silver nanoparticles misting - an innovative method of...

Silver nanoparticles misting

- an innovative method

of archaeological object disinfection

Katarzyna Pietrzak, M.Sc., Eng.

Institute of Fermentation Technology and Microbiology

Lodz University of Technology

Research co-funded by the International Visegrad FundModern approach for biodeterioration assessment and disinfection of historical book collections

Silver nanoparticles application

• filters for air purification• filters for water

purification• stomatology• dietary supplements• implants• textiles• cosmetics, detergents• home appliance• computer hardware• coatings, grouts, adhesives• food packaging• papermaking• disinfection of historical

objects

Chaloupka et al., 2010; Gutarowska et al., 2012

Silver nanoparticles (AgNPs)

Synthesis

▫ chemical

▫ physical

▫ biological

Feng et al., 2000; Kim et al., 2007

triangular spherical rods

viruses moulds yeastsGram-

negativebacteria

Gram-positive bacteria

sensitivity

Effectiveness

• shape

• form

• size

• concentration

• microorganisms

Mechanism of antibacterial action of AgNPs

disfunction of transport to the cell

inhibition of DNA replication

loss of the biological activity of the amino acid

structural and functional changes in the cell membrane

disturbance of the cell membrane electric potential

outflow of protons and certain metabolites throughthe cytoplasmic membrane

A

B

C

D

E

F

F

E

Wzorek, Konopka, 2007; Feng et al., 2000

Mechanism of antifungal action of AgNPsYeasts

• changes of cell wall functions and structure

• creation of "pits" in the cell membrane• disturbances of electric potential• inhibition of budding process

Candida albicans and Cryptococcus neoformansafter AgNPs treatment

Moulds• inhibition of sporulation process• changes in cell ultrastructure• changes in metabolome (acids, enzymes,

proteins)

Aspergillus niger after AgNPs treatment

Pinto et al., 2013: Ishida et al., 2014; Lara et al., 2015; Pietrzak et al., 2016

Aim and scope

Optimalization of AgNPs misting disinfection and its influence on microorganisms and disinfectedmaterials.

1. Determination of the sensitivity of pure culture collection microorganisms and isolatesfrom historical objects surfaces on the silver nanoparticles preparation.

2. Parameters optimization of the of effective disinfection of technical and historical materialswith silver nanoparticles misting.

3. Assessment of the effectiveness and durability of disinfection dependingon the microorganism, technical or historical material.

4. Analysis of the disinfection influence on mechanical and optical parameters of technicalmaterials before and after artificial ageing.

MaterialsTechnical:

- paper* (Sa, Hmp, Sy, CTMP, GW)

- leather (cowhide, dyed)

- textiles (wool, silk, cotton, linen)

- wood (beech, oak, pine)

*Sa - bleached pine kraft pulp; Hmp - bleached hemp kraft pulp; Sy - bleached spruce sulphite pulp; CTMP - chemi–thermomechanical pulp;GW - bleached groundwood from spruce (GW)

Historical:

- canvas (historical painting)- parchment (conservation materials)- wood (church floor; 17th c.)- paper (map; 18th - 19th c.)- textiles (wool, cotton, sisal; 13th – 15th c.)- ceramics (6th – 13th c.)

WoolCeramics Cotton

Silver nanoparticles

SEM image of nanosilver 1

TEM image of nanosilver 2

0

20

40

60

80

100

<3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 >12nu

mb

er o

f n

an

op

art

icle

s

nanoparticles 2 dimension (nm)

chemical reductionAgNO3; reductor: sodium citrate; stabilizer: PVP

concentration: 90 ppm

particle size: 10-80 nm

(10-15 nm – 60-70%; 50-80 nm –30-40%)

Mennica Polska S.A.

AgNPs 1 thermaldecompositionof silvercompounds; stabilizer: paraffins

concentration: 1000 ppm

particle size: 3-8 nm

Amepox sp. z o.o.

AgNPs 2

Microorganisms

ATCC – American Type Culture Collection; NCAIM - National Collection of Agricultural and Industrial Microorganisms; ŁOCK – Łódzki Ośrodek Czystych Kultur (Pure Culture Collection at Institute of Fermentation Technology and Microbiology, Lodz University of Technology)

•Aneurinibacillus aneurinilyticus•Brevibacillus laterosporus•Bacillus subtilis•Bacillus megaterium•Bacillus pumilus•Bacillus licheniformis•Sphingomonas paucimobilis•Micrococcus sp.•Micrococcus flavus•Pseudomonas aeruginosa•Staphylococcus aureus•Staphylococcus lentus•Staphylococcus xylosus•Nocardia sp.•Escherichia coli (ATCC 10536)•Staphylococcus aureus (ATCC 6538)•Bacillus subtilis (NCAIM 01644)

BACTERIA

•Alternaria alternata•Aspergillus versicolor•Aspergillus niger•Cladosporium cladosporioides•Cladosporium herbarum•Cladosporium macrocarpum•Mucor racemosus•Penicillium digitatum•Penicillium carneum•Penicillium crustosum•Penicillium radicola•Rhizopus nigricans•Candida sphaerica•Rhodotorula sp.•Rhodotorula mucilaginosa•Aspergillus niger (ATCC 16404)•Penicillium chrysogenum (ŁOCK 0531)•Candida albicans (ATCC 10231)

FUNGI

MethodsAnalysis Method / Standard

Silver nanoparticles

Characterization of preparation SEM; TEM

Determination of AgNPs content FAAS; LA-ICP-TOF-MS

Microorganisms

Determination of microbial sensitivity (MIC and MBC) ASTM E2149 – 01

Choosing the conditions for disinfectionEvaluation of the effectiveness and durability of the disinfection technical materials and historical objects

AATCC Test Method 100-2012

Materials

Assessment of mechanical parameters (breaking, elongation, tear, compression)

ISO 13934-1:1999 (textiles); ISO 3376:2005 (leather); ISO 20187:1993 (paper); ISO 1924-1:1998 (paper); BS EN 21974:2002 (paper); BS EN 384:2004 (wood)

Estimation of colour parametersMetoda Tappi T 524 om-94 (paper); ISO 2470:1999 (paper); ISO 105-J01:2002 (leather, textiles); Metoda SCI (wood)

SEM – Scaning Electron Microscopy; TEM – TransmissionElectron Microscopy; AATCC - American Association of Textile Chemists and Colorists; ASTM - American Society for Testing and Materials;FAAS – Flame Atomic Absorbance Spectrometry; LA-ICP-TOF-MS - Laser Ablation Inductively Coupled Plasma Time-of- Flight Mass Spectrometry; MIC – Minimnal Inhibitory Concentration;MBC – Minimal biocidal Concentration; SCI - Specular Component Included

Microbial sensitivity to AgNPs

Microorganisms Origin MIC (ppm) MBC (ppm)

Staphylococcus aureusATCC 11.25 22.50

Museum 1 22.50 45.00

Bacillus subtilis

ATCC 22.50 > 45.00

Archives 22.50 > 45.00

Museum 2 45.00 > 45.00

Escherichia coli ATCC 11.25 22.50

Nocardia sp. Library 11.25 22.50

Aspergillus nigerATCC 22.50 45.00

Museum 2 45.00 45.00

Candida albicans ATCC 11.25 22.50

Candida sphaerica Library 22.50 22.50

MIC – Minimal Inhibitory Concentration; MBC - Minimal Biocidal Concentration; ATCC – American Type Culture Collection Gutarowska et al., 2012

moulds: Aspergillus sp., Rhizopus sp., Penicillium sp.;

G+ bacilli: Bacillus sp.;

G+ cocci: Micrococcus sp., Staphylococcussp.

moulds: Cladosporium sp., Alternaria sp., Mucor sp.;

yeasts: Rhodotorula sp., Candida sp.;

actinomycetes: Nocardia sp.

G- rods: Sphingomonas sp., Escherichia sp.

MIC 45.00 ppm 22.50 ppm 11.25 ppm

Microbial sensitivity to AgNPs

Viruses Mould YeastGram

negativebacteria

Gram positivebacteria

Gutarowska et al., 2012

AgNPs misting chamber

Wasiak R., Laskowski Z., Czyżyk J. The microbiological protectionmethod of archive and museum objects and installation for themicrobiological protection of archive and museum objects. PatentPL399507, Poland 2012

INSTAL WARSZAWA S.A.

Siennicka 29, 04-394 Warsaw, Polandwww.instalwaw.com.pl

Capacity: 1.73 m3 36-42 books A4

Choosing the conditions for AgNPs misting

Misting

Relativehumidity

90%

Temperature

25ºC

Airflow

1 m/s

Nozzle position

horizontal; horizontally -

vertical

Materialhumidity

20 – 70%

AgNPs concentration

45 - 90 ppm

Misting cycles

1 - 8


Nozzle position and AgNPs distribution

Nozzle position(horizontally – vertical)

Sampling point AgNPs amount (ppm/g)*

1 2.8

2 2.2

3 3.3

4 2.7

5 2.9

6 2.3

Sample pointsdistribution

* FAAS – Flame Atomic Absorbance Spectrometry

0

20

40

60

80

100

1 3

Re

du

cti

on

of

B.

su

bti

lis

(%)

Misting cycles

horizontally horizontally-vertically


Misting cycles, AgNPs concentration,

material humidity

0

20

40

60

80

100

1 2 3 6 8

Re

du

ctio

no

f A

. nig

er(%

)

Misting cycles

45 ppm 90 ppm

Gutarowska et al., 2012; 2014

10% - dry cotton

1,0E+00

1,0E+02

1,0E+04

1,0E+06

1,0E+08

1,0E+10

E. coli S. aureus B. subtilis A. niger

Nu

mb

er o

f m

icro

org

an

ism

s (C

FU

/sa

mp

le)

control 10%* 30% 50% 65%

0

1

2

3

4

5

6

7

8

Leather Wool Linen Cotton Silk GW Hmp CTMP Sy Sa Pine Oak Beech

Ag

NP

s a

mo

un

t (p

pm

/g)

Technical material

Silver nanoparticles amount by FAAS

FAAS – Flame Atomic Absorbance Spectrometry; Sa - bleached pine kraft pulp; Hmp - bleached hemp kraft pulp; Sy - bleached spruce sulphitepulp; CTMP - chemi–thermomechanical pulp; GW - bleached groundwood from spruce (GW)

8 misting cycles


0

20

40

60

80

100

Bawełna Len Wełna Jedwab CTMP GW Hmp Sy Sa

Red

uc

tio

n (

%)

A. niger S. aureus E. coli B. subtilis

Cotton Linen Wool Silk

Disinfection effectiveness on technical materials

A. nigerS. aureusE. coliB. subtilis

*Sa - bleached pine kraft pulp; Hmp - bleached hemp kraft pulp; Sy - bleached spruce sulphite pulp; CTMP - chemi–thermomechanical pulp;GW - bleached groundwood from spruce (GW) Gutarowska et al., 2014

AgNPs misting durability

0

20

40

60

80

100

2 4 6 9 14

Re

du

ctio

n (

%)

Storage time (days)

E. coli A. niger

Storage conditions: 28°C, RH 80%

Inhibition of Pseudomonas aeruginosa growth

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

0 3 5Nu

mb

er

of

mic

roo

rgan

ism

s(C

FU/c

m2)

Storage time (days)

control AgNPs

Pseudomonas aeruginosa on historical textile

Storage conditions: 28°C, RH 80%Pietrzak et al., 2016

97.1%

Disinfection effectiveness on historical materials

1,0E+00

1,0E+02

1,0E+04

1,0E+06

1,0E+08

1,0E+10

1,0E+12

canvas parchment paper map wooden floor

Nu

mb

er

of

mic

roo

rga

nis

ms

(CF

U/c

m2)

control AgNPs


79.7%67.6%

99.7%

99.9%

Disinfection effectiveness on archaeological

textiles

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

Cotton Sisal Wool 1 Wool 2 Wool 3 Wool 4 Wool 5 Ceramics

Nu

mb

er

of

mic

roo

rgan

ism

s (C

FU/g

)

control AgNPs

Pietrzak et al., 2016

99.4%

100%

99.9%

Mechanical parameters of paper

Sa - bleached pine kraft pulp; Sy - bleached spruce sulphite pulp; GW - bleached groundwood from spruce (GW)

PaperControl AgNPs Aged control Aged AgNPs

Elongation(%)

Tear index(mN×m2/g)

Elongation(%)


Elongation(%)


Elongation(%)


GW 2.4±0.2 8.1±0.3 2.8±0.2 8.1±0.5 2.2±0.1 7.5±0.2 2.1±0.2 7.2±0.8

Sy 2.0±0.1 9.3±0.8 2.4±0.2 8.6±0.4 1.9±2.2 7.6±0.4 1.8±0.2 7.2±0.3

Sa 3.0±0.2 13.5±0.9 3.1±0.2 13.9±0.4 2.6±0.1 12.9±0.3 2.6±0.1 12.8±0.4

Optical parameters of paper

R457 – ISO Brightness; Sa - bleached pine kraft pulp; Sy - bleached spruce sulphite pulp; GW - bleached groundwood from spruce

PaperControl AgNPs Aged control Aged AgNPs

R457 R457 ΔE R457 ΔE R457 ΔE

GW 82.9±0.1 82.1±0.1 0.41 78.6±0.5 1.02 78.5±0.1 0.94

Sy 89.8±0.5 89.4±0.3 1.06 85.4±0.6 0.37 85.5±0.7 1.27

Sa 90.5±0.3 89.6±0.1 0.91 87.7±0.2 0.57 87.7±0.1 0.61

0 < ∆E < 1 - observer does not notice the difference1 < ∆E < 2 - only experienced observer can notice the difference2 < ∆E < 3.5 - unexperienced observer also notices the difference3.5 < ∆E < 5 - clear difference in colour is noticed5 < ∆E - observer notices two different colours

∆𝐸 = ∆𝐿∗ 2 + ∆𝑎∗ 2 + (∆𝑏∗)2

Disinfection of historical book collections

2 books Disinfection

Lowtemperature

plasma

Thymeessential oil

AgNPs misting

Nanosilver concentration

90 ppm

Misting cycles

8

Airflow

1 m/s

Temperature25ºC

RH

90%

Duration

520 min

AgNPs misting

Disinfection effectiveness: culture-dependent

analysis

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

BA BB BC BD BA BB BC BD

Nu

mb

er

of

ba

cte

ria

(CF

U/s

am

ple

)

Item (place)

Control AgNPs EOs LTP

1 4BA – front cover; BB - front free endpaper; BC – fore edge; BD – page inside the book; sample = 25 cm2

Disinfection effectiveness: culture-dependent

analysis

1,0E+00

1,0E+02

1,0E+04

1,0E+06

1,0E+08

1,0E+10


Nu

mb

er

of

fun

gi

(CF

U/s

am

ple

)

Item (place)

Control AgNPs EOs LTP

1 4

BA – front cover; BB - front free endpaper; BC – fore edge; BD – page inside the book; sample = 25 cm2

Disinfection effectiveness: culture-

independent analysis

1 4

0

200

400

600

800

1000

1200

1400

1600


RN

A c

on

ce

ntr

ati

on

(ng/µl)

Item (place)

control AgNPs EOs LTP

BA – front cover; BB - front free endpaper; BC – fore edge; BD – page inside the book; sample = 25 cm2

1,0E+00 1,0E+01 1,0E+02 1,0E+03

Staphylococcus succinus

Staphylococcus saprophyticus

Staphylococcus pasteuri

Staphylococcus epidermidis

Psychrobacillus psychrodurans

Microbacterium aerolatum

Bacillus licheniformis

Bacillus cereus

1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+00 1,0E+01 1,0E+02 1,0E+03

control

disinfection

AgNPs EOs LTP

Bacterial sensitivity

Number of microorganisms (CFU/sample)

sample = 25 cm2

Fungal sensitivity

1,0E+00 1,0E+03 1,0E+06 1,0E+09

Rhodotorula mucilaginosa

Penicillium spinulosum

Penicillium chrysogenum

Myxotrichum deflexum

Chaetomium murorum

Chaetomium globosum

Chaetomium elatum

Aspergillus niger

1,0E+00 1,0E+03 1,0E+06 1,0E+09 1,0E+00 1,0E+03 1,0E+06 1,0E+09

control

disinfection

Number of microorganisms (CFU/sample)

AgNPs EOs LTP

sample = 25 cm2

Conclusions

1. Silver nanoparticles misting, low temperature plasma and thyme essential oilmicroatmosphere were efficient methods of growth inhibition of bacteria andfungi.

2. The disinfection effectiveness depended on the inhabiting microbiota,disinfected book and the sampling place.

3. The reduction of microorganism numer equalled on Book no. 1 to: AgNPs andLTP (0–100%), EOs (0–97 %); and on Book no. 4 to: AgNPs 78–100%, LTP(25–100%) EOs 23–100%).

4. RNA concentration is a good marker to assess the disinfection effectiveness.

5. Aspergillus niger, Chaetomium globosum, Bacillus cereus andMicrobacterium aerolatum were insensitive to all disinfection methods.

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