Combining submerged membrane technology with anaerobic and ...

Materials and methods2.4.4. Extracellular polymeric substances (EPS) and soluble microbialproducts (SMP)To determine the concentration of extracellular polymeric substances (EPS), whichmainly consist of polysaccharides and proteins, the sample must be analysed according tothe protocol proposed by the members of AMEDEUS & EUROMBRA during the meetingwhich took place in Berlin, 1 of June 2006. The method of extraction consists of amodification of the method used by Zhang et al. (1999). The sample of 200 mL of biomassis centrifuged at 5000 rpm during 20 minutes. The supernatant is removed. Its content incarbohydrates and proteins is analyzed in order to obtain SMP concentration. Next, 200mL of deionised water is added to the remaining biomass and carefully shaken (manually)and the sample is placed in the oven at 80 °C, during 10 minutes. The tubes, still warm,are centrifuged at 5000 rpm during 20 minutes. The supernatant is filtered with thefiberglass filter. Its content in carbohydrates and proteins is analyzed in order to obtainEPS concentration.2.4.4.1. CarbohydratesCarbohydrate concentration was analysed using a modified phenol–sulphuric acidmethod proposed by Dubois et al. (1956).Reagents preparationThe following reagents are necessary in order to carry out the procedure:Reagent A: Phenol solution 5 % (v/v)Reagent B: Sulphuric acid (97 %)Determination procedureA sample of 1.0 mL is thoroughly mixed with 1.0 mL of reagent A and left for 10minutes at room temperature. Then 5.0 mL of reagent B are added rapidly (in stream) andleft for 5 minutes at room temperature for cooling. The test tube is then mixed again. After25 minutes, the absorbance at 490 nm is measured with a spectrophotometer Cecil CE7200. A blank with reagents must be also measured as a reference. The quantification isdone with 6-8 points calibration curve in the range of 0-100 mg·L -1 , using D-glucosemonohydrate (figure 2.7).79