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We developed a new method for stable carbon and nitrogen isotopic (δ 13 C and δ 15 N) analysis of underivatized amino acid (AA) enantiomers simultaneously, based on high-performance liquid chromatography (HPLC) separation and off-line isotopic measurement. l- and d-Enantiomers of each AA were isolated using a ReproSil Chiral-AA column, purified by wet chemical procedure, and analyzed for δ 13 C and δ 15 N values with a nanomol-scale elemental analyzer/isotope-ratio mass spectrometry (nano-EA/IRMS) system. We successfully achieved the separation of l- and d-enantiomers of 15 proteinogenous AAs, with all l-enantiomers eluting before respective d-enantiomers. The δ 13 C and δ 15 N values of AA enantiomers were consistent before and after HPLC separation, demonstrating that this analytical method conserves isotopic information. By coupling this column with a multidimensional HPLC system for isolating individual AAs, we analyzed l- and d-AAs in a natural sample, peptidoglycan isolated from Gram-positive bacterium Bacillus subtilis . Results show a surprisingly large 15 N-depletion, up to 20‰, in d-glutamic acid relative to its l-counterpart. The first example, to our knowledge, of δ 13 C and δ 15 N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.

Returned samples from the carbonaceous asteroid (162173) Ryugu provide pristine information on the original aqueous alteration history of the Solar System. Secondary precipitates, such as carbonates and phyllosilicates, reveal elemental partitioning of the major component ions linked to the primordial brine composition of the asteroid. Here, we report on the elemental partitioning and Mg isotopic composition ( 25 Mg/ 24 Mg) of breunnerite [(Mg, Fe, Mn)CO 3 ] from the Ryugu C0002 sample and the A0106 and C0107 aggregates by sequential leaching extraction of salts, exchangeable ions, carbonates, and silicates. Breunnerite was the sample most enriched in light Mg isotopes, and the 25 Mg/ 24 Mg value of the fluid had shifted lower by ~0.38‰ than the initial value (set to 0‰) before dolomite precipitation. As a simple model, the Mg 2+ first precipitated in phyllosilicates, followed by dolomite precipitation, at which time ~76-87% of Mg 2+ had been removed from the primordial brine. A minor amount of phyllosilicate precipitation continued after dolomite precipitation. The element composition profiles of the latest solution that interacted with the cation exchange pool of Ryugu were predominantly Na-rich. Na + acts as a bulk electrolyte and contributes to the stabilization of the negative surface charge of phyllosilicates and organic matter on Ryugu., (© 2024. The Author(s).)

We report primordial aqueous alteration signatures in water-soluble organic molecules from the carbonaceous asteroid (162173) Ryugu by the Hayabusa2 spacecraft of JAXA. Newly identified low-molecular-weight hydroxy acids (HO-R-COOH) and dicarboxylic acids (HOOC-R-COOH), such as glycolic acid, lactic acid, glyceric acid, oxalic acid, and succinic acid, are predominant in samples from the two touchdown locations at Ryugu. The quantitative and qualitative profiles for the hydrophilic molecules between the two sampling locations shows similar trends within the order of ppb (parts per billion) to ppm (parts per million). A wide variety of structural isomers, including α- and β-hydroxy acids, are observed among the hydrophilic molecules. We also identify pyruvic acid and dihydroxy and tricarboxylic acids, which are biochemically important intermediates relevant to molecular evolution, such as the primordial TCA (tricarboxylic acid) cycle. Here, we find evidence that the asteroid Ryugu samples underwent substantial aqueous alteration, as revealed by the presence of malonic acid during keto-enol tautomerism in the dicarboxylic acid profile. The comprehensive data suggest the presence of a series for water-soluble organic molecules in the regolith of Ryugu and evidence of signatures in coevolutionary aqueous alteration between water and organics in this carbonaceous asteroid., (© 2024. The Author(s).)

Ammonia (NH 3 ) is a simple and essential nitrogen carrier in the universe. Its adsorption on mineral surfaces is an important step in the synthesis of nitrogenous organic molecules in extraterrestrial environments. The nitrogen isotopic ratios provide a useful tool for understanding the formation processes of N-bearing molecules. In this study, adsorption experiments were conducted using gaseous NH 3 and representative clay minerals. The strongly adsorbed NH 3 was 15 N-enriched in a state of chemical equilibrium between the adsorption and desorption on the siliceous host surface. The nitrogen K-edge X-ray adsorption near-edge structure spectroscopy study revealed that these initial ammonia gases were chemically adsorbed as ammonium ions (NH 4 + ) on clay minerals., (© 2024. The Author(s).)

Polycyclic aromatic hydrocarbons (PAHs) contain ≲20% of the carbon in the interstellar medium. They are potentially produced in circumstellar environments (at temperatures ≳1000 kelvin), by reactions within cold (~10 kelvin) interstellar clouds, or by processing of carbon-rich dust grains. We report isotopic properties of PAHs extracted from samples of the asteroid Ryugu and the meteorite Murchison. The doubly- 13 C substituted compositions (Δ2× 13 C values) of the PAHs naphthalene, fluoranthene, and pyrene are 9 to 51‰ higher than values expected for a stochastic distribution of isotopes. The Δ2× 13 C values are higher than expected if the PAHs formed in a circumstellar environment, but consistent with formation in the interstellar medium. By contrast, the PAHs phenanthrene and anthracene in Ryugu samples have Δ2× 13 C values consistent with formation by higher-temperature reactions.

Samples from the carbonaceous asteroid (162173) Ryugu provide information on the chemical evolution of organic molecules in the early solar system. Here we show the element partitioning of the major component ions by sequential extractions of salts, carbonates, and phyllosilicate-bearing fractions to reveal primordial brine composition of the primitive asteroid. Sodium is the dominant electrolyte of the salt fraction extract. Anions and NH 4 + are more abundant in the salt fraction than in the carbonate and phyllosilicate fractions, with molar concentrations in the order SO 4 2-  > Cl -  > NO 2 O 3 2-  > NO 3 -  > NH 4 + . The salt fraction extracts contain anionic soluble sulfur-bearing species such as S n -polythionic acids (n < 6), C n -alkylsulfonates, alkylthiosulfonates, hydroxyalkylsulfonates, and hydroxyalkylthiosulfonates (n < 7). The sulfur-bearing soluble compounds may have driven the molecular evolution of prebiotic organic material transforming simple organic molecules into hydrophilic, amphiphilic, and refractory S allotropes., (© 2023. Springer Nature Limited.)

Rationale: Isolation of underivatized amino acids (AAs) using high-performance liquid chromatography (HPLC) is becoming a popular method for carbon (δ 13 C) and nitrogen isotope (δ 15 N) analyses of AAs because of the high analytical precision and for performing dual-isotope analysis. However, some AAs in natural samples, especially small, hydrophilic AAs, are not suitably separated using reversed-phase columns (e.g., C18) and ion-exchange columns (e.g., Primesep A)., Methods: We developed a new method for HPLC using a porous graphitic carbon column for the separation of nine hydrophilic AAs. After purification, δ 13 C and δ 15 N values of AAs were determined using elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). We demonstrated the application of this method by determining δ 13 C and δ 15 N values of individual hydrophilic AAs in a biological sample, the muscle of blue mackerel (Scomber australasicus)., Results: Chromatographically, the baseline separation of hydrophilic AAs was achieved in both the standard mixture and the biological sample. We confirmed that δ 13 C and δ 15 N values of AA standards remained unchanged during the whole experimental procedure. The δ 13 C values of AAs in mackerel muscle are also in good agreement with the values obtained using another verified method for δ 13 C analysis., Conclusions: The good separation performance of hydrophilic AAs and the reliability of δ 13 C and δ 15 N analyses of individual AAs using the porous graphite column offer a significant advantage over conventional settings. We suggest that, in the future, the HPLC × EA/IRMS method can be used for reliable δ 13 C and δ 15 N analyses of AAs in natural samples., (© 2023 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Phagocytosis is one of the methods used to acquire symbiotic bacteria to establish intracellular symbiosis. A deep-sea mussel, Bathymodiolus japonicus , acquires its symbiont from the environment by phagocytosis of gill epithelial cells and receives nutrients from them. However, the manner by which mussels retain the symbiont without phagosome digestion remains unknown. Here, we show that controlling the mechanistic target of rapamycin complex 1 (mTORC1) in mussels leads to retaining symbionts in gill cells. The symbiont is essential for the host mussel nutrition; however, depleting the symbiont's energy source triggers the phagosome digestion of symbionts. Meanwhile, the inhibition of mTORC1 by rapamycin prevented the digestion of the resident symbionts and of the engulfed exogenous dead symbionts in gill cells. This indicates that mTORC1 promotes phagosome digestion of symbionts under reduced nutrient supply from the symbiont. The regulation mechanism of phagosome digestion by mTORC1 through nutrient signaling with symbionts is key for maintaining animal-microbe intracellular nutritional symbiosis.

Tailoring the surface of biodegradable microparticles is important for various applications in the fields of cosmetics, biotechnology, and drug delivery. Chitin nanofibers (ChNFs) are one of the promising materials for surface tailoring owing to its functionality, such as biocompatibility and antibiotic properties. Here, we show biodegradable polymer microparticles densely coated with ChNFs. Cellulose acetate (CA) was used as the core material in this study, and ChNF coating was successfully carried out via a one-pot aqueous process. The average particle size of the ChNF-coated CA microparticles was approximately 6 μm, and the coating procedure had little effect on the size or shape of the original CA microparticles. The ChNF-coated CA microparticles comprised 0.2-0.4 wt% of the thin surface ChNF layers. Owing to the surface cationic ChNFs, the ζ-potential value of the ChNF-coated microparticles was +27.4 mV. The surface ChNF layer efficiently adsorbed anionic dye molecules, and repeatable adsorption/desorption behavior was exhibited owing to the coating stability of the surface ChNFs. The ChNF coating in this study was a facile aqueous process and was applicable to CA-based materials of various sizes and shapes. This versatility will open new possibilities for future biodegradable polymer materials that satisfy the increasing demand for sustainable development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The pristine sample from the near-Earth carbonaceous asteroid (162173) Ryugu collected by the Hayabusa2 spacecraft enabled us to analyze the pristine extraterrestrial material without uncontrolled exposure to the Earth's atmosphere and biosphere. The initial analysis team for the soluble organic matter reported the detection of wide variety of organic molecules including racemic amino acids in the Ryugu samples. Here we report the detection of uracil, one of the four nucleobases in ribonucleic acid, in aqueous extracts from Ryugu samples. In addition, nicotinic acid (niacin, a B 3 vitamer), its derivatives, and imidazoles were detected in search for nitrogen heterocyclic molecules. The observed difference in the concentration of uracil between A0106 and C0107 may be related to the possible differences in the degree of alteration induced by energetic particles such as ultraviolet photons and cosmic rays. The present study strongly suggests that such molecules of prebiotic interest commonly formed in carbonaceous asteroids including Ryugu and were delivered to the early Earth., (© 2023. The Author(s).)

The Hayabusa2 spacecraft collected samples from the surface of the carbonaceous near-Earth asteroid (162173) Ryugu and brought them to Earth. The samples were expected to contain organic molecules, which record processes that occurred in the early Solar System. We analyzed organic molecules extracted from the Ryugu surface samples. We identified a variety of molecules containing the atoms CHNOS, formed by methylation, hydration, hydroxylation, and sulfurization reactions. Amino acids, aliphatic amines, carboxylic acids, polycyclic aromatic hydrocarbons, and nitrogen-heterocyclic compounds were detected, which had properties consistent with an abiotic origin. These compounds likely arose from an aqueous reaction on Ryugu's parent body and are similar to the organics in Ivuna-type meteorites. These molecules can survive on the surfaces of asteroids and be transported throughout the Solar System.

Abyssal plains cover more than half of Earth's surface, and the main food source in these ecosystems is phytodetritus, mainly originating from primary producers in the euphotic zone of the ocean. Global climate change is influencing phytoplankton abundance, productivity, and distribution. Increasing importance of picoplankton over diatom as primary producers in surface oceans (especially projected for higher latitudes) is projected and hence altering the quantity of organic carbon supplied to the abyssal seafloor as phytodetritus, consequences of which remain largely unknown. Here, we investigated the in situ responses of abyssal biota from viruses to megafauna to different types of phytoplankton input (diatoms or cyanobacteria which were labeled with stable isotopes) at equatorial (oligotrophic) and temperate (eutrophic) benthic sites in the Pacific Ocean (1°N at 4277 m water depth and 39°N at 5260 m water depth, respectively). Our results show that meiofauna and macrofauna generally preferred diatoms as a food source and played a relatively larger role in the consumption of phytodetritus at higher latitudes (39°N). Contrarily, prokaryotes and viruses showed similar or even stronger responses to cyanobacterial than to diatom supply. Moreover, the response of prokaryotes and viruses was very rapid (within 1-2 days) at both 1°N and 39°N, with quickest responses reported in the case of cyanobacterial supply at higher latitudes. Overall, our results suggest that benthic deep-sea eukaryotes will be negatively affected by the predicted decrease in diatoms in surface oceans, especially at higher latitudes, where benthic prokaryotes and viruses will otherwise likely increase their quantitative role and organic carbon cycling rates. In turn, such changes can contribute to decrease carbon transfer from phytodetritus to higher trophic levels, with strong potential to affect oceanic food webs, their biodiversity and consequently carbon sequestration capacity at the global scale., (© 2021 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Estimating stable isotopic offset values is crucial for dietary reconstructions. Although research into stable isotope ecology of wild nonhuman primates is increasing overall, only a minority of studies involve laboratory experiments. This study is the first to report the carbon and nitrogen stable isotopic offset values in hair and feces of orangutans. During an experiment lasting 1 week, the weight of each consumed food item was recorded for each of six captive Bornean orangutan (Pongo pygmaeus) individuals. The food, hair, and fecal samples were collected for a few days, and their stable carbon and nitrogen isotope ratios were measured using an elemental analyzer/isotope ratio mass spectrometer. Subsamples of feces were treated with ethanol during the preservation process. Monte Carlo analyses showed that the 95% confidence intervals (CIs) of the carbon and nitrogen offset values between hair and diet were +0.9‰ to +3.9‰ and +2.3‰ to +4.5‰, respectively. The 95% CIs of the carbon and nitrogen offset values between feces and diet were -3.7‰ to -0.9‰ and +0.3‰ to +2.7‰, respectively. The effect of ethanol treatment on the stable isotope ratios of feces was unclear and inconclusive. The computed offset values of hair in captive orangutans are similar to those reported in other nonhuman primates, although those of feces showed greater interspecies variations. The offset values estimated in this study contribute to isotopic studies into the feeding ecology of free-ranging orangutans who are critically endangered in most wild settings., (© 2021. Japan Monkey Centre.)

Rationale: To achieve better precision and accuracy for δ 13 C analysis of individual amino acids (AAs), we have developed a new analytical method based on multi-dimensional high-performance liquid chromatography (HPLC) and elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). Unlike conventional methods using gas chromatography, this approach omits pre-column chemical derivatization, thus reducing systematic errors associated with the isotopic measurement., Methods: The separation and isolation of individual AAs in a standard mixture containing 15 AAs and a biological sample, spear squid (Heterololigo bleekeri) were performed. AAs were isolated using an HPLC system equipped with a reversed-phase column and a mixed-mode column and collected using a fraction collector. After the chromatographic separation and further post-HPLC purification, the δ 13 C values of AAs were measured by EA/IRMS., Results: The complete isolation of all 15 AAs in the standard mixture was achieved. The δ 13 C values of these AAs before and after the experiment were in good agreement. Also, 15 AAs in the biological sample, H. bleekeri, were successfully measured. The δ 13 C values of AAs in H. bleekeri varied by as much as 30‰ with glycine being most enriched in 13 C., Conclusions: The consistency between the δ 13 C values of reference and processed AAs demonstrates that the experimental procedure generates accurate δ 13 C values unaffected by fractionation effects and contamination. This method is therefore suitable for δ 13 C analysis of biological samples with higher precision than conventional approaches. We propose this new method as a tool to measure δ 13 C values of AAs in biological, ecological and biogeochemical studies., (© 2020 John Wiley & Sons, Ltd.)

Heme B is an iron-coordinated tetrapyrrole molecule that acts as a cofactor in hemoproteins. It is expected to be ubiquitous in the environment, as b -type hemoproteins catalyze a variety of essential biochemical reactions. In this study, we developed an analytical method to quantify heme B in biological and environmental samples using high-performance liquid chromatography (HPLC) coupled to a photodiode array detector. The applicability of our method was further extended by the use of liquid chromatography/mass spectrometry (LC/MS; detection limit: ∼1 fmol), which enabled the quantification of a trace amount of dissolved heme B in filtered seawater and sedimentary heme B coexisting with an abundant interfering organic matrix. For compound-specific carbon and nitrogen isotopic measurements, heme B was successfully isolated and purified from biological and environmental samples by a combination of anion-exchange column chromatography, methyl esterification, and dual-step HPLC. While carbon and nitrogen isotopic compositions of heme B in phototrophs were mostly comparable to those of chlorophyll a , heme B in suspended particulate materials in coastal water and an intertidal sediment was 13 C-depleted and 15 N-enriched relative to chlorophyll a , suggesting that nonphototrophic microorganisms are also a significant source of heme B in natural environments.

The Aptian-Albian boundary is marked by one of the major oceanic perturbations during the Cretaceous, called Oceanic Anoxic Event (OAE) 1b. Extensive volcanic episodes at the Southern Kerguelen Plateau has been suggested as the trigger of OAE1b, but compelling evidence remains lacking. Here, we reconstructed the temporal variations of marine Os isotopic ratios across the Aptian-Albian boundary in the Tethyan and Pacific pelagic sedimentary records to elucidate the causal links between OAE1b, the biotic turnover, and volcanic episodes. Our new Os isotopic records show two negative spikes that correlate with a period of planktonic foraminiferal turnover across the Aptian-Albian boundary during OAE1b and suggest multiple submarine volcanic events. By comparing our Os isotopic profile with carbon isotopic compositions of carbonate, CaCO 3 content, and the relative abundances of agglutinated foraminifera, we conclude that ocean acidification caused by the massive release of CO 2 through extensive volcanic episodes could have promoted the major planktonic foraminiferal turnover during OAE1b.

All orchids and pyroloids are mycoheterotrophic at least in the early stage. Many species are predisposed to mycoheterotrophic nutrition even in the adult stage, due to the initial mycoheterotrophy during germination. Although other green plants, such as gentian species, also produce numerous minute seeds, whose germination may depend on fungal associations to meet C demands, physiological evidence for partial mycoheterotrophy in the adult stage is lacking for most candidate taxa. Here, we compared the natural abundances of 13 C and 15 N isotopes in the AM-associated gentian species Pterygocalyx volubilis growing in high-light-intensity habitats with those of co-occurring autotrophic C 3 and C 4 plants and AM fungal spores. We found that P. volubilis was significantly enriched in 13 C compared with the surrounding C 3 plants, which suggests the transfer of some C from the surrounding autotrophic plants through shared AM networks. In addition, the intermediate δ 15 N values of P. volubilis, between those of autotrophic plants and AM fungal spores, provide further evidence for partial mycoheterotrophy in P. volubilis. Although it is often considered that light deficiency selects partial mycoheterotrophy, we show that partial mycoheterotrophy in AM-forming plants can evolve even under light-saturated conditions. The fact that there have been relatively few descriptions of partial mycoheterotrophy in AM plants may not necessarily reflect the rarity of such associations. In conclusion, partial mycoheterotrophy in AM plants may be more common than hitherto believed.

Compound-specific isotope analyses of geoporphyrins, which are derivatives of chloropigments possessed by phototrophs, provide essential records of the biogeochemical cycle of past aquatic environments. Here, we evaluated uncertainties in carbon and nitrogen isotopic compositions (δ 13 C and δ 15 N) associated with high-performance liquid chromatography (HPLC) purification and isotopic measurements of geoporphyrins. Evaluation of total blank carbon and nitrogen for the HPLC and our sensitivity-improved elemental analyzer/isotope ratio mass spectrometer (nano-EA/IRMS) analysis confirmed that blank carbon can be corrected and that blank nitrogen is negligible compared to the mass of geoporphyrins required for the isotopic measurement. While geoporphyrins exhibited substantial in-peak carbon and nitrogen isotopic fractionations, no systematic changes in δ 13 C and δ 15 N values were observed during reversed- and normal-phase HPLC isolation of Ni- and VO-porphyrin standards, with the changes in δ 13 C and δ 15 N values being within ±0.6‰ and ±1.2‰ (2σ), respectively. These values are comparable to the instrumental precision of the nano-EA/IRMS system (±1.3‰ for 0.70 μgC and ±1.1‰ for 0.08 μgN, 2σ), confirming that no substantial artifact in the δ 13 C and δ 15 N values would be expected during the reversed- and normal-phase HPLC purification. The sensitivity and precision of our method enable us to determine δ 13 C and δ 15 N values of both major and minor geoporphyrins found in ancient sediments, which would provide detailed information on the photosynthetic primary producers and the carbon and nitrogen cycles in the past.

Sugars are essential molecules for all terrestrial biota working in many biological processes. Ribose is particularly essential as a building block of RNA, which could have both stored information and catalyzed reactions in primitive life on Earth. Meteorites contain a number of organic compounds including key building blocks of life, i.e., amino acids, nucleobases, and phosphate. An amino acid has also been identified in a cometary sample. However, the presence of extraterrestrial bioimportant sugars remains unclear. We analyzed sugars in 3 carbonaceous chondrites and show evidence of extraterrestrial ribose and other bioessential sugars in primitive meteorites. The 13 C-enriched stable carbon isotope compositions (δ 13 C vs. VPDB ) of the detected sugars show that the sugars are of extraterrestrial origin. We also conducted a laboratory simulation experiment of a potential sugar formation reaction in space. The compositions of pentoses in meteorites and the composition of the products of the laboratory simulation suggest that meteoritic sugars were formed by formose-like processes. The mineral compositions of these meteorites further suggest the formation of these sugars both before and after the accretion of their parent asteroids. Meteorites were carriers of prebiotic organic molecules to the early Earth; thus, the detection of extraterrestrial sugars in meteorites establishes the existence of natural geological routes to make and preserve them as well as raising the possibility that extraterrestrial sugars contributed to forming functional biopolymers like RNA on the early Earth or other primitive worlds., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The biogeochemistry of hypersaline environments is strongly influenced by changes in biological processes and physicochemical parameters. Although massive evaporation events have occurred repeatedly throughout Earth history, their biogeochemical cycles and global impact remain poorly understood. Here, we provide the first nitrogen isotopic data for nutrients and chloropigments from modern shallow hypersaline environments (solar salterns, Trapani, Italy) and apply the obtained insights to δ 15 N signatures of the Messinian salinity crisis (MSC) in the late Miocene. Concentrations and δ 15 N of chlorophyll a, bacteriochlorophyll a, nitrate, and ammonium in benthic microbial mats indicate that inhibition of nitrification suppresses denitrification and anammox, resulting in efficient ammonium recycling within the mats and high primary productivity. We also suggest that the release of 15 N-depleted NH 3(gas) with increasing salinity enriches ammonium 15 N in surface brine (≈34.0‰). Such elevated δ 15 N is also recorded in geoporphyrins isolated from sediments of the MSC peak (≈20‰), reflecting ammonium supply sufficient for sustaining phototrophic primary production. We propose that efficient nutrient supply combined with frequent bottom-water anoxia and capping of organic-rich sediments by evaporites of the Mediterranean MSC could have contributed to atmospheric CO 2 reduction during the late Miocene.

We have improved a method for isolation and purification of individual amino acids for compound-specific radiocarbon analysis (CSRA). To remove high-performance liquid chromatography (HPLC) eluent blanks from isolated amino acid fractions prior to the radiocarbon (Δ 14 C) measurement, each fraction was filtered through a membrane filter and then washed with diethyl ether twice. Radiocarbon measurements on standard amino acids processed and purified with the above method using elemental analyzer-accelerator mass spectrometry resulted in Δ 14 C values that were in strong agreement ( R 2 = 0.998) with the original Δ 14 C value of each amino acid standard. From these measurements, we calculate dead and modern carbon contamination contributions as 1.2 ± 0.2 and 0.3 ± 0.1 μgC, respectively, which are consistent with direct assessments of HPLC procedural blanks of 1.0 ± 0.8 μgC per sample. These contamination constraints allow correction of measured Δ 14 C values for accurate and precise CSRA and are widely applicable to future archeological and biogeochemical studies.

Oceanic methane from global deep-sea sediment is largely consumed through microbially mediated sulfate-coupled oxidation, resulting in 13 C-depleted cell biomass of anaerobic methanotrophic archaea (ANME). The general ecological importance of subseafloor ANME has been well recognized in the last two decades. However, the crucial biochemical pathways for the overall anaerobic oxidation of methane (AOM) still remain enigmatic. Here, methanotrophic pathways were analyzed to trace 13 C-depleted amino acid biosynthesis in two clades of ANME (ANME-1 and ANME-2) from the Black Sea. Compound-specific analysis of ANME-dominated microbial mats showed a significant 13 C-depletion trend in association with increasing carbon numbers in protein-derived amino acid families (e.g., the pyruvate family in the order of alanine, valine, isoleucine and leucine was down to -114‰). This result indicates a stepwise elongation of 13 C-depleted carbon during amino acid biosynthesis. The overall results suggest that intracellular protein amino acids and the most 13 C-depleted signature of leucine, which has a specific branched-chain structure, are potentially propagated as isoprenoid precursor molecules into archaeal biosynthesis, resulting in the extremely 13 C- and 14 C-depleted nature of ANME cells in the deep microbial oasis.

The separation and quantification of underivatized naturally occurring amino acids (AAs) by high-performance liquid chromatography (HPLC) is advantageous for reducing preparation time, running costs, and analytical errors, and is compatible with the isolation of intact AAs. This study establishes and validates an analytical method for the separation and quantification of underivatized AAs and taurine (Tau) in the sub- to several-nanomolar range (ca. 0.1-1.6 nmol) by optimizing ion-pair HPLC coupled to a corona charged aerosol detector (corona CAD). Chromatographic separation of 19 AAs and Tau was achieved using a porous graphite carbon (PGC) column and nonafluoropentanoic acid (NFPA) as a volatile ion-pair reagent. The response of the corona CAD to the AAs was highly dependent on the eluate composition, whereas these response factors were similar for AAs in eluate with similar compositions. Regression curves and coefficients (r 2 ) >0.998 were obtained for plots of injection amount versus peak area, except for Arg which co-eluted with a background peak. On the other hand, all plots of injection amount versus peak height regressed to curves with r 2  > 0.997. Repeat quantification based on peak area showed lower relative standard deviations (RSDs) (typically better than 5%) than those based on peak height. The present method is useful for quantifying AAs from proteins and Tau in the sub- to several-nanomolar range without derivatization, and constant repeatability can be maintained by quantification using peak areas., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bats perform important ecosystem services, but it remains difficult to quantify their dietary strategies and trophic position (TP) in situ. We conducted measurements of nitrogen isotopes of individual amino acids (δ 15 N AA ) and bulk-tissue carbon (δ 13 C bulk ) and nitrogen (δ 15 N bulk ) isotopes for nine bat species from different feeding guilds (nectarivory, frugivory, sanguivory, piscivory, carnivory, and insectivory). Our objective was to assess the precision of δ 15 N AA -based estimates of TP relative to other approaches. TPs calculated from δ 15 N values of glutamic acid and phenylalanine, which range from 8.3-33.1‰ and 0.7-15.4‰ respectively, varied between 1.8 and 3.8 for individuals of each species and were generally within the ranges of those anticipated based on qualitative dietary information. The δ 15 N AA approach reveals variation in TP within and among species that is not apparent from δ 15 N bulk data, and δ 15 N AA data suggest that two insectivorous species (Lasiurus noctivagans and Lasiurus cinereus) are more omnivorous than previously thought. These results indicate that bats exhibit a trophic discrimination factor (TDF) similar to other terrestrial organisms and that δ 15 N AA provides a reliable approach for addressing questions about variation in the TP of bats that have heretofore proven elusive.

Long-term monitoring of ecosystem succession provides baseline data for conservation and management, as well as for understanding the dynamics of underlying biogeochemical processes. We examined the effects of deforestation and subsequent afforestation of a riparian forest of Japanese cedar (Cryptomeria japonica) on stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) and natural abundances of radiocarbon (Δ¹⁴C) in stream biota in the Mt. Gomadan Experimental Forest and the Wakayama Forest Research Station, Kyoto University, central Japan. Macroinvertebrates, periphytic algae attached to rock surfaces (periphyton), and leaf litter of terrestrial plants were collected from six headwater streams with similar climate, topography, and bedrock geology, except for the stand ages of riparian forests (from 3 to 49 yr old in five stands and > 90 yr old in one reference stand). Light intensity and δ¹³C values of both periphyton and macroinvertebrates decreased synchronously with forest age in winter. A Bayesian mixing model indicates that periphyton contributions to the stream food webs are maximized in 23-yr-old forests. Except for grazers, most macroinvertebrates showed Δ¹⁴C values similar to those of terrestrial leaf litter, reflecting the influence of modern atmospheric CO₂ Δ¹⁴C values. On the other hand, the Δ¹⁴C values of both periphyton and grazers (i.e., aquatic primary consumers) were significantly lower than that of modern atmospheric CO₂, and were lowest in 23-yr-old forest stands. Previous studies show that root biomass of C. japonica peaks at 15-30 yr after planting. These evidences suggest that soil CO₂ released by root respiration and dispersed by groundwater weathers carbonate substrata, and that dissolved inorganic carbon (DIC) with low Δ¹⁴C is incorporated into stream periphyton and some macroinvertebrates. The ecological response in the studied streams to clear-cutting and replanting of Japanese cedar is much slower (~20 yr) than the chemical response (< 5 yr). More than 50 yr is required for the food web structure to completely recover from clear-cutting. The ecological delay is attributed to several biogeochemical factors, the understanding of which is critical to integrated management of forest-stream continuum and the prediction of ecosystem resilience in response to environmental change.

In the Seto Inland Sea of western Japan, metacercariae of three species of trematodes, Lepotrema clavatum Ozaki, 1932, Cephalolepidapedon saba Yamaguti, 1970, and Opechona olssoni (Yamaguti, 1934), were found in the mesoglea of the jellyfish Aurelia aurita s.l., Chrysaora pacifica, and Cyanea nozakii. Moreover, these jellyfish frequently harbored juveniles of the fish species Psenopsis anomala, Thamnaconus modestus, and Trachurus japonicus. The former two fish species are well-known medusivores. We investigated seasonal changes in the prevalence and intensity of these metacercariae in their host jellyfish from March 2010 to September 2012 and presumed that infection by the trematodes of the definitive host fish occurs through these associations. The mean intensity of metacercariae in A. aurita s.l. clearly showed seasonality, being consistently high in June of each year. The intensity of metacercariae in C. nozakii was highest among all jellyfish hosts and appeared to be enhanced by medusivory of this second intermediate, and/or paratenic host. Trophic interactions between jellyfish and associated fish were verified using both gut content and stable isotope analyses. The detection of trematodes and nematocysts in the guts of P. anomala and T. modestus juveniles, in addition to stable isotope analysis, suggests that transmission of the parasites occurs via prey-predator relationships. In addition, the stable isotope analysis also suggested that P. anomala is more nutritionally dependent on jellyfish than Th. modestus and Tr. japonicus., (© Y. Kondo et al., published by EDP Sciences, 2016.)

Food webs are known to have myriad trophic links between resource and consumer species. While herbivores have well-understood trophic tendencies, the difficulties associated with characterizing the trophic positions of higher-order consumers have remained a major problem in food web ecology. To better understand trophic linkages in food webs, analysis of the stable nitrogen isotopic composition of amino acids has been introduced as a potential means of providing accurate trophic position estimates. In the present study, we employ this method to estimate the trophic positions of 200 free-roaming organisms, representing 39 species in coastal marine (a stony shore) and 38 species in terrestrial (a fruit farm) environments. Based on the trophic positions from the isotopic composition of amino acids, we are able to resolve the trophic structure of these complex food webs. Our approach reveals a high degree of trophic omnivory (i.e., noninteger trophic positions) among carnivorous species such as marine fish and terrestrial hornets.This information not only clarifies the trophic tendencies of species within their respective communities, but also suggests that trophic omnivory may be common in these webs.

Coenzyme F430 is a nickel hydrocorphinoid and is the prosthetic group of methyl-coenzyme M reductase that catalyzes the last step of the methanogenic reaction sequence and its reversed reaction for anaerobic methane oxidation by ANME. As such, function-specific compound analysis has the potential to reveal the microbial distribution and activity associated with methane production and consumption in natural environments and, in particular, in deep subsurface sediments where microbiological and geochemical techniques are restricted. Herein, we report the development of a technique for high-sensitivity analysis of F430 in environmental samples, including paddy soils, marine sediments, microbial mats, and an anaerobic groundwater. The lower detection limit of F430 analysis by liquid chromatography/mass spectrometry is 0.1 femto mol, which corresponds to 6 × 10(2) to 1 × 10(4) cells of methanogens. F430 concentrations in these natural environmental samples range from 63 × 10(-6) to 44 nmol g(-1) and are consistent with the methanogenic archaeal biomass estimated by microbiological analyses.

Cyanide compounds are amongst the most important molecules of the origin of life. Here, we demonstrate the importance of mid-size (0.1-1 km in diameter) hence frequent meteoritic impacts to the cyanide inventory on the early Earth. Subsequent aerodynamic ablation and chemical reactions with the ambient atmosphere after oblique impacts were investigated by both impact and laser experiments. A polycarbonate projectile and graphite were used as laboratory analogs of meteoritic organic matter. Spectroscopic observations of impact-generated ablation vapors show that laser irradiation to graphite within an N2-rich gas can produce a thermodynamic environment similar to that produced by oblique impacts. Thus, laser ablation was used to investigate the final chemical products after this aerodynamic process. We found that a significant fraction (>0.1 mol%) of the vaporized carbon is converted to HCN and cyanide condensates, even when the ambient gas contains as much as a few hundred mbar of CO2. As such, the column density of cyanides after carbon-rich meteoritic impacts with diameters of 600 m would reach ~10 mol/m(2) over ~10(2) km(2) under early Earth conditions. Such a temporally and spatially concentrated supply of cyanides may have played an important role in the origin of life.

What eel larvae feed on in the surface layer of the ocean has remained mysterious. Gut contents and bulk nitrogen stable isotope studies suggested that these unusual larvae, called leptocephali, feed at a low level in the oceanic food web, whereas other types of evidence have suggested that small zooplankton are eaten. In this study, we determined the nitrogen isotopic composition of amino acids of both natural larvae and laboratory-reared larvae of the Japanese eel to estimate the trophic position (TP) of leptocephali. We observed a mean TP of 2.4 for natural leptocephali, which is consistent with feeding on particulate organic matter (POM) such as marine snow and discarded appendicularian houses containing bacteria, protozoans and other biological materials. The nitrogen isotope enrichment values of the reared larvae confirm that the primary food source of natural larvae is consistent only with POM. This shows that leptocephali feed on readily available particulate material originating from various sources closely linked to ocean primary production and that leptocephali are a previously unrecognized part of oceanic POM cycling.

Early Palaeoproterozoic (2.5-2.0 billion years ago) was a critical phase in Earth's history, characterized by multiple severe glaciations and a rise in atmospheric O(2) (the Great Oxidation Event). Although glaciations occurred at the time of O(2) increase, the relationship between climatic and atmospheric transitions remains poorly understood. Here we report high concentrations of the redox-sensitive element Os with high initial (187)Os/(188)Os values in a sandstone-siltstone interval that spans the transition from glacial diamictite to overlying carbonate in the Huronian Supergroup, Canada. Together with the results of Re, Mo and S analyses of the sediments, we suggest that immediately after the second Palaeoproterozoic glaciation, atmospheric O(2) levels became sufficiently high to deliver radiogenic continental Os to shallow-marine environments, indicating the synchronicity of an episode of increasing O(2) and deglaciation. This result supports the hypothesis that climatic recovery from the glaciations acted to accelerate the Great Oxidation Event.

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound-specific nitrogen isotopic compositions of amino acids. We employed cation-exchange chromatography of protein and non-protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation-exchange chromatography. We applied the procedure to various environmental samples including 'hard' (calcareous, siliceous, rock and sediment samples) and 'soft' materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation-exchange chromatography is a pre-treatment procedure which should be widely useful for the determination of compound-specific nitrogen isotopic compositions of amino acids., (2010 John Wiley & Sons, Ltd.)

Nitrogenous compounds with high delta(15)N values were recently found in human-dominated small rivers in the Lake Biwa area. A detailed survey was performed to determine the distribution and variation of delta(15)N values in nitrogenous compounds in a representative small river (Hebisuna River) that flows into Lake Nishinoko, an inner bay of Lake Biwa. A high delta(15)N value was detected in the lower reaches of the river and the inner bay, most likely due to denitrification. These results strongly suggest that denitrification in small river systems such as the Hebisuna watershed has contributed to (15)N enrichment in the Lake Biwa ecosystem during the past 40 years. We also observed a clear, stepwise, positive correlation between population density and delta(15)N values for particulate organic matter or sediments. These results demonstrate that delta(15)N (POM) and delta(15)N (sediment) are helpful indicators for assessing nitrogen loading from domestic sewage. Moreover, they will aid in the development of new concepts in the environmental capacity of river ecosystems and its relationship to redox conditions. Finally, our data suggest that a population density of 100-200 persons per km(2) is the upper limit for a watershed in which only simple conventional sewage treatment is in effect.

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

Although analyses of chlorophyll d (Chl d)-dominated oxygenic photosystems have been conducted since their discovery 12 years ago, Chl d distribution in the environment and quantitative importance for aquatic photosynthesis remain to be investigated. We analyzed the pigment compositions of surface sediments and detected Chl d and its derivatives from diverse aquatic environments. Our data show that the viable habitat for Chl d-producing phototrophs extends across salinities of 0 to 50 practical salinity units and temperatures of 1 degrees to 40 degrees C, suggesting that Chl d production can be ubiquitously observed in aquatic environments that receive near-infrared light. The relative abundances of Chl d derivatives over that of Chl a derivatives in the studied samples are up to 4%, further suggesting that Chl d-based photosynthesis plays a quantitatively important role in the aquatic photosynthesis.

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ(13)C, δ(15)N, and δ(18)O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n=1), Japan (n=12), and USA (n=1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2-40.0% (C content), 0.8-1.4% (N content), -27.1 to -25.4% (δ(13)C), +0.4 to +9.0% (δ(15)N), and +18.8 to +22.9% (δ(18)O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ(13)C, δ(15)N, and δ(18)O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice., (Copyright © 2008 Elsevier Ltd. All rights reserved.)

Stable carbon and nitrogen isotopic compositions were determined for individual photosynthetic pigments isolated and purified from the saline meromictic Lake Kaiike, Japan, to investigate species-independent biogeochemical processes of photoautotrophs in the natural environment. In the anoxic monimolimnion and benthic microbial mats, the carbon isotopic compositions of BChls e and isorenieratene related to brown-coloured strains of green sulfur bacteria are substantially ( approximately 10 per thousand) depleted in (13)C relative to those found in the chemocline. In conjunction with 16S rDNA evidence reported previously, it strongly suggests that Pelodyctyon luteolum inhabited and photosynthesized in the anoxic monimolimnion and benthic microbial mats by using (13)C-depleted regenerated CO(2). By contrast, both Chl a and BChl a in the monimolimnion and microbial mats have similar isotopic compositions as they do in the chemocline, implying that the source organisms live only in the chemocline. In the chemocline, the nitrogen isotopic compositions of BChl e homologues ranges from -7.7 to-6.5 per thousand, whereas that of BChl a is -2.1 per thousand. These isotopic compositions suggest that green sulfur bacteria Chlorobium phaeovibrioides would conduct nitrogen fixation in the chemocline, whereas purple sulfur bacteria Halochromatium sp. and cyanobacteria Synechococcus sp. may assimilate nitrite.

We determined hydrogen, carbon and nitrogen isotopic compositions of chlorophylls a and b isolated from leaves of five C3 higher plant species (Benthamidia japonica, Prunus japonica, Acer carpinifolium, Acer argutum and Querus mongloica), and hydrogen and carbon isotopic compositions of phytol and chlorophyllides in the chlorophylls to understand isotopic fractionations associated with chlorophyll biosynthesis in these species. Chlorophylls are depleted in D relative to ambient water by approximately 189 per thousand and enriched in (13)C relative to bulk tissue by approximately 1.6 per thousand. These data can be explained by the contribution of isotopic fractionations during phytol and chlorophyllide biosyntheses. Phytol is more depleted in both D (by approximately 308 per thousand) and (13)C (by approximately 4.3 per thousand), while chlorophyllides are less depleted in D (by approximately 44 per thousand) and enriched in (13)C (by approximately 4.8 per thousand). Such inhomogeneous distribution of isotopes in chlorophylls suggests that (1) the phytol in chlorophylls reflects strong D- and (13)C-depletions due to the isotopic fractionations during the methylerythritol phosphate pathway followed by hydrogenation, and (2) the chlorophyllides reflect D- and (13)C-enrichments in tricarboxylic acid cycle. On the other hand, chlorophylls are slightly ( approximately 1.2 per thousand) depleted in (15)N relative to the bulk tissue, indicating that net isotopic fractionation of nitrogen during chlorophyll biosynthesis is small compared with those of hydrogen and carbon.