Supply of O2 regulates demand for O2 and uptake of malate by N2-fixing bacteroids from soybean nodules

Bacteroids, prepared anaerobically from soybean root nodules by fractional centrifugation or by sucrose or Percoll density-gradient methods, were retained within a stirred, flow-through reaction chamber and used to determine rates of respiration and N-2 fixation at various rates of O-2 supply. Liquid reaction solutions containing malate, oxyleghaemoglobin, dissolved N-2 and various levels of dissolved O-2 were passed through the reaction chamber at measured rates of flow. The relative oxygenation of leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O-2 ([O-2 free]) and the rates of O-2 consumption were calculated. N-2 fixation was measured by analysis of fractions of effluent. The principal finding was that stepwise increases in the flow rate (increasing the supply of O-2 and malate) induced an increase in O-2 demand (respiration) resulting in a decrease in [O-2 free] and increased N-2 fixation. In some experiments, samples of bacteroids were withdrawn from the flow chamber during steady states and the rates of malate uptake were measured in standard, microaerobic assays. Progressive taking of samples from the flow chamber whilst maintaining constant flow rates (increasing the supply of O-2 and malate per bacteroid) also resulted in increased O-2 demand and declines in [O-2 free]. With increased bacteroid respiration, transport of malate into bacteroids (linear with time between 1 and 5 min after starting each assay) increased proportionally. This suggests that the rate of malate transport is tightly coupled with bacteroid respiration. Thus, bacteroid respiration, coupled with malate uptake, must be regulated by the rate of O-2 supply, rather than by the [O-2 free] prevailing in the stirred chamber as found or assumed in previous work. These features are discussed in relation to N-2 fixation by anaerobically isolated bacteroids.