The willow microbiome is influenced by soil petroleum-hydrocarbon concentration with plant compartment-specific effects

International audienceThe interaction between plants and microorganisms, which is the driving force behind the decontamination of petroleum hydrocarbon (PHC) contamination in phytoremediation technology, is poorly understood. Here, we aimed at characterizing the variations between plant compartments in the microbiome of two willow cultivars growing in contaminated soils. A field experiment was set-up at a former petrochemical plant in Canada and after two growing seasons, bulk soil, rhizosphere soil, roots, and stems samples of two willow cultivars (Salix purpurea cv. FishCreek, and Salix miyabeana cv. SX67) growing at three PHC contamination concentrations were taken. DNA was extracted and bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) regions were amplified and sequenced using an Ion Torrent Personal Genome Machine (PGM). Following multivariate statistical analyses, the level of PHC-contamination appeared as the primary factor influencing the willow microbiome with compartment-specific effects, with significant differences between the responses of bacterial, and fungal communities. Increasing PHC contamination levels resulted in shifts in the microbiome composition, favoring putative hydrocarbon degraders, and microorganisms previously reported as associated with plant health. These shifts were less drastic in the rhizosphere, root, and stem tissues as compared to bulk soil, probably because the willows provided a more controlled environment, and thus, protected microbial communities against increasing contamination levels. Insights from this study will help to devise optimal plant microbiomes for increasing the efficiency of phytoremediation technology

AsChip:A High-Throughput qPCR Chip for Comprehensive Profiling of Genes Linked to Microbial Cycling of Arsenic

Arsenic (As) is a ubiquitous toxic element adversely affecting human health. Microbe-mediated cycling of As is largely mediated by detoxification and energy metabolism in microorganisms. We here report the development of a novel high-throughput qPCR (HT-qPCR) chip (AsChip) for comprehensive profiling of genes involved in microbial As cycling (here collectively termed “As genes”). AsChip contained 81 primer sets targeting 19 As genes and the 16S rRNA gene as a reference gene. Gene amplicon sequencing showed high identity (>96%) of newly designed primers corresponding to their targets. AsChip displayed high sensitivity (plasmid template serial dilution test; r = −0.99), with more than 96% of all PCR assays yielding true positive signals. R2 coefficients for standard curves and PCR amplification efficiencies averaged 0.98 and 0.99, respectively. A high correlation between CT values obtained by AsChip and conventional qPCR was obtained (r = 0.962, P < 0.001). Finally, we successfully applied AsChip on soil samples from a chromium–copper–arsenic-contaminated field site and identified diverse As genes with total abundance average of 0.4 As gene copies per 16S rRNA. Our results indicate that AsChip constitutes a robust tool for comprehensive quantitative profiling of As genes in environmental samples

Factors governing the solid phase distribution of Cr, Cu and As in contaminated soil after 40 years of ageing

The physico-chemical factors affecting the distribution, behavior and speciation of chromium (Cr), copper (Cu) and arsenic (As) was investigated at a former wood impregnation site (Fredensborg, Denmark). Forty soil samples were collected and extracted using a sequential extraction technique known as the Chemometric Identification of Substrates and Element Distributions (CISED) and a multivariate statistical tool (redundancy analysis) was applied. CISED data was linked to water-extractable Cr, Cu and As and bioavailable Cu as determined by a whole-cell bacterial bioreporter assay. Results showed that soil pH significantly affected the solid phase distribution of all three elements on site. Additionally, elements competing for binding sites, Ca, Mg and Mn in the case of Cu, and P, in the case of As, played a major role in the distribution of these elements in soil. Element-specific distributions were observed amongst the six identified soil phases including residual pore salts, exchangeable, carbonates (tentative designation), Mn-Al oxide, amorphous Fe oxide, and crystalline Fe oxide. While Cr was strongly bound to non-extractable crystalline Fe oxide in the oxic top soil, Cu and notably, As were associated with readily extractable phases, suggesting that Cu and As, and not Cr, constitute the highest risk to environmental and human health. However, bioavailable Cu did not significantly correlate with CISED identified soil phases, suggesting that sequential extraction schemes such as CISED may not be ideally suited for inferring bioavailability to microorganisms in soil and supports the integration of receptor-specific bioavailability tests into risk assessments as a complement to chemical methods

Characterization and manipulation of the willow microbiome for the phytoremediation of petroleum hydrocarbon- contaminated soil

Phytoremediation, a leading pioneer of green remediation, is an attractive low-cost alternative to conventional soil remediation approaches. However, it is unable to effectively compete in the market due to its lengthy decontamination rates. As such, a detailed understanding of the microbiome dynamics associated with phytoremediation systems is essential in order to optimize degradation processes. The objective of this research was to characterize the influence of compartment, contamination, cultivar type and inoculation on microbiomes associated with willow (Salix spp.) cultivars growing in petroleum hydrocarbon (PHC)-contaminated soil. Next generation sequencing of bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region was used in combination with in-depth multivariate statistics to identify the microbial communities in the soil and in the roots and stems of Salix species. In the first phase of this study, the microbiomes of S. purpurea and S. miyabeana growing in soils from a petrochemical site at different contamination concentrations were characterized. The level of PHC-contamination significantly influenced the microbiome with compartment-specific effects, while significant differences were observed between bacterial and fungal community profiles. Root bacterial communities were made up of a subset of rhizosphere bacteria and consequently, largely shaped by the surrounding environment. Increasing contamination resulted in shifts in the microbiome composition, favoring hydrocarbon degraders and microorganisms associated with plant health. These shifts were less drastic in the rhizosphere and in the root and stem tissues as the plant provided a protective buffer zone against increasing contamination. Isolation and characterization of endophytic bacterial strains living in the roots of S. purpurea and S. miyabeana growing in high concentrations of PHCs was then performed in the second phase of this study. Candidate strains showing polycyclic aromatic hydrocarbon (PAH) degradation and plant growth promotion potential were tested in a 4-month long single and mixed strain inoculation study. Plant growth parameters were monitored over time for individual pots, in addition to the microbial community structures and decontamination percentages, which were examined at the beginning and end of the growing season. An average decontamination of 65% of the PHC-contamination was achieved across all treatments. Inoculation treatments significantly modified the rhizosphere microbial communities, an effect that remained visible after 4 months of growth. Although this inoculation did not have the expected effect on plant growth and decontamination, a legacy effect of treatment inoculation was observed on bacterial and fungal soil and root microbial communities, suggesting that the willow microbiome can be modified by inoculation with bacterial endophytes.La phytoremédiation est une alternative intéressante aux approches classiques de décontamination des sols car cette technologie est moins destructrice envers l’environnement et moins dispendieuse. Par contre, cette technologie verte est incapable de rivaliser sur le marché en raison de ses taux de décontamination trop long. Une connaissance détaillée du microbiome associé aux systèmes de phytoremédiation est essentielle pour optimiser cette technologie. L'objectif de cette recherche était de caractériser l'influence du compartiment, de niveau de contamination, du type de cultivar et de l'inoculation sur les microbiomes associés aux cultivars Salix poussent dans un sol contaminés avec des hydrocarbures pétroliers. Du séquençage à haut débit visant le gène ribosomique 16S des bactéries et la région ITS des champignons a été utilisé en combinaison avec des statistiques multivariées pour identifier les communautés microbiennes dans le sol et dans les racines et les tiges des espèces de Salix. Dans la première phase de cette étude, les microbiomes associés aux cultivars S. purpurea et S. miyabeana croissant sous différents niveaux de contamination sur le site d’une ancienne raffinerie de pétrole ont été caractérisés. Cette étude a démontrée que le compartiment et le niveau de contamination en hydrocarbures pétroliers influencent le microbiome. Les communautés bactériennes des racines ont été constituées d'un sous-ensemble de bactéries de la rhizosphère. La contamination croissante a entrainée des changements dans la composition de microbiome, favorisant les dégradeurs d'hydrocarbures et microbes liés à la santé des plantes. Ces changements étaient moins prononcés dans la rhizosphère et dans les racines et les tiges car la plante fournissait une zone tampon de protection contre la contamination. Ensuite, des travaux d’isolation et de caractérisation de souches bactériennes endophytes vivant dans les racines de S. purpurea et S. miyabeana exposé à des concentrations élevées d’hydrocarbure ont été réalisés. Plusieurs souches d’intérêt ayant démontré un potentiel à dégrader des HAP et à améliorer la croissance des plantes, ont été testées dans une étude d’inoculation de 4 mois. Cette étude a mesuré la croissance des plantes tout au long de l’expérience et la concentration en hydrocarbures pétroliers ainsi que la composition des communautés microbiennes au début et à la fin de l’expérience. Aucune différence significative n’a été observée entre les traitements quant à la croissance des plantes et le pourcentage de décontamination avec une décontamination moyenne de 65%. Par contre, les traitements d'inoculation ont modifiés de manière significative les communautés microbiennes de la rhizosphère, un effet qui est resté visible après quatre mois de croissance. Un effet résiduel des traitements d’inoculation a été observé sur les communautés bactériennes et fongiques de la rhizosphere et des racines, suggérant que le microbiome de saule peut être modifié par inoculation avec des souches endophytes bactériennes

Assessment of biochar and zero-valent iron for in-situ remediation of chromated copper arsenate contaminated soil

Chromated copper arsenates (CCA) have been extensively used as wood impregnation agents in Europe and North America. Today, CCA contaminated sites remain abundant and pose environmental risks that need to be properly managed. Using a TRIAD approach that combined chemical, ecotoxicological and ecological assessment of soil quality, we investigated the abilities of biochar and zero-valent iron (ZVI) to remediate CCA contaminated soil in a microcosm experiment. Soil samples from a highly contaminated CCA site (1364, 1662 and 540 μg g−1 of As, Cu and Cr, respectively) were treated with two different biochars (fine and coarse particle size; 1% w w−1) and ZVI (5% w w−1), both as sole and as combined treatments, and incubated for 56 days at 15 °C. In general, bioavailable As (Asbio) and Cu (Cubio) determined by whole-cell bacterial bioreporters corresponded well to water-extractable As and Cu (Aswater and Cuwater). However, in biochar treatments, only Cubio and not Cuwater was significantly reduced. In contrast, under ZVI treatments only Cuwater and not Cubio was reduced, demonstrating the value of complementing analytical with bacterial bioreporter measurements to infer bioavailability of elements to soil microorganisms. The combined fine particle size biochar and ZVI treatment effectively reduced water extractable concentrations of Cr, Cu, and As on site by 45%, 45% and 43% respectively, and led to the highest ecological recovery of the soil bacterial community, as measured using the [3H]leucine incorporation technique. We conclude that the combined application of biochar and ZVI as soil amendments holds promise for in-situ stabilization of CCA contaminated sites

Transplanting soil microbiomes leads to lasting effects on willow growth, but not on the rhizosphere microbiome

Plants interact closely with microbes, which are partly responsible for plant growth, health, and adaptation to stressful environments. Engineering the plant-associated microbiome could improve plant survival and performance in stressful environments such as contaminated soils. Here, willow cuttings were planted into highly petroleum-contaminated soils that had been gamma-irradiated and subjected to one of four treatments: inoculation with rhizosphere soil from a willow that grew well (LA) or sub-optimally (SM) in highly contaminated soils or with bulk soil in which the planted willow had died (DE) or no inoculation (CO). Samples were taken from the starting inoculum, at the beginning of the experiment (T0) and after 100 days of growth (TF). Short hypervariable regions of archaeal/bacterial 16S rRNA genes and the fungal ITS region were amplified from soil DNA extracts and sequenced on the Illumina MiSeq. Willow growth was monitored throughout the experiment, and plant biomass was measured at TF. CO willows were significantly smaller throughout the experiment, while DE willows were the largest at TF. Microbiomes of different treatments were divergent at T0, but for most samples, had converged on highly similar communities by TF. Willow biomass was more strongly linked to overall microbial community structure at T0 than to microbial community structure at TF, and the relative abundance of many genera at T0 was significantly correlated to final willow root and shoot biomass. Although microbial communities had mostly converged at TF, lasting differences in willow growth were observed, probably linked to differences in T0 microbial communities.Peer reviewed: YesNRC publication: Ye