Microbial Deterioration of Ancient South Indian Mural Paintings

A Study on Microbial Deterioration of Ancient South Indian Mural Paintings and
Fabrication of Biobased Natural Protective Coating for its Conservation

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2. REVIEW OF LITERATURE
2.1 Introduction
We are very well aware that there are thousands of wall paintings existing in
various part of India dating from the prehistoric times to the present date. Wall paintings of
Ajanta, Ellora and Bagh are world famous. Besides these well known examples, there are
thousands of other mural paintings which are extremely important but are less
known (Agrawal and Pathak, 2001).
Murals are paintings made on walls of caves and palaces while miniatures are
small-sized, colorful, intricate handmade illumination. Murals are considered to be the
earliest evidence of Indian paintings, which are unearthed as the symbol of ancient
civilization. The term mural is derived from the Latin word “murus” which means wall.
The paintings carried out on the walls are known as mural paintings (Mini, 2010). Mural
paintings reflect the feelings of Indian spirituality prismed through the age-old mythologies
and spirituality.
2.2 History of wall and mural painting
2.2.1 History of wall painting
The history of art of painting in India can be traced back to pre historic and proto
historic times. The earliest specimen was found in caves and rock shelters found admits
rocky hills. These paintings are dated from 1200 BC to the tenth century AD (Mathpal
1984; Misra et al., 1977). Artifacts found at Mohenjodaro and Harappa has traces of
natural pigments suggestive of the use of colors in pre-Aryan art. These cave paintings has
quite a wide distribution though all the paintings do not belong to one period. Rock shelter
paintings are usually seen on back walls arranged in horizontal lines and often with many
super impositions of successive styles. The earliest paintings go back to at least 550 BC.
Ajantan art, the tradition of which extends more than two millennia from the second
century BC to the present, represents the flowering of Indian mural paintings. History of
wall paintings in India is studied through its evolution from Ajanta to Kerala. Ajanta
paintings considered to have been executed between the 2nd century BC and 5th centaury
AD. Murals of 6th centaury AD was found at Badami (Karnataka) and Sittannavasal Jain
cave temple (Tamilnadu).Murals of Narthamala Vijayalaya Chozeswara belongs to
9th century AD and murals of Brihadeeswara temple, Tanjore, (Tamilnadu) belongs to
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Review of Literature
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11th century AD. Murals seen in Kerala are dated between 16th, 17th and 19th century AD,
(Sasibhooshan, 2006).
The most important sites of rock paintings in North India are at Bimbetka, Gwalior,
Tikla, Ayra, Bhanpura, Rampura, Singhauli, Bhojpur, Adamgrh, Delware, and Mirzapur.
In south India, they are found at Mehaboob Nagar (Andhrapradesh), Edakkal, Marayoor
and Perimkadavila (Kerala), Mallampadi (Tamilnadu) and Bellary (Karnataka).These
paintings were considered as the early models of mural paintings. The mural painting of
Sittannavasal, Badami, Lepakshi, Tanjore, Vijayanagar and Kerala has a lot of similarities.
Style, colour, theme and technique of mural painting varied with different dynasties, their
ruling period and important painting sites are given below.The important mural painting
site, Ajantha cave 9 &10 was made during the ruling period from 2nd BC to 2nd AD of
Satavahana dynasty. Vaishnava cave in Badami in the ruling period from 6th -8th AD
belongs to early western Chalukyas. During 7th-9th AD Pallava dynasty in Kailasanatha
temple, Kancheepuram was considered to be as one of the dominant mural painting site in
Southern India. Similarly, other South Indian dynasties, early Pandya between 7 th and
9th AD and early Chera from 8th to 9th AD highlighted their mural paintings at
Sittannavasal cave temple and Thirunandikkara cave temple respectively. Mural paintitngs
at Kailasanatha temple, Ellora from 8th - 10th AD was made by the dynasties of
Rashtrakuta. The familiar mural paintings at Brihadeeswara temple, Thanjavur and
Vijayalaya Choliswaram temple, Narthamalai was carried out by the chola dynasty
between the period of 9th and 13th AD. Another biggest dynasty called Vijayanagara
betweeen 14th and 17th AD made numerous mural paintings at Vidharbha temple,
Lepakshi, Varadaraja Perumal temple Kancheepuram, Vitthala temple, Hampi and Sri
Ranganatha tenple, Srirangam. Nayaka’s dynasty ruled inthe period from 17th to 18th AD
established the mural paintings in Kapardisvara temple, Tiruvanjuli and later murals of
Thanjavur and Kancheepuram. Finally, medivial Kerala (blend of Hoysala, Chalukya and
Vijayanagara paintings) in 16th and 18th AD carried out the mural paintings at Tripayar
temple, Ettumanur temple, Thiruvanchikulam temple, Panayanarkavu temple,
Mattancherry, Padmanabhapuram and Krishnapuram palace. (Sivaramamurthi, 1994)
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2.2.2 History of mural painting
The first paintings were Cave Paintings. Ancient peoples would decorate walls of
protected Caves with paint made from dirt or charcoal mixed with spit or animal fat. In
Cave paintings, the pigments (often carbon black or ochre) stick to the wall partially
because the pigment gets trapped in the porous wall, and partially because the binding
media (the spit or fat) dries, adhering the pigment to the wall. Over the years, countless
graves unearthed by archaeologists, exposed bodies covered in red pigment or chunks of
pigment buried alongside the bodies. Red, associated with blood, the most life sustaining
of body fluids, was the appropriate color to symbolize life's meaning and end. The word
Hematite (the source of many iron oxide pigments) is derived from the Greek word "hema"
meaning blood (Chakarabarti, 1980).
Wall paintings have been cultural expressions of human creation throughout

history, from the earliest beginnings, such as rock art, extending up to present day murals.
Throughout history, humans have used the most beautiful and durable stones for
monumental buildings. Beginning in the sixth century BC marbles extracted from the
Pentelikon quarry became a common building material. Abundant limestone and marble
quarries enabled the ancient Greek architects to build the Acropolis and Roman architects
to build the Forum (Saiz, 2002).
Figure-2.1: Cave painting
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Figure-2.2: Mural painting

The oldest known Maya murals are also the most recently discovered. Murals
found at the Preclassic site of San Bartolo, Guatemala, date to 100 BC. The church of San
Pietro and the “oratorio” of San Benedetto are believed by most scholars to date from the
third quarter of the 11th century. During the last decade of the same century that some
changes were made to the edifice of the church and it was decorated with mural paintings
and stucco reliefs, deep in the jungle of southeastern Mexico, archaeologists were surprised
that the murals are about 1,350 years old.
St. Agatha’s Complex was one of the preferred sites since it consists of the
hypogean environment embellished by a significant number of wall paintings, some dating
back to the fourth century AD. The Catacombs of St. Paul’s constitute a complex of
interconnected, underground Roman cemeteries that were in use up to the 4th century AD.
There are a few surviving murals and ochre inscriptions from late Roman and early
medieval periods. Decorated tablets and reliefs adorn the graves (Zammit et al., 2008). The
uncovering of paintings was made from the middle of the 19th century until the 1960s.
2.3 Indian murals
History of wall paintings in India is studied through its evolution from Ajanta to
Kerala. Ajanta paintings considered to have been executed between the 2nd century BC and
5th century AD. Murals of 6th century AD was found at Badami (Karnataka) and
Sittannavasal Jain cave temple (Tamilnadu). Murals of Narthamala Vijayalaya Chozeswara
belong to 9th century AD and murals of Brihadeeswara temple, Tanjore, (Tamil Nadu)
belong to 11th century AD. Murals seen in Kerala are dated between 16th, 17th and
19th century AD (Sivaramamurthy, 1994).
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India has one of the greatest traditions of painting of the ancient world. Murals and
miniature are the two different forms of arts, which intrinsically related to Indian painting
tradition. The earliest evidence of murals is the beautiful frescoes painted on the caves of
Ajanta and Ellora, also on the Bagh caves and Sittanvasal. The oldest mural paintings date
is about 3000 years BC (Sasibhooshan, 2006).
The number of wall paintings in each state of India is very large and they are
suffering from all types of deterioration and damage, but in comparison facilities for their
preservation are much too small. This is a very serious problem. The main objective of the
present study is to develop a protective bio-based gel (Rajagopalan, 2008).
2.4 North Indian murals
North India has a rich heritage of Mural paintings even before the Mughal period.
The murals at the Vishnu Temple located at Madanpur in Lalitpur district of Uttar Pradesh
of 12th century AD reveals the skillful hands of the painters. In Eastern India there are
many evidences of wall and panel paintings describing Buddhist and non- Buddhist
themes. In Arunachal Pradesh and Tripura sublime mural works have been found which
needs further study. Ladakh is known for its wall paintings in Alchi and Hemis
monasteries, made on 11th-12th century and the Spiti Vally in Himachal Pradesh is known
for its Buddhist paintings in the Gomphas of Tabo Monastery (Havell, 1982).
Ajanta is known to be the fountain head and inspiration of Buddhist paintings

across the whole of Asia. There are fragments of paintings, of the time of Ajanta, which
survive at many Buddhist cave sites, including Pitalkhora near Ellora, in Maharashtra. The
magnificent Kailashnath temple had been hewn out of a mountain at Ellora in the 8th
century.The Kashmiri style was mainly responsible for the lovely wall paintings still seen
in the beautiful monasteries at Alchi, Mangyu and Sumda in Ladakh, in the Tabo
monastery in the Spiti valley and in the Nako monastery in Kinnaur district, Himachal
Pradesh. On the western edge of the trans-Himalayan plateau in Spiti is the monastery
complex of Tabo. This appears to be one of the first among the 108 monasteries built by
Yeshe. It is dated around 996 AD. There are also paintings of the late 9th century in the
Jain caves at Ellora (Spink, 2009).
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Figure-2.3: Ajanta caves
Figure-2.4: Mural Painting of Ajanta caves
Ellora Caves, Maharashtra, belong to the highest achievements of mankind both

regarding the engineering skills and arts. Some 2000 years ago Pitalkhora Caves
(Maharashtra) were among the largest temple complexes in India. These 11 - 14 caves
were created in 250 BC - 4th century AD. In 4th century AD, temples were adorned with
beautiful murals; especially fine paintings have been preserved on columns of Cave 3.
Traces of murals are in Cave 6 as well. Already the oldest group of caves here - Buddhist
caves from 630 - 700 AD - contain paintings of Buddhist panteon. Remnants of paintings
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are seen in the most beautiful temple - Cave 16, Kailasanatha, circa 760 - 860 AD. Some
Jain temples - Cave 32 (Indra Sabha), Cave 33 - contain remnants of valuable murals from
800 - 1000 AD - this artwork marks the end of ancient period in the Indian art and
beginning of medieval art (Baskaran, 1975).
Figure-2.5: Ellora caves
There are surviving mural paintings from the 17th century onwards in Rajasthan.
The verdant Pahari hills saw the finest continuation of the tradition of murals in India. The
18th and 19th century paintings on the walls of the Rang Mahal in Chamba are among the
best surviving examples of Pahari murals. The 18th century paintings on the walls of the
Viranchinarayan Temple at Buguda are some of the finest surviving murals of that period
in India.
Figure-2.6: Paintings on the walls of the Rang Mahal in Chamba
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The most important sites of rock paintings in North India are at Bimbetka, Gwalior,
Tikla, Ayra, Bhanpura, Rampura, Singhauli, Bhojpur, Adamgrh, Delware, and Mirzapur.
In South India, they are found at Mehaboob Nagar (Andhrapradesh), Edakkal, Marayoor,
Perimkadavila (Kerala), Mallampadi (Tamilnadu) and Bellary (Karnataka).These
paintings were considered as the early models of mural paintings.The mural painting of
Sittannavasal, Badami, Lepakshi, Tanjore, Vijayanagar and Kerala has a lot of similarities
(Sasibhoosan, 1987).
2.5 South Indian murals
South India also got rich tradition of mural paintings. In the reign of Cholas,
Vijayanagaras and Nayakas, this art reached the climax. The Deccan art of Bijapur,
Hyderabad, and Golconda schools were influenced by the Mughal traditions and later by
European idiom. Maratha murals are also shaped under the Mughal traditions and
employed oil as medium. The Mural art of Kerala vividly depicted on the walls of temples
and monuments show the traces of European affinity. In Tamil Nadu almost all the extent
examples of frescos have been done on rock surfaces and stone walls. This is a crucial
factor in determining the quality of frescos (Havell, 1982). The ceiling of the Virupaksha
temple in Hampi is covered with paintings of the 15th century. The paintings on the ceiling
of the mandapa of Lepakshi temple are some of the finest mural paintings of the medieval
period in India. Legends associated with Siva and Parvati, Krishna and Rama were painted
on the walls of palaces and temples in Kerala from the 16th to 19th century. Mural
paintings were made on the walls of all the palaces within the magnificent Orccha fort. The
Raj Mahal was completely adorned with mural paintings of the 17th century.
Figure-2.7: Virupaksha temple in Hampi

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Figure-2.8: Ceilings of Virupaksha temple, Hampi
The murals on the walls of the navaranga (the central pillared hall) depict stories
from Bhagavatha and are excellent examples of 19th century Mysore paintings. The
Mallikarjunaswamy temple on the hill is one of the Panchalingas of Talakad and the
temple has a beautiful mural painting on the wall of the temple. The Dariya Daulat Bagh
has excellent mural painting (Sasibhoosan, 1987).
Figure-2.9: Paintings on the walls of Jagan mohan palace, Mysore
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Figure-2.10: Painting of Raja woodeyar era
Kerala mural paintings are the frescos depicting mythology and legends, which are
drawn on the walls of temples and churches in South India, principally in Kerala. Ancient
temples, churches and palaces in Kerala, South India, display an abounding tradition of
mural paintings mostly dating back between the 9th to 12th centuries. The murals of
Thirunadhikkara Cave Temple (now ceded to Tamilnadu) and Tiruvanchikulam are
considered the oldest relics of Kerala’s own style of murals. Fine mural paintings are
depicted in temples at Trikodithanam, Ettumanur, Vaikom, Pundarikapuram,
Udayanapuram, Triprangode, Guruvayoor, Kumaranalloor, Aymanam, the Vadakkunathan
temple in Trichur and the Sri Padmanabhaswamy temple at Thiruvananthapuram. Other
mural sites are in the churches at Edappally, Vechur, Cheppad and Mulanthuruthy, and at
palaces such as the Krishnapuram Palace near Kayamkulam and the Padmanabhapuram
Palace.
Figure-2.11: Kerala murals
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Figure-2.12: Murals paintings of Kerala temples

There are wall paintings in Badami in the Bijapur district, Karnataka. Some
excellent paintings exist in the Jain caves at Sitanavassal in Puddukkottai District of Tamil
Nadu. Tamil Nadu gave exuberant and glorious expression to themes relating to Siva in the
temples of Panamalai and Kailashanatar in Kancheepuram. In the 9th century, Jain cave of
Sittannavasal in Tamil Nadu, there is a marvellous lotus pond painted on the ceiling. . In
the heart of the Brihadeeswara temple in Thanjavur, Tamil Nadu, protected by massive
walls of stone, are the finest paintings of the theme of Siva ever painted, towards the end of
the 10th century (Sivaramamurthi, 1992)
Figure-2.13: Kancheepuram Mural paintings
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Figure-2.14: Sculpture of Mahabalipuram
2.6 Methods of mural painting
In the history of mural, several methods have been used: The Buon fresco technique
consists of painting in pigment mixed with water on a thin layer of wet, fresh, lime mortar
or plaster. The pigment is then absorbed by the wet plaster; after a number of hours, the
plaster dries and reacts with the air: it is this chemical reaction which fixes the pigment
particles in the plaster. After this the painting stays for a long time up to centuries in fresh
and brilliant colors. A Secco painting is done on dry plaster. The pigments thus require a
binding medium, such as egg (tempera), glue or oil to attach the pigment to the wall.
Mezzo-fresco, is painted on nearly-dry plaster, which is defined by the sixteenth-century
author Ignazio Pozzo as “firm enough not to take a thumb-print”, so that the pigment only
penetrates slightly into the plaster. Ancestral Hopi village archaeologists found that
fourteen kivas had painted murals and multiple layers of murals on the walls, some with up
to twenty-seven layers of murals (Saturno, 2006).
The colors used in painting are generally obtained from plants and minerals.
Among them only such colors which are lime resistant are suitable for mural painting. The
selection of proper colors for a mural painting is an important part of the work as the
longevity the painting depends on it. Only mineral colors are preserved for a long time
which could be the reason for use of those colors in early days. As per “Shilpa ratna” and
“Agnipurana” the colors used in mural painting are white, black, red, yellow, blue and
green, Yellow and red are from yellow ochre and red ochre. Blue is generally indigo or
china blue. White is the prepared wall and black is the carbon soot. Mural paintings have
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survived centuries only on account of the identification and use of those colors which does
not have any reaction with lime (Miraseth, 2006).
Originally, three types of brushes were made and used: flat, medium and fine. Flat
brushes were made from hairs behind calf’s ear, the medium from the bottom of goats
belly and the fine brush from delicate blades of grass. However in due course of time and
change, use of easily available brushes from commercial market and shading of the
original colours can be distinctively noticed. The use of blue colour is also noticed over the
passage of time. The paintings have been brought down from the walls to plywood, cloth,
paper and other frames for easy to make and use purposes (Chakarabarti, 1980).
2.7 Themes of mural painting
The subjects and themes in the murals are centered on the Hindu pantheon of Gods
and Goddesses drawn from description in the invocatory verses or Dhyana Slokas. More
common representations are of Vishnu in different incarnations, Ganesha, and
manifestations of Siva. We see murals in the Pallimanna Siva Temple devoted to Siva-
Parvati’s marriage, the Ramayana Story being narrated on the walls of the Mattancheri
palace in the Sankara Narayana, the Mahabharata is retold. Murals are various scenes from
the Hindu epics and also the frolics of Lord Krishna.
It would be interesting to bring into account the influence of the mural paintings on
Christianity. On various churches like the St. George’s Orthodox Church has Christian
murals bringing out biblical characters and narrating stories from the scriptures. Mar
Sabore and Afroth Church at Akapparambu in the outskirts of Ernakulam also have murals
of scenes from the bible. Interestingly St.Mary’s Church at Kanjoor has on both sides of its
entrance a battle scene between the armies of Tipu Sultan and of the English East India
Company. It is also apparent that the history of Kerala Murals has its origin in the temple
walls and thus a study of Kerala Murals is not complete without pondering on the
architecture of Kerala its association with the murals (Warrier, 2011).
2.8 Technique used in mural paintings
Indian murals executed on walls of caves, structural temples, walls of palaces were
based on highly developed techniques. Mural painting is a composite structure of these
three basic layers, the support, the ground and the pigment layer. In case of wall painting,
the wall itself is the support. This is followed by the layer or layers of plaster which
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constitute the ground. The pigment layer is the one which contains the picture. The support
is the wall which may be rock surface in a cave or a structural one. These stone surfaces
which serve as carriers are rough or uneven. This unevenness is itself an advantage as it
provides “tooth” to hold the plaster on the surface. The uneven surface of the wall has to be
made uniform with layers of plaster in successive gradation of fineness. There is generally
one layer of rough plaster followed by one or more layers of fine plaster. Ground or plaster
is a term used to mean the surface material of a wall or building which is applied as a
plastic mass made by mixing certain materials with water letting the mass set by drying,
carbonation or hydration. The active or setting components of plaster are commonly clay
which sets by drying, lime which sets by carbonation, gypsum and pozzolana which sets by
hydration (Mini, 2010).
There are different mixtures for rough plaster. In all the mixtures, clay, limestone,
lime and shells in powdered form serve as the matrix. The particles of which are held
together by the organic materials like gum, glue, extracts from barks, etc are in fact
adhesives. The fine plaster layer requires more careful preparation because it is the layer on
which the pigments are laid to make the picture. “Shilparatna” suggest a mixture of conch
Oyster shells or white clay with gum of neem or slaked lime and coconut water. Here the
consolidation is effected by the carbonation of slaked lime i.e. it absorbs carbon dioxide
from air and becomes carbonate. Mural paintings were lime-washed from the 17th until the
beginning of 19th century. A few paintings in vaults were never lime-washed, for example
at Taby and Floda, painted by the medieval painter Albertus Pictor (Mini, 2010).
All the paintings have certain basic common characteristics. There has to be a wall
or rock surface called support over which the artist goes to work. A plaster base called
ground of varied thickness is laid on the support. The ground may consist of one or more
layers. The strength and life of the whole mural depended up on this ground over which the
painting was done. It is the process of applying the final painting on the ground determines
the character of the mural, whether it is fresco or tempera. The mural painting is done
usually by creating wall surface free of any previous oil or paint by using solvent or by
sand blasting. The brick wall is first plastered with mixture of lime and sand in the ratio of
1:2. A mixture of egg yolk, vinegar, oil colour is used in dry plaster.
There are at least six stages before a Kerala Mural Painting is complete. Lekhya
Karma is the first stage where sketching by pencil or crayon is done. The second stage is
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the Rekha Karma which enhances the outlines of the sketching done. The third stage called
Varna Karma breathes life into the painting with its colours. Here all colours are
appropriately filled in. The colouring of the human figures is done according to the
characters and their common virtues. The divine and noble characters (Saatvik) are painted
in green, those inclined towards power and wealth (Raajasik) is painted in shades of red,
lowly mean characters (Tamasik) in white and demons in black. In the fourth stage or the
Vartana Karma, shading is done. Fifth stage called the Lekha Karma brings us to the final
outline of the painting which is usually in black. The final and the finishing touches stage
are called Devika Karma (Warrier, 2011).
2.9 Dyes used in mural painting
Indians have been considered as fore runners in the art of natural dyeing. Natural
dyes find use in the colouring of textiles, drugs, cosmetics, etc. Owing to their non-toxic
effects, they are also used for colouring various food products. In India, there are more
than 450 plants that can yield dyes. In addition to their dye-yielding characteristics, some
of these plants also possess medicinal value. Though there is a large plant resource base,
little has been exploited so far. Due to lack of availability of precise technical knowledge
on the extracting and dyeing technique, it has not commercially succeeded like the
synthetic dyes (Siva, 2007).
The color materials on the mural paintings in ancient India were derived from the
natural materials like terracotta, chalk, red ochre and yellow ochre mixed with animal fat.
The dyes for painting is prepared such as, white from lime, Black from carbon soot of
lamps, Red and yellow from minerals, Blue from indigo ferra, Deep red from lac. A fine
coating of resin is done after the painting in order to make a shining appearance. Egg yolk
and vinegar was mixed with oil colors and painted on dry plaster (Krishnakumar, 1999).
Although plants exhibit a wide range of colours, not all of these pigments can be
used as dyes. Some do not dissolve in water, some cannot be adsorbed onto fibres, whereas
others fade when washed or exposed to air or sunlight. It remains a mystery, why plants
reward us with vibrant dyes. India has a rich biodiversity and it is not only one of the
world’s twelve mega diversity countries, but also one of the eight major centres of origin
and diversification of domesticated taxa. It has approximately 490,000 plant species of
which about 17,500 are angiosperms; more than 400 are domesticated crop species and
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almost an equal number their wild relatives. Thus, India harbours a wealth of useful
germplasm resources and there is no doubt that the plant kingdom is a treasure-house of
diverse natural products. One such product from nature is the dye (Siva, 2007).
Many natural dyestuff and stains were obtained mainly from plants and dominated
as sources of natural dyes, producing different colours like red, yellow, blue, black, brown
and a combination of these. Almost all parts of the plants like root, bark, leaf, fruit, wood,
seed, flower, etc. produce dyes. It is interesting to note that over 2000 pigments are
synthesized by various parts of plants, of which only about 150 have been commercially
exploited. Nearly 450 taxa are known to yield dyes in India alone, of which 50 are
considered to be the most important; ten of these are from roots, four from barks, five from
leaves, seven from flowers, seven from fruits, three from seeds, eight from wood and three
from gums and resins. Some important dye-yielding plant habitats, their distribution and
colouring pigment (Siva, 2007).
The dye is generally prepared by boiling the crushed powder with water, but
sometimes it is left to steep in cold water. The solution then obtained is used generally to
dye coarse cotton fabrics. Alum is generally used as a mordant. Flowers of Butea
monosperma (Lam) Taubert yield an orange-coloured dye, which is not fast and is easily
washed away. For the purpose of colouring, the material is steeped in a hot or cold
decoction of the flowers. A more permanent colour is produced either by first preparing the
cloth with alum and wood ash, or by adding these substances to the dye-bath. The dye
indigo is produced by steeping the plant in water and allowing it to ferment. This is
followed by oxidation of the solution with air in a separate vessel. Mallotus philippinensis
muell yields an orange colour, used for dyeing silk and wool. To prepare the annatto dye
from B. orellena, the fruits are collected when nearly ripe. The seeds and pulp are removed
from the mature fruit and macerated with water. Thereafter, they are either ground up into
an ‘annatto paste’ or dried and marketed as annatto seeds. Sometimes when the seeds and
pulp are macerated with water, the product is stained through a sieve and the colouring
matter which settles out is collected and partially evaporated by heat and finally dried in
the sun (Sowbhagya, 2002).
The predominant colors used in rock art and cave art are black (from charcoal, soot,
or manganese oxide), yellow ochre (limonite), red ochre (hematite or baked limonite), and
white (kaolin clay, burnt shells, powdered gypsum, or powdered calcium carbonate). All of
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theses pigments may be used on leather or raw hide as well. Early cave art in Seminole
Canyon was monochromatic, in red, and as culture grew more sophisticated, the color
palette expanded. Blue, Orange, Brown, Green and Purple are the most common colours
used in Ajantha murals. White, Pink, Gold, are occasionally represented mineral earths
containing ferric oxide and copper carbonates were used as source of red, yellow and green
colours. There is also evidence to establish that root of Rubia cardifolia and leaves of
Indigofera tinctoria were used as source for red and blue colours (Nayer et al., 1999).
Many colors of pigments were obtained from both organic and inorganic sources in
ancient times, and used to paint various surfaces such as braintan leather clothing and
accoutrements, rawhide, the human body, etc. Blue color was obtained from powdered
azurite and lapis, duck manure, and certain clays. Fine Blue-green clay was obtained from
deposits near Mankato, Michigan. Organic sources of blue include Turkey's Tail, a
greenish blue polypore that grows on dead decaying wood (not tannish ones). Native
Americans get Indigo from either Guatemala or Ecuador, which was traded well up onto
Kansas, long before the Spanish ever set foot here. American Indigo has purple tones to it,
but Indian/Japanese indigo has green tones. Berries and crushed flowers were also used to
obtain blue color, by rubbing the plant material directly on the leather.
An organic source of red was Cochineal. It was native west of the Mississippi, and
is produced from the bodies of small insects that grow on the prickly pear cactus. Lady's
Bedstraw gives a good vermillion red color. Harvest the roots, dry them out, clean the mud
etc. off of them and then grind the roots really fine, then soak them to extract the red
coloring. The key here is drying the root first. Blood was also used as red pigment. Pucoon
roots also yield a red pigment (Smith, 2002). Red ochre the reverse original red colours are
due to variations in iron oxide (or) calcite content and to redox conditions in the firing
produced. Brown coloured ceramics owe their colour to maghemite and must be
considered as a fabrication defect due to the position of the ceramic piece in a zone where a
reducing atmosphere prevailed in the course of firing.Red pigments were produced from
iron oxides, like hematite or limonite, and from ochre. Hematite was the most frequently
used mineral. It occurs in the form of aggregates of small red crystals, or in the form of
bigger black crystals, which are difficult to crush; white was obtained mostly from
kaolinite or illite (Clottes et al., 1990).
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2.10 Biodetoriation of mural paintings
Historic items often contain natural, biodegradable materials and have histories of
exposure to moist conditions. These two conditions make them amenable to microbial
attack. Microbial colonization may damage the building or the cultural asset
(Salonen, 2001). Biodeteriogens are organisms involved in deterioration of artifacts. They
are very specific for each type of artifact in accordance with its chemical structure and
environment. They also have different nutritional requirements and act directly or
indirectly on the substrate. The adhesion of microorganisms to a substrate is the result of
cell hydrophobicity and of excreted polymeric substances contained by sheaths, capsules
and slimes (Colao, 2005).
The surviving wall paintings and decorations in subterranean environments around

Malta are rather scarce (Buhagiar, 1986, 1993, 1998). These include those at St. Paul’s
Catacombs (Zammit, 2008), St Agatha’s Crypt and Catacombs (Camilleri, 2001),
San Catald Catacombs, Tac-Cghaqi, Abbatija tad-Dejr (Buhagiar, 1993), all in Rabat, the
cave-sanctuary in Mellieħa (Buhagiar, 1987) and the Ħal Saflieni Hypogeum in Paola
(Pace, 2000). Previous studies conducted on wall paintings and decorations in Maltese
Palaeo-Christian Catacombs have involved their manufacturing techniques (Muscat, 2005).
However, to date, limited systematic studies have been conducted into the biodeterioration
in Maltese Palaeo- Christian Catacombs. The only such studies ever conducted in Malta
concerned the biodeterioration of works of art in catacombs have been carried out at St.
Agatha’s Crypt and Catacombs in Rabat (Zammit et al., 2008, Psaila, 2006).
Microorganisms can be responsible for the destruction of monuments, buildings

and cultural heritage assets, including the decay of concrete, cement, stones, mural
paintings, etc. These microorganisms are not only related with biodeterioration but water-
damaged indoor building materials are frequently colonized by complex bacterial
communities and may emit mixed bioaerosols into the indoor air, (Jimenez, 2000). It is
well known that mural paintings are colonized by micro-organisms and can be severely
damaged by such growths (Ciferri, 1999). A large number of fungal (>50) and bacterial
taxa (70) have been detected in indoor environments. Over 50 bacterial species and 14
genera novel to science were reported in the past 10 years from indoor air and historic
items, indicating bacterìa in these enviroments are as yet only fragmentarily known
(Salonen, 2001).
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Traditionally, the study of microflora which causes this biodeterioration, was based
on classical cultivation methods, that were mainly useful to study the physiological
potential of microorganisms but do not provide information on all microbial communities
to be found in excavated artifacts. Indeed, many studies using culture-independent
techniques, which cultivate microorganisms from different environments, may often
represent only minor components of the microbial community as a whole. It is generally
accepted that cultivation methods recover less than 1% of the total microorganisms present
in environmental samples (Ward et al., 1990; Amann et al., 1995). Hence microbial
investigations based only on cultivation strategies cannot be regarded as reliable in terms
of reflecting the microbial diversity present in objects of art (Osburn et al., 2011).
Early workers suggested that the presence of living microorganisms on stone

increased its susceptibility to damage through their water-binding capacity. It has recently
been reported that the mineralogy, porosity, surface roughness and capacity to collect
water and organic materials control its bioreceptivity and tendency to biodeterioration
(Diakumaku, 1995). In addition, environmental factors as temperature, light, pH and
relative humidity affect the number and type of colonising species (Tiano et al., 1995) and
hence the progress of colonisation. Biofilms on decayed stones consist on mixed microbial
populations embedded in a polymeric matrix. Fungi, algae, filamentous and unicellular
bacteria are found in these complex communities. A variety of bacteria, have been isolated
from different stone samples, confirming the dominance of Gram-positive organisms,
especially spore-forming bacteria such as Bacillus and filamentous actinomycetes
(Ripka, 2005).
In cultivation studies of deteriorated mural and rock paintings, bacterial isolates

have frequently been attributed to the genus Bacillus (Weirich, 1989; Tate and Rebrikova,
1991; Gonzalez et al., 1999; Heyrman et al., 1999; Jimenez et al., 2000). Other studies that
have included DNA-based techniques to characterize the bacterial community involved in
biodeterioration have shown that a substantial part of the bacterial population is made up of
so far unknown species (Altenburger et al., 1996; Rolleke et al., 1996; Gurtner et al., 2000;
Heyrman and Swings, 2001), (Heymann et al., 2003).
Many potential mycotoxin producers have been detected in air and items from
indoor environments with histories of exposure to moisture, for example species of
Acremonium, Alternaria, Aspergillus, Chateomium, Fusarium, Eurotium, Paecilomyces,
24
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Penicillium, Scopulariopsis, Stachybotrys, Trichoderma, Verticillium and Wallemia

(Peltola et al., 2001; Pieckova et al., 2001). Not all strains of one species produce
mycotoxins, and different strains may produce different toxins. Whereas toxin producing
strains are frequent in crops and outdoor vegetation, toxin producing isolates from indoor
environments and non living assets have only been reported of the species Aspergillus
versicolor (Nielsen et al., 1998), Stachybotrys chartarum, Nikulin, (1997) and
Trichoderma harzianum (Peltola et al., 2001). Streptomyces griseus strains producing
valinomycin occur in environments where humans can get exposed
(Andersson et al., 2002). The biological properties and toxicity mechanisms of cereulide
were identical to those by valinomycin (Mikkola et al., 1999; Salonen et al., 2001).
The genus Paenibacillus was first proposed by Ash et al., (1993) and its description
was emended by Shida et al., (1997). The genus Paenibacillus is under intensive taxonomic
investigation, with ten species having been formally described in the last year. Heyrman &
Swings (2001) isolated a strain, originally classified as Paenibacillus sp. LMG 19508,
from biodeteriorated mural paintings in the Servilia tomb at the Roman necropolis of
Carmona in Seville, Spain (Smerda et al., 2006). Building stone and wall paintings contain
a variety of hydroscopic salts, including carbonates, chlorides, nitrates, sulfates, etc,
dispersed within the porous material or locally concentrated. These salts are solubilised
and migrate with the water in and out of the stone. Due to changes in physical parameters,
the so called salt efflorescences appear on the surfaces providing extremely saline
environments and building niches for settlement of halophilic microorganisms. Halite, and
other hygroscopic salts, can retain considerable quantities of moisture when entrapped in
the building materials and thus favouring the bacterial growth (Ripka, 2005).
It is demonstrated that water-damaged indoor building materials contained

substances of microbial origin extremely toxic to eukaryotic cells and large quantities of
several microbial species known to be potentially toxic or immunopotentiating. These
include peptidoglycans, Gram-negative endotoxin, β-1,3-D glucans, lipoglycans and
teichoic acids and mycotoxins (Jimenez, 2000). In turn these microorganisms can mediate
the crystallization process. These crystallizations can produce destructive effects. Some
salts may crystallize or recrystallize to different hydrates, which occupy a larger space and
exert additional pressure producing cracking, powdering and flaking
(Jimenez and Laiz, 2000).
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2.11 Types of deterioration on mural paintings
The frescos represent a variety of ecological niches where primary colonization is

due to photoautotrophic and chemolithoautotrophic microorganisms, followed by
secondary colonization due to heterotrophic bacteria that obtain nutrients from various
sources (metabolites of autotrophic bacteria, airborne organic contamination, animal faeces
and organic compounds in the paint layers themselves). Microbial-induced deterioration
processes cause structural as well as aesthetic damage to wall paintings such as the
discoloration of materials, the formation of crusts on surfaces and the loss of material
(Sarro et al., 2006, Osburn et al., 2011). Halophilic bacteria and archaea were thought to
be found only in extreme environments like salt lakes or black smokers. However, it has
been shown that mural paintings and stones are a common habitat for extremely salt
tolerant and moderate halophilic (Ripka, 2005). Microbial induced deterioration processes
can cause different kinds of alterations such as discolouration of materials, the formation of
crusts on surfaces and the loss of material, which leads to structural damage (Ripka, 2005).
First of all, microorganisms can cause severe problems due to the excretion of aggressive
metabolic products such as organic or inorganic acids. Secondly, material components can
be used as substrates for microbial metabolism. Exoenzymes such as cellulases, proteases,
tannases, beta-glucosidase produced by fungi and bacteria are responsible for hydrolysis of
cellulose and proteins. Further examples are mechanical processes caused by swelling and
shrinking of slime or mycelia penetrating inside plaster of the painting resulting in loss of
cohesion and detachment of the paint layer (Rolleke, 1996; Gurtner et al., 2000).
Table-2.1: Microbial damage on mural paintings (Rolleke, 1996)

S. No. Damage Microbial activity
Green, brown, red, grey Pigments produced by microorganisms (intra-or
1
surface layers (patinas) extracellular) as chlorophyll and carotinoids
Use of organic binders as nutrient and energy
2 Dislodging of painting layers
source
3 Acid attack of carbonates Acid produced by fungi or bacteria
Redox-reactions induced by fungi and
4 Discolouration of pigments
bacteria
5 Loss of paint layer Growth of microorganisms deep into the plaster
Hydrolysis of synthetic
6 Chemoorganotrophic activity of bacteria and fungi
polymers
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Discolorations are caused by excretion of pigments and microbiologically induced

reduction/oxidation-processes (Krumbein et al., 1990). Degradation of the bindings is
followed by reduction of stability. Oxidation of ammonium and reduced sulphur
compounds results in acidification of the environment such as Sulphuric acid and the
variety of organic acids produced by the heterotrophs cause solubilization of calcareous
binding material and chelation of metal ions (Bock et al., 1993). It is now well recognised
that wall paintings can be deteriorated by microorganisms (Ciferri, 1999).The growth of
microorganisms essentially results in two types of damage: aesthetic and structural.
Aesthetic damage implies pigment discoloration, stains, and formation of a biofilm on the
painted surface. Structural damage is observed as cracking and disintegration of the paint
layer, formation of paint blisters, and degradation of support polymers or of glues and
binders resulting in detachment of paint layer from the support. The colonization of mural
paintings by bacteria and other microorganisms can have different causes. Provided that
favorable environmental conditions occur, nutrients for heterotrophic bacteria and fungi
can be available from metabolites of autotrophic bacteria, from airborne contamination and
dripping water, from animal faeces, but also from organic compounds in the paint layer
itself.
2.12 Methods of studying biodeterioration
It is now well recognized that wall paintings may be deteriorated by

microorganisms, as established by several studies that linked the observed damage with the
microbial community present, as reviewed by Ciferri (1999). Despite this knowledge, a
reliable characterization of bacteria associated with such damage is often lacking. To
obtain greater insight into the bacterial community involved in biodeterioration of mural
paintings, three sites (from the necropolis at Carmona, Spain, and the Saint-Catherine
chapel of the castle at Herberstein, Austria) were sampled. In previous studies from these
sites, 385 isolates were analysed by fatty acid methyl ester GC and representatives of
different fatty acid clusters were further studied by 16S rRNA gene sequencing
(Heyrman and Swings, 2001). Several of these representative strains showed the highest
sequence similarity with members of the genus Arthrobacter (Heyrman et al., 2005).
Jimenez and Samson (1981) analysed the microbial flora of a large fresco painted
in the late 1920s in the monastery “Santa Maria de la Rabida” (Huelva, Spain) exhibiting
two types of deterioration: white efflorescence and green-to-black stains.
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Tate and Rebrikova (1990) thought autotrophic, nitrifying bacteria to be the first colonisers
of damaged frescoes and building materials in the cathedral of the nativity of the Virgin in
the Pafnutii-Borovskii monastery (Russia). The bulk of research on biodeterioration of
mural paintings has focused on explaining the observed phenomena by analysing the
microflora present. Following this approach, a precise characterisation of the
microorganisms using a polyphasic approach was never the first goal.
Only a few studies included genomic fingerprinting (16S rDNA sequence analysis)
for the characterisation of bacteria associated with damage on mural paintings
[Rolleke, (1996); Pinar et al., (2001)]. Previous studies based on culture-dependent
techniques showed that the flora present on frescos are fungi (predominant species of
Penicillium, Aspergillus, Cladosporium and Engyodontium) (Guglielminetti et al., 1994),
bacteria (predominant species of Bacillus, Arthrobacter, Micrococcus, Streptomyces and
Pseudomonas) (Heyrman et al., 1999) cyanobacteria and eukaryotic algae (species of
Nostoc, Lyngbya and Chlorophyceae) (Arino et al., 1996; Calvo et al., 1993). The killing
effect of an antimicrobial agent on a microorganism can be assessed in several ways:
(1) Lethal activity may be expressed as the rate of killing by a fixed concentration
of drug under controlled conditions. This rate is determined by measuring the number of
viable bacteria at various time intervals. The resulting graphic depiction is known as the
time-kill curve. Bacterial killing rates are, in part, dependent on the class of antibiotic and
the concentration of this agent. With certain classes of antibiotics (e.g., aminoglycosides
and fluoroquino-lones), the rate of killing increases with increased drug concentrations up
to a point of maximum effect. This is termed concentration-dependent bactericidal activity.
In contrast, the killing rates of ß-lactam agents and vancomycin are relatively slow and
continue only as long as the concentrations are in excess of the MIC. This rate of killing is
termed time-dependent bactericidal activity.
(2) The minimal concentration of drug needed to kill most of the viable organisms
after incubation for a fixed length of time (generally 24 hours) under a given set of
conditions is the most common estimation of bactericidal activity and is known as either
the minimal bactericidal concentration (MBC) or the minimal lethal concentration (MLC).
It will be referred to in this document as the MBC. Unfortunately, the definition of the
MBC (99.9% killing of the final inoculum) is somewhat arbitrary and separates the
bacteria into two populations – a segrega-tion which might not have biological relevance.
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The determination of the MBC, moreover, is so subject to methodologic variables that the
clinical relevance of MBCs is nearly impossible to assess, particularly for certain pathogen
and drug combinations (e.g., Staphylococci and beta-lactam agents).
(3) The serum of a patient receiving an antibiotic may be tested against the
infecting microorganism. This can be done using time-kill curve methodology (i.e., serum
bactericidal rate) or using dilution methodology (i.e., serum bactericidal titer). The
principles of these methods as well as the influence of biological and technical factors are
similar (Barry et al.,, 1999).
2.13 Microbial flora associated with deteriorated mural paintings
A great deal of research has been done in this field mainly by using cultivation dependent
methods. However, by using culture-dependent methods, only a very small proportion of
microorganisms can be obtained. Far less than 1% of organisms in the environment can be
cultivated by standard cultivation techniques (Pace, 1996). Recent studies have shown that
culture-dependent techniques yielded mainly sporeforming bacteria suggesting that plating
leads to an over estimation of the number of spore-forming bacteria. A wide number of
investigations have shown that in addition to chemical and physical processes,
microorganisms play a major role in degradation of objects of art, such as paintings, stone-
works, wood, paper, masonry, leather, parchment, glass and metal (Ripka, 2005).
A) Bacteria
In general growth of diverse organisms on mural paintings is supported by

humidity, slight alkaline pH values and the presence of organic and inorganic nutrient
sources (Weirich, 1989). In mural paintings, pigments are suspended in water or oil, often
binders such as casein and milk are used, and they are supplied on the damp lime plaster
(Ciferri, 1999). The calcium carbonate formed on contact with air consolidates the
pigments. Thus frescos contain mainly inorganic components. While chemoautotrophic
nitrifiers and thiobacilli obtain their energy for growth by oxidizing ammonium and
reducing sulphur-compounds respectively, heterotrophic bacteria and fungi depend on
organically bound carbon [Tate et al., (1991); Lazar, (1971); Petushkova et al., (1986);
Jimenez et al., (1981)]. Thus primary colonization of photoautotrophic Cyanobacteria,
chemolithoautotrophic nitrifying bacteria and sulphuric acid producing thiobacilli represent
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the first supply of organic matter. The death and lysis of such bacteria promotes the
secondary settlement of heterotrophic bacteria and fungal populations (Calvo et al., 1991).
Bacteria may derive from plants or pollen as many sequences obtained in this study
gave high score similarities with bacteria isolated from plants, like Moraxella osloensis,
Enterobacter cloace, Enterobacter agglomerans and Pantoea endophytica. Bacteria may
also derive from animal faeces like flies, Bacillus, Pseudomonas, Aeromonas and Panteoa
(Ripka, 2005). As Lazar and Dumitru (1973) reported studying mural paintings in a
Romanian monastery, the genera Bacillus, Arthrobacter and Pseudomonas have been
proved to produce salt efflorescences and were even considered to cause the blanching
phenomenon of frescoes (Jimenez et al., 2000). Predominant bacterial genera are Bacillus,
Streptomyces, Arthrobacter, Micrococcus and Pseudomonas [Weirich, (1989);
Tate et al., (1990); Arroyo et al., (1997); Gonzalez et al., (1999); Groth et al., (1999);
Jimenez et al., (2000)].
B) Fungi
It may be concluded that there is wide variety of fungal forms present on mural
paintings but Aspergillus and Alternaria were found dominating from all the experimental
sites, next to them were Cladosporium, Drechestera, Fusarium, Curvularia, Chaetomium,
Memnoniella, Mycelia sterelai, Pencillium, Emericella, Stachybotrytis, Trichoderma,
Phoma, Monodictys, Humicola and Epicoccum (Garg et al., 1995).
The fungal genera most commonly found in lists of isolated taxa given in literature
are Alternaria, Aspergillus, Chaetomium, Cladosporium, Fusarium, Penicillium and
Verticullum [Weirich, (1989); Tate et al., (1990); Guglielminetti et al., (1994);
Garg et al., (1995); Berner et al., (1997); Ciferri, (1999)] Some fungi having hard fruiting
bodies represented by sclerotia, pycnidia, perithecia, cleistothecia and stroma, could
organize themselves inside the painted layer. Many fungal forms having such structures
were identified from our experimental sites viz., Emericella nidulans, A. flavus,
Chaetomium sp. and Phoma sp. etc. These structures with little increase in humidity may
cause swelling in the paint film (Garg, 1995).
Strains of species of Alternaria and Cladosporium, both of them identified on the

mural paintings of the Palace of Schönbrunn, are the most common genera isolated from
stone and mural paintings in indoor environments with standard cultivation methods
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(Ventosa, 2004). Saprophytic species of the genus Cladosporium are among the most
abundant fungi isolated in saltern samples. Commonly isolated Cyanobacteria belong to
Nostoc and eukaryotic algae to Lyngbya and members of the Chlorophyceae such as
Chlorella, Pseudococcomyxa and Pseudopleurococcus [Gorbushina et al., (1987);
Calvo et al., (1993); Arino et al., (1996)].
They grow optimally on media with low water activity due to high salt or sugar
concentrations, seem to propagate 70 vegetatively in the salterns, and are thus
halotolerant/osmotolerant species rather than simple flush-through occurrences.
Cladiosporum herbarum was frequently isolated from hypersaline waters, dead and dying
plant material, air, soil, food, fruit, cereals, paint and textiles (Ventosa, 2004).
C) Algae
Most of the painted areas in temperature climate became covered with green algae
(Pleurococcus, Protococcus and Trentopholia), while under tropical conditions blue green
algae of the genera Oscillatoria and Sytonema predominated (Garg, 1995). The main
factors which contribute to the growth of algae on wall paintings are the presence of
moisture in the plaster as well as availability of visible light. These conditions are quite
prevalent in the tropical climate but in temperature regions also algal growth on wall
paintings have been reported [Tonolo et al., (1961); Pietrini et al., (1991)].
D) Actinomycetes
Usually, literature refers to specific aspects, e.g., the occurrence of cyanobacteria

and algae in caves with natural openings to sunlight or those with artificial lighting, fungi
and so forth. Actinomycetes have been considered to be mainly confined to soils, where
they find their natural habitat, but despite intensive studies there are still many gaps in our
knowledge of the role played by actinomycetes in soil processes and biodegrations of
murals cave paintings (Groth et al., 1999). In addition, halophilic bacteria, actinomycetes;
and cyanobacteria are also capable of growing on salt-affected mural paintings
(Karbowska, 2003).
Monte and Ferrari (1993) reviewed the mechanisms of biodeterioration in tombs

and underground churches. The role of actinomycetes in the deterioration of frescoes in
hypogean environments was stressed by Agarossi et al., (1985), Monte and Ferrari (1993).
Giacobini et al., (1987) described the serious damage suffered by a whole series of famous
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frescoes in underground environments. Actinomycetes were isolated from these mural

paintings located in crypts, caves, and tombs. About 200 strains of actinomycetes were
isolated, 60 of which were identified. The most frequent species were Streptomyces albus,
S. cinereoruber, S. griseolus, S. vinaceus, and some members of the Nocardia genus.
Heterotrophic bacteria and actinomycetes were the most abundant microorganisms

on the frescoes of Etruscan tombs (Agarossi et al., 1985). Bettini et al., (1977) studied
some of the oldest Etruscan tombs (7th to 4th century BC) and found white-grey patinas
and spots from streptomycetes in the mural paintings. They related the biodeterioration to
the microclimatic conditions (e.g., lower temperatures, high RH). Monte and Ferrari (1993)
considered that colonization of the mural paintings was the result of selective, competitive
and inhibitory dynamics among different microorganisms under different and changing
environmental conditions. The pigments excreted by some actinomycetes were responsible
for the violet stains found on the walls of tombs in Tarquinia. Similar stains were observed
in one of the Roman tombs, the Circular Mausoleum, the Necropolis of Carmona, Spain,
from which a Streptomyces sp. producing violet pigments in culture media was isolated.
2.14 Identification of biodeteriogens
The possibility of pigments and biodeteriogens identification in frescos and stone

monitoring is offered mostly by vibrational spectroscopy, infrared thermography, laser
induced fluorescence (LIF), laser induced breakdown spectroscopy (LIBS), XRF (X-Ray
Fluorescence), IR and UV emission. Among them, laser based methods, allowing for in
situ or remote characterization of artwork surface, were applied successfully for
prospecting , diagnosis and maintenance (Colao et al., 2005). The rep-PCR fingerprinting
of the isolates allowed determination of their genotypic diversity. Studies that have
compared rep-PCR genomic fingerprint analysis with DNA–DNA relatedness studies have
shown that the two techniques yield results that are in close agreement [Nick et al., (1999);
Rademaker et al., (2000)]. These studies therefore suggest that rep-PCR fingerprinting can
be used as a genomic screening method to differentiate at the species level and to select
representatives for DNA–DNA reassociation experiments (Heymann et al., 2003).
The characterisation of bacteria was for example often limited to isolation from
group-specific media followed by microscopy [Sorlini et al., (1987); Weirich, (1989);
Tate et al., (1990) and in more recent studies to morphological and physiological
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characteristics including miniaturised and automated systems such as API and BIOLOG
[Arroyo et al., (1997); Jimmenez et al., (2000) and Laiz et al., (2000)], or to
chemotaxonomic characteristics [Gonzalez et al., (1999); Groth et al., (1999);
Jimenez et al., (2000)].
Wagner et al., (1994) demonstrated that conventional microbiological efforts can

fail to isolate all microorganisms present in natural samples. DNA-based techniques used
to identify microorganisms have revealed only a smaller fraction of bacteria isolated from
artworks (Ward, et al., 1990; Rolleke et al., 1996). Bacterial identification using molecular
techniques, especially those including the sequencing of genes coding for ribosomal
16S rRNA, is very important to study bacterial communities found on artefacts. One such
method is Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified gene
fragments coding for rRNA (Muyzer et al., 1993) that allows the separation of partial
16S rDNA amplified fragments of identical length but different sequence due to their
different melting behaviour in a gel system containing a gradient of denaturants
(Jimenez, 2003).
Total genomic DNA was purified for 16S rRNA gene sequencing and rep-PCR
using a slight modification of the method of Pitcher et al., (1989), as described by
Heyndrickx et al., (1996). rep-PCR genomic fingerprinting was performed with the REP-
and (GTG)5 primers (Versalovic et al., 1994) using the PCR conditions described
previously by Rademaker et al., (1997). Electrophoresis and pattern analysis were further
performed as described by Heyrman et al., (2003). 16S rRNA gene sequencing and
phylogenetic analysis were performed as described by Heyrman and Swings (2001).
Phylogenetic trees were constructed using the BioNumerics software (Applied Maths) by
applying the neighbour-joining and maximum-parsimony methods on a multiple alignment
similarity matrix. The stability of relationships was assessed by a bootstrap analysis of
1000 datasets. For determination of the G+C content and levels of DNA–DNA
hybridization, approximately 1 g biomass was harvested from agar plates. DNA was
purified by using a combination of the protocols of Marmur (1961) and
Pitcher et al., (1989), as described by Logan et al., (2000). The G+C content of the DNA
was determined by HPLC (Mesbah et al., 1989) using the further specifications given by
Logan et al., (2000). DNA–DNA hybridization experiments were performed using a
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modification of the microplate method of Ezaki et al., (1989), as described by

Willems et al., (2001).
2.15 Preservation and conservation
Nowadays, the conservation and preservation of our cultural heritage is one of the
world main concerns, particularly in Europe. The increasing need for non-destructive
investigation tools has become a major issue, as sampling is in most cases restricted in
view of the value or the uniqueness of the object. Artworks, as wall paintings in buildings
and monuments of historical interest, are affected by environment physical (temperature
and humidity) and biological factors (biodeteriogens), and by human (pollutant releases)
impact and therefore modified over the time. The term “conservation” may have many
different meanings in different fields. Every intervention directed to the restoration and
safeguard of works of art is commonly considered as a conservation treatment. For
example, conservation can involve the cleaning procedures set up by chemists, or the photo
documentation of the preservation of historical memory. For the appropriate conservation
of works of art, even the lighting systems in a museum or the control of the visitor’s
movements in a site should be monitored. Chemical degradation, which induces the flaking
of paintings and deterioration from rain, wind, dust, and other environmental causes, is
mainly responsible for the weakening of the porous structure and the surface layers of the
materials used in works of cultural heritage (Colao et al., 2005).
2.16 Conservation history
Wall paintings are an integral part of monuments and sites and should be preserved
in situ. Many of the problems affecting wall paintings are linked to the poor condition of
the building or structure, its improper use, lack of maintenance, frequent repairs and
alterations. Also frequent restorations, unnecessary uncovering, and use of inappropriate
methods and materials can result in irreparable damage. Substandard and inadequate
practices and professional qualifications have led to unfortunate results. It is for this reason
that an appropriate document covering the principles of proper conservation-restoration of
wall paintings is necessary.
The Venice Charter (1964) has provided general principles for the conservation-
restoration of cultural heritage. The Amsterdam Declaration (1975) introducing the
concept of integrated conservation, and the Nara Document on Authenticity (1994) dealing
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with cultural diversity, have expanded these principles. Taking into account these and
additional relevant contributions, such as the ICOM-CC Code of Ethics (1984), Document
of Pavia (1997), and E.C.C.O. Professional Guidelines (1997), the aim of this document is
to provide more specific principles for the protection, preservation and the conservation
restoration of wall paintings. This document, therefore, reflects basic and universally
applicable principles and practices, and does not take into account particular problems of
regions or countries, which can be supplemented at regional and national level by
providing further recommendations where necessary.
2.17 Preservation techniques of the mural paintings
The removal of fat, salts, varnishes, and pollutants from the surfaces of the works
of art are mostly done by chemical and mechanical methods. At the end of the 19th
century, the practices of restoration methods that do not inhibit further future interventions
was considered acceptable. During the 1960s several chemicals such as polymeric resins
were synthesized and applied as a consolidation or protection agents. The accepted idea
was that these substances could be removed at any time, leaving a completely unaltered
substrate. On the basis of origin, archaeological objects can be classified as follows
(Carretti, 2005)
1. Inorganic: It includes siliceous and nonsiliceous materials. Archaeological materials

produced from stones, terracotta, ceramics and glass can be included under siliceous
materials whereas objects made of metals can be included under nonsiliceous inorganic
materials. Again metal objects can be classified as ferrous metals and non-ferrous metals.
Objects made of iron are termed as ferrous metal objects whereas metal objects made from
other than iron is termed as non-ferrous metal objects.
2. Organic: It includes archival and anthropological materials. Again archival materials

include paper, bhojapatra, tadapatra, textiles, wooden objects, miniature paintings whereas
anthropological objects include bone and ivory, leather, feather and fur etc.
In the past, restoration of paintings and other old artwork often involved application
of acrylic resins to consolidate and protect them. One of the most important tasks for
modern restorers is thus to remove these layers, because it turns out that acrylic resins not
only by drastically changing the optics of the treated artwork, but in many cases they
accelerate their degradation. The scientists use an oil-in-water microemulsion with the
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organic solvent para-xylene as the oil component. The matrix makes the cleaning agent
viscous, so that it cannot enter very far into the pores of a painting (Carretti, 2005).
Nanoparticle treatment is the logical evolution of the Ferroni-Dini method.

Dispersions of kinetically stable Ca (OH)2 nanoparticles in non-aqueous solvents solved
most of the drawbacks of the microsized powders. Stable dispersions of calcium hydroxide
have been successfully applied (replacing polymers) as fixatives to re-adhere lifted paint
layers during many restoration workshops in Italy and in Europe, as a consolidant. The
Ferroni–Dini method (two steps: the application of a saturated solution of ammonium
carbonate, (NH4)2CO3, and the treatment with a barium hydroxide solution, Ba(OH)2, also
called the 'barium' method, has long been the acknowledged method for the removal of
salts that threaten paintings, reinforcing at the same time the porous structure. However,
commercially available carbonates and hydroxide powders have dimensions of several
micrometres, much larger than the pores on the paint surface. This means they don’t
penetrate the painting well and there is also a risk of damaging the artwork by a white
glaze forming on the surface (Berger, 2006). Restoration should provide the reinforcement
of the porous structure and the consolidation of the surface layer of arte facts. A few
simple principles can be considered to define the most appropriate restoration method:
1) The treatment should be reversible so that one can revert to the original status of the
work of art at any desired time;
2) All the applied chemicals must ensure the maximum durability and the chemical
inertness;
3) The applied chemicals must invert the degradation processes without altering the
chemical composition of the artefacts and their physico-chemical and mechanical
properties (Baglioni, 2006)
2.18 Commercial preservative used for preservation of mural paintings
Cleaning was done using soft brushes / vacuum cleaner and Gumma paine (dough)
a natural latex sponge. Areas near the window were very dirty with black soot and were
cleaned with micro fibrous cloths moistened with synperonic N (surfactant; alkyl phenol
ethoxylate, based on nonyl phenol) and neutralized with clean water. In several places the
paint layers had separated from the substrate (exfoli-ation). In that cases loose paint flakes
were stuck back using Paraloid B72 (10% solution in ethanol) as sparingly as possible.
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Paraloid was injected with syringe behind the exfoliated area twice, pushed and held back
with Melinex (polyester film) until fixed on the third application (Bionda et al., 2002).
In a period of significant growth of molds and intensive efforts to stop the mold
contamination, some of the lines of certain paintings were obscured or faded. Solutions of
ethanol: formalin, 9:1, and Thiabendazole (TBZ) were used, but they were not very
effective. Para-formaldehyde fumigation was adopted beginning in 1981; to establish an
emergency treatment, sensitivity of microorganisms from studied samples was tested
against Desogen by adding 1-3 % (v / v) in serial dilutions. The best concentration
3% (v/v) was applied with a brush to prevent spreading of the spores. In order to overcome
the problem of fungal attack suitable fungicides, carbendizeme or kiroxy-I or 5% solute of
N formaldehyde can be used. Shellac solution in spirit or 2% solution of polyvinaycletate
in toluene can be used as preservative and consolidants.To control insect and fungal attack,
fumigants like pyrethrum, DDT or 5% solution of sodium penta chlorophenate in kerosene
oil, 2 to 5% solution of PVA in toluene or 2 to 5% solution of paraloid B-72 in toluene or
acetone be used (Miura, 2009). Microclimatic conditions constitute a critical factor in the
conservation of cave paintings. The unstable microclimatic condition is one of the factors
that always affect the state of conservation of mural paintings. In the environmental-
monument system knowledge of the functional environment and climatic conditions of the
surroundings are necessary for the identification of causes of decay (Singh, 2011).
Furthermore, the risk of formation of indoor bioaerosols in residential and public

buildings (offices, schools, museums, etc) has attracted interest in the last years due to
socio-economic as well as sanitary reasons (Ripka, 2005). Thus it is important in aspects of
health and maintenance of Cultural Heritage to analyse the complete microflora to further
develop biocides for harmful microorganisms and methods for restoration of damaged
objects of art. The biocidal testing was carried out on one representative algal species of
the two classes of algae, viz. Chlorococcum sp. (green) and Chroococcus sp. (blue-green),
found to be common in all the samples. Efficiency tests were carried out both in liquid as
well as solid media containing algal suspensions by adding a biocide solution of known
concentration and determining the growth of the microorganism after the incubation
period. The biocides used for the past studies were:
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1) Benzalkonium chlorides (M/S Loba-chime Indo-Australian Co., Bombay)

2) 2-Chloro-4, ethyl amino-6-isopropylamino-s-triazine, commercially known as
Atrazine (M/S Rallis India Ltd., Bombay).
3) chloro-4, 6-bis (ethylamino)-s-triazine commercially known as Simazine (M/S
Bharat Pulverising Mills Ltd. Bombay)
4) [3-(3,4-dichlorophenyl)-1,1-dimethyl urea], commercially known as Diuron (M/S
Bharat Pulverising Mills Ltd., Bombay)
Traditional conservators confront a challenging set of issues that must be resolved

prior to the restoration of any community mural. In some cases, the special nature of
community murals creates conflicts with conservators’ normal practices, so it is useful to
begin by considering briefly the differences between community murals and other forms of
painting. To do that, it is necessary to better understand the nature of community murals.
The biofilm was removed where possible with a low concentration of hydrogen peroxide
solution; then the area was treated with about 70% isopropyl alcohol. The conservation
techniques in Italy are barium hydroxide starts a reaction which converts the calcium
carbonate (lime) into calcium hydroxide (slaked lime) which means a new carbonation
process will take place and create adhesion to the wall. The ammonium and water
evaporates. The crystals of barium sulfate act as filler in the pores of the mural and can
also be washed away (Garreau, 2007).
Another choice for protecting the mural is to use some of the various graffiti-
resistant finishes that are commercially available. These range from protective wax
coatings that are removed with hot water to the 2-component solvent-based polyurethane
coatings. Breath ability is important to the successful adhesion of acrylic products. If a
mural encompasses an entire wall made of a masonry product, it is advisable to apply thin
coats of MSA Varnish. This will allow interior condensation and evaporating solvents and
out gassings to escape. A mixture of shellac and alcohol in ratio of 50:50 is also used.
Readhesion of the paint layer was done by application of acrylic resin emulsion (primal
AC 33.5%).the emulsion was applied by brush along the border with water and alcohol
(Agrawal and Pathak, 2001).
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2.19 Conservation agents
Indian Association for Study of Conservation of Cultural Property, IASC was

founded in the year 1966 as a spin off of the UNESCO's Regional Seminar at New Delhi.
The Association provided a National forum for the conservators practicing in isolation to
come together, and share their experience among the peers and also to seek answers to
their problems and keep the professionals informed of the difficulties in carrying out their
work. The forum took the responsibility of information dissemination about the new
developments in the area of conservation. Formation of such a specialized professional
Association gave a big boost to the conservation research activity in the country
(Singh, 1998)
The Archaeological Survey of India (ASI), under the Ministry of Culture, is the
premier organization for the archaeological researches and protection of the cultural
heritage of India. Maintenance of ancient monuments and archaeological sites and remains
of national importance is the prime concern of the ASI. For the maintenance of ancient
monuments and archaeological sites and remains of national importance the entire country
is divided into 24 Circles. The Archaeological Survey of India’s Science Branch is
responsible mainly for the chemical conservation treatment and preservation of some three
thousand five hundred ninety three protected monuments besides chemical preservation of
museum and excavated objects countrywide.
Nanotechnology has recently found practical applications in the Conservation and

Restoration of the World’s Cultural Heritage (CAR-WHC). Baglioni's group was among
the first to synthesize nanoparticles in non-aqueous solvents with the optimal properties for
application to cultural heritage conservation. Nanoparticles of calcium and magnesium
hydroxide and carbonate have been used to restore and protect wall paints, such as Maya
paintings in Mexico or 15th century Italian masterpieces (Berger, 2006).
A few years back the INTACH Indian Council of Conservation Institutes (ICCI)
conducted a survey of wall paintings in various state of India and found that a large
number of wall paintings exist in temples, palaces, havelies, churches, forts and so on. The
number of wall paintings in each state is very large but the researchers could locate and
examine only a few of them. From some wall-paintings, they collected samples of plasters
and paints and analyzed them. They could get funding for conservation of some of them
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and they were conserved by their teams (Agrawal, 2007). A state-wise break-up of their
survey work is as given below table.
Table-2.2: Status of Indian mural paintings (Agrawal, 2007)
No of murals sites Status of sites

S.No Name of the State
surveyed conserved
1 Bihar 8 Nil
2 Himachal Pradesh 4 2
3 Jammu & Kashmir 17 Nil
4 Tamil Nadu 3 2
5 Madhya Pradesh 36 8
6 Maharashtra 1 1
7 Kerala 61 3
8 Rajasthan 72 18
9 Haryana 1 1
10 Punjab 2 Nil
11 Karnataka 18 4
12 Sikkim 1 Nil
13 Uttar Pradesh 41 4
14 Uttarakhand 11 1
15 Orissa 79 5
2.20 Recent scenario of preservation
The scanning lidar fluorosensor experimental apparatus was designed by keeping in

mind that very often artworks and especially frescos on tombs are placed in small
chambers with a narrow entrance, where only a limited space is available. In order to
facilitate field operations the optical and mechanical supports have been assembled on a
small size optical bench contained in a box for easy transportation. The compact LIF
system is designed to be coupled with another laser scanning device used for
characterization of surfaces: the amplitude modulated LFR, developed in the same
laboratory for 3D model reconstruction of object and environments (e.g. whole chambers)
40
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.The mechanical mountings are then conceived to offer a rapid and stable hookup to the
LRF apparatus in the case of simultaneous operation (Giovagnoli, 2008)
2.21 Data recording
In order to facilitate the input and retrieval of data from the text fields, a guide for
the compilation of the data record card was drawn up. For each entry the guide explains the
type of data considered essential, the technical vocabulary to adopt and cites a series of
illustrative examples showing the type of syntax and punctuation required. The program
used was File Maker Pro 7.1 produced by the Claris Corporation. The data record card is
divided up into six sections:
1. Reference data. This contains basic information relating to the work in question
(geographical location, description of object/site, measurements, author, period,
etc.). The section ends with a paragraph on the methods of inspecting the painting
(visual, tactile, examinable support), which is of fundamental importance for
assessing the integrity and completeness of the survey (Giovagnoli, 2008).
2. Documentation. This section contains the necessary documentary references

relating to the work of a graphic, photographic or written (bibliographical or
archival) nature.
3. Position/display characteristics. The information contained in this section is brief

and acquired through simple visual examination as it will be presented in greater
detail on the environmental data record card.
4. Technical data and conservation condition. This is the most technical and analytical
section of the sheet, in that it presents all the visually deducible data on the
constituent materials, execution techniques and conservation condition of the
painting. The section is divided into three parts presenting data that refers to the
individual layers or elements ideally comprising the work: support structure,
preparatory layers and paint layers. The collection of data for each individual
section is carried out in the same order. First, the constituent materials and
execution techniques are examined, and then the alterations linked to factors of
deterioration of a physico-chemical and biological nature, and lastly the restoration
treatments carried out in the past. An “Other” entry is always included to provide
for those less common aspects not specified in the other fields. As far as the
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indicated factors of alteration are concerned, an assessment of the extent of the

damage is recorded and a calculation, expressed in percentage, of its distribution in
relation to the overall surface (Giovagnoli, 2008).
5. Indication of treatment programmes. Based on the previously recorded

documentary, technical and conservation elements, this section proposes the
treatment programmes that need to be carried out, indicating the type and degree of
urgency. Five different time related categories were specified for this purpose:
• Emergency treatment:
• Short-term treatment;
• Medium-term treatment;
• Long-term treatment;
• Maintenance and inspection.
2.22 Conclusion
From the above supporting articles the present study was designed in such a way to
develop a natural preservative gel for the conservation of the mural painting. The present
study focuses on development of bio-based natural preservative coating for mural painting
and monuments. The microbial flora of air from selected archeological sites was studied.
The dye pigment from the plant sources and mineral sources used in traditional mural
painting were extracted and their antimicrobial property was studied. The physico –
chemical characteristics of the extracted dyes were studied using SEM-EDAX, FTIR and
GC-MS analysis. Various combinations of these dyes and their antibacterial property were
studied. Field trials for assessing the antimicrobial durability of developed natural
preservative were studied by coating it on mural paintings at Institute of Mural Paintings,
Guruvayoor, Kerala.
42

Microbial Deterioration of Ancient South Indian Mural Paintings

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A Study on Microbial Deterioration of Ancient South Indian Mural Paintings and

Fabrication of Biobased Natural Protective Coating for its Conservation

2.2 History of wall and mural painting

2.2.1 History of wall painting

2.2.2 History of mural painting

Wall paintings have been cultural expressions of human creation throughout

Figure-2.1: Cave painting

Figure-2.2: Mural painting

2.3 Indian murals

2.4 North Indian murals

Ajanta is known to be the fountain head and inspiration of Buddhist paintings

Figure-2.3: Ajanta caves

Figure-2.4: Mural Painting of Ajanta caves

Ellora Caves, Maharashtra, belong to the highest achievements of mankind both

Figure-2.5: Ellora caves

Figure-2.6: Paintings on the walls of the Rang Mahal in Chamba

2.5 South Indian murals

Figure-2.7: Virupaksha temple in Hampi

Figure-2.8: Ceilings of Virupaksha temple, Hampi

Figure-2.9: Paintings on the walls of Jagan mohan palace, Mysore

Figure-2.10: Painting of Raja woodeyar era

Figure-2.11: Kerala murals

Figure-2.12: Murals paintings of Kerala temples

Figure-2.13: Kancheepuram Mural paintings

Figure-2.14: Sculpture of Mahabalipuram

2.6 Methods of mural painting

2.7 Themes of mural painting

2.8 Technique used in mural paintings

2.9 Dyes used in mural painting

2.10 Biodetoriation of mural paintings

The surviving wall paintings and decorations in subterranean environments around

Microorganisms can be responsible for the destruction of monuments, buildings

Early workers suggested that the presence of living microorganisms on stone

In cultivation studies of deteriorated mural and rock paintings, bacterial isolates

Penicillium, Scopulariopsis, Stachybotrys, Trichoderma, Verticillium and Wallemia

It is demonstrated that water-damaged indoor building materials contained

2.11 Types of deterioration on mural paintings

The frescos represent a variety of ecological niches where primary colonization is

Table-2.1: Microbial damage on mural paintings (Rolleke, 1996)

Discolorations are caused by excretion of pigments and microbiologically induced

2.12 Methods of studying biodeterioration

It is now well recognized that wall paintings may be deteriorated by

2.13 Microbial flora associated with deteriorated mural paintings

In general growth of diverse organisms on mural paintings is supported by

Strains of species of Alternaria and Cladosporium, both of them identified on the

Usually, literature refers to specific aspects, e.g., the occurrence of cyanobacteria

Monte and Ferrari (1993) reviewed the mechanisms of biodeterioration in tombs

frescoes in underground environments. Actinomycetes were isolated from these mural

Heterotrophic bacteria and actinomycetes were the most abundant microorganisms

2.14 Identification of biodeteriogens

The possibility of pigments and biodeteriogens identification in frescos and stone

Wagner et al., (1994) demonstrated that conventional microbiological efforts can

modification of the microplate method of Ezaki et al., (1989), as described by

2.15 Preservation and conservation

2.16 Conservation history

2.17 Preservation techniques of the mural paintings

1. Inorganic: It includes siliceous and nonsiliceous materials. Archaeological materials

2. Organic: It includes archival and anthropological materials. Again archival materials

Nanoparticle treatment is the logical evolution of the Ferroni-Dini method.

2.18 Commercial preservative used for preservation of mural paintings

Furthermore, the risk of formation of indoor bioaerosols in residential and public

1) Benzalkonium chlorides (M/S Loba-chime Indo-Australian Co., Bombay)

Traditional conservators confront a challenging set of issues that must be resolved