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Mérida, Yucatán Octubre 12 pquint@cinvestav.mx

Departamento de Física Aplicada Cinvestav- Mérida

Nanomateriales para la conservación de

bienes patrimoniales con actividad

antimicrobiana

Nanomaterials with antimicrobial activity

to preserve cultural heritage assets

Dra. Patricia Quintana

Tzompantlí, Chichén itza Lichens

Tzompantli Templo de las calaveras Chichen Itzá

Juego de Pelota de Chichen Itza

Algae: Trentepohlia sp

cyanobacteria

The breakdown of materials by

microbial action (Hueck, 1965)

The biodeterioration processes are

favored during the growth of

microorganisms, therefore the

decomposition and disintegration of

stones is due to the formation of

biominerals.

Rock: porous material

Environmental factors : weather, light,

humidity, solar radiation cycles, etc.

Biodeterioration

pitting

mosses

Biodeterioration by Microorganisms

Interpenetration of hyphae

Fungal growth

Meteorization cycles due to microorganisms Chemical, physical and biological interactions between the stone-atmosphere-microorganisms. (GADD Mycologist, 2004)

Fungal hyphae penetrates the surface of the stone to obtain nutrients and excreted small amounts of acid, which cause superficial damage to limestone

HYPHAE

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Biomineralization: The formation of hard mineral

deposits through a living organism

H2CO3 Ca+ + (HCO3)-

H2C2O4 Ca+ + (C2O4)2-

CaC2O4·2H2O Weddellite

Hydrated Calcium Oxalates

Tetragonal: a=b=12.33Å, c=7.35Å =β= γ= 90°

Monoclinic: a= 6.29Å, b=14.58Å, c=10.11Å =γ= 90°, β=109.47°

CaC2O4·H2O Whewellite

H2O + CO2 H2CO3 o H2C2O4 organic acids (carbonic)

CaCO3 +

Biomineralization: The synthesis of inorganic crystalline or amorphous minerals by living organisms often forms hard mineralized tissues.

These organisms produce inorganic solids with specific chemical and morphologic characteristics with organized crystals.

Minerals Biominerals

Calcium

carbonates

Calcite

Aragonite

Vaterite

Calcium oxalates Whewellite

Weddellite

Amorphous Silica SiO2 nH2O

Iron oxide Ferrihydrite

Sulfates Gypsum

Calcium

phosphates HAP

Microorganisms

a) Fungi b) Bacteria

c) Cyanobacteria and

microalgaes

d) Líchens

Mainly microorganisms which are involved in deterioration

processes of calcareous material

Prevention and remediation methods

When biological growths are found to be damaging

historical or modern constructions, requires removal

or management. The following actions may be

considered

Type of microorganisms

Weather conditions

Type of stone (composition, porosity,

density, surface, etc.)

Methods Advantages Disadvantages

Physical

Mechanical, washing

(water or vapor pressure)

and brushing the

biological material

Traditional method (usually

applied) good to remove algae,

lichens and mosses

The area is clean for a short period

of time, it only removes the

superficial mycelium, however

water goes into internal porous

favors the microorganisms to grow

again.

UV Energy penetration is very

effective on the surface

The work area is small and can

react with other mineral compounds

Chemical

Biocides Diluted in water or organic

solvents. Health dangerous

Fumigation Is fast removing fungi

microorganisms, insects, etc.

Gases are toxic, only applies in

close spaces

Anoxic atmosphere Fungi are susceptible with the

absence of oxygen

Large exposition period,

Expensive equipment

P. Fernández (2006)

OBJECTIVE

Preserve and protect the cultural heritage and modern

buildings, developing new coatings and biocides in order to

avoid the deterioration of petrous material

Develop new coatings compatible with the construction materials to preserve and

extend the useful life.

Evaluate In vitro the growth of microorganisms (antimicrobial and bactericide

activity) in calcareous rocks with the coatings

Stablish the mechanism of biocide or biostatic action of the coatings (CMF, CMB,

CMI; minimum concentration fungicidal, bactericidal, inhibitory) and the action

mode to prevent the organism to growth, and analysis the structural damage, etc.

Evaluate the effectivity against deterioration in situ of the calcareous rocks with

the coatings, when are exposed to different environments.

In Mexico, polymer coatings were applied to frescoes at 1990s; after 10 years it cause mechanical stresses and crystallization of salts leading to accelerated disintegration. also, the polymers became discolored and brittle. In 2008, Baglioni started to work with the Mexican INAH to come up with a removal treatment for murals at the Mayan site Mayapan

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• Synthesis of protective antifungal coatings of Ca(OH)2 + ZnO

(TiO2)

• Characterization of the natural limestone rocks collected from

quarries of Yucatan state

• Microbial and bacterial colonization analysis on the surface of

the rocks

• S. De la Rosa García, O.Ortega-Morales, Ch. Gaylarde, M. Beltrán-García, P. Quintana, M. Reyes-Estebanez,Influence of fungi in the weathering of limestone of Mayan monuments, Rev. Mx Micología 33 (2011) 43-51 (Uxmal)

• O. Ortega-Morales, S. Nakamura, G. Montejano-Zurita, J. C. Camacho-Chab, P. Quintana, S.de la Rosa García. Implications of colonizing biofilms and microclimate on west stucco masks at north Acropolis, Tikal, Guatemala. Heritage Sc. J 1:32 (2013) 2-8

Commercial Ca(OH)2 nanoparticles for the consolidation of immovable works of art, P. Baglioni, et al, Appl. Phys A (2014), 114 (3), 723

Chamber tomb on Ixcaquixtla (Puebla, Mexico). Painted layer before the application of Ca(OH)2 nanoparticles. The consolidation treatment allowed the removal of salts, recovering and revealing larger portions of the painted layer.

Optical microscope images and SEM of the red pigment painted layer in Calakmul (Mexico) Maya archaeological site. Bar is 100 μm

Before treatment After treatment

Before treatment After treatment Before treatment After treatment

Nanorestore® formulation Ca(OH)2 NPs in 2-propanol, 5 g/L Nps size 250 nm

Agitation cte

24°C 15 h

Precipitate

Washing/centrifugate (Acetone-H2O 6:3 mL)

Dry

Coating synthesis by SOL-GEL

1 11

2

3

45 6

7

8

9

1 10

2

3

45 6

7

8

9

1 10

70 °C

Titanium butoxide

H2O, tert-butanol, pH

60-90°C

Ti- butoxide Zn(ac)2 (NaOH) CaCl2.2H2O

Mix alcohol + H2O (5h)

Reflux system

Ca(OH)2-xZnO (x=10,20,30,40 y 50%) Ca(OH)2-xTiO2 (x=10,20 y 30%)

Start in 2010

SEM image of nanoparticles of: (a) Ca(OH)2, (b) ZnO and (c) TiO2

2m

(a)

100nm

(b)

100nm

(c)

15-30 nm spherical particles

10-20 nm nanoparticles

500 nm Hexagonal flakes

Culture and fungi growth Dra. Susana de la Rosa-García

Depto. Microbiología Ambiental y Biotecnología, UAC - Inoculation

- Evaluate microorganisms growth

In vitro, In situ,

- Light/darkness, UV

Stablish the action mode

Fungicidal/fungistatic

Bactericidal/bacteriostatic

Fungi and bacteria

- glass

- Limestone rocks

Specific medium Fungi:

Papa Dextrosa Agar (PDA)

Specific medium:

Bacteria Tripticaseína

Soya Agar (TSA)

Yeast (PDA)

Adjusted concentrations for

culture of microorganisms

Lab. de microbiología ambiental y biotecnología

UAC

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Aspergillus niger

Penicillium oxalicum

Fungi

Fungi colonization on the coatings on glass slides

and on the surface of the rocks

Control Aspergillus niger

Control Penicillium oxalicum

Coating

composition

A. niger P. oxalicum

Time (h) 24 72 120 24 72 120

Control

(untreated)

(–) (–) (–) (–) (–) (–)

Ca (OH)2 (++) (++) (++) (++) (++) (++)

Ca (OH)2 TiO2 10% (++) (+) (–) (++) (++) (++)

Ca (OH)2 TiO2 20% (++) (+) (+) (++) (++) (++)

Ca (OH)2 TiO2 30% (++) (+) (+) (++) (++) (++)

ZnO (+++) (+++) (+++) (+++) (+++) (+++)

Ca (OH)2 ZnO 10% (++) (++) (++) (+++ ) (+++) (+++)

Ca (OH)2 ZnO 30% (+++) (+++) (++) (+++) (+++) (+++)

Ca (OH)2 ZnO 50% (+++) (+++) (+++) (+++) (+++) (+++)

(+++) effective (++) moderate, (+) poor (–) ineffective

Antifungal activity of antifungal coatings (in vitro) on glass slides under natural illumination conditions

DRX a) before and b) after inoculation

1) Without coating; 2) Ca(OH)2-50%TiO2 3) Ca(OH)2-50%ZnO

A. niger P. oxalicum

Semi-quantitative XRD of different nanosystem coatings on glass slides after 120 days. Before and after inoculation with two fungi and two types of treatments: in darkness (D) and under simulated photoperiod conditions (D/L).

Ca(OH)2 Ca(OH)2-xZnO

Ca(OH)2-xZnO P. oxalicum

Ca(OH)2-xZnO A. niger

DRX analysis on coatings Glass slide

A.Niger (NL)

A.Niger (D)

P. oxalicum (NL)

P. oxalicum (D)

Coating without fungi

photoperiod (NL) darkness (D)

Characterization of the natural

limestone rocks

collected from quarries of Yucatan state

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Dry density: material mass (volume) including empty spaces.

Effective porosity: connected porous volume in a volume unity of rock.

Water content: amount of water content inside de rock.

Water absorption: percentage of water that a rock can retain before the measurement.

Physical properties of limestones

PROPERTY FORMULA NORM

Density ISRM 1979

Porosity ISRM 1981

TSE 1987

Water

content

UNE-EN

1936:1999

RILEM 1980.

Water

absorption

UNE-EN

13755:2001;

NORMA 7/81

V

Md S

%100XV

VV

%100XM

MM

S

sub

%100XM

MMWA

S

Ssat

Ms = Dry mass; V = total volume;

Vv = Empty space volume

Msub= mass submerged; Msat= saturated mass

Rock Density

(g/cm3)

% Water

Content

% Effective

Porosity

% Water

Absorption

A 2.178 0.12 6.16 6.59

B 2.547 0.03 1.27 1.22

C 2.477 0.27 1.71 1.01

White flagstone from Ticul: carbonated rock with a micritic matrix (59%) and cemented sparitic calcite (18%) with a few bivalve mollusks and echinoderms Biomicrita (Folk, 1962) o Grainstone (Wright, 1992)

Porosity of ~ 5%

Ticimul: carbonated rocks mainly with bioclasts (80%) with a micritic matrix (9%), and patches of microsparite and sparite (6%). Biomicrita (Folk, 1962) o Rudstone (Wright, 1992)

Porosity of ~ 10%

A C B

Pisté: Micritic calcite matrix (16%) with partially sparitic cement (67%), few bioclasts and fragments of bivalves (11%) , with interlayer of iron oxides patches (4%)

Intramicrita (Folk, 1962) o Rudstone (Wright, 1992)

Porosity of ~18%

Petrographic Analysis

5 10 15 20 25 30 35 40 45 50 55 60

M

HFe

2

C

T+C

A

Q

T+C

con HCl

sin HCl

T = Tosudita

C = Caolinita

Q = Cuarzo

M = Microclina

H = Hematita

A = Anastasa

Fe = Hidroxido

oxido de Fe

MUESTRA (A)

In

ten

sid

ad

Re

lati

va

(a

.u.)

5 10 15 20 25 30 35 40 45 50 55 60

H

MMo

2

Mo

A

T+C

MUESTRA (B)T = Tosudita

C = Caolinita

Mo = Montmorillonita

Q = Cuarzo

M = Microclina

A = Anatasa

H = Hematita

C

Q T+C

con HCl

sin HCl

In

ten

sid

ad

Re

lati

va

(a

.u.)

Str

uctu

ral cha

racte

rizatio

n

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

% T

rans

mita

ncia

(u. a

.)

longitud de onda (cm-1)

a)

OH

Si-O-Si

H-O-H

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

Longitud de onda (cm-1)

% T

rans

mita

ncia

(u.

a.)

a)

OH

C-O-O

(O=)PO-H

CH3

CO3=

MgAlOH

CO=

SI-O-SI

Antifungal activity (in situ) on limestone rocks

after removing the biofilm

P. oxalicum A. niger Natural stone

White flagstone Ticul

Piste

Limestone

coupons A B

Coating

composition A. niger P. oxalicum A. niger P. oxalicum

Control

(untreated) (–)1 (–)1 (–)1 (–)1

Ca(OH)2-50%TiO2 (+)14 (+++)6 (++)6 (+++)6

TiO2 (++)14 (++)12 (++)6 (+++)6

Ca(OH)2-50%ZnO (+++)6 (+++)6 (+++)6 (+++)6

ZnO (+++)6 (+++)6 (+++)6 (+++)6

Antifungal activity of coatings applied to two different limestone coupons after 21 days of exposure under simulated photoperiod

(+++) effective (++) moderate, (+) poor (–) ineffective.

The superscript shows for how many days after inoculation the antifungal activity decreased with time

Laboratory hood near to a window

XRD analysis comparison between different coatings before and after the inoculation (21 days) with two fungi under photoperiod conditions.

a & b Coupon A Pisté

c & d Coupon B White flagstone (1) Control

(2) Ca(OH)2-50%TiO2 (3) Ca(OH)2-50%ZnO

A. niger P. oxalicum

Gomez-Ortiz, N., De la Rosa-Garcia, S.C., Gonzalez-Gomez, W.S., Soria-Castro, M., Quintana, P., Oskam, G., Ortega-

Morales, B.O. Antifungal coatings based on Ca(OH)2 mixed with ZnO/TiO2 nanomaterials for protection of limestone

monuments. ACS Applied Materials & Interfaces 5 (2013) 1556-1565

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Transversal cut side view of the surface of inoculated limestones

(e) Coupon A Ca(OH)2-50%TiO2 P. oxalicum

(b) Coupon B weddellite (a) Coupon A TiO2 A.niger (c) B whewellite P Oxalicum

(d) B TiO2 A. niger inhomogeneous layer, showed larger pores and cracks, fragile coating

(f) Coupon A Ca(OH)2-50%ZnO A. niger. Homogeneous layer and better adhered to the rock

B Ca(OH)2–50%ZnO

Limestone (2.3mm) cross-sectional images showing hyphae penetration with and without coating

Coupon A A niger

Coupon B A niger

B TiO2

No coating

Coating thickness

0.00

20.00

40.00

60.00

80.00

100.00

10% 20% 30% 40% 50%

Síntesis de Ca(OH)2-xZnO S/C

Calcita

Portlandita

Zincita

Hidróxido doble de Ca y Zinc

0.00

20.00

40.00

60.00

80.00

100.00

10% 20% 30% 40% 50%

Síntesis Ca(OH)2-xZnO Penicillium sp. D/N

Calcita

Vaterita

Zincita

Whewelita

Weddelita

0.00

20.00

40.00

60.00

80.00

100.00

10% 20% 30% 40% 50%

Síntesis Ca(OH)2-xZnO Aspergillus sp. D/N

Calcita

Vaterita

Zincita

Semi-quantitative XRD of different nanosystem coatings on glass slides

Sample A Sample B

Elements

Zone 1

10m

Zone 2

41.3m

Zone 3

48m Zone 4

Zone 1

4m

Zone 2

9m

Zone 3

16m Zone 4

C 17.35 16.32 14.82 14.03 19.28 17.80 15.62 18.44

O 50.94 47.14 45.76 43.20 52.24 50.30 45.61 48.36

Ca 30.49 31.71 29.91 40.41 23.98 26.46 30.86 33.20

Zn 1.22 4.83 9.51 ─ 4.50 5.44 7.91 ─

Al ─ ─ ─ 1.06

Si ─ ─ ─ 1.30

Total 100 100 100 100 100 100 100 100

Rock type A Piste Rock type B

48μm

18μm

ZnO

cross-sectional images showing coating thickness

White flagstone Ticul

Rock

Sample A Sample B

Elements

Zone 1

7m

Zone 2

21.8m

Zone 3

32m

Zone 1

4m

Zone 2

13m Zone 3

C 19.44 18.72 14.02 18.61 16.92 18.44

O 54.42 43.99 43.25 50.60 42.80 48.36

Ca 23.98 26.64 40.41 28.62 33.70 33.20

Zn 1.58 9.50 ─ 2.17 6.58 ─

Al 0.26 0.56 1.03 ─ ─ ─

Si 0.32 0.59 1.30 ─ ─ ─

Total 100 100 100 100 100 100

Sample A Sample B

17 μm 21.8 μm

Ca(OH)2-18%ZnO

Muestra A Muestra B

Elementos

Zona 1

6m Zona 2

Zona 1

5m

Zona 2

15m Zona 3

C 12.94 14.02 16.30 14.23 18.44

O 36.66 43.20 45.97 39.25 48.36

Ca 29.50 40.41 22.60 23.59 33.20

Zn 20.90 ─ 15.13 22.93 ─

Al ─ 1.03

Si ─ 1.30

Total 100 100 100 100 100

Muestra A Muestra B

12.6μm 18.5μm

Ca(OH)2-50%ZnO

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A. niger P. oxalicum ZnO

Rock A

Rock B

Coating thickness interlayer between fungy and stone

22.5m

21.2m

1.69m 1.16m

fungi

25.3 μm

Rock A

Rock B

12.2μm 8.5μm

Thickness layer of Ca(OH)2-18%ZnO coating

42.4 μm

fungi

13μm

12μm

Thickness layer of Ca(OH)2-50%ZnO coating

8.68μm 7.1μm

Rock A

Rock B fungi

Cross-section showing hyphae penetration A. niger

Roca A Roca B

A con Ca(OH)2 - 50%ZnO B con Ca(OH)2 - 50%ZnO

7.1 μm 13 μm

Gomez-Ortiz, N., De la Rosa-Garcia, S.C., Gonzalez-Gomez, W.S., Soria-Castro, M., Quintana, P., Oskam, G.,

Ortega-Morales, B.O. Antifungal coatings based on Ca(OH)2 mixed with ZnO/TiO2 nanomaterials for protection of

limestone monuments. ACS Applied Materials & Interfaces 5 (2013) 1556-1565

without/coating

with/coating

Microbial and bacterial

colonization analysis on the

surface of the rocks

Different concentrations of the coatings

10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.781 mg,

are added in 8 mm of gel perforations

Sterilized plate of 10 x10 cm

Biotest was applied according to CLSI (Clinical & Laboratory Standards Inst).

Diffusion method in agar

3.5 mL of adjusted liquid

suspension are deposited in a

flask with agar PDA or Agar

Mueller Hinton (AMH).

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Microdilution method

Minimum inhibitory concentration (MIC)

Incubation in a

oven at 24°C

Microplates

Control de crecimiento Itraconazol

5 2.5 1.25 0.62 0.31 0.15 0.07 0.03 0.015 0.007 0.003 0.0015

CZ-H MIC

MIC

MIC

MIC

MIC

MIC

ZnO

CZ-ZnO H

CMF and CMB:

Is the minimum

concentration that

prevents the growing

of microorganisms

after been cultivated

in a free medium of

PDA (fungi) or

AMH (bacteria) and

without the coating

(< 3 colonies)

Microbiologic

Replicator Sterilized plate

with the culture

medium

Minimum concentration fungicide (CMF)

Minimum concentration bactericide (CMB)

Fungi and bacteria cultivated with the coating taken from the microplate

Stablish the action mode

fungicidal or fungistatic

bactericidal or bacteriostatic

Structural damage on

Aspergillus niger and

Penicillium oxalicum, when

caused by the coating applied

on the surface of the

calcareous rocks.

The procedure was the same

for MIC

Sterilized plate 24 wells

Glutaraldehyde

Structural damage on fungi

Aspergillus niger

Penicillium oxalicum

Fungi

Bacteria

Levaduriforme

Candida albicans

Escherichia coli Staphylococcus aureus

Microbial and bacterial colonization analysis on the coatings and on the surface of the rocks

Yeast

10 15 20 25 30 35 40 45 50 55 60

CZCZ

5h

4h

2h

P P

c)

b)

PP

P

ZZ Z

P

C

C

CZ

CZCZ

Inte

ns

ida

d R

ela

tiv

a (

u.a

)

CZ

C

Pa)

CZ=Calcium zincate C=Calcite P=Portlandite Z= Zincite

CaCl2 2H2O + 2 ZnO + 2 H2O + 2 NaOH Ca[Zn(OH)3]2 2H2O + 2 NaCl

CZ: retardation of cement hydration; passivation of galvanized steel corrosion in cement pastes or in alkali solutions; slowing anode degradation in Zn/NiOOH batteries, has higher lifecycle decreases precipitation and dissolution of Zn during charge-discharge; as solid base catalyst for the

methanolysis of sunflower for biodiesel.

Hydrated calcium zincate

MC a = 6.384Å, b = 10.967Å, c = 5.759Å; β = 101.92°

Zn (OH)4 Ca(OH)6

Limestone coupons A (Pisté) B (White flagstone Ticul) C (Ticimul)

A. niger P. oxalicum A. niger P. oxalicum A. niger P. oxalicum

Control (untreated) (–)2 (–)2 (–)2 (–)2 (–)2 (–)2

Ca[Zn(OH3)]2·2H2O (+++) (+++) (+++) (+++) (+++) (+++)

Antifungal activity of coatings applied to three different limestone coupons after 21 days of exposure under simulated photoperiod (D/UVL).

Antifungal activity (+++) effective (++) moderate, (+) poor (–) ineffective; superscript indicates the number of days after which the samples were completely covered with fungal colonies.

coated limestone coupons inoculated with A. niger & P. oxalicum.

without coating and before inoculation

Optical microscopy images of the limestone coupons

uncoated control coupons after inoculation with fungi strains

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Antifungal activity of the coatings and the biofilm

formation on the rock surfaces

without/coating Biofilm/coating CZ P. oxalicum +CZ

Biofilm growth on the rock without CZ

(1) Natural limestone without antifungal coating and inoculated with A. niger (2) without antifungal coating and inoculated with P. oxalicum; (3) with antifungal coating and inoculated with A. niger; (4) with antifungal coating and inoculated with P. oxalicum.

X-ray diffraction analysis of the limestone substrates, before and after 21 days of inoculation, with two fungi

A C B

The presence of weddelite is an indicator of the fungal activity

Limestone coupons A, B, C inoculated with A. niger, without and with CZ coatings.

Limestone coupons A, B, C inoculated with P. oxalicum, without and with CZ coatings.

Gómez-Ortíz, N.M., González-Gómez, W.S., De la Rosa-García S.C., Oskam, G., Quintana, P. Soria-Castro, M., Gómez-Cornelio, S., Ortega-Morales, B.O. Antifungal activity of Ca[Zn(OH)3]2 ·2H2O coatings for the preservation of limestone monuments: An in vitro study. Int. Biodeter & Biodegr 91(2014).1-8

Tyoe of

pathogen

Microorganisms Replication MIC (mg/mL) CMF (mg/mL)

CZ ZnO CZ ZnO

Fungi

P. oxalicum

0.312-1.25

Fgt

0.039-0.625

Fgt

-

1.25

Fgc

A. niger 0.156-1.25

Fgt 0.039-1.25

Fgt - -

Yeast C. albicans

0.312-.0.625

Fgt 5.0

Fgt 1.25-10

Fgc 10.0

Fgc

Measurements of CMI and CMF and the action mode

Indicating the initial growing concentration

Fgc = Fungicidal Microorganisms die Fgt = Fungistatic Preventing the growth of fungi

Type of

pathogen Microorganisms

CMI (mg/mL) CMB (mg/mL)

CZ ZnO CZ ZnO Bacteria

Gram

positive

Escherichia coli 1.25

0.156-

1.25

Bct

1.25-10

Bac 2.5-10

Bac

Bacteria

Gram

negative

Staphylococcus

aureus

0.078-

0.625

Bct

0.019-

0.78

Bct

1.25-10

Bac

0.156-10

Bac

Measurements of CMI and CMB and the action mode

Bac = Bactericidal Microorganisms die

Bct = Bacteriostatic Preventing the growth of bacteria

Indicating the initial growing concentration

Gram+ higher susceptibility one cellular membrane Gram– double membrane higher protection

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SEM

Aspergillus niger.

a) Fungal control

b) Conidia

c) CMI of ZnO

(no cell damage or

formation of

hyphaes)

d) CZ after CMI

(no changes)

Morphological effect when fungi grows due to the

presence of CZ and ZnO

SEM

Penicillium oxalicum

a) Fungal control

b) Conidia

c) CMI of ZnO

(cell damage and

conidia germination)

d) ZnO after CMI

shows unusual

protuberances that

grows in the hyphaes

Morphological effect when fungi grows due to the

presence of CZ and ZnO

PATENT Mx/a/2015/004076: Formulaciones y compositos con propiedades

antibacterianas, antimicrobianas, antimicóticas y/o antivirales

All limestone substrates without protective coating, fungal growth and

penetration into the limestone was observed. In addition, it is clearly to

observe the presence of calcium oxalate crystals, and its presence is an

indicator of the fungal activity.

Calcium zinc hydroxide dihydrate coating is well-adhered to all

substrates. The diffusion method in agar of CZ show a high antifungal,

antibacterial and no activity of yeast, also an inhibitory activity of wide

spectra against bacteria Gram-positive, in comparison to ZnO.

therefore for the three limestone substrates with the protective coating,

no fungal growth was observed, illustrating the excellent antifungal

properties of the CZ.

PATENT: Mx/a/2015/004076: Formulaciones y compositos con propiedades

antibacterianas, antimicrobianas, antimicóticas y/o antivirales

in situ (22 d)

ex situ (46d)

in vitro

Ca[Zn(OH)3]2·2H2O

ZnO

Environmental

urban and marine

(3,6,9,12 months)

20 meses

(4/12/2015) 30 meses

(6/10/2016)

Cinvestav-Mérida

Depto Física Aplicada

Dra. Patricia Quintana

Dr. Gerko Oskam

Dr. Juan José Alvarado

Friedrich Schiller Univ. Jena

Dr Carlos Guerrero

Fernando May (Posdoctorado)

Nikté Gómez (Doctorado)

Santiago González (Doctorado)

Montserrat Soria (Doctorado)

Uriel Zagada (Maestría)

Universidad Autónoma

de Campeche

Lab Microbiología

Ambiental y Biotecnología

Dr. Otto Ortega Morales

Centro Investigación

Científica de Yucatán

Unidad Académica Materiales

Dr. Francis Avilés

Collaborations Universidad Juárez Autónoma de Tabasco Lab de Microbiología

Dra. Susana de la Rosa

García y MC Sergio Gómez

Gracias!

Trompetistas, Bonampak