Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes

AIMS: To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes. METHODS AND RESULTS: The bioremediation intervention consisted of the direct application onto an artwork surface of whole bacterial cells of the Pseudomonas stutzeri A29 strain (bioaugmentation), followed by, in a final step, a purified Protease enzyme. The bioremediation was performed on a Spinello Aretino fresco that had become altered by the animal glue residues of past restoration. For the reader's interest the fresco is the 14th century Conversione di S. Efisio e battaglia (Conversion of S. Efisio and battle), size 3.5 x 7.8 m at the Pisa Camposanto Monumentale, Italy. An assessment was made of the final costs of the biological tests (whole bacterial cells, enzymes) so as to compare them with other intervention techniques. CONCLUSIONS: A successful innovative biological approach to recover valuable frescoes was set up, and the best conditions for treatment efficiency were identified. Furthermore the cost of the biological cleaning using viable bacterial cells and enzymes (P. stutzeri, Protease, Collagenase, 1 : 3 : 10, ratio respectively) was much lower than that of other conventional methods, making this biotechnology not only very interesting but also very competitive. SIGNIFICANCE AND IMPACT OF THE STUDY: New biotechnologies with an innovative, soft approach to the 'biocleaning' and 'biorestoration' of cultural heritage are in constant demand, and our results are clear evidence that such an approach has been achieved; the technique could be of significant importance towards developing other goals.     

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings.     

The bacterial aetiology of rosy discoloration of ancient wall paintings

The inventory of microorganisms responsible for biological deterioration of ancient paintings has become an integral part of restoration activities. Here, the microbial agent of rosy discoloration on medieval frescoes in the Crypt of the Original Sin (Matera, Italy) was investigated by a combination of microscopic, molecular and spectroscopic approaches. The bacterial community from three rosy-discoloured painting sites was characterized by 16S rRNA gene-based techniques. The eubacterial population was prevalently composed of Actinobacteria, among which Rubrobacter radiotolerans-related bacteria accounted for 63-87% of the 16S rRNA gene pool per sampled site. Archaea, with prevalence of Haloarchaea-related species, were detected in one of the three sites where they accounted for < 0.1% of the total 16S rRNA gene pool. Raman spectroscopy confirmed the identity between R. radiotolerans carotenoids (bacterioruberins) and pigments responsible for colour alteration of frescoes. This investigation provides the first evidence of a causal relationship between heavy contamination by Rubrobacter-related bacterioruberin-producing bacteria and rosy discoloration of ancient wall paintings.     

Immobilization of Bacteria on to Polyester Cloth

Escherichia coli 3300 (constitutive for -galactosidase) had a greater adhesion (assayed by -galactosidase) to polyester cloth than to cotton cloth. Adhered bacteria developed immobilized cells on the cloth which can generate progeny efficiently. Similarly, four other Gram-negative bacteria and three Gram-positive bacteria had greater adhesion to polyester cloth than to cotton cloth. Since coating of polyester cloth with polyvinyl alcohol greatly decreased the bacterial adhesion, hydrophobic interaction is likely responsible for the bacterial adhesion to polyester cloth.     

Analysis and comparison of the microflora isolated from fresco surface and from surrounding air environment through molecular and biodegradative assays

The aim of this study was to find a correlation among the environmental isolated microflora and the fresco colonizators through the investigation of their biodegradative abilities and DNA characteristics. A molecular technique named RAMP (Random Amplified Microsatellite Polymorphisms) was utilized in order to analyze the DNA diversity of bacterial and fungal species isolated from fresco as well as from air samples. The RAMP-PCR results were combined with the screening of some biodegradative properties obtained through the use of specific agar plate assays detecting the proteolytic, solubilization and biomineralization abilities of the isolated microflora. This comparative analysis showed that only in few cases a direct link among the fresco and airborne isolates of specific microbial group existed. The investigation clearly evidenced that colonization of surface of Ladislav’s fresco occurred in different time and by different strains than those observed at the moment of sampling campaign. Furthermore, the microflora investigation permitted the identification of taxonomically interesting bacteria with particular biodegradative properties, which had been less studied until now.     

Preliminary Raman microscopic analyses of a lichen encrustation involved in the biodeterioration of renaissance frescoes in Central Italy

Raman microscopic analysis has proved effective in the interpretation and characterization of both the physical and chemical nature of the lichen/substratum system. In the case of Dirina massiliensis forma sorediata, a lichen causing extensive damage to Italian frescoes, the lichen-generated encrustation has been proved to be composed essentially of calcium oxalate. This particular biodeterioration process may be slowed down, or indeed inhibited, by the presence of certain compounds, as yet undertermined, used in the preparation of surfaces and the painting of frescoes. From a conservator's point of view, Raman microscopic analysis uses microscopic samples in the nanogram-picogram range and is non-destructive of the sample.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

Abstract We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Eschericia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occured in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement, Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis iluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

Application of fluorescence internal transcribed spacer-PCR (f-ITS) for the cluster analysis of bacteria isolated from air and deteriorated fresco surfaces

The aim of this work was the evaluation of fluorescence ITS-PCR (f-ITS) as a molecular tool to analyze the microbial community involved in the biodeterioration of cultural heritage surfaces. As a case study we analyzed by f-ITS ninety-two bacterial strains isolated from a medieval fresco and the surrounding air environment. The internal transcribed spacer between the 16S and 23S rRNA genes was amplified, and then the fluorescently labeled PCR products were separated by capillary electrophoresis. Bacterial strains were identified by 16S rDNA sequencing. The f-ITS electropherograms showed different profiles coherent with the affiliation of the strains at the genus and species levels. Among the isolates obtained from the fresco surface, those belonging to the genus Bacillus were the most prevailing exhibiting 8 different f-ITS profiles. The airborne bacilli exhibited only 2 of these 8 profiles. Staphylococcus were mostly isolated from air and produced 4 different profiles. Pseudomonas isolates presented 3 different profiles, and one of them was typical of Pseudomonas putida. Members of the other genera produced their distinctive profiles. Our results show that f-ITS is a promising molecular tool for the rapid selection and clustering of strains isolated from different sources.     

Expression and distribution of very late antigen-5 in mouse peritoneal macrophages upon ingestion of fibronectin-bound Staphylococcus aureus

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to fibronectin. Of these adhesion molecules, fibronectin-binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that fibronectin-bound S. aureus is efficiently phagocytosed by thioglycolate-induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen-5 (VLA-5; alpha5beta1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA-5 is restricted to thioglycolate-induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, alpha5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, fibronectin-mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin-bound S. aureus, alpha5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin-bound S. aureus, VLA-5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.     

A study of the fungi occurring on 15th century frescoes in Florence,Italy

The role of fungi in the biodeterioration of frescoes has been investigated by means of transparent adhesive tape and by swabbing the surface of selected areas as well as by a study of the materials used in restoration. In the Ognissanti church at Florence a large fungal population was found on two 15th century frescoes, one by Botticelli of ‘St Augustine’ and the other by Ghirlandaio of ‘St Jerome’. Twenty-three species were identified, 15 on the Botticelli where the majority of the colony forming units (cfu) were Penicillium brevi-compactum Dierckx (61%) and P. purpurogenum Stoll (18%), and 13 on the Ghirlandaio where Aspergillus versicolor (Vuill.) Tiraboschi was the most common (74%), together with Cladosporium sphaerospermum Penz. (22%). The materials often used in restoration namely calcium caseinate, masonite and animal glue were tested and found to be suitable substrates for species of Aspergillus and Penicillium but not for Cladosporium cladosporioides and C. sphaerospermum.     

Streptococcus mutans and oral streptococci in dental plaque

The human oral microbial biota represents a highly diverse biofilm. Twenty-five species of oral streptococci inhabit the human oral cavity and represent about 20 % of the total oral bacteria. Taxonomy of these bacteria is complex and remains provisional. Oral streptococci encompass friends and foes bacteria. Each species has developed specific properties for colonizing the different oral sites subjected to constantly changing conditions, for competing against competitors, and for resisting external agressions (host immune system, physico-chemical shocks, and mechanical frictions). Imbalance in the indigenous microbial biota generates oral diseases, and under proper conditions, commensal streptococci can switch to opportunistic pathogens that initiate disease in and damage to the host. The group of "mutans streptococci" was described as the most important bacteria related to the formation of dental caries. Streptococcus mutans, although naturally present among the human oral microbiota, is the microbial species most strongly associated with carious lesions. This minireview describes the oral streptococci ecology and their biofilm life style by focusing on the mutans group, mainly S. mutans. Virulence traits, interactions in the biofilm, and influence of S. mutans in dental caries etiology are discussed.     

Repellency of deet and SS220 applied to skin involves olfactory sensing by two species of ticks

Responses of host-seeking nymphs of the blacklegged tick, Ixodes scapularis Say and lone star tick, Amblyomma americanum (Linnaeus) (Acari: Ixodidae) to the repellents N,N-diethyl-3-methylbenzamide (deet) and (1S, 2'S)-2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) were studied using fingertip laboratory bioassays. Ethanol solutions of both compounds applied to the skin strongly repelled both species of ticks at 0.8 and 1.6 micromole of compound/cm2 skin. The ticks were also repelled when two layers of organdie cloth covered the portion of a finger treated with either deet or SS220. Gas chromatographic analyses of the outer layer of cloth that had covered skin treated with 1.6 micromole compound/cm2 skin revealed only 0.1 nmole SS220/cm2 cloth and 2.8 nmole deet/cm2 cloth. However, in bioassays in which a single layer of cloth was treated with a dose of deet or SS220 equivalent to the amount found in the outer layer of cloth, ticks were not repelled. Results unequivocally demonstrated that these ticks responded to the repellents in the vapour phase when repellent treated skin was covered with cloth to obviate tactile contact with them, and made it clear that the ticks detect the repellents by olfactory sensing. Heretofore, the mode of action of deet and SS220 was unclear.     

Progression of addiction careers in young adult solvent misusers.

Four case histories of a representative sample of young adult glue sniffers who had progressed to using illicit drugs or abusing alcohol or both contained several common factors that may have been relevant in the transition. For example, all had suffered parental deprivation or rejection in childhood leading to a state of chronic stress, and all had progressed from sniffing glue to inhaling heroin and thence to "snorting" and injecting heroin. The exact reason for the transition from glue sniffing to misusing other substances was not clear. Nevertheless, although these cases represent only a minority of glue sniffers, the problem of transition may be more common than hitherto supposed.     

Altamira cave Paleolithic paintings harbor partly unknown bacterial communities

Since it has been reported that microorganisms can affect painting pigments, Paleolithic painting microbiology deserves attention. The present study is the first report on the bacterial colonization of the valuable Paleolithic paintings in the famous Altamira cave (Spain). One sample taken from a painting area in the Polychromes Hall was analyzed culture-independently. This was the first time microbiologists were allowed to take sample material directly from Altamira paintings. Identification methods included PCR amplification of 16S rRNA genes (16S rDNA) and community fingerprinting by denaturing gradient gel electrophoresis (DGGE). The applied approach gave insight into a great bacterial taxonomic diversity, and allowed the detection of unexpected and unknown bacteria with potential effects on the conservation of the painting. Regarding the number of 29 visible DGGE bands in the community fingerprint, the numbers of analyzed clones described about 72% of the phylogenetic diversity present in the sample. Thirty-eight percent of the sequences analyzed were phylogenetically most closely related to cultivated bacteria, while the majority (62%) were most closely related to environmental 16S rDNA clones. Bacteria identified in Altamira were related with sequence similarities between 84.8 and 99.4% to members of the cosmopolitan Proteobacteria (52.3%), to members of the Acidobacterium division (23.8%), Cytophaga/Flexibacter/Bacteroides phylum (9.5%), green non-sulfur bacteria (4.8%), Planctomycetales (4.8%) and Actinobacteria (4.8%). The high number of clones most closely related to environmental 16S rDNA clones showed the broad spectrum of unknown and yet to be cultivated bacteria in Altamira cave.     

Enumeration and Localization of N(2)-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N(2)-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N(2)-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N(2)-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N(2)-fixing bacteria recoverable from the rhizoplane, although high numbers of N(2)-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N(2) fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

Controlled enzymatic removal of damaging casein layers on medieval wall paintings

A new, gentle enzymatic method was developed for a controlled removal of casein layers from medieval wall paintings. These casein layers were applied over the last 60 years on wall paintings in order to decrease substantial damage due to a peeling off of the frescoes from the roughcast surface due to environmental effects. However, due to the aging of the casein layers (at 40-50 years), a more drastic peeling occurred and the danger of total destruction of the wall paintings is severe. Thus, screening was performed to find the most suitable enzyme for casein digestion. Alcalase 2.5 DX L was the most appropriate enzyme for an effective proteolysis reaction. The enzyme was immobilized on functionalized cellulose membrane. A membrane pad system with immobilized enzymes was developed which could be pressed on the casein layers on the wall painting. A controlled removal of the casein layers by proteolytic digestion was observed and it was possible to continuously wash off the hydrolyzed casein fragments from the wall painting surface by an aqueous carbonate buffer flowing through the membrane pad. The removal and the digestion was monitored by reverse HPLC. Additionally, an on-line monitoring system was set up in order to continuously follow the casein layer removal and the digestion procedure directly on the wall painting. This technique is based on noninvasive 2D-fluorescence monitoring. Optical fiber systems were used to continuously monitor the fluorescence intensity of casein-bound tryptophan. The off-line data were verified with the on-line 2D-fluorescence data. Based on the scientific result an appropriate technique for the controlled enzymatic removal of damaging casein layers on the surface of medieval wall paintings using immobilized enzyme is now available. It is now applied to remove such casein layers from medieval wall paintings in the Allerheiligen-Kapelle Cloister, Wienhausen, Germany, and the St. Alexander Kirche, Wildeshausen, Germany.     

铍对口腔链球菌生长及粘附性能的影响

目的 研究铍离子(Be2+)对口腔链球菌的生长及粘附性能的影响,探讨口腔修复治疗后牙周损伤的微生物机制.方法 含有不同浓度Be2+(5、10、20和40 mg/L)的培养液厌氧培养口腔链球菌24h.检测不同浓 度Be2作用后细菌形态和菌落形成单位(CFU),以及口腔链球菌在唾液包被羟基磷灰石微粒表面的粘附抑制率.结果 铍离子作用后口腔链球菌长链缩短,菌体出现集结趋势.20 mg/L时,菌体表面出现“触角样”变化.随Be2+浓度增加,CFU值减小(P＜0.05),口腔链球菌生长抑制.各实验组口腔链球菌粘附抑制率高于阳性对照组(P＜0.05),但各Be2+浓度组之间粘附抑制率差异无统计学意义(P＞0.05).结论 铍离子抑制口腔链球菌的生长和在牙面的粘附,可能会导致修复体周围正常微生态环境失衡,引起牙周疾病.提示临床应尽量选用理化性能稳定的修复材料.     

Selective outgrowth of fimbriate bacteria in static liquid medium

Competitive mixed cultures were grown from inocula of a large number of bacteria of a genotypically nonfimbriate (fim(-)) strain of Salmonella typhimurium and a small number of a genotypically fimbriate (fim(+)) variant strain that formed type 1 fimbriae and had been derived from the fim(-) strain by phage transduction. The fim(+) strain differed from the fim(-) strain in fermenting l-rhamnose (rha(+)), and the viable fim(+) and fim(-) bacteria present in the cultures after different periods at 37 C were counted differentially in platings on rhamnose media. When the cultures were grown under aerobic static conditions in tubes of nutrient broth, the fim(+) bacteria rapidly outgrew the fim(-) bacteria, so that, although starting as a small minority (e.g., 1 in 10(7)), they approached or surpassed the number of the fim(-) in 48 hr. A pellicle consisting of fimbriate bacteria was formed on the surface of the broth between 6 and 24 hr, and it is thought that the advantage of access to atmospheric oxygen enjoyed by these bacteria in the pellicle enabled them to outgrow the fim(-) bacteria confined in the oxygen-depleted broth. The fim(+) bacteria did not show selective outgrowth in mixed cultures grown in broth aerated by continuous shaking, in static broth incubated anaerobically in hydrogen, and on aerobic agar plates, i.e., under conditions not allowing an advantage from pellicle formation. The outgrowth of fim(+) bacteria in aerobic static broth was prevented by the addition of alpha-methylmannoside, a substance that inhibits the adhesive and early pellicle-forming properties of bacteria with type 1 fimbriae. A motile flagellate (fla(+)) variant of a fim(-)fla(-) strain of S. typhimurium outgrew its parent strain in mixed cultures in aerobic static broth, but the selective advantage conferred by motility was weaker than that conferred by fimbriation.     

Therapy with saffron and the goddess at Thera

This paper presents a new interpretation of a unique Bronze Age (c. 3000-1100 BCE) Aegean wall painting in the building of Xeste 3 at Akrotiri,Thera. Crocus carturightianus and its active principle, saffron, are the primary subjects at Xeste 3. Several lines of evidence suggest that the meaning of these frescoes concerns saffron and healing: (1) the unusual degree of visual attention given to the crocus, including the variety of methods for display of the stigmas; (2) the painted depiction of the line of saffron production from plucking blooms to the collection of stigmas; and (3) the sheer number (ninety) of medical indications for which saffron has been used from the Bronze Age to the present.The Xeste 3 frescoes appear to portray a divinity of healing associated with her phytotherapy, saffron. Cultural and commercial interconnections between the Therans, the Aegean world, and their neighboring civilizations in the early 2nd millennium BCE indicate a close network of thematic exchange, but there is no evidence that Akrotiri borrowed any of these medicinal (or iconographic) representations. The complex production line, the monumental illustration of a goddess of medicine with her saffron attribute, and this earliest botanically accurate image of an herbal medication are all Theran innovations.     

Comparative Surface Structure of 16S Ribosomal Ribonucleic Acid of 30S Ribosomes of Procaryotic Cells

Ribonuclease T(1) treatment of 30S ribosomes of Escherichia coli converts a large region at the 3' OH end of 16S ribosomal ribonucleic acid (rRNA) to low-molecular-weight RNA. The final 25 nucleotides at the 3' terminus of the molecule emerge relatively intact, whereas most of the region "upstream," for about 150 nucleotides, is converted to oligonucleotides. Identical enzyme treatment generates a fragment of about 60 nucleotides from the middle of 16S rRNA (section D'). To determine whether there are similar sequences in other bacteria, which occupy similar accessible surface locations, we treated 30S ribosomes from Azotobacter vinelandii and Bacillus stearothermophilus with RNase T(1). In each case, a fragment of RNA about 25 nucleotides in length containing the 3' OH end of 16S rRNA and a fragment of about 60 nucleotides in length similar, but not identical, in oligonucleotide composition to section D' of E. coli 16S rRNA were obtained from nuclease-treated 30S ribosomes. These data indicate that, although the primary structure at the 3' end and the middle (section D') of the various 16S rRNA's is not completely conserved, their respective conformations are conserved. A number of identical oligonucleotides were found in the low-molecular-weight fraction obtained from RNase T(1)-treated E. coli, A. vinelandii, and B. stearothermophilus 30S ribosomes. These results show that identical RNase T(1)-sensitive sequences are present in all three bacteria. Hydrolysis of these regions leads to the production of the fragments 25 and 60 nucleotides in length.