Akiko Makabe

Microbial denitrification dominates nitrate losses from forest ecosystems

Significance Nitrogen (N) losses from terrestrial ecosystems can occur as inert forms or heat-trapping greenhouse gases, and via nitrate (NO3−) leaching to drainage waters, which can contribute to eutrophication and anoxia in downstream ecosystems. Here, we use natural isotopes to demonstrate that microbial gaseous N production via denitrification is the dominant pathway of NO3− removal from forest ecosystems, with gaseous N losses that are up to ∼60-fold higher than those based on traditional techniques. Denitrification becomes less efficient compared with NO3− leaching in more N-polluted ecosystems, which has important implications for assessing the connections between terrestrial soils and downstream ecosystems under rising anthropogenic N deposition. Denitrification removes fixed nitrogen (N) from the biosphere, thereby restricting the availability of this key limiting nutrient for terrestrial plant productivity. This microbially driven process has been exceedingly difficult to measure, however, given the large background of nitrogen gas (N2) in the atmosphere and vexing scaling issues associated with heterogeneous soil systems. Here, we use natural abundance of N and oxygen isotopes in nitrate (NO3−) to examine dentrification rates across six forest sites in southern China and central Japan, which span temperate to tropical climates, as well as various stand ages and N deposition regimes. Our multiple stable isotope approach across soil to watershed scales shows that traditional techniques underestimate terrestrial denitrification fluxes by up to 98%, with annual losses of 5.6–30.1 kg of N per hectare via this gaseous pathway. These N export fluxes are up to sixfold higher than NO3− leaching, pointing to widespread dominance of denitrification in removing NO3− from forest ecosystems across a range of conditions. Further, we report that the loss of NO3− to denitrification decreased in comparison to leaching pathways in sites with the highest rates of anthropogenic N deposition.

Hadal biosphere: Insight into the microbial ecosystem in the deepest ocean on Earth

Significance Although many microbial explorations for hadal sediments began in the 1950s, the hadal water is the least-explored microbial biosphere. In this study, unexpected microbial ecosystems associated with the hadal trench water were discovered down to a 10,257-m water depth in the Challenger Deep of the Mariana Trench, which is the deepest ocean on Earth. We found the enrichment of heterotrophic population in the hadal water (6,000 ∼10,257 m) microbial communities, whereas the chemolithotrophic populations were more abundant in the upper abyssal waters. This observation suggested that the hadal microbial biosphere was supported by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology. Hadal oceans at water depths below 6,000 m are the least-explored aquatic biosphere. The Challenger Deep, located in the western equatorial Pacific, with a water depth of ∼11 km, is the deepest ocean on Earth. Microbial communities associated with waters from the sea surface to the trench bottom (0 ∼10,257 m) in the Challenger Deep were analyzed, and unprecedented trench microbial communities were identified in the hadal waters (6,000 ∼10,257 m) that were distinct from the abyssal microbial communities. The potentially chemolithotrophic populations were less abundant in the hadal water than those in the upper abyssal waters. The emerging members of chemolithotrophic nitrifiers in the hadal water that likely adapt to the higher flux of electron donors were also different from those in the abyssal waters that adapt to the lower flux of electron donors. Species-level niche separation in most of the dominant taxa was also found between the hadal and abyssal microbial communities. Considering the geomorphology and the isolated hydrotopographical nature of the Mariana Trench, we hypothesized that the distinct hadal microbial ecosystem was driven by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology.

Low δ 18 O Values of Nitrate Produced from Nitrification in Temperate Forest Soils

Analyses of δ(18)O of nitrate (NO(3)(-)) have been widely used in partitioning NO(3)(-) sources. However the δ(18)O value of NO(3)(-) produced from nitrification (microbial NO(3)(-)) is commonly estimated using the δ(18)O of environmental water and molecular oxygen in a 2:1 ratio. Here our laboratory incubation of nine temperate forest soils across a 1500 m elevation gradient demonstrates that microbial NO(3)(-) has lower δ(18)O values than the predicted using the 2:1 ratio (by 5.2-9.5‰ at low elevation sites), in contrast to previous reports showing higher δ(18)O values (up to +15‰) than their predicted values. Elevated δ(18)O values of microbial NO(3)(-) were observed at high elevation sites where soil was more acidic, perhaps due to accelerated O-exchange between nitrite, an intermediate product of nitrification, and water. Lower δ(18)O of microbial NO(3)(-) than the predicted and from previous observations suggests that the contribution of anthropogenic N inputs, such as fertilizer and atmospheric deposition, to a given ecosystem and the progress of denitrification in nitrogen removal are greater than we know. More than half of the δ(18)O of stream NO(3)(-) lower than the predicted value along the elevation gradient also indicate the impropriety using the 2:1 ratio for differentiating NO(3)(-) sources.

Ammonium first: natural mosses prefer atmospheric ammonium but vary utilization of dissolved organic nitrogen depending on habitat and nitrogen deposition

Mosses, among all types of terrestrial vegetation, are excellent scavengers of anthropogenic nitrogen (N), but their utilization of dissolved organic N (DON) and their reliance on atmospheric N remain uncharacterized in natural environments, which obscures their roles in N cycles. Natural (15) N abundance of N sources (nitrate (NO(3)(-)), ammonium (NH(4)(+)) and DON in deposition and soil) for epilithic and terricolous mosses was analyzed at sites with different N depositions at Guiyang, China. Moss NO(3)(-) assimilation was inhibited substantially by the high supply of NH(4)(+) and DON. Therefore, contributions of NH(4)(+) and DON to moss N were partitioned using isotopic mass-balance methods. The N contributions averaged 56% and 46% from atmospheric NH(4)(+), and 44% and 17% from atmospheric DON in epilithic and terricolous mosses, respectively. In terricolous mosses, soil NH(4)(+) and soil DON accounted for 16% and 21% of bulk N, which are higher than current estimations obtained using (15) N-labeling methods. Moreover, anthropogenic NH(4)(+) deposition suppressed utilization of DON and soil N because of the preference of moss for NH(4)(+) under elevated NH(4)(+) deposition. These results underscore the dominance of, and preference for, atmospheric NH(4)(+) in moss N utilization, and highlight the importance of considering DON and soil N sources when estimating moss N sequestration and the impacts of N deposition on mosses.

Evaluation of wastewater nitrogen transformation in a natural wetland (Ulaanbaatar, Mongolia) using dual-isotope analysis of nitrate.

The Tuul River, which provides water for the daily needs of many residents of Ulaanbaatar, Mongolia, has been increasingly polluted by wastewater from the citys sewage treatment plant. Information on water movement and the transformation of water-borne materials is required to alleviate the deterioration of water quality. We conducted a synoptic survey of general water movement, water quality including inorganic nitrogen concentrations, and isotopic composition of nitrogen (δ(15)N-NO(3)(-), δ(18)O-NO(3)(-), and δ(15)N-NH(4)(+)) and water (δ(18)O-H(2)O) in a wetland area that receives wastewater before it enters the Tuul River. We sampled surface water, groundwater, and spring water along the two major water routes in the wetland that flow from the drain of the sewage treatment plant to the Tuul River: a continuous tributary and a discontinuous tributary. The continuous tributary had high ammonium (NH(4)(+)) concentrations and nearly stable δ(15)N-NH(4)(+), δ(15)N-NO(3)(-), and δ(18)O-NO(3)(-) concentrations throughout its length, indicating that nitrogen transformation (i.e., nitrification and denitrification) during transit was small. In contrast, NH(4)(+) concentrations decreased along the discontinuous tributary and nitrate (NO(3)(-)) concentrations were low at many points. Values of δ(15)N-NH(4)(+), δ(15)N-NO(3)(-), and δ(18)O-NO(3)(-) increased with flow along the discontinuous route. Our results indicate that nitrification and denitrification contribute to nitrogen removal in the wetland area along the discontinuous tributary with slow water transport. Differences in hydrological pathways and the velocity of wastewater transport through the wetland area greatly affect the extent of nitrogen removal.

Nitrogen isotope ratios of nitrate and N* anomalies in the subtropical South Pacific

Nitrogen isotopic ratios of nitrate (δ15N– NO3−) were analyzed above 1000 m water depth along 17°S in the subtropical South Pacific during the revisit WOCE P21 cruise in 2009. The δ15N– NO3− and N* values were as high as 17‰ and as low as −18 μmol N L−1, respectively, at depths around 250 m east of 115°W, but were as low as 5‰ and as high as +1 μmol N L−1, respectively, in subsurface waters west of 170°W. The relationships among NO3− concentrations, N* values, δ15N– NO3− values, and oxygen and nitrite concentrations suggest that a few samples east of 90°W were from suboxic and nitrite-accumulated conditions and were possibly affected by in situ water column denitrification. Most of the high-δ15N– NO3− and negative-N* waters were probably generated by mixing between Subantarctic Mode Water from the Southern Ocean and Oxygen Deficit Zone Water from the eastern tropical South Pacific, with remineralization of organic matter occurring during transportation. Moreover, the relationship between δ15N– NO3− and N* values, as well as Trichodesmium abundances and size-specific nitrogen fixation rates at the surface, suggest that the low-δ15N– NO3− and positive-N* subsurface waters between 160°E and 170°W were generated by the input of remineralized particles created by in situ nitrogen fixation, mainly by Trichodesmium spp. Therefore, the δ15N values of sediments in this region are expected to reveal past changes in nitrogen fixation or denitrification rates in the subtropical South Pacific.

Patterns of foliar δ15N and their control in Eastern Asian forests

Foliar δ15N has been used increasingly in research on ecosystem nitrogen (N) cycling, because it can serve as an integrator of ecosystem N cycling and thus has a potential to reveal temporal and spatial patterns of N cycling as well as how the N cycle is altered by disturbances. However, the current understanding on controls of foliar δ15N is based principally on studies from America, Europe, Australia and Africa. Here we compiled data from 65 forests at 33 sites across East Asia to explore regional patterns and what controls foliar δ15N by linking it to climate, species composition, soil depth, slope position, N deposition, and soil N availability. In East Asia, foliar δ15N ranged from −7.1 to +2.7‰. Mean foliar δ15N values for tropical, subtropical and temperate forests were all −3.1‰, which was unexpected. The patterns of foliar δ15N with precipitation, temperature and altitude were not clear. The variation in foliar δ15N among species and between different slope positions appeared to be small within a given forest. The δ15N for both bulk soil N and extractable inorganic N generally increased with soil depth as expected, strengthening the idea that deep-rooted trees may have access to 15N-enriched N. Different from the positive correlations reported across America and Europe, in East Asia we found that foliar δ15N decreased with increasing N deposition and did not relate to soil N availability. These discrepancies deserve more research to elucidate the mechanisms by which foliar δ15N is affected by ecosystem N availability at a regional scale.

A Novel Pyrroloquinoline Quinone-Dependent 2-Keto-d-Glucose Dehydrogenase from Pseudomonas aureofaciens

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream

ABSTRACT Ammonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in the in situ quantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ15NNO2− and δ18ONO2−, respectively) are geochemical tracers for evaluating the sources and the in situ rate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilic Thaumarchaeota populations composed almost entirely of “Candidatus Nitrosocaldus.” The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ18O value of nitrite produced from ammonia oxidation varied with the δ18O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilic Thaumarchaeota could be estimated using δ18ONO2− in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments. IMPORTANCE Because ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilic Thaumarchaeota and elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

WHATS-3: An improved flow-through multi-bottle fluid sampler for deep-sea geofluid research

Deep-sea geofluid systems, such as hydrothermal vents and cold seeps, are key to understanding subseafloor environments of Earth. Fluid chemistry, especially, provides crucial information towards elucidating the physical, chemical and biological processes that occur in these ecosystems. To accurately assess fluid and gas properties of deep-sea geofluids, well-designed pressure-tight fluid samplers are indispensable and as such they are important assets of deep-sea geofluid research. Here, the development of a new flow-through, pressure-tight fluid sampler capable of four independent sampling events (two subsamples for liquid and gas analyses from each) is reported. This new sampler, named WHATS-3, is a new addition to the WHATS-series samplers and a major upgrade from the previous WHATS-2 sampler with improvements in sample number, valve operational time, physical robustness, and ease of maintenance. Routine laboratory-based pressure tests proved that it is suitable for operation up to 35 MPa pressure. Successful field tests of the new sampler were also carried out in five hydrothermal fields, two in Indian Ocean and three in Okinawa Trough (max. depth 3,300 m). Relations of Mg and major ion species demonstrated bimodal mixing trends between a hydrothermal fluid and seawater, confirming the high-quality of fluids sampled. The newly developed WHATS-3 sampler is well-balanced in sampling capability, field usability, and maintenance feasibility, and can serve as one of the best geofluid samplers available at present to conduct efficient research of deep-sea geofluid systems.