Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp.

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed.     

Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed.     

Investigation on the regulation of aniline utilization in Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 was isolated from forest soil after enrichment in a medium containing 0.1% aniline as the sole source of carbon and energy. Increasing aniline concentrations increasingly inhibited bacterial growth. At pH 7, aniline concentrations greater than 16mM were toxic enough to completely arrest growth. The optimal pH for growth on aniline was 6.5.On binary mixtures of aniline and additional carbon sources, diauxic growth was observed. The addition carbon sources caused various degrees of repression of the aniline catabolizing enzyme system. The fastest induction of this system occurred at pH 4, suggesting that protonization of the aniline molecule is crucial.     

Numerical taxonomy of Pseudomonas based on published records of substrate utilization

Data published by R. Y. Stanier, N. J. Palleroni, M. Doudoroff and their colleagues on Pseudomonas have been analysed by numerical taxonomy. Records on 401 strains were used, representing 155 characters, mostly utilization of substrates as carbon-energy sources. Twenty-nine phenons were recognized, which included 394 strains: the remaining 7 remained unclustered. The results were in very good accord with the conclusions of these authors. Almost all phenons were well separated with very little overlap. Many of them corresponded to distinct species, and others corresponded to recognized biotypes. Some small groups may represent unnamed new species.Analyses by Gower's Coefficient showed five major groupings: A) the fluorescent pseudomonads; B) biochemically active species (Pseudomonas cepacia, P. pseudomallei and allies); D) P. solanacearum and allies; and E) P. mallei. P. diminuta does not appear to be clearly distinct from P. vesicularis, nor does P. alcaligenes appear clearly distinct from P. pseudoalcaligenes. There may, however, be some difference between P. multivorans and P. cepacia.Analyses using the Pattern Coefficient differed mainly in the relationships shown by a few of the metabolically active species. Of the two coefficients, the Pattern Coefficient gave results that were in somewhat better agreement with evidence from nucleic acids, but it showed an unexpectedly close relationship between P. solanacearum and P. cepacia.     

L-arginine utilization by Pseudomonas species

The utilization of arginine was studied in several different Pseudomonas species. The arginine decarboxylase and agmatine deiminase pathways were found to be characteristic of Pseudomonas species of group I as defined by Palleroni et al. (1974). Pseudomonas putida strains had three distinct arginine catabolic pathways initiated by arginine decarboxylase, arginine deiminase and arginine oxidase, respectively. The two former routes were also present in P. fluorescens and P. mendocina and in P. aeruginosa which also used arginine by a further unknown pathway. None of these pathways occurred in P. cepacia strains; agmatine catabolism seemed to follow an unusual route involving guanidinobutyrate as intermediate.     

Growth and phenol activity of Pseudomonas aeruginosa strain 101/1 in batch cultures

Strain 101/1, isolated from petroleum wastewater sediment was classified as Pseudomonas aeruginosa. In wild type condition the strain tolerated phenol in concentration 1,000 mg/L under aerobic conditions and 800 mg/L under denitrifying conditions. As a result of adaptation to phenol the resistance of the strain to the compound increased to 1,600 and 1,400 mg/L, respectively. Maximum phenol activity under aerobic and denitrifying conditions was 350 and 65 mg/L x day-1, respectively. Under denitrifying conditions a reduction in incubation temperature from 30 degrees C to 20 degrees C resulted in two-fold drop in phenol activity of the adapted strain and reduction in tolerance to phenol by 400 mg/L.     

Peptide utilization by Pseudomonas putida and Pseudomonas maltophilia.

Pseudomonas putida assimilates peptides and hydrolyses them with intracellular peptidases. Amino acid auxotrophs (his, trp, thr or met) grew on a variety of di- and tripeptides up to twice as slowly as with free amino acids. Pseudomonas putida has separate uptake systems for both dipeptides and oligopeptides (three or more residues). Although the dipeptide system transported a variety of structurally diverse dipeptides it did not transport peptides having either unprotonatable N-terminal amino groups, blocked C-terminal carboxyl groups, D-residues, three or more residues, N-methylated peptide bonds, or beta-amino acids. Oligopeptide uptake lacked amino acid side-chain specificity, required a free N-terminal L-residue and had an upper size limit. Glycylglycyl-D,L-p-fluorophenylalanine inhibited growth of P. putida. Uptake of glycylglycyl[I-14C]alanine was rapid and inhibited by 2,4-dinitrophenol. Both dipeptide and oligopeptide uptake were constitutive. Dipeptides competed with oligopeptides for oligopeptide uptake, but oligopeptides did not compete in the dipeptide system. Final bacterial yields were 5 to 10 times greater when P. putida his was grown on histidyl di- or tripeptides rather than on free histidine because the histidyl residue was protected from catabolism by L-histidine ammonia-lyase. Methionine peptides could satisfy the methionine requirements of P. maltophilia. Generation times on glycylmethionine and glycylmethionylglycine were equal to those obtained with free methionine. Methionylglycylmethionylmethionine gave a generation time twice that of free methionine. Growth of P. maltophilia was inhibited by glycylglycyl-D,L-p-fluorophenylalanine.     

Nitrogen control of atrazine utilization in Pseudomonas sp. strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas(-) mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas(-) mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.     

Propachlor removal by Pseudomonas strain GCH1 in an immobilized-cell system

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.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Comparison of substrate utilization and growth kinetics between immobilized and suspended Pseudomonas cells

A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (K(g)) for the toluene system increased by 30-fold and the maximum specific growth rate (mu(max)) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed. (c) 1993 John Wiley & Sons, Inc.     

Growth substrate effects on acetate and methanol catabolism in Methanosarcina sp. strain TM-1.

When Methanosarcina sp. strain TM-1 is grown in medium in which both methanol and acetate are present, growth is biphasic, with methanol used as the primary catabolic substrate during the first phase. To better understand this phenomenon, we grew cells on methanol or on acetate or on both and examined the abilities of anaerobically washed cells to catabolize these substrates. Washed acetate-grown cells incubated with 10 mM acetate, 10 mM methanol, or both substrates together produced methane at initial rates of 325, 3, and 315 nmol min-1 mg of protein-1, respectively. Although the initial rate of methanogenesis from both substrates was nearly identical to the rate for acetate alone, after several hours of incubation the rate was greater for cells provided with both substrates. Studies with 14C-labeled methanol indicated that methanol was catabolized to methane at increasing rates by acetate-grown cells in a manner reminiscent of an induction curve, but only when cells were provided with acetate as a cosubstrate. Acetate was presumably providing energy and carbon for induction of methanol-catabolic enzymes. Methanol-grown cells showed a pattern of substrate utilization significantly different from that of acetate-grown cells, producing methane from 10 mM acetate, 10 mM methanol, or both substrates at initial rates of 10, 280, and 450 nmol min-1 mg of protein-1, respectively. There was significant oxidation of the methyl group of acetate during metabolism of both substrates. Cells grown on methanol-acetate and harvested before methanol depletion (methanol phase) showed catabolic patterns nearly identical to those of methanol-grown cells, including a low rate of methanogenesis from acetate. Cells harvested from methanol-acetate cultures in the acetate phase were capable of significant methanogenesis from either methanol or acetate alone, and the rate from both substrates together was nearly equal to the sum of the rates for the single substrates. When both 10 mM methanol and 10 mM acetate were presented to the acetate-phase cells, there was a preference for the methanol. These results are consistent with a model for regulation in Methanosarcina sp. strain TM-1 in which methanol represses acetate catabolism while methanol catabolism is inducible.