2. Antibacterial Effect of Chitosan Flakes: Application to Water Treatment Yves ANDRES, Laurence GIRAUD and Pierre Le CLOIREC Ecole des Mines de Nantes, GEPEA, UMR CNRS 6144 4 rue Alfred Kastler, BP 20722, 44307 Nantes cedex 03 France e-mail: yves.andres@emn.fr GEnie des Procédés Environnement Agroalimentaire UMR-CNRS 6144

3. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

4. Chitin O O O NH O O HO CH 2 OH HO O CH 2 OH HO O CH 2 OH OC CH 3 NH CH 3 CO NH CH 3 CO Chitosan CH 2 OH O NH 2 HO CH 2 OH O HO CH 2 OH HO NH 2 O O 2 O O O 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

5. Infrared spectrum of chitosan and chitin Deacetylation level Chitin 35 % (Aldrich) Chitosan 80 % (France Chitine) Chitosan 60 % (this study) 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

6. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment 0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 071517192326293745 NaOH 0.2M (mL) pH -10,00 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 dpH/dV Chitosan pK a near 6.5 ± 0.2 Chitin pK a near 7.3 ± 0.2 Potentiometric titration of 80 % chitosan

7. Bacterial species were grown in nutrient broth up to the stationary state The biomass was harvested and washed by centrifugation (7 200 g) Bacterial cells were starved one night at +4°C The survival curves were realized in 500 mL bottle with 200 mL of sterile demineralized water at pH near 6.5 150 mg of chitin or the chitosan flakes (355-500 µm) were sterilized with the medium before use The remaining living bacterial cells were measured by counting plate method 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

8. Survival curves of Escherichia coli (CIP 54.8 = ATCC 11775) (  ), Pseudomonas aeruginosa (CIP A 22) (  ), Enterococcus faecalis (CIP 76.117) (  ) and Staphylococcus saprophyticus (CIP 76.125) (  ) in demineralized water pH 6.5 ± 0.3. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

9. Survival curves of Escherichia coli (CIP 54.8 = ATCC 11775) (  ), Pseudomonas aeruginosa (CIP A 22) (  ), Enterococcus faecalis (CIP 76.117) (  ) and Staphylococcus saprophyticus (CIP 76.125) (  ) in demineralized water with 0.75 mg/L of chitosan (80%) flakes at pH 6.5 ± 0.3. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

10. The curves show an exponential decrease and a mortality rate constant (k) could be calculated Mortality rate constant (min –1 ) for bacteria in four experimental conditions : demineralized water, suspension with chitosan at 80 % and 60 % of deacetylation, and with chitin. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

11. E. coli mortality rate constant in relation to the chitosan (80%) flakes concentration 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

12. Potassium ion release from E. coli bacterial cells in relation to the concentration of chitosan 80%. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

13. Survival curves of E. coli in a 0.75 mg/L suspension of chitosan 80% flakes. Each 2 h the medium was inoculated with a new bacterial suspension containing 4.5 10 +04 CFU/mL. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

14. Comparison of the mortality rate constants of E. coli in spring water and in demineralized water containing chitosan 80% flakes. 1. Chitin and chitosan 2. Material and methods 3. Survival curves 4. Chitosan concentration effect 5. Application Antibacterial Effect of Chitosan Flakes: Application to Water Treatment

15. Conclusions The antimicrobial effect of chitosan onto Gram positive and negative bacteria depends on the deacetylation level and the chitosan concentration Two times potential reuse of chitosan The antimicrobial activity seems to be in relation with a polyelectrolytic effect confirmed by the potassium release Exploration of the use of chitosan in drinking water process treatment