MicroSensor Measurement of Photosynthesis and Respiration in a Biofilm

Group 3

Cleide O. A. Møller¹, David Sabourin² and Florian Berner³

¹DTU-Food²DTU-Nanotech³ZHAW Zurich University of Applied Sciences

Purpose:

• Hands-On Use of O2 Microelectrode

• Quantify oxygenic photosynthesis and consumption in a photosynthetic biofilm.

• Construction and interpretation of the obtained O2 profiles

Methods: Clark Oxygen Electrode

Silicone Membrane

Measuring Electrode Gold-Coated Pt

Guard Electrode - Pt

Tapered Glass

filled with Electrolyte Solution

Reference Electrode – Chlorinated Ag Wire – ”Ideal”

– Current generated proportional to oxygen

– Small oxygen consumption – less than a single bacteria

– Linear, stable and fast response

• At dimensions of sensor, O2 diffusion is rapid

Methods – O2 Profile:

DBL

Water

Sediment

BULK•Turbulent Flow

•Assume constant concentrations

•Laminar Flow, Vertical Transport by Diffusion Only

•Flow Changes Thickness

PHOTIC

APHOTIC

•Oxygen Production via photosynthesis

•Depth dependent on light penetration

•Oxygen Consumption

•Diffusion Controlled

ANAEROBIC

Methods: Experimental Set-Up

Methods: Layout

Work Bench

HIGH FLOW

LOW FLOW

Sunshine

Sunshine

Sunshine

Sunshine

Sunshine

WINDOW

Results: Dark Profiles-1000

-500

0

500

1000

1500

2000

2500

3000

3500

4000

0 50 100 150 200 250 300 350 400

High Flow - Position 1

High Flow - Position 2

High Flow - Position 3

Low Flow - Position 1

Low Flow - Position 2

Low Flow - Position 3

•Heterogeneity within and between samples

•”Dark”?

-800

-600

-400

-200

0

200

400

600

800

1000

0 50 100 150 200 250 300 350 400

High Flow - Position 1

High Flow - Position 2

High Flow - Position 3

Low Flow - Position 1

Low Flow - Position 2

Low Flow - Position 3

Results: Profiles - DBL

LOW FLOW DBL

~ 500 μm

HIGH FLOW DBL

~ 400 μm

Results: Dark and Light Profiles

-1000

-500

0

500

1000

1500

2000

2500

3000

3500

4000

0 100 200 300 400 500 600 700 800 900 1000

High Flow - Position 3 - Dark

High Flow - Position 3 - Light

Low Flow - Position 3 - Dark

Low Flow - Position 3 - Light

Low Flow:

DBL should not change with dark and light

High Flow:

5X Increase in O2 consumption, 2.9e-2 vs 6.3 e-3 nmol / (cm2 s)

Gross Photosynthetic Rate

•If illuminate sample for long time, steady state in/at a layer between oxygen supplying process and oxygen removal processes by diffusion and respiration

•If illumination stopped/blocked, removal processes continue without change and oxygen concentration decreases at the rate generated prior to light blocking

•Gross photosynthesis rates estimated by blocking light for short periods of time while microsensor at different depths

Results: Gross Photosynthetic Rate

0

100

200

300

400

500

600

700

800

900

1000

0 200 400 600 800 1000 1200 1400 1600 1800 2000

Depth (µm)

O2

(nm

ol/

cm

3 )

0

2

4

6

8

10

12

Ph

oto

sy

nth

eti

c r

ate

(n

mo

l/(c

m3 s)

)

High Flow - Oxygen Content

Low Flow - Oxygen Content

High Rate - Photosynthetic Rate

Low Flow - Photosynthetic Rate

High:

Photic ~ 420 μm

Low:

Photic ~ 800 μm

Net Consumption Production Transition

EvaluationSensor:• In situ measurements possible• Small oxygen consumption – less than a single bacteria• Linear, stable and fast response • Point measurements, not necessarily representative of population

• Invasive / Disruptive• Fragile• Reduction of other compounds, bubbles• Fouling of membrane

Set-up• Not completely dark, bulk flow rate, light intensity, etc not quantified• Wish List – fully automated probing and shutter system and data

analysis/report generation

Free Exercise

PSEUDOMONAS:

Not just for Cystic Fibrosis and Ear in fections in Deep Sea Divers any

more!!!

Free Exercise: Pseudomonas

But also

METAL WORKING FLUIDS:

Pseudomonas Pseudoalcaligenes

Non- Pathogenic Naturally inhabits metalworking fluid and

dominates the culture, driving out other strains

Unless it gets kicked out by them

From my previous experiments: Suspected to be a poor biofilm builder

compared to Ps. Aeruginosa

Comparison

• Ps. Aeruginosa Biomass: 4.85 μm3/ μm2

Average Thickness: 2.88 μm Max. Thickness: 6.21 μm

Ps. Pseudoalcaligenes

Biomass: 0.62 μm3/ μm2

Average Thickness: 0.50 μm

Max. Thickness: 10.45 μm

Development of Salmonella biofilm from minced pork

meat with natural microflora

3 Salmonella strains:

• S. Typhimurium DT104;• S. Typhimurium DT12;• S. Derby.

Medical Biofilm Techniques 2009

Analysis:

- Inoculation in flow-chamber channels with LB media;- CLSM image acquisition;- Treatment of images with Imaris;- Comparision of samples using COMSTAT;- Adhesion assay;- Swimming, swarming and twitching plates.

Minced porkmeat

Results

Medical Biofilm Techniques 2009

Swimming, swarming, twitching plates

ImarisCOMSTAT Comparision of samples

Adhesion assay

Does Salmonella really lack the ability to form biofilms?

Total Count Salmonella

BiofilmBiomass

(µm3/µm2)

Avg colony volume

of colonies at substratum (µm3)

Avg thickness

(µm)

Salmonella 3.53 35085.89 6.63

Total Count 1.05 1704.63 1.24

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

1

Ab

sorb

ance S

TC M

S

TC

+++ ++ +

++ +++ +

Collaborations?Polymeric Flow Cell with adhesive-free interconnections

Small Dead and System Volumes

Adhesive Free

Unobstructed Microscopic Observation

12 independent channels

Integrate Pump/Tubing

Interchangeable Chips

IB

PI

Polymeric Chip

30 mm