Azolla-Anabaena azollae Relationship

Abstract

The water ferm, Azolla caroliniana Willd., containing the symbiotic, heterocystous blue-green alga, Anabaena azollae, has been studied uder various growth conditions to characterize its light-dependent production of H2. The response of H2 production to N2 and C2H2 and the absence of a differential effect of m-chlorocarbonyl cyanide phenylbydrazone on H2 production and C2H2 reduction, coupled with the paralel ihibition of both processes by DCMU imply that the production of H2 is nitrogenase-catalyzed and ATP-dependent.H2 was produced by fronds grown under air-CO2 in the presence or absence of combined nitrogen. When cultured under argon-02-C02, only those fronds provided with combined nitrogen remained viable and produced H2. Fronds grown on nitrate under air plus 2% CO also produced H2. In comparison to fronds grown on N2 alone, fronds grown on nitrate had an increased rate of H2 production reltive to C2H2 reduction, and the inhibition of H2 production by air was less.CO in argon ± CO2 resulted in a partial inhibition of H2 production, whereas CO in argon-CO2-C2H2 enhanced H2 production in fronds grown without combined nitrogen. Our studies strongly indicate that H2 production is nitrogenase-catalyzed but the possibility that the symbiont contains a hydrogenase cannot be totally exduded. This paper reports on H2 production by the water fern, Azolla caroliniana Willd. which contains the symbiotic, heterocystous blue-green alga, Anabaena azollae within specialized leaf cavities (29). The symbiont is capable of N2 fixation when isolated from the fern and, in the intact association, the alga can supply the host plant with its total nitrogen requirement (23,24,26). Azolla freed of the symbiont is incapable of N2 fixation (C2H2 reduction) and must be provided with combined nitrogen (26,30).Azolla plants containing the symbiont and grown under air on nitrate or urea for 35 days retained 70% or more of their capacity for C2H2 reduction and appreciable nitrogenase activity was still present after 6 to 7 months of such growth (26). When fronds were grown on nitrate or urea under 1 % C02, 20% 02 in argon, growth and the capability for C2H2 reduction were comparable to those of controls grown under air. Preliminary studies indicated that H2 production followed the same pattern as C2H2 reduction (23,24 Growth Conditions. Fronds were usually grown under air with or without combined nitrogen. In experiments in which atmospheres of 1% Co2, 20% 02 in Ar, or 1% CO2 in air + 2% CO were employed, the growth flasks were vigorously purged at the onset of the experiment and then daily. Between purgings, the medium was continuously bubbled to maintain the desired gas atmosphere. The cultures were maintained at 24 C under either continuous illumination or, in the case of routine maintenance, with a 16-hr light, 8-hr dark period. Illumination at 400 ft-c was provided by a mixture of cool white and Gro-lux fluorescent lamps.C2H4, H2 and 02 Determinations. C2H2 reduction assays were carried out essentially as described previously (...

Azolla-Anabaena azollae Relationship

“…1) demonstrated that the maximum rate of C2H4 production occurred at 0.1 atm C2H2 in this association and, in accord with previous observations (11), 0.1 atm C2H2 inhibited H2 production by 95 to 97%. Although the inhibition of H2 production at any pC2H2 is somewhat greater than reported for in vitro studies (16), the pattern of inhibition is very similar.…”

“…In the intact association, the alga can provide the Azolla plant with its total nitrogen requirement. Previous studies showed that the symbiont contained nitrogenase and was capable of C2H2 reduction, ATP-dependent H2 evolution, and excretion of ammonia (10,11,13).Although C2H2 reduction is a simple and sensitive assay of nitrogenase activity, C2H2 is not the biologically important substrate, and the assay is an indirect measurement of nitrogen fixation. Since the reduction of N2 to 2NH3 requires six electrons while reduction of C2H2 to C2H4 requires two electrons, a theoretical conversion factor of 3C2H2 reduced per N2 fixed is frequently used in estimating nitrogen fixation from C2H2 reduction assays.…”

“…In the intact association, the alga can provide the Azolla plant with its total nitrogen requirement. Previous studies showed that the symbiont contained nitrogenase and was capable of C2H2 reduction, ATP-dependent H2 evolution, and excretion of ammonia (10,11,13).…”

“…C2H2 Reduction and H2 Evolution. These assays were conducted as described previously (10,11) except that various pC2H2 or pN2 were employed. For assays with the isolated alga, the final resuspension was in a modified Kratz and Myers medium (9).…”

“…All cylinder gases were obtained from Matheson Gas Products. The actual pN2 in individual flasks was monitored as described previously for H2 and O2 (11). Room air (pN2 = 0.78 atm, PO2 = 0.21 atm) was used as a standard.…”