Use of 16S-rRNA to investigate microbial population dynamics during biodegradation of toluene and phenol by a binary culture

Interspecies interactions and changes in the rate and extent of biodegradation in mixed culture-mixed substrate studies were investigated. A binary mixed culture of Pseudomonas putida F1 and Burkholderia sp. JS150 degraded toluene, phenol, and their mixture. Both toluene and phenol can serve as sole sources of carbon and energy for both P. putida F1 and strain JS150. To investigate the population dynamics of this system, a fluorescent in-situ hybridization method was chosen because of its ability to produce quantitative data, its low standard error, and the ease of use of this method. When the binary mixed culture was grown on toluene or phenol alone, significant interactions between the species were observed. These interactions could not be explained by a pure-and-simple competition model and were substrate dependent. Strain JS150 growth was slightly inhibited when grown with P. putida F1 on phenol, and P. putida F1 grew more rapidly than expected. Conversely, when the two species were grown together on toluene alone, P. putida F1 was inhibited while strain JS150 was unaffected. During growth of the mixed culture on a combination of toluene and phenol, the interactions were similar to that observed during growth on phenol alone; P. putida F1 growth was enhanced while strain JS150 was unaffected. Because of the observed interspecies interactions, monoculture kinetic parameters were not sufficient to describe the mixed culture kinetics in any experiment. This is one of the first reports of microbial population dynamics in which molecular microbial ecology and mathematical modeling have been combined. The use of the 16S-rRNA-based method allowed for observation and understanding of interspecies interactions that were not observable with standard culture-based methods. These results suggest the need for more investigations that account for both substrate and microbial interactions when predicting the fate of organic pollutants in real systems.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

Lack of Capsular Exopolymer Effects on the Biodegradation of Organic Compounds by Pseudomonas sp. Strains JS1 and JS150

Abstract Due to the increasing interest in the effects of exopolymers on microbial activities, two Pseudomonas sp. strains, JS1 (possesses capsular exopolymer) and JS150 (unencapsulated), were compared for their ability to degrade a variety of organic compounds under a number of different conditions. Degradation kinetics for citrate, salicylate, phenol, toluene, and 2,4,6-trinitrotoluene were identical for both strains in liquid media, regardless of cell density. JS1 and JS150 grew on citrate at the same rate in sand, sterile surface soil, and sterile subsurface sediments. The biodegradation curves for toluene by cells pregrown on citrate in any of the above matrices were indistinguishable. Twofold differences in water content and carbon-to-nitrogen ratios of 0.032 or 320 did not result in any apparent differences in phenol degradation by either encapsulated or unencapsulated cells in sand. These results indicate that generalizations about the effects of exopolymers on microbial processes may not be possible without further research. Received: 14 August 1996; Accepted: 19 November 1996     

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, mu(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.     

Genetic and functional analysis of the tbc operons for catabolism of alkyl- and chloroaromatic compounds in Burkholderia sp. strain JS150

Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated tbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that the tbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.     

Comparison of substrate utilization and growth kinetics between immobilized and suspended Pseudomonas cells

A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (K(g)) for the toluene system increased by 30-fold and the maximum specific growth rate (mu(max)) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed. (c) 1993 John Wiley & Sons, Inc.     

Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in Burkholderia sp. Strain JS150

Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designated tbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate that tbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that the tbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.     

Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150

Aims: To test whether bioaugmentation with genetically modified Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efficiency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) profiling obtained directly from soils were examined. Bioaugmentation significantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (5·0 mg g^?1 soil) was completely degraded in clay soil (FC) within 65 days, whereas in sand soil (FS) within 72 days. In comparison, phenol biodegradation proceeded for 68 and 96 days in nonbioaugmented FC and FS soils, respectively. The content of humic carbon remained at the same level at the beginning and the end of incubation time in all soil treatments. The number of introduced bacteria (2·50 × 10⁹ g^?1 soil) markedly decreased during the first 4 or 8 days depending on contamination level and type of soil; however, inoculant survived over the experimental period of time. Analysis of FAME patterns indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy and 19:0 cyω10c and branched fatty acids might be useful markers for monitoring the progress of phenol removal from soil. Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp. JS150 significantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Significance and Impact of the Study: In future, genetically modified Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol‐contaminated areas.     

Studies on biodegradation of a mixture of toxic and nontoxic pollutant using Arthrobacter species

The effect of a nontoxic easily degradable substrate, glucose, on the biodegradation of toxic pollutant, phenol, was studied in batch reactors using a phenol degrading culture (Arthrobacter species). The effect of glucose on phenol degradation was determined at different glucose concentrations. The effect of different inoculum on substrate removal in a phenol and glucose mixture was also studied. Results indicated that when a mixed substrate (phenol and glucose) was used, phenol acclimated population showed an initial preference for phenol and utilised glucose after phenol removal. However phenol degradation rate was reduced in the presence of glucose. It was also observed that phenol degradation was completely inhibited when the glucose concentration exceeds 2 g/l. The substrate removal pattern changed completely when inoculum was drawn from mixed substrate acclimatised culture. The glucose utilisation started immediately and the rate of glucose utilisation was not affected by the presence of phenol. The phenol degradation also started simultaneously. In presence of phenol only, the rate of phenol degradation for the culture acclimatised to mixed substrates was lower than that of phenol acclimatised culture. These results indicate that nontoxic substrate can affect the biodegradation of toxic pollutants is suitable and acclimatisation may be necessary for biodegradation of mixed substrate.     

Effect of ethanol, acetate, and phenol on toluene degradation activity and tod-lux expression in Pseudomonas putida TOD102: evaluation of the metabolic flux dilution model

The reporter strain Pseudomonas putida TOD102 (with a tod-lux fusion) was used in chemostat experiments with binary substrate mixtures to investigate the effect of potentially occurring cosubstrates on toluene degradation activity. Although toluene was simultaneously utilized with other cosubstrates, its metabolic flux (defined as the toluene utilization rate per cell) decreased with increasing influent concentrations of ethanol, acetate, or phenol. Three inhibitory mechanisms were considered to explain these trends: (1) repression of the tod gene (coding for toluene dioxygenase) by acetate and ethanol, which was quantified by a decrease in specific bioluminescence; (2) competitive inhibition of toluene dioxygenase by phenol; and (3) metabolic flux dilution (MFD) by all three cosubstrates. Based on experimental observations, MFD was modeled without any fitting parameters by assuming that the metabolic flux of a substrate in a mixture is proportional to its relative availability (expressed as a fraction of the influent total organic carbon). Thus, increasing concentrations of alternative carbon sources "dilute" the metabolic flux of toluene without necessarily repressing tod, as observed with phenol (a known tod inducer). For all cosubstrates, the MFD model slightly overpredicted the measured toluene metabolic flux. Incorporating catabolite repression (for experiments with acetate or ethanol) or competitive inhibition (for experiments with phenol) with independently obtained parameters resulted in more accurate fits of the observed decrease in toluene metabolic flux with increasing cosubstrate concentration. These results imply that alternative carbon sources (including inducers) are likely to hinder toluene utilization per unit cell, and that these effects can be accurately predicted with simple mathematical models.     

An isolated Candida albicans TL3 capable of degrading phenol at large concentration

An isolated yeast strain was grown aerobically on phenol as a sole carbon source up to 24 mM; the rate of degradation of phenol at 30 degrees C was greater than other microorganisms at the comparable phenol concentrations. This microorganism was further identified and is designated Candida albicans TL3. The catabolic activity of C. albicans TL3 for degradation of phenol was evaluated with the K(s) and V(max) values of 1.7 +/- 0.1 mM and 0.66 +/- 0.02 micromol/min/mg of protein, respectively. With application of enzymatic, chromatographic and mass-spectrometric analyses, we confirmed that catechol and cis,cis-muconic acid were produced during the biodegradation of phenol performed by C. albicans TL3, indicating the occurrence of an ortho-fission pathway. The maximum activity of phenol hydroxylase and catechol-1,2-dioxygenase were induced when this strain grew in phenol culture media at 22 mM and 10 mM, respectively. In addition to phenol, C. albicans TL3 was effective in degrading formaldehyde, which is another major pollutant in waste water from a factory producing phenolic resin. The promising result from the bio-treatment of such factory effluent makes Candida albicans TL3 be a potentially useful strain for industrial application.     

Biodegradation kinetics of select polycyclic aromatic hydrocarbon (PAH) mixtures by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> EPA505

Many contaminated sites commonly have complex mixtures of polycyclic aromatic hydrocarbons (PAHs) whose individual microbial biodegradation may be altered in mixtures. Biodegradation kinetics for fluorene, naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene were evaluated in sole substrate, binary and ternary systems using Sphingomonas paucimobilis EPA505. The first order rate constants for fluorene, naphthalene, 1,5-dimethylnaphthalene, and 1-methylfluorene were comparable; yet Monod parameters were significantly different for the tested PAHs. S. paucimobilis completely degraded all the components in binary and ternary mixtures; however, the initial degradation rates of individual components decreased in the presence of competitive PAHs. Results from the mixture experiments indicate competitive interactions, demonstrated mathematically. The generated model appropriately predicted the biodegradation kinetics in mixtures using parameter estimates from the sole substrate experiments, validating the hypothesis of a common rate-determining step. Biodegradation kinetics in mixtures were affected by the affinity coefficients of the co-occurring PAHs and mixture composition. Experiments with equal concentrations of substrates demonstrated the effect of concentration on competitive inhibition. Ternary experiments with naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene revealed delayed degradation, where depletion of naphthalene and 1,5-dimethylnapthalene occurred rapidly only after the complete removal of 1-methylfluorene. The substrate interactions observed in mixtures require a multisubstrate model to account for simultaneous degradation of substrates. PAH contaminated sites are far more complex than even ternary mixtures; however these studies clearly demonstrate the effect that interactions can have on individual chemical kinetics. Consequently, predicting natural or enhanced degradation of PAHs cannot be based on single compound kinetics as this assumption would likely overestimate the rate of disappearance.     

Biodegradation of aromatic compounds and TCE by a filamentous bacteria-dominated consortium

The Michaelis-Menten biodegradation kinetics (k and Ks) of aromatic compounds and trichloroethene (TCE) by an aerobic enrichment culture grown on phenol and dominated by a unique filamentous bacterium were measured. The average k and Ks values for phenol, benzene (B), toluene (T), ethylbenzene (E), o-xylene (oX), p-xylene (pX), naphthalene and TCE in g per g VSS-d and mg L-1 were 5.72 and 0.34, 1.20 and 0.51, 2.09 and 0.47, 0.77 and 0.23, 0.61 and 0.16, 0.73 and 0.23, 0.17 and 0.18, and 0.16 and 0.18, respectively. Significant variability in these measured kinetics was noted between tests conducted over the 5-month period during which the fed-batch culture with a 5-day solids retention time was maintained; the coefficient of variation of the k and Ks values ranged from 11–43% and 4–50%, respectively. This variation was significantly greater than the method measurement error on a given date. Degradation of BTEoXpX mixtures could be described by a basic competitive inhibition model.Batch tests during which the culture was fed individual BTEX compounds showed the culture grew poorly on the xylenes and had poor subsequent xylene degradation rates. This work indicates the potential to simultaneously treat a mixture of volatile organic compounds using this consortium, and the ability to predict the mixture biodegradation rates on the basis of the individual compound biodegradation kinetics.     

Effect of a mixed culture on co-oxidation during the degradation of saturated hydrocarbon mixture

Two bacterial strains, Pseudomonas aeruginosa K1 and Rhodococcus equi P1, were used to degrade cyclo-alkanes (such as decalin) by a co-oxidation mechanism. Both strains possessed the capacity to degrade a broad range of n-alkane mixtures (C7 to C28) within 24 h of incubation. Strain P1 rapidly degraded 10 gl-1 pristane within 24 h of incubation (mu = 0.36 h-1 and Yx/s = 0.6). The addition of hexadecane as a growth substrate (above 0.5%, v/v) resulted in complete degradation of 1% (v/v) decalin by strain P1 via a co-oxidation mechanism. Co-oxidation to degrade decalin or pristane by strain K1 proved unsuccessful. Strain P1 was able to degrade decalin totally in a saturated hydrocarbon mixture. Strain K1 was only able to degrade hexadecane from the hydrocarbon mixture, but its degradation rate was higher than that of strain P1. Therefore, there was competition for the hexadecane needed to co-oxidize decalin. As a result, degradation of the hydrocarbon mixture, especially decalin, was incomplete in a mixed culture of strain P1 and K1. Serial addition of hexadecane (twice) allowed complete degradation of the remaining decalin by strain P1. Also, the biodegradation rate of the hydrocarbon mixture by a microbial population from gasoline-contaminated soil was delayed by addition of strain K1 to the population, while the addition of strain P1 resulted in an increase in the biodegradation rate.     

Multisubstrate biodegradation kinetics of naphthalene, phenanthrene, and pyrene mixtures.

Biodegradation kinetics of naphthalene, phenanthrene and pyrene were studied in sole-substrate systems, and in binary and ternary mixtures to examine substrate interactions. The experiments were conducted in aerobic batch aqueous systems inoculated with a mixed culture that had been isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Monod kinetic parameters and yield coefficients for the individual compounds were estimated from substrate depletion and CO(2) evolution rate data in sole-substrate experiments. In all three binary mixture experiments, biodegradation kinetics were comparable to the sole-substrate kinetics. In the ternary mixture, biodegradation of naphthalene was inhibited and the biodegradation rates of phenanthrene and pyrene were enhanced. A multisubstrate form of the Monod kinetic model was found to adequately predict substrate interactions in the binary and ternary mixtures using only the parameters derived from sole-substrate experiments. Numerical simulations of biomass growth kinetics explain the observed range of behaviors in PAH mixtures. In general, the biodegradation rates of the more degradable and abundant compounds are reduced due to competitive inhibition, but enhanced biodegradation of the more recalcitrant PAHs occurs due to simultaneous biomass growth on multiple substrates. In PAH-contaminated environments, substrate interactions may be very large due to additive effects from the large number of compounds present.     

Evaluation of different phenol hydroxylase-possessing phenol-degrading pseudomonads by kinetic parameters

Phenol-degrading pseudomonads possessing different phenol hydroxylases (PH) were evaluated by the values of apparent half-saturation constant for phenol-oxygenating activity (K ( S )), maximum specific growth rate (mu (max)), lag-time length (lambda), inhibition constant (K ( I )) and growth yield factor (Y ( X/S )). Strains of the same PH type showed similar kinetic parameters: single-component PH (sPH) harbouring strains had higher values of K ( S ) and lower values of mu (max) than the strains having multicomponent PH (mPH). However, the values of K ( I ) and the dependencies of the lag-time length on initial phenol concentration were strain-specific. The elevated ratio between specific activities of catechol 1,2-dioxygenase (C12O) and muconate cycloisomerase in sPH-strains caused irreversible accumulation of a high amount of exogenous cis,cis-muconate (CCM) which resulted in decreased Y ( X/S ) values. Co-presence of sPH and mPH genes did not give the strains PC16 and P69 any extra advantage and according to determined kinetic parameters only one PH was active during phenol degradation. At the same time simultaneous functioning of catechol ortho and meta cleavage pathways (strain PC20) resulted in higher mu (max) and Y ( X/S ) values. Evaluation of strains showed that the type of PH determined the efficiency of phenol degradation, whereas the tolerance to elevated phenol concentrations was strain-specific.