Introduction

The protection of cultural heritage is a crucial activity that ensures the preservation and transmission of information from various areas of human history to future generations. In addition to natural ageing, all objects of cultural heritage, including those found on paper carriers, are exposed to several factors, which can significantly accelerate degradation processes leading to a change in chemical composition, a decrease in mechanical strength, and even the destruction of the object itself. Furthermore, as a result of improper storage conditions and insufficient preventive measures, these objects are often located in an environment that provides suitable conditions for potential microbial contamination. An important source of contamination in archives, libraries, and other memory institutions is a group of microorganisms called microscopic filamentous fungi (Fig. 1). Besides the degradation of works of art, fungal spores may affect the health of memory institution employees and visitors [1].

Fig. 1
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A set of contaminated paper-based cultural heritage objects from the collection of the Slovak National Archive

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The reasons for microbial contamination are various. It is generally known that filamentous fungi are ubiquitous and cultural heritage objects are therefore constantly exposed to the risk of microbial deterioration [2]. Fungi are able to get into archives and libraries through dust particles, which contain fungal spores [3]. Another way how micromycetes can get into the indoor environment of memory institutions is through newly admitted contaminated objects or human interactions [4]. Low-quality ventilation systems combined with different surface temperature lead to the formation of local micro-climates with higher water availability, consequently creating favourable conditions for fungal growth [5].

A wide variety of fungi can be found in archives and libraries, most often from the genera Penicillium, Aspergillus, Cladosporium, Trichoderma and Chaetomium [6, 7]. Paper, as a hygroscopic substrate made of cellulose fibres containing various additives (glues, binders, etc.), represents an excellent source of nutrients for filamentous fungi as they are chemoheterotrophs, requiring organic compounds as energy and carbon source [3, 8].

Microscopic filamentous fungi are able to deteriorate paper-based objects by the action of extracellular cellulolytic enzyme complex (cellulase) that is capable of hydrolysing β-1,4-glycosidic bonds in cellulose. Enzymatic degradation results in a transformation of cellulose into glucose molecules—the primary carbon and energy source for fungi [9]. At the same time, paper heritage objects are exposed to mechanical damage associated with the penetration of fungi hyphae in the substrate [9]. The enzymatic activity of filamentous fungi can affect the chemical, physical and aesthetic properties of contaminated artefacts [10]. This phenomenon is known as biodeterioration, defined as “any undesirable change in the properties of a material caused by the vital activities of organisms” [11]. As a result of the irreversible degradation of cellulose fibres, paper-based heritage objects become brittle and cracked [12].

Moreover, certain types of filamentous fungi are characterised by the production of pigments and weak organic acids, making the documents unreadable and diminishing its aesthetic value. The stains on the substrate caused by the activity of fungi may have different colouration (e.g. yellow, brown, black) [9, 13]. A special case of fungal deterioration, appearing as local changes in a paper's colour, is called foxing [14]. Foxing spots are described as “random circular and irregular yellowish to brownish-red stains on the surface of old paper” [15].

To inhibit biodeterioration processes, memory institutions use various methods. The most available method is applying UVC irradiation, but due to its poor penetration, it is mainly used to sterilise directly exposed surfaces and air. Its germicidal effect is based on damaging microbial RNA and DNA, making replication of genetic information impossible and thus preventing reproduction [16, 17]. Despite the fact that microwave radiation is not traditionally used for decontaminating heritage objects, some authors [18, 19] demonstrated the potential of the method for the devitalisation of microorganisms. However, the mechanism is not yet fully understood. It is not entirely clear whether the devitalisation is caused only by thermal effects, i.e. the heat generated as a result of the absorption of microwave radiation by water, or by complex organic systems with permanent or induced polarization, or it occurs due to a non-thermal microwave effect, i.e. by the direct transfer of energy from the electromagnetic field to the vibrational states, which leads to a change in their conformation [20]. Another widely used method for a long time is the application of ethylene oxide as an effective biocidal agent [10, 21, 22]. Although it belongs to one of the most effective decontamination methods, it has a huge disadvantage—it is carcinogenic and mutagenic [4]. Its biocidal effects on microorganisms completely stop their biological processes through the alkylation reaction of cellular constituents (nucleic acids, proteins). The application of electron beam irradiation is a promising and effective alternative to ethylene oxide [23]. Interaction between accelerated electrons and microorganisms leads to the cleavage of their DNA chains. The method's advantages include a short exposure time and no residues after the sterilization process [24].

Cultivation techniques have been conventionally used to detect and identify microorganisms inhabiting cultural heritage objects and materials [25]. However, the main drawback of culture-dependent methods is that they provide limited information, as the majority of microorganisms is not cultivable in laboratory conditions [26]. In recent years, interest in new methods has been growing, and other methods of detecting and identifying microorganisms have been created. Among the most used are DNA amplification by polymerase chain reaction (PCR) or DNA sequencing. However, these methods are often expensive, time-consuming and performed ex-situ [27, 28].

In order to choose the most adequate and effective ways of protection, it is necessary to know all the essential information about microbial communities inhabiting objects of cultural value, i.e. type of microorganisms, their characteristics, relationship to the contaminated objects and activity level (viable or devitalised form). Early detection and characterisation of fungal contamination make it possible to apply relatively non-invasive methods for its removal before visible and permanent damage of attacked artefacts becomes apparent [29]. Information about viability enables the selection of an appropriate conservation method, and in addition, it can help to check the effectiveness of the conservation intervention.

With a focus on determining the viability of filamentous fungi, a few techniques are currently used. In addition to the cultivation techniques, the method based on the measurement of ATP by bioluminescence showed its potential [30]. Other techniques, such as solid-phase cytometry [31], fluorometric assays or dye exclusion assays [32] are used in various fields (medicine, microbiology, etc.), but they have not yet been applied in the field of heritage protection. Ongoing and intense research aims to develop new, time-saving, easier, non-destructive, and non-invasive methods that could be used directly where microbial contamination is present. In this regard, UV-Vis-NIR and NIR Fibre Optics Reflection Spectroscopy (FORS) seems to be a promising method to fulfil these requirements. The devices are quite small, portable, and the measurements take only a few seconds. They can be used directly in collections-keeping institutions. Furthermore, measurements with optical fibre devices are not limited by the size and shape of the analysed objects. The method has already been successfully used in the cultural heritage field to identify dyes and pigments [33, 34] or iron-gall inks [35]. On the other hand, the method has not yet been sufficiently investigated to detect, characterise or identify microbial contamination of heritage objects. However, several studies from different fields (e.g. microbiology of food, environment, agriculture or medicine) demonstrate the method's applicability and other spectroscopic techniques for this intent [36,37,38,39].

The aim of this paper is to apply UV-Vis-NIR and NIR FORS to study the fungal contamination of paper substrates and evaluate the possibility of using the method to monitor the state of contaminated objects of cultural value.

Materials and methods

Model systems

Model systems mimicking microbial contamination of paper consisted of two main components—a paper substrate and filamentous fungi, which were inoculated, either in the form of conidia or mycelia. The filamentous fungi used in the experiment were selected based on a literature review as representatives of the fungi genera most often found in the environment of memory institutions. Three groups of model systems were prepared—viable, devitalised and reference. Reference model systems were exposed to the same conditions as viable and devitalised ones, but they were not inoculated with filamentous fungi. Four paper carriers inoculated with conidia or mycelia of filamentous fungi were prepared for each investigated model system. Two samples were intended for spectra measurement, and two were used for viability control.

Model substrate

Chromatographic paper Whatman 1 CHR with smooth surface, 0.18 mm thick, basis weight 87 g/m2, surface pH 7, manufactured without additives. A sheet of paper was cut into squares 2 × 2 cm and sterilized by autoclaving (120 °C, 120 kPa, 20 min).

Model microorganisms

Alternaria alternata CCM F-128 (AA), Aspergillus niger CCM 8189 (AN), Cladosporium herbarum CCM F-159 (CH), Penicillium chrysogenum ATCC 9480, CCM F-362 (PCH) and Trichoderma atroviride CCM F-534 (TV) (Czech Collection of Microorganisms, Masaryk University in Brno, Czech Republic) were cultivated on malt extract agar (MEA; Biolife, Italy).

Inoculation of paper substrates

A few millilitres of sterilized distilled water were added to the 10 day-old cultures of filamentous fungi, and the conidia were released using a sterile L-shaped spreader. The fungal spore suspensions were transferred into a Falcon-type test tubes (50 mL) and filtered through a sterile polyamide/nylon sieve (opening size 0.43 µm). Fungal spores were separated by centrifugation (10 min at 2500 rpm) and washed with water three times. A diluted solutions of the spore suspensions were prepared in a sterile Eppendorf tubes. The Bürker chamber was used for counting spores. The concentration of spores was adjusted to 106 conidia/50 µl, except for Alternaria alternata – 105 conidia/50 µl. Model systems consisting of paper carriers and conidia of filamentous fungi were prepared by inoculating the paper substrates with a 50 µl of spore suspension using a pipette.

A model systems consisting of a paper carrier and mycelia of filamentous fungi were prepared as follows: 1.5 mL of Czapek-Dox broth (CZDB; Biolife, Italy) in sterile Eppendorf microtubes were inoculated by 50 µL of spore suspension and cultivated for 48 to 72 h on a reciprocal shaker. Mycelia were separated from the medium by centrifugation (15 min at 13.400 rpm) and washed three times with water. Subsequently, the solid mycelium was quantitatively transferred to a paper carrier using a pipette.

Devitalisation of model systems

Five available methods for the devitalisation of samples were selected, whose goal was to capture the properties of the widest possible range of devitalised sample products. A model system consisting of a paper substrate and conidia or mycelia of individual filamentous fungi was prepared for each devitalisation method. Namely, UVC irradiation (UVC), microwave radiation (MW), ethylene oxide (EO), accelerated electrons (AE), and anaerobic conditions (-O2) were used as devitalisation methods.

UVC irradiation (UVC)

The devitalisation process based on applying UVC irradiation was carried out using the germicidal lamp BakMed LB-301.1 (BakMed; Poland), emitting ultraviolet rays with a wavelength of 253.7 nm. The distance between the samples and the light source was 8 cm, and the irradiation of samples lasted 15 h.

Microwave radiation (MW)

The devitalisation method based on the biocidal effects of microwave radiation was carried out using the Candy CMW 2070 M microwave oven, which has a power of 700 W. The samples were exposed to microwave radiation with a high power level (100% power output) for 15 min.

Ethylene oxide (EO)

Ethylene oxide sterilization took place in cooperation with the Slovak National Gallery (Zvolen, Slovakia). The ethylene oxide fumigation chamber (DE LAMA; Italy) has several programs, which differ in the humidity, temperature and pressure used, depending on the material and the degree of damage. The selected sterilization program P4 lasts 22 h—from when the object is inserted into the chamber until the end of sterilization and ventilation. The parameters of the P4 program are summarized in Table 1.

Table 1 P4 program used for ethylene oxide devitalisation
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Accelerated electrons (AE)

Devitalisation of model systems using a linear electron accelerator UELR-5-1S (FGUP NIIEFA, Russia) took place in cooperation with the Progresa Final SK in the Centre of electron accelerator (Trenčín, Slovakia). The process parameters are in Table 2.

Table 2 Parameters of accelerated electrons devitalisation process
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Anaerobic conditions (-O2)

The anaerobic box with AnaeroGen™ sachet (Oxoid, UK) was used to create anaerobic conditions. After inserting the sachet into the anaerobic box, oxygen is absorbed, and carbon dioxide CO2 is produced. When used correctly, the system is able to reduce the amount of oxygen below 1% in 30 min. The resulting amount of CO2 ranges from 9 to 13%. The active ingredient is ascorbic acid. A moisture absorber—a sachet with silica gel—was used to reduce the humidity. It can reduce humidity to 40% in a closed system. The samples were subjected to anaerobic conditions for four weeks.

Viability control

The effectiveness of the selected devitalisation processes was verified by the cultivation of samples. Samples intended for viability control were aseptically transferred to a sterile CZDB medium (Biolife, Italy), put on a rotary shaker, and left to culture for 72 h. After this time interval, a visual check of the viability of filamentous fungi followed. Insufficient devitalisation of conidia was observed as germination and mycelium formation. In case of unsuccessful devitalisation of the mycelia, we could observe their growth on the paper substrate.

Spectroscopy methods

Before the actual measurement of UV-Vis-NIR and NIR spectra, all prepared model systems were conditioned in a climatic chamber for 24 h (23 °C, 50% RH). Spectral data of samples in the UV-Vis-NIR and NIR spectral regions have been obtained using Ocean Optics fibre optics spectrophotometric devices (Ocean Optics, Dunedin, FL, U.S.):

UV-Vis-NIR region—spectrometer HR40000CG, light source DH-200-BAL and standard reflection accessory with 45°/45° geometry; NIR region—spectrometer NIR256-2.5, light source HL-2000-FHSA and standard reflection accessory with 45°/45° geometry. The measurements were performed with the OceanView 1.6.7 (Ocean Optics, Dunedin, FL, U.S.) software package with the following settings: integration time, automatic; the number of averaged spectra, 5; boxcar, 10.

Two parallel samples were measured at five different spots to limit the impact of uneven inoculation. The result of the measurements of each sample was a set of 10 spectra, which were statistically processed.

Processing of data

  1. 1.

    The original spectral data were interpolated in the wavelength range of 300–1050 nm (UV-Vis-NIR) and 1000–2200 nm (NIR), 150 points.

  2. 2.

    Subtraction of the average spectrum of reference samples from each spectrum of viable or devitalised samples.

  3. 3.

    Normalization of each spectrum in the interval 0–1.

  4. 4.

    Calculation of the average normalized spectrum for each sample, calculation of the standard deviation SDO of the set of spectra.

In the case of a high standard deviation value (above 0.10), possible outlier spectra of the set were identified using the Principal Component Analysis (PCA) and excluded from the data set. Spectra were displayed graphically and analysed by the PCA method.

Principal component analysis (PCA)

Modern spectrometric systems generate enormous amounts of data. Therefore, a simple evaluation is not always possible without losing valuable information. In such a situation, it is helpful to use chemometric-numerical data processing. The methods based on factor analysis are used most often.

Among the factor analysis methods, the multivariate statistical method Principal Component Analysis (PCA) is an especially useful tool for processing spectral data sets. The PCA method uses a matrix decomposition of spectral data and a corresponding reduction in the number of variables to describe the spectra of the studied samples in a reduced space. The PCA allows to detect the tendency of samples with comparable properties to cluster into groups and reveal the latent internal structure of the data. The Unscrambler X 10.5 programme (CAMO Softwares AS., Oslo, Norway) was used for the analysis.

Results and discussion

The aim of this work is to evaluate the possible application of UV-Vis-NIR and NIR FORS combined with the multivariate statistical method PCA as an effective approach to monitor microbial contamination. Several spectra were measured and analysed to evaluate the potential of Principal Component Analysis of UV-Vis-NIR and NIR spectra as a tool for the differentiation of viable and devitalised forms of filamentous fungi.

Despite several repeated attempts, we did not achieve the devitalisation of Alternaria alternata (mycelium) using UVC irradiation and Aspergillus niger (conidia and mycelium) using anaerobic conditions. Alternaria alternata has multi-celled spores with thick, melanised cell walls that can create a way of protection against UV irradiation [40]. Moreover, UVC radiation is characterised by low penetration, and it is possible that not all cells were exposed to its germicidal effects. Aspergillus species are obligate aerobic microorganisms. However, decreasing oxygen level increase sporulation. Therefore, it is possible to survive adverse conditions in this dormant form without losing viability.

The results of the PCA method—two-dimensional score scatter diagrams—allow observing the distribution of samples into clusters. It is important to note that samples located in one cluster are similar to each other, and at the same time, they are different from samples located in other clusters. Table 3 shows in which scatter score diagrams a noticeable and clear difference of the spectra of viable and devitalised forms was found (Yes), in which no/poor distinction of the spectra of viable and devitalised forms was observed (No) and in which the distinction is only partial (Partially). It turns out that the difference between viable and devitalised forms was found in almost all studied systems, at least in one studied spectral range, except for Aspergillus niger mycelia.

Table 3 The principal component analysis (PCA) results—a noticeable and clear difference of the spectra of viable and devitalised forms—Yes, no/poor distinction of the spectra of viable and devitalised forms—No, the distinction is only partial—Partially
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Figures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21 show UV–Vis-NIR and NIR spectra of viable and devitalised forms (conidia, mycelium) of examined fungi and corresponding two-dimensional score scatter diagrams. Different groups of samples have different colours: viable samples (VIT)—black; samples devitalised using UVC irradiation (UVC)—red; microwave radiation (MW)—blue; ethylene oxide (EO)—green; accelerated electrons (AE)—cyan and anaerobic conditions (-O2)—magenta.

Fig. 2
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Alternaria alternata conidia samples

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Fig. 3
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Alternaria alternata conidia samples

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Fig. 4
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Alternaria alternata mycelium samples

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Fig. 5
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Alternaria alternata mycelium samples

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Fig. 6
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Aspergillus niger conidia samples

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Fig. 7
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Aspergillus niger conidia samples

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Fig. 8
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Aspergillus niger mycelium samples

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Fig. 9
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Aspergillus niger mycelium samples

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Fig. 10
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Cladosporium herbarum conidia samples

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Fig. 11
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Cladosporium herbarum conidia samples

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Fig. 12
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Cladosporium herbarum mycelium samples

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Fig. 13
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Cladosporium herbarum mycelium samples

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Fig. 14
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Penicillium chrysogenum conidia samples

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Fig. 15
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Penicillium chrysogenum conidia samples

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Fig. 16
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Penicillium chrysogenum mycelium samples

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Fig. 17
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Penicillium chrysogenum mycelium samples

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Fig. 18
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Trichoderma atroviride conidia samples

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Fig. 19
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Trichoderma atroviride conidia samples

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Fig. 20
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UV-Vis-NIR spectra and two-dimensional score scatter diagram—viable and devitalised Trichoderma atroviride mycelium samples

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Fig. 21
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NIR spectra and two-dimensional score scatter diagram—viable and devitalised Trichoderma atroviride mycelium samples

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UV-Vis-NIR spectra of viable conidia and mycelium of Alternaria alternata (Figs. 2, 4) form separated clusters clearly distinguishable from the spectra of samples devitalised by selected methods. In the case of Alternaria alternata conidia (Fig. 2), PCA analysis in the UV-Vis-NIR spectral region led to the separation of several groups corresponding to viable and devitalised samples. Viable samples are clearly differentiated from devitalised ones and are characterised by a negative score value of both components (PC-1, PC-2). Most devitalised samples have a positive score value of component PC-1, except for samples devitalised by anaerobic conditions. UV-Vis-NIR spectra of Alternaria alternata mycelium (Fig. 4) show similar behaviour, but in this case, viable samples are characterised by a positive score value of the first component, while most devitalised samples have a negative score value, except for samples devitalised with ethylene oxide. NIR spectra and score scatter diagrams revealed differences between viable and devitalised Alternaria alternata conidia (Fig. 3) as well. The cluster corresponding to NIR spectra of viable conidia is separated by the positive score value of PC-1 and negative score value of PC-2 from the spectra of devitalised samples. However, in the case of mycelium (Fig. 5), the differentiation of viable and devitalised samples was not shown.

In the case of Aspergillus niger conidia, PCA analysis in the UV-Vis-NIR spectral region led to the separation of viable samples (Fig. 6). In the NIR spectral region (Fig. 7), the spectra of viable conidia are relatively close to the spectra of samples devitalised by microwave radiation and the distinction of the viable samples is only partial. The PCA analysis of UV-Vis-NIR and NIR spectral data of Aspergillus niger mycelium (Figs. 8, 9) could not differentiate the spectra of viable and devitalised samples, and samples remained unresolved from each other.

The analysis of spectral data of Cladosporium herbarum conidia and mycelium results in the complete separation of viable conidia samples in both spectral regions (Figs. 10, 11) and of viable mycelium in the NIR spectral region (Fig. 13). The UV-Vis-NIR spectra of viable conidia samples (Fig. 10) have a positive PC-2 score value and negative PC-1 score value what makes them distinguished from devitalised samples. PCA of NIR spectra corresponding to Cladosporium herbarum conidia samples (Fig. 11) shows that viable and devitalised samples are clearly distinguished along the PC-1. In this case, most devitalised conidia samples are characterised by negative PC-1 score values. As can be seen in the score scatter diagram (Fig. 12), points corresponding to viable mycelium spectra in the UV-Vis-NIR region overlap with the points of devitalised mycelium spectra. PCA did not reveal differences between the spectra of viable and devitalised samples.

The situation is very similar for Penicillium chrysogenum samples. The spectra corresponding to viable conidia samples form clusters clearly separated from the spectra of devitalised samples in both studied spectral regions (Figs. 14, 15). In the score scatter diagram of conidia samples in the UV-Vis-NIR region (Fig. 14), spectra of viable samples created a separated group with positive score values, both PC-1 and PC-2. In the case of the NIR spectral region (Fig. 15), viable samples are characterised by negative score values and well distinguished from devitalised samples. Separation of viable samples can be observed as well for Penicillium chrysogenum mycelium in the UV-Vis-NIR spectral region (Fig. 16). Differentiation between the spectra of viable and devitalised mycelium samples in the NIR spectral region is only partial (Fig. 17), and the points corresponding to viable samples' spectra overlap with the points of samples devitalised using microwave and ethylene oxide devitalisation.

The UV-Vis-NIR and NIR spectra of viable Trichoderma atroviride conidia were distinguished from the spectra of devitalised conidia samples (Figs. 18, 19). UV-Vis-NIR spectra of viable conidia samples (Fig. 18) and samples devitalised using ethylene oxide are both characterised by positive score values, but they are still quite well distinguishable from each other. NIR spectra of viable conidia (Fig. 19) form a separate group that is very compact and clearly differentiated from the devitalised samples. The viable and devitalised mycelium samples in the UV-Vis-NIR spectral region remained undifferentiated from each other (Fig. 20). However, in the NIR spectral region, as seen in Fig. 21, the viable mycelium samples clearly distinguish and form a separate cluster.

In cases where it is impossible to clearly confirm the differences between the spectra of viable and devitalised fungi, based on a simple visual assessment of scatter diagrams, it will be helpful to extend the data processing with more sophisticated statistical analysis, for example Linear discriminant analysis (LDA), Partial least squares discriminant analysis (PLS-DA), Cluster analysis or Multivariate analysis of variance (MANOVA), that are used in other fields (e.g. forensic science) [41,42,43].

Conclusions

The results of this study suggest that UV-Vis-NIR and NIR Fibre Optics Reflection Spectroscopy (FORS) coupled with Principal Component Analysis (PCA) can be used as the basis of the method for monitoring the microbial contamination of paper substrates. The PCA analysis has been applied to the UV-Vis-NIR and NIR spectral data of viable and devitalised filamentous fungi in two different forms—conidia and mycelium. The obtained results prove differences between the spectra of viable and devitalised forms at least in one studied spectral region, except for one sample—Aspergillus niger mycelium. A combination of spectroscopic techniques and statistical data processing seems to be a promising, alternative, relatively quick and non-destructive tool, which may help memory institutions characterise microbial contamination and evaluate the effectiveness of sterilization processes.