A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1

A two-step model is developed for the aerobic biodegradation of benzene,toluene, and p-xylene (BTX) by Pseudomonas putida F1. The model contains three unique features. First, an initial dioxygenation step transforms BTX into their catechol intermediates, but does not support biomassgrowth. Second, the benzene or toluene intermediates are mineralized, which supports biomass synthesis. Third, BTX exhibit competitive inhibition on each other's transformation, while toluene and benzenenoncompetitively inhibit the mineralization of their catechol intermediate. A suite of batch and chemostat experiments is used to systematically measure the kinetic parameters for the two-step transformations and the substrate interactions.     

Recovery of trichloroethylene removal efficiency through short-term toluene feeding in a biofilter enriched withPseudomonas putida F1

Trichloroethylene (TCE) is an environmental contaminant provoking genetic mutation and damages to liver and central nerve system even at low concentrations. A practical scheme is reported using toluene as a primary substrate to revitalize the biofilter column for an extended period of TCE degradation. The rate of trichloroethylene (TCE) degradation byPseudomonas putida F1 at 25°C decreased exponentially with time, without toluene feeding to a biofilter column (11 cm I.D.×95 cm height). The rate of decrease was 2.5 times faster at a TCE concentration of 970 μg/L compared to a TCE concentration of 110 μg/L. The TCE itself was not toxic to the cells, but the metabolic intermediates of the TCE degradation were apparently responsible for the decrease in the TCE degradation rate. A short-term (2 h) supply of toluene (2,200 μg/L) at an empty bed residence time (EBRT) of 6.4 min recovered the relative column activity by 43% when the TCE removal efficiency at the time of toluene feeding was 58%. The recovery of the TCE removal efficiency increased at higher incoming toluene concentrations and longer toluene supply durations according to the Monod type of kinetic expression. A longer duration (1.4∼2.4 times) of toluene supply increased the recovery of the TCE removal efficieny by 20% for the same toluene load.     

New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites

The influence of toluene concentration on the specific growth rate, cellular yield, specific CO₂, and metabolite production by Pseudomonas putida F1 (PpF1) was investigated. Both cellular yield and specific CO₂ production remained constant at 1.0 ± 0.1 g biomass dry weight (DW) g⁻¹ toluene and 1.91 ± 0.31 g CO₂ g⁻¹ biomass, respectively, under the tested range of concentrations (2–250 mg toluene l⁻¹). The specific growth rate increased up to 70 mg toluene l⁻¹. Further increases in toluene concentration inhibited PpF1 growth, although inhibitory concentrations were far from the application range of biological treatment processes. The specific ATP content increased with toluene concentration up to toluene concentrations of 170 mg l⁻¹. 3-Methyl catechol (3-MC) was never detected in the cultivation medium despite being an intermediary in the TOD pathway. This suggested that the transformation from toluene to 3-MC was the limiting step in the biodegradation process. On the other hand, benzyl alcohol (BA) was produced from toluene in a side chain reaction. This is, to the best of our knowledge, the first reported case of methyl monoxygenation of toluene by PpF1 not harboring the pWW0 TOL plasmid. In addition, the influence of 3-MC, BA, and o-cresol on toluene degradation was investigated respirometrically, showing that toluene-associated respiration was not significantly inhibited in the presence of 10–100 mg l⁻¹ of the above-mentioned compounds.     

Co-metabolic degradation of trichloroethylene by Pseudomonas putida in a fibrous bed bioreactor

Co-metabolic degradation of trichloroethylene (TCE) by Pseudomonas putida F1 was investigated in a novel bioreactor with a fibrous bed. A pseudo-first-order rate constant for TCE degradation was 1.4 h^–1 for 2.4 to 100 mg TCE l^–1. Competitive inhibition of toluene on TCE removal could be prevented in this bioreactor. 90% TCE was removed over 4 h when 95 mg toluene l^–1 was presented simultaneously.     

Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites

Benzene, toluene, and o-xylene (BTX) degradation by immobilized Pseudomonas putida F1 of postconsumer agave-fiber/polymer foamed-composites (AFPFC) and suspended cultures was studied under controlled conditions. Analyses using FTIR-ATR and SEM showed that P. putida F1 adhered onto the composite surface and developed a biofilm. In this sense, the AFPFC were successfully used as a support for bacterial immobilization. Both systems, immobilized and suspended cells of P. putida F1, were able to completely degrade benzene and toluene from initial concentrations of 15, 30, 60, and 90 mg l⁻¹. An inhibitory effect of the intermediary catechol from benzene degradation was observed in suspended cultures but it was not presented in the immobilized system. The degradation of o-xylene was partially accomplished in both systems. The Monod equation was used to model the experimental data obtained from the biodegradation kinetics, and they were adequately described with this model.     

The uptake of 2-ketogluconate by Pseudomonas putida

The uptake of 2-ketogluconate is inducible in Pseudomonas putida: 2-ketogluconate, glucose, gluconate, glycerol and glycerate were each good nutritional inducers of this ability. 2-Ketogluconate uptake obeyed saturation kinetics (apparent K _min 2-ketogluconate-grown cells was 0.4 mM). 2-Ketogluconate was transported against a concentration gradient, apparently in an unchanged state, and the process required metabolic energy, all of which indicate an active transport system.A number of independently isolated mutants with deranged activity of a common glucose-gluconate uptake system were found to be also defective in 2-ketogluconate transport. Strains unable to transport 2-ketogluconate which grew readily on glucose and gluconate were also isolated. These results suggest that 2-ketogluconate transport is governed by at least two genetic elements: one which is also required to take up glucose and gluconate and another which appears to be specific for 2-ketogluconate transport. Similarly glucose and gluconate transport appears to require at least one factor which is not necessary for 2-ketogluconate transport, as suggested by the lack of induction of the common glucose-gluconate uptake system by glycerol and glycerate, substrates which are good inducers of 2-ketogluconate uptake.Abbreviations CCCP carbonyl-cyanide-m-chlorophenyl-hydrazone - cpm radioactivity counts per minute - GGU glucose-gluconate uptake - PFU plaque forming units - U.V. ultraviolet Dedicated to Prof. Roger Y. Stainer on the occasion of his 60th birthday     

Toluene biodegradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1: targeting culture stability in long-term operation

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h⁻¹). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO₂ produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.     

Biodegradation of crystal violet by Pseudomonas putida

Crystal violet (CV), which has been extensively used as a biological stain and a commercial textile dye, is a recalcitrant molecule. A strain of Pseudomonas putida was isolated that effectively degraded CV: up to 80% of 60 μM CV as the sole carbon source, was degraded in liquid media within 1 week. Nine degradation products were isolated and identified. We propose that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.     

Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p-xylene mixture

For the complete biodegradation of a mixture of benzene, toluene, and p-xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate-cis-glycol dehydrogenase in the tol pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis-glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis-glycol compounds by the action of toluate-cis-glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p-xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. (c) 1994 John Wiley & Sons, Inc.     

Response of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1 cultures to fluctuating toluene loads and operational failures in suspended growth bioreactors

The response of Pseudomonas putida F1 to process fluctuations and operational failures during toluene biodegradation was evaluated in a chemostat suspended growth bioreactor. The ability of P. putida F1 to rapidly increase its specific toluene degradation capacity resulted in no significant variation in process removal efficiency when toluene load was increased from 188 to 341 g m⁻³ h⁻¹. Likewise, bacterial activity rapidly reached steady state performance (in less than 1.5 h after the restoration of steady state operational conditions) following an 8 h process shutdown, or after episodes of toluene or mineral nutrients deprivation. Process performance was however highly sensitive to pH, as pH levels below 4.5 dramatically inhibited bacterial activity, decreasing severely process robustness and inducing a cycle of periodic process collapses and recoveries. This pH mediated deterioration of bacterial activity was confirmed by further respirometric tests, which revealed a 50–60% reduction in the O₂ consumption rate during the degradation of both toluene and 3-methyl catechol when pH decreased from 5.05 to 4.55. Finally, process robustness was quantified according to methods previously described in literature.     

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.     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

The effect of a non-metabolizable analog on mandelate catabolism in Pseudomonas putida

Dl-2,3,4,5,6-pentafluoromandelic acid (PFM) specifically inhibits the growth of Pseudomonas putida (ATCC 12633) on medium containing mandelate as sole carbon and energy source by competitive inhibition of mandelate dehydrogenase. PFM is not metabolized and is neither an inducer of the mandelate catabolic enzymes nor an antagonist of induction. Mutants resistant to the inhibitory effects of PFM (PFM^r) were isolated; most prove to be superinducible, i.e. synthesize coordinately the mandelatespecific catabolic enzymes at elevated levels following induction. In at least one case the PFM^r mutation maps very near the structural genes that encode the enzymes functional in the first two steps of mandelate catabolism. It is reasoned that the PFM^r mutation is of the promotor type. Resistance to substrate analogs such as PFM offers a general method for isolation of regulatory mutants in catabolic metabolism.Dedicated to Prof. Roger Y. Stanier on the occasion of his 60th birthday     

The response of Pseudomonas putida CP1 cells to nutritional, chemical and environmental stresses

Summary The response of a pollutant-degrading bacterium P. putida CP1 to stresses was investigated. The growth on the mono-chlorophenols resulted in a decrease in dry weight of the organism, although there was an increase in cell number. There was a change of bacterial shape from rod to round as well as the reduction of cell size when grown on phenol and chlorophenols. Changes in cell shape and size were also evident in glucose-free medium, which suggested that alteration of cell shape from rod to round as well as reduction of cell size were due to nutritional stress. The increase in cell number but a drop in dry weight correlated with the reduction of cell size and shape. The organism flocculated with chlorophenols but not with phenol. The cause of flocculation was due to the toxicity of chlorophenol. Isomerization of cis to trans forms of the unsaturated fatty acids in P. putida CP1 occurred under conditions of environmental stress. Trace amounts of the polyunsaturated fatty acid linoleic acid (cis-9, cis-12-octadecadienoic acid) rarely found in bacterial membranes and oleic acid (cis-9-octadecanoic acid), which is a typical product of aerobic fatty acid synthesis, were found in P. putida CP1.     

Removal of caffeine in sewage by Pseudomonas putida: Implications for water pollution index

A strain of Pseudomonas putida (biotype A) capable of growing on caffeine (1,3,7-trimethylxanthine) was isolated from a domestic wastewater processing operation. It used caffeine as the sole carbon source with a mean growth rate constant (k) of 0.049 h^-1 (approximately 20 h per generation), whereas k for glucose utilization under similar incubation conditions was 0.31 (3.3 h per generation). The isolate contained at least two plasmids, and the increased expression of a 40 kDa protein was attributable to growth on caffeine. Degradation byproducts of caffeine metabolism by the bacterial isolate included other xanthine derivatives. The slow bacterial catabolism of caffeine in sewage has implications for the effectiveness of wastewater purification, re-use and disposal.The author is with the Laboratory for Molecular Ecology, Department of Environmental Analysis and Design, University of California at Irvine, Irvine, CA 92717-5150 U.S.A.     

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.     

Effects of inorganic nutrient levels on the biodegradation of benzene, toluene, and xylene (BTX) by Pseudomonas spp. in a laboratory porous media sand aquifer model

The effect of inorganic nutrients (sulfate, phosphate, and ammonium chloride) on the aerobic biodegradation of benzene, toluene, and xylene (BTX) by Pseudomonas spp. was studied in the laboratory using a glass sand tank. The increase of nutrient levels resulted in enhanced bacterial growth and BTX degradation. Sulfate and phosphate serve as key electron acceptors in the microbiological processes degrading BTX. The observed bacterial morphological changes during BTX degradation reveal that the filamentous bacteria were the dominant species at low temperatures about 20 degrees C. The spherical and rod-shaped cells became dominant at higher temperatures ranging from 25 degrees C to 28 degrees C. When the BTX mixture was allowed to be biodegraded for longer incubation periods of 21-42 h at high phosphate concentrations, large amounts of rod-shaped cells were clustered. The morphological adaptation appears to be controlled by the temperature and nutrient levels in the sandy medium where Pseudomonas spp. thrives.     

Characterization of a benzoate permease mutant of Pseudomonas putida

A spontaneous mutant of Pseudomonas putida (PRS 2017) has been isolated which is incapable of growth on benzoate, does not induce the enzymes of the catechol branch of the -ketoadipate pathway when grown in the presence of benzoate, cannot accumulate radioactively labeled benzoate, yet grows well with mandelate as sole source of carbon and energy. This strain apparently lacks a benzoate permease, which in the wild type shows a K _mof about 0.1 mM for benzoate, is inducible, and is not under the control of the regulatory system which governs the induction of the enzymes of the catechol branch of the -ketoadapate pathway. The lesion in PRS 2017 is apparently single site and maps near other genes governing benzoate dissimilation.Dedicated to R. Y. Stanier on the occasion of his 60th birthday     

Unusual traits of the pyoverdin-mediated iron acquisition system in Pseudomonas putida strain BTP1

Fluorescent Pseudomonas species are characterized by the production of pyoverdin-type siderophores for Fe³⁺ acquisition in iron-limited environments. Since it produces a structurally specific pyoverdin, Pseudomonas putida strain BTP1 could represent a valuable tool in an attempt to correlate the structural features of these compounds with some specificity in their two main properties i.e. affinity for iron and recognition rate by other Pseudomonas strains. An uncommonly high affinity for iron of the pyoverdin synthetized by P. putida BTP1 was observed by comparing both the apparent stability constant and the decomplexation kinetic of its ferric complex with those of ferripyoverdins from other strains. On another hand, results from growth stimulation experiments and labeled ferripyoverdin uptake assays highlighted the very low recognition rate of BTP1 isopyoverdins by membrane receptors of foreign strains. By contrast, P. putida BTP1 was able to utilize a broad spectrum of structurally unrelated exogenous pyoverdins by means of multiple receptors that are likely constitutively expressed in its outer membrane. The unusual traits of its pyoverdin-mediated iron acquisition system should contribute to enlarge the ecological competence of Pseudomonas putida BTP1 in terms of colonization and persistence in the rhizosphere.