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DIVISION S-4-SOIL FERTILITY & PLANT NUTRITION

Using an Ion Sink to Extract Microbial Phosphorus from Soil

^a Dep. of Agronomy, Throckmorton Plant Sciences Center, Kansas State Univ., Manhattan, KS 66506-5501, USA

rgm{at}ksu.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 
Two ion sinks (FeO-coated paper and anion exchange resin membrane) were tested to improve accuracy and precision in analysis of microbial P (P_m) in soil biomass. We used a noncarcinogenic biocide, hexanol. Ion-sink extracted FeO-P_m [P_m(FeO)] and resin-P_m [P_m(Res)] were compared with bicarbonate extracted P_m [P_m(Bic)]. Fractional recovery of P_m (K_p) was determined with 50 µg P_m from five different organisms, first, from preparations without soil, giving K_p of 0.49 for P_m(Bic), 0.63 for P_m(FeO), and 0.66 for P_m(Res), and second, from soil-organism mixes, giving K_p of 0.36 for P_m(Bic), 0.49 for P_m(FeO), and 0.52 for P_m(Res). This indicated ion sinks can improve accuracy of P_m recovery. We tested 18 Kansas soils with three different levels of C + N substrates to provide varying levels of microbial activity. For unamended soils, P_m values (mg kg^-1) were 5.9 for P_m(Bic), 8.2 for P_m(FeO), and 6.3 for P_m(Res). Corresponding values in soils with low levels of C + N amendments were 6.0 for P_m(Bic), 18.2 for P_m(FeO), and 18.7 for P_m(Res). In soils with high levels of C + N amendments plus alfalfa (Medicago sativa L.), corresponding values were 12.7 for P_m(Bic), 30.9 for P_m(FeO), and 37.3 for P_m(Res). Above 10.0 mg kg^-1, P_m(FeO) and P_m(Res) were at least twice as much as P_m(Bic). In addition, P_m(FeO) and P_m(Res) both had lower CVs than P_m(Bic), indicating greater precision by the ion-sink methods.

Abbreviations: P_i, inorganic P • P_m, microbial P • P_m(Bic), microbial P extracted with 0.5 M NaHCO₃ • P_m(FeO), microbial P extracted with an FeO-coated paper • P_m(Res), microbial P extracted by strips of anion exchange resin membrane • K_p, fractional recovery of organism P_m • OM, organic matter

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 
SOIL MICROBIAL BIOMASS is a dynamic force driving soil phosphorus (P) cycling in soils (Perrott and Sarathchandra, 1989; Tiessen et al., 1994). Estimating microbial P (P_m) in soil biomass is important for understanding these cycles and characterizing bioavailable P fractions (Parfitt et al., 1994; Selles et al., 1995; Huffman et al., 1996).

For many years, efforts have continued to improve the estimation of P_m in soils (Brookes et al., 1982; Martin and Correll, 1989; Thien et al., 1994; Kouno et al., 1995). Most workers analyze duplicate soil samples, one treated with a biocide and the other not treated (Hedley and Stewart, 1982; Brookes et al., 1984; Perrott and Sarathchandra, 1989). The difference between extractable P in the two samples is an estimate of P_m.

A recovery factor (K_p) often is used to improve the accuracy of P_m estimates (Clarholm, 1993; Bardgett et al., 1994; Joergensen et al., 1995). Values for K_p may vary among soils. McLaughlin et al. (1986) reported K_p of 0.33, 0.40, and 0.57 for three soils. Similarly, Hedley and Stewart (1982) reported K_p ranging from 0.32 to 0.47 on different soils, and noted that ideally, calibration should be made for each soil. Even after determining K_p for a given soil, uncertainty may remain whether the types of fungi and bacteria used for calibration actually represent the indigenous microbes in the soil. Variability in K_p between different soils and different microbes led Selles et al. (1995) to omit use of any K_p values in reporting P_m.

Fixation of P_m by soil during the extraction period can reduce the precision of P_m estimates. Sometimes corrections for P fixation are made by spiking a reference sample with P at the beginning of the extracting period, and then using the portion of the spike recovered after extraction to estimate the amount of P_m fixed by the soil during extraction (Brookes et al., 1982; McLaughlin et al., 1986). Soils with high P sorption capacities may have lower K_p values (Hedley and Stewart, 1982). When bicarbonate is used as a soil extractant, erratic P_m values obtained from acidic and highly weathered soils have been attributed to high P sorption capacities (Potter et al., 1991). Because of the variation of P sorption in different soils, some authors have omitted attempts to correct for this factor (Selles et al., 1995).

Soil preparation for P_m determination has varied among different procedures. Sometimes the soil has been dried, sometimes ground, and sometimes used in the original field-moist state (Brookes et al., 1982; Hedley and Stewart, 1982; Martin and Correll, 1989; Selles et al., 1995). Air drying or grinding soils may cause quantitative and qualitative changes in biota and biomass (Brookes et al., 1982; McLaughlin et al., 1986).

Chloroform often is used to lyse microbial cells in soil (Clarholm, 1993; Joergensen et al., 1995; Selles et al., 1995). McLaughlin et al. (1986) found that hexanol is just as effective as chloroform and is safer because chloroform is a suspected carcinogen.

A solution of 0.5 M NaHCO₃ is most often used for extracting P_m from soil biomass (Brookes et al., 1984; Potter et al., 1991; Joergensen et al., 1995). In comparing various extractants in soil biomass studies, Brookes et al. (1982) reported that 0.5 M NaHCO₃ adjusted to pH 8.5 was most satisfactory. Other studies have found that ion-sink extraction of P_m has advantages over bicarbonate extraction (Thien et al., 1994; Kouno et al., 1995).

Extraction times have varied in different procedures designed to measure soil P_m; 30-min and 16-h extractions have both been used with bicarbonate (Brookes et al., 1982; Hedley and Stewart, 1982). McLaughlin et al. (1986) reviewed the merits and weaknesses of the different extraction times and elected to use a 30-min bicarbonate extraction period in their study of P_m methods.

Studies have been made to evaluate the improvement in P_m estimates by using total P (P_t ) instead of only P_i. Hedley and Stewart (1982) reported improvement in P_m estimates by using P_t instead of only P_i; however, Brookes et al. (1984) found in the soils they used that the differences in P_m estimated by using P_t compared with using only P_i were usually within the range of analytical error.

The numerous variations in methods to measure soil P_m indicate that no standard method has yet been established and accepted. Many of the variations in procedure may result in variations in measured P_m. When correction factors for P fixation and P_m recovery are used to improve P_m estimates, the accuracy of the P_m estimates is dependent to some extent on the accuracy of the correction factors themselves.

	Mechanisms of Phosphorus Extraction: Ion Sinks vs. Chemicals

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 
Ion sinks in the form of FeO-coated paper and anion resin have been used extensively for the analysis of inorganic P in soils (Fixen and Grove, 1990; Sagger et al., 1990; Menon et al., 1997), and they have advantages over chemical extractants such as those used in Bray-1, Mehlich, and the Olsen procedures (Bray and Kurtz, 1945; Watanabe and Olsen, 1965; Mehlich, 1984). Ion sinks adsorb P onto the surface of the resin or the FeO paper and do not react with soil. In contrast, the function of chemical extractants is to react with soil components, thus freeing a portion of P_i into soil solution; however, the chemical reaction of extractants may vary across different soils with different chemical characteristics. Consequently, ion sinks can be used more reliably over soils with a wider range of chemical characteristics than can chemical extractants.

Although ion sinks have not been used widely to extract P_m from soils, their extraction mechanism of P adsorption has an advantage over the mechanism of chemical extractants for analyzing soil P_m. This advantage involves the shifts in solution P equilibrium that occur during the extraction period. Because ion sinks continuously adsorb solution P onto sink surfaces during extraction, the solution P is always kept at low levels. In this way, solution-P equilibrium shifts in the direction favoring release of P from microbial cells into solution, from which it will be adsorbed onto sink surfaces. This mechanism is in contrast to that occurring with chemical extractants, which results in a continual increase in solution P concentration as P is extracted from the soil into solution. As this occurs, the equilibrium is shifted in the direction which depresses further release of P from microbial cells into solution. Consequently, the equilibrium aspect of extraction mechanisms for analysis of soil P_m favors ion-sink methods over chemical extraction methods.

In this present study, we designed an experiment using ion sinks to improve the accuracy and precision of analyzing P_m in soil biomass. To do that, we employed anion resin membrane and FeO-coated paper as ion sinks. These were used with a noncarcinogenic biocide, hexanol. We compared this approach with a standard method of P_m analysis using chloroform as a biocide and bicarbonate as an extracting agent.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 
Soil Characteristics
We collected soil samples from the top 15 cm of 18 Kansas soils which had a wide range of chemical and physical properties (Table 1) . The soils were Mollisols except for Haynie which was an Entisol (Table 1). Tully-A was from a virgin native prairie that never had been pastured or cut for hay. Tully-B was from a pasture of native prairie grass continuously grazed by cattle. Wymore, Burchard, and Pawnee were cropped soils currently in native grass. The rest of the soils were tilled and being used for growing grains or hay. Field samples were air dried, sieved to 1.0 mm or less, and then stored at ambient room temperature. We used air-dried soils to help assure a relatively constant microbial biomass development when the soils were moistened and incubated.

Soil Sample Preparation
For each P_m analysis we prepared six samples; three samples were treated with a biocide, and three were not. The difference in extractable P between the samples with and without biocide was considered to be P_m.

We analyzed soils with three different levels of C and N substrates to provide varying levels of microbial activity. The first set of soils was unamended. The second set of soils was amended with low amounts of C and N, and a third set was amended with high amounts of C and N plus alfalfa.

For unamended treatments, we added 1.00 g soil and 0.38 mL water to a 17 by 100 mm polystyrene culture tube for all soils except Haynie to which we added only 0.19 mL of water because of its coarse texture. For the second treatment, we amended the soils with low amounts of C and N by adding to the culture tubes 1.00 g soil and 0.38 mL of a solution composed of 0.15 M dextrose [CH₂OH(CHOH)₄CHO] and 0.08 M NH₄Cl, supplying 4.1 mg C (g soil)^-1 and 0.42 mg N (g soil)^-1 except for the Haynie for which we doubled the C and N concentration but reduced the volume to 0.19 mL. The amounts of C and N were chosen to supply ample substrates for microbes for at least 24 h, but without a large excess remaining after that time. For the third set of soils amended with high amounts of C and N plus alfalfa, finely ground alfalfa was mixed thoroughly with dry soil at the rate of 10 mg g^-1. The C, N, and P concentrations for alfalfa were 429.0 mg g^-1 for C, 34.7 mg g^-1 for N, and 3.22 mg g^-1 for P. One gram of the soil–alfalfa mixture was added to culture tubes, then amended with 0.38 mL of a solution of 0.30 M dextrose and 0.16 M NH₄Cl supplying 8.2 mg C (g soil)^-1 and 0.85 mg N (g soil)^-1. Again for Haynie, we doubled the solution concentrations, but added only 0.19 mL. In these soils, the C and N substrates were doubled on the basis of the rationale that the alfalfa amendment would increase microbial populations enough to require a larger supply of nutrients.

After the samples were prepared, the tubes were capped loosely, set in racks, and incubated in a water bath at 35°C for 24 h.

Methods for Determining Microbial Phosphorus
Bicarbonate Method
At the end of the 24-h incubation period, we determined P_m in each soil using CHCl₃ as a biocide, and bicarbonate as an extractant (Brookes et al., 1982; Hedley and Stewart, 1982). The difference between P_i in nonbiocide-treated samples and P_m + P_i in biocide-treated samples was considered to be P_m.

Iron Oxide Method
We used the procedure of Myers et al. (1995; 1997) for making and using FeO-coated paper to extract P. Each of the FeO-coated papers has adequate adsorption capacity to adsorb 98 to 100% of the P in a solution containing 500 µg P. This adsorption capacity far exceeded the P available from any soil (Table 1).

Microbial P was determined following incubation of soil by first adding 1.0 mL of water to each of the three incubated soil samples and thoroughly mixing on a vortex mixer. These three samples did not have biocide added. The mixture then was transferred to a wide-mouthed glass jar (118 mL and 10.1 cm high). Eighty milliliters of 0.01 M CaCl₂ was added, some of which was used to rinse the soil from the culture tube. One FeO paper stabilized between Spectra-Mesh screens was added (Myers et al., 1995; 1997). Under a fume hood, one drop of toluene was added to each jar which then was sealed immediately. Toluene was added to repress microbial activity during the shaking period.

The samples were shaken horizontally end-to-end on a reciprocating shaker for 24 h at a speed of about 125 to 135 excursions min^-1. At the end of the shaking period, the P adsorbed on FeO papers was determined (Myers et al., 1995, 1997).

We used hexanol to lyse microbial cells in the additional three incubated soil samples. We added 1.0 mL water and 0.25 mL 1-hexanol (C₆H₁₃OH) to each of the three samples (McLaughlin et al., 1986). The tubes were capped, mixed thoroughly on a vortex mixer, and shaken horizontally end-to-end on a reciprocating shaker for 1 h at about 180 excursion min^-1. This was done to assure good contact between soil and biocide. Tubes were weighted down to hold them in place while shaking. The tubes then were placed upright in racks and left standing for 23 h to make a total fumigation time of 24 h. The soil mixture was then transferred from the culture tubes to glass shaking jars as described above for samples without biocide, but no toluene was added. Extraction and P analysis were performed the same as described above for samples without added biocide.

Because P_i and P_m were both present in biocide-treated samples, P_m was calculated by finding the difference between P_i of each sample without biocide and P_i + P_m for the corresponding biocide-treated sample. This difference was considered to estimate P_m(FeO) in the soil biomass.

Resin Method
A resin membrane, BDH product no. 55164 2S (BDH Laboratory Supplies, BH15 1TD, Poole, England) was used in resin extraction of P_m (A U.S. distributor: Gallard-Schlesinger Industries, Inc., 584 Mineola Ave., Carle Place, NY 11514, identifies this product as no. 551642 but advises that either identification is correct.). The resin membrane comes in 12.5 x 12.5 cm sheets. We cut the sheets into strips 2.08 x 4.15 cm to avoid any waste and used two strips per 1.0-g soil sample. This gave a total surface area of 34.5 cm², which is similar to 31.2 cm² reactive surface area used by Saggar et al. (1990).

We saturated the resin strips with bicarbonate ions by shaking about 50 strips in 250 mL of 0.5 M NaHCO₃ (pH 8.5) for 30 min (Schoenau and Huang, 1991). The 500-mL polystyrene wide-mouthed bottles used for shaking were placed horizontally on a reciprocating shaker, and shaken end-to-end. After decanting the solution, we repeated the procedure for another 30 min, then rinsed the strips four times by shaking them in the bottles with deionized water on the shaker 5 min. We stored the strips in deionized water in closed containers. One of these resin strips has the capacity to adsorb 98 to 100% of the P in a solution containing 500 µg P. This adsorption capacity was similar to that of one FeO-coated paper described above.

Microbial P extraction by resin was similar to the FeO procedure described above, except for the extracting bottles used and the extracting matrix. We used 125-mL wide-mouthed polystyrene bottles to which we transferred each soil sample and added 80-mL deionized water, some of which was used to rinse soil from the culture tube. Two resin strips were added. Otherwise, until the end of the 24-h shaking period, we followed the same procedure with the resin strips as with FeO paper described above.

At the end of the 24-h shaking period, we retrieved the resin strips from the jars and rinsed each strip under a stream of deionized water for a few seconds. We placed the two strips from each sample in a 125-mL wide-mouthed bottle and added 50 mL of 0.5 M HCl to elute P from the strips. We sealed the bottles, laid them horizontally and end-to-end on a reciprocating shaker and shook them 90 min. We neutralized the pH of an aliquot before analyzing P (Murphy and Riley, 1962). The same calculations were used to determine P_m(Res) as used to determine P_m(FeO) above.

Estimating the Fraction of Biomass Phosphorus Made Extractable by Biocides (K_p)
We used organisms with known amounts of P_m to estimate the fraction of biomass P made extractable by biocides (Table 2) . We used lyophylized bacteria (Sigma, St. Louis) Aerobacter aerogenes, Azotobacter vinelandii, and Bacillus subtilis and dried fungi (Sigma, St. Louis) including type I and type II of Saccharomyces cervisiae. The percentage P in the organisms was determined by dry ashing (Brookes et al., 1982; Table 2).

To determine K_p with organisms incorporated into soil, we selected the following soils: Kennebec-B (no. 2), Tully-B (no. 6), Kahola (no. 10), and Wymore (no. 16), because these soils represented a wide range of adsorbed P values, a wide range in extractable P_i values, and differences in pH levels from 5.1 to 7.1 (Table 1). We added 50 µg P_m from each of the organisms to 1.0 g dry soil. We added 1.0 mL of deionized water and the appropriate biocide for each of the three methods P_m(Bic), P_m(FeO), and P_m(Res). We also included a soil sample without the addition of any organism. Then we proceeded with fumigation and extraction of all the samples according to the three different methods P_m(Bic), P_m(FeO), and P_m(Res) as described above, and calculated K_p from the difference between the P_m extracted from the soil amended with organism P_m and the unamended soil. Duplicate analyses were performed for all K_p.

Determining the Effect of Phosphorus Fixation during Extraction
A spike of 25 µg P in the form of KH₂PO₄ was added to 1.0 g soil in the extracting containers used in each of the three methods for P_m(Bic), P_m(FeO), and P_m(Res). These soil samples then were extracted along with duplicate samples to which no P was added. Extraction was carried out as described above for each of the three methods. The difference between P values of spiked and unspiked samples was used to find the effect of P fixation during extraction (Brookes et al., 1982). Duplicate analyses were performed for all P fixation values.

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 
Microorganism K_p
The three different extraction methods were used to find K_p when extracting five organisms with P contents ranging from 8.4 to 48.0 g P kg^-1 (Table 2). When the organisms were not incorporated into soil, there was no difference in K_p between the different methods for Aerobacter aerogenes, Azotobacter vinelandii, and Bacillus subtilis (Table 3) . There were differences in K_p between the ion-sink methods and P_m(Bic) when fungi were used, and those differences demonstrated improved K_p accuracy for the ion-sink methods. When Saccharomyces cerevisiae (Type I) was used, K_p for P_m(Bic) was about 10 to 20% less than that for the ion sink methods, and when Saccharomyces cerevisiae (Type II) was used, K_p for P_m(Bic) was about 50% less than that for the two ion-sink methods (Table 3).

View this table: [in this window] [in a new window]   Table 3 Proportion of microbial P recovered (Kp) by three methods after fumigation and extraction of five different organisms containing 50 µg Pm. These organisms were not incorporated into soil

View this table: [in this window] [in a new window]   Table 4 Proportion of microbial P recovered (Kp) by three extraction methods from four soils amended with 50 µg Pm (g soil)-1 from one of five different organisms

The similarity between K_p(Feo) and K_p(Res) and the difference between K_p(FeO) and K_p(Bic) can be seen in Fig. 1 . The K_p data showed that the regression for K_p(FeO) and K_p(Res) was highly significant (P = 0.0001); the intercept was not significantly different from zero; and the slope, estimated to be 0.984, was not significantly different from 1.0 (P = 0.05). In contrast, the K_p data for K_p(FeO) and K_p(Bic) showed the slope, estimated to be 0.613, was significantly different from 1.0 (P = 0.05) (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1 Comparison of Kp (fractional recovery of microbial P) for FeO and anion-resin extraction methods (bottom); and comparison of Kp for FeO and bicarbonate extraction methods (top). Values are means of duplicate analyses from four Kansas soils amended with 50 µg microbial P from five microorganisms. Dashed lines are 95% confidence intervals. recovery by anion-resin strips; recovery by bicarbonate

Fixation of Phosphorus during Extraction
To evaluate the extent of P fixation occurring in the three different extraction methods, we added a spike of 25 µg P to soil prior to the extraction period, and then tested recovery of that spike at the end of the extraction period (Table 5) . Although P recovery for ion-sink methods averaged about 6.0% more than that for bicarbonate, differences between methods in each soil usually were not significant.

Comparison of P_m(Bic) with P_m(FeO) and P_m(Res)
Extraction method did not affect amounts of P_m in six of the 18 unamended soils (Table 6) . Most P_m values for unamended soils were below 10.0 mg kg^-1. For all 18 unamended soils the average value was 5.9 mg kg^-1 for P_m(Bic), 8.2 mg kg^-1 for P_m(FeO), and 6.3 mg kg^-1 for P_m(Res). It appears that at these low P_m levels all three extractants performed similarly.

A calibration study of the data comparing P_m(FeO) with P_m(Res) showed that the regression was highly significant (P = 0.0001); the intercept was not significantly different from 0; and the slope, estimated to be 1.123, was not significantly different from 1.0 (P = 0.05) (Fig. 2) . In contrast, a calibration study comparing P_m(FeO) with P_m(Bic) showed that the regression was highly significant (P = 0.0001); the intercept was not significantly different from 0; but the slope, estimated to be 0.344, was significantly different from 1.0 (P = 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 2 Comparison of microbial P (Pm) obtained by FeO and anion-resin extraction methods (bottom); and comparison of FeO and bicarbonate extraction methods (top). Values are means of triplicate analyses from 18 Kansas soils: unamended, amended with low levels of C and N, and amended with high levels of C and N plus alfalfa. Dashed lines are 95% confidence intervals. extracted by anion-resin strips; extracted by bicarbonate

	Conclusion

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

Lower CV values by the ion-sink methods indicated greater precision than for the bicarbonate method. The average CV values for P_m(FeO) were always lower than those for P_m(Bic), and average CV values for P_m(Res) were lower than those for P_m(Bic) for soils amended with low C + N.

The method using resin strips is more convenient than the FeO-paper method because the resin-membrane product is commercially available, whereas the FeO-coated papers have to be produced in individual laboratories. Comparing the two ion-sink methods for P_m extraction from soils with a wider range of chemical, geographical, and morphological derivation may help to more clearly define the relative strengths and weaknesses of each of the two sink methods.

	ACKNOWLEDGMENTS

 
We thank Phil Brookes, Andrew Sharpley, and Dave Whitney for discussions and information regarding the analysis of microbial P in soils.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 
Contribution no. 96-226-J from the Kansas Agric. Exp. Stn.

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TOP NOTES ABSTRACT INTRODUCTION Mechanisms of Phosphorus... Materials and methods Results and discussion Conclusion REFERENCES

 

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