Significance of phosphorus deficiency for the mitigation of mercury toxicity in the Robinia pseudoacacia L.- rhizobia symbiotic association.

Nitrogen (N2)-fixing legumes can be used for phytoremediation of toxic heavy metal Mercury (Hg) contaminated soil, but N2-fixation highly relies on phosphorus (P) availability for nodule formation and functioning. Here, we characterized the significance of P deficiency for Hg accumulation and toxicity in woody legume plants. Consequences for foliar and root traits of rhizobia inoculation, Hg exposure (+Hg) and low P (-P) supply, individually and in combination were characterized at both the metabolite and transcriptome levels in seedlings of two Robinia pseudoacacia L. provenances originating from contrasting climate and soil backgrounds, i.e., GS in northwest and the DB in northeast China. Our results reveal that depleted P mitigates the toxicity of Hg at the transcriptional level. In leaves of Robinia depleted P reduced oxidative stress and improved the utilization strategy of C, N and P nutrition; in roots depleted P regulated the expression of genes scavenging oxidative stress and promoting cell membrane synthesis. Rhizobia inoculation significantly improved the performance of both Robinia provenances under individual and combined +Hg and -P by promoting photosynthesis, increasing foliar N and P content and reducing H2O2 and MDA accumulation despite enhanced Hg uptake. DB plants developed more nodules, had higher biomass and accumulated higher Hg amounts than GS plants and thus are suggested as the high potential Robinia provenance for future phytoremediation of Hg contaminated soils with P deficiency.

Biologically mediated release of endogenous N2O and NO2 gases in a hydrothermal, hypoxic subterranean environment.

The migration of geogenic gases in continental areas with geothermal activity and active faults is an important process releasing greenhouse gases (GHG) to the lower troposphere. In this respect, caves in hypogenic environments are natural laboratories to study the compositional evolution of deep-endogenous fluids through the Critical Zone. Vapour Cave (Alhama, Murcia, Spain) is a hypogenic cave formed by the upwelling of hydrothermal CO2-rich fluids. Anomalous concentrations of N2O and NO2 were registered in the cave's subterranean atmosphere, averaging ten and five times the typical atmospheric backgrounds, respectively. We characterised the thermal conditions, gaseous compositions, sediments, and microbial communities at different depths in the cave. We did so to understand the relation between N-cycling microbial groups and the production and transformation of nitrogenous gases, as well as their coupled evolution with CO2 and CH4 during their migration through the Critical Zone to the lower troposphere. Our results showed an evident vertical stratification of selected microbial groups (Archaea and Bacteria) depending on the environmental parameters, including O2, temperature, and GHG concentration. Both the N2O isotope ratios and the predicted ecological functions of bacterial and archaeal communities suggest that N2O and NO2 emissions mainly depend on the nitrification by ammonia-oxidising microorganisms. Denitrification and abiotic reactions of the reactive intermediates NH2OH, NO, and NO2- are also plausible according to the results of the phylogenetic analyses of the microbial communities. Nitrite-dependent anaerobic methane oxidation by denitrifying methanotrophs of the NC10 phylum was also identified as a post-genetic process during migration of this gas to the surface. To the best of our knowledge, our report provides, for the first time, evidence of a niche densely populated by Micrarchaeia, which represents more than 50% of the total archaeal abundance. This raises many questions on the metabolic behaviour of this and other archaeal phyla.

Indications for enzymatic denitrification to N2O at low pH in an ammonia-oxidizing archaeon.

Nitrous oxide (N2O) is a key climate change gas and nitrifying microbes living in terrestrial ecosystems contribute significantly to its formation. Many soils are acidic and global change will cause acidification of aquatic and terrestrial ecosystems, but the effect of decreasing pH on N2O formation by nitrifiers is poorly understood. Here, we used isotope-ratio mass spectrometry to investigate the effect of acidification on production of N2O by pure cultures of two ammonia-oxidizing archaea (AOA; Nitrosocosmicus oleophilus and Nitrosotenuis chungbukensis) and an ammonia-oxidizing bacterium (AOB; Nitrosomonas europaea). For all three strains acidification led to increased emission of N2O. However, changes of 15N site preference (SP) values within the N2O molecule (as indicators of pathways for N2O formation), caused by decreasing pH, were highly different between the tested AOA and AOB. While acidification decreased the SP value in the AOB strain, SP values increased to a maximum value of 29‰ in N. oleophilus. In addition, 15N-nitrite tracer experiments showed that acidification boosted nitrite transformation into N2O in all strains, but the incorporation rate was different for each ammonia oxidizer. Unexpectedly, for N. oleophilus more than 50% of the N2O produced at pH 5.5 had both nitrogen atoms from nitrite and we demonstrated that under these conditions expression of a putative cytochrome P450 NO reductase is strongly upregulated. Collectively, our results indicate that N. oleophilus might be able to enzymatically denitrify nitrite to N2O at low pH.

Improvement of the 15 N gas flux method for in situ measurement of soil denitrification and its product stoichiometry.

RATIONALE: Field measurement of denitrification in agricultural ecosystems using the 15 N gas flux method has been limited by poor sensitivity because current isotope ratio mass spectrometry is not precise enough to detect low 15 N2 fluxes in the presence of a high atmospheric N2 background. For laboratory studies, detection limits are improved by incubating soils in closed systems and under N2 -depleted atmospheres. METHODS: We developed a new procedure to conduct the 15 N gas flux method suitable for field application using an artificially N2 -depleted atmosphere to improve the detection limit at the given precision of mass spectrometry. Laboratory experiments with and without 15 N-labelling and using different flushing strategies were conducted to develop a suitable field method. Subsequently, this method was tested in the field and results were compared with those obtained from the conventional 15 N gas flux method. RESULTS: Results of the two methods were in close agreement showing that the denitrification rates determined were not biased by the flushing procedure. Best sensitivity for N2 + N2 O fluxes was 10 ppb, which was 80-fold better than that of the reference method. Further improvement can be achieved by lowering the N2 background concentration below the values established in the present study. CONCLUSIONS: In view of this progress in sensitivity, the new method will be suitable to measure denitrification dynamics in the field beyond peak events.

Nitrous oxide effluxes from plants as a potentially important source to the atmosphere.

The global budget for nitrous oxide (N2 O), an important greenhouse gas and probably dominant ozone-depleting substance emitted in the 21st century, is far from being fully understood. Cycling of N2 O in terrestrial ecosystems has traditionally exclusively focused on gas exchange between the soil surface (nitrification-denitrification processes) and the atmosphere. Terrestrial vegetation has not been considered in the global budget so far, even though plants are known to release N2 O. Here, we report the N2 O emission rates of 32 plant species from 22 different families measured under controlled laboratory conditions. Furthermore, the first isotopocule values (δ15 N, δ18 O and δ15 Nsp ) of N2 O emitted from plants were determined. A robust relationship established between N2 O emission and CO2 respiration rates, which did not alter significantly over a broad range of changing environmental conditions, was used to quantify plant-derived emissions on an ecosystem scale. Stable isotope measurements (δ15 N, δ18 O and δ15 Nsp ) of N2 O emitted by plants clearly show that the dual isotopocule fingerprint of plant-derived N2 O differs from that of currently known microbial or chemical processes. Our work suggests that vegetation is a natural source of N2 O in the environment with a large fraction released by a hitherto unrecognized process.

Preliminary assessment of stable nitrogen and oxygen isotopic composition of USGS51 and USGS52 nitrous oxide reference gases and perspectives on calibration needs.

RATIONALE: Despite a long history and growing interest in isotopic analyses of N2 O, there is a lack of isotopically characterized N2 O isotopic reference materials (standards) to enable normalization and reporting of isotope-delta values. Here we report the isotopic characterization of two pure N2 O gas reference materials, USGS51 and USGS52, which are now available for laboratory calibration (https://isotopes.usgs.gov/lab/referencematerials.html). METHODS: A total of 400 sealed borosilicate glass tubes of each N2 O reference gas were prepared from a single gas filling of a high vacuum line. We demonstrated isotopic homogeneity via dual-inlet isotope-ratio mass spectrometry. Isotopic analyses of these reference materials were obtained from eight laboratories to evaluate interlaboratory variation and provide preliminary isotopic characterization of their δ15 N, δ18 O, δ15 Nα , δ15 Nß and site preference (SP ) values. RESULTS: The isotopic homogeneity of both USGS51 and USGS52 was demonstrated by one-sigma standard deviations associated with the determinations of their δ15 N, δ18 O, δ15 Nα , δ15 Nß and SP values of 0.12 mUr or better. The one-sigma standard deviations of SP measurements of USGS51 and USGS52 reported by eight laboratories participating in the interlaboratory comparison were 1.27 and 1.78 mUr, respectively. CONCLUSIONS: The agreement of isotope-delta values obtained in the interlaboratory comparison was not sufficient to provide reliable accurate isotope measurement values for USGS51 and USGS52. We propose that provisional values for the isotopic composition of USGS51 and USGS52 determined at the Tokyo Institute of Technology can be adopted for normalizing and reporting sample data until further refinements are achieved through additional calibration efforts.

Influence of Lumbricus terrestris and Folsomia candida on N2 O formation pathways in two different soils - with particular focus on N2 emissions.

RATIONALE: The gaseous N losses mediated by soil denitrifiers are generally inferred by measuring N2 O fluxes, but should include associated N2 emissions, which may be affected by abiotic soil characteristics and biotic interactions. Soil fauna, particularly anecic earthworms and euedaphic collembola, alter the activity of denitrifiers, creating hotspots for denitrification. These soil fauna are abundant in perennial agroecosystems intended to contribute to more sustainable production of bioenergy. METHODS: Two microcosm experiments were designed to evaluate gaseous N emissions from a silty loam and a sandy soil, both provided with litter from the bioenergy crop Silphium perfoliatum (cup-plant) and inoculated with an anecic earthworm (Lumbricus terrestris), which was added alone or together with an euedaphic collembola (Folsomia candida). In experiment 1, litter-derived N flux was determined by adding 15 N-labelled litter, followed by mass spectrometric analysis of N2 and N2 O isotopologues. In experiment 2, the δ18 O values and 15 N site preference of N2 O were determined by isotope ratio mass spectrometry to reveal underlying N2 O formation pathways. RESULTS: Lumbricus terrestris significantly increased litter-derived N2 emissions in the loamy soil, from 174.5 to 1019.3 µg N2 -N kg-1 soil, but not in the sandy soil (non-significant change from 944.7 to 1054.7 µg N2 -N kg-1 soil). Earthworm feeding on plant litter resulted in elevated N2 O emissions in both soils, derived mainly from turnover of the soil mineral N pool during denitrification. Folsomia candida did not affect N losses but showed a tendency to redirect N2 O formation pathways from fungal to bacterial denitrification. The N2 O/(N2  + N2 O) product ratio was predominantly affected by abiotic soil characteristics (loamy soil: 0.14, sandy soil: 0.26). CONCLUSIONS: When feeding on S. perfoliatum litter, the anecic L. terrestris, but not the euedaphic F. candida, has the potential to cause substantial N losses. Biotic interactions between the species are not influential, but abiotic soil characteristics have an effect. The coarse-textured sandy soil had lower gaseous N losses attributable to anecic earthworms. Copyright © 2016 John Wiley & Sons, Ltd.

Interlaboratory assessment of nitrous oxide isotopomer analysis by isotope ratio mass spectrometry and laser spectroscopy: current status and perspectives.

RATIONALE: In recent years, research and applications of the N2O site-specific nitrogen isotope composition have advanced, reflecting awareness of the contribution of N2O to the anthropogenic greenhouse effect, and leading to significant progress in instrument development. Further dissemination of N2O isotopomer analysis, however, is hampered by a lack of internationally agreed gaseous N2O reference materials and an uncertain compatibility of different laboratories and analytical techniques. METHODS: In a first comparison approach, eleven laboratories were each provided with N2O at tropospheric mole fractions (target gas T) and two reference gases (REF1 and REF2). The laboratories analysed all gases, applying their specific analytical routines. Compatibility of laboratories was assessed based on N2O isotopocule data for T, REF1 and REF2. Results for T were then standardised using REF1 and REF2 to evaluate the potential of N2O reference materials for improving compatibility between laboratories. RESULTS: Compatibility between laboratories depended on the analytical technique: isotope ratio mass spectrometry (IRMS) results showed better compatibility for δ(15)N values, while the performance of laser spectroscopy was superior with respect to N2O site preference. This comparison, however, is restricted by the small number of participating laboratories applying laser spectroscopy. Offset and two-point calibration correction of the N2O isotopomer data significantly improved the consistency of position-dependent nitrogen isotope data while the effect on δ(15)N values was only minor. CONCLUSIONS: The study reveals that for future research on N2O isotopocules, standardisation against N2O reference material is essential to improve interlaboratory compatibility. For atmospheric monitoring activities, we suggest N2O in whole air as a unifying scale anchor.

Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification--a pure culture study.

RATIONALE: The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil has not yet been sufficiently investigated. The intramolecular (15)N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. METHODS: To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under denitrifying conditions, with either NO3(-) or NO2(-) as the electron acceptor, and measured the isotopic signature (δ(18)O, δ(15)Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. RESULTS: Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO2(-) only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of (15)Nbulk were associated with larger N2O production. The δ(18)O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. CONCLUSIONS: Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO2(-) and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ(18)O values of the N2O produced.

Fungal oxygen exchange between denitrification intermediates and water.

RATIONALE: Fungi can contribute greatly to N2O production from denitrification. Therefore, it is important to quantify the isotopic signature of fungal N2O. The isotopic composition of N2O can be used to identify and analyze the processes of N2O production and N2O reduction. In contrast to bacteria, information about the oxygen exchange between denitrification intermediates and water during fungal denitrification is lacking, impeding the explanatory power of stable isotope methods. METHODS: Six fungal species were anaerobically incubated with the electron acceptors nitrate or nitrite and (18)O-labeled water to determine the oxygen exchange between denitrification intermediates and water. After seven days of incubation, gas samples were analyzed for N2O isotopologues by isotope ratio mass spectrometry. RESULTS: All the fungal species produced N2O. N2O production was greater when nitrite was the sole electron acceptor (129 to 6558 nmol N2O g dw(-1) h(-1)) than when nitrate was the electron acceptor (6 to 47 nmol N2O g dw(-1) h(-1)). Oxygen exchange was complete with nitrate as electron acceptor in one of five fungi and with nitrite in two of six fungi. Oxygen exchange of the other fungi varied (41 to 89% with nitrite and 11 to 61% with nitrate). CONCLUSIONS: This is the first report on oxygen exchange with water during fungal denitrification. The exchange appears to be within the range previously reported for bacterial denitrification. This adds to the difficulty of differentiating N2O producing processes based on the origin of N2O-O. However, the large oxygen exchange repeatedly observed for bacteria and now also fungi could lead to less variability in the δ(18)O values of N2O from soils, which could facilitate the assessment of the extent of N2O reduction.

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils.

N2O gas is involved in global warming and ozone depletion. The major sources of N2O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N2O produced by soil AOA and associated N2O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N2O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ(15)N(bulk) and δ(18)O -N2O of soil AOA strains were 13-30%, -13 to -35% and 22-36%, respectively, and strains MY1-3 and other soil AOA strains had distinct isotopic signatures. A (15)N-NH4(+)-labeling experiment indicated that N2O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N2O from AOA may be attributable to the relative contributions of these two processes. The highest N2O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N2O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N2O emissions from soil nitrification may be attributable to AOA.

Soil denitrification potential and its influence on N2O reduction and N2O isotopomer ratios.

RATIONALE: N2O isotopomer ratios may provide a useful tool for studying N2O source processes in soils and may also help estimating N2O reduction to N2. However, remaining uncertainties about different processes and their characteristic isotope effects still hamper its application. We conducted two laboratory incubation experiments (i) to compare the denitrification potential and N2O/(N2O+N2) product ratio of denitrification of various soil types from Northern Germany, and (ii) to investigate the effect of N2O reduction on the intramolecular (15)N distribution of emitted N2O. METHODS: Three contrasting soils (clay, loamy, and sandy soil) were amended with nitrate solution and incubated under N2 -free He atmosphere in a fully automated incubation system over 9 or 28 days in two experiments. N2O, N2, and CO2 release was quantified by online gas chromatography. In addition, the N2O isotopomer ratios were determined by isotope-ratio mass spectrometry (IRMS) and the net enrichment factors of the (15)N site preference (SP) of the N2O-to-N2 reduction step (η(SP)) were estimated using a Rayleigh model. RESULTS: The total denitrification rate was highest in clay soil and lowest in sandy soil. Surprisingly, the N2O/(N2O+N2) product ratio in clay and loam soil was identical; however, it was significantly lower in sandy soil. The IRMS measurements revealed highest N2O SP values in clay soil and lowest SP values in sandy soil. The η(SP) values of N2O reduction were between -8.2 and -6.1‰, and a significant relationship between δ(18)O and SP values was found. CONCLUSIONS: Both experiments showed that the N2O/(N2O+N2) product ratio of denitrification is not solely controlled by the available carbon content of the soil or by the denitrification rate. Differences in N2O SP values could not be explained by variations in N2O reduction between soils, but rather originate from other processes involved in denitrification. The linear δ(18)O vs SP relationship may be indicative for N2O reduction; however, it deviates significantly from the findings of previous studies.

An enhanced technique for automated determination of 15N signatures of N2, (N2 +N2O) and N2O in gas samples.

RATIONALE: An enhanced analytical approach for analyzing gaseous products from (15)N-enriched pools has been developed. This technique can be used to quantify nitrous oxide (N2O) and dinitrogen (N2) fluxes from denitrification. It can also help in distinguishing different N2- and N2O-forming processes, such as denitrification, nitrification, anaerobic ammonium oxidation or co-denitrification. METHODS: The measurement instrumentation was based on a commercially available automatic preparation system allowing collection and separation of gaseous samples. The sample transfer paths, valves, liquid nitrogen traps, gas chromatography column and open split of the original system were modified. A reduction oven (Cu) was added in order to eliminate oxygen and measure N2O-N as N2. Gases leaving the separation system entered an isotope ratio mass spectrometer where masses (28)N2, (29)N2 and (30)N2 were measured. RESULTS: The enhanced technique enabled rapid simultaneous measurement of stable isotope ratios (29)N2/(28)N2 and (30)N2/(28)N2 originating from dinitrogen alone (N2) and from the sum of the denitrification products (N2 +N2O) as well as the determination of (15)N enrichment in N2O. The (15)N fraction in the N pool undergoing N2 and N2O production ((15)X(N)) and the contribution of N2 and N2O originating from this pool (d) were determined with satisfactory accuracy of better than 3.3% and 2.9%, respectively. CONCLUSIONS: The precision and accuracy of this method were comparable with or better than previously reported for similar measurements. The proposed method allows for the analysis of all quantities within one run, thus reducing the measurement and sample preparation time as well as increasing the reliability of the results.

Novel laser spectroscopic technique for continuous analysis of N2O isotopomers--application and intercomparison with isotope ratio mass spectrometry.

RATIONALE: Nitrous oxide (N(2)O), a highly climate-relevant trace gas, is mainly derived from microbial denitrification and nitrification processes in soils. Apportioning N(2)O to these source processes is a challenging task, but better understanding of the processes is required to improve mitigation strategies. The N(2)O site-specific (15)N signatures from denitrification and nitrification have been shown to be clearly different, making this signature a potential tool for N(2)O source identification. We have applied for the first time quantum cascade laser absorption spectroscopy (QCLAS) for the continuous analysis of the intramolecular (15)N distribution of soil-derived N(2)O and compared this with state-of-the-art isotope ratio mass spectrometry (IRMS). METHODS: Soil was amended with nitrate and sucrose and incubated in a laboratory setup. The N(2)O release was quantified by FTIR spectroscopy, while the N(2)O intramolecular (15)N distribution was continuously analyzed by online QCLAS at 1 Hz resolution. The QCLAS results on time-integrating flask samples were compared with those from the IRMS analysis. RESULTS: The analytical precision (2σ) of QCLAS was around 0.3‰ for the δ(15)N(bulk) and the (15)N site preference (SP) for 1-min average values. Comparing the two techniques on flask samples, excellent agreement (R(2)= 0.99; offset of 1.2‰) was observed for the δ(15)N(bulk) values while for the SP values the correlation was less good (R(2 )= 0.76; offset of 0.9‰), presumably due to the lower precision of the IRMS SP measurements. CONCLUSIONS: These findings validate QCLAS as a viable alternative technique with even higher precision than state-of-the-art IRMS. Thus, laser spectroscopy has the potential to contribute significantly to a better understanding of N turnover in soils, which is crucial for advancing strategies to mitigate emissions of this efficient greenhouse gas.

Follow-up of stable isotope analysis of organic versus conventional milk.

Analysis of the stable isotope ratio of carbon (delta (13)C) and alpha-linolenic acid (C18:3omega3) content in milk fat is a useful indicator of organic milk production. Referring to corresponding measurements, further analyses of stable isotope ratios were performed in 120 samples of conventionally and organically produced whole milk collected from German retailers during a period of 18 months. Conventional milk predominantly exhibited higher delta (15)N values than organic milk, the latter of which never exceeded a maximum delta (15)N threshold value of 5.50 per thousand. Measurements of delta (34)S did not differ significantly between organic and conventional milk. Because delta (13)C, in general, is related to maize consumption, delta (13)C in milk protein and delta (13)C in milk fat were equally suited for authentication of organic milk. Thus, a high correlation (r = 0.99) was established between delta (13)C in milk protein and lipids. Although occurring on different levels in organic and conventional milk, the relatively constant fractionation of carbon isotopes between protein and fat will allow for the advanced detection of adulteration in processed milk products, such as fraudulent combinations of organic milk fat and conventional skim milk. In addition to the strong correlation between C18:3omega3 and delta (13)C(protein) (r = -0.91), a mutual dependence was identified between both delta (13)C(protein) and delta (15)N (r = 0.66) and C18:3omega3 and delta (15)N (r = -0.61). Thus, multi-variable analyses are useful to increase robustness and reduce the number of exceptions in organic milk authentication. Future work involving multivariate statistical analysis can possibly further improve milk authentication in various respects including differentiating between brands of retail milk.

Soil carbon isotopic composition and soil carbon content in an agroecosystem during six years of Free Air Carbon dioxide Enrichment (FACE).

The Free Air Carbon dioxide Enrichment (FACE) experiment conducted at the Federal Agricultural Research Centre (FAL) in Braunschweig in an arable crop rotation (total duration six years) allowed us to trace carbon (C) input in the soil C pool, as the CO(2), used in the experiment to increase the atmospheric CO(2) concentration, was depleted in (13)C. Accurate assessment of the C input by means of stable C isotope analysis requires detailed knowledge on the spatial distribution of both the C isotopic composition and the C content in the soil C. Assumed changes in these parameters were examined. CO(2) enrichment treatment over a six year period resulted in a clear trend towards an increase of soil C content in the uppermost 10 cm of soil. About 4.9% of the soil C present under ambient air conditions, and 10.7% present under elevated CO(2) conditions were determined as new input. However, the results are not statistically significant yet.

Thermal stability of soil organic matter pools and their turnover times calculated by delta(13)C under elevated CO(2) and two levels of N fertilisation.

Soil from Free-Air Carbon dioxide Enrichment (FACE) plots (FAL, Braunschweig) under ambient air (375 ppm; delta(13)C-CO(2)-9.8 per thousand) and elevated CO(2) (550 ppm; for six years; delta(13)C-CO(2)-23 per thousand), either under 100% nitrogen (N) (180 kg ha(-1)) or 50% N (90 kg ha(-1)) fertilisation treatments, was analysed by thermogravimetry. Soil samples were heated up to the respective temperatures and the remaining soil was analysed for delta(13)C and delta(15)N by Isotope Ratio Mass Spectrometry (IRMS). Based on differential weight losses, four temperature intervals were distinguished. Weight losses in the temperature range 20-200 degrees C were connected mostly with water volatilisation. The maximum weight losses and carbon (C) content were measured in the soil organic matter (SOM) pool decomposed at 200-360 degrees C. The largest amount of N was detected in SOM pools decomposed at 200-360 degrees C and 360-500 degrees C. In all temperature ranges, the delta(13)C values of SOM pools were significantly more negative under elevated CO(2) versus ambient CO(2). The incorporation of new C into SOM pools was not inversely proportional to its thermal stability. 50% N fertilisation treatment gained higher C exchange under elevated CO(2) in the thermally labile SOM pool (200-360 degrees C), whereas 100% N treatment induced higher C turnover in the thermally stable SOM pools (360-500 degrees C, 500-1000 degrees C). Mean Residence Time of SOM under 100% N and 50% N fertilisation showed no dependence between SOM pools isolated by increasing temperature of heating and the renovation of organic C in those SOM pools. Thus, the separation of SOM based on its thermal stability was not sufficient to reveal pools with contrasting turnover rates of C.

Differentiation of organically and conventionally produced milk by stable isotope and fatty acid analysis.

Increasing sales of organic milk mean intensified tests for authenticity are required. In addition to comprehensive documentation, analytical methods to identify organic milk, and thus to differentiate it from conventional milk, are needed for consumer protection. Because the composition of milk is fundamentally dependent on the feeding of the cows, thirty-five samples from both production systems in Germany, including farm and retail milk, were collected within 12 months, to reflect seasonal variation, and appropriate properties were analysed. Fatty acid analysis enabled organic and conventional milk to be completely distinguished, because of the higher alpha-linolenic acid (C18:3omega3) and eicosapentaenoic acid (C20:5omega3) content of the former. Organic milk fat contained at least 0.56% C18:3omega3 whereas the maximum in conventional milk was 0.53%. Because of the parallel seasonal course of the C18:3omega3 content of organic and conventional retail samples, however, time-resolved comparison at the five sampling dates resulted in a clearer difference of 0.34+/-0.06% on average. Analysis of stable carbon isotopes (delta13C) also enabled complete distinction of both types of milk; this can be explained by the different amounts of maize in the feed. For conventional milk fat delta13C values were -26.6 per thousand or higher whereas for organic milk fat values were always lower, with a maximum of -28.0 per thousand. The time-resolved average difference was 4.5+/-1.0 per thousand. A strong negative correlation (r=-0.92) was found between C18:3omega3 and delta13C. Analysis of a larger number of samples is required to check the preliminary variation ranges obtained in this pilot study and, probably, to adjust the limits. Stable isotopes of nitrogen (delta15N) or sulfur (delta34S) did not enable assignment of the origin of the milk; in cases of ambiguity, however, some trends observed might be useful in combination with other properties.

Changes in soil C-isotopic composition in an agroecosystem under Free Air Carbon dioxide Enrichment (FACE) treatment during a crop rotation period.

FACE (Free Air Carbon dioxide Enrichment) has been used since 1999 to evaluate the effects of future atmospheric CO(2) concentrations on an arable crop agroecosystem. The experiment conducted at the Institute of Agroecology at the Federal Research Centre in Braunschweig consists of a typical local crop rotation of winter barley, a cover crop, sugar beet and winter wheat. The atmospheric CO2 concentration of ambient air is about 375 ppm with a delta13C value of -7 to -9 per thousand, and 550 ppm (delta13C value = -20.2 per thousand) during daylight hours in the rings fumigated with additional CO2. Thus, the surplus C can be traced in the agricultural system. Over the course of the first experimental period (3-year crop rotation period), the C-isotopic composition and the C concentration in soil were monitored monthly. Plant samples were analysed according to the relevant developmental stages of the crop under cultivation. A 13C depletion was observed in plant parts, as well as in soil samples from the FACE rings under CO2 enrichment, indicating that labelled C has reached both respective ecosystem compartments. Albeit farming management practice (especially ploughing) leads to a mixing of 'old' and 'new' C compounds throughout all soil horizons down to the end of the ploughing layer and resulted in a heterogeneous distribution of newly formed C compounds in the soil, isotope analysis of soil C reflected where the surplus C went.