The development of bacterial biofilms in natural environments may alter important functions, such as pollutant bioremediation by modifying both the degraders' physiology and/or interactions within the matrix. The present study focuses on the influence of biofilm formation on the metabolism of a pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), by Cupriavidus necator JMP134. Pure cultures were established in a liquid medium with 2,4-D as a sole carbon source with or without sand grains for 10 days. Bacterial numbers and 2,4-D concentrations in solution were followed by spectrophotometry, the respiration rate by gas chromatography and the surface colonization by electron microscopy. In addition, isotopic techniques coupled with Fatty Acid Methyl Ester (FAME) profiling were used to determine possible metabolic changes. After only 3 days, approximately 80% of the cells were attached to the sand grains and microscopy images showed that the porous medium was totally clogged by the development of a biofilm. After 10 days, there was 25% less 2,4-D in the solution in samples with sand than in control samples. This difference was due to (1) a higher (+8%) mineralization of 2,4-D by sessile bacteria and (2) a retention (15%) of 2,4-D in the biofilm matrix. Besides, the amount of carbohydrates, presumably constituting the biofilm polysaccharides, increased by 63%. Compound-specific isotope analysis revealed that the FAME isotopic signature was less affected by the biofilm lifestyle than was the FAME composition. These results suggest that sessile bacteria differ more in their anabolism than in their catabolism compared to their planktonic counterparts. This study stresses the importance of considering interactions between microorganisms and their habitat when studying pollutant dynamics in porous media.

Environmental context The greenhouse gas nitrous oxide is produced by bacteria and emitted from terrestrial and aquatic environments; the origin of this compound can be determined by its 15N intramolecular distribution (site preference). The site preference of nitrous oxide was characterised experimentally in bacterial denitrifying communities under controlled conditions. This study shows the importance of the last step of denitrification on the site preference values, and that complementary methods are necessary to identify the sources of nitrous oxide. Abstract Site preference values of nitrous oxide emitted during different steps of benthic denitrification were determined. Compared to that of nitrous oxide as end product, the site preference during complete denitrification presents a large variation, due to the final step, and is highly correlated with nitrate reduction rate. The nitrous oxide reduction step appears decisive on the site preference values.

Increasing diffuse nitrate loading of surface waters and groundwater has emerged as a major problem in many agricultural areas of the world, resulting in contamination of drinking water resources in aquifers as well as eutrophication of freshwaters and coastal marine ecosystems. Although empirical correlations between application rates of N fertilizers to agricultural soils and nitrate contamination of adjacent hydrological systems have been demonstrated, the transit times of fertilizer N in the pedosphere-hydrosphere system are poorly understood. We investigated the fate of isotopically labeled nitrogen fertilizers in a three-decade-long in situ tracer experiment that quantified not only fertilizer N uptake by plants and retention in soils, but also determined to which extent and over which time periods fertilizer N stored in soil organic matter is rereleased for either uptake in crops or export into the hydrosphere. We found that 61-65% of the applied fertilizers N were taken up by plants, whereas 12-15% of the labeled fertilizer Nwere still residing in the soil organic matter more than a quarter century after tracer application. Between 8-12% of the applied fertilizer had leaked toward the hydrosphere during the 30-y observation period. We predict that additional exports of 15N-labeled nitrate from the tracer application in 1982 toward the hydrosphere will continue for at least another five decades. Therefore, attempts to reduce agricultural nitrate contamination of aquatic systems must consider the long-term legacy of past applications of synthetic fertilizers in agricultural systems and the nitrogen retention capacity of agricultural soils.

ABSTRACT Combining lipid biomarker profiling with stable isotope probing (SIP) is a powerful technique for studying specific microbial populations responsible for the degradation of organic pollutants in various natural environments. However, the presence of other easily degradable substrates may induce significant physiological changes by altering both the rate of incorporation of the target compound into the biomass and the microbial lipid profiles. In order to test this hypothesis, Cupriavidus necator JMP134, a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium, was incubated with [13C]2,4-D, [13C]glucose, or mixtures of both substrates alternatively labeled with 13C. C. necator JMP134 exhibited a preferential use of 2,4-D over glucose. The isotopic analysis showed that glucose had only a small effect on the incorporation of the acetic chain of 2,4-D into the biomass (at days 2 and 3) and no effect on that of the benzenic ring. The addition of glucose did change the fatty acid methyl ester (FAME) composition. However, the overall FAME isotopic signature reflected that of the entire biomass. Compound-specific individual isotopic analyses of FAME composition showed that the 13C-enriched FAME profiles were slightly or not affected when tracing the 2,4-D acetic chain or 2,4-D benzenic ring, respectively. This batch study is a necessary step for validating the use of lipid-based SIP methods in complex environments.

Stable Isotope Probing (SIP) is a powerful tool for analysing the fate of pesticides in soil. Together with FAME (Fatty Acid Methyl Esters), it can help identify biodegradation pathways and recycling into the microbial biomass. The fate of ring-labelled 13C-2,4-dichlorophenoxyacetic acid or 2,4-D (C2,4-D) was determined in soil during a 6-month incubation. The distribution of 13C among the microbial biomass, the CO2 respired, the water, methanol and dichloromethane soluble fractions, and the residual non-extracted bulk soil was measured. Molecular analyses were carried out on the lipid and the non-extractable fractions. After 8 days, about half of the initial amount of C2,4-D was mineralised; the other half remained in soil as non-extractable residues (NER). C2,4-D continued to be mineralised, suggesting that NER were still bioavailable. Analysis of C2,4-D-enriched FAME contained in the lipid fraction suggested that a succession of microbial populations was involved in 2,4-D biodegradation. This is possibly due to the change of 2,4-D availability. The C2,4-D yield coefficient and degrader diversity evolved during the incubation, providing corroboratory evidence that different physiological groups were active during the incubation. The 13C-labelled microbial community was always less diverse than the total community, even at the end of the incubation, suggesting that the cross-feeding community is also a specific part of the total community. This work shows that molecular analysis of 13C-labelled pesticides is a useful tool for understanding both chemical and biological aspects of their fate in soil.

Restoration of the nitrogen cycle is an important step in the recovery of an ecosystem after mining. Carbon and nitrogen in rehabilitated lignite containing mine soils can be derived from plant material as well as from lignite inherent to the parent substrate. We assessed the use elemental and stable carbon and nitrogen isotope measurements to trace the orgin of soil nitrogen and applied these techniques to elucidate the origin of mineral N in the soil and the soil solution. The conceptual approach of this study included physical fractionation in addition to sampling of vegetation and soil from a lignite-containing mine site rehabilitated in 1985 with Pinus Nigra. We studied the elemental and isotopic composition of bulk samples as well as isolated fractions and soil solution. Our data indicate that the stable carbon and nitrogen isotopic composition of the soil samples are the result of mixing between plant material and substrate inherent lignite. delta15N isotopes may be used as indicators of nitrogen contribution from plants to solid samples as well as soil solution. N-isotope composition of ammonia shows low spatial and interannual variability, despite strong concentration changes. Plant-derived nitrogen contributes in higher amounts to the soil solution compared to the bulk mineral soil.

Recent in situ 13C studies suggest that lignin is not stabilised in soil in its polymerised form. However, the fate of its transformation products remains unknown. The objective of the present research was to provide the first comprehensive picture of the fate of lignin-derived C across its transformations processes: (1) C remaining as undecomposed lignin molecules, (2) C in newly formed humic substances, i.e. no longer identifiable as lignin-polymer C, (3) C in microbial biomass, (4) C mineralised as CO2, and (5) dissolved organic C. To achieve this objective, we designed an incubation experiment with 13C-labelled lignin where both elementary and molecular techniques were applied. Lignin was isolated from 13C labelled maize plants (13C-MMEL) and incubated in an agricultural soil for 44weeks. Carbon mineralisation and stable isotope composition of the released CO2 were monitored throughout the incubation. Microbial utilisation of 13C-MMEL was measured seven times during the experiment. The turnover rate of the lignin polymer was assessed by 13C analysis of CuO oxidation products of soil lignin molecules. After 44 incubation weeks, 6.0% of initial 13C-MMEL carbon was mineralised, 0.8% was contained in the microbial biomass, and 0.1% was contained in dissolved organic C form. The compound-specific 13C data suggest that the remaining 93% were overwhelmingly in the form of untransformed lignin polymer. However, limited transformation into other humic substances potentially occurred, but could not be quantified because the yield of the CuO oxidation method proved somewhat variable with incubation time. The initial bacterial growth yield efficiency for MMEL was 31% and rapidly decreased to plateau of 8%. A two-pool first-order kinetics model suggested that the vast majority (97%) of MMEL lignin had a turnover time of about 25years, which is similar to field-estimated turnover times for soil-extractable lignin but much longer than estimated turnover times for fresh plant-residue lignin. We conclude that natural lignin structures isolated from plants are rather unreactive in soil, either due to the lack of easily available organic matter for co-metabolism or due to enhanced adsorption properties. The data also suggest that fairly undecomposed lignin structures are the main reservoir of lignin-derived C in soils.

The mine soils and sediments in the Lusatian open-cast lignite mining district (Germany) contain substantial amounts of lignite in addition to recent organic matter derived from plant litter. Knowledge of organic matter (OM) types and their transformation in mine soils and sediments is essential for the evaluation of ecosystem functioning in this region. The aim of the study was to use the isotopic signature of OM to: (1) determine lignite C contribution and (2) elucidate the contribution of OM derived from plant litter to three physical fractions in order to deduce its degree of degradation in three contrasting environments. We used stable and radioactive C isotopes to quantify the contribution of lignite C and to characterize humification processes occurring under contrasting vegetation and redox conditions. The upper mineral soil/sediment, the litter layer and the vegetation of three plots were sampled along a transect covering a forest soil, a partially submerged sediment at the land-water interface characterized by fluctuating water levels, and a constantly submerged sediment. The soil/sediment samples were fractionated into a macro (> 250 mu m), a humus (material separated by flotation) and a sand (mainly mineral material) fraction to follow the transformation processes of plant and lignite.Radiocarbon analysis of the humus fraction showed a lignite C contribution ranging from 20% to 80% of total organic carbon (OC). The C-14 activity was correlated with the delta C-13 value (r(2) = 0.95). Even if not precise, in this case the correlation was used to get an idea about the lignite contribution in coal mining-impacted freshwater sediment and soil. The C-13 data showed that lignite C contribution increases with depth in every fraction of the forest and partially submerged plots. At the submerged plot, the humus and macro fraction were almost free of lignite C, suggesting high amounts of fresh plant material at this plot. This was confirmed by the highest contribution (up to 40%) of the macro fraction, which is mainly composed of fresh plant material, to the total OC content in this plot. In the first 5 cm of all plots, most OC was found in the humus fraction. Combined elemental and isotopic analysis of the three physical fractions indicated that high amounts of humified OM were present in the constantly submerged sediment, whereas most intensive degradation of OM was occurring in the partially submerged sediment at the land-water interface. Here, delta C-13 values show that lignite C contributes to the macro fraction, which could suggest that it may have been incorporated into fresh plant material. thus being part of the active C cycle. (c) 2006 Elsevier Ltd. All rights reserved.

Downstream from metropolitan Paris (France), a large amount of ammonium is discharged into the Seine River by the effluents of the wastewater treatment plant at Achères. To assess the extent of nitrification and denitrification in the water column, concentrations and isotopic compositions of ammonium (δ15N–NH 4 + ) and nitrate (δ15N–NO 3 − , δ18O–NO 3 − ) were measured during summer low-flow conditions along the lower Seine and its estuary. The results indicated that most of the ammonium released from the wastewater treatment plant is nitrified in the lower Seine River and its upper estuary, but there was no evidence for water-column denitrification. In the lower part of the estuary, however, concentration and isotopic data for nitrate were not consistent with simple mixing between riverine and marine nitrate. A significant departure of the nitrate isotopic composition from what would be expected from simple mixing of freshwater and marine nitrates suggested coupled nitrification and denitrification in the water, in spite of the apparent conservative behavior of nitrate. Denitrification rates of approximately 0.02 mg N/L/h were estimated for this part of the estuary.

This chapter presents the results of several isotopic studies carried out on few cultivated watersheds with steep slopes located in tropical regions. Erosion of soil organic carbon (SOC) was monitored under natural rainfall conditions at various space and time scales by collecting runoff samples generated on experimental field plots and in stream flows during flood events. The tropical climate conditions prevailing in the region are highly influenced by altitude, ranging 200 to 4000 meter above sea level within the watershed. Most of the fallout occurred between 1954 and 1976 with a maximum supply in 1963. Sorting effects related to erosion intensity suggest that only fine-size organic fractions transported in stream flows will accurately reflect the contribution of soil fine fractions. Monitoring the composition of organic carbon in soils and suspended sediments of stream flows with isotopic tracers provides significant information that may help to better constrain SOC erosion budgets.

Lignin transformation and decomposition products are generally considered a major source of stable soil organic matter (SOM). Nevertheless this process remains poorly understood in part because lignin is a heterogeneous biopolymer composed of several types of phenol monomers, which potentially display specific and contrasting decomposition kinetics in soils. Here, we compared the specific in situ turnover kinetics of individual lignin monomers in a Paris-basin loamy soil through natural C-13 labeling of SOM generated in a series of 1-9-year chronosequences of maize monoculture (C4, delta C-13 - 12 parts per thousand) replacing wheat (C3, - 27 parts per thousand). Lignin monomers were released by CuO oxidation, quantified by gas chromatography (GC)/flame ionization detection (FID) and their C-13 content was determined by GC coupled via a combustion interface to isotope ratio mass spectrometry (GC/C-IRMS). We calculated the proportion of C4-derived OC in lignin monomer pools by applying the isotopic mass balance equation to each lignin monomer. Individual C4-derived phenols displayed contrasting accumulation rates in soils over time, confirming the monomer-specific nature of their transformation kinetics. In proportion to total lignin phenols in soils, syringyl (S) and cinnamyl (C) phenols from maize accumulated faster than their vanillyl (V) counterparts. Consequently, the turnover kinetics of lignin-derived V-moieties may be slower than those of S and C ones. Incorporation of maize-derived carbon was faster in the aldehyde than in the acid pool for V-type units, while the opposite was observed for S-units. These in situ trends for phenol monomers and V-, S- and C-moieties were remarkably similar to the trends described in the literature with laboratory incubation or litterbag studies. None of the observed kinetics had a linear shape. Using only the extreme points of the chronosequence to calculate the kinetic parameters would result, for all the lignin monomers, in underestimating the turnover kinetics at the beginning of the kinetics and overestimating it for longer times. This observation underlines the importance of comprehensive C-13 time-sequence labelling experiments such as provided at the Closeaux site, to accurately compute the kinetic parameters of SOM for the 1st years after the vegetation change. (c) 2006 Elsevier Ltd. All rights reserved.

Des methodes de tracage isotopique basees sur l'utilisation de mesures d'activite de traceurs radioactifs ou de teneurs en isotopes stables peuvent nous aider a identifier et quantifier la contribution de sources differentes de carbone organique particulaire dans les ecoulements generes par l'erosion hydrique sur les bassins versants ou ameliorer les bilans d'erosion du carbone organique des sols. Ainsi, sur le bassin versant experimental de Houay Pano (Laos), la redistribution le long des pentes du 137Cs provenant des retombees atmospheriques des essais atomiques des annees 1960-1970, est correlee avec la quantite de carbone organique contenue dans les premiers 30 cm des sols cultives. Processus d'erosions, hydrique et aratoire, se combinent pour redistribuer le long des pentes simultanement matieres organiques et particules argileuses des horizons superficiels des sols. Sur le bassin versant du Rio Bocono dans les Andes venezueliennes, les mesures de concentration en carbone organique particulaire, azote particulaire total, d13C et d15N effectuees sur les fractions fines (<50 µm) des matieres en suspensions prelevees sur des parcelles experimentales sont constantes au cours du ruissellement au-dela de « valeurs seuils » (30 ml.s-1 et 0.5 g.l-1 sur sol nu et sol sous culture de cafe). Les valeurs moyennes refletent la composition moyenne des horizons de surface des sols (0-20 cm). Ces resultats indiquent qu'il est theoriquement possible (au-dela d'un seuil donne et pour une granulometrie de faible taille) d'estimer dans les ecoulements la contribution respective en carbone organique particulaire de 2 sols de composition isotopique (d13C et d15N) suffisamment contrastee l'une par rapport a l'autre en utilisant des equations de melange. La mesure de la teneur en 15N des matieres organiques fines (<50 µm) en suspension, transportees par les cours d'eau pendant les episodes de crues, peut nous permettre de mettre en evidence la contribution d'azote particulaire provenant de l'erosion incisive des roches du substratum geologique (shales, schistes et pelites). Sur le bassin versant du Rio Bocono ou ce type de roches constitue une grande partie du sous-bassement geologique, l'ampleur de cette contribution depend de l'intensite de l'episode de crue. Ainsi, pendant un episode de crue de forte intensite (periode de retour : 10 ans) la contribution des matieres organiques d'origine lithique (estimees a partir de l'azote particulaire) se trouve en proportion equivalente a celle fournie par l'erosion des sols s.s.

Environmental Context.Nitrogen is an essential element for all living organisms, and its biogeochemical cycle is connected to the cycling of carbon, sulfur, phosphorous, oxygen, and trace metals. Measurement of the isotopic composition of ammonium (NH4+) and nitrate (NO3−) containing samples provides a better understanding of the nitrogen cycle. While the established ‘ammonium diffusion’ measurement has many advantages, it is not easy for inexperienced people to prepare samples. This paper shows how the method can be simplified, ideally for samples freshly collected from the field. Abstract.Several methods have been developed for nitrogen isotope measurements on ammonium (NH4+) and nitrate (NO3−) in solid or aqueous samples. We have tested the accuracy and reproducibility of the ammonium diffusion method for δ15N measurements on NH4+ and NO3− and compared this technique to other established methods. Our results show that the ammonium diffusion technique is capable of generating accurate and reproducible δ15N values for minute quantities of NH4+-N and NO3−-N in aqueous samples, if sufficient care is taken to minimize nitrogen blanks and to optimize the extraction procedure. Hence, the ammonium diffusion method offers an attractive alternative to more labour-intensive and costly methods for determining nitrogen isotope ratios of NH4+ and NO3− in aqueous samples.