Semicontinuous and continuous degradation of phenol by Pseudomonas putida P8 adsorbed on activated carbon

Summary The semicontinuous and continuous degradation of phenol by Pseudomonas putida P8 which was immobilized on activated carbon was investigated. The amount of bacteria immobilized on the activated carbon surface dependend on the cell concentration in the suspension and on the type of activated carbon. In a continuous process running for four weeks the biomass, which accumulated in the activated carbon fixed bed, was removed periodically. The average phenol degradation rate in this process was 360 mg/1 h. The degradation activity of the bacteria for phenol, measured by the activity of the catechol-2,3-dioxygenase, was stimulated by the activated carbon. During the fermentation processes the carbon particles were covered with a biofilm. The bacteria grew, especially in the caverns and the entrances of the macropores, whereby the phenol adsorption by the activated carbon was decreased.     

Dynamics of phenol degradation by Pseudomonas putida

Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h(-1) and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l(-1). These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. (c) 1993 John Wiley & Sons, Inc.     

Aerobic batch degradation of phenol using immobilized Pseudomonas putida

Alginate concentrations between 2 and 4% had little effect on the degradation rate of phenol by alginate-immobilized Pseudomonas putida. Ten-degree shifts from 25°C resulted in approximately 30% slower degradation. Maximal degradation rates were favored at pH 5.5–6.0. The response of degradation rate to increased air flow in the bubble column used was almost linear and an optimal higher than 16 vol vol⁻¹ was indicated, although free cells appeared in the reaction medium above 12 vol vol⁻¹. When the initial phenol concentration was raised, degradation rate was not significantly affected until levels higher than 1200 mg ml⁻¹ where performance was markedly reduced. Increasing the ratio of total bead volume to medium volume gave progressively smaller increases in degradation rate. At a medium volume to total bead volume ratio of 5:1, the maximum degradation rate was 250 mg L⁻¹ h⁻¹. Received 24 November 1998/ Accepted in revised form 27 January 1999     

Kinetics of phenol degradation using Pseudomonas putida MTCC 1194

Pseudomonas putida (MTCC 1194) has been used to degrade phenol in water in the concentration range 100–1000?ppm. The inhibition effects of phenol as substrate have become predominant above the concentration of 500?ppm (5.31?mmoles/dm³). The optimum temperature and initial pH required for maximum phenol biodegradation were 30?°C and 7.00 respectively. From the degradation data the activation energy (E _a ) was found to be equal to 13.8?kcal/g mole substrate reacted. The most suitable inoculum age and volume for highest phenol degradation were 12?hrs and 7% v/v respectively. Surfactants had negligible effect on phenol biodegradation process for this microorganism. Monod model has been used to interpret the free cell data on phenol biodegradation. The kinetic parameters have been estimated upto initial concentration of 5.31?mmoles/dm³. μ _max and K _S gradually increased with higher concentration of phenol. However, beyond the phenol concentration of 5.31?mmoles/dm³, the inhibition became prominant. The μ _max has been to be a strong function of initial phenol concentration. The simulated and the experimental phenol degradation profiles have good correspondence with each other.     

Double inhibition model for degradation of phenol by Pseudomonas putida Q5

A semiempirical model, based on the presence of an inhibitory intermediate metabolite excreted to the broth, was developed to better predict the dynamic responses to shock loadings of Pseudomonas putida Q5 degrading phenol. Compared to the Haldane equation, the new model exhibited better prediction capabilities for a broad range of inlet concentration and dilution rate step changes. The experiments were performed at 10 degrees and 25 degrees C and ranged from stable responses to washouts. The time delays observed experimentally were successfully predicted with the dual-inhibition model and a very good agreement with the observed phenol profile also was found in a pulse experiment. A possible intermediate metabolite was detected by HPLC analyses based on the high correlation shown with the predicted inhibitory intermediate metabolite in the model.     

Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors

The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.     

Adsorption and bio-degradation of phenol by chitosan-immobilized Pseudomonas putida (NICM 2174)

Biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different conditions. Pseudomonas putida (NICM 2174) cells immobilized in chitosan were used to degrade phenol. Adsorption of phenol by the chitosan immobilized matrix played an important role in reducing the toxicity of phenol. In the present work, results of the batch equilibrium adsorption of phenol on chitosan from its aqueous solution at different particle sizes (0.177 mm, 0.384 mm, 1.651 mm) and initial concentration of phenol (20, 40, 60, 80, 100, 120, 140, 160, 180, 200 mg/l) have been reported. The adsorption isotherms are described by Langmuir, Freundlich and Redlich-Peterson types of equations. These indicate favourable adsorption with chitosan. From the adsorption isotherms, the adsorption capacity, energy of adsorption, number of layers and the rate constants were evaluated. In batch kinetic studies the factors affecting the rate of biodegradation of phenol, were initial phenol concentration (0.100 g/l, 0.200 g/l, 0.300 g/l), temperature (30v°C, 34v°C, 38v°C) and pH (7.0, 8.0, 9.0). Biodegradation kinetic data indicated the applicability of Lagergren equation. The process followed first order rate kinetics. The biodegradation data generally fit the Lagergren equation and the intraparticle diffusion rate equation from which adsorption rate constants, diffusion rate constants and diffusion coefficients were determined. Intraparticle diffusion was found to be the rate-limiting step. Cell growth contributed significantly to phenol removal rates especially when the degradation medium was supplemented with a utilizable carbon source.     

Box-Behnken design in the development of optimized complex medium for phenol degradation using Pseudomonas putida (NICM 2174)

The biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different operating conditions. Box-Behnken design has been employed to study the effect of different experimental variables. Four variables of maltose (0.25, 0.5, 0.75?g/l), phosphate (3, 12.5, 22?g/l), pH (7, 8, 9) and temperature (30?°C, 32?°C, 34?°C) were used to identify the significant effects and interactions in the batch studies. A second order polynomial regression model, has been developed using the experimental data. It was found that the degrading potential of Pseudomonas putida (NICM 2174) was strongly affected by the variations in maltose, phosphate, pH and temperature. The experimental values were in good agreement with the predicted values, the correlation coefficient was found to be 0.9980. Optimum conditions of the variables for the growth of Pseudomonas putida (NICM2174) and for maximum biodegradation of phenol are maltose (0.052?g/l), phosphate (8.97?g/l), pH (7.9) and temperature (31.5?°C).     

Oxidation of an inhibitory substrate by washed cells (oxidation of phenol by Pseudomonas putida)

The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. Then the equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.     

Solid matrix characterization of immobilized Pseudomonas putida MTCC 1194 used for phenol degradation

Characterization studies of calcium alginate beads with encapsulated Pseudomonas putida MTCC 1194, used for the biodegradation of phenol, were carried out to investigate the reactivity, reusability and structural strength of the solid matrix. Various techniques were employed to improve the structural stability of the immobilized solid necessary for its use in commercial reactors like packed bed flow reactor, fluidized bed and CSTR systems. Experiments were performed to establish the optimum conditions for durability, strength and steady biochemical reactivity. During a batch run of 40 h a gradual decline in the rate of phenol degradation was observed with the immobilized system. The calcium alginate beads with high structural strength yielded decreased activity. Treatment with a hardening agent like glutaraldehyde for different concentrations and treatment times led to variations in structural stability, reusability and the extent of phenol degradation. Scanning electron microscope studies of the immobilized solid indicated the internal distribution pattern of the cells encapsulated in a calcium alginate bead. Received: 13 November 1998 / Received revision: 27 January 1999 / Accepted: 31 January 1999     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

Uptake rate of phenol by Pseudomonas putida grown in unsteady state

The uptake rate of phenol by washed cells of Pseudomonas putidagrown on phenol in fermenter in an un steady state, caused by the step increase of dilution rate and/or phenol concentration in the feed, was studied. The Monod-Haldane type equation was applied to fit the data and the best kinetic parameters were calculated by nonlinear least-square techniques. It was found that the minimum period of unsteady state required for induction of the phenol metabolic pathway was approximately 30 min. The values of kinetic parameters in an unsteady state varied according to each parameter. The values of u(m) first monotonically increased to reach their highest value after about 120 min and then monotonically decreased to equal the u(m) in new steady state after about five residence times. No regularity in changing of K(s) and K(i), in unsteady state was observed. However, the greatest change in the values of K(i), was 45% while the change in values of K(s) was as much as two times compared to K(i) and K(s) in steady state prior to disturbance.     

The cyo operon of Pseudomonas putida is involved in carbon catabolite repression of phenol degradation

A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation. Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified. In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon. The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain. In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate. During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged. The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.     

Degradation of phenol and phenolic compounds by Pseudomonas putida EKII

Summary The phenol-degrading strain Pseudomonas putida EKII was isolated from a soil enrichment culture and utilized phenol up to 10.6 mM (1.0 g·1 ^-1) as the sole source of carbon and energy. Furthermore, cresols, chlorophenols, 3,4-dimethylphenol, and 4-chloro-m-cresol were metabolized as sole substrates by phenol-grown resting cells of strain EKII. Under conditions of cell growth, degradation of these xenobiotics was achieved only in co-metabolism with phenol. Phenol hydroxylase activity was detectable in whole cells but not in cell-free extracts. The specificity of the hydroxylating enzyme was found during transformation of cresols and chlorophenols: ortho- and meta-substituted phenols were degraded via 3-substituted catechols, while degradation of para-substituted phenols proceeded via 4-substituted catechols. In cell-free extracts of phenol-grown cells a high level of catechol 2,3-dioxygenase as well as smaller amounts of 2-hydroxymuconic semialdehyde hydrolyase and catechol 1,2-dioxygenase were detected. The ring-cleaving enzymes were characterized after partial purification by DEAE-cellulose chromatography.     

Phenol degradation by a psychrotrophic strain of Pseudomonas putida

Summary Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, S_o, ranging from 14 to 1000 mg/1. The experimental data for 14<=S_o<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: _m=0.119 h^–1, K _S=5.27 mg/1 and K _I=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.Nomenclature K _S saturation constant (mg/l) - K _I substrate inhibition constant (mg/l) - specific growth rate (h^–1) - _m maximum specific growth rate without substrate inhibition (h^–1) - _max maximum achievable specific growth rate with substrate inhibition (h^–1) - S substrate (phenol) concentration (mg/l) - S_o initial substrate concentration (mg/l) - S_max substrate concentration corresponding to _max (mg/l) - t time (h) - X cell concentration, dry basis (mg DW/l) - X_f final cell concentration, dry basis (mg DW/l) - X_o initial cell concentration, dry basis (mg DW/l) - Y yield factor (mg DW cell produced/mg substrate consumed)     

Substrate inhibition kinetics: Phenol degradation by Pseudomonas putida

A pure culture of Pseudoinonas putida was grown in both a batch and continuous culture using phenol as the limiting substrate. Of two substrate inhibition models examined, the Haldane function was found to statistically best describe the kinetics. The applicable kinetic constants were either measured (μ_M, K_I) or estimated (K_S) from the experimental data. Particularly in the continuous culture, wall growth was found to exert significant effects on the broth biomass concentration and phenol conversion, both of which decreased with increasing amounts of wall growth. These effects are opposite to those predicted by wall growth models and to experimental results of others using mixed culture (activated sludge) systems.