Biotransformation kinetics of Pseudomonas putida for cometabolism of phenol and 4-chlorophenol in the presence of sodium glutamate

A kinetic model to describe the degradation of phenol and cometabolictransformation of 4-chlorophenol (4-cp) in the presence of sodium glutamate(SG) has been developed and validated experimentally. The integrated modelaccounts for cell growth, toxicity of 4-cp, cross-inhibitions among the threesubstrates, and the different roles of the specific growth substrate (phenol)and the conventional carbon source (SG) in the cometabolism of 4-cp. In thisternary substrate system, the overall phenol degradation and 4-cp transformation rates are greatly enhanced by the addition of SG since SG is able to attenuate the toxicity of 4-cp and therefore increase the cell growth rate. Model analysis indicates that the maximum specific degradation rate of phenol (0.819 mg (mg.h)^-1) is lowered by SG by up to 46% whereas the specific transformation rate of 4-cp is notdirectly affected by the presence of SG. The competitive inhibition coefficient of 4-cp to phenol degradation (K_i,cp) and that of phenol to 4-cp transformation (K_i,ph) were determined to be 6.49 mg l^-1 and 0.193 mg l^-1, respectively, indicatingthat phenol imposes much larger competitive inhibition to 4-cp transformation than the converse. The model developed can simultaneously predict phenol degradation and 4-cp transformation, and is useful for dealing with cometabolism involving multiple substrates.     

Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources

The enhancement of biodegradation of phenol and 4-chlorophenol (4-cp) as a cometabolised compound by Pseudomonas putida ATCC 49451 was accomplished by augmenting the medium with conventional carbon sources such as sodium glutamate and glucose. Compared with phenol as the sole carbon source, the addition of 1 gl(-1) sodium glutamate increased the toxicity tolerance of cells toward 4-cp and significantly improved the biodegradation rates of both phenol and 4-cp even when the initial concentration of 4-cp was as high as 200 mgl(-1). On the other hand, supplementation of glucose caused a significant drop in the medium pH from 7.2 to 4.3 resulting in a reduction of degradation rate, leaving a considerable amount of 4-cp undegraded when the initial concentration of 4-cp was higher than 100 mgl(-1). By regulating the pH of the medium, however, enhancement of degradation rates of phenol and 4-cp in the presence of glucose was achieved with a concomitant complete degradation of phenol and 4-cp.     

Enhancement of biodegradation of phenol and a nongrowth substrate 4-chlorophenol by medium augmentation with conventional carbon sources

The enhancement of biodegradation of phenol and4-chlorophenol (4-cp) as a cometabolised compound byPseudomonas putida ATCC 49451 was accomplishedby augmenting the medium with conventional carbonsources such as sodium glutamate and glucose. Comparedwith phenol as the sole carbon source, the addition of1 gl^-1 sodium glutamate increased the toxicitytolerance of cells toward 4-cp and significantlyimproved the biodegradation rates of both phenol and4-cp even when the initial concentration of 4-cp wasas high as 200 mgl^-1. On the other hand,supplementation of glucose caused a significant dropin the medium pH from 7.2 to 4.3 resulting in areduction of degradation rate, leaving a considerableamount of 4-cp undegraded when the initialconcentration of 4-cp was higher than 100 mgl^-1.By regulating the pH of the medium, however,enhancement of degradation rates of phenol and 4-cp inthe presence of glucose was achieved with aconcomitant complete degradation of phenol and 4-cp.     

Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source

Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy. The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p-nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P. putida KT2442. The gene encoding the native OPH (opd), with and without the secretory leader sequence, was cloned into broad-host-range plasmids under the control of tac and taclac promoters. Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity. A plasmid-harboring operons encoding enzymes for p-nitrophenol transformation to beta-ketoadipate was transformed into P. putida allowing the organism to use 0.5 mM PNP as a carbon and energy source. Transformation of P. putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 mM parathion as a source of carbon and energy. Degradation studies showed that parathion formed a separate dense, non-aqueous phase liquid phase but was still bioavailable.     

FAME profiles in Pseudomonas vesicularis during catechol and phenol degradation in the presence of glucose as an additional carbon source.

The aim of this study was to evaluate the impact of catechol and phenol added to culture media separately and with glucose as an additional, easily-degradable carbon source on fatty acid methyl ester (FAME) composition in Pseudomonas vesicularis. Simultaneously, the degradation rates of aromatic substrates used were investigated in single and binary substrate systems. Both catechol and phenol treatments caused changes in the distribution of tested groups of fatty acids. The most noticeable changes included an increase in degree of fatty acid saturation, the appearance of branched and disappearance of hydroxy fatty acids as compared to the control sample with glucose. Under catechol or phenol treatment sat/unsat ratio showed the values of 8.63 and 11.38, respectively, whereas in control cells it reached the value of 2.66. The high level of saturation comes from the high content of cyclopropane fatty acids in bacteria under exposure to aromatic substrates, regardless of the presence of glucose. In these treatments their content was more than 3-fold higher compared to the control. It has been demonstrated that glucose supplementation of culture media containing single aromatic substrate extended the degradation rates of catechol and phenol by P. vesicularis, caused an increase in number of cells but did not significantly change the fatty acid profiles in comparison with bacteria growing on catechol and phenol added to the media individually.     

Degradation of phenol by a mixed culture of Pseudomonas putida and Cryptococcus elinovii adsorbed on activated carbon

Summary Phenol degradation by a defined mixed culture of Pseudomonas putida P8 and Cryptococcus elinovii H 1, which were immobilized by adsorption on activated carbon, was studied.The immobilized mixed culture was able to degrade phenol up to 17 g/l and degraded it faster than the pure cultures, depending on a complementary metabolism of the two microorganisms.Storage experiments revealed an excellent longterm storage capability of the biocatalyst: activated carbon with adsorbed cells of Pseudomonas putida P8 and Cryptococcus elinovii H1 could be stored up to 12 months without decrease on degradation capacity.Scanning electron micrographs showed that Pseudomonas putida P8 had grown through the pore system of the activated carbon into the inside of the carbon particles.     

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.     

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.     

Modeling the kinetics of vinyl chloride cometabolism by an ethane-grown Pseudomonas sp

Pseudomonas sp strain EA1 was isolated under aerobic conditions using ethane as the sole organic carbon and electron donor source, with an observed yield of 0.99 mg total suspended solids/mg ethane (0.85 mg volatile suspended solids / mg ethane) and a maximum specific growth rate of 0.015 d(-1). When grown on ethane, EA1 cometabolizes vinyl chloride (VC) at a maximum rate of 0.350 micromol/mg volatile suspended solids/d and with a half saturation constant of 0.62 microM VC. The rate of VC use by EA1 is twice as high when ethane is also provided, even though consumption of ethane is almost completely inhibited until VC is consumed. The presence of ethane also reduces the total amount of VC cometabolized. A model was developed that adequately describes the batch kinetics of VC cometabolism in the presence and absence of ethane, as well as ethane metabolism in the presence and absence of VC. Terms are included that increase the initial rate of VC use in the presence of ethane (according to the ratio of initial ethane concentration to the half saturation coefficient) but decrease the total amount of VC cometabolized. Parameter estimates for the model were obtained using a step-wise experimental approach, with varying initial concentrations of VC and ethane. Strain EA1 completely dechlorinates VC in the presence and absence of ethane. Measurements of soluble chemical oxygen demand indicate that approximately 50% of the VC consumed is mineralized, with the balance released as soluble, nonchlorinated products. Ethene is not used as a substrate by EA1 but it does inhibit ethane metabolism and VC cometabolism. In mixtures containing all three compounds, more VC is degraded and at a faster rate compared to VC plus ethene. The results suggest that ethane-enhanced biodegradation of VC may contribute to VC removal at the aerobic fringe of groundwater plumes undergoing reductive dechlorination.     

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.     

Biodegradation kinetics of a mixture containing a primary substrate (phenol) and an inhibitory co-metabolite (4-chlorophenol)

Batch experiments on the simultaneous utilization of phenol (primary substrate) and 4-chlorophenol (cometabolic secondary substrate) demonstrated two critical substrate interactions. First, the cometabolic degradation of 4-chlorophenol was proportional to the rate of phenol oxidation, which provided the electrons for the initial monooxygenase reaction. Second, 4-chlorophenol inhibited the oxidation of the primary substrate, phenol. Modeling analyses of the degradation of phenol alone and of phenol and 4-chlorophenol together showed that the proportionality between phenol and 4-chlorophenol degradation rates averaged 0.1 mg 4-CP/mg phenol, which corresponds to 0.5% of the electrons generated by phenol oxidation being used as a cosubstrate for the monooxygenase reaction of 4-chlorophenol. In addition, modeling analyses suggest that 4-chlorophenol was a noncompetitive inhibitor of phenol oxidation for high phenol concentrations, but a competitive inhibitor for low phenol concentrations.Abbreviations GC gas chromatography - FID flame-ionization detector - DO dissolved oxygen - 4-CP 4-chlorophenol - Ph phenol - RLS relative least squares criterion - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate     

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.     

Metabolic profiling analysis of the degradation of phenol and 4-chlorophenol by Pseudomonas sp. cbp1-3

To investigate the enhancement of phenol on the biodegradation of 4-chlorophenol (4-cp), metabolic profiling approach was performed for the first time to analyze metabolite changes of Pseudomonas sp. cbp1-3 using single substrate (succinate, phenol, and 4-cp) and dual substrate (mixtures of phenol and 4-cp). Phosphoric acid, γ-aminobutyric acid, 4-cp, 4-chlorocatechol, and catechol were shown to change significantly. Results indicated that phenols, especially 4-cp, depressed cell growth by inhibiting its primary metabolic pathway. In addition, the addition of phenol into the 4-cp-containing medium had a global influence on cells including the accumulation of amino acids, amines, saturated fatty acids, and monoacylglycerols as well as the concentration changes of metabolite participating in phenols biodegradation, thus enhancing the degradation of 4-cp. This study provided novel insights into the biodegradation of mixed phenolic compounds and the method could be used to investigate the biodegradation of complicated multi-pollutants.     

Molecular cloning and sequencing of the phenol hydroxylase gene from Pseudomonas putida BH

A genomic library of the phenol-degrading bacterium Pseudomonas putida BH was constructed in the broad host range cosmid pVK100 and introduced into Escherichia coli HB101. One of the recombinant cosmids recovered from catechol- and/or 2-hydroxymuconic semialdehyde-accumulating clones, pS10–45, had a 19.6-kb insert fragment which allowed P. putida KT2440 to grow on phenol as a sole carbon and energy source. Subcloning and expression studies indicated that the phenol hydroxylase gene cluster (pheA) is located on a 6.1-kb SacI fragment. The results of DNA sequencing of the SacI fragment revealed that the pheA gene cluster encodes a multicomponent phenol hydroxylase.     

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.