# Foams in Wastewater Treatment Plants: From Causes to Control Methods
## 4 Evaluation Method
### Indirect Methods
#### 4.2.2 EPS Concentration  
The abbreviation EPS indicates different classes of macromolecules such as polysaccharides, proteins, nucleic acids, lipids and other polymeric compounds that can be found in the intracellular zone of microorganisms, in particular near or outside the cell surface [84]. EPSs are produced by bacteria during the biological process as a result of stress conditions (e.g., nutrient deficiency and high sludge age) and make the cell walls of the filamentous microorganisms present more or less hydrophobic. Therefore, EPSs take on a crucial role in the formation of biological foaming [85]. EPSs can be divided into EPS\({}_{\text{b}}\), which is the fraction contained inside the cell or attached to it, and EPSs\({}_{\text{s}}\), which is the fraction secreted into the mixed liquor [2; 21; 86; 23]. As suggested by [50] EPSs can be considered as the generic SMP, therefore, the total EPSs are calculated in accordance with Equation (7):  
\[EPS=EPS_{b}+SMP \tag{7}\]  
The first method described to evaluate the concentration of EPS is the heating method, which was applied by [PERSON] et al., [PERSON] et al., [PERSON] et al. and [PERSON] et al. [87; 88; 89; 90] to extract EPSs from activated sludge. According to the procedure proposed by [PERSON] et al. [2], after concentrating the sample by centrifugation, the supernatant was filtered to obtain the SMP fraction. The residue was resuspended in ultra-pure, heated water. The separation of EPS\({}_{\text{b}}\) by the heated sludge mixture was carried out by ultracentrifugation and following filtration. The total EPS concentration was calculated as the sum of EPS\({}_{\text{b}}\) and SMP. The following quantification methods were then applied to the aqueous phases: for both SMP and EPS\({}_{\text{b}}\) analysis, the \"Lowry's method\" [91] to determine the total concentration of proteins and the \"Anthrone method\" [92] to quantify carbohydrates. Therefore, EPS\({}_{\text{b}}\) and SMP were calculated with Equations (8) and (9):  
\[EPS_{b}=EPS_{P}+EPS_{C} \tag{8}\]  
\[SMP=SMP_{P}+SMP_{C} \tag{9}\]  
where:  
\(EPS_{P}\): protein fraction of bound extracellular polymeric substances;  
\(EPS_{c}\): carbohydrate fraction of bound extracellular polymeric substances;  
\(SMP_{P}\): protein fraction of soluble microbial product;  
\(SMP_{c}\): carbohydrate fraction of soluble microbial product. [PERSON] and [PERSON] [22] proposed a steaming extraction method, which was a modification of that proposed by [PERSON] and [PERSON] [93]. In their work, [PERSON] and [PERSON] [93] stated that the steaming treatment was the most effective extraction method for the activated sludge since it released a significant quantity of extracellular polymers from the flocs and caused less cellular disruption than other treatments described by them. The procedure implemented by [PERSON] and [PERSON] [22], in their experiments, is as follows: a volume of 5-10 mL of activated sludge sample was placed in a centrifuge tube and centrifuged at 3000 rpm for 10 min. The solid residue was suspended in 30 mL of distilled water. The centrifuge tube was placed in an autoclave at 105 \({}^{\circ}\)C for 30 min, then it was cooled, centrifuged at 14,000 rpm for 10 min and the supernatant was filtered. Protein and carbohydrate quantification in this EPS extract was carried out using the \"Lowry's method\" and the \"Anthrone method\", respectively. Given the importance attributed to EPSs, the simplest method for detecting the excess presence in cases of viscous bulking is the India ink reverse stain method proposed by [PERSON] et al. [5] and a similar procedure has also been implemented by [PERSON] et al. [2] ten years later. The procedure applied by [PERSON] et al.