Growth of Rhodobacter sphaeroides

Rba. sphaeroides wild-type strain 2.4.1 was grown in M22+ medium⁴⁴ supplemented with 0.1% casamino acids. Growth was measured by monitoring the optical density at 680 nm (OD₆₈₀). Starter cultures were grown semi-aerobically in the dark in a rotary shaker at 34°C. Photosynthetic growth under anaerobic conditions was performed at room temperature in completely full and sealed 17 ml sterile glass tubes inoculated to a starting OD₆₈₀ of 0.03–0.05. Illumination at light intensities of 1, 10, 30, 100, 250, 500 and 1000 μmol m⁻² s⁻¹ was provided using Osram 116 W Halogen bulbs. These light intensities were chosen to span a likely range of irradiation by sunlight. These values vary according to wavelength, position on the Earth’s surface, time of year, and other factors, and lie between 200–500 W m⁻² in the 750–950 nm wavelength range for standard incoming solar spectral irradiance distribution. We chose a conservative value of 200 W m⁻², equivalent to 1000 μmol m⁻² s⁻¹²⁰ as our measure of 100% full sunlight. Light intensity was monitored using a LI-250A Light Meter equipped with a LI-190 Quantum Sensor (LI-COR Biosciences). Using thin glass tubes (~ 1 cm across) and culture mixing with magnetic stirrers ensured that self-shading was minimized during photosynthetic growth. Notable for the energy use estimates discussed below, the cells did not exhibit flagellar motility; nor did they have access to an energy source other than photosynthesis for metabolic activity, although the growth medium does supply sources of carbon for photoheterotrophic growth. The observed cell populations denoted by OD₆₈₀ at each illumination are shown in Fig. 3A,B.

The growth rate and chromatophore content of Rba. sphaeroides as a function of illumination. (A) Growth rate under anaerobic conditions at room temperature determined from OD₆₈₀ for the illuminations 1 μmol m⁻² s⁻¹, 10 μmol m⁻² s⁻¹, 30 μmol m⁻² s⁻¹, 100 μmol m⁻² s⁻¹; for clarity, illumination values above 100 μmol m⁻² s⁻¹ are shown separately in (B). The exponential growth phase for each illumination is indicated by a linear fit, the inverse slope of which determines the cell doubling time (shown in Fig. 4). (B) Same as (A) but for the illuminations 100 μmol m⁻² s⁻¹, 250 μmol m⁻² s⁻¹, 500 μmol m⁻² s⁻¹, 1000 μmol m⁻² s⁻¹. It is seen that above 100 μmol m⁻² s⁻¹, the growth rate of Rba. sphaeroides saturates, resulting in near-identical doubling times (Fig. 4). (C) Whole cell absorbance spectra were obtained in order to determine the chromatophore content of Rba. sphaeroides at each illumination (see Methods). (D) Absorbance spectra of pigments from (C), separated for clarity. (E) The number of chromatophore vesicles per cell N_chrom (circles) estimated from the spectra shown in (C) and the nonlinear fit (solid line) used to interpolate N_chrom at intermediate illuminations. (F) The chromatophore mass ratio η_chrom of the cell estimated from the chromatophore count shown in (E) based on Eq. (15).