Linking soil's volatilome to microbes and plant roots highlights the importance of microbes as emitters of belowground volatile signals

Plants and microbes release a plethora of volatiles that act as signals in plant-microbe interactions. Characterizing soil's volatilome and microbiome might shed light on the nature of relevant volatile signals and on their emitters. This hypothesis was tested by characterizing plant cover, soil's volatilome, nutrient content and microbiomes in three grasslands of the Swiss Jura Mountains. The fingerprints of soil's volatiles were generated by solid-phase micro-extraction gas chromatography/mass spectrometry, whereas high-throughput sequencing was used to create a snapshot of soil's microbial communities. A high similarity was observed in plant communities of two out of three sites, which was mirrored by the soil's volatilome. Multiple factor analysis evidenced a strong association among soil's volatilome, plant and microbial communities. The proportion of volatiles correlated to single bacterial and fungal taxa was higher than for plants. This suggests that those organisms might be major contributors to the volatilome of grassland soils. These findings illustrate that key volatiles in grassland soils might be emitted by a handful of organisms that include specific plants and microbes. Further work will be needed to unravel the structure of belowground volatiles and understand their implications for plant health and development.

Effects of Subordinate Plant Species in Plant and Soil Community Structure and Ecosystem Functioning

Pierre Rémi Mariotte

The relevance of biodiversity to human health is an increasing international political issue as it causes concern for ethical and aesthetic reasons, but also has a strong impact on ecosystem properties and ecological goods and services utilised by humanity. However, humans have applied increasing pressure on worldwide biodiversity through pollution, land-use and climate change, over exploitation and the introduction of invasive species which can lead to major alterations of biological communities and a consequent decrease in biodiversity. When communities are assembled at random from a pool of species, more diverse mixtures have a higher probability to contain species or species-groups with high capacity to drive ecosystem processes. Therefore, it is relevant to ask "how may we classify species in a community in terms of functionality?", and "which species-group is important to maintain the productivity and stability of ecosystems?" In this thesis, I chose to classify species according their frequency and their cumulative relative cover in a plant community (i.e. plant hierarchy) and to differentiate three species-groups: dominant, subordinate and transient, which contribute towards biodiversity. Dominant species are clearly very important for ecosystem functioning, due to the large amount of biomass they produce, but there is growing evidence that subordinate species, which represent a low amount of plant biomass in grassland ecosystems, are also of considerable importance for ecosystem functioning. Given this recognition, a further objective was to explore the ecosystem level effects of these subordinate plant species, which is currently not well known. This research focused on two aspects: the persistence of subordinate species in the community, and their role in grassland ecosystems. The approach to satisfy these objectives encompassed four years of experimental field studies and glasshouse microcosm experiments. In order to explain the persistence of subordinate species in semi-natural grasslands, we performed two greenhouse experiments to test the effects of root competition exclusion and the inoculation of arbuscular mycorrhizal fungi (AMF) on the competitiveness of dominant and subordinate species, directly measured by their biomass production. The effects of subordinate species on ecosystem functioning were assessed through long-term field experiment in two sites in the Swiss Jura Mountains, where dominant, subordinate and transient species were preliminarily determined. At both site (Les Amburnex, La Frétaz), a removal experiment was carried out in randomly replicated plots with three different treatments: control without perturbations, removal of all subordinate species and partial removal of dominant biomass. In one site, a summer drought treatment was added using rainout shelters to simulate an extreme climatic event. Biomass production, litter decomposition and soil respiration were monitored during four years in each plot and complemented in some cases by measurements of soil dissolved inorganic nitrogen concentrations, soil microbial communities (bacteria and AMF) and carbon and nitrogen isotopes in plant leaves. The results of the different experiments conducted in this thesis highlight the importance of cattle activity and AMF on the persistence of subordinate plant species in semi-natural grasslands. Indeed, cattle activities (i.e. trampling) provide spatial heterogeneity, through gap creation (areas of root competition reduction), and thus favours the growth of less competitive subordinate species. While AMF acted as a parasite in the greenhouse experiment, the current findings suggest that the competitiveness of dominant species was reduced through the action of the fungus, which indirectly enhanced subordinate species. The interactions between subordinate species and soil organisms were confirmed in the field as bacterial and AMF communities shifted in plots where subordinate species were removed compared to the control treatments. Moreover, the absence of subordinate species had many negative effects on ecosystem functioning by reducing litter decomposition, soil respiration, nitrogen mineralization and community above-ground production. These findings suggest that plant-soil feedbacks explain the importance of subordinate species on ecosystem functioning despite only representing a very low quantity of biomass in plant communities. During the summer drought, subordinate species increased the resistance of the plant community and maintained productivity. Therefore, these outcomes confirm the role of drought-resistant subordinates in the functioning of grassland ecosystems threatened by climate change. The synthesis of this thesis has important implications for the way grasslands should be managed for biodiversity and confirms that extensive grazing must be maintained as it promotes species coexistence and the persistence of key subordinate species. Moreover, this work demonstrates the important role of subordinate species in ecosystem functioning, showing that not only dominant species determine ecosystem properties. This thesis serves as a contribution to the advances in our understanding of ecosystem functioning and above and below-ground linkages, and provides basis for future research in this domain.

EPFL2012

Land management trumps the effects of climate change and elevated CO2 on grassland functioning

Pierre Rémi Mariotte, Aurélie Thébault

Grasslands cover similar to 30% of the Earth's terrestrial surface and provide many ecosystem services. Many grasslands are heavily managed to maximize these services for human benefit, but the outcome of management is anticipated to be increasingly influenced by various aspects of climate change and elevated atmospheric CO2. The relative importance of global change vs. land management on grasslands is largely unknown. 2. A meta-analysis is used here to examine drivers at both scales primarily targeting services provided by grasslands relating to plant productivity (above- and below-ground biomass) and soil processes (nutrients and soil respiration) in 38 manipulative experiments published in the last decade. We specifically target effects of (i) single and combined land management practices (LMs), (ii) single and combined factors relating to broad-scale climate change and elevated CO2, and (iii) combined management practices and changes to climate and CO2. Collectively, this examines the general efficacy of global change models in predicting changes to grassland functioning. 3. We found that combinations of management practices had approximately double the explanatory power for variation in grassland services compared with individual or interactive effects of factors associated with climate change and CO2. These interacting management practices such as nutrient additions and defoliation predominantly influenced functions associated with productivity or biomass both below and above ground. The effects of interacting factors of climate and CO2 influenced a wider range of ecosystem functions, but the magnitude of these effects was relatively smaller. 4. Interactions between management practices or between climate change/CO2 factors always had higher explanatory power than any factor in isolation indicating that multivariate synergistic models of environmental change can better describe impacts on ecosystem function in plant communities (e.g. relative to univariate climate-based models). Given that the magnitude and direction (positive or negative) of the interactions varied widely, this also implies that the outcomes of these multivariate interactions can vary spatially, temporally or by immediate context (e.g. management prescriptions). 5. Synthesis. Although our work confirms how climate change and CO2 can affect many ecosystem-based functional attributes, it suggests that combinations of LMs remain the dominant set of factors in determining the performance of grassland plant communities. Land management may thus be critical for influencing projected responses to future climate change and elevated CO2 in models of grassland function at least for factors relating to primary production.

Wiley-Blackwell2014

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