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Soil Science Society of America Journal 65:780-787 (2001)
© 2001 Soil Science Society of America

DIVISION S-4 - SOIL FERTILITY & PLANT NUTRITION

Organic Phosphorus Mineralization Studies Using Isotopic Dilution Techniques

F. Oehl, A. Oberson, S. Sinaj and E. Frossard

Group of Plant Nutrition, Inst. of Plant Sciences, Swiss Federal Inst. of Technology (ETH), P.O. 185, CH-8315 Lindau, Switzerland

Corresponding author (astrid.oberson{at}ipw.agrl.ethz.ch)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil organic P (Po) mineralization is an important process in P cycling. No accurate method for its quantification is available because any mineralized inorganic P (Pi) may be rapidly sorbed onto the soil solid phase where it cannot be separated from already present Pi. A method for measuring soil Po mineralization is explored using isotopic dilution techniques under conditions of constant soil respiration rates. First, the specific activity (SA) as affected by physicochemical processes was extrapolated from an isotopic exchange kinetics batch experiment. Second, the SA was assessed during incubation after labeling soil with 33PO4. Lower SA measured during incubation than extrapolated from the batch experiment was attributed to the release of nonlabeled Pi due to mineralization of nonlabeled Po. In order to separate biological from biochemical mineralization processes, one set of samples was {gamma}-irradiated to stop the microbial activity while maintaining phosphatase activity. The {gamma}-irradiated soil revealed higher mineralization rates than the corresponding nonirradiated soil. This was explained by an increase of the amount of easily mineralizable Po derived from killed microbial cells by {gamma}-irradiation. Consequently, a gross, but overestimated, biochemical P mineralization can be assessed. In the nonirradiated soil, mineralization not only of nonlabeled, but also of recently synthesized labeled Po resulting from microbial turnover, may occur. Thus, in the nonirradiated soil, after several days a gross, biologically and biochemically mediated mineralization is increasingly underestimated. During the first 7 d, the mineralization rate in the nonirradiated soil was 1.7 mg P kg-1 d-1, which is an amount approximately equivalent to soil solution P in this soil, indicating that soil P mineralization is a significant process in delivering available Pi.

Abbreviations: cP, Pi concentration in the soil solution (mg P L-1) obtained at a 1:10 soil/water ratio • {Delta}E(t), difference between mesE(t) and modE(t): amount of P mineralization at time t • E value, amount of isotopically exchangeable P • mesE(t), isotopically exchangeable P measured in the incubation experiment (mg P kg-1) • mesSA, specific activity of the soil solution (33PO4/31PO4 in Bq µg-1 P) measured in the incubation experiment • modE(t), isotopically exchangeable P extrapolated from the batch experiment (mg P kg-1) • modSA, specific activity of the soil solution (33PO4/31PO4 in Bq µg-1 P) extrapolated from the short-term batch experiment • n, parameter obtained from the batch experiment usually calculated using linear regression between log [r(t)/R] and log (t) • Pi, inorganic soil phosphorus • Pmin, organic phosphorus mineralization • Po, organic soil phosphorus • r(t)/R, radioactivity remaining in the soil solution at the corresponding time (t) in relation to the radioactivity added at time zero • SA, specific activity of the soil solution (33PO4/31PO4 in Bq µg-1 P) • {Delta}SA(t), difference between mesSA and modSA at time t


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
PHOSPHORUS MINERALIZATION may play an important role in plant nutrition in eco- and low input agrosystems (Harrison, 1987; Verhoeven et al., 1990; Yanai, 1992). While P release from recently added organic materials can be assessed during decomposition studies (Palm et al., 1997; George et al., 1999), no accurate method for the quantification of soil P mineralization has been established so far because of the high chemical reactivity of Pi (Frossard et al., 1996). In recent years, new analytical techniques have been proposed to handle the problem of immediate Pi sorption. Zou et al. (1992) used anion exchange resins to extract Pi recently released from Po. Frossard et al. (1996) used isotopic exchange kinetics and changes in resin-extractable Pi to evaluate the quantities of P mineralized after the addition of specific Po compounds. Walbridge and Vitousek (1987) and López-Hernandez and Niño (1993) applied isotopic dilution techniques to study gross soil P mineralization assuming that Pi release from nonlabeled Po decreases the SA of Pi in acid fluoride or resin extracts, respectively.

López-Hernandez et al. (1998) used a short-term (100 min) isotopic exchange kinetics batch experiment (Fardeau, 1993) to determine the changes in SA of Pi present in the soil solution resulting from physicochemical processes. The SA extrapolated from this batch experiment was the baseline for a concomitant incubation experiment where SA resulting from both physicochemical and biological processes was measured. Measured and extrapolated SA values were converted into amounts of isotopically exchangeable P (E values). Measured E values were greater than extrapolated E values, which was explained by Pi release from nonlabeled Po during incubation. The P mineralization rates were deduced from the difference between measured and extrapolated E values. Using the baseline extrapolated from the batch experiment, López-Hernandez et al. (1998) overcame one of the major constraints of former P mineralization studies using isotopic dilution (Walbridge and Vitousek, 1987), which were based on the common but erroneous assumption (Fardeau, 1993) that an isotopic equilibrium between the added P isotope and all exchangeable P fractions would be rapidly achieved, and that any dilution observed afterwards would have been caused by the Pi release from nonlabeled Po. However, López-Hernandez et al. (1998) did not consider all the conditions imposed by Sheppard (1962) for studying isotopic exchange processes in multicompartment systems. First, they did not measure SA in the compartment in which the isotope was introduced, that is, in the soil solution, but they used exchange resins without knowing the relationship between SA in the soil solution and SA of resin Pi. Second, they conducted the batch experiments on nonincubated, air-dried soils without showing if the baseline of SA in air-dried soil correctly describes the physicochemical processes in the incubated soils. Since they started the incubation experiment during a phase of unsteady respiration rates, the correct assignment of measured isotopic dilution to any defined process of mineralization was not possible.

The concept of soil N mineralization used by Mary and Recous (1994) was adopted to design this soil P mineralization study. Net mineralization consists of different processes: flush effects, basal mineralization, remineralization (these three fluxes constitute gross mineralization), and biological immobilization (Mary and Recous, 1994). Flush effects are caused by sequences of drying–wetting or freezing–thawing (Chapin et al., 1978; Mary and Recous, 1994). Basal P mineralization can be defined as the gross P mineralization in the absence of flush effects, that is, at constant soil respiration rate. It presents the basal potential of a soil to deliver Pi from Po to the soil solution. Remineralization takes place due to recycling of microbial P during microbial death and predation and signifies mineralization of recently synthesized Po. Biological P immobilization corresponds with the assimilation of P by soil microbes.

All three P mineralization processes include biological and biochemical processes. Biological P mineralization occurs when cell Pi from decaying cells is released to the soil solution and when Po compounds are hydrolyzed on the outer surface of the living cell membranes (Burns, 1982). The latter is driven by the microorganisms' need for energy (McGill and Cole, 1981) and for P (Lughtenberg, 1987; Halvorson and Nakata, 1987). Biochemical P mineralization is driven by phosphatase exoenzymes (Burns, 1982; Sinsabaugh et al., 1993). Since the functionality of the exoenzymes may be kept through stabilization by sorption and association on soil compounds (Leprince and Quiquampoix, 1996; Burns, 1982), biochemical P mineralization may occur in the absence of biological activity (Zou et al., 1992; Seeling and Zasoski, 1993).

The aim of this study was to measure basal P mineralization using the approach of López-Hernandez et al. (1998) by comparing the SA of Pi in the soil solution obtained in a batch experiment examining physicochemical processes, and an incubation experiment additionally including biological processes. A preliminary experiment had to be conducted to assess the preincubation time needed to reach constant (= basal) respiration rate and constant isotopic exchange kinetics parameters. Then, differences between SA extrapolated from the batch experiment (modSA) and SA determined in the incubation experiment (mesSA) were investigated. Amounts of mineralized Po were deduced from the differences between the respective E values. In order to quantify the mineralization of Po due to phosphatase exoenzymes, P mineralization was quantified in nonirradiated and {gamma}-irradiated soils.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Soil
Soil samples were taken from a long-term field experiment near Basel, Switzerland, established in 1978 (Besson and Niggli, 1991) on a loamy silt Typic Hapludalf developed on loess. Soil was collected from a treatment managed according to bioorganic farming. This treatment was chosen to explore the methodology because long-term organic fertilization had increased overall soil biological activity, including C mineralization, soil phosphatase activity, and microbially bound P (Fliessbach and Mäder, 1997; Oberson et al., 1993; Oehl et al., 1998). At the same time, available inorganic P content was sufficient for plant growth (Oberson et al., 1993). The ploughed horizon of the four field replicates (0–20 cm) was sampled in March 1998 and mixed to form a composite sample. The sample was cooled immediately after sampling and kept at 4°C. Before starting any other experiments, the previously cooled soil was sieved at 3 mm and preincubated. A part was air-dried and sieved at 2 mm prior to chemical analysis (pH, C content, total Pi and Po; Table 1).


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  		Table 1. Soil characteristics including P data

 
Preincubation
Soil was preincubated for 28 d at 20°C, 80 to 85% atmospheric humidity, and 50% water holding capacity. Preincubation was done in order to reach conditions where basal respiration (expressed in mg CO2 kg-1 d-1) and isotopic exchange kinetics parameters remained constant for several weeks. Soil respiration was measured in 500-mL flasks on four replicates (Alef, 1995). Carbon dioxide liberated from 10 g of soil and absorbed by 20 mL of 0.05 M NaOH was determined by back titration with 0.05 M HCl after addition of 5 mL of 0.5 M BaCl2 and phenolphthalein indicator solution. During the first 10 d, CO2 release was measured every other day, and thereafter twice a week until the end of the experiment.

Batch Experiment
A batch experiment was carried out to assess the changes in SA of soil solution Pi due to physicochemical processes (modSA) after labeling a soil suspension with 33PO4 (Fardeau, 1993; Frossard et al., 1995; López-Hernandez et al., 1998). When a quantity R of carrier-free 33PO4 ions is added to a soil–solution system at steady state, isotopic exchange between ions in the soil solution and ions of the soil solid phase takes place while the P concentration in the soil solution (cP) remains constant. Usually, four samples of the soil solution are taken between 1 and 100 min (= short-term kinetics) after the addition of the radioactive tracer (Fardeau, 1993; Frossard et al., 1994). It is assumed that, during this short duration, the influence of any other processes than physicochemical on the decrease of radioactivity in the soil solution can be ignored (López-Hernandez et al., 1998; Frossard et al., 1995) as no biological 33PO4 immobilization into soil organic matter was observed during 1 d (Fares et al., 1983).

In this study, an amount of soil equivalent to 10 g soil dry weight was shaken in 99 mL of deionized water for 16 h to reach steady state. Then, 1 mL of 33PO4 tracer solution of 0.04 MBq was added at time zero and mixed with a magnetic stirrer. About 2 mL of suspension were removed with a polyethylene syringe after 1, 20, 40, and 60 min and the solution immediately separated from soil particles using a membrane filter (0.2-µm pore size). The radioactivity r(t) remaining in the solution after each period of isotopic exchange (t) was measured by liquid scintillation. The Pi concentration in the soil solution (cP in mg P L-1) was determined colorimetrically (Tiessen and Moir, 1993) on centrifuged (3500 g, 10 min) and filtered (0.2- or 0.025-µm pore size) aliquots obtained at the end of the short-term kinetics. No differences in cP were found between the two filter pore sizes (Sinaj et al., 1998).

The radioactivity r(t) remaining in solution decreases with time t according to the following equation (Fardeau et al., 1985)

(1)
where R denotes the initial quantity of 33PO4 added to the soil–water system, r1 and r({infty}) are the radioactivity remaining in the solution after 1 min and infinity, respectively, and n a parameter calculated from the batch experiment using linear regression between log[r(t)/R)] and log(t) (Fardeau, 1993). The ratio r({infty})/R is the maximum possible dilution of the isotope. It is approximated by the ratio of the water soluble P to the total Pi (Fardeau, 1993).

The quantity of isotopically exchangeable P, E(t) in mg P kg-1 soil, that can move from the soil solid phase into the soil solution represents the plant-available P (Fardeau, 1993; Frossard et al., 1994). It is calculated, modE(t), assuming that (i) 31PO4 and 33PO4 ions have the same fate in the system and (ii) that the SA in the soil solution is identical to the SA of the isotopically exchanged ions in the whole system

(2)

(3)
where the factor 10 accounts for the 1:10 soil/water ratio and R/modr(t) is calculated using Eq. [1]. The isotopic composition of soil solution Pi at any time t may also be extrapolated

(4)

The method of Fardeau (1993) was carried out several times during the preincubation (Table 2) to estimate the variability of the isotopic exchange parameters with time. It was also applied to {gamma}-irradiated soil.


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  		Table 2. Isotopic exchange kinetic parameters obtained in the short-term batch experiment (60 min) after different preincubation times

 
Isotopic Composition during the Incubation Experiment
Labeling was carried out on 600 g soil dry weight after 28 d of preincubation. Exchangeable soil P was labeled by thoroughly mixing 0.014 MBq carrier-free 33PO4 per gram of soil. The water added with 33PO4 raised the water content to 54% of water holding capacity. Subsamples (12 g soil dry wt.) were placed into sealed polyethylene bags (15 cm3) permeable to air. The {gamma}-irradiation (20 kGy within 40 h) of one set of sealed samples was started within 1 to 15 min after labeling.

The experiment was designed to destructively sample four replicates at 11 dates for SA determination. Soil equivalent to 10 g dry weight was shaken at 1:10 soil/solution ratio. The specific activity

(5)
was determined on filtrates as described previously for the isotopic exchange kinetics experiment after decay correction for r(t). The quantity of isotopically exchanged P in the incubation experiment, mesE(t), was calculated as described for modE(t) using Eq. [3].

Sterility of {gamma}-Irradiated Soils
Sterility of {gamma}-irradiated soils was controlled at the end of the incubation. Sterile tryptic soy and malt agars were inoculated with nonirradiated and {gamma}-irradiated soil extracts and incubated for 14 d (20°C). The agars contained cyclohexamide or tetramycin in order to prevent either fungal or bacterial growth in the corresponding agar.

Phosphorus Mineralization
Organic P mineralization may be detected if significant dilution of the isotopic composition in the soil solution (SA) occurs in the incubation experiment due to biological and/or biochemical processes. This should be indicated by a difference, {Delta}SA(t), between SA extrapolated from the isotopic exchange experiment (modSA; Eq. [4] and [1] using t of the respective sampling dates and the activity R used for labeling in the incubation experiment) and SA measured in the incubated sample on the same sampling date (mesSA). From this difference the amount of mineralized Po can be calculated by considering {Delta}E(t), which is the difference between mesE(t) and modE(t).

Biological Soil Properties
Soil respiration was measured as explained above throughout the whole experiment (Fig. 1) . Acid phosphatase activity (phosphomonoesterase) was measured using the method of Tabatabai (1982) on four replicates of {gamma}-irradiated and nonirradiated, air-dried soils that had been preincubated for 13 d before irradiation and air-drying.



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  		Fig. 1. Changes in soil respiration rates (mg CO2 kg-1 d-1) and the P concentration in the soil solution (cP in mg L-1) during preincubation (Days 0–28) and incubation (Days 28–70)

 
Statistics
Each parameter was analyzed on four replicates per soil and sampling date. Significant differences between different sampling dates were tested using Duncan's multiple range test after analysis of variance (SAS Institute, 1989). Significant differences between modE(t) and mesE(t) were tested using a t test (SAS Institute, 1989).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Significance of Preincubation
Soil respiration and, consequently, C mineralization showed two distinct phases (Fig. 1). During the first 13 d, initially high respiration rates decreased rapidly, indicating exhaustion of readily decomposable C substrates. Afterwards, the decrease in respiration rate was much slower. During this second phase, respiration rates are not constant in the strict sense, but changes are small. At this time the soil had already reached constant isotopic exchange parameters (Table 2). This shows that basal P mineralization could be assessed starting the batch and the incubation experiment after 13 d. For our main study, the soil was preincubated during two further weeks. Isotopic exchange parameters obtained from the batch experiment carried out on Day 28 (i.e., on the same day when the incubation experiment was started by soil labeling) were used for constructing the baseline.

Use of the Batch Experiment to Estimate the Baseline
The baseline is obtained by extrapolating SA from the short-term kinetics batch experiment to the duration of the concomitant incubation experiment. It describes SA resulting from physicochemical processes, which is a prerequisite to separate the effects of biological and physicochemical processes on mesSA obtained in the incubation experiment.

The SA obtained from short-term kinetics can be extrapolated to longer times for soils having low to medium P sorption capacity (Fardeau, 1993). According to the isotopic exchange kinetics parameters (Tables 2 and 3), both the nonirradiated and the {gamma}-irradiated soil fulfill this condition (Fardeau, 1993) (Fig. 2) .


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  		Table 3. Isotopic exchange kinetic parameters and phosphatase activity assessed after 28 d of preincubation in the nonirradiated and {gamma}-irradiated soil

 


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  		Fig. 2. Changes in the specific activities (SA) modeled from the isotopic exchange kinetics batch experiment (modSA) and assessed in the incubation experiment (mesSA) for the nonirradiated (left) and {gamma}-irradiated (right) soil

 
At the beginning of the incubation experiment, modSA and mesSA were similar (not significantly different) (Fig. 2) for the nonirradiated soil despite different experimental conditions. The rapid initial decrease of mesSA is dominated by physicochemical processes (Fardeau, 1993). Almost identical initial decreases in incubation experiments as predicted from batch experiments were found in further incubation studies (Oberson, 1998, unpublished data; Oehl, 1999). This observation strongly supports the use of modSA as a baseline. Thereafter, mesSA became lower than modSA. Under the conditions of the incubation experiment, processes such as release of nonlabeled Pi from disrupted soil structural units (Sinaj et al., 1997) can be excluded. Therefore, this dilution can be attributed to the Pi release from nonlabeled soil Po.

Isotopic exchange parameters of the soil were affected by {gamma}-irradiation (Table 3). This shows that it is important to establish a separate baseline for the {gamma}-irradiated soil; that is, batch and incubation experiments have to be carried out on soils that have been subjected to the same treatments (Fig. 2). Increased r1/R and cP and decreased n values suggest that some Pi was released from microbial cells during irradiation, as observed by Zou et al. (1992) and Seeling and Zasoski (1993). Frossard et al. (1996) found similar changes of the isotopic exchange parameters after addition of Pi and readily mineralizable Po to soils. In addition, {gamma}-irradiation can affect extractable Mn, Al, and Fe contents (Wolf et al., 1989), which in turn can affect exchange parameters (Frossard et al., 1995). A batch experiment repeated after 13 d showed that the modifications induced by {gamma}-irradiation did not result in any further changes of isotopic exchange kinetics parameters (data not shown). Therefore, results of the batch experiment carried out immediately after {gamma}-irradiation could be used to establish a baseline (Fig. 2).

Seeing the practical conditions underlying the incubation experiment, the question about the validity of the baseline for the {gamma}-irradiated soil might arise. Soils of the incubation experiment had to be labeled and sealed before {gamma}-irradiation. Therefore, during the first minutes, isotopic exchange followed the same kinetics as for the nonirradiated soil. Because of a lower r1/R in nonirradiated than {gamma}-irradiated soils (Table 3), the contribution of physicochemical processes to the decrease of mesSA was initially stronger than suggested by modSA extrapolated from short-term kinetics parameters assessed on {gamma}-irradiated soils. This might result in an overestimation of mineralization in {gamma}-irradiated soils during the initial stage of the experiment. However, as R was identical for both incubation experiments, similar modSA at t = 3 d for {gamma}-irradiated and nonirradiated soils (Fig. 2) show that this error is minor. The same indication results from {Delta}E(t) at t = 3 d (discussed below).

Biological activity had been largely eliminated by {gamma}-irradiation. In a few replicates only, single colonies of microbes appeared after 10 d, while both test agars inoculated by nonirradiated soil extracts were completely occupied within a few days.

Organic Phosphorus Mineralization in {gamma}-Irradiated vs. Nonirradiated Soils
The Po mineralization resulted in significant isotopic dilution of the SA (Fig. 2). The dilution was higher in the {gamma}-irradiated than in the nonirradiated soil (Fig. 2). This, in turn, resulted in higher {Delta}E(t), differences between mesE(t) and modE(t), and consequently higher estimates for P mineralization in the sterile than the nonsterile soil (Table 4).


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  		Table 4. Daily P mineralization rates (Pmin rates) obtained from the difference [{Delta}E(t)] of isotopically exchangeable P extrapolated from the batch experiment [modE(t)] and isotopically exchangeable P measured in the incubation experiment [mesE(t)]{ddagger}

 
Higher {Delta}E(t) in the {gamma}-irradiated than in the nonirradiated soil could not be explained by the slightly increased phosphatase activity in the {gamma}-irradiated soil (Table 3), because higher {Delta}E(t) in {gamma}-irradiated than in nonirradiated soils was also found for other soils, where phosphatase activity was either not affected or lowered by {gamma}-irradiation (Oehl, 1999). Neither was it due to an overestimation of {Delta}E(t) that might have resulted from the validity of the baseline mentioned previously: the difference between {Delta}E(t) obtained at t = 3 d on nonirradiated and {gamma}-irradiated soils shows that any overestimation would at most have been 3.1 mg P kg-1. After subtraction of this difference from the {Delta}E(t) values of the {gamma}-irradiated soil, the Pmin rates would still be higher in the sterile than the nonsterile soil. Hence, higher {Delta}E(t) in the {gamma}-irradiated soil is attributed to the release to the solution of easily mineralizable organic P compounds derived from killed microbial cells that became accessible to exoenzymes (Seeling and Zasoski, 1993).

In the nonirradiated soil a fraction of P mineralization may be masked by the release of 33PO4 from organic P compounds synthesized after labeling the soil. The extent of this remineralization process was estimated from a concomitant study carried out on the same soil to measure the SA of microbially bound P (Oehl, 1999). The daily 33PO4 release from microbial P back to the soil solution between 5 and 22 d after soil labeling was only 0.2 to 0.5% of the radioactivity remaining in the soil solution on Day 7 (Oehl, 1999). Although the assessment did not include the total microbial P because only part of it is released upon chloroform fumigation, the significance of Pi release from labeled organic P compounds seems to be minor in this incubation experiment. The conclusion that isotope release from labeled Po was low was also drawn by Fares et al. (1983), Martin (1985), and Walbridge and Vitousek (1987), but as acid extractants were used, these observations might have been biased by some Po hydrolysis or low extractability of Po (Oberson et al., 1997). Results of Seeling and Zasoski (1993) show that soil solution Po turns over rapidly and that it is replenished continually after removal. Although it is not clear from their data whether Po was derived from microbial synthesis or solubilization of soil Po, the extent by which mineralization of labeled Po might occur must be considered. With increasing incubation time, the most labile C sources will increasingly be depleted, as indicated by the decreasing trend of soil respiration (Fig. 1) and slightly decreasing differences between modSA and mesSA in the nonirradiated soil (Fig. 2). In addition, it is not known if, during the experiment, the C/P ratios of the remaining substrates increased. As biological P mineralization is partly linked to C mineralization (Gressel et al., 1996), P mineralization could in this case decrease more rapidly than C mineralization.

For all these reasons, to have relevant information on basal Po mineralization, {Delta}SA and resulting {Delta}E(t) obtained early during the incubation experiment should be used. The following discussion of quantities and processes of P mineralization is based on the results obtained during the first 10 d of the experiment.

Quantities and Processes of Phosphorus Mineralization
On Days 7 and 10, the differences between mesE(t) and modE(t), {Delta}E(t), were highly significant (Table 4). Within the first week of the experiment, the daily Pmin rates were 3.3 mg kg-1 d-1 in the {gamma}-irradiated and 1.7 mg kg-1 d-1 in the nonirradiated soil, which is, for this soil, an amount similar to soil solution P obtained in the batch experiment at a 1:10 soil/water ratio (Table 3). As mentioned above, higher {Delta}E(t) in the {gamma}-irradiated than in the nonirradiated soil may mainly be attributed to the release of easily mineralizable organic P compounds derived from lysed microbial cells (Seeling and Zasoski, 1993). This signifies that, in the {gamma}-irradiated soil, a gross, but overestimated, biochemical P mineralization can be measured using combined {gamma}-irradiation and isotopic labeling techniques. In the nonirradiated soil, a gross, biologically and biochemically mediated basal mineralization will be increasingly underestimated because of 33PO4 release from labeled Po compounds. Because of the overestimation in {gamma}-irradiated soils, it is impossible to separate biological and biochemical mineralization for the nonirradiated soil.

The daily Pmin rates determined after 1 wk are similar to those derived for several Northern American Mollisols (0.9–4.2 mg kg-1 d-1; López-Hernandez et al., 1998), but comparison with their results is limited because of the constraints discussed above. Similar data was also presented by Zou et al. (1992), who used a nonisotopic method. These authors measured 0.6 to 3.8 mg kg-1 d-1 mineralized P in {gamma}-irradiated soils, which consequently represents a biochemical, but overestimated, gross P mineralization mediated by the catalytic activity of phosphatase exoenzymes.

Limits of the Proposed Approach
Extrapolation of SA from short-term kinetics to longer times is not valid for P deficient, strongly P sorbing soils (Fardeau et al., 1985; Salcedo et al., 1990; Oehl, 1999). This means that for such soils the establishment of a baseline poses a serious problem. Long-term kinetics were tested as a means to obtain a correct description of isotopic exchange (Fardeau et al., 1985; Oehl, 1999). However, the addition of biocides in long-term kinetics experiments cannot be recommended, because either after a few days they are degraded by microorganisms (e.g., toluene; Davis and Madson, 1996), or they affect soil pH (e.g., NaN3; Trevors, 1996), or they are extremely toxic (HgCl2; Trevors, 1996). Consequently, long-term kinetics are inappropriate as a baseline exclusively describing physicochemical processes, and the P mineralization method described in this publication cannot be applied on strongly P sorbing and P deficient soils. This presents a shortcoming of the method, as on such soils, organic P turnover is assumed to play a much more significant role than on soils well supplied with P (Gijsman et al., 1996; Oberson et al., 1999).


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Basal P mineralization in soils can be measured using isotopic dilution techniques. Soils have to be preincubated for a sufficient time to stabilize basal soil respiration and obtain constant isotopic exchange parameters. The principle of the method is the comparison of the isotopic composition of soil solution Pi resulting from physicochemical processes (= the baseline) with the isotopic composition obtained under the influence of physicochemical, biological, and biochemical processes. The presented method cannot be applied to highly sorbing P deficient soils because the assessment of a correct baseline is impeded on these soils.

The existence of biochemical P mineralization was confirmed in {gamma}-irradiated soil. Greater amounts of mineralized P were measured in the {gamma}-irradiated than in nonirradiated soil, which was attributed to easily mineralizable organic P compounds released from microbial cells as a result of {gamma}-irradiation. Additionally, in nonirradiated soils, a fraction of P mineralization may be masked by the release of Pi from newly labeled Po compounds. Therefore, a gross, but overestimated, biochemical mineralization may be obtained using irradiated soils, while biologically and biochemically driven basal mineralization obtained from nonirradiated soils may be increasingly underestimated with time. Therefore, results obtained after a few days of incubation should be used to calculate P mineralization rates. After 7 d, the basal daily Pmin rate of the nonirradiated soil was 1.7 mg kg-1 d-1, which is an amount approximately equivalent to soil solution P in this soil. This shows that Pi release by mineralization may deliver significant amounts of plant-available P to the soil solution.


   ACKNOWLEDGMENTS
 
We are grateful to the Swiss Federal Research Station FAL in Zurich-Reckenholz and Liebefeld-Bern, and the Research Institute FiBL in Frick for providing the soil. We thank H.J. Zehnder for conducting the {gamma}-irradiation at the Swiss Federal Research Station FAW in Wadenswil and F. Mascher from Plant Pathology group of ETH Zurich for doing the sterilization tests of the soils. We acknowledge every person who assisted in the realization of this study, especially Dr. A. Fliessbach (FiBL) for helpful discussions, V. Hemmeler and H.U. Tagmann (both ETH Zurich) for technical assistance, Drs. H.R. Roth and M. Hürzeler (Statistic Group, ETH) for advice in statistics. Drs. L.M. Condron (Lincoln University, Canterbury, New Zealand), M. McLaughlin (CSIRO, Glen Osmond, Australia) and the anonymous reviewers are warmly acknowledged for their helpful comments on earlier drafts of the manuscript.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Sponsoring organization: Swiss Federal Inst. of Technology (ETH), Zurich.

Received for publication July 29, 1999.


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




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