Galactosamine and mannosamine are integral parts of bacterial and fungal extracellular polymeric substances.

Extracellular polymeric substances (EPS) are produced by microorganisms and interact to form a complex matrix called biofilm. In soils, EPS are important contributors to the microbial necromass and, thus, to soil organic carbon (SOC). Amino sugars (AS) are used as indicators for microbial necromass in soil, although the origin of galactosamine and mannosamine is largely unknown. However, indications exist that they are part of EPS. In this study, two bacteria and two fungi were grown in starch medium either with or without a quartz matrix to induce EPS production. Each culture was separated in two fractions: one that directly underwent AS extraction (containing AS from both biomass and EPS), and another that first had EPS extracted, followed then by AS determination (exclusively containing AS from EPS). We did not observe a general effect of the quartz matrix neither of microbial type on AS production. The quantified amounts of galactosamine and mannosamine in the EPS fraction represented on average 100% of the total amounts of these two AS quantified in cell cultures, revealing they are integral parts of the biofilm. In contrast, muramic acid and glucosamine were also quantified in the EPS, but with much lower contribution rates to total AS production, of 18% and 33%, respectively, indicating they are not necessarily part of EPS. Our results allow a meaningful ecological interpretation of mannosamine and galactosamine data in the future as indicators of microbial EPS, and also attract interest of future studies to investigate the role of EPS to SOC and its dynamics.

Intracellular carbon storage by microorganisms is an overlooked pathway of biomass growth.

The concept of biomass growth is central to microbial carbon (C) cycling and ecosystem nutrient turnover. Microbial biomass is usually assumed to grow by cellular replication, despite microorganisms' capacity to increase biomass by synthesizing storage compounds. Resource investment in storage allows microbes to decouple their metabolic activity from immediate resource supply, supporting more diverse microbial responses to environmental changes. Here we show that microbial C storage in the form of triacylglycerides (TAGs) and polyhydroxybutyrate (PHB) contributes significantly to the formation of new biomass, i.e. growth, under contrasting conditions of C availability and complementary nutrient supply in soil. Together these compounds can comprise a C pool 0.19 ± 0.03 to 0.46 ± 0.08 times as large as extractable soil microbial biomass and reveal up to 279 ± 72% more biomass growth than observed by a DNA-based method alone. Even under C limitation, storage represented an additional 16-96% incorporation of added C into microbial biomass. These findings encourage greater recognition of storage synthesis as a key pathway of biomass growth and an underlying mechanism for resistance and resilience of microbial communities facing environmental change.

Evidence of considerable C and N transfer from peas to cereals via direct root contact but not via mycorrhiza.

Intercropping of legumes and cereals is an important management method for improving yield stability, especially in organic farming systems. However, knowledge is restricted on the relevance of different nutrient transfer pathways. The objective of the study was to quantify nitrogen (N) and carbon (C) transfer from peas to triticale by (1) direct root contact (= R), (2) arbuscular mycorrhizal fungi (AMF; = A), and (3) diffusion (= D). Pea (Pisum sativum cv. Frisson and P2) and triticale (Triticum × Secale cv. Benetto) plants as intercrop were grown for 105 days. Treatment ADR enabled all transfer paths between the two crops. Treatment AD with root exclusion enabled AMF and diffusion transfer between peas and triticale. Treatment A with a diffusion gap barrier only allowed AMF transfer. Pea plants were labelled every 14 days with a 13C glucose and 15N urea solution, using the cotton wick technique. Direct root contact resulted in the highest pea rhizodeposition and thus the largest absolute amounts of N and C transfer to triticale. Root exclusion generally changed composition of rhizodeposits from fine root residues towards root exudates. Pea plant-N consisted of 17% N derived from rhizodeposition (NdfR) in treatment ADR but only 8% in the treatments AD and A, independently of pea variety, whereas pea plant-C consisted of 13% C derived from rhizodeposition (CdfR), without pea variety and transfer path treatment effects. Averaging all transfer path treatments, 6.7% of NdfR and 2.7% of CdfR was transferred from Frisson and P2 to triticale plants. Approximately 90% of this NdfR was transferred by direct root contact from Frisson to triticale and only 10% by AMF, whereas only 55% of CdfR was transferred to triticale by direct root contact, 40% by AMF and 5% by diffusion. Similar percentages were transferred from mutant P2 to triticale. Root exclusion generally changed RD composition from fine root residues towards root exudates.

Nitrite isotope characteristics and associated soil N transformations.

Nitrite (NO2-) is a crucial compound in the N soil cycle. As an intermediate of nearly all N transformations, its isotopic signature may provide precious information on the active pathways and processes. NO2- analyses have already been applied in 15N tracing studies, increasing their interpretation perspectives. Natural abundance NO2- isotope studies in soils were so far not applied and this study aims at testing if such analyses are useful in tracing the soil N cycle. We conducted laboratory soil incubations with parallel natural abundance and 15N treatments, accompanied by isotopic analyses of soil N compounds (NO3-, NO2-, NH4+). The double 15N tracing method was used as a reference method for estimations of N transformation processes based on natural abundance nitrite dynamics. We obtained a very good agreement between the results from nitrite isotope model proposed here and the 15N tracing approach. Natural abundance nitrite isotope studies are a promising tool to our understanding of soil N cycling.

Nitrogen isotope analysis of aqueous ammonium and nitrate by membrane inlet isotope ratio mass spectrometry (MIRMS) at natural abundance levels.

RATIONALE: Existing methods for the measurement of the 15 N/14 N isotopic composition of ammonium and nitrate are either only suitable for labelled samples or require considerable sample preparation efforts (or both). Our goal was to modify an existing analytical approach to allow for natural abundance precision levels. METHODS: Published reaction protocols were used to convert ammonium into N2 by NaOBr and nitrate into N2 O by TiCl3 . A membrane inlet system was developed and coupled to an isotope ratio mass spectrometer to allow precise determination of the analytes. RESULTS: Concentrations of ≥35 µmol/L N for both ammonium or nitrate could be analysed for δ15 N values with precisions of better than 0.9 mUr. While ammonium analyses exhibited a small concentration dependency and an offset of 2.7 mUr at high ammonium concentrations irrespective of the standard isotopic composition, nitrate analysis showed no offset but a blank contribution visible at very low concentrations. CONCLUSIONS: The presented method is capable of fast measurement of δ15 N values in ammonium and nitrate from aqueous samples with reasonable accuracy at natural abundance levels. It will thus facilitate the application of isotopic methods to studies of nitrogen cycling in ecosystems.

Contribution of the Fenton reaction and ligninolytic enzymes to soil organic matter mineralisation under anoxic conditions.

Mechanisms of carbon dioxide (CO2) release from soil in the absence of oxygen were studied considering the Fenton process, which encompasses the reaction of H2O2 with Fe(II) yielding a hydroxyl radical (OH), in combination with manganese peroxidase (MnP) and lignin peroxidase (LiP). This study aimed to explain the high rate of soil organic matter (SOM) mineralisation and CO2 release from humid temperate rainforest soils under oxygen-limited conditions. The investigated mechanisms challenge the traditional view that SOM mineralisation in rainforest is slow due to anaerobic (micro)environments under high precipitation and explain intensive CO2 release even under oxygen limitation. We hypothesised that the Fenton reaction (FR) greatly contributes to the CO2 released from SOM mineralised under anaerobic conditions especially in the presence of ligninolytic enzymes. We used a novel technique that combines labelled H218O2 and Fe(II) to induce the FR and measured CO18O, Fe(II) solubilisation, and peroxide consumption in a closed gas circulation system for 6 h. Maximal CO2 amount was released when the FR was induced in combination with LiP addition. The CO2 efflux with LiP was 10-fold that of abiotic FR reactions without enzymes, or in soils amended with MnP. This was consistent with i) the contribution of 18O from peroxide to CO2 release, ii) peroxide consumption, and iii) Fe(II) solubilisation by FR. The amount of consumed peroxide was closely correlated with the CO18O derived from soil without enzyme addition or with LiP addition. Concluding, abiotic Fenton Reaction coupled with oxidative enzymes, such as LiP, are crucial for SOM oxidation under anaerobic conditions, e.g. in temperate rainforest soils.

NO Reduction to N2O Improves Nitrate 15N Abundance Analysis by Membrane Inlet Quadrupole Mass Spectrometry.

Earlier an automated sample preparation unit for inorganic nitrogen (SPIN) coupled to a membrane inlet quadrupole mass spectrometer (MIMS) was developed for automated and sensitive determination of the 15N abundances and concentrations of nitrate, nitrite and ammonium of aqueous solutions without any sample preparation. Here we describe an alternative analytical protocol to convert NO3- to N2O instead of NO before measurement. This is advantageous because NO strongly interacts with surfaces, requires long purge times, and still shows considerable carryover between samples, all of which is avoided when N2O is used as analyte. The sensitivity of the measurement of NO3- as N2O is comparable to the earlier measurements with NO as analyte.

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.

A closer look into the nitrogen blank in elemental analyser/isotope ratio mass spectrometry measurements.

RATIONALE: One important limitation for the precise measurement of minute amounts of nitrogen (N) in solid samples by elemental analyser/isotope ratio mass spectrometry (EA/IRMS) is the accurate determination of the analyser blank value. This study was performed to identify different sources, amounts and isotopic composition of N blanks in EA/IRMS in order to identify measures for minimising the effect of the N blank on N isotopic data quality. METHODS: Different types of autosamplers, with and without zero-blank functionality, were tested by analysing different amounts of substances of varying isotopic composition by EA/IRMS. RESULTS: Using zero-blank autosamplers reduces the atmospheric N2 blank from 60 nmol to between 4 and 5 nmol depending on the autosampler type. This blank is derived from atmospheric N2 leaking into the elemental analyser, trapped in the sample tin capsules or contained in the oxygen added for combustion. Another source of blank is the reaction tube. As the sources of the blank differ, the isotopic composition of the blank is very variable. In addition, cross-contamination from previous samples may contribute up to 3.3 nmol N. CONCLUSIONS: For precise measurements of minute amounts of N in solid samples, reduction of the N blank is the most promising strategy. Correcting for the remaining N blank is only meaningful if the sample isotopic composition is very different from that of the N blank, because the precise determination of the isotopic composition of the N blank is not possible.

Measuring 15N Abundance and Concentration of Aqueous Nitrate, Nitrite, and Ammonium by Membrane Inlet Quadrupole Mass Spectrometry.

An automated sample preparation unit for inorganic nitrogen (SPIN) coupled to a membrane inlet quadrupole mass spectrometer (MIMS) was developed for automated and sensitive determination of the 15N abundances and concentrations of nitrate, nitrite, and ammonium in aqueous solutions without any sample preparation. The minimum N concentration for an accurate determination of the 15N abundance is 7 µmol/L for nitrite and nitrate, with a relative standard deviation (RSD) of repeated measurements of <1%, and 70 µmol/L with an RSD < 0.4% in the case of ammonium. The SPIN-MIMS system provides a wide dynamic range (up to 3500 µmol/L) for all three N species for both isotope abundance and concentration measurements. The comparison of parallel measurements of 15N-labeled NH4+ and NO3- from soil extracts with the denitrifier method and the SPIN-MIMS system shows a good agreement between both methods.

Automated system measuring triple oxygen and nitrogen isotope ratios in nitrate using the bacterial method and N2 O decomposition by microwave discharge.

RATIONALE: Triple oxygen and nitrogen isotope ratios in nitrate are powerful tools for assessing atmospheric nitrate formation pathways and their contribution to ecosystems. N2 O decomposition using microwave-induced plasma (MIP) has been used only for measurements of oxygen isotopes to date, but it is also possible to measure nitrogen isotopes during the same analytical run. METHODS: The main improvements to a previous system are (i) an automated distribution system of nitrate to the bacterial medium, (ii) N2 O separation by gas chromatography before N2 O decomposition using the MIP, (iii) use of a corundum tube for microwave discharge, and (iv) development of an automated system for isotopic measurements. Three nitrate standards with sample sizes of 60, 80, 100, and 120 nmol were measured to investigate the sample size dependence of the isotope measurements. RESULTS: The δ17 O, δ18 O, and Δ17 O values increased with increasing sample size, although the δ15 N value showed no significant size dependency. Different calibration slopes and intercepts were obtained with different sample amounts. The slopes and intercepts for the regression lines in different sample amounts were dependent on sample size, indicating that the extent of oxygen exchange is also dependent on sample size. The sample-size-dependent slopes and intercepts were fitted using natural log (ln) regression curves, and the slopes and intercepts can be estimated to apply to any sample size corrections. When using 100 nmol samples, the standard deviations of residuals from the regression lines for this system were 0.5‰, 0.3‰, and 0.1‰, respectively, for the δ18 O, Δ17 O, and δ15 N values, results that are not inferior to those from other systems using gold tube or gold wire. CONCLUSIONS: An automated system was developed to measure triple oxygen and nitrogen isotopes in nitrate using N2 O decomposition by MIP. This system enables us to measure both triple oxygen and nitrogen isotopes in nitrate with comparable precision and sample throughput (23 min per sample on average), and minimal manual treatment. Copyright © 2016 John Wiley & Sons, Ltd.

Stable isotope analysis (δ (13)C and δ (15)N) of soil nematodes from four feeding groups.

Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis ((13)C/(12)C and (15)N/(14)N, expressed as δ (13)C and δ (15)N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis-isotopic ratio mass spectrometry (µEA-IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in δ (15)N (p = 0.290) or δ (13)C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (δ (15)N = 1.08 to 3.22 mUr‰, δ (13)C = -29.58 to -27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in δ (15)N between the plant feeder and the predator group (δ (15)N = 9.89 to 12.79 mUr, δ (13)C = -27.04 to -25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr(2)) and the predators (1.73 mUr(2)), but largest for omnivores (3.83 mUr(2)). These data may reflect more preferential feeding by the plant feeder and predators, as assumed by classical morphology-based feeding groups, and indicate that omnivory may be more widespread across detritivore groups i.e. bacterial feeders (3.81 mUr(2)). Trophic information for soil nematodes derived from stable isotope analysis, scaled as finely as species level in some cases, will complement existing indices for soil biological assessment and monitoring, and can potentially be used to identify new trophic interactions in soils. The isotopic technique used here, to compare nematode feeding group members largely confirm their trophic relations based on morphological studies.

Photoautotrophic microorganisms as a carbon source for temperate soil invertebrates.

We tested experimentally if photoautotrophic microorganisms are a carbon source for invertebrates in temperate soils. We exposed forest or arable soils to a (13)CO2-enriched atmosphere and quantified (13)C assimilation by three common animal groups: earthworms (Oligochaeta), springtails (Hexapoda) and slugs (Gastropoda). Endogeic earthworms (Allolobophora chlorotica) and hemiedaphic springtails (Ceratophysella denticulata) were highly (13)C enriched when incubated under light, deriving up to 3.0 and 17.0%, respectively, of their body carbon from the microbial source in 7 days. Earthworms assimilated more (13)C in undisturbed soil than when the microbial material was mixed into the soil, presumably reflecting selective surface grazing. By contrast, neither adult nor newly hatched terrestrial slugs (Deroceras reticulatum) grazed on algal mats. Non-photosynthetic (13)CO2 fixation in the dark was negligible. We conclude from these preliminary laboratory experiments that, in addition to litter and root-derived carbon from vascular plants, photoautotrophic soil surface microorganisms (cyanobacteria, algae) may be an ecologically important carbon input route for temperate soil animals that are traditionally assigned to the decomposer channel in soil food web models and carbon cycling studies.

Comparison of methods to determine triple oxygen isotope composition of N2O.

RATIONALE: The oxygen isotope anomaly, Δ(17) O, of N2 O and nitrate is useful to elucidate nitrogen oxide dynamics. A comparison of different methods for Δ(17) O measurement was performed. METHODS: For Δ(17) O measurements, N2 O was converted into O2 and N2 using microwave-induced plasma in a quartz or corundum tube reactor, respectively, or conversion was carried out in a gold wire oven. In each case, isotope ratios were measured by isotope ratio mass spectrometry. RESULTS: All the tested methods showed acceptable precision (coefficient of variation <2.4 % at 160 nmol N2 O) with high sample size but the sample size dependence was lowest when using microwave-induced plasma in a corundum tube reactor. CONCLUSIONS: The use of microwave-induced plasma in a corundum tube yields best results for Δ(17) O measurement on N2 O gas samples.

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.

Combined ¹³C and ¹5N isotope analysis on small samples using a near-conventional elemental analyzer/isotope ratio mass spectrometer setup.

RATIONALE: A high sensitivity elemental analyzer/isotope ratio mass spectrometer setup was developed to allow analysis of (13)C and (15)N isotopic composition on microgram amounts of C and N, respectively. METHODS: Increased sensitivity of a conventional elemental analyzer equipped with a low blank autosampler was obtained by decreased carrier gas flow of 35 mL/min. The diameters of the oxidation and reduction reactors and water trap were reduced to 7.8, 7.8 and 4 mm i.d., respectively, to obtain sharp sample peaks in the mass spectrometer. To increase the lifetime of the reduction reactor, a 1:1 He/O2 mixture was used as oxidizing agent in the elemental analyzer. RESULTS: Sample amounts of 0.6 µg N and 1 µg C were sufficient for accurate isotopic analysis with <1 ‰ standard error after blank correction. One major advantage of the setup is the easy switching between conventional EA and µEA as only consumable parts need to be exchanged. CONCLUSIONS: The proposed setup proved to be suitable to analyze minute amounts of C and N in one analytical run simultaneously.

Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity.

Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide.