Self-referencing sensors for characterizing microbial biofilm physiology

Eric S. McLamore1,5, D.M. Porterfield2,3,4,5, and M.K. Banks1

1School of Civil Engineering2Department of Agricultural and Biological Engineering3Department of Horticulture & Landscape Architecture

4Weldon School of Biomedical Engineering 5Bindley Bioscience Center, Discovery Park: Physiological Sensing Facility

• Function• Ion/nutrient flux• Oxygen flux

• Structure– Shared analyses

• Atomic force microscopy

• Why is studying biofilm physiology important?– Wastewater treatment– Toxicity studies

Biofilm Physiology

Biophysical(physiological)

Biochemical(functional)

Morphological(structural)

• Respirometry• Invasive concentration (activity) sensors

– Oxygen, ammonium, nitrate, pH• Classic biochemical techniques

– In vitro detectors and assays

Techniques for characterizing biofilm physiology

Non-invasive techniques with greater sensitivity and temporal resolution are required

• Real time biophysical flux – Fixed excursion distance– Near pole– Far pole

• Corrects for mechanical motion through liquid solution– Reference measurement

• ≈0.4 cm from surface

Self-referencing sensors

drdCDJ −=

dC = difference in concentration between near pole and far pole

dr = excursion distance

N. europaea biofilm immobilized on silicon membrane

200 µm

• Very low detection limit (fmol/cm2-sec)• Non-invasive (non-destructive)• Real time temporal resolution• Direct quantification of analyte flux

– Dynamic measurement

Benefits of self-referencing technique

SEM of electrode tip

• Multiple sensor approach for measuring flux – cumulative signal error

• One sensor, one source of error

10 µm

Self-referencing sensor hardware

sample

video/zoomscope

vibration isolation table

faraday cage

amplifier

• Step-back experiments on N. europaea biofilmSensor calibration (dynamic)

N. europaea biofilm immobilized on silicon membrane

ε=dynamic efficiency

0 200 400 600 800 1000

Am

mon

ium

Influ

x [p

mol

-NH

4/cm

2 -se

c]

0

200

400

600

800

NH4 influxPredicted

ε=0.92

Distance from source [µm]0 200 400 600 800 1000

Nitr

ite E

fflu

x [p

mol

-NO

2/cm

2 -se

c]

0

100

200

300

400

500

600

700

NO2 effluxPredicted

ε=0.94

NH3 +O2 +2H+ → NH2OH +H2O → NO2- +5H+ + 4e-

• Ammonia oxidizing bacteria– Nitrosomonas europaea

• Ammonia monooxygenase (AMO)– membrane bound protein– copper at active site

• Hydroxylamine oxidoreductase (HAO)– Produces electrons required by AMO

Classic physiological study

AMO HAO

pKa = 9.2

NH4 → NH3 +H+

• Alternative substrates– AMO can oxidize over 40 substrates

• Mechanism-based (e.g., acetylene)• Reversible inhibition

– Visible light– Copper chelating agents

• Thiourea, carbon disulfide

AMO inhibition

Mixed culture nitrifying biofilm immobilized on silicon membrane

200 µm

• Microbes immobilized on oxygen-permeable silicon membranes

• Upflow membrane-aerated bioreactor– Lumen-side air flow

• Membranes transferred to flow cell for measurement

Cell harvesting and immobilization

MABR

N. europaea biofilmgrown in MABR

MABR limit the formation of anoxic zones

Experimental design

position 2

position 1

position 3

position 4

position 5

position 6

AMO inhibition study: Proton flux

Nitrosomonas europaea biofilm

pH at biofilm surface was 6.71 ± 0.10 c

Time [minutes]0 20 40 60 80 100

Prot

on F

lux

[pm

ol/c

m2 -s

ec]

-10

0

10

20

30

effluxinflux

CuCl2 addition

3 mM thiourea

6 mM thiourea

Time [minutes]0 2 4 6 8 10 12 14

Prot

on F

lux

[pm

ol/c

m2 -s

ec]

-10

0

10

20

30

effluxinflux

3 mM thiourea

6 mM thiourea

Time [minutes]20 30 40 50

Prot

on F

lux

[pm

ol/c

m2 -s

ec]

-10

0

10

20

30

efflux

influx

30 µM CuCl2

55 µM CuCl2

85 µM CuCl2

110 µM CuCl2 140 µM CuCl2

168 µM CuCl2

...from 6mM thiourea

195 µM CuCl2

Time [minutes]50 60 70 80 90 100

Prot

on F

lux

[pm

ol/c

m2 -s

ec]

-10

0

10

20

30

efflux

influx

250 µM CuCl2275 µM CuCl2

300 µM CuCl2

1500 µM CuCl2

...from 195µM CuCl2

Position 1 2 3 4 5 6

Prot

on F

lux

[pm

ol/c

m2 -s

ec]

0

10

20

30

40

50

steady statepost thiourea dosingpost copper relief

effluxinflux

Real time data collected at position 1

AMO inhibition study: Ammonium flux

Nitrosomonas europaea biofilm

Time [minutes]0 20 40 60 80

NH

4 inf

lux

[pm

ol/c

m2 -s

ec]

0

100

200

300

400

500

600

700thiourea dosing copper dosing

Real time data collected at position 6

100 µM CuCl2sufficient for

restoring steady stateNH4 influx

Time [minutes]0 5 10 15 20 25 30

Am

mon

ium

influ

x [p

mol

/cm

2 -sec

]

0

100

200

300

400

500

600

700

1.5 mM thiourea

3.0 mM thiourea4.5 mM thiourea 6.0 mM thiourea

(b)

Time [minutes]40 50 60 70 80

Am

mon

ium

influ

x [p

mol

/cm

2 -sec

]

0

100

200

300

400

500

600

700

28 µm CuCl2

...from 6.0 mM thiourea

56 µm CuCl2

85 µm CuCl2

112 µm CuCl2

168 µm CuCl2

195 µm CuCl2

140 µm CuCl2222 µm CuCl2

(c)

Position2 4 6

NH

4 flu

x [ µ

mol

/cm

2 -sec

]

-100

0

100

200

300

400

500

600steady statepost thiourea dosingpost copper relief

effluxinflux

• Successfully demonstrated reversible inhibition of AMO by copper chelation in a biofilm• Non-invasive • Real time response

• More copper required to restore steady state oxygen and proton flux levels than required to restore NH4 influx • Oxygen and copper are required for many cellular

processes other than AMO activity

• Use of self-referencing sensors will allow non-invasive investigation of real time biofilm stress response to chemical toxin exposure

Conclusions

• Chemical toxicity in pure and mixed culture biofilms– Uncoupling of aerobic respiration– Glutathione gated-potassium efflux

• Associated with deflocculation– Additionally measuring efflux of cross-linking ions (Ca2+,

Mg2+)

– Aerobic biodegradation of respiratory-inhibiting compounds

– Heavy metal exposure

Ongoing studies

• Advisors– M. K. Banks– D.M. Porterfield

• Purdue School of Civil Engineering• Discovery Park• Al Shipley (Applicable Electronics, Inc.)• Jon Shaff (Cornell, USDA)• Aeraj ul Haque, Rameez Chatni (Electric. and Comp. Engr.)

Acknowledgements

Also currently investigating effect of laminar bulk flow on sensor behavior

AMO inhibition study: Oxygen fluxNitrosomonas europaea biofilm

Time [minutes]0 50 100 150 200 250

Oxy

gen

influ

x [p

mol

/cm

2 -sec

]

-10

-5

0

5

10

15

20

25

30thiourea dosing copper dosing

Time [minutes]0 20 40 60 80

Oxy

gen

influ

x [p

mol

/cm

2 -sec

]

-10

-5

0

5

10

15

20

25

302.5 µM thiourea

3.5 µM thiourea 5.0 µM thiourea

6.2 µM thiourea 7.5 µM thiourea

8.7 µM thiourea

10 µM thiourea

Time [minutes]120 140 160 180 200

Oxy

gen

influ

x [p

mol

/cm

2 -sec

]

-10

-5

0

5

10

15

20

25

30

...from 10µM thiourea

0.3 mM CuCl2

0.6 mM CuCl2

0.9 mM CuCl2 1.1 mM CuCl2

1.3 mM CuCl2

1.6 mM CuCl2

1.8 mM CuCl2

2.0 mM CuCl2

Spatial location [µm]1 2 3 4 5 6

Oxy

gen

influ

x [p

mol

/cm

2 -sec

]

0

5

10

15

20

25

steady statepost thiourea dosingpost Cu dosing

Real time data collected at position 1