“Enzymatic decolorization of bacterial pigments from culturally significant marble”

Published in the Journal of Cultural Heritage Vol. 10 No. 02 July-Sept. 2009, pgs. 362-366

Authored By:

Joe Sembrat, Mark Rabinowitz
Conservation Solutions Inc., 833, E Palace Avenue, NM 87501, Santa Fe, USA

Nick Konkol, Chris McNamara, Ralph Mitchell
Laboratory ofApplied Microbiology, School of Engineering and Applied Sciences, Harvard University, 40, Oxford Street, MA 02138, Cambridge, USA

Received 27 August 2008; accepted 15 October 2008

Abstract

Marble monuments and facades are susceptible to microbial colonization. Microbial growth on a marble surface can develop into unsightly red stains whose removal has proven problematic. The purpose of this study was to determine if the red-brown stains on Isamu Noguchi’s marble sculpture Slide Mantra (1991) could be caused by pigment-producing microorganisms and to assess the potential of enzymatic stain remediation. Traditional cell culture methods were used to isolate a pigmented bacterium from a stained area of the sculpture. Sequencing and analysis of the 16S rRNA gene identified the organism as a strain of Serratia marcescens, and FT-IR spectroscopy demonstrated that the pigment produced by the bacteria was most likely a prodigiosin. Decolorization of the pigment in solution demonstrated that the enzyme laccase from the fungus Trametes versicolor has potential as a decolorizing agent. This study suggests that enzymatic decolorization may be applicable to stains on culturally significant marble caused by microbial colonization.

© 2009 Published by Elsevier Masson SAS.

Keywords: Microorganisms; Marble; Pigment; Enzymes; Decolorization

1. Research aims

Marble monuments and facades are susceptible to micro­bial colonization. Microbial growth on a marble surface can develop into an unsightly red stain [1] that is not easily removed. The aim of this study was to demonstrate the presence of pigment-producing microorganisms within stained marble and to investigate the potential of the enzyme laccase to decolorize pigments isolated from the surface of a stained marble sculpture.

2. Introduction

Open-air marble facades and statuary bear the damaging effects of humidity, rain, wind, pollution, and microbi­ological colonization [2]. The growth of microorganisms can alter the appearance of marble and often results in unwanted aesthetic changes to the marble surface. One such alteration is the appearance of stains. Typically orange to dark red [3], these stains plague the Carrara marble of Italian monuments and have been documented on marble facades and statuary throughout Italy [4]. The stained marble was found to contain red-pigmented het­erotrophic bacteria belonging to the genera Micrococcus, Halococcus, and Flavobacterium, as well as the red yeast Rhodotorula minuta, and some photosynthetic microorganisms [1,2,4].

Similar stains appear on the marble facades and statuary of North America. One prominent example is Isamu Noguchi’s Slide Mantra in Bayfront Park, Miami, Florida (Fig. 1A). Carved from eight irregularly-shaped blocks of white Carrara marble joined by silicone adhesive, the sculpture stands 3 m tall and weighs 29 tons. Since its instillation in 1991, large swaths of the white Carrara marble have acquired dark red stains (Fig. 1B) that have penetrated the marble matrix. The stains are thought to be the result of pigments contained within, or released by, microor­ganisms that have colonized the marble surface (Fig. 1C).

An efficient method of pigment decolorization was needed to restore Slide Mantra to its pristine condition. Although cleaning with water spray or air abrasion could be used, these mechan­ical methods may induce structural and surface damage to the marble [5]. The surface of Slide Mantra underwent multiple CB-4 biocidal detergent (Droycon Bioconcepts Inc., Regina,

Saskatchewan) and steam rinse treatments during an extensive restoration in early 2007. The treatments removed all visible biological growth and eliminated the stain from some areas of the sculpture. Enzymatic cleaning of marble has the potential to target the remaining sources of discoloration and clean Slide Mantra and other marble sculptures and artifacts with little risk to their structure or aesthetics.

Enzymes from plants (e.g. olives and soybeans) such as lipox­idase and the white-rot fungi (e.g. Phanerochaete chrysospotium and Trametes versicolor) such as laccase and manganese peroxi­dase have long been employed in the food [6], pulp and paper [7], and textile [8] industries to decolorize a variety of pigments and dyes. The enzyme laccase has recently been employed in biore­mediation, biofuel, biosensor, and organic synthesis applications [9] and may have cultural heritage applications. Noguchi’s Slide Mantra presents an opportunity to test enzymes on an item of cultural significance. If the red-brown discoloration of the sculp­ture is the result of a biological pigment, then treatment with an enzyme such as laccase may provide an acceptable means of cleaning conservation.

3. Experimental

3.1. Isolation of pigmented bacteria

Samples were collected from 12 distinct areas of Isamu Noguchi’s Slide Mantra in Bayfront Park, Miami, Florida. A 1-cm² area was sampled with sterile BBL^TM CultureSwabs^TM (Becton-Dickinson and Co., Sparks, MD). Samples were shipped overnight to Harvard University, Cambridge, Mas­sachusetts, plated onto Difco^TM Nutrient Agar (Becton-Dickinson and Co., Sparks, MD), and incubated at 22°C for 5 to 7 days. Pigmented colonies were transferred to fresh nutri­ent agar plates and streaked for isolation. A strain that displayed reddish brown pigmentation (Fig. 1C) was selected for further study and designated Noguchi Red Bacterium (NRB).

3.2. Identification of NRB

Nucleic acids were extracted from a 1.5 ml culture of the NRB with an Ultraclean^TM Microbial DNA Isolation Kit according to the manufacturer’s instructions (MoBio Laboratories, Inc., Solona Beach, CA). The 16S rRNA gene was amplified from the extracted nucleic acids using the Polymerase Chain Reac­tion (PCR). The PCR mixture contained 10X PCR buffer without Mg (10 l), 10mM dNTP mixture (2 l), 50mM MgCl2 (3 l), 10 M primer mixtures (2 l each), 100 ng/ l template DNA (1 l) and 1 unit of Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA). Bacterial domain specific primers 27F (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′) [10] were used for the reaction. An additional reaction without template DNA served as a neg­ative control. The PCR was carried out with a 95°C (3 min) denaturation, followed by 30 cycles of denaturation at 94°C (45 sec), annealing at 55°C (30 sec), and extension at 72°C (1 min 30 sec). An additional 72°C (10 min) extension was per­formed. The PCR products were cleaned using the QIAquick^® PCR Purification Kit according to the manufacturer’s instruc­tions (Qiagen, Valencia, CA). The cleaned PCR product was sequenced at the Dana Farber/Harvard Cancer Center High Throughput DNA Sequencing Facility (Cambridge, MA) using the 16S primers 27F and 1492R. A consensus sequence assem­bled from the forward and reverse sequences was constructed with the AlignX feature of the Vector NTI Suite (Invitrogen Corp., Carlsbad, CA) and edited manually. The sequence was compared to the National Center for Biotechnology Information Database using the BLAST search program [11].

3.3. Pigment extraction

The NRB was cultured on the surface of Modified Luria­Bertani (MLB) agar [12] at 22°C for 24-48 hours. Cells were scraped from the surface with a sterile spatula and washed once in sterile RO/DI H2O. Washed cells were extracted twice with 0.5 ml methanol by agitation at room temperature for 15 minutes. The cell pellet that remained after the sec­ond extraction was nearly colorless. Pooled pigment extracts were filtered through an Acrodisc^® Syringe Filter 0.2 m Supor^® Membrane (Pall Gelman Laboratory, Ann Arbor, MI) and stored at 4^◦C in the dark for no longer than 12 hours. A pigment extract of the Serratia marcescens type strain 13880^TM (American Type Culture Collection, Manassas, VA) was also prepared according to this method and used for com­parison. Both pigment extracts displayed a vivid shade of magenta.

3.4. Pigment analysis

Pigment extracts from the NRB and S. marcescens were evap­orated to dryness in a laminar flow hood. Fourier Transform Infrared (FT-IR) spectroscopy of the dried pigments were con­ducted at the Straus Center for Conservation (Cambridge, MA) with a VERTEX 70 FT-IR spectrometer connected to a Hyperion 3000 microscope (Bruker Optics, Billerica, MA).

3.5. Decolorization of NRB pigment extract

A pigment solution was created by dissolving 36 l of Tween 80 in 1 ml of the pigment extract and evaporating the mixture to near dryness [13]. The residue was dissolved immediately in 0.5 ml sterile RO/DI H2O. Decolorization reactions were con­ducted in 96-well microtiter plates at room temperature. The total reaction volume was 180 l. The decolorization solution contained the pigment solution, 50mM malonic acid (ph 4.5), 0.2mM MnSO4, 0.1mM _H2O2, and 2.5U of laccase. An equal volume of sterile RO/DI H2O was substituted for the laccase enzyme in control reactions. Prior to the addition of laccase the absorbance spectrum (250-800 nm) of the pigment solution was measured with a SpectraMax^® Plus³⁸⁴ plate reader (Molecular Devices, Sunnyvale, CA). Immediately after the addition of lac-case, the decolorization of the pigment solution was determined by measuring the absorbance (570 nm) every 5m for 240 m. An additional absorbance spectrum (250-800 nm) was performed at 240 m.

4. Results and discussion

Isolation of the bacterium NRB from a stained area of Isamu Noguchi’s Slide Mantra verified the presence of a pig­mented microorganism on a discolored section of the sculpture. Attempts to culture pigmented microorganisms from three visi­bly clean areas of the sculpture failed, indicating that pigmented bacteria are restricted to stained areas of Slide Mantra. An analysis of the NRB’s 16S rRNA gene sequence revealed a close similarity (98% maximum identity) to a strain of Serra­tia marcescens (National Center for Biotechnology Information accession number EF194094). S. marcescens are Gram-negative bacteria of the family Enterobacteriaceae that are found in a wide variety of habitats including water and plants [14]. Many strains of S. marcescens produce the distinctive red pigment 5-2-pyrryl)-2, 2′-dipyrrylmethane (prodigiosin) as a secondary metabolite [15].

Our analysis showed that the FT-IR absorption spectrum of the NRB pigment extract was nearly identical to the S. marcescens pigment extract, sharing all major peaks across the spectrum. Absorbance spectra from both pigment extracts displayed the strong aromatic peak at 2928 cm^-1 and NH peak from 3250-3500cm^-1 (not pictured) that are character­istic of prodigiosin [16]. Peaks within the fingerprint region of the spectrum (1900-600cm^-1) at 1723 cm^-1, 1125 cm^-1, and 817 cm^-1 (Fig. 2) were observed in both spectra and can be attributed to the presence of prodigiosin [16]. These FT­IR results suggest that the NRB pigment extract is prodigiosin. The presence of other pigments such as pyrimine (pink) or 2-hydroxy-5-carboxymethylmuconic acid semialdehyde (yellow) is unlikely as they are synthesized by strains of S. marcescens that do not produce prodigiosin [14].

As a secondary metabolite, the physiological function of prodigiosin remains enigmatic. The pigment is a potent antibi­otic [17], and is believed to enhance bacterial dispersal and participate in surface adherence [18]. It is possible prodigiosin facilitated the colonization of Slide Mantra by the bacterium and, once established, may have helped it secure its position within the microbial community present on the surface of the sculpture. Difficulties removing the red-brown discoloration from Slide Mantra may also reflect the qualities imparted upon the NRB by prodigiosin.

An absorption spectrum of the NRB pigment solution con­tained a peak from 485 to 635 nm with a maximum at 570 nm (Fig. 3A). After a 240-minute exposure to laccase the absorbance at all, wavelengths decreased with the largest decrease at the maximum. The reduction in absorbance was attributed to the degradation of the pigment and demonstrated that the enzyme laccase was capable of decolorizing the pigment produced by theNRB.

Decolorization of the pigment began immediately after the addition of laccase (Fig. 3B). A rapid reduction in absorbance was observed in the first 60 minutes of treatment with the reac­tion slowing as time progressed. Nearly all of the visible color, except that attributed to the enzyme itself, was eliminated after 240 minutes of exposure to laccase. Pristine Carrera marble treated with concentrated laccase (25 U/ml) for 240 minutes acquired some of this color, but was easily cleaned with a cotton applicator soaked in RO/DI H2O. Reproduction of such a rapid decolorization on the surface of marble would permit conser­vators to remediate discolored sculptures and facades on public display with limited disruption.

The number of enzymes tested will need to expand to meet the needs of individual cases of deterioration. Manganese perox­idase, another metabolite of the white-rot fungi, can decolorize a variety of azo and anthraquinone pigments [19]. Decolorization of the NRB pigment extract with manganese peroxidase proved unsuccessful. However, the enzyme may be employed when azo or anthraquinone pigments are the source of discoloration. Experimentation has also been conducted on a lipoxidase iso­lated from the soybean plant. Lipoxidase decolorizes pigments through a coupled oxidation of pigments and polyunsaturated fats [13]. Decolorization of the pigment extracted from the NRB with lipoxidase was successful (data not shown), but the rec­ommended substrate, linoleic acid, is chemically unstable [20] and might complicate the use of lipoxidase beyond a laboratory setting.

Not all red stains on historic marble are caused by pigments. Some may be the result of minium, a lead oxide that can form after lead leaches into the marble from lead support structures and accoutrement [3]. Enzymatic treatment of such a stain would not achieve the desired results. Conservators would need to prop­erly diagnose the source of any red discoloration before choosing a method of remediation. Use of purified laccase appears to provide a rapid, easy to use, and environmentally sound method of decolorizing microbially produced pigments on historic marble. Used in conjunction with commercially available biocidal detergents, an enzymatic treat­ment of Slide Mantra may eliminate the red-brown stains and microorganisms such as S. marcescens on the marble surface. Long recognized for its ability to decolorize chemical pulps and textiles, laccase from white-rot fungi may now have applications in cultural heritage where conventional cleaning techniques may cause more damage or are simply cost prohibitive.

5. Conclusions We show evidence that the red-brown discoloration of Noguchi’s Slide Mantra is microbial in origin. A microorgan­ism (NRB) related to the bacterium Serratia marcescens was isolated from the surface of the sculpture, and cultured in the laboratory. The NRB produced a red-brown pigment that was identified by FT-IR analysis as prodigiosin. Our data indicate that this pigment is the source of the discoloration observed on Slide Mantra. Decolorization of the pigment with the enzyme laccase was demonstrated under laboratory conditions. The practical­ity and feasibility of this type of enzymatic treatment of Slide Mantra or other marble statues with similar discoloration awaits field studies.

Acknowledgements

The authors thank the staff of the Straus Center for Conser­vation at Harvard University, particularly Narayan Khandek and Jens Stenger for their analysis of the pigment extracts, and Kristen Bearce of Harvard’s School of Engineering and Applied Sciences for her assistance on this project. We also thank Cyn­thia Silva of Conservation Solutions, Inc. (CSI) for collecting and documenting the samples used for this study.

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