Introduction

Library documents can be deteriorated by different organisms, including insects, rodents and microorganisms, generally called biodeteriogens (Canhoto et al. 2004; Pinzari et al. 2006). Because the paper is an organic material, it is a food source for heterotroph organisms. It is chiefly made of cellulose of various origin (cotton, linen, hardwood, softwood), but in addition paper usually contains traces of metallic elements (such as iron, copper) and other substances (such as inks, pigments, etc.) which make it a complex and heterogeneous medium (Pinzari et al. 2006). In particular, fungal species causes significant disfigurement of library materials, especially books and paper. Apart from the undesirable aesthetic aspects, including discoloration by weak acids produced by fungi and/or the accumulation of pigments that may stain the paper in a phenomenon known as foxing (Mesquita et al. 2009), the presence of these microorganisms can lead to the irreversible degradation of the paper.

Environmental factors such as relative humidity (RH), temperature, light and gas composition (Magan 1997) favor the germination and growth of fungal species on paper. To prevent fungal growth, books and archives are commonly stored in controlled environments, with temperature and humidity maintained in a defined range, and periodically cleaned of dust (Pinzari et al. 2003).

In literature, are reported numerous works on the identification of fungal species causing foxing (Rakotonirainy et al. 2007; Szczepanowska and Cavaliere 2000; Montemartini Corte et al. 2002; Florian and Manning 2000) such as Penicillium, Aspergillus and Chaetomium.

Identification of fungal species was based, in the past, on morphological and cultural methods, using a variety of standardized media. Today this scenario is shifting in favor of modern molecular techniques supported by various studies demonstrating that morphology alone may not be a sufficiently objective method for species determination (Balajee et al. 2005; Hong et al. 2005).

Currently, comparative sequence-based identification strategies can be considered a new method for fungal species identification (Summerbell et al. 2005). This method is based on PCR amplification of a selected region of genomic DNA, followed by sequencing. The Internal Transcribed Spacers (ITS regions), which are nested in the nuclear rDNA repeat, have been selected to investigate the fungal diversity. This regions possess a high variation between taxonomically distinct fungal species and even within the species (Sterflinger 2010), and has been used for species complex-level identification of Aspergillus (Hinrikson et al. 2005) and most other species. The identification of fungal species is of great relevance since allows to choose the best conservative approach to the restoration. Among physical methods, microwave (MW) heating is safe, pollution free (Riminesi and Olmi 2016) and has been successfully used on wooden cultural heritage objects for disinfection from woodworm (Hylotrupes bajulus L., Oligomerus pitilinoides Wollaston and Drosophila melanogaster) (Andreuccetti et al. 1994; Plaza et al. 2007; Cuzman et al. 2013). The effectiveness of MW is due to the ability of inactivating microbial contaminants not only on the exposed surfaces but inside them as well (Gorny et al. 2007).

In this work, we analyzed a book of 18th century characterized by a consistent foxing deterioration. The isolation of fungal species was based on culture, morphological and molecular identification. A. versicolor was used as biodeteriogen to contaminate paper samples exposed two degradation processes (exposure to wet atmosphere and to acidic attack) simulating storage conditions of 18th century book.

Finally, we investigated the use of MW as physical method to protect biological attack due to fungi in paper artworks. MW heating at three different temperatures (30, 58 and 63 °C) was applied on different types of paper samples affected by spots originating from A. versicolor. Cellulose degree of polymerization (DP) of paper samples and scanning electron microscopy (SEM) were evaluated after prefixed times of fungal attack, respectively.

Materials and methods

Materials

The private collector asked our laboratory to examine the book “Trattato delle acque minerali” printed 1783 on which foxing stains had been found, in order to investigate the cause of this damage. This book was left in a room with high relative humidity. The book did not appear to be in good condition as shown in Fig. 1a. In fact, all pages are characterized by irregular shaped, rusty red and uniformly discoloured spots. Figure 1b reports several observed foxing spots, as confirmed by Wood light (CTS ART lux 70, mod. 808).

Fig. 1
figure 1

a “Trattato delle acque minerali” dating from 18th century b foxing spots observed by Wood light

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  • Isolation and molecular identification of fungal species from 18th century book damaged by foxing

Sample isolation and culture

Fungi were isolated by sterile swab rubbed on the fluorescent area to allow a sufficient amount of material and then placed directly into liquid medium MEB (Malt Extract Broth). Incubation was carried out at 25 °C. Liquid fungal cultures were then inoculated in solid medium MEA (Malt Extract Agar) and incubated at room temperature.

According to Urzì and De Leo (2001), sampling of the fungal species from the book for microscopic observation has been done by adhesive strips (Fungi Tape DID, Calsystem, CS, Italy).

Microscopy analysis

Light microscopy

Adhesive strips were gently applied to the surface of paper and were then immediately placed on sterile glass microscope slides. A drop of sterile water, Malachite Green solution (Calsystem, DID, CS, Italy) was poured between the glass slide and the tape. A cover glass was placed on the top in order to keep the tape as flat as possible (Urzì and Albertano 2001). Direct observations of samples were carried out using a light microscope Leitz Laborlux 12 POL (Germany). Petri dishes with MEA were also observed with a light microscope equipped with phase contrast (Leica, Germany) to see the typical fungal morphology.

Scanning electron microscopy (SEM)

For SEM observations, a piece of adhesive tape (Fungi Tape DID, Calsystem, CS, Italy) was mounted face up on a stub with a biadhesive tape (Assing, Monterotondo, Italy). Observations were carried out using a SEM Leo 420 (Leo electron microscope Ltd, Cambridge, England) at 15 kV under high vacuum conditions.

DNA extraction, PCR amplification and DNA sequencing

Before proceeding with DNA extraction, a 7 day culture grown in liquid medium (MEB) was repeately washed in distilled water to remove any residual culture medium. DNA extraction was performed using CTAB (cetyltrimethylammonium bromide) combined with enzymatic (proteinase K) and mechanical steps (freeze and thaw cycles), followed by organic extractions and isopropanol precipitation of DNA. The result of the extraction was visualized on agarose 0.8% gels by ethidium bromide staining and UV illumination.

Amplification of the ribosomal internal transcribed spacer regions ITS1 and ITS2 and the 5.8S rRNA gene situated between them, was done using universal primer ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (White et al. 1990). PCR were performed in a TPersonal Thermal Cycler (Biometra, Germany). The cycling profile consisted of an initial step at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 45 s and with a final extension step of 10 min at 72 °C. PCR product was electrophoresed on 1.5% agarose gel with 100 bp DNA ladder, stained with ethidium bromide and photographed with a Kodak EDAS 290 camera.

Sample was then purified with the kit for purification of Qiagen PCR and sequenced by the BMR Genomics, Padova (Italy) following the manufacturer’s protocol.

The sequence was analyzed using BioEdit software (Hall 1999) and run against NCBI’s BLAST (Basic Local Alignment Search Tool) database (http://www.ncbi.nlm.nih.gov/blast/) in order to assess the similarity with published sequences, belonging to identified fungal species.

  • Preparation of paper samples simulating storage conditions of 18th century book and contaminated with A. versicolor

Given the high value of the book and in order to assess the effect of MW heating on artworks, two degradation processes (exposure to wet atmosphere and to acidic attack) contaminated by A. versicolor isolated from 18th century book on commercial MOTIFCOPY paper were simulated.

Exposure to acidic attack

The exposure to acidic attack was simulated by treating paper samples (2 × 7 cm2) with H2SO4 solution 72% for 5 s. Paper samples were then washed with deionized water (pH 5.0) and then dried. Morphological analysis by SEM and DP were performed. UNI 8282 method was used to determine the cellulose DP in cupriethylenediamine (CED).

Exposure to wet atmosphere

The exposure to moisture was simulated as reported by Cerchiara et al. (2009) exposing samples to vapors deriving from a water bath at 60 °C and 70% relative humidity (R.H.) for 15 days. Morphological analysis by SEM and DP were performed. UNI 8282 method was used to determine the cellulose DP in cupriethylenediamine (CED).

Fungal inoculation

Paper samples exposed to acidic attack and to wet atmosphere respectively were placed in Petri dishes with no addition of any nutritional substance, in order to ensure microbial growth at the expence of the paper (De Filpo et al. 2016). Inoculation was performed by deposition of conidia solution over the sample surface. All samples in petri dishes were covered by plastic lids and kept at 30 °C in a thermostatic cell with a relative humidity of 80% for 5, 15 and 25 days. DP and morphological analysis were performed.

  • Microwave heating on paper samples contaminated with A. versicolor

Both paper samples with fungal inoculation were directly placed flat in a commercial microwave oven (Moulinex—Ultimys Duo Grill, 5140—modello AFW1) at 300, 600 and 900 W for 2 min and 50 s. Paper samples were observed by optical microscopy after 10 and 30 days. Temperature of paper samples was carry out using Fluke 52 K/J Thermometer and DP was determined according to viscosimetric technique with respect the standard UNI 8282. The DP was measured for cellulose extracted from paper samples before and after microwave heating. Morphological analysis by SEM was performed.

Statistical analysis

All experiments were performed at least in triplicate. Results are expressed as mean ± SD. t tests were used to determine statistical significance of results. Differences were considered significant for values of P < 0.05.

Results and discussion

Isolation and molecular identification of fungal species from 18th century book damaged by foxing.

Microscopy analysis

Light microscopy

Figure 2a shows chains of spores growth on the samples and isolated by adhesive tape from the book and coloured with green malachite. In addition, we observed colonies on MEA at 25 °C grew rapidly (Fig. 2b), reaching 24–26 mm in diameter by the 7th day, centrally rising, floccose, radially sulcate. They showed regular margins, and exudates are absent. The variation in color was as follows: white at first, later becoming grayish yellow to blue green with age, reverse yellowish. Conidiophores from substrate and aerial hyphae were colorless, smooth, and thick-walled. At first observation with light microscopy the conidial heads (Fig. 2c) appear radiated and vesicles ellipsoid. Morphological observations by light microscopy lead to the morphology of the genus Aspergillus.

Fig. 2
figure 2

a Spores of Aspergillus coloured with Green Malachite solution (20×) b colonies of Aspergillus on MEA c light microscopic examination of Aspergillus conidiophores (50×)

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SEM

SEM was used to examine the conidia and in Fig. 3 is reported the SEM micrograph of isolated conidia. The conidia are jar shaped with low ridged ornamentation in a cerebellate pattern and are usually isolated or in small clusters, not in chains.

Fig. 3
figure 3

SEM micrograph of Aspergillus conidia

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The observed conidia were conspicuously roughened. Based on the surface texture of conidium, the conidium was found to be echinulate, which is a characteristic of the echinulate category of Aspergillus genus (Kozakiewicz 1989).

Molecular identification

Amplification of ITS regions generated a PCR product of 564 bp and the sequence obtained was compared in GenBank database. The sequence was also deposited in GenBank (accession numbers JN997427). Identification of the unknown sample was made using the highest bit score of listed species with analogy of 100 and 99%. The following species: Aspergillus versicolor, A. carneus, A. sydowii and A. flavus, all biodeteriogens paper, were chosen.

Alignment of ITS regions showed that the unknown sample had high homology with respect to A. versicolor.

The ITS region has been frequently used, and with good results, for the identification of fungal organisms that contaminate documents and art objects (Michaelsen et al. 2006). This region is widely used for the identification of organisms and for phylogeny studies. In fact, it satisfies many of the demands of universal marker since this region can be amplified for many fungi, is present as multiple copies in the fungal genome and has the additional advantage that a large number of sequences for this locus are deposited in GenBank (http://www.ncbi.nlm.nih.gov) making easy a quick comparison of the sequence from an unknown isolate (Balajee et al. 2009).

There is considerable consensus regarding the use of ITS sequencing as the initial step in mold identification. An international Aspergillus working group recommended the use of the ITS region for subgenus/section-level identification for the genus Aspergillus (Balajee et al. 2007). Also, the International Subcommission on Fungal Barcoding has proposed the ITS region as the prime fungal barcode or the default region for species identification (http://www.allfungi.com/its-barcode.php).

In this work, the sequences we obtained from the total ITS region, together with the morphological analysis, allowed the identification of the species A. versicolor. This species is usually found as biodeterioration agent in many different habitats and materials, including those considered as representative of historical and cultural heritage (Abrusci et al. 2005). In particular A. versicolor is an indicator organism of moisture problems in cultural heritage confirming the storage conditions where the book of 18th century was left for many years.

  • Characterization of paper samples after simulation of storage conditions of 18th century book

Two different degradative processes (exposure to wet atmosphere and to acidic attack) have been considered for simulating storage conditions of 18th century book. The deterioration state of the samples was monitored with cellulose DP and SEM.

Figure 4 shows DP of paper samples before and after degradation process. DP of paper samples exposed to degradation processes (exposure to wet atmosphere and to acidic attack) was lower than control. In particular, DP of paper samples exposed to wet atmosphere was slightly lower than exposed to acidic attack. Probably, it can be attributed to difference of exposure time of paper sample to degradation processes: 5 s to sulphuric acid for acidic attack and 15 days at 60 °C with 70% RH for wet atmosphere.

Fig. 4
figure 4

DP of paper samples exposed to degradative processes

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Moreover, the deterioration state of paper samples was also analyzed by SEM. The SEM image of the control paper is shown in Fig. 5a. Results indicated closely packed cellulose fibers with an interwoven network without any deformation. Cellulose fibers of different size are made from cotton linters. The effect of acidic attack on paper sample is shown in Fig. 5b. Fibers after exposure to acidic attack show several transversal breaks, indicating an effective degradation process induced on cellulose by acid environments (Cerchiara et al. 2009). Conversely, no degradation of the fiber structure have been observed on paper samples after exposure to wet atmosphere (Fig. 5c).

Fig. 5
figure 5

Scanning electron microscopy (SEM) micrograph of a MOTIFCOPY paper samples, b paper samples exposed to acidic attack, c paper samples exposed to wet atmosphere

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  • Characterization of paper samples exposed to degradative processes and contaminated with A. versicolor

Even in this case, DP determination and morphologic analysis using SEM were carried out to characterize paper samples degraded and contaminated with A. versicolor. DP was determined on paper samples after 5, 15 and 25 days of A. versicolor inoculum. As seen in Fig. 6, DP of paper samples decreased with increasing time indicating enzymatic hydrolysis of cellulose caused by evident fungal growth. In fact, Fig. 7 shows conidial germination of A. versicolor on paper samples exposed to acidic attack. Similar results were obtained on the paper samples exposed to wet atmosphere (data not reported).

Fig. 6
figure 6

DP of paper samples exposed to degradative processes and contaminated with A. versicolor after 5, 15 and 25 days

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Fig. 7
figure 7

Scanning electron microscopy (SEM) micrograph of conidial germination of A. versicolor on paper samples exposed to acidic attack

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  • MW heating on paper samples exposed to acidic attack and contaminated with A. versicolor

In the conservation context, MW heating is one of the physical methods proposed to ensure protection against fungal growth. So, we studied the use of MW heating at three different temperatures (30, 58 and 63 °C) on paper samples exposed to acidic attack and contaminated with A. versicolor setting MW power to 300, 600 and 900 W, respectively.

MW heating effects on paper samples were evaluated through DP determination as shown in Fig. 8. In fact, DP of paper samples exposed to 30 °C for 2 min and 50 s was lower than exposed to 58 and 63 °C indicating a clear influence of MW heating on the fungal vitality. As noted, when paper samples were exposed to low temperature (30 °C) an increase of viability of spores was observed, improving fungal growth. The growth of fungi was stopped without causing significant damage of paper such as cellulose depolymerization at higher temperature (58 and 63 °C, respectively).

Fig. 8
figure 8

DP of paper samples exposed to acidic attack and contaminated with A. versicolor after microwave heating at different temperatures

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Figure 9 shows images of inhibition fungal growth resulting from MW heating on paper samples. Five days after inoculation, the typical coloured spots were shown on the paper surface suggesting the presence of fungal colonization. After 10 days of MW heating (30 °C), it was observed on paper samples a biostatic/biocidal activity, while after 30 days there was evident fungal re-growth as shown in Fig. 10. In fact, on paper samples there are many coloured spots.

Fig. 9
figure 9

Images of paper samples exposed to acidic attack and contaminated with A. versicolor after microwave heating at different temperatures

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Fig. 10
figure 10

DP of paper samples exposed to wet atmosphere and contaminated with A. versicolor after microwave heating at different temperatures

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The effects of MW heating at 58 °C after 10 and 30 days respectively leaded a biocidal activity (no fungal re-growth was observed) or at least, biostatic activity (no spore development) after the treatment (Fig. 9). Similar results were obtained exposing paper samples at 63 °C. In addition, no spots originating from A. versicolor were observed on paper samples suggesting the biocidal activity of MW.

  • MW heating on paper samples exposed to wet atmosphere and contaminated with A. versicolor

Even in this case, results obtained for MW effects on paper samples exposed to wet atmosphere and contaminated with A. versicolor are similar to those above described for paper samples exposed to acidic attack and contaminated with A. versicolor.

Fungal re-growth was observed exposing to MW heating at 30 °C after 10 and 30 days respectively, as confirmed by DP reported in Fig. 10. Whereas, no fungal re-growth was evident after MW heating at 58 and 63 °C respectively, suggesting the ability of MW to stop growth of fungi. Paper samples exposed to 58 and 63 °C after 10 and 30 days exhibit higher DP than exposed to MW heating at 30 °C as shown in Fig. 10 indicating no cellulose degradation occurred.

Images of paper samples after the MW treatment show no fungal re-growth on paper samples exposed to MW heating at 58 and 63 °C after 10 and 30 days (data not reported).

Conclusions

The book of 18th century “Trattato delle acque minerali” written by Niccola Andria was biodeteriorated by A. versicolor as confirmed the morphological analysis and molecular biology techniques.

Molecular studies have demonstrated that a method where the ITS region is sequenced and the result is analyzed by phylogenetic methods, is a robust strategy for fungal species identification.

Identification of Aspergillus species using nucleic acid sequence analysis of the ITS 1 and 2 regions in combination with a BLAST analysis is a reliable and efficient method that provides earlier identification than standard culture methods. Indeed, to confirm there is a reduction in analysis time and the opportunity to use cultural small portions.

A. versicolor is widespread in terrestrial ecosystems from polar to southern latitudes (Domsch et al. 1993), it can survive for a long time in natural environments also in unfavorable condition (Fomicheva et al. 2006). Finally, our research is supported by several articles in the literature that confirm the presence of A. versicolor as a ubiquitary paper biodeteriogen. Taken into account it, we used A. versicolor as biodeteriogen to contaminate paper samples exposed two degradation processes (exposure to wet atmosphere and to acidic attack) simulating storage conditions of 18th century book.

Finally, we investigated the application of MW as physical method to protect biological attack due to fungi in paper artworks. MW heating at three different temperatures (30, 58 and 63 °C) was applied on different types of paper samples affected by spots originating from A. versicolor. DP of paper samples and SEM microscopy were evaluated after prefixed times of fungal attack. The best results in stopping fungi growth’s were obtained with MW heating paper samples at 58 and 63 °C for few minutes ensuring that there was no damage to the cellulose. Experiments performed proved that the use of MW heating could help to effectively protect artworks contaminated with A. versicolor.