EPS Extraction Procedure

Total Estimated Time: 8 hours

EPS Extraction Estimated Time: 4.5 hours

Method: Optimized method developed in Kirisits lab

Make Buffer: 10 mM Tris, 10 mM EDTA, 2.5% NaCl, pH 8

1. 500 mL DDI
2. 7882 g Tris-HCl
3. 861 g EDTA*2H₂O
4. 5 g NaCl
5. Adjust to pH 8 with 5 N NaOH
6. Autoclave

Do Extraction:

1. 15-mL conical
2. 2 ± 0.05 g GAC (wet weight)
3. 10 mL extraction buffer
4. Vortex for 1 min at max intensity
   1. Attach tubes to vortex adapter
5. Incubate horizontally at 35 °C for 4 h with shaking at 200 rpm
   1. Use shaker-incubator and secure with tape
6. Centrifuge for 10 min at 10,000xg
7. Filter supernatant through 0.45-mm nylon filter into fresh 15-mL conical
   1. Pre-soak the nylon filters in ultrapure water in a glass beaker
   2. Use a plastic transfer pipette to transfer the supernatant to a plastic syringe with a filter attached

At this point, the supernatant may be saved in 20^oC refrigerator for up to 5 days before polysaccharide and protein processing.

Polysaccharides/Carbohydrates Estimated Time: 2 hours

Method: Phenol-sulfuric acid method, modified from Masuko et al. 2005

Application: Pure culture and Mixed culture GAC.

Linear Range: ~5-100 mg glucose/L

Location: UNDER FUME HOOD!!

Standards: Store at 4 °C in sterile bottle.

1. Prepare 1 g/L stock solution (SS). Add 0.25 g glucose to a 250-mL volumetric flask. Dilute to volume with DDI.
2. Run a standard curve every time

Phenol Solution:

1. Make 5% (w/v) phenol solution
2. Add 0.5577 mL of concentrated (89%) phenol to 10 mL DI

Procedure:

1. Turn on small water bath to 90 °C
   1. Check the water level in the bath so that water does not enter the tubes
2. Add 150 mL of sample to 13-mm glass test tube.
3. Move to the hood
4. Add 450 mL of concentrated H₂SO₄.
5. Quickly add 90 mL of 5% (w/v) phenol.
6. Cap and vortex gently.
7. Place samples in 90 °C water bath for 10 min.
8. Cool at room temperature for 5 min in a water bath.
9. Vortex gently. Transfer 200 mL into a 96-well plate in duplicate.
10. Measure absorbance at 490 nm on the plate reader.
11. Dispose of the plate in the black waste bucket.

Proteins Estimated Time: 1.5 hours

Method: Pierce BCA Protein Kit

Linear range: 5-250 mg BSA/L

Standards: Make according to manufacturer’s instructions.

Procedure:

1. Turn on large water bath and set to 60 °C
2. Make mixed reagent.
   1. Multiply # of samples to be analyzed by 2. This is the volume (mL) of Reagent A you will use.
   2. Measure Reagent A in a graduated cylinder.
   3. Pour into a 100-mL wide-mouth bottle.
   4. Divide volume of Reagent A by 50. This is the volume (mL) of Reagent B to add.
   5. Add Reagent B to Reagent A. Swirl gently to mix.
3. Add 100 mL of sample to a 13-mm glass test tube.
4. Add 2 mL of the mixed reagent to each test tube and cap.
5. Vortex gently to mix.
6. Place samples in the 60 °C water bath and incubate for 30 min.
7. Vortex gently. Transfer 200 mL to a 96-well plate in triplicate.
8. Measure absorbance at 562 nm on the plate reader.
9. Dispose of the plate in the black waste bucket.

Total Solids Estimated Time: 0.5 hours

1. Label and weigh aluminum weigh pan
2. Scrape media from bottom of extraction tube into the weigh pan using a metal spatula
3. Dry overnight in 105 C oven
4. Transfer to desiccator to cool
5. Weigh pan + total solids (TS)

Calculations