disturbance of soil structure and calculations based on mineralization of 15N and 14N from both intact and disturbed samples. The mechanisms for protection of N were studied by use of different methods for physical or biological disturbance of the intact soil samples. For example protected N was released by perturbation of the microbial biomass by chloroform fumigation of intact samples. This release was interpreted as coming from a biological protected N pool, which for example, may be protected from degradation inside fungal hyphae with low activity. The method used by Kristensen et al. (2000), however, should be combined with field experiments. This would show if the detailed laboratory study gives
meaningful results for short-term effects of tillage on mineralization of organic matter. The purpose of this study was to investigate the shortterm effects of soil structure disturbance by tillage on N and C mineralization in a plough-tillage soil. To better understand the origin of released N, mineralization from active and protected pools of soil organic N was related to mechanisms of protection such as protection inside macroaggregates and the microbial biomass. The approach involved a field experiment with two types of tillage and disturbance of soil structure in a laboratory experiment.
2. Materials and methods 2.1. Field site and experiment The field experiment was conducted in autumn 1998 on Rugballega˚rd Experimental Station, Denmark (098470 E, 558520 N) on sandy loam developed on diluvial clay, sand and gravel with 13% clay, 13% silt, 38% fine sand, and 33% coarse sand (Munkholm et al., 2001). An in situ analysis of field moist soil after performance of the two tillage types found that large macroaggregates were distributed with 19, 17 and 65% in size classes of , 2, 2– 4 and . 4 mm, respectively (Per Schjønning, unpublished data). The organic matter content was 3%, C/N 11 and pHCaCl2 5.8. The experimental field was converted to organic farming practices in 1995 and was under conventional plough-tillage to a depth of 20 cm. The last time plough-tillage was performed was in the autumn 1997 before initiation of this study. After harvest and removal of the preceding crop of oat (Avena sativa L.), treatments of non-inverting deep loosening tillage (NIT) and conventional plough-tillage (PT) were performed to prepare the soil for sowing of winter wheat. The tillage treatments were applied in a randomised block design with three replicate blocks of field plots (10 £ 40 m) each containing three replicate sampling sites as a block effect was expected. The tillage treatment of NIT was performed with a tillage implement composed of subsoiler tines combined with a rotovator and a drill. The depth of subsoil loosening was 35 cm. The PT treatment included mouldboard ploughing to a depth of 20 cm, followed by secondary tillage and drilling in one pass by a combined implement. Details on the tillage treatments are given in Munkholm et al. (2001). The tillage was performed on 30 Sept. and 1 Oct. 1998. The soil was sampled at three occasions: before tillage (23 Sept. 1998), one day after tillage (2 Oct. 1998), and twenty days after tillage (20 Oct. 1998). Intact as well as composite bulk soil samples were taken from 8 – 12 cm depth from the soil surface at each sampling site. The 8– 12 cm depth was chosen because the sample would approximately be the same soil before and after tillage with both tillage types. No differences were found for bulk density between treatments after performance of the two
H.L. Kristensen et al. / Soil Biology & Biochemistry 35 (2003) 979–986
tillage types (Munkholm et al., 2001). The soil sampling was done by excavation of soil to make an even soil surface in the wanted sampling depth. Then stainless steel tubes (6 cm diameter, 4 cm length) were hammered into the soil and dug out. The intact samples in the sampling tubes were brought to the laboratory where they remained in the tubes throughout the experiment. The tubes were covered by a special flange during hammering and all handling of the samples were done carefully to minimize disturbance of soil structure. The average gravimetric soil water content at the 8 –12 cm depth was 16.8% of soil dry weight at 23 Sept.; 18.4% at 2 Oct.; and 23.3% at 20 Oct. 1998. 2.2. Mineralization and microbial biomass in the field experiment Net N and C mineralization in intact samples were determined according to Debosz et al. (1996). This involved immediate extraction of six replicate intact samples (two 2 from each block) for analysis of NHþ 4 and NO3 content as well as aerobic incubation of six other replicate samples (still in tubes and intact) for 14 days at 20 8C. Incubation was performed in 1 l bottles sealed with septa for gas sampling for CO2 analysis and containing a beaker with 20 ml of water for humidity control. Gas sampling was conducted from the bottles including blanks without soil samples biweekly during incubation after which the soil samples were extracted 2 for analysis of NHþ 4 and NO3 content. After each gas sampling, incubation bottles were purged with moistened air. Microbial biomass N and C content were determined on a composite soil sample from each treatment and block using a modification of the fumigation-extraction procedure with kC ¼ 0:45 and kN ¼ 0:54 (Vance et al., 1987; Kaiser et al., 1992). Soil from each sample was fumigated with ethanolfree CHCl3 for 18 h at 25 8C. The fumigated soil samples together with unfumigated samples were extracted in 0.5 M K2SO4 for total C and N analysis. 2.3. Nitrogen-15 labelling in the disturbance experiment The laboratory study of simulated tillage effects on protected and active N release involved incubation of a total of 63 intact soil samples taken from the nine sampling sites (three in each block) in the field experiment before tillage (23 Sept. 1998). In the laboratory the intact samples were left uncovered overnight for adjustment of water content by evaporation (avg. loss of 1.9 ml/sample) to allow injection of a 15N labelling solution without altering soil moisture. Each sample was then cut to an approximate dry weight of 125 g (3.0 –3.7 cm length) by removal of soil from the top end of the sampling tube. The dry weight obtained was based on bulk density measurements of three extra samples collected from each block. The active N pool in the intact samples was labelled with 15N. This was done by injection of a solution containing (15NH4)2SO4 (5 mg N g21 dry weight soil, 98% 15N) and glucose (150 mg C g21 dry
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weight soil) followed by incubation for 6 days at 20 8C. The incubation allowed incorporation of the label into the active soil N pool participating in mineralization – immobilization turnover within this period (Kristensen et al., 2000). The injections were performed in series of nine injections of the labelling solution in each sample by use of a 1-ml hypodermic syringe fitted with a needle (0.5 mm o.d., 30 mm length). The depth of injection was 2.5 –3.0 cm and the needle was pulled out evenly as the solution was dispensed. A total of 1.8 ml of solution was injected into each sample giving a final soil water content of 15.7% of soil dry weight. The treated soil samples were placed in random order together with wet paper towel into three plastic boxes (36 L) and incubated for 6 days at 20 8C. The boxes were opened for aeration every third day. 2.4. Incubation series in the disturbance experiment After 15N labelling of the active organic N pool, one sample from each of the 9 replicate sampling sites was extracted for analysis of initial content of inorganic 15N and 14 N. The remaining replicates were subjected to different treatments of disturbance of soil structure/soil microbial biomass or left intact. The treatments were: (a) left intact; (b) sieving through a 2-mm mesh followed by repacking to original bulk density to simulate tillage; (c) crushing to pass a 425-mm mesh to disrupt larger macroaggregates followed by repacking to original bulk density; (d) left intact followed by incubation in acetylene atmosphere; (e) chloroform fumigation of intact samples for 24 h to perturb microbial biomass followed by incubation in acetylene atmosphere. All samples were then incubated for 14 days at 20 8C with wet paper towels for moisture control to allow for mineralization of soil organic N. The samples from (a), (b) and (c) were incubated in 36 l boxes with aeration every third day as described above for the labelling procedure. The samples from (d) and (e) were incubated in a vacuum desiccator which had acetylene injected through a septum to a concentration of 1% of the atmosphere. The desiccator was opened for aeration followed by injection of acetylene every third day. The incubation with acetylene was to inhibit nitrification activity in the intact samples as chloroform fumigation is known to inhibit nitrification in soil. The accumulation of NHþ 4 as end product of mineralization in the soil may stimulate NHþ 4 immobilization and thus influence net mineralization of soil organic N. 2.5. Sample analysis After termination of the net N and C mineralization incubation and the disturbance experiment, each sample was sieved (mesh size 2 mm), mixed, and subsampled for 2 analysis of the soil inorganic N pool. Soil NHþ 4 and NO3 were extracted in 1 M KCl, the extracts were centrifuged and the supernatant was analysed by standard colorimetric methods. Net N mineralization was calculated as
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the difference between final and initial inorganic N contents of samples. Gas samples (1 ml) were withdrawn from incubation bottles and the CO2 content was determined by gas chromatography with thermal conductivity detection (Microlab, Denmark). The C mineralization was calculated as the difference between CO2 –C measured in bottles containing samples and in blanks. The K2SO4 soil extracts of microbial biomass C and N pools were analysed for total organic C content by automated ultraviolet persulphate oxidation using a Dohrmann DC-180 carbon analyser. Total organic N in the extracts was determined by the Kjeldahl method, on a Tecator total N-analyser. The KCl extracts from the disturbance experiment were prepared for analysis of isotopic content of inorganic N þ (NO2 3 þ NH4 ) by use of the diffusion method by Brooks et al. (1989) slightly modified according to Rackwitz (unpublished data) where Eppendorf cups instead of stainless steel wire were used for placement of the acidified filter disk during diffusion. Analysis of 15N in diffused samples as well as in dried and ground soil samples was performed using an isotope mass spectrometer (Europa Scientific Ltd.) interfaced with an automated N –C analyser. The analysis by the mass spectrometer also provided measures of total N and C content in dried soil samples. The average soil water contents in the samples were 15.4 and 15.0% of dry weight in the intact and sieved/crushed treatments, respectively, at the end of the disturbance experiment. At this point recovery of 15N was found to be 87 – 94% of the amount measured after the labelling incubation.
Ax ¼ atom percent 15N in the active N pool assumed to be the atom percent 15N measured in the mineralized NO2 3 pool in each intact soil sample Y ¼ amount of protected N mineralized to NO2 3 in each disturbed soil sample Ay ¼ atom percent 15N in the protected N pool (assumed to be natural abundance 0.366%) Amin ¼ atom percent 15N in the mineralized NO2 3 pool in each disturbed soil sample. In the field experiment, effects of tillage treatment and sampling date on soil C and N pools and mineralization rates were examined by analysis of variance using a general linear mixed model with subsamples nested within treatments. No block effects were found. Significant interaction was found between tillage system and sampling dates. Accordingly, a repeated-measures model was constructed including tillage and sampling dates as fixed factors taking into account the different variances from sampling dates and the covariances between sampling dates. Statistical significance of differences in release of N pools between treatments in the laboratory experiment were tested by analysis of variance (F-test) with subsamples nested within treatments. The test was followed by pairwise comparisons by Tukey’s studentized range test (Proc GLM). All statistical tests were performed by use of the SAS system for data analysis (SAS Institute Inc., Cary, NC, USA). Statistical significant differences between results were considered at p , 0:05:
2.6. Data analysis
3. Results
The contributions from active and protected pools of soil organic N to the mineral N pool formed during incubation of the disturbed soil samples were calculated according to Kristensen et al. (2000). The calculation was based on measurements of the dilution of 15N label by 14N in the pool of mineralized N in disturbed relative to intact samples. The 15 N and 14N already present in the samples after the labelling incubation and prior to the performance of disturbance treatments was accounted for by subtraction of the amounts found in the intact samples extracted at this point. The principle of isotope dilution (Jansson, 1958) can be expressed in the following equation:
3.1. The field experiment
Amin ¼ ðAx X þ Ay YÞ=ðX þ YÞ
ð1Þ
Mineral N pools and net N mineralization showed no significant differences between tillage systems in the soil from 8 –12 cm depth at the two sampling dates after tillage (Table 1). The level of the mineral N pool increased in both Table 1 2 Effect of tillage system on soil inorganic N content (NHþ 4 þ NO3 ) and net N mineralization for three sampling dates. Numbers in parentheses are standard errors ðn ¼ 6Þ: Different letters behind means denote significant differences between tillage treatments and between sampling dates Tillage
After harvest, 23 Sept. 1998
Post-tillage 2 Oct. 1998
20 Oct. 1998
which can be solved for both X and Y when: Z ¼XþY
ð2Þ
where Z ¼ amount of N mineralized to NO2 3 in each disturbed soil sample X ¼ amount of active N mineralized to NO2 3 in each disturbed soil sample
Inorganic N content (mg N g21 dry weight soil) PTa 7.5 (0.4)b 13.2 (1.0)a NITb 10.8 (1.0)a
5.4 (1.0)b 5.6 (1.0)b
Net N mineralization (mg N g21 dry weight soil day21) PTa 0.3 (0.1)b 0.3 (0.1)b NITb 0.3 (0.1)b
0.6 (0.1)a 0.7 (0.1)a
a b
PT: plough-tillage. NIT: non-inverting tillage.
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Table 2 Effect of tillage system on CO2 production for three sampling dates. Numbers in parentheses are standard errors ðn ¼ 6Þ: Different letters behind means denote significant differences between tillage treatments and between sampling dates for each incubation period Tillage
After harvest
Post-tillage
23 Sept. 1998
2 Oct. 1998
Day 0–4
Day 4–10
CO2 production (mg C g21 dry weight soil day21) PTa 5.6 (1.3)b 4.4 (0.6)c NIT a
Day 0– 4
Day 4 –13
Day 0–6
Day 6– 15
7.2 (1.8)ab 9.0 (0.8)a
4.5 (0.7)c 5.3 (0.7)c
6.4 (1.1)ab 8.4 (1.1)a
3.0 (1.0)c 4.0 (1.0)c
Abbreviation as in Table 1.
tillage systems immediately after tillage and decreased after 3 weeks. Net N mineralization was at the same level before and the day after tillage. Three weeks later the rate increased to approximately double the rate just after tillage. The CO2 production during the first 4 days of incubation was higher the day after NIT tillage than before (Table 2). Three weeks after NIT tillage the CO2 production during the first 6 days of incubation was also elevated. Soil tillage had no effect on microbial biomass C and N content in the 8– 12 cm depth (Table 3). 3.2. The disturbance experiment In general a substantial amount of inorganic 15N and 14N was present in the samples after labelling the active biomass, and the pools increased in all treatments during the next 14 days of incubation (Table 4). In incubations without chloroform or acetylene treatment, NHþ 4 pools were found to be small and stayed more or less at the same level (app. 0.5 mg N kg21) during the experimental period. Where chloroform and/or acetylene were applied the NO2 3 pool remained at the same level (7 – 8 mg N kg21) during the incubation which indicated an effective inhibition of nitrification during the experiment (results not shown). The results presented in Table 4 were used for calculation of contributions of active and protected N pools to mineral N release as shown in Fig. 1. In the laboratory experiment Table 3 Effect of tillage system on microbial biomass C and N content for three sampling dates. Numbers in parentheses are standard errors ðn ¼ 3Þ: Different letters behind means denote significant differences between tillage treatments and between sampling dates Tillage
After harvest, 23 Sept. 1998
Table 4 þ Total pools of inorganic 15N and 14þ15N(NO2 3 þ NH4 ) used for calculation of net mineralization of protected and active N during incubation of soil samples in the disturbance experiment. Numbers in parentheses are standard errors ðn ¼ 9Þ: Different letters behind means denote significant differences between disturbance treatments Treatment
15
N (mg g21 dry weight soil)
N (mg g21 dry weight soil)
Start Intact Sieved Crushed
0.82 (0.13)a 0.97 (0.14)a 0.97 (0.13)a 1.26 (0.14)ab
9.24 (1.54)a 13.03 (3.51)a 14.17 (2.17)a 23.86 (4.00)a
Start þ acetylene Intact þ acetylene Intact þ chloroform fumigation þ acetylene
0.71 (0.11)a 0.82 (0.14)a 1.91 (0.11)b
8.86 (2.37)a 11.43 (2.17)a 47.82 (4.86)b
14þ15
20 Oct. 1998
Microbial biomass C (mg g21 dry weight soil) PTa 197 (11)a 185 (9)a NIT 199 (9)a
181 (17)a 200 (17)a
Microbial biomass N (mg g21 dry weight soil) PT 37 (2)a 36 (1)a NIT 40 (1)a
39 (4)a 39 (4)a
Abbreviation as in Table 1.
sieving through 2 mm mesh size was used to simulate soil structure disturbance by tillage and this treatment did not significantly influence net N mineralization (Table 4, Fig. 1). The rates found in intact and sieved samples were similar to the rates of net mineralization measured just before and after tillage in the field experiment (0.3 –0.4 mg N g21 day21). Any differences between the results obtained from the field and the laboratory studies were, however, not tested statistically due to major differences in experimental conditions (e.g. field or lab conditions, 15N/glucose injection). The total mineral N release caused by soil sieving was 4.9 mg N g21 of which 1.1 mg N g21 was released as protected N and constituted 22% of net N mineralization (Fig. 1). This amount was, however, not significantly different from zero. The more extreme physical disturbance caused by soil crushing to pass a 425 mm mesh increased net mineralization by a factor three to 14.6 mg N g21. The contribution from protected N pools amounted to 3.8 mg N g21 and made up 26% of total net N mineralization after crushing. The intact cores that had been incubated in acetylene atmosphere had a net N mineralization rate at the same level as those incubated without acetylene (Fig. 1). Perturbation of the microbial biomass by chloroform fumigation increased net mineralization by a factor of 12 amounting to 39.0 mg N g21 compared to
Post-tillage 2 Oct. 1998
a
20 Oct. 1998
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Fig. 1. The amount of inorganic N released from active (black columns) or protected (grey columns) N pools during incubation of soil samples in which soil structure or the microbial biomass had been disturbed. The soil samples were Intact; Sieved (2 mm mesh size); Crushed (.425 mm); Intact þ C2H2 (incubated in acetylene atmosphere) or Chloroform þ C2H2 (fumigated with chloroform and incubated in acetylene atmosphere). Bars indicate standard errors ðn ¼ 9Þ:
the intact þ acetylene treatment. Protected N release was increased to 10.5 mg N g21 and made up 27% of net mineralization. The percentage of protected N contributions was not significantly different between the sieved, crushed, and chloroform fumigated treatments (Fig. 1).
4. Discussion 4.1. Effect of tillage on N and C mineralization and microbial pools Net N mineralization rates were not significantly influenced by NIT and PT treatments in the field or by simulation of tillage by soil sieving in the laboratory experiment (Table 1, Fig. 1). This result is comparable to that of laboratory studies that found that net N mineralization was not influenced by soil structure disturbance in PT soils (Beare et al., 1994a; Calderon et al., 2000; Kristensen et al., 2000). In the present study the NIT soil was comparable to a PT soil as the NIT treatment was performed for the first time after several years of conventional ploughtillage management. A few laboratory studies have reported a stimulating effect on net N mineralization rates of soil structure disturbance by mixing of PT soil (Nordmeyer and Richter, 1985; Stenger et al., 1995). By contrast, stimulating
effects are generally observed in no-tillage and grassland soils when disturbed by mixing or sieving (Hassink, 1992; Grace et al., 1993; Kristensen et al., 2000); although decrease of net N mineralization has also been reported (Franzluebbers, 1999). The increase in CO2 production in the NIT soil after tillage indicated release of a small amount of readily available C (Table 2). Others have instead found a decrease in CO2 production after tillage or tillage simulation (Petersen and Klug, 1994; Calderon et al., 2000; 2001). Any effects on CO2 mineralization did not, however, influence the pools of microbial C biomass. Both the NIT and PT treatments did not significantly affect the microbial N pool (Table 3). Also Jenkinson (1988) and Calderon et al. (2000) have reported minimal effects of soil structure disturbance by sieving on microbial pools in plough-tillage soils, whereas Calderon et al. (2001) found short-term effects of tillage on the microbial N pool and microbial community structure. 4.2. Protected N release by simulation of tillage The lack of effects of soil structure disturbance on net N mineralization rates found both in the field and laboratory experiments indicates that disturbance of soil structure by sieving through 2 mm mesh and by the two field tillage
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treatments were comparable with regard to effects on net N mineralization. The lack of effects, however, forms only a weak basis for judging the general usefulness of sieving through 2 mm mesh for simulation of tillage as sieving also can be said to simulate undisturbed conditions as the treatments gave similar results. The protected N release of 22% for total mineral N in the soil with sieving treatment was comparable to results obtained from a PT silt loam by use of the same 15N labelling methodology and sieving through 2 mm mesh (Kristensen et al., 2000). In the silt loam the kinetics of N release were studied and all protected N was found to be released within 3 weeks after sieving. Within the first 2 weeks, 92% of this was released amounting to 3.4 mg N g 21. With a total mineral N release of 15.2 mg N g21 during the 2 weeks the protected N release made up 23% of the total. Thus similar amounts of protected N relative to total mineral N were released as in the present study even though rates of net mineralization and microbial N pools were three times higher in the silt loam. For comparison, the protected N release was 33% after sieving when the silt loam was under no-tillage management which indicated a higher protecting ability compared to the PT soils (Kristensen et al., 2000). 4.3. Protected N release by soil crushing Protected N release was increased by the crushing treatment. This suggests that the larger macroaggregates and other matter (. 425 mm) that were disrupted by the treatment contained a pool of N which was released by net mineralization. The pool released by crushing had, however, the same relative contribution from protected N as the N released by sieving. The matter that was crushed seemed therefore to have the same protecting ability as the wholesoil when disturbed by the sieving treatment. This is surprising as the crushing treatment was a more extreme disturbance event than tillage as also indicated by the increase of net N mineralization due to crushing. Also Franzluebbers and Arshad (1997) found crushing of smaller aggregates (0.25 – 1.00 mm) to release more protected C than crushing of larger macroaggregates (1.0 –5.6 mm) in a PT soil. Macroaggregates in PT soils have, however, been found to have relatively low stability (Beare et al., 1994b). Furthermore results by Six et al. (1999) have indicated that the turnover of macroaggregates in PT soils is high due to the continuous disruption of soil structure by tillage. This may decrease the formation of microaggregates inside macroaggregates and thereby decrease the stabilization of organic matter in the soil (Six et al., 1999). If such an effect have caused a low degree of protection of organic matter in the PT soil, it may explain why the crushing treatment did not release relatively more protected N compared to that of sieving.
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4.4. Origin of protected N release Comparison of results obtained from the sieving and crushing treatments may point in another direction for interpretation when also considering the results from the microbial biomass perturbation treatment by chloroform fumigation. In this treatment the release of N by net mineralization was further increased and again the amount of released protected N was equivalent to that of the sieved and crushed treatments when calculated on a relative basis to total N release. This suggests that N released by the three treatments may have originated from the same pool of organic N. The chloroform fumigation treatment followed by incubation for mineralization is used widely as a method for release of microbial biomass N (e.g. Franzluebbers et al., 1995). If the N released by soil structure disturbance originated from the same pool of organic N involved in the release caused by biomass perturbation, then this suggests that N mineralization flush associated with tillage originates from the microbial biomass N pool. This is supported by findings of Kristensen et al. (2000) who studied the kinetics of N release over a 5 week period after soil structure disturbance. Their results showed that any stimulating effect of sieving on net N mineralization was seen already within the first week after sieving. This indicates that the released N originated from a readily available N pool, which could be the microbial biomass. Similarly Gupta and Germida (1988) suggested that microbial biomass constitutes the primary source of organic matter released by tillage. Other physical and chemical disturbance treatments like soil crushing, drying– rewetting and freeze –thaw cycles may also cause a short-term release of organic N or C by mineralization, typically within less than 10 days (Kieft et al., 1987; DeLuca et al., 1992; Franzluebbers and Arshad, 1997; Franzluebbers, 1999). These studies also suggested that the release mainly originated from the microbial biomass in the soil. The three treatments in the present study: sieving, crushing and chloroform fumigation may possibly be regarded as three degrees of treatments to perturb the soil organic N pool representing a low, intermediate, and high degree of disturbance, respectively. It is possible that the treatments also represent three degrees of disturbance of the microbial biomass, being the main contributor to short-term mineral N release.
5. Conclusions Short-term effects of plough- and non-inverting tillage on net N and C mineralization and microbial pools were few in plough-tillage soil. The use of soil sieving through 2 mm mesh for simulation of soil structure disturbance by tillage gave similar results as obtained by tillage in the field. Despite the lack of effects of tillage on net N mineralization, simulation of tillage was found to cause a small release of N
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from an organic pool which was previously protected from microbial degradation. The use of soil crushing for disruption of larger macroaggregates (. 425 mm) and chloroform fumigation for perturbation of the microbial biomass were more extreme treatments for disturbance of the soil organic N pool, and caused an increased release of both active and protected pools. The pools released by the three treatments were characterized by similar degree of protection. This suggests that the mineral N released by the treatments originated from the same organic N pool in the soil, that is the soil microbial biomass. Thus the microbial biomass seemed to contain a significant pool of protected (inactive) N. Aggregates did not give more protection against microbial degradation than the whole-soil when under plough-tillage management.
Acknowledgements This work was supported by the Danish Research Centre for Organic Farming and the OECD Cooperative Research Programme: Biological Resource Management for Sustainable Agriculture Systems. We thank Dr K. Kristensen for ˚ en and statistical support and Anette Clausen, Karen A Karen K. Jakobsen for skilful technical assistance.
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