Determination of intracellular oxygen concentration in E. coli. E. coli cells expressing FluBO were batch cultured in Overnight Express™ Instant TB medium in 48-well flowerplates using the microcultivation system BioLector. The development of biomass (black), dissolved oxygen tension (blue) and FP-mediated fluorescence (FbFP: green, YFP: yellow) was online-monitored in each well of the flowerplate. Cell density was monitored by scattered light (I-Io) at an excitation wavelength of 620 nm (a.u.: arbitrary units). Time points of changing DOT of the growth medium are labeled (t0 to t3). Arrowheads indicate changes of donor and acceptor fluorescence which is caused by a temporary shift of DOT during the logarithmic growth phase. (A) Fluorescence emission of donor (FbFP) and acceptor (YFP) was recorded at 492 nm and 532 nm, respectively, with an excitation wavelength of 380 nm. (B) Development of cyan-to-yellow fluorescent ratio (emYFP/emFbFP) of the FRET-based oxygen biosensor is shown in red. The pictograms symbolize the state of fluorescence activity of the FbFP donor domain (circle) and YFP acceptor domain (rectangle). (C) Background fluorescence of E. coli during cell growth. E. coli cells carrying the empty expression vector were batch cultured in Overnight Express™ Instant TB medium in 48-well flowerplates using the BioLector. Dissolved oxygen tension is shown in blue. According to emFbFP and emYFP, cell-dependent background fluorescence was online recorded at 492 nm and 532 nm that, respectively, at an excitation wavelength of 380 nm and the fluorescence ratio was calculated (red curve). All parallel fermentation experiments in the microwells were performed in triplicates. These results were in excellent agreement (see Additional File 4). Maximum time shifts of 10 minutes occurred due to unavoidable slight differences of cell density at the beginning of the cultivation. Therefore, a representative set of data from these three parallel independent measurements instead of the corresponding mean values is shown in the figures.