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NUTRIENT MANAGEMENT & SOIL & PLANT NUTRITION

Effects of Biological Nitrogen Reactions on Soil Lime Requirement Determined by Incubation

^a Kuo Testing Labs, Inc., 337 S. 1st Ave., Othello, WA 99344
^b Agricultural and Environmental Services Labs, 2400 College Station Rd., Univ. of Georgia, Athens, GA 30602
^c Dep. of Crop and Soil Sciences, 3111 Plant Sciences, Univ. of Georgia, Athens, GA 30602

^* Corresponding author (dkissel{at}uga.edu).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Incubation of soil with CaCO₃ is generally considered a reliable method to determine the lime requirement (LR) of acid soils. Because of their considered reliability, these incubations are often used to calibrate buffer methods; however, one study reported that the use of room temperature incubation with CaCO₃ overestimated the actual LR determined by field testing. The objective of this study was to compare the pH change following CaCO₃ incubations for 60 d with those following 3-d incubations with Ca(OH)₂ and to determine the possible role of soil N reactions causing any differences in pH change. Seventeen soils were incubated with either CaCO₃ for 60 d at approximately 85% field capacity or for 3 d with an equivalent amount of Ca(OH)₂ solution plus water to maintain a 1:1 soil/solution ratio. Both were incubated at room temperature (23 ± 2°C), followed by measurement of pH (1:1 in water). Ammonium-N and NO₃^––N were analyzed at Days 0 and 60 of the incubation. Soil pH was lower following the 60-d CaCO₃ incubation than after the 3-d incubation with Ca(OH)₂. The analysis of N transformations indicated that positive values of H⁺ (more H⁺ was produced than consumed) were generated from nitrification after 60 d of incubation in 14 out of 17 soils. Furthermore, incubations with soils that have been air dried produced a flush of nitrification that increased the ionic strength and decreased pH even further. These effects from long-term incubation would erroneously increase the LR. Incubation with Ca(OH)₂ for 2 to 4 d avoids these errors.

Abbreviations: CEC, cation exchange capacity • DI, deionized • EC, electrical conductivity • LR, lime requirement • MLRA, Major Land Resource Area

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The lime requirement of acid soils is the amount of limestone needed to increase the pH of the plow layer to a desired level (McLean, 1970). Incubation with CaCO₃ is considered by many soil scientists to be a reliable method to determine the LR of acid soils, and it is often used to calibrate buffer methods (Tran and van Lierop, 1981; Loynachan, 1981; Barrow and Cox, 1990). Baker and Chae (1977) reported, however, that laboratory incubation at room temperature of incremental mixtures of CaCO₃ and soil overestimated the actual LRs determined by field testing, primarily because soil acidity increased under laboratory incubation due to environmental conditions more favorable for biological activity. Alabi et al. (1986) noted that incubation with CaCO₃ may be subject to some errors due to long incubation times, changing moisture content, CO₂ levels, and air pollutants. The potential source of errors in long-term incubations with CaCO₃ was also discussed by Black (1993, p. 647–728), who cited data by Haynes and Swift (1989) showing that the rewetting of air-dried soils followed by 10-wk incubation resulted in major increases in pH in soils that primarily favored the process of ammonification and major decreases in pH in soils that primarily favored the process of nitrification. He noted that an increase in soluble salts from biological N reactions can have a pronounced effect on pH, but soluble salts have little effect on the titratable acidity in the soil.

Incubation with Ca(OH)₂ is also often used as a reference method to test the accuracy of other methods of LR prediction (Bradfield, 1941; Dunn, 1943; McConnell et al., 1990). Because Ca(OH)₂ reaction with soil acidity does not depend on dissolution of a mineral of low solubility (as is the case with CaCO₃), long incubation times are not required. Therefore, acidity produced by biological reactions such as nitrification will be minimal. In addition, any increases in soil solution ionic strength, discussed by Black (1993, p. 647–728), will be minimal during a few days of incubation, avoiding any confounding effects on the measurement of soil pH. Also, as pH is raised by neutralization of H⁺ by a base such as Ca(OH)₂, a negative charge is developed for each H⁺ neutralized, making it available to adsorb the accompanying Ca²⁺ ion without any increase in ionic strength. Furthermore, at pH <8, hydroxides will neutralize soil acidity in amounts identical to those neutralized by chemically equivalent amounts of carbonate ions, as shown by Kissel et al. (1988).

Because a reliable standard method is needed to independently evaluate new or modified LR methods, our objective was to compare these two incubation methods and to determine the reasons for any differences in LR by short-term Ca(OH)₂ incubation vs. those measured by longer term CaCO₃ incubation.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Seventeen soil samples with a wide range of clay and soil organic C contents were collected from five of the Major Land Resource Areas (MLRAs) of Georgia (Table 1 ). The soils were oven dried at 35°C, crushed, and sieved (2 mm) to remove small rocks and undecayed crop residue, which comprised <1% of the soil by weight. Then the soils were stored in sealed Ziploc bags until analysis. A subsample of each soil was analyzed for C and N with a LECO CNS 2000 Analyzer (LECO Corp., St. Joseph, MI) (Nelson and Sommers, 1996). Particle size distribution was determined by the pipette method described by Kilmer and Alexander (1949). Electrical conductivity (EC) was measured in each soil by equilibrating soil for 1 h with deionized (DI) water at a soil/water ratio of 1:2, filtering, and then measuring conductance of the filtrate using an Orion 162A conductivity meter (Thermo Fisher Scientific, Waltham, MA). Initial soil pH was measured at a 1:1 soil/water ratio. For purposes of calculating the total ionic strength at 60 d of CaCO₃ incubation, the measured values in the 1:2 soil/water ratio were doubled for estimating the ionic strength of the suspension used for the measurement of pH in water.

The samples were incubated in 500-mL polyethylene containers with lids. Five 2-mm openings were drilled through each lid for air exchange. A glass stirring rod was inserted through one opening of each container to mix the soil. The soils were incubated for 60 d at room temperature (23 ± 2°C), and were moistened every 5 d to keep the water content at about 85% of field capacity. At Days 30 and 60, 30-g subsamples were taken from each container for the measurement of soil pH. The soil samples were air dried and the soil pH was measured at a 1:1 soil/water ratio in a 150-mL beaker while being stirred, using equipment and procedures described by Liu et al. (2004).

On Day 60, after the pH was measured, 120 mL of 1 mol L^–1 KCl was added to each soil suspension previously measured for pH, which was then transferred to a 250-mL flask. The flasks were stoppered and shaken for 30 min at 400 strokes min^–1 in a reciprocating shaker with a stroke length of 15 cm. They were then allowed to stand for several minutes and filtered through Whatman no. 42 filter paper. The filtrates were frozen at –4°C until analysis for NO₃^––N and NH₄⁺–N. Nitrate-N was analyzed with the Griess–?losvay technique after reduction of NO₃^– to NO₂^– through a Cd column (Keeney and Nelson, 1982) and then measured using a Flow Solution 3000 autoanalyzer (OI Analytical, College Station, TX). Ammonium was analyzed using the automated phenate colorimetric procedure (USEPA, 1983). Nitrate and NH₄⁺ were also measured in the original soil samples following the above procedures.

Calcium Hydroxide Incubation
Three rates of Ca(OH)₂ solution were added to each soil equivalent to 0.5, 1.0, and 1.5 times the LR to pH 6.5 based on the full titration results. The incubations were performed at a 1:1 soil/solution ratio using the following procedure. Thirty grams of each of the 17 soil samples was weighed into 120-mL polypropylene beakers and the volume of 0.022 mol L^–1 Ca(OH)₂ to satisfy the treatment requirement was added to DI water to make a 30-mL volume. The soil and the diluted Ca(OH)₂ solution were then thoroughly mixed, along with three drops of chloroform to depress microbial activity. The samples were then covered with Parafilm to reduce evaporation. A 10-mm slit was cut in the film for air exchange. A glass stirring rod was inserted through the opening for mixing the soil periodically. The soil samples were incubated for 4 d at room temperature (23 ± 2°C). The pH was measured at 24, 48, 72, and 96 h while being stirred. For purposes of comparison with the long-term CaCO₃ incubation, 72 h (3 d) was selected because soil pH appeared to have stabilized on the third day, as evidenced by only a small increase on Day 4 (96 h) (Liu et al., 2004). Approximately half of the soil treatments were duplicated to determine any obvious discrepancies. The relationship of soil pH vs. Ca(OH)₂ added (expressed as the CaCO₃ equivalent) was fitted for each soil by nonlinear regression using Table Curve 2D (Systat Software, Richmond, CA) and the Ca(OH)₂ incubation LR to pH 6.5 was calculated from this equation. The nonlinear equation was used because, for most soils, the data of pH vs. lime added becomes nonlinear above pH 6.5 and the 1.5LR treatment gave pH values >6.5 for all soils. Ammonium and NO₃–N were not determined on these samples because we considered the time frame to be too short for significant impact by N transformations due to the short incubation time.

Statistical Analysis
The values of soil pH following incubation were compared among lime sources and incubation times using a general linear model in PROC GLM of SAS (SAS Institute, 1985). Differences were declared significant at the 0.05 probability level. Differences among means were determined by pairwise comparisons made with the DIFF option of the LSMEANS statement. The DIFF option is used to separate means for unbalanced data. It is equivalent to the LSD procedure for balanced data, which also uses the pairwise error rate. The Tukey adjustment option of the LSMEANS statement was used to protect the experimentwise error rate, which was obtained from dividing the pairwise error rate by the number of pairs compared.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The soil samples used had large variability in clay, total C, and soil pH buffering capacity, as well as pH. Four samples contained >300 g kg^–1 clay, five had clay contents in the range from 100 to 200 g kg^–1, and eight samples contained <100 g kg^–1 clay (Table 1). Three of the soil samples contained >20 g kg^–1 total C, seven ranged from 10 to 20 g kg^–1 total C, and the others had <10 g kg^–1 total C. Soil pH ranged from 4.02 to 5.80 and the soil pH buffering capacities varied from 0.70 to 5.79 cmol_c kg^–1 (pH unit) ^–1.

Incubation pH as a function of soil, lime source, and incubation time are shown in Table 2 for the 1.0LR rate of Ca(OH)₂ with a target pH of 6.5. Each variance source including soil, lime source, incubation time, and each two-way or three-way interaction were significant, with P values <0.0001. If no other reactions affected soil pH except neutralization of soil acidity (and assuming complete dissolution of the CaCO₃), the soil pH values should be 6.50 if the titration curves gave an accurate measure of soil acidity. In the 3-d incubation with Ca(OH)₂, the 17 soils had an average pH of 6.36 on Day 3 (Table 2). Soil 9 from the Ridge and Valley MLRA had the highest pH of 6.63 and soil 13 from the Atlantic Coast Flatwoods MLRA had the lowest value of 6.01 (data not shown). For those soils incubated with CaCO₃, the average soil pH for the 17 soils on Day 30 was 5.96, nearly 0.4 pH units lower than the 3-d Ca(OH)₂ incubation (Table 2). The PROC Contrast with one degree of freedom in SAS showed that the pH values following the 30-d CaCO₃ incubation were less than those following the 3-d Ca(OH)₂ incubation, with a P value <0.0001. For the CaCO₃ incubation, only Soil 17 from the Blue Ridge Mountains MLRA exceeded pH 6.5 [its pH was 6.60, nearly identical to the pH after 3 d of incubation with Ca(OH)₂]. Soil 12 had a pH value of 6.46; most of the others had values around 6.0 (data not shown). The lower pH from the 30-d CaCO₃ incubation than from the 3-d Ca(OH)₂ incubation may have been due to incomplete dissolution and reaction of the CaCO₃. The soil pH values for all 17 soils, however, decreased from Day 30 to Day 60; therefore, it is also possible that some acidifying reactions may have contributed to the lower pH. The average soil pH decreased from 5.96 on Day 30 to 5.67 on Day 60. This difference was significant, with a P value <0.0001. If incomplete reaction of CaCO₃ was the sole reason for the lower pH on Day 30 compared with the 3-d Ca(OH)₂, then pH should have increased from Day 30 to Day 60 due to further dissolution of CaCO₃ particles. To determine if CaCO₃ dissolution limited the rise in pH at 30 and 60 d in the main experiment, a second experiment was performed in which eight of the 17 original soils were treated with either CaCO₃ or Ca(OH)₂ to increase the soil pH in water to 6.5. The pH in water of soils treated with Ca(OH)₂ minus those treated with CaCO₃ for 3, 15, 30, and 60 d after treatment are shown in Fig. 1 . As noted in Fig. 1, even at Day 3, most of the CaCO₃ had reacted with the acidity because the difference in pH was approximately 0.11 at 3 d after treatment. At 60 d after lime application, the difference had decreased to about 0.02 pH units. Because the average rise in pH at 3 d with Ca(OH)₂ was 0.77 pH units (it was 0.66 with CaCO₃ at 3 d) and since the difference in pH in Fig. 1 was about 0.02 at 60 d, it can be estimated that approximately (0.02/0.77)100 = 2.6% of the CaCO₃ had not dissolved by 60 d. Even at 3 d, only 14% of the CaCO₃ was unreacted. Because almost all CaCO₃ was dissolved by 60 d, and because pH decreased from Day 30 to Day 60, factors other than CaCO₃ dissolution were affecting soil pH.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Difference in pH in water between soil incubated with Ca(OH)2 and soil incubated with CaCO3 vs. time of incubation.

[1]

where R¹ and R represent organic groups (Conyers et al.,1995).

Following their formation in Eq. [1], NH₄⁺–N ions may be used as an energy source by the chemoautotrophic bacteria Nitrosomonas and be transformed to NO₂^–, with the formation of 2H⁺, thereby lowering soil pH:

[2]

Then, Nitrobacter oxidizes NO₂^– further to NO₃^– without the formation of additional H⁺ (Conyers et al., 1995):

[3]

The concentrations of NH₄⁺–N and NO₃^––N at Day 60 were analyzed to test the hypothesis that N transformations that produce and consume H⁺ affect incubation results. Net H⁺ in Table 3 was calculated by the following:

[4]

where

[5]

[6]

Denitrification was assumed not to occur because the incubation was performed under aerobic conditions. In addition to the N transformations, other soil chemical and biochemical reactions may also change soil pH. These reactions may also have been partly responsible for the pH differences. According to Conyers et al. (1995), the alkali-producing reactions other than ammonification in a well-aerated soil may include oxidation of organic anions and SO₄^2– adsorption.

The NO₃^––N + NH₄⁺–N determined at Day 60 for both control soils and those receiving CaCO₃ are shown in Table 3. It is of interest to note the effect of NO₃^– accumulation on the pH of the control soils. For those eight control soils with increases of >0.7 mmol NO₂^– kg^–1 soil, the soil pH decreased in six (data not shown).

The net H⁺ produced from the N transformations, calculated from the changes in NH₄⁺–N and NO₃^––N and using the H⁺ consumption and production described by Eq. [4], [5], and [6], are also listed in Table 3. In the control soils, 12 of the 17 soils had positive values of net H⁺ produced. Of the 17 CaCO₃–treated soils, 14 had positive values of H⁺ produced, due to nitrification during the laboratory incubation (Table 3). These results were consistent with those of Baker and Chae (1977) for seven western Washington soils. They also concluded that laboratory incubation with CaCO₃ at room temperature increased soil acidity.

In cases with positive values of H⁺ produced, the H⁺ will lower soil pH for two reasons: (i) production of H⁺ in the soil will itself decrease pH, the extent of which depends not only on the amount of H⁺ produced, but also on the capacity of the solid phase to consume H⁺, i.e., the pH buffering capacity of the soil; and (ii) as H⁺ reacts with the soil pH buffering capacity, it reduces the pH-dependent charge, thereby reducing the cation exchange capacity (CEC) in an amount equal to the H⁺ produced, which increases the ionic strength of the soil solution as exchangeable cations equivalent to the reduction in CEC are forced into the soil solution. The measured pH will therefore be depressed further because of the increased salt content of the soil solution.

An attempt was made to test if newly generated H⁺ contributed to the pH differences. For the 14 cases in which the CaCO₃–treated soils had positive H⁺ values, pH differences between the 60-d CaCO₃ incubation and the 3-d Ca(OH)₂ incubation were related to the amount of net H⁺ gained during the 60-d CaCO₃ incubation (Fig. 2 ). The generated linear equation was y = 0.477 + 0.223x, r² = 0.370. Although the r² was not high, the relationship was significant, with a P value of 0.021. These results indicate that a significant portion of the pH differences between the two incubation methods was due to an increase in net H⁺ produced by nitrification during the 60-d CaCO₃ incubation.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Difference in pH in water between the 3-d Ca(OH)2 and 60-d CaCO3 incubations vs. net H+.

[7]

where buffer slope is the slope of the linear regression of soil pH vs. Ca(OH)₂ added from the titration. The pH due to newly generated H⁺ from N transformations was in the range from 0.07 to 0.26 pH unit (Table 3). The average pH difference between the 60-d CaCO₃ incubation and the 3-d Ca(OH)₂ incubation was 0.69 (Table 2). Obviously pH due solely to an increase in acid cations (from H⁺ generated by N transformations) did not explain the entire pH difference due to incubation methods.

An additional influence on soil incubation results is the effect of soil solution ionic strength when pH is measured in DI water (pH_w) (Sumner, 1994). As an illustration of this effect, the value of the pH in water minus the pH in CaCl₂ is plotted vs. EC of the soil solution in a 1:2 soil/water mixture for the 17 soils in this study before incubation. The data were fit with an exponential function, with a resulting r² = 0.38 (Fig. 3 ). This exponential drop was expected and is similar to the results shown by Sumner (1994).

View larger version (8K): [in this window] [in a new window]   Fig. 3. Effects of initial electrical conductivity on pH depression by 0.01 mol L–1 CaCl2.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Difference between in pH in water and pH in 0.01 mol L–1 CaCl2 vs. electrical conductivity.

[8]

We then added the salt from nitrification (in mmol_c L^–1 as net H⁺ from Table 3) to the initial value. Because net H⁺ was expressed as millimoles per kilogram of soil and because pH_w was measured in a 1:1 soil/water ratio, then because 1 kg of soil would be suspended in 1 L of water, the numbers remain the same. For example, Soil 1 with 0.53 mmol net H⁺ would have 0.53 mmol_c L^–1 of salt added to the soil solution. The total salt in the soil solution (initial + that added from nitrification) was then converted to EC (in dS m^–1) using the same relationship as before (Eq. [8]). Those values of estimated total EC were then plotted vs. the difference in ending pH_w between the 60-d CaCO₃ incubation and the 3-d Ca(OH)₂ incubation (Fig. 5 ). These data were fit by linear regression, showing that the pH difference was significant with an r² = 0.58, suggesting that the change in ionic strength of the soil solution due to nitrification was significantly reducing pH_w in the 60-d CaCO₃ incubations. We noted in this relationship that two soils appeared as outliers in the relationship. When these two soils were removed from the regression, the relationship was y = 1.14x + 0.23 with r² = 0.84. This indicates that an increase in ionic strength of the soil solution from the 60-d incubation was the primary cause of the difference in pH between the 3-d Ca(OH)₂ incubation and the 60-d CaCO₃ incubation. This conclusion is further supported by the data in Fig. 1, which show that 97% of the CaCO₃ had reacted with soil acidity by 60 d of incubation. Furthermore, CaCO₃ and Ca(OH)₂ contribute very little to increasing the ionic strength of the soil solution as they neutralize soil acidity. For example, in an incubation of an acid soil with both CaCO₃ and Ca(OH)₂, the pH_w increased from 5.5 to 6.2 and 6.3 with CaCO₃ and Ca(OH)₂, respectively, by Day 8 of an incubation in our lab. By Day 8, the EC of the control soil was 0.051 dS m^–1 whereas the Ca(OH)₂ soil was 0.056 dS m^–1 and the CaCO₃ soil was 0.054 dS m^–1. This average increase of 0.004 dS m^–1 is only about 2% of the increase described above that was responsible for a decrease of 0.4 pH units in a soil very low in ionic strength (Fig. 4).

View larger version (10K): [in this window] [in a new window]   Fig. 5. Difference in pH in water between the 3-d Ca(OH)2 and 60-d CaCO3 incubations vs. electrical conductivity after the 60-d CaCO3 incubation.

In addition to the N transformations (and the associated changes in ionic strength of the soil solution) that may change soil pH, other soil chemical and biochemical reactions may also change soil pH. These reactions may also have been partly responsible for the pH differences. According to Conyers et al. (1995), the alkali-producing reactions other than ammonification may include reduction of Mn oxides, oxidation of organic anions, and SO₄^2– adsorption.

	CONCLUSIONS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
We found that a 60-d incubation of acid soils with CaCO₃, at approximately 85% of field capacity, resulted in lower pHs (corresponding to higher LRs) than 3-d incubations with an equivalent amount of Ca(OH)₂. Differences in the resulting pHs from the two methods of incubation were related to the amounts of NH₄⁺–N and NO₃^––N formed during the 60-d incubation, including the effects of these N cycle reactions on the production and consumption of H⁺ ions. Based on the NH₄⁺–N and NO₃^––N results, we interpreted the pH differences between the 3-d and 60-d incubations as being partly due to two causes. First, a high rate of ammonification and then nitrification in some soils of the 60-d incubation produced considerable H⁺ ions, thereby dropping pH. A flush of mineralization normally occurs during the first approximately 30 d when air-dried soils are rewet in incubation. By 60 d, much of the NH₄⁺–N formed was nitrified, especially in soils treated with CaCO₃ to raise pH, probably because higher pHs favored faster rates of nitrification. Second, the H⁺ ions produced by nitrification will decrease the CEC in amounts equivalent to the net H⁺ ions produced, which increases the ionic strength of the soil solution, thereby decreasing pH even more.

The effect of the H⁺ ions consumed and produced by ammonification and nitrification, respectively, would have a limited effect in changing pH on more highly buffered soils. The increase in soil solution ionic strength from nitrification in long-term CaCO₃ incubations, however, probably affects all soils regardless of their buffering, provided that the soils taken from the field for incubation are initially low in ionic strength. One solution to this problem would be to measure pH in 0.01 mol L^–1 CaCl₂, which would minimize the effect of any changes in the ionic strength of the soil solution that would occur during long-term CaCO₃ incubations. Another solution would be to use short-term (2–4-d) incubations with Ca(OH)₂, which would minimize the effect of nitrification on the soil solution ionic strength and the value of pH_w measured.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
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Received for publication August 23, 2006.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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