Protocol

 

Authors

Timothy George, Alan Richardson

Author affiliations

Tim George – SCRI, UK; Alan Richardson – CSIRO, Australia

Background

Enzyme-labile organic P is the portion of P in a soil extract or soil suspension which is amenable to hydrolysis by a specific phosphatase enzyme. In soil the process is termed mineralization and results in the release of P as orthophosphate.

Ranges of values to be expected

H₂O Pphos = 0-10 μg P g^-1soil

NaHCO₃Pphos = 0-20 μg P g^-1soil

NaOH Pphos = 0-50 μg P g^-1soil

SoilSus Pphos = 0-20μg P g^-1soil

These measurements are based on highly weathered soils of Australia and Kenya with additions of phytase as reported by George et al., 2005, 2006, 2007a; 2007b. The range may be greater in other soils. A meta-analysis performed for various soil extracts and water samples (Bünemann, 2008), has shown that for the majority of studies (75^thpercentile), availability of organic phosphorus to enzymatic hydrolysis is up to 60% of the total extracted Po, with crude phytase preparations generally showing the lowest substrate specificity and greatest release of phosphorus.

If a given added enzyme does not release any P from a given sample (soil extract or suspension), this can only be interpreted as absence of hydrolysable substrate if controls for the activity of the enzyme in the sample (against a model substrate) and for the recovery of P (especially in soil suspensions) have been included.

Units, terms, definitions

Pi = molybdate/malachite reactive P

Pt = molybdate/malachite reactive P following sulphuric acid/persulphate digestion

Po = Pt – Pi Pphos = molybdate/malachite reactive P following incubation with a phosphatase enzyme

H₂O-P = P extractable in H₂O (1 hour, 1:10 w:v) and measured as either Pi, Pt, Po or Pphos

NaHCO₃-P = P extractable in 0.5M NaHCO₃(1 hour, 1:10 w:v) and measured as either Pi, Pt, Po or Pphos

NaOH – P, = P extractable in 0.1M NaOH (16 hours, 1:10 w:v) and measured as either Pi, Pt, Po or Pphos

SoilSus – Pphos = P released by enzymatic hydrolysis in a soil suspension (24 hour, 1:20 w:v, 37^∘C) and measured by difference in Pi

Procedure

1. Phosphatase-labile P in P fractionation schemes

One gram of air dried soil is extracted with 10mL of distilled water on a reciprocal shaker for 1 h and supernatants collected after centrifugation at 5500g for 10 min. Residual soil is then resuspended in 0.5 M NaHCO₃(pH 8.5) and extracted for 1 h (Olsen and Sommers, 1982) and supernatants are again collected. Residual soil is then resuspended for a third time in 0.1 M NaOH (pH 8.5) and extracted for 16 h (Tiessen and Moir, 1993) prior to centrifugation. Inorganic P (H₂O-Pi, NaHCO₃-Pi, NaOH-Pi) is determined on an aliquot of all supernatants, which in the case of NaHCO₃and NaOH extracts are first acidified (0.15 M H₂SO₄) to flocculate organic material and centrifuged (15000g for 2 min) to remove organic colloids from solution. Total P in extracts (H₂O-Pt, NaHCO₃-Pt, NaOH-Pt) is determined after digestion of an aliquot of the supernatants by autoclaving (120 kPa, 121 C, 60 min) with 1.8 M H₂SO₄and 3.3% ammonium persulfate. Organic P (H₂O-Po, NaHCO₃-Po, NaOH-Po) in each fraction is determined by difference between Pt and Pi. Total extractable soil P (Pi, Po, or Pt) is determined by summing all fractions.

The component of each extract that is amenable to hydrolysis by phosphatase (H₂O-Pphos, NaHCO₃-Pphos and NaOH-Pphos) is determined by incubation with an excess of phosphatase activity. For example, phytase from Aspergillus (Sigma phytase; Sigma-Aldrich Ltd, St Louis, MI, USA) can be used as a non-specific phosphatase, as this preparation has been shown to be active against not only myo-inositol hexakisphosphate, but also a wide range of other monoester and diester forms of organic P (Hayes et al., 2000; George et al., 2007a). Assays are conducted in microtitre plates using 100 L of extract made up to 300 L with buffer (15mM MES, 1mM EDTA, pH 5.5) and phosphatase added to a final activity of 10 nKat mL^-1(George et al., 2007b) Samples are then incubated at 37 C for 24 h to allow the reaction to run to completion (Hayes et al., 2000). Reactions are terminated at either time zero or after the 24 h by addition of an equal volume of 10% trichloroacetic acid, and phosphatase-labile organic P is determined by difference in orthophosphate Pi between Tand T₂₄. Alternatively to the approach of calculating with the difference of T24 and T0, parallel assays with a deactivated enzyme preparation (e.g. by autoclaving) or a subtraction approach of Pi(sample+enzyme) – Pi(sample) – Pi(enzyme) can also be used. In these latter cases, measurements are only done when the reaction has run to completion, unless a change in Pi(sample) over time is of interest to see the activity of soil enzymes.

Phosphate content of all extracts and digests is determined spectrophotometrically after reaction with either molybdate blue (Murphy and Riley (1962) or malachite green (Van Veldhoven and Mannaerts, 1987; Irving and McLaughlin 1990). The malachite green method offers advantage over the molybdate blue method in being more sensitive (~2 to 3-fold) and with greater stability of the colorometric product. Total P may also be measured by inductively coupled plasma mass spectrometry (ICP-MS), however this cannot be done for Pi alone, as the ICP procedure simultaneously measures both Pi and Po.

A more detailed outline of these procedures can be found in George et al., (2005; 2006; 2007a; 2007b).

The component of extracts that was amenable to enzyme hydrolysis can be measured using various sources of phosphatase that may differ in their specificity toward different forms of organic P. For example, phytase collected as an exudate from the roots of transgenic Arabidopsis thaliana plants that express an Aspergillus phytase gene have been used. This plant-derived heterologous phytase shows high specificity for inositol hexakisphosphate (George et al., 2005) and as such can be used to differentiate between different components of labile organic P within extract. Detailed analysis of different phosphatases, their relative specificities and sources can be found in Bünemann (2008) and in Annaheim et al. (2010). Care has to be taken that buffering to different pH-values is needed for different enzymes.

2. Phosphatase-labile P in soil suspensions

The capacity of different phosphatases to directly hydrolyse organic P in soil can also be determined in soil suspensions. In these assays, excess phosphatase (e.g., 120 nKat g^-1soil) are added to soil suspensions (1:10 soil/water; pH 5.5) with constant mixing and reactions are allowed to run to apparent completion by incubation for 24 h (George et al., 2005; George et al., 2007a; Giaveno et al., 2010). Under these assay conditions (ie., with soil suspensions and colloidal material present) it is important to measure the potential for re-adsorption of released orthophosphate by inclusion of controls containing a known addition of orthophosphate (e.g., 1 to 20 g P g^-1soil) in the absence of phosphatase enzyme. Inorganic P released to solution over the period can similarly be measured following reaction with molybdate blue or malachite green and, where necessary, be corrected for possible re-adsorption of released orthophosphate. However, in many cases this may be negligible with the use of wide soil to solution ratios.

A more detailed outline of these procedures can be found in George et al., (2005) and George et al., (2007a; 2007b).

Notes and troubleshooting tips

Related techniques

Methods for P extraction and analysis in soils as detailed by Olsen and Sommers (1982) and Tiessen and Moir (1993).

A protocol for “Organic phosphorus characterization by enzyme hydrolysis” has been published in “Soil sampling and methods of analysis”, edited by M.R. Carter and E.G. Gregorich, 2nd ed., 2008, p. 283 ff. It describes the addition of four different enzymes to NaHCO₃ soil extracts.

The phytase preparation from Aspergillus (Sigma phytase; Sigma-Aldrich Ltd, St Louis, MI, USA) seems to be no longer available. For alternatives see also Annaheim, K.E., Frossard, E., Bünemann, E.K., 2010. Characterisation of organic phosphorus compounds in soil by phosphatase hydrolysis, 19th World Congress of Soil Science, Soil Solutions for a Changing World. 1 – 6 August 2010, Brisbane, Australia. Published on DVD. Available at http://www.iuss.org/19thWCSS/WCSS_Main_Page.html.

Termination of the reaction with TCA can lead to great increases in Pi in some soils (e.g. calcareous or rich in soil organic matter), especially in soil suspensions. Alternatively, the reaction can also be terminated by addition of the first (acid) colour reagent of the malachite green method. Indeed, termination is not crucial if the reaction has run to completion.

Recovery of added inorganic P to soil suspensions can be much lower than reported by George et al. (2005). In some cases, it may help to increase the concentration of EDTA in the buffer to increase the recovery of the P spike.

Necessary controls include 1) enzyme blanks 2) Pi addition to the sample (esp. for soil suspensions) 3) model substrate alone 4) model substrate plus enzyme 5) model substrate plus sample (to check for hydrolysis through soil enzymes) 6) model substrate plus enzyme plus sample.

Addition of a microbial inhibitor may be required in some soils to suppress microbial P immobilization during incubation.

Links to resources and suppliers

Sigma-Aldrich Ltd, St Louis, MI, USA

Literature references

Annaheim, K.E., Frossard, E., Bünemann, E.K., 2010. Characterisation of organic phosphorus compounds in soil by phosphatase hydrolysis, 19th World Congress of Soil Science, Soil Solutions for a Changing World. 1 – 6 August 2010, Brisbane, Australia. Published on DVD.

Bünemann, E.K. (2008) Enzyme additions as a tool to assess the potential bioavailability of organically bound nutrients. Soil Biology & Biochemistry 40;2116-2129.

Giaveno C., Celi, L., Richardson, A.E., Simpson, R.J., Barberis E. (2010) Interaction of phytases with minerals and availability of substrate affect the hydrolysis of inositol phosphates. Soil Biology & Biochemistry 42;491-498.

George, T.S., Richardson, A.E., Simpson, R.J., (2005) Behaviour of plant-derived extracellular phytase upon addition to soil. Soil Biology & Biochemistry 37;977-978.

George, T.S., Simpson, R.J., Gregory, P.J., Richardson, A.E. (2007a) Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates. Soil Biology & Biochemistry 39;793-803.

George, T.S., Simpson, R.J., Hadobas, P.A., Marshall, D.J., Richardson, A.E. (2007b) Accumulation and phosphatase-lability of organic phosphorus in fetilised pasture soils. Australian Journal of Agricultural Research 58;47-55.

George, T.S., Turner, B.L., Gregory, P.J., Cade-Menun, B.J., Richardson, A.E. (2006) Depletion of organic phosphorus from oxisols in relation to phosphatase activities in the rhizosphere. European Journal of Soil Science 57;47-57.

Hayes, J.E., Richardson, A.E., Simpson, R.J. (2000) Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase. Biology and Fertility of Soils 32;279-286.

Irving, G.C.J., McLaughlin, M.J. (1990) A rapid and simple field-test for phosphorus in Olsen and Bray No. 1 extracts of soil. Communications in Soil Science and Plant Analysis 21;2245-2255.

Murphy, J., Riley, J.P. (1962) A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27;31-36.

Olsen, S.R., Sommers LE (1982) Phosphorus. In -Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties. Vol. 2′. (Eds., A.L. Page, R.H. Miller, D.R. Keeney) pp. 403-430. American Society for Agronomy: Madison, WI.

Tiessen, H., Moir, J.O. (1993) Characterization of available P by sequential extraction. In -Soil Sampling and Methods of Analysis’. (Ed., M.R. Carter) pp. 75-86. Lewis Publishers: Ann Arbor, MI.

Van Veldhoven, P.P., Mannaerts, G.P. (1987) Inorganic and organic phosphate measurements in the nanomolar range. Analytical Biochemistry 161;45-48.

Health, safety & hazardous waste disposal considerations

1. Chemical Extraction of Soil

Recommended Personal Protection Equipment (PPE) should be worn including dust mask, fastened laboratory coat, safety glasses, closed foot wear and gloves. Instruction on safe use of chemicals involved in the procedures, and assessment of recommended Material Safety Data Sheets (MSDS) should be sought. There is a recommendation to rest frequently while pipetting and to use electronic multi-channel pipettes to avoid repetitive strain injury.

2. Handling Malachite Green

Substance is supplied as a crystalline powder, which should be clearly labelled as harmful and stored at room temperature. Stock solutions should be made up while wearing respiratory protection (mask), along with lab coat, gloves and safety specs.

When used in assaying samples, care should be taken not to spill or splash stock solutions or samples containing the reagent. In the case of an incident were malachite green is ingested individuals should receive medical attention as a matter of urgency. Waste stock solution and sample waste after assay should be disposed down the drain with plenty of water to dilute.

In case of powder spillage, ensure adequate PPE (mask, goggles, lab coat, gloves) before sweeping up with minimal dust disturbance, then ventilate the affected area. Any remaining residue should be wiped up with dampened tissue. Retain solid waste for specialised disposal.

In case of liquid spillage, ensure PPE (as above) before using disposable tissues/paper towels to absorb the spill. Place in closed containers for disposal and wash spill site after material removal is complete. Dispose of cleaning materials in normal waste.

If inhaled, move to fresh air and consult doctor if breathing difficulties are experienced. In case of contact with skin/eyes, flush with copious amounts of water for at least 15 mins and consult a doctor. Remove contaminated clothing. After ingestion, give large amounts of water to drink (unless person is unconcious) and consult doctor.