sigmaaldrich.com). To equalize the cell densities, the cells

were collected by centrifugation, washed once in basal salts (medium Transferase inhibitor without carbon source) and resuspended in basal salts to yield an OD600 nm of 0.375. An inoculum of 20 μL was added to 130 μL of prewarmed medium in 96-well microtiter plates (round-bottom plates; Sarstedt, Nümbrecht, Germany). Thereby, the starting OD600 nm of all cultures was set to 0.05. Cultures were grown on a Heidolph Titramax 1000 rotary shaker (Heidolph, Schwalbach, Germany), which has an orbit of 1.5 mm, with 1100 r.p.m. at 42 °C. The OD600 nm was measured at the time points indicated in the respective Figures using the microtiter plate photometer Spectramax 340 (Molecular Devices, Ismaning,

Germany). Optimization of the cultivation of H. volcanii buy DZNeP in microtiter plates and the optimized protocol are described in Materials and methods. If not otherwise stated, strain H. volcanii H26 (Allers et al., 2004) was used for all the experiments, which is auxotrophic for uracil, but wild type in terms of all the features tested in the experiments described below. The strain was chosen because it is widely used by many groups for the generation of deletion mutants. As a first application, growth in microtiter plates was used to characterize the dependence of the growth yield on the glucose concentration. Eight different glucose concentrations were used in the presence of two different vitamin sources, 0.01% w/v yeast extract and 0.1% v/v of a commercially available mixture of nine vitamins (the vitamin dependence is discussed below). The growth curves are shown in Supporting Information, Fig. S1a and S1b. The dependence of the growth yield on the glucose concentration for both sets of experiments is shown in Fig. 1a. In all three figures, the average values of six independent cultures and their variations are shown. As can be seen, the growth of H. volcanii under the optimized conditions in 96-well microtiter plates is extremely reproducible. The dependence of the growth yield on the glucose

concentration was very similar in the presence of 0.01% yeast extract ioxilan and the vitamin mixture, respectively. The biggest difference is in the negative control, i.e. in the absence of glucose. As expected, 0.01% yeast extract can also be used to a small extent as a carbon and energy source. Therefore, all the following experiments were conducted in the presence of the vitamin mixture, except for the analysis of vitamin dependence. In a next experiment, the dependence of the growth yield of H. volcanii on the concentration of casamino acids as the sole carbon and energy source was clarified. The growth curves are shown in Fig. S2 and the dependence of the growth yield on the casamino concentration is shown in Fig. 1b.