**2. Materials and Methods**

#### *2.1. Cultivation of Microorganisms*

For experimental work, two extremophilic strains were used, the moderately halophilic strain *Halomonas halophila* (CCM 3662) obtained from Czech Collection of Microorganisms, Brno, Czech Republic, and the moderately thermophilic strain *Schlegelella thermodepolymerans* (DSM 15344) purchased from Leibnitz Institute DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

The first cultivation step, preparation of inoculum, was performed in 100 mL Erlenmeyer flasks with 50 mL of media. Inoculum culture of *H. halophila* was prepared in a medium consisting of yeast extract (3 g/L), peptone (15 g/L), glucose (1 g/L), and sodium chloride (66 g/L) during 24 h in 30 ◦C, permanent shaking 180 rpm. Inoculum of *S. thermodepolymerans* was prepared in 25 g/L Nutrient Broth medium (10 g/L peptone, 10 g/L beef extract and 5 g/L NaCl) during 24 h in 50 ◦C, permanent shaking 180 rpm.

After 24 h lasting cultivation, mineral salts media were inoculated by 10% (*v*/*v*) of inoculum, cultivations were performed in 250 mL Erlenmeyer flasks with 100 mL of medium. The basic mineral salt medium for *H. halophila* cultivation consisted of (NH4)2SO4 (3.0 g/L), KH2PO4 (1.02 g/L), Na2HPO4·12H2O (11.1 g/L), MgSO4·7H2O (0.2 g/L), NaCl

(66 g/L), glucose (20 g/L) and 1mL/L of microelements solution (FeCl3 (9.7 g/L), CaCl2 (7.8 g/L), CuSO4 (0.156 g/L),CoCl2 (0.119 g/L), NiCl2 (0.118 g/L) and CrCl2 (0.062 g/L) dissolved in 0.1 M HCl) and 20 g/L of glucose. The mineral salt medium for cultivations of *S. thermodepolymerans* consisted of Na2HPO4·12 H2O (9.0 g/L), KH2PO4 (1.5 g/L), NH4Cl (1.0 g/L), MgSO4·7 H2O (0.2 g/L), CaCl2·2 H2O (0.02 g/L), Fe(III)NH4citrate (0.0012 g/L), yeast extract (0.5 g/L), 20 g/L xylose and 1 mL/L of microelements solution (EDTA (50.0 g/L), FeCl3·6 H2O (13.8 g/L), ZnCl2 (0.84 g/L), CuCl2·2 H2O (0.13 g/L), CoCl2·6 H2O (0.1 g/L), MnCl2·6 H2O (0.016 g/L), H3BO3 (0.1 g/L), dissolved in distilled water) (all used chemicals including hydrochloric acid, salts, glucose and xylose were purchased from LachNer, Neratovice, CZE; yeast extract, peptone and Nutrient Broth were purchased from HiMedia, Brno, CZE). Inoculated media were cultivated during 72 h under permanent shaking at 180 rpm at the optimal growth temperature, 30 ◦C for *H. halophila* and 50 ◦C for *S. thermodepolymerans*, respectively.

### *2.2. Basic Characterization of Cultures*

After 72 h, cultivations were finished. For determination of growth of culture, optical density of cultures was measured using a spectrophotometer (P300, Implen, Munich, Germany) at 630 nm against distilled water as a blank. Gravimetrical determination of cell dry matter (CDM) was performed by centrifugation of 10 mL of culture at 3460× *g*, 5 min (EBA 200, Hettich, Spenge, Germany). The generated supernatant was discarded and the pellet was washed with distilled water and centrifuged again. The pellet was dried to constant weight at 70 ◦C in drying chamber (IP60, LTE Scientific, Oldham, UK) and then the amount of biomass (g/L) was obtained by weighing on an analytical scale (Pioneer PA224C, Ohaus, Parsippany, NJ, USA). Moreover, the content of PHA in dried biomass was measured by gas chromatography with flame ionization detection (GC-FID; gas chromatograph Trace 1300, Thermo Scientific, column: DB-WAX 30 m by 0.25 mm, Thermo Scientific, Waltham, MA, USA) as reported previously in Brandtl et al. (1988) [27]. All determinations were performed in dublicate.
