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 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, O 2 diffusion is rapid

Methods – O 2 Profile: DBL WaterWater SedimentSediment 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 WINDOWWINDOW

Results: Dark Profiles Heterogeneity within and between samples ”Dark”?

Results: Profiles - DBL LOW FLOW DBL ~ 500 μm HIGH FLOW DBL ~ 400 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 / (cm 2 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 High: Photic ~ 420 μm Low: Photic ~ 800 μm Net Consumption Production Transition

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 μm 3 / μm 2 Average Thickness: 2.88 μm Max. Thickness: 6.21 μm Ps. Pseudoalcaligenes Biomass: 0.62 μm 3 / μ m 2 Average Thickness: 0.50 μ m Max. Thickness: μ m

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 pork meat

Results Medical Biofilm Techniques 2009 Swimming, swarming, twitching plates Imaris COMSTAT Comparision of samples Adhesion assay Does Salmonella really lack the ability to form biofilms? Total Count Salmonella Biofilm Biomass (µm 3 /µm 2 ) Avg colony volume of colonies at substratum (µm 3 ) Avg thickness (µm) Salmonella Total Count S TC M S

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