1. And now to something not so medical.... …but with the same biofilm techniques

2. Diversity in Pseudomonas putida biofilm Biofilm as an industrial capacity Exercise 4 Anne-Mette Juel Christensen PhD Student, Infection Microbiology group August 2014

3. Biofilm as the bad guy…

4. Biofilm as the bad guy… • Surfaces – a space to be occupied! • Localizing cells in close proximity • Communication • Exhange of genetic material -> establish niche growth • Protection from environmental challenges e.g. antibiotics, host immune response, toxic compounds • Extracellular polymeric substances (EPS) forms a barrier • Creation of dorment cells • Differentiation of cells into subpopulations e.g. non-motile -> heterogeneity -> Robust community of cells that causes problems for the host

5. Biofilm as the good guy… -> Robust community of cells that can be… • Resilient to variety of environmental stress • Potent biocatalyst of toxic and insoluble products In industry: • Microbial subpopulations in industrial fermentations • Undesirable bi-products • Alter substrate uptake • Growth reduction • Alter metabolic activities

6. Exercise 4: Diversity in P. putida biofilm • Industrial relevant production organism – Pseudomonas putida • Non-pathogenic • Biofilm capability • Produce compounds interesting for biofuel production -> isobutanol • Isobutanol toxic to the cell • Pseudomonas sp. VLB120 • originally isolated from forest soil • has shown to be able to grow on a wide range of carbon sources • grow in the presence of different solvents e.g. toluene and styrene • GFP tagged

7. Aim of the exercise • Study diversification and change of biofilm structure of Pseudomonas sp. VLB120 biofilm when exposed to 2.5 % isobutanol during growth in FB medium with citrate as carbon source in the flow chamber system.

8. Experimental procedure Assembly of the biofilm system, preparation of medium +/- isobutanol Sterilization and washing. Preparation of cultures Inoculation of flow chamber channels CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Done for you  COMSTAT and Imaris analysis Phenotypic characterization: Stability, growth, CV staining of biofilm, motility Select morphology variants for phenotypic characterization

9. Biofilm set up FB medium + 1 mM citrate FB medium + 1 mM citrate + 2.5 % isobutanol • Each team has one flow cell with. • 2 channels treated with FB medium + citrate + 2.5 % isobutanol and 1 channel treated with FB medium + citrate • OR • - 1 channel treated with FB medium + citrate + 2.5 % isobutanol and 2 channels treated with FB medium + citrate

10. Confocal laser scanning microscopy

11. Identification of variants • Sample from effluent tubes • Plating on selective media

12. Day 2 – Monday 11th august • Morning: • CLSM • Prepare LB PIA plates • Afternoon: • CLSM cont. • Collect effluent in epp. tubes – keep sterile! • Make dilutions in 0.9 % NaCl and plate on LB PIA plates • Incubate ON at 30 °C Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

13. Slight change of experimental set-up • Flow cell 1 • VLB120 wt • VLB120 wt • VLB120 + isobutanol from t=0 • Flow cell 2 • VLB120 wt • VLB120 + isobutanol from t=0 • VLB120 + isobutanol from t=0 • Flow cell 3 • VLB120 wt • VLB120 wt • VLB120 + isobutanol from t=0 • Flow cell 4 • VLB120 wt + isobutanol from day 4 • VLB120 + isobutanol from t=0 • VLB120 + isobutanol from t=0 • Flow cell 5 • VLB120 wt + isobutanol from day 4 • VLB120 wt + isobutanol from day 4 • VLB120 + isobutanol from t=0 Only microscopy and effluent collection on VLB120 wt and VLB120 wt + isobutanol from day 4

14. Day 4 (day 3 in manual) • Team 1-3 -> Pseudomonas sp. VLB120 wt • Team 4-5 -> Pseudomonas sp. VLB120 treated with 2.5 % isobutanol at day 4 • Look for variants on plates • Take pictures (1.st floor, lab 101 – Leica) • Select 4 variants and re-streak on LB PIA plates • Incubate ON at 30 °C • Keep original plates at room temperature Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

15. Day 5 (day 4 in the manual) Stability: • Compare original plates (pictures) with re-streak. Stable? • Leave plates at room temperature (to be used for motility) Growth study and crystal violet biofilm staining (CV staining) • If stable inoculate single colony in 10 mL LB • Incubate ON at 30 °C • Prepare FB medium + 1mM NaCitrate Motility (Swimming, swarming & twitching): • Prepare 5 plates for each motility type Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

16. Day 6 (day 5 in the manual) Growth study: • 50 mL LB medium in 100 mL flasks • Measure OD600 on ON culture -> inoculate to a conc. Of OD600=0.01 • Measure OD600 at appropriate intervals e.g. every hour • Calculate doubling time and compare with wt Crystal violet staining: • Dispense 140 uL in all wells. • Add 10 uL ON cultures • Remember blanks Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

17. Day 6 (day 5 in the manual) Motility: • Twitching (bottom) Swarming (top) • Swimming (middle) • Incubate ON at 30 °C Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

18. Day 7 (day 6 in the manual) Crystal violet staining: • Wash away planktonic cells • Stain with 0.1 % CV • Wash wells • Dissolve stained biofilm with 96 % EtOH • Measure OD Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

19. Day 7 (day 6 in the manual) Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis