# A Green Approach for Preparing High-Loaded Sepiolite/Polymer Biocomposites
## Materials and Methods
### Morphological and Chemical Characterization of Colloidal Dispersion of SepX and NRL  
To assess the hydrodynamic diameter of NRL particles, dynamic light scattering (DLS) was performed by a Malvern Zetasizer Nano Series ZS90 instrument (Malvern Panalytical Ltd., Malvern, UK). (Figure S1 in Supporting Information, SI). The NRL sample was diluted approximately 1:1 with Milli-Q water. Morphology of SepX samples (Figure S2 of SI) was investigated by Scanning Electron Microscopy (SEM) analysis, performed by a Vega TSS136 XM Tescan microscope (Tescan Brno s.r.o., Kohoutovice, Czech Republic). The electron beam excitation was 30 kV at a beam current of 25 pA, and a working distance of 12 mm. In this configuration, the beam spot was 38 nm sized. Prior to SEM analysis, samples were gold-sputtered. ImageJ processing program (Image Processing and Analysis in Java, Version No 2, National Institutes of Health, Bethesda, MD, USA) was utilized to measure manually the Sep fibers dimensions. The \(\zeta\)-potential of the NRL particles was determined with Malvern Zetasizer Nano Series ZS90 in order to investigate the colloids stability. The NRL sample was diluted with deionized water until the pH value was approximately that of Milli-Q water (pH = 6.5). The \(\zeta\)-potential measurement on pristine NRL was performed at 25 \({}^{\circ}\)C at different pH by adding a suitable amount of HCl or NH\({}_{4}\)OH solution to NRL, maintaining the concentration at a constant level. \(\zeta\)-potential measurements were carried out also on 100 mgL\({}^{-1}\) SepX dispersions. The concentration of the suspension was the same as in the procedure of the preparation of Sep/NR biocomposite (see Paragraph 2.2). In detail, SepB5 aqueous dispersion was stirred for 10 min and sonicated for other 10 min, while SepS9 was stirred at different times (10 min, 40 min, 3 h, 24 h, Figure S3 of SI) before sonication. In such conditions, the SepS9 suspension after 10 min reached a pH = 8, which remains almost constant reaching the equilibrium (pH = 8.41) after 24 h. In addition, the \(\zeta\)-potential measurements of SepX suspensions were performed at different pH by adding HCl or NH\({}_{4}\)OH at 25 \({}^{\circ}\)C.  
The magnitude of the adsorption/ion exchange of ammonium ions on SepS9 was studied by using the Nessler method [23]. 10 g of SepS9 were dispersed in water (200 mL) under mechanic stirrer at 200 rpm for 60 min, successively sonicated for 10 min and mixed again for 10 min. In a second vessel, 0.1 g of dried NH\({}_{4}\)Cl (drying at 150 \({}^{\circ}\)C for 2 h) was dissolved in 50 mL of distilled water. The resultant NH\({}_{4}\)Cl solution was added to the SepS9 dispersion and stirred at a constant temperature (25 \(\pm\) 2 \({}^{\circ}\)C). The pH of all solutions was adjusted by adding HCl 37% until pH was in the 6-8 range. The concentration of the ammonium ions, in the aqueous phase containing SepS9, was analysed using the standard Nessler reagent method, employing a Varian Cary 4000 Spectrophotometer (Varian Inc., Palo Alto, CA, USA) under the following conditions: 300-600 nm wavelength range, 600 nm min\({}^{-1}\) scan rate, 0.1 s time response, and 2 nm spectral band. 20 mL of sample aliquots were withdrawn at intervals of 3, 15, 30, 60, 120 min and separated by centrifugation. Then, the ammonium content in 0.1 mL of supernatant was analysed after adding 2.5 mL of distilled water and potassium sodium tartrate tetrahydrate to avoid the magnesium and calcium interference with the Nessler reagent. The corresponding kinetic data for SepS9 are depicted in Figure S4. SepS9 dispersion was also investigated by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES), using a PerkinElmer OPTIMA7000 DV spectrophotometer (Perkin Elmer, Waltham, MA, USA), in order to investigate the potential release of metal ions. A dispersion of 50 g L\({}^{-1}\) of SepS9 in Milli-Q water was mixed for 30 min and sonicated for 10 min.