International Biodeterioration & Biodegradation: Seon Hye Jeong, Hyun Ju Lee, Dae Woon Kim, Yong Jae Chung

International Biodeterioration & Biodegradation xxx (2017) 1e10
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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod
New biocide for eco-friendly biofilm removal on outdoor stone

monuments
Seon Hye Jeong a, Hyun Ju Lee a, Dae Woon Kim b, Yong Jae Chung a, *
a
Dept. of Heritage Conservation & Restoration, Graduate School of Cultural Heritage, Korea National University of Cultural Heritage, Buyeo, Republic of
Korea
b
Con-Tech, School Based Enterprise, Korea National University of Cultural Heritage, Buyeo, Republic of Korea
a r t i c l e i n f o a b s t r a c t
Article history: In this study, an eco-friendly biocide was prepared from natural medicinal ingredients that are tradi-
Received 21 October 2016 tionally used to preserve and maintain outdoor stone monuments in South Korea, and its effectiveness
Received in revised form was verified through laboratory testing and on-site application. Eugenol, which was isolated and iden-
7 March 2017
tified from volatile clove extracts, was mixed together with eco-friendly emulsifiers from the Tween and
Accepted 7 March 2017
Available online xxx
Span series at different ratios to prepare an optimized biocide. Strong antifungal activities were
confirmed when the biocide was tested against 19 types of molds that were isolated and identified from
the biofilms of two World Heritage Sites: the Royal Tombs of the Joseon Dynasty in South Korea and the
Keywords:
Natural biocide
Vat Phou temple in Laos. When applied on-site, the lichen became decolorized and discolored, and was
Eugenol ultimately eliminated from the surface as the mycelium in its epithelial layer disintegrated. Furthermore,
Stone monument an average of 80% reduction in microorganisms was observed from the applied surface based on ATP
Biofilm measurements before and after biocide treatment. The developed biocide shows high prospects for
Cleaning commercial use as part of an eco-friendly technology applicable to outdoor stone monuments.
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction A 5-year investigation on the biological weathering of stone

monuments in South Korea was initiated in 2000 in order to assess
Stone cultural heritages that are placed outdoors are susceptible the degree of weathering on stone monuments designated as Na-
to environmental factors such as temperature, humidity, precipi- tional Treasures. The monuments were categorized into 5 grades
tation, storms, atmospheric pollution, and organisms. The effects of depending on the extent of their damage. The results revealed that
these factors are not singular but rather compound, and therefore approximately 10% of the important stone monuments (53 out of
they accelerate the degradation of such monuments to severe 533) were classified as showing grade-5 biological damage, signi-
levels. These weathering conditions can be categorized into phys- fying serious biological damage (Min, 2007). Similar phenomena
ical, chemical, biological, and artificial. In biological weathering, a have been observed in other countries. The main causes of
biofilm is formed on the surface of stone monuments, which ac- weathering for stone cultural heritages at Angkor, Cambodia were
celerates the weathering process of stonework by promoting severe determined to be chemical and biological weathering (Uchida et al.,
surface pollution, causing secondary physical damage (Chung et al., 1999), and weathering of stone cultural heritages in Spain was also
2003). The growth of organisms on stone monuments can be found to be accelerating due to ecological succession (Go mez-
regarded from two perspectives. Although the growth of various n et al., 1995). Furthermore, a study was conducted on the
Alarco
organisms illustrates the passage of time by creating an antique production of biofilms and the types of organisms growing on the
atmosphere, these organisms are also responsible for the weath- surface of stone cultural assets in Serbia composed of granite and
ering effects mentioned above; therefore, it is necessary to find an sandstone (Ljaljevic Grbic et al., 2010). Therefore, many studies are
adequate balance between the desired esthetics of a monument actively being conducted in South Korea and overseas on the bio-
and the preservation of its materials (Doehne and Price, 2010). logical weathering and preservation of stone cultural heritages.
Research on mechanical and chemical methods for biofilm
removal has significantly progressed in recent years, and these
* Corresponding author. methods are currently being applied on-site. Moreover, ultraviolet
E-mail address: iamchung@nuch.ac.kr (Y.J. Chung).
http://dx.doi.org/10.1016/j.ibiod.2017.03.004
0964-8305/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jeong, S.H., et al., New biocide for eco-friendly biofilm removal on outdoor stone monuments, International
Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/j.ibiod.2017.03.004
2 S.H. Jeong et al. / International Biodeterioration & Biodegradation xxx (2017) 1e10
radiation and biocides combining benzalkonium chloride and components. In brief, 100 g of each of the five dried medicinal in-
quaternary ammonium compounds are frequently used (Caneva, gredients was extracted by refluxing for 3 h using 125 ml of
2007). The main goal of such treatments has not been to methylene chloride (CH2Cl2) that was redistilled with simultaneous
completely remove the biofilms, but rather to reduce the concen- steam distillation and a solvent extraction device. To dehydrate the
tration of contaminants with regard to preserving the stability of extracts, they were filtered through a filter paper after adding so-
stone quality (Young, 2007). However, it is globally recommended dium sulfate (Na2SO4), and subsequently condensed with N2 gas.
to avoid the use of methods involving polishing and chemical
treatment, as they can cause mid-to long-term damage to the 2.2. Isolation and identification of the fungal constituents of the
constituent stone (Cho and Xia, 2002). Although chemical products biofilm
with NA2CO3 and (NH4)2CO3 as the main component have also been
used to remove biofilms from stone monuments in South Korea, 2.2.1. Collection and cultivation
their use has been associated with problems such as the need for The fungal constituents of the biofilms were collected from the
distilled water treatment after the application of chemicals and biofilms formed on the surfaces of the stone monuments at the
worker safety concerns, which have sharply reduced their following World Heritage Sites: the Royal Tombs of the Joseon
application. Dynasty in South Korea (Yeoju, Yeongneung), and the Vat Phou and
With the problems of chemical substances becoming more Associated Ancient Settlements within the Champasak Cultural
apparent, the approach of using natural substances and herbs has Landscape (Vat Phou Temple). Small quantities of the biofilms were
been gaining prominence in the field of cultural heritage and scraped off with a scalpel, and sterilized blades were replaced and
conservation sciences since the 2000s. Based on a study of the used for each collection. The collected specimens were pulverized
properties of insecticidal and antifungal substances isolated from and shaken in a sterilized 0.001% Tween 80 compound for 1 h. After
aromatic plants found in Bokjang (a traditional re-burial site) shaking, 100 ml of the suspension was inoculated in Czapek Dox
(Chung et al., 2001), several researchers have screened various trees agar (CZA) and malt extract agar via the spread plate method. The
and herbs to examine the antifungal and insecticidal activities of inoculated medium was incubated for 5 days in a 28 C incubator.
their extracts for potential use in the conservation of cultural her-
itages composed of organic materials (Kang et al., 2007; Lim et al., 2.2.2. Classification and identification
2008; Hong and Jung, 2009; Choi et al., 2010; Kim et al., 2011). The fungi grown on the culture medium were classified ac-
However, the studies conducted on natural substances to date have cording to factors such as the shape, observable color, and reverse
mainly focused on cultural heritages made of organic materials color of the colony, and the fungal spores, mycelia structure, and
such as wood and paper, and the scope has been limited to those other morphological characteristics were examined under a light
that are preserved indoors. microscope (Nikon LV100D, Japan) for accurate classification.
To date, extensive research efforts have been focused on char- Sequence analysis of the 18S rRNA region was conducted to classify
acterizing the effect of biofilms, including those formed by lichens, the fungi. Genomic DNA was extracted using the I-genomic BYF
on outdoor stone monuments. Lichens have been shown to cause DNA Extraction Mini Kit (iNtRON), and the ITS1 (50 -TCCGTAGGT-
weathering through either physically pressurizing the stone by GAACCTGEGG-30 )/ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) primer
sending roots into the stone cleavage, or decolorizing and dissolv- set was used to amplify the target regions. An automatic DNA
ing the stone via lichen-produced metabolites. Moreover, biofilms sequencer (Perkin-Elmer) was used for sequence analysis, and the
have been shown to have some protective effects, such as making GenBank (National Center for Biotechnology Information) BlastN
the surface of the stone more resistant to weathering, or reducing Search program was used for identification.
the thermo-hygro mechanical stress by controlling the humidity,
heat transparency, and steam diffusion (Daniela, 2014). Therefore, 2.3. Assessment of antifungal activity
the role of biofilms has been actively studied, as it is directly related
to the issue of its removal. However, formation of a biofilm can After the culture was suspended in distilled sterile water, it was
make it difficult to determine the structure of a relief or inscription smeared onto 100 ml of CZA medium via the spread plate method. A
on a tombstone, thereby necessitating biofilm removal in such paper disk was placed on top of the culture medium, and 50 ml of
cases. the biocide was applied onto it, whereas an emulsifier and distilled
Therefore, in this study, we examined substances from natural water were applied on the disks of the control group. After 5 days of
medicinal ingredient extracts that could potentially be used as incubation at 28 C, antifungal activity was assessed by measuring
biocides for the preservation and maintenance of outdoor stone the clear zone (mm) formed around the disk. Antifungal activity
monuments. The ultimate aim of this study was to develop a sub- was categorized into 4 grades (-, þ, þþ, þþþ) depending on the
stance and technique that is stable and effective for the treatment diameter of the clear zone, with each grade signifying a quarter of
of stone monuments, and to verify the effect of the developed the total diameter, ranging from a minimum of 0 mm to a
biocide through laboratory testing and on-site application. maximum of 22 mm. Natural medicinal ingredient extracts were
tested against fungi collected at Yeongneung only, whereas the
2. Materials and methods developed biocide was tested against fungi collected at both
Yeongneung and the Vat Phou temple.
2.1. Natural medicinal ingredients and preparation
2.4. Component analysis of biologically active substances
Five types of dried natural medicinal ingredients, including
Illicium verum, the flower buds of Syzygium armoaticum, Quercus Component analysis of the volatile extracts with confirmed
infectoria, Coptis chinensis, and Phellodendron amurense, were pur- antifungal activity was conducted with gas chromatography/mass
chased and used for this study. The dried specimens were used after spectrometry (GC/MS, Agilent Technologies 7890A/5975C, USA)
they were cut into 1e3-cm pieces, pulverized with a universal using a DB-5ms column (30 m 0.250 mm 0.25 mm). The oven
cutting mill (FRITSCH pulverisette 19, Germany), and filtered program was set to increase from 40 C (3 min) to 320 C at 2 C/
through a 60-mesh screen (Table 1). min; the inlet temperature was set to 150 C, and the detector
Volatile compounds were extracted among the effective temperature was set to 250 C. The inlet mode was set to spilt mode
S.H. Jeong et al. / International Biodeterioration & Biodegradation xxx (2017) 1e10 3
Table 1
Natural medicinal ingredients evaluated in this study.
Name Scientific name
1 Star anise Illicium verum
2 Clove Syzygium aromaticum Merrill et Perry
3 Gallnut Rhus javanica
4 Golden thread Coptis chienesis
5 Amur cork-tree Phellodendron amurense RUPR
(split ratio 20:1), and helium (1.0 ml/min) was used as the carrier Korea, by examining the change of its surface and weight. In
gas. addition, the components of rock-forming minerals were analyzed
to assess the material stability; that is, the amount of substances
2.5. Reagents and emulsifiers used for preparation of the natural applied on a site that could remain intact without being washed
biocide away by rain. Each stone specimen (3e4 g) was placed in 10 ml of
Biocide A, and stirred continuously at 25 C for 15 days (376 h and
Eugenol (ReagentPlus®, 99%) was purchased from Sigma-Aldrich 24 min), corresponding to the 10-year average number of days
(Seoul, Republic of Korea) for the preparation of a natural biocide without continuous rain recorded in the last 10 years in South
(hereinafter referred to as Biocide A), and emulsifiers were used to Korea. Granite specimens treated with the emulsifier and distilled
emulsify the eugenol with water. Tween 20, 40, and 80, and Span water alone were set as the control groups. Macroscopic exami-
20, 40, and 80 (Daejung Chemicals & Metals Co., Ltd., Republic of nation was conducted by rinsing the specimen with distilled water
Korea) were used as the emulsifiers. and studying its surface condition and color via photography with a
digital camera (Powershot G16, Canon, Japan). The change in
weight (DW) was measured using a precision electronic balance
2.6. Identifying the optimal ratio
(AW320, Shimadzu, Japan) to 0.0001 g. The DW value was calcu-
lated as the percentage of the weight before to that after treatment,
Eugenol, emulsifiers, and distilled water were mixed according
and was compared with that of the control group. X-ray diffraction
to the volumetric ratio, and the resulting change in color, formation
analysis (D/max-2200, Rigaku, Japan) was conducted on the spec-
of precipitate, etc. were observed. A red dye, Safranin O, was
imen before and after treatment with the following parameters:
included in the mixture to accurately determine the degree of
CuKa for the X-ray, 40 kV and 40 mA accelerating voltage and
emulsification. The concentration of eugenol was fixed at 5%, and
filament current at the anode, respectively, and 2Ɵ set at 5 /min for
the mixture was stirred for 1 h at emulsifier concentrations of 3%,
5 e80 .
5%, 7%, and 10%. The solution was assessed after being left for
24 h at room temperature (20e22 C). Moreover, Tween 20 and
Span 20 were prepared at 5 ratios (1:9, 3:7, 5:5, 7:3, 9:1), and their 2.8. On-site application
hydrophilic/lipophilic balance (HLB) was assessed in the same
manner as described above. The on-site application of Biocide A was conducted on stone
statues at the Korea National University of Cultural Heritage in
2.7. Assessment of material stability South Korea, and on stone components of the Hong Nang Sida
Temple in Laos. In South Korea, 50 ml of Biocide A was sprayed over
The optimally prepared eugenol-based material, termed Biocide the entire stone statue (width 32 cm, length 25 cm, height 90 cm)
A, was tested on granite, the most common rock type in South with a compression sprayer. In Laos, 10 ml of Biocide A was sprayed
in the same manner over a 10 10-cm area. The effectiveness of the lipophilic (Yeon et al., 2014). As Tween 20, 40, and 80 have an HLB of
biocide was assessed by comparing the extent of biofilm cover 16.7, 15.6, and 15.0, respectively, they appear to have been able to
through macroscopic (PowerShot G16, Canon, Japan) and micro- effectively emulsify the eugenol with the dispersion media of water
scopic (DG-3X, Scalar, Japan) observations. Furthermore, the ATP through their hydrophilic properties as opposed to the Span series.
levels before and after treatment were compared using an ATP Upon fixing the eugenol concentration at 5% and mixing it with
meter (Clean-Trace™, 3M, USA) to assess the microbial activity. emulsifier concentrations of 3%, 5%, 7%, and 10%, the emulsifying
capacity was shown to be greater with higher concentrations.
3. Results and discussion Evaluation of individual emulsifiers showed that the W/O type, low
HLB Span emulsifiers were not appropriate for preparation of a
3.1. Isolation and identification of fungi at Yeongneung and biocide for application on cultural assets, in which the dispersion
evaluation of the antifungal activities of natural medicinal medium is water. However, the Tween emulsifiers, which are of the
ingredient extracts O/W type, were appropriate. Nevertheless, as precipitates formed
over time, their HLB values were determined to be too high for
A total of 21 fungal cultures were originally isolated from the emulsifying eugenol.
specimen collected from the stone supporting pillar at Yeongneung. Tween 20 and Span 20 were mixed to reduce the HLB of the
In the second round of isolation, through morphological examina- Tween series to prepare emulsifiers with an HLB ranging from 9.4
tion, 11 fungi were isolated, including Penicillium sp., Trichoderma to 15.9 depending on the ratio. Stable emulsion was formed be-
sp., Aspergillus sp., and unidentified colonies. DNA analysis of the 11 tween eugenol and water at a mixing ratio (v/v) of 3:7 and an HLB
pure fungal cultures isolated ultimately identified a total of 7 as- of 14.31; the degree of emulsification and dispersion, colloid par-
comycetes, including Trichoderma viride, Trichoderma harzianum, ticles, and the absence of layers were found to be subsequently
Aspergillus aculeatus, Penicillium oxalicum, Diaporthe elonis, Pesta- stable (Table 4).
lotiopsis microspora, and Purpureocillium lilacinum (Table 2). Among
the volatile extracts of medicinal ingredients, strong antifungal 3.4. Isolation and identification of fungi in the biofilm and
activity against all 7 colony types was observed for clove extract assessment of antifungal activity
(Table 2). No colonies on the plate media showed growth, and þþþ
antifungal activity was confirmed in all cases. However, no anti- Seven (YF 1e7) and 12 (LF 1e12) types of fungi were isolated and
fungal activities were observed for the remaining four volatile ex- identified in the cultures collected from the supporting pillar at
tracts of the medicinal ingredients tested. Yeoju Yeongneung in Gyeonggi-do, South Korea, and from the
corridor of Vat Phou Temple in Laos, respectively (Table 5). The
3.2. Identification of biologically active substances antifungal activity of Biocide A, which was prepared with mixed
emulsifiers, was examined. All 19 fungi showed growth in the
The components of volatile clove extract, which showed strong control groups of emulsifier and distilled water only, whereas a
antifungal activity, were identified through GC/MS (Fig. 1). Four clear zone appeared after treatment with Biocide A (Table 5).
components, including eugenol, were detected in the analysis.
Eugenol, which was present at high concentrations accounting for 3.5. Evaluation of material stability
an area of 89.92%, was confirmed to be related to the observed
antifungal activity (Table 3). These findings of the antifungal ac- The macroscopic images of the stone specimens after treatment
tivity of eugenol from volatile clove extract support the results of with Biocide A are shown in Fig. 2. The surface condition of the
previous studies (Ha, 2000; Kwon, 2006; Chung et al., 2001), sug- stone specimen immersed in Biocide A, such as a change in color,
gesting the potential of eugenol as an effective compound for bio- showed no difference to that of the control groups (no treatment
film removal. Therefore, eugenol was further developed as a and treatment with distilled water only). There was no substantial
potential biocide for evaluations. change in weight, with a decrease in weight of less than 0.1%
observed compared to the weight of the specimen treated with
3.3. Preparation of biocide a through determination of the optimal distilled water (Fig. 3). X-ray diffraction analysis was conducted to
ratio study the changes in rock-forming minerals after the treatment
with Biocide A, which can be used to interpret the changes in the
Comparison of three emulsifiers from the Tween series (Tween constituent materials of granite due to weathering. Granite mainly
20, 40, 80) and three from the Span series (Span 20, 40, 80) used to consists of quartz, orthoclase, plagioclase, and mica (Yang et al.,
emulsify eugenol with water revealed that the Tween series 2008), and these components were commonly found in the stone
resulted in superior emulsification and dispersion. Emulsifiers with specimens, with no new substances identified after treatment with
a high HLB are hydrophilic, whereas those with a low HLB are Biocide A. In addition, no secondary weathering or damage was
Table 2
Identification of fungi from Yeongneung and evaluation of the antifungal activity of five natural medicinal ingredients.
Fungi Natural Medicine (volatile extract)
Inhibition zone(mm) Activity
1 2 3 4 5 CH2Cl2 1 2 3 4 5 CH2Cl2
Trichoderma viride 0 20 0 0 0 0 e þþþ e e e e

Diaporthe Elonis 0 19 0 0 0 0 e þþþ e e e e
Pestalotiopsis microspora 0 20 0 0 0 0 e þþþ e e e e
Purpureocillium lilacinum 0 21 0 0 0 0 e þþþ e e e e
Aspergillus aculeatus 0 19 0 0 0 0 e þþþ e e e e
Penicillium oxalicum 0 19 0 0 0 0 e þþþ e e e e
Trichoderma harzianum 0 19 0 0 0 0 e þþþ e e e e
Fig. 1. GC/MS chromatogram of the volatile flavor components in clove.
Table 3 3.6. On-site application results

Volatile compounds identified in clove.
Peak No. Retention time Compound name Peak area (%) 3.6.1. Stone monument in South Korea
1 1.55 Methylene Chloride 2.66
Biocide A was applied on the stonework in Korea National
2 41.39 Eugenol 89.92 University of Cultural Heritage in South Korea, and the results were
3 42.92 Caryophyllene 1.34 monitored for 3 weeks after its application (Fig. 5). Based on ex-
4 49.81 Acetyleugenol 2.39 aminations under a digital microscope at a magnification of 50 ,
algae at the medullary layer were partially exposed with clear
disintegration of the mycelium on the white epithelial layer.
observed, as the peak intensity and pattern were identical for Moreover, algae in the medullary layer had withered and became
Biocide A and the control groups (Fig. 4). discolored. The overall dry-out of the isidium resulted in expanded
cracks. The disintegration of the soredium at the surface of the
Table 4
Preparation of Biocide A according to different mixing ratios between Tween 20 and Span 20 emulsifiers.
Mixed ratio (by volume)
Tween 20 (HLB 16.7) 9 7 5 3 1

Span 20 (HLB 8.6) 1 3 5 7 9
HLB 15.9 14.3 12.6 11.0 9.4
Immediately after mixing
24 h after mixing
Table 5
Identification of fungi in the biofilm and assessment of antifungal activity.
Name Description Identities Activity (inhibition zone, mm)
GENE AC Pct.(%) D.W. Emulsifier Biocide A
YF1 Trichoderma atroviride JX119036.1 99 0 0 16

() () (þþþ)
YF2 Diaporthe melonis AB107888.1 100 0 0 20
() () (þþþ)
YF3 Pestalotiopsis sp. JN198530.1 99 0 0 18
() () (þþþ)
YF4 Purpureocillium lilacinum KJ415586.1 99 0 0 15
() () (þþþ)
YF5 Aspergillus aculeatus JX501412.1 99 0 0 8
() () (þþ)
YF6 Penicillium oxalicum KF465777.1 99 0 0 15
() () (þþþ)
YF7 Trichoderma harzianum KC847190.1 99 0 0 8
() () (þþ)
LF1 Penicillium citrinum KF530863.1 99 0 0 7
() () (þþ)
LF2 Fusarium sp. JQ364975.1 100 0 0 6
() () (þ)
LF3 Neosartorya fischeri AB674772.2 99 0 0 15
() () (þþþ)
LF4 Aspergillus sp. JN393254.1 100 0 0 7
() () (þþ)
LF5 Trichoderma harzianum KM079611.1 99 0 0 8
() () (þþ)
LF6 Phoma sp. JN207261.1 99 0 0 9
() () (þþ)
LF7 Fusarium solani KF060154.1 99 0 0 6
() () (þ)
LF8 Penicillium citrinum KF986248.1 99 0 0 7
() () (þþ)
LF9 Fusarium oxysporum KC254033.1 100 0 0 7
() () (þþ)
LF10 Penicillium simplicissimum JN246043.1 99 0 0 8
() () (þþ)
LF11 Lasiodiplodia theobromae KF814726.1 99 0 0 1
() () (þ)
LF12 Aspergillus fumigatus JX231005.1 100 0 0 8
() () (þþ)
Fig. 2. Macroscopic observation results (left: before Biocide A treatment, right: after Biocide A treatment).
isidium led to exposure of the epithelial layer below. Red algae the number of bacteria, ATP measurements are considered an
formed between the rock-forming minerals had also withered and adequate and valid index of microbial contamination (Choi et al.,
disintegrated over time. 2012). Therefore, the observed decrease in ATP indicated that the
In addition to the optical examination of surface conditions, treatment with Biocide A reduced the number of constituent or-
three ATP measurements (measured in relative light units [RLU]) ganisms in the biofilm.
were taken at 5 points before and after treatment, and the results
are shown in Table 6. An overall sharp decrease in the ATP level was
observed, with an 89% decrease at point 1, 98% at point 2, 96% at 3.6.2. Stone components in Laos
point 3, 44% at point 4, and 95% at point 5. Given that there is a Biocide A, emulsifier, and distilled water were applied on the
positive correlation between ATP bioluminescence assay data and stone components in the Hong Nang Sida temple in Laos, and the
results were monitored for 3 months after the application (Fig. 6).
Fig. 3. Weight variation.
Fig. 4. X-ray diffraction graphs of granite treated with Biocide A and controls.
Laos was in a monsoon season at the time of treatment, and decreased by 61% from 5306 RLU to 1777 RLU, whereas the area
therefore the stone surface was completely covered in green bio- treated with the emulsifier had decreased by only 21%. Moreover,
film; however, the biofilm on the area treated with Biocide A had the ATP level increased by 114% in the area treated with distilled
disintegrated after 3 months. Although this could be attributed to water and by 110% in untreated areas, demonstrating the effec-
the fact that Laos was in a dry season at the time of examination, tiveness of Biocide A for biofilm removal in the tropical monsoon
which could have naturally dried-out the biofilm, the green biofilm climate of Laos.
was still present in non-treated areas, which confirmed the effec-
tiveness of Biocide A. Despite the overall lower growth activity of 4. Conclusion
the biofilm after the transition from the wet to dry season, a clear
difference was observed between the area treated with Biocide A As stone monuments exposed to the natural environment are
and the control group. susceptible to the influx of organisms, the monuments become
ATP measurements before and after treatment with Biocide A weathered owing to environmental and biological factors and their
are shown in Fig. 7. The ATP level in the area treated with Biocide A interactions. Analysis of the organic species within the biofilm on
Fig. 5. Comparison of the stonework surface before and after treatment with Biocide A.
Table 6
Comparison of ATP measurements before and after treatment with Biocide A.
Before Biocide A treatment ATP After Biocide A treatment ATP ATP reduction ratio (%)
(Unit: RLU) (Unit: RLU)
1st 2nd 3rd Average 1st 2nd 3rd Average
1 57,593 57,561 56,375 57,176 6689 7945 4599 6411 89

2 51,413 50,159 48,683 50,085 1092 950 943 995 98
3 35,193 35,194 34,676 35,021 1426 1283 1278 1329 96
4 45,075 44,350 43,292 44,239 24,843 24,854 24,646 24,781 44
5 30,453 30,418 30,025 30,299 1491 1313 1319 1376 95
Fig. 6. Comparison of the stonework surface before and after treatment with Biocide A.
Fig. 7. Comparison of ATP measurements before and after treatment with Biocide A (unit: RLU).
stonework showed that the biofilm consisted of mostly algae and Acknowledgements
fungi, and molds forming on the exterior and interior parts of
stonework have been reported to be the principal factors respon- This research was supported by 2013&2014 Academic Research
sible for biological weathering (Ascaso et al., 1998). In this respect, Funds through Korea National University of Cultural Heritage and
the aim of the present study was to develop a new biocide for stone Korea Cultural Heritage Foundation. The research team deeply ap-
monuments by emulsifying essential oils that have thus far been preciates the full support of Korea National University of Cultural
used for indoor biological control. The goal was to formulate a Heritage and Korea Cultural Heritage Foundation.
product that could also be effectively applied outdoors, which was
verified through laboratory testing and on-site application.
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International Biodeterioration & Biodegradation: Seon Hye Jeong, Hyun Ju Lee, Dae Woon Kim, Yong Jae Chung

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International Biodeterioration & Biodegradation xxx (2017) 1e10

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation

New biocide for eco-friendly bioﬁlm removal on outdoor stone

1. Introduction A 5-year investigation on the biological weathering of stone

Name Scientiﬁc name

1 Star anise Illicium verum

2 Clove Syzygium aromaticum Merrill et Perry

3 Gallnut Rhus javanica

4 Golden thread Coptis chienesis

5 Amur cork-tree Phellodendron amurense RUPR

Fungi Natural Medicine (volatile extract)

Inhibition zone(mm) Activity

Trichoderma viride 0 20 0 0 0 0 e þþþ e e e e

Fig. 1. GC/MS chromatogram of the volatile ﬂavor components in clove.

Table 3 3.6. On-site application results

Mixed ratio (by volume)

Tween 20 (HLB 16.7) 9 7 5 3 1

Name Description Identities Activity (inhibition zone, mm)

GENE AC Pct.(%) D.W. Emulsiﬁer Biocide A

YF1 Trichoderma atroviride JX119036.1 99 0 0 16

Fig. 3. Weight variation.

1st 2nd 3rd Average 1st 2nd 3rd Average

1 57,593 57,561 56,375 57,176 6689 7945 4599 6411 89

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