# Biohealing through biocalcification by urolytic bacteria Bacillus subtilis ATCC 6633 on marble surfaces

## TL;DR

Ureolytic bacteria can heal marble surfaces by forming calcium carbonate, with live bacteria and calcium chloride promoting stable crystal formation.

## Contribution

Demonstrates how bacterial viability and calcium source influence biocalcification for sustainable marble restoration.

## Key findings

- Live cells and calcium chloride promote calcite and aragonite formation, while dead cells and calcium acetate favor vaterite.
- CO₂ pre-treatment enhances uniform CaCO₃ nucleation and reduces pore depth on marble surfaces.
- Biocalcification partially fills cracks and micropores, offering a sustainable method for marble conservation.

## Abstract

This study investigates the biocalcification potential of Bacillus subtilis ATCC 6633, a ureolytic bacterium, for the biohealing of marble surfaces through calcium carbonate (CaCO₃) precipitation. Comparative experiments were conducted using live and dead bacterial cells on CO₂-pre-treated and untreated marble samples, with calcium chloride and calcium acetate employed as calcium sources, to evaluate their effects on crystal polymorphism and surface modification. The results show that bacterial viability and calcium source jointly influence mineral phase formation, with live cells predominantly promoting the formation of stable calcite and aragonite, whereas dead cells and calcium acetate favor the formation of metastable vaterite. Microstructural and mineralogical analyses using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and atomic force microscopy (AFM) confirmed substantial CaCO₃ deposition on marble surfaces. AFM measurements indicated a reduction in maximum pore depth, defined as the vertical height difference between pore bottoms and the surrounding marble surface, from 35.00 ± 7.07 μm in control samples to 22.50 ± 8.20 μm in biocalcified samples, reflecting partial filling of pores and cracks. In addition, micropores (0.02–0.03 mm) were fully filled, while macropores (3–5 mm) were partially occluded by crystalline deposits. CO₂ pre-treatment enhanced surface carbon availability and promoted more uniform CaCO₃ nucleation, as supported by SEM-EDX and XRD analyses. Overall, these findings indicate that microbially induced carbonate precipitation (MICP), combined with appropriate surface preconditioning and calcium source selection, represents a potential and sustainable strategy for marble conservation and related bio-construction applications.

Microbial calcite seals microcracks, redefining marble conservation.

Calcium source dictates crystal fate: calcite, aragonite, or vaterite.

Sustainable biocalcification transforms marble restoration practices.