Graduate Thesis Or Dissertation
 

Measurement of nitrogenase activity of intact legume symbionts in situ using the acetylene reduction assay

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  • The process of biological nitrogen fixation involves the enzymic reduction of atmospheric nitrogen (N₂) to ammonia (NH₃). The enzyme which catalyzes this reduction, nitrogenase, does not exhibit a high degree of substrate specificity. It also catalyzes the reduction of acetylene (C₂2H₂) to ethylene (C₂H₄). The reduction of C₂H₂ to C₂H₄ coupled with gas chromatography has been widely used as a method of measuring nitrogenase activity. In this investigation, the acetylene reduction assay was adapted for the measurement of nitrogenase activity of intact nodulated cultures of legumes. An advantage of the method is that cultures may be assayed for nitrogenase activity in situ without serious damage to the plants. Entire cultures were placed in polyethylene wastebaskets and then were exposed to acetylene after sealing the wastebaskets with plastic lids. Time course experiments showed that rates of acetylene reduction were linear for periods of at least two hours. The correlation coefficient between rates of acetylene reduction and nodule fresh weight was 0. 79. Acetylene reduction rates of either intact nodulated plants in Per lite or nodulated root systems removed from the Per lite were approximately 40% greater than acetylene reduction rates of detached nodules from comparable cultures. No consistent diurnal variation was observed in nitrogenase activity of nodulated plants grown under controlled environmental conditions. The method is useful for the assessment of nitrogenase activity of legume cultures in a porous medium under standardized conditions, but its application to legumes in soil is complicated by factors, such as soil moisture content, that influence rates of gaseous diffusion.
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