The overall goal of this procedure is to classify enzyme hydrolyzed organic phosphorus in extracts of soil or manure samples. This is accomplished by first extracting samples with sodium hydroxide EDTA solution and adjusting the pH of the extracts. Then incubate these adjusted extracts with two different enzyme solutions.
On a 96 well plate, the final step is to measure enzyme released orthophosphate using molybdenum blue colorimetry. Ultimately, most native orthophosphate mono ester phosphorus and diaster phosphorus forms can be classified in soil or manure samples based on the differential release of orthophosphate by the enzyme hydrolysis method. This method can be performed inexpensively in any standard laboratory.
The main advantage it has over existing methods like phosphorus 31 nuclear magnetic resonance spectroscopy, is it can measure the potential for native soil enzymes to release orthophosphate from organic pea compounds in the future. Therefore, data obtained by this method can help forecast soil fertility or the potential for soil to release harmful amounts of algal available orthophosphate. Add 30 milliliters of the 0.25 molar sodium hydroxide solution to two grams of wet or dried sample in a 50 milliliter conical tube.
Incubate with agitation for 16 hours at room temperature. Centrifuge the samples 3000 times G for 30 minutes, then transfer 100 microliters to a 1.5 milliliter micro centrifuge tube that already contains acetate buffer. The adjusted sample has a total volume of one milliliter and has a pH of 5.0.
Prepare two stock solutions containing 0.5 units per milliliter of acid phosphatase in 10 milliliters of freshly prepared 0.1 molar sodium acetate pH five. Now add reconstituted lyophilized nuclease NP one from Penicillium Citron into one of the 10 milliliter tubes of acid phosphatase stocks to a final concentration of 2.5 units NP one per milliliter gently mixed by inverting several times, centrifuge the two enzyme solutions at 3000 times G for 30 minutes. In order to construct a calibration curve, begin by adding one milliliter of one millimolar potassium phosphate stock solution to a 1.5 milliliter centrifuge tube and perform seven 0.5 milliliter cereal dilutions discard the first tube so that final dilution range from 20 al to 0.625 nm phosphorus.
Now transfer the ORTHOPHOSPHATE standards to the last two columns on the plate, beginning with zero NM orthophosphate in row A and ending with 20 nm orthophosphate in row H to the first three wells of column 10. Add 40 microliters PP plus GP enzyme solution to the next three wells of column 10. Add 40 microliters PP plus GP plus NP one enzyme solution.
Finally, add 40 microliters of 100 millimolar acetate buffer to all six wells. Now each of the first six wells in column 10 should contain 80 microliters of solution to the next two wells. Add 40 microliters sodium acetate solution containing 10 M glucose, six phosphate, and 40 microliters PP plus GP enzyme solution.
For each sample being tested, distribute 40 microliters of pH adjusted sample extracts to wells one to nine in up to eight rows after all samples have been distributed. Use a multi-channel pipette to distribute PP plus GP enzyme solution to columns one to three PP plus GP plus NP one to columns four to six and 0.1 molar sodium acetate buffer to column seven to nine. Perform this step rapidly to assure all samples get equivalent incubation time.
Cover the 96 well plate with a lid and incubate samples, enzyme solutions, controls, and calibration curve exactly one hour at 37 degrees Celsius in deionized water. Prepare 50 milliliters of each of four stock solutions solution. A must be prepared daily using a multi-channel pipette rapidly.
Add 25 microliters of SDS 100 microliters of solution a 20 microliters of solution B, and 50 microliters of solution C to all wells in the 96. Well plate cover the plate and incubate for 30 minutes at room temperature. Finally, measure the absorbance at 850 nanometers in any T tuneable microplate reader using a spreadsheet application.
First, plot the inorganic phosphorus calibration curve with zero to 20 nanomoles phosphorus on the x axis and mean of duplicate absorbance measurements on the Y axis, perform a linear regression and find the equation of the line of best fit. Next, calculate the background inorganic phosphorus by averaging the values in columns one to three, and then applying the calibration curve to the dataset. Next to determine phosphorus derived from hydrolyzed phosphorus mono esters, average the triplicate sample values from columns four to six and subtract out control enzyme solution plus background inorganic phosphorus.
Then to calculate the phosphorus content from hydrolyzed phosphorus, DERs average the triplicate sample values from column seven to nine and subtract control enzyme solution plus background inorganic phosphorus plus hydrolyzed mono ester phosphorus from columns four to six. A typical 96 well plate shows results for eight samples and a calibration curve. Controls are in column 10.
Color intensity increase between columns one to three and four to six is due to hydrolyzed mono ester Phosphorus compounds between columns four to six and seven to nine is due to hydrolyzed dia eter phosphorus compounds. Here the method of high throughput enzymatic hydrolysis was used to successfully analyze distribution of the phosphorus classes in a 0.25 molar sodium hydroxide 0.05 molar EDTA extracts of a Vermont soil sample. After watching this video, you should have a good understanding of how to measure native orthophosphate, mono ester, and diaster pea compounds in soil or manure extracts using enzymatic hydrolysis.
This technique may help pave the way for researchers in the fields of environmental chemistry and nutrient management to explore organic P behavior in soil or manure systems.
