Raw date from Filipigh et al., (2024). "Fractional recovery of proteins and carbohydrates from secondary sludge from urban wastewater treatment plants" 
https://doi.org/10.1016/j.ceja.2024.100686

Biomass composition
Experiment scheme and solid, Protein, NKT, Carbohydrates determinations

Five single hydrolysis methods (AH, HT, ALK, AC and US) and four combined methods (US-AH, HT+ALK, HT+AC, and US+ALK) were compared for the solubilization of proteins and carbohydrates with the aim of maximizing their recovery as peptides and monosaccharides from Sludge biomass.
In this study, all experiments were performed in duplicate using 50 mL RS biomass suspension at 3.7% concentration (w/w dry mass). To compare the different hydrolysis treatment, trying to maximize solubilization, but considering the possible degradation of the solubilized components due to excessive treatment time, two treatment durations were used depending on the type of hydrolysis. The experiments applying mild conditions were performed at 50°C for 180 min (AH, US-AH, ALK and AC). The experiments including thermal treatment at 120°C (HT, HT+ALK and HT+AC) were carried out for 60 min . The tests applying ultrasonication (US and US+ALK) were also conducted for 60 minutes, with the aim of establishing a balance between maximum sludge solubilizations and acceptable cost.
After each treatment, the hydrolysate was centrifuged at 10,000 rpm for 10 min at 4 °C to separate liquid and solid fractions. Weight and Total Nitrogen Kjeldahl (TKN) were determined in both fractions and used to verify mass balances. TSS and VSS and carbohydrate content were measured in the exhausted solid fraction after the treatments applied. In the liquid fractions, total amino acids, monosaccharide composition, peptide size and peptide purity were analyzed. All analyses were performed in duplicate.

Calculations
olubilization yields (SY, %) were calculated by weighing the biomass and analysing the volatile solids, proteins and carbohydrates in the solid residues after each hydrolysis (HD) experiment . The recovery yields (RY, %), as peptides or amino acids from proteins and as soluble monosaccharides from complex carbohydrates, were determined from the analysis of the liquid phases after hydrolysis and the difference between the two parameters (SY-RY) are the component losses due to treatment severity or active bacterial metabolism. The degradation of the solubilized components was calculated. The ratio between the amount of solubilized glucose and the amount of solubilized xylose at the end of each experiment. The purity of the target component of this research and the most valuable component of the studied biomass, the peptides, was defined according to mass of protein/g of VSS solubilized. The content of each component in the initial biomass (RS) was used in all calculations.

Analytical determination
Biomass characterization was carried out according to the Standard Methods. TSS and VSS contents were measured in the initial RS biomass and in the exhausted solid fraction after hydrolysis by the gravimetric method. Humic acid was determined in the RS by gravimetric method after alkali extraction and acid precipitation (pH < 2). The lipid content in the initial RS was determined according by a chloroform: methanol (2:1 v/v) extraction method, which is commonly used for other biomasse.

The total nitrogen content of the initial RS and of the solid and liquid fractions after the hydrolysis experiments was determined using the TKN method [36] to check the N mass balances. The mass content of proteins in the raw sludge, as total amino acids, was determined by HPLC (Section 2.5.2). The content of proteins of the exhausted solid fraction after hydrolysis was calculated from the TKN content applying the ratio between total amino acid content: TKN calculated for the raw sludge (proteins: TKN = 4)
The amino acid profile of the initial RS and the amino acid composition of the hydrolysates (liquid fraction) were determined by HPLC according to internal analytical standards from the Instrumental Techniques Laboratory (LTI-UVA) based on Moore and Stein. The samples (RS and liquid fraction after hydrolysis tests) were hydrolyzed with 6 N HCl in an inert atmosphere. Then, the acidic medium was removed using N2 until they were completely dried. Subsequently, the samples were reconstituted in 0.1 M HCl, and the final solution was filtered through 0.22 μm nylon membrane filters. Finally, the samples were subjected to pre-column derivatization using ortho-phthalaldehyde (OPA) and fluorenylmethyloxycarbonyl chloride (FMOC). The analysis was performed by HPLC using a Zorbax Eclipse AAA column (4.6 × 150 mm, 3.5 µm) with a mobile phase gradient formed of A (Buffer NaH2PO4·H2O pH 7.8) and B (AcN:MetOH:dH2O 45:45:10), and a UV detector.

The carbohydrate content in the initial RS and in the exhausted solid fractions obtained after the applied hydrolysis was determined as total monosaccharides after concentrated acid hydrolysis (H2SO4, 72% w/w) according to the NREL protocol and the HPLC-RID method described below.
The monosaccharides were quantified in the liquid fractions (the liquid fractions obtained after the applied hydrolysis experiments and the liquid fractions from the acid digestion of the exhausted solids) using an Aminex® HPX-87H ion exclusion column (Bio-Rad) installed in a Shimadzu LC-2050C separation module. A refractive index detector (RID) was used to quantify the monosaccharide concentration using a mobile phase of 25 mM sulphuric acid solution (HPLC quality) at a flow rate of 0.6 mL/min. The column and the RID were maintained at 50°C. A multistandard calibration solution was prepared by diluting single standards (glucose, xylose, arabinose and cellobiose) commercially available with a purity > 95% (Sigma, Aldrich, Spain).