Documentation:Soil Labile Polysaccharides

From UBC Wiki

This is a draft Protocol.

What are soil polysaccharides and why are they important?

Soil polysaccharides can be an effective indicator of short-term changes of SOM (Lui et al., 2005) and have been linked to soil quality indicators including aggregate stability (Tisdall and Oades, 1982; Lui et al., 2005). Soil polysaccharides are difficult to measure because they are heterogeneously spread throughout soil and complete hydrolysis recoveries during the hydrolysis of polymers are difficult to obtain (typically resulting in monomeric units which can be then be determined individually or as a group).

How are soil labile polysaccharides measured?

Two methods that are able to determine the concentration of individual monosaccharides are:

1.     Gas liquid chromatography (Oades, 1967)

2.     High performance liquid chromatography (Hounsell, 1986)

Two methods that use colorimetry and a spectrophotometer are:

3.     Dilute acid (Lowe, 1993)

4.     Hot water (Haynes and Francis, 1993)

The Dilute Acid Extractable Polysaccharides (DAEPs) method does not give an accurate and absolute measure of polysaccharides due to variations in colours produced by sugar monomers and because sugar ‘yields’ vary by variations in hydrolysis procedures. However, the cost to determine polysaccharides this way is relatively inexpensive and has been proven to be a useful comparative approach for soil polysaccharides (Lowe, 1993).

Materials

Chemicals

  • Phenol (solid crystals)
  • Concentrated H₂SO₄ (18.0 M)
  • Glucose (dextrose anhydride)

Supplies

  • 2.5 L glass jug with lid and volume of 2.5 L is labelled (not needed if 0.5 M H₂SO₄ solution is pre-made)
  • * - 200 mL or 250 mL erlenmeyer flasks (labelled from 1 to n number of samples
  • 1 - 100 mL volumetric flask (not needed if phenol solution is pre-made)
  • 1 - 100 mL volumetric flask (not needed if glucose solution is pre-made)
  • 6 - 100 mL volumetric flasks
  • * - 200 mL volumetric flasks (labelled from 1 to n number of samples)
  • * - Funnels
  • 1 - Buret
  • * - Cuvettes (labelled from 1 to n number of samples, 7 for the glucose standards, 1 for a water blank)
  • 1 - Cuvette stand
  • 1 - Glass pipette
  • * - Whatman 42 ashless filter paper
  • * - Filter Stand
  • 1 - 96 well microplate, clear
  • Pipette tips for the 100 - 1000 µL automatic pipette
  • *  One of each item need for each sample being analyzed

Equipment

  • Fume Hood
  • Dispensette
  • Autoclave (MCML 302 or 230a)
  • 100 - 1000 µL automatic pipette
  • Drying Oven
  • TECAN Spectrophotometer (MCML 240 - FNH Teaching Lab)

Reagent Preparation

5% Phenol Solution (remake every month)
  1. Make sure you read through the SDS, as phenol is a very dangerous chemical that can potentially have harmful side effects.
  2. Under a fume hood, dissolve 2.5 g of phenol crystals in a beaker with approximately 50 mL of distilled water.
  3. Transfer to a 100 mL volumetric flask, rinse the beaker thoroughly, and make to volume. Cap with parafilm and invert 3 times to mix.
  4. Transfer the solution to a glass bottle with a lid (for longer term storeage) or a beaker with parafilm for short-term storage (< 1 week).
  5. Label with a workplace label (toxic, corrosive, health hazard, environmental contaminant – wear glove, goggles (or face shield), labcoat) and store in the refrigerator at 4°C. Remake every month.
0.5 M H₂SO₄ (remake every 6 months)
  1. Under a fume hood, wearing black rubber gloves, goggles (or face shield), and a rubber apron, add concentrated H₂SO₄ to deionized water (Always add acid to water) in a glass jug with a lid in the volumes indicated in the table below depending on your final volume.
  2. Add deionized water to make to the final volume.
  3. Label with a workplace label (toxic, corrosive, gloves, goggles, labcoat) and store under the fumehood with the concentrated sulfuric acid. Remake every 6 months
Final Volume of Solution 2 L 2.5 L
Volume of concentrated H₂SO₄ to add 55.52 mL 69.40 mL
1000 µg/mL Stock Glucose Solution (remake every 3-4 days)
  1. Dissolve 0.1 g of glucose in a beaker with approximately 50 mL of deionized water. Use a stir bar if necessary.
  2. Transfer to a 100 mL volumetric flask, rinse the beaker thoroughly, and make to volume.
  3. Cap with parafilm and invert 3 times to mix.
  4. Label appropriately with workplace label (no hazards) and store in the refrigerator at 4°C. Remake every 3-4 days.
Glucose Standard Solutions (remake every 3-4 days)
  1. Label seven, 100 mL volumetric flasks for glucose standards with the following: 20, 30, 30, 40, 60, 80, 100, 120 µg/mL
  2. Add the following amounts of stock glucose solution (at room temperature) to each of the volumetric flasks using the glass pipette (using table below)
  3. Make each volumetric flask to volume, cap with parafilm, and invert 3 times to mix.
  4. Label appropriately and store in the refrigerator at 4°C. Remake every 3-4 days.
Final Standard Concentration Amount of Glucose Stock Standard (1000 µg/mL) to add to each Volumetric Flask to make the Final Standard Concentration
20 µg/mL 2 mL
30 µg/mL 3 mL
40 µg/mL 4 mL
60 µg/mL 6 mL
80 µg/mL 8 mL
100 µg/mL 10 mL
120 µg/mL 12 mL
Procedure
  1. Prepare a microplate map with the samples (labelled 1 to n) and the standards to be run on the spectrophotometer. (Do not load samples into wells B1-3, C1-3, E1-3, G1-3 because they have been giving abnormally high readings on the TECAN_
  2. Weigh 0.75 g (± 0.02 g) of soil sample into an Erlenmeyer flask. (Soil sample should be air-dried, sieved, and ground to 2 mm.)
  3. Record the weight of the soil sample and the flask number.
  4. Repeat steps 1 and 2 for all of the samples being processed.
  5. Add 100 mL of 0.5 M H₂SO₄ solution to each of the flasks using the dispensette. Ensure that the dispensette has been properly calibrated by setting the dispensette to the desired volume. Tare a beaker on the scale and add the volume of solution to the beaker. Weigh the beaker and solution then adjust the dispensette if necessary. Repeat until the dispensette has been calibrated.
  6. Place tinfoil over the opening of each of the flasks and place the flasks on a tray or in a container.
  7. Transfer the flasks to the autoclave for 1 hour at 103 kPa (121°C), then allow to cool for 30 minutes. * See the autoclave instructions * While the flasks are in the autoclave, you can complete steps 8 and 9.
  8. Set up filter stands, fold filter paper (in half, then again, and open like a cup), and position the 200 mL volumetric flasks under the filters.
  9. Remove the glucose standards and the 5% phenol solution from the fridge, if applicable, to allow them to come to room temperature (at least 30 minutes). The 5% phenol solution should be placed under the fume hood while it is coming to room temperature.
  10. Filter the sample solution into the volumetric flask, ensuring that the flask numbers match one another. Rinse the Erlenmeyer flask thoroughly with deionized water to transfer all of the soil solution to the volumetric flask. While the solutions are filtering, you can complete steps 12 and 13. Once all of the solution has passed through the filter, make the filtrate to volume with deionized water. Seal the opening of each of the volumetric flasks with parafilm and invert 3 times to mix.
  11. The solution in the volumetric flask can be stored in the refrigerator at 4°C for up to 24 hours if the remainder of the analysis can not be completed on the same day.
  12. For each glucose standard, add 1 mL of the glucose standard into the cuvette with the corresponding label using the automatic pipette. Use a new pipette tip for each of the glucose standards. The glucose standards should be at room temperature. Before pipetting, mix the glucose standard by inverting the volumetric flask 3 times. You can pour the glucose standards into separate beakers to make the pipetting easier.
  13. Create a water blank by adding 1 mL of deionized water to a cuvette labelled water blank.
  14. For each filtrate in the volumetric flask, add 1 mL of the filtrate into the cuvette with the corresponding label using the automatic pipette. Use a new pipette tip for each of sample. If the filtrate was stored in the fridge, allow for it to come to room temperature before pipetting. Before pipetting, mix the solution by inverting the volumetric flask 3 times.
  15. Under the fume hood, add 1 mL of the 5% phenol solution to each cuvette using the automatic pipette.
  16. Under the fume hood while wearing rubber gloves and a rubber apron, add 5 mL of concentrated sulfuric acid to each cuvette using the buret.This causes an exothermic reaction, where the cuvettes can get very hot, so be careful with handling the cuvette during this step. Try to add the concentrated sulfuric acid while the cuvette is still in the stand.
  17. Place the cuvettes in the oven at 30°C for 25 minutes to incubate.
  18. Following the prepared microplate map, pipette triplicates of 150 µL of solution from each cuvette into the 96 well microplate using the automatic pipette. Use a new pipette tip for each sample. Place a lid or parafilm on the microplate when finished.
  19. Bring the microplate to MCML 240 to read on the TECAN spectrophotometer. Contact Lewis Fausak (lewis.fausak@ubc.ca) or Imelda Cheung ([[1]]) and for computer login credentials.
  20. Open the TECAN SPARKCONTROL application, found on the desktop. Run the “Labile Polysaccharides” method. Follow prompts to load the plate (lid/parafilm should be off).
  21. When finished, save the data in the excel file to a folder on the computer then save a copy on your own USB stick or email the file to yourself.
  22. To calculate the polysaccharide content use the “Labile Polysaccharides Calculation Template”. Copy the data from the Tecan excel file to the blank table in the calculation template labelled “Results from Tecan Spectrophotometer (copy and paste)”.
  23. Enter the recorded soil weights from step 3 to cells G59 to G82.
  24. The polysaccharide content of the samples can be expressed as % Total Labile Polysaccharides or as Total Polysaccharides in g per kg of soil (g/kg).

Chemical and Soil Disposal

  • Filter paper and soil should be thrown in the garbage
  • Glucose solution and standards can safely be disposed of down the drain with plenty of water
  • Waste 5% phenol should be added to the polysaccharide waste container for proper disposal
  • Waste concentrated H₂SO₄ should be added to the polysaccharide waste container for proper disposal
  • Waste 0.5 M H₂SO₄ should be added to the polysaccharide waste container for proper disposal
  • Diluted 0.5 M H₂SO₄ in the volumetric flasks – gain access to the UBC Hazardous Waste Inventory System or ask someone with access (Lewis Fausak – [[2]]) (https://srs.ubc.ca/environment/hazardous-waste-management/hazardous-waste-inventory-system-hwis/?login)
  • The solution in the cuvettes and microplate should be added to the polysaccharide waste container for proper disposal

References

  1. G. Gregorich (Eds.), Soil Sampling and Methods of Analysis (373-376), Boca Raton, FL: CRC Press
  2. Haynes, R.J., Francis, G.S., 1993. Changes in microbial biomass C, soil carbohydrate composition and aggregate stability induced by growth of selected crop and forage species under field conditions. Journal of Soil Science 44, 665–675.
  3. Lewis, D.B., J.P. Kaye, R. Jabbour, and M.E. Barbercheck. (2011). Labile carbon and other soil quality indicators in two tillage systems during transition to organic agriculture. Renewable Agriculture Food Systems, 1, 1–12.
  4. Liu, A., B.L. Ma, and A.A. Bomke. (2005). Effects of cover crops on soil aggregate stability, total organic carbon, and polysaccharides. Soil Science Society of America Journal, 69(6), 2041–2048. http://doi.org/10.2136/sssaj2005.0032
  5. Lowe, L. E. (1993). Total and Labile Polysaccharide Analysis of Soils. In M. R. Carter and E.
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