microsensor measurement of photosynthesis and respiration in a biofilm group 3 cleide o. a....
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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