The effect of concentration of phenols on their batch methanogenesis

The biodegradability of phenol and six other phenolic compounds (o-, m-, and p-cresol, 2-, 3-, and 4-ethylphenol) was examined in batch methanogenic cultures. The effect of concentration of these alkyl phenols on the anaerobic biodegradation of phenol was also evaluated. The inoculum used in this study was cultivated in a continuous flow laboratory fermenter with phenol as the primary substrate. Phenol, at initial concentrations as high to 1400 mg/L was completely degraded to methane and carbondioxide after 350 hours incubation. Complete degradation of m- and p-cresol was also observed while the ethylphenols and o-cresol were not significantly degraded.At initial concentrations exceeding 600 mg/L, phenol inhibited the phenol-degrading microorganisms but not the methanogens. At about 600 mg/L, cresols reduced the rate of phenol degradation to 50% of that observed in a control culture containing only 200 mg/L phenol. Ethylphenols were more inhibitory than cresols. Phenol degrading microorganisms were more susceptible to inhibition by cresols and ethylphenols than were the methanogens. The inhibitory effects of the three isomers of cresol and ethylphenol did not vary with the isomer but rather with the substituted functional group.     

Anaerobic biodegradation of phenolic compounds in digested sludge.

We examined the anaerobic degradation of phenol and the ortho, meta, and para isomers of chlorophenol, methoxyphenol, methylphenol (cresol), and nitrophenol in anaerobic sewage sludge diluted to 10% in a mineral salts medium. Of the 12 monosubstituted phenols studied, only p-chlorophenol and o-cresol were not significantly degraded during an 8-week incubation period. The phenol compounds degraded and the time required for complete substrate disappearance (in weeks) were: phenol (2), o-chlorophenol (3), m-chlorophenol (7), o-methoxyphenol (2), m- and p-methoxyphenol (1), m-cresol (7), p-cresol (3), and o-, m-, and p-nitrophenol (1). Complete mineralization of phenol, o-chlorophenol, m-cresol, p-cresol, o-nitrophenol, p-nitrophenol, and o-, m-, and p-methoxyphenol was observed. In general, the presence of Cl and NO2 groups on phenols inhibited methane production. Elimination or transformation of these substituents was accompanied by increased methane production, o-Chlorophenol was metabolized to phenol, which indicated that dechlorination was the initial degradation step. The methoxyphenols were transformed to the corresponding dihydroxybenzene compounds, which were subsequently mineralized.     

Metabolism of phenol and cresols by Bacillus stearothermophilus.

An obligate thermophilic strain of Bacillus stearothermophilus, strain PH24, isolated from industrial sediment by elective culture, grew readily at 55 C on phenol or on one of the isomers of cresol as the major carbon source. Intact cells grown in the presence of phenol, o-cresol, m-cresol, or p-cresol were induced to oxidize, without lag, these substrates together with catechol, 3-methylcatechol, and 4-methylcatechol. Cell extracts prepared from B. stearothermophilus PH24 after growth in the presence of phenol converted phenol to catechol with a concomitant uptake of 1 mol of oxygen per mol of substrate in reaction mixtures supplemented with reduced nicotinamide adenine dinucleotide. These preparations also catalyzed the oxidation of o-cresol to 3-methylcatechol and of m-cresol and p-cresol to 4-methylcatechol. Enzyme activity was inhibited by 1 mM p-chloromercuribenzoate and by 0.1 mM 0-phenanthroline. Catechol and the corresponding methylcatechol intermediates were further dissimilated by cell extracts of phenol-grown cells via the meta-cleavage route to yield 2-hydroxymuconic semialdehyde and the respective methylated derivatives.     

Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene

Pseudomonas cepacia G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho-monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes which were blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase, and TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol, and phenol hydroxylation.     

The metabolism of cresols by species of Pseudomonas

1. A comparison of rates of oxidation of various compounds by whole cells indicated that protocatechuate was a reaction intermediate when a non-fluorescent species of Pseudomonas oxidized p-cresol. In contrast, a fluorescent Pseudomonas oxidized 3-methylcatechol and 4-methylcatechol when grown with p-cresol, but did not oxidize protocatechuate. 2. Heat-treated extracts of the fluorescent Pseudomonas oxidized catechol, 3-methylcatechol and 4-methylcatechol to ring-fission products, the spectroscopic properties of which were recorded. Rates of enzymic degradation of these products were also measured. 3. Acetic acid and formic acid were obtained by the action of a Sephadex-treated extract on 3-methylcatechol and 4-methylcatechol respectively. In each case 0.8mol. of the carboxylic acid was formed from 1.0mol. of substrate. 4. Dialysed extracts converted 3-methylcatechol into acetaldehyde and pyruvate, with 4-hydroxy-2-oxovalerate as a reaction intermediate. 4-Methylcatechol was converted first into 4-hydroxy-2-oxohexanoate and then into propionaldehyde and pyruvate. 5. The ring-fission product of catechol was formed from phenol by a fluorescent Pseudomonas, that of 3-methylcatechol was formed from o-cresol and m-cresol, and the ring-fission product of 4-methylcatechol was given from p-cresol. Propionate was readily oxidized by these cells after growth with p-cresol, but this compound was not attacked when phenol, o-cresol or m-cresol served as source of carbon. 6. Cell extracts appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxohexanoate.     

Anaerobic degradation of phenol and phenol derivatives by Desulfobacterium phenolicum sp. nov.

A new sulfate-reducing bacterium was enriched and isolated from marine sediment with phenol as sole electron donor and carbon source. Strain Ph01 grew well in defined media without growth factors. Further aromatic compounds oxidized by strain Ph01 were benzoate, phenylacetate, 2-hydroxybenzoate, 4-hydroxybenzoate, 4-hydroxyphenylacetate, p-cresol, indole, anthranilic acid, and phenylalanine. Various fatty acids, alcohols and dicarboxylic acids were also utilized by strain Ph01. Sulfate and thiosulfate served as electron acceptors and were reduced to H₂S. Stoichiometric measurements with strain Ph01 showed complete oxidation of phenol to CO₂. Cytochromes and menaquinone MK-7(H₂) were present; desulfoviridin could not be detected. Strain Ph01 is described as type strain of the new species Desulfobacterium phenolicum.In further marine enrichments with 4-hydroxybenzoate, 4-hydroxyphenylacetate, p-cresol or o-cresol as substrates and sulfate as electron acceptor a variety of morphologically different sulfate-reducing bacteria developed. However, since the new isolate strain Ph01 was able to degrade all these aromatic compounds (except o-cresol) no further studies with the enrichment cultures were carried out.     

Biodegradation of ortho-cresol by a mixed culture of nitrate-reducing bacteria growing on toluene.

A mixed culture of nitrate-reducing bacteria degraded o-cresol in the presence of toulene as a primary growth substrate. No degradation of o-cresol was observed in the absence of toluene or when the culture grew on p-cresol and 2,4-dimethylphenol. In batch cultures, the degradation of o-cresol started after toluene was degraded to below 0.5 to 1.0 mg/liter but continued only for about 3 to 5 days after the depletion of toluene since the culture had a limited capacity for o-cresol degradation once toluene was depleted. The total amount of o-cresol degraded was proportional to the amount of toluene metabolized, with an average yield of 0.47 mg of o-cresol degraded per mg of toluene metabolized. Experiments with [ring-U-14C]o-cresol indicated that about 73% of the carbon from degraded o-cresol was mineralized to CO2 and about 23% was assimilated into biomass after the transient accumulation of unidentified water-soluble intermediates. A mathematical model based on a simplified Monod equation is used to describe the kinetics of o-cresol degradation. In this model, the biomass activity toward o-cresol is assumed to decay according to first-order kinetics once toluene is depleted. On the basis of nonlinear regression of the data, the maximum specific rate of o-cresol degradation was estimated to be 0.4 mg of o-cresol per mg of biomass protein per h, and the first-order decay constant for o-cresol-degrading biomass activity was estimated to be 0.15 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial metabolism of 2-chlorophenol, phenol and rho-cresol by Rhodococcus erythropolis M1 in co-culture with Pseudomonas fluorescens P1

Chlorophenolic waste most often contains phenol and rho-cresol along with chlorophenols. A Rhodococcus erythropolis strain M1 was isolated with the ability to degrade 2-chlorophenol, phenol and p-cresol (100 mgl(-1), each) in 18, 24 and 20 h, respectively, with negligible lag. However, Rhodococcus sp. characterized by low growth rate, pose a threat to be outgrown by bacteria occurring in natural habitats. In the present study, interaction of R. erythropolis M1 with another isolated bacteria generally encountered in activated sludge for water treatment like Pseudomonas fluorescens P1 was studied. 2-chlorophenol, phenol and p-cresol were selected as the substrates for the study. Viable cell counts showed competitive interaction between the species on 2-chlorophenol and phenol. Specific growth rate of pure culture of R. erythropolis M1 was higher than P. fluorescens P1 on 2-chlorophenol. However, in mixed culture, P. fluorescens P1 showed higher growth rate. Degradation of phenol showed higher growth rate of R. erythropolis M1 both in pure and in mixed culture form. Degradation of p-cresol had shown similar counts for both populations indicating neutral type of interaction. This observation was substantiated by detecting the growth rate, where both cultures had similar growth rate in pure and in the mixed culture form. Rate of 2-chlorophenol degradation was higher when R. erythropolis M1 was used as the pure culture as compared to the degradation rates observed with the P. fluorescens P1 or with the mixed culture. However, in case of phenol and p-cresol, degradation by the mixed culture had resulted in higher degradation rates as compared to the degradation of the substrates by both the axenic cultures.     

Laccase-mediated detoxification of phenolic compounds.

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Inhibition of prostaglandin synthesis in mouse 3T3 fibroblasts and human platelets by substituted phenols.

Several substituted phenols with antioxidant properties were potent reversible inhibitors of prostaglandin synthesis in 3T3 cell cultures. The ID50's for prostaglandin (PG) E2 synthesis in these cells were 0.1 muM for 2,6-xylenol, 5 muM for tricresol, 6 muM for p-cresol, 7 muM for o-cresol, 15 muM for 3,5-xylenol, 30 muM for m-cresol and 100 muM for phenol. The corresponding values for aspirin and indomethacin were 4 muM and 0.02 muM, respectively. The substituted phenols also inhibited serotinin release, aggregation and prostaglandin synthesis in human platelets induced by arachidonic acid but not by PGG2.     

Biodegradation of p-Cresol by Bacillus sp. Strain PHN 1

A bacterium capable of utilizing p-cresol as sole source of carbon and energy was isolated from soil and identified as a Bacillus species. The organism also utilized phenol, o-cresol, m-cresol, 4-hydroxybenzoic acid, and gentisic acid as growth substrates. The organism degraded p-cresol to 4-hydroxybenzoic acid, which was further metabolized by a gentisate pathway, as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extract. Such a bacterial strain can be used for bioremediation of environments contaminated with phenolic compounds.     

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.     

Anaerobic biodegradation of phenol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 in the presence of 4-chlorophenol

Biodegradation of phenol and 4-chlorophenol (4-cp) using pure culture of Candida albicans PDY-07 under anaerobic condition was studied. The results showed that the strain could completely degrade up to 1,800 mg/l phenol within 68 h. The capacity of the strain to degrade phenol was higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Comparatively, low-concentration phenol from 25 to 150 mg/l supplied a carbon and energy source for Candida albicans PDY-07 in the early phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in that 50 mg/l 4-cp was degraded within less time than that without phenol. While the biodegradation of 50 mg/l 4-cp was inhibited in the presence of 200 mg/l phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and dual substrates in batch cultures. The results demonstrated that the models adequately described the dynamic behaviors of biodegradation by Candida albicans PDY-07.     

Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.     

Biodegradation of cresol isomers in anoxic aquifers

The biodegradation of o-, m-, and p-cresol was examined in material obtained from a shallow anaerobic alluvial sand aquifer. The cresol isomers were preferentially metabolized, with p-cresol being the most easily degraded. m-Cresol was more persistent than the para-isomer, and o-cresol persisted for over 90 days. Biodegradation of cresol isomers was favored under sulfate-reducing conditions (SRC) compared with that under methanogenic conditions (MC). Slurries that were acclimated to p-cresol metabolism transformed this substrate at 18 and 330 nmol/h per g (dry weight) for MC and SRC, respectively. Inhibition of electron flow to sulfate reduction with 2.0 mM molybdate reduced p-cresol metabolism in incubations containing sulfate. When methanogenesis was blocked with 5 mM bromoethanesulfonic acid in incubations lacking sulfate, p-cresol catabolism was retarded. Under SRC 3.4 mol of sulfate was consumed per mol of p-cresol metabolized. The addition of sulfate to methanogenic incubations stimulated p-cresol degradation. Simultaneous adaptation studies in combination with spectrophotometric and chromatographic analysis of metabolites indicated that p-cresol was oxidized under SRC to p-hydroxybenzoate via the corresponding alcohol and aldehyde. This series of reactions was inhibited under sulfate-limited or aerobic conditions. Therefore, the primary catabolic event for p-cresol decomposition under SRC appears to involve the hydroxylation of the aryl methyl group.     

Biodegradation of cresol isomers in anoxic aquifers.

The biodegradation of o-, m-, and p-cresol was examined in material obtained from a shallow anaerobic alluvial sand aquifer. The cresol isomers were preferentially metabolized, with p-cresol being the most easily degraded. m-Cresol was more persistent than the para-isomer, and o-cresol persisted for over 90 days. Biodegradation of cresol isomers was favored under sulfate-reducing conditions (SRC) compared with that under methanogenic conditions (MC). Slurries that were acclimated to p-cresol metabolism transformed this substrate at 18 and 330 nmol/h per g (dry weight) for MC and SRC, respectively. Inhibition of electron flow to sulfate reduction with 2.0 mM molybdate reduced p-cresol metabolism in incubations containing sulfate. When methanogenesis was blocked with 5 mM bromoethanesulfonic acid in incubations lacking sulfate, p-cresol catabolism was retarded. Under SRC 3.4 mol of sulfate was consumed per mol of p-cresol metabolized. The addition of sulfate to methanogenic incubations stimulated p-cresol degradation. Simultaneous adaptation studies in combination with spectrophotometric and chromatographic analysis of metabolites indicated that p-cresol was oxidized under SRC to p-hydroxybenzoate via the corresponding alcohol and aldehyde. This series of reactions was inhibited under sulfate-limited or aerobic conditions. Therefore, the primary catabolic event for p-cresol decomposition under SRC appears to involve the hydroxylation of the aryl methyl group.     

Metabolism of hydroquinone by human myeloperoxidase: mechanisms of stimulation by other phenolic compounds.

Hydroquinone, a metabolite of benzene, is converted by human myeloperoxidase to 1,4-benzoquinone, a highly toxic species. This conversion is stimulated by phenol, another metabolite of benzene. Here we report that peroxidase-dependent hydroquinone metabolism is also stimulated by catechol, resorcinol, o-cresol, m-cresol, p-cresol, guaiacol, histidine, and imidazole. In order to gain insights into the mechanisms of this stimulation, we have compared the kinetics of human myeloperoxidase-dependent phenol, hydroquinone, and catechol metabolism. The specificity (Vmax/Km) of hydroquinone for myeloperoxidase was found to be 5-fold greater than that of catechol and 16-fold greater than that of phenol. These specificities for myeloperoxidase-dependent metabolism inversely correlated with the respective one-electron oxidation potentials of hydroquinone, catechol, and phenol and suggested that phenol- and catechol-induced stimulation of myeloperoxidase-dependent hydroquinone metabolism cannot simply be explained by interaction of hydroquinone with stimulant-derived radicals. Phenol (100 microM), catechol (20 microM), and imidazole (50 mM) did, however, all increase the specificity (Vmax/Km) of hydroquinone for myeloperoxidase, indicating that these three compounds may be stimulating hydroquinone metabolism by a common mechanism. Interestingly, the stimulation of peroxidase-dependent hydroquinone metabolism by other phenolic compounds was pH-dependent, with the stimulating effect being higher under alkaline conditions. These results therefore suggest that the interaction of phenolic compounds, presumably by hydrogen-bonding, with the activity limiting distal amino acid residue(s) or with the ferryl oxygen of peroxidase may be an important contributing factor in the enhanced myeloperoxidase-dependent metabolism of hydroquinone in the presence of other phenolic compounds.     

Substrate-substrate interaction of resorcinol and catechol in upflow anaerobic fixed film – fixed bed reactors in mono and multisubstrate matrices

Biodegradation of resorcinol and catechol was studied in upflow anaerobic fixed film-fixed bed (FFFB) reactors of uniform dimensions in mono and multisubstrate matrices. Cross feeding studies have revealed that phenol was poorly degraded in resorcinol acclimated reactor whereas it was readily degraded in catechol acclimated reactor. Addition of resorcinol along with phenol in a COD ratio 1:3 in resorcinol reactor increased phenol removal efficiency to 95% indicating that resorcinol induces phenol degradation. When both resorcinol and catechol were fed to the resorcinol acclimated reactor, it was observed that resorcinol degradation was inhibited by catechol. Catechol acclimated reactor could degrade phenol readily when added as mono substrate indicating that it may be an intermediate in catechol degradation. In binary mixture studies also catechol reactor could degrade phenol, resorcinol and hydroquinone to 90%. Catechol acclimated reactor exhibits relaxed substrate specificity whereas resorcinol acclimated reactor exhibits rigid substrate specificity for phenol as well as other isomers.     

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for synthesizing 4-methylresorcinol, methylhydroquinone, and pyrogallol

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.