Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields.

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH4) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH4 oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and 13CH4-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH4 oxidation in supporting denitrification. Our results reveal positive relationships between CH4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13CH4-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of 13C-metagenome-assembled genomes and 13C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH4 oxidation, for the coupling of CH4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.

First Synthesis of Ergotamine-13CD3 and Ergotaminine-13CD3 from Unlabeled Ergotamine.

Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.

Local circulation of elites punctuated by transregional mobility enabled steppe political consolidation in the Xiongnu nomadic state.

The Xiongnu polity (ca. 200 BC- 150 AD) emerged out of indigenous community-centered socio-political structures to forge a powerful state that commanded the Mongolian steppe and beyond. Underpinned by a highly mobile pastoralist population, accustomed to seasonally rhythmic moves and embedded in an equestrian culture that facilitated rapid transport over long-distances, it remains unclear precisely how the movement of commoners, local aristocrats and regional elites abetted the formation and organization of Xiongnu state structures. Here, we evaluate Xiongnu movement and dietary intake through multi-stable isotopic analyses of tooth enamel from directly dated Xiongnu intermediate elites recovered from the mortuary center of Baga Gazaryn Chuluu-a prominent granite outcrop set in the Gobi Desert. Carbon isotope (δ13C) analysis indicates millet was consumed by some individuals, but whether or not this C4 cultivar contributed to the diets of most elites remains ambiguous in this C3/C4 desert-steppe environment. The effectiveness of oxygen isotopes (δ18O) to establish mobility appears much reduced in steppe environments, where geospatially sensitive information appears disrupted by extraordinary seasonality in meteoric water oxygen isotopes, pronounced oxygen isotopic variation in potential drinking water sources, and culturally mediated drinking practices. Most revealing, strontium isotopes (87Sr/86Sr) indicate circulation of local elites around this central place and beyond, a mobility format that helped leaders cement their own position through political consolidation of spatially dispersed mobile pastoralist communities. The consistent presence at Baga Gazaryn Chuluu of extra-local intermediate elites also points toward the importance of transregional mobility in binding together the Xiongnu polity over the vast distances of the eastern steppe.

Shepherding the past: High-resolution data on Neolithic Southern Iberian livestock management at Cueva de El Toro (Antequera, Málaga).

The feeding strategies of the first domesticated herds had to manage the risks arising from the novelty of livestock practices in territories often distant from the animals' primary habitats. The Iberian Peninsula is characterised by a great diversity of environments, which undoubtedly influenced these dynamics. At the beginning of the Neolithic period these led the possibility to combine diverse livestock farming practices based on different animal feeding habits. This variability is also consistent with the rythms of adoption of domesticated animals, being later on the northern area. In order to address this issue, this work focuses on the dietary regimes of early sheep herds from southern Iberia, an area for which information is currently scarce. This study utilises high-resolution radiocarbon dating and stable isotope data on teeth to investigate sheep husbandry management strategies in Cueva de El Toro (Antequera, Málaga). The radiocarbon dates on the analysed remains evidenced they were deposited at the site over a short period, supporting the recurrent use of the cave. The sequential analysis of oxygen and carbon isotopes in tooth enamel reveals distinct livestock management strategies, reproduction patterns, feeding habits, and mobility during this short period. This variability demonstrates that livestock management practices in the western Mediterranean are more diverse than previously considered. Furthermore, these findings support the hypothesis that early Neolithic communities in the southern Iberian Peninsula were able to adopt different feeding strategies within the same herd, depending on their ecological and productive needs.

Amino acid-specific isotopes reveal changing five-dimensional niche segregation in Pacific seabirds over 50 years.

Hutchison's niche theory suggests that coexisting competing species occupy non-overlapping hypervolumes, which are theoretical spaces encompassing more than three dimensions, within an n-dimensional space. The analysis of multiple stable isotopes can be used to test these ideas where each isotope can be considered a dimension of niche space. These hypervolumes may change over time in response to variation in behaviour or habitat, within or among species, consequently changing the niche space itself. Here, we use isotopic values of carbon and nitrogen of ten amino acids, as well as sulphur isotopic values, to produce multi-isotope models to examine niche segregation among an assemblage of five coexisting seabird species (ancient murrelet Synthliboramphus antiquus, double-crested cormorant Phalacrocorax auritus, Leach's storm-petrel Oceanodrama leucorhoa, rhinoceros auklet Cerorhinca monocerata, pelagic cormorant Phalacrocorax pelagicus) that inhabit coastal British Columbia. When only one or two isotope dimensions were considered, the five species overlapped considerably, but segregation increased in more dimensions, but often in complex ways. Thus, each of the five species occupied their own isotopic hypervolume (niche), but that became apparent only when factoring the increased information from sulphur and amino acid specific isotope values, rather than just relying on proxies of δ15N and δ13C alone. For cormorants, there was reduction of niche size for both species consistent with a decline in their dominant prey, Pacific herring Clupea pallasii, from 1970 to 2006. Consistent with niche theory, cormorant species showed segregation across time, with the double-crested demonstrating a marked change in diet in response to prey shifts in a higher dimensional space. In brief, incorporating multiple isotopes (sulfur, PC1 of δ15N [baselines], PC2 of δ15N [trophic position], PC1 and PC2 of δ13C) metrics allowed us to infer changes and differences in food web topology that were not apparent from classic carbon-nitrogen biplots.

Using Carbon Stable Isotopes to Study C3 and C4 Photosynthesis: Models and Calculations.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

Growth and carbon turnover of Piaractus mesopotamicus Holmberg, 1887 (Osteicthyes: Characidae): contribution of extruded feed and natural food.

Piaractus mesopotamicus, is a fish usually farmed in semi-intensive systems with access to natural food and supplementary feed. This study evaluates effects of feed allowance on the productive performance, carbon turnover and proportions of nutrient (carbon) contribution of feed and natural food for the growth of pacu. Juvenile fish were stocked in fiberglass tanks and fed to 100, 75, 50, 25, 0% apparent satiety (ApS), with a practical, extruded (C4 photosynthetic pathway) feed in a randomized design trial (n=3); plankton production for simulated semi-intensive farming system condition was induced by chemical fertilization. A control treatment was set up in tanks devoid of natural food. Data on muscle stable carbon isotope ratios were used to study carbon turnover using a relative growth-based model. Low variation of the δ13C impaired fitting a turnover model curve for the 0 and 25 % ApS treatments. Fish of the 100% and 75% ApS treatments reached circa 95% and 82.85% of the carbon turnover, respectively. Extruded feed was the main nutrient source for the growth of pacu in the semi-intensive, simulated farming condition. The current study contributes to the knowledge of the relationship between feeding rates and carbon turnover rates in the pacu muscle.

Species-specific responses of C and N allocation to N addition: evidence from dual 13C and 15N labeling in three tree species.

Soil nitrogen (N) availability affects plant carbon (C) utilization. However, it is unclear how various tree functional types respond to N addition in terms of C assimilation, allocation, and storage. Here, a microcosm experiment with dual 13C and 15N labeling was conducted to study the effects of N addition (i.e., control, 0 g N kg-1; moderate N addition, 1.68 g N kg-1; and high N addition, 3.36 g N kg-1 soil) on morphological traits, on changes in nonstructural carbohydrates (NSC) in different organs, as well as on C and N uptake and allocation in three European temperate forest tree species (i.e., Acer pseudoplatanus, Picea abies and Abies alba). Our results demonstrated that root N uptake rates of the three tree species increased by N addition. In A. pseudoplatanus, N uptake by roots, N allocation to aboveground organs, and aboveground biomass allocation significantly improved by moderate and high N addition. In A. alba, only the high N addition treatment considerably raised aboveground N and C allocation. In contrast, biomass as well as C and N allocation between above and belowground tissues were not altered by N addition in P. abies. Meanwhile, NSC content as well as C and N coupling (represented by the ratio of relative 13C and 15N allocation rates in organs) were affected by N addition in A. pseudoplantanus and P. abies but not in A. alba. Overall, A. pseudoplatanus displayed the highest sensitivity to N addition and the highest N requirement among the three species, while P. abies had a lower N demand than A. alba. Our findings highlight that the responses of C and N allocation to soil N availability are species-specific and vary with the amount of N addition.

[Changes in protein requirements of elderly people over 5 years assessed using the indicator amino acid oxidation method].

OBJECTIVE: To evaluate the changes in protein requirements of the elderly during the past five years. METHODS: Based on the previous study of protein requirements of 14 elderly in 2017, 4 of these elderly(70-80 y) were included as study participants and protein requirements were re-evaluated using the indicator amino acid oxidation method. There were seven protein levels: 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 g/(kg·d). Maintenance diets were given for the first two days of each protein level. A stable isotope study was conducted on the day 3, using L-~(13)C-phenylalanine as an indicator on the basis of an amino acid rationed diet, which was orally ingested into the body along with the amino acid rationed diet, and breath and urine samples were collected when the metabolism of L-~(13)C-phenylalanine reached steady state in the body. By measuring the kinetic parameters of labeled amino acids in the samples, a nonlinear mixed-effects model was constructed for the protein intake to be tested and the oxidation rate of labeled amino acids. The mean protein requirement of the study population was determined by the protein intake corresponding to the inflection point of the curve. RESULTS: Based on the production rate of ~(13)CO_2 in exhaled breath of four elderly people at different protein levels, the mean protein requirement was 1.05(95%CI 0.51-1.60) g/(kg·d). The protein recommended nutrient intake was 1.31(95%CI 0.64-2.00) g/(kg·d) was estimated by applying the coefficient of variation of the mean protein requirement to derive the recommended nutrient intake. CONCLUSION: Protein requirements in the elderly have increased over a five-year period and sarcopenia may be the main cause of increased protein requirements.

Sources and pathways of carbon and nitrogen of macrophytes and sediments using stable isotopes in Al-Kharrar Lagoon, eastern Red Sea coast, Saudi Arabia.

Elemental ratios (δ13C, δ15N and C/N) and carbon and nitrogen concentrations in macrophytes, sediments and sponges of the hypersaline Al-Kharrar Lagoon (KL), central eastern Red Sea coast, were measured to distinguish their sources, pathways and see how they have been influenced by biogeochemical processes and terrestrial inputs. The mangroves and halophytes showed the most depleted δ13C values of -27.07±0.2 ‰ and -28.34±0.4 ‰, respectively, indicating their preferential 12C uptake, similar to C3-photosynthetic plants, except for the halophytes Atriplex sp. and Suaeda vermiculata which showed δ13C of -14.31±0.6 ‰, similar to C4-plants. Macroalgae were divided into A and B groups based on their δ13C values. The δ13C of macroalgae A averaged -15.41±0.4 ‰, whereas macroalgae B and seagrasses showed values of -7.41±0.8 ‰ and -7.98 ‰, suggesting uptake of HCO3- as a source for CO2 during photosynthesis. The δ13C of sponges was -10.7±0.3 ‰, suggesting that macroalgae and seagrasses are their main favoured diets. Substrates of all these taxa showed δ13C of -15.52±0.8 ‰, suggesting the KL is at present a macroalgae-dominated lagoon. The δ15N in taxa/sediments averaged 1.68 ‰, suggesting that atmospheric N2-fixation is the main source of nitrogen in/around the lagoon. The heaviest δ15N (10.58 ‰) in halophytes growing in algal mats and sabkha is possibly due to denitrification and ammonia evaporation. The macrophytes in the KL showed high C %, N %, and C/N ratios, but this is not indicated in their substrates due possibly to a rapid turnover of dense, hypersaline waters carrying most of the detached organic materials out into the Red Sea. The δ13C allowed separation of subaerial from aquatic macrophytes, a proxy that could be used when interpreting paleo-sea level or paleoclimatic changes from the coastal marine sediments.

The influence of biopsy site and pregnancy on stable isotope ratios in humpback whale skin.

RATIONALE: Stable isotope analysis (SIA) of free-swimming mysticetes using biopsies is often limited in sample size and uses only one sample per individual, failing to capture both intra-individual variability and the influence of demographic and physiological factors on isotope ratios. METHODS: We applied SIA of δ13C and δ15N to humpback whale (Megaptera novaeangliae) biopsies taken during the foraging season along the western Antarctic Peninsula to quantify intra-individual variation from repeatedly sampled individuals, as well as to determine the effect of biopsy collection site, sex, and pregnancy on isotope ratios. RESULTS: There was substantial variability in δ13C from multiple biopsies taken from the same individuals, though δ15N was much more consistent. Side of the body (left versus right) and biopsy location (dorsal, anterior, ventral, and posterior) did marginally affect the isotopic composition of δ15N but not δ13C. Pregnancy had a significant effect on both δ13C and δ15N, where pregnant females were depleted in both when compared to non-pregnant females and males. CONCLUSIONS: These results indicate that isotopic signatures are influenced by multiple endogenous and exogenous factors and emphasize value in accounting for intra-individual variability and pregnancy status within a sampled population. Placed within an ecological context, the endogenous variability in δ13C observed here may be informative for future isotopic analyses.

Effects of particle size, storage conditions, and chemical pretreatments on carbon and oxygen isotopic measurements of modern tooth enamel.

Isotopic analysis of human tooth enamel can provide life history information useful in forensic identification. These applications depend on the availability of reference data documenting isotopic values for individuals with known life history and on the comparability of data from reference and case work samples. Here we build on previous methodological research, which has largely focused on paleontological and archaeological samples, and conduct experiments using enamel from modern human teeth targeting three sample preparation variables (sample particle size, storage conditions, and chemical pretreatments). Our results suggest that differences in particle size affect the efficiency of sample reactions during pretreatment and analysis, with coarse particles giving reduced loss of enamel carbonate during acid pretreatments but producing slightly higher oxygen isotope values than fine particles during analysis. Data for samples stored in dry and ambient environments following pretreatment were indistinguishable, suggesting no exchange of oxygen between samples and ambient water vapor. Finally, chemical pretreatments with a range of commonly used reactants and conditions showed a pervasive, moderate oxygen isotope shift associated with acetic acid treatment, which may be caused by exchange of enamel hydroxyl groups with reagents or rinse waters. Collectively, the results emphasize the importance of methodological standardization to improve comparability and reduce potential for bias in the forensic application of tooth enamel isotope data.

Preserved particulate organic carbon is likely derived from the subsurface sulfidic photic zone of the Proterozoic Ocean: evidence from a modern, oxygen-deficient lake.

Biological processes in the Proterozoic Ocean are often inferred from modern oxygen-deficient environments (MODEs) or from stable isotopes in preserved sediment. To date, few MODE studies have simultaneously quantified carbon fixation genes and attendant stable isotopic signatures. Consequently, how carbon isotope patterns reflect these pathways has not been thoroughly vetted. Addressing this, we profiled planktonic productivity and quantified carbon fixation pathway genes and associated organic carbon isotope values (δ13 CPOC ) of size-fractionated (0.2-2.7 and >2.7 µm) particulate matter from meromictic Fayetteville Green Lake, NY, USA. The high-O2 Calvin-Benson-Bassham (CBB) gene (cbbL) was most abundant in the <2.7 µm size fraction in shallow oxic and deep hypoxic waters, corresponding with cyanobacterial and eukaryote algal populations. The low-O2 CBB gene (cbbM) was most abundant near the lower oxycline boundary in the larger size fraction, coincident with purple sulfur bacteria populations. The reverse citric acid cycle gene (aclB) was equally abundant in both size fractions in the deepest photic zone, coinciding with green sulfur bacteria populations. Methane coenzyme reductase A (mcrA), of anaerobic methane cyclers, was most abundant at the lower oxycline boundary in both size fractions, coinciding with Methanoregula populations. δ13 CPOC values overlapped with the high-O2 CBB fixation range except for two negative excursions near the lower oxycline boundary, likely reflecting assimilation of isotopically-depleted groundwater-derived carbon by autotrophs and sulfate-reducers. Throughout aphotic waters, δ13 CPOC values of the large size fraction became 13 C-enriched, likely reflecting abundant purple sulfur bacterial aggregates. Eukaryote algae- or cyanobacteria-like isotopic signatures corresponded with increases in cbbL, cbbM, and aclB, and enrichment of exopolymer-rich prokaryotic photoautotrophs aggregates. Results suggest that δ13 CPOC values of preserved sediments from areas of the Proterozoic Ocean with sulfidic photic zones may reflect a mixture of alternate carbon-fixing populations exported from the deep photic zone, challenging the paradigm that sedimentary stable carbon isotope values predominantly reflect oxygenic photosynthesis from surface waters.

Constraining activity and growth substrate of fungal decomposers via assimilation patterns of inorganic carbon and water into lipid biomarkers.

Fungi are among the few organisms on the planet that can metabolize recalcitrant carbon (C) but are also known to access recently produced plant photosynthate. Therefore, improved quantification of growth and substrate utilization by different fungal ecotypes will help to define the rates and controls of fungal production, the cycling of soil organic matter, and thus the C storage and CO2 buffering capacity in soil ecosystems. This pure-culture study of fungal isolates combined a dual stable isotope probing (SIP) approach, together with rapid analysis by tandem pyrolysis-gas chromatography-isotope ratio mass spectrometry to determine the patterns of water-derived hydrogen (H) and inorganic C assimilated into lipid biomarkers of heterotrophic fungi as a function of C substrate. The water H assimilation factor (αW) and the inorganic C assimilation into C18:2 fatty acid isolated from five fungal species growing on glucose was lower (0.62% ± 0.01% and 4.7% ± 1.6%, respectively) than for species grown on glutamic acid (0.90% ± 0.02% and 7.4% ± 3.7%, respectively). Furthermore, the assimilation ratio (RIC/αW) for growth on glucose and glutamic acid can distinguish between these two metabolic modes. This dual-SIP assay thus delivers estimates of fungal activity and may help to delineate the predominant substrates that are respired among a matrix of compounds found in natural environments.IMPORTANCEFungal decomposers play important roles in food webs and nutrient cycling because they can feed on both labile and more recalcitrant forms of carbon. This study developed and applied a dual stable isotope assay (13C-dissolved inorganic carbon/2H) to improve the investigation of fungal activity in the environment. By determining the incorporation patterns of hydrogen and carbon into fungal lipids, this assay delivers estimates of fungal activity and the different metabolic pathways that they employ in ecological and environmental systems.

Effect of Chronic Ethanol Consumption on Exogenous Glucose Metabolism in Rats Using [1-13C], [2-13C], and [3-13C]glucose Breath Tests.

The C3 carbon of glucose molecules becomes the C1 carbon of pyruvate molecules during glycolysis, and the C1 and C2 carbons of glucose molecules are metabolized in the tricarboxylic acid (TCA) cycle. Utilizing this position-dependent metabolism of C atoms in glucose molecules, [1-13C], [2-13C], and [3-13C]glucose breath tests are used to evaluate glucose metabolism. However, the effects of chronic ethanol consumption remain incompletely understood. Therefore, we evaluated glucose metabolism in ethanol-fed rats using [1-13C], [2-13C], and [3-13C]glucose breath tests. Ethanol-fed (ERs) and control rats (CRs) (n = 8 each) were used in this study, and ERs were prepared by replacing drinking water with a 16% ethanol solution. We administered 100 mg/kg of [1-13C], [2-13C], or [3-13C]glucose to rats and collected expired air (at 10-min intervals for 180 min). We compared the 13CO2 levels (Δ13CO2, ‰) of breath measured by IR isotope ratio spectrometry and area under the curve (AUC) values of the 13CO2 levels-time curve between ERs and CRs. 13CO2 levels and AUCs after administration of [1-13C]glucose and [2-13C]glucose were lower in ERs than in CRs. Conversely, the AUC for the [3-13C]glucose breath test showed no significant differences between ERs and CRs, although 13CO2 levels during the 110-120 min interval were significantly high in ERs. These findings indicate that chronic ethanol consumption diminishes glucose oxidation without concomitantly reducing glycolysis. Our study demonstrates the utility of 13C-labeled glucose breath tests as noninvasive and repeatable methods for evaluating glucose metabolism in various subjects, including those with alcoholism or diabetes.

Why aquatic scientists should use sulfur stable isotope ratios (ẟ34S) more often.

Over the last few decades, measurements of light stable isotope ratios have been increasingly used to answer questions across physiology, biology, ecology, and archaeology. The vast majority analyse carbon (δ13C) and nitrogen (δ15N) stable isotopes as the 'default' isotopes, omitting sulfur (δ34S) due to time, cost, or perceived lack of benefits and instrumentation capabilities. Using just carbon and nitrogen isotopic ratios can produce results that are inconclusive, uncertain, or in the worst cases, even misleading, especially for scientists that are new to the use and interpretation of stable isotope data. Using sulfur isotope values more regularly has the potential to mitigate these issues, especially given recent advancements that have lowered measurement barriers. Here we provide a review documenting case studies with real-world data, re-analysing different biological topics (i.e. niche, physiology, diet, movement and bioarchaeology) with and without sulfur isotopes to highlight the various strengths of this stable isotope for various applications. We also include a preliminary meta-analysis of the trophic discrimination factor (TDF) for sulfur isotopes, which suggest small (mean -0.4 ± 1.7 ‰ SD) but taxa-dependent mean trophic discrimination. Each case study demonstrates how the exclusion of sulfur comes at the detriment of the results, often leading to very different outputs, or missing valuable discoveries entirely. Given that studies relying on carbon and nitrogen stable isotopes currently underpin most of our understanding of various ecological processes, this has concerning implications. Collectively, these examples strongly suggest that researchers planning to use carbon and nitrogen stable isotopes for their research should incorporate sulfur where possible, and that the new 'default' isotope systems for aquatic science should now be carbon, nitrogen, and sulfur.

Anaerobic biotransformation of hexachlorocyclohexane isomers in aqueous condition: Dual CCl isotope fractionation and impact on microbial community compositions.

Hexachlorocyclohexane (HCH) isomers are persistent organic pollutants (POPs) with high toxicity, lipid solubility, chemical stability. Despite the current ban on usage of Lindane, residual contamination cannot be ignored, and HCH are frequently detected in groundwater and threaten human health. Cultures capable of degrading α-HCH, ß-HCH, γ-HCH, and δ-HCH individually have been enriched in anoxic aqueous conditions. Compound-Specific Isotope Analysis (CSIA) was applied to examine the transformation mechanisms of different HCH isomers by the four enrichment cultures. 16S rRNA sequencing techniques were employed to examine the community composition of the enrichment cultures and detect changes in these communities resulting from adding individual HCH isomers. The results indicated that the ability of the enrichment cultures for dichloroelimination of HCH isomers was inconsistent. During dichloroelimination, different bond cleavage mode of ß- and δ-HCH led to distinct isotopic effects. HCH isomers had significant impact on the microbial community, while different microbial communities showed comparable isotopic effects during the transformation of a specific HCH isomer. In addition, bacteria in the phyla Proteobacteria and Firmicutes were proposed as the dominant dechlorinators. This study provides a novel perspective on the mode of bond cleavage during HCH dichloroelimination and the effect of HCH on microbial communities, which could potentially support the evaluation of HCH transformation by CSIA and their effects on the microecosystems of groundwater.

Tracking the transformation of persistent organic pollutants in food webs using multi element isotope and enantiomer fractionation.

In order to track the transformation of persistent organic pollutants (POPs) in food webs, field experiments were conducted at two sites using stable isotope and enantiomer fractionation concepts. The enantiomers of α-hexachlorocyclohexane (α-HCH) were selected as representative compounds for POPs. Isotope and enantiomer fractionation allowed the characterization of α-HCH enantiomer biotransformation processes along trophic levels of the food web - from soil and plants to animal livers, fat tissues and milk. The enrichment of heavy isotopes in soils, plants and sediments as well as the changes of enantiomer fractionation indicate that the biotransformation of α-HCH occurred in these compartments. Moreover, the increase of carbon and chlorine isotopic compositions as well as the changes of enantiomer fractionation of liver, fat tissues and milk demonstrated that the overall HCH exposure was much higher than estimates based on concentration levels, while the isotope and enantiomer fractionation revealed the enantiomer specific enantiomer uptake across the blood-brain barriers. Dual element isotope analysis suggested that complex transformation processes have occurred along the potential food web from the HCH sources over different environmental compartments to animal livers, fat tissues and milk. The results imply that the analyses of stable isotope compositions and concentrations has potential to reconstruct the exposure of higher organisms to POPs.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

Effects of HCO3- and CO2 conversion rates on carbon assimilation strategies in marine microalgae: Implication by stable carbon isotope analysis of fatty acids.

Marine microalgae are an essential component of marine plankton and critical primary producers, playing a vital role in marine ecosystems. The seawater carbonate system is a dynamic equilibrium system, and changes in any component can alter the carbonate balance. In CO2-concentrating mechanisms (CCMs), carbonic anhydrase (CA) regulates CO2 concentration by catalyzing the interconversion between CO2 and HCO3-. Therefore, limiting the activity of extracellular carbonic anhydrase (exCA) alters the rate at which carbonate equilibrium is reached and further affects the carbon assimilation process in microalgae. In this study, two different microalgae, Phaeodactylum tricornutum and Nannochloropsis oceanica, were selected to investigate the effects of changes in the carbonate system on photosynthetic carbon assimilation in microalgae by inhibiting exCA activity with acetazolamide (AZ). Inhibition of exCA activity reduces specific growth rates and photosynthetic efficiency of microalgae. The total alkalinity, HCO3- concentration, and CO2 concentration of the cultures increased with the decrease of pH, but the changes of the ribulose 1,5- bisphosphate carboxylase/oxygenase (Rubisco) activities of the two microalgae were different. In addition, the two microalgae possessed different lipid and carbohydrate synthesis strategies, but both restricted triacylglycerol (TAG) synthesis. Meanwhile, the microalgal cells had to utilize more 13CO2 when HCO3- and CO2 conversion rates were limited and restricted. This led to the continuous accumulation of 13C in fatty acids and the elevation of δ13CFAs. In conclusion, our study provides a new perspective on the role of microalgae in correlating carbonate changes with photosynthetic carbon assimilation strategies under mechanistic constraints on inorganic carbon utilization.