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NEW APPROACHES TO DETECT AND REMEDIATE MICROBIAL DETERIORATION

Nick Konkol, Chris McNamara, Joseph Sembrat1, Mark Rabinowitz1, and Ralph Mitchell

Harvard University: School of Engineering and Applied Sciences

Microbial growth is frequently a factor in the deterioration of stone cemetery materials, particularly in damp or humid environments. Metabolically diverse bacteria and fungi attack the stone statuary, monuments and facades through enzymatic activity and through mechanical weathering. The detection and elimination of these growths can be difficult. Removal of their residues is even more difficult as the organisms are frequently embedded deep within fissures and surface crevices. Recently, microbiologists and conservators have begun using enzymes both to safeguard and restore stone artifacts. We have developed two new enzyme-based techniques that can be applied to cemetery materials.

Mold detection

Protecting outdoor stonework from mold is a challenge to conservators. Molds are ubiquitous components of the air we breathe and have the capacity to infect a variety of materials. These fungal infections are often overlooked until they attain enough biomass to be visible to the human eye. A great deal of damage can be done in the time between infection and detection. Current methods used for detecting and measuring fungal biomass on surfaces, such as microscopic biovolume estimates and chemical analysis of the mold, are expensive and time-consuming. In order to facilitate early fungal detection, we have developed a rapid, inexpensive, and non-destructive means of identifying fungal growth.

Figure 1. Fungal growth on marble.  Fungal biomass measured over 96 hours on a Carrera marble substratum.

Figure 1. Fungal growth on marble. Fungal biomass measured over 96 hours on a Carrera marble substratum.

Fungal cell walls are composed of the rigid biopolymer chitin that fungi must continually deconstruct and rebuild in order to grow. Deconstruction of chitin is carried out with chitinase, an enzyme that catalyzes the break-down of N-acetyl-β-D-glucosamine (NAG). Fluorogenic 4-methylumbelliferyl (MUF)-labeled NAG can be used as a substrate to detect chitinase activity during mold growth. Originally developed to detect and quantify fungal chitinase activity in soil1 it was quickly demonstrated that MUF-NAG could be used to detect fungi on building surfaces2. We used this assay to quantify fungal biomass on a variety of materials, including marble (Fig. 1) with sensitivity comparable to that of chemical techniques (lower detection limit near 40 µg dried mycelium). Our assay can be used to detect early mold growth on stone surfaces as an aid to conservators in identifying “at risk” materials.

Stain removal

Remediation of outdoor sculptures, monuments and facades that have suffered from microbial deterioration also poses a challenge to conservators. Treatments developed to clean them are often time-consuming, expensive, risk further damage, and are not always effective. Enzymes may overcome many of these limitations, and have already found use in the remediation of frescoes and easel paintings3. We have begun to expand the use of enzymes for remediation by conducting experiments on open-air marble sculptures. Many marble sculptures, monuments, and facades throughout Europe and North America suffer from biodeterioration. It often manifests itself as unsightly red stains whose removal has been problematic.

Figure 2.  Decolorization of prodigiosin.  Absorbance –ΔE570 of the prodigiosin solution from 0 to 240 m after the addition of laccase.  Data are the averages of three reactions.

Figure 2. Decolorization of prodigiosin. Absorbance –ΔE570 of the prodigiosin solution from 0 to 240 m after the addition of laccase. Data are the averages of three reactions.

We determined that red-brown stains on Isamu Noguchi’s marble sculpture Slide Mantra in Bayfront Park, Miami, Fl, were probably caused by pigment-producing microorganisms. Traditional cell culture methods were first used to isolate a red-pigmented bacterium from a stained area of Slide Mantra. Sequencing and analysis of the 16S rRNA gene identified the organism as a strain of Serratia marcescens. Fourier transform infrared spectroscopy demonstrated that the pigment produced by the Bacteria was most likely a prodigiosin. Our analysis found that the enzyme laccase, isolated from the fungus Trametes versicolor, effectively decolorized pure prodigiosin (Fig 2). Tests of the effects of this enzyme on the bacterium that stained Slide Mantra similarly decolorized the stain. This study suggests that enzymatic decolorization may be applicable to stains on culturally significant marble caused by microbial colonization. We are currently investigating the use of this enzyme and others to remediate the surfaces of Slide Mantra and other marble sculptures exposed to outdoor air.

1. Reeslev, M., M. Miller, and K.F. Nielsen. 2003. Quantifying Mold Biomass on Gypsum
Board: Comparison of Ergosterol and Beta-N-Acetylhexosaminidase as Mold Biomass Parameters. Appl. Environ. Microbiol. 69: 3996-3998.

2. Miller, M., A. Palojärvi, A. Rangger, M. Reeslev, and A. Kjøller. 1998. The Use of
Fluorogenic Substrates to Measure Fungal Presence and Activity in Soil. Appl. Environ. Microbiol. 64: 613-617.

3. Ranalli, G., G. Alfano, C. Belli, G. Lustrato, M.P. Colombini, I. Bonaduce, E. Zanardini,
P. Abbruscato, F. Cappitelli, and C. Sorlini. 2005. Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes. J. Appl. Micro. 98: 73-83.

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4 Responses to Nationwide Cemetery Preservation Summit Abstracts and Video

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  2. Robert Wrigley says:

    My wife’s gr-gr-grandfather is a Union soldier buried there. When we visited this grave site last October we were quite dismayed at the condition and damage done to the stones by lawn mowers running over and chipping it. At the time we did not know the full history of the markers being altered in the 1930s. We would fully support the efforts to restore them.
    Would it be possible for a private citizen to be able to contribute to the restoration of their family’s marker or replacement? Perhaps a volunteer effort of those with relatives there could help accomplish some of this initiative to restore to their original condition.

    • Jason Church says:

      Dear Robert,
      The best person to talk with will be Betsy Dinger Elizabeth_Dinger@nps.gov who is a historian for Petersburg National Battlefield and Cemetery.

    • Bryan Cheeseboro says:

      Mr. Wrigley,
      I don’t know if you’ll get this message but I would be very interested to know which of the men buried at Battleground Cemetery is your wife’s gr-gr-grandfather. I am a historian and I have spent the last several years researcing the battle of Fort Stevens and Battleground National Cemetery. Does your wife have a photograph of her ancestor? That would be a goldmine. I have never seen any picture of any of the men killed at the battle.

      If you can be of help to me, my e-mail address is bryanac625@yahoo.com. Thank you.

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