Antibacterial Effect of Chitosan Flakes: Application to Water Treatment Yves ANDRES, Laurence GIRAUD and Pierre Le CLOIREC Ecole des Mines de Nantes,

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



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




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)







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




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




1,0E+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.




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.




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)




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

)




1,0E+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.




Calcium 62,27 Bicarbonates 219Magnesium 14,3 Sulfates 29,8

Sodium 10,52 Chloride 37,4Potassium 1,02

Spring water ionic composition (mg/L)


E. coli 6,00E-04 8,00E-04 1,70E-03 2,90E-03


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.




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

Documents

Antibacterial Effect of Chitosan Flakes: Application to Water Treatment Yves ANDRES, Laurence GIRAUD and Pierre Le CLOIREC Ecole des Mines de Nantes,