Kinetics of pentachlorophenol degradation by a Flavobacterium species

The kinetics of pentachlorophenol (PCP) degradation by a Flavobacterium sp. ATCC 39723 has been investigated. Sodium glutamate was supplied as an additional carbon source to increase the rate of cell growth and PCP degradation. A kinetic model including PCP toxicity for cell growth and PCP inhibition of its own degradation was developed. This model was also applied to 2,4-dichlorophenol (DCP) degradation by the same organism. Although PCP and DCP are degraded by different pathways, the model describes these two degradation processes very well.     

Cytotoxic effects of environmentally relevant chlorophenols on L929 cells and their mechanisms

The chlorophenol chemicals (CPs) are a major class of widely distributed and frequently occurring persistent environmental pollutants. Pentachlorophenol (PCP) has been proposed to be procarcinogen in rodents and in possibly human beings. Human beings also easily expose to other chlorophenol chemicals, including 4-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,3,4-trichlorophenol (TCP), prompting this investigation of their comparative cytotoxic effects and cell death mechanisms, assayed in fibroblast L929 cells. The effective concentration for half-maximal response (EC50 values at 24 h for CP, DCP, TCP, and PCP are 2.18, 0.83, 0.46, and 0.11 mmol/L respectively and the EC50 values at 48 h are 1.18, 0.13, 0.08, and 0.06 mmol/L respectively by using 3-(4,5-dimethylthiazd-2-yl)-2,5-diphenyltentrazolium bromide (MTT) reduction assay. A clear structure-activity relationship was observed between toxicity of CPs and their octanol-water partition coefficients. The further studies indicate that CP, DCP, and TCP induce apoptosis in L929 cells in a concentration or time-dependent manner, but PCP mediates cell death more characteristic of necrosis than apoptosis. These results not only demonstrate that L929 cell growth inhibition bioassay may be useful to provide the comparative evaluation of toxicity of CPs in vitro, but also implicate that CP, DCP, TCP, in comparison with PCP, can induce L929 cell death by apoptosis, resulting in lower procarcinogensis, which may help to elucidate the molecular basis for the adverse health effects associated with CPs exposure.     

Multisubstrate monod kinetic model for simultaneous degradation of chlorophenol mixtures

Chlorophenols (CPs) are persistent and highly toxic compounds rated as priority pollutants by the Environmental Protection Agency (EPA). Frequently, these compounds are present as mixtures of CPs in industrial wastewaters. Therefore the study of biodegradation on mixed pollutants is an important aspect of biodegradation and wastewater treatment. In this work, we studied the multisubstrate degradation of CPs by a mixed culture of Pseudomonas aeruginosa and a novel Acromobacter sp. capable of using pentachlorophenol (PCP), 2,4,6 trichlorophenol (2,4,6 TCP) and 2,3,5,6 tetrachlorophenol (2,3,5,6 TeCP) as the sole sources of carbon and energy. The main objective of this work was to evaluate the effect of substrate mixtures on the degradation kinetics of PCP. Batch experiments were conducted with each CP separately and in mixtures of PCP + 2,4,6 TCP, PCP + 2,3,5,6 TeCP, and PCP + 2,4,6 TCP + 2,3,5,6 TeCP. Based upon our results we have concluded that the simultaneous degradation of CPs is a key factor contributing to the improvement of PCP degradation. The kinetic parameters for PCP and 2,4,6 TCP were obtained by fitting the data to a Monod kinetics model. Using such parameters, the model was able to predict simultaneous multisubstrate degradation of PCP with others CPs.     

Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat.

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with ¹⁴C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to ¹⁴CO₂ by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium.

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Physiological properties and substrate specificity of a pentachlorophenol-degradingPseudomonas species

A bacterial strain capable of utilizing pentachlorophenol (PCP) as sole source of carbon and energy for growth was isolated from enrichment cultures containing 100 mg/l PCP in a mineral salts medium inoculated with contaminated soil from a lumber treatment waste site. The isolate, designated strain SR3, was identified as a species ofPseudomonas by virtue of its physiological and biochemical characteristics. Mineralization of PCP byPseudomonas sp. strain SR3 was demonstrated by loss of detectable PCP from growth medium, stoichiometry of chloride release (5 equivalents of chloride per mole of PCP), and formation of biomass consistent with the concentration of PCP mineralized. PCP-induced cells of strain SR3 showed elevated rates of oxygen consumption in the presence of PCP, and with different chlorinated phenols, with complete degradation of 2,3,5,6-, 2,3,6-, 2,4,6-, 2,4-, and 2,6-chloro-substituted phenols. Concentrations of PCP up to 175 mg/liter supported growth of this organism, but maximal rates of PCP removal were observed at a PCP concentration of 100 mg/liter. Based on its degradative properties,Pseudomonas sp. strain SR3 appears to have utility in bioremediation of soil and water contaminated with PCP.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol Contribution No. 750 from the United States Environmental Protection Agency Environmental Research Laboratory, Gulf Breeze, FL32561, USA. A preliminary report of this work has appeared in abstract form (Resnick & Chapman 1990; Abstr. Annu Meet Amer Soc Microbiol Q-70, p. 300).     

Effect of adsorbents on degradation of toxic organic compounds by coimmobilized systems

The use of coimmobilized systems for treatment of toxic organic compounds has been proposed. The proposed approach combines the use of adsorbents and laboratory identified microorganisms immobilized in a protective permeable barrier to achieve a greater degree of control over the remediation process. This study was launched to understand the effect of adsorbents and changes in adsorption on the degradation of toxic compounds by coimmobilized systems. The specific case studied involved the degradation of pentachlorophenol (PCP) by Arthrobacter (ATCC 33790) coimmobilized with powdered activated carbon within calcium alginate capsules.The design parameters studied included adsorbent content and type as well as the effect of solution pH and surfactant concentration on adsorption and biodegradation. It was found that the equilibrium adsorption behavior of PCP was strongly influenced by solution pH and surfactant concentration. A mathematical model was developed that combined the physical processes of mass transfer and adsorption with biological degradation of PCP. The model was used to predict the effect of various parameters on the degradation of PCP. Based on model predictions, the degradation of PCP. Based on model predictions, the degradation of PCP was strongly dependent on variations in adsorbent capacity and affinity for this contaminant.     

Biodegradation of phenol and cresol isomer mixtures by Arthrobacter

The Arthrobacter species can degrade phenol, o-cresol and p-cresol much faster (as reflected in high specific growth rates) than other microbes which are reported to degrade toxic compounds. In mixtures, phenol and p-cresol mutually inhibited each other; the inhibition constants show that phenol degradation is strongly inhibited in the presence of p-cresol rather than reverse. o-Cresol enhanced phenol degradation marginally but o-cresol degradation was not affected by the presence of phenol.     

The treatment of chlorophenols with laccase immobilized on sol–gel-derived silica

Laccase, a so-called “blue-copper” oxidase, is able to oxidize a variety of organic compounds. Sol–gel derived silica glasses are frequently adopted as an immobilization method to improve the stability of enzymes and make them reusable. In this study, immobilization conditions were optimized to achieve improved embedding results. The thermal stability, reaction stability and storage stability were improved with the immobilized enzyme when compared to the free enzyme. 2,4-Dichlorophenol (DCP) and 2,4,6-trichlorophenol (TCP) were chosen as model compounds. The treatment of chlorophenols (CPs) by immobilized laccase demonstrated excellent removal and response stability. The affinity of TCP for immobilized laccase was higher than that of DCP. This finding leads to different removal efficiencies under variable conditions (reaction time, initial concentration, dosage of immobilized laccase and removal rate in mixed solution). By fitting the experimental data with the diffusion model of the degradation process, the degradation of CPs by immobilized laccase matches an intraparticle diffusion-controlled model.     

Aerobic secondary utilization of a non-growth and inhibitory substrate 2,4,6-trichlorophenol by Sphingopyxis chilensis S37 and sphingopyxis-like strain S32

This paper reports 2,4,6-trichlorophenol (246TCP) degradation bySphingopyxis chilensis S37 and Sphingopyxis chilensis-like strain S32,which were unable to use 246TCP as the sole carbon and energy source. In R2A broth, the strainsdegraded 246TCP up to 0.5 mM. Results with mixtures of different 246TCP and glucose concentrations in mineral salt media demonstrated dependence on glucose to allow bacterial growth and degradation of 246TCP. Strain S32 degraded halophenol up to 0.2 mM when 5.33 mM glucose was simultaneously added, while strain S37 degraded the compound up to 0.1 mM when 1.33 mM glucose was added. These 246TCP concentrations were lethal for inocula in absence of glucose. Stoichiometricreleases of chloride and analysis by HPLC, GC-ECD and GC-MS indicated 246TCP mineralisation by both strains. To our knowledge, this is the first report of bacteriaable to mineralize a chlorophenol as a non-growth and inhibitory substrate. The concept of secondary utilization instead of cometabolism is proposed for this activity.     

Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus

Summary An actinomycete, Rhodococcus chlorophenolicus, isolated from a pentachlorophenol-degrading mixed bacterial culture is a polychlorophenol degrader. It was shown to oxidize pentachlorophenol into carbon dioxide and to metabolize also 2,3,4,5-,2,3,4,6-, and 2,3,5,6-tetrachlorophenol, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,6-, and 2,4,5-trichlorophenol, 2,5-, and 2,6-dichlorophenol and tetrachloro-p-hydroquinone in an inducible manner. Pentachlorophenol set on the synthesis of enzymes required for the metabolism of all these chlorophenols and of tetrachloro-p-hydroquinone. 2,4,5-, and 2,4,6-trichlorophenol and 2,5-, and 2,6-dichlorophenol were degraded by R. chlorophenolicus cells only if these had previous contact to pentachlorophenol. Other chlorophenols mentioned were able to set on the synthesis of enzymes for their own degradation. 2,3,4,5-, and 2,3,4,6-tetrachlorophenol, and 2,3,5-, 2,4,5-, and 3,4,5-trichlorophenol were more toxic to R. chlorophenolicus than the other chlorophenols, but nevertheless 2,3,4,5-, and 2,3,4,6-tetrachlorophenol and 2,3,5-trichlorophenol were readily degraded by the bacteria.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - TeCH tetrachloro-p-hydroquinone An example of numeration: 2345-TeCP, 2,3,4,5-tetrachlorophenol     

Isolation and characterization of a pentachlorophenol-degrading bacterium.

With a new enrichment protocol, pentachlorophenol (PCP)-degrading bacteria were isolated from soil, water, and sewage. When characterized, all isolates were related and shared characteristics of the genus Arthrobacter. Growth rates for strain NC were determined for a number of substrates, including PCP and 2,4,6-trichlorophenol. Changes in PCP concentration affected growth rate and length of the lag phase but not cell yield. Increasing the pH from 6.8 to 7.8 decreased the length of the lag phase for growth on PCP. Cessation of growth, upon incremental addition of PCP, was found to be related to a decrease in pH rather than to a buildup of a toxic metabolite. Degradation of PCP by strain NC was shown to be complete.     

Metabolism of mono- and dihalogenated C1 and C2 compounds by <Emphasis Type="Italic">Xanthobacter autotrophicus</Emphasis> growing on 1,2-dichloroethane

The conversion of and toxic effects exerted by several mono- and dihalogenated C1 and C2 compounds on cultures of Xanthobacter autotrophicus GJ10 growing on 1,2-dichloroethane were investigated. Bromochloromethane, dibromomethane and 1-bromo-2-chloroethane were utilized by strain GJ10 in batch culture as a cosubstrate and sole carbon source. The rate of degradation of dihalomethanes by whole cells was lower than that of 1,2-dichloroethane, but a significant increase of the rate of dihalomethane biodegradation was observed when methanol or ethanol were added as a cosubstrate. Products of the degradation of several tested compounds by haloalkane dehalogenase were analyzed and a new metabolic pathway based on hydrolytic conversion to formaldehyde was proposed for the dihalomethanes. Strain GJ10 growing on 1,2-dichloroethane converted 2-fluoroethanol and 1-chloro-2-fluoroethane to 2-fluoroacetate, which was tolerated up to a concentration of 2.5 mM. On the basis of the results from batch cultures an inert (dichloromethane), a growth-supporting (dibromomethane) and a toxic (1,2-dibromoethane) compound were selected for testing their effects on a continuous culture of strain GJ10 growing on 1,2-dichloroethane. The compounds were added as pulses to a steady-state chemostat and the response of the culture was followed. The effects varied from a temporary decrease in cell density for dibromomethane to severe toxicity and culture washout with 1,2-dibromoethane. Our results extend the spectrum of halogenated C1 and C2 compounds that are known to be degraded by strain GJ10 and provide information on toxic effects and transformation of compounds not serving as a carbon source for this bacterium.     

Flocculation behavior of Sphingobium chlorophenolicum in degrading pentachlorophenol at different life stages

The cell flocculation behavior of degrading pentachlorophenol (PCP) by using Sphingobium chlorophenolicum (Flavobacterium sp., ATCC 39723) is investigated in the present paper. It is found that these Sphingobium cells can efficiently degrade PCP when the concentration of this toxic compound is below 150 ppm. These degradation rates of PCP can be facilitated with the additions of three supplementary carbons: glutamate (4.0 g/L), glucose (3.2 g/L), and cellobiose (3.05 g/L), among which the highest specific growth rate of cells is obtained when glucose is added. More importantly, in these biodegradation experiments described herein, in order to investigate the cell flocculation behavior, the temporal variations of cell size, zeta potential, and stability ratio of the cell suspension are also measured. It is found that, no matter what kind of supplementary carbon source is added, the highest stability ratio of the cell suspension can be always obtained at the end of the exponential growth phase, which can be well explained by using the results of cell size and zeta potential measurements according to the DLVO theory.     

Degradation kinetics of pentachlorophenol by Phanerochaete chrysosporium

The extracellular enzymes and cell mass from the pregrown Phanerochaete chrysosporium cultures were used for the degradation of PCP. The use of both extracellular enzymes and cell mass resulted in extensive mineralization of PCP, while the action of only the crude extracellular enzymes led to the formation of a degradation intermediate (TCHD). A kinetic model, which describes the relationship among PCP degradation, initial PCP concentration, dosage of extracellular enzymes, and cell mass concentration, was developed. Based on this model, various effects of initial PCP concentration, dosage of extracellular enzymes, and cell mass concentration were evaluated experimentally. It was found that when initial PCP concentration is lower than 12 mumol/L, the model of a parallel-series first-order reaction is sufficient to describe the degradation process. PCP disappearance and mineralization were enhanced by increasing either the extracellular enzyme concentration or the cell mass concentration. As high as 70% of PCP mineralization could be obtained by using a higher dosage of extracellular enzymes and cell mass. Various parameters of the kinetic model were determined and the model was verified experimentally. Simulation using this model provided the criteria needed to choose rational dosages of extracellular enzymes and cell mass for the degradation of PCP. Data reported allow some insight into the function of the extracellular enzymes and cell mass of P. chrysosporium in degradation processes of toxic pollutants and assist in the design and evaluation of practical bioremediation methods.     

Dual substrate biodegradation of a nonionic surfactant and pentachlorophenol by Sphingomonas chlorophenolica RA2

The simultaneous biodegradation of the nonionic surfactant Tween 20 (Tw20) and pentachlorophenol (PCP) by Sphingomonas chlorophenolica sp. Strain RA2 (RA2) was measured. As a sole substrate, Tw20 biodegradation was best described by the Contois kinetic model. During concurrent biodegradation of Tw20 and PCP, the biodegradation rates of Tw20 were not significantly affected by 50 or 100 mg/L PCP, but were significantly inhibited by 500 mg/L PCP. Decreases in cell yield in the presence of PCP suggest that PCP was acting as an uncoupler. Cultures were pre-grown on PCP or Tw20 before degradation of PCP to evaluate enzyme induction effects, and long lags before PCP biodegradation after growth on Tw20 occurred. Although biokinetic models could accurately describe some of the data sets of RA2 growth and Tw20 and PCP degradation, finding a single set of kinetic parameters that predicted all dual substrate tests was not achieved. The complicating factors to modeling PCP and Tw20 interactions are described and may be more widely applicable to the biodegradation of toxic organic compounds in the presence of a biodegradable surfactant.