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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-Food ²DTU-Nanotech ³ZHAW Zurich University of Applied Sciences. Purpose:. Hands-On Use of O 2 Microelectrode
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MicroSensor Measurement of Photosynthesis and Respiration in a BiofilmGroup 3 Cleide O. A. Møller¹, David Sabourin² and Florian Berner³ ¹DTU-Food ²DTU-Nanotech ³ZHAW 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 • ”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 Reference Electrode – Chlorinated Ag Wire Guard Electrode - Pt Tapered Glass filled with Electrolyte Solution Measuring Electrode Gold-Coated Pt Silicone Membrane
Methods – O2 Profile: Water • Turbulent Flow • Assume constant concentrations BULK • Laminar Flow, Vertical Transport by Diffusion Only • Flow Changes Thickness DBL PHOTIC • Oxygen Production via photosynthesis • Depth dependent on light penetration Sediment APHOTIC • Oxygen Consumption • Diffusion Controlled ANAEROBIC
Methods: Layout Sunshine Sunshine Sunshine Sunshine Sunshine Work Bench WINDOW LOW FLOW HIGH FLOW
Results: Dark Profiles • Heterogeneity within and between samples • ”Dark”?
Results: Profiles - DBL HIGH FLOW DBL ~ 400 μm LOW FLOW DBL ~ 500 μm
Results: Dark and Light Profiles 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 Net Consumption Production Transition High: Photic ~ 420 μm Low: Photic ~ 800 μm
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. Minced pork meat • Analysis: • - Inoculation in flow-chamber channels with LB media; • CLSM image acquisition; • Treatment of images withImaris; • Comparision of samples using COMSTAT; • Adhesionassay; • Swimming, swarming and twitchingplates. Medical Biofilm Techniques 2009
Results Swimming, swarming, twitching plates Adhesion assay S +++ ++ + S ++ +++ + TC TC M Imaris COMSTAT Comparision of samples Salmonella Total Count DoesSalmonellareallylack the ability to form biofilms? Medical Biofilm Techniques 2009
Polymeric Chip IB 30 mm PI 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