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Gross nitrogen transformation rates in soil at a surface coal mine site reclaimed for prime farmland use
PII:
Soil Biol. Biochem. Vol. 30, No. 8/9, pp. 1099±1106, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(97)00202-2 0038-0717/98 $19.00 + 0.00
GROSS NITROGEN TRANSFORMATION RATES IN SOIL AT A SURFACE COAL MINE SITE RECLAIMED FOR PRIME FARMLAND USE M. S. COYNE,1* Q. ZHAI,1 C. T. MACKOWN2 and R. I. BARNHISEL1 Department of Agronomy, University of Kentucky, N-122 Agricultural Science Building, Lexington, KY 40546-0091, U.S.A. and 2USDA-ARS, Grazinglands Research Laboratory, 7207 W. Cheyenne St. El Reno, OK 73036, U.S.A. 1
(Accepted 29 July 1997) SummaryÐOrganic wastes were used to increase N fertility at a surface mine reclamation site, with the ultimate goal to stimulate microbial activity and improve the reclaimed soil's chemical and physical properties. Gross N transformation rates are indicators of microbial activity but are undocumented in such reconstructed ecosystems. We measured gross nitri®cation, N mineralization and N immobilization in waste-amended and unamended soil using 15 N pool dilution techniques. Measurements were made in June, July and November 1993 at the reclamation site in western Kentucky, and compared to net N transformation rates. The premise that organic waste amendment stimulates microbial activity in reclaimed soils was supported by the data. Gross N mineralization, nitri®cation and immobilization rates were as much as 4.5 times greater in waste-amended soil than unamended soil. Gross N mineralizÿ ation and nitri®cation rates and gross NH+ 4 and NO3 immobilization rates were signi®cantly greater ÿ than net rates in waste-amended and unamended soil. There was net immobilization of NH+ 4 and NO3 in waste-amended soil, whereas there was net N mineralization in unamended soil. This was consistent with using substrates containing high C-to-N ratios. Reclamation practices created soil environments in which gross N transformation rates were of the same magnitude as those measured for less disturbed soil ecosystems. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Adverse physical, chemical and biological conditions (for example, low organic matter content, poor structure, de®cient N and P and lack of microbial activity) often limit restoration at surface mine reclamation sites. Restoring N cycling is one of the major goals of ecosystem recovery in these disturbed environments (Whitford, 1988). Adding organic materials to soil promotes N transformation and nutrient cycling and speeds ecosystem recovery (Whitford, 1988). Most ecosystem comparisons of N transformation are based on net rates. The magnitude of net change may not necessarily coincide with the magnitude of microbial activity that produced it because of assimilative and dissimilative N loss. Nitrogen dynamics, nutrient cycling and ecosystem recovery at mine reclamation sites would be better understood if gross rather than net rates of N mineralization, nitri®cation and immobilization were measured. Gross rates are dicult to measure because many microbial processes are involved in N turnover. Nitrogen-15 pool dilution (determination of any *Author for correspondence.
gross rate by measuring the rate at which an isotopically-enriched product pool is diluted by unlabeled product) obviates some of these diculties because it permits distinction between gross transformation processes depending on the labeled product used. Pool-dilution techniques have been used to measure gross N transformation rates in anoxic marine sediment (Blackburn, 1979), forest soils (Davidson et al., 1992; Tietema and Wessel, 1992), annual grasslands (Davidson et al., 1990) and sieved agricultural soils (Myrold and Tiedje, 1986; Nishio and Fujimoto, 1989). However, the measurement of gross N transformations at reclaimed surface mine sites is undocumented, particularly with respect to those reclaimed sites that are also amended with organic wastes. Surface mining has important environmental eects in Kentucky and is a major contributor to point- and non point-source pollution. The unique environment of soils at surface mine reclamation sites, their potential environmental eects, and the underlying rationale that adding organic wastes will stimulate microbial activity prompted us to investigate N transformation processes in these sites. We used 15 N isotope dilution methods to study gross N transformations in an unamended and an organic
1099
1100
M. S. Coyne et al.
waste-amended soil laid down during surface mine reclamation activities. Our objective was to test the premise that adding organic waste stimulates microbial activity in soil at a surface mine reclamation site. Our test was a comparison of gross and net rates of N mineralization, nitri®cation and immobilization in soil amended with organic waste compared to rates in similar, but unamended soil.
Table 1. Soil properties in waste-amended and unamended soils at a surface mine reclamation site sampled on 5 June 1993 (0±10 cm) Soil property Bulk density (g cmÿ3) Water holding capacity (kg kgÿ1) pH (1:1 water) Total carbon (mg kgÿ1)$ Total nitrogen (mg kgÿ1) C to N ratio
Waste-amended mine soil
Unamended mine soil
1.4 0.21
1.5 0.20
7.2 9600 250 38
6.8 4000 210 19
$
The inorganic C was assumed to be negligible.
MATERIALS AND METHODS
Study site and preparation The study site (45 ha total area) was a non-replicated demonstration plot at a surface coal mine near Central City in Muhlenberg County, Kentucky. Prime farmland was reconstructed on spoils consisting of mixed acid sandstone, shale, mudstone and siltstone during the summer of 1992 (Allen, 1992). Soil used to reconstruct the topsoil and subsoil was free of any contaminating coal or spoil. The soil used for reconstruction (a Sadler silt loam, ®ne, silty, mixed, mesic Glossic Fragiudalf) had been stockpiled for several years before replacement. It was used to replace the subsoil to a depth of 90 cm. A 50 cm layer of silt loam A horizon material from the Sadler series was placed over the subsoil using scraperpans. All but 2 ha of the reconstructed prime farmland was seeded on 16 Sept 1992 with a forage seed mixture of 10.2 kg haÿ1 alfalfa (Medicago sativa L. cv. Cimmeron), 10.2 kg haÿ1 red clover (Trifolium hybridum L. cv. Common), and 18.2 kg haÿ1 tall fescue (Festuca arundinacea Schreb. cv. Ky 31). This area was the unamended soil used for comparison in our study. The topsoil of the remaining 2 ha of reconstructed prime farmland was amended with a mixture of 25 Mg poultry manure haÿ1 and 40 Mg sawdust haÿ1 on 16 Sept 1992. This material was incorporated by disc into the upper 20 cm of the surface soil. The waste-amended area was contiguous on three sides with the unamended soil. On 16 September 1992, 34 kg haÿ1 of hairy vetch seed (Vicia villosa L.) was broadcast on the soil surface of the waste-amended soil to serve as a winter cover crop. Baseline soil samples were taken on 5 June 1993. Representative properties of the wasteamended and unamended soils are given in Table 1. On 18 May 1993 the hairy vetch was killed by a mixture of atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) and glyphosate (N-[phosphonomethyl]glycine). Corn (Zea mays L. cv. Pioneer 3245) was planted on 19 May 1993 in 76 cm wide rows at a seeding rate of 59,000 kernels haÿ1. Corn stands were severely damaged by birds and were replanted on 7 June 1993 at the same seeding rate and row spacing. No corn was planted
on the unamended soil and the forage cover was not killed.
Labeling of intact cores Nitrogen transformation rates were measured on 10 June, 29 July and 25 November 1993. These dates corresponded to planting, silking and harvest in the area seeded to corn. Three, 1 m2 areas were randomly selected in both the waste-amended and the unamended soils and used for all subsequent measurements during the study. Areas within the waste-amended site were located so that soil samples were equidistant from corn rows. Sampling areas were at least 50 m apart and 50 m from any border. The method of Davidson et al. (1991) was used for 15 N pool dilution of intact soil cores. Four plastic cylinders (4.25 cm inner dia 10 cm height) were driven into the soil and a larger metal cylinder (20 cm inner dia 10 cm height) was driven into the soil around them. The plastic cylinders were removed and the soil between them was placed in a plastic bag, mixed and subsampled immediately by extraction in 2 M KCl (about 15 g dry-weight-equivalent in 75 ml). Gravimetric water content was determined after drying the soil at 1058C for 24 h. Caps were placed on the ends of each plastic cylinder and either (15 NH4)2SO4 or K15 NO3 solutions (72 and 86 at.% 15 N enrichment, respectively) were injected into six equally spaced holes on the side. 1 ml was injected in each hole to the center of the soil core with a side-port spinal needle. The total N injected in each core was 200 mg. The holes were sealed with plastic tape immediately after injection to minimize loss of added N. Four cores on each sample date were labeled for each of the three sampling areas in the wasteamended and unamended soils. Two cores received 15 15 NH+ NOÿ 4 and two received 3 . One core of each labeled pair was removed and sampled immediately after 15 N injection. The soil was mixed in a plastic bag and subsampled by extraction with 2 M KCl. The time between injection and extraction was 15 min or less. The other core of each labeled pair, at each location, was immediately reburied in the spot from which it came and extracted 24 h later.
N transformation rates at a surface mine reclamation
Analysis of
15
N in KCl extracts
All KCl extracts were cleared by centrifugation. ÿ The NH+ 4 and NO3 were measured colorimetrically using a Technicon II autoanalyser. A diusion procedure was used for 15 NH+ 4 -N analysis (Brooks et al., 1989). Aliquots (1-ml, measured gravimetrically) of each KCl extract of labeled samples were placed in 120-ml acid-washed plastic urine specimen containers. Because the N content of most extracts was low, a 1-ml aliquot (measured gravimetrically) of (NH4)2SO4 carrier solution (0.366 at.% 15 N) containing 100 mg N mlÿ1 was added to each container to provide sucient N for subsequent 15 N analysis. The solution was made alkaline by adding MgO, causing NH3 vapor to be released and subsequently captured on two ®lter paper discs acidi®ed with 2.5 M KHSO4. The discs were dried in a vacuum drier and analyzed for 15 N enrichment by an automated ¯ash-combustion mass spectrometer (Europa Scienti®c, Franklin, OH). Nitrate 15 N enrichment was analyzed directly on a CEC 21-614 mass spectrometer. Duplicate aliquots (1-ml, measured gravimetrically) of each KCl extract of labeled samples were placed in 12 75mm tubes. A 1-ml aliquot (measured gravimetrically) of KNO3 carrier solution containing 280 mg N mlÿ1 (0.366 at.% 15 N) was added to each tube to provide sucient N for subsequent 15 N analysis. The tubes were placed in aluminum blocks and dried at 958C. The NOÿ 3 was then converted to NO and 15 N enrichment was measured (Volk et al., 1979). Nitrogen-15 pool dilution calculations were based on the equations of Kirkham and Bartholomew (1954) with modi®cations by Hart et al. (1994). Measurement of net N mineralization and net nitri®cation On all sampling dates, one additional soil core in a plastic cylinder was removed from each location, capped and reburied in the remaining mine soil from within the large cylinders. These cores were unvented and did not have additional water added to them. The soil cores were retrieved, mixed and immediately subsampled by KCl extraction after 49 d for the 10 June samples, 119 d for the 18 July samples and 54 d for the 25 Nov samples. Net nitri®cation was calculated as the change in NOÿ 3 content in the core after retrieval and net N mineralization was calculated as the change in total and NOÿ inorganic-N after retrieval. The NH+ 4 3 pool sizes were estimated with a Technicon II autoanalyser after KCl extraction of subsamples from the intact cores. ÿ Estimation of NH+ 4 and NO3 immobilization by pool dilution and chloroform fumigation
The NH+ 4 immobilization rate was assumed to be the dierence between the gross NH+ 4 consumption
1101
and gross nitri®cation rates (Davidson et al., 1991). The NH+ 4 immobilization rate was also calculated as the 15 N ¯ush following chloroform fumigation± incubation based on a non-linear model described in detail by Davidson et al. (1991). Brie¯y, the immobilization rate is estimated by the equation: i vt1 y0 =x 0 1 ÿ eÿk =k
1
15
where vt = 1 is the excess N content of the chloroform labile N pool (mg N gÿ1 soil) after the 7-d incubation. (y0/x0) is the initial enrichment of the ÿ NH+ 4 or NO3 pool (depending on which immobilization rate is being determined) in which y0 is the ÿ 15 15 1415 NH+ NH+ 4 ( NO3 ) pool size and x0 is the 4 ÿ ÿ1 1415 ( NO3 ) pool size (mg N g soil). k is a rate constant calculated from equation (2) using the initial (y0, x0) and ®nal (yt, xt) pool sizes for 15 NH+ 4 1415 1415 (15 NOÿ NH+ NOÿ 3 ) and 4 ( 3 ), respectively. k ÿln yt x 0 = y0 x t t
2
immobilization rate measured by The NH+ 4 chloroform fumigation was only measured on waste-amended and unamended soils sampled on 29 July. After labeled and incubated soils were subsampled in the ®eld by KCl extraction, as described above for the pool dilution method, an additional subsample was used to estimate microbial biomassN by the chloroform fumigation±incubation method (Horwath and Paul, 1994). The soil samples were fumigated for 24 h, then incubated for 7 d. Soil samples were extracted by 0.5 M K2SO4 after incubation (Davidson et al., 1989). The K2SO4 extracts were digested (Brookes et al., 1985) during which the NOÿ 3 was removed. Nitrogen-15 labeled NH+ 4 was recovered by diusion. The NOÿ 3 immobilization rate was equated with the gross NOÿ 3 consumption rate, based on the suggestion by Davidson et al. (1991) that dissimilative ÿ fates of NOÿ 3 such as dissimilatory NO3 reduction and denitri®cation were negligible in the to NH+ 4 short term. Analyses of soil physical, chemical and biological properties Soil bulk density (Black and Hartage, 1986), water holding capacity (Klute, 1986), pH, total carbon (Nelson and Sommers, 1982) and total N (Bremner and Mulvaney, 1982) were measured by standard laboratory techniques. Soil microbial biomass C and soil respiration were only measured when samples for initial soil properties were taken (5 June). Microbial biomass C was measured by the chloroform fumigation ÿ incubation method (Horwath and Paul, 1994). Erlenmeyer ¯asks with 100 g moist soil were placed in an evacuated desiccator along with a beaker of chloroform and incubated for 24 h.
immobilization
15
ÿwaste 2.1 (0.2) 2.5 (1.2) 1.0 (0.1) +waste 3.7 (0.3) 5.0 (0.4) 2.4 (0.3)
Gross immobilization Gross
ÿwaste 1.2 (0.7) 1.6 (1.0) 0.6 (0.3) +waste ÿ0.07 (0.01) ÿ0.03 (0.004) ÿ0.03 (0.07) ÿwaste 0.4 (0.3) 1.6 (1.0) 0.6 (0.3)
Gross nitri®cation
+waste 1.8 (0.4) 2.9 (0.3) 0.8 (0.2)
Net nitri®cation
ÿwaste 0.03 (0.01) 0.03 (0.01) 0.01 (0.004)
+waste 4.6 (0.2) 4.0 (1.4) 0.4 (0.4)
NOÿ 3 Net N mineralization
ÿwaste 0.03 (0.01) 0.04 (0.01) 0.01 (0.01) $Values in parentheses are 21 SD. %Negative values indicate net immobilization. n = 3.
Gross N mineralization rates were signi®cantly greater (P < 0.01) in waste-amended soil than in unamended soil at every sample date (Table 2). The highest rates were in July. The net N mineralization rates in unamended soil were always less than 3% of the gross N mineralization rates. The net N mineralization rates in waste-amended soil were always signi®cantly less (P < 0.01) than in unamended soil (with the exception of the 25 November sample) and indicated that N had been immobilized.
10 June 29 July 25 November
Nitrogen mineralization and nitri®cation
+waste ÿ0.08 (0.01)% ÿ0.06 (0.03) ÿ0.04 (0.10)
Waste-amended and unamended soil from the reclamation site had similar bulk densities, water holding capacities and pH (Table 1). Gravimetric water content (kg water kgÿ1 soil) was virtually identical between treatments, ranging (in the case of the waste-amended treatment) from 0.13 kg kgÿ1 on 10 June to 0.19 kg kgÿ1 on 29 July. Gravimetric water content was 0.17 kg kgÿ1 on 25 November. There were obvious treatment dierences in terms of total C and N (Table 1). Sawdust and manure addition doubled the C:N ratio. Approximately 9 months after the incorporation of organic wastes, the microbial biomass C was 439 mg C kgÿ1 in waste-amended soil and 302 mg C kgÿ1 soil in unamended soil. Soil respiration was 49 mg CO2 kgÿ1 hÿ1 in waste-amended soil and 43 mg CO2 kgÿ1 hÿ1 in unamended soil.
ÿwaste 1.3 (0.3) 1.5 (0.5) 0.6 (0.1)
Comparison of soil chemical and biological properties
Gross N mineralization
RESULTS
+waste 3.7 (0.4)$ 5.2 (0.1) 1.4 (0.4)
Statistical analyses were performed using the statistical analysis system (SAS/STAT, 1990). The general linear models (GLM) procedure was used to obtain the analysis of variance based on a repeated measures design to assess the eect of amendment and time on net and gross N transformation rates.
Date (1993)
Statistical analyses
NH+ 4
Fumigated soil was amended with approximately 1 g of unfumigated soil after 24 h and mixed thoroughly. A glass vial containing 1 ml of 1 N NaOH was inserted into each ¯ask and incubated at 238C for 7 d. The NaOH was titrated after incubation with 1 N HCl to determine CO2 evolution. A Kc of 0.41 was used to convert CO2 evolution to a measure of microbial biomass C (the value of Kc is de®ned as the fraction of biomass C mineralized to CO2) (Horwath and Paul, 1994). Erlenmeyer ¯asks with 100 g moist soil and a glass tube containing 5 ml of 1 N NaOH were incubated at 238C for the soil respiration measurements. Carbon dioxide was trapped in 1 N NaOH and determined by titration with 1 N HCl every 2 d for a total of 6 d.
N pool dilution
M. S. Coyne et al.
ÿ ÿ1 Table 2. Gross and net nitrogen mineralization, nitri®cation and NH+ soil dÿ1) in waste-amended and unamended mine soils (0±10 cm) measured by 4 and NO3 immobilization rates (mg N kg
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N transformation rates at a surface mine reclamation
Correlation of selected soil properties
ÿ Table 3. Soil NH+ 4 and NO3 pools on each sample date ÿ1 NH+ 4 (mg kg )
Date (1993)
Wasteamended
Unamended
ÿ1 NOÿ 3 (mg kg )
Wasteamended
1103
Unamended
10 June 1.462 0.10 0.712 0.04 1.312 0.12 0.88 20.04 29 July 1.422 0.06 1.292 0.07 0.492 0.01 0.47 20.03 25 November 0.672 0.04 0.702 0.05 1.692 0.08 1.17 20.03 n = 3.
Gross nitri®cation rates, with the exception of the 25 November sample, were signi®cantly greater (P < 0.01) in waste-amended soil than unamended soil (Table 2). As with N mineralization, the highest rates were in July. Gross nitri®cation rates were also greater than net nitri®cation rates in both waste-amended soil and unamended soil. There was net NOÿ 3 immobilization in waste-amended soil and net nitri®cation in unamended soil (Table 2).
Microbial immobilization The inorganic N pools in the waste-amended and unamended soils prior to each pool dilution assay are shown in Table 3. Waste-amended soils routinely had more inorganic N than unamended soils, although these dierences were minimal on the 29 July date. This date corresponds to the silking period for corn at this site. In both soils, NOÿ 3 -N but not NH+ 4 -N was at a minimum. Microbial immobilization rates measured by chloroform fumigation or pool dilution were not statistically dierent in the one sample on 29 July for which they were compared. Ammonium immobilization in waste-amended mine soil was 5.7 2 2.1 mg N kgÿ1 soil dÿ1 when determined by chloroform fumigation and 4.0 2 1.4 mg N kgÿ1 soil dÿ1 when measured by pool dilution. In unamended mine soil, the corresponding rates were 1.5 2 0.5 and 1.6 2 1.0 mg N kgÿ1 soil dÿ1 for the chloroform fumigation and pool dilution methods, respectively. immobilization rates Ammonium and NOÿ 3 measured by pool dilution were typically highest in the 29 July sample and in almost all cases were signi®cantly greater in waste-amended than in unamended mine soil (P < 0.01) (Table 2).
Net N mineralization and nitri®cation were correlated with each other but not with any other property (Table 4). In contrast, gross N mineralization + and nitri®cation and gross NOÿ 3 and NH4 immobilization, were all positively correlated with each other. Soil respiration was correlated with all measures of gross transformation but was not as well correlated with gross transformation rates as was microbial biomass (Table 4). DISCUSSION
The waste-amended and unamended soils had dierent crop management, but for the purpose of this study, a direct comparison was warranted. The reclamation process at each site was the same except for the addition of organic waste; soil type, placement and depth were identical. Soil physical properties at the two sites were similar; bulk density and water holding capacity were virtually identical. The dierent crops were in place for less than 9 months before the assays were performed. It seems unlikely that short-term dierences in the plant community would have been signi®cant compared to the eect of the waste addition, although this would likely change as the site aged. Gross transformation rates and organic waste addition One could have concluded that N mineralization and nitri®cation were more dynamic in unamended than waste-amended soil because net transformation rates were greater in the former. However, the gross N mineralization and nitri®cation rates indicated just the opposite. Furthermore, it is important to observe what the gross transformation rates indicate about microbial activity in these soils, but that the ambient N pool sizes do not. Namely, that N mineralization, nitri®cation and immobilization occur at rates capable of turning over the KCL extractable-inorganic N pool several times a day. Low inorganic N concentrations do not necessarily re¯ect low biological activity. Organic C and total soil N were increased by waste-amendment, and these altered soil properties often stimulate microbial activity in disturbed soils (Whitford, 1988). There is a close relationship
Table 4. Pearson correlation coecients (n=18)
Net N mineralization (NM) Net nitri®cation (NN) Gross N mineralization (GM) Gross nitri®cation (GN) NH4 + immobilization (AI) NO3 ÿ immobilization (NI) Soil respiration (SR) Microbial biomass-C (MB)
NN
Gm
0.98**
ÿ0.51 ÿ0.48
*Signi®cant at P<0.05; **Signi®cant at P<0.01.
GN ÿ0.35 ÿ0.29 0.80**
AI ÿ0.40 ÿ0.37 0.97** 0.84**
NI 0.55 0.50 0.84** 0.92** 0.83**
SR ÿ0.07 ÿ0.05 0.74** 0.67* 0.76* 0.64*
MB ÿ0.41 ÿ0.38 0.91** 0.78** 0.92** 0.77* 0.68* Ð
1104
M. S. Coyne et al.
between gross N transformation and microbial activity in agricultural soils (Nishio et al., 1993; Pilabeam et al., 1993). In our study, N transformation rates were faster in waste-amended soil. However, the NH+ and NOÿ 4 3 produced during gross N mineralization and nitri®cation did not subsequently accumulate (i.e. they were assimilated). The high gross immobilization rates that we observed are presumably due to elevated C-to-N ratios in the organic amendments, which is consistent with the results of Nishio et al. (1993). Net rates re¯ect an integration of N mineralization and nitri®cation, or of immobilization processes. They can be either positive or negative depending on the respective rates of each process and are usually controlled by the C-to-N ratio (Keeney, 1982). In waste-amended soil, incorporating sawdust and the subsequent elevation of the Cto-N ratio undoubtedly promoted immobilization due to microbial assimilation of available inorganic N during waste decomposition. Net N mineralization and nitri®cation rates on a 1-d basis, calculated by dierence from the gross rates (not shown), are similar in magnitude to the gross rates (but of dierent sign). This is quite dierent from net rates calculated from long-term incubations of undisturbed soil cores and may re¯ect the perturbation of soil unavoidable in the pool dilution technique. Nevertheless, they also demonstrate the greater potential for immobilization in the waste-amended soils compared to the unamended site. Ammonium consumption in soil cores was dominated by microbial immobilization and nitri®cation. Davidson et al. (1991) concluded that the 15 N pool dilution technique may overestimate consumption stimulates rates because the addition of 15 NH+ 4 NH+ 4 assimilation when initial pool sizes are low. For every sample date and treatment, the initial ÿ ÿ1 NH+ and 4 - and NO3 -N pools were <2 mg kg ÿ1 usually <1.5 mg kg . This may explain why gross immobilization rates on two of the three NH+ 4 sampling dates were greater than, or equal to, the corresponding gross N mineralization rates. Overestimating consumption rates due to signi®cant increases in the inorganic N substrate can also be signi®cant, particularly for NOÿ 3 assimilation at low ambient NOÿ 3 concentrations (Hart et al., 1994). The ratio of gross NOÿ 3 immobilization-tonitri®cation was frequently >1 indicating that microbial assimilation of NOÿ 3 may have been stimulated by NOÿ 3 addition. Davidson et al. (1991) suggest that estimates of NH+ 4 immobilization by pool dilution and chloroform fumigation bracket the true immobilization rate: the former providing an upper limit and the latter a lower limit of immobilization. In this highly-modi®ed environment, chloroform fumigation and pool dilution gave similar results for
NH+ 4 immobilization. The dierences we observed between the two methods were not signi®cant. Correlation of gross transformation rates to other microbial measurements Gross transformation rates were always higher in June and July than in November and were routinely highest on the July sampling date. This trend corresponds well with the climatic conditions for those dates. Average air temperatures in June and July were >278C, considerably warmer than on the 25 November sample date (108C), so one would expect greater microbial activity. The July samples also had the highest water content of the three dates (0.19 kg water kgÿ1 soil). Microbial biomass C and respiration were both greater in the waste-amended than unamended soil. They were also signi®cantly correlated with gross N mineralization and nitri®cation rates. Microbial biomass C and soil respiration were correlated with N turnover rates in reclaimed mine soils amended with organic wastes by Zhai (1995). The contrast between waste-amended and unamended soils was best revealed by microbial biomass measurements rather than by soil respiration. This was probably because the most readily-metabolizable C in the waste-amended soil had been utilized during the 9months between application and respiration measurements. Comparisons at an earlier date might have shown greater dierences between amended and unamended soils. Signi®cant correlations between gross rates and microbial biomass and activity also appear in forest soils (Davidson et al., 1992; Tietema and Wessel, 1992). Thus, with respect to undisturbed ecosystems, the reclaimed mine soil manifested similar positive correlations between overt net measures of microbial activity and gross transformation rates. Comparison of reclaimed mine soils to other soil ecosystems Our results support the thesis that organic wastes stimulate microbial N transformation rates in reclaimed soil. But are they comparable to those observed for other soil systems? At one extreme are the gross transformation rates measured in forest litter layers. Gross N mineralization rates ranging from 36.5 to 72.1 mg N kgÿ1 soil dÿ1 have been reported (Davidson et al., 1992; Tietema and Wessel, 1992). Davidson et al. (1992) found that than in a mature conifer forest gross N mineralization rates were 2- to 3-fold greater than gross N mineralization rates in a 10-y-old conifer plantation, although net N mineralization rates were somewhat higher in the plantation. In contrast to these rates, Davidson et al. (1990) found that gross N mineralization and nitri®cation in a central California grassland were 1.37 and 0.89 mg N kgÿ1 soil dÿ1, respectively. Our rates for
N transformation rates at a surface mine reclamation
unamended reconstructed prime farmland under forage species (Table 2) are comparable to those values. They are also comparable (though somewhat lower) to gross transformation rates that have been reported for agricultural soils (Nishio et al., 1985; Myrold and Tiedje, 1986; Bjarnason, 1988). Gross N mineralization rates in these soils ranged from 0.6 to 4.0 mg N kgÿ1 soil dÿ1. Myrold and Tiedje (1986) measured gross nitri®cation rates ranging from 0.7 to 4.6 mg N kgÿ1 soil dÿ1 in a sandy loam soil from a corn±soybean rotation. The gross nitri®cation rates we measured from both wasteamended and unamended reconstructed soil often fell within this range. From the standpoint of gross N mineralization and nitri®cation, even though the surface soil was stockpiled and replaced during reclamation, N transformation rates remained remarkably similar to those of other agricultural soils that have not been so thoroughly disturbed. This is an interesting ®nding since it implies that the basic N transformation properties of some soils may resist anthropomorphic in¯uences. This is in contrast to Ross et al. (1982) who observed that when topsoil was mined, the biochemical activity of the underlying soil had not recovered to control values after 3 yr. CONCLUSIONS
There was bene®t to adding organic wastes in terms of the magnitude of gross activity. However, when good-quality topsoil was used during reclamation (even when stockpiled for a period), such amendment was clearly not necessary to ensure that N transformations at this site occurred at rates comparable to other agricultural soils. This is in contrast to abandoned minesites with exposed acidic spoils in which 50 yr or more may be necessary before the original biological activity of the soil is recovered (Insam and Domsch, 1988). The thesis that adding organic wastes to disturbed soil ecosystems stimulates microbial N transformations would have been discounted if measurements relied only on net changes. The pool dilution technique used in this study indicates that microbial activity was stimulated by adding organic wastes to soil. However, the addition of wastes with a relatively high C-to-N ratios (such as sawdust) appear to have defeated the purpose of increasing the immediate availability of N availability to plants since the N was immobilized. Thus, the type of organic amendments used in surface mine reclamation is a factor, particularly where yield and bond release are important considerations in the reclamation process. AcknowledgementsÐThe investigation in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the director.
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REFERENCES
Allen M. (1992) A review of procedures OSM uses to evaluate and improve state regulatory programs regarding prime farmland reclamation of mined soils. In Proceedings of the 1992 National Symposium on Prime Farmland Reclamation, eds. R. E. Dunker, R. I. Barnhisel and R. G. Darmody, pp. 169±172. Department of Agronomy, University of Illinois, Urbana. Bjarnason S. (1988) Calculation of gross nitrogen immobilization and mineralization in soil. Journal of Soil Science 39, 393±406. Blackburn T. H. (1979) Method for measuring rate of ammonium turnover in anoxic marine sediments using 15 N dilution technique. Applied and Environmental Microbiology 37, 760±765. Black G. R. and Hartage K. H. (1986) Soil bulk density. In Methods of Soil Analysis, Part 1. Physical and Mineralogical Methods, ed. A. Klute, pp. 363±376. American Society of Agronomy, Madison. Bremner J. M. and Mulvaney C. S. (1982) Soil total nitrogen. In Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties, ed. A. L. Page, pp. 595±622. American Society of Agronomy, Madison. Brookes P. C., Kragt J. F., Powlson D. S. and Jenkinson D. S. (1985) Chloroform fumigation and the release of soil nitrogen: The eects of fumigation time and temperature. Soil Biology & Biochemistry 17, 831±835. Brooks P. D., Stark J. M., McInteer B. B. and Preston T. (1989) Diusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society of America Journal 53, 1707±1722. Davidson E. A., Eckert R. W., Hart S. C. and Firestone M. K. (1989) Direct extraction of microbial biomass nitrogen from forest and grassland soils of California. Soil Biology & Biochemistry 21, 773±778. Davidson E. A., Hart S. C. and Firestone M. K. (1992) Internal cycling of nitrate in soils of a mature coniferous forest. Ecology 73, 1148±1156. Davidson E. A., Hart S. C., Shanks C. A. and Firestone M. K. (1991) Measuring gross nitrogen mineralization, immobilization and nitri®cation by 15 N isotopic pool dilution in intact soil cores. Journal of Soil Science 42, 335±349. Davidson E. A., Stark J. M. and Firestone M. K. (1990) Microbial production and consumption of nitrate in an annual grassland. Ecology 71, 1968±1975. Hart S. C., Stark J. M., Davidson E. A. and Firestone M. K. (1994) Nitrogen mineralization, immobilization, and nitri®cation. In Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties, ed. R. W. Weaver, pp. 985±1018. American Society of Agronomy, Madison. Horwath W. R. and Paul E. A. (1994) Microbial biomass. In Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties, ed. W. Weaver, pp. 753±774. American Society of Agronomy, Madison. Insam H. and Domsch K. H. (1988) Relationship between soil organic carbon and microbial biomass on chronosequences of reclaimed sites. Microbial Ecology 15, 177± 188. Keeney D. R. (1982) Nitrogen availability indices. In Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties, ed. A. L. Page, pp. 711±730. American Society of Agronomy, Madison. Kirkham D. and Bartholomew W. V. (1954) Equations for following nutrient transformation in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33±34. Klute A. (1986) Water holding capacity. In Methods of Soil Analysis, Part 1: Physical and Mineralogical
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Methods, ed. A. Klute, pp. 789±886. American Society of Agronomy, Madison. Myrold D. D. and Tiedje J. M. (1986) Simultaneous estimation of several nitrogen cycle rates using 15 N: Theory and application. Soil Biology & Biochemistry 18, 559± 568. Nelson D. W. and Sommers L. E. (1982) Total carbon, organic carbon, and organic matter. In Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties, A. L. Page, pp. 539±577. American Society of Agronomy, Madison. Nishio T. and Fujimoto T. (1989) Mineralization of soil organic matter in upland ®elds as determined by a 15 NH+ 4 dilution technique and absorption of nitrogen by maize. Soil Biology & Biochemistry 21, 661±665. Nishio T., Kanamori T. and Fujimoto T. (1985) Nitrogen transformations in an aerobic soil as determined by a 15 NH+ 4 dilution technique. Soil Biology & Biochemistry 17, 149±154. Nishio T., Sekiya H. and Kogano K. (1993) Estimate of nitrogen cycling in 15 N-amended soil during long-term submergence. Soil Biology & Biochemistry 25, 785±788. Pilabeam C. J., Mahapatra B. S. and Wood M. (1993) Soil matric potential eects on gross rates of nitrogen
mineralization in an orthic ferralsol from Kenya. Soil Biology & Biochemistry 25, 1409±1413. Ross D. J., Speir T. W., Tate K. R., Cairns A., Meyrick K. F. and Pansier E. A. (1982) Restoration of pasture after topsoil removal: Eects on soil carbon and nitrogen mineralization, microbial biomass, and enzyme activities. Soil Biology & Biochemistry 14, 575±581. SAS/STAT (1990) SAS/STAT User's Guide, Version 6 edn. SAS Inst., Cary, NC. Tietema A. and Wessel W. W. (1992) Gross nitrogen transformations in the organic layer of acid forest ecosystems subjected to increased atmospheric nitrogen input. Soil Biology & Biochemistry 24, 943±950. Volk R. J., Pearson C. J. and Jackson W. A. (1979) Reduction of plant tissue nitrate to nitric oxide for mass spectrometric 15 N analysis. Analytical Biochemistry 97, 131±137. Whitford W. G. (1988) Decomposition and nutrient cycling in disturbed arid ecosystems. In The Reconstruction of Disturbed Arid Lands: An Ecological Approach, ed. E. B. Allen, pp. 136±161. Westview Press, Boulder. Zhai Q. (1995) Unpublished Ph.D. dissertation, University of Kentucky.
Soil Biol. Biochem. Vol. 30, No. 8/9, pp. 1099±1106, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(97)00202-2 0038-0717/98 $19.00 + 0.00
GROSS NITROGEN TRANSFORMATION RATES IN SOIL AT A SURFACE COAL MINE SITE RECLAIMED FOR PRIME FARMLAND USE M. S. COYNE,1* Q. ZHAI,1 C. T. MACKOWN2 and R. I. BARNHISEL1 Department of Agronomy, University of Kentucky, N-122 Agricultural Science Building, Lexington, KY 40546-0091, U.S.A. and 2USDA-ARS, Grazinglands Research Laboratory, 7207 W. Cheyenne St. El Reno, OK 73036, U.S.A. 1
(Accepted 29 July 1997) SummaryÐOrganic wastes were used to increase N fertility at a surface mine reclamation site, with the ultimate goal to stimulate microbial activity and improve the reclaimed soil's chemical and physical properties. Gross N transformation rates are indicators of microbial activity but are undocumented in such reconstructed ecosystems. We measured gross nitri®cation, N mineralization and N immobilization in waste-amended and unamended soil using 15 N pool dilution techniques. Measurements were made in June, July and November 1993 at the reclamation site in western Kentucky, and compared to net N transformation rates. The premise that organic waste amendment stimulates microbial activity in reclaimed soils was supported by the data. Gross N mineralization, nitri®cation and immobilization rates were as much as 4.5 times greater in waste-amended soil than unamended soil. Gross N mineralizÿ ation and nitri®cation rates and gross NH+ 4 and NO3 immobilization rates were signi®cantly greater ÿ than net rates in waste-amended and unamended soil. There was net immobilization of NH+ 4 and NO3 in waste-amended soil, whereas there was net N mineralization in unamended soil. This was consistent with using substrates containing high C-to-N ratios. Reclamation practices created soil environments in which gross N transformation rates were of the same magnitude as those measured for less disturbed soil ecosystems. # 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
Adverse physical, chemical and biological conditions (for example, low organic matter content, poor structure, de®cient N and P and lack of microbial activity) often limit restoration at surface mine reclamation sites. Restoring N cycling is one of the major goals of ecosystem recovery in these disturbed environments (Whitford, 1988). Adding organic materials to soil promotes N transformation and nutrient cycling and speeds ecosystem recovery (Whitford, 1988). Most ecosystem comparisons of N transformation are based on net rates. The magnitude of net change may not necessarily coincide with the magnitude of microbial activity that produced it because of assimilative and dissimilative N loss. Nitrogen dynamics, nutrient cycling and ecosystem recovery at mine reclamation sites would be better understood if gross rather than net rates of N mineralization, nitri®cation and immobilization were measured. Gross rates are dicult to measure because many microbial processes are involved in N turnover. Nitrogen-15 pool dilution (determination of any *Author for correspondence.
gross rate by measuring the rate at which an isotopically-enriched product pool is diluted by unlabeled product) obviates some of these diculties because it permits distinction between gross transformation processes depending on the labeled product used. Pool-dilution techniques have been used to measure gross N transformation rates in anoxic marine sediment (Blackburn, 1979), forest soils (Davidson et al., 1992; Tietema and Wessel, 1992), annual grasslands (Davidson et al., 1990) and sieved agricultural soils (Myrold and Tiedje, 1986; Nishio and Fujimoto, 1989). However, the measurement of gross N transformations at reclaimed surface mine sites is undocumented, particularly with respect to those reclaimed sites that are also amended with organic wastes. Surface mining has important environmental eects in Kentucky and is a major contributor to point- and non point-source pollution. The unique environment of soils at surface mine reclamation sites, their potential environmental eects, and the underlying rationale that adding organic wastes will stimulate microbial activity prompted us to investigate N transformation processes in these sites. We used 15 N isotope dilution methods to study gross N transformations in an unamended and an organic
1099
1100
M. S. Coyne et al.
waste-amended soil laid down during surface mine reclamation activities. Our objective was to test the premise that adding organic waste stimulates microbial activity in soil at a surface mine reclamation site. Our test was a comparison of gross and net rates of N mineralization, nitri®cation and immobilization in soil amended with organic waste compared to rates in similar, but unamended soil.
Table 1. Soil properties in waste-amended and unamended soils at a surface mine reclamation site sampled on 5 June 1993 (0±10 cm) Soil property Bulk density (g cmÿ3) Water holding capacity (kg kgÿ1) pH (1:1 water) Total carbon (mg kgÿ1)$ Total nitrogen (mg kgÿ1) C to N ratio
Waste-amended mine soil
Unamended mine soil
1.4 0.21
1.5 0.20
7.2 9600 250 38
6.8 4000 210 19
$
The inorganic C was assumed to be negligible.
MATERIALS AND METHODS
Study site and preparation The study site (45 ha total area) was a non-replicated demonstration plot at a surface coal mine near Central City in Muhlenberg County, Kentucky. Prime farmland was reconstructed on spoils consisting of mixed acid sandstone, shale, mudstone and siltstone during the summer of 1992 (Allen, 1992). Soil used to reconstruct the topsoil and subsoil was free of any contaminating coal or spoil. The soil used for reconstruction (a Sadler silt loam, ®ne, silty, mixed, mesic Glossic Fragiudalf) had been stockpiled for several years before replacement. It was used to replace the subsoil to a depth of 90 cm. A 50 cm layer of silt loam A horizon material from the Sadler series was placed over the subsoil using scraperpans. All but 2 ha of the reconstructed prime farmland was seeded on 16 Sept 1992 with a forage seed mixture of 10.2 kg haÿ1 alfalfa (Medicago sativa L. cv. Cimmeron), 10.2 kg haÿ1 red clover (Trifolium hybridum L. cv. Common), and 18.2 kg haÿ1 tall fescue (Festuca arundinacea Schreb. cv. Ky 31). This area was the unamended soil used for comparison in our study. The topsoil of the remaining 2 ha of reconstructed prime farmland was amended with a mixture of 25 Mg poultry manure haÿ1 and 40 Mg sawdust haÿ1 on 16 Sept 1992. This material was incorporated by disc into the upper 20 cm of the surface soil. The waste-amended area was contiguous on three sides with the unamended soil. On 16 September 1992, 34 kg haÿ1 of hairy vetch seed (Vicia villosa L.) was broadcast on the soil surface of the waste-amended soil to serve as a winter cover crop. Baseline soil samples were taken on 5 June 1993. Representative properties of the wasteamended and unamended soils are given in Table 1. On 18 May 1993 the hairy vetch was killed by a mixture of atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) and glyphosate (N-[phosphonomethyl]glycine). Corn (Zea mays L. cv. Pioneer 3245) was planted on 19 May 1993 in 76 cm wide rows at a seeding rate of 59,000 kernels haÿ1. Corn stands were severely damaged by birds and were replanted on 7 June 1993 at the same seeding rate and row spacing. No corn was planted
on the unamended soil and the forage cover was not killed.
Labeling of intact cores Nitrogen transformation rates were measured on 10 June, 29 July and 25 November 1993. These dates corresponded to planting, silking and harvest in the area seeded to corn. Three, 1 m2 areas were randomly selected in both the waste-amended and the unamended soils and used for all subsequent measurements during the study. Areas within the waste-amended site were located so that soil samples were equidistant from corn rows. Sampling areas were at least 50 m apart and 50 m from any border. The method of Davidson et al. (1991) was used for 15 N pool dilution of intact soil cores. Four plastic cylinders (4.25 cm inner dia 10 cm height) were driven into the soil and a larger metal cylinder (20 cm inner dia 10 cm height) was driven into the soil around them. The plastic cylinders were removed and the soil between them was placed in a plastic bag, mixed and subsampled immediately by extraction in 2 M KCl (about 15 g dry-weight-equivalent in 75 ml). Gravimetric water content was determined after drying the soil at 1058C for 24 h. Caps were placed on the ends of each plastic cylinder and either (15 NH4)2SO4 or K15 NO3 solutions (72 and 86 at.% 15 N enrichment, respectively) were injected into six equally spaced holes on the side. 1 ml was injected in each hole to the center of the soil core with a side-port spinal needle. The total N injected in each core was 200 mg. The holes were sealed with plastic tape immediately after injection to minimize loss of added N. Four cores on each sample date were labeled for each of the three sampling areas in the wasteamended and unamended soils. Two cores received 15 15 NH+ NOÿ 4 and two received 3 . One core of each labeled pair was removed and sampled immediately after 15 N injection. The soil was mixed in a plastic bag and subsampled by extraction with 2 M KCl. The time between injection and extraction was 15 min or less. The other core of each labeled pair, at each location, was immediately reburied in the spot from which it came and extracted 24 h later.
N transformation rates at a surface mine reclamation
Analysis of
15
N in KCl extracts
All KCl extracts were cleared by centrifugation. ÿ The NH+ 4 and NO3 were measured colorimetrically using a Technicon II autoanalyser. A diusion procedure was used for 15 NH+ 4 -N analysis (Brooks et al., 1989). Aliquots (1-ml, measured gravimetrically) of each KCl extract of labeled samples were placed in 120-ml acid-washed plastic urine specimen containers. Because the N content of most extracts was low, a 1-ml aliquot (measured gravimetrically) of (NH4)2SO4 carrier solution (0.366 at.% 15 N) containing 100 mg N mlÿ1 was added to each container to provide sucient N for subsequent 15 N analysis. The solution was made alkaline by adding MgO, causing NH3 vapor to be released and subsequently captured on two ®lter paper discs acidi®ed with 2.5 M KHSO4. The discs were dried in a vacuum drier and analyzed for 15 N enrichment by an automated ¯ash-combustion mass spectrometer (Europa Scienti®c, Franklin, OH). Nitrate 15 N enrichment was analyzed directly on a CEC 21-614 mass spectrometer. Duplicate aliquots (1-ml, measured gravimetrically) of each KCl extract of labeled samples were placed in 12 75mm tubes. A 1-ml aliquot (measured gravimetrically) of KNO3 carrier solution containing 280 mg N mlÿ1 (0.366 at.% 15 N) was added to each tube to provide sucient N for subsequent 15 N analysis. The tubes were placed in aluminum blocks and dried at 958C. The NOÿ 3 was then converted to NO and 15 N enrichment was measured (Volk et al., 1979). Nitrogen-15 pool dilution calculations were based on the equations of Kirkham and Bartholomew (1954) with modi®cations by Hart et al. (1994). Measurement of net N mineralization and net nitri®cation On all sampling dates, one additional soil core in a plastic cylinder was removed from each location, capped and reburied in the remaining mine soil from within the large cylinders. These cores were unvented and did not have additional water added to them. The soil cores were retrieved, mixed and immediately subsampled by KCl extraction after 49 d for the 10 June samples, 119 d for the 18 July samples and 54 d for the 25 Nov samples. Net nitri®cation was calculated as the change in NOÿ 3 content in the core after retrieval and net N mineralization was calculated as the change in total and NOÿ inorganic-N after retrieval. The NH+ 4 3 pool sizes were estimated with a Technicon II autoanalyser after KCl extraction of subsamples from the intact cores. ÿ Estimation of NH+ 4 and NO3 immobilization by pool dilution and chloroform fumigation
The NH+ 4 immobilization rate was assumed to be the dierence between the gross NH+ 4 consumption
1101
and gross nitri®cation rates (Davidson et al., 1991). The NH+ 4 immobilization rate was also calculated as the 15 N ¯ush following chloroform fumigation± incubation based on a non-linear model described in detail by Davidson et al. (1991). Brie¯y, the immobilization rate is estimated by the equation: i vt1 y0 =x 0 1 ÿ eÿk =k
1
15
where vt = 1 is the excess N content of the chloroform labile N pool (mg N gÿ1 soil) after the 7-d incubation. (y0/x0) is the initial enrichment of the ÿ NH+ 4 or NO3 pool (depending on which immobilization rate is being determined) in which y0 is the ÿ 15 15 1415 NH+ NH+ 4 ( NO3 ) pool size and x0 is the 4 ÿ ÿ1 1415 ( NO3 ) pool size (mg N g soil). k is a rate constant calculated from equation (2) using the initial (y0, x0) and ®nal (yt, xt) pool sizes for 15 NH+ 4 1415 1415 (15 NOÿ NH+ NOÿ 3 ) and 4 ( 3 ), respectively. k ÿln yt x 0 = y0 x t t
2
immobilization rate measured by The NH+ 4 chloroform fumigation was only measured on waste-amended and unamended soils sampled on 29 July. After labeled and incubated soils were subsampled in the ®eld by KCl extraction, as described above for the pool dilution method, an additional subsample was used to estimate microbial biomassN by the chloroform fumigation±incubation method (Horwath and Paul, 1994). The soil samples were fumigated for 24 h, then incubated for 7 d. Soil samples were extracted by 0.5 M K2SO4 after incubation (Davidson et al., 1989). The K2SO4 extracts were digested (Brookes et al., 1985) during which the NOÿ 3 was removed. Nitrogen-15 labeled NH+ 4 was recovered by diusion. The NOÿ 3 immobilization rate was equated with the gross NOÿ 3 consumption rate, based on the suggestion by Davidson et al. (1991) that dissimilative ÿ fates of NOÿ 3 such as dissimilatory NO3 reduction and denitri®cation were negligible in the to NH+ 4 short term. Analyses of soil physical, chemical and biological properties Soil bulk density (Black and Hartage, 1986), water holding capacity (Klute, 1986), pH, total carbon (Nelson and Sommers, 1982) and total N (Bremner and Mulvaney, 1982) were measured by standard laboratory techniques. Soil microbial biomass C and soil respiration were only measured when samples for initial soil properties were taken (5 June). Microbial biomass C was measured by the chloroform fumigation ÿ incubation method (Horwath and Paul, 1994). Erlenmeyer ¯asks with 100 g moist soil were placed in an evacuated desiccator along with a beaker of chloroform and incubated for 24 h.
immobilization
15
ÿwaste 2.1 (0.2) 2.5 (1.2) 1.0 (0.1) +waste 3.7 (0.3) 5.0 (0.4) 2.4 (0.3)
Gross immobilization Gross
ÿwaste 1.2 (0.7) 1.6 (1.0) 0.6 (0.3) +waste ÿ0.07 (0.01) ÿ0.03 (0.004) ÿ0.03 (0.07) ÿwaste 0.4 (0.3) 1.6 (1.0) 0.6 (0.3)
Gross nitri®cation
+waste 1.8 (0.4) 2.9 (0.3) 0.8 (0.2)
Net nitri®cation
ÿwaste 0.03 (0.01) 0.03 (0.01) 0.01 (0.004)
+waste 4.6 (0.2) 4.0 (1.4) 0.4 (0.4)
NOÿ 3 Net N mineralization
ÿwaste 0.03 (0.01) 0.04 (0.01) 0.01 (0.01) $Values in parentheses are 21 SD. %Negative values indicate net immobilization. n = 3.
Gross N mineralization rates were signi®cantly greater (P < 0.01) in waste-amended soil than in unamended soil at every sample date (Table 2). The highest rates were in July. The net N mineralization rates in unamended soil were always less than 3% of the gross N mineralization rates. The net N mineralization rates in waste-amended soil were always signi®cantly less (P < 0.01) than in unamended soil (with the exception of the 25 November sample) and indicated that N had been immobilized.
10 June 29 July 25 November
Nitrogen mineralization and nitri®cation
+waste ÿ0.08 (0.01)% ÿ0.06 (0.03) ÿ0.04 (0.10)
Waste-amended and unamended soil from the reclamation site had similar bulk densities, water holding capacities and pH (Table 1). Gravimetric water content (kg water kgÿ1 soil) was virtually identical between treatments, ranging (in the case of the waste-amended treatment) from 0.13 kg kgÿ1 on 10 June to 0.19 kg kgÿ1 on 29 July. Gravimetric water content was 0.17 kg kgÿ1 on 25 November. There were obvious treatment dierences in terms of total C and N (Table 1). Sawdust and manure addition doubled the C:N ratio. Approximately 9 months after the incorporation of organic wastes, the microbial biomass C was 439 mg C kgÿ1 in waste-amended soil and 302 mg C kgÿ1 soil in unamended soil. Soil respiration was 49 mg CO2 kgÿ1 hÿ1 in waste-amended soil and 43 mg CO2 kgÿ1 hÿ1 in unamended soil.
ÿwaste 1.3 (0.3) 1.5 (0.5) 0.6 (0.1)
Comparison of soil chemical and biological properties
Gross N mineralization
RESULTS
+waste 3.7 (0.4)$ 5.2 (0.1) 1.4 (0.4)
Statistical analyses were performed using the statistical analysis system (SAS/STAT, 1990). The general linear models (GLM) procedure was used to obtain the analysis of variance based on a repeated measures design to assess the eect of amendment and time on net and gross N transformation rates.
Date (1993)
Statistical analyses
NH+ 4
Fumigated soil was amended with approximately 1 g of unfumigated soil after 24 h and mixed thoroughly. A glass vial containing 1 ml of 1 N NaOH was inserted into each ¯ask and incubated at 238C for 7 d. The NaOH was titrated after incubation with 1 N HCl to determine CO2 evolution. A Kc of 0.41 was used to convert CO2 evolution to a measure of microbial biomass C (the value of Kc is de®ned as the fraction of biomass C mineralized to CO2) (Horwath and Paul, 1994). Erlenmeyer ¯asks with 100 g moist soil and a glass tube containing 5 ml of 1 N NaOH were incubated at 238C for the soil respiration measurements. Carbon dioxide was trapped in 1 N NaOH and determined by titration with 1 N HCl every 2 d for a total of 6 d.
N pool dilution
M. S. Coyne et al.
ÿ ÿ1 Table 2. Gross and net nitrogen mineralization, nitri®cation and NH+ soil dÿ1) in waste-amended and unamended mine soils (0±10 cm) measured by 4 and NO3 immobilization rates (mg N kg
1102
N transformation rates at a surface mine reclamation
Correlation of selected soil properties
ÿ Table 3. Soil NH+ 4 and NO3 pools on each sample date ÿ1 NH+ 4 (mg kg )
Date (1993)
Wasteamended
Unamended
ÿ1 NOÿ 3 (mg kg )
Wasteamended
1103
Unamended
10 June 1.462 0.10 0.712 0.04 1.312 0.12 0.88 20.04 29 July 1.422 0.06 1.292 0.07 0.492 0.01 0.47 20.03 25 November 0.672 0.04 0.702 0.05 1.692 0.08 1.17 20.03 n = 3.
Gross nitri®cation rates, with the exception of the 25 November sample, were signi®cantly greater (P < 0.01) in waste-amended soil than unamended soil (Table 2). As with N mineralization, the highest rates were in July. Gross nitri®cation rates were also greater than net nitri®cation rates in both waste-amended soil and unamended soil. There was net NOÿ 3 immobilization in waste-amended soil and net nitri®cation in unamended soil (Table 2).
Microbial immobilization The inorganic N pools in the waste-amended and unamended soils prior to each pool dilution assay are shown in Table 3. Waste-amended soils routinely had more inorganic N than unamended soils, although these dierences were minimal on the 29 July date. This date corresponds to the silking period for corn at this site. In both soils, NOÿ 3 -N but not NH+ 4 -N was at a minimum. Microbial immobilization rates measured by chloroform fumigation or pool dilution were not statistically dierent in the one sample on 29 July for which they were compared. Ammonium immobilization in waste-amended mine soil was 5.7 2 2.1 mg N kgÿ1 soil dÿ1 when determined by chloroform fumigation and 4.0 2 1.4 mg N kgÿ1 soil dÿ1 when measured by pool dilution. In unamended mine soil, the corresponding rates were 1.5 2 0.5 and 1.6 2 1.0 mg N kgÿ1 soil dÿ1 for the chloroform fumigation and pool dilution methods, respectively. immobilization rates Ammonium and NOÿ 3 measured by pool dilution were typically highest in the 29 July sample and in almost all cases were signi®cantly greater in waste-amended than in unamended mine soil (P < 0.01) (Table 2).
Net N mineralization and nitri®cation were correlated with each other but not with any other property (Table 4). In contrast, gross N mineralization + and nitri®cation and gross NOÿ 3 and NH4 immobilization, were all positively correlated with each other. Soil respiration was correlated with all measures of gross transformation but was not as well correlated with gross transformation rates as was microbial biomass (Table 4). DISCUSSION
The waste-amended and unamended soils had dierent crop management, but for the purpose of this study, a direct comparison was warranted. The reclamation process at each site was the same except for the addition of organic waste; soil type, placement and depth were identical. Soil physical properties at the two sites were similar; bulk density and water holding capacity were virtually identical. The dierent crops were in place for less than 9 months before the assays were performed. It seems unlikely that short-term dierences in the plant community would have been signi®cant compared to the eect of the waste addition, although this would likely change as the site aged. Gross transformation rates and organic waste addition One could have concluded that N mineralization and nitri®cation were more dynamic in unamended than waste-amended soil because net transformation rates were greater in the former. However, the gross N mineralization and nitri®cation rates indicated just the opposite. Furthermore, it is important to observe what the gross transformation rates indicate about microbial activity in these soils, but that the ambient N pool sizes do not. Namely, that N mineralization, nitri®cation and immobilization occur at rates capable of turning over the KCL extractable-inorganic N pool several times a day. Low inorganic N concentrations do not necessarily re¯ect low biological activity. Organic C and total soil N were increased by waste-amendment, and these altered soil properties often stimulate microbial activity in disturbed soils (Whitford, 1988). There is a close relationship
Table 4. Pearson correlation coecients (n=18)
Net N mineralization (NM) Net nitri®cation (NN) Gross N mineralization (GM) Gross nitri®cation (GN) NH4 + immobilization (AI) NO3 ÿ immobilization (NI) Soil respiration (SR) Microbial biomass-C (MB)
NN
Gm
0.98**
ÿ0.51 ÿ0.48
*Signi®cant at P<0.05; **Signi®cant at P<0.01.
GN ÿ0.35 ÿ0.29 0.80**
AI ÿ0.40 ÿ0.37 0.97** 0.84**
NI 0.55 0.50 0.84** 0.92** 0.83**
SR ÿ0.07 ÿ0.05 0.74** 0.67* 0.76* 0.64*
MB ÿ0.41 ÿ0.38 0.91** 0.78** 0.92** 0.77* 0.68* Ð
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between gross N transformation and microbial activity in agricultural soils (Nishio et al., 1993; Pilabeam et al., 1993). In our study, N transformation rates were faster in waste-amended soil. However, the NH+ and NOÿ 4 3 produced during gross N mineralization and nitri®cation did not subsequently accumulate (i.e. they were assimilated). The high gross immobilization rates that we observed are presumably due to elevated C-to-N ratios in the organic amendments, which is consistent with the results of Nishio et al. (1993). Net rates re¯ect an integration of N mineralization and nitri®cation, or of immobilization processes. They can be either positive or negative depending on the respective rates of each process and are usually controlled by the C-to-N ratio (Keeney, 1982). In waste-amended soil, incorporating sawdust and the subsequent elevation of the Cto-N ratio undoubtedly promoted immobilization due to microbial assimilation of available inorganic N during waste decomposition. Net N mineralization and nitri®cation rates on a 1-d basis, calculated by dierence from the gross rates (not shown), are similar in magnitude to the gross rates (but of dierent sign). This is quite dierent from net rates calculated from long-term incubations of undisturbed soil cores and may re¯ect the perturbation of soil unavoidable in the pool dilution technique. Nevertheless, they also demonstrate the greater potential for immobilization in the waste-amended soils compared to the unamended site. Ammonium consumption in soil cores was dominated by microbial immobilization and nitri®cation. Davidson et al. (1991) concluded that the 15 N pool dilution technique may overestimate consumption stimulates rates because the addition of 15 NH+ 4 NH+ 4 assimilation when initial pool sizes are low. For every sample date and treatment, the initial ÿ ÿ1 NH+ and 4 - and NO3 -N pools were <2 mg kg ÿ1 usually <1.5 mg kg . This may explain why gross immobilization rates on two of the three NH+ 4 sampling dates were greater than, or equal to, the corresponding gross N mineralization rates. Overestimating consumption rates due to signi®cant increases in the inorganic N substrate can also be signi®cant, particularly for NOÿ 3 assimilation at low ambient NOÿ 3 concentrations (Hart et al., 1994). The ratio of gross NOÿ 3 immobilization-tonitri®cation was frequently >1 indicating that microbial assimilation of NOÿ 3 may have been stimulated by NOÿ 3 addition. Davidson et al. (1991) suggest that estimates of NH+ 4 immobilization by pool dilution and chloroform fumigation bracket the true immobilization rate: the former providing an upper limit and the latter a lower limit of immobilization. In this highly-modi®ed environment, chloroform fumigation and pool dilution gave similar results for
NH+ 4 immobilization. The dierences we observed between the two methods were not signi®cant. Correlation of gross transformation rates to other microbial measurements Gross transformation rates were always higher in June and July than in November and were routinely highest on the July sampling date. This trend corresponds well with the climatic conditions for those dates. Average air temperatures in June and July were >278C, considerably warmer than on the 25 November sample date (108C), so one would expect greater microbial activity. The July samples also had the highest water content of the three dates (0.19 kg water kgÿ1 soil). Microbial biomass C and respiration were both greater in the waste-amended than unamended soil. They were also signi®cantly correlated with gross N mineralization and nitri®cation rates. Microbial biomass C and soil respiration were correlated with N turnover rates in reclaimed mine soils amended with organic wastes by Zhai (1995). The contrast between waste-amended and unamended soils was best revealed by microbial biomass measurements rather than by soil respiration. This was probably because the most readily-metabolizable C in the waste-amended soil had been utilized during the 9months between application and respiration measurements. Comparisons at an earlier date might have shown greater dierences between amended and unamended soils. Signi®cant correlations between gross rates and microbial biomass and activity also appear in forest soils (Davidson et al., 1992; Tietema and Wessel, 1992). Thus, with respect to undisturbed ecosystems, the reclaimed mine soil manifested similar positive correlations between overt net measures of microbial activity and gross transformation rates. Comparison of reclaimed mine soils to other soil ecosystems Our results support the thesis that organic wastes stimulate microbial N transformation rates in reclaimed soil. But are they comparable to those observed for other soil systems? At one extreme are the gross transformation rates measured in forest litter layers. Gross N mineralization rates ranging from 36.5 to 72.1 mg N kgÿ1 soil dÿ1 have been reported (Davidson et al., 1992; Tietema and Wessel, 1992). Davidson et al. (1992) found that than in a mature conifer forest gross N mineralization rates were 2- to 3-fold greater than gross N mineralization rates in a 10-y-old conifer plantation, although net N mineralization rates were somewhat higher in the plantation. In contrast to these rates, Davidson et al. (1990) found that gross N mineralization and nitri®cation in a central California grassland were 1.37 and 0.89 mg N kgÿ1 soil dÿ1, respectively. Our rates for
N transformation rates at a surface mine reclamation
unamended reconstructed prime farmland under forage species (Table 2) are comparable to those values. They are also comparable (though somewhat lower) to gross transformation rates that have been reported for agricultural soils (Nishio et al., 1985; Myrold and Tiedje, 1986; Bjarnason, 1988). Gross N mineralization rates in these soils ranged from 0.6 to 4.0 mg N kgÿ1 soil dÿ1. Myrold and Tiedje (1986) measured gross nitri®cation rates ranging from 0.7 to 4.6 mg N kgÿ1 soil dÿ1 in a sandy loam soil from a corn±soybean rotation. The gross nitri®cation rates we measured from both wasteamended and unamended reconstructed soil often fell within this range. From the standpoint of gross N mineralization and nitri®cation, even though the surface soil was stockpiled and replaced during reclamation, N transformation rates remained remarkably similar to those of other agricultural soils that have not been so thoroughly disturbed. This is an interesting ®nding since it implies that the basic N transformation properties of some soils may resist anthropomorphic in¯uences. This is in contrast to Ross et al. (1982) who observed that when topsoil was mined, the biochemical activity of the underlying soil had not recovered to control values after 3 yr. CONCLUSIONS
There was bene®t to adding organic wastes in terms of the magnitude of gross activity. However, when good-quality topsoil was used during reclamation (even when stockpiled for a period), such amendment was clearly not necessary to ensure that N transformations at this site occurred at rates comparable to other agricultural soils. This is in contrast to abandoned minesites with exposed acidic spoils in which 50 yr or more may be necessary before the original biological activity of the soil is recovered (Insam and Domsch, 1988). The thesis that adding organic wastes to disturbed soil ecosystems stimulates microbial N transformations would have been discounted if measurements relied only on net changes. The pool dilution technique used in this study indicates that microbial activity was stimulated by adding organic wastes to soil. However, the addition of wastes with a relatively high C-to-N ratios (such as sawdust) appear to have defeated the purpose of increasing the immediate availability of N availability to plants since the N was immobilized. Thus, the type of organic amendments used in surface mine reclamation is a factor, particularly where yield and bond release are important considerations in the reclamation process. AcknowledgementsÐThe investigation in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the director.
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