Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.Respiration rates of an aerobic bacterial population can be used as a gauge of the metabolic state. For metabolic modes not involving primary oxygenases (e.g., methane monooxygenase) (9), changes in oxygen uptake reflect alterations in respiratory chain activity due to a phenotypic response or genetic manipulation (11). Given the tight coupling between energy metabolism and a cell's metabolic network (14), changes in respiration rates can reflect shifts in overall metabolism and how a specific metabolic state adapts to change (11).A number of methods are available to detect oxygen concentrations, such as the use of Clark electrodes, electrochemical cells, electrochemical microscopy, and paramagnetic cells (7,15). One of the most commonly performed techniques is the use of a Clark electrode. However, the caveats of this method are low sensitivities, signal drift, probe fragility, electrode consumption of oxygen, and the ability to only measure the immediate microenvironment (15,17,22). In addition, high-throughput analysis requires a number of individual devices, increasing the cost and decreasing reproducibility. One method that has seen a rapid increase in use recently is the application of optical sensors, such as phosphorescent dyes (4, 12), which impart greater signal-to-noise ratios, signal independence of the dye concentration and photobleaching, rapid response characteristics, and functionality while imbedded in a variety of materials (15, 23). Additionally, optical methods are amenable to high-throughput screening using high-density well formats (1), but existing systems tend to be custom designed and not broadly available.Recently, commercially available polystyrene beads doped with a platinum(Pt)-porphyrin dye and inexpensive off-theshelf components have become available for O 2 measurements. We examined this system to demonstrate its utility in measuring respiration rates of Methylobacterium extorquens AM1 cultures. M. extorquens AM1 has the ability to grow on C 1 substrates, e.g., methanol, as a sole source of carbon and energy and is an inexpensive renewable biofeedstock, which can reduce production costs of value-added products (5, 13). A broad range of biochemical and genetic tools along with a metabolic flux balance model has allowed a comprehensive mapping of central metabolism during C 1 and multicarbon growth (19,20).In addition to the wild type, two mutant strains were analyzed. The first mutant (20) was null for NADH-ubiquinone oxidoreductase subunit B (NADH-UOR; NADH-UOR subunit B::Tet r ), which couples NADH oxidation to the respiratory chain during multicarb...