Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C1-C6) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring-fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ring-cleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11-0.74 h-1), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a 1-m3 bioreactor supplied with 1 g nitrogen l-1 for removal of toluene was 57 g day-1 and for removal of trichloroethylene was 3.4 g day-1. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts.     

The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis

BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols. RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction. CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.     

Protein phosphatase inhibitors induce the release of serotonin from rat basophilic leukemia cells (RBL-2H3)

Oxidation capacities of laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) from Phlebia radiata were compared using non-phenolic (veratryl alcohol and ABTS) and phenolic (syringaldazine, vanillalacetone and Phenol red) compounds as reducing substrates. The effect of Mn(II) on enzyme reactions was also studied. Highest specific activities were recorded with laccase in the oxidation of phenolic compounds or ABTS and irrespective of Mn(II) concentration. LiP and MnP oxidized all these substrates but only the catalysis of MnP was dependent upon Mn(II). Only LiP clearly oxidized veratryl alcohol. However, Mn(II) interfered with this reaction by repressing veratraldehyde formation. These results point to multiple participation of manganese ions, either as a reducing (Mn(II)) or oxidizing (Mn(III)) agent in the enzymatic reactions.     

Reactor Models for Horseradish Peroxidase–Catalyzed Aromatic Removal

Horseradish peroxidase catalyzes the oxidation of aqueous aromatic compounds by hydrogen peroxide, resulting in the formation of polymers, which spontaneously precipitate from solution. This process is being investigated as a means of removing toxic phenols and aromatic amines from industrial wastewaters. Models of plug-flow reactors and continuous-flow stirred tank reactors (CFSTR) were developed for the horseradish peroxidase–peroxide-aromatic substrate system as an aid for reactor design and process optimization. The models were verified for phenol removal at pH 7 and 25°C, both in the presence and absence of high molecular weight polyethylene glycol, a protective additive. Modeling suggests that the rate of enzyme inactivation is lower in a CFSTR than in a plug-flow reactor. Nevertheless, no single optimal reactor configuration can be identified, because the best configuration depends on the initial phenol concentration, the desired effluent quality, and the selected retention time. The CFSTR performance could be improved further by engineering a system that returns effluent active enzyme to the treatment process.     

A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.     

Kinetic Model for Removal of Phenol by Horseradish Peroxidase with PEG

A kinetic model has been developed to simulate horseradish peroxidase-catalyzed polymerization of phenol in the presence of polyethylene glycol based on the following kinetics. The phenol conversion expression is a second-order Michaelis–Menten equation with respect to the concentrations of phenol and hydrogen peroxide. The enzyme inactivation is attributed to the polymer and hydrogen peroxide simultaneously. The inactivation by polymer is an apparently second-order reaction, first-order in each of enzyme and phenol, whereas the inactivation by peroxide is also an apparently second-order reaction, first-order in each of enzyme and hydrogen peroxide. The rates of consumption of hydrogen peroxide and polyethylene glycol are directly proportional to the rate of conversion of phenol. Experimental data show that the model output can predict the phenol removal and activity depletion realistically under a variety of reaction conditions. The model has been verified by predicting some independent experimental results on reaction stoichiometry, horseradish peroxidase dose effect, and semibatch operation with respect to hydrogen peroxide.     

Cometabolic Transformation of High Concentrations of 4-Chlorophenol in an Immobilized Cell Hollow Fiber Membrane Bioreactor

The cometabolic transformation of high concentrations of 4-chlorophenol (4-cp) in the presence of phenol (growth substrate) was investigated in an immobilized cell hollow fiber membrane bioreactor. The biotransformation rates of 4-cp and phenol by free cells and immobilized cells were compared and it was found that biotransformation by immobilized cells was more efficient than that by freely suspended cells, especially at high substrate concentrations. Even when the initial substrate concentrations were both 1,000?mg?L?1, the substrates could still be transformed completely within 45?h in the hollow fiber membrane bioreactor. It was found that removal rates of the substrates were dependent not only on absolute substrate concentration, but also on the concentration ratios of 4-cp to phenol. With increased concentration ratios, phenol depletion rate decreased while 4-cp depletion rate increased. This was attributed to increased competitiveness of 4-cp in the competitive inhibition with phenol since the membranes had relatively better sorption for 4-cp over phenol. The bioreactor was operated batchwise for 18 times to investigate the feasibility of the bioreactor for long term operation. It was found that there was no significant decrease in the bioactivity, which demonstrates a potential for continuous operation.     

Purification and characterization of laccase from Chaetomium thermophilium and its role in humification

Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process. C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45 degreesC both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70 degreesC and had half-lives of 24 and 12 h at 40 and 50 degreesC, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.     

石墨烯-ZnO复合物修饰玻碳电极微分脉冲伏安法同时测定邻苯二酚和对苯二酚

通过壳聚糖（CHIT）成膜, 制备了一种新的石墨烯-ZnO复合物修饰玻碳电极（GR-ZnO/CHIT/GCE）。运用循环伏安法研究了邻苯二酚和对苯二酚在修饰电极上的电化学行为。实验结果表明, 在0.1mol/LB-R(pH4.0)缓冲液中, 修饰电极对邻苯二酚和对苯二酚的电化学氧化还原显示出较高的催化特性。在优化条件下, 利用微分脉冲伏安法测定, 邻苯二酚和对苯二酚的氧化峰电流与浓度在8.0×10^-7～5.0×10^-5mol/L范围内呈良好的线性关系, 检测限均为2.0×10^-7mol/L(S/N=3)。将该方法用于模拟水样中邻苯二酚和对苯二酚的测定, 结果较满意。     

Antioxidant efficiency of polyhydric phenols in photooxidation of benzaldehyde

An experimental system is described for the observation of the kinetics of photochemically initiated oxidation reactions; this system is based on the measurement of oxygen consumption with a polarographic oxygen electrode. The photooxidation of benzaldehyde in dilute aqueous solution was examined and appears to conform to a free radical chain mechanism. The antioxidant efficiency of some polyhydric phenols was determined kinetically and found to be catechol greather than pyrogallol greater than hydroquinone greather than resorcinol greater than n-propyl gallate for the benzaldehyde photooxidation.     

Acinetobacter radioresistens metabolizing aromatic compounds. 2. Biochemical and microbiological characterization of the strain

The metabolic potentialities of an Acinetobacter radioresistens strain, isolated from the soil adjacent to an activated sludge plant, were investigated. Among 26 aromatic substrates tested, only phenol, benzoate and catechol were metabolized. Since this strain possessed abundant plasmid DNA, the antibiotic and heavy metal resistance was examined, and the bacterial cells proved to be sensitive to all metals (Ni, Tl, Pb, Cd, Ag, Co, Zn) and antibiotics tested except for Fosfomycin and chloramphenicol. The degradation kinetics for phenol and benzoate as the sole carbon/energy source (pH 7, 30 degrees C) displayed different trends, confirmed by the bacterial growth curve. Crude extracts from phenol-grown cultures showed both phenol hydroxylating activity and catechol dioxygenating activity. Phenol hydroxylase possessed a reductase component able to reduce nitroblue tetrazolium (NBT) and cytochrome C, thus exhibiting differences from previously reported monocomponent phenol hydroxylases from the same genus. Catechol dioxygenase is an intradiol-cleaving enzyme recognizing also substituted catechols.     

Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1, 2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for DHB were 22 +/- 2 and 8 +/- 1 microM, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.     

Epstein-Barr virus and CD21 expression in gastrointestinal tumors

OBJECTIVES: Animal inhalation studies and theoretical models suggest that the pattern of formation of benzene metabolites changes as exposure to benzene increases. To determine if this occurs in humans, benzene metabolites in urine samples collected as part of a cross sectional study of occupationally exposed workers in Shanghai, China were measured. METHODS: With organic vapour monitoring badges, 38 subjects were monitored during their full workshift for inhalation exposure to benzene. The benzene urinary metabolites phenol, catechol, hydroquinone, and muconic acid were measured with an isotope dilution gas chromatography mass spectroscopy assay and strongly correlated with concentrations of benzene air. For the subgroup of workers (n = 27) with urinary phenol > 50 ng/g creatinine (above which phenol is considered to be a specific indicator of exposure to benzene), concentrations of each of the four metabolites were calculated as a ratio of the sum of the concentrations of all four metabolites (total metabolites) and were compared in workers exposed to > 25 ppm v < or = 25 ppm. RESULTS: The median, 8 hour time weighted average exposure to benzene was 25 ppm. Relative to the lower exposed workers, the ratio of phenol and catechol to total metabolites increased by 6.0% (p = 0.04) and 22.2% (p = 0.007), respectively, in the more highly exposed workers. By contrast, the ratio of hydroquinone and muconic acid to total metabolites decreased by 18.8% (p = 0.04) and 26.7% (p = 0.006), respectively. Similar patterns were found when metabolite ratios were analysed as a function of internal benzene dose (defined as total urinary benzene metabolites), although catechol showed a more complex, quadratic relation with increasing dose. CONCLUSIONS: These results, which are consistent with previous animal studies, show that the relative production of benzene metabolites is a function of exposure level. If the toxic benzene metabolites are assumed to be derived from hydroquinone, ring opened products, or both, these results suggests that the risk for adverse health outcomes due to exposure to benzene may have a supralinear relation with external dose, and that linear extrapolation of the toxic effects of benzene in highly exposed workers to lower levels of exposure may underestimate risk.     

The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries

Human recombinant CD38 catalyzes the formation of both cyclic ADP-ribose and ADP-ribose products from NAD+ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD+. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD+) were rapidly hydrolyzed by the enzyme. The kcat for NMN+ was 5-fold higher than that of NAD+ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD+ was hydrolyzed at approximately one-third the rate of NHD+ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the beta-methoxy riboside compound, with a relative nucleophilicity of MeOH to H2O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD+ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre-steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD+ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond-breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD+.     

The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis

The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.     

Michaelis-Menten analysis of immobilized enzyme by affinity capillary electrophoresis

Michaelis constant of enzymatic reaction was evaluated by affinity capillary electrophoresis using beta-galactosidase as a model enzyme and o- and p-isomers of nitrophenyl-beta-galactoside as substrates. The enzyme was immobilized on the inner surface of a fused-silica capillary by the covalent bonding through a bridging group, and the substrates were introduced into the capillary. The reaction products migrated electrophoretically toward the detection side (anodic side), while the unreacted substrates moved toward the injection side (cathodic side) on a slow electroosmotic flow generated by the weak negative charge of the immobilized enzyme. The initial velocity of the enzymatic reaction was estimated from the peak height of the product, and the Michaelis constant was calculated according to Lineweaver-Burk equation. The results (Km, 2.34 mM for o-isomer and 1.09 mM for p-isomer) were reproducible (RSD < 11.8%, n = 5). Although the estimated Michaelis constants were larger than the reported values measured in homogeneous solution, the ratio of the Michaelis constants of o-/p-isomers was in good agreement with the literature value. The present method required as low as a few microgram amount of enzyme and nanogram amount of substrate which is far smaller than those required in a conventional affinity HPLC.     

cDNA expression studies of rat liver aryl sulphotransferase

A cDNA encoding an isoenzyme of rat liver aryl sulphotransferase was isolated from a rat liver bacteriophage Lambda gt 11 library by the polymerase chain reaction technique. The resulting cDNA was functionally expressed in COS-7 cells and characterised by determining the sulphating capacity of the cells with a range of substrates. The COS-expressed enzyme catalysed the sulphation of both phenol and dopamine with Kms of the same order as those obtained for the high affinity isozyme in rat liver cytosol, while low activity was observed with tyrosine methyl ester. The common food additive vanillin was also a good substrate for sulphate conjugation. The sulphation of vanillin catalysed by the COS-expressed enzyme was consistent with a single enzyme system, in contrast, the kinetics of the reaction catalysed by cytosolic sulphotransferase indicated that vanillin was sulphated by more than one isozyme.     

Verification of Anaerobic Biofilm Model for Phenol Degradation with Sulfate Reduction

A mathematical model was developed to describe phenol degradation with sulfate reduction in an anaerobic biofilm process. The model incorporates the mechanisms of diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear's method. A pilot-scale column reactor was conducted to verify the model. The batch kinetic tests were independently conducted to determine nine biokinetic coefficients used in the model while shear loss and initial thickness of the biofilm were assumed so that the model simulated the substrate concentration results very well. The removal efficiencies for phenol and sulfate are 98 and 88%, respectively. At a steady-state condition, the experimental data of phenol and sulfate concentrations were higher than those obtained from the model. The reason was that the effect of shear loss became significant as the biofilm grew thicker. The higher shear loss resulted in the more-suspended biomass. The suspended biomass decomposed and released soluble microbial products which increased the substrate effluent concentrations. The procedures presented in this paper could be employed for the design of anaerobic biofilm reactor systems for the biodegradation of multiple substrates.     

Novel membrane bioreactor with gas/liquid two-phase flow for high-performance degradation of phenol

The use of a membrane bioreactor with cell retention to achieve high biomass concentrations has been examined for phenol degradation by the bacteria Alcaligenes eutrophus. This process is particularly interesting for toxic substrates as the hydraulic dilution rate and the growth rate are independently controlled. In the case of a transitory excess of phenol, this potentially toxic situation can be overcome by modifying the substrate concentration or the dilution rate without any loss of cells. The injection of a gas phase at the filter inlet increased both the permeate flow rate (by a factor of 1. 75) and the oxygen transfer capacity (by a factor of 1.5). This has enabled the cell concentration to reach a maximal value of 60 g L-1 with a hydraulic dilution rate of 0.5 h-1 and a phenol feed concentration of 8 g L-1. The volumetric productivity of this process corresponds to a phenol degradation rate approaching 100 kg m-3 day-1. The on-line measurement of the characteristic yellow color of 2-hydroxymuconate semialdehyde, a metabolic intermediate of the phenol degradation pathway, in the permeate provides an interesting basis for process control of phenol supply into the reactor since the color intensity correlates directly to the specific rate of phenol degradation.