N-15 immobilization in forest soil: a sterilization experiment coupled with (15)CPMAS NMR spectroscopy

In temperate forests, soils are the main sink for atmospheric N deposition. The main processes proposed for N retention are microbial and abiotic immobilization in soil organic matter. The relative importance of these processes as well as the kind of resulting chemical compounds are not totally understood. We carried out a laboratory incubation of Hg-sterilized and non-sterilized organic and organo-mineral soil horizons, labelled with either (NO3-)-N-15 or (NH4+)-N-15. The labelled samples were incubated for 1 hour, 1 day, or 6 days, then subjected to K2SO4 extraction and analysed with N-15 CPMAS NMR spectroscopy. N immobilization was already effective in all samples and treatments after 1 hour. The corresponding NMR spectra showed that part of the immobilized N-15 was already incorporated into an amide structure. In the sterilized soils labelled with (NH4+)-N-15, the tracer was rapidly and largely immobilized by an unknown process related to the presence of Hg. In the sterilized soils labelled with (NO3-)-N-15, between one-third and one-half of the added tracer was immobilized during the first hour and only 10% more over the 6 days. These results suggest that the sterilization was incomplete at first, allowing relatively great microbial immobilization during the first hour. By contrast, over a longer time, NO3- immobilization was significantly reduced to a level corresponding to an abiotic process as Hg sterilization became more effective. Even if the low signal-to-noise ratio precluded quantitative N-15 NMR measurements, we showed that the amide-peptide signal, considered as a biotic signature, was dominant in all cases.

Retention of atmospherically deposited nitrogen in soil

Isabelle Morier Jaquet

Since a few decades, the balance of the nitrogen (N) cycle has been deeply disturbed by human activies. The global impact of these activities on the N cycle can be described as a doubling of the transfer from the vast and unreactive atmospheric pool to biologically available forms (N fixation). The main sources responsible for the increase of reactive N emissions are the use of artificial fertilisers (NH3) as well as the combustion processes (NOx). Reactive N is then transformed, transported by both the atmosphere and the hydrosphere and, finally, deposited on both the terrestrial and the aquatic ecosystems with potentially strong impacts. Amongst the terrestrial ecosystems, the temperate forests are particularly sensitive to reactive N increases for various reasons: they are located near to strongly anthropised areas and are thus subjected to strong depositions; they are naturally N-limited and their biochemical cycle can indeed be strongly influenced by additional N, which leads to eutrophication and potential impacts such as soil acidification, nitrate leaching and losses of biodiversity within microorganisms, plants and fauna communities. Previous studies carried out in temperate forest ecosystems have shown that the soil, namely the soil organic matter, acts as a main deposited N sink for the ecosystem. However, biogeochemical reactions responsible for N retention in the soil are still not fully understood. Moreover, the majority of the concerned studies were conducted in acidic or hydromorphous soils and very little is known today about the fate of N deposition under other soil physico-chemical conditions. Consequently, the present research aims at filling some of the gaps described above and deals with two main topics concerning the retention of atmospherically deposited N in soils that are (1) the characterisation of the retention of atmospherically deposited N in the soil, in terms of both duration and quantity, more specifically, in a well drained calcareous soil and (2) the mechanisms and processes responsible for such a retention. From a practical point of view, the retention of N deposition in the soil was characterised by means of a 15N field labelling experiment simulating N atmospheric deposition and conducted at the Grandvillard research site, in the riparian zone of La Sarine River (Swiss Prealps). The stand is a beech forest mixed with planted spruces. The soil consists of a well drained calcareous fluvisol with a fast organic matter turnover. We tracked the N tracers (15NH4+ or 15NO3-, corresponding to the main forms of deposition) from short-term (hours, days or weeks) to longer-term intervals (one year), by measuring the partitioning of 15N into different biochemical soil fractions (extractable N, microbial N, roots N and N immobilised in soil). Aiming at studying the mechanisms and processes responsible for N retention, we performed two laboratory experiments. The first one was an acid hydrolysis, in order to determine the proportion of N deposition retained in the hydrolysable fraction (i.e. the more labile N forms, including the most bioavailable ones) as well as in the non-hydrolysable fraction covering the more recalcitrant N compounds. Within the framework of this experiment, we compared the results obtained in Grandvillard and in an aditional constrasting site (Alptal). The second experiment was a short-term (one hour to one week) laboratory incubation of sterilised and not sterilised soils, which purpose was to highlight the relative importance of biotic and abiotic processes in N immobilisation. Soils were labelled with either 15NH4+ or 15NO3- and subjected to 15N CPMAS NMR spectroscopy analysis, which is a powerful technique to identify the N organic molecules in soils. Within the framework of the field labelling experiment, more than half of the tracers (either 15NO3- or 15NH4+) was recovered in the soil between one hour and one year after labelling. Therefore, the general assumption that non-fertilised temperate forest soils are the main sink for deposited N can be confirmed. Since previous studies showing the importance of the soil for the retention of deposited N were all conducted on either acidic or hydromorphous soils, our results allow us to extend the validity of their general conclusions to a wider range of unfertilised temperate forest soils. We further showed that the main forms of N deposition (i.e. NO3-and NH4+) were retained in the soil within the same range of magnitude, in spite of their different biochemical pathways. N retention occurred mainly in the soil organic layers and, consequently, our results confirme the importance of the organic fraction in the deposited N retention. Since deposited N was retained in the soil in the very short term, we confirmed the presence of very fast processes and demonstrated that the very short term dynamics determined the fate of deposited N in the longer term: the main processes were i) a loss of deposited N as extractable through lixiviation (and lateral fluxes) and ii) a retention as N immobilised in soil. Furthermore, we demonstrated that N immobilisation brought deposited N both into the hydrolysable and into the recalcitrant fractions of soil N within short-time range (i.e. one week) . We also demonstrated that the hydrolysability (hydrolysable N/ total N) was constant over the year. Within the framework of the laboratory incubation of soils subjected to the 15N CPMAS NMR spectroscopy analysis, we showed that all NMR spectra were dominated by a single signal corresponding to the amide-peptide structure, whatever the soil layer concerned (organic or organo-mineral), the form added (NO3- or NH4+) and whether the soil was sterilised or not. Such dominance of proteinaceous compounds was in agreement with the results obtained in humic substances and in various soils all over the world. Further to the extractable 15N dynamics during the incubation, we proposed that biotic processes were dominant in the short-term N immobilisation. However, an abiotic fixation of less importance was not excluded for 15NO3-. Considering our results, we propose two mechanisms responsible for the retention of deposited N in soil in the long term. The first mechanism is a rapid and stable immobilisation in the recalcitrant pool of soil organic matter. Our results showed that biotic and abiotic processes could be involved: the afore-mentioned process of abiotic nitrate fixation could lead to the N immobilisation in heterocycles whereas N incorporated in amides and then stabilised in soil organic matter seemed to be the major pathway. The second mechanism of retention we are proposing is the biological recycling within the soil or the soil-plant system: on a short scale, microbial recycling seemed to be very rapid because deposited N was mainly present under a biosynthesised form (amides), in spite of the low amounts of tracer found in the microbes. Within the soil profile, roots recycling was probably efficient: fine roots, which are often the major contributors of soil organic matter were an important sink for N deposition all over the year. The combination of various scales and techniques allowed the connection between the dynamics of N retention in soil organic layers observed and the mechanisms responsible for it (i.e. rapid immobilisation in a recalcitrant pool and biological recycling through micro-organisms and plants). The results of this multi-scale approach suggest further developments and impacts for the modelling of the long-term fate of deposited N.

EPFL2006

Assessment of microbial community changes and limiting factors during bioremediation of hydrocarbon-polluted soil with new miniaturized physiological methods

Karin Kaufmann

Due to human activities, organic pollutants are spilled to the environment where they threaten public health, often as contaminants of soil or groundwater. Living organisms are able to transform or mineralize many organic pollutants, and bioremediation techniques have been developed to remove pollutants from a contaminated site. However, fast and easy methods to document both the efficacy of bioremediation and the changes in soil microbial communities during bioremediation are not well developed. The major aim of this thesis was to develop miniaturized methods targeting the physiology of microbial communities during pollutant degradation in soil and to assess pollution-induced community changes. Furthermore, the methods should identify factors limiting efficient pollutant biodegradation. Petroleum hydrocarbons have been chosen as pollutants because these compounds are frequently spilled, readily water-soluble, partly toxic and carcinogenic, and therefore undesirable in soils and drinking water supplies. The influence of petroleum hydrocarbons on a microbial community in the vadose zone was assessed under field conditions. An artificial hydrocarbon mixture consisting of volatile and semi-volatile compounds similar to jet-fuel was emplaced in a previously uncontaminated vadose zone in nutrient-poor glacial melt water sand. The experiment included monitoring of microbial parameters and CO2 concentrations in soil gas over 3 months, in- and outside the hydrocarbon vapor plume that formed around the buried petroleum. Microbial and chemical analyses of vadose zone samples were performed on material from 9 cores drilled on 3 days at 3 distances from the buried petroleum mass to 3.3 m depth. Significantly elevated CO2 concentrations were observed after contamination. Total cell numbers as determined by fluorescence microscopy were strongly correlated with soil organic carbon and nitrogen content but varied little with contamination. Redundancy analysis (RDA) allowed direct analysis of effects of selected environmental variables or the artificial contamination on microbiological parameters. Variation in biomass and CO2 production was to 46 % explained by soil parameters and to 39.8 % by the duration of contamination. The microbial community structure was assessed by community-level physiological profile analysis using BiologTM EcoPlates. Only 35.9 % of variation in BiologTM data could be attributed to soil parameters and contamination, however, the samples with greatest exposure to hydrocarbons grouped together on RDA plots. It is concluded that at this nutrient-poor site, the microbial community was dominated by the natural heterogeneity and that the influence of petroleum hydrocarbon vapor was weak. BiologTM Eco-Plates combined with RDA were able to distinguish between samples with high and low hydrocarbon exposure. However, the method was not sensitive enough to produce consistent patterns in three replicates when extracts from subsurface soils with low cell numbers were inoculated. A more sensitive, MPN-based system was therefore developed and its ability to provide information about specific pollutant degraders was tested in alluvial sand. Eight dilutions of sand extracts were inoculated on medium- and substrate-containing polypropylene deep-well plates. To keep the concentrations of the volatile substrates constant over several days of incubation without intoxicating the soil community, a substrate-containing organic carrier phase was applied to each vial. The biochemically inert 2,2,4,4,6,8,8-heptamethylnonane (HMN) was appropriate for this purpose. Thus, the volatile substrates partition from HMN into the microorganism-containing soil water where they achieve relatively low concentrations. The sensitive watersoluble tetrazolium dye (WST-1) was added to each well after 7 days incubation to detect dehydrogenase activity. A dark yellow signal was developed for the substrates toluene, n-octane, n-dodecane, 1,2,4-trimethylbenzene and methylcyclohexane but not for isooctane, being strongest in n-alkane amended wells. With this method, more hydrocarbon degrading bacteria were detected in an alluvial sand exposed to kerosene for 72 hours compared to the community in the pristine sand. Both communities were mainly composed of n-alkane degraders. Although encouraging results were achieved, we noticed that the tetrazolium reduction was inhibited in some cases. Wells with high cell numbers, in which obvious growth occurred, did not produce a signal. In a next step, the two-liquid phase system with HMN was applied to modify a miniaturized respiration detection system using whole soil samples instead of soil extracts (Campbell et al., AEM 69, p. 3593). Soil was incubated in deep-well plates in presence of single volatile organic hydrocarbons. The carrier phase was added to the interstitial space in a bead layer on the bottom of the well (below the soil sample), and the hydrocarbons diffused into the soil-water. The soil activity was determined by means of an agar plate containing a pH indicator, which changes color as a function of the produced CO2. Physiological profiles specific to petroleum hydrocarbons in pristine and contaminated soils were assessed. The substrate concentrations, which induced highest microbial activity, as well as concentrations causing inhibition, were determined with effect-concentration curves. Community-level physiological profiles based on hydrocarbon degradation were obtained by applying multivariate analysis on the CO2 yields. The first measurement period of 0-6 hours after addition of relatively high hydrocarbon concentrations to the system yields values, which best separated soil types as well as pristine and contaminated soils. If low concentrations were applied, we obtained only significant separations after 24 hours incubation, probably after a growth step. The same micro-respiration system equipped with a 14CO2 detection plate was applied to study N and P limitation of aerobic mineralization of 14C-toluene in a soil previously exposed to petroleum. Significant nutrient limitation during short-term tests (24-48 hours) was identified and the optimized combination of nutrient addition was determined. Conclusively, miniaturized physiological methods have been proven to detect soil microbial community changes in petroleum hydrocarbon-contaminated soils. Adding an organic carrier phase to the miniaturized systems allowed assuring a volatile organic hydrocarbon supply in non-toxic concentrations in the soil water or the medium for several days. Thus simple, cost-effective systems to investigate the soil community during bioremediation and to address questions of nutrient shortage in soils have been provided with this thesis.

EPFL2004

Dynamics of atmospheric N deposition in a temperate calcareous forest soil

Claire Guenat, Isabelle Morier Jaquet, Jean-Claude Védy

In temperate forest ecosystems, soil acts as a major sink for atmospheric N deposition. A 15N labeling experiment in a hardwood forest on calcareous fluvisol was performed to study the processes involved. Low amounts of ammonium (15NH4 +) or nitrate (15NO3 −) were added to small plots. Soil samples were taken after periods ranging from 1 h to 1 yr. After 1 d, the litter layer retained approximately 28% of the 15NH4 + tracer and 19% of 15NO3 − The major fraction of deposited N went through the litter layer to reach the soil within the first hours following the tracer application. During the first day, a decrease in extractable 15N in the soil was observed (15NH4 +: 50 to 5%; 15NO3 −: 60 to 12%). During the same time, the amount of microbial 15N remained almost constant and the 15N immobilized in the soil (i.e., total 15N recovered in the bulk soil minus extractable 15N minus microbial 15N) also decreased. Such results can therefore be understood as a net loss of 15N from the soil. Such N loss is probably explained by NO3 − leaching, which is enhanced by the well-developed soil structure. We presume that the N immobilization mainly occurs as an incorporation of deposited N into the soil organic matter. One year after the 15N addition, recovery rates were similar and approximately three-quarters of the deposited N was recovered in the soil. We conclude that the processes relevant for the fate of atmospherically deposited N take place rapidly and that N recycling within the microbes–plants–soil organic matter (SOM) system prevents further losses in the long term.