Assessment of inhibition kinetics of the growth of strain P5 on pentachlorophenol under steady-state conditions in a nutristat

A bacterium degrading pentachlorophenol (PCP) as the only source of carbon and energy was grown in a nutristat, i.e., a continuous culture with on-line measurement and control of the substrate concentration. We improved the PCP nutristat by incorporation of a personal computer with a proportional integral derivative (PID) algorithm for controlling the medium feed pump. The controlled value deviated from the average (set-point) value by 1% maximally. In the PCP nutristat (30°C), the steadystate dilution rate, and hence, specific growth rate, showed a maximum value of 0.142±0.004 h^-1 at set-point PCP concentrations between 37 and 168 M. At PCP concentrations above 168 M, the steady-state growth rate decreased because of inhibition. The growth yield coefficient was not seriously affected by the PCP concentration, suggesting that uncoupling was not the inhibitory mechanism. It was concluded that the PCP nutristat is very useful for establishing steady-state conditions that maintain growth-inhibitory PCP concentrations and high cell concentrations, conditions for which the chemostat is not suitable.Abbreviations MCA Metabolic control analysis - NTA Nitrilotriacetic acid - PCP Pentachlorophenol - PID Proportional integral derivative     

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.     

Sensitivity of ruminal microorganisms to pentachlorophenol

Pentachlorophenol (PCP) is used extensively as a biocidal agent, and there is considerable concern about the adverse effects of this compound in biological ecosystems. The effects of PCP on the growth and fermentative activity of cultures of mixed ruminal microorganisms and the sensitivity of 14 ruminal bacterial species to PCP in pure culture were examined in this study. Increasing concentrations of PCP (9.4 to 375.4 microM) depressed growth and propionate concentrations in cultures of mixed ruminal microorganisms. Wide differences in the sensitivities of ruminal bacterial strains to various concentrations of PCP were observed. Cellulolytic strains were highly sensitive to PCP, while amylolytic, sugar-utilizing, and intermediate acid-utilizing strains were more resistant. Growth of major succinate-producing strains was depressed by PCP. Strains which depend on substrate level phosphorylation appeared to be more resistant. The data suggest that the adverse effects of PCP on ruminal microorganisms may be the result of its role as both an uncoupler of electron transport and a protonophore.     

Sensitivity of ruminal microorganisms to pentachlorophenol.

Pentachlorophenol (PCP) is used extensively as a biocidal agent, and there is considerable concern about the adverse effects of this compound in biological ecosystems. The effects of PCP on the growth and fermentative activity of cultures of mixed ruminal microorganisms and the sensitivity of 14 ruminal bacterial species to PCP in pure culture were examined in this study. Increasing concentrations of PCP (9.4 to 375.4 microM) depressed growth and propionate concentrations in cultures of mixed ruminal microorganisms. Wide differences in the sensitivities of ruminal bacterial strains to various concentrations of PCP were observed. Cellulolytic strains were highly sensitive to PCP, while amylolytic, sugar-utilizing, and intermediate acid-utilizing strains were more resistant. Growth of major succinate-producing strains was depressed by PCP. Strains which depend on substrate level phosphorylation appeared to be more resistant. The data suggest that the adverse effects of PCP on ruminal microorganisms may be the result of its role as both an uncoupler of electron transport and a protonophore.     

Kinetic evidence for pentachlorophenol-dependent growth of a dehalogenating population in a pentachlorophenol- and acetate-fed methanogenic culture

A method was developed to evaluate growth of a reductively dechlorinating bacterial population within a pentachlorophenol (PCP)- and acetate-fed, mixed, methanogenic culture. In 6- to 12-day experiments, a computer-monitored/feedback-controlled bioreactor was used to maintain constant pH, temperature, and acetate concentration, while transformation of multiple PCP additions was monitored. The potential at a platinum electrode, EPt, was not controlled externally, but was maintained constant at -0.25 +/- 0.002 V (vs. SHE) by iron sulfides in the medium and the activity of the culture. PCP was reductively dechlorinated at the ortho position, yielding 3, 4,5-trichlorophenol (3,4,5-TCP) via 2,3,4,5-tetrachlorophenol (2,3,4, 5-TeCP). Below an initial PCP concentration of 0.5 microM, PCP was transformed to 3,4,5-TCP within 3 to 6 h. Biomass concentration changes were small during this period, and PCP and 2,3,4,5-TeCP transformations were modeled as pseudo-first-order reactions. Increases in pseudo-first-order rate constants for PCP and 2,3,4, 5-TeCP were directly related to the amount of PCP transformed to 3,4, 5-TCP, suggesting enrichment of a PCP-catabolizing population. Moreover, rate constant increases were independent of the amount of acetate consumed, changes in the overall volatile suspended solids (VSS) concentration, and the experimental duration. When PCP was added to the reactor at increasingly shorter time intervals in an exponential pattern, pseudo-first-order rate constants increased exponentially. An average rate constant doubling time of 1.7 days (1. 4 to 2.3 d) was estimated. While the VSS concentration of the culture increased 60% in an 8-day period, pseudo-first-order rate constants increased by a factor of approximately 6. This large increase in transformation rate constants suggests growth of a bacterial population capable of using PCP and 2,3,4,5-TeCP as terminal electron acceptors.     

Phytoremediation of Pentachlorophenol in the Crested Wheatgrass (Agropyron cristatum × desertorum) Rhizosphere

Pentachlorophenol (PCP) is a widespread, highly toxic contaminant of soil and water that is generally recalcitrant to microbial breakdown and thus may be considered a good candidate for phytoremediation. PCP toxicity and rates of mineralization were compared in crested wheatgrass seedlings that were either sterile or root-inoculated with microbial consortia derived from soil at a PCP-contaminated site. Inoculated seedlings were more tolerant to PCP and mineralized threefold more ¹⁴C-PCP than sterile seedlings. Only 10% of the recovered radioactivity from sterile seedlings represented mineralized PCP, indicating that rhizosphere microorganisms are primarily responsible for PCP mineralization. The levels of PCP degradation exhibited by several microbial consortia and isolates in liquid culture were not correlated with their ability to protect crested wheatgrass seedlings from PCP toxicity. Most probable number estimates showed that the presence of crested wheatgrass root exudates enhanced the number of PCP-degrading microorganisms by 100-fold in liquid culture, indicating that exudate components provide some nutritive benefit, possibly as PCP co-metabolites. A close association of plants and rhizosphere microorganisms appears to be necessary for crested wheatgrass survival in PCP-contaminated soil, although understanding the molecular details of this association requires further research.     

Evaluation of pentachlorophenol-degrading potentiality of Pseudomonas sp. in a soil microcosm

Pseudomonas sp. strain IST103 obtained from a stable consortium was capable of degrading pentachlorophenol (PCP) as sole carbon and energy source. The PCP-degrading potentiality of the strain was determined by growth of bacteria in culture medium, utilization of PCP by high performance liquid chromatography (HPLC), chloride release and ring cleavage. The strain was applied in two set of soil microcosms containing 20 and 40% moisture, each having different concentrations, 0, 10, 100, 500, and 1000 mg/l, of PCP. The result showed significant utilization of PCP (77% in 45 days) and higher growth of bacterial strain when PCP was applied in 100 mg/l concentration at 40% moisture. Inhibitory effects on the growth of bacterial strain were seen in 500 and 1000 mg/l concentration.     

Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP. Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1). The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.     

Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations.

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP. Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1). The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.     

PHYTOREMEDIATION POTENTIAL OF MAIZE (ZEA MAYS L.) IN CO-CONTAMINATED SOILS WITH PENTACHLOROPHENOL AND CADMIUM

The ubiquitous coexistence of heavy metals and organic contaminants was increased in the polluted soil and phytoremediation as a remedial technology and management option is recommended to solve the problems of co-contamination. Growth of Zea mays L and pollutant removal ability may be influenced by interactions among mixed pollutants. Pot–culture experiments were conduced to investigate the single and interactive effect of cadmium (Cd) and pentachlorophenol (PCP) on growth of Zea mays L, PCP, and Cd removal from soil. Growth response of Zea mays L is considerably influenced by interaction of Cd and PCP, significantly declining with either Cd or PCP additions. The dissipation of PCP in soils was notably affected by interactions of Cd, PCP, and plant presence or absence. At the Pentachlorophenol in both planted and non-planted soil was greatly decreased at the end of the 10-week culture, accounting for 16–20% of initial extractable concentrations in non-planted soil and 9–14% in planted soil. With the increment of Cd level, residual pentachlorophenol in the planted soil tended to increase. The pentachlorophenol residual in the presence of high concentration of Cd was even higher in the planted soil than that in the non-planted soil.     

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)     

Delftia acidovorans MC1 resists high herbicide concentrations--a study of nutristat growth on (RS)-2-(2,4-Dichlorophenoxy)propionate and 2,4-dichlorophenoxyacetate

Delftia acidovorans MC1 was continuously cultivated under nutristat conditions with elevated concentrations of the herbicides (RS)-2-(2,4-dichlorophenoxy)propionate [(RS)-2,4-DP] and 2,4-dichlorophenoxyacetate (2,4-D). The presence of 1-5 mM of either of these compounds did not essentially inhibit growth. Moreover, substrate consumption was not essentially affected at pH values of 7.0-9.0 selected by reason of alkaline in situ conditions found e.g. on contaminated building rubble but was decreased at pH 9.3. The adenylate energy charge declined to some degree as the herbicide concentration rose, the extent of this increasing as the pH rose. This was caused by an increase in the concentration of ADP and in particular AMP, in contrast to the fairly constant ATP level of around 4 nmol/mg dry mass with (RS)-2,4-DP and 2 nmol/mg with 2,4-D. Comparison of the individual growth parameters with theoretical data taking into account maintenance coefficients of 0.48 mmol (RS)-2,4-DP/g*h and 0.6 mmol 2,4-D/g*h revealed that the culture followed purely kinetic rules. This excludes the necessity of using substrate to a significant extent to satisfy extra efforts in energy for homeostasic work under these accentuated conditions.     

Anaerobic mineralization of pentachlorophenol (PCP) by combining PCP-dechlorinating and phenol-degrading cultures

The dechlorination and mineralization of pentachlorophenol (PCP) was investigated by simultaneously or sequentially combining two different anaerobic microbial populations, a PCP-dechlorinating culture capable of the reductive dechlorination of PCP to phenol and phenol- degrading cultures able to mineralize phenol under sulfate- or iron-reducing conditions. In the simultaneously combined mixture, PCP (about 35 microM) was mostly dechlorinated to phenol after incubation for 17 days under sulfate-reducing conditions or for 22 days under iron-reducing conditions. Thereafter, the complete removal of phenol occurred within 40 days under both conditions. In the sequentially combined mixture, most of the phenol, the end product of PCP dechlorination, was degraded within 12 days of inoculation with the phenol degrader, without a lag phase, under both sulfate- and iron-reducing conditions. In a radioactivity experiment, [14C-U]-PCP was mineralized to 14CO2 and 14CH4 by the combined anaerobic microbial activities. Analysis of electron donor and acceptor utilization and of the production and consumption of H2, CO2, and CH4 suggested that the dechlorinating and degrading microorganisms compete with other microorganisms to perform PCP dechlorination and part of the phenol degradation in complex anoxic environments in the presence of electron donors and acceptors. The presence of a small amount of autoclaved soil slurry in the medium was possibly another advantageous factor in the successful dechlorination and mineralization of PCP by the combined mixtures. This anaerobic-anaerobic combination technology holds great promise as a cost-effective strategy for complete PCP bioremediation in situ.     

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.     

Pentachlorophenol removal from slightly acidic mineral salts,commercial sand,and clay soil by recovered Arthrobacter strain ATCC 33790

Arthrobacter strain ATCC 33790, a pentachlorophenol (PCP)-metabolizer isolated by the author, has been recovered after 10 years of storage. The freeze-dried preparation grown on half-strength Trypticase Soy Broth adapted to utilize PCP within 1 week. Cultures grown on PCP-nutrient agar were found to utilize PCP in mineral salts medium within 2–3 days. The culture was prepared for continuous growth at pH 6.5 by successive feeding of 100–110 mg solid aliquots of PCP to a 1-l culture initially grown at pH 7.4. Continuous culture growth at pH 6.5 was possible on a mineral salts feed containing 1800 ppm PCP. Continuous cultures grown at pH 6.7 on mineral salts feeds containing 500 and 340 mg PCP/l were especially efficient in removing PCP. Less than 4 mg PCP/l were detected in the effluent at dilution rates near washout. In batch culture studies at pH 6.5 the PCP utilization kinetics were found to be similar at low PCP concentration to those at pH 7.4 for the approximately same inoculum size. Utilization of 35 mg PCP/l was very slow at pH 6.0. Growth rates at pH 6.5 at controlled PCP concentration ranges of 5–35 and 75–115 mg/l were 0.09 h^–1 and 0.05 h^–1, respectively. The ability of strain ATCC 33790 to utilize PCP in mineral salts media containing naphthalene, methylnaphthalenes, and cresols was examined. Naphthalene, 1-, and 2-methylnaphthalenes at their solubility limit, and o- and m-cresols at 900–1000 mg/l prevented utilization of 80–90 mg PCP/l. PCP was rapidly removed from both commercial sand at 30°C and from clay soil at room temperature. Estimated inoculum sizes of 6.6 × 10⁶, 6.6 × 10⁴, and 656 cells/g were found to be effective in removing approximately half the starting amount of PCP from sand in 3, 19, and 42 h, respectively. Nearly complete disappearance of extractable PCP was observed after 1 day in clay soil inoculated with 6 × 10⁶ cells/g.     

Dynamics and control of continuous microbial propagators to subject substrate inhibition

Inhibitory substrate levels are common in industrial fermentations and in biological waste-water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate-limiting and growth-limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X /YD )[dμ/dS]s }, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition. The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.     

Aniline oxidation by microorganisms in chemostat cultivation

Under the conditions of chemostat cultivation, a mixed culture of microorganisms oxidized aniline at a gradually increasing concentration (up to 2.5 g/litre) as a sole source of carbon and nitrogen. The specific rate of aniline oxidation was as high as 160 mg per 1 g of dry biomass when aniline concentration in the growth medium was 2.5 g/litre. As aniline concentration in the growth medium was gradually raised, selection of the microorganisms took place and the number of the strains decreased from ten to two. The growth rate of the microorganisms fell down abruptly when phosphorus concentration in the medium was below 35 mg per 1 g of aniline.     

Biodegradation and ecotoxicity of soil contaminated by pentachlorophenol applying bioaugmentation and addition of sorbents

Biodegradation of pentachlorophenol (PCP) in soil by autochthonous microorganisms and in soil bioaugmented by the bacterial strain Comamonas testosteroni CCM 7530 was studied. Subsequent addition of organomineral complex (OMC) or lignite as possible sorbents for PCP immobilization has been investigated as well. The OMC was prepared from humic acids (HAs) isolated from lignite by binding them onto zeolite. Biodegradation of PCP and number of colony forming units (CFUs) were determined in the three types of soil, Chernozem, Fluvisol, and Regosol, freshly spiked with PCP and amended separately with tested sorbents. The enhancing effect of sorbent addition and bioaugmentation on PCP biodegradation depended mainly on the soil type and the initial PCP concentration. Microbial activity resulted in biotransformation of PCP into certain toxic substances, probably lower chlorinated phenols that are more soluble than PCP, and therefore more toxic to present biota. Therefore, it was necessary to monitor soil ecotoxicity during biodegradation. Addition of the OMC resulted in a more significant decrease of soil toxicity in comparison with addition of lignite. Lignite and OMC appear to be good traps for PCP with potential application in remediation technology.