A Highly Sensitive, Flow Through H₂ Gas Analyzer for Use in Nitrogen Fixation Studies

Abstract

ABSTRACrStudies of H2 evolution by N2 fixing systems are frequently limited by an inability to accurately measure H2 gas concentrations of less than about 10 microliters per liter. In this study, a H2 gas analyzer is described which is able to accurately and reproducibly detect up to 100 times lower H2 concentrations than most thermal conductivity gas chromatographs or other conventional instruments used for the measurement of H2 gas. This high level of sensitivity (maximum of about 0.02 microliter per liter H2 per millivolt output) and the ability to continuously monitor H2 concentration directly in a flowing gas stream, makes this instrument well suited for use in an open gas exchange system.Since the sensor used in the instrument was also sensitive to other combustible gases, it was necessary to demonstrate that H2 was the only combustible gas produced by the N2 fixing system being studied. When an air stream was passed through a pot containing nodulated soybean (Glycine max L.) roots, gas chromatographic analysis of the effluent ps stream revealed that H2 was the only combustible gas present. These results were supported by other studies in which no combustible gases were detected in the effluent gas stream from soybean roots nodulated with USDA 110, a Rhizobium strain which displays active uptake hydrogenase activity.Traditionally, rates of C2H2 reduction and H2 evolution in N2 fixing legumes have been determined in sealed vessels of known volume in which changes in gas concentration are measured with time. H2 accumulation in the vessels equivalent to a change of 10 Al I' or more, can be detected using thermal conductivity gas chromatography. The closed system, however, does not permit the maintenance of a constant gas composition around the nodules and small leaks or removal of samples can introduce further errors. These latter problems are largely alleviated in open gas exchange systems where gas composition is constant and sampling or minor leaks do not affect pressure or composition of the atmosphere. However, since the gas of interest is not allowed to accumulate within the assay vessel, much higher levels of sensitivity are required for its detection. This is generally not a problem for the measurement of C2H2 reduction, as evidenced by recent studies in which open gas exchange systems have been used (3,5). However, the best current instruments would usually not be sensitive enough to detect the H2 produced.In this study, we describe a highly sensitive H2 analyzer which is capable of continuously monitoring a change of as little as 0

Comparing Symbiotic Efficiency between Swollen versus Nonswollen Rhizobial Bacteroids

“…Here, we measured the efficiency of each of two strains as swollen bacteroids in one host and nonswollen bacteroids in another. We measured nitrogenase activity as hydrogen (H 2 ) production in an N 2 -free atmosphere (Layzell et al, 1984;Witty and Minchin, 1998), and compared it to carbon dioxide (CO 2 ) respiration to estimate return on nodule operation cost. We also compared host biomass growth per total nodule mass growth to estimate return on nodule construction cost.…”

Effect of Increases in Oxygen Concentration during the Argon-Induced Decline in Nitrogenase Activity in Root Nodules of Soybean

“…The gas exchange system contained computer-controlled mass flow controllers (model FMA-100 series, Omega Engineering, Stamford, CT) which allow linear changes in gas concentrations at a constant total flow rate. CO2 evolution was measured by infrared gas analyzers (model 225, Mark III, Analytical Development Corp., Hoddeson, UK), 02 evolution with an O2 electrode (model CB 1 D, Hansatech, King's Lynn, UK), and H2 evolution with custom-built H2 analyzers (11). The complete system has been described previously (6,8).…”

Bacteroid Proline Catabolism Affects N2 Fixation Rate of Drought-Stressed Soybeans

“…The N 2 fixation rate was calculated from the rate of H 2 evolution (Layzell et al, 1984) as given in Equations 1 and 2 in ''Results.'' N 2 fixation rate is a function of H 2 fixation rate and EAC (Eq.…”

“…Because exposure to acetylene results in decreasing nitrogenase activity (Minchin and Witty, 1989), we chose to assess the rate of N 2 fixation based on H 2 evolution (Layzell et al, 1984) in plants whose seeds had been inoculated with ProDH 1 and ProDH 2 strains of B. japonicum in a hup 2 background (Straub et al, 1996). It was necessary to use hup 2 strains because strains with uptake hydrogenase activity (hup 1 ) can reutilize H 2 produced by nitrogenase.…”