Bioavailability of Cd, Zn and Hg in Soil to Nine Recombinant Luminescent Metal Sensor Bacteria
Abstract
:1. Introduction
- To investigate the effect of host bacterium (Gram-positive or -negative), genetic metal-response element and location of the metal-response element (plasmid or chromosome) on sensitivity (limit of determination and toxicity) of sensor bacteria towards target heavy metals
- To evaluate total bioavailable and water-extracted bioavailable fractions of Cd, Zn and Hg in spiked soils using sensor bacteria belonging to both Gram-negative (Pseudomonas fluorescens and Escherichia coli) as well as Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial families thus representing organisms of different natural habitats, physiology and cell wall structure.
- To compare the total and water-extracted bioavailable fractions measured by different recombinant bacterial sensors in order to investigate whether the bioavailability of metals depends on the type of bacterial cell or nature of the metal-response element used for the construction of the sensors.
- Using Cd as a model, to monitor the changes in bioavailability of Cd as a result of bacterial metabolic activity during 2-hour incubation of sensor bacteria with soil-water extracts and suspensions.
2. Results and Discussion
2.1. Characterization of bacterial sensors
2.2. Response of the sensor strains to Hg, Cd and Zn
2.3. Bioavailability of Cd, Zn and Hg in soil
2.4. Differences in bioavailability of Cd, Zn and Hg in soil to different sensor bacteria
2.5. Time-dependent changes in bioavailability of Cd in soil
3. Materials and Methods
3.1 Bacterial strains
3.2. Cultivation of bacteria
3.3. Soil samples and their preparation
3.4. Bioavailability tests and calculations
3.5. Fluorescence microscopy
3.6. Measurement of mobile Cd from soil
Conclusions
- The limit of determination of the sensors was determined mainly by the type of the genetic metal-response element used for the construction of the sensor bacteria. At the same time, toxicity of the Cd, Zn and Hg standard solutions was mostly dependent on the host bacterium, Gram-positive bacteria being in general more sensitive to all the metals than Gram-negative.
- Bioavailability of Cd, Zn and Hg in soil did not depend neither on the limit of determination (determined according to standard calibration curve) of the used sensor nor on the metal-response elements expressed in these sensor cells.
- The water-extracted bioavailable fractions of Zn, Cd and Hg were low (making 0.24 – 0.37%, 0.19 – 0.46 % and 1.7 – 4.9 % of the total Zn Cd and Hg, respectively) and similar to all the used sensor strains.
- The total bioavailable fraction of Cd and Zn (2.6 – 5.1% and 0.32 – 0.61%, of the total Cd and Zn, respectively) was almost comparable for all the sensors whereas the bioavailability of Hg in soil-water suspensions was about 10 fold higher for Gram-negative sensor cells (30.5% of total Hg) compared to Gram-positive ones (3.2% of the total Hg).
- In the case of Zn, the water-extracted and total bioavailable fractions were equal indicating that no additional Zn could be mobilized by the sensor bacteria upon direct contact with soil matrix in suspension assay.
- The bioavailable fraction of Cd and Hg (only in the case of Gram-negative sensor strains) in soil-water suspensions exceeded the water-extracted bioavailable fraction about 14-fold indicating that upon direct contact, additional fraction of Cd and Hg was mobilized by those sensor bacteria
- Using Cd as a model we showed that in the used test conditions, 2-hour incubation (standard incubation time for the test with sensor bacteria) was enough for all the used bacterial strains to access all potentially available Cd in the soil-water suspensions.
Acknowledgments
References
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Host bacterium and sensor strain | Genetic metal-response element a | Location of metal-response elements | Inducing metals b | Limit of determination (LOD), μg·L-1±SD | |||
---|---|---|---|---|---|---|---|
Cd2+ | Zn2+ | Hg2+ | |||||
Gram-negative | |||||||
Pseudomonas fluorescensOS8 | |||||||
OS8::KncadRPcadAlux c | CadR/PcadA | chromosome | Cd, Zn, Hg,Pb | 7 ± 2.5 | 400 ± 110 | 4 ± 1.3 | |
OS8(pDNcadRPcadAlux)d | CadR/PcadA | plasmid | Cd, Zn, Hg,Pb | 8 ± 1.4 | 500 ± 40 | 15 ± 1.7 | |
OS8::KnzntRPzntAluxc | ZntR/PzntA | chromosome | Cd, Zn, Hg,Pb | 20 ± 4.7 | 5000 ± 580 | 60 ± 19 | |
OS8::KnmerRBSBPmerluxc | MerB/MerR/Pmer | chromosome | Hg, MeHg, Cd | 4500 ± 1230 | not induced | 0.8 ± 0.2 | |
OS8(pDNmerRBSBPmerlux)d | MerB/MerR/Pmer | plasmid | Hg, MeHg, Cd | 650 ± 220 | not induced | 0.2 ± 0.05 | |
Escherichia coli MC1061 | |||||||
MC1061(pSLzntR/pDNPzntAlux)d | ZntR/PzntA | plasmid | Cd, Zn, Pb | 2 ± 0.5 | 700 ± 170 | 20 ± 5 | |
MC1061 (pmerRBSBPmerlux)d | MerB/MerR/Pmer | plasmid | Hg, MeHg, Cd | 40 ± 13 | not induced | 0.03 ± 0.009 | |
MC1061(pmerGFP)f | MerR/Pmer | plasmid | Hge | 0.6e | |||
Gram-positive | |||||||
Bacillus subtilisBR151 | |||||||
BR151(pcadCPcadAlux)d | CadC/PcadA | plasmid | Cd, Zn, Hg,Pb | 2 ±0.3 | 1000 ± 150 | 10 ± 1.5 | |
Staphylococcus aureusRN4220 | |||||||
RN4220(pcadCPcadAlux)d | CadC/PcadA | plasmid | Cd, Zn, Hg,Pb | 7 ± 2 | 1500 ± 210 | 2 ± 0.7 |
Metal | Host bacterium (species) | Strain | Metal-response elements (location)a | Viable cells in test | Total bioavailable b,%of total | Water-extracted bioavailablec,%of total |
---|---|---|---|---|---|---|
Cdd | Gram-negative | |||||
Pseudomonas fluorescens | OS8::KncadR PcadAlux | CadR/PcadA (C) | 3×107 | 3.5 ± 1.8 | 0.46 ± 0.19 | |
OS8(pDNcadR PcadAlux) | CadR/PcadA (P) | 4×106 | 4.4 ± 2.5 | ND | ||
OS8::KnzntR PzntAlux | ZntR/PzntA (C) | 6×106 | 4.8 ± 0.7 | 0.23 ± 0.0011 | ||
OS8::KnmerRBSB Pmerlux | MerR/Pmer (C) | 2×107 | 2.6 ± 0.4 | 0.41 ± 0.039 | ||
OS8(pDNmerRBSB Pmerlux) | MerR/Pmer (P) | 5×106 | 3.7 ± 1.5 | ND | ||
Escherichia coli | MC1061(pSLzntR/ pDNPzntAlux) | ZntR/PzntA (P) | 1×107 | 5.1 + 0.51 | 0.24 ± 0.18 | |
MC1061(pmerRBSB Pmerlux) | MerR/Pmer (P) | 4×107 | 3.7 ± 1.5 | 0.44 ± 0.18 | ||
AVERAGE for Gram-negative bacteria | 4.2±0.71 | 0.36±0.11 | ||||
Gram-positive | ||||||
Bacillus subtilis | BR151(pcadC PcadAlux) | CadC/PcadA (P) | 3×106 | 3.2 ± 1.1 | 0.19 ± 0.08 | |
Staphylococcus aureus | RN4220(pcadC PcadAlux) | CadC/PcadA (P) | 8×106 | 2.6 ± 0.3 | 0.38 ± 0.054 | |
AVERAGE for Gram-positive bacteria | 2.9±0.36 | 0.28±0.13 | ||||
Hg e | Gram-negative | |||||
Pseudomonas fluorescens | OS8::KncadR PcadAlux | CadR/PcadA (C) | 3×107 | 27.0 ± 7.7 | 2.4 ± 0.73 | |
OS8(pDNcadR PcadAlux) | CadR/PcadA (P) | 4×106 | 28.1 ± 14.0 | ND | ||
OS8::KnzntR PzntAlux | ZntR/PzntA (C) | 6×106 | 26.7 ± 1.1 | 2.6 ± 0.16 | ||
OS8::KnmerRBSB Pmerlux | MerR/Pmer (C) | 2×107 | 31.9 ± 12.2 | 2.6 ± 0.58 | ||
OS8(pDNmerRBSB Pmerlux) | MerR/Pmer (P) | 5×106 | 18.7 ± 7.3 | ND | ||
Escherichia coli | MC1061(pmerRBSB Pmerlux) | MerR/Pmer (P) | 1×107 | 27.9 ± 5.3 | 1.9 | |
MC1061(pSLzntR/ pDNPzntAlux) | ZntR/PzntA (P) | 4×107 | 38.9 ± 5.6 | 1.67 ± 0.40 | ||
AVERAGE for Gram-negative bacteria | 30.5±5.17 | 2.2±0.43 | ||||
Hg e | Gram-positive | |||||
Bacillus subtilis | BR151(pcadC PcadAlux) | CadC/PcadA (P) | 3×106 | 3.8 ± 2.8 | 4.9 ± 1.3 | |
Staphylococcus aureus | RN4220(pcadCPcadAlux) | CadC/PcadA (P) | 8×106 | 2.6 ± 1.2 | 2.8 ± 1.3 | |
AVERAGE for Gram-positive bacteria | 3.22±0.81 | 3.92±1.51 | ||||
Znf | Gram-negative | |||||
Pseudomonas fluorescens | OS8::KncadR PcadAlux | CadR/PcadA (C) | 3×107 | 0.35 ± 0.17 | 0.34 ± 0.18 | |
OS8(pDNcadR PcadAlux) | CadR/PcadA (P) | 4×106 | 0.32 ± 0.045 | ND | ||
OS8::KnzntR PzntAlux | ZntR/PzntA (C) | 6×106 | 0.42 ± 0.19 | 0.37 + 0.11 | ||
Escherichia coli | MC1061(pSLzntR/ pDNPzntAlux) | ZntR/PzntA (P) | 4×107 | 0.61 ± 0.35 | 0.27 ± 0.037 | |
AVERAGE for Gram-negative bacteria | 0.36±0.05 | 0.37±0.04 | ||||
Gram-positive | ||||||
Bacillus subtilis | BR151(pcadC PcadAlux) | CadC/PcadA (P) | 3×106 | 0.44 ± 0.20 | 0.24 ± 0.11 | |
Staphylococcus aureus | RN4220(pcadCPcadAlux) | CadC/PcadA (P) | 8×106 | 0.33 ± 0.18 | 0.25 ± 0.0018 | |
AVERAGE for Gram-positive bacteria | 0.39±0.07 | 0.25±0.004 |
Bacterium | Time of incubation, min | |||
---|---|---|---|---|
0 | 30 | 60 | 120 | |
Pseudomonas fluorescens 0S8::KncadRPcadAlux | 1.7 | 2.21 | 2.19 | 2.21 |
Escherichia coli MCI061 (pSLzntR/pDNPzntAlux) | 1.7 | 3.18 | 3.20 | 3.19 |
Staphylococcus aureus RN4220(pcadCPcadAlux) | 1.7 | 2.60 | 2.50 | 2.56 |
Bacillus subtilis BR151 (pcadCPcadAlux) | 1.7 | 2.70 | 2.85 | 3.41 |
Soil | Cd | Zn | Hg |
---|---|---|---|
Total, mg·kg1dwt of soil | |||
Non-spiked soila | 0.45 | 219 | 0.14 |
Spiked soil samples | Added metalb, mg·_kg-1dwt of soil | ||
1 | 1.5 | 0 | 0 |
2 | 15 | 0 | 0 |
3 | 150 | 0 | 0 |
4 | 1500 | 0 | 0 |
5 | 15000 | 0 | 0 |
6 | 0 | 900 | 0 |
7 | 0 | 9000 | 0 |
8 | 0 | 90000 | 0 |
9 | 0 | 0 | 0.28 |
10 | 0 | 0 | 2.8 |
11 | 0 | 0 | 17 |
12 | 0 | 0 | 28 |
13 | 0 | 0 | 280 |
Permitted limit values for soilc | |||
1-3 | 150-300 | 1-1.5 |
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Bondarenko, O.; Rõlova, T.; Kahru, A.; Ivask, A. Bioavailability of Cd, Zn and Hg in Soil to Nine Recombinant Luminescent Metal Sensor Bacteria. Sensors 2008, 8, 6899-6923. https://doi.org/10.3390/s8116899
Bondarenko O, Rõlova T, Kahru A, Ivask A. Bioavailability of Cd, Zn and Hg in Soil to Nine Recombinant Luminescent Metal Sensor Bacteria. Sensors. 2008; 8(11):6899-6923. https://doi.org/10.3390/s8116899
Chicago/Turabian StyleBondarenko, Olesja, Taisia Rõlova, Anne Kahru, and Angela Ivask. 2008. "Bioavailability of Cd, Zn and Hg in Soil to Nine Recombinant Luminescent Metal Sensor Bacteria" Sensors 8, no. 11: 6899-6923. https://doi.org/10.3390/s8116899