Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria

Abstract

A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.

Isolation and Characterization of a Nonheterocystous Cyanobacterium,Lyngbyasp. Isolate No. 108, for Large-quantity Hydrogen Production

“…In seven days, this strain produced 770 ml hydrogen/g dry cells (0.205,umol/mg cells/hr). Anabaena eylindriea, 13) Mastigoẽ ladus laminosus 14 ) and Oseil/atoria sp. Miami BG-7 9 ) produce hydrogen in closed vessels.…”

“…This may be a consequence of the fact that the ferric ion is a component of Fe-protoporphyrine, which is an intermediate in the phycocyanin system. 13 ) Table III shows the optimum conditions for hydrogen production by Lyngbya sp. (No.…”

The Hydrogen Metabolism of Cyanobacteria (Blue‐green Algae)

“…Other workers have reported H, formation from non-heterocystous species (Weare & Benemann, 1974;Mitsui & Kumazawa, 1977), marine (Lambert & Smith, 1977;Mitsui & Kumazawa, I 977) and thermophilic (Miyamoto, Hallenbeck & Benemann, I 979 a ) cyanobacteria. Anabaena CA, a rapidly growing and rapidly nitrogen-fixing thermophilic cyanobacterium (Stacey et al,) does not produce significant H, in either an argon/CO, or an air/CO/C,H,/CO, gas phase (G. D. S., unpublished observation), while Anabaena sp.…”

“…Some variability is seen in the level of CO, used for the growth of A . cylindrica, frequently being either 0.3 yo (Benemann & Weare, 1 9 7 4~; Lambert et al, 1979~) or 5 % Tel-Or et al, 1978) by volume. However, detailed studies revealed this variation not to be a factor responsible for variable rates of H, formation reported by these various workers .…”

“…For example, a sodium requirement for nitrogenase activity in cyanobacteria has been demonstrated (Brownwell & Nicholas, 1967; Apte & Thomas, 1980). Magnesium may play an important role in nitrogenase regulation (Davis & Kotake, 1980).…”