ΔιερεύνησηΗ της αντιμικροβιακή επίδρασης του

φυσικού και του τροποιημένου με άργυρο ζεόλιθου

στην επιβίωση του βακτηρίου E.coli

Κεφάλαιο 1: Ζεόλιθος
1.1 Προέλευση και γεωγραφική κατανομή του ζεόλιθου
1.2 Δομή του ζεόλιθου
1.3 Φυσικές και χημικές ιδιότητες των ζεολίθων
1.4 Συνθετικοί ζεόλιθοι
1.5 Χημικά τροποποιημένοι ζεόλιθοι
1.6 Χρήσεις ζεόλιθου
Κεφάλαιο 12: E. coli Μικροβιολογία υδάτων
2.1 Μικροοργανισμοί – δείκτες μόλυνσης νερού
2.2 Ε.coli
12.2.1 Δομή και ιδιότητες
12.2.2 Επιδημιολογία
1.3 Παθοφυσιολογία
1.4 Θεραπεία και διαχείριση
Κεφάλαιο 2: Ζεόλιθος
2.3 Η E.coli ως δείκτης κοπρανώδους μόλυνσης των νερών
2.4 Κλασικές μέθοδοι απολύμανσης νερού και υγρών
αποβλήτων
2.1 Χημικές ιδιότητες και βιολογική εφαρμογή του φυσικού
ζεόλιθου
2.2 Χρήση του ζεόλιθου στην ιατρική του ανθρώπου
2.3 Τοξικολογία του ζεόλιθου σε ζώα και ανθρώπους
Κεφάλαιο 3: O Χημικά xημικά τροποποιημένος με άργυρο
ζεόλιθος ως αντιμικροβιακος παράγοντας
3.1 Μηχανισμός αντιμικροβιακής δραστηριότητας του
αργύρου
3.2 Αντιμικροβιακή δράση του χημικά τροποποιημένου με
άργυρο ζεόλιθου
 3.3 Ο χημικά τροποποιημένος με άργυρο ζεόλιθος στην
απολύμανση νερού και αποβλήτων
Κεφάλαιο 4: Η χρήση φυσικών, συνθετικών και
τροποποιημένων ζεόλιθων για την απομάκρυνση κατιόντων
και ανιόντων από υδατικά συστήματαστην επεξεργασία
νερού και αποβλήτων
4.1 Απομάκρυνση βαρέων μετάλλων
4.2 Απομάκρυνση χρωστικών
4.3 Απομάκρυνση οργανικών μικρορρύπων
4.4 Απομάκρυνση θρεπτικών και άλλων ανιόντων
4.5 Απομάκρυνση σκληρότητας
4.6 Απολύμανση
Κεφάλαιο 5: Πειραματική Διαδικασία
5.1 Υλικά και μέθοδοι
5.2 Φυσικός ζεόλιθος (ΟΛΥΜΠΟΣ ΑΕ)
5.3 Τροποποίηση του φυσικού ζεόλιθου με Άργυρο
5.4 Προετοιμασία αιωρήματος βακτηρίων E.coli και
πρωτόκολλο μέτρησης
5.5 Πειράματα διαλείποντος έργου αδρανοποίησης E.coli
Κεφάλαιο 6: Αποτελέσματα και Συζήτηση
Κεφάλαιο 7: Συμπεράσματα
Βιβλιογραφία

Materials and Methods

Natural zeolite

Natural zeolite is a low cost mineral found worldwide in high amounts. The

natural Greek zeolite (Olympus Industrial Minerals S.A.) is collected from a

deposit in the northern part of Greece. The major component (>85%) is

clinoptilolite in multicationic form [(Na,K,Ca) 6(Si,Al)36O72∙20H2O] with some

impurities such as mica/illite, plagioclase feldspar and quartz. Note that, the

Na+ cation in the structure of natural clinoptilolite due to the similar hydrated
ionic radii is more preferable to exchange with hydrated Ag + ion than Ca2+, K+,

and Mg2+ cations (Koyama and Takeuchi, 1977; Arcoya et al., 1996). Natural

Greek zeolite (NZ) was ground and then sieved to different fractions, of which

0–1 (fine) and 1-3 mm (coarse) were used in this study, after being thoroughly

washed with deionized water (DIW) in order to eliminate dust eventually

present. After drying at 60 °C, the sample was stored in a desiccator.

Modification of natural zeolite with silver

Natural zeolite was modified with silver nitrate (AgNO 3) by ion exchange

method described by Boschetto et al. (2012). Briefly, 3 g of natural zeolite

were added to 50 mL of AgNO 3 aqueous solution (0.25% and 0.1 w/v) at pH

5±0.2 (to prevent metal precipitation) and shaken at 300 rpm for 24 h in a dark

room (due to the light sensitivity of silver) to achieve maximum exchange of

silver onto the zeolites. Note that, if the pH of exchange is greater than 7.5–8,

the samples become dark because Ag+ in the zeolite turned to Ag 0. The Ag-

exchanged zeolite solution was centrifuged, washed repeatedly with DIW and

the zeolite was dried at 60 °C for 24 h to obtain the silver-modified natural

zeolites (0.25%Ag-MNZ, 0.1%Ag-MNZ).

Bacterial suspensions preparation and enumeration

The two model bacteria used in the batch experiments were the Gram-

negative E. coli (NCTC 9001) and the Gram-positive E. faecalis (NCTC 775).

Cultures were maintained as a frozen stock (−80 °C) in growth media

supplemented with 80% glycerol. Prior to each experiment, the bacteria were

cultured on Nutrient Agar (non-selective growth medium) at 37°C for 48 h.

Subsequently, some of the sufficiently formed colonies of the microbial

cultures were isolated and transferred to a test-tube of sterilized deionized

water (DIW) and bacterial concentration in the suspension was quantified

based on the 0.5 McFarland turbidity scale, according to which, 0.1 optical

absorbance at 600 nm equals approximately to a concentration of 10 8 cfu/mL.

Optical density measurements were conducted using a UV-visible

spectrophotometer (U-2001, Hitachi). The dense bacterial suspensions were

diluted to obtain the initial bacterial concentrations for the batch experiments.

The bacteria concentrations were determined by serial dilution of the samples

and plating out (in duplicates) the aliquots (100 μL) on the surface of selective

growth media: Harlequin Chromogenic Coliform Agar (NEOGEN NCM 1005A)

and Slanetz & Bartely Agar (LAB166) for the growth of E. coli and E. faecalis,

respectively. The plates were incubated at 37 °C for 48 h and the total number

of colonies was counted. Note that reliable dilutions for quantification were

considered to be the ones resulting in formation of 30-300 distinct colonies.

The concentration of bacteria in the media was calculated by taking into

account dilution of the sample and the sample-volume plated out on the solid

media, and was reported as colony-forming units per milliliter (cfu/mL).

Batch inactivation experiments

For bacteria inactivation experiments with ΝΖ and Ag-MNZ, a standard

procedure in batch process was used. In the batch process a solution of

synthetic water inoculated with bacteria (E. coli, E. faecalis) containing a

certain amount of NZ or Ag-MNZ (5 g/L) was used. Samples were taken at

certain interval times. Bach procedure was carried out at constant room
temperature and hydrodynamic conditions. 50 mL tubes were gently filled

completely with the above synthetic water inoculated with bacteria of initial

concentration range 103-106 cfu/mL, so that no air remained in the tubes upon

their closure with caps. The tubes were attached onto a rotator that enabled

the tubes to be rotated at 12 rpm.

For each experiment, 20 tubes were employed, which were divided into two

sets. The first set of 10 tubes (controls) contained 50 mL suspension of

bacteria without NZ or Ag-MNZ were used in order to monitor the time

depended bacterium inactivation. The second set of 10 tubes (reactors)

contained 50 mL mixed suspension of bacteria with NZ or Ag-MNZ in order to

monitor any changes in bacterium inactivation provoked by presence of

zeolites. At various time intervals over a 6-h time period, a tube from each set

of tubes was taken for sampling. The supernatant was sampled (2mL

collected sample) to determine the remaining bacteria concentration.

The log reduction of bacterial concentrations after the experimental time

period was obtained by applying the following expression:

Ctotal,0
Log(Reduction)=log10 =log10C total,0 -log10C total  t 
Ctotal  t 

(1)

where Ctotal [cfu/mL] is the total bacterial concentration in the suspension at

timet and Ctotal,0= Ctotal(t=0)is the initial total concentration of bacteria.

Also, the log reduction was converted to percent reduction (P) as follows:

P(%)  (1  10 log(Reduction) )  100

(2)