Degradation of nitriles and amides by the immobilized cells of Pseudomonas putida

Pseudomonas putida, capable of utilizing acetonitrile as a sole source of C and N, was immobilized in calcium alginate and the rates of degradation of nitriles, including acetonitrile, and their respective amides were studied. All the organic nitriles and amides tested were converted into NH₃ and CO₂.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

Degradation of 2-chloroethanol by free and immobilized Pseudomonas putida US 2

The degradation of 2-chloroethanol by Pseudomonas putida US 2 was investigated in shaking flasks, air-bubble columns and packed-bed fermenters by free cells, calcium-alginate-entrapped cells and on cells on granular clay adsorbed. Entrapped cells tolerated increasing concentrations of 2-chloroethanol better than free cells. Their maximum degradative activity could be observed at 34°C and pH 7.0. The degradation of 2-chloroethanol leads to a decrease of pH and to a stagnation of mineralization, particularly with free or entrapped cells. Following the stabilization of pH, supplementation with succinate resulted in a complete degradation of higher 2-chloroethanol concentrations. Less 2-chloroethanol was degraded in air-bubble columns and larger amounts in packed-bed fermenters. 2-Chloroethanol was mineralized faster by free or entrapped P. putida US 2 than by adsorbed cells, which, on the other hand, were able to remove higher concentrations of the compound. The results with P. putida US 2 are a good indication that this microorganism could be used in waste-water treatment and soil-decontamination systems.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Degradation of Acetonitrile by Pseudomonas putida

A bacterium capable of utilizing high concentrations of acetonitrile as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas putida. This bacterium could also utilize butyronitrile, glutaronitrile, isobutyronitrile, methacrylonitrile, propionitrile, succinonitrile, valeronitrile, and some of their corresponding amides, such as acetamide, butyramide, isobutyramide, methacrylamide, propionamide, and succinamide as growth substrates. Acetonitrile-grown cells oxidized acetonitrile with a K_m of 40.61 mM. Mass balance studies with [¹⁴C]acetonitrile indicated that nearly 66% of carbon of acetonitrile was released as ¹⁴CO₂ and 14% was associated with the biomass. Metabolites of acetonitrile in the culture medium were acetic acid and ammonia. The acetate formed in the early stages of growth completely disappeared in the later stages. Cell extracts of acetonitrile-grown cells contained activities corresponding to nitrile hydratase and amidase, which mediate the breakdown of actonitrile into acetic acid and ammonia. Both enzymes were intracellular and inducible and hydrolyzed a wide range of substrates. The specific activity of amidase was at least 150-fold higher than the activity of the enzyme nitrile hydratase.     

Degradation of dibenzothiophene by Pseudomonas putida

The microbial degradation of dibenzothiophene (DBT) and other organosulphur compounds such as thiophene-2-carboxylate (T2C) is of interest for the potential desulphurization of coal. The feasibility of degradation of DBT and T2C by Pseudomonas putida and other bacteria was analysed. Pseudomonas putida oxidized sulphur from DBT in the presence of yeast extract, but it did not when DBT was the sole source of carbon.     

Degradation of 1,4-naphthoquinones by Pseudomonas putida

Pseudomonas putida J1 and J2, enriched from soil with juglone, are capable of a total degradation of 1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, and 2-chloro-1,4-naphthoquinone. Naphthazerin and plumbagin are only converted into the hydroxyderivatives 2-hydroxynaphthazerin and 3-hydroxyplumbagin, respectively, whereas 2-amino-1,4-naphthoquinone is not attacked at all. The degradation of 1,4-naphthoquinone begins with a hydroxylation of the quinoid ring, yielding 2-hydroxy-1,4-naphthoquinone (lawsone). Lawsone is reduced to 1,2,4-trihydroxynaphthalene with consumption of NADH. The fission product of the quinol could not be detected by direct means because of its instability. However, the presence of 2-chromonecarboxylic acid, a secondary product of lawsone degradation, leads to the conclusion, that the cleavage of the quinol takes place in the meta-position. The resulting ring fission product is converted into salicylic acid by removal of the side chain, presumably as pyruvate. Further degradation of salicyclic acid leads to the formation of catechol, which is then cleaved in the ortho-position and then metabolized via the 3-oxoadipate pathway. The initial steps in the degradation of 2-chloro-1,4-naphthoquinone, namely, the hydroxylation of the quinone to 2-chloro-3-hydroxy-1,4-naphthoquinone, followed by the elimination of the chlorine substituent lead to lawsone, which is further degraded through the pathway described. The degradation steps could be verified by the accumulation products of mutant strains blocked in different steps of lawsone metabolism. Generation of mutants was carried out by chemical and by transposon mutagenesis. The regulation of the first steps of the pathway catalysed by juglone hydroxylase and lawsone reductase, was investigated by induction experiments.     

Degradation of aromatic compounds by Pseudomonas putida

The influence of different process kinetics on the course of phenol degradation has been studied as well as the influence of axial dispersion in the liquid phase on the reactor height with relatively large biofilm thickness in a conventional fluidized bed and air-lift bioreactor. The object of this was to achieve a high conversion of substrate in a device of real size in real process time. For calculating the mathematical model, the method of orthogonal collocation with the STIFF integration routine has been used.     

Degradation of 2-methylnaphthalene by free and immobilized cells of Pseudomonas sp. strain NGK1

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of utilizing 2-methylnaphthalene (2-MN) was immobilized in various matrices namely, polyurethane foam (PUF), alginate, agar and polyvinyl alcohol (PVA) (1.5 × 10¹² c.f.u. g^–1 beads). The degradation rates of 25 and 50 mM 2-MN by freely suspended cells (2 × 10¹¹ c.f.u. ml^–1) and immobilized cells in batches, semi-continuous with shaken culture and continuous degradation in a packed-bed reactor were compared. The PUF-immobilized cells achieved higher degradation of 25 and 50 mM of 2-MN than freely suspended cells and the cells immobilized in alginate, agar or PVA. The PVA- and PUF-immobilized cells could be reused for more than 30 and 20 cycles respectively, without losing any degradation capacity. The effect of dilution rates on the rate of degradation of 25 and 50 mM 2-MN with freely suspended and immobilized cells were compared in the continuous system. Increase in dilution rate increased the degradation rate only up to 1 h^–1 in free cells with 25 mM 2-MN and no significant increase was observed with 50 mM 2-MN. With immobilized cells, the degradation rate increased with increase in dilution rate up to 1.5 h^–1 for 25 mM and 1 h^–1 for 50 mM 2-MN. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for biodegradation of 2-MN.     

Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028

Polyacrylamide-gel-immobilized cells of Pseudomonas strain EPS 5028 were effective in the removal of uranium (U) from synthetic effluents. Metal accumulation was performed in an open system in columns filled with immobilized cells that were challenged with continuous flows containing U. Possible variables of the system were studied. Uranium uptake by the immobilized cells of this microorganism was affected by pH but not by temperature or flow rate. In addition, U binding could be interpreted in terms of the Freundlich adsorption isotherm indicating single-layer adsorption. The feasibility of reusing the immobilized cells was suggested after the recovery of U with a solution of 0.1 m sodium carbonate. Correspondence to: M. C. Fusté     

Degradation of methoxylated aromatic acids by Pseudomonas putida

J.E. TURNER AND N. ALLISON. 1995. A newly-isolated strain of Pseudomonas putida (HVA-1) utilized homovanillic acid as sole carbon and energy source. Homovanillate-grown bacteria oxidized homovanillate and homoprotocatechuate but monohydroxylated and other methoxylated phenylacetic acids were oxidized poorly; methoxy-substituted benzoates were not oxidized. Extracts of homovanillate-grown cells contained homoprotocatechuate 2,3-dioxygenase but the primary homovanillate-degrading enzyme could not be detected. No other methoxylated phenylacetic acid supported growth of the organism but vanillate was utilized as a carbon and energy source. When homovanillate-grown cells were used to inoculate media containing vanillate a 26 h lag period occurred before growth commenced. Vanillate-grown bacteria oxidized vanillate and protocatechuate but no significant oxygen uptake was obtained with homovanillate and other phenylacetic acid derivatives. Analysis of pathway intermediates revealed that homovanillate-grown bacteria produced homoprotocatechuate, formaldehyde and the ring-cleavage product 5-carboxymethyl 2-hydroxymuconic semialdehyde (CHMS) when incubated with homovanillate but monohydroxylated or monomethoxylated phenylacetic acids were not detected. These results suggest that homovanillate is degraded directly to the ring-cleavage substrate homoprotocatechuate by an unstable but highly specific demethylase and then undergoes extradiol cleavage to CHMS. It would also appear that the uptake/degradatory pathways for homovanillate and vanillate in this organism are entirely separate and independently controlled. If stabilization of the homovanillate demethylase can be achieved, there is potential for exploiting the substrate specificity of this enzyme in both medical diagnosis and in the paper industry.     

Degradation of lawsone by Pseudomonas putida L2

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.     

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.     

Degradation of Ethylbenzene by Pseudomonas putida Harboring OCT Plasmid

We have investigated the utilization of a variety of alkylbenzenes by P. putida strains and found that a strain harboring the OCT plasmid assimilated ethylbenzene. The linkage between the determinant for the degradation of ethylbenzene (Etb⁺ phenotype) and the OCT plasmid was inferred from conjugation experiments. The growth characteristics of the strains carrying mutations in the alk genes of the OCT plasmid which determine the assimilation of /t-alkanes indicated that alkB, alkA, and alkR should be responsible for the degradation of ethylbenzene. The exposure of ethylbenzene to the P. putida strain harboring the CAM-OCT plasmid resulted in the accumulation of β-phenylethyl alcohol. A possible degradation pathway for ethylbenzene including the terminal oxidation of the alkyl side chain was proposed.     

Degradation of naphthalene by cells of Pseudomonas sp. strain NGK 1 immobilized in alginate, agar and polyacrylamide

A Pseudomonas sp. strain NGK 1 (NCIM 5120) was immobilized in various matrices, namely, alginate, agar (1.8 × 10¹¹ cfu g⁻¹ beads) and polyacrylamide (1.6 × 10¹¹ cfu g⁻¹ beads). The degradation of naphthalene was studied, by freely suspended cells (4 × 10¹⁰ cfu ml⁻¹) and immobilized cells in batches, with shaken culture and continuous degradation in a packed-bed reactor. Free cells brought about the complete degradation of 25 mmol naphthalene after 3 days of incubation, whereas, a maximum of 30 mmol naphthalene was degraded by the bacteria after 3–4 days of incubation with 50 mmol and 75 mmol naphthalene, and no further degradation was observed even after 15 days of incubation. Alginate-entrapped cells had degraded 25 mmol naphthalene after 3.5 days of incubation, whereas agar- and polyacrylamide-entrapped cells took 2.5 days; 50 mmol naphthalene was completely degraded by the immobilized cells after 6–7 days of incubation. Maximum amounts of 55 mmol, 70 mmol and 67 mmol naphthalene were degraded, from an initial 75 mmol naphthalene, by the alginate-, agar- and polyacrylamide-entrapped cells after 15 days of incubation. When the cell concentrations were doubled, 25 mmol and 50 mmol naphthalene were degraded after 2 and 5.5 days of incubation by the immobilized cells. Complete degradation of 75 mmol naphthalene occurred after 10 days incubation with agar- and polyacrylamide-entrapped␣cells, whereas only 60 mmol naphthalene was degraded by alginate-entrapped cells after 15 days of␣incubation. Further, with 25 mmol naphthalene, alginate-, agar- and polyacrylamide-entrapped cells (1.8 × 10¹¹ cfu g⁻¹ beads) could be reused 18, 12 and 23 times respectively. During continuous degradation in a packed-bed reactor, 80 mmol naphthalene 100 ml⁻¹ h⁻¹ was degraded by alginate- and polyacrylamide-entrapped cells whereas 80 mmol naphthalene 125 ml⁻¹␣h⁻¹ was degraded by agar-entrapped cells. Received: 21 October 1997 / Received revision: 15 January 1998 / Accepted: 18 January 1998     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.