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Microbiological survey for analysis of the brown spots on the walls of the tomb of King Tutankhamun

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  • Getty Conservation Institute

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This paper presents the preliminary findings from our investigation into the possible microbial origin of brown spots on the walls of Tutankhamun's tomb. GC/MS analysis of the brown spots indicated that they contained 16% (by weight) of malic acid, suggesting microbial involvement in their formation. However, no microbial structures associated with the brown spots were detected using scanning electron microscopy. Our observations indicate that the organism that created the spots is not active. We undertook an investigation of the current microbial communities on the walls in Tutankhamun's tomb and two other nearby tombs. There were no significant differences in the numbers or types of culturable microorganisms among the three tombs sampled. Using pyrosequencing, no statistically significant differences in community composition and structure were observed. Fungal communities were composed primarily of Penicillium whereas the abundant bacterial taxa were members of the Firmicutes, Actinobacteria and Bacteroidetes phyla. Penicillium chrysogenum isolated in this study produced malic acid in vitro, suggesting that they or other microorganisms may be responsible for the malic acid detected in the brown spots. Findings from this study were compared to those from previous studies, and a possible scenario for the formation of the brown spots was developed.
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Microbiological survey for analysis of the brown spots on the walls of the tomb of
King Tutankhamun
Archana Vasanthakumar
a
,
*
, Alice DeAraujo
a
, Joy Mazurek
b
, Michael Schilling
b
, Ralph Mitchell
a
a
Laboratory of Applied Microbiology, School of Engineering and Applied Sciences, Harvard University, 58 Oxford St., Cambridge, MA 02138, USA
b
Getty Conservation Institute, Getty Center Drive, Los Angeles, CA 90049, USA
article info
Article history:
Received 18 October 2012
Received in revised form
22 January 2013
Accepted 25 January 2013
Available online
Keywords:
Tutankhamun
Brown pigment
Microbial
Penicillium
Malic acid
Pyrosequencing
abstract
This paper presents the preliminary ndings from our investigation into the possible microbial origin of
brown spots on the walls of Tutankhamuns tomb. GC/MS analysis of the brown spots indicated that they
contained 16% (by weight) of malic acid, suggesting microbial involvement in their formation. However,
no microbial structures associated with the brown spots were detected using scanning electron micro-
scopy. Our observations indicate that the organism that created the spots is not active. We undertook an
investigation of the current microbial communities on the walls in Tutankhamuns tomb and two other
nearby tombs. There were no signicant differences in the numbers or types of culturable microor-
ganisms among the three tombs sampled. Using pyrosequencing, no statistically signicant differences in
community composition and structure were observed. Fungal communities were composed primarily of
Penicillium whereas the abundant bacterial taxa were members of the Firmicutes, Actinobacteria and
Bacteroidetes phyla. Penicillium chrysogenum isolated in this study produced malic acid in vitro, sug-
gesting that they or other microorganisms may be responsible for the malic acid detected in the brown
spots. Findings from this study were compared to those from previous studies, and a possible scenario for
the formation of the brown spots was developed.
Ó2013 Elsevier Ltd. All rights reserved.
1. Introduction
The more than 3000 year-old tomb of King Tutankhamun,
a Pharaoh of the 18th Dynasty (c. 1323 BCE), perhaps the most
famous tomb in Egypt, is located in the Valley of the Kings on the
West Bank of the Nile, near Luxor, Egypt. As is known, the tomb was
discovered in 1922 and nearly all of its funerary contents were
found intact. In 2008, the Getty Conservation Institute entered into
ave-year partnership with Egypts Supreme Council of Antiquities
to collaborate on a project for the conservation and management of
the tomb. One of the project objectives was to identify the origins
and causes of extensive brown spots on the walls in the tomb. If the
spots are of microbiological origin, then it would be important to
ascertain whether the organisms are active, thereby posing a fur-
ther threat to the wall paintings and visitors.
The tomb is small and contains the main burial chamber,
a treasury, an annex and the antechamber. The brown spots were
observed when the tomb was rst opened. They partially obscure
the wall paintings in the burial chamber (Fig. 1a, b). They are also
present on unpainted rock surfaces in the rest of the tomb (Wong,
2010; unpublished GCI report).
Attempts to isolate microorganisms from the spots immediately
after the discovery of the tomb were unsuccessful. The brown spots
were described as fungoidgrowths and their shape and mor-
phology supported a microbiological origin (Lucas, 1923;Lucas,
1963;Carter unpublished manuscript;Scott, 1963). In spite of
multiple investigations over the past 50 years, there is still no
consensus on the origin and nature of the brown spots (Fahd,1994;
Szczepanowska and Cavaliere, 2004,2008).
The microbiological objective of this study was two-fold: 1) to
investigate the possible microbial origin of the brown spots and 2)
to describe the microbial communities associated with samples
from the tomb and two other nearby tombs that also contain
substantial wall paintings, but do not contain brown spots. All three
tombs are open to the public. Brown spots were analyzed using
microscopy and gas chromatography/mass spectrometry. In
*Corresponding author. Laboratory of Applied Microbiology, School of Engi-
neering and Applied Sciences, Harvard University, 58 Oxford St., Room 301, Cam-
bridge, MA 02138, USA. Tel.: þ1 617 495 3307; fax: þ16174961471.
E-mail address: archanav@seas.harvard.edu (A. Vasanthakumar).
Contents lists available at SciVerse ScienceDirect
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http://dx.doi.org/10.1016/j.ibiod.2013.01.014
International Biodeterioration & Biodegradation 79 (2013) 56e63
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addition, culture-based and DNA-based methods were used to
compare the microbial communities of the tombs.
2. Materials and methods
2.1. Sampling
Sampling was undertaken by GCI staff with permission of the
SCA. Samples for microbiological analysis were collected using
sterile swabs, which were moistened in sterile de-ionized water
just before sampling. Some swabs were also moistened with sterile
water amended with a surfactant (Tween 80, Sigma Aldrich, St.
Louis, MO) before sample collection. Brown spots and areas close to,
but outside, brown spots were sampled in multiple locations in the
burial chamber, treasury and antechamber of the tomb. In addition,
swab samples were collected from two nearby tombs, those of
Rameses III and Thutmes IV (Table 1). Collected samples were
maintained at 4
C and were analyzed at Harvard University within
ve to seven days. In addition, minute samples of the brown spots
were taken from the burial chamber and treasury.
2.2. Microscopic analysis
2.2.1. Light microscopy
Minute samples of brown spots were placed on a drop of sterile
water on a clean microscope slide, a cover slip placed over them
and the slide viewed under bright light and phase contrast objec-
tives at magnications ranging from 100to 1000.
2.2.2. Scanning electron microscopy
Minute samples from brown spots were mounted on stubs and
sputter-coated with a mix of gold/palladium. The coated samples
were viewed using the Zeiss EVO Environmental SEM (Zeiss Inc.,
Thornwood NY).
2.3. Growth on microbiological media
One to 1.5 ml of sterile water was placed into the swab con-
tainers and thoroughly mixed using a vortex mixer for w3 min.
Swabs used on the brown spots had clearly become discolored by
the brown pigment, which diffused into the water. A dilution series
was spread plated onto a wide range of microbiological culture
media, including tryptic soy agar, malt extract agar, corn meal agar,
potato dextrose agar, Rose Bengal agar, Sabourauds Dextrose Agar,
all from Difco (Difco, Sparks, MD), a halophilic medium (10% (w/v)
MgSO
4
.7H
2
O added to tryptic soy agar), a modied alkaliphilic
medium (Horikoshi, 1998) and a minimal salts medium containing
gum Arabic (an acacia gum which was identied as the paint
binding medium) as the sole carbon source. Inoculated growth
media were maintained for two to eight weeks before analysis.
2.4. Identication of cultivable isolates by ribosomal RNA gene
sequencing
Pure cultures of representative microbial isolates from various
samples were identied by ribosomal RNA gene sequencing. To
summarize, DNA was extracted from representative isolates and
regions of their ribosomal RNA genes amplied using primers
specic for bacteria or fungi (White et al., 1990;Lane, 1991). The
amplied fragment was gel puried using the Qiaquick Gel puri-
cation kit (Qiagen Inc., Valencia, CA). Dilutions of the puried
fragments were directly sequenced using the eubacterial 16S rRNA
primer, 27F or the fungal intergenic spacer primer, ITS1. Sequences
were trimmed using FinchTV (Geospiza Inc. Seattle, WA). Phylo-
genetic placement of sequences was performed using the software
ARB (Ludwig et al., 2004) and closest matches found using RDP
Classier (Ribosomal Database Project, Wang et al., 2007) and
UNITE databases (Abarenkov et al., 2010).
2.5. Molecular analysis of microbial communities
2.5.1. Community DNA isolation and PCR amplication
Total DNAwas extracted from pooled samples from brown spots
as well as areas outside brown spots using the UltraClean Soil DNA
kit (MoBio, Carlsbad, CA). ITS regions of the fungal ribosomal
operon were successfully amplied using a semi-nested PCR
Fig. 1. a) Brown spots on a painting on the wall of King Tutankhamun's tomb. b) Close-up of brown spots. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of this article.)
Table 1
Description of sampling sites and analysis performed on samples from the three
tombs.
Sample
ID
Sample
source
Location/Tomb Culturing (C),
Sequencing (S)
BCA Brown spot Burial chamber/Tutankhamun C, S
BCD Non-brown spot Burial chamber/Tutankhamun C, S
ACA Brown spot Antechamber/Tutankhamun C
ACD Non-brown spot Antechamber/Tutankhamun C
TA Brown spot Treasury/Tutankhamun C, S
TD Non-brown spot Treasury/Tutankhamun C, S
Thutmes Non-brown spot Thutmes IV C
RAM Non-brown spot Rameses III C, S
A. Vasanthakumar et al. / International Biodeterioration & Biodegradation 79 (2013) 56e63 57
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approach. Primers NS5 (AACTTCCCGGAATTGACGGAAG) and ITS4
(TCCTCCGCTTATTGATATGC) were used for the rst PCR and ITS1
(TCCGTAGGTGAACCTGCGG) and ITS4 for the second PCR. Bacterial
16S ribosomal RNA genes were amplied using a nested PCR
approach. Primers27F/1492R were used for the rst PCR and 357F/
907R for the second PCR (Lane, 1991). All amplied fragments were
gel puried using the QiaQuick Gel Extraction kit (Qiagen Inc.
Valencia, CA) before they were sent for 454 pyrosequencing.
2.5.2. 454 pyrosequencing
Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)
was performed as described previously for the 16S rRNA gene
(Dowd et al., 2008). The sequencing library was generated using
a one-step PCR for 30 cycles containing Hot Start high delity Taq
polymerases and the forward primer. Tag-encoded FLX amplicon
pyrosequencing analyses utilized a Roche 454 FLX instrument with
Titanium reagents. Sequencing procedures were performed at the
Research and Testing Laboratory (Lubbock, TX) based upon RTL
protocols (www.researchandtesting.com).
2.5.3. Sequence analysis
A 454 pipeline in the software program mothur v.1.25.0 (Schloss
et al., 2009;Schloss and Westcott, 2011) was used to extract, clean
up and trim sequences so that only high quality sequences were
obtained. A Phylip-based distance matrix was constructed and an
operational taxonomic unit (OTU)-based approach was employed
to analyze communities (Schloss et al., 2009). Bacterial 16S rRNA
sequences were aligned against a database containing 14,956 rRNA
sequences (www.mothur.org). Fungal ITS sequences were aligned
using a pairwise alignment approach incorporated in mothur.
Representative sequences from each OTU were compared against
the RDP and UNITE databases to obtain information about taxo-
nomic placement. Rarefaction analysis was performed. In addition,
commonly used richness and diversity indices such as the Chao1
index and the Simpsons inverse diversity index were computed for
each sample. Microbial community diversity was compared among
brown spots and areas outside brown spots using beta diversity
statistics. Finally, statistical tests were performed to determine the
signicance of any differences among the communities.
2.6. Chemical analysis
Preliminary ndings are given below. The brown spot and the
plaster from the wall were analyzed by an Agilent 6890N 5973 Gas
Chromatograph/Mass Spectrometer. Samples were subjected to
acid hydrolysis using 6N HCl. An internal standard,
L
-norleucine,
was added to the samples. The hydrolyzate was evaporated and
dissolved in 25 mM HCl, and ethyl chloroformate was added as
a derivatization reagent (Schilling et al., 1996). The derivatives were
evaporated, reconstituted in chloroform, and injected into the GC/
MS instrument. In calibration, the use of quadratic curve ts forced
through the origin gave correlation coefcients of 0.995 or better
for most analytes over the calibration range of 2e50 ppm. Fungal
cultures isolated from the tombs were also investigated for malic
acid production using the same method.
3. Results
3.1. Analysis of the brown spots
3.1.1. Microscopic analysis
Light microscopic observation of minute samples of the brown
spot revealed a homogeneous mass with no dened boundaries.
The surface of the brown spot was irregular. The color varied from
dark brown in the center to a lighter yellowish-brown on the
outside. The brown spots were easily aked off from the rock using
a scalpel. No microbiological material efungal hyphae, spores or
bacterial cells ewas observed directly on the surface of the brown
spot or within the stratigraphy.
Scanning electron microscopy revealed that the brown spot was
free of any microorganisms. The brown spots had dust particles
associated with them but there was no evidence of hyphae, spores
or bacterial cells embedded in them (Fig. 2).
3.1.2. Chemical analysis
A signicant amount of malic acid (16% by weight) was found
associated with the brown spots but not the plaster (Fig. 3a). Small
amounts of two amino acids, aspartic and glutamic acid, were also
detected in the brown spot (Fig. 3a).
3.2. Microbial community analysis in samples from three tombs
3.2.1. Identication of cultivable microorganisms
In our analysis of the modern microbial communities on the
walls of the chambers in the three tombs, bacterial and fungal
growth was detected on all media tested. Tryptic soy agar, malt
extract agar and the halophilic medium supported the highest di-
versity of microorganisms. Population sizes were low in all sam-
ples. The largest population of bacteria recovered was 600 cfu/
square cm of area sampled. This was from the antechamber in the
Tutankhamun tomb, which had maximum exposure to tourists.
A representative subset of the isolates was chosen for ribosomal
RNA gene sequencing. Gram positive bacteria, represented by
members of the Firmicutes and Actinobacteria, dominated among
bacterial isolates (Table 2). Red-pigmented isolates from the acti-
nobacterial genus Kocuria were isolated from brown spots as well
as areas outside them (from this point on called non-brown spots/
samples) in all the chambers in the Tutankhamun tomb as well as
the tombs of Rameses III and Thutmes IV. Bacillus and Staphylo-
coccus species were also represented in multiple samples from both
brown and non-brown samples. Penicillium was isolated from
Rameses III and Tutankhamun tombs. Other commonly occurring
fungi such as Cladosporium and Epicoccum were also isolated from
Tutankhamuns tomb.
3.2.2. Malic acid production by Penicillium chrysogenum
Since malic acid was detected in the brown spots, the most
abundant fungal genus in the tomb, Penicillium, was tested for malic
Fig. 2. Scanning electron micrograph of a small portion of a brown spot, in which no
microbial structures are evident. Cracking of the sample was caused by the SEM
electron beam.
A. Vasanthakumar et al. / International Biodeterioration & Biodegradation 79 (2013) 56e6358
Author's personal copy
acid production using the same GC/MS method used for analysis of
the brown spots. P. chrysogenum isolates produced malic acid
in vitro (Fig. 3b).
3.2.3. Molecular analysis of microbial communities in brown spots
and areas outside brown spots
Total DNA was extracted from swab samples taken from tomb
walls in the tombs of Tutankhamun and Rameses III in order to
determine the nature of the current microbial community. DNA
from replicate samples from the same sampling area was pooled
together. Microbial communities in ve pooled DNA samples iso-
lated from swab samples were analyzed by pyrosequencing. Each of
these ve samples was analyzed separately for fungal and bacterial
communities. There were two brown spot samples, one each from
the burial chamber and the treasury (BCAITS/BC16S and TAITS/
TA16S) and similarly, two non-brown samples (BCDITS/BCD16S and
TDITS/TD16S), as well as a sample from the Rameses III tomb
(RamITS/Ram16S).
At 3% difference among sequences, fungal communities in the
samples BCAITS, TAITS, BCDITS and TDITS could be divided into 13,
8, 13 and 3 operational taxonomic units (OTUs) respectively while
the Rameses sample contained 4 OTUs (Table 3). When the taxo-
nomic afliation of the representatives of each of these OTUs was
investigated, however, the same genus and species combination
could be represented in multiple OTUs. For ease of visualization,
relative abundance data were combined in order to represent only
genera. The relative abundance of Penicillium in the samples ranged
from 28% in the non-brown sample from the burial chamber to 96%
in the Rameses samples. Aspergillus was represented by 20e40% of
sequences in the burial chamber samples but was almost unde-
tectable in the treasury sample (Fig. 4a). Only one fungal OTU,
Penicillium, was shared among all four Tutankhamun samples
analyzed. In fact, this was the only OTU shared among all ve
samples in this study, accounting for 70% of the total number of
sequences in all ve samples. In our investigation, we detected
Aspergillus in the burial chamber samples but not in the samples
from the treasury or the other two tombs.
At 3% sequence difference, there were no fungal OTUs that were
unique to either brown or non-brown spots. However, the OTU
corresponding to the Cladosporium and Amorphotheca group was
represented only in the non-brown samples (Fig. 4a). The only OTU
that was unique to the burial chamber samples was represented by
the fungus Epicoccum (Fig. 4a).
At 3% difference among sequences, bacterial communities in
brown spot samples BCA16S and TA16S were composed of 90 and
678
Brown Spot
Aspartic Glutamic
67 8
Plaster
I.S. Nor
Malic Acid
I.S. Nor
Retention Time ->
0.E+00
2.E+05
4.E+05
6.E+05
8.E+05
1.E+06
1.E+06
Malic Acid
2.3 6.0
Retention Time
Fig. 3. Comparison of brown spot and plaster by GC/MS. a) A large peak corresponding
to malic acid is seen in the brown spot sample but is absent from the plaster sample. b)
Malic acid production by Penicillium chrysogenum isolated from Tutankhamuns tomb.
The other peaks shown here could not be identied when compared against the NIST
mass spectral library.
Table 2
Phylogenetic placement of a representative subset of cultivable microbial isolates from the tombs of Tutankhamun, Rameses III and Thutmes IV.
Closest match % identity GenBank accession # Tomb Location Brown spot Non-brown spot
Bacillus aeris 99 EF451042 Tut Treasury U
Bacillus jeotgali 100 GU474983 Tut Burial chamber U
Bacillus cereus 99 HQ833025 Tut Treasury U
Kytococcus sp. 99 HM196770 Tut Burial Chamber U
Kocuria rosea 99 HQ202871 Tut Burial chamber U
Kocuria rosea 99 HQ202871 Tut Treasury U
Kocuria rosea 98 EU977667 Rameses III Chamber 8 U
Kocuria rosea 99 EU660350 Thutmes IV Burial chamber U
Kocuria turfanensis 99 HQ671070 Tut Treasury U
Kocuria turfanensis 99 DQ531634 Tut Burial chamber U
Micrococcus luteus 99 FJ999946 Tut Treasury U
Micrococcus luteus 99 FJ999946 Tut Burial chamber U
Staphylococcus epidermidis 99 AB617572 Tut Treasury U
Penicillium chrysogenum 99 JQ861220 Rameses III Chamber D1 U
Penicillium chrysogenum 100 JN986786 Tut Treasury U
Penicillium chrysogenum 99 JN986786 Tut Treasury U
Cladosporium cladosporioides 99 HQ380770 Tut Burial chamber U
A. Vasanthakumar et al. / International Biodeterioration & Biodegradation 79 (2013) 56e63 59
Author's personal copy
87 OTUs, respectively, while non brown samples BCD16S and TD16S
contained 59 and 70 OTUs respectively. The Rameses sample con-
tained 59 bacterial OTUs. No OTUs were shared among the four
Tutankhamun samples at this level of sequence difference. Samples
were also analyzed at 10% difference among sequences, which
corresponded to a taxonomic class level. Members of the class
Bacilli were detected in all samples and were the most abundant
group, represented by 33e91% (Fig. 4b). Bacteroidia was the next
abundant class in four samples, ranging from 9 to 57% of the total
number of sequences in those samples (Fig. 4b). At 10% sequence
difference, two OTUs were shared among all ve samples, the
classes Bacilli and Actinobacteria. Both of these classes were also
represented in our culture-based study by Bacillus and Kocuria
species. There was only one OTU unique to the brown spot samples
(the class Clostridia) and this was represented by only 2 sequences.
Rarefaction analysis indicated that the fungal communities have
been adequately sampled at a distance of 3% (Fig. 5). All ve rar-
efaction curves were already at a plateau or fast approaching one.
Fungal diversity in the burial chamber samples was higher than in
the treasury (Fig. 5a). Rarefaction curves of the bacterial commu-
nities also showed a high degree of similarity among the ve
samples (Fig. 5b). In addition, the coverage of the samples was 98%
and above for all samples (Table 3).
Multiple beta diversity statistics were computed to detect sim-
ilarities among communities. Dendrograms were generated to
compare communities. Parsimony and unifrac tests revealed that
there were no signicant differences in clustering within the den-
drograms (data not shown). Since limited samples were available, it
was not possible to perform AMOVA, the test for molecular var-
iance, which requires replicates from a single type of sample.
4. Discussion
Scanning electron microscopy revealed no evidence of micro-
organisms associated with the brown spots. It is therefore apparent
that the original organism(s) responsible for these spots are no
longer alive. The detection of malic acid only in the brown spots but
not in the plaster indicated microbial involvement in the formation
of the spots. Many microorganisms, including fungi such as Asper-
gillus spp. (Peleg et al., 1988) and bacteria such as Arthrobacter and
Pseudomonas (Vyas and Gulati, 2009), can produce malic acid in the
environment; therefore, these ndings suggest a microbiological
cause for the brown spots. In addition, P. chrysogenum isolated in
this study produced malic acid in vitro. It is likely that other mi-
croorganisms from the tomb can also produce malic acid under
similar conditions. This nding provides further evidence that the
brown spots are microbiological in origin. It is conceivable that
malic acid detected in the brown spots was produced by
microorganisms on the tomb walls under conditions in the tomb at
the time the brown spots were produced. Malic acid has been
detected in wine residues from ca. 2000 years ago, suggesting that
it can survive for many years (Pecci et al., 2013).
GC/MS analysis of the brown spots also demonstrated the
presence of amino acids such as glutamic acid and aspartic acid,
both of which were detected in previous studies on the brown spots
(Porta, 1992,2009;Arai, 2000). These amino acids are common
microbial metabolites (Chiou, 1997;Arai, 2000).
There were no signicant differences in cultivable communities
from brown spots or outside of their periphery. The occurrence of
fungi such as Penicillium and Cladosporium and Gram positive
bacteria such as Bacillus has been recorded in previous studies
(Fahd, 1994;Szczepanowska and Cavaliere, 2004). In addition,
these genera are ubiquitous and have adapted to various lifestyles.
Molecular analysis of the fungal communities on the tomb walls
indicate that they are comprised of fungi such as Penicillium,
Aspergillus and Cladosporium, with Penicillium being the most
abundant genus. Penicillium is a ubiquitous fungus and its sapro-
phytic lifestyle has been studied in a variety of environments (Pitt,
2000). Its abundance in the tombs suggests that this organism has
adapted its lifestyle to the dry conditions existing in these tombs. In
an earlier study, Arai, who has extensively studied formation of
brown spots (foxing) on many materials (Arai, 1992,2000), suc-
ceeded in isolating a xerophilic microorganism, Aspergillus pen-
icilloides from brown spot samples in Tutankhamuns tomb by using
a low water activity medium. He suggested that this and other
xerophilic microorganisms can cause foxing to occur by a non-
enzymatic browning reaction (Arai, 2000). In our investigation,
we detected Aspergillus in the burial chamber samples but not in the
samples from the treasury or the other two tombs. The fungal
communities in the burial chamber samples were more diverse
than in the treasury. This is likely due to the presence of visitors in
the antechamber and their proximity to the burial chamber as
compared with the treasury. There were no fungal OTUs that were
unique to either brown or non-brown spots, suggesting that fungal
communities are similar across both types of samples.
Among the bacterial communities on the tomb walls, the classes
Bacilli and Actinobacteria, were shared among all ve bacterial
samples at 10% sequence difference. The former contains spore-
forming bacteria, which are usually able to withstand harsh condi-
tions such as drying and heat. Both of these classes were also rep-
resented in our culture-based study by Bacillus and Kocuria species.
DNA analysis demonstrated the presence of a diverse microbial
community on the walls. It is possible that some of the DNA is
ancient; however, it is likely that ancient DNA has been degraded to
the extent of being intractable using the methods employed in the
current study (Pääbo et al., 2004).
Table 3
Microbial community richness and diversity indices at 3% difference among sequences. Fungal and bacterial communities in pooled DNA samples were analyzed by pyro-
sequencing the 16S rRNA gene or the ITS region. Samples were separated according to location/tomb from which they were collected and the type of sample (brown/non-
brown).
Sample Sample Location/Tomb Library coverage Observed OTUs Chao richness Invsimpson
diversity index
BCAITS Brown spot Burial chamber/Tut 0.9935 13 23.5 1.9565
BCDITS Non-brown spot Burial chamber/Tut 0.9972 13 13.6 3.6671
RAMITS Non-brown spot Rameses III 1 4 4 1.0746
TAITS Brown spot Treasury/Tut 0.9972 8 11 1.9770
TDITS Non-brown spot Treasury/Tut 0.9991 3 3 1.1245
BCA16S Brown spot Burial chamber/Tut 0.9832 91 230 2.6635
BCD16S Non-brown spot Burial chamber/Tut 0.9895 59 144 2.4744
RAM16S Non-brown spot Rameses III 0.9892 59 149 1.4866
TA16S Brown spot Treasury/Tut 0.9856 87 171 2.2418
TD16S Non-brown spot Treasury/Tut 0.9898 70 105 3.4291
A. Vasanthakumar et al. / International Biodeterioration & Biodegradation 79 (2013) 56e6360
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No signicant differences were observed in the current micro-
bial communities associated with brown spots and areas outside
brown spots. This study also revealed that microbial communities
in nearby tombs are not different from those in Tutankhamuns
tomb. The methods described herein facilitated the isolation of
a representative community of bacteria and fungi from the tombs
in the Valley of the Kings. Cultivable populations were low in all
these samples. It is possible that the hot and dry conditions in the
Valley of the Kings contribute to low populations.
Although this investigation provided information about the
dominant members of the modern microbial community, the
ability of specic isolates to produce brown spots similar to those
found in the tomb was not analyzed. Therefore, additional research
is required, including detailed chemical and microbial analysis to
further elucidate the origin and nature of the spots.
Because Tutankhamuns tomb is the only one known to contain
the brown spots, it is very likely that they were formed under
conditions specic to the tomb. Howard Carter, who discovered the
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
BCAITS BCDITS RamITS TAITS TDITS
Trametes
Eucasphaeria
Epicoccum
Amorphotheca and Cladosporium
Aspergillus
Malassezia et al.
Penicillium
Oidiodendron
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
BCA16S BCD16S RAM16S TA16S TD16S
Erysipelotrichia
Clostridia
Thermolithobacteria
Flavobacteria
Deinococci
Deltaproteobacteria
Actinobacteria
Gammaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Bacilli
Bacteroidia
Fig. 4. a: Composition of different genera of fungi based on partial sequencing of the ITS region of the ribosomal operon. Five samples are represented: two brown spot samples in
Tutankhamuns tomb (BCAITS, TAITS), two non-brown samples (BCDITS, TDITS) and one sample from the Rameses tomb (RAMITS). b: Composition of different classes of bacteria
based on partial sequencing of the 16S rRNA gene. Five samples are represented: two brown spot samples in Tutankhamuns tomb (BCA16S, TA16S), two non-brown samples
(BCD16S, TD16S) and one sample from the Rameses tomb (RAM16S).
A. Vasanthakumar et al. / International Biodeterioration & Biodegradation 79 (2013) 56e63 61
Author's personal copy
tomb, reported that some of the deterioration observed is indica-
tive of the existence of high humidity in the tomb following the
burial, and suggested that infrequent ooding had occurred in the
tomb (Carter and Mace, 1963). In addition, the tomb remained
almost entirely sealed until 1922, creating conditions that were
unlike those in any of the other tombs. With further collaborative
studies, it may be possible to develop a more denitive character-
ization of the conditions within the tomb that led to development
of the brown spots.
Acknowledgments
This research was carried out as a collaborative agreement be-
tween the Getty Conservation Institute and Egypts Supreme
Council of Antiquities. Scanning electron microscopy was per-
formed at the Center for Nanoscale Systems (CNS), a member of the
National Nanotechnology Infrastructure Network (NNIN), which is
supported by the National Science Foundation under NSF award
number ECS-0335765. CNS is part of Harvard University. The au-
thors wish to thank Nick Konkol, Chris McNamara, Marc Mittelman
and Federica Villa for insightful discussions, Conor OHerin and
Felix Wu for technical assistance and the anonymous reviewers for
useful suggestions.
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... Fungi are typical heterotrophic organisms able to survive under extreme conditions, especially low humidity, by adapting morphologically, structurally and metabolically (Trovão et al. 2019). Severe fungal biodeterioration on wall paintings has been widely reported, including the prehistoric rock painting at the Lascaux caves, the Altamira caves in Spain, King Tutankhamun's tomb in Egypt, the Takamatsuzuka and Kitora Tumuli in Japan, as well as the Maijishan and Mogao Grottoes in China (An et al. 2009;Bastian et al. 2010;Saiz-Jimenez et al. 2011;Vasanthakumar et al. 2013;Ma et al. 2015;Duan et al. 2017). Most of these fungi thrive in the painted layers or in the spaces between the paint and plaster layers, and sometimes penetrate micro cracks of paint layers (Fig. 3). ...
... Wall paintings on the Santa Clara church (Porto, Portugal) darkened due to the oxidation of the lead-based pigments and the formation of plattnerite (Rosado et al. 2016). Similarly, several microbial contaminations, specifically with Penicillium and Cladosporium, were detected with the development of brown and black spots (Vasanthakumar et al. 2013;Rosado et al. 2016;Ma et al. 2020). An examination of the biocolonization on yellow and red ochres and ultramarine blue pigments shows filamentous fungi (in particular, Aspergillus niger), yeasts and bacteria. ...
... of cultural heritage (Vasanthakumar et al. 2013;Gadd 2017;Liu et al. 2020;Ma et al. 2020). ...
Chapter
Murals or wall paintings are important cultural heritage, but are both valuable and vulnerable. Because of the intrinsic interactions among living organisms, nutritious materials and the surrounding environmental conditions, most of murals are facing severe and irreversible deterioration and damages from aesthetic alteration to material degradation of the paint layers, leading to a substantial loss of the artistic aesthetics to historical authenticity and cultural values. Microorganisms including algae, cyanobacteria, lichens, fungi, bacteria, and archaea are known for their abilities to colonize murals under different microhabitats, resulting in undesirable biodeterioration, and even destruction of wall paintings. By combining non-destructive sampling methods with traditional culture-dependent and culture-independent ones, DNA/RNA-based and/or multi-omics, the characteristics of metabolically active and biochemical functional microbial communities of the whole microbiome associated with murals have been revealed in greater depth providing new information for a better knowledge. Mechanical and/or physical and biocides are commonly used methods for controlling of microbial growth, but none of them is effective for the long term. Environmental conditions of the micro-niches, surroundings and local environments, including temperature, water activity (relative humidity) and light (natural or artificial) are the key factors for biodeterioration and selective of them could be regulated in heritage protection. The active microorganisms and destructive biochemical reactions shall be the high priority of focus so that the control measures and management can be selected accordingly. Under the scenario of climate change, anthropogenic disturbance, application of restoration materials and tourism are the main challenges for the sustainable conservation of murals in the future. The most effective approach can be identified with a better understanding of the mural materials under the specific environmental conditions for a scientifically protection strategy for implementation with minimum or no intervention directly onto the mural, which requires contributions from biologists, chemists, materials scientists and geologists collective to achieve the goals for protection of mural and paintings.
... The metabolic activity and degradation products of microbes cause severe deterioration of the aesthetic value of the ancient and invaluable wall paintings, the structural integrity of the pigment layers, and the disintegration of painting materials worldwide (Garg et al., 1995). Reports are available on the microbial colonization on the paleolithic paintings in the Lascaux Cave in France (Alonso et al., 2019;Bastian et al., 2010;Saiz-Jimenez et al., 2011) and Altamira Cave in Spain (Cuezva et al., 2012;Saiz-Jimenez et al., 2011), the wall paintings of the tomb of King Tutankhamun in Egypt (Vasanthakumar et al., 2013), the Japanese tumuli of Takamatsuzuka (Sugiyama et al., 2017), brick mural of Han and Jin dynasty (Ma et al., 2020) and the wall paintings of caves at Mogao Grottoes, Tiantishan Grottoes, Maijishan Grottoes and Beishiku Temple in China (Duan et al., 2018;Ma et al., 2015). ...
... Using culturedependent and culture-independent methods, studies have shown that the wall painting surfaces are dominated by the fungal groups such as Penicillium spp., Aspergillus spp., Cladosporium spp., and Engyodontium spp. (Bastian et al., 2009b, c;Guglielminetti et al., 1994;Leplat et al., 2017;Ma et al., 2020;Ma et al., 2015;Vasanthakumar et al., 2013), and the bacterial groups such as Bacillus spp., Arthrobacter spp., Micrococcus spp., Pseudomonas spp., Geobacillus spp., Lactococcus spp., Streptomyces spp., Rubrobacter spp., and Pseudonocardia spp. (Abdel-Haliem et al., 2013;Altenburger et al., 1996;Duan et al., 2017Duan et al., , 2018Heyrman et al., 1999;Imperi et al., 2007;Ma et al., 2015). ...
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The Maijishan Grottoes, located on the ancient Silk Road in West China, was constructed from the 4th centuries onward for more than 1500 years and became a UNESCO World Cultural Heritage in 2014. A severe microbe outbreak occurred on the wall paintings in the caves of the Maijishan Grottoes in 2018. To prevent and control microbial damage to the wall paintings, we analyzed the bacterial and fungal communities in the normal wall samples and those with microbial plaques using high-throughput sequencing and the environmental factors associated with the microbe outbreak. The results showed that the normal wall samples were dominated by bacterial phyla Actinobacteria (71.7%), Proteobacteria (17.8%), Firmicutes (6.5%), Chloroflexi (2.4%), and Bacteroidetes (0.9%), and fungal families Trichocomaceae (43.4%), unclassified Capnodiales (23.2%), unclas-sified Ascomycota (12.1%), Teratosphaeriaceae (10.2%), and unclassified Eurotiomycetes (6.2%). The wall samples with microbial plaques were dominated by bacterial Actinobacteria (92.9%), Proteobacteria (5.3%), Firmicutes (1.1%), and Chloroflexi (0.3%) and fungal families unclassified Eurotiomycetes (93.3%), unclassified Ascomycota (3.3%), and Microascaceae (2.9%). These results indicated the great changes in bacterial and fungal communities during the microbe outbreak. The dominant fungus in the wall samples with microbial plaques was isolated and identified as a potential novel species which has 92% ITS sequence similarity with Arachnomyces sp. Canonical correspondence analysis (CCA) revealed that the microbe outbreak was primarily attributed to the excessive air humidity inside and outside of the caves. Thus, air humidity management is important for protecting the ancient wall paintings from microbial colonization and attack.
... 、西班 牙阿尔塔米拉洞穴壁画 [6][7] 、埃及图坦卡蒙墓壁 画 [8] 、日本高松冢古坟壁画 [9] 、意大利原罪地下墓 室壁画 [10] 以及我国敦煌莫高窟壁画 [11][12] 等多处世 界遗产地及古迹遗址地壁画都曾遭到或仍面临着 包括细菌、真菌和藻类的侵蚀和破坏。近些年,随 Tel: 010-64807511; E-mail: tongbao@im.ac.cn; http://journals.im.ac.cn/wswxtbcn 着微生物分子生态学研究的发展,对于微生物侵蚀 壁画机理的相关研究也在不断深入 [13] 。 就不同环境下保存的壁画而言,墓室环境壁画 所面临的微生物侵蚀与退化问题则更为严重和复 杂 [14][15] 。究其原因,土壤本身是微生物资源的储藏 库,地下高湿度和低空气交换等环境特点也非常有 利于墓室壁画中微生物的滋生和侵染。为了阻止或 减缓病害微生物对珍贵壁画的进一步劣化,多种杀 灭剂曾被广泛应用到壁画病害菌的防治中,然而防 护现状却依然不容乐观 [16][17] ,这在很大程度上被归 因于当前对于壁画病害微生物群落本底水平及其 附存环境的调查和研究依然不够全面和细致 [18] 。 自 20 世纪 90 年代开始,我国研究人员使用基 于培养的手段对甘肃酒泉丁家闸十六国墓壁画和嘉 峪关新城魏晋墓壁画 [19] 、河南密县汉墓壁画 [20][21] 、 陕西长安唐墓壁画 [22] 、西安曲江翠竹园西汉墓壁 画 [23] 等几处墓室壁画病害微生物进行了分离和鉴 定;然而,利用分子生物学技术研究墓室壁画微生 物的报道截至目前还较少,仅见对我国北方 5 世纪 墓室壁画 [24] 和嘉峪关魏晋墓壁画 [25][26] [4][5] ...
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... The former was one of the most frequently isolated genera, with A. flavus and A. niger among the more frequently recorded species. As reported in the literature, even from the first older papers in this field, these two genera, along with Alternaria, Fusarium, Cladosporium, Mortierella, Chaetomium, and Acremonium, are among the most common deteriogens of such paintings [1,11,15,31,39,46,62,78,87,91]. These taxa are ubiquitous, and their frequent occurrence is due to the production of numerous conidia, which are widespread in the environment because they are easily dispersed by air. ...
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... Lines of evidence have shown that many species in Aspergillus and Penicillium genera have the ability to produce a large amount of organic acids [62,63]. The production of acids can promote fungal proliferation and fade the painted colors, which further causes damage to mural painting materials and subsequently deterioration [69][70][71]. The ability to form calcium oxalate of the Aspergillus genera on agar was also reported [72]. ...
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Representing countless societies and cultures, rock imagery and cultural stone are diverse and valuable resources that are, despite their perceived resilience, incredibly fragile and vulnerable to copious geomorphological processes. This article discusses various multidisciplinary theoretical frameworks and field assessment research tools that have been employed to better understand the dynamic nexus of heritage sciences, geomorphology, archeology, material sciences, and more. Additional information on ethical considerations scholars must take when researching sensitive materials is also provided.
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Gas chromatography has long been used as a means of identifying proteins in acid hydrolysates of paints and adhesives on the basis of their amino acid composition. Existing procedures for amino acid derivatization can, however, take up to 4 hours per analysis, whereas a new procedure employed at the Getty Conservation Institute can be carried out in less than 20 minutes. This procedure involves the formation of N(O,S)-ethoxycarbonyl ethyl esters of amino acids using ethyl chloroformate and then a separation on an HP-INNOWAX capillary column. This procedure was employed to analyze samples from the binding media library at the Getty Conservation Institute, and amino acid compositions were tabulated for various proteins that could be used for identification purposes.
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Full-text available
Gas chromatography has long been used as a means of identifying proteins in acid hydrolysates of paints and adhesives on the basis of their amino acid composition. Existing procedures for amino acid derivatization can, however, take up to 4 hours per analysis, whereas a new procedure employed at the Getty Conservation Institute can be carried out in less than 20 minutes. This procedure involves the formation of N(O,S)-ethoxycarbonyl ethyl esters of amino acids using ethyl chloroformate and then a separation on an HP-INNOWAX capillary column. This procedure was employed to analyze samples from the binding media library at the Getty Conservation Institute, and amino acid compositions were tabulated for various proteins that could be used for identification purposes.