Water and soil contamination by chlorinated and recalcitrant compounds has recently become of increasing concern. The relatively new concept of bioremediation provides a potentially cheap alternative to traditional disposal techniques in addition to representing a genuine removal of contaminants by microbial degradation as compared with the relocation of contaminants in such processes as landfilling. The efficient application of bioremediation projects remains to understand the behaviours of microorganisms involved in the degradation or removal of the target pollutants at the remediation sites. A pilot-scale bioreactor was designed to improve oxadiazon (ODZ) biotransformation efficiency by a soil isolated Pseudomonas fluorescens strain CG5. The reactor was installed in a ornamental plant nursery (OPN), and the modeling of the process was considered.

Molecular techniques and modelling are presented as powerful tools required in the performance of efficient soil and water bioremediation systems. An Escherichia coli CC118-D strain was constructed by inserting the Klebsiella pneumoniae hpa B gene, coding for the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase, into its chromosome. When the constructed strain was immobilized, both enzyme stability and viability increased along the studied period, in absence of antibiotic. We proposed this strategy as an approach to overcoming plasmid instability and to enhance enzyme activity and stability, avoiding antibiotic utilization. A model was developed to understand and predict the behaviour of bacteria and pollutants in a bioreactor system, considering: fluid dynamics, molecular/cellular scale processes and biofilm formation.

Based on 3,4-dihydroxyphenylacetate (3,4-DHPA) dioxygenase amino acid sequence and DNA sequence data for homologous genes, two different oligonucleotides were designed. These were assayed to detect 3,4-DHPA related aromatic compound—degrading bacteria in soil samples by using the FISH method. Also, amplification by PCR using a set of ERIC primers was assayed for the detection ofPseudomonasGCH1 strain, which used in the soil bioremediation process. A model was developed to understand and predict the behavior of bacteria and pollutants in a bioremediation system, taking into account fluid dynamics, molecular/cellular scale processes, and biofilm formation.

A set of ERIC primers was assayed for the detection of some aromatic compound-degrading bacteria in environmental samples. Pseudomonas GCH1 strain was used in contaminated soil bioremediation, and amplification by PCR using the DNA isolated from soil gave a specific DNA band pattern corresponding to the GCH1 strain. Based on 3,4-dihydroxyphenylacetate (3,4-DHPA) dioxygenase amino acid sequence and DNA sequence data of homologous genes, two different oligonucleotides were designed which have been assayed for detecting 3,4-DHPA related aromatic-compounds degrading bacteria in soil samples. The results indicated that these molecular techniques were highly specific and may represent a powerful tool for efficient remediation of xenobiotics pollutants by natural microbial communities.

Columns, pilot-scale and industrial bioreactors have been used for the decontamination and recycling of watering effluents from a farm nursery. Chlorinated herbicide removal rates of 90-98% were reached. The immobilized cell system has been applied to remediate agricultural soils with a history of propachlor contamination. The bioremediation system significantly increased the herbicide removal from contaminated soils.

A bacterial strain capable of growing on propachlor (2-chloro-N-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genus Pseudomonas. Washed-cell suspensions of strain GCH1 accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 +/- 0.15 micromol h(-1). Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 +/- 0.02 mg of protein micromol(-1). GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.

Propachlor (2-chloro-N-isopropylacetanilide) is an acetamide herbicide used in preemergence. In this study, we isolated and characterized a soil bacterium, Acinetobacter strain BEM2, that was able to utilize this herbicide as the sole and limiting carbon source. Identification of the intermediates of propachlor degradation by this strain and characterization of new metabolites in the degradation of propachlor by a previously reported strain of Pseudomonas (PEM1) support two different propachlor degradation pathways. Washed-cell suspensions of strain PEM1 with propachlor accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain PEM1 grew on propachlor with a generation time of 3.4 h and a Ks of 0.17 +/- 0.04 mM. Acinetobacter strain BEM2 grew on propachlor with a generation time of 3.1 h and a Ks of 0.3 +/- 0.07 mM. Incubations with strain BEM2 resulted in accumulation of N-isopropylacetanilide, N-isopropylaniline, isopropylamine, and catechol. Both degradative pathways were inducible, and the principal product of the carbon atoms in the propachlor ring was carbon dioxide. These results and biodegradation experiments with the identified metabolites indicate that metabolism of propachlor by Pseudomonas sp. strain PEM1 proceeds through a different pathway from metabolism by Acinetobacter sp. strain BEM2.

Establishing if immobilized cells have greater tolerance to aromatic pollutant chemicals than cells in suspension, is an important target in modelling and designing cell bioreactors. In this paper 4-hydroxyphenylacetic acid mineralization; the specific 4-hydroxyphenylacetic acid hydroxylase activity; and the viability in freely suspended and immobilized monocultures of two different strains, Escherichia coli W21 (pGA260) and Klebsiella pneumoniae are compared. The immobilized cells, growing on a ceramic support, exhibit viability and catalytic activity higher than suspended cells. By using a kinetic model for describing CO2 formation, it is demonstrated that immobilized K. pneumoniae cells are biocatalytically more active than the other systems studied.

Simazine is one of the most heavily used herbicides for weed control in the production of a variety of agricultural crops. Few microorganisms have been isolated that metabolize S-triazines such as simazine at rates that are suitable for environmental remediation.DSZ1 strain cells were immobilized by adsorption onto ceramic supports. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between immobilized and suspended cells. The effect of substrate concentration and inoculum size/support ratio on kinetic parameters was investigated. Physiological status of immobilized cells was assessed by measuring their capacity for degrading the herbicide and this capacity was compared to that of free-living cells under different experimental conditions. (C) 1998 Elsevier Science Ltd. All rights reserved.

The soil isolated Pseudomonas strain PEM1 has the ability to degrade propachlor (2-Cl-N-isopropylacetanilide) and alachlor (2-Cl-N-(2,6-diethylphenyl)N-methoxymethyl-acetamida). These cells were immobilized by adsorption onto ceramic support. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between immobilized and suspended Pseudomonas PEM1. The effect of inoculum/support ratio on kinetic parameters was investigated. Implications of these results for developing an industrial process with immobilized cells are discussed.

Gentisate 1,2-dioxygenase (E.C.I.14.13) was purified to homogeneity from Klebsiella pneumoniae M5a1, a soil bacterium able to degrade a great variety of aromatic compounds. The molecular mass of the purified holoenzyme was 159 kDa and its structure was deduced to be a tetramer with 38 kDa per subunit. Gentisate 1,2-dioxygenase appears to contain Fe2+ in its active site. The optimum temperature for enzyme activity was estimated to be 30 °C, the optimum pH values varied between 8 and 9 and the isoelectric point was 4.7. Gentisate dioxygenase exhibited typical saturation kinetics and had an apparent Km of 52 μM for gentisate. Its amino acid content was determined to be very similar to that of the enzyme from Pseudomonas acidovorans.

Soil from a herbicide disposal site was used to enrich for microorganisms that degraded 2-Chloro-N-isopropylacetanilide (propachlor). A bacterium, PEM1, able to mineralize the herbicide was isolated. A product of the microbial metabolism of propachlor was identified as N-isopropylacetanilide, suggesting that the first step in the mineralization process is dehalogenation. Strain PEM1 could grow on N-substituted acylanilides, but was incapable of growth on aniline and phenol. Rates of degradation of propachlor were monitored in low-organic-matter soil at field capacity, amended with 0·1–100 μg of herbicide per g of soil and inoculated with two different amounts of strain PEM1. Rates of degradation were comparable, and removal of propachlor close to 90% by 8 days.

The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin.

We isolated 3-hydroxybenzoate-6-hydroxylase (E.C.1.14.13.), an inducible enzyme that catalyzed the para-hydroxylation of 3-hydroxybenzoate (3-HBA) to 2,5-dihydroxybenzoate, from Klebsiella pneumoniae. Although the enzyme was found to be mainly induced by its substrate, a coordinated induction of 3-hydroxybenzoate hydroxylase and gentisate dioxygenase was also observed in the presence of the product of the reaction. The purified enzyme was a monomer with a molecular mass of 42,000. It contained FAD as a prosthetic group, utilized NADH or NADPH with similar efficiencies and its activity was inhibited by Cu2+, Fe2+ and Hg2+. Other properties, such as induction mechanism and kinetic parameters were also studied. Moreover, for the first time the amino acid composition of a 3-hydroxybenzoate-6-hydroxylase was determined.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase, an extradiol-ring-cleavage dioxygenase, has been purified from Klebsiella pneumoniae to homogeneity. The enzyme has an M(r) of 102,000 in its tetrameric form with an M(r) of 25,500 for each subunit. Unlike most other dioxygenases, the enzyme reported here contains Mg2+, as determined by atomic-absorption spectrophotometry and plasma emission metal analysis. The enzyme was shown to contain approx. 1 g-atom of Mg2+/mol of protein and we suggest an alpha 4 Mg2+ quaternary structure. This is the first report of a dioxygenase containing Mg2+ in its structure.

Klebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via metacleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrate specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.

Klebsiella pneumoniae M5a1 grows readily on two compounds, 4-hydroxyphenylacetate and 4-aminobutyrate, whose catabolism produces succinic semialdehyde. A single succinic semialdehyde dehydrogenase was detected, native molecular weight 52000, that has NAD as the preferred cofactor and is induced by succinic semialdehyde functions in the oxidation of succinic semialdehyde during growth on both 4-hydroxyphenyl-acetate and 4-aminobutyrate. This contrasts with the situation for Escherichia coli and Pseudomonas putida where two distinct forms of succinic semialdehyde dehydrogenase have been observed.

A cloned gene specifying the 2-oxo-hept-3-ene-1,7-dioate (OHED) hydratase of Escherichia coli C was used to produce large amounts of the hydratase enzyme. The enzyme was purified to homogeneity by a simple two-step procedure and some of its properties investigated. The first 34 residues at the amino terminus were sequenced and a mixture of oligonucleotides corresponding to the first six amino acid residues was constructed. This mixture was used as a probe to look for sequence homology in DNA obtained from various related organisms that had OHED hydratase activity. Strong hybridization was seen for E. coli strains B and C but E. coli strain W and Klebsiella pneumoniae M5a1 showed no detectable hybridization.

The partition of chick-embryo and young-chick erythrocytes in dextran-poly-(ethylene glycol) two-phase systems depends on the interfacial tension and electrical potential differences between the phases. Counter-current distribution with charged 5% dextran-poly(ethylene glycol) systems has proved to be an adequate method for the separation of primitive and definitive erythrocytes present in chick embryos when a phase settling time of 20 min is used. The computer-aided numerical resolution of experimental curves has shown the existence of subpopulations which could not have been detected by using conventional methods.

The presence of two red cell populations in young chicks has been demonstrated after increasing the settling time used during counter-current distribution with charged 5% Dextran—4% poly(ethylene glycol) two-phase systems. A Fortran program using statistical methods was applied to show the resolution in two peaks (with two or three subpopulations assumed) of the counter-current distribution curves.