Phytotoxicity of weathered petroleum hydrocarbons in soil to boreal plant species.

Canada has extensive petroleum hydrocarbon (PHC) contamination in northern areas and the boreal forest region from historical oil and gas activities. Since the 2013 standardization of boreal forest species for plant toxicity testing in Canada, there has been a need to build the primary literature of the toxicity of weathered PHCs to these species. A series of toxicity experiments were carried out using fine-grained (<0.005-0.425 mm) background (100 total mg/kg total PHCs) and weathered contaminated soil (11,900 mg/kg total PHCs) collected from a contaminated site in northern Ontario, Canada. The PHC mixture in the contaminated site soil was characterized through Canadian Council of Ministers of the Environment Fractions, as indicated by the number equivalent normal straight-chain hydrocarbons (nC). The soil was highly contaminated with Fraction 2 (>nC10 to nC16) at 4790 mg/kg and Fraction 3 (>nC16 to nC34) at 4960 mg/kg. Five plant species (Elymus trachycaulus, Achillea millefolium, Picea mariana, Salix bebbiana, and Alnus viridis) were grown from seed in 0%, 25%, 50%, 75%, and 100% relative contamination mixtures of the PHC-contaminated and background soil from the site over 2-6 weeks. All five species showed significant inhibition in shoot length, shoot weight, root length, and/or root weight (Kruskal-Wallis Tests: p < 0.05, df = 4.0). Measurements of 25% inhibitory concentrations (IC25) following PHC toxicity experiments revealed that S. bebbiana was most significantly impaired by the PHC-contaminated soil (410-990 mg/kg total PHCs), where it showed <35% germination. This study indicates that natural weathering of Fraction 2- and Fraction 3-concentrated soil did not eliminate phytotoxicity to boreal plant species. Furthermore, it builds on the limited existing literature for toxicity of PHCs on boreal plants and supports site remediation to existing Canadian provincial PHC guidelines.

Assessment of the toxicity of weathered petroleum hydrocarbon impacted soils to native plants from a site in the Canadian Subarctic.

Remedial guidelines for petroleum hydrocarbons (PHCs) in soil aid in the mitigation of risks to human health and the environmental. However, some remediation guidelines may overestimate the potential for adverse effects to native plant species, contributing to unnecessary remedial efforts in attempts to meet the guidelines. At sites where PHC-contaminated soils undergo weathering, some PHCs may persist but with decreased bioavailability to organisms. In this study, the toxicity of both coarse and fine-grained subarctic soils, contaminated with weathered PHCs were assessed using five native plant species (Picea mariana, Achillea millefolium, Alnus viridis, Elymus trachycaulus and Salix bebbiana). Soil toxicity tests were conducted in a growth chamber with parameters set to simulate the site's subarctic climate conditions. Reference toxicant tests using boric acid were conducted to provide confidence in the interpretation of the results for the PHC-contaminated soils, and also provide new information on the sensitivities of the four boreal species to boric acid. All plants exhibited reduced growth and germination rates as boric acid concentrations increased. Despite exceeding the Canada-wide standard guidelines for Fraction 3 PHCs, field-collected contaminated soils had no significant negative impacts on the growth (i.e., length, dry weight and emergence) of any of the plant species tested.

Identification of degrader bacteria and fungi enriched in rhizosphere soil from a toluene phytoremediation site using DNA stable isotope probing.

Improved knowledge of the ecology of contaminant-degrading organisms is paramount for effective assessment and remediation of aromatic hydrocarbon-impacted sites. DNA stable isotope probing was used herein to identify autochthonous degraders in rhizosphere soil from a hybrid poplar phytoremediation system incubated under semi-field-simulated conditions. High-throughput sequencing of bacterial 16S rRNA and fungal internal transcribed spacer (ITS) rRNA genes in metagenomic samples separated according to nucleic acid buoyant density was used to identify putative toluene degraders. Degrader bacteria were found mainly within the Actinobacteria and Proteobacteria phyla and classified predominantly as Cupriavidus, Rhodococcus, Luteimonas, Burkholderiaceae, Azoarcus, Cellulomonadaceae, and Pseudomonas organisms. Purpureocillium lilacinum and Mortierella alpina fungi were also found to assimilate toluene, while several strains of the fungal poplar endophyte Mortierella elongatus were indirectly implicated as potential degraders. Finally, PICRUSt2 predictive taxonomic functional modeling of 16S rRNA genes was performed to validate successful isolation of stable isotope-labeled DNA in density-resolved samples. Four unique sequences, classified within the Bdellovibrionaceae, Intrasporangiaceae, or Chitinophagaceae families, or within the Sphingobacteriales order were absent from PICRUSt2-generated models and represent potentially novel putative toluene-degrading species. This study illustrates the power of combining stable isotope amendment with advanced metagenomic and bioinformatic techniques to link biodegradation activity with unisolated microorganisms. Novelty statement: This study used emerging molecular biological techniques to identify known and new organisms implicated in aromatic hydrocarbon biodegradation from a field-scale phytoremediation system, including organisms with phyto-specific relevance and having potential for downstream applications (amendment or monitoring) in future and existing systems. Additional novelty in this study comes from the use of taxonomic functional modeling approaches for validation of stable isotope probing techniques. This study provides a basis for expanding existing reference databases of known aromatic hydrocarbon degraders from field-applicable sources and offers technological improvements for future site assessment and management purposes.

Toluene biodegradation in the vadose zone of a poplar phytoremediation system identified using metagenomics and toluene-specific stable carbon isotope analysis.

Biodegradation is an important mechanism of action of phytoremediation systems, but performance evaluation is challenging. We applied metagenomic molecular approaches and compound-specific stable carbon isotope analysis to assess biodegradation of toluene in the vadose zone at an urban pilot field system where hybrid poplars were planted to remediate legacy impacts to an underlying shallow fractured bedrock aquifer. Carbon isotope ratios were compared spatio-temporally between toluene dissolved in groundwater and in the vapor phase. Enrichment of 13C from toluene in the vapor phase compared to groundwater provided evidence for biodegradation in the vadose zone. Total bacterial abundance (16S rRNA) and abundance and expression of degradation genes were determined in rhizosphere soil (DNA and RNA) and roots (DNA) using quantitative PCR. Relative abundances of degraders in the rhizosphere were on average higher at greater depths, except for enrichment of PHE-encoding communities that more strongly followed patterns of toluene concentrations detected. Quantification of RMO and PHE gene transcripts supported observations of active aerobic toluene degradation. Finally, spatially-variable numbers of toluene degraders were detected in poplar roots. We present multiple lines of evidence for biodegradation in the vadose zone at this site, contributing to our understanding of mechanisms of action of the phytoremediation system.

Transcriptome analysis of a springtail, Folsomia candida, reveals energy constraint and oxidative stress during petroleum hydrocarbon exposure.

Petroleum hydrocarbon (PHC) contamination in soil is ubiquitous and poses harmful consequences to many organisms. The toxicity of PHC-impacted soil is difficult to predict due to variations in mixture composition and the impacts of natural weathering processes. Hence, high-throughput methods to assess PHC-impacted soils is required to expedite land management decisions. Next-generation sequencing is a robust tool that allows researchers to investigate the effects of contaminants on the transcriptome of organisms and identify molecular biomarkers. In this study, the effects of PHCs on conventional endpoints (i.e., survival and reproduction) and gene expression rates of a model springtail species, Folsomia candida were investigated. Age-synchronized F. candida were exposed to ecologically-relevant concentrations of soils spiked with fresh crude oil to calculate the reproductive EC25 and EC50 values using conventional toxicity testing. Soils spiked to these concentrations were then used to evaluate effects on the F. candida transcriptome over a 7-day exposure period. RNA-seq analysis found 98 and 132 differentially expressed genes when compared to the control for the EC25 and EC50 treatment groups, respectively. The majority of up-regulated genes were related to xenobiotic biotransformation reactions and oxidative stress response, while down-regulated genes coded for carbohydrate and peptide metabolic processes. Promotion of the pentose phosphate pathway was also found. Results suggest that the decreased reproduction rates of F. candida exposed to PHCs is due to energy constraints caused by inhibition of carbohydrate metabolic processes and allocation of remaining energy to detoxify xenobiotics. These findings provide insights into the molecular effects in F. candida following exposure to crude oil for seven days and highlight their potential to be used as a high-throughput screening test for PHC-contaminated sites. Adverse molecular effects can be measured as early as 24 h following exposure, whereas conventional toxicity tests may require a minimum of four weeks.

Quantification of toluene phytoextraction rates and microbial biodegradation functional profiles at a fractured bedrock phytoremediation site.

This field study evaluated the efficacy of a mature hybrid poplar phytoremediation system for the remediation of toluene in a fractured bedrock aquifer site. Phytoextraction activity of the trees and the ecology and biodegradation potential of root-colonizing bacteria that ultimately influence how much toluene is transported from the roots and phytoextracted to the aboveground point of measurement were explored. Peak-season toluene mass removal rates ranging from 313 to 743 µg/day were quantified using passive in planta contaminant sampling techniques and continuous heat dissipation transpiration measurements in tree stems. Root bacterial microbiome structure and biodegradation potential were evaluated via high-throughput sequencing and predictive metagenomic functional modelling of bacterial 16S rRNA genes in roots. Poplar roots were colonized mostly by Proteobacteria, Actinobacteria, and Bacteroidetes. Distinct, more uniform communities were observed in roots associated with trees planted in the toluene source area compared to other areas, with differences apparent at lower taxonomic levels. Significant enrichment of Streptomyces in roots was observed in the source area, implicating that genus as a potentially important poplar endophyte at toluene-impacted sites. Moreover, significantly greater aerobic toluene biodegradation capacity was predicted in these roots compared to other areas using taxonomic functional modelling. Together with passive sampling, the molecular results provided supporting evidence of biodegradation activity in the source area and contextualized the detected phytoextraction patterns. These results support the application of phytoremediation systems for aromatic hydrocarbons in environments with complex geology and demonstrate field-validated monitoring techniques to assess phytoextraction and biodegradation in these systems.

Siderophore sorption to clays.

Siderophores are low molecular weight organic ligands exuded by some aerobic organisms and plants to acquire Fe under Fe-limited conditions. The hydroxamate siderophores may sorb to aluminosilicate clays through a variety of mechanisms depending upon the nature of the clay and of the siderophore along with solution conditions such as pH, ionic strength, and presence of metal cations. They may also affect metal binding to clays. Here, we review previous studies of siderophore sorption to aluminosilicate clays; briefly discuss how the techniques of X-ray diffractometry, Fourier-transform infrared spectroscopy, and X-ray absorption spectroscopy may be applied to such studies; review effects of siderophores on metal sorption to clays; and highlight some areas for future research.

Assessing toluene biodegradation under temporally varying redox conditions in a fractured bedrock aquifer using stable isotope methods.

In complex hydrogeological settings little is known about the extent of temporally varying redox conditions and their effect on aromatic hydrocarbon biodegradation. This study aims to assess the impact of changing redox conditions over time on aromatic hydrocarbon biodegradation in a fractured bedrock aquifer using stable isotope methods. To that end, four snapshots of highly spatio-temporally resolved contaminant and redox sensitive species concentrations, as well as stable isotope ratio profiles, were determined over a two-years time period in summer 2016, spring 2017, fall 2017 and summer 2018 in a toluene contaminated fractured bedrock aquifer. The concentration profiles of redox sensitive species and stable isotope ratio profiles for dissolved inorganic carbon (DIC) and sulfate (δ13CDIC, δ34SSO4, δ18OSO4) revealed that the aquifer alternates between oxidising (spring 2017/summer 2018) and reducing conditions (summer 2016/fall 2017). This alternation was attributed to a stronger aquifer recharge with oxygen-rich meltwater in spring 2017/summer 2018 compared to summer 2016/fall 2017. The temporally varying redox conditions coincided with various extents of toluene biodegradation revealed by the different magnitude of heavy carbon (13C) and hydrogen (2H) isotope enrichment in toluene. This indicated that the extent of toluene biodegradation and its contribution to plume attenuation was controlled by the temporally changing redox conditions. The highest toluene biodegradation was observed in summer 2016, followed by spring 2017 and fall 2017, whereby these temporal changes in biodegradation occurred throughout the whole plume. Thus, under temporally varying recharge conditions both the core and the fringe of a contaminant plume can be replenished with terminal electron acceptors causing biodegradation in the whole plume and not only at its distal end as previously suggested by the plume fringe concept. Overall, this study highlights the importance of highly temporally resolved groundwater monitoring to capture temporally varying biodegradation rates and to accurately predict biodegradation-induced contaminant attenuation in fractured bedrock aquifers.

Effects of the microbial siderophore DFO-B on Pb and Cd speciation in aqueous solution.

This study investigates the complexation environments of aqueous Pb and Cd in the presence of the trihydroxamate microbial siderophore, desferrioxamine-B (DFO-B) as a function of pH. Complexation of aqueous Pb and Cd with DFO-B was predicted using equilibrium speciation calculation. Synchrotron-based X-ray absorption fine structure (XAFS) spectroscopy at Pb L(III) edge and Cd K edge was used to characterize Pb and Cd-DFO-B complexes at pH values predicted to best represent each of the metal-siderophore complexes. Pb was not found to be complexed measurably by DFO-B at pH 3.0, but was complexed by all three hydroxamate groups to form a totally "caged" hexadentate structure at pH 7.5-9.0. At the intermediate pH value (pH 4.8), a mixture of Pb-DFOB complexes involving binding of the metal through one and two hydroxamate groups was observed. Cd, on the other hand, remained as hydrated Cd2+ at pH 5.0, occurred as a mixture of Cd-DFOB and inorganic species at pH 8.0, and was bound by three hydroxamate groups from DFO-B at pH 9.0. Overall, the solution species observed with EXAFS were consistent with those predicted thermodynamically. However, Pb speciation at higher pH values differed from that predicted and suggests that published constants underestimate the binding constant for complexation of Pb with all three hydroxamate groups of the DFO-B ligand. This molecular-level understanding of metal-siderophore solution coordination provides physical evidence for complexes of Pb and Cd with DFO-B, and is an important first step toward understanding processes at the microbial- and/or mineral-water interface in the presence of siderophores.

Biofilm hydrous manganese oxyhydroxides and metal dynamics in acid rock drainage.

Biofilms in shallow, tailings-associated acid rock drainage (ARD) accumulated metals from May to September, indicating scavenging is stable within these biological solids over seasonal time frames. Results indicate a doubling (Mn, Cr) to over a 6-fold increase (Ni, Co) in biofilm metal concentrations. Biofilm oxygen and pH gradients measured over diel time scales with microelectrodes were observed to be both spatially and temporally variable, indicating that biofilms are highly dynamic geochemical environments. Biofilm metal retention and affinities were element specific indicating different processes control their sequestration. Metals were specifically scavenged by the organic constituents of the biofilm itself (Ni, Co) and associated biominerals of amorphous Mn oxyhydroxides (HMO; Ni, Co, and Cr). Results are consistent with sorption and coprecipitation processes controlling Ni and Co biofilm association, while Cr dynamics appear linked to those of Mn through redox processes. Biofilm HMO concentrations increased seasonally but showed significant diel fluctuations, indicating that both formation and dissolution processes occurred over rapid time scales in these biofilms. Biofilm HMO concentrations increased nocturnally but decreased during daylight hours to late afternoon minima. Under the geochemical conditions of the streams, observed HMO formation rates can only be explained by microbial catalysis. These results are the first to quantitatively examine microbial biofilm metal dynamics using microscale, geochemical techniques at both diel and seasonal time scales. They provide strong evidence for the significant role that microbial activity can play in metal geochemistry in natural environments.