Deterioration-Associated Microbiome of Stone Monuments: Structure, Variation, and Assembly

Abstract

Research on the microbial communities that colonize stone monuments may provide a new understanding of stone biodeterioration and microbe-induced carbonate precipitation. This work investigated the seasonal variation of microbial communities in 2016 and 2017, as well as its effects on stone monuments. We determined the bacterial and fungal compositions of 12 samples from four well-separated geographic locations by using 16S rRNA and internal transcribed spacer gene amplicon sequencing. Cyanobacteria and Ascomycota were the predominant bacterial and fungal phyla, respectively, and differences in species abundance among our 12 samples and 2 years showed no consistent temporal or spatial trends. Alpha diversity, estimated by Shannon and Simpson indices, revealed that an increase or decrease in bacterial diversity corresponded to a decrease or increase in the fungal community from 2016 to 2017. Large-scale association analysis identified potential bacteria and fungi correlated with stone deterioration. Functional prediction revealed specific pathways and microbiota associated with stone deterioration. Moreover, a culture-dependent technique was used to identify microbial isolates involved in biodeterioration and carbonatogenesis; 64% of 85 bacterial isolates caused precipitation of carbonates in biomineralization assays. Imaging techniques including scanning electron microscopy with energy-dispersive spectroscopy, X-ray diffraction, and fluorescence imaging identified CaCO₃ crystals as calcite and vaterite. Although CaCO₃ precipitation induced by bacteria often has esthetically deleterious impacts on stone monuments, this process may potentially serve as a novel, environmentally friendly bacterial self-inoculation approach to the conservation of stone.IMPORTANCE Comprehensive analyses of the microbiomes associated with the deterioration of stone monuments may contribute to the understanding of mechanisms of deterioration, as well as to the identification of potentially beneficial or undesirable microbial communities and their genomic pathways. In our study, we demonstrated that Cyanobacteria was the predominant bacterial phylum and exhibited an increase from 2016 to 2017, while Proteobacteria showed a decreasing trend. Apart from esthetic deterioration caused by cyanobacteria and fungi, white plaque, which is composed mainly of CaCO₃ and is probably induced by Crossiella and Cyanobacteria, was also considered to be another threat to stone monuments. We showed that there was no significant correlation between microbial population variation and geographic location. Specific functional genes and pathways were also enriched in particular bacterial species. The CaCO₃ precipitation induced by an indigenous community of carbonatogenic bacteria also provides a self-inoculation approach for the conservation of stone.

Keywords: biomineralization; cyanobacteria; fluorescent imaging; microbiome; stone monuments.

FIG 1

SEM images of microbial communities on different stone monuments. (A) Lichens and cyanobacterial cells on a stone surface. (B) Extensive fungal hyphal aggregates associated with diatom cells. (C) A porous structure is likely caused by microbial deterioration in QT1. (D) FL3 was increasingly colonized by actinomycete hyphae. (E, F) Signs of microbial colonization and spheroidal or hemispheroidal calcium carbonate precipitation and EPS. (G to I) Samples FL3 and QX3 were mostly composed of calcium carbonate, while calcium was absent from QX1 tested by EDS analysis.

FIG 2

Distribution patterns of bacterial (a) and fungal (b) taxa in 2016 and 2017.

FIG 3

Co-occurrence network of significantly interacting bacterial families (a) and genera (c and d). Positively and negatively interacting bacterial families and genera are connected by green and red lines, respectively. The thickness of each line is proportional to the significance of the interaction (q value), and the size of the circle is proportional to the average relative abundance of bacterial families and genera. Panel b represents the bacterial families that were assigned to betweenness centrality and closeness centrality.

FIG 4

KEGG pathways enriched in epilithic bacterial communities. The relative abundances of pathways were compared among 12 samples in both 2016 and 2017.

FIG 5

Guild assignments of 138 identified OTUs (a) and variation in fungal community composition (b, c). OTUs and sequences not assigned to guilds were placed in the unassigned group.

FIG 6

Bacterial and fungal strains were isolated from 12 samples.

FIG 7

Relationship between bacterial isolates and CaCO₃ precipitation. (A, B) Spheroidal CaCO₃ crystals (black arrows) formed on the surface of B-4 agar incubated in the presence of B. cereus (FL4-T2) after 10 days. (C to F) Relationships among bacterial cells (black arrows), CaCO₃ precipitation (red arrows), and EPS (blue arrows).

FIG 8

Observation of calcein incorporation into CaCO₃ precipitate with a fluorescence microscope (blue light excitation, green fluorescence emission). CaCO₃ crystals imaged by calcein staining appear green. (A) Abiotic CaCO₃ crystals observed with an optical microscope as an unstained control. (B) Fluorescent image of the position shown in panel A after staining with calcein. (C) Optical microscopic image of bacterial isolates after incubation on B-4 agar for 10 days. The white arrow indicates CaCO₃ crystals. The white ellipse indicates bacterial isolates. (D) Fluorescent image of the position shown in panel C after staining with calcein. Most CaCO₃ crystals were stained with calcein (green), although calcein cannot be incorporated into some bacterial cells (white ellipse). (E, F, H) Local observation of CaCO₃ precipitation before and after staining with calcein. (G) Fluorescence microscopic image of bacterial isolates incubated on modified B-4 agar (without Ca²⁺) for 10 days without CaCO₃ precipitate formation.

FIG 9

PCoA of bacterial (a) and fungal (b) communities.