Source: UNIV OF WISCONSIN submitted to
MINERALIZATION OF SOIL ORGANIC PHOSPHORUS BY PHOSPHATASE AND PHYTASE AND ITS RELATION TO PLANT NUTRITION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0186999
Grant No.
(N/A)
Project No.
WIS04450
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2000
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Project Director
Helmke, P. A.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
SOIL SCIENCE
Non Technical Summary
The reactions of organically bound forms of soil phosphorus and their relation to plant nutrition are unknown. This project develops our new hypothesis that the efficacy of enzymes in soils that mineralize phosphorus bound to soil humic substances is greatly increased if the metals normally bound to soil humic substances are removed by plant-secreted metal complexing agents.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110200080%
1022499100020%
Goals / Objectives
This research will quantify and study the mineralization of phosphorus associated with soil humic substances. The effects of various metals bound to humic substances and concentrations and types of plant-exuded metal complexing agents on the mineralization of organically bound soil phosphorus by plant secreted acid phosphatase and phytase will be determined. We will also characterize the types of soil-organic phosphorus compounds from soils and other sources that can be mineralized by enzymes.
Project Methods
The effects of metals on the ability of phosphatase and phytase to mineralize soil organic phosphorus compounds will be determined with whole soils treated with metal complexing agents and with soil humic substances extracted from soils by reaction with metal-chelating ion exchange resins or citrate buffers. The rhizosphere soil of sweet white lupin (Lupinus albus L.) grown in soils deficient in phosphorus will be analyzed for metal complexing agents, phosphatase and phytase activity, and chemical forms of phosphorus to confirm the hypothesis in a soil-water-plant system. The soil organic phosphorus compounds will be characterized by several techniques including size fractionation on Sephadex gels, FTIR and 31P NMR spectroscopy.

Progress 10/01/00 to 09/30/04

Outputs
Low-molecular-weight-organic-acids (LMWOAs) are secreted into soils by many plant species and microorganisms. This activity is most intense in the root rhizosphere of plants. The LMWOAs can enhance the plant uptake of metals and phosphorus from soils but little information is known about the reactions that occur. Our earlier studies have shown that plant secreted LMWOAs form complexes with metals bound to soil humic substances, which greatly enhances the ability of plant and microbial secreted phytase and phosphatase to mineralize organically bound forms of soil phosphorus. A major stumbling block in this research was the interfering effects of citrate and other metal complexing agents on the classical determination of orthophosphate by the molybdenum blue method. Determination of phosphorus by inductively coupled plasma emission spectroscopy gives highly accurate results for total dissolved phosphorus but does not distinguish orthophosphate from organically bound forms of phosphorus. We have developed analytical methods for the accurate and rapid determination of orthophosphate, citrate, and phytic acid in complex natural samples such as soils and manure using capillary electrophoresis. Confirmation of the accuracy of the analyses and the absence of interferences was determined by comparing the results for orthophosphate with those obtained by nuclear magnetic spectroscopy (NMR). Analysis by nuclear magnetic spectroscopy requires removal of most of the iron from the sample because the ferromagnetic properties of iron degrades the NMR signal. This requirement makes NMR too tedious for routine analyses of orthophosphate in the complex matrices typical of soil solutions. We have used these improved analytical techniques to show that phytase releases much more orthophosphate from soil humic substances than does phosphatase. Iron and aluminum are the dominant metals blocking the action of enzymes on the soil humic substances.These studies were extended to determine the ability of phosphate hydrolyzing enzymes to mineralize organically bound phosphorus in animal manures and the effects of calcium, iron, and aluminum and various trace elements on the hydrolysis reactions. The addition of calcium to the dairy ration increases the content of phytic acid in the manure. The key to identifying phytic acid in manure is that the metals, especially Ca added to the dairy diet as dicalcium phosphate, must be complexed with a metal complexing agent, such as EDTA or citrate to increase the efficacy of the enzymes. These results support the hypothesis that removing metal bridging ions from natural organic phosphorus compounds greatly improves the hydrolysis efficacy of phytase and phosphatase in soils and manures.

Impacts
This research is showing the importance of organically bound forms of soil phosphorus in soils and manures for plant nutrition. Up to one-half of the total phosphorus in soils is chemically bound to soil organic matter, but this portion of soil phosphorus is ignored in current soil tests and fertilizer recommendations. We have shown that two mechanisms are important so that plants and microorganisms can utilize phosphorus from soil organic matter: 1) The plants or microorganisms must exude organic acids which complex metals to remove them from the organic matter and 2) they must also exude phosphate hydrolyzing enzymes. This organic portion of the total soil phosphorus is an underutilized natural resource that contributes to phosphorus loads in natural waters from soil runoff.

Publications

  • Boerth, T.J., X.He, and P.A. Helmke. 2005. Citrate increases mineralization of soil organic phosphorus by acid phosphatase and phytase from Lupinus albus. Soil Sci. Soc. Am. J. (In revision).
  • Julie Howe. August, 2004. Speciation of trace metals in soils. PhD Thesis. University of Wisconsin-Madison. 109 pp.
  • J.A. Howe and P.A. Helmke. Cadmium, copper, and zinc speciation effects on phosphorus in a cultivated soil amended with a single sewage sludge application 27 years ago. Annual Meeting Abstracts, Soil Science Society of America. CD ROM. November, 2004.


Progress 01/01/03 to 12/31/03

Outputs
Low-molecular-weight-organic-acids (LMWOAs) are secreted into soils by many plant species and microorganisms. This activity is most intense in the root rhizosphere of plants. The LMWOAs can enhance the plant uptake of metals and phosphorus from soils but little information is known about the reactions that occur. Our earlier studies have shown that plant secreted LMWOAs form complexes with metals bound to soil humic substances, which greatly enhances the ability of plant and microbial secreted phytase and phosphatase to mineralize organically bound forms of soil phosphorus. A major stumbling block in this research was the interfering effects of citrate and other metal complexing agents on the classical determination of orthophosphate by the molybdenum blue method. Determination of phosphorus by inductively coupled plasma emission spectroscopy gives highly accurate results for total dissolved phosphorus but does not distinguish orthophosphate from organically bound forms of phosphorus. We have developed analytical methods for the accurate and rapid determination of orthophosphate, citrate, and phytic acid in complex natural samples such as soils and manure using capillary electrophoresis. Confirmation of the accuracy of the analyses and the absence of interferences was determined by comparing the results for orthophosphate with those obtained by nuclear magnetic spectroscopy (NMR). Analysis by nuclear magnetic spectroscopy requires removal of most of the iron from the sample because the ferromagnetic properties of iron degrade the NMR signal. This requirement makes NMR too tedious for routine analyses of orthophosphate in complex matrices. We have used these improved analytical techniques to show that phytase releases much more orthophosphate from soil humic substances than does phosphatase. Iron and aluminum are the dominant metals blocking the action of enzymes on the soil humic substances. Metal complexing agents remove the blocking action of the metals. These studies were extended to determine the ability of phosphate hydrolyzing enzymes to mineralize organically bound phosphorus in animal manures and the effects of calcium, iron, and aluminum and various trace elements on the hydrolysis reaction. The most significant finding is that bovine manure contains much more phytic acid susceptible to hydrolysis by phytase than indicated by previous studies. The addition of calcium to the dairy ration increases the content of phytic acid in the manure. The key to identifying phytic acid in manure is that the metals, especially Ca added to the dairy diet as dicalcium phosphate, must be complexed with a metal complexing agent, such as EDTA or citrate during the analysis. This chemical masking of the metals greatly enhances the efficacy of the enzymes.

Impacts
This research is showing the importance of organically bound forms of soil phosphorus in soils and manures for plant nutrition. Up to one-half of the total phosphorus in soils is chemically bound to soil organic matter, but this portion of soil phosphorus is ignored in current soil tests and fertilizer recommendations. We have shown that two mechanisms are important so that plants and microorganisms can utilize phosphorus from soil organic matter: 1) The plants or microorganisms must exude organic acids which complex metals to remove them from the organic matter and 2) they must also exude phosphate hydrolyzing enzymes. This organic portion of the total soil phosphorus is an underutilized natural resource that contributes to phosphorus loads in natural waters from soil runoff.

Publications

  • T.J. Boerth, and P.A. Helmke. 2003. Enzymatic hydrolysis of organic phosphorus in soils. Paper Number 529433. Annual Meeting Abstracts, Soil Science Society of America. CD ROM.
  • Howe, J.A., W.F. Bleam, and P.A. Helmke. 2003. A carbon and nitrogen XANES study of copper and nickel binding to fulvic acid. Paper Number 401429. Annual Meeting Abstracts, Soil Science Society of America. CD ROM.
  • Boerth, Thomas J. 2003. Enzymatic hydrolysis of organic phosphorus in soils and dairy feces. PhD Thesis. University of Wisconsin-Madison. 94 pages.
  • Boerth, T.J., and P.A. Helmke. 2003. Effect of supplemental inorganic phosphorus on content of organic phosphorus in dairy feces. Proceedings of the 2003 Wisconsin Fertilizer, Aglime & Pest Management Conference. pp. 126-130.


Progress 01/01/02 to 12/31/02

Outputs
Low-molecular-weight-organic-acids (LMWOAs) are secreted into soils by many plant species and microorganisms. This activity is most intense in the root rhizosphere of plants. The LMWOAs can enhance the plant uptake of metals and phosphorus from soils but little information is known about the reactions that occur. Our earlier studies have shown that plant secreted LMWOAs form complexes with metals bound to soil umic substances, which greatly enhances the ability of plant and microbial secreted phytase and phosphatase to mineralize organically bound forms of soil phosphorus. A major stumbling block in this research was the interfering effects of citrate and other metal complexing agents on the classical determination of orthophosphate by the molybdenum blue method. Determination of phosphorus by inductively coupled plasma emission spectroscopy gives highly accurate results for total dissolved phosphorus but does not distinguish orthophosphate from organically bound forms of phosphorus. We have developed analytical methods for the accurate and rapid determination of orthophosphate, citrate, and phytic acid in complex natural samples such as soils and manure using capillary electrophoresis. Confirmation of the accuracy of the analyses and the absence of interferences was determined by comparing the results for orthophosphate with those obtained by nuclear magnetic spectroscopy (NMR). Analysis by nuclear magnetic spectroscopy requires removal of most of the iron from the sample because the ferromagnetic properties of iron degrade the NMR signal. This requirement makes NMR too tedious for routine analyses of orthophosphate in complex matrices. We have used these improved analytical techniques to show that phytase releases much more orthophosphate from soil humic substances than does phosphatase. Iron and aluminum are the dominant metals blocking the action of enzymes on the soil humic substances. These studies were extended to investigate the ability of these phosphate hydrolyzing enzymes to mineralize organically bound phosphorus in animal manures and the effects of calcium, iron, and aluminum and various trace elements on the hydrolysis reaction. The most significant finding is that bovine manure contains much more phytic acid susceptible to hydrolysis by phytase than indicated by previous studies. The key to identifying phytic acid in manure is that the metals, especially Ca added to the dairy diet as dicalcium phosphate, must be complexed with a metal complexing agent, such as EDTA or citrate during the analysis. This chemical masking of the metals greatly enhances the efficacy of the enzymes.

Impacts
This research is showing the importance of organically bound forms of soil phosphorus in soils and manures for plant nutrition. Up to one-half of the total phosphorus in soils is chemically bound to soil organic matter, but this portion of soil phosphorus is ignored in current soil tests and fertilizer recommendations. We have shown that two mechanisms are inportant so that plants and microorganisms can utilize phosphorus from soil organic matter: 1) The plants or microorganisms must exude organic acids which complex metals to remove them from the organic matter and 2) they must also exude phosphate hydrolyzing enzymes. This organic portion of the total soil phosphorus is an underutilized natural resource that contributes to phosphorus loads in natural waters from soil runoff. We are exploring various mechanisms of how this portion of the soil phosphorus can be used by plants so that the total concentration of phosphorus in soils can be kept at lower levels to reduce environmental contamination.

Publications

  • Boerth, T.J., and P.A. Helmke. 2003. Effect of supplemental inorganic phosphorus on content of organic phosphorus in dairy feces. Proceedings of the 2003 Wisconsin Fertilizer, Aglime & Pest Management Conference. pp. 126-130.
  • Howe, J.A., and P.A. Helmke. 2002. Effect of pH on copper complexation by fulvic acid. Paper Number 1933. Annual Meeting Abstracts, Soil Science Society of America. CD ROM. Boerth, T.J., and P.A. Helmke. 2002. Phytate determination in dairy feces. Paper Number 2036. Annual Meeting Abstracts, Soil Science Society of America. CD ROM.


Progress 01/01/01 to 12/31/01

Outputs
Low-molecular-weight-organic-acids (LMWOAs) are secreted into soils by many plant species and microbes. This activity is most intense in the root rhizosphere. The LMWOAs can enhance the plant uptake of metals and phosphorus from soils but little information is known about the reactions that occur. Our earlier studies have shown that plant secreted LMWOAs form complexes with metals bound to soil humic substances, which greatly enhances the ability of plant and microbial secreted phytase and phosphatase to mineralize organically bound forms of soil phosphorus. These studies have been extended to investigate the ability of these phosphate hydrolyzing enzymes to mineralize organically bound phosphorus in animal manures and the effects of calcium, iron, and aluminum and various trace elements on the hydrolysis reaction. The most significant finding is that bovine manure contains much more phytic acid susceptible to hydrolysis by phytase than indicated by previous studies. The key to identifying phytic acid in manure is that the metals, especially Ca added to the dairy diet as dicalcium phosphate, must be complexed with a metal complexing agent, such as EDTA or citrate during the analysis. This chemical masking of the metals greatly enhances the efficacy of phosphatase enzyme. A major stumbling block in this research was the interfering effects of citrate and other metal complexing agents on the classical determination of orthophosphate by the molybdenum blue method. Determination of phosphorus by inductively coupled plasma emission spectroscopy gives highly accurate results for total dissolved phosphorus but does not distinguish orthophosphate from organically bound forms of phosphorus. Significant time was spent developing analytical methods for the accurate and rapid determination of orthophosphate, citrate, and phytic acid in complex natural samples such as soils and manure. We developed a method that uses capillary electrophoresis to determine simultaneously all three of these components, other LMWOAs in addition to citrate, and several metals. Confirmation of the accuracy of the analyses and the absence of interferences was determined by comparing the results for orthophosphate with those obtained by nuclear magnetic spectroscopy (NMR). Analysis by nuclear magnetic spectroscopy requires removal of most of the iron, whose ferromagnetic properties degrades the signal, from the sample. This requirement makes NMR too tedious for routine analyses of orthophosphate.

Impacts
This research is showing the importance of organically bound forms of soil phosphorus in soils and manures for plant nutrition. Up to one-half of the total phosphorus in soils is chemically bound to soil organic matter, but this portion of soil phosphorus is ignored in current soil tests and fertilizer recommendations. We have shown that plants can acquire phosphorus from soil organic matter by exuding organic acids, that react with and complex metals, and phosphate hydrolyzing enzymes. This organic portion of the total soil phosphorus is an underutilized natural resource that contributes to phosphorus loads in natural waters from soil runoff. We are exploring various mechanisms of how this portion of the soil phosphorus can be used by plants so that the total concentration of phosphorus in soils can be kept at lower levels to reduce environmental contamination.

Publications

  • Escosteguy, P.A.V. 2001. Exchangeability and formation constants of copper, zinc, and cadmium with humic subtances at indigenous concentrations. PhD Thesis. 196 pp.
  • Howe, J.A., P.A. Helmke, L.M. Diener, and W.F. Bleam. 2001. Binding of metals to humic substances using Donnan membrane and XANES analysis. Annual Meeting Abstracts (CD ROM), ASA, CSA, SSSA, Madison, WI.
  • Boerth, T.J., and P.A. Helmke. 2001. Factors affecting hydrolysis of phytate in soils and manures. Annual Meeting Abstracts (CD ROM), ASA, CSA, SSSA, Madison, WI.