Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

The overall goal of the following experiment is to show an extraction methodology to obtain structural extracellular polymeric substances or EPS, from biofoams, specifically from aerobic granular sludge. This method can have help answer key questions in the granular sludge field. Such as understanding the granulation process, and analyzing the EPS composition.

The main advantage of this technique is that the presence of gelfoaming polymers in EPS can be tested in a short time. Centrifuge the granular sludge at 4, 000 times G and four degrees Celsius for 20 minutes, and decant the supernatant. Collect the granules in the pellet for the extraction.

Preheat 150 milliliters of tap water in a 1, 000 milliliter glass beaker on a magnetic stirrer to 80 degrees Celsius. Transfer three grams of granules in a 250 milliliter baffled flask and fill up the flask to 50 milliliters with demineralized water. Add 0.67 grams of sodium carbonate decahydrate into the flask to obtain a 0.5%weight per volume sodium carbonate concentration.

Put the flask containing the mixture into the water bath. Cover the flask and the beaker glass separately with aluminum foil, to prevent evaporation. Stir the mixture for 35 minutes at 400 RPM and 80 degrees Celsius.

Transfer the mixture into a 50 milliliter centrifugation tube. Centrifuge the tube containing the mixture at 4, 000 times G and four degrees Celsius for 20 minutes. Collect the supernatant and discard the pellet.

The dialysis step is optional to obtain structural EPS from aerobic granular sludge. But it has several advantages which are described in the protocol. Pipette the supernatant into a dialysis bag, with the molecular weight cut off of 3, 500 daltons, and dialyze for 24 hours against 1000ml of ultra-pure water.

Change the dialysis water after 12 hours to enhance the effect of the dialysis. Transfer the dialyzed extract into a 250ml glass beaker and slowly stir the extraction at 100 rpm and room temperature. Constantly monitor the PH changes with a PH electrode, while adding 1 molar hydrochloric acid to a final PH of 2.2 05 to obtain ale in the acidic form.

After adjusting the PH to 2.2, transfer the extract into a 50ml centrifugation tube, and centrifuge at 4000 times G and four degrees Celsius, for twenty minutes. Discard the supernatant and collect the gel-like pellet. The gel-like pellet is ale in the acidic form.

To obtain the sodium form of ale, slowly add 0.5 molar sodium hydroxide to the gel, while mixing the gel slowly with a glass stick by hand until PH 8.5 is reached. Using a pasteur pipette, slowly drip the sodium ale extract into a calcium chloride solution. If the extracted EPS has ionic hydrogel gel-forming properties, drop-shaped sperical beads will be formed.

Alternatively, if the extracted EPS has no ionic hydrogel gel-forming properties, the extract will disperse in the calcium chloride solution. Keep the hydrogel beads and the calcium chloride solution for 30 minutes. Use a spoon to take out the hydrogel beads from the calcium chloride solution and transfer them to an EDTA solution.

Store the hydrogel beads in 10ml of EDTA solution for three hours at four degrees Celsius. This test demonstrates one example of the stability tests described in the protocol. Monitor if there is visible disintegration of the beads during this storage to evaluate if the beads can withstand these conditions.

If the extraction was successful, the aerobic granular sludge will be solubulized after the alkaline extraction step, and a pellet will be obtained after acidifying and centrifuging the extracted EPS. If the extracted EPS are structural EPS and have the ability to form hydrogels with calcium ions, gel beads will be formed when dripping the extracted EPS in the calcium solution. If EPS that do not have this gel-forming ability are extracted, the EPS solution will disperse in the calcium solution.

After watching this video, you should be able to perform the extraction of structural EPS from aerobic granular sludge. Also you should be able to test the gel-forming ability of the obtained EPS with calcium ions. We chose to show this extraction methodology to illustrate the impact of the extraction technique on the obtained EPS from aerobic granular sludge.

Besides that, we wanted to show that no EPS extraction technique is universally applicable.