Natural Inhibitory Treatment of Fungi-Induced Deterioration of Carbonate and Cellulosic Ancient Monuments: Isolation, Identification and Simulation of Biogenic Deterioration

Abstract

Fungi play a significant role in the deterioration of various types of monuments. Therefore, the protection of ancient monuments from fungal attacks is an important goal that must attract the attention of researchers worldwide. A total of 69 fungal isolates were recovered from 22 deteriorated objects compromising paper, textiles, wood, and stone in the National Museum of Egyptian Civilization (NMEC) storeroom, Cairo, Egypt. The isolates were identified as 12 different species categorized into three different genera, namely, Aspergillus (9 species), Penicillium (2 species) and Trichoderma (1 species). Among them, Aspergillus fumigatus was the most prevalent species. Three essential oils were assessed for antifungal activity and compared with the antifungal effects of five synthetic microcides to identify a natural inhibitory treatment. Thyme oil and sodium azide were found to be the most active growth inhibitors, with minimum inhibitory concentrations (MICs) of 625 and 100 ppm, with inhibition zone diameters of 19.0 ± 0.70 - 23.76 ± 1.15 and 13.30 ± 0.35 - 19.66 ± 0.54 mm, respectively. An in vitro simulation of the biodeterioration process was conducted using spores of the A. fumigatus strain NMEC-PSTW.1 on model cubes made of paper, textile, wood, and stone materials. The changes in the characteristics of the artificially deteriorated materials were analyzed using environmental scanning electron microscopy/energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. The results revealed changes in the morphology, physical properties, and chemical composition induced by A. fumigatus NMEC-PSTW.1. Overall, thyme oil is recommended as a natural inhibitor to protect carbonate and cellulosic monuments in NMEC against fungal attack.

Keywords: Ancient monuments; Aspergillus fumigatus; NMEC; biogenic deterioration; thyme oil.

Fig. 1. Distribution of fungal isolates among the examined archaeological objects.

Fig. 2. The identified fungal species recovered from the deteriorated archaeological objects under study.

(A) A. fumigatus, (B) A. flavus, (C) A. oryzae, (D) A. flavipes, (E) A. japonisas, (F) A. parasiticus, (G) A. terreus, (H) A. aureus, (I) A. unguis, (J) P. simplicissium, (K) P. canescens, and (L) T. harzianum. In this figure, the right photograph (I) shows the macroscopic characteristics of the culture features, while the left photograph (II) shows the micromorphology of the vegetative and reproductive fungal structures.

Fig. 3. The phylogenetic tree of the ITS sequences of the twelve deteriorating fungal species and corresponding reference sequences retrieved from NCBI was inferred using the neighbor-joining method and Kimura 2-parameter phylogenetic analysis in MEGA 11.0 software.

Fig. 4. Phylogenetic tree of the twelve deteriorating fungal species using NCBI analysis of the β-tub and TEF- 1α regions inferred using the neighbour-joining method and Kimura's 2-parameter phylogenetic analysis in MEGA 11.0 software.

Fig. 5. Biodeterioration signs caused by spores of A. fumigatus NMEC-PSTW.1 for model cubes of different materials:

(I) paper, (II) stone, (III) textile, and (IV) wood. In this figure, the left photograph (A) is the control (noncolonized), and the right (B) is the treated (colonized) cube.

Fig. 6. ESEM micrographs of stone cubes.

(A) noncolonized stone (control), (B) stone inoculated with A. fumigatus NMEC-PSTW.1; (I) magnification scale 100× (scale bar =1 mm); (II) magnification scale 800× (scale bar = 100 μm). In this figure, the signs of deterioration are marked by yellow arrows (for surface cracking), blue arrows (for the fungal hyphae within limestone granules), and red arrows (for the fungal biofilm and exopolymeric substances).

Fig. 7. EDXS microanalysis of the stone elemental content; (A) control, (B) treatment (deteriorated) cubes.