Bacteroids from soybean root nodules: accumulation of poly- β -hydroxybutyrate during supply of malate and succinate in relation to N ₂ fixation in flow-chamber reactions

A liquid reaction medium containing dissolved air and oxyleghaemoglobin was supplied to bacteroids confined in a stirred reaction chamber with no gas phase. The relative oxygenation of leghaemoglobin, dissolved CO$_{2}$ and NH$_{3}$ from N$_{2}$ fixation were monitored before, during (when N$_{2}$ fixation was diminished) and after the supply of [$^{14}$C]malate and succinate. The amounts and location of [$^{14}$C]labelled products were determined in bacteroids recovered from the chamber and $^{14}$CO$_{2}$ was measured in the effluent. During the supply of [$^{14}$C]succinate, the bacteroids accumulated most $^{14}$C from C$_{2}$ and C$_{3}$ whereas $^{14}$CO$_{2}$ arose primarily from C$_{1}$ and C$_{4}$. This suggested that production of pyruvate from malate via malic enzyme was a central feature. For up to 80 min after removal of [$^{14}$C]malate and succinate, CO$_{2}$ arose from stored carbon and N$_{2}$ fixation was enhanced; during this period $^{14}$C continued to accumulate in poly-$\beta $-hydroxybutyrate, until this accounted for about 90% of bacteroid $^{14}$C. Thereafter CO$_{2}$ was evolved with steady, low radioactivity and enhanced N$_{2}$ fixation continued. These results supported the proposal that poly-$\beta $-hydroxybutyrate, which is present in large amounts (50-70% of dry mass) in these bacteroids, is a mobilizable, energy-yielding reserve that provides endogenous substrates for support of N$_{2}$ fixation when exogenous substrates are not available.