Sébastien Roy

Biofiltration of air contaminated with toluene on a compost-based bed

Many studies have focused on problems created by emissions to the atmosphere of gaseous effluents containing volatile organic compounds (VOCs). Over the more recent decades, such studies have led to the development of various bioreactors such as the bioscrubber, the biotrickling filter and the biofilter. This paper presents the results of a study on the biofiltration of airborne toluene, the biofilter employed being operated at the laboratory-scale for a continuous period of 3 months. The focus of this particular study has been the development of a new compost-based filter-bed material, which consists of an association between matured compost and a proprietary organic binder that is intended to prolong the period of the beds efficient operations. No inoculum was added to the filter-bed material. During the experimental program, the performance of two different bed irrigation solutions was examined, the most effective nutrient supply solution then being used, along with toluene input levels varying from 0.6–2.6 g/m3, and toluene polluted air flow rates ranging from 0.4–1 m3/h, equivalent to empty bed residence times of 65–165 s. The results of this program have demonstrated removal efficiencies approaching 95%, while maximum elimination capacities of 55 g/m3 h, for an inlet load of 65 g/m3 h, have been achieved, supporting the view that the compost-based filter material tested in this work functions as a promising biofilter medium in this application. Finally, in order to present the biofilter performance observed under the best operating conditions, a simplified representation based on Ottengrafs model has been developed from the experimental results and is included here.

Combining alders, frankiae, and mycorrhizae for the revegetation and remediation of contaminated ecosystems

Alder shrubs and trees that are capable of forming symbioses with mycorrhizal fungi and the nitrogen-fixing ac- tinomycete Frankia sp. are particularly hardy species found worldwide in harsh and nutrient-deficient ecosystems. The my- corrhizal symbiosis may assist alders in nutrient and water uptake, while the actinorhizal symbiosis provides assimilable nitrogen. It is through these highly efficient symbioses, in which microsymbionts benefit from plant photosynthates, that actinorhizal plants such as alders colonize poor substrates, enrich soil, and initiate plant succession. These natural capabil- ities, combined with careful screening of microsymbionts and host plants, may prove useful for the rehabilitation of dis- turbed ecosystems. Although alders have been used extensively at industrial scales in forestry, nurse planting, and contaminated land revegetation, relatively little research has focussed on their actinorhizal and mycorrhizal plant-microbe interactions in contaminated environments. To study such a topic is, however, critical to the successful development of phytotechnologies, and to understand the impact of anthropogenic stress on these organisms. In this review, we discuss two alder-based phytotechnologies that hold promise: the stimulation of organic contaminant biodegradation (rhizodegrada- tion) by soil microflora in the presence of alders, and the phytostabilization of inorganic contaminants. We also summarize the plant-microbe interactions that characterize alders, and discuss important issues related to the study of actinorhizal and (or) mycorrhizal alders for the rehabilitation of disturbed soils.

In vitro selection and characterization of RNA aptamers binding thyroxine hormone.

RNA possesses the ability to bind a wide repertoire of small molecules. Some of these binding interactions have been shown to be of primary importance in molecular biology. For example, several classes of mRNA domains, collectively referred to as riboswitches, have been shown to serve as RNA genetic control elements that sense the concentrations of specific metabolites (i.e. acting as direct sensors of chemical compounds). However, to date no RNA species binding a hormone has been reported. Here, we report that the use of an appropriate SELEX (systematic evolution of ligands by exponential enrichment) strategy results in the isolation of thyroxine-specific aptamers. Further biochemical characterization of these aptamers, including mutational studies, the use of transcripts with site-specific modified nucleotides, nuclease and chemical probing, binding-shift assays and CD, demonstrated that these RNA structures included a G-rich motif, reminiscent of a guanine quadruplex structure, adjacent to a helical region. The presence of the thyroxine appeared to be essential for the formation of the structural motifs scaffold. Moreover, the binding is shown to be specific to thyroxine (T4) and tri-iodothyronine (T3), the active forms of the hormone, whereas other inactive derivatives, including thyronine (T0), do not support complex formation. These results suggest that this aptamer specifically binds to the iodine moieties of the thyroxine, a previously unreported ability for an RNA molecule.

Uncovering the Prevalence and Diversity of Integrating Conjugative Elements in Actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their hosts chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.

Effect of chitin waste-based composts produced by two-phase composting on two oomycete plant pathogens

Biphasic composts were prepared by first mixing peat moss and sawdust with a nitrogen-rich biomass such as chitinous waste or cow manure and composting them until termination of the thermophilic phase. These partially stabilized composts were then amended with shrimp waste inducing a second thermophilic phase. Filter-sterilized water extracts obtained from two mature biphasic composts (SP2W2+S and MPW+S) reduced the growth of two oomycete plant pathogens, Phytophthora fragariae var. rubi and Pythium ultimum. Both SP2W2+S and MPW+S composts significantly reduce the incidence of cucumber damping-off caused by Pythium ultimum as compared to a commercial brand of compost made from shrimp waste and peat moss. Hydrolysis products of chitin were unlikely to be responsible for growth inhibition since no oligomeric forms of chitin were detected in SP2W2+S. The shrimp waste amendment carried out after the first thermophilic phase modified the microbial populations of biphasic composts. Following the amendment, the proportion of branched-chain microbial fatty acids typical of Gram-positive bacteria increased considerably suggesting that this group of bacteria became more prevalent within the total microbial population. These data suggests that the two-phase composting process promotes the proliferation of Gram-positive bacteria antagonistic to oomycete plant pathogens.

High-throughput screening of microbial adaptation to environmental stress

We developed a microwell plate, high-throughput, screening method aimed at quantitating the tolerance of a panel of Gram-positive and Gram-negative bacteria to metals (Frankia sp., Escherichia coli, Cupriavidus metallidurans, Rhizobium leguminosarum, and Streptomyces scabies). Microbial viability was quantified using MTS; a tetrazolium salt converted to a water-soluble formazan through microbial reduction. In this paper, we present the stepwise development of the method, highlighting the main elements underlying its reliability, and compare results obtained with literature. We conclude the method is well suited to efficiently screen bacteria, including those that are filamentous and slow-growing, without the need for large amounts of inoculum which may not always be available. The method allows testing of compound gradients with sufficient replicates to generate statistically robust results, and is transposable to other types of cell proliferation assays such as those for antimicrobial susceptibility, and chemoresistance.

Comparison of dioxin and furan TEQ determination in contaminated soil using chemical, micro-EROD, and immunoassay analysis

High resolution mass spectrometry gas chromatography (GC/MS) is the standard method for dioxin and furan analysis in environmental matrices. Considered as very accurate, this method is however time consuming and expensive. Methods based on biological interactions have the necessary sensitivity but began only recently to be investigated in the context of environmental applications. We have compared dioxin and furan toxicity levels (expressed as toxic equivalent quantities (TEQs)) in soil samples by three analytical approaches: the micro-ethoxyresorufin-o-deethylase (EROD) bioassay (a receptor-based method), an immunoassay (antibody-based method) and GC/MS analysis (used as a reference) using a shortened extraction-purification method. Both biological methods were sensitive to interferences from compounds co-extracted from samples. Most samples were underestimated by the immunoassay and, at a greater extent, overestimated by the EROD bioassay. The average accuracy of TEQ estimation (86 +/- 45% of values established by GC/MS) and the absence of false-negatives showed by the immunoassay suggest the usefulness of this method for semi-quantitative, preliminary characterization of potentially contaminated sites.

A Novel Two-Phase Composting Process Using Shrimp Shells as an Amendment to Partly Composted Biomass

Shrimp and crab shells as well as other seafood industry residues contain chitin as a major component. Partly degraded (oligomeric) chitin is recognized as an elicitor of plant defense mechanisms and a growth suppressor of pathogenic fungi. A method of composting shrimp shells was developed with the objective of obtaining a compost of good quality and characterized by a significant content of oligomeric chitin. The method consists in mixing peat moss, sawdust and a nitrogen rich biomass such as cow manure and composting them until the thermophilic phase is terminated. The immature compost is then amended with 30 percent of shrimp waste on a dry weight basis, inducing a second thermophilic phase. An enzymatic method was developed to monitor the process of chitin degradation and to determine the most appropriate moment to stop the composting process. It was also shown that the peak value of oligomeric chitin content is accompanied by a decisive drop in ammonium content. The shrimp waste-based composts produ...

The Symbiosis between Frankia alni and Alder Shrubs Results in a Tolerance of the Environmental Stress Associated With Tailings from the Canadian Oil Sands Industry

Alders are well recognized for their ability to colonize the harsh environments created by either natural processes or human activity. They establish symbiosis with the actinomycete Frankia which supplies 70 to 100 % of the plant’s nitrogen requirements. An important challenge facing the oil sands industry in Alberta, Canada, is management of the toxicity of Tailing Sands (TS) that are alkaline, saline and contain Naphthenic Acids (NA).In order to begin to understand how alders, Frankia and their symbiosis perform and adapt to these challenging environmental conditions, the tolerance of the microsymbiont (Frankia), the host plants (alders) and their symbiosis to specific compounds found in TS was studied. In addition, the metabolic response of Frankiaalni ACN14a to the presence of NA was characterized. The Frankia strains tested tolerated both high pH and NA levels in addition to salts near or at the concentrations found in TS. Furthermore, actinorhizal symbiosis can establish itself under these conditions. NaCl was observed to exert the greatest stress on the establishment of symbiosis, decreasing the efficiency of the actinorhizal symbiosis. Inoculation of the alder plants with Frankia induced a significant increase in aerial biomass allocation. Finally, intracellular proteins in Frankiaalni ACN14a whose expression level were influenced by naphthenic acids were identified. Together, these results demonstrate that actinorhizal alders show significant promise for use in the revegetation of lands affected by the mine tailings of the Canadian petroleum industry.

Actinorhizal Alder Phytostabilization Alters Microbial Community Dynamics in Gold Mine Waste Rock from Northern Quebec: A Greenhouse Study.

Phytotechnologies are rapidly replacing conventional ex-situ remediation techniques as they have the added benefit of restoring aesthetic value, important in the reclamation of mine sites. Alders are pioneer species that can tolerate and proliferate in nutrient-poor, contaminated environments, largely due to symbiotic root associations with the N2-fixing bacteria, Frankia and ectomycorrhizal (ECM) fungi. In this study, we investigated the growth of two Frankia-inoculated (actinorhizal) alder species, A. crispa and A. glutinosa, in gold mine waste rock from northern Quebec. Alder species had similar survival rates and positively impacted soil quality and physico-chemical properties in similar ways, restoring soil pH to neutrality and reducing extractable metals up to two-fold, while not hyperaccumulating them into above-ground plant biomass. A. glutinosa outperformed A. crispa in terms of growth, as estimated by the seedling volume index (SVI), and root length. Pyrosequencing of the bacterial 16S rRNA gene for bacteria and the ribosomal internal transcribed spacer (ITS) region for fungi provided a comprehensive, direct characterization of microbial communities in gold mine waste rock and fine tailings. Plant- and treatment-specific shifts in soil microbial community compositions were observed in planted mine residues. Shannon diversity and the abundance of microbes involved in key ecosystem processes such as contaminant degradation (Sphingomonas, Sphingobium and Pseudomonas), metal sequestration (Brevundimonas and Caulobacter) and N2-fixation (Azotobacter, Mesorhizobium, Rhizobium and Pseudomonas) increased over time, i.e., as plants established in mine waste rock. Acetate mineralization and most probable number (MPN) assays showed that revegetation positively stimulated both bulk and rhizosphere communities, increasing microbial density (biomass increase of 2 orders of magnitude) and mineralization (five-fold). Genomic techniques proved useful in investigating tripartite (plant-bacteria-fungi) interactions during phytostabilization, contributing to our knowledge in this field of study.