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MicroSensor Measurement of Photosynthesis and Respiration in a Biofilm Group 3

Explore the hands-on use of an O2 microelectrode to quantify oxygenic photosynthesis and consumption in photosynthetic biofilms. Learn about the construction and interpretation of O2 profiles using a Clark Oxygen Electrode method with a reference electrode and measuring electrode with gold-coated Pt silicone membrane. Discover results from experimental set-ups with different flow rates and light intensities. Evaluate the sensor with in situ measurements and explore the potential of fully automated probing systems. Lastly, delve into Free Exercises on Pseudomonas and its role in metalworking fluids, alongside a study on Salmonella biofilm development in minced pork meat.

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MicroSensor Measurement of Photosynthesis and Respiration in a Biofilm Group 3

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  1. 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

  2. Purpose: • Hands-On Use of O2 Microelectrode • Quantify oxygenic photosynthesis and consumption in a photosynthetic biofilm. • Construction and interpretation of the obtained O2 profiles

  3. 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

  4. 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

  5. Methods: Experimental Set-Up

  6. Methods: Layout Sunshine Sunshine Sunshine Sunshine Sunshine Work Bench WINDOW LOW FLOW HIGH FLOW

  7. Results: Dark Profiles • Heterogeneity within and between samples • ”Dark”?

  8. Results: Profiles - DBL HIGH FLOW DBL ~ 400 μm LOW FLOW DBL ~ 500 μm

  9. 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)

  10. 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

  11. Results: Gross Photosynthetic Rate Net Consumption Production Transition High: Photic ~ 420 μm Low: Photic ~ 800 μm

  12. 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

  13. Free Exercise PSEUDOMONAS: Not just for Cystic Fibrosis and Ear in fections in Deep Sea Divers any more!!!

  14. Free Exercise: Pseudomonas But also METAL WORKING FLUIDS:

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

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