Lab 6 Soil Nitrogen – Use of Colorimetric Assays

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Intro Soils – Lab 6 Soil Nitrogen – Use of Colorimetric Assays

Lecture Materials: Soil Nitrogen (Chapter 13)

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Lab 6 – Plant Available Nitrogen and Colorimetric Assays

Nitrogen is the most complex and studied of the nutrient cycles in soil due to its importance as a plant macronutrient and its complex cycle mediated by microbial transformations. Nitrogen is often the most limiting of the macronutrients and is needed in the largest supply to maximize yield.

The cycling of nitrogen is largely mediated by the microbial population which facilitates the fixation of atmospheric nitrogen into the soil system, mineralizes nitrogen from the organic to inorganic forms, then transforms the inorganic ammonium to nitrate in nitrification, and finally returns the nitrogen back to gaseous forms in denitrification. Nitrogen mineralization is the conversion of organic nitrogen sources into inorganic sources in a process also called ammonification.

Ammonium (NH4+) is liberated from organic nitrogenous compounds such as proteins, nucleic acids, and the breakdown of soil organic matter. A wide variety of heterotrophic organisms including bacteria, actinomycetes, and fungi can carry out this process under a wide variety of environmental conditions.

The microbial community gains energy from the reduction of nitrogen compounds. The ratio of carbon to nitrogen in the soil system dictates whether soil nitrogen will be mineralized or immobilized. Immobilization is the opposite of mineralization and is the conversion of inorganic nitrogen back to organic nitrogen. Soil conditions especially the amount of carbon dictate whether nitrogen is mineralized or immobilized.

If C:N ratios are less than 20 and all other things are in good order like aeration and water status, net mineralization will occur as there is ample carbon and nitrogen in the system to mineralize organic nitrogen into microbial biomass and plant available ammonium. If the ratio is above 30, net immobilization can occur where the microbial community actually scavenges nitrogen from the system at the expense of plant available nitrogen.

The next step in the transformation of nitrogen is the microbial conversion of the inorganic nitrogen between ionic forms in a process called nitrification. Nitrification is the conversion of ammonium (NH4+) first to nitrite (NO2-) and then to nitrate (NO3-). This process is also microbially mediated in a two-step process.

First, chemoautotrophs, Nitrosomonas spp. oxidize ammonium to nitrite and then Nitrobacter spp. oxidize nitrite into nitrate. If conditions are favorable, including reactants and bacterial species are present, this reaction occurs quickly in soils. Thus, ammonium is a relatively transient nitrogen ion in soils. Plants can utilize both ammonium and nitrate but generally prefer nitrate. Unfortunately, nitrate is readily leached out of the soil profile or under certain conditions can also be lost to denitrification. For this reason, products called nitrification inhibitors have been developed to slow this process.

Denitrification is the anaerobic transformation of nitrate into gaseous forms of nitrogen gas. The nitrogen gas is lost to the atmosphere and no longer plant available. In this process, nitrate replaces oxygen as a terminal electron acceptor and can be carried out by a number of facultative anaerobic soil bacteria including Pseudomonas, Bacillus, and Micrococcus.

When wet waterlogged conditions occur even over short periods of time and at microsites in soil, nitrogen can be lost from the soil system. Agronomically, this is an important loss of nitrogen, but environmentally it can be a positive. If nitrogen lost in overland flow from runoff or erosion events can make its way through a wetland where anaerobic conditions persist prior to reaching surface water, it can be significantly decreased in wetland environments.

Denitrification in this case is an absolute positive and is very helpful in decreasing the load of nitrogen in the downstream surface water body. The denitrification process though is what is called a ‘leaky pipe’ in that in some situations the conversion of nitrate all the way to dinitrogen gas can be shunted and the intermediary nitrogen gases including nitric and nitrous oxide gas can be produced which are considered greenhouse gases and contribute to the warming of the earth’s atmosphere.

Intro Soils – Lab 6 Soil Nitrogen – Use of Colorimetric Assays

Soil sampling is the gold standard for testing levels of macro and micronutrients in soil as well as pH, CEC, and even SOM. But even though nitrogen is typically the nutrient recommended at the highest rates of application, nitrogen is actually not included in the routine analysis of extracted nutrients in a soil test panel. Nitrogen recommendations are typically made based on previous crop and management, i.e. whether legumes were utilized and manure was applied, as well as crop and target crop yield to be planted in the coming season.

Ammonium is generally transient in soils due to the rapid microbial conversion to nitrate. So nitrate is most relevant for soil analysis. Nitrate levels in soils fluctuate tremendously based on time of year, temperature, water conditions, and other soil variables due to the nature of its cycle. Thus, nitrate testing in humid regions is especially problematic. Nitrate also is very easily leached out of the soil system with a rain event.

Due to this fluctuation, nitrogen testing is not very informative especially when tested long before planting season. Corn is the row crop with highest demand for nitrogen and is the center of a large portion of the nitrogen utilization research including nitrate testing. A nitrate test has been developed called the presidedress soil nitrate test (PSNT) to help in determining potential nitrogen available for crop uptake during the growing season.

The PSNT is sampled at twelve inches depth when the corn is approximately 12 inches tall, tested just prior to adding a second application of nitrogen prior to when nitrogen needs are highest. Each state has developed their own standards for testing and generally, if there is greater than 25 ppm of nitrate available additional nitrogen may not be needed to maximize yields.

If the corn is not following a legume (soybeans), cover crop (also with legumes), soil is high in organic matter, or had a recent manure addition, soil nitrogen tend to still be recommended, i.e. needed, to maximize yield.

But in cases where soils do have ample nitrogen to meet plant and yield goals, this test can be utilized to reduce the amount of additional nitrogen added which reduces both cost and environmental burden. UT soil testing labs and others in the region offer this service, but utilization of this tool has been somewhat slow by producers.

Research efforts to characterize the nitrogen cycle in the lab, in research trials on small and very large scales, and even by producers in fact test all of the various forms of nitrogen routinely. Total nitrogen, nitrate, and ammonium can be readily tested using analytical methods while ammonium and nitrate can also be quantified using colorimetric assays as well as test kit methods in the field.

Analytical methods are much more accurate but the test kits are easy to utilize, can be done quickly in the field, and offer a general range of the amount of nitrate in a soil or water sample. The field kit strips measure both nitrate and nitrite. The strips contain indicators which in the presence of nitrate cause a color change. The darker the color, the more nitrate in the sample.

Figure 1 below is a picture of the color change and their corresponding amounts of nitrate from a Hatch Water Quality test strip canister. Again, the larger the color change, i.e. the darker the color, the more product is in the sample. A video demonstration of this procedure is included in the link for testing for nitrate using this strip test

 

 

Figure 1: Hatch, Water Quality Test Strips for Nitrate/Nitrate-N. Color block on the side of the bottle. (Note, this is one of many of these type kits on the market and is not a product endorsement.) The use of color change to quantify a product is can be quantified in what is called a colorimetric assay. Many, many variables have indicator tests designed for color change analysis including pH, phosphorus, and many enzymes in soils just to name a few.

These results can be quantified utilizing a spectrometer and the basic principles of the Beer-Lambert Law which states that the absorbance of light is directly proportional to the concentration of absorbing species when the path length is fixed. A colorimeter or spectrometer is used to quantify the color change.

These instruments are relatively simple in that a specific wavelength of light is directed through a sample. If the sample contains a species that absorbs that particular wavelength of light, the intensity of the light that can pass through the sample will be diminished. The absorbance of the light can then be related to the concentration of the chemical species in the sample. The darker the color, the more absorbance (or less transmittance) which equates to a higher concentration of the species in the sample.

A dilution series is created with a standard, known quantity of the species to be tested which used to make what is called a calibration curve. A range of concentrations is created to fit the range of potential concentrations in the samples to be tested including the maximum concentration and minimum (zero) made of the diluent. The known concentrations and the absorbance values for each can then be used as x, y pairs to create a regression line. The absorbance is plotted on the y axis and concentration on the x axis. The equation of the line can readily be calculated by hand or using a computing program like Excel.

Concentrations of unknown samples can then be quantified using the absorbance values (y) and solving for concentration (x) based on the equation of the line from the calibration standards. There are several reagents and extractants commonly utilized in colorimetric assays to test for ammonium and nitrate.

Nessler’s reagent is commonly used in soil microbiology labs to detect ammonium which yields a yellow to orange-brown color while Griess’ reagent can be used to detect nitrite and nitrate and yields a pink to red-violet color. See below an image of a dilution set used to develop a standard curve, an example of standard concentrations and absorbance values in table form, and finally the creation of a standard curve plot

https://www.youtube.com/watch?v=4gMVoLTRMvY

line with the equation included (y = mx + b where y = absorbance, x = nitrate concentration, and b = slope of the line.)

Low Nitrate Concentration High Nitrate Concentration No color change Dark Purple Figure 2. Dilution series in cuvettes (standard tubes utilized for spectrometer) for the creation of a standard curve for nitrate-nitrogen. Nitrate concentration increases from left to right with no color change on the left to a dark purple on the right. (Photo credit:

Example Nitrate Standard Curve Data

Nitrate (ppm) Absorbance (540 nm)

X axis Known Values

Y axis Determined with

Spectrometer

0 0

2.5 0.03

5 0.06

10 0.15

25 0.4

50 0.9

http://public.iorodeo.com/docs/colorimeter/lab_3.html

To calculate the concentration of unknown samples, utilize the equation of the line with the absorbance (y) values determined via spectrometer and solve for nitrate concentration (x). For instance, if the unknown absorbance value was 0.7 (y), plug that into the equation of the line and solve for x. (The R2 value is how well the data ‘fit a line’ so a perfect fit would be 1.0)

Equation of the line: y = 0.02x – 0.02 Insert your absorbance value for y: 0.7 = 0.02x – 0.02 Solve for x: (0.7 + 0.2) = 0.02x

0.72 = 0.02x x = 0.72/0.02 x = 36

You can also get an estimated value by locating the absorbance value on the y-axis, moving over to the line, and then moving straight down to the x axis to see the corresponding nitrate concentration. For this example, an absorbance value of 0.7 would yield an approximate concentration of 40 ppm of nitrate.

The known absorbance and concentration values for the standard curve are simply x,y pairs plotted on a graph and then utilizing the equation of that line as the more exact relationship between the two variables. To put this into perspective, the 50 ppm standard sample had just over double the absorbance value than the 25 ppm sample (50 ppm, 0.9 absorbance vs 25 ppm, 0.4 absorbance). The standard sample with no nitrate had an absorbance value of zero; the sample had no nitrate, so no color change and thus no absorbance.

The 50 ppm sample was just over double the intensity of the dark pink color than the 25 ppm sample; the more color, the more absorbance, the more concentrated the nitrate is in the sample. This is the exact same concept as the color wheel utilized in the test kit strips just using actual data to calculate the concentration rather than a visual determination. Colorimetric assays are relatively easy to conduct in the laboratory with standardized extraction procedures and indicators for many, many chemical ions seem in soil and water. Producing a standardized curve with known quantities of the ion in question allows an easy determination of the

y = 0.02x – 0.02 R² = 0.99

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60

A b

so rb

an ce

( 5

4 0

n m

)

Nitrate (ppm)

Nitrate Standard Curve

concentration of unknown samples by utilizing the calculated relationship of absorbance and concentration described by the equation of the standard regression line.

Intro Soils – Lab 6 – Assignment Questions Nitrogen Cycle – Colorimetric Assays

Utilize Lab, Lecture and Text Materials: N (Ch. 16) as well as review questions for P, K, S, and the micronutrients (Ch. 16 thru 18) Nitrogen Cycle Review (3 points each, 12 points total)

1.) Utilize the example dataset in the lab to calculate nitrate concentration (ppm) in the following samples: a.) Sample A Absorbance: 0.09 b.) Sample B Absorbance: 0.25 c.) Sample C Absorbance: 0.54

2.) Why is nitrogen not included in routine soil test analysis? How are nitrogen recommendations

determined?

3.) Nitrogen is generally most important macronutrient needed in high quantities to achieve even modest yield goals. Describe at least two ways nitrogen can be lost from the soil system and some management practices producers can utilize to decrease this loss.

4.) How can denitrification be both a positive and a negative? Matching Review Section with Answer Bank Below (2 points each, 28 points total)

  1. Bacteria spp. which converts NH4+ to NO2- 2. Bacteria spp. which converts NO2- to NO3- 3. Enzymatic catalyst for the biological fixation of N2 to NH3 4. Bacteria spp. involved in biological N fixation 5. Conversion of organic to inorganic forms 6. Conversion of inorganic to organic forms 7. Name of the human induced process by which nitrogen gas is fixed to ammonia 8. Type of plants best known for their ability in concert with bacteria to fix their own nitrogen 9. Macronutrient where fixation is a major limitation to plant availability 10. Macronutrient needed to combat environmental stress 11. Secondary macronutrient that can be sorbed through the plant leaves
  2. Bacteria spp. which facilitates acid mine drainage problems 13. Only micronutrient that becomes more available with increased pH 14. Complexation with organic compounds, common with cationic micronutrients

Answer Bank for Matching

  1. Molybdenum
  2. Sulfur
  3. Nitrobacter
  4. Phosphorus
  5. Bradyrhizobium
  6. Haber-Bosh
  7. Chelation
  8. Nitrosomonas
  9. Potassium
  10. Mineralization
  11. Thiobacillus
  12. Nitrogenase
  13. Legumes
  14. Immobilization

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