Phenol Degradation Pathway Research Articles

Enhanced degradation of phenoxyacetic acid in soil by horizontal transfer of the tfdA gene encoding a 2,4-dichlorophenoxyacetic acid dioxygenase

Few studies have investigated the possible impact of in situ gene transfer on the degradation of xenobiotic compounds in natural environments. In this work we showed that horizontal transfer of the tfdA gene, carried on plasmid pRO103, to phenol degrading recipient strains significantly increased the degradation rate of phenoxyacetic acid in sterile and non-sterile soil microcosms. The tfdA gene encodes a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase and by complementation with the phenol degradation pathway an expanded catabolic substrate range, now including phenoxyacetic acid, is evolved. Presence of selective pressure had a positive effect on the emergence of transconjugants. However, even in the absence of phenoxyacetic acid transconjugant populations were detected and were kept at a constant level throughout the experimental period. The residuesphere (interface between decaying plant material and soil matrix) of dry leaves of barley was shown to be a hot-spot for gene transfer and presence of barley straw increased the conjugation frequencies in soil microcosms to the same extent as presence of organic nutrients. The results of this study indicate that dissemination of catabolic plasmids is a possible mechanism of genetic adaptation to degradation of xenobiotic compounds in natural environments, and that complementation of catabolic pathways possibly plays an important role in the evolution of new degradative capabilities. The application of horizontal gene transfer as a possible tool in bioremediation of contaminated sites is discussed.

Role of oxygen in the utilization of phenol by Pseudomonas CF600 in continuous culture

The dissolved oxygen (DO) level is the key factor which decides the rate of degradation of the organic load in aerobic growth conditions. In this study the role of DO levels on the utilization of phenols has been reported using the continuous culture system. A phenol-utilizing strain, Pseudomonas CF600 has been used as a model. Its phenol-degrading capacity was studied using continuous cultivation for a period of 60 days. The bioreactor was kept at a dilution rate of 0.006 h−1 with DO levels maintained at 2, 3, and 4 ppm keeping all the other cultivation conditions constant. Physiological variations under the cultivation conditions were studied by monitoring off-line phenol utilization and respirometric analysis of harvested culture against different substrates. It was observed that the accumulation of 2-hydroxymuconate semialdehyde (HMS), an intermediate in the phenol degradation pathway, depends on the DO level. The maximum level of HMS in the medium observed was 3.92 μM when DO was maintained at 2 ppm whereas with 3 ppm of DO, HMS level was below 0.4 μM. Oxygen uptake data of the cells harvested from cultures grown at different DO levels showed that the uptake was highest at 3 ppm DO for all the substrates tried. When phenol was used as substrate, the oxygen uptake rate was 42.66, 66.36 and 35.55 nM/min/mg dry weight of cells at 4, 3 and 2 ppm DO respectively. Results show that DO levels influence the rate of phenol utilization in Pseudomonas CF600.

Generation of novel bacterial regulatory proteins that detect priority pollutant phenols.

The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. We have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.

Degradation of phenol under meso- and thermophilic, anaerobic conditions

Based on the results of preliminary studies on phenol degradation under mesophilic conditions with a mixed methanogenic culture, we proposed a degradation pathway in which phenol is fermented to acetate: Part of the phenol is reductively transformed to benzoate while the rest is oxidised, forming acetate as end product. According to our calculations, this should result in three moles of phenol being converted to two moles of benzoate and three moles of acetate (3phenol+2CO2+3H2O→3acetate+2benzoate): To assess the validity of our hypothesis concerning the metabolic pathway, we studied the transformation of phenol under mesophilic and thermophilic conditions in relation to the availability of hydrogen. Hence, methanogenic meso- and thermophilic cultures amended with phenol were run with or without an added over-pressure of hydrogen under methanogenic and non-methanogenic conditions. Bromoethanesulfonic acid (BES) was used to inhibit methanogenic activity. In the mesophilic treatments amended with only BES, about 70% of the carbon in the products found was benzoate. During the course of phenol transformation in these BES-amended cultures, the formation pattern of the degradation products changed: Initially nearly 90% of the carbon from phenol degradation was recovered as benzoate, whereas later in the incubation, in addition to benzoate formation, the aromatic nucleus degraded completely to acetate. Thus, the initial reduction of phenol to benzoate resulted in a lowering of H2levels, giving rise to conditions allowing the degradation of phenol to acetate as the end product. Product formation in bottles amended with BES and phenol occurred in accordance with the hypothesised pathway; however, the overall results indicate that the degradation of phenol in this system is more complex.During phenol transformation under thermophilic conditions, no benzoate was observed and no phenol was transformed in the BES-amended cultures. This suggests that the sensitivity of phenol transformation to an elevated partial pressure of H2is higher under thermophilic conditions than under mesophilic ones. The lack of benzoate formation could have been due to a high turnover of benzoate or to a difference in the phenol degradation pathway between the thermophilic and mesophilic cultures.

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.

Cloning and sequencing of a gene cluster for the Meta-cleavage pathway of aniline degradation in Acinetobacter sp. strain YAA

A 17.0-kb SacI fragment, which contains a large region just downstream of the aniline dioxygenase genes ( atdA1–A5) of Acinetobacter sp. strain YAA, was cloned from YAA plasmid DNA. We determined the nucleotide sequence of a 10.5-kb segment within the fragment, in which eleven genes were found. Eight of the genes, designated atdSBCDEFGH, were homologous to the meta-cleavage pathway genes dmpQBCDEFGH and xylTEGFJQKI the products of which are involved in the phenol and toluene degradation pathways, respectively. The G+C content of the atdSBCDEFGH genes ranges from 51.1% to 59.5% and is unusual for Acinetobacter genes (38–47%), indicating that the genes originated from other bacteria. In addition, these values are quite different from those of the upstream atdA1–A5 genes (40.1–47.7%). This result suggests that this aniline degradation gene cluster is a hybrid structure.

Carbon and energy flux constraints in continuous cultures of Alcaligenes eutrophus grown on phenol.

The growth behaviour of Alcaligenes eutrophus on phenol was investigated in continuous cultures to identify the phenomena limiting both growth efficiency and substrate degradation rates. It was shown that the fixed stoichiometry of the meta pathway of phenol degradation, leading to equimolar quantities of pyruvate and acetate, and the structure of the central pathways, which do not allow gluconeogenesis of acetate during growth on phenol, provoke the accumulation of polyhydroxybutyrate (PHB) under certain growth conditions. Acetate is predominantly used as an energy source and PHB accumulates when the cells are carbon-limited rather than energy-limited. The maximum rates of phenol degradation can be attributed to the expression of the enzymes of the catabolic pathway. This is particularly true of phenol hydroxylase and 2-hydroxymuconate semialdehyde (2-hms) dehydrogenase, whose substrates accumulated to physiologically significant concentrations at high growth rates. Indeed the concentration of 2-hms that accumulated in the medium indicated that this enzyme was substrate-saturated at maximum growth rates. However, the specific activity profiles of other catabolic enzymes associated with phenol degradation were close to the estimated flux through the pathway. This suggests a complex control structure in which several enzymes contribute to the control of pathway flux, as would be expected in a catabolic pathway.

Biological, thermal and photochemical transformation of 2-trifluoromethylphenol

This is the first report on the metabolism of trifluoromethyl aromatics in a thermophilic bacterium, Bacillus thermoleovorans A2. Enzymes of the phenol degradation pathway are induced when cultivating Bacillus thermoleovorans A2 on complex medium. Direct measurements of fluorinated xenobiotics in cell suspensions using 19F-NMR made it possible to follow quantitatively the biotransformation routes. During the biotransformation of 2-CF3-phenol by B. thermoleovorans A2, a fluorinated metabolite, 2-hydroxy-6-oxo-7,7,7,-trifluorohepta-2,4-dienoate (7-TFHOD), accumulated. This metabolite is transformed non-enzymatically when exposed to sunlight. The accumulation of 7-TFHOD as an intermediate in the 2-CF3-phenol pathway was rationalized by calculating molecular properties of a series of meta-cleavage products.

Catalytic wet oxidation of phenol over a Pt/TiO catalyst

Wet oxidation of phenol by air or oxygen over a Pt/TiO 2 catalyst is studied in a batch reactor in the temperature range 150–200°C, pressure range 34–82 atm, and a catalyst loading range of 0–4 g catalyst L −1. The catalyst was powdered 4.45% Pt/TiO 2 with a maximum particle size of 105 μm. Results show complete oxidation of phenol and almost complete total organic carbon (TOC) removal. Small amounts of stable organic acids are formed in side reactions of the phenol degradation pathway and are not readily degraded. Experimental results show that the reaction rate decreases by increased oxygen concentration. Theoretical rate expressions are derived, based on postulated oxidation and TOC reduction mechanisms.

Localization and organization of phenol degradation genes of Pseudomonas putida strain H.

The genetic organization of the DNA region encoding the phenol degradation pathway of Pseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via the meta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase and meta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.

Cloning and sequences of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putida P35X

Pseudomonas putida P35X (NCIB 9869) metabolises phenol and cresols via a chromosomally encoded meta-cleavage pathway. A 13.4-kb fragment of the chromosome involved in encoding phenol catabolism was cloned and characterized. Deletion analysis and nucleotide sequencing of a 6589-bp region, in conjunction with enzyme assays, were used to identify the phhKLMNOP genes encoding the phenol hydroxylase, the phhB gene encoding catechol 2,3-dioxygenase (EC 1.13.11.2) and the phhQ gene that encodes a small ferredoxin-like protein. The genes are organised in an operon-like structure, in the order phhKLMNOPQB, and the deduced amino-acid sequences share high homology (68.3–99.7%) with those of the plasmid-encoded genes dmpKLMNOPQB of Pseudomonas sp. strain CF600. Genetic evidence is presented that the difference in the growth substrate ranges of Pseudomonas P35X and CF600 are due to the effector activation specificities of the regulators of these systems, rather than the substrate specificities of the catabolic enzymes.

Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85

In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.

Kinetics of growth and catechol production byBacillus stearothermophilus BR321

A continuous bioprocess for the specialty chemical catechol has been developed usingBacillus stearothermophilus BR321. Themeta pathway for phenol degradation has been blocked at the catechol 2,3-dioxygenase enzyme using transposon mutagenesis. As a result, the organism grows on a dilute growth medium supplemented with phenol and accumulates catechol. In batch culture, catechol concentrations up to 1 mM were obtained. A kinetic model has been developed to describe batch growth for different initial phenol concentrations. In continuous culture conducted at a diluation rate of 0.12 hr−1, steady states were obtained for feed phenol concentrations up to 0.24 mM, resulting in catechol concentrations up to 0.08 mM. Above this phenol concentration, oscillatory dynamics were observed.

Fluorophenols and 3-fluorobenzoate in phenol-degrading methanogenic cultures

3-Fluorobenzoate and all three isomers of fluorophenol were used as analogues and inhibitors of phenol degradation in a methanogenic consortium. 3-Fluorobenzoate was not transformed by phenol-degrading cultures, but it facilitated the detection of the formation of 4-hydroxybenzoate and benzoate from phenol. The effects of the fluorophenols depended on their concentration in the cultures. When added at 0.90 mM, all fluorophenols prevented phenol transformation. At concentrations of 0.45 to 1.8 mM, 2-fluorophenol was transformed to 3-fluoro-4-hydroxybenzoate which accumulated in the medium. When both 2-fluorophenol and phenol were added to cultures at concentrations of 1 mM each, 3-fluoro-4-hydroxybenzoate, 4-hydroxybenzoate, 3-fluorobenzoate and benzoate were detected. 4-Fluorophenol was never transformed, and when it was present at ≦0.22 mM, it had no effect on phenol degradation. At concentrations ≦0.09 mM, 2-fluorophenol was mineralized by the phenol-degrading cultures to methane, carbon dioxide, and fluoride. The release of fluoride was also observed from 3-fluorophenol when it was initially present at ≦0.09 mM. These results support the proposed pathway for phenol degradation involving an initial para-carboxylation to 4-hydroxybenzoate followed by dehydroxylation to benzoate and further metabolism to carbon dioxide and methane. They also demonstrate defluorination of 2- and 3-fluorophenols under methanogenic conditions.

Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600

The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.

Molecular cloning and mapping of phenol degradation genes from Bacillus stearothermophilus FDTP-3 and their expression in Escherichia coli.

Two genes of the meta pathway of phenol degradation were cloned from a phenol-utilizing strain of Bacillus stearothermophilus and were mapped by subcloning and by use of a Tn5 insertion mutation. They code for phenol hydroxylase and catechol 2,3-dioxygenase, respectively. The gene encoding catechol 2,3-dioxygenase, which is more thermostable than catechol 2,3-dioxygenase encoded by the other gene, shares rather limited homology with that from Pseudomonas putida.

Sequence of the plasmid-encoded catechol 1,2-dioxygenase-expressing gene, pheB, of phenol-degrading Pseudomonas sp. strain EST1001

Phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12O), the two first enzymes of the phenol-degradation pathways, are encoded by a 3.4-kb DNA fragment cloned from Pseudomonas sp. EST1001 plasmid DNA. We have previously shown that activation of the cloned genes in Pseudomonas putida PaW85 is controlled by insertion of the 17-kb transposon, Tn4652, from the host chromosome into the plasmid carrying these genes [Kivisaar et al. Plasmid 24 (1990) 25–36]. Transcription of the DNA encoding PMO ( pheA) and C12O ( pheB) is activated by a promoter located on a 0.2-kb SacI- ClaI fragment from Tn4652. We have determined the nucleotide sequence of pheB. The 906-bp gene encodes a protein product with a deduced M r of 33362. The relationship between the pheB gene and other C12O-encoding genes has been shown: comparison of the pheB sequence with sequences of catA of Alcaligenes calcoaceticus, tfdC of A. eutrophus and clcA of P. putida demonstrated that there are conserved residues in all the four protein products of these genes.

Methanogenic Degradation of Phenol and Benzoate in Acclimated Sludges

Anaerobic phenol and benzoate degrading consortia were cultivated by acclimation of methanogenic sludges to be capable of degrading completely to CO2 and CH4 1,000 mg/l of phenol within 5–7 days, and 3,000 mg/l of benzoate within 5–7 days, respectively. By using the acclimated sludges, the effect of gaseous atmospheres (H2:CO2/80:20 and N2:CO2/80:20) on the biodegradability and the degradation pathways of phenol and benzoate were examined. Although the anaerobic degradation of phenol was accelerated in the H2/CO2 atmosphere compared with the N2/CO2 atmosphere, benzoate was accumulated. Degradations of benzoate and butyrate were inhibited in the H2/CO2 atmosphere under stirred conditions, but not under static conditions. Through a series of biodegradation tests by using several intermediates in phenol degradation reported previously, the anaerobic degradation pathway of phenol in the N2/CO2 atmosphere was suggested to be phenol → benzoate → cyclohexane carboxylate (or 1-cyclohexene carboxylate) → fatty acids → CO2,CH4.

Degradation of phenolic compounds by the yeast Candida tropicalis HP 15. II. Some properties of the first two enzymes of the degradation pathway.

The first two enzymes of the phenol degradation pathway were determined and characterized in crude extracts from Candida tropicalis HP 15. The phenol hydroxylase (EC 1.14.13.7) was a stable NADPH2- and FAD-dependent enzyme with a pH-optimum of 7.6 to 8.0 and a broad substrate specificity. Influence of ultrasound rapidly reduced the enzyme activity. The catechol 1,2-oxygenase (EC 1.13.1.1) had a broad pH-optimum between 7.5 and 9.6 and a limited substrate specificity. Two active protein bands indicating the presence of two isofunctional enzymes were detectable after electrophoretic separation of crude and partially purified extracts on polyacrylamide gels and specific staining for catechol 1,2-oxygenase activity.