byPseudomonas Putida Research Articles

Cleaning and Disinfection of Biofilms Composed of Listeria monocytogenes and Background Microbiota from Meat Processing Surfaces.

Surfaces of food processing premises are exposed to regular cleaning and disinfection (C&D) regimes, using biocides that are highly effective against bacteria growing as planktonic cells. However, bacteria growing in surface-associated communities (biofilms) are typically more tolerant toward C&D than their individual free-cell counterparts, and survival of pathogens such as Listeria monocytogenes may be affected by interspecies interactions within biofilms. In this study, Pseudomonas and Acinetobacter were the most frequently isolated genera surviving on conveyor belts subjected to C&D in meat processing plants. In the laboratory, Pseudomonas, Acinetobacter, and L. monocytogenes dominated the community, both in suspensions and in biofilms formed on conveyor belts, when cultures were inoculated with eleven-genus cocktails of representative bacterial strains from the identified background flora. When biofilms were exposed to daily C&D cycles mimicking treatments used in food industry, the levels of Acinetobacter and Pseudomonas mandelii diminished, and biofilms were instead dominated by Pseudomonas putida (65 to 76%), Pseudomonas fluorescens (11 to 15%) and L. monocytogenes (3 to 11%). The dominance of certain species after daily C&D correlated with high planktonic growth rates at 12°C and tolerance to C&D. In single-species biofilms, L. monocytogenes developed higher tolerance to C&D over time, for both the peracetic acid and quaternary ammonium disinfectants, indicating that a broad-spectrum mechanism was involved. Survival after C&D appeared to be a common property of L. monocytogenes strains, as persistent and sporadic subtypes showed equal survival rates in complex biofilms. Biofilms established preferentially in surface irregularities of conveyor belts, potentially constituting harborage sites for persistent contamination.IMPORTANCE In the food industry, efficient production hygiene is a key measure to avoid the accumulation of spoilage bacteria and eliminate pathogens. However, the persistence of bacteria is an enduring problem in food processing environments. This study demonstrated that environmental bacteria can survive foam cleaning and disinfection (C&D) at concentrations used in the industrial environment. The phenomenon was replicated in laboratory experiments. Important characteristics of persisting bacteria were a high growth rate at low temperature, a tolerance to the cleaning agent, and the ability to form biofilms. This study also supports other recent research suggesting that strain-to-strain variation cannot explain why certain subtypes of Listeria monocytogenes persist in food processing environments while others are found only sporadically. The present investigation highlights the failure of regular C&D and a need for research on improved agents that efficiently detach the biofilm matrix.

Roles of Cyclic Di-GMP and the Gac System in Transcriptional Control of the Genes Coding for the Pseudomonas putida Adhesins LapA and LapF

LapA and LapF are large extracellular proteins that play a relevant role in biofilm formation by Pseudomonas putida. Current evidence favors a sequential model in which LapA is first required for the initial adhesion of individual bacteria to a surface, while LapF participates in later stages of biofilm development. In agreement with this model, lapF transcription was previously shown to take place at late times of growth and to respond to the stationary-phase sigma factor RpoS. We have now analyzed the transcription pattern of lapA and other regulatory elements that influence expression of both genes. The lapA promoter shows a transient peak of activation early during growth, with a second increase in stationary phase that is independent of RpoS. The same pattern is observed in biofilms although expression is not uniform in the population. Both lapA and lapF are under the control of the two-component regulatory system GacS/GacA, and their transcription also responds to the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP), although in surprisingly reverse ways. Whereas expression from the lapA promoter increases with high levels of c-di-GMP, the opposite is true for lapF. The transcriptional regulator FleQ is required for the modulation of lapA expression by c-di-GMP but has a minor influence on lapF. This work represents a further step in our understanding of the regulatory interactions controlling biofilm formation in P. putida.

Effect of Enhanced Conditions (C, N and Ultrasonic Power) on the Biodegradation of Mixed Endocrine Disrupting Compounds by Pseudomonas putida

Endocrine disrupting compounds (EDCs), such as estrone (E1), estrodiol (E2), estriol (E3), 17α-Ethynylestradiol (EE2) and bisphenol A (BPA) were biodegraded byPseudomonas Putida. Under the fixed conditions of inoculums size and substrate concentration, the influences of enhanced conditions, such as carbon source, nitrogen source and ultrasonic power, had been investigated. The biodegradation rates of EDCs have increased by 16.98-40.15% and 18.37-24.86% after adding carbon source and nitrogen source, respectively. Ultrasonic power can improve the biodegradation rate by 14.09-24.66%. Meanwhile, the biodegradation rate could be up to about 90% under the external conditions. The study provided a basis for the enhance control of EDCs and other contaminants.

Biodegradation of direct golden yellow, a textile dye byPseudomonas putida

Biodegradation of direct golden yellow, a textile dye byPseudomonas putida

Influence of the chemical structure on the biodegradability of acids yellow 17, violet 7 and orange 52 byPseudomonas putida

In the present study, the textiles azo dyes acids orange 52 (AO52), yellow 17 (AY17) and violet 7 (AV7) were degraded byPseudomonas putida mt-2 in mineral medium at concentration up to 100 mg/l. The culture media were completely (case of AO52) or partially (case of AY17 and AV7) decolourised under static incubation, this faster than under continuous shaking incubation. Decolourisation kinetic by intact cells indicated that AO52 disappeared completely and faster than the two other azo dyes. To understand the differentiated action of this bacterium on the three dyes, cell-free extracts activity was assessed and compared for each couple of dyes “inductor-substrate”. Results showed the same halfsaturation constant Km for a given dye as a substrate whatever the dye inductor, from where the assumption of a same protein with nonspecific azoreductase activity. The enzyme showed slightly more affinity for AO52 than for AY17 and AV7, but especially weaker specific activities against these two last dyes independently of the inductor. Finally, AO52 would be the best azoreductase inductor among the three tested dyes. These results indicated that the level as well the induction as the specificity of the azoreductase activity seems to be strongly dependent on the chemical structure of the dyes.

TheppuI-rsaL-ppuRQuorum-Sensing System Regulates Biofilm Formation ofPseudomonas putidaPCL1445 by Controlling Biosynthesis of the Cyclic Lipopeptides Putisolvins I and II

Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C(10)-N-acyl homoserine lactone (3-oxo-C(10)-AHL) or 3-oxo-C(12)-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.

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.

Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors.

The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.

Synthesis of peptide-modified pyoverdins by a fluorescent Pseudomonas strain grown in isoleucine-supplemented medium

The pattern of pyoverdin-type siderophores produced byPseudomonas putida BTP16 in iron-limited succinate medium supplemented or not with isoleucine was characterized by electrospray ionization mass spectrometry. Two new compounds were observed in culture supernatants from BTP16 grown in the presence of isoleucine. For both molecules, mass differences with pyoverdins originally synthesized by the strain were only interpreted considering the incorporation of Ile into the peptide chain. Further evidence supporting this phenomenon was provided by radioactivity incorporation in pyoverdins produced in the presence of14C-labeled Ile. Our results are discussed in relation to the non-ribosomal mechanism supposed to be involved in the synthesis of these molecules.

Isotopic composition of inorganic carbon as an indicator of benzoate degradation by pseudomonas putida: temperature, growth rate and pH effects.

Degradation experiments of benzoate by Pseudomonas putida resulted in enzymatic carbon isotope fractionations. However, isotopic temperature effects between experiments at 20 and 30 degrees C were minor. Averages of the last three values of the CO(2) isotopic composition (delta(13)C(CO2(g))) were more negative than the initial benzoate delta(13)C value (-26.2 per thousand Vienna Pee Dee Belenite (VPDB)) by 3.8, 3.4 and 3.2 per thousand at 20, 25 and 30 degrees C, respectively. Although the maximum isotopic temperature difference found was only 0.6 per thousand, more extreme temperature variations may cause larger isotope effects. In order to understand the isotope effects on the total inorganic carbon (TIC), a better measure is to calculate the proportions of the inorganic carbon species (CO(2)(g), CO(2)(aq) and HCO(3)(-)) and to determine their cumulative delta(13)C(TIC). In all three experiments delta(13)C(TIC) was more positive than the initial isotopic composition of the benzoate at a pH of 7. This suggests an uptake of (12)C in the biomass in order to match the carbon balance of these closed system experiments.

Effect of temperature on the inhibition kinetics of phenol biodegradation by Pseudomonas putida Q5.

The temperature-dependent performance of mixed-culture wastewater treatment processes may be strongly influenced by their content of psychrotrophic bacteria. In this work, the effect of temperature on cell growth and phenol biodegradation kinetics of the psychrotrophic bacterium Pseudomonas putida Q5 were determined using both batch and continuous cultures in the range of 10-25 degrees C. The Haldane equation was found to be the most suitable substrate-inhibition model for the specific growth rate. The Haldane parameters mu(max) and K(I) were best modeled by a square-root dependency on temperature. However, the Arrhenius model provided a better prediction of the temperature dependence of K(S). The variation of the yield constant with temperature also was studied experimentally. Comparisons with results of previous workers are presented.

Closed-loop control of bacterial high-cell-density fed-batch cultures: Production of mcl-PHAs byPseudomonas putida KT2442 under single-substrate and cofeeding conditions

Pseudomonas putida KT2442 is able to accumulate medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) as intracellular inclusions on a variety of fatty acids and many other carbon sources. Some of these substrates, such as octanoic acid, alkenoic acids, and halogenated derivatives, are toxic when present in excess. Efficient production of mcl-PHAs on such toxic substrates therefore requires control of the carbon source concentration in the supernatant. In this study, we develop a closed-loop control system based on on-line gas chromatography to maintain continuously fed substrates at desired levels. We used the graphical programming environment LABVIEW to set up a flexible process control system that allows users to perform supervisory process control and permits remote access to the fermentation system over the Internet. Single-substrate supernatant concentration in a high-cell-density fed-batch fermentation process was controlled by a proportional (P) controller (P = 50%) acting on the substrate pump feed rate. Na-octanoate concentrations oscillated around the setpoint of 10 mM and could be maintained between 0 and 25 mM at substrate uptake rates as high as 90 mmol L(-1) h(-1). Under cofeeding conditions Na-10-undecenoate and Na-octanoate could be individually controlled at 2.5 mM and 9 mM, respectively, by applying a proportional integral (PI) controller for each substrate. The resulting copolymer contained 43.5 mol% unsaturated monomers and reflected the ratio of 10-undecenoate in the feed. It was suggested that both substrates were consumed at similar rates. These results show that this control system is suitable for avoiding substrate toxicity and supplying carbon substrates for growth and mcl-PHA accumulation.

Effects of pH and ionic strength on the adsorption of Cs, Sr, Eu, Zn, Cd and Hg byPseudomonas putida

Bacterial metal accumulation may influence the mobility and chemical form (speciation) ofmetals in the environment. The passive adsorption of six metals (Cs, Sr, Eu, Zn, Cd and Hg) by asoil bacterium, Pseudomonas putida, was studied in the present work, using a radiotracerbatch-distribution technique. To replicate natural conditions, the adsorption was considered as afunction of pH (4-10) and ionic strength (0.01 M and 0.1 M KCl) at a low metal concentration(10-8 M). P. putida exhibited a total metal accumulating capacity of 200-1000 meq kg-1 bacteria(dry weight) (measured in 0.01 M KCl at pH 6.4). This capacity is comparable to that of manyorganic soil components and it is above the capacity of most inorganic constituents. The followingaffinity order of adsorption was observed: Hg>Eu>Cd,Zn,Sr>Cs. The results indicatethat the bacterial surface carries different sites that exhibit varying affinity and capacity for bindingmetal ions. It can be concluded that the overall adsorption of metals by P. putida is determined byseveral interacting processes related to the properties of both the metals and the bacterial surfaceand to the composition of the solution phase (pH as well as ionic strength).

A structured approach to design and operation of biotransformation processes

A structured approach to design and operation of biotransformation (bioconversion) processes, based on previous case studies, has been developed. This requires knowledge of the key characteristics of a biotransformation which determine the constraints on process selection. The approach is illustrated for five biotransformations, two enzymic and three microbial. Some generic problems such as low water solubility and volatility of reactants, reactant and product toxicity have been identified. The microblal oxidations of toluene and fluorobenzene to toluenecis-glycol and fluorocatechol respectively byPseudomonas putida have been used to illustrate how these constraints may be overcome by addition of tetradecane as a second liquid phase, use of a membrane oxygenator and introduction ofin situ product removal.

Determination of reactor operation for the microbial hydroxylation of toluene in a two-liquid phase process

Application of biotransformations to the synthesis of industrial chemicals is in part limited by a number of process challenges. We discuss the conversion of toxic, poorly water-soluble organic substrates by whole cells, using as an illustrative example the specific hydroxylation of toluene to toluenecis-glycol byPseudomonas putida UV4. Toxic effects may be eliminated through the introduction of tetradecane, to partition toluene away from the biocatalyst, to give product concentrations of 30–60 gL−1, in a two-liquid-phase reactor. The operational limits of this system have been experimentally determined and are presented in the form of windows of operation.

Effects of culture conditions on molecular weights of poly(3-hydroxyalkanoates) produced byPseudomonas putida from octanoate

Time-dependent changes in the molecular weights of poly(3-hydroxyalkanoates) (P(3HA)) produced byPseudomonas putida from octanoate were investigated. The mole ratio of carbon to nitrogen sources (C/N), incubation temperature, concentration of octanoate, and pH of culture solution were varied to study the effects of culture conditions on the yield and molecular weights of P(3HA). The molecular weights of P(3HA) decreased with time to reach a constant value during incubation. When the incubation temperature and the concentration of octanoate were low, P(3HA) polymers of high molecular weights were produced.

Penetration of poultry meat byPseudomonas andLactobacillus spp.

The penetration of poultry muscle strips byPseudomonas putida, P. fragi, Lactobacillus plantarum andL. casei was studied at 37°C. The first three bacteria penetrated along the muscular septa whileLactobacillus casei did not invade the poultry muscle strips at all. The penetration by both pseudomonads occurred throughout the 3 cm muscle strip butL. plantarum showed minor penetration up to 2 cm only.

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 μM succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix+ and fix-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a14C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of14C in the medium than two species ofPseudomonas and two species ofArthrobacter.

Synthesis of the Enzymes of the Mandelate Pathway by Pseudomonas putida II. Isolation and Properties of Blocked Mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. II. Isolation and properties of blocked mutants. J. Bacteriol. 91:1155-1160. 1966.-Mutants of Pseudomonas putida blocked in early reactions of the pathway for oxidation of d-mandelate were isolated and partially characterized. The specific genetic lesions in these mutants made normal inducer-metabolites of the pathway nonmetabolizable. Under the conditions of gratuitous enzyme synthesis so obtained, it could be shown that the d and l isomers of mandelate are equipotent inducers, and that the synthesis of the first five enzymes of the mandelate pathway is coordinate. Further experiments with the blocked mutants showed that benzoylformate, the third intermediate of the pathway, acts as an inducer without prior conversion to mandelate, and that there is no inducible, concentrating permease for mandelate.

Synthesis of the Enzymes of the Mandelate Pathway by Pseudomonas putida III. Isolation and Properties of Constitutive Mutants

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. III. Isolation and properties of constitutive mutants. J. Bacteriol. 91:1161-1167. 1966.-Mutants of Pseudomonas putida constitutive for the synthesis of l(+)-mandelate dehydrogenase were obtained after mandelate- or benzoylformate-limited growth in a chemostat. When grown in media noninducing for the wild type, the mutants are capable of coordinate, constitutive synthesis of the first five enzymes of the mandelate pathway. Later enzymes of the pathway that were examined are normally repressed. The constitutive mutants have two other noteworthy properties: they are superinducible by some compounds which induce the mandelate group enzymes in the wild type, or as a result of exhaustion of the carbon and energy source of the medium in which they are grown; and they exhibit a decreased specificity of induction, being inducible by a wide range of compounds devoid of inductive function for the wild type. These results, together with other evidence indicating that the five mandelate group enzymes comprise a regulatory unit, are discussed and evaluated in the context of the general problem of the regulation of complex dissimilatory pathways.