La relazione tra i sali e la crescita delle colonie di streptomiceti isolati dagli affreschi di alcune antiche tombe egiziane
10.6092/issn.1973-9494/4190
SummaryEight out of the forty-six samples of Streptomyces representing the most potent deteriorating isolates were taken from scaled and stained mural paintings in the tombs at Tell Basta and Tanis, Lower Egypt. These isolates were identified using conventional and 16S rDNA sequencing methods and attributed to S. albidofuscus, S. ambofaciens, S. canaries, S. chibaensis, S. coelicolor, S. corchorusii, S. nigrifaciensand S. parvullus. The different salts occurring on the ancient Egyptian tombs, such as sodium chloride, potassium chloride and magnsium sulphates enhanced the growth of Streptomyces isolates till 10% of these salts. Streptomyces isolates adapted/adopted different defense mechanisms such as pigmentation, osmotic balance and amino acidproduction. The salts had a synergistic effect in the deterioration of the mural paintings through the cooperation of the mycelium of Streptomyces with salts in the mechanical deterioration of the stone surfaces. RiassuntoOtto dei quarantasei campioni di streptomiceti che rappresentano i più potenti biodeteriogeni sono stati isolati dagli affreschi deteriorati all’interno delle tombe a Tell Basta e Tanis, nel basso Egitto. Questi isolati sono stati identificati usando metodi convenzionali e del sequenziamento 16S rDNA e sono stati attribuiti alle specie S. albidofuscus, S. ambofaciens, S. canaries, S. chibaensis, S. coelicolor, S. corchorusii, S. nigrifaciens and S. parvullus. I diversi sali ritrovati sulle antiche tombe egizie, come il cloruro di sodio, clorurodi potassio e il solfato di magnesio hanno accelerato la crescita degli streptomiceti isolati fino ad una concentrazione del 10% di questi sali. Gli streptomiceti isolati hanno adottato diversi meccanismi di difesa come la pigmentazione, l’equilibrio osmotico e la produzione di aminoacidi. I sali hanno svolto un effetto sinergico sul deterioramento degli affreschi attraverso l’interazione con il micelio degli streptomiceti ed i sali, incrementando il deterioramento meccanico delle superfici lapidee.RésuméHuit des quarante-six échantillons de streptomycètes, qui représentent les biodétériogènes les plus puissants, ont été isolés des fresques détériorées situées à l’intérieur des tombes à Tell Basta et Tanis, en Basse-Égypte. Ces isolats ont été identifiés à l’aide de méthodes conventionnelles et du séquençage 16S ADNr, et ils ont été attribuésaux espèces S. albidofuscus, S. ambofaciens, S. canaries, S. chibaensis, S. coelicolor, S. corchorusii, S. nigrifaciens et S. parvullus. Les différents sels retrouvés sur les anciennes tombes égyptiennes, tels que le chlorure de sodium, le chlorure de potassium et le sulfate de magnésium, ont amplifié la croissance des streptomycètesisolés jusqu’à 10 % de ces sels. Les streptomycètes isolés ont adopté différents mécanismes de défense tels que la pigmentation, l’équilibre osmotique et la production d’acides aminés. Les sels ont eu un effet synergique sur la détérioration des fresques, à cause de leur interaction avec le mycélium des streptomycètes : la détériorationmécanique des surfaces en pierre a été ainsi augmentée. ZusammenfassungAcht der sechsundvierzig Proben von Streptomyzeten, die die schlimmste Verrottung hervorrufen, wurden in den verrotteten Fresken im Inneren der Grabstätten in Tell Basta und Tanis in Niederägypten isoliert. Sie wurden anhand herkömmlicher Methoden und durch Sequenzierung des 16S rDNA isoliert und den Spezies S. albidofuscus, S. ambofaciens, S. canaries, S. chibänsis, S. cölicolor, S. corchorusii, S. nigrifaciens und S. parvullus zugeordnet. Die verschiedenen Salze, die in den antiken ägyptischen Gräbern vorgefunden wurden, wie beispielsweise Natriumchlorid, Kaliumchlorid und Magnesiumsulfat, haben das Wachstum der isolierten Streptomyzeten bis auf 10% dieser Salze gesteigert. Die isolierten Streptomyzeten bedienten sich verschiedener Verteidigungssysteme, wie beispielsweise Pigmentierung, Osmose-Gleichgewicht und Erzeugung von Aminosäuren. Die Salze waren durch usammenwirkung mit dem Myzelium der Streptomyzeten synergisch an der Verrottung der Fresken beteiligt und haben die mechanische Schädigung der Steinflächen verstärkt.ResumenOcho de las cuarenta y seis muestras de estreptomicetos, que constituyen uno de los biodeteriógenos más potentes, han sido aislados en los frescos deteriorados dentro de las tumbas de Tell Basta y Tanis, en el bajo Egipto. Estos agentes aislados han sido identificados utilizando métodos convencionales y mediante el secuenciamientodel 16S rDNA y han sido atribuidos a las especies S. albidofuscus, S. ambofaciens, S. canaries, S. chibaensis, S. coelicolor, S. corchorusii, S. nigrifaciens y S. parvullus. Las distintas sales encontradas sobre las antiguas tumbas egipcias, como el cloruro de sodio, cloruro de potasio y el sulfato de magnesio, han aumentado el crecimiento de losestreptomicetos aislados hasta el 10% de dichas sales. Los estreptomicetos aislados han adoptado distintos mecanismos de defensa como la pigmentación, el equilibrio osmótico y la producción de aminoácidos. Las sales han tenido un efecto sinérgico en el deterioro de los frescos mediante la interacción con el micelio de los estreptomicetos, incrementando el deterioro mecánico de las superficies lapídeas. РезюмеВосемь из сорока шести образцов стрепромицитов, которые являются самыми мощными биоразрушителями, были изолированы от поврежденных фресок внутри гробниц в Тель Баста и Танисе в Нижнем Египте . Они были идентифицированы при использовании традиционных методов и последовательности 16S рДНК ибыли отнесены к видам S. albidofuscus, S. ambofaciens, S. canaries, S. chibaensis, S. coelicolor, S. corchorusii, S. nigrifaciens и S. parvullus . Различные соли, найденные на древнеегипетских могилах, такие как хлорид натрия, хлорид калия и сульфата магния увеличили рост изолированных стрептомицитов до 10% этих солей . Изолированные стрептомициты приняли различные защитныемеханизмы такие, как пигментация, осмотическое равновесие и производство аминокислот. Соли оказали синергетический эффект на ухудшение фресок, при взаимодействии мицелия стрептомицитов с солями увеличивался механический износ каменных поверхностей. 摘要代表最强效生物清洗剂的四十六种链霉菌标本中的八种是从位于埃及南部的布巴斯提斯和塔尼斯的墓穴内部受损壁画上提取出来的。这些提取物通过16SrDNA排序和常规方法被确认，归属于物种微白褐链霉菌、生二素链霉菌、黄雀链霉菌黄雀变种、千叶链霉菌、天蓝色链霉菌、黄麻链霉菌、生黑链霉菌和小小链霉菌。在古埃及墓中所发现的多种盐，比如氯化钠、氯化钾和硫酸镁都增加了10%的提取链霉菌的生长。提取的链霉菌已采取了不同的防御机制，比如色素、渗透平衡和氨基酸的产生。这些盐通过与链霉菌菌丝体的相互作用已经对壁画损坏起到了协助作用，同时还提高了石材表面的机械化变质。
application/pdf
1973-9494
4190
en
Mona Fouad Ali
Conservation Science in Cultural Heritage
carotenoid
The relationship between salts and growth of Streptomyces isolated from mural paintings in some ancient Egyptian tombs
Text.Serial.Journal
https://conservation-science.unibo.it/
13
Articles
2013-12-30
2014-02-09
2013-12-30
2015-11-02
313-330
CSCH
Mona Fouad Ali
2013/12/30
10.6092/issn.1973-9494/4190
313
1973-9494
carotenoid
en
330
by Streptomyces fradiae 1, J. Bacteriol, 75, pp. 161-166
The relationship between salts and growth of Streptomyces isolated from mural paintings in some ancient Egyptian tombs
https://conservation-science.unibo.it/article/download/4190/3636
13
T    HE RELATIONSHIP BETWEEN SALTS AND GROWTH OF
     STREPTOMYCES COLONIES ISOLATED FROM MURAL PAINTINGS
     IN ANCIENT EGYPTIAN TOMBS



   Akmal Ali Sakr●1 ·
   Botany Department, Faculty of Science, Zagazig University, Zagazig, Egypt
   Conservation Department, National Museum of Egyptian Civilization (NMEC),
   Cairo, Egypt

   Mohammed Farouk Ghaly
   Botany Department, Faculty of Science, Zagazig University, Zagazig, Egypt

   Mona Fouad Ali
   Conservation Department, Faculty of Archaeology, Cairo University, Cairo, Egypt

   Keywords: tombs, Egypt, Streptomyces, salts, carotenoid


  1. Introduction

    Mural paintings in ancient Egyptian tombs are often deteriorated by microbial colo-
nization and, particularly by Streptomyces. These bacterial species commonly colo-
nize hypogea environments, such as caves and tombs, characterized by darkness,
low temperature and nutrients [1], this may be attributed to the ability of Streptomyces
to utilize a wide range of carbon sources and to produce a wide range of enzymes.




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
Enzymatic activities that could decompose complex polymers of chopped straw used
as filler, or organic matters present in limestone or dead fungal hyphae, could be used
by microorganisms as a carbon source [2-3].
    Streptomyces colonies on mural paintings and stone surfaces are considered as
indicators of an advanced deterioration stage and are the most dominant in the micro-
biota colonizing these paintings [4]. Each Streptomyces species seems to play a spe-
cific role in the deterioration of mural paintings and stone supports [5], causing discol-
oration and enhancement of mechanical deterioration in relationship to salt cycles [6].
    Identification of microorganisms colonizing mural paintings is an important step in
putting forward sound conservation strategies, but due to problems imposed by mor-
phological methods in identification, molecular methods based on 16S rDNA sequence
analysis have been used to identify Actinomycetes isolated from deteriorated stones
and mural paintings, confirming the morphological and biochemical identification keys
[7-12].
    Streptomyces are involved significantly in the deterioration of mural paintings in
cooperation with other deterioration agents, salts in particular. Several salts were de-
tected in deteriorated mural paintings in ancient Egyptian tombs from different sources.
The sources of these salts are the composition of building stone and leakage of un-
derground water containing salt ions from the soil to the walls of the tombs, since NaCl

● ·Corresponding author: akmlsakr@yahoo.com

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                                                                                                          concentrations in the soil of investigated tombs reached up to 11574 ppm/g [13].
                                                                                                             The most common salts detected in ancient Egyptian mural paintings were sodium
                                                                                                          chloride, potassium chloride, magnesium sulfate and sodium nitrate. Sodium chloride
                                                                                                          (NaCl) is a highly soluble and hygroscopic salt that absorbs humidity from the air and
                                                                                                          retains it in the wall surface, thus producing dark batches [14, 16].
                                                                                                             Actinomycetes and halotolerant bacteria could thrive in hostile environments (solar
                                                                                                          energy, hypersalinity and radiation) where there is a little microbial competition. Micro-
                                                                                                          coccus, Arthrobacter and Streptomyces were the most dominant in salted deteriorated
                                                                                                          mural paintings [4,6].
                                                                                                             Streptomyces isolates were halotolerant NaCl, KCl and Mg SO4. 7 H2O, the most
                                                                                                          common salts occurring in deteriorated mural paintings, as well as S. rishiriensis colo-
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                          nizing mural paintings in the Altamira Cave, Spain and could grow up to 15 % NaCl [16].
                                                                                                          Moreover, Streptomyces diastaticus, S.cyaneus, S.antimycoticus, S.chromofuscus, S.
                                                                                                          exfoliatus, S.rochei, S. albidoflavus, S.anulatus, S. roseflavus, S.badius, S. violace-
                                                                                                          us, S. griseoflavus, S. macrosporus, S. phaeochromogenes, S. griseostramines and
                                                                                                          Streptomyces sp. isolated from the tomb of Maat Tell Basta, were halotolerant to 7-13
                                                                                                          and 10 % for NaCl and N2SO4- 10 H2O respectivley [17].
                                                                                                             Saiz-Jimenez and Laiz [16] reported that the optimum salt concentration for the
                                                                                                          growth of Farnkia, S. griseus and Pseudonocardiaceae isolated from mural paintings
                                                                                                          at Herberstein Castle, Austria, was 10 % of magnesium sulfate and 5 % sodium chlo-
                                                                                                          ride, showing a rosy color, while higher salinity, 10-15 %, reduced the counts of micro-
                                                                                                          bial cells.
                                                                                                             Furthermore, Halococcus salifodinae was isolated from white efflorescence of so-
                                                                                                          dium nitrate and sodium chloride from the chapel of St. Virgil, Vienna, Austria, and their
                                                                                                          optimal growth occurred at 20-25% NaCl [18].
                                                                                                             Streptomyces have a synergistic mechanical action with salts, where it was reported
                                                                                                          that salts enhance the mechanical deterioration of stone surfaces since the penetrat-
                                                                                                          ing Streptomyces mycelium enlarge voids and cracks and resulted in the dissolving
                                                                                                          and recrystallization of salts. Moreover, sodium chloride is a hygroscopic salt and has
                                                                                                          high water retention, increasing the volume of Streptomyces mycelium that are mainly
                                                                                                          composed of extracellular-polymeric substances (EPS); whereas wetting and drying
                                                                                                          cycles cause the disintegration of stone surfaces [3,6,16,19]. On the other hand, salt
                                                                                                          action can roughen the stone surface that is the preferential site for colonization by
                                                                                                          various communities of microorganisms [20, 21].
                                                                                                             It is reported that the presence of sulfate salts can lead to irreversible damage to
                                                                                                          historic constructions; in the presence of humidity it can dilute and withdraw the pig-
                                                                                                          ment layers of mural paintings and this high water content could dissolve the binding
                                                                                                          media that can also serve as nutrients for some micro-organisms [22].
                                                                                                             In addition, there was an interrelation between the detected salts, and the growth
                                                                                                          and metabolic activity of isolated Streptomyces colonies, whereas salts obligate
                                                                                                          Streptomyces to choose different adaption mechanisms such as producing protec-
                                                                                                          tive molecules for example melanin and carotenoids [23-27]. These pigments cause
                                                                                                          aesthical damage and irreversible staining of colonized paintings and plaster layers
                                                                                                          [28, 29].  
                                                                                                             In addition to pigment production Streptomyces have an osmotic balance mecha-
                                                                                                          nism of organic osmolytes responsible for osmotic balance (glycine betaine, ectoines,
                                                                                                          praline, N-acetylated diamino acids, N-derivatized glutamine amides and other amino
                                                                                                          acid derivatives), that have been found in all microbial colonies isolated from salt ef-


                                                                                                    314
florescence [16, 25]. The main function of osmolytes could be to protect the protein
conformations and so prevent their denaturation, or influence the conformational sta-
bility of nucleic acids induced by high salt concentrations [30].
    The purpose of this research is to isolate and identify Streptomyces colonies from
deteriorated mural paintings in ancient Egyptian tombs and to determine the effect of
salts detected in these mural paintings on the growth and metabolic activity of Strep-
tomyces strains, in order to put forward the most suitable conservation strategies for
these paintings.

  2. Experimental

  2.1. Location and Sampling

   Eight samples of Streptomyces were collected from deteriorated mural paintings in
the tombs of Ankh m b3st, Ist, Ankh h.f and Ihi at Tell Basta and the tomb of Oserkon II
at Tanis, Lower Egypt (Figure 1), using a sterile cotton swab method. These samples
were cultivated onto starch-nitrate-agar (SNA) medium (agar 20 g, starch 20 g;
KH2PO4 1 g; MgSO4 0.5 g; NaCl 0.5 g; KNO3 2 g; CaCO3 3 g in 1L distilled water),
supplemented with an antifungal molecule (Dermatine 10-50 μg / l), to inhibit the
growth of competitive fungi. Plates were incubated for 45 days at 30°C.

  2.2. Identification of Streptomyces

   Bacterial isolates were identified morphologically and biochemically according to
the identification keys of Kämpfer, [31] and confirmed by analyzing the 16S rDNA se-
quences.




                                                                                                 CONSERVATION SCIENCE IN CULTURAL HERITAGE
  2.3. 16S rDNA sequencing

   16S rDNA sequencing is one of the most promising techniques for identification
of Streptomyces, whereas total DNA was extracted from eight isolated colonies of
Actinobacteria [32]. The gene coding for 16S rRNA was amplified from each isolate by
PCR with universal primers (forward primer, F27, 5’-AGAGTTTGATCCTGGCTCAG-3’
[33] and reverse primer, R1492, 5’-GGTTACCTTGTTACGACTT-3’) [34]. These prim-
ers bind to universally conserved regions and permit the amplification of a DNA frag-
ment, approximately 1500 bp in length. The PCR amplification was carried out by a
Gene-Amp PCR system 9600 thermocycler (Perkin Elmer). The amplification condi-
tions were as follows: 94 °C for 10 min and 35 cycles of denaturation at 95 °C for 30
s, annealing at 56 °C for 1 min, extension at 72 °C for 1 min; completed by a final
extension step at 72 °C for 10 min. Presence and yield of specific PCR products (16S
rRNA gene) were analyzed on 1% agarose gel. Before sequencing PCR product was
cleaned up by using GeneJET™ PCR Purification Kit (Fermentas).
   Amplified DNA fragments were partially sequenced at GATC Biotech AG (Konstanz,
Germany) using an ABI 3730xl DNA sequencer, using forward primer (F27). The 16S
rDNA sequences determined, were deposited into NCBI web server (www.ncbi.nlm.
nih.gov). Sequence analysis and comparison were performed using the Basic Local
Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/blast) [35].



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                                                                                                              a)                                                 b)
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                              c)                                                d)




                                                                                                              e)                                                f)




                                                                                                          Figure 1. Location of sampling: (a) Azurite blue in tomb of Oserkon II with white crust, Tanis.
                                                                                                          (b): Red ochre, tomb Ankh m b3st. (c) Azurite blue, tomb of Ihi, Tell Basta. (d) Red and yellow
                                                                                                          paintings, tomb Ankh h3 f, Tell Basta (e) Black color from tomb of Ankh h3 f, Tell Basta. (f) White
                                                                                                          crust of soduim chloride, tomb of Ist, Tell Basta.




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  2.4. Salts analysis

   White crust, sampled from the blue color in the ceiling room of the King Oserkon II
at Tanis, was analyzed by SEM-EDS (Energy Dispersive Spectrometry) microscopy,
using a Philips XL 30 ESEM scanning electron microscope; National Research Centre,
Dokky, Cairo, Egypt.

  2.5. Effect of different concentrations of salts NaCl, KCl and MgSO4. 7 H2O
  (2.5, 5 - 10 %) on metabolic activity

  To investigate the mechanical destruction caused by Streptomyces, limestone discs
were inoculated with S. coelicolor and S. canarius, incubated for 3 weeks at 30°C.

  2.6. Scanning Electron Microscope (SEM) investigations

     Limestone discs (diameter 1.5 cm), initially treated with different concentrations of
NaCl, KCl and MgSO4. 7 H2O, were inoculated under aseptic conditions with Strepto-
myces isolates and incubated for 6 months at 30°C, in beakers containing 50 ml dis-
tilled water to establish a humid environment. Samples of inoculated limestone discs
were prepared for SEM investigations according to Milanesi et al., [21], whereas these
samples were fixed in 4% glutaraldehyde for 24 h and then dehydrated by ethyl alcohol
series 25, 50, 75 and 100 %, (one hour per step). Samples were then coated with gold
microparticles and investigated by using the Scanning Electron Microscopy, National
Research Centre, Dokky, Cairo, Egypt.

  2.7. Extraction and purification of red pigment




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
   Extracellular red pigment produced by S. canaries under stress conditions (differ-
ent NaCl concentrations) was extracted and purified according to Sterflinger, [36] and
Baskar et al. [37]. The S. canaries strain was cultured on broth starch-nitrate medium,
Erlenmeyer flasks (250 ml), containing 50 ml of medium supplemented with 10 % NaCl
and incubated for 7 days at 30 °C.
   5 grams of biomass were added to a mixture of n-hexane and acetone (92/8 v/v),
then centrifuged for 10 min. at 5000 rpm. The volume of the organic mixture where
the red pigment was dissolved, was reduced by up to 0.1 ml. The resulting volume
of pigment was purified on TLC plates and the fractions of pigment were analyzed by
JASCo. FT / IR 61000, National Research Centre, Dokky, Cairo, Egypt.

  2.8. Determination of amino acids

    Amino acids produced by Streptomyces strains under stress of salinity were valued in
broth medium, by using a HPLC amino acid analyzer, LC300 Eppendorf-Germany, con-
nected to a flourescence detector, National Research Centre, Dokky, Egypt, according to
Callejon et al. [38]. Streptomyces strains were cultured on broth starch-nitrate-agar me-
dium in Erlenmeyer flasks 250 ml, and incubated at 30°C for 7 days. 20µL of filtrate was
injected in the column (Luna C18,5 µm, 250 × 4.6 mm and guard column 4.0 × 3.0 mm).
Detection was performed by fluorescence excitation at 250 nm and emission at 395 nm.
Fractions were obtained at a flow rate of 1 mL min-1 at 34 °C. Results were normalized
against standard a solution of 22 amino acid.

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                                                                                                             3. Results

                                                                                                             3.1. Identification of Streptomyces

                                                                                                              Our finding indicated that all identified isolates were of Streptomyces genus and
                                                                                                          were attributed to S. albidofuscus, S. ambofaciens, S. canaries, S. chibaensis, S. coe-
                                                                                                          licolor, S. corchorusii, S. nigrifaciens and S. parvullus. The authors’ accession num-
                                                                                                          bers in the International Gene Bank were illustrated in Table 1.

                                                                                                          Table 1. Phylogenetic affiliation of inoculated strains (Homology of 16S r DNA and similarity in
                                                                                                          comparison with NCBI Data)
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                                                                      Similarity         Authors‫ۥ‬
                                                                                                                                  Homology                                                   G+C
                                                                                                               Location                             enter genes         accession
                                                                                                                                 Approximately                                              content
                                                                                                                                                     16S r DNA           number
                                                                                                                                                                      Later name is
                                                                                                               Azurite blue,
                                                                                                                                                                    S. pyridomyceticus
                                                                                                            tomb of Ihi, Tell    S. albidofuscus       99 %                                  58 %
                                                                                                                                                                     BankIt1507621
                                                                                                                Basta
                                                                                                                                                                        JQ625331

                                                                                                             Yellow color of
                                                                                                                                                                      BankIt1507642
                                                                                                            Southern wall of     S. ambofaciens        99 %                                  60.6 %
                                                                                                                                                                        JQ625332
                                                                                                            Ankh h3 f tomb

                                                                                                           Blue color, ceiling
                                                                                                                                                                      BankIt1507650
                                                                                                             burial tomb of       S. canaries          99 %                                 58.9 %
                                                                                                                                                                        JQ625337
                                                                                                           Oserkon II, Tanis
                                                                                                           Black color, tomb
                                                                                                                                                                      BankIt1507649
                                                                                                             Ankh h.f, Tell      S. chibaensis         100%                                  58.9 %
                                                                                                                                                                        JQ625336
                                                                                                                Basta
                                                                                                           Red color, tomb
                                                                                                                                                                      BankIt1507648
                                                                                                           Ankh m b3st, Tell      S. coelicolor          99 %                                59.2 %
                                                                                                                                                                        JQ625335
                                                                                                                Basta
                                                                                                           Limestone, tomb
                                                                                                                                                                      BankIt1507647
                                                                                                                  of             S. corchorusii        98 %                                  58.9 %
                                                                                                                                                                        JQ625334
                                                                                                           Oserkon II, Tanis
                                                                                                                                                                     Later name is S.
                                                                                                           North wall, tomb of                                         flavovirens
                                                                                                                                 S. nigrifaciens       98 %                                  ~56 %
                                                                                                             Ist, Tell Basta                                         BankIt1507149
                                                                                                                                                                        JQ625330
                                                                                                             Yellow color of
                                                                                                                                                                      BankIt1507645
                                                                                                            Southern wall of      S. parvullus         99 %                                  56.3 %
                                                                                                                                                                        JQ625333
                                                                                                            Ankh h3 f tomb



                                                                                                             3.2. SEM-EDX spectra of white crust

                                                                                                               SEM-EDX spectra of white crusts covering the blue pigment in the tomb of Oserkon
                                                                                                          II, revealed that sodium chloride (NaCl) was in approximately pure needle form as illus-
                                                                                                          trated in Figure 2 a. Moreover, SEM micrograph in Figure 2 b pointed out that halite in
                                                                                                          needle shape was formed, before reaching the cubic crystals, the typical form of halite


                                                                                                    318
and veins of halite within the limestone blocks in the burial chamber of King Oserkon II,
Tanis (Figure 2 c). Figure 2 d showed dissolving and precipitation of calcite by under-
ground water in the form of stalactites. The EDX pattern of white crusts on the surface of
the tomb of Ihy in Figure 2 e showed that crusts were of nearly pure halite.


                               a




 b                                   c                                d




                                                                                                                  CONSERVATION SCIENCE IN CULTURAL HERITAGE
        e




Figure 2. (a) SEM-EDX spectra of white efflorescence from ceiling room, tomb of Oserkon II, Tanis.
(b) needle form of halite from the ceiling room tomb of Oserkon II, Tanis, (c) veins of halite within the
limestone blocks, burial chamber of King Oserkon II. (d) dissolving and precipitation of calcite, tomb
of King Oserkon II (e) EDX pattern of white crust from limestone surface, tomb of Ihy, Tell Basta.


                                                                                                            319
                                                                                                             3.3. Effect of sodium chloride

                                                                                                              With regard to the effect of Nacl on the growth of Streptomyces, Figure 3 and Table
                                                                                                          2 showed that S. coelicolor, S. ambofaciens, S. canarius grew very well at 10% NaCl.
                                                                                                          S. parvullus had good growth, S. chibaensis, S. corchorusii, S. nigrifaciens had moder-
                                                                                                          ate growth, while S. albidofuscus had reduced growth at the same NaCl concentration.
                                                                                                          It was found that the growth rate decreased with the increase of NaCl concetntration
                                                                                                          up to 10 %, and S. canaries showed a high tolerance to NaCl 10% and produced red
                                                                                                          pigment, not produced under normal culture conditions (Figure 4).


                                                                                                                             4.5
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                                               4
                                                                                                                             3.5
                                                                                                                               3                                                                  Na Cl 2.5 %
                                                                                                                             2.5                                                                  Na Cl 5%
                                                                                                                               2
                                                                                                                             1.5                                                                  Na Cl 10%
                                                                                                                               1
                                                                                                                             0.5
                                                                                                                               0
                                                                                                                       S. fa s
                                                                                                                             rc ns
                                                                                                                      S. r ifa us




                                                                                                                                        r
                                                                                                                      S. do f iu s




                                                                                                                        S. ae s
                                                                                                                        am ar v ii




                                                                                                                               el is
                                                                                                                           bo u llu




                                                                                                                                     lo
                                                                                                                                      s


                                                                                                                             ib n
                                                                                                                            co n s
                                                                                                                          co cie




                                                                                                                          ch cie
                                                                                                                         S. oru
                                                                                                                            g sc




                                                                                                                                 ico
                                                                                                                                   r
                                                                                                                        al a na

                                                                                                                                u


                                                                                                                               h
                                                                                                                     S. p
                                                                                                                             c
                                                                                                                          bi
                                                                                                                         S.


                                                                                                                         ni
                                                                                                                          S.




                                                                                                              Figure 3. Effect of different NaCl concentrations on the growth of isolated Streptomyces


                                                                                                            Streptomyces                                                 Salts concentrations (%)

                                                                                                                                                       NaCl                             KCl              MgSO4∙7 H2O
                                                                                                                                                 2.5    5   10                  2.5      5      10      2.5   5    10

                                                                                                           S. albidofuscus                   +++    +++       +            ++       ++      +++        +++    +++    +++

                                                                                                           S. ambofaciens                    ++++   +++     +++            ++++   +++     +++          +++    +++    +++

                                                                                                           S. albidofuscus                   +++      +++      +           ++      ++      +++         +++    +++    +++
                                                                                                           S. ambofaciens                    ++++     +++     +++          ++++   +++     +++          +++    +++    +++
                                                                                                           S. canarius                       +++      +++     ++++ ++      ++      +++                 ++       ++     +++

                                                                                                           S. chibaensis                      +++     +++      ++          ++++   +++    +++           ++++    +++    +++

                                                                                                           S. coelicolor                     ++++    +++      +++          ++++    +++    +++           ++        ++       +
                                                                                                           S. corchorusii                    +++       ++       ++         +++     +++    +++          +++     +++    +++
                                                                                                           S. nigrifaciens                   +++       +++     ++          ++       ++     ++          ++++   +++    +++
                                                                                                           S. parvullus                      ++++    +++      +++          ++++   +++    +++           ++++   +++   +++

                                                                                                                     (++++) very good growth (+++) good growth (++) moderate growth (+) low growth.



                                                                                                    320
        a                                            b




                Figure 4. Effect of NaCl on S. canaries: (a) 5 % (b) 10 % NaCl

  3.4. Effect of potassium chloride

   The second most common salt identified in deteriorated mural paintings was KCl
(Sylvite). Figure 5 and Table 1 showed that S. coelicolor, S. chibaensis, S. ambofa-
ciens, S. parvullus, S. corchorusii have very good growth in the presence of 10% KCl;
good growth for S. canarius, S. albidofuscus; reduced growth for S. nigrifaciens, at the
same KCl concentration. Streptomyces could grow up to 10%, whereas growth was
poor at 15% and impaired at 20% KCl. Our finding indicated that KCl up to 10% en-
hanced S. ambofaciens to produce a rosy pigment (Figure 7 c) and enhanced sporula-
tion of S. coelicolor after 14 days of incubation.




                                                                                                    CONSERVATION SCIENCE IN CULTURAL HERITAGE
                  4.5
                    4
                  3.5
                    3                                                KCl 2.5 %
                  2.5
                    2                                                KCl 5 %
                  1.5                                                KCl 10 %
                    1
                  0.5
                    0
                S. ae s


                              or
                       el is
               S. ofa s
              S. dof ius


                    r c ns

               am rv ii
              S. rifa us




                     ib n
             S. pa us
                   b ullu


                   co n s
                            ol
                  ch cie
                 ni usc

                 co cie
                 bi ar




                S. hor




                         ic
               al an
             S. . c


                   g
                 S




    Figure 5. Effect of different KCl concentrations on the growth of isolated Streptomyces

  3.5. Effect of magnesium sulfate

   Figure 6 shows that S. nigrifaciens, S. corchorusii, S. parvullus, S. ambofaciens and
S. chibaensis have very good growth with 10% magnesium sulfate (MgSO4 .7 H2O), but

                                                                                              321
                                                                                                          S. canarius, S. albidofuscus have good growth, and S. coelicolor had reduced growth
                                                                                                          with the same salt concentration. Current results indicated that different concentrations
                                                                                                          2.5, 5 and 10% of NaCl, KCl and MgSO4 -7 H2O, enhanced sporulation of S. coelicolor,
                                                                                                          increasing the pigmentation of S. ambofaciens (Figure 7).



                                                                                                                          4.5
                                                                                                                            4
                                                                                                                          3.5
                                                                                                                            3                                                    MgSO4 2.5 %
                                                                                                                          2.5
                                                                                                                                                                                 MgSO4 5 %
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                                            2
                                                                                                                          1.5                                                    MgSO4 10 %
                                                                                                                            1
                                                                                                                          0.5
                                                                                                                            0
                                                                                                                       S. bae s


                                                                                                                                     or
                                                                                                                              el is
                                                                                                                      S. ofa lus
                                                                                                                     S. ido rius



                                                                                                                       S. hor s
                                                                                                                      am arv ii
                                                                                                                     S. rifa cus




                                                                                                                                    n
                                                                                                                    S. p us
                                                                                                                           rc n




                                                                                                                          co ns
                                                                                                                                   ol
                                                                                                                         ch cie
                                                                                                                        co cie



                                                                                                                          b ul
                                                                                                                        b a
                                                                                                                        ni fus




                                                                                                                                ic
                                                                                                                      al an
                                                                                                                    S. S. c




                                                                                                                            i
                                                                                                                          g




                                                                                                          Figure 6. Effect of different MgSO4 7 H2O concentrations on the growth of isolated Streptomyces



                                                                                                           a)                                 b)                               c)




                                                                                                          Figure 7. Effect of KCl on growth of Streptomyces strains: (a) control, (b) effect of KCl 2.5% on the
                                                                                                          growth of Streptomyces coelicolor. (c): pigmentation of S. ambofaciens

                                                                                                             3.6. IR pattern of red pigment

                                                                                                             Red pigment was extracted by a mixture of n-hexane and acetone (92/8 v/v) on
                                                                                                          TLC plates (Figure 8 a). On the one hand, Figure 8 b showed that organic solvents
                                                                                                          have different dissolvability for red pigment, since acetone has high dissolvability,
                                                                                                          n-Hexan, chloroform and methanol have moderate dissolvability, but ethanol has low
                                                                                                          dissolvability. On the other hand, the IR pattern of extracted red pigment indicated
                                                                                                          the presence of quinon (ON-O-R) group at 3457 cm-1, carbonyl compounds (R2C =
                                                                                                          O) at 1638 cm-1 and alkanes (CH3)2 CH at 957 cm-1 (Figure 8 c). The red pigment is


                                                                                                    322
non pH sensitive, its red color did not change with the changing of pH values. Finally,
HPLC data indicate that all Streptomyces strains produced glutamic acid at NaCl
10% (unpublished data).




 a)                                                         b)




                                                                                                          CONSERVATION SCIENCE IN CULTURAL HERITAGE
                                                      (R2C=O)
                                                                       (CH3)2CH




                     ON-O-R




  c)


Figure 8. (a) Extraction of red pigment, (b) Extractability of red pigment with different organic
solvents, (c) IR spectra of red pigment produced by S. canaries under hyper-salinity induced by
NaCl.




                                                                                                    323
                                                                                                             3.7. Mechanical deterioration

                                                                                                             Figure 9, showed Streptomyces mycelia, enlarging the fissures and cracks caused
                                                                                                          by salt, penetrating within inter-granular spaces of calcite crystals, in deteriorated lime-
                                                                                                          stone discs.

                                                                                                                  a)                                          b)
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                          Figura 9. SEM micrographs illustrate the synergistic action of Streptomyces and salts, in the
                                                                                                          deterioration of limestone discs, Tomb of Maat, Tell Basta (Magnitude X 1500).

                                                                                                             Figure 10 showed that Streptomyces isolates varied in their productivity of amino
                                                                                                          acids under stress of hypersalinity of sodium chloride, since S. albidofuscus was the
                                                                                                          main producer, S. canarius, S. ambofaciens, S. nigrifaciens and S. chibanensis showed
                                                                                                          moderate productivity, but S. coelicolor, S. corchorusii and S. parvullus showed low
                                                                                                          productivity of amino acids.




                                                                                                                         Figura 10. Productivity of amino acids by Streptomyces isolates


                                                                                                    324
  4. Discussion

    16S rDNA data showed that all isolates had a similarity of more than 98% with
Streptomyces genus, the most dominant in the microbiota colonizing deteriorated mu-
ral paintings [4,36]. On the other hand, it was reported that Streptomyces are the first
colonizers of deteriorated mural paintings and an indicator of an advanced phase of
deterioration [28,39]. From the survey of the salts detected in the investigated tombs, it
was found that the most common salts in the deteriorated mural painting were sodium
chloride, potassium chloride and magnesium sulfate, this is in agreement with the re-
sults of Arnold [41]. Sodium chloride commonly occurs as impurities in gypsum precipi-
tations used as plaster layers or in limestone supports, as veins within the limestone
blocks or infiltrated from underground water to a stone support and paintings since this
salt is not produced by microorganisms [13]. Sodium chloride is a hygroscopic salt with
high solubility, so it is usually observed on the higher zones of paintings in tombs, in
the form of a white margin [41].
    The relation between the salts and Streptomyces colonization is a close one, since
the obtained results indicated that Streptomyces isolates varied in their tolerance pro-
file to NaCl, so different Streptomyces isolates could grow and colonize specific zones
of deteriorated mural paintings according to the occurring salts [42]. Our findings in-
dicated that most Streptomyces isolates were halotolerant to sodium chloride up to
10%, and it was reported that the optimum of NaCl, for most Streptomyces strains,
was 5-10 % [43,44]. Moreover, Streptomyces ssp., S. amctimycoticus and S. etfoliatus
isolated from the tomb of Maat at Tell Basta could grow in cultures supplemented with
NaCl up to 13% [17]. On the other hand, Milanesi et al. [20] showed that Streptomyces
isolated from deteriorated fresco paintings had a high tolerance to NaCl concentra-
tions in reality and under laboratory conditions. Moreover, Saiz-limenez and Samson
[46] studied the efflorescence of the frescoes of the monastery of Santa Maria de la




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
Rabida (Spain), and identified gypsum, calcium chloride, sodium chloride, potassium
chloride, quartz and silicates, where Micrococcus luteus, Bacillus, Pseudomonas and
Streptomyces were identified.
    The current results indicated that S. canarius was halotolerant and could grow up
to 10 % of NaCl; at this concentration, this isolate produced reddish pigment of carote-
noid that was not produced under normal cultures. S. canarius was isolated from white
efflorescence, nearly pure sodium chloride, covering the ceiling of the burial room of
King Oserkon II᾿s tomb, at Tanis and the tomb of Ihy at Tell Basta. Pigment production
corresponds to a defense mechanism of Streptomyces, against adverse environmen-
tal conditions such as hyper salinity, where this pigment acts as a shield between the
microbial cells and surrounding adverse environmental conditions, so these pigments
were called protective pigments [25,35].
    The second common salt detected in the investigated tombs was potassium chlo-
ride, identified on the paintings of Ankh h3 f tomb, at Tell Basta, probably due to the
surrounding cultivated lands, where a consistent amount of nitrogen fertilizers were
utilized [47]. Our results indicated that potassium chloride (KCl Sylvite) enhanced the
production of carotenoid pigment and sporulation, corresponding to a Streptomyces
mechanism against hyper-salinity.
    Our results indicated, in the tombs’ mural painting surfaces, the presence of mag-
nesium sulfate, frequently found in deteriorated mural paintings [48], indicating that
isolated Streptomyces strains could grow up to 10% MgSO4·7H2O, detected in Ibsmite


                                                                                             325
                                                                                                          present on the paintings of Ankh h3f at Tell Basta [47]; Magnesium sulfate crystalized
                                                                                                          in different forms such as Schonite (MgSO4.6H2O); Keiserite (MgSO4.H2O); Hexahy-
                                                                                                          drate (MgSO4.6H2O), and Ibsmite [40]. The action of crystallization and hydration of dif-
                                                                                                          ferent salts was linked with the biodeterioration of paintings colonized by Streptomyces
                                                                                                          in the investigated tombs [19,40].
                                                                                                              SEM micrographs showed the penetration of Streptomyces mycelium into the inter-
                                                                                                          granular structure of calcite crystals, at a considerable depth and forming a microbial
                                                                                                          network, mainly composed of polymeric substances. These polymers are able to ex-
                                                                                                          pand and shrink in relationship to wetting and drying cycles, and to the presence of
                                                                                                          sodium chloride (high water retention). This in agreement with the results of Suihko
                                                                                                          et al., [3] pointed out that the growth of Streptomyces colonizing the stone surfaces
Ali Sakr, Farouk Ghaly, Fouad Ali - The relationship between salts and growth of streptomyces colonies




                                                                                                          enhanced mechanical deterioration, caused by the penetration of biofilm within the
                                                                                                          pores, related to salt cycles, penetration that increases the width and depth of the
                                                                                                          pores [40]. Streptomyces sp. colonizing limestone discs from the Maat tomb at Tell
                                                                                                          Basta, increased the porosity of these discs up to 7%, after salt treatment (50 cycles)
                                                                                                          with 10% NaCl [16].
                                                                                                              IR spectra of the extracted red pigment, related to the presence of the quinon
                                                                                                          group (ON-O-R) that appeared at 3457 cm-1, allow us to attribute the red pigment
                                                                                                          produced by S. canarius under stress of 10% NaCl to carotenoid. Carotenoids (C40H50)
                                                                                                          are lipophilic pigments with a yellow-red color, including three molecules: ß-carotene,
                                                                                                          γ-carotene and rhodoxanthin. These pigments act as membrane stabilizers, protecting
                                                                                                          the microbial cells against adverse environmental conditions such as hypersalinity,
                                                                                                          irradiation and nutrient depletion [36], so this pigment is termed a protective pigment
                                                                                                          [26,27]. The problem imposed by carotenoid production by Streptomyces colonizing
                                                                                                          mural paintings causes chromatic alteration to these paintings, stone surfaces and
                                                                                                          plaster layers with irreversible stains, in particular if this pigment is exolithic in nature
                                                                                                          [48,50]. This phenomenon was well documented in the fresco paintings from the Cha-
                                                                                                          pel of St. Virgil, Vienna (Austria) since Rubrobacter sp. isolates showed a moderate
                                                                                                          halophilic behaviour to NaCl up to 6% and caused discoloration of the fresco paintings
                                                                                                          with rosy stains [18].
                                                                                                              Furthermore, HPLC data indicated that all identified Streptomyces isolates were
                                                                                                          able to produce glutamic acid at 5-10% NaCl and KCl concentrations, this in agree-
                                                                                                          ment with Killham and Firestone [51]. Amino acids production by Streptomyces is con-
                                                                                                          sidered a defense mechanism against hypersalinity. This was attributed to the prefer-
                                                                                                          ence of Streptomyces for amino acid groups including glutamic, proline and arginine
                                                                                                          acids for supporting their growth; the productivity of amino acids varied even if be-
                                                                                                          tween closely related strains [52].

                                                                                                             5. Conclusions

                                                                                                             In conclusion Streptomyces were found to be the main bacterial species on de-
                                                                                                          teriorated mural paintings in the tombs of Tanis and Tell Basta. These bacteria were
                                                                                                          halotolerant up to 10% NaCl, KCl and MgSO4. 7 H2O. Moreover, 10 % NaCl enhanced
                                                                                                          the production of red carotenoid pigment in S. canarius.
                                                                                                             Glutamic acid and other amino acid derivatives, produced by Streptomyces isolates
                                                                                                          as defense mechanisms against hypersalinity, were also found.




                                                                                                    326
      Acknowledgements

   Authors wish to thank Professor Gamal El Didamony, Botany Department, Faculty
of Science, Zagazig University for revising this paper and his helpful discussion.

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                                                                                                            Biographical notes

                                                                                                              Akmal Ali Sakr has a PhD in the conservation of mural paintings with a thesis
                                                                                                          on the role of Actinomycetes in chromatic alterations of paintings in Egyptian tombs.
                                                                                                          He now works in the Conservation Department at the National Museum of Egyptian
                                                                                                          Civilization,Cairo, Egypt. He has published some papers on the role of Streptomyces
                                                                                                          in the biodeterioration of cultural heritage and methods of treatment. He has a book in
                                                                                                          press entitled “The role of Actinomycetes in deterioration of cultural heritage objects”.
                                                                                                          He is involved in international projects: joint project with the Bulgarian Academy of Sci-
                                                                                                          ence on the Modification of polymers used in conservation of mural paintings by antibi-
                                                                                                          otics produced by Streptomyces; project financed by Zagazig University, “Using antibi-
                                                                                                          otics produced by Streptomyces in the biocontrol of microorganisms colonizing mural
                                                                                                          paintings”; an Egypt–Italy project, “Application of microbial enzymes and bactericides
                                                                                                          in the conservation of deteriorated mural paintings”; Egypt-France project, “Elucida-
                                                                                                          tion of the link between storage lipids and antibiotic production in Streptomyces” and
                                                                                                          another, “Using essential oils in combating microorganisms colonizing mural paintings
                                                                                                          in ancient Egyptian tombs” in the Faculty of Science, Zagazig University.

                                                                                                             Mohammed Farouk Ghaly has a PhD in Taxonomy of Actinomycetes and he is
                                                                                                          vice dean of the Faculty of Science, Zagazig University for environmental affairs and
                                                                                                          Professor of Microbiology in the Botany Department.

                                                                                                             Mona Fouad Ali has a PhD in conservation and is a professor of Conservation in
                                                                                                          the Conservation Department at the Faculty of Archaeology, Cairo University, Egypt.




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