Vol. 59: 197–206, 2010 AQUATIC MICROBIAL ECOLOGY Published online April 8 doi: 10.3354/ame01407 Aquat Microb Ecol Hydrogen production by Trichodesmium erythraeum Cyanothece sp. and Crocosphaera watsonii Samuel T. Wilson1, 2,*, Rachel A. Foster1, 3, Jonathan P. Zehr1, 3, David M. Karl1, 2 1Center for Microbial Oceanography: Research and Education, Honolulu, Hawaii 96822, USA 2Department of Oceanography, 1000 Pope Road, University of Hawaii, Honolulu, Hawaii 96822, USA 3Ocean Sciences, University of California, Santa Cruz, California 95064, USA ABSTRACT: Diazotrophic cyanobacteria are important components of marine ecosystems, where they contribute to primary production and provide a source of fixed nitrogen (N). During biological fixation of atmospheric nitrogen (N2), hydrogen is produced as an obligate by-product. The present study investigated the potential contribution of 4 marine diazotrophs to the pool of dissolved H2 in the oceans. N2 fixation, as measured by acetylene reduction, and H2 production rates were monitored throughout the diel period in cultures of the filamentous Trichodesmium erythraeum strain IMS101, and the unicellular organisms Cyanothece sp. strain ATCC 51142 and Crocosphaera watsonii strains WH8501 and WH0002. H2 production coincided with diel variations in N2 fixation for each strain regardless of whether N2 fixation peaked during the day or night. Chlorophyll-normalized rates of H2 production ranged 100-fold from a maximum of 3 nmol µg chl a–1 h–1 in T. erythraeum IMS101 cul- –1 –1 tures to 0.03 nmol µg chl a h in Crocosphaera watsonii WH0002. Overall, the ratio of net H2 pro- duced to N2 fixed varied from 0.05 to 0.003 in the unicellular cyanobacteria, compared to 0.3 in the filamentous T. erythraeum IMS101, indicating that unicellular cyanobacteria produce less, or alterna- tively, re-assimilate more of the H2 produced during N2 fixation. Crocosphaera watsonii has recently been identified as a significant source of fixed N in the marine environment, and an efficient recy- cling of H2 would provide a valuable source of energy to their respiratory electron transport chain. Furthermore, the magnitude of H2 produced by T. erythraeum IMS101 strongly implicates this organism in the production of H2 in the upper ocean. KEY WORDS: N2 fixation · Hydrogen · Cyanobacteria Resale or republication not permitted without written consent of the publisher INTRODUCTION 3 nmol l–1, followed by sharp decreases in concentra- tion with depth, becoming under-saturated typically The ocean is a net source of hydrogen (H2) to the within 100 m of the surface (e.g. Conrad & Seiler 1988, atmosphere (Schmidt 1974, Hauglustaine & Ehhalt Moore et al. 2009). 2002), where it acts as an indirect greenhouse gas by There is conflicting evidence of a diel cycle associ- influencing the concentrations of methane in the tro- ated with H2 concentrations in the upper water column posphere and water vapor in the stratosphere (Ehhalt of the open ocean. A clear increase during the daytime & Prather 2001). Although the global ocean is typically was reported in the oligotrophic South Atlantic (Herr classified as being 2 to 3 times supersaturated with et al. 1984), compared to a weak daytime peak in the respect to atmospheric H2 concentrations (Seiler & equatorial Atlantic (Conrad & Seiler 1988), and no diel Schmidt 1974), the net production of H2 in surface sea- variation in the California Current System (Setser et al. water is restricted to tropical and subtropical biomes 1982). The longer-term temporal variability of dis- (Herr et al. 1981). Within the upper water column, solved H2 concentrations over several months has been vertical profiles of H2 display the highest concentra- reported for high-latitude coastal waters (Punshon et tions within the top 50 m, where values range from 1 to al. 2007). *Email: [email protected] © Inter-Research 2010 · www.int-res.com 198 Aquat Microb Ecol 59: 197–206, 2010 The most important environmental parameter influ- flicting, as laboratory-maintained cultures showed a encing ambient H2 concentrations was suggested by positive correlation between H2 production and N2 fix- Conrad (1988) to be the biological fixation of nitrogen ation (Punshon & Moore 2008), yet field-collected (N2). H2 evolution is an obligate by-product of the colonies showed no correlation with N2 fixation rates enzymatic N2 fixation reaction, summarized in Eq. (1): (Scranton 1983). Resolving this discrepancy is of par- ticular importance to the marine H cycle, given that N + 8 H+ + 8 e– + 16 ATP → 2 NH + H + 16 ADP + 16 Pi 2 2 3 2 Trichodesmium spp. is found throughout warm oligo- (1) trophic waters and is estimated to contribute 25 to 50% where Pi is inorganic phosphate. H2 is formed during of the new production in the North Pacific Subtropical the binding of a N2 molecule to the molybdenum-iron Gyre (Karl et al. 1997). (MoFe) subunit of the nitrogenase enzyme complex, To better understand the role of N2 fixing Cyanobac- prior to the reduction of N2 to ammonia (Lowe & Thor- teria in H2 cycling in the upper ocean, cultures of 3 neley 1984, Howard & Rees 2006). According to Eq. (1), model marine Cyanobacteria were analyzed for their which represents the least energetically costly stoi- N2-fixing and H2-production capabilities. The test chiometry for N2 fixation, the theoretical production of organisms included Trichodesmium erythraeum strain H2 under N2-saturating conditions should be equimo- IMS101, Cyanothece sp. strain ATCC 51142, and Cro- lar to the rate of N2 fixation (Burns & Hardy 1975). cosphaera watsonii strains WH8501 and WH0002. However, diazotrophs contain uptake hydrogenase, which recycles the H2 produced during N2 fixation, and measured rates of net production of H2 are typi- MATERIALS AND METHODS cally much less than the theoretical maximum (Bothe et al. 1980). Culture conditions. The Cyanobacteria strains used A recent oceanographic cruise across the equatorial in this work are listed in Table 1 and were grown using Pacific revealed a strong correlation between the rate fixed N-free media: YBC II medium at pH 8.0 and of N2 fixation and H2 supersaturation (Moore et al. salinity of 34 for Trichodesmium erythraeum IMS101 2009). Further evidence for N2 fixation as the mecha- (Chen et al. 1996), and SO medium (pH 8.0, salinity 28) nism responsible for dissolved H2 comes from labora- (Waterbury & Willey 1988) for Cyanothece sp. ATCC tory-based research on photobiological H2 production 51142 and both strains of Crocosphaera watsonii. All (reviewed by Asada & Miyake 1999, Prince & Kheshgi cultures were maintained at 26°C using a 12:12 h 2005, Rupprecht et al. 2006). A large and diverse range square-wave light:dark cycle with a light intensity of –2 –1 of microbes have been analyzed for their H2 produc- 44 µmol photons m s . This light intensity is approx- tion rates, and the more extensively examined N2 fix- imately equivalent to a depth of 110 m at Stn ALOHA. ing cyanobacteria include Anabaena sp. (Masukawa et Growth of all cultures was monitored by daily mea- al. 2002), Nostoc punctiforme (Schütz et al. 2004), and surements of in vivo fluorescence with a TD-700 Synechococcus sp. (Mitsui & Suda 1995). Turner Designs fluorometer. Chlorophyll a (chl a) mea- Despite the fact that N2 fixing microorganisms have surements were made at the beginning of each exper- been studied for their ability to produce H2, it remains iment. Triplicate aliquots of cultures were filtered onto unclear how different diazotrophs contribute to dis- 25 mm Whatman GF/F filters and the chl a extracted in solved H2 dynamics in the marine environment. The 5 ml of 90% acetone for 24 h at –20°C before being majority of simultaneous measurements of N2 fixation analyzed using a Turner Designs Model 10-AU fluo- and H2 production in the marine environment have rometer (Strickland & Parsons 1972). been conducted on Trichodesmium spp., a filamentous The cultures were grown in 500 ml plastic culture non-heterocystous cyanobacterium that fixes N2 dur- flasks and transferred to borosilicate glass vials for ing the daytime while evolving oxygen via photosyn- experimental analysis. Typical volumes for sample thesis (Capone et al. 1997). However, results are con- analysis were 200 ml (for the unicellular diazotrophs) Table 1. Cyanobacteria strains examined in the present study Strain Location isolated Year isolated Source Trichodesmium erythraeum IMS101 North Carolina coast 1992 Prufert-Bebout et al. (1993) Crocosphaera watsonii WH8501 Tropical South Atlantic 1984 Waterbury & Rippka (1989) Crocosphaera watsonii WH0002 Hawaii 2000 Webb et al. (2009) Cyanothece sp. ATC51142 Gulf of Mexico 1993 Reddy et al. (1993) Wilson et al.: Hydrogen production by marine Cyanobacteria 199 and 40 ml (for Trichodesmium erythraeum IMS101), (Airgas). One unavoidable feature of the analytical set- which were analyzed in 240 ml and 76 ml glass vials, up is the inherent delay in purging the sample of H2; respectively. The smaller sample volume for T. ery- i.e. if H2 production were to cease, this would not be in- thraeum IMS101 was due to the magnitude of the H2 stantly recorded by the analyzer. Controlled measure- produced. The glass vials were crimp-sealed with stan- ments showed that it took 45 min to completely purge dard gray butyl rubber stoppers and aluminum seals to H2 from the larger 240 ml glass vial used for the unicel- ensure the incubations were gas-tight. For each organ- lular diazotrophs and 15 min to purge the 76 ml glass ism, the measurements of H2 and ethylene (C2H4) were vial used for Trichodesmium erythraeum IMS101. run on separate experimental flasks due to the residual H2 concentrations were measured using a reduced H2 in the cylinder of acetylene (C2H2) (Praxair) contam- gas analyzer that incorporated a mercuric oxide bed inating the samples and inundating the H2 analyzer. coupled to a reducing compound photometer (Peak The analytical method was designed to measure Laboratories).