Jean-Guy Bisaillon

Isolation and characterization of Desulfitobacterium frappieri sp. nov., an anaerobic bacterium which reductively dechlorinates pentachlorophenol to 3-chlorophenol

An anaerobic bacterium, strain PCP-1T (T = type strain), which dechlorinates pentachlorophenol (PCP) to 3-chlorophenol, was isolated from a methanogenic consortium. This organism is a spore-forming rod-shaped bacterium that is nonmotile, asaccharolytic, and Gram stain negative but Gram type positive as determined by electron microscopic observations. Inorganic electron acceptors, such as sulfite, thiosulfate, and nitrate (but not sulfate), stimulate growth in the presence of pyruvate and yeast extract. The optimum pH and optimum temperature for growth are 7.5 and 38 degrees C, respectively. The dechlorination pathway is: PCP-->2,3,4,5-tetrachlorophenol -->3,4,5-trichlorophenol-->3,5-dichlorophenol-->3-chlorophenol. This bacterium dechlorinates several different chlorophenols at ortho, meta, and para positions; exceptions to this are 2,3-dichlorophenol, 2,5-dichlorophenol, 3,4-dichlorophenol, and the monochlorophenols. The time course of PCP dechlorination suggests that two enzyme systems are involved in dehalogenation in strain PCP-1T. One system is inducible for ortho dechlorination, and the second system is inducible for meta and para dechlorinations. A 16S rRNA analysis revealed that strain PCP-1T exhibits 95% homology with Desulfitobacterium dehalogenans JW/IU-DC1, an anaerobic bacterium which can dehalogenate chlorophenols only in ortho positions. These results suggest that strain PCP-1T is a member of a new species and belongs to the recently proposed genus Desulfitobacterium. Strain PCP-1T differs from D. dehalogenans JW/IU-DC1 by its broader range of chlorophenol dechlorination. Strain PCP-1 is the type strain of the new species, Desulfitobacterium frappieri.

Optimization of high-molecular-weight polycyclic aromatic hydrocarbons‘ degradation in a two-liquid-phase bioreactor

A microbial consortium degrading the high‐molecular‐weight polycyclic aromatic hydrocarbons (HMW PAHs) pyrene, chrysene, benzo[a]pyrene and perylene in a two‐liquid‐phase reactor was studied. The highest PAH‐degrading activity was observed with silicone oil as the water‐immiscible phase; 2,2,4,4,6,8,8‐heptamethylnonane, paraffin oil, hexadecane and corn oil were much less, or not efficient in improving PAH degradation by the consortium. Addition of surfactants (Triton X‐100, Witconol SN70, Brij 35 and rhamnolipids) or Inipol EAP22 did not promote PAH biodegradation. Rhamnolipids had an inhibitory effect. Addition of salicylate, benzoate, 1‐hydroxy‐2‐naphtoic acid or catechol did not increase the PAH‐degrading activity of the consortium, but the addition of low‐molecular‐weight (LMW) PAHs such as naphthalene and phenanthrene did. In these conditions, the degradation rates were 27 mg l−1 d−1 for pyrene, 8·9 mg l−1 d−1 for chrysene, 1·8 mg l−1 d−1 for benzo[a]pyrene and 0·37 mg l−1 d−1 for perylene. Micro‐organisms from the interface were slightly more effective in degrading PAHs than those from the aqueous phase.

Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste

The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques. The temperature increased quickly in the first hours of the treatment cycles and values up to 72°C were reached. Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles. A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60°C. Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found. In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%). The other Bacilli could not be assigned to a known species. Schineria larvae was the second most abundant species (14%) in the clone library. Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40°C and 60°C. Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated.The microflora of a self-heating aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste was investigated by a combination of culture and culture-independent techniques. The temperature increased quickly in the first hours of the treatment cycles and values up to 72°C were reached. Denaturing gradient gel electrophoresis of the PCR-amplified V3 region of 16S rDNA (PCR-DGGE) revealed important changes in the bacterial community during 3-day cycles. A clone library was constructed with the near-full-length 16S rDNA amplified from a mixed-liquor sample taken at 60°C. Among the 78 non-chimeric clones analysed, 20 species (here defined as clones showing more than 97% sequence homology) were found. In contrast to other culture-independent bacterial analyses of aerobic thermophilic wastewater treatments, species belonging to the Bacilli class were dominant (64%) with Bacillus thermocloacae being the most abundant species (38%). The other Bacilli could not be assigned to a known species. Schineria larvae was the second most abundant species (14%) in the clone library. Four species were also found among the 19 strains isolated, cultivated and identified from samples taken at 40°C and 60°C. Ten isolates showed high 16S rDNA sequence homology with the dominant bacterium of a composting process that had not been previously isolated.

Hydrolysis of 4-Hydroxybenzoic Acid Esters (Parabens) and Their Aerobic Transformation into Phenol by the Resistant Enterobacter cloacae Strain EM

ABSTRACT Enterobacter cloacae strain EM was isolated from a commercial dietary mineral supplement stabilized by a mixture of methylparaben and propylparaben. It harbored a high-molecular-weight plasmid and was resistant to high concentrations of parabens. Strain EM was able to grow in liquid media containing similar amounts of parabens as found in the mineral supplement (1,700 and 180 mg of methyl and propylparaben, respectively, per liter or 11.2 and 1.0 mM) and in very high concentrations of methylparaben (3,000 mg liter−1, or 19.7 mM). This strain was able to hydrolyze approximately 500 mg of methyl-, ethyl-, or propylparaben liter−1 (3 mM) in less than 2 h in liquid culture, and the supernatant of a sonicated culture, after a 30-fold dilution, was able to hydrolyze 1,000 mg of methylparaben liter−1 (6.6 mM) in 15 min. The first step of paraben degradation was the hydrolysis of the ester bond to produce 4-hydroxybenzoic acid, followed by a decarboxylation step to produce phenol under aerobic conditions. The transformation of 4-hydroxybenzoic acid into phenol was stoichiometric. The conversion of approximately 500 mg of parabens liter−1 (3 mM) to phenol in liquid culture was completed within 5 h without significant hindrance to the growth of strain EM, while higher concentrations of parabens partially inhibited its growth.

Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP → 2,3,5,6-tetrachlorophenol → 2,3,5-trichlorophenol → 3,5-dichlorophenol → 3-chlorophenol → phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 μmol (1 reactor volume)−1 day−1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.

Quantitative flow cytometric detection of specific microorganisms in soil samples using rRNA targeted fluorescent probes and ethidium bromide

Specific detection and accurate enumeration of microorganisms in the environment have been hampered by the lack of suitable techniques. A three-parameter flow cytometric method (FCM) was developed to detect quantitatively Sphingomonas sp. strain 107 inoculated into soil samples. By combining light scattering profiles (i.e., morphological properties), ethidium bromide (EtBr) influx (i.e., wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could accurately discriminate the bacterium of interest from the indigenous microflora and soil debris. EtBr was used, first, to determine the optimal cell wall permeabilization treatment to allow oligonucleotide probes to enter the bacterial cells and, second, to achieve clear discrimination of fixed cells from debris in soil samples. This method allowed effective qualitative and quantitative analysis by fluorescence in situ hybridization. The results showed that the detection threshold by FCM was 3 x 10(4) cells/g of dry soil. Cell counts deduced from FCM analysis were similar to those obtained by the colony forming unit assay when soils contained fewer than 3 x 106 cells/g dry soil. This method should be useful for either quantitative monitoring of microorganisms inoculated in contaminated soil samples during bioremediation or detecting known bacterial strains in environmental samples.

Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Desulfitobacterium frappieri strain PCP-1.

Abstract Anaerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil from a wood-treating industrial site was studied in soil slurry microcosms inoculated with a PCP-degrading methanogenic consortium. When the microcosms containing 10%–40% (w/v) soil were inoculated with the consortium, more than 90% of the PCP was removed in less than 30 days at 29 °C. Less-chlorinated phenols, mainly 3-chlorophenol were slowly degraded and accumulated in the cultures. Addition of glucose and sodium formate to the microcosms was not necessary, suggesting that the organic compounds in the soil can sustain the dechlorinating activity. Inoculation of Desulfitobacterium frappieri strain PCP-1 along with a 3-chlorophenol-degrading consortium in the microcosms also resulted in the rapid dechlorination of PCP and the slow degradation of 3-chlorophenol. Competitive polymerase chain reaction experiments showed that PCP-1 was present at the same level throughout the 21-day biotreatment. D. frappieri, strain PCP-1, inoculated into the soil microcosms, was able to remove PCP from soil containing up to 200 mg PCP/kg soil. However, reinoculation of the strain was necessary to achieve more than 95% PCP removal with a concentration of 300 mg and 500 mg PCP/kg soil. These results demonstrate that D. frappieri strain PCP-1 can be used effectively to dechlorinate PCP to 3-chlorophenol in contaminated soils.

Degradation of phenolic compounds by microorganisms indigenous to swine waste

Abstract Degradation of phenol and p-cresol by mixed microorganisms indigenous to swine manure was investigated. Resting-cell suspensions catalyzed complete degradation of both compounds under aerobic conditions. The stoichiometry of the phenol-oxidase system yielded a value of 6 mol of CO2 produced when 7 mol of O2 and 1 mol of phenol were consumed, while the p-cresol-oxidase system yielded a value of 7 mol of CO2 produced when 8·5 mol of O2 and 1 mol of p-cresol were consumed. Although phenol and p-cresol were completely degraded by intact, mixed cells under aerobic conditions, these compounds were not metabolized under the anaerobic conditions used. Since complete anaerobic conditions exist in the stored swine manure, these phenolic compounds accumulate during storage. Since these compounds were degraded completely by the mixed microflora in the presence of oxygen, large-scale aeration of stored wastes might be used for the degradation of such compounds present in the wastes.

Quantification of Desulfitobacterium frappieri strain PCP-1 and Clostridium-like strain 6 in mixed bacterial populations by competitive polymerase chain reaction

Abstract Competitive polymerase chain reaction (cPCR) was used to quantify two anaerobic, Gram-positive bacteria, Desulfitobacterium frappieri strain PCP-1 and Clostridium -like strain 6 in mixed bacterial populations. cPCR was done with specific primers targeting the respective 16S rRNA genes of these strains and an internal standard (IS). The IS for each strain had the same primer binding sites, size and sequence as the target DNA, except that a unique restriction site was introduced by PCR-mediated site-directed mutagenesis. To distinguish between the IS and the target DNA, appropriate restriction digestion was done. cPCR was performed on genomic DNA of both strains, from which, a close value of the number of genomes added in the PCR mixtures was obtained. cPCR was also carried out with DNA extracted from a soil inoculated with a known amount of strain PCP-1 cells (determined by a plating method). The cell number of strain PCP-1 in the soil, determined by the cPCR, was similar to the number of CFU inoculated. Evaluation of the strain PCP-1 cell concentration was achieved in a pentachlorophenol (PCP)-degrading methanogenic consortium, from which strain PCP-1 was isolated. This consortium was used in a continuous flow activated sludge reactor to treat PCP-contaminated industrial effluents. cPCR revealed that strain PCP-1 was present at a concentration of 1.8×10 5 cells/mL in the reactor. The cell concentration of strain 6 was evaluated in a phenol-degrading methanogenic consortium, from which strain 6 was isolated. Strain 6 was estimated by cPCR to be 6.5×10 5 cells/mL in a 20-day-old consortium culture. This is the first time that these two anaerobic Gram-positive bacteria were quantified in mixed bacterial populations.

Isolation of an anaerobic bacterial consortium degrading phenolic compounds—Assay in swine waste

Abstract A microbial culture able to degrade phenol and p-cresol under anaerobic conditions was isolated from a mixture of swamp water, sewage sludge, swine waste and soil after an acclimation period of 5 weeks. The culture was enriched by periodic transfer in mineral salts medium containing phenol or p-cresol as the sole source of carbon. After several months of enrichment, the degradative activity of some cultures was lost. Addition of proteose peptone to the minimal medium maintained the activity, whereas the addition of acetate, glucose or nitrate or the presence of silica gel as a solid matrix to hold the cells, had no effect. The degradation of the phenolic compounds appeared to be carried out via methanogenic fermentation by a bacterial consortium. Optimal degradation by the enriched culture was at pH between 7 and 8 and at 37°C. The bacterial culture was adapted to grow in swine waste and complete degradation of 100–120 mg liter −1 of phenolic compounds was obtained in swine waste seeded with this culture.