Natural Abundance Stable Isotope Research Articles

Mode of carbon gain and fungal associations of Neuwiedia malipoensis within the evolutionarily early-diverging orchid subfamily Apostasioideae.

The earliest-diverging orchid lineage, Apostasioideae, consists only of two genera: Apostasia and Neuwiedia. Previous reports of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages. We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundances (δ13C, δ15N, δ2H, δ18O) of different developmental stages with those of autotrophic reference plants. Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and the 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults were predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition. Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon. The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows various habitat options in the earliest-diverging orchid lineage, Apostasioideae. While A. nipponica relies on the heterotrophic carbon gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic carbon gain as an adult, allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur.

Soil carbon stabilization of mining-degraded, reforested lands in southern Ontario

We took advantage of the northern forest near Sudbury, Ontario, Canada where logging and metal-mining pollution had degraded the landscape prior to it being restored and reforested to investigate how soil organic matter is stabilized with stand age in highly ‘eroded’ sites with minimal residual soil and in ‘stable’ sites with residual soil. Soil organic matter in sandy, acidic soils of the northern forest is thought to be stabilized in organo-mineral particles (<20 μm in size); however, forest disturbance and soil erosion might help to promote less-decomposed plant detritus being stabilized within aggregate structures (53 μm in size). We use a combination of physical fractionation (size) and chemical analysis (carbon to nitrogen ratio, natural abundance of stable isotopes of carbon and nitrogen) of the fractions. The composition of bulk soils with young trees (15-yr old) was 25% microaggregates (53 to 250 μm in size) and 47% in a smaller silt + clay fraction (<53-μm in size). Bulk soil with older trees (28- and 30- to 32-yr old) had equal proportions of macroaggregates (>250 μm in size), microaggregates, and silt + clay fraction. The C density was much greater and less decomposed for macroaggregates than for the silt + clay fraction. Untreated sites, with trees but not restored with added lime, had a smaller proportion of macroaggregates in bulk soil. Even though aggregate structures typically are not associated with sandy, acidic forest soils, the results for these Regosols in the northern forest of the Greater Sudbury region show that stabilized soil C involves formation of macroaggregates along with microaggregates, both of which help to stabilize particulate organic matter.

Linking Water Age, Nitrate Export Regime, and Nitrate Isotope Biogeochemistry in a Tile‐Drained Agricultural Field

AbstractAccurately quantifying and predicting the reactive transport of nitrate () in hydrologic systems continues to be a challenge, due to the complex hydrological and biogeochemical interactions that underlie this transport. Recent advances related to time‐variant water age have led to a new method that probes water mixing and selection behaviors using StorAge Selection (SAS) functions. In this study, SAS functions were applied to investigate storage, water selection behaviors, and export regimes in a tile‐drained corn‐soybean field. The natural abundance stable nitrogen and oxygen isotopes of tile drainage were also measured to provide constraints on biogeochemical transformations. The SAS functions, calibrated using chloride measurements at tile drain outlets, revealed a strong young water preference during tile discharge generation. The use of a time‐variant SAS function for tile discharge generated unique water age dynamics that reveal an inverse storage effect driven by the activation of preferential flow paths and mechanically explain the observed variations in isotopes. Combining the water age estimates with isotope fingerprinting shed new light on export dynamics at the tile‐drain scale, where a large mixing volume and the lack of a strong vertical contrast in concentration resulted in chemostatic export regimes. For the first time, isotopes were embedded into a water age‐based transport model to model reactive transport under transient conditions. The results of this modeling study provided a proof‐of‐concept for the potential of coupling water age modeling with isotope analysis to elucidate the mechanisms driving reactive transport.

Water sources for street trees in mesic urban environments

Street trees support climate resiliency through a variety of pathways, such as offsetting urban heat and attenuating storm water runoff. While urban trees in arid and semiarid ecosystems have been shown to take up water from irrigation, it is unknown where street trees in mesic cities obtain their water. In this study, we use natural abundance stable isotopes to estimate the proportional sources of water taken up by Acer platanoides street trees in Boston, Massachusetts, United States, including precipitation, irrigation, groundwater, and wastewater. We use Bayesian multisource mixing models to estimate water sources by comparing the natural abundance isotopic ratios of hydrogen and oxygen across potential water sources with water extracted from tree stem samples. We find that during the summer of 2021, characterized by anomalously high rainfall, street trees predominantly utilized water from precipitation. Precipitation accounted for 72.3 % of water extracted from trees sampled in August and 65.6 % from trees sampled in September. Of the precipitation taken up by street trees, most water was traced back to large storm events in July, with July rainfall alone accounting for up to 84.0 % of water found within street trees. We find strong relationships between canopy cover fractions and the proportion of precipitation lost to evapotranspiration across the study domain, supporting the conclusion that tree planting initiatives result in storm water mitigation benefits due to utilization of water from precipitation by urban vegetation. However, while the mature trees studied here currently support their water demand from precipitation, the dependency of street trees on precipitation in mesic cities may lead to increased water stress in a changing climate characterized by a higher frequency and severity of drought.

Sewer subsidies from overflows and pipe leaks dominate urban stream solute loads in all storm events

Stream channel burial drastically alters watershed flowpaths by routing surface waters underground and increasing the potential for interactions between stream water and urban infrastructure such as storm and sanitary sewers. While numerous studies have investigated storm event solute loads from urban watersheds, the influences of stream channel burial and sewer overflows are often overlooked. This study uses grab samples and natural abundance stable isotope tracers to quantify the event dynamics of solute concentration-discharge relationships as well as cumulative loads in a buried urban stream. Our results demonstrate that different solutes, as well as different sources of the same solute (atmospheric NO3− and sewer-derived NO3− differentiated by the Δ17O tracer), are delivered via separate watershed flowpaths and thus have different timings within the event and contrasting relationships to flow. This inter-event variability reveals dynamics that result from temporal and spatial heterogeneity in infiltration, exfiltration, and pipe overflows. These results can help guide system-wide infrastructure maintenance as cities seek to meet challenges in sustaining and improving water quality as infrastructural systems age.

The natural abundance of stable water isotopes method may overestimate deep-layer soil water use by trees

Abstract. Stable water isotopes have been used extensively to study the water use strategy of plants in various ecosystems. In deep vadose zone (DVZ) regions, the rooting depth of trees can reach several meters to tens of meters. However, the existence of roots in deep soils does not necessarily mean the occurrence of root water uptake, which usually occurs at a particular time during the growing season. Therefore, quantifying the contribution of deep-layer soil water (DLSW) in DVZ regions using the natural abundance of stable water isotopes may not be accurate because this method assumes that trees always extract shallow- and deep-layer soil water. We propose a multi-step method for addressing this issue. First, isotopic labeling in deep layers identifies whether trees absorb DLSW and determines the soil layer depths from which trees derive their water source. Next, we calculate water sources based on the natural abundance of stable isotopes in the soil layer determined above to quantify the water use strategy of trees. We also compared the results with the natural abundance of stable water isotopes method. The 11- and 17-year-old apple trees were taken as examples for analyses on China's Loess Plateau. Isotopic labeling showed that the water uptake depth of 11-year-old apple trees reached 300 cm in the blossom and young fruit (BYF) stage and only 100 cm in the fruit swelling (FSW) stage, whereas 17-year-old trees always consumed water from the 0–320 cm soil layer. Overall, apple trees absorbed the most water from deep soils (&gt;140 cm) during the BYF stage, and 17-year-old trees consumed more water in these layers than 11-year-old trees throughout the growing season. In addition, the natural abundance of stable water isotopes method overestimated the contribution of DLSW, especially in the 320–500 cm soil layer. Our findings highlight that determining the occurrence of root water uptake in deep soils helps to quantify the water use strategy of trees in DVZ regions.

Mineralogical control on physically protected soil organic matter in a neotropical moist forest

Context Minerals and organic matter physically associate in many soils, yet the precise nature, either via a hierarchy of aggregate particles or by build-up of organo–mineral associations, remains obscure, especially in tropical forest environments. Aims We investigated physically protected organic matter by comparing soils with contrasting parent material, topography, and pedogenesis, but with similar tropical moist forest on Barro Colorado Island in the lowlands of Panama. Methods Bulk soil from 10 sites was separated by size into free-floating particles, macroaggregates (&gt;250 μm), microaggregates (53–250 μm), and &lt;53-μm particles. A subsample of macroaggregates was disintegrated and separated into coarse particles (&gt;250 μm), occluded microaggregates, and occluded &lt;53-μm particles. Concentrations and natural abundance of stable isotopes for carbon (C) and nitrogen (N) were determined for each fraction, and ratios (C:N, stable isotopes) were used to characterise organic matter for each fraction. Key results Macroaggregates were the largest fraction of bulk soil (71%) and were 15% greater in kaolinite- than smectite-dominated soils. Macroaggregates were composed of coarse particles (14%), occluded microaggregates (62%), and occluded &lt;53-μm particles (24%). Concentrations of C and N widely varied among fractions but the variation was not related to clay mineralogy. The C:N ratio and stable N isotope ratio indicated more decomposed organic matter in kaolinite- than smectite-dominated soils. Conclusions and implications Macroaggregates composed of plant detritus and microaggregates in the Barro Colorado Island soils imply that the aggregate hierarchy route ultimately protects soil organic matter in this tropical forest environment.

Relationships between stable isotope natural abundances (δ13C and δ15N) and water use efficiency in rice under alternate wetting and drying irrigation in soils with high clay contents.

Natural abundance of the stable isotope (δ13C and δ15N) in plants is widely used to indicate water use efficiency (WUE). However, soil water and texture properties may affect this relationship, which remains largely elusive. Therefore, the purpose of this study was to evaluate δ13C as affected by different combinations of alternate wetting and drying irrigation (AWD) with varied soil clay contents in different organs and whole plant and assess the feasibility of using δ13C and δ15N as a physiological indicator of whole-plant water use efficiency (WUEwhole-plant). Three AWD regimes, I100 (30 mm flooded when soil reached 100% saturation), I90 (30 mm flooded when reached 90% saturation) and I70 (30 mm flooded when reached 70% saturation) and three soil clay contents, 40% (S40), 50% (S50), and 60% (S60), were included. Observed variations in WUEwhole-plant did not conform to theoretical expectations of the organs δ13C (δ13Corgans) of plant biomass based on pooled data from all treatments. However, a positive relationship between δ13Cleaf and WUEET (dry biomass/evapotranspiration) was observed under I90 regime, whereas there were no significant relationships between δ13Corgans and WUEET under I100 or I70 regimes. Under I100, weak relationships between δ13Corgans and WUEET could be explained by (i) variation in C allocation patterns under different clay content, and (ii) relatively higher rate of panicle water loss, which was independent of stomatal regulation and photosynthesis. Under I70, weak relationships between δ13Corgans and WUEET could be ascribed to (i) bigger cracks induced by water-limited irrigation regime and high clay content soil, and (ii) damage caused by severe drought. In addition, a negative relationship was observed between WUEwhole-plant and shoot δ15N (δ15Nshoot) across the three irrigation treatments, indicating that WUEwhole-plant is tightly associated with N metabolism and N isotope discrimination in rice. Therefore, δ13C should be used cautiously as an indicator of rice WUEwhole-plant at different AWD regimes with high clay content, whereas δ15N could be considered an effective indicator of WUEwhole-plant.

Quantifying Isotopologue Reaction Networks (QIRN): A modelling tool for predicting stable isotope fractionations in complex networks

Natural-abundance stable isotope compositions are powerful tools for understanding complex processes across myriad scientific disciplines. However, quantitative interpretation of these signals often requires equally complex models. Previous stable isotope models have treated isotopic compositions as intrinsic properties of molecules or atoms (e.g. δ13C, 13R, etc.). This has proven to be a computationally efficient but inflexible approach. Here, we present a new isotope modelling software tool that combines computational strategies used in metabolic modeling with an understanding of natural isotope fractionations from the geosciences, called Quantifying Isotopologue Reaction Networks (QIRN, “churn”). QIRN treats isotopic properties as distributions of discrete isotopologues, i.e. molecules with different numbers and distributions of isotopic substitutions. This approach is remarkably generalizable and computationally tractable, enabling models of reaction networks with unprecedented complexity. QIRN parameterizes reactions as rate law equations with distinct isotopologues as the reactants and products. Isotope effects are implemented as small changes to the relevant isotopologues’ rate constants. Running this model forward in time gives the numerical solution for steady state isotopologue abundances. Different subsets of the isotopologue population can then be sampled to quantify numerous isotopic proprieties simultaneously (i.e. compound-specific, site-specific, and multiply-substituted isotope compositions). Furthermore, QIRN can model any physical, chemical or biological process as reversible or irreversible. As such, it incorporates both kinetic and equilibrium isotope effects. It can be readily applied to any isotope system (i.e. C, N, O, etc.), though at present can only track two isotopes of one element at a time. Given its generalizability, QIRN has a diverse range of applications. To demonstrate the flexibility and efficiency of QIRN, we reconstructed previous (intrinsic-property) models of sulfate reduction, abiotic amino acid synthesis, lipid biosynthesis, and photosynthesis. In these examples, QIRN consistently reproduced outputs from prior models and predicted isotopic anomalies that have been measured in nature. With its new approach to isotope modelling, QIRN will expand the potential complexity of modelled reaction networks, help predict isotopic signals that can direct experimental efforts, and provide a more efficient means of modeling emerging isotopic properties such as ‘clumped isotopes’.

Stable isotope mixing models demonstrate the role of an invasive plant in wetland songbird food webs.

PremiseInvasive plants in wetlands are often ecosystem engineers, mediating changes in ecosystem functions like trophic support. We documented the impacts of Lepidium latifolium, an invasive plant, on the food web of omnivorous birds (Suisun song sparrows, Melospiza melodia maxillaris) in a tidal wetland of northern California, USA.MethodsWe used analysis of natural abundance stable isotopes of 13C and 15N in song sparrow blood, invertebrate food sources, L. latifolium seeds, and other marsh plant seeds to inform Bayesian, concentration‐dependent mixing models that predicted average song sparrow diets.ResultsSeason and plant phenology influenced food source incorporation and isotopic signatures. Song sparrows showed higher isotopic variability in the summer. The observed changes in song sparrow diets were driven by altered invertebrate communities related to seasonal L. latifolium presence and by shifts from seeds to consumption of invertebrate food sources during the breeding season in the spring and summer.DiscussionThis study used stable isotope tools and modeling to demonstrate two mechanisms of isotopic influence by L. latifolium on omnivorous song sparrows. This study can inform site‐ and species‐specific management strategies by demonstrating how changes to the plant community can impact entire trophic systems.

Using the natural abundance of nitrogen isotopes to identify cattle with greater efficiency in protein-limiting diets

The difficulty in selecting cattle for higher feed and nitrogen use efficiency (NUE) is an important factor contributing to poor growth and reproductive performance in dry-tropics rangelands. Therefore, the objectives were to examine the cattle variation in retaining nitrogen in a protein-deficient diet and the natural abundance of stable isotopes in body tissues as a practical alternative for the detection of more efficient cattle. In experiment 1, feed efficiency parameters were determined in 89 Brahman steers fed a protein-limiting diet for 70 days, followed by 7 days in metabolism crates for total collection of urine and faeces and calculation of nitrogen retention and NUE. The diet-animal fractionation of nitrogen isotopes (Δ15N) was quantified in tail hair and plasma proteins using isotope-ratio MS. There was a large variation in growth performance, feed efficiency and nitrogen losses among steers. Quantifying Δ15N in tail hair (Δ15Ntail hair) resulted in stronger correlations with feed efficiency and nitrogen metabolism parameters than when quantified in plasma proteins. Δ15Ntail hair was positively correlated with nitrogen losses in urine (r = 0.31, P < 0.01) and faeces (r = 0.25, P = 0.04), leading to a negative correlation with NUE (r = −0.40, P < 0.01). The group of steers with lower Δ15Ntail hair had greater feed efficiency, lower nitrogen losses, and greater NUE. In experiment 2, for evaluation of isotope fraction as a predictor of reproductive performance, 630 Brahman-crossed cows were classified for reproductive performance for 2 years. From this group, 25 cows with poor reproductive performance and 25 cows with good reproductive performance were selected. Tail hair representing 7 months of growth were segmented and analysed for carbon (δ13C) and nitrogen (δ15N) isotope enrichment. Reproductive performance was not associated with diet selection, as there was no difference in tail hair δ13C between groups. However, more productive cows had lower (P < 0.05) tail hair δ15N during the dry season, indicating differences in N metabolism and possibly lower N losses. In addition, cows with better reproductive performance and, therefore, greater nutrient demands, had similar body condition scores and a tendency (P = 0.09) for higher live weight at the end of the trial. In conclusion, the findings of the present study confirm that nitrogen isotope fractionation in tail hair can be used as a predictor of nitrogen losses, NUE, and reproductive performance of Brahman cattle on low-protein diets.

Verifying the relationship between δ13C isotope profile variables and individual feed conversion ratio in large rainbow trout (Oncorhynchus mykiss)

The study aimed to verify the relationship found between individual indicator traits, derived from an isotope ratio of 13C/12C in various tissues (muscle, liver, and adipose), and individual feed efficiency. A 50-day experiment was performed with 46 rainbow trout (Oncorhynchus mykiss) kept in individual tanks after a dietary switch, using an experimental feed based on maize gluten with an enhanced level of 13C, during the grow-out phase. Individual growth, feed intake, and isotope profiles (muscle, liver, fin, and adipose tissue) were recorded. Using a Lasso-regression model, feed efficiency was found to be positively associated with visceral adipose tissue synthesis, meaning that feed efficiency was worsened when visceral adipose synthesis increased. The results indicated that deposition efficiency through lipid metabolism (δ13C in adipose tissue) was the most important determinant of feed efficiency of rainbow trout at this life stage. The results suggest that phenotyping of lipid deposition efficiency is possible through the use of feed ingredients with a natural abundance of stable isotopes.

Fungal association and root morphology shift stepwise during ontogenesis of orchid Cremastra appendiculata towards autotrophic nutrition.

The chlorophyllous, terrestrial orchid Cremastra appendiculata from East Asia is unique concerning its fungal mycorrhiza partners. The initially mycoheterotrophic protocorms exploit rather specialized non-rhizoctonia saprotrophic Psathyrellaceae. Adult individuals of this orchid species are either linked to Psathyrellaceae being partially mycoheterotrophic or form mycorrhiza with fungi of the ubiquitous saprotrophic rhizoctonia group. This study provides new insights on nutrition mode, subterranean morphology and fungal partners across different life stages of C. appendiculata. We compared different development stages of C. appendiculata to surrounding autotrophic reference plants based on multi-element natural abundance stable isotope analyses (δ13C, δ15N, δ2H, δ18O) and total N concentrations. Site- and sampling-time-independent enrichment factors of stable isotopes were used to reveal trophic strategies. We determined mycorrhizal fungi of C. appendiculata protocorm, seedling and adult samples using high-throughput DNA sequencing. We identified saprotrophic non-rhizoctonia Psathyrellaceae as dominant mycorrhizal fungi in protocorm and seedling rhizomes. In contrast, the roots of seedlings and mature C. appendiculata were mainly colonized with fungi belonging to the polyphyletic assembly of rhizoctonia (Ceratobasidium, Thanatephorus and Serendipitaceae). Mature C. appendiculata did not differ in isotopic signature from autotrophic reference plants suggesting a fully autotrophic nutrition mode. Characteristic of orchid specimens entirely relying on fungal nutrition, C. appendiculata protocorms were enriched in 15N, 13C and 2H compared to reference plants. Seedlings showed an intermediate isotopic signature, underpinning the differences in the fungal community depending on their subterranean morphology. In contrast to the suggestion that C. appendiculata is a partially mycoheterotrophic orchid species, we provide novel evidence that mature C. appendiculata with rhizoctonia mycobionts can be entirely autotrophic. Besides an environmentally driven variability among populations, we suggest high within-individual flexibility in nutrition and mycobionts of C. appendiculata, which is subject to the ontogenetic development stage.

Distinguishing carbon gains from photosynthesis and heterotrophy in C3-hemiparasite-C3-host pairs.

Previous carbon stable isotope (13C) analyses have shown for very few C3-hemiparasites utilizing C4- or CAM-hosts the use of two carbon sources, autotrophy and heterotrophy. This 13C approach, however, failed for the frequently occurring C3-C3 parasite-host pairs. Thus, we used hydrogen stable isotope (2H) natural abundances as a substitute for 13C within a C3-Orobanchaceae sequence graded by haustoria complexity and C3-Santalaceae. Parasitic plants and their real or potential host plants as references were collected in Central European lowland and alpine mountain meadows and forests. Parasitic plants included the xylem-feeding holoparasite Lathraea squamaria parasitizing on the same carbon nutrient source (xylem-transported organic carbon compounds) as potentially Pedicularis, Rhinanthus, Bartsia, Melampyrum and Euphrasia hemiparasites. Reference plants were used for an autotrophy-only isotope baseline. A multi-element stable isotope natural abundance approach was applied. Species-specific heterotrophic carbon gain ranging from 0 to 51 % was estimated by a 2H mixing-model. The sequence in heterotrophic carbon gain mostly met the morphological grading by haustoria complexity: Melampyrum- < Rhinanthus- < Pedicularis-type. Due to higher transpiration and lower water-use efficiency, depletion in 13C, 18O and 2H compared to C3-host plants should be expected for tissues of C3-hemiparasites. However, 2H is counterbalanced by transpiration (2H-depletion) and heterotrophy (2H-enrichment). Progressive 2H-enrichment can be used as a proxy to evaluate carbon gains from hosts.

Enhancing protein perdeuteration by experimental evolution of Escherichia coli K-12 for rapid growth in deuterium-based media.

Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild‐type laboratory strain of E. coli K‐12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi‐generation adaptive laboratory evolution‐based approach, we have isolated strains that show increased fitness in deuterium‐based growth media. Whole‐genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium‐based media. By screening a collection of single‐gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.

Stable Isotope Tracer Addition Reveals the Trophic Status of Benthic Infauna at an Intertidal Area Adjacent to a Seagrass Bed

Stable isotope tracer addition can enhance the isotopic differences of primary producers for a wider food-web resolution than the use of natural abundance stable isotopes (NASIs) alone, particularly in ecosystems where primary producers have similar NASI values. To investigate the food sources and the trophic status of benthic infauna in an intertidal area near a Halophila minor seagrass bed within inner Tai Tam Bay, Hong Kong, China, a 15N addition experiment was conducted, and the results were compared with those from NASI data. Only benthic microalgae (BMA) were labeled by applying 15N-enriched NH4Cl to the sediment daily for the first 7 days during a 21-day study. In contrast to the NASI results, Bayesian mixing models based on the isotope tracer experiment suggested a larger dietary contribution of BMA for nematode Daptonema sp. and copepods, whereas a higher reliance on phytoplankton and seagrass detritus was noted for polychaete Neanthes sp. However, both NASI and isotope tracer addition demonstrated that seagrass detritus was a major food source for nematode Spilophorella sp. The present isotope tracer experiment also revealed a contrasting result of the relatively lower contribution of meiofauna in the diets of carnivores/omnivores as compared to the results of NASIs. This finding suggested that the isotope values in these consumers may have not reached an equilibrium with the added isotope in the study period. Thus, there is a need for applying NASI coupled with isotope tracer addition in the investigation of ecosystems in which primary producers have similar isotope values, especially in ecosystems with lower tissue turnover rates, in order for a more accurate determination of dietary contribution and trophic status of consumers in the food-web study.

Differences in the isotopic signature of activated sludge in four types of advanced treatment processes at a municipal wastewater treatment plant

The natural abundance of stable isotopes is a powerful tool for evaluating biological reactions and process conditions. However, there are few stable isotope studies on the wastewater treatment process. This study carried out the first investigation on variations in natural abundance of carbon and nitrogen stable isotope ratios (δ13C and δ15N) of activated sludge in four types of advanced treatment process (extended aeration activated sludge (EAAS), aerobic-anoxic-aerobic (A2O), recycled nitrification-denitrification (RND), and modified Bardenpho (MB)) at a municipal wastewater treatment plant. The δ13C and δ15N values of influent suspended solids settled in the primary sedimentation tank (i.e., primary sludge) ranged from −25.4‰ to −24.6‰ and 0.5‰–2.9‰, respectively, during monitoring periods. The δ13C values of the activated sludge were −24.6‰ to −23.6‰ (EAAS), −25.4‰ to −24.3‰ (A2O), −25.7‰ to −24.9‰ (RND), and −25.7‰ to −24.3‰ (MB). The δ13C values of the activated sludge were similar to those of influent suspended solids. However, the δ13C values of activated sludge in EAAS was significantly higher than in A2O, RND, and MB. Meanwhile, the δ15N values of activated sludge were obviously higher than influent suspended solids; 5.8‰–7.5‰ (EAAS), 6.6‰–8.1‰ (A2O), 5.5‰–7.5‰ (RND), and 5.3‰–7.6‰ (MB). Changes in δ13C and δ15N values of the activated sludge within the treatment system were also found. These findings indicate that changes in δ13C and δ15N values of the activated sludge rely on important function for biological wastewater treatment such as nitrification, denitrification, and methane oxidation through wastewater treatment over time.

Natural attenuation of large anthropogenic nitrate loads in a subtropical stream revealed by δ15N and δ18O

Nitrogen pollution in subtropical waters is rapidly increasing due to land-use change, but specific sources, transformations, and attenuation rates remain understudied compared to cooler temperate catchments. Here, we quantify high-resolution nitrate (NO3−-N) loads, sources and natural attenuation in a subtropical creek in Australia over contrasting hydrological conditions. We observed large creek NO3−-N loads (ranging from 44 to 2938 μmol m−2 catchment d−1) exceeding the bottom-up estimates of nitrogen input to the catchment at the most upstream sites. Stable isotope natural abundances (δ15N and δ18O in NO3−-N) and Bayesian analysis revealed that greywater was the dominant source accounting for ~55% of NO3−-N in the upper creek, but fertilisers (~29%) and rainfall (~16%) were also relevant NO3−-N sources. NO3−-N loads at the most downstream site were only 0.2–9.7% of loads at the most upstream site. The resulting NO3−-N attenuation efficiency (mainly via denitrification) was 52–84% of original upstream load per km of creek, depending on hydrological conditions. This large capacity to attenuate NO3−-N during dry and first-flush events exceeds the attenuation found in temperate creeks subject to several decades of pollution in the northern hemisphere. During periods of high water flow and saturated soils, high NO3−-N loads were exported downstream, turning the creek from a natural bioreactor to a system resembling a flow-through pipe. In spite of effective natural nitrogen attenuation providing a valuable ecosystem service, concentrations and loads remained well above expected for natural systems and water quality guidelines. Overall, our results highlight the need for modifying fertiliser use, capturing nitrogen on farms and reducing greywater nitrogen to prevent significant losses to subtropical waterways.

Diet Analysis of Hawai‘i Island's Blackburnia hawaiiensis (Coleoptera: Carabidae) Using Stable Isotopes and High-Throughput Sequencing1

Determining the diet of arthropods can be difficult due to their small size and complex food webs, especially in Hawai‘i, where knowledge of arthropod predator–prey interactions is sparse. The diet of the Hawai‘i Island-endemic carabid beetle, Blackburnia hawaiiensis Sharp (Coleoptera: Carabidae) is of particular interest because of its peculiar arboreal behavior and metathoracic flight wings. Our study objective was to determine the diet of B. hawaiiensis in replicated, geographically separated locations by using two different yet complementary laboratory techniques: natural abundance stable isotope analysis (SIA) and high-throughput sequencing (HTS). Overall, B. hawaiiensis had a greater average δ15N and similar δ13C compared to the other arthropods sampled in this study and HTS data revealed Diptera and Lepidoptera sequences in the beetle's gut contents. These results are consistent with B. hawaiiensis being classified as a generalist predator. The combination of SIA and HTS are important methods for determining the diet of species within complex food webs, particularly for species that are difficult to observe in nature.

氮稳定同位素自然丰度法在森林生态系统氮循环中的应用

氮稳定同位素自然丰度法在森林生态系统氮循环中的应用