Keisuke Koba

Intermittent denitrification: The application of a 15N natural abundance method to a forested ecosystem

Abstract The 15N natural abundance method was used to assess the intermittent occurrence of denitrification in the Kiryu watershed in Shiga Prefecture, Japan. The concentration and isotopic composition (° 15N) of NO3− −N, as well as some physical and chemical variables that potentially affect denitrification, were measured along the flow path from precipitation to stream water via soil solution and groundwater. High maximum groundwater level promoted a decrease in N03−−N concentration that was associated with an increase in the δ 15N of N03−−N with soil depth; i.e., denitrification occurred in the soil due to increasing soil moisture. While the maximum level of groundwater fell, no such changes were observed. Dissolved oxygen (DO) and Mn2+ concentrations in soil solutions indicated that strong anaerobic condition did not occur during the study period. These results suggested that denitrification was occurring temporarily in anaerobic microsites such as waterlogged soil aggregates. Upward expansion of groundwater zone thus appeared to play an important role in promoting such microanaerobic sites, resulting in the intermittent occurrence of denitrification. Based on these data, a schematic model for assessing denitrification was proposed based on a N03−−N/Cl− − δ 15S N of NO3−−N map for soil-water systems. Our data showed that variations of NO3−-N concentration and δ 15N value are useful indicators for elucidating nitrogen dynamics as affected by water mixing, plant uptake, nitrification, and denitrification in forested ecosystems.

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.

Natural 15 N Abundance of Plants and Soil N in a Temperate Coniferous Forest

Measurement of nitrogen isotopic composition (δ15N) of plants and soil nitrogen might allow the characteristics of N transformation in an ecosystem to be detected. We tested the measurement of δ15N for its ability to provide a picture of N dynamics at the ecosystem level by doing a simple comparison of δ15N between soil N pools and plants, and by using an existing model. δ15N of plants and soil N was measured together with foliar nitrate reductase activity (NRA) and the foliar NO3– pool at two sites with different nitrification rates in a temperature forest in Japan. δ15N of plants was similar to that of soil NO3– in the high-nitrification site. Because of high foliar NRA and the large foliar NO3– pool at this site, we concluded that plant δ15N indicated a great reliance of plants on soil NO3– there. However, many δ15N of soil N overlapped each other at the other site, and δ15N could not provide definitive evidence of the N source. The existing model was verified by measured δ15N of soil inorganic N and it explained the variations of plant δ15N between the two sites in the context of relative importance of nitrification, but more information about isotopic fractionations during plant N uptake is required for quantitative discussions about the plant N source. The model applied here can provide a basis to compare δ15N signatures from different ecosystems and to understand N dynamics.

Nitrogen Fixation in Surface Soils and Vegetation in an Arctic Tundra Watershed: A Key Source of Atmospheric Nitrogen

Abstract Atmospheric nitrogen (N) fixation is a key N input to arctic ecosystems, but relatively few estimates of annual N-fixation rates are available. We measured N-fixation of plant-soil cores by the acetylene reduction technique at different topographic positions in an upland tundra watershed, Imnavait Creek, through two growing seasons in order to evaluate spatial and temporal variation in N-fixation. We also examined the effects of light and temperature on N-fixation to estimate annual N-fixation rates of surface soil in this watershed using field meteorological data. Surface soil at Imnavait Creek had significant acetylene reduction potential throughout the watershed (generally 6 to 10 μmol C2H4 m−2 h−1), indicating that N-fixing organisms were present everywhere. Although acetylene reduction potential was roughly constant through the growing season, moisture, temperature and light intensity strongly affected the measured acetylene reduction rates in laboratory incubations. In addition, the relatively few samples that included the lichen, Peltigera apthosa, had significantly greater acetylene reduction potential, although the overall influence of Peltigera on N-fixation in this watershed seems to be small. The N input via N-fixation at Imnavait Creek was estimated at 80 to 131 mg N m−2 yr−1, indicating that N-fixation contributed 85 to 90% of total watershed N inputs.

Nitrogen and phosphorus enrichment and balance in forests colonized by cormorants: Implications of the influence of soil adsorption

Although much concern has been directed at nitrogen (N) cycling in terrestrial ecosystems with bird colonies, little has been clarified on the processes of phosphorus (P) cycling itself, and few comparisons between P and N cycling in bird colonies have been made. On the Isaki Headland and Chikubu Island, which are located on or near the shore of Lake Biwa, Central Japan, a dramatic increase in the population of cormorants has occurred since the 1980s. There has been a concomitant increase in the transport of nutrients from the lake to the waterside ecosystems. We compared the pools and dynamics of N and P in the cormorant-colony forests in order to clarify the effects of differences in soil N and P dynamics on the N–P balance of these colony forests. The total N concentration in the forest floor at excrement-influenced sites was not significantly different from that at sites without such influence, in spite of the heavy load of cormorant-derived N. In contrast to N, forest floor P concentration at the sites with excrement influence was significantly higher compared to sites without such influence, resulting in the lower forest floor N/P ratio at the excrement-influenced sites even after colony abandonment. The site pattern of total N and P concentrations and N/P ratio for mineral soil was similar to that for the forest floor. It seems that the leaky character for N and the accumulative character for P are due to the high mobility of nitrate in soils and the tight absorption of inorganic P to clay minerals, respectively. The site pattern of N/P ratios observed for Chamaecyparis obtusa Sieb. et Zucc. leaves is consistent with that for the forest floor and/or mineral soil, suggesting that the soil geochemical property was reflected in the foliar N/P ratio. The chemistry of throughfall and soil solution was also changed due to deposition of cormorant excrement, and the changes continued for a few years after abandonment of the colony. The quantitative analyses for N and P suggested that the major part of N and P transported by cormorants was not retained in plant matter and the surface soil beneath the colony but instead leached into deeper soil layers. The influence of cormorant excrement on nutrient balance of the whole colony ecosystem is also discussed.

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.

WATER UTILIZATION OF NATURAL AND PLANTED TREES IN THE SEMIARID DESERT OF INNER MONGOLIA, CHINA

We used stable isotope techniques to investigate water utilization of two native trees, Sabina vulgaris Ant. and Artemisia ordosica Krasch., and one introduced tree, Salix matsudana Koidz., in the semiarid Mu-Us desert, Inner Mongolia, China. The study site was in a region where there has been a decline in agricultural productivity, caused by severe desertification over the past several decades. S. matsudana is used extensively for reforestation to protect farms and cultivated lands from shifting sand dunes. We identified water sources for each tree species by comparing the stable isotopes δD and δ18O in water in stems, soil, and groundwater. We also measured δ13C levels in leaves to evaluate the intrinsic water-use efficiency (WUE) of each plant. Comparison of isotopes showed that S. vulgaris and S. matsudana consume relatively deep soil water as well as groundwater, whereas A. ordosica uses only shallow soil water. The δ13C measurements indicated that S. vulgaris has exclusively high WUE, whereas that of the other species was typical of temperate-region C3 plants. The water source data plus WUE data suggest that planted S. matsudana uses groundwater freely, whereas native plants conserve water. Thus, reforestation with S. matsudana might cause irreversible groundwater shortages. Corresponding Editor: E. A. Holland.

The natural abundance of 15N in plant and soil‐available N indicates a shift of main plant N resources to NO 3− from NH 4+ along the N leaching gradient

To investigate which of ammonium (NH(4)(+)) or nitrate (NO(3)(-)) is used by plants at gradient sites with different nitrogen (N) availability, we measured the natural abundance of (15)N in foliage and soil extractable N. Hinoki cypress (Chamaecyparis obtusa Endlicher) planted broadly in Japan was selected for use in this study. We estimated the source proportion of foliar N (NH(4)(+) vs. NO(3)(-)) quantitatively using mass balance equations. The results showed that C. obtusa used mainly NH(4)(+) in N-limited forests, although the dependence of C. obtusa on NO(3)(-) was greater in other NO(3)(-)-rich forests. We regarded dissolved organic N (DON) as a potential N source because a previous study demonstrated that C. obtusa can take up glycine. Thus we added DON to our mass balance equations and calculated the source proportion using an isotope-mixing model (IsoSource model). The results still showed a positive correlation between the calculated plant N proportion of NO(3)(-) and the NO(3)(-) pool size in the soil, indicating that high NO(3)(-) availability increases the reliance of C. obtusa on NO(3)(-). Our data suggest the shift of the N source for C. obtusa from NH(4)(+) to NO(3)(-) according to the relative availability of NO(3)(-). They also show the potential of the foliar delta(15)N of C. obtusa as an indicator of the N status in forest ecosystems with the help of the delta(15)N values of soil inorganic and organic N.

Abundance, diversity, and species composition of fungal communities in a temperate forest affected by excreta of the Great Cormorant Phalacrocorax carbo

The possible effects of excreta of the Great Cormorant Phalacrocorax carbo on abundance, diversity, and species composition of fungal communities were investigated in a temperate evergreen coniferous forest near Lake Biwa in central Japan. Samples were collected at three study sites that had the same vegetation composition, but which had been influenced by different stages of breeding colony establishment: Site C (control site), Site 2 (colonizing site), and Site 3 (post-colony site). In forest floor samples and in needles and twigs of Chamaecyparis obtusa, total hyphal length was lowest at Site 3, and clamp-bearing hyphal length (biomass of basidiomycetous fungi) was lower at Sites 2 and 3 than at Site C. Dark-pigmented hyphal length was highest at Site 2. Dilution plating of forest floor samples and mineral soil revealed: (i) species richness was higher at Sites 2 and 3 than at Site C, (ii) diversity was higher at Site 3 than at Sites C and 2, and that (iii) species composition differed among the sites. Surface sterilization of needles and twigs of C. obtusa revealed (i) with the exception of species richness in twigs, species richness and diversity were higher at Site 3 than at Sites C and 2, and that (ii) species composition differed markedly among the sites. In twig samples white rot Marasmius-like fungus and Geniculosporium sp. 1 were dominant at Site C and reduced at Sites 2 and 3. A coprophilous species, Sordaria sp. 1, showed a marked increase at Site 2 in needle and twig samples.

Carbon autonomy of reproductive shoots of Siberian alder (Alnus hirsuta var. sibirica)

Carbon autonomy of current-year shoots in flowering, and of current-year shoots plus 1-year-old shoots (1-year-old shoot system) in fruiting of Siberian alder (Alnus hirsuta var. sibirica) was investigated using a stable isotope of carbon, 13C. The current-year shoot and 1-year-old shoot systems were fed 13CO2 and the atom% excess of 13C in flowers and fruits was determined. The majority of photosynthate allocated to flower buds was originally assimilated in the leaves of the flowering current-year shoots. Of all the current-year shoots on fruiting 1-year-old shoots, only those nearest to the fruits allocated the assimilated photosynthate to fruit maturation. These results indicate that the current-year shoots and 1-year-old shoot systems are carbon-autonomous units for producing flowers and maturing fruits, respectively.