MicroSensor Measurement
of Photosynthesis and
Respiration in a Biofilm
Cleide O. A. Møller¹, David Sabourin² and Florian Berner³
¹DTU-Food
²DTU-Nanotech
³ZHAW Zurich University of Applied Sciences
Group 3
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
Reference Electrode –
Chlorinated Ag Wire
Guard
Electrode - Pt
Tapered
Glass
filled with
Electrolyte
Solution
Measuring Electrode
Gold-Coated Pt
Silicone
Membrane
– ”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:
W
a
t
e
r
S
e
d
i
m
e
n
t
BULK
DBL
PHOTIC
APHOTIC
•Turbulent Flow
•Assume constant concentrations
•Laminar Flow, Vertical Transport by Diffusion Only
•Flow Changes Thickness
•Oxygen Production via photosynthesis
•Depth dependent on light penetration
•Oxygen Consumption
•Diffusion Controlled
ANAEROBIC
Methods: Experimental Set-Up
Methods: Layout
Sunshine
Sunshine
Work Bench
HIGH
FLOW
LOW
FLOW
W
I
N
D
O
W
Sunshine
Sunshine
Sunshine
Results: Dark Profiles
-1000
-500
0
50
100
150
200
250
300
0
500
1000
High Flow - Position 1
High Flow - Position 2
High Flow - Position 3
Low Flow - Position 1
1500
2000
Low Flow - Position 2
Low Flow - Position 3
2500
3000
3500
4000
•Heterogeneity within and between samples
•”Dark”?
350
400
Results: Profiles - DBL
-800
-600
-400
HIGH FLOW DBL
LOW FLOW DBL
-200
~ 400 μm
~ 500 μm
0
50
100
150
200
250
300
350
400
0
200
400
600
800
1000
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: Dark and Light Profiles
-1000
-500
0
100
200
300
400
500
600
700
800
900
1000
0
500
1000
1500
2000
Low Flow:
2500
DBL should not change with dark and light
3000
3500
4000
High Flow - Position 3 - Dark
High Flow - Position 3 - Light
Low Flow - Position 3 - Dark
Low Flow - Position 3 - 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
Net Consumption
Production Transition
12
High Flow - Oxygen Content
Low Flow - Oxygen Content
High Rate - Photosynthetic Rate
Low Flow - Photosynthetic Rate
900
O2 (nmol/cm 3)
800
700
High:
600
Photic ~ 420 μm
10
8
500
6
400
Low:
300
4
Photic ~ 800 μm
200
2
100
0
0
200
400
600
800
1000
1200
Depth (µm)
1400
1600
1800
0
2000
Photo synthetic rate (nmol/(cm 3 s) )
1000
Evaluation
Sensor:
• 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.
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.
Medical Biofilm Techniques 2009
Minced pork
meat
Results
Adhesion assay
Swimming, swarming, twitching plates
S
+++
++
+
TC
++
+++
+
0.50
0.45
0.40
Absorbance
0.35
S
0.30
0.25
0.20
0.15
0.10
TC
0.05
M
0.00
Imaris
COMSTAT Comparision of samples
Biomass
Biofilm
(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
Total Count
Does Salmonella really lack the ability to form biofilms?
Medical Biofilm Techniques 2009
Salmonella
Collaborations?
Polymeric Flow Cell with adhesive-free interconnections
Polymeric
Chip
IB
Small Dead and System
Volumes
Adhesive Free
Unobstructed
Microscopic Observation
12 independent channels
Integrate Pump/Tubing
Interchangeable Chips
PI
30 mm