In Situ Determination of the Reduction Levels of Cytochromes b and c in Growing Bacteria: A Case Study with N2-Fixing Azorhizobium caulinodans

Abstract

The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth rate of 0.1 h−1 are recorded. This is accomplished by transporting the emitted light from the spectrophotometer via glass fibers to one side of the chemostat vessel and detecting the transmitted light via a photomultiplier at the other side. The vessel itself is enclosed in a dark box, which contains mirrors on the inside surfaces. The reduction levels of cytochromes b and c during steady state in chemostat cultures are expressed as percentage absorbance of fully reduced cytochromes in the α-region of the spectrum. Steady state spectra are recorded in N2-fixing, succinate-limited continuous cultures of Azorhizobium caulinodans at dissolved oxygen tensions in the range between 0.1 and 3.5% O2 Spectra of fully reduced cytochromes are obtained on the basis of spectra recorded after having reached anoxic conditions by sparging pure nitrogen gas through the culture. These spectra of cytochromes b and c reduced by endogenous substrates are corrected as to give the spectrum of fully reduced cytochromes. The respective contributions of cytochromes b and c to spectra in the α-region are estimated by deconvolution using best-fit analysis. Using this in situ technique it is observed that at each dissolved oxygen tension the reduction level of the cytochromes b is higher than that of the cytochromes c. The reduction level of cytochromes b is constant (52%) in the range of 0.1 to 2.0% O2 and increases gradually to 64% in the range from 2.0 to 3.5% O2. The reduction level of cytochromes c decreases from 34 to 28% over the range 0.1-0.5% O2, increases from 28 to 42% in the range between 0.5 and 2.0% O2, and remains constant (42%) from 2.0 to 3.5% O2.