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Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
Yves ANDRES, Laurence GIRAUD and Pierre Le CLOIRECEcole des Mines de Nantes, GEPEA, UMR CNRS 6144
4 rue Alfred Kastler, BP 20722, 44307 Nantes cedex 03 Francee-mail: [email protected]
GEnie des ProcédésEnvironnementAgroalimentaire
UMR-CNRS 6144
1. Chitin and chitosan
2. Material and methods
3. Survival curves
4. Chitosan concentration effect
5. Application
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
Chitin
O
OO
NH
O
OHO
CH 2OH
HOO
CH 2OH
HO
O
CH 2OH
OC
CH 3
NH
CH 3
C O
NH
CH 3
C O
Chitosan
CH 2OH
O
NH 2
HO
CH 2OH
O HO
CH 2OH
HO
NH 2
O
O
NH 2
O
OO
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
0,8
1,3
1,8
2,3
2,8
3,3
3,8
1302603904120515061807210824092710301133123613
cm-1
Ab
s
00,10,20,30,40,50,60,70,80,91
Chitosan
Chitin
Ab
s
Infrared spectrum of chitosan and
chitin
Amide 1 bands (N-H amine)
Amide 2 bands (protonated amine)
Amide 3 bands ( C-N from amines)
in cm-1 in cm-1 in cm-1
Chitosan 1633 1590 1300Chitin 1655-1626 1561 1320
Deacetylation level
• Chitin 35 % (Aldrich)
• Chitosan 80 %(France Chitine)
• Chitosan 60 %(this study)
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. 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
0 7 15 17 19 23 26 29 37 45
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 pKa near 6.5 ± 0.2
Chitin pKa near 7.3 ± 0.2
Potentiometric titration of 80 % chitosan
• 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 chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
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.
1,0E+00
1,0E+01
1,0E+02
1,0E+03
1,0E+04
1,0E+05
0 20 40 60 80 100 120 140
Time (h)
CF
U/m
L
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
1,0E+00
1,0E+01
1,0E+02
1,0E+03
1,0E+04
1,0E+05
1,0E+06
0 100 200 300 400 500Time (min)
CF
U/m
L
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 chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
ktNNLn 0
Chitin Chitosan (80%) Chitosan (60%)
E. coli 5,50E-04 -5,82E-04 9,05E-02 2,27E-02
P. aeruginosa 1,20E-03 -3,70E-04 1,32E-02 ndE. faecalis 2,50E-04 1,60E-04 3,67E-02 ndS. saprophyticus (NaCl) 1,60E-03 2,65E-04 2,81E-02 5,00E-03
Demineralized waterDemineralized water with polymer
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 chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
E. coli mortality rate constant in relation to the chitosan (80%) flakes concentration
0,00
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0 0,2 0,4 0,6 0,8 1
chitosan (mg/mL)
Mor
talit
y co
nsta
nt (
min
-1)
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
Potassium ion release from E. coli bacterial cells in relation to the concentration of chitosan 80%.
0,00
0,05
0,10
0,15
0,20
0,25
0 0,2 0,4 0,6 0,8 1
Chitosan (mg/L)
Pot
assi
um
(m
g/L
)
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
1,0E+00
1,0E+01
1,0E+02
1,0E+03
1,0E+04
1,0E+05
0 1 2 3 4 5 6 7
Time (h)
CF
U/m
L
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 chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
Calcium 62,27 Bicarbonates 219Magnesium 14,3 Sulfates 29,8
Sodium 10,52 Chloride 37,4Potassium 1,02
Spring water ionic composition (mg/L)
Chitin Chitosan (80%) Chitosan (60%)
E. coli 6,00E-04 8,00E-04 1,70E-03 2,90E-03
Chitin Chitosan (80%) Chitosan (60%)
E. coli 6,00E-04 -5,82E-04 9,05E-02 2,27E-02
Demineralized waterDemineralized water with polymer
Spring waterSpring water with polymer
Comparison of the mortality rate
constants of E. coli in spring water and in demineralized water containing chitosan
80% flakes.
1. Chitin and chitosan2. Material and methods3. Survival curves4. Chitosan concentration effect5. Application
Antibacterial Effect of Chitosan Flakes:
Application to Water Treatment
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