A Chronology of 

Middle Missouri Plains 

Village Sites

By Craig M. Johnson

with contributions by

Stanley A. Ahler, Herbert Haas, and Georges Bonani

Smithsonian Institution

Scholarly Press

Smithsonian Institution

Scholarly Press

smithsonian contributions to museum conservation • number 2

Biocolonization of Stone:

Control and Preventive

Methods

Proceedings from the MCI Workshop Series

Edited by

A. Elena Charola, 

Christopher McNamara, 

and Robert J. Koestler
Series Publications of the Smithsonian Institution

Emphasis upon publication as a means of “diffusing knowledge” was 

expressed by the first Secretary of the Smithsonian. In his formal plan 

for the Institution, Joseph Henry outlined a program that included 

the following statement: “It is proposed to publish a series of reports, 

giving an account of the new discoveries in science, and of the changes 

made from year to year in all branches of knowledge.” This theme of 

basic research has been adhered to through the years by thousands of 

titles issued in series publications under the Smithsonian imprint, commencing with Smithsonian Contributions to Knowledge in 1848 and 

continuing with the following active series: 

Smithsonian Contributions to Anthropology

Smithsonian Contributions to Botany

Smithsonian Contributions to History and Technology

Smithsonian Contributions to the Marine Sciences

Smithsonian Contributions to Museum Conservation 

Smithsonian Contributions to Paleobiology

Smithsonian Contributions to Zoology

In these series, the Institution publishes small papers and full-scale 

monographs that report on the research and collections of its various 

museums and bureaus. The Smithsonian Contributions Series are 

distributed via mailing lists to libraries, universities, and similar institutions throughout the world. 

Manuscripts submitted for series publication are received by the 

Smithsonian Institution Scholarly Press from authors with direct affiliation with the various Smithsonian museums or bureaus and are subject 

to peer review and review for compliance with manuscript preparation 

guidelines. General requirements for manuscript preparation are on the 

inside back cover of printed volumes. For detailed submissions requirements and to review the “Manuscript Preparation and Style Guide for 

Authors,” visit the Submissions page at www.scholarlypress.si.edu.
smithsonian contributions to museum conservation • number 2

Biocolonization of Stone: 

Control and Preventive 

Methods

Proceedings from the MCI Workshop Series

Edited by

A. Elena Charola, 

Christopher McNamara, 

and Robert J. Koestler

washington d.c.

2011
ABSTRACT

Charola, A. Elena, Christopher McNamara, and Robert J. Koestler, editors. Biocolonization of Stone: Control and Preventative Methods, Proceedings from the MCI Workshop Series. Smithsonian Contributions to Museum Conservation, 

number 2, 116 pages, 87 figures, 5 tables, 2011. — The Smithsonian Museum Conservation Institute Workshop on Biocolonization of Stone was the second workshop in a series and was dedicated to research on removal and control of biocolonization in stone objects. Twelve presentations were made, and the workshop ended with a roundtable discussion open to 

the 71 attendees. The goal was to provide a discussion forum for biologists, material scientists, and conservators interested 

in stone biodeterioration. Seven papers were presented, ranging from microbiological laboratory studies to combination 

of on-site testing and laboratory evaluation for World Heritage Sites such as Angkor Wat, to a literature overview. Five 

case studies were also presented, covering control of biodeterioration at Veterans Affairs cemeteries, experience gathered 

from the installation of zinc strips at the Stanford Mausoleum in San Francisco, the red staining found on the marble of 

the Memorial Amphitheater at Arlington National Cemetery, problems posed by deer stones in Mongolia, and the site test 

installed at San Ignacio Miní Jesuit mission in Misiones, Argentina. The roundtable and discussions drew attention to the 

importance of exploring new methods to prevent microbial colonization of stone. Finally, in a closed session, suggestions 

were offered for developing criteria to evaluate microbial growth and determine when treatment is necessary. It was recommended that a database be prepared on stone biocolonization and its control.

Cover images, from left to right: Details of Figure 5b by Warscheid and Leisen and Figure 1b by Delgado et al.; Figure 8 

by DePriest and Beaubein. 

Published by 

SMITHSONIAN INSTITUTION SCHOLARLY PRESS

P.O. Box 37012

MRC 957

Washington, D.C. 20013-7012

www.scholarlypress.si.edu

Library of Congress Cataloging-in-Publication Data

Smithsonian Museum Conservation Institute Workshop on Biocolonization of Stone: Control and Preventive Methods 

(Washington, D.C. : 2009)

Biocolonization of stone : control and preventive methods : proceedings from the MCI workshop series / edited by 

A. Elena Charola, Christopher McNamara, and Robert J. Koestler.

 p. cm. — (Smithsonian contributions to museum conservation, ISSN 1949-2359 ; no. 2)

Includes bibliographical references.

1. Museum conservation methods—Congresses. 2. Stone—Biodegradation—Congresses. 3. Microbiologically 

influenced corrosion—Congresses. 4. Cleaning—Environmental aspects—Congresses. 5. Museums—Collection management—Congresses. I. Charola, A. Elena. II. McNamara, Christopher J. III. Koestler, Robert J. (Robert John), 1950– 

IV. Museum Conservation Institute. V. Title.

AM145.S648 2009

069'.53—dc22

2010052916

ISSN (print): 1949-2359

ISSN (online): 1949-2367

∞ The paper used in this publication meets the minimum requirements of the American National Standard for 

Permanence of Paper for Printed Library Materials Z39.48–1992.
Contents

Preface vii

Acknowledgments viii

Microbiological Studies on Stone Deterioration and Development 

of Conservation Measures at Angkor Wat 1

Thomas Warscheid, LBW Bioconsult, Wiefelstede, Germany

Hans Leisen, Cologne University of Applied Sciences, Cologne, 

Germany

Microbial Community Diversity and the Complexity of Preserving 

Cultural Heritage 19

M. Carmen Portillo, Instituto de Recursos Naturales y 

Agrobiologia, Seville, Spain

Juan M. Gonzalez, Instituto de Recursos Naturales y 

Agrobiologia, Seville, Spain

Characterization of Bacterial Colonization of Stone at Global 

and Local Scales 29

Christopher McNamara, Harvard School of Public Health, 

Boston, Massachusetts

Nick R. Konkol, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Brandon P. Ross, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Ralph Mitchell, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Methods to Prevent Biocolonization and Recolonization: 

An Overview of Current Research for Architectural and 

Archaeological Heritage 37

Ornella Salvadori, Soprintendenza Speciale per il Polo Museale 

Veneziano, Venice, Italy

A. Elena Charola, Smithsonian Museum Conservation Institute, 

Suitland, Maryland
i v • smithsonian contributions to museum conservation

New Environmentally Friendly Approaches against Biodeterioration 

of Outdoor Cultural Heritage 51

Francesca Cappitelli, Dipartimento di Scienze e Tecnologie 

Alimentari e Microbiologiche, Università degli Studi di Milano, 

Milan, Italy

Federica Villa, Dipartimento di Scienze e Tecnologie Alimentari e 

Microbiologiche, Università degli Studi di Milano, Milan, Italy

Claudia Sorlini, Dipartimento di Scienze e Tecnologie Alimentari e 

Microbiologiche, Università degli Studi di Milano, Milan, Italy

Bioremediation of Algal Contamination on Stone 59

Eric May, School of Biological Sciences, University of Portsmouth, 

Portsmouth, UK

Dania Zamarreño, School of Biological Sciences, University of 

Portsmouth, Portsmouth, UK

Sarah Hotchkiss, Cybercolloids, Carrigaline, Ireland

Julian Mitchell, School of Biological Sciences, University of 

Portsmouth, Portsmouth, UK

Robert Inkpen, Department of Geography, University of 

Portsmouth, Portsmouth, UK

Recolonization of Marble Sculptures in a Garden Environment 71

José Delgado Rodrigues, Laboratório Nacional de Engenharia 

Civil, Lisbon, Portugal

Marta Vale Anjos, Laboratório Nacional de Engenharia Civil, 

Lisbon, Portugal

A. Elena Charola, World Monuments Fund-Portugal, Lisbon, 

Portugal

Case Study: Red Staining on Marble 87

Claire Gervais, Smithsonian Museum Conservation Institute, 

Suitland, Maryland

Carol Grissom, Smithsonian Museum Conservation Institute, 

Suitland, Maryland

Christopher McNamara, Harvard School of Public Health, 

Boston, Massachusetts

Nick R. Konkol, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Ralph Mitchell, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Case Study: Biocontrol Testing at the San Ignacio Miní JesuitGuaraní Mission, Misiones, Argentina 91

Marcelo L. Magadán, Magadán y Asociados, Buenos Aires, 

Argentina

Gisela M. A. Korth, Magadán y Asociados, Buenos Aires, 

Argentina

Marcela L. Cedrola, Granado-Campinas, São Paulo, Brazil

A. Elena Charola, Buenos Aires, Argentina

José L. Pozzobon, Programa Misiones Jesuíticas de la Provincia 

de Misiones, Misiones, Argentina 
number 2 • v

Case Study: Comparative Study of Commercially Available Cleaners 

for Use on Marble Veterans Affairs Headstones 99

Jason Church, National Center for Preservation Technology 

and Training, Natchitoches, Louisiana

Mary Striegel, National Center for Preservation Technology 

and Training, Natchitoches, Louisiana

Christopher McNamara, Harvard School of Public Health, 

Boston, Massachusetts

Kristen Bearce Lee, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Ralph Mitchell, Harvard School of Engineering and Applied 

Sciences, Cambridge, Massachusetts

Case Study: Deer Stones of Mongolia after Three Millennia 103

Paula T. DePriest, Smithsonian Museum Conservation Institute, 

Suitland, Maryland 

Harriet F. Beaubien, Smithsonian Museum Conservation Institute, 

Suitland, Maryland

Case Study: Field Observations on the Effectiveness of Zinc Strips 

to Control Biocolonization of Stone 109

David P. Wessel, Architectural Resources Group, San Francisco, 

California

Discussions, Conclusions, and Recommendations 113

A. Elena Charola, Museum Conservation Institute, Suitland, 

Maryland

Christopher McNamara, Harvard School of Public Health, Boston, 

Massachusetts

Robert J. Koestler, Museum Conservation Institute, Suitland, 

Maryland

Preface

This volume brings together the papers that were presented at the 

Smithsonian Museum Conservation Institute (MCI) Workshop “Biocolonization of Stone: Control and Preventive Methods” that was 

held at the MCI on 20–22 April 2009. Seven papers and five case 

studies were presented. The papers dealt with such diverse topics as microbiological laboratory studies; combination of on-site testing and laboratory evaluation for World Heritage Sites, such as Angkor Wat; and a literature overview. 

The five case studies presented practical experiences from work with a diverse 

range of subjects: Veterans Affairs cemeteries, the Stanford Mausoleum in San 

Francisco, the Memorial Amphitheater at Arlington National Cemetery, the 

deer stones in Mongolia, and the San Ignacio Miní Jesuit mission in Misiones, 

Argentina.

An objective of the workshop was to obtain answers to the following three 

questions:

1. What should be the criteria for determining when microbial colonization 

and growth on stone heritage materials is problematic?

2. Are there new methods with minimum environmental impact that will prevent microbial deterioration and colonization and control recolonization of 

stone after a conservation intervention?

3. What future research directions can help achieve these goals?

The answers to these questions were discussed during the meeting and are 

summarized in the conclusions included in this volume.

A. Elena Charola Robert J. Koestler

Research Associate Director

Museum Conservation Institute Museum Conservation Institute

Smithsonian Institution Smithsonian Institution
viii • smithsonian contributions to museum conservation

Acknowledgments

This publication would not have been possible without the enthusiasm and support provided by Dr. Paula T. 

DePriest. Many others contributed indirectly through 

their support during the organization of the workshop. 

Among them we thank MCI’s Beverley Smith, Francine 

Lewis, Vernetta Williams, and, in particular, Ann N’Gadi.
Thomas Warscheid, LBW Bioconsult, Schwarzer 

Weg 27, D-26215 Wiefelstede, Germany. Hans 

Leisen, Cologne University of Applied Sciences, 

Ubierring 40, D-50678 Cologne, Germany. Correspondence: T. Warscheid, lbw.bb@gmx.de.

Microbiological Studies on Stone 

Deterioration and Development of 

Conservation Measures at Angkor Wat

Thomas Warscheid and Hans Leisen

ABSTRACT. Microbiological studies at Angkor Wat and surrounding temples in the 

Angkor site have shown that the natural microflora on the stones is composed of a complex and stable microbial community of algae, bacteria, fungi, and lichens. The microbial 

biofilms are mainly located in the uppermost layers of rocks, but certain microbes can 

penetrate deeper into the stone profile. The metabolic activity of the microflora, i.e., 

respiration and photosynthesis, is at a very high level, especially during the rainy season, and results in potentially biocorrosive and bio-oxidative activities. Nevertheless, in 

mature biofilms, such as those resulting from lichen colonization, the biodeteriorating 

activities reach a balanced state. This equilibrium should not be interfered with unless 

a thoroughly considered conservation program can be developed. This is fundamental 

since the microbial biofilm regulates the moisture and thermal absorption of the stones at 

the Angkor site. Cleaning and biocidal treatments should be applied only where needed 

for stone consolidation treatments or in order to achieve better visibility and readability 

of details in the historic structure. Organic- and chloride-containing biocides should be 

avoided because of their toxicity, short-term effectiveness, and possible nutritive value for 

microflora. Any conservation activity needs to be complemented by constructive water 

management. The long-term effect of conservation treatment has to be monitored during 

ongoing maintenance of the monuments and temples at the Angkor site.

INTRODUCTION: BIODETERIORATION PROCESSES

The importance of microbial impacts in the alteration and deterioration of 

porous inorganic materials used in cultural artifacts has been widely acknowledged in the course of many recent investigations (May et al., 1993; Warscheid 

and Braams, 2000). In the past, biodeterioration problems on cultural artifacts 

were not thoroughly analyzed before being subjected to biocidal treatments. 

However, a deeper interdisciplinary understanding of the environmental factors and material properties regulating the biogenic damage will permit a morepractical control strategy. The contamination, growth, and metabolic activity of 

individual or complex microorganism communities, such as algae, bacteria, cyanobacteria, fungi, and lichens, will influence the complex interaction between 

the various types of materials present leading to their physical as well as chemical damage (Koestler et al., 1994).

Biofouling can be defined as the presence of colloidal microbial biofilms on 

or inside materials. In the course of its development by the microflora, it leads to 
2 • smithsonian contributions to museum conservation

an unaesthetic discoloration by biogenic pigments, such as 

green chlorophyll, brownish melanin, or red carotenoids, 

and to the alteration of physicochemical characteristics of 

the materials with regard to their mechanical properties, 

superficial absorbency/hydrophobicity, diffusivity, and 

thermohygric behavior (Warscheid, 1996). Subsequently, 

the microbial consortia may cause a biocorrosive attack 

leading to the alteration of the structure and stability of 

materials by (1) the phototrophic enrichment of organic 

biomass, (2) the selective cellular enrichment and redox 

processes of cations and anions, such as iron, manganese, 

and nitrates, (3) the excretion of immediate corrosive metabolic products, such as organic and inorganic acids, and 

(4) the enzymatic mineralization of the respective organic 

materials (Warscheid and Krumbein, 1996).

It is important that during the survey and documentation process the environmental conditions that are favorable for allowing the development of microbial infection, 

contamination, and biodeterioration processes are recorded. This record allows the basic parameters for effective countermeasure strategies to be established. Microbial 

contamination on and in materials is basically determined 

by the availability of water (provided by rainwater, rising 

damp, and condensation) and depends on the sorption isotherms of the material. Two parameters are critical: water 

activity, aw, the ratio of the vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure 

at the same temperature of pure water, and time of wetness 

(TOW). Fungal growth is enabled at aw > 0.6 and TOW > 

0.5, e.g., more than 12 h during the day, and optimal conditions for fungal growth are aw ~ 0.75 (Adan, 1994). Other 

microorganisms, such as algae or bacteria, usually need 

higher moisture levels (aw > 0.9), but when a biofilm is present, the microorganisms may survive under less-favorable 

moisture conditions because of the ability of the biofilm to 

retain moisture (Flemming and Schaule, 1994).

Overall material characteristics, such as surface roughness, absorbency/hydrophobicity, porosity, and inner surface, determine the adhesion, colonization, and spreading 

of the microorganisms on and within the material (Warscheid et al., 1993). In addition, the chemical composition 

of the material may support the microbial succession by 

providing inorganic and organic nutrients. Further decomposable nutrient sources may be offered by exposure 

to light, leading to the enrichment of the photosynthetic 

biomass, as well as by the deposition of natural and anthropogenic aerosols, such as ammonia, nitrate, or combustion of biogenic hydrocarbons (Warscheid et al., 1991, 

1993; Saiz-Jimenez, 1995; Mitchell and Ji-Dong, 2000). 

When evaluating the nutrition conditions for particular 

microbial consortia, it is important to consider that microorganisms settling on material surfaces are able to survive 

or even grow under oligotrophic conditions, i.e., with low 

concentrations of nutrients (May et al., 1993). The contamination process will even be extended when buffering 

capacities for biogenic metabolic compounds with acidic 

properties are in the material since the optimum pH for 

most of the microorganisms studied on cultural artifacts 

falls around the neutral point.

The optimal temperature for most of the microorganisms involved in the biodeterioration of cultural artifacts 

ranges between 16°C and 35°C. The rate of oxygen supply 

will not exclude microbial activity, but it will determine 

the type of the metabolic pathway, whether oxidative or 

fermentative. Finally, the possible pathways of contamination, e.g., airborne transmission, have to be considered as 

potential causes of biodeterioration processes on historical 

objects.

Control of biodeterioration processes on materials can 

be achieved basically by measures limiting the aforementioned growth conditions for microflora. Therefore, “good 

housekeeping” and climate control are the basic means of 

reducing the biocolonization problem. Furthermore, the 

application of appropriate, especially microbial-resistant, 

protective products is preferable to the application of ecotoxic, questionable, and unhealthy biocides (Warscheid, 

1999, 2003).

THE ANGKOR SITE: THE GERMAN 

APSARA CONSERVATION PROJECT

The temple complex of Angkor, built between the 

ninth and fourteenth centuries, is located near the town 

of Siem Reap, close to the Tonle Sap Lake in the center of 

Cambodia. The region lies in a tropical climate with alternating dry and rainy seasons. The temple complex constitutes the largest religious monument in the world, with 

more than 100 temples over an area of some 400 km2. The 

largest temple of the Angkor complex is Angkor Wat (Figure 1). The temples are decorated with bas-reliefs depicting 

mythological scenes and Apsaras, celestial nymphs; these 

bas-reliefs are badly deteriorating through flaking and scaling of the sandstone. Figure 2 shows a detail of one of the 

reliefs and the losses that have occurred over time.

In order to prevent total loss of this important heritage, it was necessary to develop an effective conservation strategy. This in turn required analysis of the causes 

for stone deterioration and an evaluation of the microbial impact within this process. In 1995, studies for the 
number 2 • 3

FIGURE 1. View of the Angkor Wat temple (a) around 2004 and (b) in 2009. The color variation is mostly the result 

of different lighting and of the damp stone in the later photograph. Note that incipient biological recolonization is 

becoming evident.
4 • smithsonian contributions to museum conservation

FIGURE 2. Detail of one of the Apsara reliefs showing increased loss over time, induced by scaling and flaking of the sandstone: (a) 

1959, (b) 1995, and (c) 1996.
number 2 • 5

conservation of the Apsara reliefs began at Angkor Wat. 

This initiative, the German Apsara Conservation Project 

(GACP), was undertaken by the Restoration and Conservation Department of the Cologne University of Applied 

Sciences (Leisen et al., 2000, 2008). 

PRELIMINARY STUDIES

On the basis of the experiences and results of former 

and recent microbiological studies by French and Japanese 

scientists (Pochon et al., 1960; Hyvert, 1968; Fusey, 1991; 

Japanese Government Team for Safeguarding Angkor, 

1995, 1996a, 1996b, 1998, 1999, 2000, 2002a, 2002b, 

2003a, 2003b) the most typical sites for biodeterioration 

processes at Angkor Wat were selected for detailed and 

long-term microbiological studies. Additional microbiological investigations were performed at Preah Ko, Preah 

Khan, Bayon, and Banteay Srei in order to obtain a comprehensive view of the biodeterioration processes at the 

Angkor Site. The microbiological studies during 1997 and 

2004 included the assessment of both quantity and quality 

of microbial infestation, i.e., algae, lichens, fungi, bacteria, 

and actinomycetes, within the stone profile; the monitoring of the microbial metabolic activity over time and climatic conditions; the analysis of the possible impact of 

microorganisms in the deterioration of stones; the evaluation of previous conservation treatments; and the development and testing of conservation and biocidal treatments 

in regard to possible biodeterioration control strategies.

FIELD STUDIES

Site Studies: Locations and Tests

The microbiological investigations were carried out 

at different sites in the Angkor Wat temple complex over 

a five-year period (1997–2001). Forty sites in seven different areas were established and monitored regularly for 

microflora metabolic activity. These ranged from areas 

that had practically no biocolonization to those with algae 

and lichens and others with scaling of enriched iron oxide 

crusts to areas that had been treated with an acrylic resin 

(Figure 3).

In addition, eight stone samples were analyzed in detail for composition and quantity of microorganisms. Also, 

different biocide test treatment areas were established in 

the third enclosure of the temple: two with a northern exposure on the North Library, in 1997 and 1998 (biocide 

test field [BT] I); two with a southern exposure in the both 

the North and the South Libraries; one additional northern exposure on the North Library in 2001 (BT II); and 

the roofs and facades of the central East Gopura, in 2004 

and 2005 (BT III).

The application of biocides was performed by spraying (100 mL/m2), once with and once without pretreatment of medicinal alcohol (i.e., 70% ethanol), without 

prior brushing or any other mechanical cleaning so as to 

avoid destroying the original stone surface. The biocides 

applied included:

•	 Thymol (1% by volume in isopropanol),

•	 Aseptin A (active ingredient: quaternary ammonium 

compounds; 3% by volume in n-butanol),

•	 Barrycidal (active ingredients: quaternary ammonium 

compounds [ready-made solution]),

•	 Borax (5% w/v in water),

•	 algal wash, a specifically developed copper-complex 

formulation, later nicknamed Melange d’Angkor, 

•	 Mergal S 90 (active ingredients: carbendazim and triazin; 1.5% volume/volume (v/v) in water with 0.1% 

weight/volume (w/v) ethylenediaminetetraacetic acid 

(EDTA), Troy Inc., Hanover, Germany),

•	 Mergal S 88 (active ingredient: carbamate; 2.5% v/v 

in water with 0.1% w/v EDTA, Troy Inc., Hanover, 

Germany),

•	 Zinc Omadine (active ingredient: pyrithione 2% in 

water, Arch Chemicals, Atlanta, Georgia), 

•	 Algophase (active ingredient: tetrachloro-methylsulfonyl pyridine; 3% v/v in isopropyl alcohol + 20% 

acetone, PHASE, Florence, Italy), and

•	 Parmetol DF 12 (active ingredient: isothiazolinone; 

3% v/v in water, Schülke & Mayr, Norderstedt, 

Germany).

Nondestructive methods, such as adenosine triphosphate (ATP) measurements, respiration measurements, 

and contact agar plates, were used to assess the microbial 

infestation at the mentioned test sites as well as on other 

areas in the Angkor temple complex that had been treated 

previously with various biocidal formulations. For example, ammonium formaldehyde (also called formamide or 

formamine) was applied by French scientists in the late 

1960s at Bayon; ammonium zinc fluosilicate, tributyltin 

oxide (TBTO), and pentachlorophenol (PCP) were used 

by Indian restorers at Angkor Wat around 1990; copper 

sulfate solutions and sodium hypochlorite applications 

were made by Frank Preusser (Getty Conservation Institute) for the World Monuments Fund Project around 

1995 at the “Preusser Hall” in Preah Khan; and Metatin 
6 • smithsonian contributions to museum conservation

FIGURE 3. Four of the seven test sites used to monitor the metabolic activity of 

the biocolonization. (a) Apsara relief with slight black patina. (b) Apsara with iron 

oxide patina and flaking. (c) Apsara with black iron oxide patina and scaling. (d) 

Apsara treated in the past with Paraloid B-72 and with different colored patinas. 

Note that the white or pale blue squares are the devices installed for measuring the 

microflora metabolic activity.
number 2 • 7

58-10 was applied by Hungarian restorers to plaster at 

Preah Ko around 1996. Also, at Banteay Srei the biological infestation on the stone carvings was investigated in 

cooperation with the Swiss Banteay Srei Conservation 

Project in 2005.

Finally, laser cleaning to remove the black patina from 

the stone was carried out in cooperation with the Los Angeles Laser Conservation Research Center. Different wavelength laser beams were used, such as ultraviolet (355 nm, 

energy of 60 mJ), green (532 nm, energy of 100–190 mJ), 

and near infrared (1064 nm, energy of 300 mJ), with the 

following characteristics: beam diameter of 5 mm, pulse 

length of 3–6 ns, and frequency of 3 Hz.

Microbiological Studies

The stone-colonizing microflorae were examined by 

(1) microscopical analyses of the biofilm formation and 

distribution, (2) enumeration of the different microorganism groups, (3) quantification of their actual metabolic 

activity, (4) culturing and identification of typical microbial strains, and (5) physiological investigations on physicochemical deterioration mechanisms. The environmental 

parameters and characteristics of the sample sites were 

visually examined and documented by detailed photographs, registration of the relevant data concerning site 

exposure, and descriptions of the weathering pattern, its 

degree, and extent. 

Microscopic Analysis

The microscopic analysis of the collected stone specimens was performed, after staining of the microbial 

biofilm with periodic acid Schiff reagent (PAS), with a stereomicroscope using magnifications up to 320× in combination with digital documentation equipment. The PAS 

staining method is suitable for staining carbohydrate-rich 

microbial biofilms since the stain reacts with organic substances such as glycogen, starch, cellulose, mucin, chitin, 

protein-carbohydrate complexes, and many glycolipids. 

The procedure was carried out in screw cap test tubes, 

and the amount of added reagent was approximately four 

or five times greater than the size of the sample under investigation. After each treatment step, the added solution 

was completely taken out with a Pasteur pipette. 

On selected samples, the microscopic investigations 

were extended to fluorescence microscopy, after the addition of fluorescent stains such as acridine orange (AO) or 

fluorescein diacetate (FDA). Scanning electron microscopy 

(SEM) was also used, and selected stone samples were 

fixed in 4% glutaraldehyde for 24 h and then dehydrated 

in an ethyl alcohol series (25%, 50%, 75%, 90%, and 

100%, each concentration applied thrice for l h). After 

critical-point treatment the specimens were gold coated.

Determination of Metabolic Activity

In order to quantify the metabolic activity of the microflora on the stone in situ, the respiration rate of the 

microflora was analyzed using respiration boxes, following the method of Isermeyer that measures the release of 

carbon dioxide from a particular stone area. The respiration rate box method is a nondestructive technique for the 

rapid and semiquantitative estimation of potentially damaging microbial activity on materials of historical buildings and monuments (Becker et al., 1994).

Metabolic activity was also assessed using a sterile 

cotton swab to nondestructively collect adherent particles 

from the material surface. These particles were then analyzed for the concentration of ATP using a luminometer.

Culture Enrichment of Microorganisms

After a careful selection of the sites that were most 

characteristic of typical weathering patterns at Angkor 

Wat, small cubes of approximately 20 × 20 × 4 mm were 

cut from the stone surface using a diamond drill. The 

specimens were removed from the stone using a chisel and 

were transferred into sterile petri dishes and transported 

to the laboratory in Germany.

In the laboratory, the collected specimens were powdered in a ceramic mortar. Then, 1 g of the powder was 

transferred into 10 mL of a 0.001% Tween 80 solution 

and shaken for 1 h. The resulting solutions were plated 

out on the selected enrichment media, applying 37 μL on 

the plate using a spiral plater device in order to quantify 

the concentration of microorganisms in the sample material under study. The inoculated enrichment media were 

incubated at 22°C for 14 days. Then the cultivated sample 

was counted, photographed, and selected for further microbiological studies (i.e., pure culturing, simulation tests). 

The counts were computed as colony forming units (CFU) 

per gram of stone (Becker et al., 1994). Subsequently, nondestructive enrichments were performed using Rodac impression agar plates, simply attached to the stone surface 

at the site and incubated in the laboratory under the same 

conditions as mentioned above.

The following solid nutrient media were used for the 

enrichment and quantification of the microflora present 

on the stone specimens:
8 • smithsonian contributions to museum conservation

•	 Czapek-Dox medium for copiotrophic fungi (MerckDiagnostica No. 5460),

•	 Bunt-Rovira medium for oligotrophic bacteria (Bunt 

and Rovira, 1955),

•	 Glycerine-nitrate-casein medium for actinomycetes 

(Küster and Williams, 1964),

•	 VM-T (synthetic full medium, Merck-Diagnostica 

No. 5450 with tyrosine) for melanin-producing 

microorganisms,

•	 BF-Mn (Bromfield agar with MnSO4) for manganeseoxidizing microorganisms (Bromfield, 1955), and

•	 BF-Fe (Bromfield agar with FeSO4) for iron-oxidizing 

microorganisms (Bromfield, 1955).

The enrichment of nitrifying, sulfur-oxidizing, and 

photosynthetic microorganisms was performed in liquid 

solutions and analyzed quantitatively by the most probable number (MPN) method using the following enrichment media:

•	 BNM or BNB media for nitrifying bacteria (Krümmel, 

1978; Bock and Engel, 1966),

•	 S5 and S6 medium after Hutchinson for sulfuroxidizing bacteria (Hutchinson et al., 1965), and

•	 BG11 for photosynthetic algae (Ripka et al., 1979).

The counts of nitrifying bacteria, sulfur-oxidizing bacteria, and algae were examined as prescribed by the MPN 

method. Nitrifying and sulfur-oxidizing bacteria were incubated for six weeks in the dark at 28°C, and the counts 

were computed as MPN/g stone. The algae and cyanobacteria were incubated for six weeks in the light at 16°C, and 

the counts were also computed as MPN/g stone.

Biochemical Investigations

The biochemical investigations on the stone specimens 

taken from the selected sites were based on determination 

of the protein content as the parameter for the concentration of microbial biomass, dehydrogenase activity with 

tritetrazoliumchloride (TTC) as the biochemical indicator 

for the extent of actual microbial metabolic activity, presence of chlorophyll as the biochemical marker for photosynthetic microorganisms, and assessment of microbial 

acid excretion as the marker for biocorrosion processes. To 

measure protein content, the analytical protocol of Lowry 

was used (Lowry et al., 1951). The dehydrogenase activity 

provides a rapid method to confirm and evaluate microbial activity on decaying stones (Warscheid et al., 1990). 

To test acid production, the isolated microorganisms (i.e., 

fungi, bacteria, and algae) were cultivated in a glucosecontaining mineral solution, referred to as ANG (Warscheid et al, 1990), for 14 days at 28°C. After this, the 

pH of the culture filtrate was measured with a pH meter 

(Warscheid, 1990). Selected stone samples, characterized 

by algal and lichen infestation, were crushed, and 200 

mg of the crushed sample were introduced into 2 mL of 

methanol and centrifuged for 10 minutes; the supernatant 

was then removed and analyzed in a UV-visible spectrophotometer for its absorption profile.

Substrate Characterization

Four in situ methods were used to characterize the 

substrate: (1) capillary water uptake with the RILEM or 

Karsten tube, (2) rebound hardness with a recently developed impact hammer, (3) surface temperature measurements, and (4) drilling resistance. The RILEM or Karsten 

tube allows determination of the time-dependent water 

uptake of the stone at the monument. The plot of the water uptake (kg/m2

) as a function of the square root of time 

is linear, except for the initial water uptake period. The 

water uptake coefficient w (kg/m2 h0.5) can be calculated 

by the slope of the initial straight line. The measurement 

takes, in general, about 1 h but has to be stopped if wet 

stains appear on the sample surface, as this indicates a 

faulty seal of the tube to the substrate. If the water penetration coefficient B (cm/s0.5) of the substrate is known 

from laboratory tests, the depth of penetration of water 

as a function of time can be calculated (Wendler and 

Snethlage, 1988).

Surface temperature measurements were carried out 

on different sunlit and shaded stone surfaces with various microbial infestation characteristics. Fixed measuring 

points were monitored by using either a surface thermal 

sensor or a thermohygrometer.

The rebound hardness, measured with an impact hammer, correlates to the compressive strength of a material 

(Bögner, 1999; Grassegger and Leisen, 2005). A calibration diagram is required to translate the reading obtained 

by the impact hammer into a compressive strength value.

The drill resistance equipment provides a plot of the 

depth of drilling as a function of time. From this plot, the 

drilling resistance is obtained from the slope dt/dx (s/mm) 

at each specific depth (Leonard and Kiessl, 1990). Calibrating in the laboratory with materials of known strength, a 

high-resolution (±1 mm) strength profile can be obtained. 

The method is very useful in detecting subsurface flaking 

or detachment. In addition, the drill powder obtained can 

be used for chemical analysis of soluble salts. 
number 2 • 9

RESULTS

The evaluation of the macroflora showed that the 

stones have a significant cover of lichens, such as the Parmelia, Caloplaca, Pyxine, Dirinaria, and Lepraria species. 

Only in very humid areas can green algae, such as Pleurococcus sp., be found. There are also some distinct red and 

black areas that could be related to green algae, such as 

the Trentepholia sp., which appear red-brown under normal conditions, and cyanobacteria, such as Gloeocapsa 

and Scytonema sp., which can appear black (Figure 4).

The natural microflora found on stone and stuccos, 

although very complex, is well balanced, so that overgrowth and domination from one specific group of microorganisms is avoided. The trees that grow in the Angkor 

enclosures shelter its monuments from rapid moisture and 

temperature changes, thus preserving the original stone 

surface and the readability of their historic testimony, despite the detrimental mechanical effects of the roots to the 

structural integrity of the temples.

In the early 1990s, a biocide-based cleaning was carried out under an Indian conservation project that removed the surface biocolonization. Subsequent years 

showed an intensive blackening of the treated stone surfaces due to uncontrolled overgrowth of black-colored cyanobacteria. The resulting thermal effect of the blackened 

surfaces, especially under tropical conditions, added to the 

normal thermohygric stress suffered by an already weakened and sensitive clay-bearing stone, leading to increased 

delamination, scaling, and flaking of the surface. Thus, the 

removal of the biopatina of lichens can be considered a 

major threat to the endangered stone material (Figure 5).

The natural microbial biofilm is located mainly on the 

uppermost layers of the stone surfaces (Figure 6), but metabolically active cells can also penetrate into deeper parts 

of the rocks (Figure 7). SEM analyses support the finding 

that photosynthetic microorganisms, i.e., lichens, algae, 

and cyanobacteria, are found mainly on the surface of the 

rock, whereas chemoorganotrophic fungi and bacteria can 

also be found in the interior (Figure 8).

Analysis and measurement of the enriched microorganisms from selected stone samples from the Angkor Wat 

temple indicate that they consist mainly of phototrophic 

algae and cyanobacteria. This was proven by the increased 

photosynthetic activity when exposed to light through 

ATP measurements and by the presence of chlorophyll in 

a methanol extract obtained from black surface samples 

as determined by UV-visible spectrophotometry. Culture 

enrichment methods showed that both actinomycetes 

and chemoorganotrophic bacteria dominate compared 

to fungi, which have been found only in small quantities, 

although their presence clearly correlates with the occurrence of black patina, as a result of the melanin-producing 

strains (Figure 9).

The isolated fungi mainly consisted of the so-called 

“black” fungi, such as Alternaria alternata, Cladosporium

cladosporoides, Cladosporium sphaerospermum, and Ulocladium chartarum. They also included “fungi imperfecti,” 

such as Aspergillus niger; ubiquitous fungi, such as Curvularia geniculata and Trichoderma viride; “dirt fungi,” such 

as Fusarium oxysporum; some biocorrosive penicillia, e.g., 

Penicillium glabrum and P. griseofulvum; and mycotoxinproducing fungal strains, such as Paecilomyces variotii, 

Stachbotrys chartarum, and Acremonium strictum.

The presence of nitrifying bacteria is scarce, and so 

far, the presence of sulfur-oxidizing bacteria cannot be established, although their presence is always referred to in 

the literature dealing with microbiological studies at the 

Angkor sites (Pochon et al., 1960). To complement this information, it should be mentioned that sulfates have often 

been found in relation to the habitats of bats around the 

towers and pillars of the galleries.

The protein content of the stone samples investigated 

was quite high (up to 4.48 mg/g), as was the metabolic 

activity (up to 0.391 μg CO2

/g). The latter was highest 

during the rainy season, but it could easily be increased by 

the addition of moisture during the dry season, as proven 

by higher respiration and photosynthesis rates.

The isolated microbial strains showed a high potential for biocorrosivity due to the significant excretion of 

organic acids that could lead to stone surface sanding. 

Finally, the presence of iron-oxidizing microorganisms 

could be established, particularly at locations that show 

a significant reddish-orange coloration resulting from the 

migration and oxidation of iron minerals in the stone (see 

Figure 3b).

From the studies carried out it is evident that the microflora on the stones of Angkor Wat and other temples 

has biocorrosive and bio-oxidative properties, affecting 

the mineral structures of stones. However, results from the 

comparative analysis of the water transport data suggest 

that the natural biofilm of a stable, long-term developed 

macroflora, i.e., lichens, has an important moisturecontrolling function for the environmentally stressed 

stones. The measurement of the capillary water uptake at 

different places on the temple (Figure 10) shows that lichens protect the stone from rapid water uptake, whereas 

certain algal and blackened cyanobacterial biofilms significantly increase it. This phenomenon is especially disadvantageous during rainy seasons, when the heated stone 
1 0 • smithsonian contributions to museum conservation

is rapidly cooled by rain, thus increasing the thermohygric 

expansion-contraction processes that lead to delamination, scale formation, and flaking (see Figure 2). It should 

be noted that the “dead” biofilm remaining on the stone 

after a biocidal treatment also slows down water uptake, 

at least initially, though presumably the stone’s surface will 

shift to that of an unpatinated stone over time. The surface temperature differences measured on stones covered 

with the dark black patina as compared to those with the 

grayish-colored lichen infestations, or even the uninfested 

stones, can be as high as 15°C.

Rebound hardness and drill resistance measurements 

taken on mature microbial biofilms, i.e., lichens, have not 

shown any evidence that they significantly affect the mechanical properties of the stones. Consequently, it can be 

stated that the mature microbial biofilms (i.e., lichen patina) regulate the humidity, thermal transmission, and water vapor diffusion, reducing thermohygric stresses to the 

stone at Angkor Wat. Any ongoing conservation activities 

FIGURE 4. Colored biofilms of different photosynthetic organisms, e.g., lichens, algae, and cyanobacteria, found on the stones at the Angkor monuments, depending 

on exposure and microclimate.
number 2 • 1 1

FIGURE 5. (a) The removal of the natural lichen biopatina at Angkor Wat resulted in an intense colonization by blackening microorganisms. (b) The increased thermohygric stresses to the clay-bearing sandstone 

resulted in severe flaking and delamination on the exposed carved surfaces.
1 2 • smithsonian contributions to museum conservation

FIGURE 6. Microbial biofilm on a sample of Angkor Wat stone 

surface visualized by PAS staining. (a) Surface is completely covered 

with a microbial film. (b) The visible biofilm is reduced in the subsurface. (c) Almost no biological biofilm is visible in the interior of 

the stone.

FIGURE 7. Microbial biofilm, mainly due to lichen infestation, on a 

rock flake at Angkor Wat visualized by fluorescent acridine orange 

staining. Fluorescent staining indicates metabolically active cells. 

(a) The surface shows round-shaped lichen thalli. (b) The biofilm 

penetrates only slightly into the sandstone. (c) A fungal infestation is 

present in the interior of the stone flake.
number 2 • 1 3

should take into account the preservation of these acceptable microbial infestations while removing those microbial 

biofilms that enhance deterioration or where the visibility 

and readability of historic artifacts is essential.

The results of the biocide test sites evaluated during 

the seven-year period (1997–2004) have shown that the 

sequenced application of disinfecting and biocidal solutions, provide an effective strategy for the long-term control of local microbial infestation. As disinfectants, medical 

ethanol was used to destabilize the microbial biofilms of 

either lichens, algae, or fungi, and this was followed by 

the application of effective but fairly ecologically sound 

biocidal compounds, such as algal wash (based on a low 

concentration copper complex solution), or the Parmetol 

DF 12 (based on an aqueous solution of a mixture of various isothiazolinones, biodegradable by bacteria).

Also investigated was the effectiveness of previous biocide treatments applied at different sites in the temple complex. The evaluation revealed that mostly weak effects were 

obtained by the application of “soft” biocides, whereas the 

use of highly toxic and environmentally critical biocides 

gave only moderate and temporary relief from microbial infestation and often resulted in recolonization by blackening 

cyanobacteria, as seen at Angkor Wat (see Figure 5).

The long-term growth-controlling effect of the algal 

wash, the copper complex formulation, could be observed 

even after seven years of exposure at the first biocide test 

field (BT I) treated in 1997. Figures 11 and 12 show two 

of the sections of the second test site (BT II) after four 

years of the application of algal wash in 2001. It should 

be noted that no mechanical cleaning had been performed 

at or after the application of the biocide.

Laser cleaning also gave positive results on test cleaning of stone fragments and shows potential for cleaning 

stone surfaces at carefully selected areas, such as very friable stone that needs to be consolidated. The portable laser 

with 532 nm (green) wavelength and an energy of 190 mJ 

gave the most promising cleaning. Nonetheless, the thermal impact on the stone needs to be further analyzed in order to avoid microstructural damage of the mineral lattice.

Finally, it has to be considered that conservation treatments, such as cleaning, consolidation, or hydrophobization, need to be evaluated with regard to their possible 

contribution toward the recurrence of microbial colonization. For example, the use of local well water for soaking 

bricks prior to their use resulted in an algal infestation 

of the finished wall constructed with them (Figure 13a), 

whereas the application of a water repellent at the temple 

of Ta Nei resulted in the selective reinfestation by Trentepholia sp., the red-colored green algae (Figure 13b).

FIGURE 8. SEM examination of a microbial biofilm on a stone 

sample from Angkor Wat. (a) Lichen thallus grows on the surface of 

the stone. (b) At the subsurface, the concentration of photosynthetic 

microorganisms diminishes while fungal and bacterial cells prevail. 

(c) Fungal contamination dominates in the interior of the sample. 
1 4 • smithsonian contributions to museum conservation

FIGURE 10. In situ capillary water absorption measurements at stone areas with and 

without visible biofilms and after three years of application of algal wash (coppercomplex-based product) to a cyanobacterial biofilm. Note that the lichen biofilm 

shows the lowest water absorption and the applied treatment nearly lowers the absorption to this level.

FIGURE 9. Cultural enrichment of bacteria, fungi, and actinomycetes, respectively, from top to bottom, on different stone samples characterized by (a) a black patina, (b) a slight black patina, (c) no 

patina, and (d) a lichen patina.
number 2 • 1 5

FIGURE 11. Comparison of an area on the northern library, northern exposure, (a) before treatment and (b) four years after 

the application of Parmetol DF 12.

FIGURE 12. Successful application of algal wash 

at the southern library, southern exposure, four 

years after treatment.
1 6 • smithsonian contributions to museum conservation

Within the GACP Project, applied products were regularly 

screened for contaminants and microbial resistance during 

the conservation work. 

CONCLUSIONS

The studies carried out have shown that although the 

microflorae present on Angkor Wat and other temples 

have biocorrosive and bio-oxidative properties, there are 

significant differences between different biofilms. Stable, 

long-term developed algal biofilms possess an important 

moisture-controlling function for the stone substrate, 

whereas certain algal and black-colored cyanobacterial 

biofilms significantly increase moisture as well as surface 

temperature. This is a particularly negative feature for the 

clay-bearing sandstone of the monuments, given its thermohygric expansion-contraction properties, which lead to 

delamination, scale formation, and flaking.

Consequently, any conservation activities should take 

into account the preservation of these acceptable microbial infestations while removing those microbial biofilms 

that enhance deterioration. For this purpose, the application of effective, long-term biocides, tested on site, should 

be used. Furthermore, it is recommended that any other 

conservation treatment, be it cleaning, consolidation, or 

the application of a water repellent, needs to be evaluated 

with an eye to the potential of applied products for enhancing or changing the existing biocolonization. 

Acknowledgments

The authors acknowledge the support received from 

the Ministry for Foreign Affairs of the German government for the research and restoration activities within the 

German Apsara Conservation Project. Thanks are also 

owed to Dr. S. Scherer, who conducted the studies on laser cleaning, and to Dr. A. E. Charola, who helped in the 

preparation of this text. 

REFERENCES

Adan, O. C. G. 1994. On the Fungal Defacement of Interior Finishes. Ph.D. 

thesis, Technical University Eindhoven, Eindhoven, Netherlands.

Becker, T. W., W. E. Krumbein, T. Warscheid, and M. A. Resende. 1994. 

“Investigations into Microbiology.” In IDEAS: Investigations 

into Devices against Environmental Attack on Stones, ed. H. K. 

Bianchi, pp. 147–190. Final report. Geesthact, Germany: GKSSForschungszentrum Geesthacht.

Bock, E., and H. Engel. 1966. Untersuchungen über die postoxidative 

CO-Fixierung bei Nitrobacter winogradskyi Buch. [Investigations 

FIGURE 13. (a) Algal infestation of a brick wall because well water was used to 

soak the brick prior to its use. (b) Selective infestation by red-colored green algae 

at the temple of Ta Nei after treatment with a water repellent.
number 2 • 1 7

on the Postoxidative Fixation of CO by Nitrobacter winogradskyi

Buch.] Archives of Microbiology, 54:191–198.

Bögner, H. 1999. Entwicklung einer einfachen Stoßsonde und Zuordnung der Stoßeigenschaften zu der mechanischen Eigenschaften 

von verwittertem und unverwittertem Naturstein. [Development of 

a Simple Impact Hammer and Correlation of the Impact Results to 

the Mechanical Properties of Weathered and Unweathered Stone.] 

Doctoral thesis, Faculty of Construction Engineering, University of 

Stuttgart, Stuttgart, Germany.

Bromfield, S. M. 1955. Oxidation of Manganese by Soil Microorganisms. Australian Journal of Biological Sciences, 9:238–254.

Bunt, J. S., and A. D. Rovira. 1955. Microbiological Studies of Some 

Subantartic Soils. Journal of Soil Science, 6:119–128.

Flemming, H. C., and G. Schaule. 1994. Mikrobielle Werkstoffzerstörung—Biofilm und Biofouling: Biofouling. Werkstoffe und Korrosion, 45(1):29–39.

Fusey, P. 1991. Alterations biologiques des grès cambodgiens et recherche 

de moyens de protection—Essaie de synthèse. [Biological Alterations 

of the Cambodian Sandstones and Studies on Protection Methods—

A Synthesis.] 91 pp. École Francaise d ́Extreme-Orient, Paris.

Grassegger, G., and H. Leisen. 2005. “Mikromechanische Messungen 

zur Festigkeit und zum Erfolg von Koservierungmaßnahmen am 

Tempel von Angkor Wat” [Micromechanical Hardness Measurements for the Evaluation of Conservation Measures at the Temple of Angkor Wat]. In Natursteinrestaurierung Ergebnisse und 

messtechnische Erfassungen, ed. G. Grassegger and G. Patitz, pp. 

15–26. Stuttgart, Germany: Fraunhofer IRB Verlag.

Hutchinson, M., K. I. Johnstone, and D. White. 1965. The Taxonomy of 

Certain Thiobacilli. Journal of General Microbiology, 41:357–366.

Hyvert, G. 1968. Altération des grés des monuments khmers. [Sandstone 

Alteration of the Khmer Monuments.] Ph.D. thesis, Université de 

Paris VI, Paris.

Japanese Government Team for Safeguarding Angkor. 1995. “Investigations of Deteriorating Organisms and Control Methods.” In Annual 

Report on the Technical Survey of Angkor Monument, pp. 417–422. 

Tokyo: Japanese Government Team for Safeguarding Angkor.

———. 1996a. “Aerial Algae Collected from Angkor Monuments.” In 

Annual Report on the Technical Survey of Angkor Monument, pp. 

430–438. Tokyo: Japanese Government Team for Safeguarding 

Angkor.

———. 1996b. “Experiments of Controlling Methods of Growth of Algae and Lichens.” In Annual Report on the Technical Survey of Angkor Monument, pp. 439–441. Tokyo: Japanese Government Team 

for Safeguarding Angkor.

———. 1998. “Deteriorating Process of Stone Materials by Lichens—

New Anti Lichens Treatments—Tests to Control Lichens and Algae 

at Angkor Materials, Methods and Results.” In Annual Report on 

the Technical Survey of Angkor Monument, pp. 395–410. Tokyo: 

Japanese Government Team for Safeguarding Angkor.

———. 1999. “Microbiological Analyses of Deteriorating Stone 

Materials—Records of Algaecidal and Lichenocidal Tests.” In Annual Report on the Technical Survey of Angkor Monument, pp. 

179–195. Tokyo: Japanese Government Team for Safeguarding 

Angkor.

———. 2000. “Investigations of Biodeterioration and Its Control.” In 

Annual Report on the Technical Survey of Angkor Monument, 

pp. 80–90. Tokyo: Japanese Government Team for Safeguarding 

Angkor.

———. 2002a. “Conservation Science Survey (II) Research on Microorganisms.” In Annual Report on the Technical Survey of Angkor 

Monument, pp. 321–327. Tokyo: Japanese Government Team for 

Safeguarding Angkor.

———. 2002b. “Conservation Science Survey (III) Bad Research of Angkor Monument.” In Annual Report on the Technical Survey of Angkor Monument, pp. 329–333. Tokyo: Japanese Government Team 

for Safeguarding Angkor.

———. 2003a. “Conservation Science Survey (I) Test for the Removal 

of Algae and Lichens.” In Annual Report on the Technical Survey 

of Angkor Monument, pp. 183–194. Tokyo: Japanese Government 

Team for Safeguarding Angkor.

———. 2003b. “Conservation Science Survey (II) Research on Microorganisms.” In Annual Report on the Technical Survey of Angkor 

Monument, pp. 195–203. Tokyo: Japanese Government Team for 

Safeguarding Angkor.

Koestler, R. J., P. Brimblecombe, D. Camuffo, W. S. Ginell, T. E. Graedel, 

P. Leavengood, J. Petushkova, M. Steiger, C. Urzi, V. Vergès-Belmin, 

and T. Warscheid. 1994. “How Do External Environmental Factors Accelerate Change?” In Durability and Change—The Science, 

Responsibility and Cost of Sustaining Cultural Heritage, ed. W. E. 

Krumbein, P. Brimblecombe, D. E. Cosgrove, and S. Staniforth, pp. 

149–163. New York: John Wiley and Sons.

Krümmel, A. 1978. Vergleichende Untersuchungen zum Einfluss organischer C-Verbindungen auf das Wachstum, das Fettsäuremuster 

und die Feinstruktur ammoniakoxidierender Bakterien. [Comparative Research on the Influence of Organic Carbon Compounds on 

the Growth, Fatty Acids Configuration and Structure of Ammonia 

Oxidizing Bacteria.] Ph.D. thesis, University of Hamburg, Hamburg, Germany.

Küster, E., and S. T. Williams. 1964. Selection of Media for Isolation of 

Streptomycetes. Nature, 202:928–929.

Leisen, H., J. Poncar, and S. Warrack. 2000. German Apsara Conservation Project—Angkor Wat. Cologne University of Applied Sciences, 

Authors’ Edition, Cologne, Germany.

Leisen, H., E. von Plehwe-Leisen, S. Warrack, and N. Long. 2008. “The 

German Apsara Conservation Project at Angkor Wat. Stone Conservation of the Khmer Heritage of Cambodia: Practical Execution and New Results.” In Interpreting Southeast Asia ́s Past, ed. 

E. Bacus, E.C.Glover , and P.D. Sharrock, pp. 391–399. Singapore: 

National University of Singapore Press.

Leonard, H., and K. Kiessl. 1990. Schnellbestimmung des Festigkeitsprofiels verwitterter Oberflächenzonen alter Gebäude durch Bohrwiderstandmessung. [Quick Method for Determining the Hardness 

Profile of Weathered Superficial Zones in Old Building by the Drill 

Resistance Measurement.]. IBP-Mitteilung 17. Stuttgart, Germany: 

Institut für Bauphysik.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. 

Protein Measurement with the Folin Phenol Reagent. Journal of 

Biological Chemistry, 193:265–275.

May, E., F. J. Lewis, S. Pereira, S. Tayler, M. R. D. Seaward, and D. Allsopp. 1993. Microbial Deterioration of Building Stone—A Review. 

Biodeterioration Abstracts, 7(2):109–123.

Mitchell, R., and G. Ji-Dong. 2000. “Interactions between Air Pollutants 

and Biofilms on Historic Limestone.” In Of Microbes and Art: The 

Role of Microbial Communities in the Degradation and Protection 

of Cultural Heritage, ed. O. Ciferri, P. Tiano, and G. Mastromei, 

pp. 143–145. New York: Kluwer Academic.

Pochon, J., P. Tardieux, J. Ajudie, M. Charpentier, J. Delvert, R. Triau, 

and M. Bredillet. 1960. Dégradation des temples d ́Angkor et processus biologiques. [Degradation of the Angkor Temples and Biological Processes.] Annales de l’Institut Pasteur (Paris), 98:457–461.

Ripka, R., J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier. 1979. Generic Assignments, Strain Histories and Properties of 

Pure Cultures of Cyanobacteria. Journal of General Microbiology,

111:1–61.

Saiz-Jimenez, C. 1995. Deposition of Airborne Organic Pollutants on 

Historical Buildings. Atmospheric Environment, Part B, 27:77–85.

Warscheid, T. 1990. Untersuchungen zur Biodeterioration von Sandsteinen unter besonderer Berücksichtigung der chemoorganotrophen 
1 8 • smithsonian contributions to museum conservation

Bakterien. [Studies on the Biodeterioration of Sandstones with Regards to Chemoorganotrophic Bacteria.] Technical dissertation 

T 2275, IRB Verlag, Stuttgart, Germany. 

———. 1996. Impacts of Microbial Biofilms in the Deterioration of 

Inorganic Building Materials and Their Relevance for the Conservation Practice. Internationale Zeitschrift für Bauinstandsetzen,

2(6):493–504.

———. 1999. “Integrated Concepts for the Protection of Cultural Artifacts against Biodeterioration.” In Of Microbes and Art: The Role 

of Microbial Communities in the Degradation and Protection of 

Cultural Heritage, ed. O. Ciferri, P. Tiano, and G. Mastromei, pp. 

185–202. New York: Kluwer Academic.

———. 2003. “The Evaluation of Biodeterioration Processes on Cultural Objects and Respective Approaches for Their Effective Control”. In Art, Biology, and Conservation 2002: Biodeterioration of 

Works of Art, ed. R. J. Koestler, V. H. Koestler, A. E. Charola, and 

F. E. Nieto-Fernandez, pp. 14–27. New York: The Metropolitan 

Museum of Art.

Warscheid, T., K, Petersen, W. E. Krumbein. 1990. “A Rapid Method to 

Demonstrate and Evaluate Microbial Activity on Decaying Sandstone.” Studies in Conservation, 35:137–147.

Warscheid, T., T. W. Becker, J. Braams, S. Brüggerhoff, C. Gehrmann, 

W. E. Krumbein, and K. Petersen. 1993. “Studies on the Temporal 

Development of Microbial Infection of Different Types of Sedimentary Rocks and Its Effect on the Alteration of the Physico-chemical 

Properties in Building Materials.” In Volume 1: Causes of Disorders 

and Diagnosis, ed. M.-J. Thiel, pp. 303–310, Conservation of Stone 

and Other Materials. London: E & FN Spon. 

Warscheid, T., and J. Braams. 2000. Biodeterioration of Stones—A Review. 

International Biodeterioration and Biodegradation, 46:343–368. 

Warscheid, T., and W. E. Krumbein. 1996. “Biodeterioration of Inorganic 

Nonmetallic Materials—General Aspects and Selected Cases.” In 

Microbially Induced Corrosion of Materials, ed. E. Heitz, W. Sand, 

and H. C. Flemming, pp. 273–295. New York: Springer-Verlag.

Warscheid, T., M. Oelting, and W. E. Krumbein. 1991. Physico-chemical 

Aspects of Biodeterioration Processes on Rocks with Special Regard 

to Organic Pollution. International Biodeterioration, 28:37–48.

Warscheid, T., K. Petersen, and W. E. Krumbein. 1990. A Rapid Method 

to Demonstrate and Evaluate Microbial Activity on Decaying Sandstone. Studies in Conservation, 35:137–147.

Wendler, E., and R. Snethlage. 1989. Die Wassereindringsprüfung nach 

Karsten—Anwendung und Interpretation der Meßwerte. [Water 

Penetration Test According to Karsten—Use and Interpretation 

of the Measurements.] Bautenschutz und Bautensanierung, 12: 

110–115.
M. Carmen Portillo and Juan M. Gonzalez,

Instituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Avenida Reina Mercedes 10, E-41012 

Sevilla, Spain. Correspondence: Gonzalez, 

jmgrau@irnase.csic.es.

Microbial Community Diversity and the 

Complexity of Preserving Cultural Heritage

M. Carmen Portillo and Juan M. Gonzalez

Abstract. In addition to physical and chemical factors, microorganisms also affect 

the conservation of stone monuments. The development of novel methods of detection 

has significantly advanced the study of microorganisms. Whereas classical microbiological techniques are based on culturing bacteria, the use of molecular methods based on 

nucleic acid technology permits the detection of numerous bacteria that previously remained unnoticed. Besides the detection of bacteria from DNA, using RNA for bacterial 

identification permits us to determine the bacteria showing active metabolism within the 

community. The metabolism of most bacteria is unknown, and the effect of bacteria on 

stone monuments can hardly be predicted. This drastic change in our understanding of 

bacterial communities and their effect on cultural heritage is leading to different perspectives in preventing, controlling, and monitoring biocolonization of stone monuments. 

The incredibly high bacterial diversity detected in monuments suggests a great complexity, thus complicating any attempt to prevent microbial colonization of cultural heritage. 

This study focuses on the bacterial diversity of caves and natural shelters with prehistoric 

paintings and the consequences of this diversity for the conservation of this cultural heritage in these specific environments.

INTRODUCTION

Stone materials, including natural structures, buildings, monuments, and 

other cultural heritage objects, are exposed to the action of physical, chemical, 

and biological factors. Among the latter, the role of microorganisms has been 

proposed to have a high deteriorating potential to stone because it is acting 

constantly over time. The small size of these living creatures (at the micrometer 

scale) makes their study difficult. Thus, different methodological strategies are 

being applied to determine which microorganisms are present and what they are 

doing. This research aims at elucidating the role and potential impact of microorganisms on stone biodeterioration.

Most of the pioneering studies on microorganisms were performed by growing these cells on culture media. This strategy allows the cells to multiply so that 

colonies of macroscopic size can be visualized. Also, culturing microorganisms 

permits the investigation of their physiological characteristics. When using these 

culturing methods, natural microbial communities appear to be composed of 

just a few microbial types. The microorganisms detected by culturing techniques 

are those that grow optimally under the conditions provided. However, recent 
2 0 • smithsonian contributions to museum conservation

studies are changing this simplistic perspective, and the 

introduction of advanced detection methods for microorganisms is drastically expanding our knowledge of the real 

scale of the problem.

By the end of twentieth century, the introduction of 

molecular methods based on the detection of specific DNA 

sequences contributed to the identification of a large number of novel microorganisms previously undetected. Presently, it is widely accepted that standard culturing methods 

in natural environments only permit the detection of 

about 1% of the total number of microorganisms present 

in the samples (Ward et al., 1990). Therefore, nearly the 

complete microbial community remained undetected. This 

scenario was drastically changed when molecular methods 

were introduced because they greatly contribute to our understanding of the actual composition of microbial communities and their potential as a deterioration factor.

In spite of being able to detect a large number of 

the microorganisms in nature, recent studies have demonstrated an even higher diversity of microorganisms in 

natural environments than were ever expected. In fact, 

microbial diversity appears to be so high that a true estimation is not possible with currently available methods 

(Curtis et al., 2002). Because of this huge diversity, how 

can we discriminate which microbes are important in the 

biogeochemical processes carried out by the community 

and what role is performed by specific microorganisms? 

Understanding which microorganisms are present and 

their role in a natural community is not a trivial subject.

In order to understand which microbes play important 

roles in biogeochemical transformations it is necessary to 

identify and study the physiology of those microorganisms 

actually carrying out significant metabolic activity within 

a microbial community (Mills et al., 2004; Portillo et al., 

2008, 2009; Portillo and Gonzalez, 2009a). This has been 

achieved by detecting those microorganisms through their 

RNA because the amount of RNA per cell is proportional 

to the cell growth and activity (Molin and Givskov, 1999). 

RNA is highly unstable, so its handling greatly increases 

experimental complexity. Furthermore, even if we know 

which microbes are active within a community, this does 

not tell us the function of those microorganisms in a given 

environment.

Because the metabolic capabilities and physiology of 

most microorganisms detected in samples through molecular techniques are unknown, their influence on stone deterioration cannot be inferred. For this purpose, it is necessary 

to rely on culturing techniques that allow evaluation of the 

metabolic capabilities and physiology of microorganisms.

This study presents some examples of the microbial diversity, activity, and metabolism detected in two culturally 

important prehistoric paintings locations in Spain: one 

in a cave and the second in a natural stone shelter. From 

these examples, the consequences, problems, and future 

perspectives of microbial deterioration are discussed.

EXPERIMENTAL METHODS

Two locations were studied. One site was the Altamira 

Cave (Cantabria, northern Spain), containing unique 

polychromatic Paleolithic paintings, and the other was a 

natural shelter, Muriecho L (Aragón, northeastern Spain), 

with Neolithic paintings. Microbial colonies that had developed in these two environments were analyzed in order 

to detect the microorganisms involved in these communities. Molecular surveys of microorganisms were based on 

both DNA and RNA. Microbial detection through DNA 

was directed at identifying those microorganisms present 

in the studied areas, whereas the microorganisms showing 

significant metabolic activity in these communities were 

detected through RNA-based molecular methods.

DNA and RNA were extracted separately using the 

NucleoSpin Food (Macherey-Nagel, Düren, Germany) 

and RNAqueos-4PCR (Ambion, Inc., Austin, Texas, 

United States) extraction kits, respectively (Portillo et al., 

2008). The complementary DNA to the extracted RNA 

was obtained by a reverse transcription reaction using the 

primer 518R (5′-ATT ACC GCG GCT GCT GG; Muyzer 

et al., 1993).

In order to obtain a visual image of the complexity of 

the microbial communities and to carry out comparisons 

among these communities, a molecular profiling technique 

was utilized. This allowed visualization of the major bacterial constituents of the communities. The protocol was 

optimized to obtain relative quantification of the contribution of major bacterial types to total DNA and RNA from 

the microbial community. The method used was the denaturing gradient gel electrophoresis (DGGE) as described 

by Gonzalez and Saiz-Jimenez (2004) with the modifications for quantitative analyses and the statistical procedure for profile comparison recently described by Portillo 

and Gonzalez (2008a).

For cloning purposes, amplification of the 16S rRNA 

genes was performed by PCR with primers 27bF (5′-AGA 

GTT TGA TYM TGG CTC AG; Zimmermann et al., 

2005) and 907R (5′-CCC CGT CAA TTC ATT TGA GTT 

T; Gonzalez et al., 2003) and 27bF and 518R for DNA- and 

RNA-based analyses, respectively. The amplification products were used to identify the bacteria represented in the 

DNA and RNA extracted from the samples under study. 

In order to identify these bacteria, 16S rRNA gene libraries 
number 2 • 2 1

were constructed using the TOPO TA cloning kit (Invitrogen, Carlsbad, California, United States). The screening 

procedure for selecting the clones in each library was previously described in detail by gonzalez et al. (2003). Selected 

clones were sequenced and analyzed as previously described 

(gonzalez et al., 2006; Portillo et al., 2008). This procedure 

represents a rapid and effi cient method to detect specifi c 

sequences within complex gene libraries.

The obtained sequences were differentiated, and accumulation curves of the number of processed clones versus 

the number of newly found different operational taxonomic units (OTUs) or bacterial phyla were constructed. 

These curves were constructed as described by Hughes and 

Hellmann (2005) and were used to approach the grade of 

coverage of the detected sequences with respect to the total bacterial community. Thus, when the curve reaches a 

horizontal asymptote, most groups in the analyzed community have been detected.

MICROBIAL DIVERSITY 

IN ALTAMIRA CAVE

Altamira Cave is located in Santillana del Mar (Cantabria, Spain) near the northern coast of Spain. It contains 

some of the most famous paintings from the Paleolithic 

period. Most of these representations were painted in two 

colors, red (mostly hematite) and black (carbon), and were 

designed to take advantage of natural three- dimensional 

structural features of the rock (Lasheras, 2002). Three major types of bacterial colonies have been detected in the cave, 

and they have been named according to their coloration as 

white, yellow, and grey (Figure 1). At present, the effect of 

microorganisms on the substrate or rock forming the cavity 

is unknown, although the phenomenon of carbonate deposition and dissolution has been suggested (Sanchez- Moral 

et al., 1999) as a potential long- term effect caused by the 

development of these communities. Nevertheless, the aesthetic effect produced by their presence on cave walls represents per se a serious biodeteriorating phenomenon. The 

risk of a potential expansion of bacterial colonies is driving 

the investigation into understanding their composition and 

ecology, with the aim of being able to control or reduce 

their presence in the cave. The present study focuses on the 

biocolonization found on the substrate.

Using the dual strategy, DNA and RNA, for molecular detection of microorganisms offers the possibility of 

differentiating those metabolically active from those just 

present (Figure 2). Microorganisms showing no detectable 

metabolic activity represent a large fraction of the communities, which are waiting for more favorable conditions to 

appear. They can become active by changes in the environmental conditions or nutrient availability as well as by the 

elimination of a fraction of the community, which could 

allow the development of those previously inactive microorganisms. The use of biocides is an example that leads to 

FIGURE 1. Photographs of the three different microbial colonies 

observed in Altamira Cave. (A) Yellow and grey colonies and (B) 

white colonies are shown. (C) One of the Altamira’s polychromatic 

paintings representing a bison is also presented.
2 2 • SMITHSONIAN CONTRIBUTIONS TO MUSEUM CONSERVATION

a modifi cation of microbial communities and their equilibria. The consequences of these changes in the communities 

are normally unpredictable. generally, any change of the 

microenvironment or its conditions results in altering the 

long- term equilibrium in that ecosystem, and so it could 

imply the development of different microorganisms into 

a series of uncontrolled unstable associations until a new 

equilibrium is reached in the system. A typical example of 

this scenario is the excessive growth of specifi c types of 

microorganisms, which usually results in undesired effects 

for the conservation of the object under study.

White colonies in Altamira Cave are formed by a large 

number of different microorganisms as detected through 

DNA- based molecular surveys (Portillo et al., 2009). To 

identify the microorganisms that are responsible for the 

major proportion of metabolic activity within the microbial community in white colonies, the use of RNA- based 

molecular techniques has been proposed. For instance, 

FIGURE 2. Microbial community fi ngerprints by DggE for (A) white, (B) yellow, and (C) grey colonies sampled in Altamira Cave analyzed by molecular 

techniques based on DNA and RNA. The microorganisms corresponding to the 

major bands on RNA- based community profi les were identifi ed. Identifi cation 

codes correspond to those in Table 1.
number 2 • 2 3

results showed that heterotrophic bacteria such as Alphaproteobacteria (e.g., Sphingomonas sp.), Gammaproteobacteria (e.g., Pseudomonas sp.), Acidobacteria, and 

Deltaproteobacteria are the major metabolically active 

bacterial groups in these white colonies (Table 1). In addition, many other microbial groups representing minor 

fractions of the total number of microorganisms present 

in these colonies were detected through RNA- and DNAbased techniques.

Another critical aspect was whether the white colonies 

located in different areas in the cave had identical or similar composition. Portillo and Gonzalez (2008a) proposed 

a statistical method for comparing molecular profiles and 

corroborated that the white colonies from different points 

of the cave were composed of the same microbial community, which had been able to spread and colonize specific 

locations along the cave (Portillo et al., 2009).

Yellow bacterial colonies have also been observed, 

although mainly at the entrance to Altamira Cave. The 

major bacterial groups constituting the metabolically 

active fraction of the community in these colonies were 

Gammaproteobacteria, Deltaproteobacteria (e.g., Desulfovibrio sp.), Acidobacteria, and Betaproteobacteria, 

(Portillo et al., 2008). The metabolism and physiology of 

this last group in the cave is practically unknown. Acidobacteria have been reported to represent a huge diversity 

in Altamira Cave, almost equivalent to that reported in 

the whole world (Zimmermann et al., 2005). The presence of active members of the Gammaproteobacteria (i.e., 

Chromatiales and Xanthomonadales) has been reported 

to be responsible for the yellow coloration of these colonies (Portillo et al., 2008).

The major components of the grey colonies in the cave 

were Gammaproteobacteria (e.g., Pseudomonas, enterobacteria), Acidobacteria, and Betaproteobacteria (e.g., 

Thauera).

In addition to finding the major bacterial groups in 

each type of colonization, it was realized that Altamira 

Cave has a huge microbial diversity. Many of these microbes had not been previously identified, but they could 

pose threats to the preservation of the cave. An example 

is the presence of a sulfate-reducing bacterium (SRB) that 

has been recently reported (Portillo and Gonzalez, 2009a). 

Sulfate-reducing bacteria, mainly belonging to the Deltaproteobacteria, have been detected as metabolically active 

bacteria in the cave. The most typical examples detected 

in this group are members of the genera Desulfovibrio and 

Desulfomicrobium. The SRB are anaerobic bacteria that 

reduce sulfates to sulfides, thus helping to maintain localized anaerobic conditions, darkening the rock surfaces by 

reacting with iron minerals present in the rock, increasing 

pH, and enhancing calcium carbonate precipitation. The 

SRB serve as an example of a microbial group that could 

be proposed as a target for monitoring efforts directed to 

detect massive colonization development in the cave.

Another unexpected microbial group detected as metabolically active is represented by the Archaea classified 

as Crenarchaeota (Gonzalez et al., 2006). The diversity 

of this group is high, and they represent practically the 

totality of the Archaea ever detected in the cave (Gonzalez 

et al., 2006). The metabolism and physiological characteristics of these temperate Crenarchaeota are practically 

Table 1. Identification of the major metabolically active bacteria detected in white, yellow, and grey colonies on the rock of 

Altamira Cave. Bands are named as in Figure 2. OTU, organizational taxonomic units.

OTU

Major accession

bands number Taxonomic affiliation

 White colonies

W1 EF188810 Pseudomonas (Gammaproteobacteria)

W2 EF188320 Alphaproteobacteria

W3 EF188427 Alphaproteobacteria

W4 EF188619 Actinobacteria

EF188426 Alphaproteobacteria

W5 EF188321 Betaproteobacteria

W6 AY960228 Acidobacteria

 Yellow colonies

Y1 AY960221 Chromatiales (Gammaproteobacteria)

AY960228 Acidobacteria

Y2 AY960248 Betaproteobacteria

AY960249 Betaproteobacteria

AY960253 Gammaproteobacteria

Y3 AY960231 Desulfovibrio (Deltaproteobacteria)

Y4 AY960236 Actinobacteria

Y5 AY960233 Acidobacteria

AY960224 Gammaproteobacteria

Y6 AY960234 Deltaproteobacteria

Y7 AY960269 Microbacterium (Actinobacteria)

Y8 AY960254 Acidobacteria

Y9 AY960257 Desulfovibrionales (Deltaproteobacteria)

Y10 AY960257 Desulfovibrionales (Deltaproteobacteria)

Y11 AY960220 Acidobacteria

 Grey colonies

G1 AY960273 Pseudomonas (Gammaproteobacteria)

G2 AY960260 Enterobacteriaceae (Gammaproteobacteria)

AY960268 Enterobacteriaceae (Gammaproteobacteria

G3 AY960261 Gammaproteobacteria

G4 AY960270 Betaproteobacteria

G5 AY960263 Pseudonocardia (Actinobacteria)

G6 AY960244 Thauera (Betaproteobacteria)

G7 AY960238 Acidobacteria

G8 AY960220 Acidobacteria
2 4 • smithsonian contributions to museum conservation

unknown. Recently, the first cultured member of the lowtemperature Crenarchaeota was reported (Konneke et al., 

2005). It has an ammonium-oxidizing metabolism, and if 

this is also their role in the cave, massive consumption of 

ammonium ions could cause a significant reduction of the 

pH, resulting in calcium carbonate dissolution.

Other microbial groups have been detected in Altamira Cave as present (on the basis of DNA analyses) 

and/or as metabolically active (from RNA-based analyses), although they represent low proportions of the total 

bacterial communities (Gonzalez et al., 2008). The accumulation curves shown in Figure 3 suggest that most large 

bacterial groups (at the phylum level; Figure 3A) present 

in Altamira Cave have been detected because the curve 

tends to an asymptote. A linear increase of the final portion of the curve in Figure 3B indicates that a great number of the bacterial OTUs remain undetected. So there is a 

huge diversity of different bacterial taxa (Figure 3), which 

introduces a large potential for response to environmental 

changes in the cave and for generating highly dynamic microbial communities.

Most of the bacteria found in Altamira Cave are understudied; little is known about their metabolism. Therefore, their potential importance for cave conservation is 

unknown. Filling this gap will be an important step for 

predicting the potential risk involved in the development 

of specific microorganisms or microbial communities, including bacteria and fungi, and their ability to respond to 

any change in the environment.

MICROBIAL DIVERSITY IN THE NATURAL 

MURIECHO L SHELTER

The region of Aragón harbors a large number of natural shelters containing prehistoric paintings dating from 

the Neolithic period. These represent numerous human 

and animal figures (Figure 4). Different areas covered by 

microorganisms have been detected in the Muriecho L 

shelter (Hameau and Painaud, 2004).

At this natural shelter three characteristic colonizations have been observed (Figure 4). Each has a specific 

microbial community. These colonizations occurred in areas where microbial development was induced by continuous runoff water, in a black crust that covers the upper 

portion of the shelter, and in a cryptoendolithic microbial 

community. These communities were studied using molecular methods based on DNA and RNA.

Results showed that the three microbial communities 

were clearly different (P < 0.001) in their microbial components even when their locations were only separated by 

about 1 m. This reflects the importance of the microenvironments for microbial development and the great capacity of these microbes to adapt to highly specific conditions. 

The phototrophic microorganisms detected in this study 

characterized these communities because they represented 

a major portion of their total DNA and RNA (Table 2). 

Also, they are likely to support the growth of other bacteria with a heterotrophic metabolism based on the consumption of photosynthates produced by cyanobacteria 

and other microscopic phototrophs (generally belonging 

to the Bryophyta).

FIGURE 3. Accumulation curves for the identification of microorganisms detected in the microbial colonies sampled at Altamira 

Cave. Plots corresponding to the (A) identified bacterial phyla and 

(B) organizational taxonomic units (OTUs) are shown. The 1:1 lines 

(dotted lines) are presented for reference.
number 2 • 2 5

The zone influenced by surface water paths showed 

the dominance of the cyanobacteria Anabaena, Nostoc, 

and Microcoleus. Bacteria associated with these phototrophs were mainly represented by Actinobacteria (e.g., 

Pseudonocardia). Chloroflexi represented a major group 

in DNA-based analyses, suggesting that this group might 

be dominant at some seasonal periods, depending on the 

environmental conditions. In addition, these chloroflexi 

were highly related to phototrophic bacteria, specifically 

to the genera Cloroflexus and Roseiflexus. These two genera are generally thermophilic photoheterotrophic bacteria, suggesting that they probably become active and 

develop mainly during the summer period when annual 

temperatures are highest (frequently over 40°C).

An area covered by a black crust was also dominated 

by cyanobacteria and was characterized by Actinobacteria 

(e.g., Crossiella) as the major bacterial component. Other 

bacteria in this area belong to the Gammaproteobacteria 

(e.g., Enterobacteria), Firmicutes (e.g., Bacillus), and Alphaproteobacteria (e.g., Sphingomonas), all presenting 

significant metabolic activity because they were detected 

through RNA analyses.

Cyanobacteria related to the genera Chroococcidiopsis, Phormidium, and Cylindrospermum represented most 

of the RNA from a cryptoendolithic microbial community. 

Bryophyta were also well represented in this community. 

Heterotrophic bacteria detected in this group included 

mainly Bacteroidetes (e.g., Hymenobacter, Chitinophaga), 

the candidate division WYO (which has no cultured representatives), and Actinobacteria (e.g., Rubrobacter). 

Table 2 summarizes the major microbial groups detected 

and quantified through DNA- and RNA-based molecular 

analyses at the three differentially colonized zones studied 

at the natural Muriecho L shelter. Some microorganisms 

were detected by RNA only. This might be a result of the 

low copy number of DNA sequences, which made their 

detection difficult. An abundance of RNA sequences from 

these microorganisms allowed an easier detection through 

RNA than DNA analysis. Results show that most of the 

major microbial components of these communities were 

detected, as suggested by the leveling off of the accumulation curve obtained for the studied shelter (Figure 5).

Clear differences can be observed between the environments of Altamira Cave and a natural shelter. The former 

is an almost-closed site, whereas the latter is in an outdoor 

environment. Thus, different factors might have different influences. Whereas a cave has no illumination (other 

than artificial lights) and generally has relatively constant 

FIGURE 4. Three characteristic types of microbial colonies observed in the natural Muriecho L shelter. (A) A black crust, (B) the development 

of a cryptoendolithic community, and (C) a zone affected by runoff water are shown. (D) Some of the paintings representing human and animal 

figures in this shelter are also presented.
2 6 • smithsonian contributions to museum conservation

humidity and temperature, the natural shelter is exposed 

to natural solar radiation and to a wide range of humidity and temperature conditions. In caves, phototrophs only 

have the opportunity to develop if artificial light is provided, usually for visitors entering the cave. This is not the 

case in Altamira Cave, where permanent illumination has 

been removed to avoid the development of phototrophic 

microorganisms. The natural shelter, however, suffers the 

consequence of large daily and seasonal environmental 

fluctuations in humidity and temperature as well as natural 

illumination. Phototrophs develop well on the natural shelter and represent the basis of the microbial trophic chain. 

Thus, environmental conditions are a critical factor that 

needs to be analyzed in order to evaluate the potential of 

specific microorganisms able to develop at different scales, 

both at the whole site under study and their microhabitats.

CONSEQUENCES OF MICROBIAL DIVERSITY 

ON BIODETERIORATION

The ecology and distribution of microorganisms and 

microbial communities are highly complex. Recent studies 

have shown a huge potential for microorganisms to disperse through different mechanisms, such as air currents 

and transportation by macroorganisms (Gorbushina et al., 

2007; Portillo and Gonzalez, 2008b). Accordingly, microorganisms live almost everywhere, and stone materials are 

no exception. Combining this fact with the existing huge diversity of microorganisms on our planet, it can be easily inferred that microorganisms are the first colonizers of almost 

any material or site available or underexploited by other organisms (plants or animals). However, it is clear that even if 

microorganisms can reach any place on Earth, the environment, by providing a given set of conditions, will constrain 

the growth to those better adapted to develop under them 

(Portillo and Gonzalez, 2008b; Martiny et al., 2006).

Furthermore, microbial communities are dynamic and 

can show alterations with climatic or seasonal changes 

FIGURE 5. Accumulation curve of OTUs for the results obtained 

during the study of the natural Muriecho L shelter. The 1:1 line (dotted line) is shown for reference.

Table 2. Identification of the major bacterial groups detected from DNA and RNA analyses, comprising the 

communities of a zone affected by runoff water, a black crust, and a cryptoendolithic colonization, from the 

natural Muriecho L shelter. The frequency of encountered clones corresponding to a specific bacterial group in 

16S rRNA gene libraries is shown as a percentage. A dash (-) indicates that data are not applicable.

Runoff water Cryptoendolithic affected area Black crust community

Bacterial group DNA RNA DNA RNA RNA

Alphaproteobacteria 20.0 - - 7.1 -

Gammaproteobacteria - - 25.0 21.4 -

Betaproteobacteria - - 5.0 - -

Deltaproteobacteria - - 5.0 - -

Actinobacteria - 22.2 - 57.1 6.7

Bacteroidetes - - 5.0 - 20.0

Firmicutes - - - 14.3 -

Candidate division WYO - - - - 13.3

Chloroplasts (Bryophyta) - - - - 20.0

Chloroflexi 40.0 - 10.0 - -

Cyanobacteria 40.0 77.8 50.0 - 40.0
number 2 • 2 7

(Tayler and May, 2000). Yet to be determined is the rate at 

which microbial colonization occurs. Results from Portillo and Gonzalez (2008b) suggest that microorganisms are 

continuously reaching uncolonized environments and that 

the arrival of a high number and variety of microorganisms can occur within the time frame of a few minutes.

Microorganisms cannot be totally eliminated, and even 

if that were possible, it is questionable whether elimination 

would be a wise decision; what is important to determine 

is the acceptable level of biocolonization, especially for the 

case of buildings and monuments. Biocolonization will be 

undesirable if the growth is excessive and, for example, 

conspicuous colonies or films are formed and if these lead 

to appreciable deterioration of the stone or discolorations. 

However, is there a possibility of controlling stone colonization by microorganisms? The use of biocides has been reported in some studies, although pre- and postapplication 

monitoring has rarely been carried out. Although biocides 

can be effective in eliminating, temporally or definitely, 

specific types of microorganisms, sooner or later, biocolonization will reoccur. If one fraction of the microbial community has been inhibited or removed by biocides, the rest 

of the community will certainly take over. If there are no 

survivors, new microorganisms will arrive. In both cases, 

the resulting microbial community will be unpredictable, 

as will be its effect on the colonized surface. Furthermore, 

most biocides can be subject to physical, chemical, or biological degradation, and so they can become a nutrient or 

result in the release of nutrients for microorganisms. An 

example is benzalkonium chloride, which can be degraded 

by a range of fairly common bacteria (Patrauchan and 

Oriel, 2003). The products of this degradation can also become carbon and energy sources for other bacteria, and the 

ammonium produced leads to changes in pH and fosters 

the growth of ammonium oxidizers or nitrogen-limited microbial species (Patrauchan and Oriel, 2003). Finally, dead 

biomass can become a nutrient source for microorganisms, 

fomenting novel microbes and leading to accelerated colonization events. The removal of microbial communities is 

generally a short-term event that may have serious consequences and, in many cases, adverse results.

The best strategy to control the development of microorganisms appears to be limiting their source of nutrients, 

or substrates for growth, as well as controlling the environmental conditions, such as light, temperature, pH, and 

humidity. By knowing which microorganisms are implicated in a specific colonization process, their physiology 

and metabolism could be investigated, allowing us to identify the specific compounds or elements needed for their 

growth so as to limit them. This is not an easy strategy but 

appears to be a promising alternative for the long-term 

success in controlling the biodeterioration of stone.

Natural or human-caused alteration in the natural 

equilibrium of microbial communities on stone may be 

the cause of undesirable microbial colonization and the 

resulting secondary effects. The interactions among microorganisms and the environment characterize a highly dynamic system representing a fascinating area to explore. 

Although some changes can occur rapidly, in some cases 

the consequences of these interactions may only be evident 

after some years. This can best be exemplified by anthropogenic air pollution and the damage it caused to stone 

materials. Climate change (Battisti and Naylor, 2009), including temperature alterations and changes in rain and 

drought events, may well represent a new factor to be 

monitored in the near future because climate change will 

certainly influence not only microorganisms but the world 

population as a whole.

FUTURE PERSPECTIVES

To ensure that future generations will have the opportunity to appreciate the monuments we enjoy today, 

a careful study of potential physical, chemical, and biological deteriorating factors is required to guarantee their 

conservation. From the microbiological perspective, the 

microbial colonization of stone may result in damage to 

monuments. Microorganisms are numerous and diverse. 

Their action on stone substrates may not be fast, but in 

the long-term their effect may be important. Because stone 

monuments cannot be sterile, at present, a promising strategy for stone conservation is to reduce or eliminate microbial unbalanced or massive growth. This objective can be 

approached in two major ways. The first and obvious line 

of research is to develop novel, rapid, and simple methods 

for the monitoring and evaluating microbial colonization 

on stone materials. This could be achieved by using rapid 

methods of detection and quantification of microorganisms and their activity. As an example, the use of fluorescent methods specific for biomolecules (e.g., Portillo 

and Gonzalez, 2009b) shows great potential. The second 

initiative involves the advancement of environmental microbiology toward closing the current gap of information 

existing between microbial phylogeny and physiology so 

that microorganisms can be identified and their metabolism easily deduced or quickly understood. A result of this 

major progress is that it will allow the development of 

strategies for limiting or eliminating the growth of specific 

microbial communities or microorganisms.
2 8 • smithsonian contributions to museum conservation

The analysis of the microbial communities found in 

the sites studied showed that although the level of diversity in these two environments may be different, the overall microbial diversity is too high to be easily understood 

by current methodological standards. Microorganisms 

can reach any environment on Earth, and removing one 

type of microorganisms will certainly bring others to replace it. Considering this scenario, the strategies of choice 

should focus on monitoring and controlling excessive microbial development. With this aim in mind, a variety of 

factors should be considered, and they range from physical and chemical aspects of the environment surrounding 

the stone materials to the microbiology of the site and specific microenvironments.

Acknowledgments

The authors acknowledge funding through projects 

31321000710T, CGL2006-11561/BTE, and PM055/2006 

from the Spanish Ministry of Culture, the Spanish Ministry of Science and Innovation, and the government of 

Aragon, respectively.

REFERENCES

Battisti, D. S., and R. L. Naylor. 2009. Historical Warnings of Future 

Food Insecurity with Unprecedented Seasonal Heat. Science, 

323:240–244.

Curtis, T. P., W. T. Sloan, and J. W. Scannell. 2002. Estimating Prokaryotic Diversity and Its Limits. Proceedings of the National Academy 

of Sciences of the United States of America, 99:10,494–10,499.

Gonzalez, J. M., A. Ortiz-Martinez, M. A. Gonzalez-del-Valle, L. Laiz, 

and C. Saiz-Jimenez. 2003. An Efficient Strategy for Screening 

Large Cloned Libraries of Amplified 16S rDNA Sequences from 

Complex Environmental Communities. Journal of Microbiological 

Methods, 55:459–463.

Gonzalez, J. M., M. C. Portillo, and C. Saiz-Jimenez. 2006. Metabolically Active Crenarchaeota in Altamira Cave. Naturwissenschaften, 

93:42–45.

———. 2008. Microbes Pose a Risk to Prehistoric Cave Paintings. Microbe, 3:72–77.

Gonzalez, J. M., and C. Saiz-Jimenez. 2004. Microbial Diversity in Biodeteriorated Monuments as Studied by Denaturing Gradient Gel 

Electrophoresis. Journal of Separation Science, 27:174–180.

Gorbushina, A. A., R. Kort, A. Schulte, D. Lazarus, B. Schnetger, H.-J. 

Brumsack, W. J. Broughton, and J. Favet. 2007. Darwin’s Dust: 

Intercontinental Transport and Survival of Microbes in the Nineteenth Century. Environmental Microbiology, 9:2911–2922.

Hameau, P., and A. Painaud. 2004. Schematic Expression in Aragon: 

Presentation and Recent Research. L’Antropologie, 108:617–651.

Hughes, J. B., and J. J. Hellmann. 2005. The Application of Rarefaction 

Techniques to Molecular Inventories of Microbial Diversity. Methods in Enzymology, 397:292–308.

Konneke, M., A. E. Bernhard, J. R. de la Torre, C. B. Walker, J. B. Waterbury, and D. A. Stahl. 2005. Isolation of an Autotrophic AmmoniaOxidizing Marine Archaeon. Nature, 437:543–546.

Lasheras, J. A. 2002. Redescubrir Altamira. Madrid: Turner.

Martiny, J. B. H., B. J. M. Bohannan, J. H. Brown, R. K. Colwell, J. A. 

Fuhrman, J. L. Green, M. C. Horner-Devine, M. Kane, J. A. Krumins, C. R. Kuske, P. J. Morin, S. Naeem, L. Ovreas, A.-L. Reysenbach, V. H. Smith, and J. T. Staley. 2006. Microbial Biogeography: 

Putting Microorganisms on the Map. Nature Reviews Microbiology, 

4:102–112.

Mills, H. J., R. J. Martinez, S. Story, and P. A. Sobecky. 2004. Identification of Members of the Metabolically Active Microbial Populations 

Associated with Beggiatoa species. Applied and Environmental 

Microbiology, 70:5447–5458.

Molin, S., and M. Givskov. 1999. Application of Molecular Tools for 

in situ Monitoring of Bacterial Growth Activity. Environmental 

Microbiology, 1:383–391.

Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of 

Complex Microbial Populations by Denaturing Gradient Gel 

Electrophoresis Analysis of Polymerase Chain Reaction-Amplified 

Genes Coding for 16S rRNA. Applied and Environmental Microbiology, 59:695–700.

Patrauchan, M. A., and P. J. Oriel. 2003. Degradation of Benzyldimethylalkylammonium Chloride by Aeromona hydrophila sp. K. Journal of Applied Microbiology, 94:266–272.

Portillo, M. C., and J. M. Gonzalez. 2008a. Statistical Differences between Molecular Fingerprints from Microbial Communities. 

Antonie van Leeuwenhoek, 94:157–163.

———. 2008b. Microbial Communities and Immigration in Volcanic 

Environments of Canary Islands (Spain). Naturwissenschaften, 

95:307–315.

———. 2009a. Sulfate-Reducing Bacteria Are Common Members of 

Bacterial Communities in Altamira Cave (Spain). Science of the Total Environment, 407:1114–1122.

———. 2009b. Fluorescent Measurements of DNA, RNA, and Proteins 

to Perform Comparative Analyses of Microbial Communities from 

the Environment. Journal of Rapid Methods and Automation in 

Microbiology, 17:398–410.

Portillo, M. C., J. M. Gonzalez, and C. Saiz-Jimenez. 2008. Metabolically Active Microbial Communities of Yellow and Grey Colonizations on the Walls of Altamira Cave, Spain. Journal of Applied 

Microbiology, 104:681–681.

Portillo, M. C., C. Saiz-Jimenez, and J. M. Gonzalez. 2009. Molecular 

Characterization of Total and Metabolically Active Bacterial Communities of “White Colonizations” in Altamira Cave, Spain. Research in Microbiology, 160:41–47.

Sanchez-Moral, S., V. Soler, J. C. Cañaveras, E. Sanz, R. Van Grieken, 

and K. Gysells. 1999. Inorganic Deterioration Affecting Altamira 

Cave: Quantitative Approach to Wall-Corrosion (Solution Etching) 

Processes Induced by Visitors. Science of the Total Environment, 

243:67–84.

Tayler, S., and E. May. 2000. Investigations of the Localisation of Bacterial Activity on Sandstone from Ancient Monuments. International 

Biodeterioration and Biodegradation Journal, 46:327–333.

Ward, D. M., D. Weller, and M. M. Bateson. 1990. 16S rRNA Sequences 

Reveal Uncultured Inhabitants of a Well-studied Thermal Community. FEMS Microbiology Reviews, 75:105–116.

Zimmermann, J., J. M. Gonzalez, W. Ludwig, and C. Saiz-Jimenez. 2005. 

Detection and Phylogenetic Relationships of a Highly Diverse Uncultured Acidobacterial Community on Paleolithic Paintings in Altamira Cave Using 23S rRNA Sequence Analyses. Geomicrobiology 

Journal, 22:379–388.
Christopher McNamara, Harvard School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA; and 

Harvard School of Public Health, Harvard University, Boston, Massachusetts 02215, USA. Nick 

R. Konkol, Brandon P. Ross, and Ralph Mitchell, 

Harvard School of Engineering and Applied Sciences, Laboratory of Applied Microbiology, Harvard University, 40 Oxford Street, Cambridge, 

Massachusetts 02138, USA. Correspondence: 

McNamara, cmcnamar@hsph.harvard.edu.

Characterization of Bacterial Colonization 

of Stone at Global and Local Scales

Christopher McNamara, Nick R. Konkol, 

Brandon P. Ross, and Ralph Mitchell

ABSTRACT. A wide range of microorganisms, including bacteria, archaea, cyanobacteria, algae, fungi, and lichens, have been found on historic stone. Data are presented 

on bacterial colonization of stone at two different scales: global, comparing communities on stone monuments in Europe and southern Mexico, and local, comparing communities on different locations of the same monument. The purpose was to determine if differences in bacterial communities on stone at these two scales could be detected. At the local 

scale, samples of bacterial communities on a headstone in a Massachusetts cemetery were 

collected and compared to 16S rDNA from different locations. The bacterial community 

from the headstone was dominated by cyanobacteria and many species were found at both 

deteriorated and undeteriorated locations. Richness of the cyanobacterial community was 

greater in the undeteriorated location. At the global scale, a meta-analysis comparison of 

published 16S rDNA sequences from bacteria on historic stone was carried out. Globally, microbial communities on historic stone were dominated by similar bacterial groups, 

such as the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria and the 

Actinobacteria. However, at a finer level of resolution (97% similarity of 16S rDNA sequences), communities from around the world appear to be quite different.

INTRODUCTION

Microbial colonization of historic stone may have two effects. First, there 

are aesthetic changes caused by the growth of pigmented microorganisms on or 

just beneath the stone surface. Cyanobacteria, algae, fungi, lichens, and some 

pigmented bacteria are responsible for these effects. Second, many microorganisms play an important role in the deterioration of stone in historic buildings, 

monuments, and archeological sites (e.g., Saiz-Jimenez, 1999), often through 

the action of organic and inorganic acids produced as metabolic by-products of 

biofilm growth.

Whereas aesthetic problems are, almost by definition, obvious, detection of 

microbial deterioration of culturally important stone objects is inherently difficult. In some cases, detection is based on macroscopic observations of pitting 

or etching of stone (Wakefield and Jones, 1998). However, attributing deterioration to chemical, physical, and/or biological agent in a specific case is extremely 

difficult (Koestler et al., 1994). There is no standard technique used to measure 

the potential of a community to cause biodeterioration. Laboratory experiments 

have been used to determine the potential for biodeterioration, and changes in 
3 0 • smithsonian contributions to museum conservation

stone have been analyzed by a number of methods. Some 

examples include measurement of changes in stone mass, 

measurement of calcium loss (from limestone or marble; 

Di Bonaventura et al., 1999; McNamara et al., 2005), 

scanning electron microscopy, nuclear magnetic resonance 

(Alesiani et al., 2000), acoustic wave velocity (Papida et 

al., 2000), and X-ray (Bentz et al., 1995; Jacobs et al., 

1995) or microcomputed X-ray tomography (McNamara 

et al., 2002, 2003).

Laboratory experiments generally require that the 

microorganisms first be cultured. However, studies from 

many environments, including cultural heritage materials, 

have demonstrated that the majority of microorganisms 

cannot be cultured (Jannasch and Jones, 1959; McNamara 

et al., 2003). Furthermore, the metabolic (and thereby 

biodeteriorative) activity of microorganisms growing on 

laboratory media may differ significantly from the activity 

of the same organisms in situ. Recent application of tools 

from molecular biology has revealed a wide range of microorganisms on historic stone, including bacteria (Sand and 

Bock, 1991), archaea (Rölleke et al., 1998), cyanobacteria 

and algae (Tomaselli et al., 2000), fungi (Gorbushina et al., 

1993), and lichens (Garcia-Rowe and Saiz-Jimenez, 1991). 

In particular, the application of molecular techniques has 

revealed the presence of an extremely diverse bacterial assemblage (Gurtner et al., 2000; Schabereiter-­Gurtner et al., 

2001a, 2001b, 2002a, 2002b; McNamara et al., 2006).

However, information is still lacking about the activity and function of these uncultured bacteria. For example, 

in one of the few studies to measure microbial activity on 

historic stone, Tayler and May (2000) found that roughly 

20%–50% of bacteria on sandstone monuments were active. However, their methodology did not provide information about the activity of specific taxonomic groups. 

New studies assessing the presence of 16S rRNA have begun to address the issue of activity in uncultured bacteria 

(Gonzalez et al., 2008) but do not provide information 

about function (i.e., metabolism, biochemistry, or deteriorative activity).

A current assessment of the situation in the field of microbial deterioration of stone suggests that there is substantial knowledge of the taxonomy of microorganisms that 

colonize stone but little information about their in situ activity and function. The purpose of this study was to test the 

observation that the microbial community on culturally important stone has been well described. Data are presented 

on bacterial colonization of stone at two different scales: 

global, comparing communities on stone monuments in Europe and southern Mexico, and local, comparing communities in different locations of the same monument.

METHODS

Local Scale

Samples were collected from a headstone at the Woodlawn Cemetery located in Ayer, Massachusetts. Samples 

from locations on the same headstone were compared to 

minimize variation in the microbial community caused 

by other factors (e.g., age, type of stone, exposure, prior 

cleaning, or conservation treatments). Collection locations 

were chosen to represent undeteriorated and deteriorated 

areas. The condition of the stone was determined by visual examination. Stone from the undeteriorated locations 

was characterized by smooth surfaces and sharp edges 

and corners. In deteriorated locations, the stone surface 

appeared rough, and edges or corners appeared weathered 

and rounded. Microbial growth was not visible to the naked eye at either location.

Microorganisms were removed nondestructively from 

stone using swabs. Swabs were immersed in a dilute detergent solution to facilitate removal of microorganisms, 

and a 1.0 cm2

 area was swabbed. After sampling, swabs 

were placed on ice and returned to the lab. DNA was extracted using the UltraClean Soil DNA Kit (MoBio Labs, 

Carlsbad, California). The 16S rDNA was amplified using the polymerase chain reaction (PCR) as previously described (Perry et al., 2005) with primers 27f and 1492r 

(Lane, 1991). PCR products were cloned into the pCR 

2.2-TOPO vector and transformed into competent Escherichia coli, as described in the manufacturer’s instructions 

(TOPO TA Cloning Kit K4500-01, Invitrogen, Carlsbad, 

California).

Clone inserts were sequenced at the Dana Farber/

Harvard Cancer Center High-Throughput DNA Sequencing Facility (Cambridge, Massachusetts) using a 

3700 DNA Analyzer (Applied Biosystems, Foster City, 

California). Unaligned sequences were compared to the 

National Center for Biotechnology Information (NCBI) 

database using the BLAST search program to find closely 

related sequences (Altschul et al., 1997). Sequences were 

aligned using ClustalX (Thompson et al., 1997). Phylogenetic analysis was performed using PAUP 4.0 beta 10 

(Swofford, 2003). Trees were constructed using neighborjoining distances with 1,000 bootstrap replicates. Groupings that occurred in less than 50% of replicates were 

excluded. The number of operational taxonomic units 

(OTUs) and the Chao statistic were calculated using 

the DOTUR and SONS software packages (Schloss and 

Handelsman, 2005, 2006). Proportional Venn diagrams 

were calculated with VBVenn (Granato, 2002) and drawn 

using Microsoft PowerPoint.
number 2 • 3 1

Global Scale

The DNA sequences were collected from the NCBI 

database using accession numbers found in the publications listed in Table 1. Sequences were aligned using the 

myRDP auto aligner feature of the Ribosomal Database 

Project (RDP) (Cole et al., 2009). Multiple sequences of 

insufficient length (<700 base pairs) or homology were 

eliminated from the alignment. Neighbor-joining trees 

were assembled using the Jukes-Cantor distance correction model and bootstrapped using the Tree Builder feature of the RDP.

The number of OTUs was calculated as described 

above. Sequences were divided into three groups for comparison: sequences from tropical locations, sequences from 

temperate locations, and sequences from caves. Threepart proportional Venn diagrams were calculated using 

DrawVenn (http://theory.cs.uvic.ca/euler/DrawVenn/) and 

drawn using Microsoft PowerPoint.

RESULTS

Local Scale

A total of 62 clones were analyzed from deteriorated 

and undeteriorated locations. Rarefaction analysis indicated that most of the taxa present in the two communities 

were identified (Figure 1). At the 97% level of similarity, 

which is the agreed upon level for delineating different 

species, 15 different OTUs were found on the stone (Figure 2). Three OTUs (20%) were shared between the two 

libraries (27% of the undeteriorated OTUs and 43% of 

the deteriorated OTUs). Using the chao statistic, it was 

estimated that 7.5 OTUs were present in the deteriorated 

sample, and 11 OTUS were present in the undeteriorated 

sample (at the 97% similarity level).

Neighbor-joining analysis demonstrated that sequences from the deteriorated and undeteriorated clone 

libraries were closely related to each other (Figure 3). In 

both locations, the microbial community was dominated 

by sequences related to Cyanobacteria, such as Gleothece, 

Chroococcidiopsis, and Nostoc.

Global Scale

Analysis of sequences obtained from the literature indicated that there were 247 OTUs at the 97% similarity 

level. However, there was minimal overlap between the 

FIGURE 1. Rarefaction analysis of clones sequenced from (A) deteriorated and (B) undeteriorated locations. The number of operational 

taxonomic units (OTUs) at the 97% similarity level was calculated 

using the chao statistic.

Table 1. Sources used in the meta-analysis of bacterial 16S 

rDNA sequences.

Source Number of sequences

Altenburger et al. (1996) 3

Eppard et al. (1996) 8

Gurtner et al. (2000) 82

Heyrman and Swings (2003) 7

McNamara et al. (2003) 10

McNamara et al. (2006) 84

Perry et al. (2003) 8

Piñar et al. (2001a) 3

Piñar et al. (2001b) 13

Rölleke et al. (1996) 10

Schabereiter-­Gurtner et al. (2001a) 27

Schabereiter-­Gurtner et al. (2001b) 23

Schabereiter-­Gurtner et al. (2002a) 41

Schabereiter-­Gurtner et al. (2002b) 22

Schabereiter-­Gurtner et al. (2003) 30

Schabereiter-­Gurtner et al. (2004) 58

Urzì et al. (2001) 6

Vlasceanu et al. (2000) 2
3 2 • smithsonian contributions to museum conservation

FIGURE 2. Venn diagram depicting the relationship between clones 

identified from deteriorated and undeteriorated locations of the same 

headstone. Total number of OTUs is 15, and distance level is 0.03.

FIGURE 3. Phylogenetic relationships based on partial 16S rDNA sequences of clones from 

deteriorated and undeteriorated locations. For neighbor-joining trees, bootstrap values 

based on 1,000 replicates are indicated for branches supported by >50% of trees. Scale bar 

represents 0.1 nucleotide changes per position.
number 2 • 3 3

communities at this level (1 OTU in common between 

temperate and tropical locations and 1 OTU in common 

between cave and temperate locations). Overlap among the 

three groups of sequences was first observed at the 95% 

similarity level, but only 7 of the 221 OTUs were found 

in more than one group (Figure 4a). It was not until similarity decreased to 80% that overlap of the communities 

reached levels similar to those seen in the local samples. At 

80% similarity there were 103 total OTUs, and 26 of these 

(25%) were found in more than one group (Figure 4b).

The neighbor-joining tree constructed with sequences 

from tropical, temperate, and cave locations presented 

a different picture of the relationship between the three 

communities. This analysis demonstrated that sequences 

from all three locations were interspersed among bacterial phyla (Figure 5). The presence of clusters exclusive to 

sequences from one location were absent.

DISCUSSION

Bacterial communities from deteriorated and undeteriorated locations of the same headstone were similar. A 

large percentage of OTUs was found in both locations. 

Furthermore, neighbor-joining analysis indicated that in 

many cases, sequences from deteriorated and undeteriorated locations were closely related. This is not surprising 

given that the sample locations were in close proximity. 

The low level of biological growth suggests that the deterioration observed in some locations was the result of 

nonbiological or, more likely, a combination of biological 

and nonbiological processes.

Differences in the richness of sequences (i.e., the number of different OTUs or taxa) found in deteriorated and 

undeteriorated locations were apparent. Higher microbial 

diversity was expected on the deteriorated stone because 

the rough surface provides for additional microniches 

that can be colonized. In fact, greater richness was observed on the undeteriorated stone. Multiple factors that 

influence richness or diversity have been reported for microbial communities, such as the supply of organic matter 

(Zhou et al., 2002; Waldrop et al., 2006) and interactions 

with other organisms (Taylor et al., 2004; Weinbauer and 

Rassoulzadegan, 2004) or the environment (Rickard et 

al., 2004). The importance of these factors on historic 

stone is unknown. If consistent differences in community richness or diversity could be demonstrated between 

deteriorated and undeteriorated stone, it is possible that 

a metric could be developed to indicate deterioration or 

biodeterioration.

Analysis of 16S rDNA sequences from bacterial communities found on heritage stone throughout the world 

revealed different results when compared by similarity of 

OTUs and by constructing a phylogenetic tree. The phylogenetic analysis (Figure 5) indicated that sequences obtained from locations around the globe were closely related. 

This is in agreement with previous work on photosynthetic 

microorganisms. Although there appear to be differences 

in the photosynthetic microorganisms that colonize various types of stone and building materials (Tomaselli et al., 

FIGURE 4. Overlap between OTUs in bacterial communities among 

cave, tropical, and temperate locations at the (A) 5% and (B) 20% 

similarity levels.
3 4 • smithsonian contributions to museum conservation

2000), the organisms found in tropical and temperate climates are quite similar (Crispim et al., 2003).

In contrast to the phylogenetic tree, comparison of 

OTUs from locations around the globe indicated that the 

communities were quite different from each other. This 

may represent true differences between the communities 

and could be the result of a biogeographic distribution of 

bacteria around the globe. Alternatively, the differences 

could be artifacts of the methodology employed.

Many factors may have contributed to the observed 

differences. For example, sequences used in the “global” 

portion of this study were obtained using different protocols and different primer sets. Also, it is not clear how 

well the communities were sampled or if all sequences were 

published. Furthermore, the sequences were grouped into 

broad categories on the basis of their general location (temperate, tropical, and cave). Grouping of the sequences in 

other categories could provide a different picture of the relationship between bacteria on stone in different locations.

Differences observed among OTUs could also reflect 

real differences among the microbial communities. Each of 

the sites studied has its own history of environmental exposure, materials, and conservation interventions. Analysis 

of the community at one point in time provides a snapshot 

of the community that, in some sense, reflects this history 

but that also may not well represent the community. Moving forward, it will be important to more thoroughly characterize microbial communities throughout the year and 

FIGURE 5. Phylogenetic relationships based on partial 16S rDNA sequences of selected clones 

from publications describing the microbial communities on historic stone (see Table 1). For neighbor joining trees, bootstrap values based on 1,000 replicates are indicated for branches supported 

by >50% of trees. Scale bar represents 0.1 nucleotide changes per position.
number 2 • 3 5

to determine which historical and environmental factors 

are important in shaping the community.

CONCLUSIONS

Sequencing of bacterial communities on stone heritage 

materials has yielded a great deal of information about 

those communities. However, a better understanding of 

the interaction between microorganisms and the stone is 

needed.

Acknowledgments

This research was supported, in part, by grant number 

MT-2210-06-NC-02 from the National Park Service National Center for Preservation Technology and Training.

REFERENCES

Alesiani, M., S. Capuani, F. Curzi, and B. Maraviglia. 2000. “Evaluation 

of Stone Pore Size Distribution by Means of NMR.” In Proceedings 

of the 9th International Congress on Deterioration and Conservation 

of Stone, ed. V. Fassina, pp. 579–585. Amsterdam: Elsevier Science.

Altenburger, P., P. Kämpfer, A. Makristathis, W. Lubitz, and H.-J. Busse. 

1996. Classification of Bacteria Isolated from a Medieval Wall 

Painting. Journal of Biotechnology, 47:39–52.

Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, 

W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: 

A New Generation of Protein Database Search Programs. Nucleic 

Acids Research, 25:3389–3402.

Bentz, D. P., N. S. Martys, P. Stutzman, M. S. Levenson, E. J. Garboczi, 

J. Dunsmuir, and L. M. Schwartz. 1995. “X-ray Microtomography 

of an ASTM C109 Mortar Exposed to Sulfate Attack.” In Microstructure of Cement-Based Systems/Bonding and Interfaces in Cementitious Materials, ed. S. Diamond, S. Mindess, F. P. Glasser, 

L. W. Roberts, J. P. Skalny, and L. D. Wakeley, pp. 77–82. Pittsburgh, Pa.: Materials Research Society.

Di Bonaventura, M. P., M. Del Gallo, P. Cacchio, C. Ercole, and 

A. Lepidi. 1999. Microbial Formation of Oxalate Films on Monument Surfaces: Bioprotection or Biodeterioration? Geomicrobiology Journal, 16:55–64.

Cole, J. R., Q. Wang, E. Cardenas, J. Fish, B. Chai, R. J. Farris, A. S. 

Kulam-Syed-Mohideen, D. M. McGarrell, T. Marsh, G. M. Garrity, 

and J. M. Tiedje. 2009. The Ribosomal Database Project: Improved 

Alignments and New Tools for rRNA Analysis. Nucleic Acids Research, 37, Suppl. 1:D141–D145, doi:10.1093/nar/gkn879.

Crispim, C. A., P. M. Gaylarde, and C. C. Gaylarde. 2003. Algal and 

Cyanobacterial Biofilms on Calcareous Historic Buildings. Current 

Microbiology, 46:79–82.

Eppard, M., W. E. Krumbein, C. Koch, E. Rhiel, J. T. Staley, and 

E. Stackebrandt. 1996. Morphological, Physiological, and Molecular Characterization of Actinomycetes from Dry Soil, Rocks and 

Monument Surfaces. Archives of Microbiology, 166:12–22.

Garcia-Rowe, J., and C. Saiz-Jimenez. 1991. Lichens and Bryophytes as 

Agents of Deterioration of Building-Materials in Spanish Cathedrals. International Biodeterioration, 28:151–163.

Gonzalez, J. M., M. C. Portillo, and C. Saiz-Jimenez. 2008. Microbes 

Pose a Risk to Prehistoric Cave Paintings. Microbe, 3:72–77.

Gorbushina, A. A., W. E. Krumbein, C. H. Hamman, L. Panina, 

S. Soukharjevski, and U. Wollenzien. 1993. Role of Black Fungi 

in Color-Change and Biodeterioration of Antique Marbles. Geomicrobiology Journal, 11:205–221.

Granato, G. E. 2002. “VBVenn—A Visual Basic Program for Calculating 

and Graphing Quantitative Two-Circle Venn Diagrams.” In Volume 2: Project Documentation, ed. G. E. Granato, S. G. Dionne, 

C. K. Tana, and T. L. King, 22 p., National Highway Runoff WaterQuality Data and Methodology Synthesis. Research Report FHWAEP-02-008. Washington, D.C.: Federal Highway Administration.

Gurtner, C., J. Heyrman, G. Piñar, W. Lubitz, J. Swings, and S. Rölleke. 

2000. Comparative Analysis of the Bacterial Diversity on Two Different Biodeteriorated Wall Paintings by DGGE and 16S rDNA 

Sequence Analysis. International Biodeterioration and Biodegradation, 46:229–239.

Heyrman, J., and J. Swings. 2003. “Description of Novel Bacterial Species Associated with Biodeteriorated Mural Paintings Using Molecular Techniques.” In Molecular Biology and Cultural Heritage, ed. 

C. Saiz-Jimenez, pp. 29–34. Lisse, Netherlands: Swets and Zeitlinger.

Jacobs, P., E. Sevens, and M. Kunnen. 1995. Principles of Computerized 

Tomography and Applications to Building Materials. Science of the 

Total Environment, 167:161–170.

Jannasch, H. W., and G. E. Jones. 1959. Bacterial Populations in Seawater as Determined by Different Methods of Enumeration. Limnology and Oceanography, 4:128–139.

Koestler, R. J., P. Brimblecombe, D. Camuffo, W. Ginell, T. Graedel, 

P. Leavengood, J. Petushkova, M. Steiger, C. Urzi, V. Verges-Belmin, 

and T. Warscheid. 1994. “How Do Environmental Factors Accelerate 

Change?” In The Science, Responsibility, and Cost of Sustaining Cultural Heritage, ed. W. E. Krumbein, P. Brimblecombe, D. E. Cosgrove, 

and S. Staniforth, pp. 149–163. New York: John Wiley and Sons.

Lane, D. J. 1991. “16S/23S rRNA Sequencing.” In Nucleic Acid Techniques in Bacterial Systematics, ed. E. Stackebrandt and M. Goodfellow, pp. 115–175. New York: John Wiley and Sons.

McNamara, C. J., T. D. Perry IV, K. Bearce, G. Hernandez-Duque, and 

R. Mitchell. 2005. Measurement of Limestone Biodeterioration Using the Ca2+ Binding Fluorochrome Rhod-5N. Journal of Microbiological Methods, 61:245–250.

———. 2006. Epilithic and Endolithic Bacterial Communities in Limestone from a Maya Archaeological Site. Microbial Ecology, 51: 

51–64.

McNamara, C. J., T. D. Perry, M. Zinn, M. Breuker, G. HernandezDuque, and R. Mitchell. 2003. “Microbial Processes in the Deterioration of Mayan Archaeological Buildings in Southern Mexico.” In 

Art, Biology, and Conservation: Biodeterioration of Works of Art, 

ed. R. J. Koestler, V. H. Koestler, A. E. Charola, and F. E. NietoFernandez, pp. 518–535. New York: The Metropolitan Museum 

of Art.

McNamara, C. J., T. D. Perry, M. Zinn, M. Breuker, and R. Mitchell. 

2002. “Biodeterioration of Concrete and Stone.” In Microbiologically Influenced Corrosion, ed. B. Little. Houston, Tex.: NACE 

International.

Papida, S., W. Murphy, and E. May. 2000. “The Use of Sound Velocity 

Determination for the Non-destructive Determination of Physical 

and Microbial Weathering of Limestones and Dolomites.” In Proceedings of the 9th International Congress on Deterioration and 

Conservation of Stone, ed. V. Fassina, pp. 609–617. Amsterdam: 

Elsevier Science.

Perry, T. D., IV, V. Klepac-Ceraj, X. V. Zhang, C. J. McNamara, M. F. 

Polz, S. T. Martin, N. Berke, and R. Mitchell. 2005. Binding of 

Harvested Bacterial Exopolymers to the Surface of Calcite. Environmental Science and Technology, 39:8770–8775.
3 6 • smithsonian contributions to museum conservation

Perry, T. D., C. J. McNamara, R. Mitchell, and G. Hernandez-Duque. 

2003. “An Investigation of Bacterial Dissolution of Maya Limestone: Biodiversity and Functional Analysis.” In Molecular Biology 

and Cultural Heritage, ed. C. Saiz-Jimenez, pp. 137–140. Lisse, 

Netherlands: Swets and Zeitlinger.

Piñar, G., C. Ramos, S. Rölleke, C. Schabereiter-­Gurtner, D. Vybiral, 

W. Lubitz, and E. B. M. Denner. 2001a. Detection of Indigenous 

Halobacillus Populations in Damaged Ancient Wall Paintings and 

Building Materials: Molecular Monitoring and Cultivation. Applied and Environmental Microbiology, 67:4891–4895.

Piñar, G., C. Saiz-Jimenez, C. Schabereiter-­Gurtner, M. T. Blanco-Varela, 

W. Lubitz, and S. Rölleke. 2001b. Archaeal Communities in Two 

Disparate Deteriorated Ancient Wall Paintings: Detection, Identification and Temporal Monitoring by Denaturing Gradient Gel 

Electrophoresis. FEMS Microbiology Ecology, 37:45–54.

Rickard, A. H., A. J. McBain, A. T. Stead, and P. Gilbert. 2004. Shear 

Rate Moderates Community Diversity in Freshwater Biofilms. Applied and Environmental Microbiology, 70:7426–7435.

Rölleke, S., G. Muyzer, C. Wawer, G. Wanner, and W. Lubitz. 1996. 

Identification of Bacteria in a Biodegraded Wall Painting by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments Coding for 16S rRNA. Applied and Environmental Microbiology, 62:2059–2065.

Rölleke, S., A. Witte, G. Wanner, and W. Lubitz. 1998. Medieval Wall 

Paintings—A Habitat for Archaea: Identification of Archaea 

by Denaturing Gradient Gel Electrophoresis (DGGE) of PCRAmplified Gene Fragments Coding for 16S rRNA in a Medieval 

Wall Painting. International Biodeterioration and Biodegradation, 41:85–92.

Saiz-Jimenez, C. 1999. Biogeochemistry of Weathering Processes in 

Monuments. Geomicrobiology Journal, 16:27–37.

Sand, W., and E. Bock. 1991. Biodeterioration of Mineral Materials by 

Microorganisms—Biogenic Sulfuric and Nitric Acid Corrosion of 

Concrete and Natural Stone. Geomicrobiology Journal, 9:129–

138.

Schabereiter-­Gurtner, C., G. Pinar, W. Lubitz, and S. Rolleke. 2001a. An 

Advanced Molecular Strategy to Identify Bacterial Communities on 

Art Objects. Journal of Microbiological Methods, 45:77–87.

Schabereiter-­Gurtner, C., G. Piñar, W. Lubitz, S. Rölleke, and C. SaizJimenez. 2003. “Acidobacteria in Paleolithic Painting Caves.” In 

Molecular Biology and Cultural Heritage, ed. C. Saiz-Jimenez, pp. 

15–22. Lisse, Netherlands: Swets and Zeitlinger.

Schabereiter-­Gurtner, C., G. Piñar, D. Vybiral, W. Lubitz, and S. Rölleke. 

2001b. Rubrobacter-Related Bacteria Associated with Rosy Discolouration of Masonry and Lime Wall Paintings. Archives of Microbiology, 176:347–354.

Schabereiter-­Gurtner, C., C. Saiz-Jimenez, G. Piñar, W. Lubitz, and 

S. Rölleke. 2002a. Phylogenetic 16S rRNA Analysis Reveals the 

Presence of Complex and Partly Unknown Bacterial Communities 

in Tito Bustillo, Spain, and on Its Paleolithic Paintings. Environmental Microbiology, 4:392–400.

———. 2002b. Altamira Cave Paleolithic Paintings Harbor Partly 

Unknown Bacterial Communities. FEMS Microbiology Letters,

211:7–11.

Schabereiter-­Gurtner, C., C. Saiz-Jimenez, G. Piñar, W. Ubitz, and 

S. Rölleke. 2004. Phylogenetic Diversity of Bacteria Associated 

with Paleolithic Paintings and Surrounding Rock Walls in Two 

Spanish Caves (Llonín and La Garma). FEMS Microbiology Ecology, 47:235–247.

Schloss, P. D., and J. Handelsman. 2005. Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and 

Estimating Species Richness. Applied and Environmental Microbiology, 71:1501–1506.

———. 2006. Introducing SONS, a Tool for OTU-Based Comparisons 

of Membership and Structure between Microbial Communities. 

Applied and Environmental Microbiology, 72:6773–6779.

Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and 

Other Methods). Version 4. Sunderland, Mass.: Sinauer Associates.

Tayler, S., and E. May. 2000. Investigations of the Localisation of Bacterial Activity on Sandstone from Ancient Monuments. International 

Biodeterioration and Biodegradation, 46:327–333.

Taylor, M. W., P. J. Schupp, I. Dahllöf, S. Kjelleberg, and P. D. Steinberg. 

2004. Host Specificity in Marine Sponge-Associated Bacteria, and 

Potential Implications for Marine Microbial Diversity. Environmental Microbiology, 6:121–130.

Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. 

Higgins. 1997. The ClustalX Windows Interface: Flexible Strategies 

for Multiple Sequence Alignment Aided by Quality Analysis Tools. 

Nucleic Acids Research, 24:4876–4882.

Tomaselli, L., G. Lamenti, M. Bosco, and P. Tiano. 2000. Biodiversity 

of Photosynthetic Microorganisms Dwelling on Stone Monuments. 

International Biodeterioration and Biodegradation, 46:251–258.

Urzì, C., L. Brusetti, P. Salamone, C. Sorlini, E. Stackebrandt, and 

D. Daffonchio. 2001. Biodiversity of Geodermatophilaceae Isolated 

from Altered Stones and Monuments in the Mediterranean Basin. 

Environmental Microbiology, 3:471–479.

Vlasceanu, L., S. M. Sarbu, A. S. Engel, and B. K. Kinkle. 2000. Acidic 

Cave-Wall Biofilms Located in the Frasassi Gorge, Italy. Geomicrobiology Journal, 17:125–139.

Wakefield, R. D., and M. S. Jones. 1998. An Introduction to Stone Colonizing Micro-organisms and Biodeterioration of Building Stone. 

Quarterly Journal of Engineering Geology, 31:301–313.

Waldrop, M. P., D. R. Zak, C. B. Blackwood, C. D. Curtis, and D. Tilman. 2006. Resource Availability Controls Fungal Diversity across 

a Plant Diversity Gradient. Ecology Letters, 9:1127–1135.

Weinbauer, M. G., and F. Rassoulzadegan. 2004. Are Viruses Driving 

Microbial Diversification and Diversity? Environmental Microbiology, 6:1–11.

Zhou, J., B. Xia, D. S. Treves, L.-Y. Wu, T. L. Marsh, R. V. O’Neill, A. V. 

Palumbo, and J. M. Tiedje. 2002. Spatial and Resource Factors Influencing High Microbial Diversity in Soil. Applied and Environmental Microbiology, 68:326–334.
Ornella Salvadori, Soprintendenza Speciale per 

il Polo Museale Veneziano, Laboratorio Scientifico, Cannaregio 3553, I-30131 Venice, Italy. 

A. Elena Charola, Smithsonian Museum Conservation Institute, Suitland, Maryland. Correspondence: O. Salvadori, ornella.salvadori@ 

beniculturali.it.

Methods to Prevent Biocolonization 

and Recolonization: An Overview of 

Current Research for Architectural 

and Archaeological Heritage

Ornella Salvadori and A. Elena Charola

ABSTRACT. The paper presents an overview of both the results of relevant case studies 

and the current trends in research that address the control and prevention of biocolonization. Recolonization of architectural and archaeological heritage depends on various 

factors, such as the biocide used and the application methodology, the nature of the stone 

and its state of conservation, the type and degree of colonization before the treatment, 

the application of other products, and, last but not least, on the micro- and macroenvironment surrounding the object. The application of preventive measures to discourage 

recolonization is especially valid for archaeological areas because of the weathered type 

of stone structures and their location. It is known that the effectiveness of biocidal treatments is limited in time if not associated with other control methods. Although the application of water repellents retards biological colonization as long as the surface retains its 

hydrophobicity, they may interfere with the biocidal application or result in the development of disfiguring patterns. The sequential application of these two products, regardless 

of the application order, may result in negative interactions. However, the application of 

mixtures of both products appears to provide a more-effective long-term protection that 

needs to be further researched. Recently developed experimental approaches to control 

microbiological growth involve the use of substances, such as pigment and polysaccharide inhibitors as well as permeabilizers, that make biofilms more vulnerable to biocides 

and the use of antifouling agents derived from plants or marine animals.

INTRODUCTION

This overview deals primarily with methods to prevent biocolonization and 

recolonization. It has focused on visible biocolonization. The presence of microorganisms and organisms on stone surfaces outdoors is generally made evident 

by the soiling, fouling, discoloration, patinas, etc., that they cause. Therefore, 

attention to biodeterioration problems, and consequently the study of them, is 

very often given only after the appearance of visually perceptible signs. It is well 

known that biological alterations may also be “atypical,” difficult to recognize 

and interpret as of biotic nature without any appreciable evidence (i.e., the presence of unpigmented or endolithic microorganisms). The presence of endoliths 
3 8 • smithsonian contributions to museum conservation

on monuments has rarely been reported, mainly because 

of the sampling and identification techniques generally 

used (Salvadori, 2000). 

It is common knowledge that biological growth on stone 

is highly dependent on climatic and microclimatic conditions, 

such as humidity, temperature, and light, and on chemical 

and biological pollution levels. In closed environments, climatic and microclimatic conditions can be managed up to a 

point through microlimate controls and what is referred to 

as “good housekeeping,” whereas the possibility of controlling them outdoors is rather limited (Nugari and Salvadori, 

2003a; Warscheid, 2003). Control of environmental factors 

becomes extremely difficult in archaeological sites, where the 

decision to install covers and roofs is a permanent dilemma. 

Since reducing the water absorption of a material is an important step in reducing the tendency for biocolonization, 

for the case of outdoor exposed stone or wood, the most 

common solution is the application of water-repellent products (Charola, 2003; Panov and Terziev, 2009).

The number of factors influencing biocolonization of 

stone is so large that it is practically impossible to make 

any generalizations. Nonetheless, the type of substrate, 

such as limestone, granite, or mortar, and its bioreceptivity, i.e., the potential of a material to be colonized by 

microorganisms, which is related to its mineralogical and 

petrographic properties, are probably the most important 

ones (Tiano et al., 1995; Shirakawa et al., 2003; Miller et 

al., 2006). Surface roughness, the presence of cavities, and 

porosity, i.e., pore sizes and pore size distribution, in a 

stone have critical importance for moisture retention and, 

consequently, colonization (Caneva et al., 2004). Next 

in importance is the location of the object, whether in a 

closed, semiclosed, or open environment, and its exposure. If outdoors, its geometry will define the areas that 

receive higher moisture input and that will be colonized 

first. When a comparison is made of objects with similar 

substrate, environment, and exposure, the more relevant 

factors are climatic conditions, previous history, and biogeographic characteristics of the site (Caneva et al., 2008).

Objects located outdoors are directly exposed to meteorological phenomena, such as rain, solar irradiation, 

thermal variations, and wind. These will depend upon the 

climate of the site, such as desert, Mediterranean, temperate, or tropical, and the geographic and topographic 

location of the site in question. A further distinction can 

be made between rural and urban environments, where 

microclimatic effects resulting from the presence of vegetation and pollution exert an important positive or negative influence over biocolonization. Weathering of stone 

exposed outdoors, added to the action of microorganisms and pollutants, results in an increase in the natural 

bioreceptivity of the material since a deteriorated surface 

presents a higher surface roughness and porosity, which 

facilitates the attachment of microorganisms to it (Guillitte, 1995). Furthermore, all conservation interventions, 

such as cleaning and the application of various products 

(e.g., biocides, consolidants, and water repellents), will 

modify the bioreceptivity of the substrate. In most cases, 

they will decrease it, but sometimes they may increase it, 

as synthetic polymers can represent potential substrates 

for the development of heterotrophic microorganisms 

(mainly fungi) that can partially degrade and modify them 

(Nugari and Salvadori, 2008a).

It is known that the deposition of particulate matter on 

surfaces is higher for sheltered areas than for those regularly washed by rain, on rougher than on smoother surfaces 

in a given environment, and, in general, proportional to the 

length of the exposure time. Particulate matter is composed 

of many different materials of inorganic, organic, or biological origin. The latter can be subdivided into those with 

a vital nature, such as cells, spores, conidia, and nonvital, 

such as vegetal and animal debris (e.g., leaves, feathers, and 

feces). Surfaces treated with water-repellent products may 

show a tendency to accumulate a higher amount of particulate matter with respect to untreated ones, with significant 

differences depending upon the nature of the applied products, e.g., silicone resins, acrylic resins, or fluoroelastomers 

(Bracci et al., 2002; Moreau et al., 2008b).

Few studies have dealt with the problem of biological 

recolonization, and most of these are recent. In cases when 

objects undergo conservation interventions, apart from 

the normal factors affecting biological colonization mentioned above, the products applied during the intervention 

and the methodology used will influence the recolonization rate. Thus, the number of factors that regulate biocolonization is even higher. Statues in parks and gardens 

present an excellent opportunity to study recolonization 

of stone objects (Charola et al., 2008; Nascimbene and 

Salvadori, 2008). In general, the presence of higher vegetation induces an increase in humidity and a decrease in 

temperature, both of which result in an increase in relative humidity. They also reduce the effects of the wind and 

chemical air pollution while providing nutrients for microorganisms, thus fostering their development. All these factors will influence the mode and timing of recolonization 

and, in general, increase biodeterioration.

Nascimbene and Salvadori (2008) have studied lichen 

recolonization of some limestone statues located in parks 

in three Venetian villas in Italy. A significant difference 

between recolonization times was observed and could 

be related to the consolidation and water-repellent products that had been applied, rather than the biocides used 
number 2 • 3 9

because thorough rinsing had eliminated most residues of 

the latter, and to the environmental conditions. For example, recolonization started after a few weeks on statues 

treated with a fluorinated polymer, whereas it took several 

years for those treated with a silane and/or polysiloxane. A 

comparative study on the durability of different products 

applied to sandstone after exposure in a natural environment for five years evidenced different performances for 

them: acrylic resins and fluoroelastomers were degraded 

in a short time, whereas silicate-based products maintain 

their good performance over time (Bracci et al., 2002). 

The time it takes for biological recolonization is fundamentally important when developing a maintenance plan or 

when planning major conservation interventions to prevent 

objects from attaining a complex biological community since, 

generally, the latter (recolonization) will induce the most significant deterioration (Nascimbene et al., 2009). Thus, the 

minimum intervention criteria should be implemented, reducing both costs and efforts (Charola et al., 2007).

ISSUES IN ARCHAEOLOGICAL AREAS

In situ conservation of archaeological structures requires 

both active and preventive conservation. Active conservation 

indicates those interventions required for the preservation of 

the site. These can be subdivided into emergency interventions, conservation, restoration, regular maintenance, and 

the installation of seasonal or permanent protection systems. 

In most cases, archaeological remains have lost their roofs 

and coverings, so that the structures and their surface finishes are exposed to climatic factors, e.g., rain, wind, and 

thermal changes. Thus, the installation of permanent coverings over archaeological sites reduces their deterioration 

rate by serving to control some of the deterioration factors. 

However, this reduction occurs only if the covering is appropriately designed and well constructed since otherwise the 

deterioration rate will be increased (Laurenti, 2006). The 

effectiveness of these systems depends on several factors, 

among which shape and material are critical. For example, 

transparent roofs increase the temperature of a protected object and allow light to reach its surface, resulting in enhanced 

biocolonization. The greater the number of transparent elements present near the surface of the structure, the higher 

the colonization by photosynthetic microorganisms and 

plants will be, as shown in Figure 1.

In tropical climates, characterized by alternating dry 

seasons and wet seasons with torrential rains, the only 

way to reduce biological recolonization is to change the 

environmental conditions and, in particular, the moisture 

Figure 1. A glass cover over a marble stairway on the archaeological site of Ding Ling, 

China. Note the moisture condensation and the thick growth of plants underneath the glass, 

completely obscuring the sculpted marbles.
4 0 • smithsonian contributions to museum conservation

content in the substrate. Some positive examples can be 

cited, such as that of the Mayan Hieroglyphic Stairway in 

the Copán (Honduras) archaeological site. This site had 

been highly colonized by microorganisms and lichens, and 

many biocidal treatments were carried out since the late 

1970s. Starting in 1985, the monument was covered with 

a tarpaulin protective shelter, initially only during the rainy 

season, but after 1987 the shelter was modified to permanently cover the structure; this improved its protective 

function and the visibility and presentation of the stairway 

(Getty Conservation Institute, 2006) (Figure 2a). In 2000, 

a study was begun to determine the result of this measure 

and showed a drastic reduction of biological colonization. 

This reduction was reflected in the complete disappearance 

of the lichens with only a relatively small amount of cyanobacteria and green algae remaining (Caneva et al., 2005), 

as shown in Figure 2b. Treatment with biocides produced 

a clean surface (Figure 3a), which remained clean if the 

surface was protected (Figure 2b) but was recolonized if 

the surface was unprotected (Figure 3b).

The implementation of the use of metallic strips of 

copper or zinc to prevent recolonization on buildings was 

carried out by Wessel (2003). The toxic effect of heavy 

metals on microorganisms is well known and was the basis 

of many biocides in the past, but because of their negative 

impact on the environment, their use has been discontinued. The effectiveness of the metallic strips relies on the 

slow and limited dissolution of metal ions when rain water 

Figure 2. (a) Protective shelter installed 

over the Hieroglyphic Stairway in Copán 

(Honduras). Nonprotected stones to the 

left of the stairway show a thick blackish 

biocolonization. (b) View of the stairway 

under the tarpaulin. Only a slight covering of cyanobacteria and algae remain, 

and all lichens have disappeared. Note 

that the stone has a natural greenish hue.
number 2 • 4 1

Figure 3. (a) Altar 41, Copán (Honduras). Appearance of a nonprotected carved stone immediately after cleaning and the 

application of a biocide. (b) Detail of the same area, 15 years later, showing intense recolonization by lichens.
4 2 • smithsonian contributions to museum conservation

washes over them. They prove effective for preventing recolonization of clean surfaces but are extremely slow in 

reducing existing biocolonization. The main problems are 

the installation on the structure, its design, and environmental conditions, such as rain frequency, exposure, and 

shading (Guillitte, 1993). Among the problems that this 

system can induce is that some of the ions released can 

stain the substrate, especially if it is a white, porous stone. 

Copper oxides and hydroxides from bronze or copper elements will stain it blue, and zinc compounds from zinc alloy objects may induce a grayish color, as commonly seen 

on the stone bases of metal statues. Eventual deterioration from the presence of these ions has also been reported 

(Gaylarde et al., 2008). However, the application of the 

system is fairly common in areas with high precipitation 

to protect wooden or masonry buildings. 

The application of this system in archaeological sites 

presents a challenge. A pilot project installed metal strips 

on one of the walls of the former houses in the JesuitGuaraní mission of San Ignacio Miní, Misiones, Argentina. The wall was partly cleaned, and three areas on the 

top were covered by a zinc mesh, a bronze mesh, and lead 

strips, respectively (Magadán et al., 2007).

Finally, soft-wall capping of grasses and other plants, 

as an alternative to hard-top capping, has been shown 

to be viable (Viles et al., 2002), but further study of this 

method is needed.

BIOFILMS AND BIOCIDES

Biocolonization results from the colonization of a 

surface by a single species of microorganism or, more frequently, from a community of them. If a community forms, 

it usually produces a biofilm. A biofilm results from the secretion of extracellular polysaccharide substances (EPS) by 

the microorganisms, which will enclose and shield the community from desiccation and other factors, such as biocides. 

The different microorganisms may be in different physiological states. Elimination of a biofilm is usually more difficult than treating a solitary species because biocides have to 

penetrate through the biofilm to reach the microorganisms.

There are three approaches for the elimination and 

prevention of biofilms: (1) regular disinfection of the surface to prevent the attachment of microorganisms and the 

formation of biofilms (Meyer, 2003); (2) elimination or 

destruction of the biofilm using biocides, oxidizing or nonoxidizing substances (Meyer, 2003); and (3) reduction and/

or retardation of the biofilm formation by modifying the 

bioreceptivity of the surface. In this last case, both the type 

of surface and the accumulation of organic matter, which 

would enhance the formation of a biofilm, especially under 

oligotrophic (i.e., low-nutrient) conditions, are crucial.

It is known that biocide effectiveness can be limited by 

the biocide’s penetration capability into a biofilm and its reaction with the EPS material. Each biofilm, although having 

common characteristics, is unique and is characterized by 

its microbial composition, amount and type(s) of EPS, and 

nutrient availability, among other factors. Even for similar 

biofilms, the effectiveness of a biocide will depend on its 

chemical composition, concentration, solvent type, and application method, as well as on the substrate of the biofilm, 

the presence of organic matter on its surface, and meteorological conditions (Nugari and Salvadori, 2003b). With 

regard to the substrate, both its mineralogical composition 

and its porosity are fundamental. Thus, to have similar biocidal effects on biocolonization, different concentrations 

should be used depending on the stone type and taking into 

consideration that some biocidal compounds, such as the 

quaternary ammonium compounds, are strongly adsorbed 

by clay minerals, particularly the expansive ones like smectites (also called the montmorillonite group of clays), which 

can retain them in variable amounts (Young et al., 1995). 

Therefore, sandstones may require higher concentrations of 

these biocides as compared to limestones, but the biocidal 

effect will last far longer for sandstones because of their 

mineralogical composition. It should be noted that the concentration at which these products are applied is not usually 

adapted to the type of substrate requiring the treatment. 

The application of biocides to biocolonized stone can 

be made with two different objectives and methods: (1) application of the biocide and, after its action has been completed, proceeding with the complete removal of any residue 

so as to avoid any negative effects on the substrate and/or 

products to be applied after it and (2) application of the 

biocide without residue removal, so as to retard as much 

as possible the recolonization of the surface. In general, the 

first approach is predominantly used, given the uncertainty 

of the recolonization rate and the usual insufficient information provided for the commercial products (Nugari, 1999).

The second approach is most often used for products 

that have low or no solubility in water and therefore will 

not be easily washed off by rain. One of the active ingredients that has been tested using the second approach is 

a derivative of pyridine (2,3,5,6-tetrachloro-4(methylsulfonyl) pyridine; Gomez-Bolea et al., 1999; Pietrini et al., 

1999). This has been shown to be effective in removing 

photosynthetic microorganisms, fungi, and lichens and in 

keeping the surface clean for five or six years after application. Eight years after the application of this product, the 
number 2 • 4 3

presence of a biological patina made up of bacteria, algae, 

and fungi was apparent but was limited to the lower part 

of the base of the object, up to a height of half a meter 

because of rising damp (Urzì et al., 2000).

It is important to mention that even after half a century 

of studies, testing of biocides is mostly limited to evaluation with regard to their effectiveness in killing target microbes. In the past, the assessment of biocide treatments 

was empirically performed in situ with the naked eye, often 

providing misleading results; today, this evaluation is carried out with laboratory and/or in situ tests with portable 

instruments (Nugari and Salvadori, 2008b; Tretiach et al., 

2008). Only rarely are any substrate interactions with these 

compounds considered (Tudor et al., 1990; Nugari and 

Salvadori, 2003b). Some studies in the past 10 years have 

clearly shown the effects that some biocides may induce on 

the stone, for example, changes in color, capillary water absorption, and mobilization of the calcium ion (Altieri et al., 

1997; Tretiach et al., 2007). Recurrent biocide treatments 

can enhance negative effects on stone, cause resistance in 

treated microorganisms, and change biofilm composition to 

favor the development of more harmful microorganisms.

Comparing the capillary absorption curves before and 

after the application of a biocide (active ingredient: alkylaminotriazine + N’-(3,4 dichlorophenyl)-N,N dimethyl 

urea) on three different stone types, i.e., limestone, sandstone, and granite, it is evident that they show a similar 

behavior to that of samples treated with a water repellent, with a significant decrease in both the absorption 

rate and the total amount of water absorbed (Tretiach et 

al., 2007). Biocides that provide some hydrophobicity to 

the stone, such as this one, have proved to slow down recolonization. For example, lichenocidal effects were still 

observable three years after treatments on some Angkor 

monuments (Uchida et al., 1999). However, it is unknown 

what produces the water-repellent action since for biocides, only the active ingredient is identified and not the 

eventual presence of other molecules that might provide 

other functions, such as adhesiveness or water repellency. 

As a matter of fact, commercial formulations include various substances, and among the most recent developments 

is the enclosing of the biocide within polymers so as to 

have it available on the surface “when needed” (Nugari 

and Salvadori, 2003b). This development, similar to the 

slow release of microencapsulated pharmaceuticals, would 

allow a reduction in the number of required treatments 

and provide a long-term protective action. For example, 

some skin disinfectants currently in use may contain, apart 

from the active biocidal principle, siloxanes or silicones 

that keep the active principle on the skin for longer times.

Among the recent compounds tested in the conservation field, titanium dioxide has been shown to act as 

both an algicide and a fungicide. Titanium dioxide, particularly in the anatase form, is a photocatalyst under ultraviolet light and is capable of oxidizing various organic 

compounds into water and carbon dioxide. On the basis 

of this property it could be expected that some materials activated by solar light could serve to destroy organic 

compounds present on their surface, such as pollutants 

and microorganisms, and would therefore act as “selfcleaners.” In the study of Maury Ramirez and De Belie 

(2009), the reduction of the algal growth on concrete surfaces treated with TiO2

 was evident when compared to the 

untreated ones. On the other hand, Pinna et al. (2009), 

experimenting with ceramic materials in outdoor environments, found only a reduction in fungal growth and practically no influence on the colonization by cyanobacteria, 

green algae, and lichens. The photolytic degradation of 

microorganisms could, perhaps, be improved by increasing the amount of light or the relevant UV component of 

the light.

INTERACTIONS BETWEEN PRODUCTS

In general, conservation interventions are completed, 

after a biocidal treatment, with the application of a water 

repellent. The aim of the latter products is to reduce the uptake of water by the stone and, consequently, its biological 

recolonization. To date, no standard procedure has been 

developed that outlines the order and methodology to be 

followed for the different conservation operations, such as

•	 elimination of the biocide residues by thorough washing,

•	 application of a biocide after the stone object has been 

completely cleaned and before the application of a 

water repellent,

•	 application of a water repellent and then a biocide, and

•	 application of a water repellent mixed with a biocide.

Furthermore, it is known that the effectiveness of a 

water repellent is limited in time, depending on various 

factors (Charola, 2003), and the surface may therefore require regular maintenance with biocides if the aim is to 

reduce recolonization of the object. Although this is a key 

point, the effectiveness of biocides and that of water repellents are, in general, evaluated individually, and the result 

of combining both treatments has only been studied sporadically and to a limited degree (Malagodi et al., 2000; 

Moreau et al., 2008a). It is of fundamental importance to 
4 4 • smithsonian contributions to museum conservation

develop guidelines for the evaluation of the combination 

of biocides with water repellents to determine how the effectiveness of the combined or sequenced application of 

products can be improved. For this purpose, an elucidation of the interaction of these products is necessary so as 

to be able to develop improved products and application 

methodologies.

Sequential application of two different products may 

result in negative interactions. For example, biocides appear to interact with water-repellent products, whether 

they are applied before or after them (Malagodi et al., 

2000). Furthermore, the application of a quaternary ammonium biocide on limestone previously treated with an 

alkylpolysiloxane showed a decrease in the superficial 

water repellency and a modification of the capillary absorption of the stone (Moreau et al., 2008a). However, 

poulticing the surface was sufficient to eliminate the 

biocide and reestablish the original water repellency of the 

surface. In any case, this type of biocide would not remain 

long on the surface of the object if exposed to rain given 

its high solubility in water. These studies only evaluated 

the performance of the water repellent and did not assess 

whether the biocidal effect might be decreased or even 

eliminated if a water repellent was applied as well.

The hydrophobization effect has a variable duration, 

in most cases years, although in other cases it may be 

limited to months or even only weeks. The duration depends on the type of product, concentration, and amount 

applied for a given type of stone. On surfaces that have 

lost their water repellency or have not been treated with a 

water repellent, biological recolonization begins on those 

areas that are directly wetted by the rain, and these are 

correlated to the geometry of the object (Nascimbene et 

al., 2009), as shown in Figure 4. On surfaces that retain 

Figure 4. Two limestone statues in different environments: 

(a) urban environment (Venice, Italy) and (b) rural environment 

in a park in Passariano, Italy, showing that the geometrical configuration of the statues favors wetting of some areas where biocolonization will develop preferentially and that the biocolonization developed is dependent on the environment.
number 2 • 4 5

water repellency, a striped pattern develops where dust 

accumulates and biocolonization develops first (Charola 

et al., 2008). As expected, these stripes develop on the 

areas that receive direct rain or water flow, as shown in 

Figure 5.

DEVELOPMENT OF NEW PRODUCTS

Over the past 10 years different products have been 

developed with the aim of retarding biological colonization for as long as possible. A few scholars have studied 

new repair mortar materials that prevent or retard biological growth, especially for application on archaeological sites. Mortars have a high bioreceptivity given their 

high porosity and surface roughness, which favors their 

colonization by microorganisms and plants, as shown in 

Figure 6.

Antimicrobial activity was obtained by adding different copper compounds, biocides, water repellents or water repellents plus biocide to mortars (Quaresima et al., 

1997; Ferone et al., 2000; Nugari and Salvadori, 2003b). 

Mortars treated with water repellents are effective in preventing algae and lichen colonization. However, the development of fungi seems to be significantly favored by 

this treatment (Mansch et al., 1999). These results were 

confirmed by Urzì and De Leo (2007): fungi, in particular, 

black fungi, were the main colonizers of mortars treated 

with water repellents as they require less water to develop 

and can grow on various polymers (Koestler, 2000).

The application of water repellents and biocides in a 

single step (the products were mixed together before the 

application) is the most effective approach to retard biological growth in culture; it has been effective up to 15 

months after the treatment, especially against algae and 

bacteria (Urzì and De Leo, 2007). However, there are no 

data to compare the effectiveness of each product separately. A commercial product, based on silane derivatives 

Figure 5. (a) Detail of a limestone sculpture 

treated with a water repellent, showing the resulting streaked pattern where biocolonization 

develops (Villa Pisani, Stra, Italy). (b) Detail 

of the garden wall of the Palace of Belem (the 

residence of the president of Portugal) in Lisbon. 

Note that only the render is water repellent, 

showing the streaking effect. On nonhydrophobized surfaces, such as the stone in the arch and 

around the plaque, colonization is more diffuse.
4 6 • smithsonian contributions to museum conservation

Figure 6. (a) A brick wall near the archaeological area of Ostia Antica (Italy) where no capping was installed. The horizontal surfaces capture the water that is retained in the wall, favoring the development of the 

whole range of colonizing species. (b) The same site some years after the installation of the capping.
number 2 • 4 7

and a mixture of biocides, has been shown to provide effective protection even after five to seven years, with little 

biological colonization developing (L. Borgioli, C.T.S., 

Altavilla Vicentina (Vicenza), Italy, personal communication). Another example of good performance of an application of a mixed solution (alkylalkoxysilane plus a 

pyridine derivative) has been reported for sandstone where 

little biocolonization had occurred after six years (GomezBolea et al., 1999). This same treatment displayed good 

results on bricks and mortars but not under continuous 

rising damp conditions (Ariño et al., 2002), confirming 

once again that the long-term effectiveness of these products depends on the microenvironmental conditions and, 

in particular, the water content of the substrate (Caneva 

et al., 2008).

The application of water dispersions of zinc- or tincontaining polymers follows the idea of the metal strips 

(Wessel, 2003). These form a polymeric film on the stone 

surface, where the slow release of the metal ions serves to 

prevent recolonization. So far, these products have only 

been tested in the laboratory, where their bactericidal and 

fungicidal properties have been demonstrated (Chernorukova et al., 2004).

A similar approach was used by the addition of metallic copper, either as fine fibers or as a powder, into the mortar mix. It was found that 0.35% by weight of this element 

was sufficient to retard colonization for more than nine 

years (Henriques et al., 2007). An important factor is the 

physical form since the fine powder was far more effective 

than the same amount of metal in fine fibers. However, it 

is important to point out that the mechanical properties of 

the mortar, such as compressive strength, flexural strength 

and, to a lesser degree, the elasticity modulus were reduced. The porosity and, consequently, the water absorption were also reduced, and there was a slight change in 

color of the mortar toward a bluer shade. 

NEW TRENDS IN PREVENTING 

BIOCOLONIZATION

The European project Biofilm Inhibitors of Damage 

on Materials (BIODAM) (Alatomi et al., 2004; Young et 

al., 2008) proposed a multiphase approach to eliminate 

biofilms or prevent their formation. For this purpose biocides were paired with other chemical substances, such as 

permeabilizing agents, pigment and exopolymer inhibitors, and photodynamic agents, that increase the vulnerability of microorganisms. This approach would reduce 

the amount of biocide required, an important point that 

affects not only conservators but the environment as a 

whole. Permeabilizing agents serve to increase the permeability of the cellular membrane, thus increasing the penetration rate of the biocide. Several permeabilizing agents, 

e.g., polyethyleneimine, meso-2,3-dimercaptosuccinic acid, 

nitrilotriacetic acid, bis(2-ethylhexyl) sulfosuccinate, and 

ethylenediaminetetraacetic acid (EDTA), have been tested 

on biocide-resistant Pseudomonas aeruginosa successfully.

Pigment inhibitors serve to block the development of 

pigments in the microorganisms without causing them to 

die. Thus, compounds such as tricyclazole, arbutin, and 

carpropamid inhibit the formation of melanin and other 

dark pigments in fungi, and cerulein inhibits the synthesis of carotenoids in bacteria. This approach addresses the 

chromatic problem posed by various microorganisms but 

does not prevent the growth of the organisms. Furthermore, the presence of melanin serves as an efficient protective barrier to several environmental factors as well as to 

biocides. Thus, reducing the amount of this pigment will 

make the microorganisms more vulnerable to these agents.

It is known that the EPS play a crucial role in the formation and survival of the biofilm and its adherence to 

the substrate. Therefore, substances such as bismuth derivatives that inhibit the synthesis of exopolysaccharide 

synthesis may serve to actually prevent the formation of 

the biofilm.

Some photodynamic agents, once activated by light, 

can produce free radicals that can destroy any nearby 

cells or damage their cellular membranes. Among these, 

nuclear fast red and methylene blue are capable of annihilating cyanobacteria on stone, and since they degrade 

with light, they do not cause any staining problems on the 

substrate. Although laboratory studies proved encouraging, outdoor pilot testing on sandstone treated with these 

chemicals proved inconclusive (Young et al., 2008).

Antifouling agents, i.e., substances that prevent the 

establishment, adhesion, and growth of microorganisms 

under water, could also prove effective for preventing 

biocolonization. Cuzman et al. (2008) tested natural antifouling agents, some derived from plants, such as capsaicin and cinnamaldehyde, and some from marine animals, 

such as 3-poly-alkyl piridium salts, Ceramium botryocarpum extract, and zosteric acid. These were applied on 

marble or glass samples immersed in water to which a 

biological mixture of cyanobacteria, algae, fungi, and 

protozoa was added. The antifouling compounds were 

mixed with a silicone water repellent and an ethyl silicate 

consolidant. A significant reduction of colonization was 

observed in comparison to that obtained by treatment 

with either the water repellent or the consolidant alone, 

even when these were mixed with a commercial biocide 

(Algophase).
4 8 • smithsonian contributions to museum conservation

CONCLUSIONS

The new products being developed show promise. 

However, until these are available for general use and their 

effectiveness has been proven and their limitations defined, 

the best approaches to reduce biocolonization and the deterioration that it induces on the substrate are (1) modification of the environmental conditions, whenever possible, 

and (2) development of a regular maintenance program after the conservation or restoration intervention. This could 

be limited to the removal of any accumulation of dust or 

soiling, even without the application of a biocide.

It is obvious that further studies are needed with regard to the interaction between biocides and water repellents and mixtures thereof. These studies should focus not 

only on the initial hydrophobization or biocidal properties 

but also on their long-term effects as well as the interactions that may develop between the products with time.

Also, it would be desirable that any compound added 

to a commercial biocidal formulation that will affect the 

performance of the active ingredient should be listed. It is 

evident that this will require the development of a code of 

practice or a code of ethics for industry and commerce as 

well as international standards for product specifications. 

For this to occur, the importance of the issue has to be 

understood.

In conclusion, it is fundamental to be able to determine when biocolonization is desirable and does not pose 

a problem, when it is merely an aesthetic issue, and when 

it is a serious deterioration factor. This is probably the 

most difficult task yet to be solved.

REFERENCES

Alatomi, H. L., N. Arrien, A. A. Gorbushina, W. E. Krumbein, I. Maxwell, C. McCullagh, P. Robertson, N. Ross, M. Saarela, J. Valero, 

M. Vendrell, and M. E. Young. 2004. “Inhibitors of Biofilm Damage on Mineral Materials (BIODAM).” In 10th International Congress on Deterioration and Conservation of Stone, Volume 1, ed. 

D. Kwiatkowski and R. Löfvendahl, pp. 399–406. Stockholm: International Council on Monuments and Sites-Sweden.

Altieri, A., M. Coladonato, G. Lonati, M. Malagodi, M. P. Nugari, and 

O. Salvadori. 1997. “Effects of Biocidal Treatments on Some Italian 

Lithotypes Samples”. In New Concepts, Technologies and Materials for the Conservation and Management of Historic Cities, Sites 

and Complexes, Volume 3, ed. A. Moropoulou, F. Zezza, E. Kollias, 

and I. Papachristodoulou, pp. 31–40. Athens: Technical Chamber 

of Greece.

Ariño, X., A. Canals, A. Gomez-Bolea, and C. Saiz-Jimenez. 2002. “Assessment of the Performance of a Water-Repellent/Biocide Treatment after 8 years.” In Protection and Conservation of the Cultural 

Heritage of the Mediterranean Cities, ed. E. Galán, and F. Zezza, 

pp. 121–125. Lisse, Netherlands: A. A. Balkema.

Bracci, S., M. J. Melo, and P. Tiano. 2002. “Comparative Study on Durability of Different Treatments on Sandstone after Exposure in 

Natural Environment.” In The Silicates in Conservative Treatments. 

Tests, Improvements and Evaluation of Consolidating Performance. 

Proceedings of the International Congress, pp. 129–135. Torino:

Fondazione per le Biotecnologie and Associazione Villa dell’Arte. 

Caneva, G., D. Di Stefano, C. Giampaolo, and S. Ricci. 2004. “Stone 

Cavity and Porosity as a Limiting Factor for Biological Colonisation: The Travertine of Lungotevere (Rome).” In 10th International 

Congress on Deterioration and Conservation of Stone, Volume 1, 

ed. D. Kwiatkowski and R. Löfvendahl, pp. 227–232. Stockholm: 

International Council on Monuments and Sites-Sweden.

Caneva, G., M. P. Nugari, and O. Salvadori, eds. 2008. Plant Biology for 

Cultural Heritage: Biodeterioration and Conservation. Los Angeles: Getty Conservation Institute.

Caneva, G., O. Salvadori, S. Ricci, and S. Ceschin. 2005. Ecological 

Analysis and Biodeterioration Processes over Time at the Hieroglyphic Stairway of Copán (Honduras) Archaeological Site. Plant 

Biosystems, 139(3):295–310.

Charola, A. E. 2003. Water Repellents and Other “Protective” Treatments: A Critical Review. Restoration of Buildings and Monuments, 9(1):3–22.

Charola, A. E., J. Delgado Rodrigues, and M. Vale Anjos. 2008. Disfiguring Biocolonization Patterns after the Application of Water Repellents. Restoration of Buildings and Monuments, 14(5):365–372.

Charola, A. E., M. Vale Anjos, J. Delgado Rodrigues, and A. Barreiro. 

2007. Developing a Maintenance Plan for the Stone Sculptures 

and Decorative Elements in the Gardens of the National Palace 

of Queluz, Portugal. Restoration of Building and Monuments, 

13(6):377–388.

Chernorukova, Z. G., N. Y. Novospasskaya, and D. N. Yemelyanov. 

2004. “New Polymer Biocides for Protection of Monuments from 

Biodestructions.” In 10th International Congress on Deterioration 

and Conservation of Stone, Volume 1, ed. D. Kwiatkowski and 

R. Löfvendahl, pp. 543–546. Stockholm: International Council on 

Monuments and Sites- Sweden.

Cuzman, O. A., P. Tiano, and S. Ventura. 2008. “New Control Methods against Biofilms’ Formation on the Monumental Stones.” In 

11th International Congress on Deterioration and Conservation of 

Stone, , Volume 2, ed. J. W. Lukaszewicz and P. Niemcewicz, pp. 

837–846. Torun, Poland: Nicolaus Copernicus University Press.

Ferone, C., M. Pansini, M. C. Mascolo, and A. Vitale. 2000. “Preliminary Study on the Set Up of Mortars Displaying Biocidal Activity.” 

In 9th International Congress on Deterioration and Conservation 

of Stone, Volume 2, ed. V. Fassina, pp. 371–378. Amsterdam: 

Elsevier.

Gaylarde, C. C., P. M. Gaylarde, and I. B. Beech. 2008. Deterioration 

of Limestone Structures Associated with Copper Staining. International Biodeterioration and Biodegradation, 62:179–185.

Getty Conservation Institute. 2006. The Hieroglyphic Stairway of 

Copán, Honduras. Study, Results and Conservation Proposals. A 

project report. Los Angeles: Getty Conservation Institute.

Gomez-Bolea, A., X. Ariño, R. Balzarotti, and C. Saiz-Jimenez. 1999. 

“Surface Treatment of Stones: Consequences on Lichenic Colonisation.” In Of Microbes and Art, an International Conference on 

Microbiology and Conservation, ed. P. Tiano and C. Feroci, pp. 

233–237. Florence: Centro Stampa Toscana Nuova.

Guillitte, O. 1993. Cinétique de la colonisation végétale de materiaux 

composites de construction. [Kinetics of the Vegetal Colonization 

on Composite Construction Materials.] Ph.D. thesis, Faculté des 

Sciences Agronomiques de Gembloux, Université de Liège, Belgium.

———. 1995. Bioreceptivity: A New Concept for Building Ecological 

Studies. Science of the Total Environment, 167:215–220.

Henriques, F., A. E. Charola, V. Moreira Rato, and P. Faria Rodrigues. 

2007. Development of Biocolonization Resistant Mortars: Pre-
number 2 • 4 9

liminary Results. Restoration of Building and Monuments, 

13(6):389–400.

Koestler, R. J. 2000. “Polymers and Resins as Food for Microbes.” In 

Of Microbes and Art: The Role of Microbial Communities in the 

Degradation and Protection of Cultural Heritage, ed. O. Ciferri, P. 

Tiano, and G. Mastromei, pp. 153–167. New York: Kluwer Academic/Plenum Publishers.

Laurenti, M. C., ed. 2006. Le coperture delle aree archeologiche. Museo 

aperto. [Covers for Archaeological Areas, Open Museums] Rome: 

Gangemi Editore.

Magadán, M. L., M. L. Cedrola, G. M. A. Korth, and A. E. Charola. 

2007. Addressing Biocolonization in the Conservation Project of 

the Portal of the Church at San Ignacio Miní, Misiones, Argentina. 

Restoration of Building and Monuments, 13(6):401–412.

Malagodi, M., M. P. Nugari, A. Altieri, and G. Lonati. 2000. “Effects of 

Combined Application of Biocides and Protectives on Marble.” In

9th International Congress on Deterioration and Conservation of 

Stone, Volume 2, ed. V. Fassina, pp. 225–233. Amsterdam: Elsevier.

Mansch, R., C. Pinck, C. Urzì, F. De Leo, and P. Salamone. 1999. 

“Microbial Colonisation of Silicone Treated Mortar at Schloss 

Weissenstein in Pommersfelden, Germany.” In Of Microbes and 

Art, an International Conference on Microbiology and Conservation, ed. P. Tiano and C. Feroci, pp. 200–205. Florence: Centro 

Stampa Toscana Nuova.

Maury Ramirez, A., and N. De Belie. 2009. Evaluation of the Algaecide Activity of Titanium Dioxide on Autoclaved Aerated Concrete. 

Journal of Advanced Oxidation Technologies, 12(1):1–5.

Meyer, B. 2003. Approaches to Prevention, Removal and Killing of Biofilms. International Biodeterioration and Biodegradation, 51:249–

253.

Miller, A., A. Dionisio, and M. F. Macedo. 2006. Primary Bioreceptivity: 

A Comparative Study of Different Portuguese Lithotypes. International Biodeterioration and Biodegradation, 57:136–142.

Moreau, C., V. Vergès-Belmin, L. Cadot-Leroux, G. Orial, G. Fronteau, 

and V. Barbin. 2008a. Water-Repellent and Biocide Treatments: Assessment of the Potential Combinations. Journal of Cultural Heritage, 9:394–400.

Moreau, C., V. Vergès-Belmin, L. Leroux, A. Kisselinskaia, G. Orial, and 

V. Barbin. 2008b. “The Influence of Protective Treatments on Limestone Soiling, Sulphation and Cleaning: Results after Ten Years of 

Outdoor Exposure.” In 11th International Congress on Deterioration and Conservation of Stone, Volume 2, ed. J. W. Lukaszewicz, 

and P. Niemcewicz, pp. 983–991. Torun, Poland: Nicolaus Copernicus University Press.

Nascimbene, J., and O. Salvadori. 2008. Lichen Recolonization on Restored Calcareous Statues of Three Venetian Villas. International 

Biodeterioration and Biodegradation, 62:313–318.

Nascimbene, J., O. Salvadori, and P. L. Nimis. 2009. Monitoring Lichen 

Recolonization on a Restored Calcareous Statue. Science of the Total Environment, 407:2420–2426.

Nugari, M. P. 1999. “Interference of Antimicrobial Agents on Stone.” In 

Of Microbes and Art, an International Conference on Microbiology and Conservation, ed. P. Tiano and C. Feroci, pp. 211–214. 

Florence: Centro Stampa Toscana Nuova.

Nugari, M. P., and O. Salvadori. 2003a. “Biodeterioration Control of 

Cultural Heritage: Methods and Products.” In Molecular Biology 

and Cultural Heritage, ed. C. Saiz-Jimenez, pp. 233–242. Lisse, 

Netherlands: Swets and Zeitlinger.

———. 2003b. “Biocides and Treatment of Stone: Limitations and Future Prospects.” In Art, Biology and Conservation: Biodeterioration 

of Works of Art, ed. R. J. Koestler, V. H. Koestler, A. E. Charola, 

and F. E. Nieto-Fernandez, pp. 518–535. New York: The Metropolitan Museum of Art.

———. 2008a. “Products Employed in Conservation.” In Plant Biology for Cultural Heritage: Biodeterioration and Conservation, 

ed. G. Caneva, M. P. Nugari, and O. Salvadori, pp. 166–170. Los 

Angeles: Getty Conservation Institute.

———. 2008b. “Stone Materials.” In Plant Biology for Cultural Heritage: Biodeterioration and Conservation, ed. G. Caneva, M. P. 

Nugari, and O. Salvadori, pp. 326–335. Los Angeles: Getty Conservation Institute.

Panov, D., and N. Terziev. 2009. Study on Some Alkoxysilanes Used for 

Hydrophobation and Protection of Wood Against Decay. International Biodeterioration and Biodegradation 63: 456-461.

Pietrini, A. M., S. Ricci, and M. Bartolini. 1999. “Long-Term Evaluation 

of Biocide Efficacy on Algal Growth.” In Of Microbes and Art, 

an International Conference on Microbiology and Conservation, 

ed. P. Tiano and C. Feroci, pp. 238–245. Florence: Centro Stampa 

Toscana Nuova.

Pinna, D., A.M. Lega, and V. Mazzotti. 2009. Monitoraggio e manutenzione di manufatti ceramici situati all’aperto: gli aspetti relativi al 

degrado biologico nel caso di alcuni monumenti faentini [Monitoring and Maintenance of Ceramic Objects in Outdoor Environment: 

Aspects of Biologidal Deterioration for some Faenza Monuments]. 

Arkos 21: 18-25.

Quaresima, R., A. Baccante, R. Volpe, and B. Corain. 1997. “Realisation and Possibility of Polymeric Metalo-organic Matrixes with 

Biocides Activity.” In New Concepts, Technologies and Materials 

for the Conservation and Management of Historic Cities, Sites and 

Complexes, Volume 3, ed. A. Moropoulou, F. Zezza, E. Kollias, and 

I. Papachristodoulou, pp. 323–335. Athens: Technical Chamber of 

Greece.

Salvadori, O. 2000. “Characterisation of Endolithic Communities of 

Stone Monuments and Natural Outcrops.” In Of Microbes and 

Art. The Role of Microbial Communities in the Degradation and 

Protection of Cultural Heritage, ed. O. Ciferri, P. Tiano, and 

G. Mastromei, pp. 89–101. New York: Kluwer Academic/Plenum 

Publishers.

Shirakawa, M. A., I. B. Beech, R. Tapper, M. A. Cincotto, and W. Gambale. 2003. The Development of a Method to Evaluate Bioreceptivity of Indoor Mortar Plastering to Fungal Growth. International 

Biodeterioration and Biodegradation, 51:83–92.

Tiano, P., P. Accolla, and L. Tomaselli. 1995. Phototrophic Biodeteriogens on Lithoid Surfaces: An Ecological Study. Microbial Ecology,

29:299–309.

Tretiach, M., S. Bertuzzi, and O. Salvadori. 2008. “In situ Vitality Monitoring of Photosynthetic Organisms by Chlorophyll a Fluorescence 

Techniques.” In In situ Monitoring of Monumental Surfaces, ed. 

P. Tiano and C. Pardini, pp. 279–286. Florence: Edifir.

Tretiach, M., P. Crisafulli, N. Imai, H. Kashiwadani, K. H. Moon, 

H. Wada, and O. Salvadori. 2007. Efficacy of a Biocide Tested on 

Selected Lichens and Its Effects on Their Substrata. International 

Biodeterioration and Biodegradation, 59:44–54.

Tudor, P. B., F. G. Matero, and R. J. Koestler. 1990. “Restoration of an 

Outdoor Marble Sculpture in New Orleans: A Case Study of the 

Compatibility of Biocidal Cleaning and Consolidation.” In Biodeterioration Research 3, ed. G. C. Llewelly, and C. E. O’Rear, pp. 

525–533. New York: Plenum Press.

Uchida, E., Y. Ogawa, N. Maeda, and T. Nakagawa. 1999. Deterioration 

of Stone Materials in the Angkor Monuments, Cambodia. Engineering Geology 55:101–112.

Urzì, C., and F. De Leo. 2007. Evaluation of the Efficiency of Water 

Repellent and Biocide Compounds against Microbial Colonization 

of Mortars. International Biodeterioration and Biodegradation,

60:25–34.

Urzì, C., F. De Leo, M. Galletta, P. Salamone, and R. Balzarotti. 2000. 

“Efficiency of Biocide in ‘in situ’ and ‘in vitro’ Treatment. Study 

Case of the ‘Templete de Mudejar,’ Guadalupe, Spain.” In 9th International Congress on Deterioration and Conservation of Stone, 

Volume 1, ed. V. Fassina, pp. 531–539. Amsterdam: Elsevier.
5 0 • smithsonian contributions to museum conservation

Viles, H., C. Groves, and C. Wood. 2002. “Soft Wall Capping Experiments.” In Stone, Volume 2, ed. J. Fidler, pp. 59–73. London: English Heritage.

Warscheid, T. 2003. “The Evaluation of Biodeterioration Processes on 

Cultural Objects and Approaches for Their Effective Control.” In 

Art, Biology and Conservation: Biodeterioration of Works of Art, 

ed. R. J. Koestler, V. H. Koestler, A. E. Charola, and F. E. NietoFernandez, pp. 14–27. New York: The Metropolitan Museum of Art.

Wessel, D. P. 2003. “The Use of Metallic Oxides in Control of Biological 

Growth on Outdoor Monuments.” In Art, Biology and Conservation: Biodeterioration of Works of Art, ed. R. J. Koestler, V. H. 

Koestler, A. E. Charola, and F. E. Nieto-Fernandez, pp. 536–551. 

New York: The Metropolitan Museum of Art.

Young, M. E., H. L. Alakomi, I. Fortune, A. A. Gorbushina, W. E. 

Krumbein, I. Maxwell, C. McCullagh, P. Robertson, M. Saarela, 

J. Valero, and M. Vendrell. 2008. Development of a Biocidal Treatment Regime to Inhibit Biological Growths on Cultural Heritage: 

BIODAM. Environmental Geology, 56:631–641.

Young, M. E., R. Wakefield, D. C. M. Urquhart, K. Nicholson, and 

K. Tonge. 1995. “Assessment in a Field Setting of the Efficacy of 

Various Biocides on Sandstone.” In Methods of Evaluating Products for the Conservation of Porous Building Materials in Monuments, pp. 93–99. Rome: International Centre for the Study of the 

Preservation and Restoration of Cultural Property.
Francesca Cappitelli, Federica Villa, and Claudia 

Sorlini, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi 

di Milano, Via Celoria 2, I-20133 Milan, Italy. 

Correspondence: F. Cappitelli, francesca.cappitelli

@unimi.it.

New Environmentally Friendly Approaches 

against Biodeterioration of Outdoor 

Cultural Heritage

Francesca Cappitelli, Federica Villa, 

and Claudia Sorlini

ABSTRACT. Microbial biofilms have a role in the weathering of stone exposed to open 

air. It is a common practice to use toxic products to control microbial fouling. The aim 

of the present research was the development of an innovative, green, and cost-effective 

technology for the prevention of biodeterioration on outdoor cultural heritage surfaces. 

Therefore, the effects of a synthetic analogue of capsaicin (from chili peppers) and zosteric acid (from eelgrass, Zostera marina) in preventing the development of detrimental 

biofilms were studied. In particular, the use of zosteric acid was an effective biocide-free 

strategy against both bacterial and fungal biofilms. Zosteric acid caused a more than 

90% reduction of Escherichia coli and Bacillus cereus adhesion, whereas coverage of 

Aspergillus niger and Penicillium citrinum was reduced by 57%. Under different environmental conditions, zosteric acid reduced Candida albicans (the model fungus for fungal 

biofilm studies) biofilm formation by at least 70%.

THE NATURE OF BIOFILMS

Bacteria have two modes of growth: planktonic and sessile. It is believed 

that planktonic cells suspended or growing in a fluid environment are important 

for rapid proliferation and spread into new habitats, whereas slow-growing sessile cells grow attached to a surface and act in a multicellular organized manner. 

Biofilms are defined as sessile microbial communities embedded in self-produced 

extracellular polymeric substances (EPS) on either organic or inorganic substrata (Costerton et al., 1995). Biofilms are ubiquitous and can be found on 

practically any surface, including those of cultural heritage, such as buildings 

and monuments.

Biofilms include bacteria, algae, and fungi that are responsible for the microbial deterioration of cultural heritage. A further problem associated with 

biofilm formation includes biofouling, the undesirable accumulation of microorganisms, plants, and animals on a surface (Whelan and Regan, 2006). Bacteria 

are generally the first organisms to foul surfaces exposed to different environments, through adhesion and subsequent biofilm formation.
5 2 • smithsonian contributions to museum conservation

The formation of microbial biofilms begins with the 

reversible adhesion of a small number of cells to a surface. 

On the abiotic surface, initial attachment between bacteria and the surface is governed by nonspecific interactions, 

such as electrostatic, hydrophobic, or van der Waals forces. 

Initial studies have proved that the rate of bacterial adhesion to a wide variety of substrata is responsive to some 

physical features, such as hydrophobicity. After binding to 

the surface through a gluelike matrix, bacterial cells begin the process of irreversible adhesion, proliferation, and 

accumulation as multilayered cell clusters. The attached 

cells synthesize new exopolysaccharide material in order to 

cement their adhesion to the surface and to other cells in 

the developing biofilm. These extracellular matrices, composed of a mixture of materials like polysaccharides, proteins, nucleic acids, and other substances, are considered to 

be essential in holding bacterial cells together in the biofilm 

structure, in helping to retain nutrients for cell growth, and 

in protecting cells from dehydration and other stresses. 

This phase is characterized by active binary division of 

attached cells and cell recruitment. Cell accumulation requires coordinated efforts from the microbial community 

to produce a well-organized structure. After having irreversibly attached to a subtratum, bacterial cells undergo 

phenotypic changes. Mature biofilms typically consist of 

differentiated mushroom-like structures of cells embedded within the extracellular polymer matrix. This contains 

voids open to the bulk fluid to allow the transport of nutrients and oxygen from the interface to the inner parts of the 

biofilm and for the removal of metabolic wastes. Bacterial 

cells detached from the biofilm reenter the planktonic state 

and may start a new biofilm formation cycle.

Microbial biofilms display discrete temporal and spatial organization (Pace et al., 2006). There are many microenvironments within a biofilm. The metabolic activities 

of the cells, together with diffusion processes, result in concentration gradients of nutrients, signaling compounds, and 

waste products within the biofilms. A variety of conditions 

are governed by local pH, oxidizing potential (redox), and 

other parameters. Cells close to the surface are exposed to 

high oxygen concentrations, whereas in deeper layers oxygen is hardly available. Gradients for either nutrients or 

metabolites from the biofilm create a heterogeneous environment even for a single-species biofilm. Within a biofilm 

community, cells with diverse genotypes and phenotypes 

follow distinct metabolic pathways, stress responses, and 

other specific biological activities, although they are located side by side (metabolic heterogeneity) (Stewart and 

Franklin, 2008). In order to develop preventive conservation strategies for biofilm control, it is necessary to fully 

understand the mechanisms involved in initial attachment, 

development of the biofilm phenotype, maturation and detachment, and the related regulatory processes.

THE ACTION OF BIOCIDES

There are no current indexes of microbial biodeteriogenic activity that are widely accepted by the scientific 

community involved in the conservation of stone objects 

or structures. Such indexes would be very helpful to plan 

a case-by-case conservation action compatible with the 

principle of minimal intervention (Gazzano et al., 2009). 

For decades, abatement of microbial growth has commonly 

been achieved by applying biocides to surfaces. Biocides are 

chemicals with an active, and in general toxic, effect on living organisms. European Community Directive 98/8/EC of 

the European Parliament addresses the issue of placing biocidal products on the market. In particular, it concerns the 

authorization for the marketing of biocidal products within 

the member states. At the European Community level this 

directive established a list of approved active substances 

that may be used in biocidal formulations. It aims to ensure that a high level of protection is provided for users, 

the public in general, and the environment since it has long 

been recognized that biocidal effects may extend beyond 

the target organism with resulting undesired adverse effects 

to man and the environment (Rasmussen et al., 1999). On 

the basis of the hazard assessment and risk characterization 

for human health, the European Commission has banned 

the use of some frequently used active substances, including 

some commonly used in conservation practice. 

In recent years, the inherent resistance of biofilm microorganisms to biocides has been found to be generalized. 

Bacteria within a biofilm display a variety of phenotypes, 

providing the community as a whole with an enormous 

capability to resist biocides. Treatments with traditional 

doses of biocides are insufficient to destroy all of the biofilm population. Bacteria embedded in the biofilm matrix 

are remarkably more tolerant to biocides, up to 1,000-

fold relative to planktonic cultures of the same bacterial 

strains, depending on the species-drug combination.

In addition, organic biocides might serve as nutrients; therefore, they must be used carefully in conservation practice (Krumbein and Gross, 1992; Leznicka, 1992; 

Warscheid, 2000). Dead cells may also provide nutrients 

for subsequent colonization; therefore, leaving dead cells 

on the surface is not advisable (Flemming, 1998). 

Finally, after exposure to the killing effects of biocides, a small surviving population of persistent bacteria 
number 2 • 5 3

can repopulate the surface immediately and can acquire 

enhanced resistance to further biocidal treatment. 

A number of biofilm characteristics contribute to the 

biocide resistance of biofilm cells, such as the presence of a 

diffusion barrier provided by the EPS, the interaction with 

the exopolymers, the slow growth mode of sessile cells, the 

multicellular nature of the biofilm, and the possible genetic 

expression of certain resistance genes. A biofilm extrapolymeric matrix has the potential to reduce the penetration 

of biocides either by physically slowing their diffusion or 

chemically reacting with them. Sessile cells generally have 

low metabolic activity, thereby resulting in a reduction of 

the antimicrobial susceptibility. Genetic studies indicated 

that, in general, more genes are expressed in biofilms than 

by planktonic cells. Finally, it is thought that persisters, a 

subpopulation of extremely resistant cells, are responsible 

for biofilm regrowth when the treatment is discontinued 

(Pace et al., 2006).

CELL-TO-CELL 

COMMUNICATION SYSTEMS

Cell-to-cell communication systems regulate the expression of a panel of genes, allowing microorganisms 

to adapt to modified environmental conditions. Bacteria 

organize themselves structurally though the synthesis of, 

and response to, intercellular signal compounds (small 

diffusible molecules). The ability to coordinate gene expression in accordance with population density and hence 

to act as a group is a process termed quorum sensing (QS) 

(Balaban, 2008). “Quorum” is the number of individual 

cells needed to induce an activity. In this process bacteria 

produce diffusible hormone-like molecules (autoinducers) 

that interact with specific receptors on themselves and on 

neighboring cells, which, in turn, regulate the expression 

of specific target genes. By integrating this with other environmental stimuli, bacteria are capable of exhibiting 

complex responses and take part in sophisticated interactions, allowing them to survive in adverse environments. 

A large number of bacterial species are known to possess 

this communication mechanism, through which bacteria 

can sense changes in their environment and coordinate 

gene expression in favor of the survival for the entire community. To date, several types of QS systems are known: 

one for gram-positive bacteria relying on polypeptides and 

another for gram-negative bacteria mediated by N-acyl 

homoserine lactone (AHL) derivatives. A third type of 

QS system, AI-2, has been proposed as a global signaling system common to all bacteria (Pace, 2006). Quorum 

sensing in many bacteria regulates a number of physiological activities, including those that involve biofilm 

formation.

NEW STRATEGIES TO PREVENT 

BIOFILM FORMATION

Although it is evident that alternatives to the use of 

biocides for biofilm treatment are needed, very few studies 

in the conservation field have investigated them. The first 

step toward this direction was the EU project “Inhibitors 

of Biofilm Damage on Mineral Materials” (BIODAM). It 

evaluated the combination of biocides with (1) permeabilizers, (2) special slime (EPS) blockers, (3) pigment inhibitors, and (4) photodynamic treatments.

However, to the best of our knowledge, no study in 

the conservation field has investigated the prevention of 

bacterial cell adhesion to the substratum with nontoxic, 

environmentally friendly compounds and the disruption of 

cell-to-cell communication involved in biofilm formation 

through physical, chemical, and biological approaches, as 

proposed for other research areas (Flemming et al., 1996). 

The need for environmentally benign antifouling technologies has led to renewed interest in the ways that organisms protect themselves against predation. Therefore, 

natural nontoxic antifouling compounds represent an attractive strategy. These antifouling compounds have been 

isolated mainly from marine organisms that are not colonized by microorganisms. An interesting antifouling agent 

is zosteric acid (p-sulfoxy cinnamic acid), a natural extract 

from eelgrass (Zostera marina) that prevents biofouling by 

some organisms, such as algae, barnacles, and tubeworms, 

at nontoxic concentrations (Barrios et al., 2005). Most recently, with the further understanding of cell-to-cell communication in microorganisms, QS has emerged as one of 

the most important mechanisms for controlling the development of highly structured and cooperative biofilm consortia, on both organic and inorganic substrata. The QS 

systems offer three points of attack: (1) signal generation, 

(2) the molecule itself, and (3) the signal receptor. Steinberg et al. (1997) isolated halogenated furanones (secondary metabolites) from the Australian macroalga (seaweed) 

Delisea pulchra. These compounds interfered with bacterial signaling and displayed remarkable anticolonization 

activity (de Nys et al., 1993). Furanones protect the alga 

from colonization by both prokaryotes and eukaryotes 

and therefore are strong deterrents of the settlement and 

growth of both micro- and macrofouling (de Nys et al, 

1993). Since this discovery, methods have been developed 
5 4 • smithsonian contributions to museum conservation

to synthesize the natural antifouling molecules and their 

synthetic derivatives in laboratory.

The research goal proposed has been the development 

of this innovative, green, and cost-effective technology for 

the prevention of biodeterioration on outdoor cultural 

heritage surfaces. The results obtained so far indicate that 

this technology may be a powerful tool in the conservation 

field (Cappitelli et al., 2006; Cappitelli and Sorlini, 2008). 

In particular, effects of synthetic analogues to capsaicin 

(Villa et al., 2009) and zosteric acid in preventing the development of detrimental biofilms were studied. This is 

particularly relevant to materials in contact with water or 

exposed to outdoor conditions. Since antifouling agents 

play a strong and specific in situ effect against both microand macrofouling, one strategy considered for preventing 

biofilm formation is to coat cultural heritage surfaces with 

compounds capable of interfering with adhesion or signaling mechanisms. Antifouling agents affixed and immobilized on surfaces should inhibit bacterial attachment and 

retain activity for extended periods. Methods for binding 

antifoulants to surfaces of protectives or consolidants or 

for incorporating them into the products themselves are 

being investigated, as described in the next section.

METHODOLOGY TO SELECT POTENTIAL 

ANTIFOULING AGENTS

Bacteria and fungi used in the experiments were either 

pure or mixed cultures. It is advisable to employ microbial 

isolates from biofilms, especially those growing on a cultural heritage substrate (Figures 1 and 2).

All organisms require a source of carbon and energy 

for growth. A good candidate for an environmentally 

friendly antifouling agent should not be a carbon and/

or energy source for the microorganisms tested. Mineral 

media that would not provide a source of carbon and energy were therefore prepared with the antifoulant added 

at different concentrations. Furthermore, the antifouling 

molecule was used at a concentration below the minimal 

biocidal concentration. To complement these studies, toxicity experiments were conducted in a rich medium to which 

the antifoulant was added at different concentrations. The 

specific growth rates with or without the antifoulant were 

determined via cultivation or by measuring the optical density spectrophotometrically. Alternatively, the viability of 

microbes was measured by staining with a LIVE/DEAD®

BacLightTM Bacterial Viability Kit (Figure 3).

FIGURE 1. Bacterial biofilm on a metal surface (image taken with a 3D HIROX KH-7700 videomicroscope).
number 2 • 5 5

To statistically validate the investigation, biofilm tests 

were carried out in 96-well microtiter plates. There are 

different kinds of microtiter plates in terms of their hydrophobic or hydrophilic character (Figure 4a).

Adhesion can be assessed quantitatively using 

fluorochrome-labeled cells incubated on various black microtiter plate test substrata. Fluorescence was measured using 

a plate reader (Figure 4b). For each experiment, a standard 

fluorescence intensity curve versus cell number (as measured 

by direct counting) and microorganism type was prepared.

FIGURE 3. A LIVE/DEAD® BacLightTM Bacterial Viability Kit was 

applied to estimate both viable and total counts of bacteria. BacLight is composed of two nucleic-acid-binding stains: SYTO 9TM 

and propidium iodide. SYTO 9 penetrates all bacterial membranes 

and stains the cells green (top), whereas propidium iodide only penetrates cells with damaged membranes, producing red fluorescent 

cells (bottom). The pictures show that the tested molecule acted as a 

biocide, killing all cells.

FIGURE 2. (Top) Phase contrast and epifluorescence images of biofilm on adhesive tape strips sampled from an outdoor wall showing 

(middle) DAPI staining and (bottom) hybridization with the universal Eukarya probe EUK 516.
5 6 • smithsonian contributions to museum conservation

There are several parameters that should be taken into 

account to evaluate the efficiency of an antifouling molecule. The most important are the following: type of microorganism, type of surface (hydrophobic or hydrophilic), 

antifoulant concentration, pH, time, and temperature. 

In the past, the assay optimization process has typically 

taken between 4 and 12 months, using traditional onefactor-at-a-time experiments (Altekar et al., 2006). However, statistically designed experiments are a powerful 

tool for improving the efficiency of experimentation. The 

technique of design of experiments (DOE) is an important 

link between the experimental and the modeling world. 

It requires only a small set of experiments where all the 

variables are analyzed simultaneously within fixed ranges 

(selected so they do not inhibit cell growth) according to 

rigorously formulated mathematical protocols. Besides 

identifying the variables that most influence the results, 

DOE serves to identify possible interactions and synergies among factors. With this multivariate approach it is 

possible to obtain a model describing the performance of 

potential antifoulants in complex scenarios. 

Although the microplate assay demonstrates the antifouling properties of a new compound, it does not predict its impact on biofilm architecture. Laboratory-scale 

biofilm reactors combined with microscopic techniques 

are extremely valuable for discovering the fundamental 

characteristics of biofilms (e.g., the spatial and temporal 

patterns of microbial colonization, specific biofilm components, interactions, and cellular activities), providing useful 

insights for developing effective antifouling technologies.

Once a compound has been proven to be effective 

against bacterial attachment, a delivery system must be 

found. This could be a protective or a consolidant, e.g., a 

synthetic polymer (e.g., Villa et al., 2009).

RESULTS

Using the above-mentioned techniques, the antifouling potential of N-vanillylnonanamide and zosteric acid 

was investigated for both bacteria and fungi. Before the 

adhesion tests, it was proved that neither the synthetic 

capsaicin nor zosteric acid was toxic or represented a carbon and energy source for all the tested microorganisms. 

Microbial attachment assays on glass and polysine slides 

(special glass microscope slides with a permanent adhesive) were carried out with N-vanillylnonanamide in dispersion and were applied onto the surfaces using a polymer 

coating (Villa et al., 2009). It required 205 μM (μmol/L) 

of N-vanillylnonanamide to inhibit Bacillus adhesion by 

48% on glass slides. The same compound blended into 

or sprayed onto a polyurethane coating at 205 μmol/kg 

showed that this capsaicinoid did not have any substantial 

impact on preventing adhesion.

As the results from N-vanillylnonanamide investigations were not completely satisfactory, attention was focused on the new potential antifoulant zosteric acid. For 

the studies, zosteric acid was successfully synthesized by 

Domenico Albanese (Dipartimento di Chimica Organica 

e Industriale, Università degli Studi di Milano). Using the 

DOE method, it could be shown that the antibiofilm activity of zosteric acid is species specific, causing a more than 

90% reduction of Escherichia coli and Bacillus cereus

adhesion, whereas Aspergillus niger and Penicillium citrinum coverage was affected by 57% (Villa et al., 2010). 

Zosteric acid reduced Candida albicans (the model fungus for fungal biofilm studies) adhesion and subsequent 

biofilm formation by at least 70%. In addition, the mathematical models revealed that the molecule successfully 

FIGURE 4. (a) The 96-well plates for (left) hydrophobic surfaces and 

(right) hydrophilic surfaces. (b) Multilabel plate readers by Perkin 

Elmer.

a

b
number 2 • 5 7

counteracted the effects of some colonization-promoting 

factors, such as time and temperature (Villa et al., 2010). 

Experiments on Candida albicans showed that zosteric acid strongly impacted biofilm thickness and morphology, resulting from the inability of fungal cells to form 

filamentous structures while maintaining metabolically active cells (Figure 5). Finally, zosteric acid enhanced their 

sensitivity to different traditional biocidal treatments, 

causing a decrease in biofilm resistance of 0.5–8 log units.

By virtue of its ability to reduce microbial adhesion, 

modify the shape of fungal biofilm architecture, and enhance the performance of antimicrobial agents, zosteric 

acid should be further tested and developed as a means of 

preventing biofilm formation, by itself or integrated into a 

more complex protective system.

CONCLUSIONS

Although more research is still needed, environmentally friendly alternatives to toxic biocides to prevent 

biofilm formation represent a very promising method to 

control biocolonization. It is hoped that conservators will 

be eager to use these alternatives to biocides when they are 

technically available. 

REFERENCES

Altekar, M., C. A. Homon, M. A. Kashem, S. W. Mason, R.M. Nelson, 

L. A. Patnaude, J. Yingling, and P. B. Taylor. 2006. Assay Optimization: A Statistical Design of Experiments Approach. Journal of the 

Association for Laboratory Automation, 11:33–41.

Balaban, N., ed. 2008. Control of Biofilm Infections by Signal Manipulation. Springer Series on Biofilms, No. 2. Berlin: Springer.

Barrios, C. A., Q. Xu, T. Cutright, and B. Z. Newby. 2005. Incorporating Zosteric Acid into Silicone Coatings to Achieve Its Slow Release 

while Reducing Fresh Water Bacterial Attachment. Colloids and 

Surfaces B: Biointerfaces, 41:83–93.

Cappitelli, F., P. Principi, and C. Sorlini. 2006. Biodeterioration of Modern Materials in Contemporary Collections: Can Biotechnology 

Help? Trends in Biotechnology, 24:350–354.

Cappitelli, F., and C. Sorlini. 2008. Microorganisms Attack Synthetic 

Polymers in Items Representing Our Cultural Heritage. Applied 

and Environmental Microbiology, 74:564–569.

Costerton, J. W., Z. Lewandowski, D. E. Caldwell, D. R. Korber, and 

H. M. Lappin-Scott. 1995. Microbial Biofilms. Annual Review of 

Microbiology, 49:711–745.

FIGURE 5. Three-dimensional projection of C. albicans biofilm (a, c) without and (b, d) with zosteric acid. The biofilm 

was visualized by confocal laser scanning microscopy after staining with the fluorescence probe FUN1. Images were 

analyzed with IMARIS software. Top is the view as seen from above; bottom is the view as seen sideways.
5 8 • smithsonian contributions to museum conservation

de Nys, R., A. D. Wright, G. M. Konig, and O. Sticher. 1993. New Halogenated Furanone from the Marine Alga Delisea pulchra (cf. fimbriata). Tetrahedron, 49:11,213–11,220.

Flemming, H.-C. 1998. Relevance of Biofilms for the Biodeterioration of 

Surfaces of Polymeric Materials. Polymer Degradation and Stability, 59:309–315.

Flemming, H.-C., T. Griebe, and G. Schaule. 1996. Antifouling Strategies 

in Technical Systems—A Short Review. Water Science and Technology, 34:517–524.

Gazzano, C., S. E. Favero-Longo, E. Matteucci, A. Roccardi, and R. Piervittori. 2009. Index of Lichen Potential Biodeteriogenic Activity (LPBA): 

A Tentative Tool to Evaluate the Lichen Impact on Stonework. International Biodeterioration and Biodegradation, 63:836–843. 

Krumbein, W. E., and M. Gross. 1992. “Interactions of Biocides with 

Cultures of Biodeteriorating Microbiota in Agar Diffusion and 

Rock Tube Tests.” In Proceedings of the Fifth International Congress on Deterioration and Conservation of Stone, ed. J. Delgado 

Rodrigues, F. Henriques, and F. Telmo Jeremias, pp. 501–509. Lisbon: Laboratorio Nacional de Engenharia Civil.

Leznicka, S. 1992. “Antimicrobial Protection of Stone Monuments with 

Phydroxybenzoic Acid Esters and Silicone Resin.” In Proceedings 

of the Seventh International Congress on Deterioration and Conservation of Stone, ed. J. Delgado Rodrigues, F. Henriques, and 

F. Telmo Jeremias, pp. 481–490. Lisbon: Laboratorio Nacional de 

Engenharia Civil.

Pace, J. L., M. E. Rupp, and R. G. Finch, eds. 2006. Biofilms, Infection, 

and Antimicrobial Therapy. Boca Raton, Fla.: CRC Press.

Rasmussen, K., P. Chemin, and P. Haastrup. 1999. Regulatory Requirements for Biocides on the Market in the European Union according 

to Directive 98/8/EC. Journal of Hazardous Materials, 67:237–251.

Steinberg, P. D., R. de Nys, and S. Kjelleberg. 1997. Chemical Defenses 

of Seaweeds against Microbial Colonization. Biodegradation, 

8:211–220.

Stewart, P. S., and M. J. Franklin. 2008. Physiological Heterogeneity in 

Biofilms, Nature Reviews, 6:199–210.

Villa, F., L. D. Albanese, B. Giussani, P. Stewart, D. Daffonchio, and 

F. Cappitelli. 2010. Hindering Biofilm Formation with Zosteric 

Acid. Biofouling, 26:739–752.

Villa, F., L. Giacomucci, A. Polo, P. Principi, L. Toniolo, M. Levi, S. Turri, 

and F. Cappitelli. 2009. N-vanillylnonanamide Tested as a Nontoxic Antifoulant, Applied to Surfaces in a Polyurethane Coating. 

Biotechnology Letters, 31:1407–1413.

Warscheid, T. 2000. “Integrated Concepts for the Protection of Cultural 

Artefacts against Biodeterioration.” In Of Microbes and Art: The 

Role of Microbial Communities in the Degradation and Protection 

of Cultural Heritage, ed. O. Ciferri, P. Tiano, and G. Mastromei, 

pp. 185–202. Dordrecht: Kluwer Academic Publishers.

Whelan, A., and F. Regan. 2006. Antifouling Strategies for Marine and 

Riverine Sensors. Journal of Environmental Monitoring, 8:880–

886.
Eric May and Dania Zamarreño, School of 

Biological Sciences, University of Portsmouth, 

King Henry I Street, Portsmouth PO1 2DY, 

Hampshire, UK. Sarah Hotchkiss, Cybercolloids, Carrigaline Industrial Park, Carrigaline, 

County Cork, Ireland. Julian Mitchell, School of 

Biological Sciences, University of Portsmouth, 

King Henry I Street, Portsmouth PO1 2DY, 

Hampshire, UK. Robert Inkpen, Department 

of Geography, University of Portsmouth, Buckingham Building, Lion Terrace, Portsmouth, 

Hampshire PO1 2HE, UK. Correspondence: 

E. May, eric.may@port.ac.uk.

Bioremediation of Algal Contamination 

on Stone

Eric May, Dania Zamarreño, Sarah Hotchkiss, 

Julian Mitchell, and Robert Inkpen

Abstract. Green algae are common colonizers of stone throughout the world. Research suggests that algae do not cause biodeterioration directly, but the growth of such 

organisms facilitates colonization by other organisms. Standard treatment methods to 

date have centered on abrasive and chemical treatments rather than biologically based 

solutions. It is widely accepted that risk assessments for proposed treatments should include the potential for regrowth or accelerated growth and any impact upon the structure 

of the stone. In addition to these side effects, the use of chemical treatments is increasingly 

subject to tighter control from an environmental aspect. Significant research has been 

carried out on naturally occurring algicides for control of aquatic algal species. Work in 

the aquatic field suggests that some effective treatments for dealing with growths of algae 

exist, but caution and extensive testing are likely to be needed to assess the viability of 

these treatments to cross over to terrestrial heritage environments. In recent years, ecological studies of naturally occurring biological control of aquatic algae have focused on 

viruses, which cause lysis and subsequent death of the host organism. Viruses are found 

in sediments and are believed to be important in natural control of algal populations. 

This paper will describe pilot studies to assess the relative effectiveness and impacts of 

bioremediation by viruses for removal of growths of algae from Portland limestone.

INTRODUCTION

Green algae are common colonizers of stone throughout the world, and 

reports of the growth of this group of organisms on buildings and monuments 

come from across Europe (Rifón-Lastra and Noguerol-Seoane, 2001; Darienko 

and Hoffmann, 2003), southeast Asia (Uchida et al., 2000; Lee et al., 2005), 

Central America (Ortega-Morales et al., 2000), North America (Koestler et al. 

1985), and South America (Crispim, 2004; Gaylarde and Gaylarde, 2005). Standard treatment methods to date have centered on abrasive and chemical solutions rather than biologically based solutions. 

The factors affecting growth of algae and other phototrophic organisms on 

monuments were reviewed by Ortega-Calvo et al. (1995). They found no proof 

of biodeterioration directly caused by algae but point out that the growth of such 

organisms represents a significant input of organic matter to the stone, which 

would, in turn, facilitate colonization by other organisms. They stressed the importance of understanding the potential for synergistic interactions among the 
6 0 • SMITHSONIAN CONTRIBUTIONS TO MUSEUM CONSERVATION

microbial communities within a stone ecosystem. Young 

(1997), in a study examining algal growth on sandstone, 

found that the change in composition of a “natural” soiling layer was leading to a change in the biological community colonizing the stone as resident pollution products 

such as sulfate were removed. Differences in algal growth 

were found to be related to the chemical cleaner used; in 

some cases a higher growth rate and greater algal growth 

were found on chemically cleaned surfaces than on untreated surfaces. MacDonald (1993) noted the retention 

of chemicals in the pores of the stone following cleaning. 

Young (1997) compared pore size and phosphate residues after treatment and concluded that the latter was responsible for increased algal growth but that the effect of 

phosphate varied between sandstones, depending on their 

mineral composition (see also Young at al., 1995). This 

suggests that any proposed treatment needs to be assessed 

over an extended period (two years or more) to assess the 

potential for both regrowth and accelerated growth. Additionally, any proposed treatment will need to be assessed 

for its impact upon the structure and subsequent behavior 

of the stone microfl ora (Tayler and May, 1994).

Signifi cant research has been carried out on naturally 

occurring algicides for control of aquatic algal species. 

Sources for these algicides have included higher plants 

such as barley straw (Ball et al., 2001), Juncus acutus

(Dellagreca et al., 2003), and Potamogeton natans (Cangiano et al., 2001); marine algae (König et al., 1999); and 

fungi (Jenkins et al., 1998). With respect to bacteria, two 

separate Bacillus species have been identifi ed with antimicrobial activity against cyanobacteria (Reim et al., 1974; 

Nakamura et al., 2003). Work in this fi eld suggests that 

existing effective treatments for dealing with algae growth 

exist, but caution and extensive testing are likely to be 

needed to assess the ability of these treatments to cross 

over to terrestrial environments. Some work has been carried out already on such “crossover” treatments. Choi et 

al. (2005) reported the use of Streptomyces neyagawaensis

in suppressing the growth of cyanobacteria and a wide 

range of algae, including the epilithic green algae Chlorella, several species of which were found on granite walls 

in northwest Spain (Rifón-Lastra and Noguerol-Seoane, 

2001) and on building facades by Schumann et al. (2005). 

Such studies are, however, limited, and no standard methodology for testing has been developed. In recent years, 

ecological studies of naturally occurring biological control 

of aquatic algae have focused on viruses, which cause lysis 

and subsequent death of the host organism. Viruses are 

found in sediments and are believed to be important in 

natural control of algal populations (Bratbak et al., 1996; 

Suttle, 2002). Numerous viruses have been identifi ed 

that infect algae, including the chloroviruses that infect 

Chlorella- like green algae (Kang et al., 2005). 

This paper describes attempts to investigate the relative effectiveness and impacts of bioremediation by viruses 

for the removal of algae growths on Portland limestone 

gravestones. The research strategy had three components: 

(1) to identify the nature of the problem on gravestones 

and other Portland Stone surfaces, (2) to isolate viruses 

with antialgal activity from mature biofi lms, (3) to test the 

effectiveness of different viral treatments in laboratory pilot studies. 

METHODS

study site And sAmpling regime

The study site was Brookwood Military Cemetery in 

Surrey, United Kingdom, which is administered and in 

the care of the Commonwealth War graves Commission. 

Headstones were chosen from sections 11 and 12 (British 

and Australian) of the cemetery (Figure 1), where algal contamination was known to be a serious problem during the 

winter months. All headstones were of Portland limestone 

and had a similar aspect (facing northwest) but were infl uenced to varying degrees by overhanging or shading vegetation. Most headstones had splash plants in front of them, 

but these varied in size, shape, and form. All headstones 

FIGURE 1. Study headstones at Brookwood Military Cemetery. All 

headstones are of similar aspect and known age, but environmental 

conditions vary. The oak tree to the right and stand of trees to the left 

were considered very likely to have an effect on algal growth.
number 2 • 6 1

were originals and therefore of a known age, but they were 

a mix of first and second World War headstones. 

Image Analysis

High-resolution digital photographs were taken of 

the front and back of each gravestone (Figure 2). Three 

zones of biological contamination were identified on most 

stones, at the crown, central area, and base of the headstone (Figure 2). Using the Corel Photopaint v11 software 

package, the surface area of each zone of contamination 

was measured, and percent cover for crown, central area, 

base, and total area of contamination was calculated.

Biological Sampling

Biological material was removed from representative 

headstones using sterile razor blades and then placed in 

sterile containers for transportation to the laboratory. 

Small surface scrapes were taken where possible in all 

three algal contamination zones on back and front of 

headstones. Every effort was made to ensure that minimal 

physical damage was inflicted on the surface of the headstone and that no unsightly marks were left. 

In the laboratory, a portion of each sample was suspended in sterile water and observed under 40× and 100× 

magnification using a Leitz Laborlux S microscope. Digital images were taken using a Leica IM1000 image capture 

system. Images were collected for all observed taxa from 

each sample in order to estimate the biological diversity of 

each sample. A digital record of diversity was prepared.

Culturing

Biological samples were cultured in the laboratory using 

modified Knops medium and an ambient temperature and 

FIGURE 2. Image analysis of a representative second World War headstone. (left) A digital image of the back of the 

headstone with three zones of algal contamination at the crown, central area, and base. (right) Extracted zones of algal 

cover (using Corel Photopaint) from which the percentage area cover of algal contamination was calculated.
6 2 • smithsonian contributions to museum conservation

light regime. The medium was chosen for its suitability for 

culturing both algae and cyanobacteria and the growth regime, as it represented the most natural scenario. Algal cultures appeared to be reaching a level of growth suitable for 

experimental manipulation in approximately one month. 

Portland limestone test surfaces were inoculated with algal 

cultures for use in subsequent phases of the project. Solid 

and liquid cultures containing small Portland limestone 

discs approximately 15 mm in diameter and larger discs approximately 4 cm diameter and 1 cm thick were inoculated 

with purified algal cultures and virus dilutions.

Assessment of Algal Cover

The algal contamination percentage cover and the 

biological diversity data were analyzed using the PRIMER 

v6 software package. PRIMER is a multivariate statistical 

package specifically developed for the analysis of complex 

ecological data (Clarke and Warwick, 2001; Clarke and 

Gorley, 2006). The underlying strategy was to highlight 

any patterns of similarity or dissimilarity across samples 

of data on the basis of their biotic composition. The results were then compared with known or hypothesized 

interrelations between predetermined groups of samples. 

In this research, the predetermined groups were samples 

taken from groups of headstones influenced by different 

environmental factors. Age of headstone, front versus 

back of headstone, splash plant, proximity of tree cover 

and/or shading, and whether headstones were placed in 

singles, doubles, or triples were all investigated.

A typical output of this kind of analysis is an ordination (Figure 3) based on multidimensional scaling (MDS). 

The ordination gives a two-dimensional visual representation of how similar or dissimilar the groups of samples are 

and thus provides a tool for rapid analysis of biological 

patterns. The example given in Figure 3 shows two distinct groups of samples; in this case it shows a subsample 

of the complete Brookwood data set. Group 1 represents 

first World War headstones, and group 2 represents second World War headstones. 

RESULTS

Surveys to Determine the Extent of Algal Cover

The aim of the surveys was to assess the effectiveness 

of image analysis as a tool for monitoring patterns of algal 

colonization on headstones and the effects of any treatment process. The technique proved to be extremely accurate and rapid, and high-quality digital images of selected 

headstones were archived. These were used for comparative studies with images taken during future surveys with 

the aim of monitoring any changes to seasonal patterns of 

algal contamination and also for comparing headstones 

that have and have not been treated using bioremediation. 

During September 2006 there were distinct differences 

in patterns of algal growth on selected headstones, and 

these varied over broad scales throughout the study area. 

However, headstones experiencing similar environmental 

conditions had similar levels of algal contamination. The 

percentage cover analysis revealed distinct groups of headstones that reflected known environmental patterns. Figure 

4 shows an MDS ordination in which groups of headstones 

are evident and have been highlighted by colored circles.

FIGURE 3. MDS ordination from PRIMER percentage cover analysis of algal contamination on headstones. Two distinct groups of 

samples can be seen in this ordination, although there are some outliers. Each number represents either a first or second World War 

headstone. The closer the samples group are together in the ordination, the more similar they are in biotic composition.

FIGURE 4. MDS ordination showing similarity in percentage cover 

of algal contamination between groups of headstones in September 

2006. The four color-coded groups represent groups of headstones 

influenced by varying levels of tree cover.
number 2 • 6 3

As would be expected with any study of this kind, 

there was much variability, and some samples did not necessarily group with others, but essentially, the samples fell 

into groups representing headstones affected by varying 

levels of tree cover along a gradient from no tree cover 

(green group) to full tree cover (red group). The other 

groups (dark and light blue) were situated at the edge of a 

tree canopy and thus are intermediate in their physical environment and do not separate out into distinct groups but 

form a continuum. It must be noted that the groups highlighted in Figure 4 are preselected and represent groups of 

headstones with expected differences. Nevertheless, they 

clearly fall along a known environmental gradient that 

suggests that tree cover has a strong effect on patterns of 

algal cover on headstones.

Although proximity of tree cover was expected to 

have a major effect on patterns of algal colonization, further analysis of the data showed a number of other environmental factors could be influencing the patterns of 

algal growth on smaller scales. Age of headstone and aspect, i.e., front versus back of headstone, also appeared to 

have some influence. 

A second survey confirmed that broad-scale patterns 

of algal contamination were evident in October 2006 (Figure 5) and that the effect of tree cover was still the most 

influential factor. Unlike in the initial survey, an additional 

group of headstones were investigated that were situated 

close to a stand of trees but not directly beneath the canopy. These headstones were shaded for parts of the day and 

were in full sunlight for parts of the day. Figure 5 clearly 

shows a gradient in patterns of algal cover that directly 

corresponds with a gradient in tree cover. Algal cover on 

headstones under a tree canopy (red group) is different 

from that on headstones not affected by tree cover (dark 

blue). Headstones situated at the edge of the canopy (light 

blue group) are somewhat intermediate in algal cover. The 

group of headstones situated close to a stand of trees has 

variable patterns of algal cover, and these are similar to 

a range of other headstones placed throughout the study 

area. Given that these headstones would be influenced to 

varying degrees by tree cover, this result is to be expected.

Further analysis showed there was little influence 

from the age of a headstone (Figure 6a) or the front versus 

back aspect (Figure 6b) of a headstone as suggested by 

the first survey. The presence of a splash plant (Figure 6c) 

also appears to have little effect on patterns of algal cover, 

although this would be expected to affect only the base 

and possibly the central area of the headstone, and further 

analysis is required to fully assess this. 

Characterization of Algae

Biological samples were taken from Brookwood and 

Portsmouth limestone structures to assess the diversity 

of microorganisms present. In order to develop remedial 

FIGURE 6. MDS ordinations of percentage algal cover data in October 2006. (a) Age of headstone, (b) front or back aspect, and (c) 

presence and type of splash plant appear to have little effect on patterns of algal contamination.

FIGURE 5. MDS ordination showing similarity in percentage cover 

of algal contamination in October 2006 between groups of headstones influenced to varying degrees by tree cover.
6 4 • smithsonian contributions to museum conservation

treatments, information was required on the number of 

different organisms present, their relative abundances, and 

their spatial variability. Microscopic analysis suggested 

the algal growth on the Brookwood headstones to be diverse. However, the microorganisms found in the three 

contamination zones on each headstone were different, 

but this difference was not quantified. The crown, which 

is the area that receives annual remedial treatment, appears to host the least diverse biological community and 

comprises mainly single-celled and colonial green algae, 

predominantly Chlorococcales, including Chlorella spp., 

Chlorococcum, and Nitzchia. The central and basal areas host far more diverse communities comprising green 

algae, cyanobacteria, diatoms, and golden brown algae. 

Isolation of Algae and Detection of Viruses

Viral assay and growth requires individually pure cultures of significant density, and these were very difficult to 

obtain from the mixed algal communities isolated from 

stone. Stone algae grew very slowly in the laboratory, both 

in pure liquid culture and on stone, which impeded isolation and experimentation with viruses. Contamination of 

the algal cultures obtained from stone with bacteria and 

fungi was also a major technical problem and clearly made 

assay and amplification of viruses very difficult. To prevent contamination, antibiotics, ampicillin, streptomycin, 

and nystatin were added to the growth medium. Using 

this approach, environmental samples could be processed 

and any algae present could be grown successfully under 

laboratory conditions in Bold’s Basal Medium (BBM) 

liquid and on solid media. Thirty purified algal cultures 

were isolated from only 1 of 70 mixed cultures obtained 

from Brookwood headstones growing under laboratory 

conditions. 

In order to detect the presence of viruses in Brookwood samples, 1 mL of different algal suspensions, grown 

in Knop’s liquid medium, was spread onto Knop’s agar 

and incubated until an algal lawn developed, as shown in 

Figure 7. In some lawns, zones of clearance, or plaques, 

were detected, indicating the presence of virus. Some viruses were identified in the laboratory from plate cultures, 

producing obvious clearing, in algal lawns. This evidence 

further suggested that this might be a viable method for 

destroying algal biofilms on stone. When agar culture 

plates did show signs of plaques indicative of virus attack, 

the cleared zone was removed with a sterile razor blaze, 

suspended in sterile saline, microcentrifuged, and then filter sterilized (pore size of 0.2 μm) to remove cells. Suspensions were then stored at 4oC in the dark until needed.

Fluorescence Microscopy

Work with solid plate cultures of algae was the most 

promising in terms of recognition of virus activity. However, although no clear evidence of viral presence was detected in many of the mixed liquid cultures obtained from 

Brookwood samples, four pure cultures denominated K, 

I, L, and M showed some signs of viral presence in liquid 

cultures, indicated by reduced algal growth. Viral assay 

through the solid plate technique, in which the suspected 

liquid was added to a suspension of algal culture and 

poured over a solid agar surface, was unsuccessful with 

these four samples. However, the presence of virus particles was confirmed by fluorescence microscopy by staining with SYBR Green I stain, which targets nucleic acid 

and stains virus (Figure 8). By this means, virus presence 

was also identified in 9 out of 70 of the mixed algal cultures after centrifugation and filter sterilization, although 

the concentration of virus detected in the different samples 

seemed to be very low.

Viral Assays Using Aquatic Viruses

Since Chlorella was one of the predominant colonists 

on headstones and a common isolate from samples, pure 

cultures of three Chlorella viruses and their corresponding hosts, which had been isolated from aquatic environments, were obtained from James L. van Etten (University 

of Nebraska). The three different Chlorella viruses, PBCV1, MT325, and ATCV-1, had Chlorella strains, NC64A, 

Pbi, and Sag 3.83, respectively, as algal hosts. The paired 

combinations made it possible to further develop methods 

for virus testing to test using these viruses against the algal 

isolates obtained from Brookwood headstones and investigate the concept of inhibition of algae on building stone.

In order to detect whether particular algal isolates were 

sensitive to virus, 100 μL of three mixed algal cultures, 

FIGURE 7. A typical view of (left) an uninfected algal lawn and 

(right) an infected lawn developed on Knop’s agar.
number 2 • 6 5

designated SAM1, SAM2, and SAM3, were plated onto 

Knop’s agar, from which single colonies were developed 

after approximately three weeks. Eight single colonies 

were isolated from SAM1, denominated A to G; seven 

single colonies were isolated from SAM2, denominated H 

to N; and 12 single colonies were isolated from SAM3, denominated Nˇ to Y. All algal samples were inoculated into 

10 mL of BBM liquid medium in plastic universal bottles 

and grown for one month. Viral assays to isolate the viral 

particles and detect sensitive algae were carried out with 

samples A–G and pure cultures of Chlorella. The viral assay carried out with Chlorella Sag 3.83 showed that algae 

did not grow at all on the plates with the highest viral concentration, producing confluent lysis, but with intermediate dilutions (8.1 × 106 plaque-forming units (pfu)/mL), 

viral plaques could be seen clearly (Figure 9).

Action of Viruses against Algae on Stone Discs

Small-diameter (1 cm) limestone discs were soaked 

in 9 mL of different viral dilutions (10–1–10–13) for three 

days. They were then inoculated with 100 μL of Chlorella

NC64A and incubated in natural daylight at room temperature (25°C). Figure 10 shows the effects of virus on the 

growth of the algae. Whole plates were photographed 22 

days after inoculation for NC64A. Algal growth could be 

detected in the positive control and on those discs treated 

with a low virus concentration. However, algae did not 

grow on those discs treated with high virus concentration 

even 22 days after inoculation.

A second series of experiments was done where largediameter (4 cm) limestone discs were simultaneously inoculated with a growing culture of Chlorella NC64A and 

a series of dilutions of the appropriate virus and incubated 

on a bed of moist vermiculite without extra nutrients. 

It was observed that on those limestone discs that were 

treated with the highest concentration (dilutions 1–4) of 

viruses, algal growth was not established. However, when 

higher dilutions were applied, algal growth was established. Algal growth was not established on those discs 

treated with high virus concentration, even three weeks 

after treatment (Figure 11). On the first, second, and third 

week after the experiment started, the limestone discs were 

retreated with the corresponding virus dilutions. At this 

time, one of the positive control discs (showing untreated 

algal growth, disc g) was treated with virus (Figure 11). 

By comparing Figure 11a and 11b, it can be seen that (1) 

no algal growth occurred on discs h–k, (2) algal growth 

decreased in discs l–o, and (3) no effect was seen on discs 

FIGURE 8. Viral particles (green dots) detected by fluorescence 

microscopy (SYBR Green I stain under immersion oil, × 400) in 

sample I3.

FIGURE 9. Viral assay plate prepared with Chlorella Sag 3.83 after 

four days of incubation at room temperature and natural light.
6 6 • smithsonian contributions to museum conservation

FIGURE 11. NC64A growing on stone discs at room temperature (25°C) under natural light for (A) 2, (B) 3, and (C) 6.5 weeks 

after initial treatment. Discs a–c are the negative controls where no algal growth occurred, and discs d–f are positive controls. 

Discs h–k were treated with high virus concentration, discs l–o with intermediate virus concentration, and discs p–t with low 

virus concentration. The arrow corresponds to the limestone disc that was treated with a high virus concentration after algal 

growth was established.

FIGURE 10. Chlorella growth on stone discs pretreated by submersion 

with different viral dilutions (PBCV-1) on Modified Bold’s Basal Medium (MBBM) medium at 25°C: (a) control discs with (bottom) and 

without (top) algal inoculation; (b) after high virus concentration treatment, showing lack of algal growth; (c) disc treated with low virus concentration, showing algal growth on stone disc; and (d) disc treated with 

high virus concentration, showing complete inhibition of algal growth 

on stone disc. All discs were inoculated with Chlorella NC64A and incubated for 22 days after virus treatment.
number 2 • 6 7

p–t. Three and a half weeks after the last treatment, it was 

evident that algal growth had been completely inhibited 

and Chlorella NC64A had disappeared. Algal growth had 

also disappeared on discs k–m (high levels of virus) and 

was reduced in discs n–p and r (intermediate levels of virus). No effect was seen on discs q, s, and t, which had 

received low levels of virus.

Search for Viral Effect and Viruses on

Algal Cultures from Brookwood Cemetery

Treatment of Chlorella growing on stone and pretreatment with viruses showed that they successfully damaged 

algal growth; research was directed to find out if Chlorella 

viruses were able to affect Brookwood algae. A microtiter 

plate technique was used to assess 25 pure or semipure algal cultures that had been isolated from Brookwood Cemetery headstones. This approach showed that six natural 

algal cultures were affected by the Chlorella viruses. When 

a two-phase separation technique based on PEG 6000 was 

used to assay 56 algal cultures from Brookwood Cemetery 

for viral samples, 48 samples formed an interphase, indicating the presence of virus. The different interphases 

were dialyzed, and the dialysis product was spread plated 

onto BBM agar plates to check for viral activity. Ten of 

these samples showed viral plaques, and these were collected and stored in Tris-HCl (pH 7.8) buffer at 4oC until 

use. Any sensitive algal cultures were grown in 10 mL of 

BBM liquid medium in 20 mL Universal plastic tubes. One 

of the Universal tubes was inoculated with 100 μL of the 

stored viral plaques. All the algal cultures grew without 

showing signs of viral attack.

Isolation of Viruses from Other Sources

The search for viruses was extended to pond waters 

taken from parks within Portsmouth that suffer algal 

blooms during the year. Water samples were tested for the 

presence of viruses that might attack algae on stone. Water 

samples were filtered through a 0.45 μm pore size Nucleopore filter to remove bacteria, algae, and other large particles. The different filtrates were dialyzed and stored at 

4oC for further use. The filters were placed onto BBM agar 

plates and incubated at 25oC at constant illumination for 

one week. The heaviest algal growth was obtained from 

Victoria Park, and the lowest growth was obtained from 

Canoe Lake (Figure 12).

A microtiter plate technique was used to test the different water samples for the presence of algal viruses using algal isolates obtained from Brookwood headstones 

(Figure 13). A dilution series (tenfold) of each water type 

was inoculated with different algal species that had been 

purified from Brookwood samples. Plates from Victoria 

Park did not show signs of algal attack or damage, and for 

the microtiter plate set up with Baffins Pond filtrate, only 

sample K showed change or damage. However, plates prepared from Canoe Lake filtrate showed that two different 

algal samples, K and I, were sensitive, and dialyzed samples 

FIGURE 12. Algae growing on 0.45 μm nucleopore filters placed on BBM agar after one month of incubation at 25oC at continuous illumination from (right) Victoria Park, (center) Baffins Pond, and (left) Canoe Lake.
6 8 • smithsonian contributions to museum conservation

showed that samples N, O, R, S, and T were sensitive to 

varying extents. Eight days after the initial treatment, it 

was found that algal growth was still being inhibited in 

some of the wells, notably samples N, O, and S.

DISCUSSION

Two surveys were conducted at the Brookwood Military Cemetery in Surrey, and data were collected on patterns of algal cover on headstones. An image analysis 

technique was designed and tested for use in capturing 

data relating to patterns of algal contamination on headstones. The technique proved to be extremely accurate and 

rapid and will be used for any future surveys of headstone 

contamination. High-quality digital images of selected 

headstones have been archived for future use.

The image analysis technique chosen proved to be very 

suitable for surveys of algal contamination on headstones, 

and the data collected form a baseline study for future surveys. Such surveys of the headstones are essential in order 

to monitor the in situ effects of treatment and subsequent 

recolonization by microorganisms. Broad-scale patterns 

of algal contamination are evident, and a number of environmental factors that potentially affect these patterns 

have been identified. Algal cover is highly variable within 

the sections of cemetery sampled. Proximity of tree cover 

or shading vegetation, age of headstone, and front versus 

back aspect of headstone all appear to influence algal contamination. By preselecting groups of headstones for the 

second survey it was possible to highlight the influential 

environmental factors using statistical analysis. Although 

the results of the first survey suggested that age and aspect 

of headstone may influence patterns of algal cover, this 

was not the case in the second survey. However, growth 

conditions were very different between the two surveys, 

and it is possible that differences in seasonal conditions 

may have affected the observed patterns of algal cover. 

The first survey was carried out after a period of very little 

rain and high temperatures, whereas the second survey 

followed a period of heavy rainfall and a time when daylight hours were decreasing.

Algae and cyanobacteria will grow under a wide 

range of environmental conditions but tend to be sensitive 

to high light (i.e., high UV) levels. It is possible that algae 

growing on the backs (facing SE) of the headstones were 

experiencing high UV conditions during the summer and 

hence that their growth was affected. Observation (not 

quantified) of algal growth in laboratory cultures showed 

a rapid increase in algal growth as light levels and temperature fell. It was possible that by the second survey, environmental conditions were approaching those necessary 

FIGURE 13. Microplate inoculated with purified cultures of environmental algae isolated from Brookwood headstones. Each row is a dilution series from left (high) to right (low), with the first two wells as alga-only control. The top row is an uninoculated, medium-only control, 

and subsequent rows (from second row to bottom) contain different algae samples N to T, treated with dilutions of dialyzed Canoe Lake 

water after (a) four days and (b) eight days. Samples N, O, R, S, and T were sensitive, showing clearing of growth (example well shown 

by small arrow) in wells, relative to the alga-only controls. Clearing occurred over a greater dilution range for N and, to some extent, O; 

it was very limited for R and T; a limited effect with S only became apparent after eight days; no effects were found for Q. Alga P showed 

poor growth, including the controls.
number 2 • 6 9

for optimal algal growth, and an increase in cover would 

have obscured any small-scale differences in patterns of 

algal contamination, i.e., between headstones of different ages and aspects. These observations highlight the 

importance of seasonal effects on biological communities 

and their consideration when developing treatments for 

biological control of populations that potentially change 

throughout the year. 

Three zones of algal cover are evident on headstones, 

at the crown, the base, and the central area of the headstone, and these zones are common to both faces. The 

analysis of microorganism diversity suggested that the 

diversity of each of these zones is different. Patterns of 

microorganism diversity suggested that a number of different taxa are common across groups of headstone samples from Brookwood. Obviously, this is encouraging as a 

lower diversity and variability of microorganisms present 

will potentially reduce the number of antialgal microorganisms required for the remediation treatment. The pure 

cultures of algae obtained from the Brookwood samples 

have a high incidence of Chlorella species.

The initial work carried out to detect viral particles 

in the natural environment with fluorescence microscopy 

proved to be a successful technique. Viral particles were 

detected not only by plaque development in agar plates 

but also by microscopy. A two-phase separation technique 

was successfully used to concentrate viral particles. Algal 

growth, actively growing or established, was then treated 

with virus suspensions to test whether algal growth on 

limestone could be inhibited or cleared. All the different 

experiments showed that viral attack could decrease or inhibit algal growth on different media or surfaces. On agar 

plates, limestone discs, and slabs, high virus concentration 

inhibited algal growth over an extended period of time. 

The most marked effect was found when the highest virus 

concentration was used, as in the experiments carried out 

with Chlorella NC64A. 

The experimental design also showed that viral treatment could be applied before, after, and during the period 

of algal growth. The number of applications will be further investigated by fieldwork. Treatment of environmental samples with the algal viruses acquired from James L. 

van Etten showed the high specificity of a particular virus 

for the algal host. A high viral concentration is required 

for elimination of algal growth, and this fact, taken together with the high specificity, could explain why early 

experiments carried out with environmental samples were 

unsuccessful. In addition, the concentration of viruses in 

the environmental samples, as detected by fluorescence 

microscopy, seemed to be quite low. The high specificity 

of virus for the host makes this work more challenging. 

Any treatment process will be based on a cocktail of viruses against the predominant algae, together with the 

application of chemical synergists to facilitate virus infection. As algal populations fluctuate during the year, virus 

concentrations in the natural environment are changing 

naturally; thus, there is a higher probability of finding appropriate viruses as the populations decline, as in aquatic 

ecosystems. The laboratory trials have shown that it is 

possible to eliminate a particular species of algae using viral remediation, but a much wider range of viruses needs 

to be isolated so that field trials tests against the natural 

algal populations identified can be done.

CONCLUDING REMARKS

Much of the work that has been done on algal viruses 

has been for aquatic systems. Our research was concerned 

with testing the feasibility of using viruses that pose no 

health threat to humans to control natural algal populations on stone. The presence of naturally occurring viruses 

in isolation plates obtained from Brookwood headstones 

illustrates the impact they can have on the algal communities. Although we were able to identify viruses using 

fluorescence microscopy, we were not able to isolate and 

amplify these viruses in the laboratory. Nevertheless, using 

paired algal hosts and viruses from aquatic systems, we 

obtained evidence that they can inhibit algal populations 

on stone in laboratory culture. 

Isolation of the components of the algal populations 

proved to be very difficult and slowed progress on isolating natural viruses from stone:

•	 Stone algae grow very slowly in the laboratory in pure 

liquid culture and on stone, and this has impeded 

experimentation.

•	 Viral assay and growth requires pure cultures of significant densities, and these were very difficult to obtain from stone algal communities.

•	 Contamination with bacteria and fungi was a major 

technical problem.

Our results demonstrate proof of principle that algal 

types that are commonly found on the stones can be inhibited by viruses on stone in the laboratory. Clearly, at 

this stage, although it has been possible to show limited 

effects in the laboratory, the essential components of any 

treatment system, i.e., a range of viruses reflecting the variety of algal types, have not been obtained so far. They 
7 0 • smithsonian contributions to museum conservation

were clearly present and were detectable but could not 

be isolated into laboratory culture. The answer may lie 

in our ability to control the different factors that affect 

the expression of activity of those viruses that affect algal 

populations on stone.

Acknowledgments

This work was funded by a contract with the Commonwealth War Graves Commission, Maidenhead, UK, 

and we thank CWGC Technical Director Brian Davidson 

and Chris Heath, Systems and Standards Manager, for 

their support throughout the research period. We are also 

grateful to Dr. James L. van Etten at Nebraska University 

for advice and supplying the chloroviruses and their corresponding hosts.

REFERENCES

Ball, A. S., M. Williams, D. Vincent, and J. Robinson. 2001. Algal 

Growth Control by a Barley Straw Extract. Bioresource Technology, 77:177–181.

Bratbak, G., W. Wilson, and M. Heldal. 1996. Viral Control of Emiliania 

huxleyi Blooms? Journal of Marine Systems, 9:75–81.

Cangiano, T., M. DellaGreca, A. Fiorentino, M. Isidori, P. Monaco, and 

A. Zarrelli. 2001. Lactone Diterpenes from the Aquatic Plant Potamogeton natans. Phytochemistry, 56:469–473. 

Choi, H. J., B. H. Kim, J. D. Kim, and M. S. Han. 2005. Streptomyces 

neyagawaensis as a Control for the Hazardous Biomass of Microcystis aeruginosa (Cyanobacteria) in Eutrophic Freshwaters. Biological Control, 33:335–343.

Clarke, K. R., and R. N. Gorley. 2006. PRIMER v6: User Manual/

Tutorial. Plymouth, U.K.: PRIMER-E.

Clarke, K. R., and R. M. Warwick. 2001. A Further Biodiversity Index 

Applicable to Species Lists: Variations in Taxonomic Distinctness. 

Marine Ecology Progress Series, 102:153–160.

Crispim, C. A. 2004. Biofilms on Church Walls in Porto Alegre, RS, Brazil, with Special Attention to Cyanobacteria. International Biodeterioration and Biodegradation, 54(2–3):121–124.

Darienko, T., and L. Hoffmann. 2003. Algal Growth on Cultural Monuments in Ukraine. Biologia, 58(4):575–587.

DellaGreca, M., A. Fiorentino, P. Monaco, L. Previtera, F. Temussi, and 

A. Zarrelli. 2003. New Dimeric Phenantrenoids from the Rhizomes 

of Juncus acutus. Structure Determination and Anti-algal Activity. 

Tetrahedron, 59:2317–2324. 

Gaylarde, C. C., and P. M. Gaylarde. 2005. A Comparative Study of 

the Major Microbial Biomass of Biofilms on Exteriors of Buildings 

in Europe and Latin America. International Biodeterioration and 

Biodegradation, 55(2):131–139. 

Jenkins, K. M., S. G. Toske, P. R. Jensen, and W. Fenical. 1998. Solanapyrones E–G, Antialgal Metabolites Produced by a Marine Fungus. 

Phytochemistry, 49:2299–2304.

Kang, M., D. D. Dunigan, and J. L. Van Etten. 2005. Chlorovirus: A 

­Genus of Phycodnaviridae That Infects Certain Chlorella-like 

Green Algae. Molecular Plant Pathology, 6(3):212–224.

Koestler, R. J., A. E. Charola, M. Wypyski, and J. J. Lee. 1985. “Microbiologically Induced Deterioration of Dolomitic and Calcitic Stone 

as Viewed by Scanning Electron Microscopy.” In 5th International 

Congress on Deterioration and Conservation of Stone, ed. G. Felix, 

pp. 617–626. Lausanne, Switzerland: Presses Polytechnique Romandes. 

König, G. M., A. D. Wright, and A. Linden. 1999. Plocamium hamatum

and Its Monoterpenes: Chemical and Biological Investigations of 

the Tropical Marine Red Alga. Phytochemistry, 52:1047–1053.

Lee, C. H., M. S. Lee, M. Suh, and S.-W. Choi. 2005. Weathering and 

Deterioration of Rock Properties of the Dabotap Pagoda (World 

Cultural Heritage), Republic of Korea. Environmental Geology, 

47(4):547–557.

MacDonald, J. 1993. The Effects of Commercial Cleaning Processes on 

Sandstones in Scotland. Ph.D. thesis, Robert Gordon University, 

Aberdeen, U.K.

Nakamura, N., K. Nakano, N. Sugiura, and M. Matsumura. 2003. A 

Novel Cyanobacteriolytic Bacterium, Bacillus cereus, Isolated 

from a Eutrophic Lake. Journal of Bioscience and Bioengineering, 

95(2):179–184.

Ortega-Calvo, J. J., X. Ariño, M. Hernandez-Marine, and C. SaizJimenez. 1995. Factors Affecting the Weathering and Colonization 

of Monuments by Phototrophic Microorganisms. The Science of 

the Total Environment, 167:329–341.

Ortega-Morales, O., J. Guezennec, G. Hernandez-Duque, C. C. Gaylarde, and P. M. Gaylarde. 2000. Phototrophic Biofilms on Ancient Mayan Buildings in Yucatan, Mexico. Current Microbiology, 

40(2):81–85.

Reim, R. L., M. S. Shane, and R. E. Cannon. 1974. The Characterization 

of Bacillus Capable of Blue-Green Bactericidal Activity. Canadian 

Journal of Microbiology, 20:981–986.

Rifón-Lastra, A., and A. Noguerol-Seoane. 2001. Green Algae Associated with the Granite Walls of Monuments in Galicia (NW Spain). 

Cryptogamie, Algologie, 22(3):305–326.

Schumann, R., N. Haubner, S. Klausch, and U. Karsten. 2005. Chlorophyll Extraction Methods for the Quantification of Green Microalgae Colonizing Building Facades. International Biodeterioration 

and Biodegradation, 55(3):213–222.

Suttle, C. A. 2002. Viruses Causing Lysis of the Toxic Bloom-Forming 

Alga Heterosigma akashiwo (Raphidophyceae) Are Widespread in 

Coastal Sediments of British Columbia, Canada. Limnology and 

Oceanography, 47(2):545–550.

Tayler, S., and E. May. 1994. The Investigation of Biocides against Bacteria Isolated from Stone and Their Effectiveness against in situ Populations. Material und Organismen, 28:265–277. 

Uchida, E., Y. Ogawa, N. Maeda, and T. Nakagawa. 2000. Deterioration 

of Stone Materials in the Angkor Monuments, Cambodia. Engineering Geology, 55(1–2):101–112.

Young, M. E. 1997. Biological Growths and Their Relationship to the 

Physical and Chemical Characteristics of Sandstones before and after 

Cleaning. Ph.D thesis, Robert Gordon University, Aberdeen, U.K. 

Young, M. E., R. Wakefield, D. C. M. Urquhart, K. A. Nicholson, and 

K. H. Tonge. 1995. “Assessment in a Field Setting of the Efficacy 

of Various Biocides on Sandstone.” Paper presented at International 

Colloquium on Methods of Evaluating Products for the Conservation of Porous Building Materials in Monuments, Rome, 19–21 June.
José Delgado Rodrigues (retired), Laboratório 

Nacional de Engenharia Civil, Avenida do Brasil 

101, P-1700-066 Lisbon, Portugal. Marta Vale 

Anjos, Laboratório Nacional de Engenharia 

Civil, Avenida do Brasil 101, P-1700-066 Lisbon, Portugal. A. Elena Charola, World Monuments Fund-Portugal, Mosteiro dos Jerónimos, 

Praça do Império, P-1400-206 Lisbon, Portugal. 

Correspondence: J. Delgado Rodrigues, delgado

@lnec.pt.

Recolonization of Marble Sculptures 

in a Garden Environment

José Delgado Rodrigues, Marta Vale Anjos, 

and A. Elena Charola

ABSTRACT. The usual practice for eliminating biocolonization from stone statues in 

outdoor conditions has been the application of a biocide and subsequent cleaning. However, the cleaned condition is a transient state, and recolonization is inevitable. The recolonization rates can vary significantly as a function of the local and regional environmental 

conditions, and the respective patterns may exhibit strong variations at the microscale 

of the object. Given the variable time required for recolonization, the present study was 

undertaken over several years to collect data that although particularly relevant to the 

substrate considered and its environment, could shed some light as to which measures 

were the most effective in eliminating and preventing biocolonization. The paper focuses 

on the evaluation of various treatments applied in the past to the ornamental marble 

statues in the gardens of the National Palace of Queluz, Portugal.

INTRODUCTION

The National Palace of Queluz, as seen today, dates back to the seventeenth 

and eighteenth centuries. It is located some 12 km west-northwest of Lisbon, 

half way to Sintra. The baroque palace includes several buildings of high architectonic quality and is particularly known for the intimate relation between 

the buildings and the extensive contiguous gardens that are decorated with over 

100 statues and busts, bases and pedestals, vases, balustrades, and over 20 marble fountains and a large limestone cascade. The larger part of the 15 hectare 

grounds is a park. The two formal gardens, the Malta Garden and the Hanging 

Garden, where most of the statuary is concentrated, are right next to the palace, 

and many French doors open directly into them. 

The Robillion wing, which was annexed to the original building, bridges the 

level change from the Hanging Garden constructed over a water reservoir to the 

ground level, where a creek runs through the park. The west side of this wing 

faces the creek, which in this section flows in a highly decorated azulejo-lined 

canal. It has two terraces; the lower having a magnificent staircase leading down 

toward the promenade next to the canal. 

The sculptures and decorative elements are distributed throughout the gardens, many of them under or close to high trees and dense bushes, where higher 

relative humidity and lower sun exposures are to be expected. Most stone elements are in a reasonable state of conservation, particularly those carved in 
7 2 • smithsonian contributions to museum conservation

marble, although some degree of deterioration can be 

identified in most of them. Minor scaling and fissures as 

well as sporadic fractures can be seen, but the major degradation that affects these decorative elements corresponds 

to surface erosion mostly caused by direct rain flow and/

or biological activity. 

Biocolonization is widespread all over the gardens, 

and it can reach nearly total and dense coverage, which 

may completely disfigure the decorative elements (Figure 

1a). Green algae appear frequently during the wet season 

and turn dark grey when the dry season comes. In many 

places, this diffuse dark colonization constitutes the main 

colonizing component. Lichens widely proliferate on some 

of the stone elements and may constitute a cover so dense 

as to entirely conceal the stone surface, as illustrated in 

Figure 1. 

In the course of the recent multiyear conservation and 

restoration project of the gardens of the palace (Charola et 

al., 2007), the opportunity was presented to evaluate the 

condition of the statuary, mostly marble and limestone, 

in the gardens. A study was carried out to compile all the 

information dealing with the cleaning and subsequent protection treatments from the reports and records kept by 

the palace staff for the past quarter century (Vale Anjos, 

2006). In older records, some dating from 1820, it was 

confirmed that regular cleaning was considered normal 

practice in the early times of the gardens’ history. Around 

1948, some restoration actions were reported, and after 

a long period where no actions were recorded, documented interventions reappeared in 1977. Since then, the 

sculptures have been submitted to sporadic and variable 

interventions following different and even antagonistic approaches, facts that lead to a quite inconsistent situation in 

terms of appearance and conservation condition. Different methods for elimination of the dense biocolonization 

have been used, ranging from an intensive grit blasting, a 

FIGURE 1. Two marble sculptures in the Queluz gardens, both of which are heavily colonized by lichens. Note the different types of lichen 

colonization on the sculptures as a function of their local environments.
number 2 • 7 3

method that is ethically unacceptable for carved elements, 

to the application of inappropriate sodium hypochlorite, 

and to biocides such as Preventol R80. Different concentrations and numbers of applications of the biocides are 

reported, reaching the extreme of 5% volume per volume 

(v/v) concentration applied five times. 

Analyzing these data in conjunction with several years 

of photographic records, correlations could be drawn between the cleaning and hydrophobization treatments undergone by the statues and their condition after a given 

number of years. 

The study also evaluated new approaches for eliminating visible biocolonization and controlling the subsequent recolonization (Vale Anjos, 2006). Effectiveness 

of biocides and recolonization were monitored by using 

a portable fluorometer prototype (Delgado Rodrigues et 

al., 2004).

The results obtained helped to define the strategy for 

the maintenance plan that was subsequently developed and 

is described in detail elsewhere (Charola et al., 2007). In 

brief, this approach is based on using low concentrations 

of Preventol R80 (of the order of 1.5% v/v) and leaving 

the object for several months without any additional action. This approach resulted in the elimination of any dead 

organisms by rain and wind, leaving a surface that looked 

naturally aged, which is considered highly appropriate for 

attaining a coherent integration of stone surfaces and the 

surrounding vegetation-based landscape.

MONITORING BIOCOLONIZATION

The study aimed to determine how difficult it was to 

remove existing biocolonization, to find the simplest way 

to apply the biocides, to define the minimum effective 

concentration required, and to outline the protocols to be 

implemented at a large scale in the future maintenance of 

the garden sculptures and decorative elements. It focused 

on visible biocolonization as compiled from the analysis 

of the historic data and field evaluation of the condition 

of the sculptures; it was evident that the most eroded 

areas corresponded to those that had been colonized by 

visible microorganisms, such as algae and lichens. Areas 

that showed no visible colonization were far less eroded, 

and this minimum erosion could be attributed to chemical attacks from rainwater combined with any corrosion 

induced by nonvisible microorganisms, which can be considered normal weathering of the stone. Since this weathering is minor compared to the damage induced by visible 

microorganims and the subsequent attempts to remove 

them, this deterioration was not considered of practical 

importance.

For this study an on-site testing program was carried 

out where the biocidal action of the tested biocides was 

monitored with a portable handheld fluorometer, available as a prototype, called “Biofinder”, developed under 

the European research project “ONSITE—On-site monitoring of biological colonization on stone and plaster surfaces using field portable fluorescence based techniques.” 

The prototype consists of an LED light excitation source 

in a defined wave length and a CCD for photon detection of the fluorescence emission in the characteristic wave 

lengths of chlorophyll pigments. The exciting and emission 

wave lengths were optimized to detect both chlorophyceae 

algae and cyanobacteria. It had been demonstrated that 

the instrument is capable of detecting on-site colonizers 

on a surface before they can be visually observed and is 

particularly well suited for the evaluation of biocide effectiveness. This project was based on earlier work that tried 

to use quantitative field techniques, namely, color analysis 

(Young et al., 1995), laser-induced fluorescence (Cecchi 

et al., 1996), and LED-induced fluorescence (Brechet et 

al., 1997). This last technology was the base technology 

applied in the Biofinder instrument, and once it was successfully improved, it served to fill the need that existed 

between on-site detection and quantification of biodeteriogens. The instrument was successfully tested in several 

field studies and proved to be particularly helpful for monitoring the biocidal action on all the lichen species found in 

the different case studies (Delgado Rodrigues et al., 2004). 

The first phase of the study carried out at the Queluz gardens tested two biocidal products: Preventol R80, 

a biocide based on a quaternary ammonium salt, and zinc 

chloride. Concentrations of 1.5%, 2.0%, and 3.0% (v/v 

for Preventol R80 and weight per volume (w/v) for zinc 

chloride) were applied in one to four applications with 

about a one week interval between them. The biological activity was monitored with the portable fluorometer 

(Biofinder) before application and after each successive 

treatment. Figure 2 illustrates the type of data gathered 

in this study. The emission values can be used directly as 

measured or transformed as the ratio of the values measured in wet conditions versus those measured in dry conditions. The experience acquired in the ONSITE project 

showed that this ratio is especially relevant to discriminate 

biocolonization from high natural background fluorescence stones as well as live colonizations from dead ones. 

In fact, this ratio is close to 1 for noncolonized areas and 

for dead organisms, whereas its value clearly differs from 

1 when live chlorophyll-bearing specimens are present. 
7 4 • smithsonian contributions to museum conservation

For the specific conditions illustrated in Figure 2, it is clear 

that Preventol R80 is slightly more effective than the zinc 

chloride since the wet/dry ratio is very close to 1 after the 

second application. It was also interesting to note that biocides continue to be effective even after a last application, 

as seen in the zinc chloride data. This result leads to the 

conclusion that leaving the treated areas alone for some 

time might be a more efficient way of eliminating biocolonization than trying to remove it all at once. 

Figure 3 shows the plots for the wet/dry ratio obtained 

from measurements taken during and after four applications of Preventol 1.5%, one per week, to areas colonized 

with orange lichens. Figure 3 also shows the effect of the 

application of two different biocides, Preventol and zinc 

chloride, at the 1.5% concentration to two different types 

of lichens, identified as white and orange lichens.

EFFECTS OF PAST CLEANING 

INTERVENTIONS ON RECOLONIZATION

Inspection of the sculptures that had recently undergone cleaning intervention showed that this procedure is 

usually pushed to extremes, particularly when grit blasting 

FIGURE 2. Examples of measurements taken with the fluorometer in successive applications of (a) Preventol R80 1.5% v/v and (b) ZnCl2 1.5% 

w/v to areas with white lichens. Bars represent data measured in dry (white) and wet (grey) conditions. Squares represent the ratio wet/dry values. Values from left to right correspond to nontreated (NT); one, two, three, and four applications spaced one week apart; and 9 months after 

the last application (data are from Vale, 2006).

FIGURE 3. (a) Wet/dry ratio obtained from fluorometer readings for four successive applications of Preventol 1.5% to areas colonized with 

orange lichens. (b) Wet/dry ratio obtained after single applications of dilute biocide solutions (1.5%) of Preventol and zinc chloride on two different types of lichens, where the higher effectiveness of the Preventol is evident (data are from Vale, 2006).
number 2 • 7 5

is used to eliminate biocolonization. In these cases, the 

stone surface acquires an evenly rough surface as compared to the uneven and pitted surface left behind by gentler cleaning methods (Figure 4). Furthermore, the latter 

do not induce the brilliant whiteness that results from aggressive grit blasting.

The surface roughness resulting from a cleaning intervention will affect the subsequent recolonization. The 

relatively smoother surfaces left by grit blasting are more 

resistant to the installation of new colonies, but this fact 

should not convey the mistaken notion that this unacceptable cleaning method may have some advantages in terms 

of maintenance. 

The colonization rates that could be observed show a 

very high dependence on the local environmental conditions, with the fastest rates being observed on sculptures 

FIGURE 4. Surface conditions left by two different cleaning methods: (a) application of 

a biocide and gentle brushing (roughly 30 × 40 cm area) and (b) aggressive grit blasting 

(area about 40 × 50 cm).
7 6 • smithsonian contributions to museum conservation

that are under or very close to large trees. This is illustrated by four busts that decorate a balustrade surrounding a lead sculpture group and a fountain. Two of them 

in particular are directly under a large sycamore tree that 

provides a very favorable environment for biocolonization. The busts were cleaned in 1993 by grit blasting. Two 

years later, they underwent low-pressure water washing, 

and a biocide was applied. Four years later, in 1999, they 

needed to be cleaned again, this time with sodium hypochlorite poultices and the application of both a biocide 

and an aqueous dispersion of a water repellent. The repeated cleaning required clearly shows how fast recolonization can take place in this sheltered area. Figure 5 shows 

the appearance of these busts four years after cleaning.

FIGURE 5. Aspect of two of the more-shaded busts four years after the 

last cleaning. The difference in visible biocolonization between seasons 

is clearly seen between (a) the spring, after a wet winter, and (b) the fall, 

after a dry summer, in 2003.
number 2 • 7 7

When the objects are located in areas exposed to sunlight for only part of the day, their orientation influences 

the recolonization location, as observed for a group of 

four sculptures representing the arts. The statues, Painting, Music, Sculpture, and Architecture, are located in the 

Malta Garden around a small fountain with no large trees 

in the neighborhood and are right next to the palace building. Figure 6 gives the location of the statues and the palace building as well as its orientation.

These sculptures were cleaned in 1996 and 2003 

and were inspected in 2003, 2006, and 2008. The 1996 

cleaning was based on biocide poultices and low-pressure 

washing followed by the application of a water repellent. 

The first observation was carried out in 2003, seven years 

after the first cleaning and prior to the second one (Figure 

7) and showed a fairly severe recolonizaton of the surfaces.

The aggressive cleaning intervention carried out in 

2003 left all the sculptures a brilliant white. Interestingly, 

dry grit blasting was used for two statues, Painting and 

Architecture, whereas pressure washing was used for the 

other two, Music and Sculpture, but no significant differences in their appearance could be noted. Nonetheless, 

two years after this drastic cleaning, the first signs of recolonization could be observed. Five years later, green algae 

FIGURE 6. Diagram of the Malta Garden, showing the fountain in the center and the four sculptures, identified by their initials M, P, A, and S, around it. Hatched areas represent parts of the 

palace building. Black arrow points north.
7 8 • smithsonian contributions to museum conservation

were widespread on the north- and northeast-facing areas, 

and lichens were already present on the south-facing areas 

(Figures 8 and 9).

After the fall 2003 cleaning, yearly measurements 

were taken with the Biofinder on different areas of the 

statues, e.g., north and south facing, until biocolonization 

became evident three years after cleaning. The wet/dry 

ratio of selected site measurements, representative of the 

increase in recolonization that took place over those three 

years, is shown in Figure 10. These measurements confirm 

the visual evaluation of the appearance of the first signs of 

recolonization, and although the positive trend is not very 

sharp, it indicates that recolonization is well established 

in the sculptures. Nonetheless, it is worth mentioning that 

monitoring recolonization on a complex figure such as 

these sculptures presents some difficulties as colonization 

patterns are spatially heterogeneous, with areas where recolonization is dense and visible and others where virtually no recolonization is present. Consequently, no single 

graph will fully represent the reality of any sculpture, and 

Figure 10 is obviously just an illustration of the kind of 

data that can be gathered during the recolonizing phase. 

Another example is from a group of six sculptures 

(putti groups) located on the south balustrade of the Malta 

Garden near the palace building (numbered 32 to 37 in 

Figure 6). These were cleaned in 1996, with the application of a biocide (a quaternary ammonium salt formulation) and a water repellent. By 2003 their appearance had 

significantly changed, and three years later, in 2006, the 

biological cover was already very noticeable (Figure 11). 

The striped pattern of the recolonization can be attributed 

to the water repellent because of the changes this product 

induces in the way water runs down the treated surface 

(Charola et al., 2008a, 2008b).

On the other hand, the sculptures shown in Figure 12 

are located on the balustrade between the Malta and the 

Hanging gardens. This is a totally unsheltered space where 

they are exposed to direct sunlight for most of the day. 

FIGURE 7. Recolonization condition of the arts statues in 2003, seven years after the 1996 cleaning: (a) Music and (b) Painting. Cleaning had 

been carried out with biocides and low-pressure washing, followed by the application of a water repellent.
number 2 • 7 9

According to information provided by museum staff, these 

statues received a biocide spray application (a quaternary 

ammonium salt) in 1999. No written records for this intervention are available. In 2003 they already showed evident signs of recolonization that evolved in the subsequent 

three years to a particularly striking increase in the lichen 

recolonization.

Finally, the two groups of four sculptures placed on 

the upper and lower balustrades, respectively, of the west 

façade of the Robillion Pavilion illustrate yet another 

set of conditions that influence the recolonization rates. 

These sculptures were cleaned by grit blasting, leaving 

them a brilliant white and deprived of the natural patina. 

Some six years after cleaning, the sculptures showed incipient (upper balustrade) to moderate (lower balustrade) 

recolonization, so the application of a biocide was carried out. In a recent inspection, some eight years after the 

biocidal treatment, recolonization had again reached an 

incipient level in the sculptures of the upper balustrade 

and was already intense in the sculptures of the lower one 

(Figure 13).

The clear difference in the microclimatic conditions 

of the upper and lower balustrades has an evident impact 

on the recolonization rates. Whereas the upper balustrade 

has full western exposure and not much shading from the 

building on the east, the lower balustrade is shaded both 

on the east, by the palace building, and on the west, by tall 

trees. Consequently, the environment on the lower balustrade tends to favor a faster recolonization rate. The recolonization pattern is relatively independent of the exposure 

FIGURE 8. The arts statues in 2008, five years after cleaning with abrasive methods, showing recolonization of green algae in the northwardfacing areas: (a) Painting and (b) Music.
8 0 • smithsonian contributions to museum conservation

orientation, suggesting that the presence of the shadowing 

trees overrides the effect of the orientation identified in the 

unsheltered sculptures.

Table 1 summarizes the main data collected during 

this study, including two fountains that were relocated in 

the garden but were not connected to the hydraulic system. These are located in a rather sheltered area in the 

park and are referred to as “dry fountains” in Table 1.

DISCUSSION

The many stone sculptures in the gardens of the National Palace of Queluz, the diversity of environments to 

which they are subjected, and the cleaning and protective 

interventions they suffered provided a wealth of information 

that served to evaluate the effectiveness of biocides and the 

performance of past interventions. The study corroborated 

FIGURE 9. The face of Music, showing the first occurrences of lichens on the sunny south-facing areas. (a) Lichens and growth of algae on the 

relatively protected underside of the chin. (b) A detail of the lichen growth.

FIGURE 10. Wet/dry ratio of the measurements taken with Biofinder for the arts statues one, two, and three years after cleaning. The positive trend of the graphs indicates 

that recolonization is clearly in progress. The local decrease is certainly due to the spatial heterogeneity of the recolonization patterns.
number 2 • 8 1

FIGURE 11. Recolonization of one of the putti groups cleaned in 1996. Appearance is shown (a) in 2003 and 

(b) in 2006. Note the well-defined dark green stripes in (a) as a result of the water-repellent treatment and their 

subsequent evolution to heavier features over time.

FIGURE 12. Sculptures located in open, unsheltered spaces. Recolonization is shown (a) four years after cleaning and 

(b) seven years after cleaning. Note the initial relatively slow increase in biocolonization, with the subsequent appearance 

of lichen colonization.
8 2 • smithsonian contributions to museum conservation

the well-known relationship between environment and the 

type and rate of recolonization. In highly exposed and unsheltered areas, recolonization by green algae occurs within 

two years after cleaning, whereas it takes lichens between 

four and five years to make their appearance. Meanwhile, 

in shaded areas protected by large trees, it takes slightly 

over one year to trigger a significant recolonization, particularly by green algae. Occasional shade, such as provided by 

buildings and/or nearby trees, results in disparities in the colonization rates according to the orientation of the sculptures, 

as evidenced by the sculpture group of the arts. Differences 

in recolonization rates for objects located with the same orientation but with different shading were observed between 

the upper and lower balustrades of the Robillion wing. 

The application of water repellents after cleaning 

has been a common practice for sculptures in gardens, 

FIGURE 13. Appearance of the sculptures from the (a–c) upper and (d–f) lower balustrades eight years after application of a biocide. Note the 

significant increase in average colonization of the lower balustrade, where the statues are shaded on the east by the palace building and on the 

west by tall trees.
number 2 • 8 3

irrespective of their location or the methods used for 

cleaning them. The analysis carried out showed that for 

this specific situation the negative effects induced by these 

products, i.e., the development of unsightly biocolonization 

patterns, were not compensated for by the protection they 

provide against recolonization. These patterns develop on 

hydrophobic surfaces because the water repellent inhibits 

the spreading of dew or initial rain drops, thus favoring the 

creation of water stripes as the drops roll down the stone 

surface. The wet stripes, apart from accelerating soiling 

and particulate deposition, constitute a water reservoir for 

the fast-growing initial colonizers, such as green algae and 

cyanobacteria, that eventually develop into well-colonized 

stripes on the stone surface (Charola et al., 2008a, 2008b). 

In addition, water repellents may also serve as a nutrient to 

colonizers (Koestler and Santoro, 1988). 

Water repellents applied to a surface form a physical barrier between the water droplets and the stone substrate, thus preventing the droplets from dissolving and 

incorporating any traces of the applied biocides after 

the cleaning process, should they have a residual action. 

Consequently, under this hypothesis, the application of 

a water repellent after a biocidal treatment is not only 

ineffective but could also be counterproductive in terms 

of the subsequent recolonization. Furthermore, the biocide may interfere with the hydrophobicity that the water 

repellent should impart (Malagodi et al., 2000), a point 

that has been confirmed recently in a study verifying the 

negative impact of biocides on the effectiveness of a water 

repellent (Moreau et al., 2008). This result validates the 

need for a careful assessment of the interaction between 

the two types of products before any decision of their use 

can be made. 

CONCLUSIONS

The history of conservation interventions carried out in 

the Queluz gardens shows that practically all cleaning methods have been used, ranging from gentle to very aggressive 

ones, reflecting a lack of clear guidelines for cleaning interventions and the erratic approach to maintenance over time. 

Also, long periods may have elapsed where no relevant conservation interventions were carried out, whereas at other 

times interventions were repeated within short intervals. As 

a consequence, many sculptures show loss of detailing, erosion from water paths concentrated by the application of 

water repellents, and rather smoothened surfaces in the case 

of those cleaned with grit blasting. It is therefore fundamental that a regular and long-term conservation approach be 

implemented to minimize these problems.

The conservation approach suggested for the Queluz gardens is that the sculptures should be treated only 

with biocides, left untouched for several months, and then 

lightly brushed to remove any detaching vegetation remnants. In most cases, brushing is not even necessary to 

remove the dead colonizers (Charola et al., 2007). Thus, 

the intervention is reduced to a minimum, with practically 

Table 1. Summary of recolonization rates for some of the decorative marble sculptures in different locations in the garden of the 

National Palace of Queluz. WR indicates the application of a water repellent; degrees 1–5 indicate the degree of recolonization, 

with 1 being none to incipient and 5 being the highest degree.

Statues Exposure Treatment Comments

Busts Fully sheltered Grit blasting and WR One year to reach degrees 2–3.

Four years to reach degree 5.

 Green algae are the dominant type within these periods.

Dry fountains Protected Biocides Five years to reach degree 3.

Painting and Architecture Partially protected Grit blasting Five years to reach degree 2.

Algae predominate in NW and NE faces.

First lichens appear in SE and SW faces.

Music and Sculpture Partially protected Pressure washing Five years to reach degree 2.

Algae predominate in NW and NE faces.

First lichens appear in SE and SW faces.

Putti groups Exposed Biocide and WR Six years to reach degree 2.

Robillion upper balustrade Fully exposed Grit blasting Five years to reach degree 1.

Robillion lower balustrade Protected Grit blasting Two years to reach degree 2.

Sheltering effect reduces the influence of orientation.
8 4 • smithsonian contributions to museum conservation

no cleaning actions required. Since the application of the 

biocides should only be carried out when visible recolonization changes from incipient to moderate intensity, the 

number of required applications over time is reduced, thus 

diminishing the probability of inducing the appearance of 

biocide-resistant species. 

Since there are many potential new biocides being 

developed, it is obvious that further on-site studies will 

have to be carried out in the future (Nugari and Salvadori, 

2003). This study focused on finding a practical methodology to reduce the negative impact induced by cleaning 

actions while achieving an acceptable solution for the presentation of marble sculptures in historic gardens. 

So far, the sculptures treated under this new approach 

show equal if not slower recolonization compared to more 

aggressive cleaning methods. Additionally, it should be 

noted that biocolonization is inevitable in outdoor environments and that the deterioration induced by incipient biocolonization is less than that resulting from regular cleaning 

over the same time period. Finally, the overall aesthetic aspect is by far more harmonious and compatible with a garden environment. The overall analysis of the data gathered 

in this case study leads to the following conclusions:

1. It is undesirable to have all sculptures and other stone 

objects cleaned at very close intervals because of the 

inevitable damage that can be induced. 

2. The highly diverse environmental conditions, e.g., 

sheltering effects and exposure orientation, lead to an 

endless number of microenvironments and recolonization rates. 

3. A certain degree of recolonization is acceptable for the 

specific cases of sculptures in gardens since this contributes to a coherent aesthetic presentation of the site.

4. Localized biocide application is recommended whenever recolonization changes from incipient to moderate levels. 

5. The application of water repellents is not recommended for sculptures in gardens.

6. Further studies of the interaction of water repellents 

and biocides, including the order in which they are 

applied, are necessary.

The general conclusion that can be drawn from the 

present study is the importance of defining the aim of any 

intervention and of understanding the nature of the deterioration factors and their interaction with the substrate. 

This study also emphasizes the well-known fact that regular and long-term maintenance is the only way to preserve 

our heritage.

Acknowledgments

The director and staff at the National Palace of Queluz were very helpful in providing access to their records 

and in authorizing the experimental tests carried out on 

some of the decorative elements in the gardens. The National Laboratory of Civil Engineering provided technical 

assistance in the development of the Master’s thesis for 

one of the coauthors on this topic. This study is a product 

of the Conservation/Restoration Project of the Gardens of 

the National Palace of Queluz that was made possible in 

part by the World Monuments Fund’s Robert W. Wilson 

Challenge to Conserve Our Heritage. 

REFERENCES

Brechet, E., D. McStay, and R. Wakefield. 1997. “Detection of Algae on 

Stone Using a Novel Low Cost Hand-Held Fluorimeter.” In Sensors 

and Their Applications, Proceedings of the 8th Conference, ed. A. T. 

Augousi and N. M. White, pp. 3–8. Bristol: Institute of Physics.

Cecchi, G., L. Pantani, V. Raimondi, D. Tirelli, L. Tomaselli, G. Lamenti, 

M. Bosco, and P. Tiano. 1996. “Fluorescence LIDAR Technique for 

the Monitoring of Biodeteriogens on the Cultural Heritage.” In Remote Sensing for Geography, Geology, Land Planning and Cultural 

Heritage, ed. D. Arroyo-Bishop, R. Carla, J. B. Lurie, C. M. Marino, A. Panunzi, J. J. Pierson, and E. Zilioli, pp. 137–146. Bellingham, Wash.: SPIE.

Charola, A. E., J. Delgado Rodrigues, and M. Vale Anjos. 2008a. “An Unsatisfactory Case of Water Repellents Applied to Control Biodeterioration.” In Hydrophobe V, Fifth International Conference on Water 

Repellent Treatment of Building Materials, ed. H. DeClerq and A. E. 

Charola, pp. 117–128. Freiburg, Germany: Aedificatio Verlag. 

———. 2008b. Disfiguring Biocolonization Patterns after the Application of Water Repellents. Restoration of Buildings and Monuments, 

14(5):365–372.

Charola, A. E., M. Vale Anjos, J. Delgado Rodrigues, and A. Barreiro. 

2007. Developing a Maintenance Plan for the Stone Sculptures and 

Decorative Elements in the Gardens of the National Palace of Queluz, 

Portugal. Restoration of Buildings and Monuments, 13(6):377–388.

Delgado Rodrigues, J., J. Valero, R. Wakefield, E. Brechet, and I. Larrañaga. 2004. Monitoraggio della biocolonizzazione e valutazione 

dell’efficacia di un biocida. [Monitoring of Biocolonization and 

Evaluation of the Effectiveness of a Biocide.] Arkos Scienza e Restauro dell’Architettura, 7:52–58.

Koestler, R. J., and E. D. Santoro. 1988. Assessment of the Susceptibility 

to Biodeterioration of Selected Polymers and Resins. GCI Scientific 

Program Report. Los Angeles: Getty Conservation Institute.

Malagodi, M., M. P. Nugari, A. Altieri, and G. Lonati. 2000. “Effects 

of Combined Application of Biocides and Protectives on Marble.” 

In Proceedings of the 9th International Congress on Deterioration 

and Conservation of Stone, ed. V. Fassina, Volume 2, pp. 225–233. 

Amsterdam: Elsevier.

Moreau, C., V. Vergès-Belmin, L. Leroux, G. Orial, G. Fronteau, and 

V. Barbin. 2008. Water-Repellent and Biocide Treatments: Assessment of the Potential Combinations. Journal of Cultural Heritage, 

9(4) 394–400.

Nugari, M. P., and O. Salvadori. 2003. “Biocides and Treatment of 

Stone: Limitations and Future Prospects.” In Art Biology and 
number 2 • 8 5

Conservation: Biodeterioration of Works of Art, ed. R. J. Koestler, V. H. Koestler, A. E. Charola, and F. E. Nieto-Fernandez, pp. 

518–535. New York: The Metropolitan Museum of Art.

Vale Anjos, M. 2006. A estatuária de pedra dos jardins do Palácio Nacional de Queluz: Formas de degradação, enquadramento, tratamentos 

e avaliação. [The Stone Statues of the Gardens of the National Palace 

of Queluz.] Master’s thesis, University of Evora, Evora, Portugal.

Young, M. E., R. Wakefield, D. C. M. Urquhart, K. Nicholson, and 

K. Tonge. 1995. “Assessment in a Field Setting of the Efficacy of 

Various Biocides on Sandstone.” In Methods of Evaluating Products for the Conservation of Porous Building Materials in Monuments, pp. 93–99. Rome: International Center for the Study of the 

Preservation and Restoration of Cultural Property.

Claire Gervais and Carol Grissom, Museum 

Conservation Institute, Smithsonian Institution, 

Museum Support Center, 4210 Silver Hill Road, 

Suitland, Maryland 20746, USA. Christopher 

McNamara, Laboratory of Applied Microbiology 

and the Harvard School of Public Health, Harvard University, Boston, Massachusetts 02215, 

USA. Nick R. Konkol and Ralph Mitchell, Laboratory of Applied Microbiology, Harvard School 

of Engineering and Applied Sciences, Harvard 

University, 40 Oxford Street, Cambridge, Massachusetts 02138, USA. Correspondence: C. Gervais, gervais.claire@gmail.com.

Case Study: Red Staining on Marble

Claire Gervais, Carol Grissom, Christopher 

McNamara, Nick R. Konkol, and Ralph Mitchell

Red stains were observed on the marble of the Certosa of Pavia in Italy 

as early as 1844 (Realini and Sorlini, 1988), and they have since been 

found on other marble monuments, such as the Cathedral of Orvieto, 

the Fountain of Galatea at Villa Litta north of Milan, the Fountain of 

the Labyrinth in Florence, and the pedestal of an equestrian statue in Copenhagen. Microbiological analyses carried out on stained marble samples have sometimes detected carotenoids and red-pigmented organisms. On the other hand, 

the presence of minium (Pb3O4, with a bright red color) on the aforementioned 

monuments suggests that these red stains can also be caused by the corrosion 

and oxidation of lead present in gutters, fountain plumbing pipes, or between 

marble blocks. Up to now, there has been no clear explanation of how the corrosion of lead and the formation of minium occur. Hypotheses include corrosion 

of lead gutters by acidic water (Zanardini et al., 1994), oxidation of lead salts 

derived from atmospheric attack of lead building components (Realini and Sorlini, 1988; Bruni et al., 1995), attack of lead by alkaline water that percolated 

through noncarbonated mortar (Bredal-Jørgensen et al., 2008), production of 

hydrogen peroxide by microorganisms (Petushkova and Lyalikova, 1986), and 

possibly oxidation of lead by bacteria (Realini et al., 2005). 

The Memorial Amphitheater at Arlington National Cemetery, made of 

Danby Vermont marble (Mountain White grade) and constructed between 1915 

and 1920, presents another example of red staining. Preliminary microbiological analysis resulted in the isolation of a red-pigmented bacterium (Figure 1). 

However, in all cases the stains have been found to contain lead corrosion products, in particular, the bright red minium. Scanning electron microscopy (SEM) 

accompanied by energy dispersive spectrometry (EDS), X-ray diffraction (XRD) 

analyses, X-ray fluorescence spectroscopy (XRF), and Raman spectroscopy revealed considerable diversity of lead compounds in the stains as well as in their 

shapes, sizes, and distribution.

The staining at Arlington National Cemetery, which also varies significantly 

in color and appearance (Figure 2), occurs at discrete locations. Most often, it is 

found on marble paving blocks between external columns of the amphitheater, 

particularly between the east and north entrances. Located on contiguous horizontal and vertical surfaces of the same blocks, these stains generally appear as 

agglomerations of small red spots on horizontal surfaces, brown-purple stains 
8 8 • smithsonian contributions to museum conservation

on vertical edges, and yellow washes underneath. In situ 

XRF analysis confirmed that lead is present in all stained 

areas, with higher concentrations in purple areas. Staining is mainly concentrated in the middle of the blocks and 

never continues over vertical joints, as staining of biological origin likely would.

A second striking example of staining is located on 

the outer corner of a column base to the left of the amphitheater’s stage. This area features a Liesegang-ring-like 

pattern, with purple, coral red, orange, and yellow areas 

located sequentially outward from the white corner. Comparison of photographs taken in 2004 and 2008 shows a 

net progression of staining, with migration away from the 

corner and expansion of the area of yellow washes (Figure 

3). In situ XRF analysis confirmed the presence of lead in 

all pigmented areas as well as in the white area between 

the missing corner (apparently replaced with a mortar repair, now also missing) and purple stain (Figure 4). The 

largest quantity of lead was found in white and purple 

stains (regions 2 and 3).

Finally, a stairway post behind and to the left of the 

stage exhibits bright red spots around partially spalledoff corners and along crevices as well as yellow washes. 

A lead sheet was detected in the joint above the stained 

block. The XRF analysis confirmed lead in all pigmented 

areas, and both Raman spectroscopic and XRD analyses 

found minium in one of the bright orange-red corners. 

Identification of compounds in the yellow washes proved 

more difficult. Samples contained a mixture of red minium 

particles and tiny yellow particles that did not yield identification by XRD or Raman spectroscopy (possible causes 

could be a poor crystalline state or a scarce amount of corrosion products for XRD and low scattering of lead oxides 

for detection by Raman). The SEM-EDS also showed two 

lead-containing compounds with different crystal sizes 

and shapes. Although precise characterization of the two 

compounds is not possible because of topographic and 

thickness variations, one might reasonably assume that 

the compounds correspond to the red Pb3

O4 and to one 

of the two yellow polymorphs of PbO (either litharge or 

massicot) on the basis of their gray values in secondary 

electron SEM images (Aze, 2005).

Possible sources of lead in stained areas include the 

lead sheet found in the joint above the stairway block and 

FIGURE 1. A red-pigmented bacterium isolated from stained areas 

in Memorial Amphitheater. FIGURE 2. Typical colors and textures of colored stains observed at 

the Memorial Amphitheater (scale is approximate). (i) Purple stains, 

sometimes very dark and almost brown, often thickly encrusted, 

found mainly between the columns; (ii) yellow thin washes on a 

stairway corner post with red stains along fissures (note the presence 

of black Verrucaria lichens); (iii) typical staining of vertical areas on 

marble blocks between columns, with the presence of dark crusts, 

red stains, and yellow washes; and (iv) patchy orange-red stain on 

the stairway corner post.
number 2 • 8 9

FIGURE 3. Evolution of the red staining between 2004 and 2008. The dotted line indicates the inner edge of the 2004 stain, and the black arrow indicates its outer limit. Vertical black lines reflect 

the same locations in each photograph.

FIGURE 4. X-ray fluorescence analysis of the corner, showing highest concentrations of lead in 

white and purple areas. For regions 1, 2, and 3, spectra exhibit peak height ratios Lβ

/Lα of slightly 

more than 1, which indicates that the concentration of lead is higher inside the marble than at the 

surface.
9 0 • smithsonian contributions to museum conservation

a lead drip edge observed to the right of the stage on the 

interior of the amphitheater. The drip edge is heavily corroded, with minium found in red corrosion products by 

XRD analysis, and the vertical wall underneath also presented yellow washes similar to those observed on stained 

corners. In this case lead sheets were almost certainly used 

to prevent water penetration into basement rooms. Finally, 

lead sheets are likely to be present at floor level under the 

balustrade between the exterior columns, although none 

were observed. Regular water washing of the pavement 

in combination with rain has likely contributed to the severe red staining of the external paving blocks between 

the columns.

Lead sheets corrode rapidly in the presence of Portland cement and lime water (Brady, 1934). Typically, fresh 

Portland cement can give rise to pH values in the pore 

water up to 13.5 (in comparison, saturated lime water has 

a pH of 12.5). At this very high pH, PbO becomes a stable 

oxide, as does Pb3

O4 when the environment is slightly oxidizing (i.e., in the presence of oxygen). Although it has not 

yet been confirmed, it appears that Portland cement may 

have been used for both pointing and mortar repairs at 

the Memorial Amphitheater. With that in mind, it is not 

surprising to find examples of red staining consisting of 

lead corrosion products.

Despite the presence of red-pigmented bacteria isolated from the Memorial Amphitheater, the staining observed on the marble is caused by lead oxides. In this case, 

we believe that the pigmented organism comprises a relatively small percentage of the bacterial community on the 

stone and that biases introduced by culturing caused large 

numbers of this bacterium to be isolated. However, the 

etiology of red staining on stone structures does not appear limited to lead oxides. Konkol et al. (2009) found red 

stains on a marble sculpture produced by Serratia marcescens. The stains were caused by an organic pigment, and 

no lead was used in construction of the sculpture. Careful 

analysis of red stains is needed to ascertain the cause of the 

staining and to determine appropriate solutions.

Acknowledgments

The authors thank Jeff Speakman, Elyse Canosa, and 

Colby Phillips for XRF analyses, Judy Watson for SEMEDS analyses, Odile Madden for Raman spectroscopy, 

and Nicole Little for XRD analyses. Richard A. Livingston 

is thanked for very helpful insights on Pourbaix diagrams.

REFERENCES

Aze, S. 2005. Alterations chromatiques des pigments au plomb dans les 

oeuvres du patrimoine: Etude expérimentale des altérations observées sur les peintures murales. [Chromatic Alteration of Lead 

Pigments in Artworks: Experimental Study of the Alterations Observed on Mural Paintings.] Ph.D. thesis, University Aix-Marseille 

III, Aix-en-Provence, France. 

Brady, F. L. 1934. The Corrosion of Lead in Buildings. Building Research

Board Technical Paper 8. London: Her Majesty’s Stationary Office.

Bredal-Jørgensen, J., T. Seir Hansen, and M. Petersen. 2008. “Lead Salts 

on a Pedestal of Marble—A Case Study.” In Salt Weathering on 

Buildings and Stone Sculptures, Proceedings from the International 

Conference, pp. 283–298. Lyngby: Technical University of Denmark, Department of Civil Engineering.

Bruni, S., F. Cariati, C. L. Bianchi, E. Zanardini, and C. Sorlini. 1995. 

Spectroscopic Investigation of Red Stains Affecting the Carrara Marble Façade of the Certosa of Pavia. Archaeometry, 37(2):249–255.

Konkol, N., C. McNamara, J. Sembrat, M. Rabinowitz, and R. Mitchell. 

2009. Enzymatic Decolorization of Bacterial Pigments from Culturally Significant Marble. Journal of Cultural Heritage, 10(3):362–366.

Petushkova, J. P., and N. N. Lyalikova. 1986. Microbiological Degradation of Lead-Containing Pigments in Mural Paintings. Studies in 

Conservation, 31:65–69.

Realini, M., C. Colombo, A. Sansonetti, L. Rampazzi, M. P. Colombini, I. Bonaduce, W. Zanardini, and P. Abbruscato. 2005. Oxalate Films and Red Stains on Carrara Marble. Annali di Chimica, 

95(3–4):217–226.

Realini, M., and C. Sorlini. 1988. “Le ‘Macchie’ Rosse del Marmo 

Apuano.” In La Certosa di Pavia: Passato e Presente nella Facciata 

della Chiesa, ed. C. Sorlini, pp. 253–255. Rome: Consiglio Nazionale delle Ricerche.

Zanardini, E., S. Bruni, F. Cariati, G. Ranalli, and C. Sorlini. 1994. “Investigations of the Red Present on the Carrara Marble Façade of 

Orvieto Cathedral.” In Proceedings 3rd International Symposium 

on the Conservation of Monuments in the Mediterranean Basin, ed. 

V. Fassina, H. Ott, and F. Zezza, pp. 349–352. Venice: Soprintendenza ai Beni Artistici e Storici.
Marcelo L. Magadán, Magadán y Asociados, 

Sarandí 1350 4B, C1251 ADD Buenos Aires, 

Argentina. Gisela M. A. Korth, Pringles 463 

8-35, C1183 AEG Buenos Aires, Argentina. 

Marcela L. Cedrola, Rua Eliseu Teixeira De Camargo 1070, Condominio Colinas de SantanderCasa 15, Granado-Campinas, São Paulo, Brazil. 

A. Elena Charola, La Pampa 1959 12B, C1428 

EAF Buenos Aires, Argentina. José Luis Pozzobon, Programa Misiones Jesuíticas de la Provincia de Misiones, Sarmiento 1867 2o Piso, 3300 

Posadas, Misiones, Argentina. Correspondence: 

M. L. Magadán, magadan@magadanyasociados

.com.ar.

Case Study: Biocontrol Testing at the 

San Ignacio Miní Jesuit-­Guaraní Mission, 

Misiones, Argentina

Marcelo L. Magadán, Gisela M. A. Korth, 

Marcela L. Cedrola, A. Elena Charola, 

and José L. Pozzobon

I

n the early seventeenth century Jesuit missionaries established some 30 missions to settle the nomadic Guaraní tribes in the area that now corresponds 

to southwest Brazil, east Paraguay, and northeast Argentina. After expulsion of the Jesuits in 1768, the missions were taken over by different religious orders, such as the Dominicans and Franciscans. Under the Jesuits, the 

missions had worked on an association system, therefore the attempt to have 

them work as separate units by the different orders was completely unsuccessful. Thus, the missions were abandoned, suffering vandalism and neglect, and 

were in most cases taken over by the jungle. The San Ignacio Miní mission, the 

first mission established in what is now Argentina, is located some 60 km (36 

miles) northeast of Posadas, the capital of the province of Misiones. The site, 

some 70,000 m2

 (17.5 acres) in extension, was restored in the 1940s (Onetto, 

1999) and was listed by the United Nations Educational, Scientific and Cultural 

Organization as a World Heritage Site in 1984.

In 2002, the Jesuit-­Guaraní Missions Program of the province of Misiones 

was established as part of an international campaign to link the missions of the 

three countries in a tourism circuit that would help make them self-sustainable 

while developing uniform criteria for their conservation. Within this program, 

several restoration and conservation activities were carried out, among them 

that of the main portal of the church in the San Ignacio Miní mission. This intervention was carried out between 2006 and 2007 and provided an opportunity to 

test passive biocontrol systems (Magadán et al., 2007). 

Passive biocontrol systems have been applied successfully in the past (Wessel, this volume). The system relies on the slow leaching of metal ions, such as 

copper or zinc, from metal strips by rainwater that then flows over the surface 

to be controlled, a point that can be observed on any structure that has bronze, 

brass, or zinc elements present. It is best observed in cemeteries, where metal 

lettering leaves a clean stripe in an otherwise biocolonized surface. The leached 
9 2 • smithsonian contributions to museum conservation

ions can act as a long-term biocide or as a fungistat to prevent new biogrowth if the metal strips are appropriately 

placed (Wessel, 2003). Although the implementation of 

these systems can be considered relatively straightforward 

on buildings of regular geometric shape, it is a challenge 

when dealing with ruins at archaeological sites. Therefore, 

a test site was established in the San Ignacio Miní site to 

evaluate performance. 

For this purpose, a wall in one of the housing rows of 

the site was selected. The wall is easily accessible but not 

too visible to visitors, so as to avoid any tampering with 

the test site. Figure 1 identifies the location of the test area 

on a plan of the San Ignacio Miní site.

The wall, some 2 m high and nearly 1 m wide, is 6 m 

in length, and the test area occupied about half of the 

wall on its west end. Only the top and the north side were 

FIGURE 1. Site plan for the San Ignacio Miní mission showing the test wall location, circled and 

shaded, in the upper left corner. (1) Current entrance to site. (2) Interpretation center. (3) Former 

hostel for visitors. (4) Former living quarters for the Guaraní. (5) Former chapels. (6) Current exit 

from site. (7) Central plaza. (8) Living quarters row where the biocontrol tests were applied. (9) City 

Hall. (10) Home for widows and orphans. (11) Cemetery. (12) Church. (13) Cloister and living 

quarters for the Jesuits. (14) School and workshops.
number 2 • 9 3

treated. First of all, the thick colonization on the top and 

upper part of the north wall, consisting of some shrubs, 

higher plants, and a mat formed by the growth of liverworts, mosses, and ferns, was sprayed with an herbicide 

(Tordon, active ingredient picloran, i.e., 4-amino-3,5,6-trichloropicolinic acid) that is absorbed through the leaves. 

After three days, the vegetation had wilted, and it was 

removed by scraping with wooden spatulas. The larger 

bushes were cut, and any remaining intermediate plants 

were mechanically removed by pulling them out. 

Three sections, each about 1 m in length, were laid 

out on the top of the wall to test the three different metals 

selected, lead, zinc, and brass (58% copper, 40% zinc, and 

2% lead), to allow a comparative evaluation of their effectiveness as passive controls. These areas were covered, 

respectively, with (1) six lead strips (10 cm wide, 1 mm 

thick, and most of them nearly 1 m long, with some shorter 

pieces added to supplement them), (2) zinc mesh made of 

2 mm thick wire (2 × 2 cm opening), and (3) hand-braided 

brass mesh made of 2 mm thick wire (2.5 × 2.5 cm opening) topped by four brass strips, about half a meter long 

(see Figure 2). In the middle section the whole width of the 

wall was covered by the zinc mesh; in the other sections 

only half the width of the wall was covered.

FIGURE 2. View of the top of the wall with the passive system in place. From the front 

to the back are brass mesh and strips, zinc mesh, and lead strips.
9 4 • smithsonian contributions to museum conservation

The north side of the wall was divided into the three 

sections corresponding to the sections on top of the wall 

where the metals had been installed, as shown in Figure 

3. The right side of each section was left uncleaned as a 

control, whereas the left side was cleaned. First, each section was sprayed with a biocide (7.5% volume per volume 

(v/v) of the active ingredient, benzalkonium chloride, in 

water), and then the moist surfaces were brushed clean. 

A rather high concentration of biocide was used because 

the stone contains clays (a quartz arenite cemented by iron 

oxides and ferruginous clays), and it is known that quaternary ammonium salt biocides are strongly adsorbed by 

these minerals (Young et al., 1995). This work was carried 

out in the fall season in May 2007. Although the elimination of existing biocolonization with this passive control system is very slow, requiring several years (Wessel, 

2003), the purpose of having control areas was to check 

how many years it would take to remove the existing layer 

of mixed biocolonization, including algae, lichens, mosses, 

hepaticas, and ferns, and which of the three metals would 

be more effective for this purpose. 

As can be seen from the control areas in Figure 3, biocolonization of the structures in the site is a constant concern and requires regular maintenance, i.e., mowing the 

FIGURE 3. View of the north side of the wall where the passive control systems were installed on the top. The left area is below lead strips, the 

middle area is below zinc mesh, and the right area is below brass mesh (photo taken in May 2007). The left half of each area had been cleaned at 

the beginning of the experiment; the right half was left uncleaned and served as a control. The upper orange arrow points to a small plant, and 

the lower one points to a hepatica. The blue arrow points to a moss- and hepatica-covered block. The red arrow points to a detaching lichen.
number 2 • 9 5

grass and periodically cutting down bushes that start to 

grow in the masonry structures of the site, so as to prevent 

them from mechanically damaging the structures with 

their roots as they develop into trees. Therefore, the idea 

of a passive control system that would reduce the required 

maintenance was gladly accepted by the authorities who 

allowed the installation of the test site. 

Figure 4 shows the appearance of the test wall after 

16 months, in late winter (August 2008). Most apparent 

is the regrowth of shrubs that had been cut, such as the 

Manihot tweediana (known locally as “falso café”), visible near the top of the uncleaned lead test area on the 

left. On cleaned areas no new biocolonization is visible 

except for some algae on the cleaned lead section. On 

the uncleaned control areas some vegetation even seems 

to have disappeared. In the lead section, for example, 

the upper orange arrow indicates the former location of 

a small plant, and the lower orange arrow indicates the 

area covered by liverwort or hepatics (Marchantiophyta 

division), where growth has decreased. In the zinc control 

section in the center, mosses (Bryophita division) appear 

to have been lost, but liverwort coverage appears to have 

increased, indicated by the blue arrow. In addition, dead 

brush between the first two rows of blocks has fallen off. 

Figure 4. Same view as the one in Figure 3 16 months after the installation of the passive control system (photo taken in August 2008). The 

upper orange arrows point to the area where the small plant noted in Figure 3 has disappeared, and the lower orange arrow points to a decrease 

in hepatica cover. The blue arrow points to a diminished moss and hepatica cover. The red arrow points to the area where the detaching lichen 

was lost.
9 6 • smithsonian contributions to museum conservation

In the brass control area on the right, the red arrow indicates the location of the partially detached lichen present 

in 2007, which has since fallen off. However, some plants 

started to grow in the open joints (see cleaned block to the 

right of the blue area), and in contrast to the somewhat diminished growth on surfaces of the north side of the wall, 

new growth appeared on the top of the wall (see Figure 5), 

apparently unaffected by the passive controls.

One month later, as vegetation increased with the beginning of spring (September 2008), the wall was cleaned 

because it had been expected that the system would prevent this regrowth. Plants were removed manually on 

the top of the wall without disturbing the mesh or metal 

strips. On test areas on the side of the wall, smaller plants 

were removed by hand and with soft brushing on both 

previously cleaned and uncleaned control areas. The 

appearance of the wall by the beginning of summer (December 2008), three months after this cleaning, is shown 

in Figure 6. The result of the second cleaning campaign is 

best reflected in the loosening of some lichens that were 

starting to flake off, as can be seen in the top right hand 

block in the control area below the brass mesh.

Figure 7 shows the appearance of the wall in late 

summer (February 2009, five months after the second 

cleaning). The Manihot tweediana bush, ferns, and other 

smaller plants have reappeared. No obvious recolonization has occurred on the originally cleaned areas. 

To date, this experimental setup has performed as expected (Wessel, 2003). Cleaned areas may have shown a 

slight increase in blue-green algae colonization. Continued 

monitoring is important to determine the length of time 

required to eliminate existing growth from control areas 

FIGURE 5. View of the center section of the top of the wall, covered with the zinc mesh. Note the abundant growth of higher plants. 

Similar growth was found in open joints of other sections as well (photo taken in August 2008).
number 2 • 9 7

FIGURE 6. View of the side of the wall three months after hand removal of higher plants and 

brushing on all test areas (photo taken December 2008).

FIGURE 7. Same view as in Figure 6, five months after the September 2008 cleaning (photo taken 

in February 2009). Ferns, smaller plants, and the Manihot tweediana bush have regrown on the 

originally uncleaned control areas. No obvious recolonization has occurred on the originally 

cleaned areas.
9 8 • smithsonian contributions to museum conservation

and to determine which metals and which form of metals (strips or sheet) are most effective. Finally, and with 

hindsight, it is evident that some actions should have been 

implemented at the time the metals were installed. For example, an herbicide or biocide should have been injected 

into the cut stump of the bush, and there should have been 

a control area above with no metal was installed. Sealing the top of the wall with an appropriately formulated 

hydraulic mortar capping, perhaps including some metal 

powder (Henriques et al., 2007), would have prevented recolonization, as was done on top of the walls of the main 

portal and the lateral portal at the time of their restoration 

(Magadán et al., 2007; Magadán, 2008). 

Acknowledgments

The authors thank the Programa de Misiones Jesuíticas for the photographs included in this manuscript. The 

test described was carried out at the time the main portal 

of the church at San Ignacio Miní was restored through 

a World Monuments Fund Robert W. Wilson Challenge 

to Conserve Our Heritage grant and with the support of 

the government of the province of Misiones, the Bunge 

y Born Foundation, and both Acindar and the Acindar 

Foundation.

REFERENCES

Henriques, F., A. E. Charola, V. Moreira Rato, and P. Faria Rodrigues. 

2007. Development of Biocolonization Resistant Mortars: Preliminary Results. Restoration of Building and Monuments, 13(6):389–

400.

Magadán, M. L. 2008. Restauración del portal lateral este del templo. 

San Ignacio Miní. Primera parte. Habitat, XIV 54:36–44.

Magadán, M. L., M. L. Cedrola, G. M. A. Korth, and A. E. Charola. 

2007. Addressing Biocolonization in the Conservation Project of 

the Portal of the Church at San Ignacio Mini, Misiones, Argentina. 

Restoration of Buildings and Monuments, 13(6):401–412.

Onetto, C. L. 1999. San Ignacio Miní. Un testimonio que debe perdurar. 

Buenos Aires: Editorial Valero S.A.

Wessel, D. P. 2003. “The Use of Metallic Oxides in Control of Biological 

Growth on Outdoor Monuments.” In Art, Biology and Conservation, ed. R. J. Koestler, V. Koestler, A. E. Charola, and F. NietoFernandez, pp. 536–551. New York: The Metropolitan Museum 

of Art.

———. 2011 (this volume). “Case Study: Field Observations on the 

Effectiveness of Zinc Strips to Control Biological Colonization.” 

In Biocolonization of Stone: Control and Preventive Methods, 

Proceedings from the MCI Workshop Series, ed. A. E. Charola, 

C. McNamara, and R. J. Koestler, pp. 109–112. Smithsonian Contributions to Museum Conservation, No. 2. Washington, D.C.: 

Smithsonian Institution Scholarly Press.

Young, M. E., R. Wakefield, D. C. M. Urquhart, K. Nicholson, and 

K. Tonge. 1995. “Assessment in a Field Setting of the Efficacy of 

Various Biocides on Sandstone.” In Methods of Evaluating Products for the Conservation of Porous Building Materials in Monuments, pp. 93–99. Rome: International Center for the Study of the 

Preservation and Restoration of Cultural Property.
Jason Church and Mary Striegel, National Center for Preservation Technology and Training, 

645 University Parkway, Natchitoches, Louisiana 71457, USA. Christopher McNamara, 

Harvard School of Public Health, Harvard 

University, Boston, Massachusetts 02215, USA. 

Kristen Bearce Lee and Ralph Mitchell, Laboratory of Applied Microbiology, Harvard School 

of Engineering and Applied Sciences, Harvard 

University, 40 Oxford Street, Cambridge, 

Massachusetts 02138, USA. Correspondence: 

M. Striegel, mary_striegel@nps.gov.

Case Study: Comparative Study of 

Commercially Available Cleaners for 

Use on Marble Veterans Affairs Headstones

Jason Church, Mary Striegel, Christopher 

McNamara, Kristen Bearce Lee, and 

Ralph Mitchell

A variety of commercial products are available for use in cleaning stone 

surfaces contaminated with microbiological growth. The effectiveness of many of these products is questionable, however, and direct 

comparison of some commonly used products would be of significant 

interest to conservators. Therefore, a study was carried out to compare commercially available cleaners for the removal of soiling and biological growth 

from federally issued headstones. Specific goals were to test cleaning products 

for effectiveness to recommend those products and methods best suited to clean 

and preserve headstones.

The study focused on five national cemeteries: Alexandria National Cemetery in Pineville, Louisiana; Bath National Cemetery in Bath, New York; Jefferson Barracks National Cemetery in St. Louis, Missouri; San Francisco National 

Cemetery in San Francisco, California; and Santa Fe National Cemetery in Santa 

Fe, New Mexico. These cemeteries were chosen to represent the various regions 

of the National Cemetery Administration (NCA) as well as different climatic 

zones, including subtropical, temperate, continental, semiarid, and oceanic climates. Stones that were tested in the cemeteries were carved from the Colorado 

Yule marble and Georgia White Cherokee marble used for the majority of both 

modern and historic federally issued headstones.

Prior to cleaning, baseline biological activity was documented on test areas in the fall of 2005. Headstones were then evaluated 6 and 12 months after cleaning. Tap water from the site and five commercially available cleaners 

were selected for application to test areas on 48 headstones at each cemetery. 

Products were chosen to include cleaners that are frequently used, environmentally friendly, user friendly, and unlikely to damage the stone. Daybreak (NCH 

Corp., Certified Labs), based on sodium hypochlorite, is the most commonly 

used cleaner within the NCA. Kodak Photo-Flo (Kodak Corp.), a mixture of 

p-tert-octylphenoxy polyethoxyethyl alcohol and propylene glycol, has been 
100 • smithsonian contributions to museum conservation

commonly used to clean headstones following the recommendation by Strangstad (1988), presumably because it 

promotes faster drying. The three remaining cleaners were 

chosen to provide a range of compositions. H2Orange2

Grout Safe (Proven Solutions Inc.) contains hydrogen 

peroxide in a slightly acidic solution. D/2 Architectural 

Biocide (Sunshine Makers Inc.), mainly containing quaternary ammonium compounds, has antimicrobial properties. Marble Cleaner (World Environmental Group) 

has cleaning properties based on the action of surfactants 

and chelating agents. Cleaners were spray applied to test 

patches measuring approximately 6 × 6 inches (approximately 15 × 15 cm) on 20 headstones at each site. Each 

solution was applied to stones in both sun-exposed and 

shaded locations to account for possible differences arising from local environmental variations.

Headstone test patches were evaluated for changes in 

appearance after 6 months and biological activity after 6 

and 12 months. Appearance changes were documented using photography and color measurements. The color data 

were collected with a Minolta CR-400 Colorimeter (Figure 1), measuring three spots on each test area and averaged for a total of 18 measurements per headstone. The 

same areas were measured in each case and evaluated by 

calculating the number of color changes where the total 

change (DE) was greater than 5 points, which is perceptible to the human eye.

A 3 × 3 cm area of the headstone surface was sampled for microorganisms using BBL Liquid Amies Culture 

Swabs (Becton-Dickinson). To quantify microbial growth, 

heterotrophic bacteria and fungi were enumerated by 

plating samples on solid media (nutrient agar and malt 

extract agar, respectively). Plates were incubated at room 

temperature for two days, and colonies were counted. 

Cyanobacteria and algae were enumerated using a hemocytometer. Performance of test cleaners was compared on 

the basis of biological regrowth activity by ranking the 

cleaners from 1 to 6. The ranking of 1 was given to the 

cleaner that had the highest regrowth rate, and 6 was 

given to the cleaner with the lowest regrowth rate. Thus, 

lower numbers indicate more poorly performing cleaners.

Algae and cyanobacteria were not observed on any of 

the headstones prior to cleaning, but other bacteria and 

fungi were detected in almost all locations by sampling 

using the Liquid Amies Culture Swabs. Numbers of organisms varied greatly among cemeteries and headstones, and 

FIGURE 1. Jason Church takes color measurements of a headstone after cleaning.
number 2 • 101

numbers of bacteria on the headstones (~105/cm2) generally averaged one or two orders of magnitude greater than 

the numbers of fungi (~103/cm2).

After six months, the cleaned areas showed the greatest number of changes in color measurements (ΔE > 5 and 

10) on test patches cleaned with Kodak Photo-Flo (Table 

1). No algae or cyanobacteria were observed in samples 

collected after six months, but numbers of other bacteria and fungi were generally consistent with the color 

measurements for specific cleaners. The lowest levels of 

growth, for example, were often observed in samples that 

had been treated with D/2 and Daybreak, which performed best according to the color measurements. The 

numbers of bacteria (107–108/cm2) and fungi (103–104/cm2) 

were generally higher than in the initial samples, however, 

most likely because of seasonal effects. Samples collected 

in November and December could be expected to have less 

biological material than samples collected in the spring 

months of April and May. 

On the basis of the data obtained for appearance 

change and biological activity, Kodak Photo-Flo was eliminated from further testing after six months. H2

Orange2

Grout Safe cleaner performed well according to color 

measurements of biological activity after six months, but 

closer inspection of headstones indicated that biological 

staining was present on some test areas. On the edges of 

headstones at Jefferson Barracks, for example, activity is 

clearly visible near the edges away from areas measured 

for color change (Figure 2). On the basis of these observations of growth, H2

Orange2 Grout Safe was also eliminated from the study.

Biological activity evaluations after 12 months did not 

detect algae and cyanobacteria on areas treated with the 

four remaining solutions (D/2, Daybreak, Marble Cleaner, 

and water). These organisms typically provide the most visual evidence of growth on headstones, and their absence, 

even from stones treated with water, suggests that a 12 

month period may be too short for determination of the effectiveness of the cleaners’ biocidal properties in the field. 

Bacterial and fungal growth varied among cemeteries.

Santa Fe National Cemetery displayed the largest 

amount of bacterial and fungal activity of the five cemeteries, which was five times greater than any other location. Jefferson Barracks results showed small quantities of 

fungal growth on all but one headstone. Fungi were found 

on headstones in both sunny and shady locations. Bacterial counts were limited to a few headstones in Jefferson 

Barracks. In Alexandria, more bacterial and fungal activity was seen on headstones in shady locations compared 

to sunny locations. Bacteria were not detected in many 

samples from San Francisco National Cemetery but, when 

Table 1. Number of color change measurements 

(ΔE) greater than 5 and 10 for each cleaner applied 

on headstones at Alexandria, Jefferson Barracks, 

San Francisco, and Santa Fe National Cemeteries.

Cleaner ΔE > 5 ΔE > 10

D/2 5 1

Daybreak 7 2

H2Orange2 5 2

Marble Cleaner 8 1

Photo-Flo 11 3

Water 7 2

FIGURE 2. Biological growth observed on the test stone at Jefferson 

Barracks after six months: area A is treated with Kodak Photo-Flo, 

and area B is treated with H2

Orange2 Grout Safe cleaner.
102 • smithsonian contributions to museum conservation

found, were more likely to be seen in sunny locations. In 

contrast, bacteria and fungi were detected in few samples 

from Bath National Cemetery.

Initially, the presence of higher biological activity at 

Santa Fe National Cemetery seemed counterintuitive. Santa 

Fe is a drier climate, and little biological soiling had been 

observed in the cemetery. Locations such as Jefferson Barracks or Alexandria would be expected to have richer environments for biological growth because of their climates 

and higher relative humidities. It is important to note before 

evaluating results from initial biological analyses that each 

cemetery has its own regular maintenance schedule, which 

will influence the nature of the biological activity on headstones from that cemetery. For example, Santa Fe National 

Cemetery is the only one in the study where the stones have 

not been bleached as part of a regular maintenance schedule.

In terms of differences in location within the cemeteries, Daybreak showed better control of bacterial growth 

in sunny locations than the other treatments (Figure 3A); 

analysis of variance (ANOVA) confirmed a significant difference among the cleaners at a 95% confidence level, i.e., 

a probability level p < 0.05. No other significant differences were found, however, for fungi in sunny locations 

(Figure 3B), bacteria in shaded locations (Figure 3C), and 

fungi in shaded locations (Figure 3D).

Laboratory studies are planned to further evaluate the 

effectiveness of the three remaining cleaners, and accelerated studies will be carried out on stone samples in the 

Laboratory of Applied Microbiology at Harvard University. It must be noted, however, that none of the cleaners provide long-term protection against microbiological 

growth on stone. 

Acknowledgments

This work was funded by an interagency agreement 

between the U.S. Department of Veterans Affairs, the 

National Cemetery Administration (NCA), and the National Center for Preservation Technology and Training 

(NCPTT). More information about this study can be 

found in the project report at http://www.ncptt.nps.gov/

interim-report-on-commercially-available-cleaners-for

-use-on-federally-issued-headstones/.

REFERENCES

Strangstad, L. 1988. A Graveyard Preservation Primer. Walnut Creek, 

Calif.: Altamira Press.

FIGURE 3. Bacterial and fungal growth on Colorado Yule marble headstones averaged for five cemeteries in two 

types of locations: (A) bacteria in sunny locations, (B) fungi in sunny locations, (C) bacteria in shaded locations, and 

(D) fungi in shaded locations. Error bars indicate the standard error of the mean.
Paula T. DePriest and Harriet F. Beaubien, Museum Conservation Institute, Smithsonian Institution, Museum Support Center, 4210 Silver Hill 

Road, Suitland, Maryland 20746, USA. Correspondence: P. T. DePriest, depriestp@si.edu.

Case Study: Deer Stones of Mongolia 

after Three Millennia

Paula T. DePriest and Harriet F. Beaubien

Deer stones, Mongolia’s mysterious ancient monuments, are some of 

the most spectacular expressions of Bronze Age megalithic art anywhere in the world. The steppe region of central Asia is home to an 

estimated 700 deer stones; Mongolia alone has over 550. Although 

they are considered to be among the most important archaeological treasures of 

the region, very little is understood about their stylistic development, function, 

and meaning within the cultural contexts that produced them, in part because 

of their remote and isolated locations. Renewed interest in Mongolian culture 

and increased ecotourism have prompted a documentation project as part of 

a larger archaeological and ethnobotanical research endeavor, the AmericanMongolian Deer Stone Project (Fitzhugh, 2005). Since 2005, the project has 

documented over 100 deer stones, located in nearly 20 sites, photographically 

and with condition notes; over 40 have been scanned down to a submillimeter 

scale using 3D imaging technology (Beaubien et al., 2007; Beaubien and Karas, 

2008; Wachowiak and Karas, 2009).

The deer stones, carved stone monoliths that stand 1–4 m high, are found 

individually, in small groups, or concentrated in larger groupings in grassy 

steppe environments, typically as part of stone burial complexes (Figure 1). The 

burial sites have been dated from the Late Bronze to Early Iron Age, between 

2,000 and 3,300 years ago (Fitzhugh, 2005:17, 22–23). Deer stones bear elaborate depictions in bas relief of flying “spirit deer” with swept-back antlers and 

legs folded beneath their bodies, perhaps representing spirits of ancient chiefs 

and clan leaders (Figure 2).

Most of these monoliths are granite, varying in color from grey to pink and 

in quality from a very compact cohesive stone to one flawed by joints, microfractures, or foliations. The deer stones made from the more compact granites are 

extremely well preserved, with carved figures and symbols still sharply sculpted 

in the stone. Others, however, show wear from the eroding forces of wind, rain, 

and Mongolia’s unforgiving dust storms. Rapid shifts between temperature extremes experienced on the Mongolian steppe may expand microfractures and 

weaken the structure as the component minerals, quartz, feldspars, and mica, 

have different thermal expansion coefficients. Some deer stones are almost unrecognizable, with their surface features erased or their forms broken into a trail 

of blocks littering the ground. 
104 • smithsonian contributions to museum conservation

Whether standing, toppled and reclining on the steppe, 

or long buried, many deer stones are altered by biological 

organisms that have colonized their surfaces, ranging from 

microorganisms to lichens, mosses, and higher plants. Animals also leave their marks; horses, camels, and yaks use 

them as rubbing posts, birds use them for perching and 

decorate them with guano, and insect webs and cocoons 

can be found in nooks and crannies. Humans may be the 

most damaging organisms. Before the end of the Bronze 

Age some deer stones were reused for so-called square 

or slab burials where, like a four-poster bed, the stones 

formed the posts and the rails (Jacobson, 1993:145). Humans have pulled the deer stones down, scattered them, 

and moved them, including some early researchers who 

dug up deer stones and, after measuring, drawing, and 

tracing their carvings, left them scattered in the dirt (P. T. 

DePriest, personal observation; Fitzhugh, 2006:16). Some 

stones, including ones from Ulaan Tolgoi (Figures 1 and 

3), have been recently reerected, sometimes set in cement, 

often with no information about their original orientation 

and location (Fitzhugh, 2005:20). The standing stones 

are still objects of veneration, sometimes decorated with 

Buddhist khadags (prayer scarves; see Figure 4), horsehair 

bundles, etc., and occasionally sprinkled with milk and 

smeared with oils and fats.

The focus of this case study is the gentle removal of 

lichens from a 3,000-year-old deer stone. Lichens, a symbiotic association of specialized fungal and algal partners, 

often colonize substrates such as rock outcrops and stone 

FIGURE 1. The Ulaan Tolgoi site in Mongolia has several carved stone monoliths.

FIGURE 2. Carvings of flying “spirit deer” on the south side of a 

deer stone (DS4 in Beaubien et al., 2007) at the Ulaan Tolgoi site.
number 2 • 105

monuments. Their combination plantlike structures, called 

thalli, grow slowly, but in tens to hundreds of years may 

form extensive colonies on stone surfaces. At the Ulaan 

Tolgoi site (see Figure 1), the five standing deer stones 

have an average lichen cover of 10% or less, and this cover 

is concentrated on the more-humid and sheltered north 

and east sides. Since the colonizing lichens are tightly adhered to them, the surfaces do not collect sufficient dust or 

organic materials that could serve as soils for the development of higher plants. Most of the deer stones have mixed 

lichen colonies of different genera and species, apparent 

from the different colors and forms present (Figure 5). 

FIGURE 4. Khadag (prayer flag) decorated deer stone at the Ulaan Tolgoi site.

FIGURE 3. Location of the Ulaan Tolgoi site. The red square shows 

the location of the inset map in Mongolia.
106 • smithsonian contributions to museum conservation

Preliminary observations suggest that at this site lichens 

have not caused much obvious damage to the stones.

However, one stone (DS4) showed a fairly uniform 

colonization of a single type of lichen; although it is pale 

green (called “mineral green”), it has the structure of the 

crustose genus Acarospora (Figure 6). This deer stone is 

about 1.34 m high, 75 cm wide, and 23 cm thick. The colony covers over a half meter square, an area greater than 

that of other lichen colonies on similar stones at the site. 

In 2004, the lichen colony appeared healthy and thick. 

To determine if a simple treatment would remove lichens 

from deer stones, in that year a narrow strip, about 25 cm 

long and 10 cm wide, on the right side of this lichen cover 

was treated by blotting with 70% ethanol until soaked but 

without scrubbing or any attempt to mechanically remove 

the lichen thalli. Ethanol kills the lichen’s food-producing 

algae, turning them from chlorophyll green to brown when 

they are dead, and without the algae, the food-dependent 

fungal partners starve. 

After the treatment in 2004, the deer stone was examined and photographed annually, except in 2006. The first 

year after the ethanol application, 2005, the lichens in the 

treated strip turned brown, indicating that the one-time 

treatment had killed their algal partners. By the third year, 

2007, the plantlike thalli in the treated area had dropped 

off the stone. Notably, during the same period the sweep 

of the lichen on the entire north side of this deer stone also 

began to turn brown and thin out, demonstrating that the 

colony is dynamic. By 2009, all the lichen was entirely 

brown and flaking, and the untreated area was almost as 

bare as the treated area (Figure 7). Figure 8 shows a detail of the edge of the treated area, four years after the 

FIGURE 6. North side of the deer stone (DS4) at the Ulaan Tolgoi 

site with uniform lichen colonization photographed in 2004 before 

treatment. The string shown serves to mark the boundary of the area 

that was treated with ethanol.

FIGURE 5. Another deer stone at the Ulaan Tolgoi site showing 

mixed lichen colonization. Note the slanted lower edge of the biocolonization, which reflects that this stone most likely was partly 

buried to this line.
number 2 • 107

application of ethanol. Once this lichen drops off, it is possible that the surface will be recolonized, perhaps by the 

more-typical mixture of different genera and species.

Although the treatment with ethanol certainly accelerated the death of the lichens in the strip where it was 

applied, the ongoing decrease in the rest of the lichen coverage cannot be attributed to it. There are no other obvious changes in the stone or its environment that would 

account for the death of the lichen: no chemical stains, 

no exceptional input of toxic nitrogen compounds from 

bird droppings, no new shading of the area, and no ritual application of sticky materials. However, what could 

have changed was the position of the stone since the lichens originally colonized it. The site was discovered by 

Mongolian archaeologist Sanjmyatav in 1988, and in the 

early to mid-1990s the Mongolian-Russian archaeological 

team reerected some of the fallen deer stones (Fitzhugh, 

2006:15), possibly including this one. It is important to 

note that the mineral green lichen cover originally ended 

rather abruptly with a slightly angled line across the stone 

(see Figure 6), as did the mixed lichen cover on another 

stone in the site (see Figure 5). The position of the lichens 

suggests that for many years the treated stone was lying 

practically flat on the ground with its upward side (now 

the north side) half covered with the soil up to this line. 

In this case, moisture in the soil’s boundary layer and the 

sunlight’s angle of insolation would have combined to 

support a continuous cover of the lichen species, a situation also seen in outcropped rocks in the area. Consequently, the stone may have been colonized by a single 

lichen rather than by the mixed lichen colonization found 

on most of the other standing deer stones (as in Figure 

5). With a change in the stone’s position to standing, the 

moisture content in this upper area would have dropped, 

and the lichen cover would have begun a steady decline 

over the five years it was photographed.

FIGURE 7. North side of the deer stone (DS4) from Figure 4 (a) in 2007, three years after the application of ethanol, and (b) in 2009, five years 

after the treatment. Note the diminishing difference between the treated area and the rest of the stone.
108 • smithsonian contributions to museum conservation

The loss of the lichen cover on this particular stone 

reveals 3,000-year-old carvings that are still in very good 

condition in spite of the years of lichen colonization they 

suffered, most likely because of the relatively unflawed 

quality of the granite surface. Although lichens will generally accelerate deterioration of the substrate, in other 

instances it has been shown that they also may serve to 

prevent deterioration resulting from the colonization of 

other microorganisms (Warscheid and Leisen, this volume) and perhaps from wind and rain erosion. What is 

important, however, is that if their removal is required for 

documentation purposes, then only gentle methods that 

do not damage the stone surface should be employed, such 

as the ethanol used in this instance without scrubbing or 

any mechanical lichen removal, and that these methods 

are given the required time to act on the organisms (Delgado Rodrigues et al., this volume). Biocolonization takes 

time to develop, and it requires time to be eliminated.

REFERENCES

Beaubien, H. F., and B. V. Karas. 2008. “3D Scanning Deer Stones on the 

Mongolian Steppe.” In Proceedings of the Objects Specialty Group 

Session, 35th Annual Meeting 2007, ed. V. Green, P. Griffin, and 

C. Del Re, pp. 178–196. Washington, D.C.: American Institute for 

Conservation of Historic and Artistic Works. 

Beaubien, H. F., B. V. Karas, and W. W. Fitzhugh. 2007. “Documenting 

Mongolia’s Deer Stones: Application of Digital Imaging Technology 

to Preservation.” In Scientific Research on the Sculptural Arts of 

Asia: Proceedings of the 3rd Forbes Symposium at the Freer Gallery of Art, ed. J. G. Douglas, P. Jett, and J. Winters, pp. 133–142. 

London: Archetype Publications, Ltd.

Delgado Rodrigues, J., M. Vale Anjos, and A. E. Charola. 2011 (this 

volume). “Recolonization of Marble Sculpture in a Garden Environment.” In Biocolonization of Stone: Control and Preventive 

Methods, Proceedings from the MCI Workshop Series, ed. A. E. 

Charola, C. McNamara, and R. J. Koestler, pp. 71–85. Smithsonian 

Contributions to Museum Conservation, No. 2. Washington, D.C.: 

Smithsonian Institution Scholarly Press.

Fitzhugh, W. W. 2005. “The Deer Stone Project: Exploring Northern 

Mongolia and Its Arctic Connections.” In The Deer Stone Project: Anthropological Studies in Mongolia 2002–2004, ed. W. W. 

Fitzhugh, J. Bayarsaikhan, and P. K. Marsh. pp. 3–31. Washington, 

D.C.: Arctic Studies Center, National Museum of Natural History, 

Smithsonian Institution.

———. 2006. “Mongolian Field Journals 2006.” In The AmericanMongolian Deer Stone Project: Field Report 2006, ed. W. W. 

Fitzhugh, pp. 14–43. Washington, D.C.: Arctic Studies Center, National Museum of Natural History, Smithsonian Institution.

Jacobson, E. 1993. The Deer Goddess of Ancient Siberia: A Study in the 

Ecology of Belief. New York: E. J. Brill.

Warscheid, T., and H. Leisen. 2011 (this volume). “Microbiological 

Studies on Stone Deterioration and Development of Conservation 

Measures at Angkor Wat.” In Biocolonization of Stone: Control 

and Preventive Methods, Proceedings from the MCI Workshop Series, ed. A. E. Charola, C. McNamara, and R. J. Koestler, pp. 1–18. 

Smithsonian Contributions to Museum Conservation, No. 2. Washington, D.C.: Smithsonian Institution Scholarly Press.

Wachowiak, M. J., and B. V. Karas. 2009. “3D Scanning and Replication 

for Museum and Cultural Heritage Applications.” Journal of the 

American Institute for Conservation, 48(2):141–158.

FIGURE 8. Detail of the untreated (left) and treated (right) areas in 2008, four years 

after the treatment. Note that the lichen colony on the left is already thinning out.
David P. Wessel, Architectural Resources Group, 

Pier 9, The Embarcadero, San Francisco, California 94111, USA. Correspondence: david@argsf

.com.

Case Study: Field Observations on the 

Effectiveness of Zinc Strips to Control 

Biocolonization of Stone

David P. Wessel

The use of metallic strips made of copper, bronze, or zinc to control 

biocolonization has long been known, but the implementation of this 

method has not been easy. The method relies on the dissolution by 

rainwater of minimal amounts of metal ions that act as inhibitors for 

the growth of microorganisms (Ashurst and Dimes, 1999:136–137). This paper 

evaluates the effectiveness of zinc strips installed on the roof of the Stanford 

Mausoleum after 12 years.

The Stanford Mausoleum is a small neoclassical building of rectangular 

plan measuring approximately 8 × 12 m. In 1889 the entire building (including 

the roof) was constructed of gray-colored granite from Barre, Vermont (Figure 

1). In 1995, Stanford University commissioned a condition survey of the building because of severe water penetration problems that had developed over the 

previous 106 years. Apart from those problems, the roof had developed significant lichen colonization, including foliose and crustose lichens. The former were 

identified as belonging to the Parmeliaceae family and probably to the Xanthoparmelia genus since the latter is a common lichen found in the San Francisco 

Bay area (Figure 2).

A conservation intervention on the building was completed in 1997. After 

an initial cleaning of the entire building by pressure washing, the proprietary 

Heavy Duty Restoration Cleaner by ProSoCo, Inc. (a mixture of glycolic, hydrofluoric, orthophosphoric, and citric acids with a nonionic surfactant) was used 

in a 1:5 aqueous solution to facilitate removal of the lichens. After the roof was 

free of the lichens, zinc strips bent to conform to the angle of the roof’s ridge 

were installed on the ridge, and a commercial, one-part urethane sealant (Sikaflex 1a) was used to keep them in place (Figure 3) (Wessel, 2003).

Twelve years later, the roof was inspected to evaluate the effectiveness of the 

zinc strips. No biological colonization was evident, and only minor soiling, which 

could be attributed to a passing seagull, was found (Figures 4 and 5). Nevertheless, lichen colonization is slow to develop, and judging from lichen colonization of other materials, it certainly would require more than five years to develop 

(Henriques et al., 2007; Delgado Rodrigues et al., this volume). Only a small 

amount of soiling, under the roof cornice, had reappeared (Figure 6).
110 • smithsonian contributions to museum conservation

FIGURE 2. The roof of the Stanford Mausoleum in 1995, showing the heavy lichen colonization 

that had developed over 106 years.

FIGURE 1. The Stanford Mausoleum (photo courtesy of Stanford Archives).
number 2 • 111

FIGURE 3. The roof of the Stanford Mausoleum after cleaning in 1997, showing the zinc strips installed on 

the roof’s ridges.

FIGURE 4. The roof of the Stanford Mausoleum in 2009, 12 years after the cleaning and the installation of 

the zinc strips.
112 • smithsonian contributions to museum conservation

It is important to point out that the installation of 

zinc strips to prevent recolonization by lichens and other 

microorganisms can be most effective when the object to 

be protected has a regular shape and design that ensures 

even distribution of rain water over the surface. Furthermore, this approach should be used as part of an integral 

conservation intervention and not as an isolated measure. 

The condition of the Stanford Mausoleum 12 years after 

the intervention was documented to serve as reference 

for future monitoring. So far, the conservation intervention has been a major success. Not only has the building 

remained largely free of biological recolonization, but its 

maintenance cost has been significantly reduced, an important point in architectural conservation. 

REFERENCES

Ashurst, J., and F. G. Dimes. 1999. Conservation of Building and Decorative Stone. Part 2. London: Butterworth-Heinemann.

Delgado Rodrigues, J., M. Vale Anjos, and A. E. Charola. 2011 (this 

volume). “Recolonization of Marble Sculptures in a Garden Environment.” In Biocolonization of Stone: Control and Preventive 

Methods, Proceedings from the MCI Workshop Series, ed. A. E. 

Charola, C. McNamara, and R. J. Koestler, pp. 71–85. Smithsonian 

Contributions to Museum Conservation, No. 2. Washington, D.C.: 

Smithsonian Institution Scholarly Press.

Henriques, F., A. E. Charola, V. Moreira Rato, and P. Faria Rodrigues. 

2007. Development of Biocolonization Resistant Mortars: Preliminary 

Results. Restoration of Buildings and Monuments, 13(6):389–400.

Wessel, D. P. 2003. “The Use of Metallic Oxides in Control of Biological 

Growth on Outdoor Monuments.” In Art, Biology and Conservation, 

ed. R. J. Koestler, V. Koestler, A. E. Charola, and F. Nieto-Fernandez, 

pp. 536–551. New York: The Metropolitan Museum of Art.

FIGURE 5. (a) Detail of the roof in 2009, showing minor soiling. (b) Close up of the area of soiling indicated by the arrow in (a).

FIGURE 6. Detail of the cornice in 2009, showing new soiling.
A. Elena Charola, Museum Conservation Institute, Smithsonian Institution, Suitland, Maryland 20746, USA. Christopher McNamara, 

Harvard School of Public Health, Harvard 

University, Boston, Massachusetts 02215, 

USA. Robert J. Koestler, Museum Conservation Institute, Smithsonian Institution, Suitland, Maryland 20746, USA. Correspondence: 

A. E. Charola, charolaa@si.edu.

Discussions, Conclusions, and 

Recommendations

A. Elena Charola, Christopher McNamara, 

and Robert J. Koestler

ABSTRACT. The results of the discussions and the conclusions that could be drawn 

from this workshop are summarized, especially with reference to the three questions 

that had been posed to the participants. The first question addresses the identification of 

criteria to determine when the colonization on a stone surface may be problematic. This 

requires compiling information on both the substrate and the microorganisms. The second deals with new methods for biocolonization control. This entails the development of 

eco-friendly compounds (e.g., biocides) methods of biocontrol, and development of longand short-term management plans based on regular monitoring and simple maintenance 

practices. The final question aims to identify future research directions to achieve the 

two goals mentioned. Several topics were identified, ranging from rapid testing methods 

to interdisciplinary research methodology. Most significant was the recommendation to 

develop a practical database from the existing literature.

INTRODUCTION

The international workshop “Biocolonization of Stone: Control and Preventive Measures” was held at the Museum Conservation Institute in April 

2009. The goal of the workshop was to provide a discussion forum for biologists, materials scientists, and conservators interested in stone biodeterioration 

to address the following three questions:

1. How can we develop criteria to determine when microbial colonization and 

growth on stone heritage materials is problematic?

2. What new environment-friendly methods are available to prevent microbial 

deterioration and colonization and to control recolonization of stone after 

a conservation intervention?

3. What future research directions will aid in achieving these goals?

The workshop featured presentations from seven experts in the field of microbial degradation of stone monuments and buildings and five case studies 

illustrating practical examples. A discussion period served to identify some of 

the key points that needed clarification, and a subsequent panel discussion with 

the speakers aimed to address the questions listed above. Finally, a closed-door 
114 • smithsonian contributions to museum conservation

half-day meeting of all the speakers focused on developing 

criteria that can be used to evaluate microbial colonization 

and growth and to determine conditions when treatment is 

necessary. The participants also worked on identifying future research directions for the field. For this purpose, the 

speakers were divided into two groups, a largely biologically oriented group and a second that could be considered 

the “practical approach” group. Both groups addressed 

the same three questions. The interesting result was that 

both groups came up with essentially the same answers 

and recommendations. 

CONCLUSIONS AND 

RECOMMENDATIONS

The conclusions and recommendations made after the 

discussions can be summarized as follows. 

1. How can we develop criteria to determine when 

microbial colonization and growth on stone heritage materials is problematic?

Microbial colonization of outdoor stone surfaces is 

inevitable. However, problematic microbial growth (characterized by aesthetic changes and/or biodeterioration) 

does not occur on all objects. Borrowing a framework developed by food microbiologists, categories of factors that 

could affect microbial colonization and growth were proposed, and important factors within each category were 

identified. These include:

1. Factors intrinsic to the substrate. Among these, the 

chemical and mineralogical nature of the stone, and 

its porosity, surface roughness, and orientation with 

regard to bedding planes in the building, are the most 

important.

2. Present state and previous history of the building or 

monument. These factors include the presence of soluble salts or inorganic surface crusts; the type of deterioration, e.g., powdering or flaking, that the stone 

has developed; contact with the soil; and previous 

treatments that may have been applied to the object, 

such as cleaning, consolidation, water repellents, and 

biocides.

3. Extrinsic factors defining the environment of the object. These include geographic location of the object; 

the environment, i.e., rural or urban; general macroclimate, e.g., average temperature and humidity, rain 

periodicity; exposure, e.g., whether northern or southern, and light irradiation regime; and microclimate for 

the object, including thermohygric changes and rate 

of these changes. 

4. Factors describing the physiology and taxonomy of 

the microorganisms. Factors that should be considered include coloration (green or black pigments), the 

presence of biofilm or slime layers, the presence of 

filaments, the depth profile of colonization, the extent 

of colonization, comparison of the microbial community with nearby objects, and the rate of change (or 

seasonality) of the microbial growth.

2. What new environment-friendly methods are available to prevent microbial deterioration and colonization 

and to control recolonization of stone after a conservation 

intervention with a minimum environmental impact?

New methods are needed to prevent and control microbial colonization of stone. Particular areas of need include the development of eco-friendly compounds (e.g., 

biocides), methods of biocontrol, and development of 

long- and short-term management plans based on regular 

monitoring and simple maintenance practice, such as dust 

removal or periodic washing of the object.

3. What future research directions will aid in achieving these goals?

Five conclusions regarding directions for future research were reached:

1. Simple, quick, and accurate methods for measuring 

microbial colonization and growth are needed. Improved sampling techniques and a rapid biofilm sensor would be extremely useful.

2. Multidisciplinary studies of deteriorated areas that 

have biocolonization are needed. It is necessary to 

assess the contribution of biocolonization to the observed damage. The presence of microorganisms does 

not necessarily imply that they are responsible for the 

deterioration present.

3. Advances in molecular biology have improved our 

knowledge of the taxonomy of microorganisms found 

on stone. Future efforts need to make use of this information, for example, by linking taxa with function or 

biodeterioration. Possible methods for accomplishing 

this include application of genomic, proteomic, and 

metabolomic techniques.
number 2 • 115

4. Regarding scenario testing, weathering experiments 

are needed to better understand the consequences of 

microbial colonization and growth on historic stone.

5. Further studies and testing are needed to evaluate the 

interaction between two conservation products, for 

example, the influence of biocides on the effectiveness 

of water repellents and vice versa, and the long-term 

effect of the addition of biocides mixed directly into 

other conservation products, such as consolidants formulated with a biocide. 

Finally, the need for the development of an interactive 

framework to facilitate communication and collaboration 

among conservators and scientists interested in the deterioration of historic stone was discussed. Scientists and conservators interested in microbial colonization and growth 

on historic stone are spread across the globe. In addition, 

studies of microbial activities on stone are found throughout the scientific and conservation literature in a wide 

range of journals, books, and gray literature representing 

many different disciplines. So there is a need for a centralized portal that can facilitate communication and access to 

information. Development of a Web site or wiki was proposed. The site would contain features such as a database 

of publications and information about biodeterioration 

and a discussion board.

Therefore, the immediate action recommendation is to 

develop a practical database from the existing literature. 

This would serve to identify the gaps between the various 

disciplines that have to collaborate in this field. For example, 

many of the microbiological studies reported in biological 

journals do not present full information on the substrate on 

which the microorganisms develop. On the other hand, materials scientists report the existence of biocolonization but 

do not identify the species in question. Therefore, to gain a 

better understanding of the problem from the vast literature 

that has already developed, it is necessary to complete the 

existing studies. Since these are dealing with monuments, 

the information is likely to be available; what is necessary is 

a means to bring the information together.

All manuscripts are reviewed for adherence to the SISP Manuscript Preparation and 

Style Guide for Authors (available on the “Submissions” page at www.scholarlypress.si.edu). Manuscripts not 

in compliance will be returned to the author. Manuscripts intended for publication in the Contributions Series are evaluated by a content review board and undergo substantive peer 

review. Accepted manuscripts are submitted for funding approval and scheduling to the Publications Oversight Board. 

Minimum manuscript length is thirty manuscript 

pages. If a manuscript is longer than average, an appropriate 

length will be determined during peer review and evaluation 

by the Content Review Board. Authors may be asked to edit 

manuscripts that are determined to be too long. 

Text must be prepared in a recent version of Microsoft 

Word; use a Times font in 12 point for regular text; be double 

spaced; and have 1” margins. Each chapter/section must be 

saved in a separate file.

Required elements are title page, abstract page, table 

of contents, main text, and reference section. See the SISP 

Manuscript Preparation and Style Guide for Authors for the 

order of all elements.

Headings should be styled so different levels of headings are 

distinct from each other and so the organization of the manuscript is clear. Insert one line space above and one line space 

below all headings.

Front matter should include title page, abstract page, 

and table of contents. All other sections are optional. Abstracts must not exceed 300 words. Table of contents should 

include A-, B-, and C-level headings.

Tables (numbered, with captions, stubs, rules) should be 

submitted in separate MS Word files; should include footnotes, if appropriate; should have rules only at top, bottom, 

and beneath column heads. Print outs of each table should 

accompany the manuscript to ensure correct layout of data. 

Tabulations within running text should not be numbered or 

formatted like formal tables, and should be included in the 

text of the manuscript. 

Figure captions should be provided in a separate MS 

Word file.

Figures (e.g., photographs, line art, maps) should be numbered sequentially (1, 2, 3, etc.) in the order called out; be 

placed throughout text, not at end of manuscript; have all 

components of composites lettered with lowercase letters and 

described in the caption; include a scale bar or scale description, if appropriate; include any legends in or on the figure 

rather than in a caption. 

Art must not be embedded in the main text.

Figures must be original and submitted as individual TIFF 

or EPS files. Resolution for art files must be at least 300 dpi 

for grayscale and color images and at least 1200 dpi for line 

art. Electronic images should measure no more than 100% 

and no less than 75% of final size when published. JPG files 

will not be accepted. Color images significantly increase 

costs so should be included only if required. Funding for 

color art is subject to approval by SISP and the Publications 

Oversight Board.

In-text references should be used rather than bibliographic notes and should follow the author-date system in the 

following format: “(author last name, year)” or “. . . author 

(year)”; “(author, year:page used within the text)” or “. . . 

author (year:page).” A full citation should be included in a 

“References” section.

Endnotes are to be used in lieu of footnotes and should 

be keyed manually into a separate MS Word file, in a section titled “Notes”. Notes should not contain bibliographic 

information. Manually type superscript numerals in text and 

use full-sized numerals at the beginning of each note in the 

“Notes” section. SISP will determine the best placement of the 

notes section, either at the end of each chapter or at the end 

of the main text.

References should be in alphabetical order, and in chronological order for same-author entries. Each reference should 

be cited at least once in main text. Complete bibliographic 

information must be included in all citations (e.g., author/editor, title, subtitle, edition, volume, issue, pages, figures). For 

books, place of publication and publisher are required. For 

journals, use the parentheses system for volume(number):pagination [e.g., “10(2):5–9”]. Do not use “et al.”; all authors/

editors should be included in reference citations. In titles, 

capitalize first word, last word, first word after colon, and all 

other words except articles, conjunctions, and prepositions. 

Examples of the most common types of citations are provided 

in the SISP Manuscript Preparation and Author Style Guide. 

For questions regarding the guidelines, please email SISP at 

schol.press@si.edu. 

requirements for smithsonian series publication