Diversity and Biomineralization Potential of the Epilithic Bacterial Communities Inhabiting the Oldest Public Stone Monument of Cluj-Napoca (Transylvania, Romania)

Abstract

In this study, we investigated the biomineralization potential and diversity of the epilithic bacterial communities dwelling on the limestone statue of Saint Donatus, the oldest public monument of Cluj-Napoca city (Transylvania region, NW Romania). Their spatial distribution together with phylogenetic and metabolic diversity, as well as their capacity to precipitate calcium carbonate was evaluated by combining molecular and phenotypic fingerprinting methods with X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron-microscopy analyses. The results of real-time quantitative PCR, molecular fingerprinting and community-level physiological profiling showed that diverse and abundant bacterial assemblages that differ in relation to their collection site colonized the statue. The cultivation and molecular identification procedures allowed the characterization of 79 bacterial isolates belonging to Proteobacteria (73.4%), Firmicutes (19%), and Actinobacteria (7.6%). Amongst them, the 22 strains identified as being capable of calcium carbonate precipitation were found to belong mostly to Bacillus and Pseudomonas genera. We found that bacteria acted as nucleation sites, inducing the formation of nanoscale aggregates that were shown to be principally composed of vaterite. Furthermore, we expanded the current knowledge on culturable diversity of carbonatogenic bacteria by providing evidence for biogenic vaterite/calcite formation mediated by: Pseudomonas synxantha, P. graminis, Brevibacterium iodinum, Streptomyces albidoflavus, and Stenotrophomonas chelatiphaga. Overall, this study highlights the need to evaluate the carbonatogenetic potential of all the bacterial communities present on stone artwork prior to designing an efficient conservation treatment based on biomineralization.

Calcium carbonates: induced biomineralization with controlled macromorphology

“…The occurrence of carbonate-forming bacteria has been investigated with respect to different environments (Andrei et al, 2017;Gray and Engels, 2013;Horath and Bachofen, 2009;Kang and Roh, 2016;Rusznyak et al, 2012). The process has generally been linked to changes induced in the direct microenvironment of the growing bacteria through metabolic features or to providing nucleation sites for crystallization in a supersaturated environment (Roberts et al, 2013;Seifan et al, 2017;Yan et al, 2017).…”

A Novel Approach to Isolation and Screening of Calcifying Bacteria for Biotechnological Applications

“…To our knowledge, Andrei et al [60] were the first to use Biolog EcoPlates to investigate the carbonatogenic potential of bacterial communities (dwelling a limestone statue in Romania), based on their ability to metabolize amino acids since this metabolization promotes the pH conditions necessary for the biomineralization process (by ammonia released in the process of oxidative deamination). Overall, Andrei et al study highlights the need to evaluate the carbonatogenic potential of the bacterial communities present on a stone artwork prior to designing an efficient conservation treatment based on resident calcifying bacteria.…”

A Novel Approach to Isolation and Screening of Calcifying Bacteria for Biotechnological Applications

“…To our knowledge, Andrei and co-workers [60] were the first to use Biolog EcoPlates to investigate the carbonatogenic potential of bacterial communities (dwelling a limestone statue in Romania), based on their ability to metabolize amino acids since this metabolization promotes the pH conditions necessary for the 9 biomineralization process (by ammonia released in the process of oxidative deamination). Overall, Andrei and co-workers study, highlights the need to evaluate the carbonatogenic potential of the bacterial communities present on a stone artwork prior to designing an efficient conservation treatment based on resident calcifying bacteria.…”

“…Observation of pure solid bacterial cultures under light microscopy, showed that 100% of the bacterial isolates were capable of forming crystalline CaCO3 on B4 agar plates at 20° and 28°C ( Table 5). This result confirms that: i) biomineralization leading to CaCO3 deposits is a quite common phenomenon in natural habitats [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]; ii) the rhizospheric soil is a good source for efficient isolation of calcifying bacteria as karst caves and secondary carbonate soils; iii) nutrient availability is a critical factor affecting microbial metabolism and precipitation activity [27]; iv) the efficiency of the Biolog EcoPlate system as a tool for estimating calcification potentiality of environmental bacterial communities [60]; v) the importance of bacterial calcification as a detoxification process (calcification by bacterial species may be an important phenotype for survival in high calcium environments as the studied soil - Table 1). Table 5 shows the calcification parameters of the bacterial isolates: number of days required to start precipitation of CaCO3 in B4 agar cultures at 4, 20, and 28°C, position on B4 agarized medium of the deposited crystals, amount and size of the crystals visually established by the microscope, presence of bioliths aggregates and mineralization ability in liquid B4 medium.…”