Physiological and isotopic characteristics of nitrogen fixation by hyperthermophilic methanogens: Key insights into nitrogen anabolism of the microbial communities in Archean hydrothermal systems
Introduction
Deep-sea hydrothermal systems provide a variety of microbial habitats, and the focused and diffusing hydrothermal fluids contain microorganisms from reduced hot subseafloor environments (Deming and Baross, 1993, Summit and Baross, 1998, Takai and Nakamura, 2010, Takai and Nakamura, 2011). Microbiological and chemical components entrained in the discharging hydrothermal fluids and included in the subseafloor fluids are key signals for understanding the composition and function of indigenous microbial communities living in the subseafloor (Karl et al., 1989, Cowen et al., 2003, Butterfield et al., 2004, Takai et al., 2004, Orcutt et al., 2011). Many hyperthermophilic (optimal growth temperature: 70–120 °C) and thermophilic (optimal growth temperature: 50–70 °C) microorganisms have been isolated from seafloor hydrothermal environments, and these microorganisms utilise a variety of energy, carbon and nitrogen sources (e.g., Jones et al., 1983, Neuner et al., 1990, Huber et al., 1992, Nakagawa et al., 2003). Thermodynamic calculations and microbial community compositions in the hydrothermal mixing zones suggest that the chemolithotrophic energy potentials obtained from the hydrothermal fluids and the ambient seawater would control the development of chemolithotrophic microbial communities associated with hydrothermal activities (McCollom and Shock, 1997, Shock and Holland, 2004, Tivey, 2004, Takai and Nakamura, 2010, Takai and Nakamura, 2011). In addition to the chemolithotrophic energy state, the abundance and availability of biologically essential elements, such as nitrogen, phosphorus and transition metals, would significantly affect the composition and function of the microbial communities (Takai and Nakamura, 2010). However, the nitrogen sources supporting the development of chemolithotrophic microbial communities in the seafloor and subseafloor hydrothermal environments remain poorly understood.
The nitrate (NO3−) concentration in diffusing hydrothermal fluids (<120 °C) is generally lower than that expected from a simple mixing of a magnesium-zero end-member hydrothermal fluid (0 μM) and the ambient deep-sea water (40 μM) (Johnson et al., 1988, Karl et al., 1989, Bourbonnais et al., 2012a). For instance, the nitrate concentration is less than 20 μM in the low-temperature (20 °C) diffusing fluids of the Galapagos spreading centre (Johnson et al., 1988). The non-conservative nitrate depletion most likely originates from biological consumption because many microorganisms can utilise nitrate via assimilatory and/or dissimilatory reduction (Nakagawa et al., 2003, Nakagawa et al., 2005, Bourbonnais et al., 2012b). Nitrogen isotopic ratios of the nitrate in the diffusing fluids have been reported only from the Juan de Fuca Ridge, and they seem to increase from 6‰ (the value of nitrate in deep-sea water) to 10‰ as the degree of non-conservative nitrate depletion increases (Bourbonnais et al., 2012a). The ammonium (NH4+) concentration in high temperature (>150 °C) hydrothermal fluids in unsedimented systems is typically similar to that of the ambient deep-sea water (1 μM or less), but it is occasionally as high as 15 μM in certain fields (German and Von Damm, 2003, Bourbonnais et al., 2012a). The exception is hydrothermal fluids (>300 °C) venting from the Endeavour Segment on the Juan de Fuca Ridge, where decomposition of organic matter in sediments buried at an early stage of the ridge formation has been proposed to be the candidate source of ammonium (1000 μM, Lilley et al., 1993, Bourbonnais et al., 2012a). Nitrogen isotopic ratios of ammonium in hydrothermal fluids have been reported only from the Juan de Fuca Ridge, and they are 6.7 ± 1.0‰ (n = 16) at the Axial Volcano and 3.7 ± 0.6‰ (n = 37) at the Endeavour Segment (Bourbonnais et al., 2012a). Dissolved dinitrogen (N2) is more abundant in hydrothermal fluids than nitrate and ammonium (400–3400 μM in magnesium-zero end-member hydrothermal fluids and 590 μM in deep-sea water) (Charlou et al., 1996, Charlou et al., 2000, Charlou et al., 2002). Isotopic ratios of N2 in hydrothermal fluids have been reported only from the Tonga-Kermadec Arc, and they are slightly depleted in 15N relative to deep-sea water (0‰) by 2‰ (de Ronde et al., 2011).
Previous studies have shown that a limited number of hyperthermophilic and thermophilic microorganisms can assimilate N2 via a nitrogenase enzyme complex that catalyses N2 reduction to ammonia (diazotrophy) (Belay et al., 1984, Mehta and Baross, 2006, Steunou et al., 2006, Hamilton et al., 2011). Furthermore, the phylogenetic diversity of nitrogenase genes (nifH) in deep-sea hydrothermal fluids has pointed to the presence of methanogenic archaea and anaerobic bacteria (clostridia, sulphate-reducing proteobacteria) as potential nitrogen fixers in the subseafloor microbial communities (Mehta et al., 2003).
The discovery of diazotrophic hyperthermophilic methanogens (Mehta and Baross, 2006) has highlighted the potential ubiquity and important role of these organisms in H2-abundant marine hydrothermal environments throughout Earth history. Hyperthermophilic methanogens represent one of the most predominant primary producers in the deep-sea hydrothermal environments with hydrothermal fluid chemistries that are characterised by highly enriched H2 (more than approximately 1 mM) (Takai et al., 2004, Flores et al., 2011). Furthermore, hyperthermophilic methanogenesis has been theoretically and empirically predicted as one of the most ancient chemolithotrophic energy metabolisms supporting the earliest ecosystem associated with the ocean hydrothermal systems on the Hadean Earth (Russell and Martin, 2004, Ferry and House, 2006, Takai et al., 2006, Sleep and Bird, 2007, Martin et al., 2008, Russell et al., 2010). In fact, geological evidence of ancient methanogenesis in seafloor and subseafloor hydrothermal environments has been furnished by hydrothermal deposit records that date to 3.5 billion years ago (giga-annum, Ga) (Ueno et al., 2006a, Ueno et al., 2006b).
Although many studies have reported on the ecophysiology and biochemistry of methanogenesis metabolisms and functions (Garcia et al., 2000, Thauer et al., 2008), the physiology of nitrogen fixation in hyperthermophilic methanogens remains to be elucidated, including the rate and energetics of nitrogen fixation and the biological requirement of transition metals used in the nitrogenase cofactors (e.g., molybdenum (Mo) and iron (Fe)). In addition, the isotopic systematics of nitrogen fixation in hyperthermophilic methanogens should be investigated to explain the role of the global biogeochemical nitrogen cycle throughout Earth’s history.
In the present-day ocean, more than 70% of biological nitrogen compounds are provided by microbial nitrogen fixation (1–3 × 1014 gN/y) (Brandes and Devol, 2002). By contrast, on the early Earth, the potential nitrogen sources for living forms should have been produced by abiotic processes, such as atmospheric production of nitric oxide by lightning (Navarro-González et al., 2001), photochemical production of hydrogen cyanide (Zahnle, 1986), multistep conversion of nitric oxide and hydrogen cyanide to ammonium in the ocean (Zahnle, 1986, Summers and Chang, 1993, Brandes et al., 1998, Brandes et al., 2008, Summers, 2005, Singireddy et al., 2012), shock synthesis of amines and amino acids (Furukawa et al., 2009), and hydrothermal synthesis of ammonia from N2 reduction (Brandes et al., 1998, Schoonen and Xu, 2001, Smirnov et al., 2008). These prebiotic sources of biologically available nitrogen may have been sufficient immediately after the origin of life, but such abiotically produced nitrogen pools were likely drained by the early expansion of microbial populations and habitats. This process may have triggered the onset of biological nitrogen fixation. Based on the phylogenetic analyses of nitrogenase sequences, two possible hypotheses for the origin of nitrogen fixation have been proposed (Leigh, 2000, Raymond et al., 2004, Boyd et al., 2011b). One hypothesis proposes that nitrogenase was present in the last universal common ancestor (LUCA origin model) (Leigh, 2000, Raymond et al., 2004), whereas the other claims that nitrogenase was derived from the ancestral methanogens (methanogen origin model) (Boyd et al., 2011b). To trace the time and place of the possible onset of biological nitrogen fixation, researchers have used not only an approach based on molecular evolution but also an approach involving the exploration of chemical fossils (isotopic signatures) in the geological record (Beaumont and Robert, 1999, Nishizawa et al., 2007). However, because the isotopic characteristics of nitrogen fixation in methanogens have, until now, been completely unknown, the interpretation of the geological record has been equivocal.
We report, for the first time, the physiological properties and isotopic characteristics of nitrogen anabolisms, including nitrogen fixation, in hyperthermophilic and thermophilic methanogenic genera found in global hydrothermal environments (Methanocaldococcus and Methanothermococcus spp.) (Takai et al., 2004, Flores et al., 2011, Ver Eecke et al., 2012). These methanogens, together with anaerobic archaeal methanotrophs, are known to encode for nitrogenase homologs that do not cluster phylogenetically with previously characterised nitrogenases with iron–molybdenum (FeMo), iron–vanadium (FeV) or iron–iron (FeFe) cofactors (Dekas et al., 2009, Boyd et al., 2011a, Dos Santos et al., 2012). Cultivation experiments were conducted under various conditions (e.g., under varying concentrations of Mo, Fe, N2 and H2 in the culture media) to potentially reproduce present and past oceanic and hydrothermal environments. The results include the novel finding that diazotrophic methanogens produce biomass that is more depleted in 15N than diazotrophic photosynthetic prokaryotes (Minagawa and Wada, 1986, Macko et al., 1987, Carpenter et al., 1997, Beaumont et al., 2000, Zerkle et al., 2008, Bauersachs et al., 2009). The relatively large isotopic fractionation effect of the methanogens and its evolutionary implications are also discussed.
Section snippets
Isolation and phylogenetic characterisation of methanogenic strains
We used two strains of hyperthermophilic and thermophilic methanogens isolated from the Kairei field on the Central Indian Ridge. A hyperthermophilic methanogen was isolated from an in situ cultivation system (ISCS) deployed in 362 °C black smoker fluid from the Kali chimney at the Kairei Field. A slurry sample of the ISCS substratum was inoculated into a nitrogen-fixing medium (see Section 2.2 for the chemical composition) prepared in test tubes with a gas phase of N2 (0.1 MPa), CO2 (0.1 MPa) and
Rates and metal requirements of nitrogen fixation
Both Mc 1–85N and Mt 5–55N utilised N2 and ammonium as the sole nitrogen source, but not nitrate (Fig. 1a and b; Table 1). The diazotrophic growth of Mc 1–85N was observed in the presence of broad ranges of Mo and Fe concentrations (Mo = 5 nM–1 mM; Fe = 100 nM–10 mM) (Fig. 1a and Table 1). In the media with higher Mo concentrations (10–1,000 μM), growth followed a simple exponential curve until the H2 was largely consumed. In the media with lower Mo concentrations (5 nM to 1 μM), growth initially
Factors influencing isotopic fractionation during nitrogen fixation and intracellular ammonia assimilation
In the nitrogen fixation experiments, the concentrations of cellular nitrogen (i.e., PN) produced were 1–10 μg N/mL, while those of ammonium initially present in the media were ⩽ 70 ng N/mL. The cellular nitrogen produced from the assimilation of the ammonium contamination could thus alter the overall δ15N (PN) values by + 1‰ at the most, assuming that the δ15N values of the ammonium contamination are close to the NH4Cl reagent (8.4‰, Section 2.4). Furthermore, the amounts of N2 consumed in
Acknowledgments
We thank R. Senda and K. Suzuki for experimental assistance. We also thank T. Shibuya, K. Kawagucci, K. Nakamura, J. Glass, and A. Kraepiel for fruitful discussion. Comments by T. McCollom and two anonymous reviewers greatly improved the manuscript. This research was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (22540499 to M.N.).
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