Exposure of Metal Oxide Nanoparticles on the Bioluminescence Process of Pu- and Pm-lux Recombinant P. putida mt-2 Strains.

Comparison of the effects of metal oxide nanoparticles (NPs; CuO, NiO, ZnO, TiO2, and Al2O3) on different bioluminescence processes was evaluated using two recombinant (Pm-lux and Pu-lux) strains of Pseudomonas putida mt-2 with same inducer exposure. Different sensitivities and responses were observed according to the type of NPs and recombinant strains. EC50 values were determined. The negative effects on the bioluminescence activity of the Pm-lux strain was greater than for the Pu-lux strains for all NPs tested. EC50 values for the Pm-lux strain were 1.7- to 6.2-fold lower (corresponding to high inhibition) than for Pu-lux. ZnO NP caused the greatest inhibition among the tested NPs in both strains, showing approximately 11 times less EC50s of CuO, which appeared as the least inhibited. Although NPs showed different sensitivities depending on the bioluminescence process, similar orders of EC50s for both strains were observed as follows: ZnO > NiO, Al2O3 > TiO2 > CuO. More detailed in-depth systematic approaches, including in the field of molecular mechanisms, is needed to evaluate the accurate effect mechanisms involved in both bioluminescence metabolic processes.

Comparisons of the Effect of Different Metal Oxide Nanoparticles on the Root and Shoot Growth under Shaking and Non-Shaking Incubation, Different Plants, and Binary Mixture Conditions.

We evaluated the toxicity of five metal oxide nanoparticles (NPs) in single or binary mixtures based on root and shoot growth of two plant species under non-shaking and shaking conditions. The effects of NPs on root and shoot growth differed depending on the NP type, incubation condition, and plant type. The half maximal effective concentration (EC50) of NPs based on root growth were significantly lower, by 2.6-9.8 times, under shaking than non-shaking conditions (p = 0.0138). The magnitude of the effects of NPs followed the order CuO > ZnO > NiO >> Al2O3, TiO2. In addition, Lactuca sativa L. was more sensitive to the tested NPs than Raphanus sativus L., with an EC50 0.2-0.7 times lower (p = 0.0267). The observed effects of 12 combinations of binary NP mixtures were slightly, albeit non-significantly, lower than expected, indicative of an additive effect of the individual NPs in the mixtures. The results emphasize the importance of careful plant model selection, appropriate application of incubation conditions, and consideration of chemical mixtures rather than single compounds when evaluating the effects of metal oxide NPs.

Evaluation of the Effects of Particle Sizes of Silver Nanoparticles on Various Biological Systems.

Seven biological methods were adopted (three bacterial activities of bioluminescence, enzyme, enzyme biosynthetic, algal growth, seed germination, and root and shoot growth) to compare the toxic effects of two different sizes of silver nanoparticles (AgNPs). AgNPs showed a different sensitivity in each bioassay. Overall, the order of inhibitory effects was roughly observed as follows; bacterial bioluminescence activity ≈ root growth > biosynthetic activity of enzymes ≈ algal growth > seed germination ≈ enzymatic activity > shoot growth. For all bacterial activities (bioluminescence, enzyme, and enzyme biosynthesis), the small AgNPs showed statistically significantly higher toxicity than the large ones (p < 0.0036), while no significant differences were observed among other biological activities. The overall effects on the biological activities (except shoot growth) of the small AgNPs were shown to have about 4.3 times lower EC50 (high toxicity) value than the large AgNPs. These results also indicated that the bacterial bioluminescence activity appeared to be an appropriate method among the tested ones in terms of both sensitivity and the discernment of particle sizes of AgNPs.

Comparative Effects of Particle Sizes of Cobalt Nanoparticles to Nine Biological Activities.

The differences in the toxicity of cobalt oxide nanoparticles (Co-NPs) of two different sizes were evaluated in the contexts of the activities of bacterial bioluminescence, xyl-lux gene, enzyme function and biosynthesis of ß-galactosidase, bacterial gene mutation, algal growth, and plant seed germination and root/shoot growth. Each size of Co-NP exhibited a different level of toxicity (sensitivity) in each biological activity. No revertant mutagenic ratio (greater than 2.0) of Salmonella typhimurium TA 98 was observed under the test conditions in the case of gene-mutation experiments. Overall, the inhibitory effects on all five bacterial bioassays were greater than those on algal growth, seed germination, and root growth. However, in all cases, the small Co-NPs showed statistically greater (total average about two times) toxicity than the large Co-NPs, except in shoot growth, which showed no observable inhibition. These findings demonstrate that particle size may be an important physical factor determining the fate of Co-NPs in the environment. Moreover, combinations of results based on various biological activities and physicochemical properties, rather than only a single activity and property, would better facilitate accurate assessment of NPs' toxicity in ecosystems.

Toxic Effects of Metal Oxide Nanoparticles Based on the Enzymatic Activity and Biosynthesis of ß-Galactosidase Using a Mutant Strain of E. coli.

The effects of six metal oxide nanoparticles (MO-NPs) on the activity and biosynthesis of an enzyme (ß-galactosidase) were examined using a mutant strain of E. coli. Different sensitivities were observed according to the type of NP and metabolic process. The toxic effects on enzyme activity were significantly greater than on biosynthesis (p < 0.011), except in the presence of NiO. In both cases, ZnO NP caused the greatest inhibition among the tested NPs, followed by CuO. The EC50s for ZnO were 0.19 and 3.68 mg/L for enzyme activity and biosynthesis, respectively. Similar orders of toxicity were observed as follows: ZnO > CuO > NiO > Co3O4 > TiO2, Al2O3 for enzyme activity; and ZnO > CuO > NiO ≫ Al2O3, TiO2, Co3O4 for the biosynthetic process. More systematic research, including in-depth studies like investigation of the molecular mechanisms, is necessary to elucidate the detailed mechanisms of inhibition involved in both metabolic processes.

Ecotoxicity evaluation of Cu- and Fe-CNT complexes based on the activity of bacterial bioluminescence and seed germination.

The toxic effects of the composites of Fe0 and Cu0 with different percentages of CNTs were examined based on the activity of bacterial bioluminescence and seed germination. In terms of the EC50 values, the toxic effects of Cu0 on bacterial bioluminescence and seed germination were approximately 2 and 180 times greater than that of Fe0, respectively. The toxicity increased with increasing CNT content in the Cu-CNT mixtures for both organisms, whereas opposite results were observed with Fe-CNT mixtures. The mean toxic effects of Cu-CNT (6%) were approximately 1.3-1.4 times greater than that of Cu-CNT (0%), whereas the toxic effects of Fe-CNT (6%) were approximately 2.1-2.5 times lower than that of Fe-CNT (0%) for both the bioluminescence activity and seed germination. The causes of this phenomenon are unclear at this point. More research will be needed to elucidate the mechanism of the toxicity of nano-mixture materials and the causes of the different patterns of toxicity with Cu- and Fe-CNT mixtures.

Toxicity Evaluation of Individual and Mixtures of Nanoparticles Based on Algal Chlorophyll Content and Cell Count.

The toxic effects of individual and binary mixtures of five metal oxide nanoparticles (NPs) were evaluated based on changes in two endpoints of algal growth: the cell count and chlorophyll content. Various effects were observed according to the concentration tested and type of NPs, and there were no significant differences in findings for the two endpoints. In general, ZnO NPs caused the greatest inhibition of algal growth, and Fe2O3 NPs the least. The EC50 for ZnO was 2.0 mg/L for the cell count and 2.6 mg/L for the chlorophyll content, and it was 76 and 90 mg/L, respectively, for Fe2O3. The EC50 values were in the order ZnO > NiO > CuO > TiO2 > Fe2O3. Subsequently, the effects of 30 binary mixture combinations on the chlorophyll content were evaluated. Comparisons were made between the observed and the expected toxicities calculated based on the individual NP toxicities. Overall, additive action (67%) was mainly observed, followed by antagonistic (16.5%) and synergistic (16.5%) actions. These results suggest that environmental exposure to NP mixtures may cause toxicity levels similar to the sum of those of the constituent NPs.

Evaluation of the Effects of Nanoparticle Mixtures on Brassica Seed Germination and Bacterial Bioluminescence Activity Based on the Theory of Probability.

Effects of binary mixtures of six metal oxide nanoparticles (NPs; 54 combinations) on the activities of seed germination and bacterial bioluminescence were investigated using the theory of probability. The observed toxicities of various NPs combinations were compared with the theoretically expected toxicities, calculated based on individual NPs toxicities. Different sensitivities were observed depending on the concentrations and the types of NPs. The synergistic mode (67%; observed toxicity greater than expected toxicity) was predominantly observed in the bioluminescence test, whereas both synergistic (47%) and additive (50%) modes were prevalent in the activity of seed germination. With regard to overall analysis, a slightly high percentage (56%) of the synergistic mode of action was (30 out of 54 binary mixture combinations; p < 0.0392) observed. These results suggest that the exposure of an NPs mixture in the environment may lead to a similar or higher toxicity level than the sum of its constituent NPs would suggest. In addition, one organism for assessment did not always show same results as those from a different assessment. Therefore, combining results of different organisms exposed to a wide range of concentrations of binary mixture will more properly predict and evaluate the expected ecotoxicity of pollutants on environments.

Influence of Incubation Conditions on the Nanoparticles Toxicity Based on Seed Germination and Bacterial Bioluminescence.

The effects of the modified incubation conditions of a conventional bioassay on the toxicity of partially soluble nanoparticles (NPs) were evaluated based on the activity of seed germination and bacterial bioluminescence. Different levels of toxicity were observed for seed germination (CuO > ZnO > NiO) and bacterial bioluminescence (ZnO > CuO > NiO). The NP inhibition of seed germination increased strongly under modified incubation conditions: sample volume from 5 mL to 10 mL, shaking from none to 70 rpm, and working vessel from a Petri dish (+/− filter paper) to an Erlenmeyer flask (no filter paper). In the case of seed germination, the toxicity levels of NPs under the modified conditions were 1.26 to 8.49 times higher than the conventional method according to the type of NPs and modified conditions (p-values < 0.05). No significant differences in bacterial bioluminescence were observed between conventional (130 rpm) and modified (160 rpm) conditions. These findings show that for an accurate assessment of partially soluble NPs toxicity in ecosystems, the conventional bioassay method, which is designed for soluble chemicals, needs to be performed under modified conditions because of their insolubility.

Application of the freeze-dried bioluminescent bioreporter Pseudomonas putida mt-2 KG1206 to the biomonitoring of groundwater samples from monitoring wells near gasoline leakage sites.

This study examined the applicability of a freeze-dried bioluminescent bioreporter, Pseudomonas putida mt-2 KG1206 (called KG1206), to the biomonitoring of groundwater samples. Samples were collected from the monitoring wells of gas station tanks or old pipeline leakage sites in Korea. In general, the freeze-dried strain in the presence of pure inducer chemicals showed low bioluminescence activity and a different activity order compared with that of the subcultured strain. The effects of KNO3 as a bioluminescence stimulant were observed on the pure inducers and groundwater samples. The stimulation rates varied according to the type of inducers and samples, ranging from 2.2 to 20.5 times (for pure inducers) and from 1.1 to 11 times (for groundwater samples) the total bioluminescence of the control. No considerable correlations were observed between the bioluminescence intensity of the freeze-dried strain and the inducer concentrations in the samples (R 2 < 0.1344). However, samples without a high methyl tertiary butyl ether (MTBE) level and those from the gas station leakage site showed reasonable correlations with the bioluminescence activity with R 2 values of 0.3551 and 0.4131, respectively. These results highlight the potential of using freeze-dried bioluminescent bacteria as a rapid, simple, and portable tool for the preliminary biomonitoring of specific pollutants at contaminated sites.

Effects of properties of metal-contaminated soils on bacterial bioluminescence activity, seed germination, and root and shoot growth.

This study examined the effects of several factors (metal contents and soil properties) on bacterial bioluminescence activity, seed germination and root/shoot growth of Lactuca in metal-contaminated soils. Each bioassay showed different sensitivities to extractants of soil samples. Average sensitivities of the bioassay were in the following order: root growth > bioluminescence ≥ shoot growth ≥ seed germination. Both total and weak acid-extracted metal contents showed no observable correlations with the activity of any bioassays (r(2) < 0.279). However, reasonable correlations were observed between the bioluminescence activity and organics (r(2) = 0.7198) as well as between root growth and CEC (r(2) = 0.6676). Effects of soils were difficult to generalize since they were dependent on many factors, such as soil properties, metal contents, and the organism used in each test. Nonetheless, these results indicated that a battery of bioassays is an effective strategy for assessment of contaminated soils. Furthermore, specific soil factors were shown to more influence on soil toxicity, depending on the type of bioassay.

Interactive Effects of Metal Mixtures on Seed Germination and Bioluminescence Activities based on the Theory of Probabilities.

The interactive effects of metal mixtures on seed germination (36 combinations) and bacterial bioluminescence (60 combinations) activities were investigated based on the theory of probabilities. Observed effects were compared with expected effects, which were calculated based on single metal activities. Different responses and sensitivities were observed depending on the types and concentrations of mixture metals as well as the organisms tested. In general, an additive mode of action (observed and expected effects are nearly same) was mostly common in the bioluminescence assay, whereas synergistic and additive inhibition modes were mostly observed in seed germination. Therefore, the mixture effects of the different bioassays could not be generalized because they were dependent on the types and concentrations of chemicals and organisms used. The results indicated that mixture, rather than single, effects by various bioassays and wide ranges of concentration combinations may comprise a better strategy for the bioassessment of contaminated environments.

Effects of Monotypic and Binary Mixtures of Metal Oxide Nanoparticles on Microbial Growth in Sandy Soil Collected from Artificial Recharge Sites.

The potential effects of monotypic and binary metal oxide nanoparticles (NPs, ZnO, NiO, Co3O4 and TiO2) on microbial growth were evaluated in sandy soil collected from artificial recharge sites. Microbial growth was assessed based on adenosine triphosphate (ATP) content, dehydrogenase activity (DHA), and viable cell counts (VCC). Microbial growth based on ATP content and VCC showed considerable differences depending on NP type and concentration, whereas DHA did not significantly change. In general, ZnO NPs showed the strongest effect on microbial growth in all measurements, showing an EC50 value of 10.9 mg/L for ATP content. The ranking (EC50) of NPs based on their effect on microbial growth assessed by ATP content and VCC was ZnO > Co3O4 > NiO > TiO2. Upon exposure to binary NP mixtures, synergistic and additive modes of action were observed for ATP content and VCC, respectively. The ranges of observed (P(O)) and expected (P(E)) activity were 83%-92% and 78%-82% of the control (p-value 0.0010) based on ATP content and 78%-95% and 72%-94% of the control (p-value 0.8813) based on VCC under the tested conditions, respectively. The results indicate that the effects of NP mixtures on microbial growth in the sandy soil matrix were as great, or greater, than those of single NPs. Therefore, understanding the effects of single NPs and NP mixtures is essential for proper ecological risk assessment. Additionally, these findings demonstrate that the evaluation of NP effects may be profoundly influenced by the method of microbial growth measurement.

Effects of binary mixtures of inducers (toluene analogs) and of metals on bioluminescence induction of a recombinant bioreporter strain.

This paper investigated the effects of binary mixtures of bioluminescence inducers (toluene, xylene isomers, m-toluate) and of metals (Cu, Cd, As(III), As(V), and Cr) on bioluminescence activity of recombinant (Pm-lux) strain KG1206. Different responses and sensitivities were observed depending on the types and concentrations of mixtures of inducers or metals. In the case of inducer mixtures, antagonistic and synergistic modes of action were observed, whereas metal mixtures showed all three modes of action. Antagonistic mode of action was most common for mixtures of indirect inducers, which showed bioluminescence ranging from 29% to 62% of theoretically expected effects (P(E)). On the other hand, synergistic mode of action was observed for mixtures of direct and indirect inducers, which showed bioluminescence between 141% and 243% of P(E). In the case of binary metal mixtures, bioluminescence activities were ranged from 62% to 75% and 113% to 164% of P(E) for antagonistic and synergistic modes of action, respectively (p-values 0.0001-0.038). Therefore, mixture effects could not be generalized since they were dependent on both the types and concentrations of chemicals, suggesting that biomonitoring may constitute a better strategy by investigating types and concentrations of mixture pollutants at contaminated sites.

Toxic effects of nanoparticles on bioluminescence activity, seed germination, and gene mutation.

The potential environmental toxicities of several metal oxide nanoparticles (NPs; CuO, TiO2, NiO, Fe2O3, ZnO, and Co3O4) were evaluated in the context of bioluminescence activity, seed germination, and bacterial gene mutation. The bioassays exhibited different sensitivities, i.e., each kind of NP exhibited a different level of toxicity in each of the bioassays. However, with a few exceptions, CuO and ZnO NPs had most toxic for germination of Lactuca seed (EC50 0.46 mg CuO/l) and bioluminescence (EC50 1.05 mg ZnO/l). Three NPs (Co3O4, TiO2, and Fe2O3) among all tested concentrations (max. 1,000 mg/l) showed no inhibitory effects on the tested organisms, except for Co3O4 NPs on bioluminescence activity (EC50 62.04 mg/l). The sensitivity of Lactuca seeds was greater than that of Raphanus seeds (EC50 0.46 mg CuO/l versus 26.84 mg CuO /l ). The ranking of metal toxicity levels on bioluminescence was in the order of ZnO > CuO > Co3O4 > NiO > Fe2O3, TiO2, while CuO > ZnO > NiO > Co3O4, Fe2O3, TiO2 on germination. No revertant mutagenic ratio (greater than 2.0) of Salmonella typhimurium TA 98 was observed under any tested condition. These findings demonstrate that several bioassays, as opposed to any single one, are needed for the accurate assessment of NP toxicity on ecosystems.

Joint effects of heavy metal binary mixtures on seed germination, root and shoot growth, bacterial bioluminescence, and gene mutation.

This investigation was to assess the joint effects of metal binary mixtures on seed germination, root and shoot growth, bacterial bioluminescence, and gene mutation based on the one toxic unit (1 TU) approach. Different sensitivities and orders of toxicity of metal mixtures were observed among the bioassays. In general, mostly additive or antagonistic effects were observed, while almost no synergistic effects by the binary metal mixtures in all bioassays. Therefore, the combined effects of heavy metals in the different bioassays were difficult to generalize since they were dependent on both chemical type and the organism used in each bioassay. However, these results indicate that a battery of bioassays with mixture chemicals as opposed to just a single assay with single metal is a better strategy for the bioassessment of environmental pollutants.

Evaluation of the toxic effects of arsenite, chromate, cadmium, and copper using a battery of four bioassays.

The sensitivities of four different kinds of bioassays to the toxicities of arsenite, chromate, cadmium, and copper were compared. The different bioassays exhibited different sensitivities, i.e., they responded to different levels of toxicity of each of the different metals. However, with the exception of the α-glucosidase enzyme activity, arsenite was the most toxic compound towards all the tested organisms, exhibiting the highest toxic effect on the seeds of Lactuca, with an EC(50) value of 0.63 mg/L. The sensitivities of Lactuca and Raphanus were greater than the sensitivities of two other kinds of seeds tested. Therefore, these were the seeds appropriate for use in a seed germination assay. A high revertant mutagenic ratio (5:1) of Salmonella typhimurium was observed with an arsenite concentration of 0.1 µg/plate, indicative of a high possibility of mutagenicity. These different results suggested that a battery of bioassays, rather than one bioassay alone, is needed as a more accurate and better tool for the bioassessment of environmental pollutants.

Application of stimulating agents on the immobilized bioluminescence strain Pseudomonas putida mt-2 KG1206, preserved by deep-freezing, for the convenient biomonitoring.

This study was conducted to develop methods for the application of an immobilized bioluminescence strain (KG1206), preserved by deep-freezing (DF), for the monitoring of contaminated environments. The immobilized cells, preserved by DF, required approximately 2 hr for reconstitution of their activity. A large reduction in bioluminescence was observed due to the DF process; 0.07-0.58 times that of the non deep-frozen (NDF) immobilized strain. The decreased bioluminescence activity induced by the DF process was enhanced by the stimulants, sodium lactate (SL) and KNO3. However, regardless of the inducer chemical tested, the immobilized strain modified with KNO3 consistently produced greater bioluminescence than that treated with SL, in the range of 3.0-10.7 (avg. 6.7 +/- 3.69) and 1.2-4.2 (avg. 2.4 +/- 1.47) times that of control, respectively. All KNO3 treatments of contaminated groundwater samples also resulted in an increase in bioluminescence activity, but the rate of stimulation varied for each sample. Also, no strong linear correlation was observed between the bioluminescence and the total concentration of an inducer, which may related to the complex characteristics of the environmental samples. Overall, the results demonstrated the ability of immobilized genetically engineered bacteria, preserved by DF, to measure a specific group of environmental contaminants using a stimulating agent (KNO3), suggesting the potential for its preliminary application in a field-ready bioassay.

Bioassessments of anaerobically decomposing organic refuse in laboratory lysimeters with and without leachate recycling and pH adjustment.

In this paper, various microbial characteristics of degrading refuse in three lysimeters were compared to bioassess the operating conditions with and without leachate recycling and pH adjustment. Laboratory lysimeters with leachate recycling produced more gas and took less time to reach the highest methane percentage than a lysimeter without leachate recycling. Generally, lysimeters with leachate recycling showed high ATP (adenosine triphosphate) contents in the leachate. But there were no significant differences in dehydrogenase activities among the lysimeters. Leachate of all lysimeters inhibited the bioluminescence activities of the strain tested. Bioluminescence activity was more inhibited by the lysimeter with no leachate recycling (high inhibition corresponds to high toxicity of leachate). Generally, less inhibition was observed in the middle of the operation phase, which was related with the biodegradation activity.

The use of bioluminescence stimulant on the immobilized strain, P. putida mt-2 KG1206, with toluene analog inducers and environmental samples.

This study was conducted to investigate the applicability of the stimulant conditions for the bioluminescence activity of a recombinant strain of Pseudomonas putida, mt-2 KG1206, when immobilized using alginate polymer. The bioluminescence activity of the immobilized strain was generally approximately three to five times lower than the subcultured strain, and the activity was observed to slowly decrease. These facts may have been caused by several factors, such as the low biomass and the time required for diffusion into the entrapped biomass. Although different inducers produced different degrees of stimulation, immobilized bacteria modified with KNO(3) consistently produced more bioluminescence than those treated with sodium lactate, regardless of the inducer chemical tested. Cells treated with KNO(3) exhibited 2.8 times greater bioluminescence than that of the control activity. This condition also stimulated the bioluminescence activities of the immobilized bacteria exposed to contaminated groundwater samples. Based on these results, the immobilized KG1206 presented in this research can be used as a portable assay for the purpose of preliminary on-site monitoring of specific inducer contaminants, with subsequent off-site instrumental analysis, suggesting the potential of this immobilized cell for preliminary application in a field-ready bioassay.