ABSTRACTThe influence of mineral substrate composition and structure on bacterial calcium carbonate productivity and polymorph selection was studied. Bacterial calcium carbonate precipitation occurred on calcitic (Iceland spar single crystals, marble, and porous limestone) and silicate (glass coverslips, porous sintered glass, and quartz sandstone) substrates following culturing in liquid medium (M-3P) inoculated with different types of bacteria (Myxococcus xanthus,Brevundimonas diminuta, and a carbonatogenic bacterial community isolated from porous calcarenite stone in a historical building) and direct application of sterile M-3P medium to limestone and sandstone with their own bacterial communities. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD), and 2-dimensional XRD (2D-XRD) analyses revealed that abundant highly oriented calcite crystals formed homoepitaxially on the calcitic substrates, irrespective of the bacterial type. Conversely, scattered spheroidal vaterite entombing bacterial cells formed on the silicate substrates. These results show that carbonate phase selection is not strain specific and that under equal culture conditions, the substrate type is the overruling factor for calcium carbonate polymorph selection. Furthermore, carbonate productivity is strongly dependent on the mineralogy of the substrate. Calcitic substrates offer a higher affinity for bacterial attachment than silicate substrates, thereby fostering bacterial growth and metabolic activity, resulting in higher production of calcium carbonate cement. Bacterial calcite grows coherently over the calcitic substrate and is therefore more chemically and mechanically stable than metastable vaterite, which formed incoherently on the silicate substrates. The implications of these results for technological applications of bacterial carbonatogenesis, including building stone conservation, are discussed.

The deterioration of the stone built and sculptural heritage has prompted the search and development of novel consolidation/protection treatments that can overcome the limitations of traditional ones. Attention has been drawn to bioconservation, particularly bacterial carbonatogenesis (i.e. bacterially induced calcium carbonate precipitation), as a new environmentally friendly effective conservation strategy, especially suitable for carbonate stones. Here, we study the effects of an in situ bacterial bioconsolidation treatment applied on porous limestone (calcarenite) in the sixteenth century San Jeronimo Monastery in Granada, Spain. The treatment consisted in the application of a nutritional solution (with and without Myxococcus xanthus inoculation) on decayed calcarenite stone blocks. The treatment promoted the development of heterotrophic bacteria able to induce carbonatogenesis. Both the consolidation effect of the treatment and the response of the culturable bacterial community present in the decayed stone were evaluated. A significant surface strengthening (consolidation) of the stone, without altering its surface appearance or inducing any detrimental side effect, was achieved upon application of the nutritional solution. The treatment efficacy was independent of the presence of M. xanthus (which is known as an effective carbonatogenic bacterium). The genetic diversity of 116 bacterial strains isolated from the stone, of which 113 strains showed carbonatogenic activity, was analysed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR) and 16S rRNA gene sequencing. The strains were distributed into 31 groups on the basis of their REP-PCR patterns, and a representative strain of each group was subjected to 16S rRNA gene sequencing. Analysis of these sequences showed that isolates belong to a wide variety of phylogenetic groups being closely related to species of 15 genera within the Proteobacteria, Firmicutes and the Actinobacteria. This study shows that the abundant carbonatogenic bacteria present in the decayed stone are able to effectively consolidate the degraded stone by producing new calcite (and vaterite) cement if an adequate nutritional solution is used. The implications of these results for the conservation of cultural heritage are discussed.

Enhanced salt weathering resulting from global warming and increasing environmental pollution is endangering the survival of stone monuments and artworks. To mitigate the effects of these deleterious processes, numerous conservation treatments have been applied that, however, show limited efficacy. Here we present a novel, environmentally friendly, bacterial self-inoculation approach for the conservation of stone, based on the isolation of an indigenous community of carbonatogenic bacteria from salt damaged stone, followed by their culture and re-application back onto the same stone. This method results in an effective consolidation and protection due to the formation of an abundant and exceptionally strong hybrid cement consisting of nanostructured bacterial CaCO3 and bacterially derived organics, and the passivating effect of bacterial exopolymeric substances (EPS) covering the substrate. The fact that the isolated and identified bacterial community is common to many stone artworks may enable worldwide application of this novel conservation methodology.

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Ba proxies have been broadly used to reconstruct past oceanic export production. However, the precise mechanisms underlying barite precipitation in undersaturated seawater are not known. The link between bacterial production and particulate Ba in the ocean suggests that bacteria may play a role. Here we show that under experimental conditions marine bacterial biofilms, particularly extracellular polymeric substances (EPS), are capable of bioaccumulating Ba, providing adequate conditions for barite precipitation. An amorphous P-rich phase is formed at the initial stages of Ba bioaccumulation, which evolves into barite crystals. This supports that in high productivity regions where large amounts of organic matter are subjected to bacterial degradation, the abundant EPS would serve to bind the necessary Ba and form nucleation sites leading to barite precipitation. This also provides new insights into barite precipitation and opens an exciting field to explore the role of EPS in mineral precipitation in the ocean.