Isotope Tracer Method Research Articles

Construction of complete degradation pathway for nitrobenzene in Escherichia coli

Nitrobenzene is widely present in industrial wastewater and soil. Biodegradation has become an ideal method to remediate organic pollutants due to its low cost, high efficiency, and absence of secondary pollution. In the present study, 10 exogenous genes that can completely degrade nitrobenzene were introduced into Escherichia coli, and their successful expression in the strain was verified by fluorescence quantitative polymerase chain reaction and proteomic analysis. The results of the degradation experiment showed that the engineered strain could completely degrade 4 mM nitrobenzene within 8 h. The formation of intermediate metabolites was detected, and the final metabolites entered the E. coli tricarboxylic acid cycle smoothly. This process was discovered by isotope tracing method. Results indicated the integrality of the degradation pathway and the complete degradation of nitrobenzene. Finally, further experiments were conducted in soil to verify its degradation ability and showed that the engineered strain could also degrade 1 mM nitrobenzene within 10 h. In this study, engineered bacteria that can completely degrade nitrobenzene have been constructed successfully. The construction of remediation-engineered bacteria by synthetic biology laid the foundation for the industrial application of biological degradation of organic pollutants.

Diverse nirS-type Denitrifying Bacteria Contribute to Vital Nitrogen Loss in Natural Acidic Red Soils.

Denitrifying bacteria, playing a key role in nitrogen removal in ecosystem, are highly diverse and complex in their community composition. However, there were few reports on the abundance, community composition, and the contribution to nitrogen loss of denitrifiers in natural acidic red soils. In this study, we investigated the structure and function of nirS-type denitrifying bacteria in ten natural red soil samples collected from nine provinces in southern China, based on quantitative polymerase chain reaction (qPCR) and high-throughput sequencing techniques. Nitrogen loss from microbial denitrification in red soils of southern China was estimated up to 9.86 Tg N per year based on 15N isotope tracing method. The abundance of nirS-type denitrifiers varied from 8.41 × 105 to 2.55 × 109 copies per gram of dry weight. The community of nirS-type denitrifying bacterial was revealed, which contained 50 dominant OTUs assigned to 9 clusters phylogenetically related to Marinobacter, Rhodobacter, and other uncultured species. pH was the key factor affecting both denitrification rates and community composition. Our results demonstrate that nirS-type denitrifying bacteria have higher abundance, diversity, and contribution to the nitrogen loss in natural acidic red soils of southern China.

Insulin increases placental triglyceride as a potential mechanism for fetal adiposity in maternal obesity

ObjectiveMaternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus. MethodsWe used placental villous explants and isolated trophoblasts from normal weight and obese women to assess the effect of insulin and IGF-2 on triglyceride content and insulin receptor signaling. Stable isotope tracer methods were used ex vivo to determine effect of hormone treatment on de novo lipogenesis (DNL), fatty acid uptake, fatty acid oxidation, and esterification in the placenta. ResultsHere we show that placentae from euglycemic women with normal weight and obesity both have abundant insulin receptor. Placental depth and triglyceride were greater in women with obesity compared with normal weight women. In syncytialized placental trophoblasts and villous explants, insulin and IGF-2 activate insulin receptor, induce expression of lipogenic transcription factor SREBP-1 (sterol regulatory element-binding protein 1), and stimulate triglyceride accumulation. We demonstrate elevated triglyceride is attributable to increased esterification of fatty acids, without contribution from DNL and without an acceleration of fatty acid uptake. ConclusionsOur work reveals that obesity-driven aberrations in maternal metabolism, such as hyperinsulinemia, alter placental metabolism in euglycemic conditions, and may explain the higher prevalence of excess adiposity in the newborns of obese women.

Application of deuterium oxide (D2O) isotope tracing technique for encapsulated QFN failure analysis

Polymer encapsulated components are universally used in consumable applications. However, polymer encapsulants for packages commonly are non-hermetic. The polymer encapsulants can absorb moisture from atmosphere which will post a risk to the package reliability. In this study, a type of polymer encapsulated Quad Flat No-Lead (QFN) package encountered electricity leakage after Highly Accelerated Stress Tests (HAST) 96 h. Although it is suspected that penetration of moisture into the encapsulated package caused electrical leakage failure, there is no direct evidence to prove this assumption. Traditional X-ray and Scanning Acoustic Microscopy (C-SAM) techniques cannot detect any defect of the package due to their detection limit.To solve above issues, this study employed a new deuterium oxide (D2O) isotope tracing method. The encapsulated package underwent highly Accelerated Stress Tests (HAST) with D2O, and D signal of the sample was analysed by Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). The results showed that D localized at the failure area where leakage occurred. It is direct evidence to show it is a moisture-induced leakage.

Effect of Aquaculture Reclamation on Sediment Nitrates Reduction Processes in Mangrove Wetland

Sediment denitrification, anaerobic ammonium oxidation (anammox), and nitrate dissimilation to ammonium (DNRA) play an important role in controlling the dynamics of nitrates (NOx−) and their fate in estuarine and coastal ecosystems. However, the effects of land-use change on NOx− reduction processes in mangrove sediments are still unclear. Here, we used a mud experiment method combined with a 15N stable isotope tracer method to study the mechanism and ecological environment of the change of land use pattern on the sediment NOx− reduction processes in mangrove wetlands. Our study showed that most physicochemical parameters, NOx− reduction rates, and their gene abundances varied considerably. The denitrification, anammox, and DNRA rates in mangrove sediment cores were in a range of 1.04–4.24 nmol g−1 h−1, 0.14–0.36 nmol g−1 h−1, and 0–2.72 nmol g−1 h−1, respectively. The denitrification, anammox, and DNRA rates in aquaculture sediment cores were in a range of 1.06–10.96 nmol g−1 h−1, 0.13–0.37 nmol g−1 h−1, and 0–1.96 nmol g−1 h−1, respectively. The highest values of denitrification, anammox, DNRA, the contribution of denitrification and DNRA to total NOx− reduction (DEN% and DNRA%), gene abundances (nirS, Amx 16S rRNA, and nrfA), total organic carbon (TOC), total nitrogen (TN), and TOC/TN in sediments were generally found in the top layer (0–5 cm) and then decreased with depth, while the contribution of anammox to total NOx− reduction (ANA%), Fe2+, and Fe2+/Fe3+ were generally increased with sediment depth in both mangrove and aquaculture ecosystems. When mangrove wetlands are transformed into pools, some properties (including TOC, TN, and Fe3+), DNRA rates, DRNA%, and nrfA gene abundances were decreased, while some properties (including NH4+, TOC/TN, Fe2+, and Fe2+/Fe3+), denitrification rates, DEN%, nirS, and ANAMMOX 16S gene abundances were increased. Sediment organic matter (TOC and TN) content and Fe2+ both affected NO3− reduction rates, with organic matter the most prominent factor. Thus, aquaculture reclamation enhances N loss while reducing N retention in sediments of mangrove wetlands, which plays an important role in regulating the source and fate of reactive N in mangrove ecosystems.

Removal efficacy and mechanism of nitrogen and phosphorus by biological aluminum-based P-inactivation agent (BA-PIA)

In this study, aluminum-based P-inactivation agent (Al-PIA) was used as a high-efficiency microbial carrier, and the biological Al-PIA (BA-PIA) was prepared by artificial aeration. Laboratory static experiments were conducted to study the effect of BA-PIA on reducing nitrogen and phosphorus contents in water. Physicochemical characterization and isotope tracing method were applied to analyze the removal mechanism of nitrogen and phosphorus. High-throughput techniques were used to analyze the characteristic bacterial genus in the BA-PIA system. The nitrogen and phosphorus removal experiment was conducted for 30 days, and the removal rates of NH4+-N, TN and TP by BA-PIA were 81.87%, 66.08% and 87.97%, respectively. The nitrogen removal pathways of BA-PIA were as follows: the nitrification reaction accounted for 59.0% (of which denitrification reaction accounted for 56.4%), microbial assimilation accounted for 18.1%, and the unreacted part accounted for 22.9%. The characteristic bacteria in the BA-PIA system were Streptomyces, Nocardioides, Saccharopolyspora, Nitrosomonas, and Marinobacter. The loading of microorganisms only changed the surface physical properties of Al-PIA (such as specific surface area, pore volume and pore size), without changing its surface chemical properties. The removal mechanism of nitrogen by BA-PIA is the conversion of NH4+-N into NO2−-N and NO3−-N by nitrifying bacteria, which are then reduced to nitrogen-containing gas by aerobic denitrifying bacteria. The phosphorus removal mechanism is that metal compounds (such as Al) on the surface of BA-PIA fix phosphorus through chemisorption processes, such as ligand exchange. Therefore, BA-PIA overcomes the deficiency of Al-PIA with only phosphorus removal ability, and has better application prospects.

Hypoxia Enhances HIF1α Transcription Activity by Upregulating KDM4A and Mediating H3K9me3, Thus Inducing Ferroptosis Resistance in Cervical Cancer Cells.

Objective Cervical cancer (CC) is a prevalent cancer in women. Hypoxia plays a critical role in CC cell ferroptosis resistance. This study explored the mechanism of hypoxia in CC cell ferroptosis resistance by regulating HIF1α/KDM4A/H3K9me3. Methods Cultured SiHa and Hela cells were exposed to CoCl2 and treated with Erastin. Cell viability was detected by MTT assay, and concentrations of iron ion, MDA and GSH were determined using corresponding kits. Expressions of KDM4A, HIF1α, TfR1, DMT1, and H3k9me3 were detected by RT-qPCR, Western blot, and ChIP assay. The correlation of KDM4A and HIF1α was analyzed on Oncomine, UALCAN, and Starbase. CC cells were co-transfected with shKDM4A or/and pcDNA3.1-HIF1α. Iron uptake and release were assessed using the isotopic tracer method. The binding relationship between HIF1α and HRE sequence was verified by dual-luciferase assay. Results Cell viability and GSH were decreased while iron concentration, MDA, KDM4A, and HIF1α levels were increased in hypoxia conditions. The 2-h hypoxia induced ferroptosis resistance. KDM4A and HIF1α were highly-expressed in CC tissues and positively correlated with each other. KDM4A knockdown attenuated cell resistance to Erastin, increased H3K9me3 level in the HIF1α promoter region, and downregulated HIF1α transcription and translation. H3K9me3 level was increased in the HIF1α promoter after hypoxia. HIF1α overexpression abrogated the function of KDM4A knockdown on ferroptosis in hypoxia conditions. Iron uptake/release and TfR1/DMT1 levels were increased after hypoxia. Hypoxia activated HRE sequence in TfR1 and DMT1 promoters. Conclusion Hypoxia upregulated KDM4A, enhanced HIF1α transcription, and activated HRE sequence in TfR1 and DMT1 promoters via H3K9me3, thus inducing ferroptosis resistance in CC cells.

Resistant soil carbon is more vulnerable to priming effect than active soil carbon

The newly added exogenous organic matter may change the decomposition rate of native soil organic matter (SOM) by the priming effect (PE) and further impact the terrestrial carbon (C) balance. Earth system models have not yet considered the distinct responses of soil C pools with different stability and turnover rates to PE. We addressed this knowledge gap by incubating three soils (from the same site but at different stages of SOM decomposition) developed under C3 vegetation with or without the addition of maize straw (C4) and its pyrolysis product-biochar (C4) in a 180 d-incubation at 22 and 32 °C. The three soils were sampled from 15-year old-field, 15-year bare-fallow, and 23-year bare-fallow plus additional laboratory 815-d incubation, representing active (old-field soil) and relatively resistant (two bare-fallow soils, with more stable fraction and less labile fraction) soil C pools, respectively. Soil-derived CO2 was distinguished by a two-component mixing model using a natural 13C isotopic tracing method (C3 vs. C4). The PE was by 30%–239% higher in two bare-fallow soils than in old-field soil regardless of types of new C input (straw and biochar) or temperatures (22 and 32 °C), and the straw-induced PE was by 90%–297% higher than the biochar-induced PE regardless of soils or temperatures. Moreover, the straw-induced PE was not altered with warming in the three soils, but the biochar-induced PE was significantly decreased by warming, especially in old-field soil. These results suggested that the PE varies with soil C pools with different stability. The relatively resistant soil C pool is more vulnerable to PE than the active soil C pool, but the resistant soil C pool can sequestrate more exogenous C and result in higher soil C storage than the active soil C pool, at least in the short term. In addition, more biochar-C can be retained in these soils than straw-C, but this difference would decrease with warming. Overall, this study suggests that future experimental and modeling studies should pay attention to the distinct vulnerability of soil C pools with different stability to PE and warming for accurately predicting soil C dynamics.

Gasification Decoupling during Pressurized Oxy-Coal Combustion by the Isotope Tracer Method

Gasification Decoupling during Pressurized Oxy-Coal Combustion by the Isotope Tracer Method

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats, a pilot study.

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with 15N and 2H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of 13C-spirulina. Blood samples were collected 0, 1h and 3h after meal ingestion from the tail vein. The rats were euthanized 4h (n = 6) or 6h (n = 6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of 15N and 2H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal 15N or 2H/13C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

A Cycle of Inflammatory Adipocyte Death and Regeneration in Murine Adipose Tissue.

Adipose tissue (AT) expands by a combination of two fundamental cellular mechanisms: hypertrophic growth of existing adipocytes or through generation of new adipocytes, also known as hyperplastic growth. Multiple lines of evidence suggest a limited capacity for hyperplastic growth of AT in adulthood and that adipocyte number is relatively stable, even with fluctuations in AT mass. If the adipocyte number is stable in adulthood, despite well-documented birth and death of adipocytes, then this would suggest that birth may be coupled to death in a regenerative cycle. To test this hypothesis, we examined the dynamics of birth of new fat cells in relationship to adipocyte death by using high-fidelity stable isotope tracer methods in C57Bl6 mice. We discovered birth of new adipocytes at higher frequency in histological proximity to dead adipocytes. In diet-induced obesity, adipogenesis surged after an adipocyte death peak beyond 8 weeks of high-fat feeding. Through transcriptional analyses of AT and fractionated adipocytes, we found that the dominant cell death signals were inflammasome related. Proinflammatory signals were particularly evident in hypertrophied adipocytes or with deletion of a constitutive oxygen sensor and inhibitor of hypoxia-inducible factor, Egln1. We leveraged the potential role for the inflammasome in adipocyte death to test the adipocyte death-birth hypothesis, finding that caspase 1 loss of function attenuated adipocyte death and birth in murine visceral AT. These data collectively point to a regenerative cycle of adipocyte death and birth as a driver of adipogenesis in adult murine AT.

Atomic-level insight into effect of substrate concentration and relative humidity on photocatalytic degradation mechanism of gaseous styrene

Substrate concentration and relative humidity (RH) impact the photocatalytic efficiency of industrial aromatic hydrocarbons, but how they influence intermediate formation and degradation pathway remains unclear. With the help of oxygen isotope tracing method, the effects of these two environmental parameters on degradation mechanism of styrene were revealed at atomic level. Increasing styrene concentration favored product formation, which was however inhibited by RH elevation. Gaseous products were not directly formed in gaseous phase, but originated from desorption of interfacial intermediates. The volatile aldehydes and furans further exchanged their 16O with 18O in H218O. Increase of RH showed higher enhancement on 18O distribution in all products and pathways than that of substrate concentration. Low RH preferred high generation of 16O2•- and (16)1O2, dominating reaction to form 1-phenyl-1,2-ethandiol, 2-hydroxy-1-phenyl-ethanon and phenylglyoxal monohydrate in sequence. Successive production of benzyl alcohol, benzaldehyde and benzoic acid through the reaction of styrene with promoted •18OH by increasing RH became predominant. Hydration was firstly observed and confirmed as an important gaseous transformation step of aldehyde and furan products. Our findings provide a deep insight into photocatalytic degradation mechanism of aromatic hydrocarbons regulated by environmental parameters to further improve their industrial purification efficiency, and are helpful predicting environmental geochemistry fate of organics and preventing their negative impact on natural environment.

Source tracing of potentially toxic elements in soils around a typical coking plant in an industrial area in northern China

Coking plants are a substantial source of potentially toxic elements (PTEs) in soil. In this study, we examined the concentration of PTEs, the soil physicochemical properties, and the Pb isotopes in the soil inside and around a coking plant in an industrial city in northern China. We analyzed the spatial distribution of PTEs and the pollution risk areas by Igeo index, the enrichment factor (EF), and the Nemerow index, and we quantitatively identified the contribution of PTE pollution sources in the soil on a small- and medium-scale (plant and work section). Our results indicated that the Hg concentration inside the plant and the Cd concentration in the agricultural land around the plant were both relatively high. A comprehensive analysis of the soil in the study area was performed using the positive matrix factorization model and Pb isotope (206/207Pb, 208/206Pb) tracing method, based on the MixSIAR model, this analysis indicated that burning coal was the main source of Pb both inside (46.8%) and outside (26.3%) the coking plant. The pollution emission sources with significant influence on the soil outside the coking plant were diesel vehicles (12.5%), gas tanks (12.4%), and coke ovens (11.5%), while the sources inside the plant were quenching sections (11.1%), atmospheric deposition (11.0%), coke oven sections (9.6%), and diesel vehicles (6.1%). The results of PTE pollution risk zoning and Pb isotope tracing indicated that pollution is more serious in the western part of the plant, which is the area where coking and gas production takes place, and the most serious pollution outside the plant is mainly distributed to the southeast. This study provides theoretical and practical data indicating the contribution of industrial enterprises to soil pollution, and will help identify pollution responsibility and the management of pollution sources.

Editorial introductions.

Editorial introductions.

Field to laboratory comparison of metal accumulation on aged microplastics in coastal waters

The ubiquity of microplastics in the environment has attracted much attention on their risks. Though newly produced plastics were considered inert to aqueous metals, a few studies suggest aged microplastics can accumulate metals. Still, knowledge gap exists on the comparability of metal accumulation in field condition and that acquired in controlled laboratory settings. Accordingly, we comparatively assessed the field accumulation and laboratory adsorption of metals on aged microplastics in coastal waters. Microplastics of different polymeric types were aged for 8 weeks at three coastal sites with different contamination levels. Microplastics accumulated metals to substantial concentrations during ageing (median concentrations, μg g−1: Fe = 950, Mn = 94, Zn = 19, Cu = 2.8, Ni = 1.7, Pb = 1.6, and Cd = 0.005). Adsorption capacity of (aged) microplastics was evaluated in laboratory using a stable isotope tracer method. At environmentally realistic concentrations (μg L−1, 114Cd = 1.7, 65Cu = 4.4, 62Ni = 5.4, 206Pb = 0.5, and 68Zn = 13), the median concentrations of newly adsorbed isotopes on the aged microplastics were 0.01, 1.4, 0.07, 0.56, and 1.1 μg g−1, respectively, one to two orders of magnitude higher than those adsorbed on pristine microplastics. However, the composition pattern of metals accumulated on aged microplastics differed from the composition of metals newly adsorbed in laboratory: the prior one reflected the contamination status of ageing sites and varied by polymeric types; whereas the laboratory newly adsorbed metals on aged microplastics were uniformly correlated to particulate Fe and Mn concentrations, suggesting Fe and Mn mineral coatings mediated the ensuing metal adsorption. Such discrepancy unveiled the complexity of metal accumulation behavior in the real environment and highlighted that cares should be taken when translating laboratory findings to risk assessment of metal contaminated microplastics in the real environment.

Recent advances in measuring and understanding the regulation of exercise-mediated protein degradation in skeletal muscle.

Skeletal muscle protein turnover plays a crucial role in controlling muscle mass and protein quality control, including sarcomeric (structural and contractile) proteins. Protein turnover is a dynamic and continual process of protein synthesis and degradation. The ubiquitin proteasome system (UPS) is a key degradative system for protein degradation and protein quality control in skeletal muscle. UPS-mediated protein quality control is known to be impaired in aging and diseases. Exercise is a well-recognized, nonpharmacological approach to promote muscle protein turnover rates. Over the past decades, we have acquired substantial knowledge of molecular mechanisms of muscle protein synthesis after exercise. However, there have been considerable gaps in the mechanisms of how muscle protein degradation is regulated at the molecular level. The main challenge to understand muscle protein degradation is due in part to the lack of solid stable isotope tracer methodology to measure muscle protein degradation rate. Understanding the mechanisms of UPS with the concomitant measurement of protein degradation rate in skeletal muscle will help identify novel therapeutic strategies to ameliorate impaired protein turnover and protein quality control in aging and diseases. Thus, the goal of this present review was to highlight how recent advances in the field may help improve our understanding of exercise-mediated protein degradation. We discuss 1) the emerging roles of protein phosphorylation and ubiquitylation modifications in regulating proteasome-mediated protein degradation after exercise and 2) methodological advances to measure in vivo myofibrillar protein degradation rate using stable isotope tracer methods.

Microbial nitrogen loss by coupled nitrification to denitrification and anammox in a permeable subterranean estuary at Gloucester Point, Virginia

The nitrogen (N) loss processes have not been well examined in subterranean estuaries (STEs) between land and sea. We utilized a 15N isotope tracer method, q-PCR, and high-throughput sequencing to reveal the activities, abundances, and community compositions of N loss communities in a STE in Gloucester Point, Virginia, US. The highest activities, abundances and diversity of denitrifiers and anammox bacteria were detected at 50–60 cm depth in the aerobic-anaerobic transition zone (AATZ) characterized by sharp redox gradients. nirS-denitrifiers and anammox bacteria were affiliated to 10 different clusters and three genera, respectively. Denitrification and anammox played equal roles with an estimated N loss of 13.15 mmol N m−3 day−1. A positive correlation between ammonia oxidizing prokaryote abundances and DO as well as NOx− suggested that nitrification produces NOx− which supports the hotspot of denitrification and anammox within the AATZ. Overall, these results highlight the roles of N loss communities in STEs.

A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo.

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short‐term (days‐weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope‐ratio mass spectrometry (GC‐pyrolysis‐IRMS). Forty‐six male and female rats from a selectively bred model ingested D2O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T‐PRO), and distal (T‐DIS) epiphyses‐metaphyses and tibia mid‐shaft diaphyses (T‐MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein‐bound alanine via GC‐pyrolysis‐IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106–0.156]) was greater than the FSR at T‐MID (0.055 ± 0.049%/day; 95% CI [0.040–0.070]; p < 0.001). The T‐PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166–0.241]) and T‐DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022–0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site‐specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.

Habitat heterogeneity induces regional differences in sediment nitrogen fixation in eutrophic freshwater lake

Biological nitrogen fixation (BNF) in sediments is an important source of bioavailable nitrogen in aquatic systems. However, the effect of habitat change caused by eutrophication on nitrogen fixation within sediments is still unclear. In this study, nitrogen fixation rates and diazotroph diversities in sediments with heterogeneous ecological status in one eutrophic lake were investigated by using an isotope tracer method and sequencing of nitrogen-fixing (nif) genes. The results showed that both nitrogenase activity (NA) and nifH abundance in sediments of blooms area were higher than those in vegetation-dominated habitats. Correlation analysis showed that NA was correlated closely to nifH abundance, dissolved sulfide, and iron. The diazotrophic assemblage contained mainly Proteobacterial sequences belonging to Cluster I and III, and the variations of diazotrophic community could be explained by total nitrogen content, total phosphorus content, organic matters, sulfides, ammonium and iron content. Moreover, the co-occurrence network analysis showed the Alphaproteobacteria shaped the major interactions in diazotrophic community, and sediment properties had stronger effect on diazotrophic community in cyanobacteria-dominated habitat. This study revealed that habitat heterogeneity in eutrophic lakes shaped different succession of BNF in sediments and cyanobacterial blooms significantly improved the nitrogen-fixing activity in sediments, which broadened our understanding of nitrogen cycle and nutrient management in eutrophic freshwater lakes.

The dynamics and stoichiometry of dissolved organic carbon release by kelp.

Canopy-forming kelps are foundational species in coastal ecosystems, fixing tremendous amounts of carbon, yet we know little about the ecological and physiological determinants of dissolved organic carbon (DOC) release by kelps. We examined DOC release by the bull kelp, Nereocystis luetkeana, in relation to carbon fixation, nutrient uptake, tissue nitrogen content, and light availability. DOC release was approximately 3.5 times greater during the day than at night. During the day, N. luetkeana blades released an average of 16.2% of fixed carbon as DOC. Carbon fixation increased with light availability but DOC release did not, leading to a lower proportion of fixed carbon released as DOC at high light levels. We found no relationship between carbon fixation and DOC release rates measured concurrently. Rather, DOC release by N. luetkeana blades declined with marginal significance as blade tissue nitrogen content increased and with experimental nitrate addition, supporting the role of stoichiometric relationships in DOC release. Using a stable isotope (13 C) tracer method, we demonstrated that inorganic carbon is rapidly fixed and released by N. luetkeana blades as 13 DOC, within hours. However, recently fixed carbon (13 DOC) comprised less than 20% of the total DOC released, indicating that isotope studies that rely on tracer production alone may underestimate total DOC release, as it is decoupled from recent kelp productivity. Comparing carbon and nitrogen assimilation dynamics of the annual kelp N. luetkeana with the perennial kelp Macrocystis pyrifera revealed that N. luetkeana had significantly higher carbon fixation, DOC production and nitrogen uptake rates per unit dry mass. Both kelp species were able to perform light-independent carbon fixation at night. Carbon fixation by the annual kelp N. luetkeana is as high as 2.35kg C·m-2 ·yr-1 , but an average of 16% of this carbon (376g C·m-2 ·yr-1 ) is released as DOC. As kelp forests are increasingly viewed as vehicles for carbon sequestration, it is important to consider the fate of this substantial quantity of DOC released by canopy-forming kelps.