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.     

Effects of p-Nitrophenol and Organophosphorous Nitroaromatic Insecticides on the Respiratory Activity of Free and Immobilized Cells of Strains C-11 and BA-11 of Pseudomonas putida

The possibility of using the respiratory activity (RA) of microbial cells (of strains C-11 and BA-11 of Pseudomonas putida) as an instrument for quantitative determination of organophosphorous nitroaromatic insecticides, metaphos and Sumithion, and their hydrolysis product, p-nitrophenol (PNP), has been explored. The dependences of RA on the concentrations of the three compounds were linear within the range 0.5–2.5 mM. The cells of the strain BA-11 exhibited maximum selectivity in the determination of the compounds. The RAs of microbial cells differing in the modes of immobilization (adsorption to carrier surfaces vs. incorporation into gels) have been compared. Prospects of development of the microbial cell-based sensor system for determining metaphos, Sumithion, and PNP in aqueous media are discussed.     

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.     

Determination of organophosphorus aromatic nitro insecticides and p-nitrophenol by microbial-cell respiratory activity

We examined the possibility of measuring the organophosphorus aromatic nitro insecticides metaphos and sumithion as well as their hydrolysis product p-nitrophenol (PNP) by the specific respiratory activity (SRA) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122. The plots of cellular SRA against the two insecticides and PNP were linear over the ranges of 0.5-2.5 microM for P. putida C-11 and BA-11 and 0.5-1.0 microM for A. calcoaceticum A-122. P. putida BA-11 showed the greatest respiratory-response selectivity in the determination of the test substrates. We made comparison studies of the SRA of cells immobilised by two methods: carrier-surface adsorption and inclusion in various gels. We discuss the feasibility of developing a microbial sensor system for the determination of metaphos, sumithion, and PNP in aqueous media.     

A comparative study of the formation of 2-keto-D-gluconic acid by free and immobilized cells of Pseudomonas putida

The effect of glucose, oxygen and 2-keto-D-gluconic acid (2KG) concentrations on the 2KG production by free and immobilized cells of Pseudomonas putida was studied. The effect of these factors was found to be similar in case of both free and immobilized cells, but the rate of the 2KG production by the free cells was a little higher as compared to the immobilized cells.     

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.     

A comparative study on the production of amidase using immobilized and dehydrated immobilized cells of Pseudomonas putida MTCC 6809

Pseudomonas putida MTCC 6809, a plant growth promoting rhizobacteria producing amidase was isolated from the rhizosphere of Pisum sativum. The cells were immobilized in sodium alginate for the production of amidase and the effect of dehydration on immobilized beads were studied. Optimization of process parameters for amidase production was carried out to enhance enzyme production using immobilized cells. From the results it is clear that 2% and 3% (w/v) of alginate were suitable for amidase production with 12.8 and 13 U/ml activity, respectively after 36 h of incubation. Among the various substrates studied acetamide (2% w/v) was a good inducer of amidase. It was observed that immobilized catalysts could be recycled up to five batches. Amidase production was observed in both free and immobilized cells, nevertheless immobilization is much favored in comparison to free cells, as it leads to reusability of beads, lesser contamination, consistent amidase production and adaptability to wide range of culture conditions. The relative enzyme activity with the dehydrated beads was only 27% in comparison to hydrated beads, it is possible to pack considerably more into a fixed volume as the relative volume of dehydrated beads is 20%. Even though consistent amidase production was difficult to achieve using dehydrated beads, which may have certain advantages like less chances for microbial contamination and easy to transport.     

Removal and recovery of Cu(II) from industrial effluent by immobilized cells of Pseudomonas putida II-11

A copper [Cu(II)]-accumulating strain, Pseudomonas putida II-11, isolated from electroplating effluent removed a significantly high amount of Cu(II) from growth medium and buffer. A laboratory-scale fixed bed reactor with cells of P. putida II-11 immobilized in polyacrylamide gel was constructed. The adsorption of Cu(II) by the immobilized cells was pH-dependent. Maximum removal of Cu(II) by the immobilized cells was at pH 8.0. The presence of Cr(IV), Ni(II) and Zn(II) did not significantly inhibit Cu(II) uptake whereas the presence of Pb(II) reduced Cu(II) uptake by fivefold. The presence of borate, carbonate, chloride and sulphate did not significantly inhibit Cu(II) uptake. The Cu(II) removal capacity of the bioreactor with immobilized cells did not change significantly when operated at retention times greater than 3 min. More than 90% of Cu(II) adsorbed on immobilized cells could be recovered by eluting with 0.1 m HCl. The bioreactor could be used for at least five loading-elution cycles without loss of Cu(II) removal capacity. The feasibility of using this bioreactor to remove and recover Cu(II) from electroplating effluent is discussed. Correspondence to: P. K. Wong     

Surfactant-mediated permeabilization of Pseudomonas putida KT2440 and use of the immobilized permeabilized cells in biotransformation

Surfactants were used to permeabilize cells of Pseudomonas putida KT2440 so as to maximize retention of the arginine deiminase (ADI) activity within the treated cells. The surfactants cetyltrimethylammoniumbromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X100 were tested separately. Statistical models were developed for the effects on the ADI activity of the following factors: the concentration of the surfactant, the length of the treatment period and the concentration of the cells. For all surfactants, the concentration of cells was the most significant factor in influencing permeabilization. All permeabilization treatments used mild conditions (pH 7, 37 °C). The permeabilized cells were immobilized in alginate beads for the biotransformation of arginine to citrulline. The optimal conditions for immobilization and biotransformation were as follows: 2% (w/v, g/100 mL) sodium alginate, 100 g/L of treated cells, 40 mM arginine, pH 6.0, a temperature of 35 °C and an agitation speed of 150 rpm. The immobilized biocatalyst retained nearly 90% of its initial activity after nine cycles of repeated use in batch operations. In contrast, the freely suspended cells were barely active after the second use cycle.     

Glucose consumption and dehydrogenase activity of the cells of the arsenite-oxidizing bacterium Pseudomonas putida

The rates of glucose assimilation and dehydrogenase activity were studied in Pseudomonas putida oxidizing arsenite. The rate of glucose utilization by the cells decreased in the presence of arsenites in the medium at the beginning because of the microbial adaptation to arsenite. The activity of dehydrogenase fell down when the cells were cultivated in the medium with arsenite. An inverse correlation existed between the rate of glucose assimilation and arsenite oxidation. Apparently, arsenites were oxidized under the action of metabolites produced by P. putida in the process of its heterotrophous growth.     

Biodegradation of cyanides, cyanates and thiocyanates to ammonia and carbon dioxide by immobilized cells of Pseudomonas putida

Pseudomonas putida utilizes cyanide as the sole source of carbon and nitrogen. Agar, alginate, and carrageenan were screened as the encapsulating matrices for P. putida. Alginate-immobilized cells of P. putida degraded sodium cyanide (NaCN) more efficiently than non-immobilized cells or cells immobilized in agar or carrageenan. The end products of biodegradation of cyanide were identified as ammonia (NH₃) and carbon dioxide (CO₂). These products changed the medium pH. In bioreactors, the rate of cyanide degradation increased with an increase in the rate of aeration. Maximum utilization of cyanide was observed at 200 ml min⁻¹ of aeration. Immobilized cells of P. putida degraded cyanides, cyanates and thiocyanates to NH₃ and CO₂. Use of Na[¹⁴C]-CN showed that 70% of carbon of Na[¹⁴C]-CN was converted into ¹⁴CO₂ and only 10% was associated with the cell biomass. The substrate-dependent kinetics indicated that the K _m and V _max values of P. putida for the substrate, NaCN were 14 mM and 29 nmol of oxygen consumed mg protein⁻¹ min⁻¹ respectively. Received 29 January 1996/ Accepted in revised form 19 September 1997     

Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW]

Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW. After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651. The opposite situation, i.e. reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described. Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements. These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

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.     

Regulation of the utilization of glucose and aromatic substrates in four strains of Pseudomonas putida

During the oxidation of various mixtures of glucose and aromatic substrates by four strains of Pseudomonas putida, diauxic growth was not observed. Strain A3.12 grew faster on benzoate than on glucose, whereas three other strains showed faster growth on glucose than on the aromatic test substrates. Growth rates on mixtures of glucose and aromatics were intermediate between those on the single substrates.The presence of glucose in media containing aromatic substrates accelerated in the bacteria the appearance of the ability to oxidize aromatic substrates. During growth of the organisms on binary mixtures of aromatics, simultaneous utilization of these compounds occurred, the utilization ratio depending on the quality of the compounds as carbon and energy sources. Addition of glucose to dual aromatic substrate media greatly increased the utilization ratio in favour of the better aromatic substrate.With decreasing concentration of glucose in relation to that of aromatic substrates, the rate of carbon assimilation from glucose increased. Enzymological and radiochemical studies demonstrated that even in the presence of an excess of aromatic substrates, glucose was exclusively catabolized via the 2-keto-3-deoxy-6-phosphogluconate pathway. In contrast, the rate of carbon assimilation from ¹⁴C-ring-labelled benzoate and anisate was unaffected by the presence of an excess of glucose.Abbreviations KDPG 2-keto-3-deoxy-6-phosphogluconate - PP pentose-phosphate - OD optical density     

Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos

Two strains of Pseudomonas putida (epI and epII), isolated previously from ethoprophos-treated soil, were able to degrade ethoprophos (10 mg 1(-1)) in a mineral salts medium plus nitrogen (MSMN) in less than 50 h with a concurrent population growth. Addition of glucose or succinate to MSMN did not influence the degrading ability of Ps. putida epI, but increased the lag phase before rapid degradation commenced with Ps. putida epII. The degrading ability of the two isolates was lost when the pesticide provided the sole source of phosphorus. Degradation of ethoprophos was most rapid when bacterial cultures were incubated at 25 and 37 degrees C. Pseudomonas putida epI was capable of completely degrading ethoprophos at a slow rate at 5 degrees C, compared with Ps. putida epII which could not completely degrade ethoprophos at the same time. Pseudomonas putida epI was capable of degrading ethoprophos when only 60 cells ml(-1) were used as initial inoculum. In contrast, Ps. putida epII was able to totally degrade ethoprophos when inoculum densities of 600 cells ml(-1) or higher were used. In general, longer lag phases accompanied the lower inoculum levels. Both isolates rapidly degraded ethoprophos in MSMN at pHs ranging from 5.5 to 7.6, but not at pH 5 or below.