Catabolic degradation of 4-chlorobiphenyl byPseudomonas sp. DJ-12 via consecutive reaction ofmeta-cleavage and hydrolytic dechlorination

Pseudomonas sp. strain DJ-12 is a bacterial isolate capable of degrading 4-chlorobiphenyl (4CBP) as a carbon and energy source. The catabolic degradation of 4CBP by the strain DJ-12 was studied along with the genetic organization of the genes responsible for the crucial steps of the catabolic degradation. The catabolic pathway was characterized as being conducted by consecutive reactions of themeta-cleavage of 4CBP, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, andmeta-cleavage of protocatechuate. ThepcbC gene responsible for themeta-cleavage of 4CBP only showed a 30 to 40% homology in its deduced amino acid sequence compared to those of the corresponding genes from other strains. The amino acid sequence of 4CBA-CoA dechlorinase showed an 86% homology with that ofPseudomonas sp. CBS3, yet only a 50% homology with that ofArthrobacter spp. However, thefcb genes for the hydrolytic dechlorination of 4CBA inPseudomonas sp. DJ-12 showed an uniquely different organization from those of CBS3 and other reported strains. Accordingly, these results indicate that strain DJ-12 can degrade 4CBP completely viameta-cleavage and hydrolytic dechlorination using enzymes that are uniquely different in their amino acid sequences from those of other bacterial strains with the same degradation activities.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600

Summary The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and meta-cleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pV1150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.     

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 M when DO was maintained at 2 ppm whereas with 3 ppm of DO, HMS level was below 0.4 M. 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.     

Genetics and biochemistry of 1,2-dichloroethane degradation

Dichloroethane (1,2-DCE) is a synthetic compound that is not known to be formed naturally. Nevertheless, several pure microbial cultures are able to use it as a sole carbon source for growth. Degradation of 1,2-DCE proceeds via 2-chloroethanol, chloroacetaldehyde and chloroacetate to glycolate. The genes encoding the enzymes responsible for the conversion of 1,2-DCE to glycolic acid have been isolated. The haloalkane dehalogenase and an aldehyde dehydrogenase are plasmid encoded. Two other enzymes, the alcohol dehydrogenase and the haloacid dehalogenase, are chromosomally encoded. Sequence analysis indicates that the haloacid dehalogenase belongs to the L-specific 2-chloroproprionic acid dehalogenases. From the three-dimensional structure and sequence similarities, the haloalkane dehalogenase appears to be a member of the / hydrolase fold hydrolytic enzymes, of which several are involved in the degradation of aromatic and aliphatic xenobiotic compounds.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.     

Cloning, nucleotide sequence and primary biochemical characterization of esterase EstA from Ralstonia eutropha CH34

A genomic library of Ralstonia eutropha CH34 was screened in Escherichia coli S17-1 for esterase activity by using -naphthyl acetate and Fast Blue RR. A 1,711 bp DNA fragment was subcloned from an esterase-positive clone and sequenced. Esterase EstA was encoded by a 825-bp open reading frame and exhibited significant amino acid similarities with the enzymes involved in the meta-cleavage pathway. EstA is composed of 275 amino aicds with a predicted molecular mass of 30785 Da. The optimal pH for EstA was 7.0, and the enzyme retained more than 65% activity when incubated in buffers with pH 3.8–9.2 for 2 h. EstA was active at temperatures up to 80 °C and retained more than 77% activity after exposure to temperatures below 60 °C for 2 h.     

Uptake of volatilen-alkanes byPseudomonas PG-I

Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.     

Degradation of phenanthrene by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. C3: initial 1,2- and 3,4-dioxygenation and <Emphasis Type="Italic">meta</Emphasis>- and <Emphasis Type="Italic">ortho</Emphasis>-cleavage of naphthalene-1,2-diol

Burkholderia sp. C3 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Hilo, Hawaii, USA, and studied for its degradation of phenanthrene as a sole carbon source. The initial 3,4-C dioxygenation was faster than 1,2-C dioxygenation in the first 3-day culture. However, 1-hydroxy-2-naphthoic acid derived from 3,4-C dioxygenation degraded much slower than 2-hydroxy-1-naphthoic acid derived from 1,2-C dioxygenation. Slow degradation of 1-hydroxy-2-naphthoic acid relative to 2-hydroxy-1-naphthoic acid may trigger 1,2-C dioxygenation faster after 3 days of culture. High concentrations of 5,6-␣and 7,8-benzocoumarins indicated that meta-cleavage was the major degradation mechanism of phenanthrene-1,2- and -3,4-diols. Separate cultures with 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid showed that the degradation rate of the former to naphthalene-1,2-diol was much faster than that of the latter. The two upper metabolic pathways of phenanthrene are converged into naphthalene-1,2-diol that is further metabolized to 2-carboxycinnamic acid and 2-hydroxybenzalpyruvic acid by ortho- and meta-cleavages, respectively. Transformation of naphthalene-1,2-diol to 2-carboxycinnamic acid by this strain represents the first observation of ortho-cleavage of two rings-PAH-diols by a Gram-negative species.     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

Isolation and identification of antialgal substances produced byPseudomonas aeruginosa

Pseudomonas aeruginosa strongly inhibited the growth of green microalgae and cyanobacteria by the release of low molecular weight, thermoresistant factors. The antialgal substances were resistant to various enzymes and remained active in agar after a 3-months storage period at 4 °C in the absence of light. The results indicate that inhibition of algal growth was mediated by the phenazine pigments released by the bacterium. Pyocyanine and an unidentified pale blue pigment had no effect on algal growth, whereas 1-hydroxyphenazine and oxychlororaphine showed strong antialgal activity.Groupe de Recherche en Recyclage Biologique et AquicultureDépartement des Sciences et Technologie des Aliments     

Effect of alginate encapsulation and selected disinfectants on survival of and phenanthrene mineralization byPseudomonas sp UG14Lr in creosote-contaminated soil

The survival and phenanthrene-mineralizing ability of free and alginate-encapsulatedPseudomonas sp UG14Lr cells were examined in a creosote-contaminated soil. Alginate encapsulation adversely affected both survival and phenanthrene mineralization. This was postulated to be due to concentration of water-soluble toxic compounds in the alginate beads. Toxicity studies showed that the concentrated water-soluble fraction of the creosote-contaminated soil may be toxic toPseudomonas sp UG14Lr in soil with a low moisture content. Survival of alginate-encapsulated cells improved with increasing soil moisture content. Free cells survived well at a steady population of 10⁸ CFU g^–1 dry soil for 28 days in the creosote-contaminated soil. However, phenanthrene mineralization was not improved compared to the uninoculated control. This was attributed to the existence of indigenous phenanthrene-mineralizing microorganisms already present in this contaminated soil. The effect of calcium hypochlorite and Germiphene on survival of and phenanthrene mineralization by free and alginate-encapsulatedPseudomonas sp UG14Lr cells in creosote-contaminated soil was also studied. Addition of 0.1% (w/w dry soil) calcium hypochlorite reduced the introduced free cells to below detection limits (10 CFU g^–1 dry soil) within 14 days, while Germiphene had no effect on cell numbers. Phenanthrene mineralization by free cells was not adversely affected by treatment with calcium hypochlorite or Germiphene. Survival of alginate-encapsulated cells after treatment with disinfectants was as poor as that without disinfection. The results show that alginate encapsulation may not be a suitable formulation for introduction ofPseudomonas sp UG14Lr into creosote-contaminated soils.     

Degradation of highly chlorinated PCBs byPseudomonas strain LB400

Summary Congeners of polychlorinated biphenyl (PCB) differ in the number and position of chlorine substituents. Although PCBs are degraded, those congoners with five or more chlorines have been considered resistant to bacterial degradation. Metabolism byPseudomonas strain LB400 of PCBs representing a broad spectrum of chlorination patterns and having from two to six chlorines was investigated. Degradation of pure PCB congeners and synthetic congener mixes was measured in resting cell assays with biphenyl- or Luria broth-grown cells. In addition, the appearance of metabolites was followed using HPLC purification, and GC and GC-MS characterization. 2,4,5,2,4,5-[¹⁴C]hexachlorobiphenyl was also used to follow the accumulation of¹⁴C-labeled metabolites. Evidence indicates that LB400 aerobically metabolizes representatives of all major structural classes of PCB's including several congeners which lack adjacent unchlorinated carbon atoms. The mechanisms by which many of these congeners are degraded are not fully understood, but it is apparent that aerobic bacteria can degrade a broader spectrum of PCB congeners than previously believed and that this broad spectrum of degradative competence can exist in a single strain.     

Mineralization of 4-sulfophthalate by aPseudomonas strain isolated from the River Elbe

The bacteriumPseudomonas sp. strain RW31 isolated from the river Elbe utilized the ammonium salt of 4-sulfophthalate (4SPA) as sole source of carbon, sulfur, nitrogen, and energy and grew also with phthalate (PA) and several other aromatic compounds as sole carbon and energy source. The xenobiotic sulfo group of 4SPA was eliminated as sulfite, which transiently accumulated in the culture supernatant up to about 10 µM and was slowly oxidized to the stoichiometrical amount of sulfate. Biodegradation routes of 4SPA as well as of PA converged into the protocatechuate pathway and from found activities for the decarboxylation of 4,5-dihydroxyphthalate we deduce this compound the first rearomaticized intermediate after initial dioxygenation. Protocatechuate then underwentmeta-cleavage mediated by a protocatechuate 4,5-dioxygenase activity which was competitively inhibited by the structurally related compound 3,4,5-trihydroxybenzoate; protocatechuate accumulated in the medium up to an about 2 mM concentration. Indications for the presence of selective transport systems are presented.     

Short communication: Methylparathion degradation by Pseudomonas sp. A3 immobilized in sodium alginate beads

An organophosphate-degrading soil isolate of Pseudomonas sp. A3, immobilized at 5% (wet wt/v) cell mass in 3% (w/v) sodium alginate beads, detoxified 99% of 1 mm methylparathion in 48 h. The beads were re-usable for five batches, the sixth batch only giving 73% methylparathion removal.     

Metabolism of benzalphthalide by Pseudomonas sp.

A Pseudomonas sp. degraded benzalphthalide to o-phthalate and benzoate. A tentative pathway for the metabolism of benzalphthalide in this Pseudomonas sp. is proposed on the basis of isolated metabolites, oxygraphic assay and enzymatic studies.     

Kinetic studies of phenol degradation by Rhodococcus sp. P1 II. Continuous cultivation

The degradation of phenol by Rhodococcus sp. P1 was studied in continuous culture systems. The organism could be adapted by slowly increasing concentration, step by step, up to 30.0 g · 1^-1 phenol in the influent. The degradation rate reached values of about 0.3 g · g dry mass^-1 ·h^-1. Large step increases in phenol concentration and addition of further substrates (e.g., catechol) were tolerated up to a certain concentration. With increasing dilution rate and increasing inlet phenol concentration the stability of the system decreased.     

A novel EDTA-degrading Pseudomonas sp.

A bacterial strain LPM-410 capable of utilizing ethylenediaminetetraacetate (EDTA) as the sole source of energy, carbon, and nitrogen was isolated from sewage sludge and identified as a Pseudomonas sp. on the basis of its phenotypic characteristics. Suspensions of exponential-phase cells degraded EDTA, Mg–, Ca–, Ba–, and Mn–EDTA at constant specific rates ranging from 0.363 to 0.525 mmol EDTA/(g cells h). The more stable chelate, Zn–EDTA, was degraded at a lower rate (0.195 ± 0.030 mmol EDTA/(g cells h)), and here was no degradation of Co–, Cu–, Pb–, and Fe(III)–EDTA.     

Evidence that phenol phosphorylation to phenylphosphate is the first step in anaerobic phenol metabolism in a denitrifying Pseudomonas sp.

Anaerobic phenol degradation has been shown to proceed via carboxylation of phenol to 4-hydroxybenzoate. However, in vitro the carboxylating enzyme was inactive with phenol; only phenylphosphate (phosphoric acid monophenyl ester) was readily carboxylated. We demonstrate in a denitrifying Pseudomonas strain that phenylphosphate is the first detectable product formed from phenol in whole cells and that subsequent phenylphosphate consumption parallels 4-hydroxybenzoate formation. These kinetics are consistent with phosphorylation being the first step in anaerobic phenol degradation. Various cosubstrates failed so far to act as phosphoryl donor for net phosphorylation of phenol in cell extracts. Yet, cells anaerobically grown with phenol contained an enzyme that catalyzed an isotope exchange between [U-¹⁴C]phenol and phenylphosphate. This transphosphorylation activity was anaerobically induced by phenol but was stable under aerobic conditions and required Mn²⁺ and polyethylene glycol. Activity was optimal at pH 5.5 and half-maximal with 0.6 mM Mn²⁺, 0.2 mM phenylphosphate, and 1 mM phenol. It is proposed that the phenol exchange/transphosphorylation reaction is catalyzed as partial reaction by an inducible phenol phosphorylating enzyme. The isotope exchange demands that a phosphorylated enzyme was formed in the course of the reaction, which might be similar to the phosphotransferase system of sugar transport.     

Utilization and cooxidation of chlorinated phenols byPseudomonas sp. B 13

Pseudomonas sp. B13 was grown in continuous culture on 4-chlorophenol as the only carbon source. Maximum growth rate of 0.4h^-1 was observed at a substrate concentration of >0.01 mM and <0.15 mM. In addition to the enzymes of phenol catabolism, high specific 1,2-dioxygenase activities with chlorocatechols as substrates were found. The isomeric monochlorinated phenols were also totally degraded by 4-chlorophenol grown cells. (+)-2,5-Dihydro-4-methyl- and (+)-2,5-dihydro-2-methyl-5-oxo-furan-2-acetic acid were formed in high yield as dead-end catabolites from cooxidation of cresoles.Several dichlorophenols except 2,6-dichlorophenol were removed from the culture fluid by chlorophenol grown cells. Ring cleavage of chlorinated catechols were shown to be one of the critical steps in chlorophenol catabolism. A catabolic pathway for isomeric chlorophenols is discussed.Non-Standard Abbreviations HPLC High performance liquid chromatography - DHB Dihydrodihydroxybenzoate 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid