Inlab semi micro

The kinetics of complex structure changes in the Co(II)–AlaSal system was investigated by UV/Vis absorption spectroscopy (Cary 50 Bio spectrophotometer) using a fiber-optic device coupled with the Titrando 905 automatic titrator system and a InLab Semi-Micro combined polymer microelectrode (METTLER TOLEDO). The AlaSal solution (at a concentration of 2 × 10⁻³ M) in the presence of cobalt(II) ions at molar ligand–metal ratio of 2:1 was prepared in an aqueous 5 mM Tris-HCl/NaCl buffer. Then, the solution was placed in a closed thermstatted vessel at 25.0 ± 0.1 °C. Argon was constantly passed over the solution surface in the vessel to ensure the absence of oxygen and carbon dioxide, and the tested solution was adjusted to a pH of about 7.2 (with NaOH). Following this, UV/Vis spectra were recorded under anaerobic conditions at consecutive, long enough intervals, within 200–900 nm, simultaneously monitoring the pH values.

P. pastoris cells expressing UmRh1 were harvested from 500 ml culture. The medium was exchanged to an unbuffered solution (100 mM NaCl, 10 mM MgCl₂) by washing twice and subsequent centrifugation at 3,000 x g for 5 min, with more than 30 min of incubation between the two steps. Further additional washing steps were done twice to exchange to 100 mM NaCl. The final cell suspension was adjusted to OD₆₀₀ = 10. The pH of the stirred suspension was monitored by a pH meter (InLab Semi-micro, Mettler Toledo, ctd) in the dark or under continuous illumination by yellow light (Schott, ctd. 150 W Halogen lamp with 515 nm long pass and 1,500 nm short pass filters). The protonophore carbonylcyanid-m-chlorphenylhydrazon (CCCP) was used to collapse the proton gradient.

pH was measured on sieved soil, before activation and after each substrate pulse using an InLab R422 pH electrode (InLab Semi-Micro; Mettler-Toledo, Gießen, Germany). Gravimetric soil moisture content was determined by weighing the soil before and after weight constancy following drying at 60 °C.