## 2 Materials and Methods
### Chemical Transformation of Eggshell Waste by Acetic and \(o\)-Phosphoric Acid  
Chemical transformation of eggshell waste using acetic and \(o\)-phosphoric acid (Figure 2) was based on designed ESW transformation process using 5% (\(w/v\)) hydrochloric acid reported by our research group [1] but with some modifications. These included the use of higher acid (\(w/v\)) percentage, the lack of neutralization of the excess of acids and organic matter flocculation and drying of obtained Ca-salts at lower temperatures. The starting point in designing ESW transformation process using acetic and \(o\)-phosphoric acid was the selection of right percentage (\(w/v\)) of acid used. This was based on the report by [PERSON] and [PERSON] [46], in which the authors used a four times greater stoichiometric amount of acetic and \(o\)-phosphoric acid than necessary to completely dissolve ESW calcium carbonate and release the eggshell membranes completely devoid of ESW calcium carbonate. Based on the amount of calcium carbonate present in ESW collected from households and restaurants reported in our previous research [1], the desired concentration of acetic and \(o\)-phosphoric acids necessary for complete removal of ESW calcium carbonate was calculated, and preliminary study for complete dissolution of ESW calcium carbonate, using a 7.5, 10 and 12.5% (\(w/v\)) solution of acetic acid (corresponding to ESW calcium carbonate and acetic acid molar ratio of: 1:2.4; 1:3.2; and 1:4) and 10, 12.5. and 15% (\(w/v\)) solution of \(o\)-phosphoric acid (corresponding to ESW calcium carbonate and \(o\)-phosphoric acid molar ratio of 1:2.3; 1:2.9; and 1:3.4), was performed by monitoring time-dependent solvation of ESW calcium carbonate every 15 min for 4 h. Based on the preliminary results, 10% (\(w/v\)) acetic or 15% (\(w/v\)) \(o\)-phosphoric acid chemical transformation process of ESW for 3 hours was selected as most adequate, and transformation process of ESW was performed in future examinations. Chemical transformation of eggshell waste (ESW) started with washing 90 g of ESW (halves and pieces) with 900 mL of distilled water 3 \(\times\) 30 min on orbital shaker IKA KS 260 basic (IKA, Staufen, Germany) set at 250 rpm min\({}^{-1}\) in order to remove possible impurities, as well as adherent egg white proteins [1]. Washed eggshells were mixed with 1350 mL of 10% (\(w/v\)) acetic or 15% (\(w/v\)) \(o\)-phosphoric acid, and the process of ESW chemical transformation was performed by mixing on a magnetic stirrer (LLG, Meckenheim, Germany) set at 500 rpm min\({}^{-1}\) for 3 h at room temperature. After complete conversion of eggshells calcium carbonate to corresponding calcium salts, separation of ESM from salt solutions was performed by filtration through a plastic mesh screen of 1 mm pore size. Obtained calcium salt solutions were subjected to clarification by two successive steps of vacuum filtration through filter paper Whatman 114 followed by Whatman 1, and subsequent calcium acetate solution evaporation at 100-130 \({}^{\circ}\)C to the 1/10 of volume, or calcium hydrogen phosphate solution evaporation to the 1/2 of volume. Precipitation of crystals from saturated salt solutions cooled down to room temperature was performed by addition of 3 volumes of acetone. Precipitated crystals of calcium acetate and calcium hydrogen phosphate were separated from the rest of liquid by vacuum filtration through filter paper Whatman 1 and dried at 60 \({}^{\circ}\)C for 24 h. Afterwards, dried salts were ground in a mortar with a pestle and kept in sealed plastic containers until analysis. Figure 2: Schematic representation of the proposed process of eggshell waste utilization using 10% (\(w/v\)) acetic and (15% \(w/v\)) \(o\)-phosphoric acid. Eggshell membranes (ESMs), separated from calcium salt solutions, were washed, dried and milled using a procedure previously reported by [PERSON] et al. [1].