Pseudomonas Fluorescens HK44 Research Articles

Immobilized Cell Bioreactor Industrialization in the Development of an Innovative Optical Biosensor Technology

This commentary shows the development of a new optical biosensor, based on cell immobilization of Pseudomonas Fluorescens HK44, in nano and micro-tubular cellulose (TC) and a mixture of carbohydrate nanotubes (CHNTs) and carbohydrate micro-tubes (CHMTs). Methodology follows, this biocatalyst can be industrialized with the use of a single tank immobilized cell bioreactor (ICB). A techno-economic analysis was conducted within the framework of it by designing a process flow sheet with mass and energy balance. According to its case study, the investment is 227,800 euros, and the daily production cost is 1434 euros, with a maximum daily added value of 25,000 euros. The discussion revealed that novel research proposals and a novel study concept are being developed in the field of biosensors. The results are supported by papers published on ICB area development. The problem that leads to this commentary is industrialization of ICB, in the case of a simple biosensor development using immobilized cells and it is the objective.

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.

Pseudomonas fluorescens HK44: Lessons Learned from a Model Whole-Cell Bioreporter with a Broad Application History

Initially described in 1990, Pseudomonas fluorescens HK44 served as the first whole-cell bioreporter genetically endowed with a bioluminescent (luxCDABE) phenotype directly linked to a catabolic (naphthalene degradative) pathway. HK44 was the first genetically engineered microorganism to be released in the field to monitor bioremediation potential. Subsequent to that release, strain HK44 had been introduced into other solids (soils, sands), liquid (water, wastewater), and volatile environments. In these matrices, it has functioned as one of the best characterized chemically-responsive environmental bioreporters and as a model organism for understanding bacterial colonization and transport, cell immobilization strategies, and the kinetics of cellular bioluminescent emission. This review summarizes the characteristics of P. fluorescens HK44 and the extensive range of its applications with special focus on the monitoring of bioremediation processes and biosensing of environmental pollution.

Ameliorating Risk: Culturable and Metagenomic Monitoring of the 14 Year Decline of a Genetically Engineered Microorganism at a Bioremediation Field Site

In 1996, the first EPA sanctioned release of a recombinant microbe (Pseudomonas fluorescens HK44) into the subsurface soil environment was initiated in a replicated semi-contained array of soil lysimeters. With an aim to access the survivability/environmental fate of HK44, soil sampling was performed 14 years post release. Although after extensive sampling culturable HK44 cells were not found, qPCR and metagenomic analyses indicated that genetic signatures of HK44 cells still persisted in the soils, with genes diagnostic for the bioluminescent transposon carried by strain HK44 (luxA and tetA) being found at low concentrations (< 5000 copies/g). Additionally, metagenome analysis of lysimeter 2 using amplicon pyrosequencing showed that Burkholderia was more abundant in the sample extracted before storage at 4°C than after storage at 4°C (79% and 5.6% Burkholderia sequences, respectively).

Salicylic acid degradation from aqueous solutions using Pseudomonas fluorescens HK44: parameters studies and application tools

The optimal conditions for salicylic acid biodegradation by Pseudomonas fluorescens HK44 were determined in this study with the intention to create a microbial sensor. Kinetic experiments permitted a definition of 60 and 30min the time needed to achieve the maximum degradation of salicylic acid presented in a medium with and without yeast extract, respectively. The degradation in medium without yeast extract and the quantification by spectrophotometry 230 nm were selected to be used in further tests. The use of preactivated cells or on the exponential growth phase showed better salicylic acid degradation percentages when compared to nonactivated cells or on the stationary growth state. Finally, the best cellular concentration used on the salicylic acid degradation was 0,1 g.L-1. Strain HK44 shows to be capable of degrade salicylic acid presented in simple aqueous systems, making this strain a promising tool for the application on a luminescent microbial sensor.

Response of the bioluminescent bioreporterPseudomonas fluorescens HK44 to analogs of naphthalene and salicylic acid

Pseudomonasfluorescens HK44 is a lux-based bioluminescent bioreporter capable of selective luminescence in the presence of naphthalene and/or salicylic acid intermediate of its metabolism. We attempted to induce bioluminescence (BL) in this strain with 72 compounds, viz. substituted naphthalenes, naphthalene-like compounds (e.g., quinoline), substituted salicylic acids, salicylic acid-like compounds (e.g., 2-anthranilic acid), oligocyclic aromates, and intermediates of naphthalene metabolism to better discriminate response specificity. From them, 42 induced BL significantly lower as compared to naphthalene, three (viz. isoquinoline, o-cresol, and salicylamide) induced BL significantly greater than naphthalene, and 27 yielded no bioluminescent response whatsoever. Strain HK44 is therefore not prone to extensive false-positive signaling and can serve as a fairly specific indicator organism for naphthalene bioavailability. At elevated concentrations, 41 compounds inhibited BL. Thus, the inclusion of constitutive bioreporter controls as indicators of sample toxicity is vital to successful biosensing application.

Visualization and modeling of the colonization dynamics of a bioluminescent bacterium in variably saturated, translucent quartz sand

An experimental and numerical investigation was conducted to study the colonization dynamics of a bioluminescent bacterium, Pseudomonas fluorescens HK44, during growth in a porous medium under steady, variably saturated flow conditions. Experiments were conducted in a thin-slab light transmission chamber filled with uniform, translucent quartz sand. Steady, variably saturated flow conditions were established using drip emitters mounted on the top of the chamber, with glucose applied through a central dripper located directly above an inoculated region of the chamber. Periodic pulses of salicylate and a dye tracer were applied to induce bioluminescence of the bacterium to monitor colony expansion and to track changes in the hydraulic and transport properties of the sand. Changes in the apparent water saturation of the sand were quantified by monitoring light transmission through the chamber with a CCD camera. The colonized region expanded laterally by about 15 cm, and upward against the flow by 7–8 cm during the 6-day experiment while apparent saturations in the colonized region decreased by 7–9% and the capillary fringe dropped by ∼5 cm. The observed data were reproduced approximately using a numerical model that accounted for the processes of water flow, solute and bacterial transport, cell growth and accumulation, glucose and oxygen consumption, and gas diffusion and exchange. The results of this study illustrate some of the complexities associated with coupled flow, reactive transport, and biological processes in variably saturated porous media, such as localized desaturation, capillary fringe lowering effects, and upstream movement of bacterial colonization, that may not readily observable using other experimental techniques.

Experimental Observations and Numerical Modeling of Coupled Microbial and Transport Processes in Variably Saturated Sand

An experimental and numerical investigation was conducted to study interactions between microbial dynamics and transport processes in variably saturated porous media. Experiments were conducted with constant, surface‐applied water fluxes in duplicate, variably saturated, sand‐filled columns that were uniformly inoculated with the bacterium Pseudomonas fluorescens HK44. The permeability of the sand in the columns was reduced by a factor of 45 during 1 wk of growth on glucose. Pressure heads increased (became less negative) at all measured depths, but significant increases in the apparent volumetric water contents were observed in only the upper 5 cm of the columns, corresponding to the areas with the highest concentrations of attached bacteria. A numerical model was used to simulate the experiments. The model accounted for the processes of water flow, solute and bacterial transport, cell growth and accumulation, glucose and O2 consumption, and gas diffusion and exchange. Observed changes in water content and pressure head were reproduced approximately using fluid‐media scaling to account for an apparent surface‐tension lowering effect. Reasonable correspondence was obtained between observed and simulated effluent data and final attached biomass concentration distributions using first‐order reversible cell attachment and detachment kinetics. The attachment rate coefficients were based on particle‐filtration theory and time‐dependent detachment rate coefficients. The results of this study illustrate the potential importance of using fully coupled multifluid flow and multicomponent reactive transport equations to model coupled biogeochemical and transport processes in soils.

Monitoring of the Viability of Cells Immobilized by Sol-Gel Process

Three different types of cells, Pseudomonas fluorescens HK44, Saccharomyces cerevisiae strain SP4 and plant cells Nicotiana tabacum L. BY-2, were immobilized by entrapment in tetramethoxysilane prepolymer (TMOS) gel or in composite gel containing prepolymer TMOS and alginate in various ratios. Their growth and viability were monitored by bioluminescence and 2-D fluorescence spectra, which are fast and do not need the dissolution of a matrix. The resulting biocomposite gels were obtained by gelation of the mixtures of TMOS prep. or TMOS/alginate sols and the particular cells in proper media on glass supports to provide films ∼1 mm thick. The effect of the following parameters on the growth and viability of the cells was studied: (a) the composition of the biocomposites, (b) the preparation conditions of TMOS and (c) the conditions of the procedure of entrapment. All three types of cells were tested in TMOS gel and the composite TMOS/alginate = 1:1 (v/v). The sensitivity of the cells to the changes of conditions increased in the sequence: P. fluorescence HK44 < S. cerevisiae strain SP4 < N. tabacum L. BY-2. Cell viability decreased with the increasing content of Si in biocomposites. The entrapment into alginate–silica composites resulted in the leakage of microbial and yeast cells. However, it had positive effects on the growth and metabolic activity of plant cells.

Naphthalene detection by a bioluminescence sensor applied to wastewater samples

A bioluminescence detection system using Pseudomonas fluorescens HK44 was applied for naphthalene measurement in the range 0–0.5mgl−1, lower than the maximum of 1mgl−1 recommended by EPA. Diluted sample injections of naphthalene were added to 0.2 or 0.4gl−1 cellular concentrations used in the detection medium. The lowest cell density (0.2gl−1) exhibited a linear response up to naphthalene concentration of about 0.4mgl−1 being selected to be used in the subsequent experiments. The same system was applied to samples of influent and effluent streams of a biological wastewater treatment from a petroleum industry containing 2.4 and 1.1mg hydrocarbonsl−1, respectively, including naphthalene. The output signal of the biosensor system for the influent stream sample was fully compatible with the results obtained for the naphthalene solutions. Results obtained with the effluent sample showed that other contaminants than naphthalene may influence the biosensor response and are under investigations.

Effect of root-derived substrates on the expression of nah-lux genes in Pseudomonas fluorescens HK44: implications for PAH biodegradation in the rhizosphere.

The bioluminescent reporter strain Pseudomonas fluorescens HK44 with a nah-lux fusion, was used to investigate the effect of root material (from hybrid poplars, willow, kou, milo, Osage orange, mulberry, and switch grass) and potential root-derived substrates (e.g., sugars, carboxylic acids, amino acids, and phenolics) on the expression of nahG, one of the genes responsible for naphthalene dioxygenase transcription. Whereas nahG was induced by some phenolic substrates that could be released by plants (i.e., salicylate, methyl salicylate, and acetyl salicylate), no induction by root extracts was observed. Rather, increasing root extract concentrations (50 to 275 mg L(-1) as total organic carbon) inhibited nahG expression in assays with cells concurrently exposed to naphthalene. Root extracts also decreased nahG expression at the individual cell level during naphthalene degradation assays. However, treatments with root extracts exhibited significantly higher microbial growth and overall bioluminescence, indicating a higher level of nahG expression by the resulting larger microbial population. This generally resulted in faster naphthalene degradation rates, suggesting that plant-promoted proliferation of competent genotypes could compensate for the interference that labile substrates exert on the expression of genes that code for the degradation of polynuclear aromatic hydrocarbons (PAHs). This could explain the faster PAH degradation commonly reported in planted than in unplanted soils.

Bioluminescent bioreporter integrated circuits: potentially small, rugged and inexpensive whole-cell biosensors for remote environmental monitoring.

1. SUMMARYA bioluminescent bioreporter integrated circuit (BBIC) is anovel whole-cell biosensor that combines the environmentalmonitoring capabilities of genetically engineered biolu-minescent micro-organisms (bioreporters) with optical appli-cation-specific integrated circuits. A BBIC device consistsof bioreporters sustained within a micro-environment, theintegrated circuit microluminometer, and a light-tightenclosure. The bioreporter typically contains the luxCDABEreporter genes encoding the enzymes required for biolumin-escence. In the presence of a targeted analyte, a gene(transcriptional) regulatory system induces the expressionof the luxCDABE genes. Analytical benchmark data forexposure of the bioreporter Pseudomonas fluorescens 5RLto salicylate was determined using a flow-through testsystem. The detection limit (after a 45 min exposure) wasca 50 lgl

On-line detection of low naphthalene concentrations with a bioluminescent sensor

On-line tests with Pseudomonas fluorescens HK44 were carried out using a biosensor system with a flow-through cell and sample injections to detect low concentrations of naphthalene. Kinetic experiments permitted a 19 min response time and definition of a 50 min induction period for bioluminescence assays. A mathematical model showed that 0.2 g/l is the best cell concentration to be used in the detection of low naphthalene concentrations, with the highest acceleration rate of bioluminescence sensitivity increase (0.46 nA l/(g min 2)). Calibration curves with different concentrations of naphthalene showed a linear correlation up to 0.4 mg/l (3 μM) corresponding to 211 nA output signal. The lower limit of naphthalene detection by strain HK44 was in the region of 0.02 mg/l (0.16 μM), below the naphthalene health advisory limit for drinking water consumed over a lifetime suggested by EPA.

A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media

A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm 3 H 2O/cm 3 of sand, respectively), and at cell densities ranging between 1×10 6 and 8.5×10 8 cells/ml. Before O 2 availability became a limiting factor, the rate of light emission ( L) increased with the square of time ( t) and linearly with increasing cell density ( c). A nonlinear model was developed that contains a “rate of increase in light emission” constant, B′, which is determined directly from the slope of a plot of √ L/ c against t. The model predicted the behavior of lux induction in HK44 under a variety of conditions. Similar B′ values were determined [49.0–57.6×10 −10 light units/(cell min 2)] for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand. Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B′=61.6±3.2, log phase cells plus glucose, B′=63.2±7.2). The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media.

Vertical transport of a field-released genetically engineered microorganism through soil

The vertical movement of a genetically-engineered bacterium released into a sub-surface soil contained within a lysimeter was investigated. The strain used was Pseudomonas fluorescens HK44, a lux-based microbe capable of bioremediating specific polyaromatic hydrocarbons (PAHs). Strain HK44 was inoculated into a 4 m deep by 2.5 m diameter soil lysimeter outfitted with a hydraulic assembly to control groundwater levels. After 230 days, the lysimeter was subjected to a water table manipulation that resulted in significant transport of HK44 cells to depths 60 cm below the original inoculation zone within 20 days. A lysimeter not exposed to water level fluctuations exhibited no significant vertical transport. These results demonstrate the potential distribution of genetically engineered microorganisms in subsurface soil due to groundwater effects.

Controlled Field Release of a Bioluminescent Genetically Engineered Microorganism for Bioremediation Process Monitoring and Control

Pseudomonas fluorescens HK44 represents the first genetically engineered microorganism approved for field testing in the United States for bioremediation purposes. Strain HK44 harbors an introduced lux gene fused within a naphthalene degradative pathway, thereby allowing this recombinant microbe to bioluminesce as it degrades specific polyaromatic hydrocarbons such as naphthalene. The bioremediation process can therefore be monitored by the detection of light. P. fluorescens HK44 was inoculated into the vadose zone of intermediate-scale, semicontained soil lysimeters contaminated with naphthalene, anthracene, and phenanthrene, and the population dynamics were followed over an approximate 2-year period in order to assess the long-term efficacy of using strain HK44 for monitoring and controlling bioremediation processes. Results showed that P. fluorescens HK44 was capable of surviving initial inoculation into both hydrocarbon contaminated and uncontaminated soils and was recoverable from these soils 660 day...

Containment of a genetically engineered microorganism during a field bioremediation application.

A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10(14) bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low.

Physiological considerations of environmental applications of lux reporter fusions

The performance of the bioluminescent reporter bacterium Pseudomonas fluorescens HK44, which contains a nahG-luxCDABE fusion for naphthalene catabolism and bioavailability was investigated for environmental samples and mixed contaminants. For aqueous extracts from fuel hydrocarbon-contaminated soils, a reproducible bioluminescence response was obtained, which coincided with, but overestimated the presence of naphthalene. Therefore, the strain's bioluminescence response to mixed contaminants was investigated further. The strain showed a linear correlation between bioluminescence and the amount of JP-4 jet fuel present in an aqueous solution, representing a mixture of compounds including naphthalene. However, some non-inducing organic solvents such as toluene, p-xylene and acetone caused a significant bioluminescence increase as well. The analysis of nah-lux mRNA from cells exposed to toluene revealed that the bioluminescence response was not due to increased nahG-luxCDABE gene expression, whereas increased lux mRNA levels were found with exposure to naphthalene or JP-4 jet fuel. While different mixture combinations of solvents resulted in either additive or intermediate effects, the combination of naphthalene and solvent resulted in a synergistic effect on the bioluminescence response. The addition of n-decanal, a substrate for luciferase, showed that the cells were aldehyde-limited. If aldehyde was added, only the presence of naphthalene caused a significantly increased bioluminescence response over the control. The solvent effects were dependent on the physiological status of the reporter culture and were present in growing, but not in resting cell cultures. It was postulated that the increase in bioluminescence after exposure to solvents was due to changed fatty acid synthesis patterns affecting the aldehyde supply for the bioluminescence reaction. Exposure to toluene resulted in altered membrane fatty acid composition and release of fatty acids from the cells. Exposure to n-alkanes resulted in minor changes in the bioluminescence response, whereas, exposure to heavy metals or cyanide resulted in significant reductions in the overall bioluminescence. These results demonstrate the utility of bioluminescent reporter bacteria for environmental applications, as well as the need for adequate experimental controls in interpreting environmental sensing data.

Kinetics and response of aPseudomonas fuorescens HK44 biosensor

The reporter bacterium, Pseudomonas fluorescens HK44 (HK44), was characterized in an immobilized state to investigate utility for deployment as a remote sensor in the subsurface. A packed-bed reactor with alginate-immobilized HK44 simulated hydrodynamic conditions such as might be found in a subsurface environment. The reporter bacterium, HK44, harbors a reporter plasmid, pUTK21, which contains a transcriptional fusion between the nahG gene in the lower pathway of the catabolic plasmic NAH7 and a luxCDABE gene cassette. The upper nah pathway and the lux pathway in pUTK21 are induced by salicylate. The lux enzymes catalyze the light reaction. HK44 demonstrated a quantitative relationship between salicylate concentration and degradation. Light intensity mimicked salicylate concentration, whereas degradation was first order in biomass and first order in salicylate concentration, with a degradation constant of 2.23 x 10(-2) dm(3) g(-1) min(-1).

Bioluminescence induction response and survival of the bioreporter bacterium Pseudomonas fluorescens HK44 in nutrient-deprived conditions

Pseudomonas fluorescens HK44 is a bioluminescent bioreporter synthesizing light in the presence of naphthalene or salicylate. Upon immobilization, HK44 is useful as an in situ or on-line biosensor of bioavailable naphthalene and salicylate in waste streams or contaminated fields. The bioreporting efficacy of alginate/SrCl2-immobilized HK44 was investigated in simulated groundwater with different pH regimes. When induced with complex (salicylate plus auxiliary energy supplements) and simple (salicylate as the sole energy supplement) inducer solutions, the specific light response was steadier at pH 6 than at pH 7 in a 35-day study. There was no bioluminescence response from cells incubated in groundwater samples with pH below 6. The rate of the luminescence reaction was stable at pH 6 irrespective of the type of inducer solution, indicating the robust physiological status of the bioreporter bacteria. In addition, the quantity of light synthesized was at least one order of magnitude higher with complex inducer solution than with simple inducer solution. The numbers of viable and cultivable cells remained constant in groundwater at pH 6 and 7 (approx. 107 g−1 beads). The numbers decreased by four orders of magnitude (107 to 103) to zero in groundwaters with pH below 6. This study suggested that HK44 is useful for long-term biosensor applications in moderately acidic to neutral groundwater conditions.