Altered fungal communities in contaminated soils from French industrial brownfields.

Polycyclic aromatic hydrocarbons (PAHs) and metals are contaminants of industrial brownfield soils. Pollutants can have harmful effects on fungi, which are major actors of soil functioning. Our objective was to highlight fungal selection following long-term contamination of soils. Fungal diversity was assessed on 30 top-soil samples from ten sites gathered in three groups with different contamination levels and physico-chemical characteristics: 1) uncontaminated controls, 2) slag heaps displaying high PAH and moderate metal contaminations, and 3) settling ponds displaying high metal and intermediate PAH contaminations. Although fungal abundance and richness were similar among the soil groups, the diversity and evenness indices were lower for the slag heap group. Fungal diversity differed among soil groups at the phylum and OTU levels, and indicator species were identified. The relative abundance of Agaricomycetes, Saccharomycetes, Leotiomycetes and Chytridiomycota was higher in the control soils than in the two groups of contaminated soils. Cryptomycota LKM11 representatives were favoured in the slag heap and settling pond groups, and their relative abundance was correlated to the zinc and lead contamination levels. Dothideomycetes - positively linked to PAH contamination - and Eurotiomycetes were specific to the slag heap group. Pucciniomycetes and especially Gymnosporangium members were favoured in the settling pond soils.

DNA stable isotope probing reveals contrasted activity and phenanthrene-degrading bacteria identity in a gradient of anthropized soils.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous soil organic pollutants. Although PAH-degrading bacteria are present in almost all soils, their selection and enrichment have been shown in historically high PAH contaminated soils. We can wonder if the effectiveness of PAH biodegradation and the PAH-degrading bacterial diversity differ among soils. The stable isotope probing (SIP) technique with 13C-phenanthrene (PHE) as a model PAH was used to: (i) compare for the first time a range of 10 soils with various PAH contamination levels, (ii) determine their PHE-degradation efficiency and (iii) identify the active PHE-degraders using 16S rRNA gene amplicon sequencing from 13C-labeled DNA. Surprisingly, the PHE degradation rate was not directly correlated to the initial level of total PAHs and phenanthrene in the soils, but was mostly explained by the initial abundance and richness of soil bacterial communities. A large diversity of PAH-degrading bacteria was identified for seven of the soils, with differences among soils. In the soils where the PHE degradation activities were the higher, Mycobacterium species were always the dominant active PHE degraders. A positive correlation between PHE-degradation level and the diversity of active PHE-degraders (Shannon index) supported the hypothesis that cooperation between strains led to a more efficient PAH degradation.

Soil Properties and Multi-Pollution Affect Taxonomic and Functional Bacterial Diversity in a Range of French Soils Displaying an Anthropisation Gradient.

The intensive industrial activities of the twentieth century have left behind highly contaminated wasteland soils. It is well known that soil parameters and the presence of pollutants shape microbial communities. But in such industrial waste sites, the soil multi-contamination with organic (polycyclic aromatic hydrocarbons, PAH) and metallic (Zn, Pb, Cd) pollutants and long-term exposure may induce a selection pressure on microbial communities that may modify soil functioning. The aim of our study was to evaluate the impact of long-term multi-contamination and soil characteristics on bacterial taxonomic and functional diversity as related to the carbon cycle. We worked on 10 soils from northeast of France distributed into three groups (low anthropised controls, slag heaps, and settling ponds) based on their physico-chemical properties (texture, C, N) and pollution level. We assessed bacterial taxonomic diversity by 16S rDNA Illumina sequencing, and functional diversity using Biolog® and MicroResp™ microtiter plate tools. Although taxonomic diversity at the phylum level was not different among the soil groups, many operational taxonomic units were influenced by metal or PAH pollution, and by soil texture and total nitrogen content. Functional diversity was not influenced by PAH contamination while metal pollution selected microbial communities with reduced metabolic functional diversity but more tolerant to zinc. Limited microbial utilisation of carbon substrates in metal-polluted soils was mainly due to the nitrogen content. Based on these two observations, we hypothesised that reduced microbial activity and lower carbon cycle-related functional diversity may have contributed to the accumulation of organic matter in the soils that exhibited the highest levels of metal pollution.

Metal release from contaminated leaf litter and leachate toxicity for the freshwater crustacean Gammarus fossarum.

Industrialization has left large surfaces of contaminated soils, which may act as a source of pollution for contiguous ecosystems, either terrestrial or aquatic. When polluted sites are recolonized by plants, dispersion of leaf litter might represent a non-negligible source of contaminants, especially metals. To evaluate the risks associated to contaminated leaf litter dispersion in aquatic ecosystems, we first measured the dynamics of metal loss from leaf litter during a 48-h experimental leaching. We used aspen (Populus tremula L.), a common tree species on these polluted sites, and collected leaf litter on three polluted sites (settling pond of a former steel mill) and three control sites situated in the same geographic area. Then, toxicity tests were carried out on individuals of a key detritivore species widely used in ecotoxicology tests, Gammarus fossarum (Crustacea, Amphipoda), with uncontaminated and contaminated leaf litter leachates, using a battery of biomarkers selected for their sensitivity to metallic stress. Leaf litters collected on polluted sites exhibited not only significantly higher cadmium and zinc concentrations but also lower lignin contents. All leaf litters released high amounts of chemical elements during the leaching process, especially potassium and magnesium, and, in a lesser extent, phosphorus, calcium, and trace metals (copper, cadmium, and zinc but not lead). Toxicity tests revealed that the most important toxic effects measured on G. fossarum were due to leaf litter leachates by themselves, whatever the origin of litter (from polluted or control sites), confirming the toxicity of such substances, probably due to their high content in phenolic compounds. Small additional toxic effects of leachates from contaminated leaf litters were only evidenced on gammarid lipid peroxidation, indicating that contaminated leaf litter leachates might be slightly more toxic than uncontaminated ones, but in a very reduced manner. Further studies will be required to verify if these patterns are generalizable to other species and to investigate the effects of contaminated leaf litter ingestion by consumers on aquatic food webs. Nevertheless, our results do not permit to exclude potential chronic effects of an exposure to contaminated leaf litter leachates in aquatic ecosystems.

Litter chemistry prevails over litter consumers in mediating effects of past steel industry activities on leaf litter decomposition.

Soil pollution has adverse effects on the performance and life history traits of microorganisms, plants, and animals, yet evidence indicates that even the most polluted sites can support structurally-complex and dynamic ecosystems. The present study aims at determining whether and how litter decomposition, one of the most important soil ecological processes leaf, is affected in a highly trace-metal polluted site. We postulated that past steel mill activities resulting in soil pollution and associated changes in soil characteristics would influence the rate of litter decomposition through two non-exclusive pathways: altered litter chemistry and responses of decomposers to lethal and sub-lethal toxic stress. We carried out a litter-bag experiment using Populus tremula L. leaf litter collected at, and allowed to decompose in, a trace metal polluted site and in three unpolluted sites used as controls. We designed a fully-factorial transplant experimental design to assess effects of litter origin and exposure site on the rate of litter decomposition. We further determined initial litter chemistry, fungal biomass, mesofauna abundance in litter bags, and the soil macrofauna community. Irrespective of the site of litter exposure, litter originating from the polluted site had a two-fold faster decomposition than litter from the unpolluted sites. Litter chemistry, notably the lignin content, seemed most important in explaining the degradation rate of the leaf litter. Abundance of meso and macro-detritivores was higher at the polluted site than at the unpolluted sites. However, litter decomposition proceeded at similar rates in polluted and unpolluted sites. Our results show that trace metal pollution and associated soil and litter changes do not necessarily weaken consumer control on litter decomposition through lethal and sub-lethal toxic stress.

Impact of carbohydrate supply on stem growth, wood and respired CO2 delta13C: assessment by experimental girdling.

The present study examines the impact of the C source (reserves vs current assimilates) on tree C isotope signals and stem growth, using experimental girdling to stop the supply of C from leaves to stem. Two-year-old sessile oaks (Quercus petraea) were girdled at three different phenological periods during the leafy period: during early wood growth (Girdling Period 1), during late wood growth (Girdling Period 2) and just after growth cessation (Girdling Period 3). The measured variables included stem respiration rates, stem radial increment, delta(13)C of respired CO(2) and contents of starch and water-soluble fraction in stems (below the girdle) and leaves. Girdling stopped growth, even early in the growing season, leading to a decrease in stem CO(2) efflux (CO(2R)). Shift in substrate use from recently fixed carbohydrate to reserves (i.e., starch) induced (13)C enrichment of CO(2) respired by stem. However, change in substrate type was insufficient to explain alone all the observed CO(2R) delta(13)C variations, especially at the period corresponding to large growth rate of control trees. The below-girdle mass balance suggested that, during girdling periods, stem C was invested in metabolic pathways other than respiration and stem growth. After Girdling Period 1, the girdle healed and the effects of girdling on stem respiration were reversed. Stem growth restarted and total radial increment was similar to the control one, indicating that growth can be delayed when a stress event occurs early in the growth period. Concerning tree ring, seasonal shift in substrate use from reserves (i.e., starch) to recently fixed carbohydrate is sufficient to explain the observed (13)C depletion of tree ring during the early wood growth. However, the inter-tree intra-ring delta(13)C variability needs to be resolved in order to improve the interpretation of intra-seasonal ring signals in terms of climatic or ecophysiological information. This study highlighted, via carbohydrate availability effects, the importance of the characterization of stem metabolic pathways for a complete understanding of the delta(13)C signals.

Is there any 12C/13C fractionation during starch remobilisation and sucrose export in potato tubers?

The delta(13)C (carbon isotope composition) variations in respired CO(2), total organic matter, proteins, sucrose and starch have been measured during tuber sprouting of potato (Solanum tuberosum) in darkness. Measurements were carried out both on tubers and on their growing sprouts for 23 days after the start of sprout development. Sucrose was slightly (13)C-depleted compared with starch in tubers, suggesting that starch breakdown was associated with a small isotope fractionation. In sprouts, all biochemical fractions including sucrose were (13)C-enriched compared with source tuber-sucrose, suggesting that sucrose translocation from tuber to sprouts fractionated against (12)C. However, both apparent fractionations were explained by the consumption of (13)C-depleted carbon for respiration or growth that enriched in the (13)C sucrose molecules left behind. In addition, whole tuber sucrose is constantly composed of recent sucrose from starch breakdown and old sucrose associated with an inherited, slightly (13)C-depleted pool. We therefore conclude that any fractionation at either the starch breakdown or the sucrose translocation level is unlikely under our conditions.

Seasonal changes in optically assessed epidermal phenolic compounds and chlorophyll contents in leaves of sessile oak (Quercus petraea): towards signatures of phenological stage.

Seasonal patterns of dry mass invested in chlorophyll and epidermal phenolic compounds (EPhen) were investigated in vivo using optical methods, in leaves of 2-year-old oaks (Quercus petraea Matt. (Liebl.)) grown under semi-controlled conditions. The plasticity of the seasonal pattern was investigated by applying stem girdling treatment. In control young expanding leaves, leaf dry mass per area, dry mass investment in chlorophyll and abaxial EPhen content increased. In late May, at leaf maturity, these variables reached a plateau, and adaxial and abaxial EPhen contents became similar. Thereafter, as leaves aged, dry mass investment in chlorophyll gradually decreased, whereas it remained steady for EPhen. Girdling treatment impacted this seasonal pattern differently depending on the phenological stage. Treatment effects and their reversion revealed in vivo EPhen turnover. Finally, optical signatures of immature and mature leaf phenological stages with contrasting nitrogen and carbon economy were proposed, based on the relationship between the chlorophyll to EPhen ratio and the leaf nitrogen to carbon ratio.

Biochemical composition is not the main factor influencing variability in carbon isotope composition of tree rings.

From June to December, we determined the effects of variations in biochemical composition on delta(13)C of tree rings of 2-year-old oaks (Quercus petraea (Matt.) Liebl.) growing under semi-natural conditions, and the dependence of these effects of water stress during the growth season. Percent abundance, carbon concentration and delta(13)C were measured in holocellulose, lignin, extractive-free wood, starch and a water-soluble fraction. Relative concentrations of lignin and holocellulose in the extractive-free wood varied little during the season or in response to water stress, indicating that these compounds could not quantitatively explain the variations in whole-wood delta(13)C. Among all sampled tree rings, the relative concentration of each structural compound (holocellulose and lignin) accounted for less than 5% of the delta(13)C variability in whole wood. Variations in holocellulose and extractive-free wood delta(13)C between tree rings were almost identical (r > 0.95), whereas variations in lignin delta(13)C were less well correlated to these compounds. Whole-wood delta(13)C had a slightly altered isotopic signal compared with that of the structural compounds because of the presence of the extractive component. These results showed that variations in lignin delta(13)C and lignin concentration have little influence on extractive-free wood delta(13)C and whole-wood delta(13)C. Rather, holocellulose influences delta(13)C the most. Thus, we confirmed that, for climatic reconstruction from tree rings, removal of extractives by soxhlet is generally sufficient and sometimes unnecessary. Our findings also indicate that, in the case of rapid and severe water stress, the structural component did not accurately record the associated increase in delta(13)C because of dilution with previously formed organic matter and cessation of trunk growth. The effect of drought on carbon isotope ratios was more pronounced in the extractive compounds, making them good water stress indicators but only on a scale of days to months.