Determination of microbial phosphorus Kp factors in a spodosol: influence of extractant, water potential, and soil horizon

Soil Biology & Biochemistry 36 (2004) 1925–1934 www.elsevier.com/locate/soilbio

Determination of microbial phosphorus Kp factors in a spodosol: influence of extractant, water potential, and soil horizon C.M. Bliss*, N.B. Comerford, R.M. Muchovej Department of Soil and Water Science, University of Florida, P.O. Box 110290, 2169 McCarty Hall Gainesville, FL 32611-0290 USA Received 30 July 2003; received in revised form 7 May 2004; accepted 20 May 2004

Abstract The acidic, sandy soils in the southeastern US are phosphorus (P) limited for forest production and are commonly fertilized with P. There is no P retention capacity in the A horizon. However, microbial biomass may immobilize and retain P fertilizer before it is leached below seedling rooting depth making P fertilization more efficient. An accurate estimation of microbial P is dependent on measuring the Kp factor in the fumigation–extraction method. The overall purpose of this study was to examine the fumigation–extraction method for microbial P in acidic, forested, sandy soils. The three objective were: to determine which extractant was the most useful extracting microbial P by comparing the standard basic extractant, 0.5 M NaHCO3 at pH 8.5, against several acidic and oxalate extractants; to evaluate whether soil water potential influenced the Kp factor; and to test whether the Kp factor differed by soil horizon within the profile of a representative Flatwoods Spodosol. Three millimolar oxalate was determined to be the preferred extractant due to its efficient removal of microbial P and ease of analysis. The Kp factor was dependent on soil water potential and horizon. The range in Kp at different water potentials using 3 mM oxalate was from 0.31 to 0.67 in the A horizon, 0.48 to 0.91 in the E horizon, and 0.22 to 0.45 in Bh horizon. The highest Kp factors tended to be at water potentials near saturation and under the driest condition. Differences in Kp were attributed to the influence that water potential and soil horizon had on microbial assemblages and diversity. Using a literature value of Kp, instead of measuring Kp directly, caused an overestimate of K7 to 63% in the A horizon, 63–160% in the E horizon and 7–32% in the Bh horizon. The best estimate of microbial P required that Kp be evaluated for specific soil conditions. q 2004 Elsevier Ltd. All rights reserved. Keywords: Microbial phosphorus; Microbial diversity; Soil water potential; Spodosol; Flatwoods; Lower coastal plain; Forest soils

1. Introduction Microbial biomass, which is composed of bacteria, fungi, and other microbiota, is a major controlling component of nutrient transformations and cycling in soils. Phosphorus (P) transformation and cycling through the microbial biomass have been studied (Seeling and Zasoski, 1993; Gijsman et al., 1997; He et al., 1997; Grierson et al., 1998) under a variety of soil conditions. Brookes et al. (1984) estimated that total P held in the microbial biomass ranged from 1.4 to 4.7%. Subsequent studies have fallen within this range (Dı´az-Ravin˜a et al., 1995; Joergensen et al., 1995).

* Corresponding author. Tel.: C1-352-392-1951; fax: C1-352-392-3902 E-mail address: [email protected] (C.M. Bliss). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.05.012

In the Coastal Plain of the southeastern US, Spodosols cover approximately 14 million ha, with approximately 10 million ha in Florida. Phosphorus availability limits forest production on these Spodosols (Colbert et al., 1990). The sandy nature of the surface horizons limits P retention capacity (Humphreys and Pritchett, 1971; Ballard and Fiskell, 1974; Fox et al., 1990a; Harris et al., 1996; Zhou et al., 1997), resulting in P mobility. Therefore, P that is not absorbed by plants or microorganisms is leached below the A horizon (Harris et al., 1996; Nair et al., 1999). Due to the low native bioavailability of P in these Spodosols, P fertilization is commonly used in combination with weed control. Since the surface horizon has a low P retention and the associated understory vegetation is removed or reduced, the microbial population could be a significant sink for P fertilizer. Microbial P estimates, as defined by Brookes et al. (1982), Hedley and Stewart (1982) and McLaughlin et al.

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(1986), are based on extracting soil with 0.5 M NaHCO3 at pH 8.5. McLaughlin et al. (1986) investigated both basic and acidic soil extracting solutions and concluded that 0.5 M NaHCO3 at pH 8.5 removed the most P from the microbial biomass. It is important to note that the pH of the soils they tested was 6.0 and higher. However, forested soils in the southeastern Coastal Plain are acidic, with the pH often ranging between 3.8 and 4.5. Inorganic P (Pi) chemistry is dominated by aluminum oxides where present, such as in the spodic and argillic horizons. The appropriateness of using a high pH extractant under these conditions is therefore questionable. Other extractants, such as low molecular weight organic acids promote ligand exchange and aluminum oxide dissolution (Fox et al., 1990b; Lan et al., 1995) and are known to remove P from acidic soils. The fumigation–extraction method underestimates microbial P due to the incomplete release of P from the microbial cells during fumigation, microbes resistant to fumigation, and subsequent adsorption of Pi onto the mineral soil surface (Brookes et al., 1982; Hedley and Stewart, 1982; McLaughlin et al., 1986). A correction factor, Kp, takes into account microbial P that is not extractable and is needed to accurately estimate P held in the microbial biomass (Brookes et al., 1982; Hedley and Stewart, 1982; McLaughlin et al., 1986). Kp factors vary with soil condition, due to either changes in microbial communities or P sorption (Brookes et al., 1982; Hedley and Stewart, 1982; McLaughlin et al., 1986). Brookes et al. (1982) determined that inorganic Kp (Kpi) factors ranged from 0.32 to 0.38 and total Kp (Kpt) factors ranged from 0.44 to 0.49, while Hedley and Stewart (1982) reported a similar range of 0.32–0.47 for Kpt. Brookes et al. (1982) and Hedley and Stewart (1982) concluded that a Kpi of 0.40 provided a good estimate for soils with a basic pH. Studies have used 0.40 as the correction factor in microbial P estimation (Perrott et al., 1990; Tate et al., 1991; Clarholm, 1993; Gijsman et al., 1997; Lukito et al., 1998). McLaughlin et al. (1986) determined that Kp factors ranged from 0.33 to 0.57 and suggested that they must be calibrated for each soil because of different microbial populations present in different environments. Since microbial communities also vary with soil water potential (Wilson and Griffin, 1975; Lund and Goksoyr, 1980; Orchard and Cook, 1983; Skopp et al., 1990), it seems reasonable to suggest that Kp factors may also differ with this variable.

This study’s overall purpose was to examine the fumigation–extraction method for measuring microbial P in acidic, forested, sandy soils. There were three specific objectives. The first objective was to determine which extractant was most efficient at removing microbial P by comparing the standard basic extractant, 0.5 M NaHCO3 at pH 8.5, against several acidic and oxalate extractants with the hypothesis that 0.5 M NaHCO3 is not the most efficient extractant. Since soil water potential controls microbial communities and population levels, the second objective was to evaluate whether the soil water potential influenced the Kp factor. The null hypothesis was that Kp factors are not affected by water potential. The third objective was to test whether the Kp factor differed by soil horizon within a profile of a representative Flatwoods Spodosol. The hypothesis for the third objective was that Kp factors do not differ by soil horizon and that a common Kp would be appropriate for all horizons.

2. Methods 2.1. Study area and field sampling The study area was located 33 km northeast of Gainesville, FL. The site was a 16 ha managed slash pine plantation (Pinus elliottii var. elliottii Engelm.) that was clearcut in April 1994, bedded in September and November 1994, and planted with slash pine seedlings in January 1995. In February 1995, 85 g haK1 of imazypyr was applied as Arsenal in 1.5 m wide bands down the beds for weed control. The plantation was underlain by a sandy, siliceous, thermic Ultic Alaquod, which is primarily Pomona fine sand and sand. The average annual air temperature was 21 8C, and the long-term average annual rainfall was approximately 1330 mm. In an average year, the shallow water table may be less than 25 cm from the soil surface for 1–3 months, between 25 and 100 cm for 6 months, and exceeding 100 cm during the dry season (Soil Conservation Service, 1985). Soil from the A, E, and Bh horizons was sampled in July 1996 from a subsection of the study area, approximately 2 ha. Approximately 10 kg of soil from each horizon was collected from 20 random points with a 7.5 cm diameter soil auger. The samples were combined to form one combined sample for each horizon. The soil was air-dried and sieved to pass a 2 mm screen. Table 1 provides chemical and physical

Table 1 Soil characterization for the A, E and Bh horizons of a Florida flatwood Spodosol Horizon

pH

Sand (%)

Silt (%)

Clay (%)

Organic C (%)

Al (cmolc kgK1)

Fe (cmolc/kg)

Ca (cmolc/kg)

K (cmolc/kg)

Mg (cmolc/kg)

Na (cmolc/kg)

A E Bh

4.1 4.0 4.3

94 96 90

5 3 8

1 1 2

0.69 (b)a 0.16 (c) 1.23 (a)

0.15 0.00 2.24

0.01 0.01 0.02

0.14 0.04 0.04

0.33 0.12 0.17

0.27 0.16 0.28

0.01 0.01 0.03

a

Significant differences (P!0.05) between horizons within organic C.

C.M. Bliss et al. / Soil Biology & Biochemistry 36 (2004) 1925–1934

characteristics of the A, E, and Bh. Horizon depths of a typical Spodosol in this area are approximately 0–12 cm for the A horizon, 12–40, and 40–60 cm for the Bh horizon (Soil Conservation Service, 1985). 2.2. Growth and culture of microbes Five water potentials were selected to represent a dry soil (K1000 kPa), near field capacity (K15 and K8 kPa), an unsaturated condition that represents a high water table (K3 kPa), and near saturation (K0.1 kPa). These water potentials were based on results from P mineralization studies that evaluated mineralization rate versus soil water potential in a similar soil (Grierson et al., 1999). In order to culture native microbial populations representative of the different water potentials, soil samples from each horizon were incubated at each water potential for a minimum of 10 days at 28 8C. Water potential was kept constant throughout the incubation period by weighing the samples and adding double deionized water when required. The soil sample was then diluted with double deionized water to a soil to solution ratio of 1:10 and 1:1000 for fungal and bacterial growth, respectively. The native fungal populations were grown by adding 1 ml of the 1:10 dilution to a growth medium containing 660 mg NaNO3, 330 mg KH2PO4, 265 mg KCl, 165 mg MgSO 4 7H 2O, 6.6 mg FeSO 4, 165 mg yeast extract, 10 g sucrose, 100 mg streptomycin sulphate, and 5 mg tetracycline hydrochloride in 1 l of double deionized water (McLaughlin et al., 1986). Native bacterial populations were grown by adding 1 ml of the 1:1000 solution to a 0.3% typic soy broth in double deionized water containing 100 mg cycloheximide lK1 (McLaughlin et al., 1986). The microbes were grown in the dark at 28 8C for approximately 7 days. The fungal community was filtered from the nutrient solution, rinsed in double deionized water, blotted dry, and subsampled before adding back into soil samples for determination of Kp factors. The bacterial community was grown at the same temperature and time length as the fungal community. The bacterial suspension was centrifuged, rinsed with double deionized water, and resuspended in double deionized water before addition back into soil samples. Total P (Pt) was measured in the fungal and bacterial communities following a modified method of Smethurst and Comerford (1993). A known amount of sample (approximately 300 mg of fungi and 5 ml of bacterial suspension) was dried in a muffle furnace at 104 8C overnight. The samples were then ashed at 500 8C for 4 h. When cool, 7 ml of 40% HCl were added to samples and evaporated to dryness on a hot plate. Five milliliter of concentrated HCl were added and again evaporated until dry. Ten milliliter of 0.1 N HCl were added to samples and allowed to stand overnight. A known quantity of the sample in 0.1 N HCl, depending upon the P concentration, was diluted to 25 ml and analyzed for P using the method of Murphy and Riley (1962).

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2.3. Kp determination Approximately 80 mg of the fungi and 1 ml of the bacterial suspension were added to soil samples at the same water potential and horizon from which the microorganisms were grown. The samples were then fumigated with 2 ml of liquid chloroform for 24 h. The chloroform was allowed to evaporate, and the soil was extracted for P. The Kp factor, or recovery fraction of microbial P, was calculated using the equation: Kp Z ðm K nÞ=ðxyÞ

(2.1)

from Brookes et al. (1982), where m is the amount of Pi measured in the samples that contained the added microbial biomass, n is the amount of Pi measured in fumigated samples without additional microbes added, x is the percent recovery of Pi determined from a P spike, and y is the amount of Pt in the microbial biomass added to the sample. This equation is the same when measuring Kp factors for microbial Pt, except that m becomes the amount of Pt measured in the samples with added microbes and n becomes the Pt measured in sample without the added microbes. Total P was determined by digestion of the extractant as described above. In order to determine the percent recovery of Pi for the variable x above, a range of Pi concentrations from 40 to 100 mg P gK1 soil were added to a set of soil samples in the Bh horizon. This range was used because of the variable P contained in the added microbes and to determine if P adsorption was linear. Although P adsorption was assumed to be negligible in the A and E horizons (Harris et al., 1996; Zhou et al., 1997), a spike of 40 mg P gK1 soil was added. This concentration of P was used because added P in the microbes was generally 40 mg P gK1 soil. Phosphorus recovery is presented in Table 2. Inorganic P was used as the spike for both Kpi and Kpt on account of most P released from the microbial cells is Pi (Brookes et al., 1982). 2.4. Extractants The following solutions were used to extract P from the soil samples: 0.5 M NaHCO3 at pH 8.5 (Olsen et al., 1954), 0.03 N NH4F and 0.25 N HCl (Bray and Kurtz, 1945), 0.05 N HCl and 0.025 N H2SO4, or Mehlich 1, (Nelson et al., 1953), and 1, 2, and 3 mM oxalate at pH 3.2, 2.9, and 2.7, respectively. The Bray and Kurtz extractant has been used in acidic soils as an index of P availability. It removes easily acid-soluble forms of P from calcium phosphates and a portion of aluminum and iron phosphates (Bray and Kurtz, 1945). Mehlich 1 was used as another measure of P availability in acidic soils because of its ability to dissolve aluminum and iron phosphates (Nelson et al., 1953). Oxalate is a naturally occurring, low molecular weight organic acid whose presence in the soil solution is due to microbial activity and root exudation. Oxalate increases P

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Table 2 Percent recovery of Pi added as a spike to the A, E and Bh horizon soil samples for each extractant Horizon

Percentage recovery of P added 8.5 M NaHCO3

Bh 40 mg g soil Bh 60 mg gK1 soil Bh 80 mg gK1 soil Bh 100 mg gK1 soil Bh average A 40 mg gK1 soil E 40 mg gK1 soil K1

a

50 52 52 48 50 94 90

1 mM Oxalate 63 61 72 75 68 95 96

2 mM Oxalate 82 84 89 80 84 97 97

3 mM Oxalate 85 82 82 86 84 100 100

Bray and Kurtz ND ND ND ND ND 96 95

a

Mehlich 1 79 83 82 90 83 96 94

ND, not determined.

solubility through formation of stable complexes with aluminum (Martell et al., 1988) in solution, ligand exchange (Stumm, 1986; Fox et al., 1990a), and dissolution of metaloxide surfaces (Stumm, 1986). The NaHCO3 extract was used with a 1:10 soil to solution ratio and shaken for 1 h (Grierson et al., 1998). A 1:7 soil to solution ratio with a 1 min shaking time and a 1:4 soil to solution ratio with a 5 min shaking time was used for the Bray and Kurtz extract and Mehlich I, respectively (Olsen and Sommers, 1982). A 1:10 soil to solution ratio and 10 min shaking time was used with the oxalate solutions. Ten minute shaking time was determined with initial studies to be the time that extracted the most P. After extraction and filtration, Pi was measured using the Murphy and Riley (1962) method. When required, filtered samples were treated with HCl to lower the pH to 3.0. The NaHCO3 samples had to be refiltered after adding the acid because acidification caused precipitation of organic compounds. Total P was also measured for each sample as described previously. 2.5. Evaluation of water potential and soil characteristics on the Kp method Bacterial and fungal populations were grown from each horizon at K8 kPa using the same methods described above. These microbial populations were then added into soil samples maintained at each of the five different water potentials. Microbes were also added into soil samples from the other horizons maintained at K8 kPa. For instance, microbes grown from the A horizon at K8 kPa were added to soil samples from the A horizon at each water potential and into the E horizon and Bh horizon at K8 kPa. Samples were then extracted with 3 mM oxalate and analyzed for Pi. 2.6. Statistical analysis Significant differences were determined using SAS, version 8.01 (SAS Institute, Inc., 2001) using a mixed ANOVA model. The mixed model was used due to missing data. Due to significant interactions between main effects, separation of least-squared means for individual levels of

main effects were performed using the Tukey standardized range test. The separation of the least-squared means of the Kp factors in the follow up study at K8 kPa were conducted using the same procedure. All differences were deemed significant for P levels less than 0.05.

3. Results Organic carbon (C) was significantly different by horizon, with the most organic C in the Bh and the least in the E horizon. The greatest concentration of sand was in the E horizon, and the most silt and clay was in the Bh horizon. Aluminum concentration was highest in the Bh horizon (Table 1). 3.1. Recovery of P spike In the Bh horizon, more P was recovered with the 2 mM oxalate (80–89%), 3 mM oxalate (82–86%), and Mehlich 1 (79–90%) extractants (Table 2). Less P was recovered with 1 mM oxalate and even less with NaHCO3. Approximately 50% of the P spikes were recovered when NaHCO3 was used. In the A and E horizons, 3 mM oxalate recovered the entire P spike while NaHCO3 recovered 94 and 90% in the A and E horizon, respectively. The Bray and Kurtz extractant and Mehlich 1 recovered 96 and 95 and 96 and 94% in the A and E horizons, respectively. 3.2. Kp factors by extractant Inorganic Kp factors ranged from 0.14 to 0.72 in the A horizon, 0.19 to 0.99 in the E horizon, and 0.11 to 0.45 in the Bh horizon (Table 3). The Kpi factors for each extractant within each water potential were compared for each horizon. In all three horizons, at least one of the low oxalate concentrations provided similar Kpi factors to the standard extractant, 0.5 M NaHCO3 (pH 8.5), with the exception of two instances. However, within the different concentrations of oxalate, 3 mM oxalate had a tendency to be more efficient in removing Pi that was attributable to the microbial biomass.

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Table 3 Comparison of inorganic Kp factors by extractant within each water potential and by water potential within each extractant in the A, E, and Bh horizons Extractant A horizon 0.5 M NaHCO3 1 mM oxalate 2 mM oxalate 3 mM oxalate Bray and Kurtz Mehlich 1 E horizon 0.5 M NaHCO3 1 mM oxalate 2 mM oxalate 3 mM oxalate Bray and Kurtz Mehlich 1 Bh horizon 0.5 M NaHCO3 1 mM oxalate 2 mM oxalate 3 mM oxalate Bray and Kurtz Mehlich 1 a b c d

Water potential (kPa) K0.1

K3

K8

K15

K1000

0.65 (Aaab) 0.72 (Aa) 0.71 (Aa) 0.67 (Aa) 0.39 (Bb) 0.22 (Ca)

0.42 (BCb) 0.53 (ABb) 0.47 (Bbc) 0.65 (Aa) 0.29 (CDc) 0.21 (Da)

0.35 (Ab) 0.30 (ABc) 0.28 (ABc) 0.31 (ABc) 0.34 (Abc) 0.19 (Bab)

0.45 (Ab) 0.41 (Abc) 0.48 (Ab) 0.49 (Ab) 0.51 (Aa) 0.18 (Bb)

0.38 (Bb) 0.51 (ABb) 0.60 (Aab) 0.56 (Aab) 0.28 (BCc) 0.14 (Cb)

0.99 (Aa) 0.66 (Bb) 0.85 (Abab) 0.74 (Bab) 0.79 (Ba) 0.43 (Ca)

0.75 (Bb) 0.95 (Aa) 0.90 (Aa) 0.91 (Aa) 0.55 (Cb) 0.35 (Dab)

0.60 (Ac) 0.53 (Ab) 0.58 (Ac) 0.48 (Ac) 0.30 (Bc) 0.19 (Bc)

0.73 (Abc) 0.70 (Aab) 0.68 (Abc) 0.64 (Abc) 0.42 (Bbc) 0.28 (Bbc)

0.73 (ABbc) 0.71 (ABab) 0.86 (Aab) 0.83 (Aa) 0.59 (Bb) 0.28 (Cbc)

0.36 (ABb) 0.15 (Ca) 0.25 (BCa) 0.45 (Aa) NDd 0.16 (Cab)

0.27 (Ab) 0.13 (Ba) 0.17 (Bc) 0.28 (Ab) ND 0.11 (Bb)

0.33 (Ab) 0.17 (Ba) 0.23 (ABa) 0.30 (Aab) ND 0.14 (Bb)

NAc NA NA NA ND NA

0.72 (Aa) 0.13 (Ca) 0.18 (BCbc) 0.22 (Bb) ND 0.21 (BCa)

Significant differences (P!0.05) between extractants within each water potential and horizon (upper case letters down columns). Significant differences (P!0.05) between water potentials within each extractant and horizon (lower case letters across rows). NA, missing data. ND, not determined.

The in A and E horizons, the different solutions extracted similarily. The oxalate concentrations provided either similar or better Kp values. The Bray and Kurtz extractant removed less Pi from the microbial biomass, but it was not always significantly lower than oxalate or 0.5 M NaHCO3. Mehlich 1 consistently extracted less P than all other extractants. It extracted significantly less Pi than both oxalate and 0.5 M NaHCO3 but was not always significantly lower than Bray and Kurtz. Within the Bh horizon, the solutions did not extract P similarly as seen in the A and E horizons. Sodium bicarbonate was similar to 2 and 3 mM oxalate, but the lowest concentration of oxalate was clearly less able to extract P. The extraction efficiency of 3 mM oxalate was clearly shown in the Bh horizon, in which the different oxalate concentrations displayed increasing P extraction with increasing concentration. The Mehlich 1 solution extracted the least Pi from the microbial biomass, but it was not significantly lower than the other extractants in all cases. Total Kp factors ranged from 0.22 to 0.97 in the A horizon and 0.10 to 0.89 in the Bh horizon (Table 4). There was not sufficient sample to evaluate Kpt for the E horizon. As with the Kpi, the Kpt factors showed similar trends. In the A horizon, the Kpt factors for the three oxalate concentrations were similar to 0.5 M NaHCO3, with Bray and Kurtz and Mehlich 1 extracting less Pt from the microbial biomass. The Bh horizon provided similar trends. However,

2 mM oxalate was overall able to remove more Pt from the added microbes. 3.3. Kp factors by water potential When comparing Kpi factors by water potential within extractants (Table 3), the most Pi was extracted from the microbial biomass when the soil was near saturation (K0.1 kPa). Soil at saturation had the significantly highest Kpi factor or was similar to the highest Kpi factor for the majority of comparisons. In the A and E horizons, soil with the water potential closest to field capacity (K8 kPa) yielded the least quantity of P from the microbial biomass. The Kpi factors tended to decrease as the soil became drier but then increased after K8 kPa in both the A and E horizons. In the Bh horizon, the lowest Kpi occurred in soil at K3 kPa, after which the Kpi increased. When comparing the amount of Pt extracted (Table 4), the Kpt factors followed the same trend as the Kpi factors with more P extracted in soil at saturated and dry conditions and least when the soil was at an optimum water potential, near field capacity. Again, more P was extracted from the microorganisms in the saturated soil with a decrease in extractability with decreasing water potential. Extractable P increased again in the drier soils. In the A horizon, the least P was extracted from the microbial biomass in soil at K8 kPa whereas in the Bh horizon, the least P was extracted from microbes at K3 kPa.

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Table 4 Comparison of total Kp factors by extractant within each water potential and by water potential within each extractant for the A and Bh horizons. Kp factors were not determined for the E horizon Extractant

Water potential ( kPa)

A horizon 0.5 M NaHCO3 1 mM oxalate 2 mM oxalate 3 mM oxalate Bray and Kurtz Mehlich 1 Bh horizon 0.5 M NaHCO3 1 mM oxalate 2 mM oxalate 3 mM oxalate Mehlich 1 a b c

K0.1

K3

K8

K15

K1000

0.91 (Aaab) NA 0.97 (Aa) 0.74 (Ba) 0.41 (Cb) 0.22 (Da)

0.74 (Aab) 0.59 (Ba) 0.76 (Aab) 0.77 (Aa) 0.34 (Cb) 0.23 (Ca)

0.63 (Abc) 0.38 (Bbc) 0.44 (Bb) 0.43 (Bc) 0.38 (Bb) 0.22 (Ca)

0.53 (Bc) NA 0.60 (Bb) 0.59 (Bb) 0.86 (Aa) 0.29 (Ca)

NAc 0.51 (Bab) 0.82 (Aab) NA 0.44 (BCb) 0.26 (Ca)

0.43 (Ab) 0.23 (Ba) 0.55 (Ab) 0.46 (Aa) NA

0.55 (Ab) 0.17 (Ca) 0.33 (Bc) NA 0.16 (Cb)

0.40 (Ab) 0.23 (Ba) 0.43 (Abc) NA 0.17 (Bb)

NA NA NA NA NA

0.78 (Ba) 0.21 (Ca) 0.89 (Aa) 0.39 (Cab) 0.33 (Ca)

Significant differences (P!0.05) between extractants within each water potential and horizon (upper case letters down columns). Significant differences (P!0.05) between water potentials within each extractant and horizon (lower case letters across rows). NA, missing data.

3.4. Kp factors by soil horizon When comparing the Kpi factors by horizon, the highest Kpi factors tended to be in the E horizon and Table 5 Comparison of inorganic Kp factors by horizon within each water potential for each extractant Horizon

the lowest in the Bh horizon (Table 5). Although the E horizon provided the highest K pi factor for all extractants except for the Mehlich 1 extract; the value was not always statistically higher than other horizons. The Kpt factors were significantly higher for the A horizon (Table 6). This occurred for all extractants except Mehlich 1, with which no significant differences were obtained.

Water potential (kPa) K0.1

0.5 M NaHCO3 A 0.65 (ba) E 0.99 (a) Bh 0.36 (c) 1 mM oxalate A 0.72 (a) E 0.66 (a) Bh 0.15 (b) 2 mM oxalate A 0.71 (a) E 0.85 (a) Bh 0.25 (b) 3 mM oxalate A 0.67 (a) E 0.74 (a) Bh 0.45 (b) Bray and Kurtz A 0.39 (b) E 0.79 (a) Bh NDc Mehlich 1 A 0.22 (b) E 0.43 (a) Bh 0.16 (b)

K3

K8

K15

K1000

0.42 (b) 0.75 (a) 0.27 (c)

0.35 (b) 0.60 (a) 0.33 (b)

0.45 (b) 0.73 (a) NAb

0.38 (b) 0.73 (a) 0.72 (a)

0.53 (b) 0.95 (a) 0.13 (c)

0.30(b) 0.53 (a) 0.17 (b)

0.41 (b) 0.70 (a) NA

0.51 (a) 0.71 (a) 0.13 (b)

0.47 (b) 0.90 (a) 0.17 (c)

0.28 (b) 0.58 (a) 0.23 (b)

0.48 (b) 0.68 (a) NA

0.60 (b) 0.86 (a) 0.18 (c)

0.65 (b) 0.91 (a) 0.28 (c)

0.31 (b) 0.48 (a) 0.30 (b)

0.49 (a) 0.64 (a) NA

0.56 (b) 0.83 (a) 0.22 (c)

0.29 (b) 0.55 (a) ND

0.34 (a) 0.30 (b) ND

0.51 (a) 0.42 (b) ND

0.28 (b) 0.59 (a) ND

0.21 (b) 0.35 (a) 0.11 (c)

0.34 (a) 0.30 (a) 0.14 (b)

0.18 (b) 0.28 (a) NA

0.14 (b) 0.28 (a) 0.21 (ab)

a Significant differences (P!0.05) between horizons within each water potential and extractant. b NA, missing data. c ND, data not determined.

Table 6 Comparison of total Kp factors by horizon within each water potential for each extractant Horizon

Water potential ( kPa) K0.1

0.5 M NaHCO3 A 0.91 (aa) Bh 0.43 (b) 1 mM oxalate A NA Bh 0.23 2 mM oxalate A 0.97 (a) Bh 0.55 (b) 3 mM oxalate A 0.74 (a) Bh 0.45 (b) Bray and Kurtz A 0.41 Bh NDc Mehlich 1 A 0.22 Bh NA

K3

K8

K15

K1000

0.74 (a) 0.55 (b)

0.63 (a) 0.40 (b)

0.53 (a) NA

NAb 0.78

0.59 (a) 0.17 (b)

0.38 (a) 0.17 (b)

NA NA

0.51 (a) 0.21 (b)

0.76 (a) 0.33 (b)

0.44 (a) 0.33 (a)

0.60 NA

0.82 (a) 0.89 (a)

0.77 (a) 0.26 (b)

0.43 (a) 0.10 (b)

0.59 NA

NA 0.39

0.34 ND

0.38 ND

0.86 ND

0.44 ND

0.23 (a) 0.16 (a)

0.22 (a) 0.17 (a)

0.29 NA

0.26 (a) 0.33 (a)

a Significant differences (P!0.05) between horizons within each water potential and extractant. b NA, missing data. c ND, data not determined.

C.M. Bliss et al. / Soil Biology & Biochemistry 36 (2004) 1925–1934 Table 7 Inorganic Kp factors measured in the ‘test’ horizons. Microbes were grown from the original horizon, added to the test horizon, and the Kp factor was measured in the test horizon. For example, microbes were grown from the A horizon at K8 kPa and added to samples in each of the test horizons Original horizon

Test horizon

Kp factor

A (K8 kPa)

A (K0.1 kPa) A (K3 kPa) A (K8 kPa) A (K15 kPa) A (K1000 kPa) E (K8 kPa) Bh (K8 kPa) E (K0.1 kPa) E (K3 kPa) E (K8 kPa) E (K15 kPa) E (K1000 kPa) A (K8 kPa) Bh (K8 kPa) Bh (K0.1 kPa) Bh (K3 kPa) Bh (K8 kPa) Bh (K15 kPa) Bh (K1000 kPa) A (K8 kPa) E (K8 kPa)

0.35 0.30* 0.36 0.37 0.37 0.42* 0.46* 0.83 0.92 0.94 0.90 0.85 0.85 0.65* 0.26 0.22* 0.28 0.23 0.25 0.34 0.35*

E (K8 kPa)

Bh (K8 kPa)

1931

Table 8 Comparison of microbial P concentrations with determined inorganic Kp factors using 0.5 M NaHCO3 and a Kp factor of 0.40. For comparison purposes, 5 kg P haK1 of microbial P is used Water potential (kPa)

With measured Kpi (kg P haK1)

A horizon K0.1 8 K3 12 K8 14 K15 11 K1000 13 E horizon K0.1 5 K3 7 K8 8 K15 7 K1000 7 Bh horizon K0.1 14 K3 19 K8 15 K15 NAa K1000 7 a

With KpZ0.40 (kg P haK1)

Over estimation (%) (kg P haK1)

Under estimation (%) (kg P haK1)

13 13 13 13 13

63 8 – 18 0

– – 7 – 0

13 13 13 13 13

160 86 63 86 86

– – – – –

13 13 13 13 13

– – – – 86

7 32 7 – –

NA, missing data.

*Significantly different (P!0.05) from the original horizon value.

3.5. Evaluation of water potential and soil characteristics on the Kp method When comparing the Kpi factors measured within the original horizon, there was no significant difference when microbes from a water potential were added to other water potentials (Table 7). This result was consistent in all three horizons. But when comparing the Kpi factors measured when the microbes were added to different horizons, significant differences were determined. When testing microbes from the A horizon in the E and Bh horizons, there were no significant differences. Significant differences were detected when E microbes were added into the A and Bh horizons and when Bh microbes were added into the A and E horizons.

3.6. Comparison of Kp factors with the literature Kp factor Since the Kpi factors measured were significantly different by soil water potential and horizon, the differences in microbial P were calculated using the commonly used literature value of 0.40 (Brookes et al., 1982; Hedley and Stewart, 1982) and the current study’s values (Table 8). Assuming a Kp factor from the literature resulted in estimates of microbial P that were 0–160% different from the estimates using Kp values from this study.

4. Discussion 4.1. Objective 1: the most efficient extractant When comparing the ability and suitability of extracts for removing Pi from the microbial biomass, oxalate performed more consistently than the other extractants, including NaHCO3. In Spodosols of the southeastern US, the Bh horizon is dominated by amorphous Al-oxides (Ballard and Fiskell, 1974; Lee et al., 1988). Oxalate has an effect on both P and Al release from the Bh horizon because it replaces P through ligand exchange and dissolution of Al-oxide surfaces (Fox and Comerford, 1992). The A horizon in this soil has little Al (Fox and Comerford, 1992) beyond what is found on the meager cation exchange capacity, and the Pi in this horizon is nearly all water soluble (Fox et al., 1990a). Under these conditions, oxalate and NaHCO3 extract similar amounts of Pi. As shown in the recovery of the P spike in the A and E horizon, a high percentage was recovered with all extractants, but 3 mM oxalate recovered the entire spike. In acidic soils, HCOK 3 ions replaced P sorbed on the soil surface (Olsen et al., 1954), but the high pH of NaHCO3 also dissolved some organic compounds. The dissolved organic matter precipitated when the sample was acidified for the measurement of Pi by the method of Murphy and Riley (1962). Therefore extra filtration was required. The Bray and Kurtz extractant is a commonly used extractant for P on acidic soils, it did not extract microbial P well. Although Mehlich 1 recovered a high percentage of the P spike in the Bh horizon (most likely due to

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its ability to dissolve Al and Fe phosphates), it also did not perform well in extracting P from microbial cells. Due to the better extraction of P from the microbial biomass and the ease of analysis, oxalate is recommended for these Spodosols. Three millimolar oxalate was the extraction of choice due to its ability to recover more Pi and the ease of use when compared with NaHCO3. As with Kpi, 2 and 3 mM oxalate were not significantly different the majority of the time. 4.2. Objective 2: effect of water potential on Kp factors Microbial communities change with season (Smit et al., 2001), temperature (Dalias et al., 2001), pH and substrate (Yan et al., 2000), and water potential (Brockman et al., 1992; Nazih et al., 2001; Marschner et al., 2002; Treves et al., 2003). Dı`az-Ravin˜a et al. (1995) determined that differences in nutrient concentrations in the microbial biomass are significantly related to soil characteristics, including soil moisture. Since water potential affects the diversity of microbial communities, this diversity would also influence the Kp factors measured at different water potentials. Brookes et al. (1982) and Hedley and Stewart (1982) measured the Kp factor of individual microbial species and determined that different species of microorganisms had different Kp factors. Several studies determined that Kc factors are also dependent upon the diversity of organism assemblages (Jenkinson, 1976; Anderson and Domsch, 1975; Nicolardot et al., 1984). Thus, it seems reasonable to conclude that Kp factors in this study differed with water potential because of changes in microbial communities caused by the change in water potential. Since the method cancels all background factors (soil chemical and physical properties), there are only two factors left to influence the value of Kp. Those factors are changes in the microbial community and the influence of water potential on P extraction from the microbial biomass. We determined that water potential does not affect the extraction procedure by adding microbes from one water potential to all water potentials. This is evidence that P extraction from the microbial biomass is not affected by water potential as suggested by Sparling and West (1989). Therefore, the changing microbial community must be the cause of different Kp factors. When comparing our NaHCO3 study results with other studies (Brookes et al., 1982; McLaughlin et al., 1986), Kpi factors for the A horizon were within the published range (0.36–0.42) when the soil was K3 kPa or drier. When the soil was near saturation, Kpi increased to 0.65. This is in contrast to studies that showed that the microbial Kc factor (the fraction of microbial C that decomposes into CO2-C in 10 days) decreased with increasing water content (Ross, 1987; Wardle and Parkinson, 1990). Why more P is extractable from microbial populations in saturated soils cannot be clarified from this experiment,

but one may reason that it is due to differences in the microbial cells. 4.3. Objective 3: influence of soil horizon on Kp factors Microbial population diversity should occur in different horizons within a soil profile. Nutrient concentrations, water regime, quantity of and type of organic carbon as a food source, soil texture, and possibly pH change with horizon. Both organic C and the water regime in the three soil horizons of this soil are considerably different. Soil water potential is different between the A horizon and the lower horizons as shown by Philips et al. (1989). As mentioned previously, microbial communities are affected by water potential (Dı`az-Ravin˜a et al., 1995). Because the horizons have different water regimes, it seems reasonable that microbial diversity may also change with horizon due to differences in water potential regimes. Microbial activity (Bauhus et al., 1998) and microbial community composition (Sessitsch et al., 2001) also differ with soil texture. When comparing this study’s A horizon Kpi factors using NaHCO3 with the Kpi factors in similar textured soils by Brookes et al. (1982) and McLaughlin et al. (1986), the Kpi factors were similar; 0.35 (this study at K8 kPa), 0.32 (Brookes et al., 1982), and 0.33 (McLaughlin et al., 1986). McLaughlin et al. (1986) measured Kp factors on three sites with different soil textures, finding that Kp differed by site. These data support the premise of McLaughlin et al. (1986) by providing additional examples that soil texture does play a role in microbial diversity.

5. Conclusion This study has shown that using a low concentration of oxalate to extract microbial P from these soils provides similar or higher quantities of microbial P compared to sodium bicarbonate, with 3 mM oxalate able to extract the microbial P in the spodic horizon. Soil physical and chemical properties were also found to influence the value of microbial Kp factors, which in turn appear linked to changes in the microbial community. Kp factors were found to be a function of soil water potential and soil characteristics and should not be generalized for different soils. Since Kp is clearly influenced by soil characteristics, using literature values for a Kp factor will cause a significant error, as much as 62, 145, and 48% in the A, E, and Bh horizons, respectively, when using NaHCO3 as an extractant, in microbial P estimates.

Acknowledgements This research was supported by the National Council for Air and Stream Improvement. We also gratefully acknowledge the generosity of The Timber Company (now Plum

C.M. Bliss et al. / Soil Biology & Biochemistry 36 (2004) 1925–1934

Creek) for the use of their land. We wish to thank M. McLeod for technical assistance. This is Journal Series Paper No. R-XXXX of the Florida Agriculture Experiment Station.

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