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. 2019 Sep 17;14(9):e0219650. doi: 10.1371/journal.pone.0219650

Preserving cultural heritage: Analyzing the antifungal potential of ionic liquids tested in paper restoration

Kevin Schmitz 1,*, Sebastian Wagner 1,#, Manfred Reppke 1,#, Christian Ludwig Maier 2,3, Elisabeth Windeisen-Holzhauser 1, J Philipp Benz 1
Editor: Binod Bihari Sahu4
PMCID: PMC6748409  PMID: 31527882

Abstract

Early industrialization and the development of cheap production processes for paper have led to an exponential accumulation of paper-based documents during the last two centuries. Archives and libraries harbor vast amounts of ancient and modern documents and have to undertake extensive endeavors to protect them from abiotic and biotic deterioration. While services for mechanical preservation such as ex post de-acidification of historic documents are already commercially available, the possibilities for long-term protection of paper-based documents against fungal attack (apart from temperature and humidity control) are very limited. Novel processes for mechanical enhancement of damaged cellulosic documents use Ionic Liquids (IL) as essential process components. With some of these ILs having azole-functionalities similar to well-known fungicides such as Clotrimazole, the possibility of antifungal activities of these ILs was proposed but has not yet been experimentally confirmed. We evaluated the potency of four ILs with potential application in paper restoration for suppression of fungal growth on five relevant paper-infesting molds. The results revealed a general antifungal activity of all ILs, which increased with the size of the non-polar group. Physiological experiments and ultimate elemental analysis allowed to determine the minimal inhibitory concentration of each IL as well as the residual IL concentration in process-treated paper. These results provide valuable guidelines for IL-applications in paper restoration processes with antifungal activity as an added benefit. With azoles remaining in the paper after the process, simultaneous repair and biotic protection in treated documents could be facilitated.

Introduction

Private and public archives harbor myriads of historic paper documents as an invaluable treasure of cultural heritage. Numerous library, archive and museum communities around the globe have committed to the task of preserving and–if necessary–restoring these documents to prevent additional deterioration.

A large share of paper wear is caused by endogenous abiotic factors, with acidic hydrolysis as a major cause [1]. The use of acidic sizing components and pulping processes, such as the bisulfite process, which were particularly popular throughout the 20th century, left paper in an acidic state and prone to slow auto-hydrolysis of glycosidic bonds [1, 2]. Furthermore, oxidization of lignin components creates further acidification and hence contributes to progression of paper brittleness [1, 3]. As a result, industrial mass de-acidification of paper-based documents has become an important business with several players involved [4, 5]. Current paper standards promoted by library, archive & museum communities, encompassing endogenous alkaline reserves and lignin contents below 1%, may have reduced the risk of acidic auto-hydrolysis, but today’s paper remains exposed to a variety of other risk factors, warranting further research in preservation and restoration [1].

In parallel to endogenous risks, the control of exogenous factors is an important countermeasure in paper preservation [6]. Exogenous abiotic factors, such as humidity and temperature for example, not only affect abiotic deterioration processes, but also facilitate microbial attack [7]. In a recent survey among 57 institutional participants from 20 different countries, Sequeira et al. [8] have found that 79% of the participants had to deal with fungal infestations even though common preventative measures against fungal deterioration had been undertaken. According to the authors, most of these infestations were caused by unforeseen water contact (floods, leakage, fire suppression, outdoor humidity) or failure of (micro-)climate control systems, illustrating the importance of this issue beyond (sub-)tropical areas [1]. Extensive literature on deterioration-causing microorganisms, especially fungal isolates from libraries and archives, is available [6–11]. For example, Chaetomium globosum, Penicillium chrysogenum as well as other species of the respective genera were isolated from and identified as causative for dark stains in historic documents [8]. Species such as Aspergillus versicolor (among Aspergillus species) in addition to a diverse set of Trichoderma species were also reported to be ubiquitously found in archives, museums and libraries [10, 11]. The scientific literature also illustrates a continuous effort in preventing and controlling fungal deterioration of paper-based documents [12–17], underlining the significant threat for cellulosic documents imposed by fungi.

As identified in a survey by Sequeira et al. [16], available antifungal options are not yet satisfactory–especially since no high-throughput treatments for removal or prevention of fungal infestations on cellulosic documents are available to date. A novel approach, however, promises a potential for integration of antifungal components in paper documents in a combined industrial de-acidification and mechanical paper reinforcement process [5]. In this process, regenerated cellulose fibers are solubilized in an ionic liquid (IL) and dimethyl sulfoxide (DMSO) as a co-solvent, before applying the solution to cellulosic documents in an immersion bath (Fig 1). Upon solvent exchange with hexamethyldisiloxane (HMDSO), the cellulose fibers precipitate at the cellulosic surfaces [18, 19], thereby conferring mechanical reinforcement with neglectable impairment of readability [5]. Notably, residues of (co-)solvents and the IL used for cellulose solubilization get incorporated in the paper material along the way.

Fig 1. Process scheme of the paper reinforcement method.

Fig 1

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Process as described by Krupp et al. [18] and based on Maier [5]. Cellulose fibers are solubilized in IL (green triangles) and DMSO (orange circles) as co-solvent (left). Upon solvent exchange with HMDSO (blue circles; arrows indicate solvent replacement), cellulose fibers precipitate at cellulosic surfaces, thereby adding mechanical strength to the cellulosic documents while incorporating IL molecules (and other co-solvents).

ILs, also commonly referred to as liquid salts [20], consist of an organic cation or anion with an asymmetrical charge distribution (which is usually aprotic and stably charged in its nature) and a matching organic or inorganic counter ion [21]. ILs are mostly recognized as high potential solvents in green chemistry applications due to their non-volatile characteristics [22]. Interestingly, the class of ionic liquid used in the paper reinforcement process (represented by 1-butyl-3-methylimidazolium-acetate; BA) shares key structural characteristics (Fig 2) with well-known antifungal agents such as Clotrimazole [23] or Miconazole [24]. Similarities comprise a charged, polar imidazolium group, which is known to be involved in blocking of an enzyme in ergosterol biosynthesis [25, 26], as well as a nonpolar (derivatized) aliphatic molecule tail, believed to interact with fungal membranes and thereby disturbing their integrity at higher concentrations [27]. Extrapolating these findings, ILs of this class could potentially confer antifungal properties to documents treated in the aforementioned paper reinforcement process upon incorporation into the cellulosic documents. In this study, we therefore tested four ILs matching these characteristics and showing promising process compatibility properties for their antifungal potential. We could show in bioassays that BA and HC (1-hexyl-3-methylimidazolium-chloride; Fig 2) abolished growth of all tested fungi at 10% and 1% volume concentrations, respectively. Furthermore, fungal growth was significantly impaired at lower concentrations of 1% and 0.1%, respectively. While antifungal properties could not be confirmed on BA-treated paper samples, this study nevertheless provides important guidelines for future IL selection for incorporation of fungal protection in paper restoration.

Fig 2. Chemical structures of known antifungals and ILs.

Fig 2

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Clotrimazole (a) and Miconazole (b) as known antifungals with polar protic imidazolium groups (blue) and a rather nonpolar, (derivatized) aliphatic molecule region (green) causing overall asymmetrical molecule properties. Ionic liquids tested for antifungal activity in this study were: 1-butyl-3-methylimidazolium with (c) chloride (BC) and (d) acetate (BA) as counter ions as well as (e) 1-allyl-3-methylimidazolium-chloride (AC) and (f) 1-hexyl-3-methylimidazolium-chloride (HC). All four ILs contain an aprotic imidazolium group (blue) as well as nonpolar tails (green) varying in size and hydrophobicity.

Materials and methods

Growth media and ionic liquids

Malt extract media (MEM) agar was prepared as 3% malt extract (Roth) and 0.5% peptone from casein (Sigma) in 1.5% agar-agar (Sigma). Minimal medium (MM) agar for fungal spore generation was prepared as 1x Vogel’s salts [28] and 2% sucrose in 1.5% agar-agar (Sigma). MM agar with 2% carboxymethyl-cellulose (CMC low viscosity, Sigma Aldrich) instead of sucrose was used for ionic liquid dependent growth experiments for all fungal strains and was supplemented with varying amounts of different ILs provided by Nitrochemie Aschau GmbH (1-butyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, 1-allyl-3-methylimidazolium-chloride, 1-hexyl-3-methylimidazolium-chloride) for some experiments as stated in the main text. Paper samples treated in the aforementioned paper reinforcement process [19] with 1-butyl-3-methylimidazolium chloride or 1-butyl-3-methylimidazolium acetate as cellulose solving ionic liquids were provided by Nitrochemie Aschau GmbH. 1-Butyl-3-Methylimidazolium chloride was applied as a solution in DMSO as it was solid at room temperature.

Fungal strains

Cultures of Aspergillus versicolor (DSM-1943), Chaetomium globosum (DSM-62109), Penicillium chrysogenum (DSM-244) and Penicillium glabrum (DSM-2017) were retrieved from the Leibnitz Institute German Collection of Microorganisms and Cell Cultures (DMSZ) and verified via ITS sequencing of PCR products generated using primer pair ITS1 and ITS4 [29] on genomic DNA isolated via microwave treatment [30]. Retrieved ITS sequences were blasted against the fungal genome database using the National Center for Biotechnology Information (NCBI) nucleotide BLAST [31]. Perithecia formation of C. globosum indicated the presence of both mating types in the culture. Isolate Trichoderma capillare P9_B2_1 (NCBI accession number MF964230) was retrieved from an isolated stock [32].

Harvest of fungal spores

Spores were harvested after incubation on MEM agar at room temperature (RT) under light for seven days for Aspergillus versicolor and T. capillare, while Penicillium species were grown on MM agar under identical conditions for spore generation. Spores were scratched off of the mycelial biomass using a toothpick, suspended in physiological sodium chloride spore solution containing 0.05% Tween® 80 and subsequently filtered through sterile Miracloth® to eliminate hyphal remainders. Chaetomium globosum was incubated as a culture of mixed mating types on MEM agar at RT under light for five weeks until black perithecia were formed [33]. Perithecia were selectively harvested into a 1.5 ml Eppendorf tube and vortexed with 1 ml of spore solution to release spores before the suspension was filtered accordingly. Correlation curves for spectroscopic microplate reader (Tecan® Infinite M200 Pro) optical density values at 600 nm wavelength (OD600) and spore counts on a Neubauer counting chamber were determined for each spore type for quantification of spore inoculum volumes in successive experiments.

Agar-based assays for antifungal IL activity and potency determination

Fungal spores were densely plated onto MM-CMC agar plates at a density of 105 spores per cm2. Wells were punched into the inoculated agar to locally apply 20 μl of each IL. Plates were incubated at RT under light for germination and evaluated for halo formation and stability after four, seven, 14 and 28 days. Clotrimazole (in DMSO) was applied at 10,000 fold lower concentration as a positive, DMSO as a negative control. For potency evaluation, equimolar amounts of 140 μmol of each IL as well as an aqueous dilution series thereof (75%, 50%, 25% and 10%) were spotted onto inoculated plates prepared as above in duplicates. Matching molar amounts of sodium chloride and sodium acetate were also tested for fungal growth impairment in this assay to exclude specific effects of the IL counter ions. Halo radii were measured fourfold after 14 days and plotted against the applied amount of IL. For microtiter scale experiments, the outermost wells of the plate were filled with 200 μl sterile water to provide a humidity reservoir. MM-CMC was supplemented with varying volume per volume percentages of each IL (10%, 1%, 0.1%, 0.01%, 0%), respectively. Three wells per IL and dilution step were prepared with 200 μl medium per plate. Upon Inoculation of each well with 104 spores, plates were covered with a standard microtiter plate coverlid with or without aeration spacers, incubated at room temperature under light and evaluated for growth after four, seven and fourteen days.

Calculation of IL content in paper samples via elemental analysis of nitrogen (N)

Determination of the IL-content of paper treated in the reinforcement process was carried out via elemental analysis aiming at total nitrogen quantification after complete combustion. The analyses were performed on a CHN-O Rapid Elemental Analyser (Heraeus). An increase of 0.86 basis points in nitrogen content of treated compared to untreated paper was determined. Neither of the co-solvents DMSO or HMDSO contains nitrogen, and the overall nitrogen content of viscose fibers (Kelheim fibers GmbH) applied in the paper reinforcement process was assumed to be in a comparable (if not even lower) range compared with the original cellulosic document. Considering the nitrogen mass content of BA being 14.14%, this results in an estimated IL content of 6.1% in treated paper.

Growth assays on paper

Fungal spores were prepared as a spore suspension at 106 spores per ml in spore solution. 1 ml of this suspension was used to inoculate autoclaved paper samples of 2 cm x 2 cm in size. Inoculated paper samples were placed in individual sterile petri dishes and incubated at room temperature in the light under high humidity conditions to avoid paper desiccation. Growth was checked after four, seven ant fourteen days using a stereomicroscope.

Results

For our study, we selected four fungal strains known to cause paper deterioration in archives, namely Chaetomium globosum, Penicillium chrysogenum, Penicillium glabrum and Aspergillus versicolor [8, 10, 11] plus one recently isolated wild type strain of the highly cellulolytic genus Trichoderma, T. capillare [32]. All of the five selected fungal strains were reported to have good cellulolytic capabilities [34–37], making them suitable for the evaluation of the antifungal potentials of ILs on paper-decomposing molds.

General antifungal activity and comparison of antifungal potency of ILs

Principle susceptibility of all five fungal strains towards all four ILs was tested in an initial screening. To this end, all ILs were spotted onto Minimal Medium plates with carboxymethyl-cellulose as carbon source (MM-CMC) densely inoculated with fungal spores. As a representative example of all fungi, Fig 3a and 3b show susceptibility of P. glabrum to HC and BA, while BC and AC caused minimal to no observable effects. Stereomicroscopic analysis of the hyphal-free halos formed around HC and BA application sites revealed that germination was suppressed in these areas. Spores were not observed to germinate even after 28 days of incubation, thereby demonstrating persistence and durability of the antifungal effect.

Fig 3. Principal investigation and comparison of antifungal IL activity.

Fig 3

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(a) shows the exemplary result of the initial antifungal activity screening of all tested ILs including DMSO as a negative and Clotrimazole as a positive control on densely inoculated MM-CMC plates for P. glabrum, spotted following scheme (b), after seven days of incubation. c) and d) demonstrate the molar potency comparisons of the four ILs, using P. glabrum spot dilution series of HC (c) and the halo radius plot for all four ILs (d) as representative data for all tested fungi. e) MIC and MGIC concentration analysis in the microtiter scale growth assay for P. glabrum and for the most susceptible fungal strain in this study, T. capillare (f).

Quantification of fungal growth impairment by equimolar amounts of each of the four ILs was conducted for comparison of the four ILs via measurement of growth-free halo sizes on inoculated agar plates. Representative results are displayed for P. glabrum in Fig 3c and 3d. HC clearly had the strongest effect on all fungi tested, followed by BA, while no significant effect was observed for AC and BC in accordance with previous results. Notably, C. globosum and T. capillare revealed even stronger susceptibility to HC and BA than P. glabrum, which levelled equal to A. versicolor and P. chrysogenum in terms of susceptibility to the tested ILs (S1 Fig). Halos were observed to be stable and not penetrated by adjacent hyphae throughout 28 days of incubation. Halo formation was not observed on plates inoculated with corresponding molar amounts of sodium chloride or sodium acetate, indicating that the different counter ions (or the ionic strength, respectively) of the tested ILs did not facilitate growth impairment or inhibition per se.

As an important measure with regard to the paper reinforcement process, antifungal activities of the tested ILs would best be assessed as minimal inhibitory concentrations (MIC) in volume percentages. For this, a miniaturized growth assay was developed that allowed assessment of MIC and minimal growth impairing concentration (MGIC). MM-CMC with varying IL concentrations was inoculated with fungal spores in microtiter plate wells (Fig 3e and 3f). All ILs but BC caused clear inhibition of growth at 10% (v/v) concentration, while BC could only mildly impair but not abolish growth at 10% in P. chrysogenum and P. glabrum. Apart from HC, which caused strong growth impairment and reduced sporulation already at the 1% concentration level in all tested fungi (and even completely inhibited germination of T. capillare, P. glabrum and C. globosum), only BA was observed to show mild growth and sporulation impairment at the 1% concentration level in some of the tested fungi, especially in T. capillare and C. globosum (S2 Fig). In summary, BA and especially HC were determined to have the highest antifungal potential.

Testing fungal growth on IL-treated vs. non-treated paper as carbon source

Elemental analysis revealed an IL content of about 6.1% (w/w) in treated paper (see Methods). With an observed MIC value of around 1% for HC and MGIC of 1% for BA, antifungal effects should therefore, in principle, be observable in incubation experiments of fungal spores on BA- and HC-treated paper samples. Since AC and HC had shown poor cellulose solubilizing properties, the paper reinforcement process could only be established with BC and BA in pilot scale so far. Hence, no AC- or HC-treated paper could be tested for antifungal activity in this study.

When BA-treated and spore-inoculated paper samples were incubated on water agar plates, no qualitative or quantitative effect in growth impairment was observed for T. capillare, which had been found to have the strongest susceptibility to all tested ILs in previous experiments (Fig 4a). To obviate a possible dilution effect of the effective IL concentration through passive diffusion from the treated paper into the water agar, we also tested whether growth impairment could be observed during incubation of inoculated paper samples without agar but under high humidity conditions. Again, no perceptible difference in growth of T. capillare was observed on untreated versus BA-treated paper samples (Fig 4b), imposing a need for additional investigation in subsequent studies.

Fig 4. Fungal growth tests on IL-treated paper samples.

Fig 4

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T. capillare spores inoculated on BA-treated (right) or untreated (left) paper and incubated on water agar (a) or under saturated humidity conditions in a high humidity chamber (b) did not show growth impairment or inhibition effects after paper treatment.

Discussion

We were able to demonstrate that all tested ILs exhibited growth-inhibiting effects on relevant paper-deteriorating molds at high concentrations of 10% in an agar-based assay, while BC could only impair growth of the two Penicillia under these conditions. Potency experiments considering halo sizes on IL-inoculated agar plates and determining minimal inhibiting concentrations (MIC) as well as minimal growth impairment concentrations (MGIC) further revealed HC to be the strongest antifungal substance of the tested set, followed by BA, AC and BC. Detailed evaluation of the IL effects revealed that the order of their respective potency matches well with the size of their corresponding non-polar group, which is pointing towards the importance of the hydrophobic region for antifungal activity. Similar results were observed when the membrane-disturbing effects of three antifungal imidazolium drugs were tested [27], indicating that larger nonpolar groups may facilitate stronger membrane interactions and hence better antifungal activities in imidazolium-containing ILs. A systematic study focusing purely on the dependency of the antifungal effect on non-polar group sizes and structures would likely help to elucidate this correlation further.

Our study also allowed for hypothesis generation on antifungal effects of IL characteristics beyond the non-polar group constitution. Remarkably, the two ILs BA and BC only differ in their respective counter ions acetate and chloride, which by themselves were shown not to impair or inhibit fungal growth at equivalent concentrations in this study. It remains unclear, however, why the antifungal activity of BA was quite prominent while that of BC was very poor. Literature on plant cuticular membrane penetration of glyphosate salts for example indicates that the nature of the counter ion indeed influences the membrane penetration characteristics [38]. Fungal cell membrane penetration would be a crucial prerequisite for inhibition of ergosterol biosynthesis as a proposed mode of antifungal action of imidazolium ILs similar to that described for established imidazolium antifungals such as Clotrimazole [25, 26]. Additional research should address the influence of different counter ions in imidazolium ILs on antifungal activity, which could further benefit the development of highly potent antifungal drugs.

Considering an application as an antifungal component in paper restoration, the persistence of growth-free halos around IL inoculation sites throughout the observation period of four weeks indicated long-term stability and inertness of the tested ILs towards fungal decay or abiotic factors, which would be a beneficial trait for archived documents. However, while strong growth impairment was observed for T. capillare on MM-CMC containing 1% (v/v) of BA, no such growth impairment could be observed on BA-treated paper–which was found to contain about 6% (w/w) of BA in elemental analysis. Moreover, replacing the agar plates with a high-humidity chamber, as a precaution to avoid the potential dilution of the effective IL concentration from the paper samples, did not result in perceptible growth differences. Although the actual cause for the absence of growth impairment on BA-treated paper could not finally be assessed in this study, we hypothesize that the IL might be trapped inside the adsorbed cellulose fibers, rendering it poorly available for the fungus. Effective IL amounts released during fungal enzymatic cellulose degradation might thus be too low to cause notable growth impairment. However, further studies need to be conducted to examine this hypothesis.

Apart from IL characteristics like non-polar group size and the type of counter ion identified in this study as potential optimization targets for further studies on antifungal IL activity, we also recommend the consideration of adapted strategies to implement antifungals in mechanical paper restoration. For example, the antifungal drug Clotrimazole–which is structurally highly related to the tested ILs–displayed more than 1000 times higher activity compared to HC, the most potent IL tested in this study. However, Clotrimazole and related known antifungal compounds alone are not suitable for substitution of the cellulose-solubilizing ILs in the industrial process. Additional application designs should therefore also evaluate the possibility of applying highly potent antifungals as additional additives in the established organic solvent-based mechanical restoration processes to deliver them to the cellulosic material alongside the fiber repair processes.

Altogether, by demonstrating a general antifungal potential of ILs and identifying initial chemical parameters that appear to affect their activity, this study can serve as a guidance to focus ongoing research on the integration of biotic preservation substances into mechanical restoration processes for cellulosic documents–and hence contribute to biotic preservation of cultural heritage.

Ethics and consent

This article does not contain any studies with human participants or animals performed by any of the authors.

Supporting information

S1 Fig. Molar potency evaluation of ILs on remaining fungi via halo size analysis.

As seen for P. glabrum, HC had the strongest effect on all tested fungi, followed by BA, AC and BC. T. capillare (a) did show the strongest susceptibility to the tested ILs, followed by C. globosum (b), P. glabrum, A. versicolor (c) and P. chrysogenum (d).

(TIF)

Click here for additional data file. (206KB, tif)
S2 Fig. MIC and MGIC concentration analysis in the microtiter scale growth assay.

HC inhibited growth of C. globosum at 1% (v/v) and impaired growth of A. versicolor and P. chrysogenum at this concentration. BA was further shown to mildly impair growth of C. globosum at 1% (v/v). All ILs but BC were able to inhibit growth of all tested fungi at 10% concentration, stringently.

(TIF)

Click here for additional data file. (775.6KB, tif)

Acknowledgments

We want to acknowledge Dr. Klaus Langerbeins for strategic research and funding support. We furthermore want to thank Petra Arnold, Sabrina Paulus, Nadine Griesbacher and Claudia Strobel for excellent technical assistance.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was partly funded by the Nitrochemie Aschau GmbH (https://www.nitrochemie.com/en/nitrochemie_group/home.php). CLM is an employee of this company, supplied the ionic liquids used in this study and was involved in data interpretation and manuscript proof reading. Other than this, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The study design was nevertheless independent of any influence by the company. There was no additional external funding received for this study.

References

  • 1.Area MC, Cheradame H. Paper Aging and Degradation: Recent Findings and Research Methods. Bioresources. 2011;6(4):5307–37. [Google Scholar]
  • 2.Jiang ZC, Fan JJ, Budarin VL, Macquarrie DJ, Gao Y, Li TZ, et al. Mechanistic understanding of salt-assisted autocatalytic hydrolysis of cellulose. Sustain Energ Fuels. 2018;2(5):936–40. 10.1039/c8se00045j [DOI] [Google Scholar]
  • 3.Cedzová M, Gállová I, Katuščák S. Patents for Paper Deacidification. Restaurator2006. p. 35.
  • 4.Cedzová M, Gállová I, Katuščák S. Patents for Paper Deacidification. Restaurator International Journal for the Preservation of Library and Archival Material. 2008;27(1):35–45. 10.1515/REST.2006.35 [DOI] [Google Scholar]
  • 5.Maier CL. Papierverfestigung—neue Lösungswege [Dissertation]: Ludwigs-Maximilians-University Munich; 2019. 0001/UMC 26186
  • 6.Borrego S, Lavin P, Perdomo I, Gómez de Saravia S, Guiamet P. Determination of Indoor Air Quality in Archives and Biodeterioration of the Documentary Heritage. ISRN Microbiology. 2012;2012:10 10.5402/2012/680598 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Nyuksha JP. Biodeterioration and biostability of Library materials. Restaurator International Journal for the Preservation of Library and Archival Material. 1980;4(1):71–7. 10.1515/rest.1980.4.1.71 [DOI] [Google Scholar]
  • 8.Sequeira SO, Carvalho HPd, Mesquita N, Portugal A, Macedo MF. Fungal stains on paper: is what you see what you get? Conservar Património. 2019;31 10.14568/cp2018007 [DOI] [Google Scholar]
  • 9.Mesquita N, Portugal A, Videira S, Rodriguez-Echeverria S, Bandeira AML, Santos MJA, et al. Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra. Int Biodeter Biodegr. 2009;63(5):626–9. [Google Scholar]
  • 10.Skora J, Gutarowska B, Pielech-Przybylska K, Stepien L, Pietrzak K, Piotrowska M, et al. Assessment of microbiological contamination in the work environments of museums, archives and libraries. Aerobiologia (Bologna). 2015;31(3):389–401. 10.1007/s10453-015-9372-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zyska B. Fungi isolated from library materials: A review of the literature. Int Biodeter Biodegr. 1997;40(1):43–51. [Google Scholar]
  • 12.da Silva M, Moraes AML, Nishikawa MM, Gatti MJA, de Alencar MAV, Brandao LE, et al. Inactivation of fungi from deteriorated paper materials by radiation. Int Biodeter Biodegr. 2006;57(3):163–7. 10.1016/j.ibiod.2006.02.003 [DOI] [Google Scholar]
  • 13.Florian M-LE. Fungal Facts: Solving Fungal Problems on Heritage Collections in Museums and Archives. Journal of the American Institute for Conservation. 2004;43(1):114–6. [Google Scholar]
  • 14.Ricelli A, Fabbri AA, Fanelli C, Menicagli R, Samaritani S, Pini D, et al. Fungal growth on samples of paper: Inhibition by new antifungals. Restaurator. 1999;20(2):97–107. 10.1515/rest.1999.20.2.97 [DOI] [Google Scholar]
  • 15.Sanmartin P, DeAraujo A, Vasanthakumar A. Melding the Old with the New: Trends in Methods Used to Identify, Monitor, and Control Microorganisms on Cultural Heritage Materials. Microb Ecol. 2018;76(1):64–80. 10.1007/s00248-016-0770-4 [DOI] [PubMed] [Google Scholar]
  • 16.Sequeira SO, Cabrita EJ, Macedo MF. Fungal Biodeterioration of Paper: How are Paper and Book Conservators Dealing with it? An International Survey. Restaurator International Journal for the Preservation of Library and Archival Material. 2014;35(2):181–99. [Google Scholar]
  • 17.Szczepanowska H, Lovett CM. A study of the removal and prevention of fungal stains on paper. Journal of the American Institute for Conservation. 1992;31(2):147–60. [Google Scholar]
  • 18.Krupp A, Langerbeins K, Maier C, Ramin M, Sünkel K, inventors; Nitrochemie Aschau GmbH assignee. A method for processing materials comprising fibers, Luxembourg (2016).
  • 19.Ramin M, Langerbeins K, Maier C, SÜNKEL K, Krupp A, inventors; Nitrochemie Aschau GmbH assignee. Process for the treatment of materials containing fibers, Europe (2017).
  • 20.Araque JC, Hettige JJ, Margulis CJ. Modern Room Temperature Ionic Liquids, a Simple Guide to Understanding Their Structure and How It May Relate to Dynamics. J Phys Chem B. 2015;119(40):12727–40. 10.1021/acs.jpcb.5b05506 [DOI] [PubMed] [Google Scholar]
  • 21.Hayes R, Warr GG, Atkin R. Structure and Nanostructure in Ionic Liquids. Chemical Reviews. 2015;115(13):6357–426. 10.1021/cr500411q [DOI] [PubMed] [Google Scholar]
  • 22.Wilkes JS. A short history of ionic liquids—from molten salts to neoteric solvents. Green Chem. 2002;4(2):73–80. 10.1039/b110838g [DOI] [Google Scholar]
  • 23.Plempel M, Bartmann K, Buchel KH. Experimental study of a new orally effective broad-spectrum antimyocotic agent. Ger Med Mon. 1969;14(11):532–7. [PubMed] [Google Scholar]
  • 24.Godefroi EF, Heeres J, Van Cutsem J, Janssen PA. The preparation and antimycotic properties of derivatives of 1-phenethylimidazole. J Med Chem. 1969;12(5):784–91. 10.1021/jm00305a014 [DOI] [PubMed] [Google Scholar]
  • 25.Park N-H, Shin K-H, Kang MK. 34—Antifungal and Antiviral Agents In: Dowd FJ, Johnson BS, Mariotti AJ, editors. Pharmacology and Therapeutics for Dentistry; 10.1016/B978-0-323-39307-2.00034-5 Seventh Edition ed 2017. p. 488–503. [DOI] [Google Scholar]
  • 26.Shuter J. Antifungal and antiviral agents: A review. Cancer Invest. 1999;17(2):145–52. [PubMed] [Google Scholar]
  • 27.Sud IJ, Chou DL, Feingold DS. Effect of Free Fatty-Acids on Liposome Susceptibility to Imidazole Anti-Fungals. Antimicrob Agents Ch. 1979;16(5):660–3. 10.1128/Aac.16.5.660 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Vogel HJ. A Convenient Growth Medium for Neurospora crassa. Microbial Genetics Bulletin. 1956;13:42–7. [Google Scholar]
  • 29.White TJ, Bruns T, Lee S, Taylor J. Amplification and Direct Sequencing of Fungal Ribosomal Rna Genes for Phylogenetics In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols—A Guide to Methods and Applications. 1: Elsevier Inc.; 1989. p. 315–22. [Google Scholar]
  • 30.Ferreira A, Glass VB, Louise N. PCR from fungal spores after microwave treatment. Fungal Genetics Report. 1996;43:Aerticle 9. [Google Scholar]
  • 31.Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: a better web interface. Nucleic acids research. 2008;36(Web Server issue):W5–9. 10.1093/nar/gkn201 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Young D, Dollhofer V, Callaghan TM, Reitberger S, Lebuhn M, Benz JP. Isolation, identification and characterization of lignocellulolytic aerobic and anaerobic fungi in one- and two-phase biogas plants. Bioresource technology. 2018;268:470–9. 10.1016/j.biortech.2018.07.103 [DOI] [PubMed] [Google Scholar]
  • 33.Gäumann E. Familie Meanosporaceae Die Pilze—Grundzüge ihrer Entwicklungsgeschichte und Morphologie. 2: Springer Basel AG; 1964. p. 104–70. [Google Scholar]
  • 34.Rastegari AA. Molecular Mechanism of Cellulase Production Systems in Penicillium In: Gupta VK, Rodriguez-Couto S, editors. New and Future Developments in Microbial Biotechnology and Bioengineering; 10.1016/B978-0-444-63501-3.00008-9: Elsevier B.V.; 2017. p. 153–66. [DOI] [Google Scholar]
  • 35.Lakshmikant. Cellulose degradation and cellulase activity of five cellulolytic fungi. World J Microbiol Biotechnol. 1990;6(1):64–6. 10.1007/BF01225357 [DOI] [PubMed] [Google Scholar]
  • 36.Qaisar S, Zohra RR, Aman A, Qader SA. Enhanced production of cellulose degrading CMCase by newly isolated strain of Aspergillus versicolor. Carbohydr Polym. 2014;104:199–203. 10.1016/j.carbpol.2014.01.014 [DOI] [PubMed] [Google Scholar]
  • 37.Vale LHFD, Filho EXF, Miller R N.G., Ricart CAO, deSousa MV. Cellulase Systems in Trichoderma: An Overview In: Gupta VK, Schmoll M, Herrera-Estrella A, Upadhyay RS, Druzhinina I, Tuohy MG, editors. Biotechnology and Biology of Trichoderma; Elsevier B.V.; 2014. p. 229–44. [Google Scholar]
  • 38.Schönherr J. A mechanistic analysis of penetration of glyphosate salts across astomatous cuticular membranes. Pest Manag Sci. 2002;58(4):343–51. 10.1002/ps.462 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Binod Bihari Sahu

7 Aug 2019

PONE-D-19-17935

Preserving cultural heritage: Analyzing the antifungal potential of ionic liquids tested in paper restoration

PLOS ONE

Dear M.Sc. Schmitz,

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Binod Bihari Sahu, Ph.D.

Academic Editor

PLOS ONE

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This research was partly funded by the Nitrochemie Aschau GmbH (https://www.nitrochemie.com/en/nitrochemie_group/home.php).

Christian L. Maier is an employee of this company, supplied the ionic liquids used in this study and was involved in data interpretation and manuscript proof reading. The study design was nevertheless independent of any influence by the company.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The manuscript presented by Schmitz entitled " Preserving cultural heritage: Analyzing the antifungal

potential of ionic liquids tested in paper restoration " can be accepted after clarification of the following points:

1. The experiments are performed taking DMSO as the medium which itself shows zwiterionic properties and bear a appreciable value of polarity. So inorder to understand the effects of the planted Ionic liquids, other solvent mediums like ethanol, acetone, ethers may be done in presence of ionic liquids. Also blank tests in these solvents have to be done and reported with comparisons with DMSO.

2. The authors have presented a model of the spatial orientation of the ionic liquid, solvent on cellular fibres on paper in Fig.2. The logical design may be explained clearly.

Reviewer #2: Paper is good and very much applied aspects. It definitely, open a new area of research in the field of conservation work. But, personally, I feel if there is scope for modification, specially in discussion section.

**********

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Reviewer #1: Yes: Debayan Sarkar

Reviewer #2: Yes: Ramesh Sahani

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PLoS One. 2019 Sep 17;14(9):e0219650. doi: 10.1371/journal.pone.0219650.r002

Author response to Decision Letter 0

21 Aug 2019

To the editor:

We have made the amended formal changes and included required statements in the Funding and Competing Interest sections as requested (Editor Requests 1-3).

------------------

To the reviewers:

We would like to gratefully thank you for your kind appreciation of our work and for your critical review of our article "Preserving cultural heritage: Analyzing the antifungal potential of ionic liquids tested in paper restoration". We value your constructive remarks and would like to respond to them point-by-point in the following:

To reviewer #1:

"The manuscript presented by Schmitz entitled "Preserving cultural heritage: Analyzing the antifungal potential of ionic liquids tested in paper restoration" can be accepted after clarification of the following points:

1. The experiments are performed taking DMSO as the medium which itself shows zwitterionic properties and bear a appreciable value of polarity. So in order to understand the effects of the planted Ionic liquids, other solvent mediums like ethanol, acetone, ethers may be done in presence of ionic liquids. Also blank tests in these solvents have to be done and reported with comparisons with DMSO."

Answer: We highly appreciate your constructive suggestions. However, as the original process, in which our ILs are applied, involves initial solubilization and subsequent precipitation of cellulose onto printed document surfaces, we are tightly limited in terms of the solvents we can use. Ethanol and acetone, for example, do not facilitate solubilization of cellulose fibers and strongly impair legibility of the treated documents due solubilization of the ink. We would also like to clarify that DMSO was only used as a solvent for Clotrimazole and 1-Butyl-3-Methylimidazolium chloride (both solid at room temperature), while the other ionic liquids were applied as pure liquids (without the need for additional solvents during potency assessment in this study). In order to clarify the methodological sequence for the reader, we made some amendments in the methods section. Moreover, a blank test of the antifungal activity of DMSO had already been included in the initial antifungal activity screening (Figure 3 A, B). Overall, we therefore do not think that additional tests with further solvents would give relevant results that could actually be used in the process.

"2. The authors have presented a model of the spatial orientation of the ionic liquid, solvent on cellular fibers on paper in Fig.2. The logical design may be explained clearly."

Answer: We believe that the reviewer is actually referring to Figure 1 and – after reconsideration - agree with the reviewer that the initial illustration was partially misleading in terms of how the ionic liquid and DMSO entities were positioned. We have therefore now adapted the figure including its figure legend accordingly. We believe that the figure is now more accurately representing the process.

To reviewer #2:

Paper is good and very much applied aspects. It definitely open a new area of research in the field of conservation work. But, personally, I feel if there is scope for modification, especially in discussion section."

Answer: We have gladly modified the discussion section and hope to have improved it by doing so. Firstly, we have re-arranged the discussion paragraphs to better mirror the presentation sequence of the results. We believe that this allows a better orientation for the reader and hence could benefit the level of comprehensiveness.

We have rearranged the last bit of the first paragraph (i) to bring the sections discussing the correlation of IL potency and IL structure closer together in the text and (ii) to be able to discuss the long-term stability of the antifungal activity in the broader context of the paper-based application.

We furthermore included a statement in the second paragraph that points out the necessity for further studies on the contribution of non-polar group size and structure to the respective antifungal activity of an ionic liquid – as was done in the third paragraph as well.

Lastly, we extended the second-to-last paragraph. We aimed at putting stronger emphasis on the proposed future directions for research on biotic preservation implementation in paper restoration processes.

Additional minor modifications were made to improve overall readability and to clarify certain statements.

Attachment

Submitted filename: Response To Reviewers.docx

Click here for additional data file. (15.7KB, docx)

Decision Letter 1

Binod Bihari Sahu

27 Aug 2019

[EXSCINDED]

Preserving cultural heritage: Analyzing the antifungal potential of ionic liquids tested in paper restoration

PONE-D-19-17935R1

Dear Dr. Schmitz,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Binod Bihari Sahu, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The suggested corrections with details have been incorporated. The paper can be accepted for publication.

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Acceptance letter

Binod Bihari Sahu

9 Sep 2019

PONE-D-19-17935R1

Preserving cultural heritage: Analyzing the antifungal potential of ionic liquids tested in paper restoration

Dear Dr. Schmitz:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Binod Bihari Sahu

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Molar potency evaluation of ILs on remaining fungi via halo size analysis.

    As seen for P. glabrum, HC had the strongest effect on all tested fungi, followed by BA, AC and BC. T. capillare (a) did show the strongest susceptibility to the tested ILs, followed by C. globosum (b), P. glabrum, A. versicolor (c) and P. chrysogenum (d).

    (TIF)

    Click here for additional data file. (206KB, tif)
    S2 Fig. MIC and MGIC concentration analysis in the microtiter scale growth assay.

    HC inhibited growth of C. globosum at 1% (v/v) and impaired growth of A. versicolor and P. chrysogenum at this concentration. BA was further shown to mildly impair growth of C. globosum at 1% (v/v). All ILs but BC were able to inhibit growth of all tested fungi at 10% concentration, stringently.

    (TIF)

    Click here for additional data file. (775.6KB, tif)
    Attachment

    Submitted filename: Response To Reviewers.docx

    Click here for additional data file. (15.7KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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