Introduction

Applied microbiology and biotechnology are commonly associated to chemical and pharmaceutical industries, medicine, agricultural, food and feed sectors and bioremediation (Schmid et al. 2002; Gavrilescu and Chisti 2005; Whiteley and Lee 2006). Tools and methodologies available from applied microbiology and biotechnology have been barely used in other fields of knowledge, among them the conservation of cultural heritage materials such as stone, paper, paintings, textiles, or glass. Until recently, those methods were only used for the identification of biological agents that cause deterioration of such materials. The introduction of DNA-based identification methods has enhanced the range of microorganisms that can be detected on relevant cultural items. The information thus gathered has further highlighted the role played by microorganisms (Bacteria, Archaea, fungi, algae, lichens), along with mosses and higher plants, in the deterioration of such diverse materials as historic stoneworks, wood, tapestries, papyrus, canvas, paper, parchment, and leather photographic/cinematographic materials, magnetic media or even optical disks (Cappitelli and Sorlini 2005; McNamara and Mitchell 2005; Ramírez et al. 2005). As there is not enough space to discuss thoroughly all of these, and given the wide array of culturally relevant items (e.g., statues, historic buildings, monuments, and mural paintings) that share stone as common component, this mini-review will focus on such stoneworks.

It must be pointed out that the biodeterioration of a given artifact commonly results from the complex interaction established by the microorganisms co-existing simultaneously (Warscheid and Braams 2000; McNamara and Mitchell 2005). On the other hand, the microbial and enzymatic potential for effective bioremediation of deteriorated stone cultural heritage materials is gradually being unveiled, and promising results of this methodology have been obtained in field sites. The present work aims to bring an up-to-date perspective on the contribution of applied microbiology and biotechnology for the preservation of such stone materials, a matter that currently gathers considerable attention and investment (Da Silva 2004).

Biodeterioration of historic stoneworks: main agents and their identification

Biodeterioration of stoneworks in mainly due to biofilm formation, biocorrosion caused by organic and inorganic acids, redox processes on cations from the mineral lattice, and physical penetration by microbial communities (Gómez-Alarcón et al. 1995; Warscheid and Braams 2000). Outdoor stone structures are particularly susceptible to these factors. Biocorrosion of stone can occur through the action of nitrous and nitric acid excreted by nitrifying bacteria (Nitrosomonas spp. and Nitrobacter spp.), leading to stone dissolution and formation of nitrate salts as well as through sulfuric acid excreted by sulfur-oxidizing bacteria (Thiobacillus spp.). The acid may react with given constituents of the stone to yield sulfate-based crusts. These sulfates can be precipitated within the pores of the stone and, upon recrystallization, exert considerable stress in the porewalls. Biocorrosion can also occur through the excretion of organic acids by chemoorganothrophic microorganisms, including fungi and lichens. Those acids can also chelate metal cations (e.g., Al, Ca, Fe, Mg, Mn, Si) from minerals to form stable complexes (Kumar and Kumar 1999; Warscheid and Braams 2000; McNamara and Mitchell 2005).

Several chemoorganotrophic bacteria and fungi (Acidithiobacillus ferrooxidans, Bacillus spp., Leptospirillum spp., Aureobasidium spp.) are also capable of removing cations, in particular, iron and manganese cations from the mineral lattice by oxidation, contributing to stone deterioration (Warscheid and Braams 2000; Rawlings 2005).

Biofilm formation results from the complex aggregation of microorganisms. Algae and cyanobacteria are common on stoneworks, and their presence is easily identifiable by the resulting stains on the stone. Given their photolithoautotrophic nature, they facilitate stone colonization by other microorganisms (e.g., fungi and bacteria), although the establishment of heterotrophic microorganisms on stone is possible even without the pioneering presence of phototrophic organisms (Warscheid and Brams; McNamara and Mitchell 2005). Biofilm bacteria often excrete exopolymer, consisting mainly of polysaccharides, but comprising also lipids, pigments, and proteins. Such complex mixture helps to consolidate the biofilm, as it provides protection against disinfectants and biocides, and also a reservoir for nutrient storage (McNamara and Mitchell 2005).

Besides growing on stone surface, microorganisms also colonize the interior of the stone. Lichens and fungi can cause serious degradation by physical penetration. Fungal hyphae are able to penetrate deeply beneath the stone surface, contributing to mechanical deterioration. This penetration simultaneously allows the transport of water and nutrients through the stone, which facilitates the colonization of the interior of the stone by bacteria and concomitantly triggers biochemical deterioration (Gómez-Alarcón and de La Torre 1994).

The identification of biological agents associated with the deterioration of stoneworks can be performed through traditional phenotypic identification methods (Saiz-Jimenez and Laiz 2000; Videla et al. 2000; May 2003; McNamara and Mitchell 2005). The use of those traditional phenotypic methods for the identification of such agents has nevertheless several disadvantages. Only less than 10% of the microorganisms, particularly bacteria, present in a given environment can be cultured on standard media. Culturing is, furthermore, a time-consuming task, and phenotypic identification methods are more subjective than genotypic identification methods. On the other hand, phenotypic identification methods are well established and are based in mature technology and easy to implement (Sutton and Cundell 2004; McNamara and Mitchell 2005). In recent years, molecular-based techniques, or genotypic identification methods, have been successfully used to examine the biological diversity in deteriorated artifacts.

These methods do not call for cultivation of the organism, or even its extraction from the environmental sample, and require smaller samples than those needed for phenotypic identification methods. The use of genotypic identification methods expanded the array of identifiable microorganisms present in samples collected from artifacts (Schabereiter-Gurtner et al. 2001; Rölleke 2003; Helms et al. 2004; Schabereiter-Gurtner et al. 2004; Milanesi et al. 2006). Until recently, molecular-based techniques were unable to discriminate living and dead organisms. In a recent paper, Wang and Lewin (2006) were, however, able to discriminate viable bacterial cells from dead cells in real-time PCR. This significantly extends the utility of molecular-based methods for the fast determination and quantification of viable microorganisms in environmental samples without prior enrichment.

Phenotypic and genotypic identification methods can be advantageously combined to provide a thorough picture of the microbial diversity responsible for the deterioration of stoneworks (Heyrman and Swings 2003; Kennedy and Clipson 2003). Molecular-based techniques have also been shown to provide information on what animal or plant a given artifact derives from (Burger et al. 2000), and were thus tentatively used in the identification of hemoglobin on prehistoric tool residues utilized for rock painting (Williamson 2000). The results, however, were not totally conclusive (Mawk et al. 2002). Along with these techniques, it is foreseeable that the use of biosensors will be implemented to screen contamination of stoneworks in a non-invasive way, given their established efficiency in the detection of contaminants, pollutants, and pathogens and in the detection of cellular metabolic activity (Nunes-Halldorson and Duran 2003).

Preservation and restoration of historic stoneworks

The key issue that has to be considered when establishing a strategy for the preservation and restoration of stoneworks is the need for doing so through methods that do not cause any damage to the artifact.

Eradication of biological agents accountable for deterioration of stoneworks

The conservation of stoneworks is based on the use of preventive and remedial methods. Preventive methods aim at inhibiting biological attack on stoneworks. Selection of suitable environmental conditions (e.g., temperature, light, humidity) that reduce the risk of microbial deterioration is only feasible indoors. Periodic cleaning of dirt and dust, deposits of various organic substances and bird droppings removes potential sources of nutrition for microorganisms (Kumar and Kumar 1999; Warscheid and Braams 2000). The use of low-frequency electric currents has been used to keep higher animals, namely, birds, from monuments (Horakova and Martinek 1984). The use of synthetic polymers and resins has been assayed as protective coating and consolidant of stone. However, such preservative treatment may induce the growth of microorganisms, by providing a source of nutrients (Kumar and Kumar 1999; Warscheid and Braams 2000; May 2003; Gu 2003).

Remedial methods aim at eradicating the biological agent(s) responsible for biodeterioration. This is mostly performed by physical and/or chemical methods, as evidenced in Table 1. Irrespective of the approach, the goal is to inhibit metabolic activity or to cause irreversible damage to the organism leading to his death, again without damaging the stonework. A thorough evaluation of this matter is out of the scope of the present work, but details can be found in Price (1996); Kumar and Kumar (1999); Warscheid and Braams (2000); Salvadori (2003); Allsopp et al. (2004).

Table 1 Methodologies for the eradication of biodegradative agents of stoneworks
Full size table

The use of biological methods relies on the use of antagonistic or parasitic organisms of the biodeteriorating agents (Salvadori 2003; Ramírez et al. 2005). Although well established in agriculture, the application of this approach in the preservation of stoneworks is to be developed (Salvadori 2003; Szewczyk et al. 2006). The use of biopesticides, which have been proven effective in the control of insects, is promising, particularly given their narrow specificity, which makes them harmless to people and wildlife (Ramírez et al. 2005; Szewczyk et al. 2006).

Restoration of deteriorated historic stoneworks

Bioremediation of artworks is based on the use of sulfate- and nitrate-reducing bacteria (SRB and NRB, respectively), hydrocarbon-degrading bacteria (HDB), biocalcifying bacteria (BCB), and in the hydrolytic activity of lipases, proteases, and carbohydrases (Wolbers 2000). SRB and NRB can be used for the reduction of sulfate and nitrate salts embedded in the mineral matrix, respectively. HDB can be used for breaking down aliphatic and aromatic hydrocarbons deposited in stone. BCB can be used for stone consolidation, and hydrolytic activity may be required to remove carbohydrates, lipidic, or proteinaceous materials from artworks (Saiz-Jimenez 1997; Tiano et al. 1999; Wolbers 2000).

Calcium sulfate dehydrate (gypsum) deposits are often found in stoneworks. The salt crusts on stone tend to accumulate soot particles, mostly originated from the consumption of fossil fuels, to yield the so-called black crust (Kumar and Kumar 1999; Warscheid and Braams 2000). SRB reduce sulfate to gaseous hydrogen sulfide, whereas NRB reduce nitrates to gaseous nitrogen or nitrous oxide (Saiz-Jimenez 1997; Castanier et al. 1999). Desulfovibrio desulfuricans and D. vulgaris are SRB effectively used for the reduction of gypsum and black crust (Gauri et al. 1992; May 2003; Ranalli et al. 1997; Cappitelli et al. 2006). Pseudomonas spp. cells were able to remove nitrates from weathered stone (Ranalli et al. 1996) and Pseudomonas spp. and Bacillus spp. have been shown to degrade phenantrene deposited in weathered stone (Saiz-Jimenez 1997).

Weathering of the calcareous matrix of stone results in increased porosity. This leads to a decay of its mechanical stability. Biomediated approaches to reverse this process, and thus promote the consolidation of stone, have been described. These are mostly based on calcite (CaCO3 with hexagonal crystallization) precipitation. Two main approaches have been put into practice to induce calcite precipitation.

  1. a)

    Calcite precipitation can result from the natural metabolic activity of some bacteria, either by passive or active bioprecipitation, as thoroughly described by Castanier et al. (1999). Bacterial activity may induce chemical changes in the environment that induce calcite precipitation, provided Ca2+ is available (Castanier et al. 1999; Stocks-Fischer et al. 1999). This passive method led to the development of an approach based on enzymatic (urease) activity. The enzyme hydrolyzes urea to CO2 and ammonia, the latter increasing the pH of the surroundings, which concomitantly induces calcite precipitation (Bachmeier et al. 2002; Nemati and Voordouw 2003). Crystals may also build up in the wall of the bacterial cell. Microbial activity may lead to the accumulation of hydrogen carbonate and carbonate ions, and ultimately to pH increase that favors CaCO3 precipitation (Castanier et al. 1999). Several microorganisms have been effectively tried for precipitation of calcite, among them Bacillus spp. (Castanier et al. 1996), in particular, B. pasteurii (Stocks-Fischer et al. 1999; Bang et al. 2001) and B. subtilis (Tiano et al. 1999; Perito and Mastromei 2003), Micrococcus sp. (Tiano et al. 1999), Myxococcus xanthus (Rodriguez-Navarro et al. 2003) and Pseudomonas spp. (Castanier et al. 1996).

  2. b)

    Calcite precipitation may be induced by the presence of organic matrix macromolecules (OMM) extracted from the shell of mollusks Mytilus californianus (Tiano 1995) and Mytilus edulis (Tiano et al. 2006), respectively, which are rich in aspartic acid. Recently the use of poly-aspartic acid to induce calcite precipitation was favored when compared to OMM. Although less efficient than OMM in inducing calcite precipitation, poly-aspartic acid is more widely available, and the process for OMM extraction is costly and time consuming (Tiano et al. 2006).

Once the strains with the required biological activity have been identified, efforts are required to develop a methodology that allows easy and effective application of the bacteria onto the stone. In pioneering work, the weathered stone was immersed in growth medium for gypsum removal (Gauri et al. 1992). This approach is not feasible for large objects and is prone to damage the stone (Cappitelli et al. 2006). Cell entrapment onto a carrier that is contacted with the surface to undergo treatment is a more feasible approach (Ranalli et al. 1997; Cappitelli et al. 2006). The work developed by BioBrush Consortium (Research Project Contract No. EVK4-CT-2001-00055, http://www.biobrush.org) suggests SRB and BCB delivery entrapped in Carbogel and NRB delivery entrapped in alginate beads.

Time length of the treatment varies from 2 (SRB) to 15 days (BCB) and 1 month (NRB) (Webster and May 2006). Cappitelli and co-workers (2006) recently published a thorough paper describing a methodology for application of SRB for the removal of black crusts from stone artworks. Besides the effectiveness of the treatment (98% removal of sulfates of the crust in a 45-h treatment), the authors also developed suitable strategies to avoid potential problems due to the application of SRB on stone, namely, sulfite precipitation and remains of the exhausted media. Bang et al. (2001) suggested immobilization of BCB in polyurethane foams, as the immobilization matrix protected the microbial cells from the aggressive environment and provided nucleation sites for calcite crystals, allowing for calcite precipitation rates similar to those observed for free cells.

Efficiency of these approaches has been tested in actual field trials aiming to remove deposited salts, degrade organic matter or consolidate stone (Da Silva 2004; Cappitelli et al. 2006; Tiano et al. 2006; Webster and May 2006).

Enzymatic activity has also recently been effectively used to remove remains of organic materials (e.g., casein and/or collagen containing materials such as animal glues), that are commonly used as part of the physical-chemical approaches to restore frescoes. As an outcome of repeated application of conventional restoration methods, frescoes are, thus, often covered with anesthetic and potentially damaging casein layers often cross-linked with glutaraldehyde, which is applied generously as anti-microbial agent. To cope with the hitherto untreatable proteinaceous material, the use of proteolytic activity has proved effective. Beutel et al. (2002) were able to remove casein layers deposited in frescoes from the 14th century using a commercial enzyme preparation, Alcalase® 2.5 DX L, immobilized onto an epoxide-functionalized cellulose acetate membrane. Ranalli et al. (2005) were able to restore a fresco altered by animal glue by treatment with Pseudomonas stutzeri cells. Cells were applied on the surface of the fresco in a cotton wool layer soaked with the microbial suspension. Antonioli et al. (2005) tried to identify the enzymatic activity of this cell preparation, as commercially available proteases failed to digest the proteinaceous material. These authors were able to identify strong collagen and casein-degrading activity and suggested cooperative action and uniqueness of the proteolytic enzymes from P. stutzeri.

Conclusions

The role of applied microbiology and biotechnology in the preservation of stone artwork is gradually being acknowledged. The powerful, environmentally and health-friendly bio-based techniques already provide effective methods to identify biodeteriorating agents. Biotechnological approaches to counter the effect of both biological and non-biological degradation agents are starting to pile up. Given the resourcefulness of the microbial world, it can easily be foreseen that contribution of the microbial world in the preservation of stoneworks still has a long road ahead. In particular, the development of biotechnological restoration methodologies feasible for field applications and dissemination are being comprehensively looked at. It is, however, clear that the success in the preservation and restoration of stone cultural heritage materials does not solely rely on biological methods. It requires strong interaction and integration with physical and chemical-based methods, as well as a better understanding and dialogue between the science and technological world and the art world.