Living History

Tourists who visit the Maya temples at Ek’ Balam in Yucatán, Mexico, aren’t allowed to pocket souvenir chunks of the intricate carvings on the centuries-old buildings. Visitors aren’t supposed to even touch the soft limestone with their hands for fear they’ll rub away the structures’ delicate sculptural details. Yet even as Ek’ Balam’s caretakers keep people from destroying the archaeological site, microbes are working in a rock-eating bacchanal that could, if unchecked, leave behind nothing but dust.

Recent research has revealed that hordes of bacteria living inside the temples’ stones are physically and chemically dismantling the structures bit by grainy bit. The onslaught is slow, so the temples will probably stand for centuries. But the buildings are gradually losing their finely chiseled designs and images of people, gods, and animals.

The keepers of Ek’ Balam used to think that deterioration of these objects was almost always the work of forces such as wind and sun or humanmade threats, including pollution. However, scientists in the past several years have been finding that bacteria and fungi living on art works and artifacts at Ek’ Balam and elsewhere often cause extensive damage.

“Lots of materials used in [cultural objects] were never meant to last forever. They’re going to deteriorate,” says Christopher McNamara, who, with Ralph Mitchell and his colleagues in Harvard University’s Laboratory of Microbial Ecology, has studied microbes at Ek’ Balam and other historic sites. “The scientist’s role is to try to understand the processes going on in deterioration so that [the objects] can be preserved with the right methods,” Mitchell explains.

Fungus among us

Some researchers have long suspected that microbial vandals were behind some of the worst deterioration in historic objects or art—for example, the black smudges on some aging documents or changes in the shades or textures in some old paintings. In the past, proving those allegations was tricky, so the first order of business for artifact microbiology has been to show that microbes indeed are to blame.

Traditionally, biologists have tried to identify what microbes might be living on a canvas, temple wall, or parchment, for instance, by collecting organisms from a deteriorating object and then growing them in the lab. To do that, scientists run swabs over damaged paintings and manuscripts or, when permitted, snip or scrape off small pieces for analysis. They then place these samples in a nutrient-rich medium in petri dishes and identify artifact-wrecking microbes from those species of bacteria and fungi that grow into colonies in the dish.

It’s a limited method, notes Guadalupe Piñar, a microbiologist at the University of Vienna. For every species that thrives in a dish of generic nutrients, many others don’t. Some of those that live on artifacts do so because the artifacts supply a nutrient that they need but that the standard medium lacks. Piñar estimates that these microbe-culture tests reveal only about 1 percent of the species present within an artifact.

That oversight has misled researchers for decades into thinking that microbes play only a minor part in an object’s deterioration, says Piñar. But with access to powerful new tools for microbiological analyses, scientists have been getting an inkling of just how much they’ve been missing.

For example, Piñar’s University of Vienna colleague Astrid Michaelsen is adapting a technique known as gel electrophoresis to quickly identify, without having to culture the microbes, which fungal species are damaging old books and manuscripts. She’s perfecting her technique on paper samples that collaborators at the Central Institute for Book Pathology in Rome inoculated with known fungal species 20 years ago. Among them are Aspergillus versicolor and Chaetomium globosum. Some of these species secrete melanin, darkening the paper. Some send tiny appendages into the paper itself, wreaking mechanical damage. Some eat the paper, thinning it over time.

When Michaelsen extracts DNA from a fungus in these stored samples and runs it through gel electrophoresis, she gets a series of bands that amount to a DNA fingerprint. The plan now is take the same kind of fingerprint from unknown fungi in artifacts. If the result matches one from the stored papers, Michaelsen will make an ID. She says she hopes to be ready to do just that sometime this fall with a number of old documents, including a 13th-century manuscript and books from the 17th and 18th centuries.

Juan Gonzalez has been applying related techniques to determine whether prehistoric paintings in several Spanish caves are under microbial attack. A microbiologist at the Spanish National Research Council in Seville, Gonzalez notes that finding the DNA of an organism rarely tells what that organism is up to. “DNA will tell us that an organism is there, but not if it’s metabolically active,” for example, deteriorating mineral pigments, he says.

To determine whether an organism is active, Gonzalez searches samples for specific bits of ribosomal RNA. Because this RNA is a go-between in the process of translating DNA into proteins, its presence indicates that the organism it came from was actively making protein. Using this approach, Gonzalez has found that certain species of bacteria are thriving and possibly damaging the cave paintings.

Tool time

Using these and other tools, researchers are making headway in understanding the deterioration of historic and cultural works.

At Ek’ Balam, for one, McNamara and his colleagues have found that damage can occur from the inside out. While other researchers had previously identified damaging bacteria and fungi living on the outside of the ancient city’s buildings, little was known about bacterial communities in the limestone’s labyrinth of pores.

To identify the microbes there, he and his colleagues chiseled off small pieces of stone from the city’s largest temple. They then collected bacteria from both the inside and outside of these sections and searched the organisms for a particular gene, known as the 16S ribosomal gene. The sequence of this gene varies in different bacterial species, so it can be used to identify the microbes.

McNamara says that he was surprised to find that the bacterial communities outside the stone differed drastically from those inside. However, many of the microbes both inside and outside could secrete acid. That acid is a probable cause of the deterioration occurring at Ek’ Balam, McNamara asserts.

Robert Blanchette, a forest pathologist at the University of Minnesota in St. Paul, also is relying on gene sequencing, as he examines fungal damage in wooden huts built in the early 1900s by Ernest Shackleton, Robert Scott, and their crews as they explored Antarctica. Although Antarctica has a cold and dry climate, which generally restricts the growth of fungi, the huts’ interiors tend to be slightly more humid and warmer than the windblown outside surfaces.

By analyzing DNA from fungi in the huts, Blanchette and his colleagues determined that the species are native to Antarctica and typically feed on moss and lichens. “The [huts] provided them with a nice, new nutrient source,” says Blanchette.

The wilds of Mexico and Antarctica are far from the carefully controlled environment of a museum. However, ongoing research by Tanya Khijniak of the Winogradsky Institute of Microbiology in Moscow and her colleagues is showing that microbial deterioration may be striking even the most-protected museum pieces.

Previous work showed that some bacterial species, including a strain of Pseudomonas chromatophila, can corrode metals present in solid crocoite, a mineral that artists, including Vincent Van Gogh and Marc Chagall, have used to make yellow or orange paint.

When chromate ions inside the mineral absorb electrons, the mineral turns green or brownish. Conservators had noticed that some paintings using crocoite have become discolored, but researchers hadn’t pinned down the cause. Khijniak and her colleagues suspected that microbes might be behind the damage.

Because they haven’t been permitted to take swab samples from any priceless paintings, the researchers devised another way to determine whether bacteria might be at work.

First Khijniak’s team prepared crocoite-based paints, mixing powdered crocoite with oil, and brushed the paint on swatches of canvas. Next, the scientists inoculated their tiny masterpieces with selected species of bacteria. Finally, they monitored the swatches for any changes in the paint’s shade. Because many of the species Khijniak’s team works with thrive under low-oxygen conditions, the researchers speed up the bacteria’s action by enclosing the swatches in airtight bottles. “People had centuries to notice changes in some of these paintings,” she says. “In my research, it’s not easy to wait for so long.”

The team has confirmed that swatches inoculated with the bacteria change to the same shades evident in the masters’ discolored paintings. Khijniak plans to present preliminary results from this ongoing project at a conference this month in Spain.

Dodging damage

Now that scientists have identified some of the microbes responsible for damaging historic structures and art, the next task is to use the information to protect these items.

Blanchette says that preventing further damage, rather than eradicating fungal colonies already in place, is often the best approach. Toward that end, he and his colleagues are looking for the best way to keep the wood in the Antarctic huts clean and dry.

Prevention also means working with conservators to make sure that their cleanup efforts aren’t causing further harm. For example, McNamara and his colleagues are working with conservators to develop a reinforcing liquid that will soak into and strengthen the stones at Ek’ Balam. Such products are called consolidants, and several are on the market. But they’re made of organic materials that many microbial species find irresistible. So, the Harvard team has suggested that the keepers of Ek’ Balam wait for a new formulation. “If you’re adding a potential food source, it could be stimulating growth and making a more-severe problem,” says McNamara.

Robert Koestler, director of the Smithsonian Center for Materials Research and Education in Suitland, Md., notes that even applying biocides to kill organisms invading artifacts can be a bad idea. First, chemicals that kill bacteria or fungi may damage delicate paints or other materials. Additionally, many biocides kill only some species of bacteria, giving other equally damaging species the chance to take over and run wild.

Koestler and his colleagues have found an alternative to biocides. Instead of spraying delicate artifacts with chemicals, they put the items in airtight bags and pump in argon. The oxygenfree environment suffocates almost anything living on or in an object.

At times, the best course of action can be to leave relics alone, says Thomas Warscheid of LBW-Bioconsult, a private consulting company in Oldenburg, Germany. He cites the troubling example of the Angkor Wat temple in Cambodia. In the early 1990s, conservators scrubbed a thick layer of moss and lichens from the temple and sprayed biocides over the structure’s sandstone surfaces. It was the first thorough cleaning the centuries-old building had ever had. When the conservators finished, the temple was gleaming white.

However, dark patches soon grew and spread on the temple sandstone. The surfaces turned from white to a musty black that was much darker than the coating that had been scrubbed away.

“What people underestimated was that microorganisms tend to recover very fast,” Warscheid adds. The first bacteria to recolonize the stones after the biocides’ effects wore off were cyanobacteria, a group of dark-colored, photosynthetic microorganisms that had been held in check by competition with other microbial species. Furthermore, since the building is now darker than it was before, it absorbs more heat, changing the physical properties of its stones and hastening some types of physical degradation.

Nevertheless, scientific developments may turn around the temple’s prospects again. Warscheid and his collaborators have developed an environmentally sound biocide that may halt the cyanobacteria’s spread over the temple of Angkor Wat. He anticipates that the increasing collaboration among microbiologists, mycologists, and conservators will find many more strategies for heading off the deterioration of historical items and valuable art.