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R Beaudet

Publications and source records attributed to R Beaudet.

At least 37 records · Page 2Linked to original sources

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

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 degrees 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.

Bacteria, Anaerobic↗

Spectrum of the reductive dehalogenation activity of desulfitobacterium frappieri PCP-1

Desulfitobacterium frappieri PCP-1 was induced for ortho- and para-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at ortho, meta, and para positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.

Journal Article↗

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.

Cell Separation↗

Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction.

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 microgram soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time.

Bacteria, Anaerobic↗

Anaerobic biodegradation of pentachlorophenol in a liquor obtained after extraction of contaminated chips and wood powder.

Recovery of 97.5% of the pentachlorophenol (PCP) in contaminated wood powder was obtained after extraction with 0.1% KOH solution at 60 degrees C for 75 min. Extraction with NaOH and Na2CO3 was less effective than KOH. The neutralized extract was treated using a methanogenic consortium in an upflow anaerobic fixed-film reactor. The reactor was operated at 29 degrees C for over 600 d. The best performance of the reactor was observed when the PCP liquor was supplemented with glucose and formate. Complete dechlorination of PCP and phenol removal was obtained for a PCP loading rate of 13.3-18.0 mg l(-1) of reactor volume d(-1) with recirculation of the effluent and a hydraulic retention time (HRT) of 0.5-0.6 d.

Bacteria, Anaerobic↗

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.

Bacteria, Anaerobic↗

Transformation of phenol into phenylalanine by a methanogenic consortium.

The conversion by a methanogenic consortium of phenol into phenylalanine, with benzoic and phenylpropionic acid as intermediates, was investigated. When (sup14)C-labelled phenol was fed to the consortium, the radioactivity was mostly transferred into methane and CO(inf2), but 4% of the radioactivity was found in the water fraction after extraction of the culture medium with an organic solvent. Utilization of labelled compounds and analysis by gas chromatography coupled with mass spectrometry revealed that a fraction of the benzoic acid produced was transformed into 3-phenylpropionic acid. When fully (sup13)C-labelled acetic acid was fed to the consortium, the labels were incorporated at the 1 and 2 positions of 3-phenylpropionic acid. When deuterium-labelled 3-phenylpropionic acid was fed to the consortium, part of the phenylalanine of the biomass was labelled. These metabolic transformations are reversible, since deuterium-labelled phenylalanine generated labelled 3-phenylpropionic acid. Cinnamic acid was also transformed into 3-phenylpropionic acid.

Journal Article↗

Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions.

A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture. Clostridium ghonii was inhibited by chloramphenicol (10 micrograms/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 micrograms/ml), without the transformation of phenol being affected. Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C hastiforme strain (strain 2) (width, 1 micron) and an unidentified strain 6 (width, 0.6 micron) which was not detected on solid medium. Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6. C hastiforme 2 was eliminated from this culture by dilution. The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone. Both of these activities are inducible. This strain is a gram- variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol. It has a cellular fatty acid composition like that of C. hastiforme. However, strain 6 does not hydrolyze gelatin or produce indole. The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86%. Tbe evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium-related species revealed that it does not emerge from any of these groups. Strain 6 most likely belongs to a new species closely related to Clostridium species.

Anaerobiosis↗

Study of the reductive dechlorination of pentachlorophenol by a methanogenic consortium.

Pentachlorophenol (PCP) dechlorination by a methanogenic consortium was observed when glucose, formate, lactate, or yeast extract was present in the mineral medium as a secondary carbon source. Acetate was not a good substrate to sustain dechlorination. The consortium was able to dechlorinate the different monochlorophenols, although the chlorine in position ortho and meta was removed more readily than in para position. Dechlorination was most efficient at 37 degrees C. At 45 degrees C, the first PCP dechlorination steps were very rapid, but 3,5-dichlorophenol (3,5-DCP) was not further dechlorinated. At 15 and 4 degrees C, dechlorination was very slow. The dechlorination of PCP to 3-chlorophenol (3-CP) was still observed after the consortium had been subjected to heat treatment (80 degrees C, 60 min), suggesting that spore-forming bacteria were responsible. The dechlorinating activity of the consortium was significantly reduced by the presence of hydrogen, 2-bromoethanosulfonic acid (BESA), or sulfate but not of nitrate. The dechlorination of 3-CP was completely inhibited by heat treatment or the presence of BESA, suggesting that a syntrophic microorganism would be involved. Vigorous agitation of the consortium stopped the dechlorination, but the presence of DEAE-Sephacel acting as a support was very efficient in restoring the activity, suggesting that association between certain members of the consortium was important.

Alkanesulfonic Acids↗

Protection of mice and swine against infection with Actinobacillus pleuropneumoniae by vaccination.

CaCl2 and LiCl cell extracts and a crude hemolysin preparation were isolated from Actinobacillus pleuropneumoniae serotype 1 strain 4074 and tested for protection against A. pleuropneumoniae serotype 1 and 5 in mice. The LiCl cell extract adsorbed on AlPO4 and the crude hemolysin preparation adsorbed on Al(OH)3 showed a highly significant protection (P < 0.01) against both serotypes. Different vaccine preparations were used to immunize pigs by intra-muscular injection at days 0 and 14; the pigs were then challenged at day 21 by intra-tracheal inoculation of 1 x 10(8) colony forming units (CFU) of a serotype 1 strain 4074. A vaccine which combined the LiCl extract and the crude hemolysin preparation adsorbed on Al(OH)3 gave the best protection with no mortality and no sign of morbidity in the vaccinated pigs. In the other experimental groups which included a group immunized with a commercial bacterin, mortality, respiratory disease and extensive pulmonary lesions were noted. This mixture shows good potential as a vaccine against pleuropneumonia in pigs.

Actinobacillus Infections↗

Production and purification of a low molecular weight haemolysin produced by Actinobacillus pleuropneumoniae serotype 1.

An unstable haemolytic activity produced by a strain of serotype 1 of Actinobacillus pleuropneumoniae was isolated when 1 per cent bovine serum albumin (BSA) was added to RPMI 1640 medium. BSA acts as a carrier molecule and stabilises activity. This haemolysin (BSA-haemolysin) was precipitated with ammonium sulphate, dialysed and lyophilised. Of the species tested, bovine erythrocytes were the most susceptible to the BSA-haemolysin while mouse and rabbit erythrocytes were the least susceptible. The haemolytic activity was dependent on the incubation temperature, no activity being observed at or below 24 degrees C. The haemolytic activity was also partly stabilised by 100 micrograms ml-1 dithiothreitol (DTT). The DTT-haemolysin was purified to homogeneity by ultrafiltration and high pressure liquid chromatography on a Protein Pak DEAE-5PW column. The molecular weight was estimated at 16 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and at 23 kDa by molecular gel filtration from the elution position of the haemolytic activity. The DTT-haemolysin activity was completely destroyed by pronase treatment suggesting that this substance could be a polypeptide. The addition of BSA to DTT-haemolysin increased its activity and stability to lyophilisation. The addition of 10 mM calcium chloride in the titration assay increased the activity of DTT-haemolysin from 220 to 476 haemolytic units ml-1. The BSA-haemolysin activity was only slightly affected by the addition of calcium chloride.

Actinobacillus pleuropneumoniae↗

Potential for carboxylation-dehydroxylation of phenolic compounds by a methanogenic consortium.

An anaerobic consortium that carboxylated and dehydroxylated phenol to benzoate, and 2-cresol to 3-methylbenzoic acid, under methanogenic conditions was studied. Phenol induced this transformation activity. Addition of 4-hydroxypyridine or an increase in the concentration of proteose peptone to 0.5% (w/v) delayed the transformation. Phenol enhanced the rate of transformation of 2-cresol whereas 2-cresol delayed the transformation of phenol. Phenols with ortho-substitutions (chloro-, fluoro-, bromo-, hydroxyl-, amino-, or carboxyl-) were transformed to meta-substituted benzoic acids. However, meta- and para-substituted phenols (cresols, fluorophenols, and chlorophenols) were not transformed. Phenol was most rapidly metabolized, followed by catechol, 2-cresol, 2-fluorophenol, 2-aminophenol, 2-chlorophenol, 2-hydroxybenzoic acid, and 2-bromophenol. The consortium O-demethylated anisole to phenol and 2-methoxyphenol to catechol, and oxidized 2-hydroxybenzyl alcohol to 2-hydroxybenzoic acid. Aniline, 2-ethylphenol, 2-hydroxypyridine, 2-acetamidophenol, 2,6-dimethylphenol, 2-phenylphenol, and 1-naphthol were not metabolized.

Aminophenols↗

Detection and purification of a potential precursor protein or a prohaemolysin produced by Staphylococcus haemolyticus.

The haemolytic substance H7 produced by Staphylococcus haemolyticus is composed of three peptides out of 44 amino acid residues each having a structure resembling a signal peptide. The cytoplasmic fraction for the presence of a protein precursor containing this signal sequence was investigated. Specific rabbit IgGs to substance H7 were isolated by affinity chromatography on Sepharose-4B-H7. These anti-H7 IgGs recognized mainly a 51 kD protein in the cytoplasmic fraction of S. haemolyticus from 2, 4, 6, 8, 10 and 23 h cultures. These results support the idea that the 51 kD protein could be either a prohaemolysin or else the precursor of a protein of unknown function with a signal sequence showing homology with the haemolytic peptides. After affinity chromatography on Sepharose-4B-anti-H7, the 51 kD protein was shown associated with an RNA-protein complex composed of four or five proteins and an RNA estimated at 300 nt. This complex could be associated with the machinery of protein secretion. The 51 kD protein was finally purified to homogeneity by HPLC on a Protein Pak DEAE-5PW column in the presence of 5 M urea.

Antibodies↗

Carboxylation of o-cresol by an anaerobic consortium under methanogenic conditions.

The metabolism of o-cresol under methanogenic conditions by an anaerobic consortium known to carboxylate phenol to benzoate was investigated. After incubation with the consortium at 29 degrees C for 59 days, o-cresol was transformed to 3-methylbenzoic acid, which was not further metabolized by the consortium. Proteose peptone in the culture medium was essential for the transformation of o-cresol. In addition, a transient compound detected in the culture was identified as 4-hydroxy-3-methylbenzoic acid. o-Cresol-6d was transformed by the consortium to deuterated hydroxy-methylbenzoic acid and deuterated methylbenzoic acid. These results demonstrate that o-cresol is carboxylated in the para position relative to the phenolic hydroxyl group and dehydroxylated by the anaerobic consortium.

Bacteria, Anaerobic↗

Microbiological aspects of aerobic thermophilic treatment of swine waste.

A thermophilic strain (D2) identified as a Bacillus sp. was isolated from an aerobic digestor of swine waste after several months of operation at 55 degrees C. Aerobic thermophilic batch treatment of swine waste inoculated with strain D2 was studied in a 4-liter fixed-bed reactor. Stabilization of the waste was achieved in less than 30 h when the original chemical oxygen demand (COD) was between 15 and 20 g/liter or in less than 48 h when the COD was around 35 g/liter. When the COD was higher than 30 g/liter, the pH of the waste reached 9.2 to 9.5 during the treatment, and periodic adjustment of the pH to 8.5 was necessary to maintain the activity of the biofilm. In this reactor, ammoniacal nitrogen was completely eliminated by desorption in less than 72 h of incubation. The different packing materials used resulted in similar rates of degradation of organic matter. The thermophilic treatment was also efficient in the 75-liter digestor, and stabilization was achieved in approximately 50 h. A bank of 22 thermophilic bacterial strains originating from different environments and adapted to the thermophilic treatment of swine waste was established. This thermophilic treatment allows, in one step, rapid stabilization of the waste, elimination of the bad smell, and complete elimination of ammonia nitrogen by stripping.

Aerobiosis↗

Comparative study of hemolytic substances produced by coagulase-negative Staphylococcus strains.

Hemolytic substances H7, H62, and E56, produced by Staphylococcus haemolyticus 7 and 62 and S. epidermidis 56, respectively, were purified. H7 and H62 are probably similar on the basis of their isoelectric focusing profiles in 8 M urea and complete immunological identity as revealed by immunodiffusion with rabbit anti-H7 and anti-E56 sera. For E56, we observed seven bands instead of three in isoelectric focusing and only partial immunological identity with H7 and H62. However, H7 and E56 were similar with regard to the following characteristics: hemolytic spectra against different erythrocytes, kinetics of erythrocyte lysis, heat stability, and inhibition by phosphatidylcholine. E56 was not active at a temperature lower than or equal to 25 degrees C, and its activity increased more rapidly with increased temperature compared with H7. For both substances, the complexes obtained by molecular filtration on Ultrogel AcA54 and the purified peptides by reverse-phase high-pressure liquid chromatography showed some hemolytic activity. These results suggest that a particular association or the presence of a given peptide could enhance the activity.

Chromatography, High Pressure Liquid↗

Nitrification of swine waste.

Complete oxidation of ammonia nitrogen (approximately 1000 mg/L) to nitrite was observed in stabilized swine waste after 49 days in incubation at 400 rpm and 29 degrees C, only if 10% (v/v) activated sludge from a wastewater treatment unit and 1.5% (w/v) CaCO3, were added. Stabilized swine waste contains less than 0.09 most probable number (MPN) per millilitre of nitrosobacteria and 2.3 MPN/mL of nitrobacteria. In activated sludge, the concentrations of these bacteria were 2.4 MPN/mL for nitrosobacteria and 4.2 x 10(5) MPN/mL for nitrobacteria. In the swine waste where ammonia was oxidized to nitrite, the nitrosobacteria growth increased to 5.5 x 10(5) MPN/mL, while the nitrobacteria growth decreased to 2.3 MPN/mL. Inoculation of a freshly stabilized swine waste with 10% (v/v) of the active nitrifying waste and addition of 1.5% (w/v) CaCO3, accelerated the oxidation of ammonia nitrogen to nitrite; the reaction was completed after only 5 days of incubation. Increasing the incubation period to 10 days resulted in the complete oxidation of the accumulated nitrite to nitrate. In the stabilized swine waste, complete nitrification without accumulation of nitrite was obtained in only 5 days of incubation when the waste was inoculated with both enriched nitrifying populations (10(6)-10(7) MPN/mL).

Animals↗

Production and partial purification of a gonococcal growth inhibitor produced by a strain of Neisseria meningitidis isolated from a homosexual man.

A Neisseria meningitidis strain isolated from the oropharynx of a homosexual man was shown to produce antigonococcal activity in vitro. A production method, on solid medium, was developed which yielded a soluble activity. The activity was detected at the end of logarithmic growth phase and the maximum activity was reached after 24 h of incubation. The antigonococcal substance was purified more than 300 times by ammonium sulphate precipitation, gel filtration on Ultrogel AcA 54, and chromatography on DEAE-Sephacel. The molecular weight of the inhibitory substance, estimated by molecular filtration, was 29 X 10(3) daltons. The partially purified inhibitor showed two major bands, 32 and less than 12.5 X 10(3) daltons by sodium dodecyl-sulphate polyacrylamide electrophoresis. The chemical nature of the inhibitor is probably proteic on the basis of trypsin sensitivity and the absorbance spectrum.

Antibiosis↗