On the α-polarization of bacterial suspensions: SIP measurements … · 2016. 7. 14. · because...
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580 585 590 595 600 605 610 615 620 1 10 100 1 000 10 000 σ'(20 °C) [μS.cm-1] Frequency (Hz) Water R. erythropolis T902.1 E. coli K12 B. subtilis Ps. putida α-polarization has been postulated to explain changes in the complex electrical conductivity observed in geological media affected by bacterial activity below 10 kHz. To date, α-polarization measurements on bacteria remain rarely reported expect in a few dielectric spectroscopy studies (e.g. Bot and Prodan, 2009; Zhang et al., 2013) because of the magnitude of the correction resulting from electrode polarization that occurs in the kHz frequency range and below (Asami, 2014). We performed complex electrical conductivity measurements on four bacterial suspensions using an impedance spectrometer at frequencies from 1 to 10 kHz. We observed small polarization for B. subtilis from 100 to 10 kHz (corresponding to a phase shift of 0.1mrad). SEM observations showed that B. subtilis was the only strain to form aggregates of tens of μm and to produce some extracellular polymeric substance, like in biofilms. Whether the cause of the polarization could be attributed to the size of the aggregates and/or to the extracellular polymeric substance is still to be determined. Future experiments will focus on bacterial growth and attachment, and biofilm formation in silica sands. On the α-polarization of bacterial suspensions: SIP measurements on four bacterial strains Tamara PILAWSKI (1)*, Wolfgang TAPPE (2) , Egon ZIMMERMANN (3) , Johan Alexander HUISMAN (2) , Frank DELVIGNE (4) , Frédéric NGUYEN (1) (1) Applied Geophysics Research Unit, Department ArGEnCo, Faculty of Applied Sciences, University of Liège, Allée de la Découverte 9 (B52), 4000 Liège, Belgium; (2) Institute of Bio- and Geosciences, Agrosphere (IBG-3), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany; (3) Central Institute for Engineering, Electronics and Analytics (ZEA-2), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany; (4) Microbial Processes and Interactions, Gembloux Agro-Bio Tech, University of Liège, Passage des Déportés 2, 5030 Gembloux, Belgium Introduction This experiment is part of a PhD thesis aiming at a better understanding of complex conductivity measurements during hydrocarbon biodegradation. The goal is to quantify different mechanisms proposed to explain the influence of bacteria on the electrical properties with lab experiments of increasing complexity. This experiment was designed to study direct bacterial polarization on bacterial suspensions at frequencies below 10 kHz, so-called α- polarization. To our knowledge, only a few studies have reported α-polarization measurements and they were performed by dielectric spectroscopy techniques relying on two electrodes and models to correct for electrode polarization at low frequencies. Objectives References • Measuring the complex conductivity of bacterial suspensions from 1 Hz to 10 kHz with an impedance spectrometer based on a four point measurement method as described in Zimmermann et al. (2008) and Huisman et al. (2015) • Determining the α-polarization associated with bacterial cells in this frequency range. Results Bacterial strains Conclusions Acknowledgements This work was supported by a Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture (F.R.I.A.) grant funded by the Belgian National Fund for Scientific Research (Fonds National de la Recherche Scientifique, FNRS) and attributed to T. Pilawski. Incubation until early stationary phase Washing Dilution SIP measurement From 1 Hz to 10 kHz Cultures in triplicates Washed bacterial suspension Optical density (O.D.) measurement Final O.D. at 570 nm = 0.4 3 times Centrifugation Removal of the supernatant Resuspension of bacteria Addition of M284 minimal medium diluted 20 times • Asami, K., 2014, Low-frequency dielectric dispersion of bacterial cell suspensions: Colloids and Surfaces B: Biointerfaces, 119. • Bot and Prodan, 2009, Probing the membrane potential of living cells by dielectric spectroscopy: European Biophysics Journal, 38 (8), 1049-1059. • Huisman ,J.A., Zimmermann, E., Esser, O., Haegel, F.-H., Treichel, A., and Vereecken, H., 2015. Evaluation of a novel correction procedure to remove electrode impedance effects from broadband SIP measurements: Journal of Applied Geophysics. • Zhang, C., Slater, L. and Prodan, C., 2013, Complex Dielectric Properties of Sulfate-Reducing Bacteria Suspensions: Geomicrobiology Journal, 30 (6), 490-496. • Zimmermann, E., Kemna, A., Berwix, J., Glaas, W., Münch, H.M., and Huisman, J.A., 2008, A high-accuracy impedance spectrometer for measuring sediments with low polarizability: Measurement Science and Technology, 18, 105603. Experimental protocol R. erythropolis E. coli B. subtilis Ps. putida (9.0±0.2)*10 7 (6.3±0.2)*10 8 (5.5±2.9)*10 7 (6.7±1.0)*10 8 Water R. erythropolis E. coli B. subtilis Ps. putida 7.2 7.0 6.6 7.0 7.0 Four bacterial strains were chosen for their different surface electrical properties. Two gram-positive bacteria Two gram-negative bacteria Bacillus subtilis GA1 Rhodococcus erythropolis T902.1 Pseudomonas putida KT2420 Escherichia coli K12 2: Real part of the complex electrical conductivity (σ’) measured on the four bacterial suspensions. They were obtained by averaging triplicates. The vertical error bars represent the standard deviation of the data. 1: Imaginary part of the complex electrical conductivity (σ’’) measured on the 4 bacterial suspensions. The vertical error bars represent the standard deviation of the data from 3 independent measurements. For comparison, σ’’ of the water used to suspend the bacteria is also shown on the graph. Only B. subtilis shows a small polarization below 10 kHz. a b c d 3: Scanning electron microscope images of (a) R. erythropolis, (b) E. coli, (c) B. subtilis and (d) Ps. putida. Only B. subtilis forms large aggregates. 4: Colony-forming units per millilitre (CFU/ml) of bacterial suspensions. These values give an estimate of the number of viable cells in each sample. 5: pH of the bacterial suspensions and of the water used to suspend the bacteria. The bacteria produced organic acids because of limited oxygen during the SIP measurements. 6: Scanning electron microscope image of B. subtilis. Red curves delineate some of the extracellular polymeric substance produced by the bacteria. *[email protected] -0.05 0.00 0.05 0.10 0.15 0.20 1 10 100 1 000 10 000 σ''(20 °C) [µS.cm-1] Frequency (Hz) Water R. erythropolis T902.1 E. coli K12 B. subtilis Ps. putida 1 2 3 4 5 6
Transcript of On the α-polarization of bacterial suspensions: SIP measurements … · 2016. 7. 14. · because...
580
585
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1 10 100 1 000 10 000
σ'(2
0 °C
) [µ
S.c
m-1
]
Frequency (Hz)
WaterR. erythropolis T902.1E. coli K12B. subtilisPs. putida
α-polarization has been postulated to explainchanges in the complex electrical conductivityobserved in geological media affected by bacterialactivity below 10 kHz. To date, α-polarizationmeasurements on bacteria remain rarely reportedexpect in a few dielectric spectroscopy studies(e.g. Bot and Prodan, 2009; Zhang et al., 2013)because of the magnitude of the correctionresulting from electrode polarization that occurs inthe kHz frequency range and below (Asami, 2014).We performed complex electrical conductivitymeasurements on four bacterial suspensions usingan impedance spectrometer at frequencies from 1to 10 kHz. We observed small polarization for B.subtilis from 100 to 10 kHz (corresponding to aphase shift of 0.1mrad ). SEM observationsshowed that B. subtilis was the only strain to formaggregates of tens of µm and to produce someextracellular polymeric substance , like inbiofilms. Whether the cause of the polarizationcould be attributed to the size of the aggregatesand/or to the extracellular polymeric substanceis still to be determined.Future experiments will focus on bacterial growthand attachment, and biofilm formation in silicasands.
On the α-polarization of bacterial suspensions: SIP measurements on four bacterial strains
Tamara PILAWSKI(1)*, Wolfgang TAPPE(2), Egon ZIMMERMANN(3), Johan Alexander HUISMAN(2), Frank DELVIGNE(4), Frédéric NGUYEN(1)
(1)Applied Geophysics Research Unit, Department ArGEnCo, Faculty of Applied Sciences, University of Liège, Allée de la Découverte 9 (B52), 4000 Liège, Belgium; (2)Institute of Bio- and Geosciences, Agrosphere (IBG-3), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany; (3)Central Institute for Engineering, Electronics and Analytics (ZEA-2), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany; (4)Microbial Processes and Interactions, Gembloux Agro-Bio Tech, University of Liège, Passage des Déportés 2, 5030 Gembloux, Belgium
IntroductionThis experiment is part of a PhD thesis aiming at abetter understanding of complex conductivitymeasurements during hydrocarbon biodegradation.The goal is to quantify different mechanismsproposed to explain the influence of bacteria on theelectrical properties with lab experiments ofincreasing complexity.This experiment was designed to study directbacterial polarization on bacterial suspensions atfrequencies below 10 kHz , so-called α-polarization . To our knowledge, only a fewstudies have reported α-polarizationmeasurements and they were performed bydielectric spectroscopy techniques relying ontwo electrodes and models to correct forelectrode polarization at low frequencies.
Objectives
References
• Measuring the complex conductivity ofbacterial suspensions from 1 Hz to 10 kHzwith an impedance spectrometer based on afour point measurement method as describedin Zimmermann et al. (2008) and Huisman et al.(2015)
• Determining the α-polarization associated withbacterial cells in this frequency range.
Results
Bacterial strains
Conclusions
AcknowledgementsThis work was supported by a Fonds pour la Formation à la Recherche dansl’Industrie et dans l’Agriculture (F.R.I.A.) grant funded by the Belgian National Fund for Scientific Research (Fonds National de la Recherche Scientifique, FNRS) and attributed to T. Pilawski.
Incubationuntil early stationary
phase
Washing Dilution SIP measurement
From 1 Hz to 10 kHz
Cultures in triplicates
Washed bacterial suspension
Optical density (O.D.) measurement
Final O.D. at
570 nm = 0.4
3 times
CentrifugationRemoval of the
supernatant
Resuspension of bacteria
Addition of M284 minimal medium diluted 20 times
• Asami, K., 2014, Low-frequency dielectric dispersion of bacterial cell suspensions: Colloids and Surfaces B: Biointerfaces, 119.
• Bot and Prodan, 2009, Probing the membrane potential of living cells by dielectric spectroscopy: European Biophysics Journal, 38 (8), 1049-1059.
• Huisman ,J.A., Zimmermann, E., Esser, O., Haegel, F.-H., Treichel, A., and Vereecken, H., 2015. Evaluation of a novel correction procedure to remove electrode impedance effects from broadband SIP measurements: Journal of Applied Geophysics.
• Zhang, C., Slater, L. and Prodan, C., 2013, Complex Dielectric Properties of Sulfate-Reducing Bacteria Suspensions: Geomicrobiology Journal, 30 (6), 490-496.
• Zimmermann, E., Kemna, A., Berwix, J., Glaas, W., Münch, H.M., and Huisman, J.A., 2008, A high-accuracy impedance spectrometer for measuring sediments with low polarizability: Measurement Science and Technology, 18, 105603.
Experimental protocol
R. erythropolis E. coli B. subtilis Ps. putida
(9.0±0.2)*107 (6.3±0.2)*108 (5.5±2.9)*107 (6.7±1.0)*108
Water R. erythropolis E. coli B. subtilis Ps. putida7.2 7.0 6.6 7.0 7.0
Four bacterial strains were chosen for theirdifferent surface electrical properties.
Two gram-positive bacteria
Two gram-negative bacteria
Bacillus subtilis GA1
Rhodococcuserythropolis T902.1
Pseudomonas putida KT2420
Escherichia coli K12
2: Real part of the complex electrical conductivity (σ’) measured on thefour bacterial suspensions. They were obtained by averaging triplicates.The vertical error bars represent the standard deviation of the data.
1: Imaginary part of the complex electrical conductivity (σ’’) measuredon the 4 bacterial suspensions. The vertical error bars represent thestandard deviation of the data from 3 independent measurements. Forcomparison, σ’’ of the water used to suspend the bacteria is also shownon the graph. Only B. subtilis shows a small polarization below 10 kHz.
a b
c d
3: Scanning electron microscope images of (a) R. erythropolis, (b) E.coli, (c) B. subtilis and (d) Ps. putida. Only B. subtilis forms largeaggregates.
4: Colony-forming units per millilitre (CFU/ml) of bacterial suspensions.These values give an estimate of the number of viable cells in eachsample.
5: pH of the bacterial suspensions and of the water used to suspendthe bacteria. The bacteria produced organic acids because of limitedoxygen during the SIP measurements.
6: Scanning electron microscopeimage of B. subtilis. Red curvesdelineate some of theextracellular polymeric substanceproduced by the bacteria.
-0.05
0.00
0.05
0.10
0.15
0.20
1 10 100 1 000 10 000
σ''
(20
°C
) [µ
S.c
m-1
]
Frequency (Hz)
Water
R. erythropolis T902.1
E. coli K12
B. subtilis
Ps. putida
1 2
3
4
5
6
