• KSBB Journal
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• v.14 no.1
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• pp.91-95
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• 1999

The objective of this work was to improve bioluminescence stability of Photobacterium phosphoreum when it stored in view of developing continuous on-line monitoring system for pullutants. Long-term experiments were made to determine the effect of immobilization and storage temperature on the maintenance and stability of bioluminescence from luminescent bacteria. The immobilized cells of P. phosphoreum were compared with free cells in terms of maintenance of bioluminescence at room temperature. The bioluminescence of cells immobilized showed higher bioluminescence intensity that free and strontium bioluminescence stability was investigated with free and immobilized cells stored at $20^{\circ}C,\; 4^{\circ}C,\; -20^{\circ}C\;and\;-70^{\circ}C$for 20 days. Both free and immobilized cells stored at $4^{\circ}C$ emitted a stable bioluminescence while the bioluminescence markedly decreased with those stored at $20^{\circ}C,\;-20^{\circ}C\;and\; -70^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.242-249
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• 1997

Stability of bioluminescence was investigated with Photobacterium phosphoreum immobilized on the strontium alginate in order to develope continuous real time monitoring of pollutants. The stability of bioluminescence emission was improved by prolonged aging time. The aging time of ${\geq}40$ min and the cell concentration of ${\leq}0.6\;of\;OD_660$ were selected for the immobilization of P. phosphoreum to give linearity between cell concentrations and bioluminescence intensity. In sensitivity tests using phenol, it was found that this compound quenched bioluminescence proportional to the concentration without lowering of cell growth. The lower value for maximum quenching ($q_s$) and higher dissociation constant ($K_s$) were observed with strontium-alginate immobilized cells compared to free cells. The response of bioluminescence to toxicants was evaluated with the immobilized luminescent bacteria. The sensitivity of the immobilized cells was found to be good in response to toxicants, 4-nitrophenol, salicylate and cadmium, when evaluated with a specific rate of bioluminescence quenching.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.94-99
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• 2000

Bioluminescence is being used as a prevailing reporter of gene expression in microorganisms and mammalian cells. Bacterial bioluminescence draws special attention from environmental biotechnologists since it has many advantageous characteristics, such as no requirement of extra substractes, highly sensitive, and on-line measurability. Using bacterial bioluminescence as a reporter of toxicity has replaced the classical toxicity monitoring technology of using fish or daphnia with a cutting-edge technology. Fusion of bacterial stress promoters, which control the transcription of stress genes corresponding to heat-shock, DNA-, or oxidative-damaging stress, to the bacterial lux operon has resulted in the development of novel toxicity biosensors with a short measurement time, enhanced sensitivity, and ease and convenient usage. Therefore, these recombinant bioluminescent bacteria are expected to induce bacterial bioluminescence when the cells are exposed to stressful conditions, including toxic chemicals. We have used these recombinant bioluminescent bacteria in order to develop toxicity biosensors in a continuous, portable, or in-situ measurement from for air, water, and soil environments. All the data obtained from these toxicity biosensors for these environments were found to be repeatable and reproducible, and the minimum detection level of toxicity was found to be ppb (part per billion) levels for specific chemicals.

• Journal of Korean Society of Environmental Engineers
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• v.30 no.1
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• pp.79-84
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• 2008

This paper describes the application of bioluminescence stimulating agents on a genetically engineered microorganism, Pseudomonas putida mt-2 KG1206, to monitor toluene analogs using in groundwater samples from petroleum hydrocarbon contaminated sites. The maximum bioluminescent response with pure chemicals followed in the order: m-methyl benzyl alchohol > m-toluate > toluene > m-xylene > benzoate > p-xylene > o-xylene. Generally, the bioluminescence production of strain mixed with groundwater samples was dependent on the contaminated total inducer concentrations. However, few samples showed opposite results, where these phenomena may be caused by the complexicity of environmental samples. Two chemicals, SL(sodium lactate) and KNO$_3$, were tested to determine a better bioluminescence stimulant. Both chemicals stimulate the bioluminescence activity of strain KG1206, however, a slightly high bioluminescence was observed with nitrogen chemical. This selected stimulant was then tested on samples collected from contaminated groundwater samples. The bioluminescence activity of all samples mixed with the strain was stimulated with KNO$_3$ amendment. This suggests that the low bioluminescence activity exhibited by the environmental groundwater samples can be stimulated by amending the culture with a proper agent, such as nitrogen compound. These findings would be useful, especially, when strain was used to monitor the groundwater samples contaminated with low inducer contaminants. Overall, the results of this study found the ability of bioluminescence producing bacteria to biosensor a specific group of environmental contaminants, and suggest the potential for more efficient preliminary application of this engineered strain in a field-ready bioassay.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.349-353
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• 2002

Two recombinant bacteria containing luxAB showed an increased tolerance to stresses associated with lyophilization, when the cells were freeze-dried in the presence of trehalose. In the case of a recombinant, UV2, only $2.5\%$ of the original bioluminescence and $2.7\%$ of the cell viability were restored after 4 h of freeze-drying without trehalose, which implies that the cells were heavily damaged during the dehydration. To improve these losses, trehalose was added before freeze-drying using different modes. Trehalose increased the bioluminescence and the viability of freeze-dried UV2 under all conditions tested, and it was also observed that the addition of trehalose to the cultures (final concentration of 0.08 M) for 15 min before the freeze-drying resulted in the restoration of $45\%$ of the original bioluminescence and $50\%$ of the cell viability. Trehalose also showed a similar efficacy with the other luminescent recombinant, YH9. Therefore, it was tentatively concluded that trehalose played a role as a protective agent in the freeze-drying of bacterial cells.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.250-257
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• 1997

A new sensing system based on the immobilization of luminescent bacteria, photobacterium phosphoreum, was proposed for continuous real-time monitoring of pollutants. The response curves demonstrate that Photobacterium phosphoreum immobilized on the strontium alginate were very sensitive to seven reference chemicals used. The significant inhibitory concentrations for bioluminescence emission were 5 ppm for Pb$(NO_3)_2$), $NiCl_2$, $CdCl_2$, 50 ppm for $NaASO_2$, 0.1 ppm for $HgCl_2$, 0.5 ppm for pentachlorophenol and less than 5 ppm for SDS, respectively. The alginate mixed-cells (AMC) retained their luminescence during experimental period (29 days) under storage condition of $-80^{\circ}C$. The variables affecting performance of continuous flow through monitoring (CFTM) was optimized in order to ensure stability and efficiency. The flow through cell with strontium-alginate immobilized luminescent bacteria was tested with salicylate and 4-nitrophenol. A rapid response of luminescence was recorded by time drive mode in bioluminescence spectrometer after exposure to both toxicants.

• Journal of Soil and Groundwater Environment
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• v.18 no.4
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• pp.32-39
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• 2013

This research focused on the effects of heavy metals on the biomonitoring activity of genetically engineered bioluminescent bacteria, Pseudomonas putida mt-2 KG1206. KG1206 was exposed to single or binary mixtures of different heavy metals as well as soils contaminated with heavy metals. In case of single exposure with different inducer pollutant, the toxicity order was as followings : As(III) > Cd, As(V) >> Cu, Cr(VI). The toxic effects of the binary mixtures was compared to the expected effect based on a simple theory of probabilities. The interactive effects were mostly additive, while in few cases antgonistic and synergistic mode of action was observed for some concentration combinations. No considerable correlation was found between the total metal contents in soils and the bioluminescence activity of each sample. However, statistically significant differences (p = 0.0102) were observed between two groups, classified based on arsenite contamination. These results demonstrate the usage of recombinant bioluminescent microorganism in biomonitoring and the complex interactive effects of metals.

• Korean Journal of Food Science and Technology
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• v.18 no.2
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• pp.88-92
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• 1986

Determination of bacterial adenosine triphosphate (ATP) based on luciferin-luciferase bioluminescene reaction was applied to the measurement of bacteria on the surface of chicken as an alternative rapid method. The light yield was proportional to the concentration of ATP giving a straight line within a range of $10^{-10}\;to\;10^{-6}M$. The bacteria isolated from the surface of chicken were identified as Escherichia coli, Hafnia alvei, Pseudomonas putida and Aeromonas hydrophila. When the ATP contents of each bacteria were determined by bioluminescence reaction and compared, there was no significant difference (r = 0.95). The Patterns of growth curves for E. coli look very similar, when the bacterial growth was monitored by ATP content and viable cell count. When bacterial ATP of each samples collected every 2 days during storage for 14 days at $4^{\circ}C$was determined and compared with viable cell count, it gave a good correlation (r = 0.95, n = 32).

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.212-215
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• 2000

Recombinant bioluminescent bacterial strains that use specific promoters fused to the bioluminescence genes (lux genes) have been applied in environmental monitoring. Advantages of using recombinant bioluminescent bacteria as blosensing cells include rapid responses, low costs, and improved reproducibility. In this study, a recombinant Escherichia coli, GC2, containing a lac::luxCDABE fusion immobilized with solid agar media and glass beads was used to estimate the effect of this soil flushing technique. This bacterium constitutively emits light under normal conditions (no toxic chemicals). When growth and metabolism of these bioluminescent bacteria is inhibited by their exposure to toxic chemicals, the bioluminescence (BL) is reduced. A biosurfactant, rhamnolipids, was used to extract phenanthrene from the soil after flushing.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.390-393
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• 2000

In this work, effects of trehalose on the recovery of bioluminescence and viability of luxAB-containing recombinant bacteria were investigated. In case of a recombinant, UV2, only 2.5% of bioluminescence and 2.7% of cell viability were restored after 3.5hr of freeze-drying without trehalose, which implies that cells were heavily damaged during the dehydration. To improve these losses, trehalose was added before freeze-drying on different modes. Trehalose increased the bioluminescence and viability of freeze-dried UV2 in all conditions tested and it was observed that addition of trehalose into the broth(final concentraion, 0.08M) for 15min before the freeze-drying resulted in 45% of bioluminescence and 50% of cell viability. For the other luminescent recombinant, YH9, trehalose showed a similar efficacy.