The toxic mode of action of cyclic lipodepsipeptide fusaricidins, produced by Paenibacillus polymyxa, toward mammalian cells.

AIMS: Toxigenic strains of Paenibacillus polymyxa were isolated from buildings connected with the symptoms of ill health. Our aim was to identify the toxic compounds of Paenibacillus polymyxa and to describe their toxic actions. METHODS AND RESULTS: The toxins of Paenibacillus polymyxa were purified and analysed by HPLC and mass spectrometry. Toxic fusaricidins A and B, and LI-F05a with mass ions at m/z 883·7, 897·6 and 897·6, respectively, were found. The cytotoxicity of purified fusaricidins A and B was measured using boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts. The ion channel forming properties of fusaricidins were studied using the black lipid membrane (BLM) technique. Fusaricidins A and B depolarized the mitochondria of boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts at concentrations of 0·5-1 µg ml-1 and caused nuclear fragmentation and induced apoptosis at concentrations of 2·5-5 µg ml-1 . Furthermore, fusaricidins A and B induced K+ permeating single channels. CONCLUSIONS: It was concluded that fusaricidins were toxic to mitochondria and induced apoptosis in mammalian cells. It was proposed that the observed toxicity of fusaricidins is due their ion channel forming properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper revealed, for the first time, the mode of action of Paenibacillus polymyxa fusaricidins toxins towards mammalian cells. Fusaricidins, due to their potassium ionophoricity and mitochondria depolarizing impacts, may have contributed to the health damage observed at sites where the producer strains were isolated at high density.

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate.

AIMS: To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms. METHODS AND RESULTS: Hyphal extracts of an indoor fungus, identified as the cycloheximide-tolerant species Acremonium exuviarum, inhibited motility of boar spermatozoa (EC(50) 5 +/- 2 microg of crude solids ml(-1)) and caused cytolysis of murine neuroblastoma cells (MNA) and feline fetal lung cells (FL). The responsible substances were purified and identified as two structurally similar, heat-stable, novel, toxic peptaibols, 1726 Da and 1740 Da, respectively, with amino acid sequences of Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Aib-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH and Acetyl-Phe-Iva/Val-Gln-Aib-Ile-Thr-Leu-Val-Pro-Aib-Gln-Pro-Aib-(X-X-X)-SerOH. Purified acrebol inhibited motility of boar sperm, depleted ATP half-content in 1 day (EC(50) of 0.1 microg ml(-1), 60 nmol l(-1)) depolarised the mitochondria after 2 days, but did not affect the cellular content in NADH. This indicates mitochondrial toxicity. Plate-grown biomass of A. exuviarum BMB4 contained 0.1-1% (w/w) of acrebol, depending on the culture medium. CONCLUSIONS: Acrebol paralysed the energy generation of mammalian cells suggesting that mitochondria were its target of action. SIGNIFICANCE AND IMPACT OF THE STUDY: Acremonium exuviarum, as an indoor fungus, is potentially hazardous to health because of the toxic peptaibols that it produces.

Proliferation of mycobacteria in a piggery environment revealed by mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization.

Pig mycobacteriosis is the most common animal mycobacterial disease in Finland with a long-term average prevalence of 0.34% and temporary peaks as high as 0.85%. In the current study Mycobacterium-specific real-time qPCR and 16S rRNA sandwich hybridization were utilized for culture-independent detection and measurement of potentially infectious mycobacteria in selected piggeries. Participating herds (n=5) were selected according to prevalence of tuberculous lesions (>4%) in slaughtered carcasses. When DNA extracted from piggery bedding materials was analyzed by Mycobacterium-targeted qPCR using the SYBR green I dye for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA were detected in unused bedding materials and 10(8) to 10(10)g(-1) dry weight in used bedding materials. When Mycobacterium-specific hybridization probes were used for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA g(-1) dry weight were detected in unused bedding materials in four out of the five piggeries studied and up to 10(8) cell equivalents in used bedding material. The results were confirmed by the Mycobacterium-specific 16S rRNA sandwich hybridization assay. The present results show, that mycobacteria occur in organic materials commonly used on pig farms, and may proliferate in bedding materials during use. We also show that DNA- and RNA-based methods may be utilized for detection of environmental reservoirs of mycobacteria causing porcine and human infection.

Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants.

Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar ribotypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.

Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes.

This paper is an overview on bacilli in industrial processes, with focus on food grade paper and paperboard production. Paperboards mainly contain sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus, usually found in quantities from < 50 to 250 cfu g(-1) homogenized paperboard. Of those frequently found, Bacillus cereus group, B. licheniformis, B. subtilis and Brevibacillus brevis are important for food hygiene because of their hydrolytic activities on food components and the ability of some strains to produce food poisoning toxins or to grow at refrigerated temperatures. We found that the phenotypic properties (lecithinase activity, nitrate reduction) used in standard methods (e.g., ISO, FDA, IDF) to recognize B. cereus, were unreliable for industrial isolates. Whole cell fatty acid composition of a group of the industrial isolates deviated so much from those in a widely used commercial database that the strains were not or only poorly recognized as B. cereus. Industrial isolates, including toxigenic ones, often missed one or more of these characters, even in cases where 100% 16S rDNA identity was found with B. cereus or with B. thuringiensis. 11-Methyldodecanoic acid and trans-9-hexadecenoic acid were found without exception in over 200 industrial B. cereus group isolates and in over 30 culture collection strains. The detection of these fatty acids is a secure method for the identification of B. cereus. Negative reaction for starch hydrolysis and for BCET-RPLA test and a specific ribotype were found in all B. cereus strains producing the emetic toxin.

Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food.

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography-ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 microg ml(-1) or g(-1) wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (> 99.5% N2), less than 0.05 microg of cereulide ml(-1) or g(-1) wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l(-1)) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16-24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food.

Bacillus cereus in a whey process.

A cheese dairy and its whey manufacturing line were examined for Bacillus cereus. Colonies typical of B. cereus were detected in 120 (17%) samples out of 720 analysed. Only 3% of the sampled raw milk contained B. cereus ( > or = 10 cfu ml(-1)) whereas in evaporated whey concentrate B. cereus was present in 76% of the samples. Nitrate reductase negative and weakly casein hydrolysis isolates were rare in raw milk and the early parts of the process but these defective biotypes became increasingly frequent towards the end of the whey process. The composition of whole cell fatty acids of B. cereus isolates originating from the whey part of the process was different from that of the type strain and of the isolates originating from the raw materials of cheese making. The B. cereus strains in concentrated whey were 100% similar to the type strain in 16S rDNA sequence (500 bp) although they were not or only poorly recognized as B. cereus by a commercial whole cell fatty acid library. All of B. cereus isolates in raw milk were sensitive to one or more of the B. cereus group phages (n = 17) whereas 43% of the isolates from the whey process were sensitive to none. None of the 23 strains originating from the whey processing lines grew at < or = 8 degrees C. although strains with minimum growth temperatures of 5.3 degrees C and 7.0 degrees C were present in the raw materials. Our results indicate that the B. cereus population of the warm ( > 30 degrees C) parts of the cheese dairy process was separate from that of cold (2 degrees C to 4 degrees C) part of the process.

A new method for in vitro detection of microbially produced mitochondrial toxins.

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus.

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

In situ polychlorophenol bioremediation potential of the indigenous bacterial community of boreal groundwater.

The composition and chlorophenol-degrading potential of groundwater bacterial community in a permanently cold, oxygen-deficient chlorophenol contaminated aquifer at Kärkölä, Finland was studied with the aim of evaluating in situ bioremediation potential. The groundwater contained from 10(4) to 10(7) microscopically counted cells/ml and up to 10(5) CFU/ml heterotrophic bacteria cultivable at 8 and 20 degrees C. Of the 102 pure cultures, of which 86% Gram-negative, from the plume area (10,000 microg of chlorophenols/l), 57% degraded 2, 3, 4, 6-tetrachlorophenol (TeCP), the main component of the wood preservative which was the source of contamination: 17% also degraded pentachlorophenol (PCP). The degraders were scattered among 16 different clusters of Gram-negatives mainly proteobacteria and members of Cytophaga/Flexibacter/Bacteroides phylum judged by the composition of whole-cell fatty acids. Only one Gram-positive degrading cluster was found containing seven actinobacteria closest to Nocardioides. Of the 88 pure cultures isolated from outside the plume (< 10 microg of chlorophenols/l) 67% were Gram-negative. Seven percent of the isolates degraded 2, 3. 4, 6-TeCP and/or PCP. Five of the Gram-positive isolates from outside the plume were Mycobacterium/Rhodococcus-related actinobacteria and O-methylated 2, 3, 4, 6-TeCP and PCP. The results show that chlorophenol degrading bacterial flora had been enriched as a result of contamination of the aquifer. This suggests significant in situ bioremediation potential of the site.

Measurement of organic halogen compounds in urine as an indicator of exposure.

The report describes the measurement of urinary organic halogen compounds. The method is an application of the adsorbable organic halogen assay which is widely used for the analysis of industrial waste water and drinking water. It was found that this assay can be applied to human urine if the urine is pretreated to hydrolyze the mucins so as to cleave the neuraminic acid residues responsible for the high viscosity of these slimy proteins. The method was found to be sensitive down to 1 microgram of organic halogen/100 ml of urine. Fifty to 260 micrograms of organic halogen was measured in the night urine of healthy, occupationally unexposed volunteers. Since many toxic chemicals to which man may be exposed environmentally or occupationally are, in fact, halogen compounds, this assay may be used to monitor for human exposure.

Evaluation of ecological disturbance and intrinsic bioremediation potential of pulp mill-contaminated lake sediment using key enzymes as probes.

A rapid protocol was developed to measure 10 different enzymic activities from a large number of 1-cm-sliced freshly collected lake sediments. Layers heavily polluted by organic halogens (4900 mg Cl kg(-1)) revealed severe depression of phosphatase, sulfatase, leucine-aminopeptidase, chitinase, acetate esterase and butyrate esterase activities as compared to layers above and below the most polluted zone. alpha-Glucosidase, beta-glucosidase, beta-xylosidase and palmitate esterase were less affected. Methane oxidation potential was dramatically depressed in the polluted strata whereas tetrachloromethane dehalogenating activity was observed in the polluted sediment only. The sediment layers formed after the chlorine discharges into the lake had diminished to 1/10, and showed restoration of the activities close to those observed in non-recipient sediment, in spite of the persisting presence of >1000 mg of organic chlorine (kg dry wt)(-1). We conclude that certain enzymic activities involved in breakdown or oxidation of organic matter in the sediments are useful probes for assessing the degree of ecological damage and its potential for restoration in recipient lakes of industrial discharges.

Impaired semen quality of AI bulls fed with moldy hay: a case report.

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay.

Sediment accumulation of organic halogens in pristine forest lakes: Tracking of a single defined discharge of pulp bleaching wastewater.

Sediment accumulation of organic halogen was studied in two forest lakes, one pristine and one which received 30 m(3) of biologically purified bleaching wastewater from a kraft pulp mill in 1979 equivalent to ca. 2 kg of adsorbable organic halogen (AOX). Lake sediments were dated with(210)Pb,(134)Cs and(137)Cs and the annual deposition rates of organic halogens and organic matter were calculated. Organic bound halogen contents of the sediment aged 150 years was 180 microg Cl g(-1) d.w. in both lakes. The concentration of organic bound halogen at the topmost 6 cm of the sediments (less than 20-years-old) ranged from 45 to 80 microg Cl g(-1) d.w. This suggests that solvent extractable halogen had enriched in the older sediment layers. The deposition of extractable organic halogen (EOX) in the lakes in 1950's was 4 to 5 mg Cl m(-2) a(-1). Since then, the deposition of EOX doubled in both lakes. The deposition of organic matter increased concomitantly from 50 g m(-2) a(-1) to 110 g m(-2) a(-1) in Lake Mustalampi and from 35 g m(-2) a(-1) to 62 g m(-2) a(-1) in Lake Pyylampi suggesting that the increase in the deposition of organic halogen followed the increase in the deposition of organic matter. Of the 2 kg of organic halogen discharged into the lake, 5% or less was detected in the sediment in tetrahydrofuran extractable form 15 years later.

Bleached kraft pulp mill discharged organic matter in recipient lake sediment: Environmental and molecular properties.

Environmental properties of organic matter contained halogen and sulfur were studied in sediments of bleached kraft pulp mill effluent (BKME) recipient lakes and 2 m(3) outdoor enclosures (mesocosms). The BKME contributed to 1% (v/v) of the total water flow in the lake downstream of the pulp mill where the sediments contained 1.7 to 4 mg of tetrahydrofuran extractable organic halogen (EOX-Cl) and 0.6 to 0.8 mg of tetrahydrofuran extractable organic sulfur (EOS-S) g(-1) of organic matter. Upstream sediment contained 0.03 mg of EOXCl and 0.7 mg of EOS-S g(-1) of organic matter. EOX was a better indicator for the influence of BKME in the recipient sediment than EOS. The polarity of BKME contained EOX corresponded to log K(ow) of < 1, and that of the downstream sediment contained EOX to > 4.5. HP-SEC analysis of the molecular weight distribution (MWD) of the EOX showed a peak between 300 to 600 g mol(-1) for the BKME and between 1000 to 2000 g mol(-1) for the downstream sediment. The MWD of the BKME contained EOS peaked at 300 to 1000 g mol(-1), and that of the downstream sediment contained EOS at 1000 to 5000 g mol(-1). These results indicate that BKME contained organic halogen and sulfur undergo major structural transformations when incorporated into sediment. The biota-to-sediment accumulation factor (BSAF) of EOX from sediments formed downstream of the mill and in the mesocosms to the lipids of Lumbriculus variegatus was 0.4 to 0.7. This is of a similar order of magnitude to the BSAF reported for 2,3,7,8-tetrachlorodibenzop-dioxin and 2,3,7,8-tetrachlorodibenzofuran.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria.

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili.

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.

Hydroxylation and Dechlorination of Tetrachlorohydroquinone by Rhodococcus sp. Strain CP-2 Cell Extracts.

A cell extract of a polychlorophenol-degrading bacterium, Rhodococcus sp. strain CP-2, isolated from chlorophenol-contaminated soil, was shown to dechlorinate tetrachlorohydroquinone, the first intermediate in pentachlorophenol and 2,3,5,6-tetrachlorophenol degradation. Degradation of tetrachlorohydroquinone was catalyzed by a soluble enzyme(s). The reaction sequence for complete dechlorination involved hydroxylation and three reductive dechlorinations, producing 1,2,4-trihydroxybenzene. All chlorines were thus removed from the polychlorinated compound before ring cleavage.

Transformation of chlorinated phenolic compounds in the genusRhodococcus.

The ability of strains of the genusRhodococcus to transform chlorinated phenolic compounds was studied. Noninduced cells of several strains ofRhodococcus, covering at least eight species, were found to attack mono-, di-, and trichlorophenols by hydroxylation at theortho position to chlorocatechols. 3-chlorophenol and 4-chlorophenol were converted to 4-chlorocatechol, 2,3-dichlorophenol to 3,4-dichlorocatechol, and 3,4-di-chlorophenol to 4,5-dichlorocatechol. The chlorocatechols accumulated to nearly stoichiometric amounts. Other mono- and dichlorophenols were not transformed. The ability of the strains to hydroxylate chlorophenols correlated with the ability to grow on unsubstituted phenol as the sole source of carbon and energy. SeveralRhodococcus strains attacked chlorophenolic compounds by both hydroxylation and O-methylation. 2,3,4-, 2,3,5- and 3,4,5-trichlorophenol were hydroxylated to trichlorocatechol and then sequentially O-methylated to chloroguaiacol and chloroveratrole. Tetrachlo-rohydroquinone was O-methylated sequentially to tetrachloro-4-methoxy-phenol and tetrachloro-1,4-dimethoxybenzene. Several of the active strains had no known history of exposure to any chloroaromatic compound. Rhodococci are widely distributed in soil and sludge and these results suggest that this genus may play an important role in transformation of chlorinated phenolic compounds in the environment.

Absorption of pentachlorophenol (PCP) by bark chips and its role in microbial PCP degradation.

A pentachlorophenol (PCP)-degrading mixed bacterial population was enriched in a biofilter filled with soft wood bark chips. We found that bark chips were essential for the degradation to proceed at PCP concentrations higher than 10µM. PCP-degrading bacteria were found to be extremely sensitive to PCP. Bark chips absorbed PCP reversibly, thus detoxifying the medium and allowing degradation to proceed at higher concentrations of PCP (beyond 200µM).