Succinate-limited continuous cultures of an Azorhizobium caulinodans strain were grown on ammonia or nitrogen gas as a nitrogen source. Ammonia-grown cells became oxygen limited at 1.7 ,uM dissolved oxygen, whereas nitrogen-fixing cells remained succinate limited even at dissolved oxygen concentrations as low as 0.9 ,uM. Nitrogen-fixing cells tolerated dissolved oxygen concentrations as high as 41 ,uM. Succinate-dependent oxygen uptake rates of cells from the different steady states ranged from 178 to 236 nmol minl mg of protein-' and were not affected by varying chemostat-dissolved oxygen concentration or nitrogen source. When equimolar concentrations of succinate and 0-hydroxybutyrate were combined, oxygen uptake rates were greater than when either substrate was used alone. Azide could also used alone as a respiratory substrate regardless of nitrogen source; however, when azide was added following succinate additions, oxygen uptake was inhibited in ammonia-grown cells and stimulated in nitrogen-fixing cells. Use of 25 mM succinate in the chemostat resevoir at a dilution rate of 0.1 h-1 resulted in high levels of background respiration and nitrogenase activity, indicating that the cells were not energy limited. Lowering the reservoir succinate to 5 mM imposed energy limitation. Maximum succinate-dependent nitrogenase activity was 1,741 nmol of C2H4 h-1 mg (dry weight)-', and maximum hydrogen-dependent nitrogenase activity was 949 nmol of C2H4 hmg (dry weight)-'. However, when concentrations of 5% (vol/vol) hydrogen or greater were combined with succinate, nitrogenase activity decreased by 35% in comparison to when succinate was used alone. Substitution of argon for nitrogen in the chemostat inflow gas resulted in "washout," proving that ORS571 can grow on N2 and that there was not a nitrogen source in the medium that could substitute.
High performance anion exchange chromatography and pulsed amperometry were used to separate and quantify peanut sugars extracted with methanol: chloroform:water (60:25:15, V/V/V), a highly polar sol vent which solubilizes other seed components including amino acids. Free sugars were separated on an anion exchange column using a sodium hydroxide gradient and detected with a pulsed amperometric detector equipped with a gold electrode. Free amino acids in the seed extract interfered with sugar analysis by causing peak shifting and co-elution of some amino acids with sugars. Free arginine co-eluted with inositol resulting in a peak area which was 42% of the actual total area for both compounds. Proline co-eluted with fructose. Serine eluted on the leading edge of sucrose. Peak areas of these interfering amino acids were either "additive" or "subtractive" to sugar peaks. Altering the gradient elution or using cation syringe filters to remove contami nant amino acids permitted the accurate identification and quantification of peanut seed sugars. Results from this study suggest applications that can be applied in other biological systems containing free amino acids and sugars.
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