Hydrogen Cycling by the Unicellular Marine Diazotroph Crocosphaera watsonii Strain WH8501

The hydrogen (H₂) cycle associated with the dinitrogen (N₂) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H₂ production and acetylene (C₂H₂) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H₂ production was coincident with the maximum rates of C₂H₂ reduction. Theoretical stoichiometry for N₂ fixation predicts an equimolar ratio of H₂ produced to N₂ fixed. However, the maximum rate of net H₂ production observed was 0.09 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹ compared to the N₂ fixation rate of 5.5 nmol N₂ μg chl a⁻¹ h⁻¹, with an H₂ production/N₂ fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H₂ production was hypothesized to be a result of H₂ reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H₂ production rates ∼40-fold to a maximum rate of 3.5 nmol H₂ μg chl a⁻¹ h⁻¹. We conclude that the reassimilation of H₂ by C. watsonii is highly efficient (>98%) and hypothesize that the tight coupling between H₂ production and consumption is a consequence of fixing N₂ at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H₂ cycle provides unique insight into N₂ fixation and associated metabolic processes in C. watsonii.The biological production of hydrogen (H₂) can occur as a by-product of photosynthesis, fermentation, and N₂ fixation (22). Of these three metabolic pathways, N₂ fixation remains a particularly enigmatic process, and to date there is no clear explanation for why H₂ evolves during the reduction of N₂ (11). The unfavorable energy cost of N₂ fixation can be mitigated by reassimilating the released H₂ via uptake hydrogenase enzyme activity (30). The coupled production and consumption of H₂ during cellular nitrogenase activity creates a H₂ cycle that can be hidden from measurements of ambient environmental H₂ concentrations and fluxes, depending upon the overall efficiency of H₂ assimilation (Fig. ​(Fig.11).Open in a separate windowFIG. 1.H₂ is formed during N₂ fixation by the binding of a N₂ molecule to the molybdenum-iron protein of the nitrogenase enzyme complex, prior to the reduction of N₂ to ammonia (11, 15). The most energetically favorable theoretical in vivo stoichiometry predicts that one mole of H₂ is produced for every mole of N₂ reduced: N₂ + 8H⁺ + 8e⁻ + 16ATP → 2NH₃ + H₂ + 16ADP + 16P_i. The production of H₂ consumes 25% of the electron flux through nitrogenase and diazotrophs mitigate this loss of potential energy by reassimilating the H₂ via uptake hydrogenase (21, 30). The electrons produced by uptake hydrogenase either generate reductant or ATP with simultaneous consumption of O₂ (3). (Adapted from reference 32a.)For most cultures of phototrophic marine diazotrophs grown under optimal conditions, complete reassimilation of H₂ is not achieved, and the excess H₂ is lost to the surrounding environment. This excess H₂ equates to the net production of H₂ and is expressed as the ratio of H₂ formed to N₂ fixed or the H₂/N₂ ratio. To date, H₂/N₂ ratios have mainly been measured on filamentous, colony-forming diazotrophs such as Anabaena spp. and Trichodesmium spp. with H₂ production rates of up to 20 nmol H₂ μg chlorophyll a (chl a)⁻¹ h⁻¹ and H₂/N₂ ratios ranging from 0.01 to 0.48 (3, 20, 24). H₂ production has also been quantified in unicellular diazotrophs (12, 16, 17, 32), although the H₂ measurements have rarely been performed in conjunction with rates of N₂ fixation. However, recent H₂ measurements of two N₂-fixing unicellular cyanobacteria species reached a maximum of 1.38 nmol H₂ μg chl a⁻¹ h⁻¹, with H₂/N₂ ratios ranging from 0.003 to 0.05, indicating an effective reassimilation of H₂ can occur under certain conditions (34).H₂ cycling in marine diazotrophs has important ecological implications both for the cell and for the marine H₂ cycle. Surface waters of low-latitude oceans are typically 200 to 300% supersaturated in dissolved H₂ with respect to atmospheric concentrations (25), implying a sustained localized production of H₂. The source of the dissolved H₂ is thought to be biological N₂ fixation (7); however, the relative contributions of diverse diazotrophic communities and in situ controls on H₂/N₂ ratios are not well constrained. N₂ fixation is performed by a suite of diazotrophs typically identified by their nitrogenase gene (nifH) sequences amplified directly from oceanic water samples (35). The importance of unicellular diazotrophs, including Crocosphaera spp., in marine N₂ fixation has recently become widely recognized (36). Size-fractionated rates of N₂ fixation indicate that in the oligotrophic ocean, <10-μm microorganisms, which include the unicellular cyanobacteria, make a substantial contribution to the daily N₂ fixation (9, 18). Correlating the species-specific production of H₂ with the activity and biomass of diazotrophs will help elucidate dissolved H₂ cycling in the upper ocean.We examined the cycling of H₂ in cultures of Crocosphaera watsonii strain WH8501, a marine unicellular diazotroph, and correlated it with other metabolic parameters, including N₂ fixation measured via acetylene (C₂H₂) reduction, O₂ production and consumption, and photosynthetic efficiency. Carbon monoxide (CO) was used as an inhibitor of intracellular H₂ reassimilation to reveal the H₂ cycling that can occur in conjunction with nitrogenase activity. H₂ reassimilation by C. watsonii was shown to be very efficient in our laboratory experiments, which is considered to be a consequence of the temporal separation between daytime photosynthetic activity and nighttime N₂ fixation. Therefore, the present study not only reveals the cell''s H₂ cycle but also provides insight into the metabolism of nitrogenase in C. watsonii.