A biosensor for the detection of gas toxicity using a recombinant bioluminescent bacterium

A whole-cell biosensor was developed for the detection of gas toxicity using a recombinant bioluminescent Escherichia coli harboring a lac::luxCDABE fusion. Immobilization of the cells within LB agar has been done to maintain the activity of the microorganisms and to detect the toxicity of chemicals through the direct contact with gas. Benzene, known as a representative volatile organic compound, was chosen as a sample toxic gas to evaluate the performance of this biosensor based on the bioluminescent response. This biosensor showed a dose-dependent response, and was found to be reproducible. The immobilizing matrices of this biosensor were stored at 4 degrees C and were maintained for at least a month without any noticeable change in its activity. The optimal temperature for sensing was 37 degrees C. A small size of this sensor kit has been successfully fabricated, and found to be applicable as a disposable and portable biosensor to monitor the atmospheric environment of a workplace in which high concentrations of toxic gases could be discharged.     

Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant

A biosensor for detecting the toxicity of polycylic aromatic hydrocarbons (PAHs) contaminated soil has been successfully constructed using an immobilized recombinant bioluminescent bacterium, GC2 (lac::luxCDABE), which constitutively produces bioluminescence. The biosurfactant, rhamnolipids, was used to extract a model PAH, phenanthrene, and was found to enhance the bioavailability of phenanthrene via an increase in its rate of mass transfer from sorbed soil to the aqueous phase. The monitoring of phenanthrene toxicity was achieved through the measurement of the decrease in bioluminescence when a sample extracted with the biosurfactant was injected into the minibioreactor. The concentrations of phenanthrene in the aqueous phase were found to correlate well with the corresponding toxicity data obtained by using this toxicity biosensor. In addition, it was also found that the addition of glass beads to the agar media enhanced the stability of the immobilized cells. This biosensor system using a biosurfactant may be applied as an in-situ biosensor to detect the toxicity of hydrophobic contaminants in soils and for performance evaluation of PAH degradation in soils.     

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.     

A cell array biosensor for environmental toxicity analysis

In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.     

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.     

Isolation and characterization of Acidithiobacillus caldus from a sulfur-oxidizing bacterial biosensor and its role in detection of toxic chemicals

A novel toxicity detection methodology based on sulfur-oxidizing bacteria (SOB) has been developed for the rapid and reliable detection of toxic chemicals in water. The methodology exploits the ability of SOB to oxidize sulfur particles in the presence of oxygen to produce sulfuric acid. The reaction results in an increase in electrical conductivity (EC) and a decrease in pH. The assay is based on the inhibition of SOB in the presence of toxic chemicals by measuring changes in EC and pH. We found that SOB biosensor can detect toxic chemicals, such as heavy metals and CN−, in the 5-2000 ppb range. One bacterium was isolated from an SOB biosensor and the 16S rRNA gene of the bacterial strain has 99% and 96% sequence similarity to Acidithiobacillus sp. ORCS6 and Acidithiobacillus caldus DSM 8584, respectively. The isolate was identified as A. caldus SMK. The SOB biosensor is ideally suited for monitoring toxic chemicals in water having the advantages of high sensitivity and quick detection.     

Toxicity monitoring of endocrine disrupting chemicals (EDCs) using freeze-dried recombinant bioluminescent bacteria

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was determined on the first four freeze-dried bacteria, while nonspecific cellular stresses were measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.     

A bioluminescent sensor for high throughput toxicity classification

A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon. The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage. In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples. As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased. This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range. In particular, each test requires only 4 h to give clear bioluminescent response signature. Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response. The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.     

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

Design and Application of a Biosensor for Monitoring Toxicity of Compounds to Eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

Glass beads as a solid matrix in in vitro study of the role of polyamines in cold hardiness of white clover

Glass beads were used as an alternative to agar in the study of the role of polyamines in cold hardiness of white clover. Plantlet growth performance, in vitro hardening and cold stress tolerance were similar on both the agar solidified medium and the liquid medium in glass beads matrix. Glass beads allowed media exchange without plant transfer and an easy monitoring of the uptake of putrescine synthesis inhibitor, ¹⁴C difluoromethylornithine from the medium. The matrix is recommended in in vitro studies of whole plant physiology, screening procedures and bioassays where media exchange and/or uniform application of a selection pressure is required. There is also 60% saving on media components and the beads can be re-used after acid wash.Abbreviations DFMA difluoremethylarginine - DFMO difluoromethylornithine - MS Murashige and Skoog salts - Kcpm thousand counts per minute     

A whole cell bioluminescent biosensor for the detection of membrane-damaging toxicity

The recombinant bacteria strain DPD2540, containing afabA::luxCDABE fusion, was used to detect the toxicity of various chemicals in this study. Membrane damaging agents such as phenol, ethanol, and cerulenin induced a rapid bioluminescent response from this strain. Other toxic agents, such as DNA-damaging or oxidative-damaging chemicals, showed a delayed bioluminescent response in which the maximum peak appeared over 150min after induction. This strain was also tested for measurement of toxicity in field samples such as wastewater and river water effluents.     

Enhancing the sensitivity of a two-stage continuous toxicity monitoring system through the manipulation of the dilution rate.

Optimization of the dilution rates has been studied to provide an enhanced sensitivity to toxicity by several recombinant bioluminescent Escherichia coli strains, TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE) and DPD2540 (fabA::luxCDABE), in the two-stage continuous toxicity monitoring system. It was found that the sensitivity of both TV1061 and DPD2794 to a pulse injection of phenol and mitomycin C increased with a decrease in the dilution rate. The sensitivity, however, for all the strains to step injections of the toxic chemicals was found to increase with an increase in the dilution rate up to a certain dilution rate and then decreased, mainly due to the rapid washing out of the injected chemicals. The response kinetics of the strains were explained by evaluating the mode of action of the recombinant bioluminescent bacteria to toxicity with the dilution rate, the operating parameter of minibioreactors under consideration in this study.     

Monitoring and classification of PAH toxicity using an immobilized bioluminescent bacteria

An immobilized recombinant bioluminescent Escherichia coli strain, harboring a lac::luxCDABE fused plasmid, which shows lower bioluminescence levels when cellular metabolism is inhibited, was used to monitor the cellular toxicity of polycyclic aromatic hydrocarbons (PAHs). PAHs, classified as pericondensed (PCPAHs) or catacondensed (CCPAHs) according to their molecular structures, were differentiable according to the response of this biosensor. Only CCPAHs were found to cause cellular toxicity, resulting in a dose-dependent decrease in the bioluminescent output. The induction of cellular toxicity by CCPAHs and PCPAHs was compared with acute toxicity predictions obtained using the quantitative structure-activity relationship (QSAR) model. A good relationship was obtained between the toxicities determined with the bioluminescent response of the immobilized bacterium GC2 and the QSAR model. It was also found that the present study offers a new method of predicting the cellular toxicities of CCPAHs or PCPAHs using this biosensor.     

A multi-channel continuous toxicity monitoring system using recombinant bioluminescent bacteria for classification of toxicity

A multi-channel system for continuous toxicity monitoring and classification of toxicity was developed based upon a previously developed two-stage minibioreactor system. The multi-channel system consists of a series of a two-stage minibioreactor systems connected by a fiber optic probe to a luminometer. Each channel was used for cultivating different recombinant bacterial strains, such as TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2540 (fabA::luxCDABE), which are induced by protein-, DNA-, and cell membrane damaging-agents, respectively. GC2 (lac::luxCDABE) is a bacterium expressing bioluminescence constitutively, which shows a reduction in its light level as cellular toxicity increases. Artificial wastewater samples were made by combining toxic chemicals, including Mitomycin C (a representative DNA damaging agent), phenol (a representative protein damaging agent), and cerulenin (a representative cell membrane damaging agent), and injecting this sample into each channel in order to simulate the detection of toxicity for mixed chemical samples. Each channel showed a specific bioluminescent response due to the toxic chemicals contained in the sample wastewater, while GC2 showed a general response to cellular toxicity. By using this multi-channel continuous toxicity monitoring system, classification of toxicity in field samples was found to be possible.     

In vitro control of de novo flower differentiation from tobacco thin cell layers cultured on a liquid medium

Thin cell layers of tobacco (three to six layers of epidermal and subepidermal cells) were allowed to float on the surface of a liquid medium. Whereas the control on an identical medium solidified by agar gave 100% of explant-forming flowers, no flowers formed in the absence of agar (100% of the explants formed buds). In order to initiate flower formation, various modifications of the liquid medium were tried: different ratios of indolyl-3-butyric acid (IBA) to kinetin (Kin) from 1 to 100, a range of pH from 4.0 to 7.0, and glass beads of different diameters (in an attempt to change the physico-chemical characteristics of the culture substrate itself). On a liquid medium with glass beads, four types of morphogenesis (flowers, buds, a mixed programme of flowers and buds, and a non-morphogenetic programme – i.e. the explants remained unchanged) were separately induced by changing one of the three factors: pH, IBA and Kin in equimolar concentrations, and diameter of the glass beads. Changing only the IBA/Kin ratio failed to provoke flower differentiation. pH of the medium was found to change after autoclaving and the effect of glass beads on morphogenesis was partly related to modification of pH.     

Chemiluminescent choline biosensor using histidine-modified peroxidase immobilised on metal-chelate substituted beads and choline oxidase immobilised on anion-exchanger beads co-entrapped in a photocrosslinkable polymer

A novel sensing layer design is presented based on the non-covalent immobilisation of enzymes on derivatized Sepharose beads subsequently entrapped in PVA-SbQ photopolymer. Two different modified Sepharose beads were used, IDA- and DEAE-Sepharose, for the immobilisation, respectively, of horseradish peroxidase (HRP) modified with histidine, and choline oxidase (Chx). The HRP-IDA-Sepharose-based sensing layer was used in a flow injection analysis chemiluminescent system as the basis of an H2O2 biosensor. It was shown that the pre-immobilisation on IDA-Sepharose beads enhanced the sensing layer stability and enabled the immobilisation of a larger amount of enzyme. A 1.8 mg charge of HRP-IDA-Sepharose beads in the sensing layer produced the most sensitive H2O2 biosensor. Such an analytical system exhibited very good performances, with a cycle time of 2 min and a detection limit of 15 pmol (detection ranging over four decades at least), and an unusual long operational stability of 200 measurements (CV, 3.5%). The HRP-IDA-Sepharose beads were then combined with Chx-DEAE-Sepharose. With this modified Sepharose-based biosensor the limit of detection for choline (S/N, 3) was equal to 0.5 pmol and the working range was 0.35 pmol-10 nmol. Moreover, the cycle time was only 2.5 min with the new sensing layer, and a long operational stability of 150 successive assays was found, with a variation coefficient of 2.6%.     

A novel continuous toxicity test system using a luminously modified freshwater bacterium

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.     

Measurement of biologically available naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor

Genetically constructed microbial biosensors for measuring organic pollutants are mostly applied in aqueous samples. Unfortunately, the detection limit of most biosensors is insufficient to detect pollutants at low but environmentally relevant concentrations. However, organic pollutants with low levels of water solubility often have significant gas-water partitioning coefficients, which in principle makes it possible to measure such compounds in the gas rather than the aqueous phase. Here we describe the first use of a microbial biosensor for measuring organic pollutants directly in the gas phase. For this purpose, we reconstructed a bioluminescent Pseudomonas putida naphthalene biosensor strain to carry the NAH7 plasmid and a chromosomally inserted gene fusion between the sal promoter and the luxAB genes. Specific calibration studies were performed with suspended and filter-immobilized biosensor cells, in aqueous solution and in the gas phase. Gas phase measurements with filter-immobilized biosensor cells in closed flasks, with a naphthalene-contaminated aqueous phase, showed that the biosensor cells can measure naphthalene effectively. The biosensor cells on the filter responded with increasing light output proportional to the naphthalene concentration added to the water phase, even though only a small proportion of the naphthalene was present in the gas phase. In fact, the biosensor cells could concentrate a larger proportion of naphthalene through the gas phase than in the aqueous suspension, probably due to faster transport of naphthalene to the cells in the gas phase. This led to a 10-fold lower detectable aqueous naphthalene concentration (50 nM instead of 0.5 micro M). Thus, the use of bacterial biosensors for measuring organic pollutants in the gas phase is a valid method for increasing the sensitivity of these valuable biological devices.     

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.