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.

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.