Conservation of ornamental stone by Myxococcus xanthus-induced carbonate biomineralization

Abstract

Increasing environmental pollution in urban areas has been endangering the survival of carbonate stones in monuments and statuary for many decades. Numerous conservation treatments have been applied for the protection and consolidation of these works of art. Most of them, however, either release dangerous gases during curing or show very little efficacy. Bacterially induced carbonate mineralization has been proposed as a novel and environmentally friendly strategy for the conservation of deteriorated ornamental stone. However, the method appeared to display insufficient consolidation and plugging of pores. Here we report that Myxococcus xanthus-induced calcium carbonate precipitation efficiently protects and consolidates porous ornamental limestone. The newly formed carbonate cements calcite grains by depositing on the walls of the pores without plugging them. Sonication tests demonstrate that these new carbonate crystals are strongly attached to the substratum, mostly due to epitaxial growth on preexisting calcite grains. The new crystals are more stress resistant than the calcite grains of the original stone because they are organic-inorganic composites. Variations in the phosphate concentrations of the culture medium lead to changes in local pH and bacterial productivity. These affect the structure of the new cement and the type of precipitated CaCO(3) polymorph (vaterite or calcite). The manipulation of culture medium composition creates new ways of controlling bacterial biomineralization that in the future could be applied to the conservation of ornamental stone.

FIG. 1.

Schematic representation of the biomineralization experiments using M. xanthus: (a) Transmission electron photomicrographs of the bacterium. (b) Inoculum culture was incubated for 48 h at 28°C (shaking conditions). Incubation of the bacterial culture was carried out in test tubes (c) and Erlenmeyer flasks (d) containing calcarenite slabs immersed in M-3 or M-3P liquid media and incubated under shaking and stationary conditions, respectively.

FIG. 2.

Weight increase (ΔWt) versus time of small (a) and large (b) calcarenite slabs following bacterially induced carbonate mineralization. Error bars, standard deviations.

FIG. 3.

pH variations versus time for M-3 and M-3P liquid culture media (controls refer to uninoculated media with calcarenite slabs). Error bars, standard deviations.

FIG. 4.

XRD patterns of calcarenite slabs subjected to bacterially induced carbonate mineralization tests (abbreviations: Cc, calcite; Vat, vaterite) (a) and solid residua formed in uninoculated culture media (both M-3 and M-3P) (b). Arrows indicate hydroxylapatite diffraction peaks. Goniometer calibration was performed using a silicon standard. Bragg peak identification was performed using JPDF files. Cu Kα radiation, λ = 1.5406 Å.

FIG. 5.

SEM photomicrographs of small samples cultured under shaking conditions. (a) Representative image of control calcarenite. Sparitic calcite (cc) crystals are indicated by arrows (note that microtextural features of sterilized calcarenite controls and of controls immersed in uninoculated M-3 or M-3P media are identical). (b) Stone sample subjected to biomineralization in the M-3 medium, showing calcified bacterial cells (cbc) and needle-like vaterite (vt) crystals (inset shows a magnified view of the upper-right corner). (c) Newly formed calcite rhombohedra (cc′) on calcarenite cultured in the M-3P medium (bacterial casts [bc] are also indicated). (d) Newly formed calcite crystals developing epitaxially (cc′e) on preexisting calcite crystals and showing preferred crystallographic orientation (bacterial casts are also indicated).

FIG. 6.

SEM photomicrographs of large samples cultured under nonshaking conditions. (a) Calcified bacterial cells (cbc) covering the pore walls of samples cultivated in the M-3 medium (after 30 days). (b) Detail of calcified bacteria linked by needle-like vaterite (vt) crystals in samples cultured in the M-3P medium (after 10 days). (c) Sample cultured in the M-3P medium (after 30 days) showing incipient development of an EPS film (the arrow indicates cracks in the film; note that no pore plugging occurred). (d) Massive needle-like vaterite crystals blanketing stone pores following 30 days of culture in the M-3P medium. Calcified bacterial cells are also observed covering the pore walls.

FIG. 7.

SEM photomicrographs of samples subjected to sonication. (a) Control (i.e., calcarenite slab not submitted to biomineralization but sonicated) showing spalling and fissuring of calcite crystals along rhombohedral {104} cleavage planes (cc_{104}). (b) Calcified bacteria (cbc) blanketing stone samples cultured in the M-3P medium. (c) Newly formed calcite (cc′) rombohedra blanketing the calcarenite. (d) Detail of a calcarenite showing a partially removed EPS film (EPS) which covered the newly formed calcite cement (cc′).

FIG. 8.

Calcarenite slab weight loss (ΔWt) versus sonication time. One set of biomineralized samples (cultured in inoculated M-3 or M-3P media for 30 days) was sonicated, and the other was not (i.e., was only immersed in deionized water). Two control sets were tested (i.e., calcarenite slabs not subjected to biomineralization): calcarenite slabs (i) not sonicated (control nonsonicated) and (ii) sonicated (control sonicated). Error bars, standard deviations.

FIG. 9.

Representative MIP plots showing both the cumulative intrusion curves (i.e., porosity) and pore size distribution curves [i.e., log of differential intrusion, or log(dv/dr), versus r, where v is the intruded volume and r is the pore radius] for fresh (solid line) and biomineralized (dotted line) stone samples.